Plant Physiology(2)

Biological and Medical Physics, Biomedical Engineering

Maria Duca

Plant Physiology

Plant Physiology

BIOLOGICAL AND MEDICAL PHYSICS, BIOMEDICAL ENGINEERING The fields of biological and medical physics and biomedical engineering are broad, multidisciplinary and dynamic. They lie at the crossroads of frontier research in physics, biology, chemistry, and medicine. The Biological and Medical Physics, Biomedical Engineering Series is intended to be comprehensive, covering a broad range of topics important to the study of the physical, chemical and biological sciences. Its goal is to provide scientists and engineers with textbooks, monographs, and reference works to address the growing need for information. Books in the series emphasize established and emergent areas of science including molecular, membrane, and mathematical biophysics; photosynthetic energy harvesting and conversion; information processing; physical principles of genetics; sensory communications; automata networks, neural networks, and cellular automata. Equally important will be coverage of applied aspects of biological and medical physics and biomedical engineering such as molecular electronic components and devices, biosensors, medicine, imaging, physical principles of renewable energy production, advanced prostheses, and environmental control and engineering.

Editor-in-Chief: Elias Greenbaum, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA

Editorial Board: Masuo Aizawa, Department of Bioengineering, Tokyo Institute of Technology, Yokohama, Japan

Judith Herzfeld, Department of Chemistry, Brandeis University, Waltham, Massachusetts, USA

Olaf S. Andersen, Department of Physiology, Biophysics and Molecular Medicine, Cornell University, New York, USA

Mark S. Humayun, Doheny Eye Institute, Los Angeles, California, USA

Robert H. Austin, Department of Physics, Princeton University, Princeton, New Jersey, USA James Barber, Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, England Howard C. Berg, Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA Victor Bloomfield, Department of Biochemistry, University of Minnesota, St. Paul, Minnesota, USA Robert Callender, Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, USA Britton Chance, University of Pennsylvania Department of Biochemistry/Biophysics Philadelphia, USA Steven Chu, Lawrence Berkeley National Laboratory, Berkeley, California, USA Louis J. DeFelice, Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, USA Johann Deisenhofer, Howard Hughes Medical Institute, The University of Texas, Dallas, Texas, USA George Feher, Department of Physics, University of California, San Diego, La Jolla, California, USA Hans Frauenfelder, Los Alamos National Laboratory, Los Alamos, New Mexico, USA Ivar Giaever, Rensselaer Polytechnic Institute, Troy, NewYork, USA Sol M. Gruner, Cornell University, Ithaca, New York, USA

Pierre Joliot, Institute de Biologie Physico-Chimique, Fondation Edmond de Rothschild, Paris, France Lajos Keszthelyi, Institute of Biophysics, Hungarian Academy of Sciences, Szeged, Hungary Robert S. Knox, Department of Physics and Astronomy, University of Rochester, Rochester, New York, USA Aaron Lewis, Department of Applied Physics, Hebrew University, Jerusalem, Israel Stuart M. Lindsay, Department of Physics and Astronomy, Arizona State University, Tempe, Arizona, USA David Mauzerall, Rockefeller University, New York, New York, USA Eugenie V. Mielczarek, Department of Physics and Astronomy, George Mason University, Fairfax, Virginia, USA Markolf Niemz, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany V. Adrian Parsegian, Physical Science Laboratory, National Institutes of Health, Bethesda, Maryland, USA Linda S. Powers, University of Arizona, Tucson, Arizona, USA Earl W. Prohofsky, Department of Physics, Purdue University, West Lafayette, Indiana, USA Andrew Rubin, Department of Biophysics, Moscow State University, Moscow, Russia Michael Seibert, National Renewable Energy Laboratory, Golden, Colorado, USA David Thomas, Department of Biochemistry, University of Minnesota Medical School, Minneapolis, Minnesota, USA

More information about this series at http://www.springer.com/series/3740

Maria Duca

Plant Physiology

123

Maria Duca University of Academy of Sciences of Moldova Chişinău Moldova

ISSN 1618-7210 ISSN 2197-5647 (electronic) Biological and Medical Physics, Biomedical Engineering ISBN 978-3-319-17908-7 ISBN 978-3-319-17909-4 (eBook) DOI 10.1007/978-3-319-17909-4 Library of Congress Control Number: 2015939679 Springer Cham Heidelberg New York Dordrecht London © Springer International Publishing Switzerland 2015 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. Printed on acid-free paper Springer International Publishing AG Switzerland is part of Springer Science+Business Media (www.springer.com)

Preface

The past decades came with tremendous advances in understanding molecular systems that lie at the core of life itself, a fact which has revolutionized biological research and the field of plant physiology was not an exception. Moreover, with the current advent of high throughput technologies in genomics and proteomics the potential appears to reveal the most subtle details regarding the molecular actors and the processes in which they are involved. But for being able to interpret and make use of such complex data, to understand its place and significance in the global context of plant metabolism, one must first hold basic knowledge of the key processes in the life of the plants, integrated across several dimensions like structure, function, ecology, etc. Plant physiology can offer such an integrated view. The subject of plant physiology is highly interdisciplinary and builds upon the knowledge derived from fields like botany, zoology, plant morphology and anatomy, cytology, biochemistry, molecular biology, etc. While at the theoretical level one of the priorities is to integrate the information from these scientific areas for a most complete understanding of the processes undergoing in living system, at the practical level this field comes with abundant experimental knowledge and wellestablished practices inherited from previous decades that allow to manipulate crop species in the desired manner, even if the theoretical aspects are not always completely elucidated. The course, presented by this book, offers the possibility to enter into the essence of the most important phenomena of the living matter—photosynthesis, respiration, growth and development, etc. By being conceived in agreement with the requirements of modern biology, Plant Physiology offers a perspective over the instruments and methods which allow the manipulation of the vegetal organism and which lie at the foundation of biotechnology as we know it today. The present book is not one that reflects only the principles and fundamental directions of plant physiology by using the scientific literature passed through the prism of own reflections, but also includes results of the personal research summarizing a big volume of experimental data.

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The presented content adheres to the principle of applicability of the provided knowledge which means that theoretical topics are accompanied by real examples of their relevance from agriculture, plant breeding, etc. A special place is left for graphical illustrations, diagrams, pictures, which occupy a significant proportion of the content and are meant to facilitate the process of assimilating the information. The author wants to thank the university professor, habilitated doctor A.I. Derendovschi for the detailed analysis of the content of the book and for the useful and constructive suggestions. I am grateful and want to thank everyone who made a contribution to the appearance of this book—PhDs in Biology Angela Port, Ana Căpăţână, Aliona Glijin, Ana Bârsan, Elena Savca, Alexei Levitchi, Victor Lupascu, Ph.D. students Lucia Ciobanu and all other students who helped me conceive this book. I would like to thank Prof. V. Ciobanu, Prof. V. Reva, PhDs Elena Muraru, Tatiana Homenco, Otilia Dandara for the important suggestions regarding the undertaken approach and the full and complex support offered in the process of preparing and editing this book. For the help provided in obtaining and consulting the most up to date scientific literature, I would like to thank my colleagues from the University of California, Riverside (USA)—Professors Isgouhi Kaloshen, Carol Lovatt, Seymour Van Gundy. I would also like to express special gratitude to my family for the patience and understanding that they showed all these years. Chişinău

Maria Duca

Contents

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Introduction to the Educational Course of Plant Physiology 1.1 The Definition and Scope of Plant Physiology . . . . . . . 1.2 Purposes of Plant Physiology as a Science . . . . . . . . . . 1.3 Research Methods Used by Plant Physiology . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Plant Cell Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 The Cell as a Structural, Morphological, Functional Unit of Living Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Structural Organization, Chemical Composition and Function of the Cell Wall . . . . . . . . . . . . . . . . . . . . . 2.3 Structure and Ultrastructure of Cell Protoplasm . . . . . . . . . 2.4 Structure and Function of Biological Membranes . . . . . . . . 2.5 Exchange of Substances Between the Cell and the Medium . 2.5.1 Ion Flow into the Cell . . . . . . . . . . . . . . . . . . . . . 2.5.2 Water Flow into the Cell . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Water Regime . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Role of Water in Plants . . . . . . . . . . . . . . . . . . . . . . . 3.2 Water Content and State in Plants . . . . . . . . . . . . . . . . 3.3 Forms of Water in the Soil. Accessible and Inaccessible Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 The Root System as a Specialized Organ for Water Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 The Influence of External Factors on Water Absorption Intensity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 Water Elimination. Physiological Importance of Plant Transpiration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6.1 Indices of Transpiration . . . . . . . . . . . . . . . . . 3.7 Structure of the Leaf as an Organ of Transpiration . . . .

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Stomatal and Cuticular Transpiration . . . 3.8.1 Stomatal Transpiration. . . . . . . 3.8.2 Cuticular Transpiration . . . . . . 3.9 Water Absorption Mechanism and Ways of Its Circulation in Plants . . . . . . . . . . 3.9.1 Water Transport in Plants . . . . 3.10 Ecology of the Water Regime in Plants . References. . . . . . . . . . . . . . . . . . . . . . . . . . 4

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Photosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Importance of Photosynthesis and the Global Role of Green Plants. . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 The Leaf as a Specialized Photosynthesis Organ . . . 4.3 The Structure, Chemical Composition, Function and Origin of Chloroplasts . . . . . . . . . . . . . . . . . . 4.4 Photosynthesis Pigments . . . . . . . . . . . . . . . . . . . 4.5 Photosynthesis Energetics . . . . . . . . . . . . . . . . . . 4.6 Photosynthesis Mechanism. . . . . . . . . . . . . . . . . . 4.6.1 Light Phase of Photosynthesis . . . . . . . . . 4.6.2 The Dark Phase of Photosynthesis . . . . . . 4.7 Photorespiration . . . . . . . . . . . . . . . . . . . . . . . . . 4.8 Endogenous Regulatory Elements of Photosynthesis 4.9 Ecology of Photosynthesis . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Respiration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General Notions of Respiration. . . . . . . . . . . . . . . . . . . Respiratory Enzymes. . . . . . . . . . . . . . . . . . . . . . . . . . A.N. Bach’s and V.I. Palladin’s Theories . . . . . . . . . . . . Respiration Mechanism . . . . . . . . . . . . . . . . . . . . . . . . 5.4.1 Genetic Link Between Respiration and Fermentation . . . . . . . . . . . . . . . . . . . . . . 5.4.2 Glycolysis—The Anaerobic Phase of Respiration 5.4.3 Krebs Cycle (Tricarboxylic Acid Cycle) . . . . . . 5.4.4 The Electron Transport Chain and the Energetic Outcome of Aerobic Respiration . . . . . . . . . . . . 5.5 Different Types of Respiratory Substrate Oxidation . . . . . 5.6 Ecology of Respiration . . . . . . . . . . . . . . . . . . . . . . . . 5.7 Regulation and Self-regulation of the Respiration Process References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Mineral Nutrition of Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1 Importance of Mineral Elements in Plant Nutrition . . . . . . . . . 6.2 Chemical Composition of the Ash. . . . . . . . . . . . . . . . . . . . .

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Plant 5.1 5.2 5.3 5.4

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Methods of Mineral Nutrition Research . . . . . . . . . . . . . . . The Root System as an Organ for Absorption and Transport of Mineral Elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5 Physiological Role of Macroelements . . . . . . . . . . . . . . . . 6.5.1 Absorption, Transport and Metabolism of Nitrogen . 6.5.2 Absorption, Transport and Metabolism of Sulfur. . . 6.5.3 Absorption, Transport and Metabolism of Phosphorus . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5.4 The Physiological Role of Other Macroelements. . . 6.6 Physiological Role of Microelements. . . . . . . . . . . . . . . . . 6.7 Mechanism of Absorption and Transport of Ions in Plants . . 6.7.1 Mineral Element Absorption. . . . . . . . . . . . . . . . . 6.7.2 Mineral Element Transport. . . . . . . . . . . . . . . . . . 6.8 Soil as a Substrate for Plant Nutrition . . . . . . . . . . . . . . . . 6.9 Influence of Various Environmental Factors on Mineral Nutrition in Plants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

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Plant Growth and Development. . . . . . . . . . . . . . . . . . . . . 7.1 The Concept of Plant Growth and Development . . . . . . 7.1.1 Dormancy in Plants (Repose) . . . . . . . . . . . . . 7.2 Types of Plant Growth . . . . . . . . . . . . . . . . . . . . . . . 7.3 Phases of Cell Growth and Development . . . . . . . . . . . 7.4 Phases of Plant Growth and Development . . . . . . . . . . 7.5 Genetic Aspects of Plant Morphogenesis . . . . . . . . . . . 7.6 Endogenous Factors of Plant Growth and Development . 7.6.1 Auxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6.2 Gibberellins . . . . . . . . . . . . . . . . . . . . . . . . . 7.6.3 Cytokinins . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6.4 Abscisic Acid. . . . . . . . . . . . . . . . . . . . . . . . 7.6.5 Ethylene . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.7 Photoperiodism and Yarovization . . . . . . . . . . . . . . . . 7.8 The Influence of Exogenous Factors on Plant Growth and Development . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.9 Plant Growth Movements—Tropism and Nasties . . . . . 7.10 Self-Regulation of Plant Growth and Development . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Plant 8.1 8.2 8.3 8.4

Biorhythms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Classification and Mechanisms of Biological Rhythms Biological Rhythms in Plants . . . . . . . . . . . . . . . . . . Circadian Rhythms in Plants . . . . . . . . . . . . . . . . . . The Molecular Mechanism of the Circadian Clock . . . 8.4.1 Environmental Signals Involved . . . . . . . . . .

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8.4.2 8.4.3 8.4.4 8.4.5 References. .

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Elimination of Substances in Plants . . . . . . . . . . . . . . . . 9.1 Classification of the Types of Substance Elimination . 9.2 Excretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3 Secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.1 Lignin, Cutin and Wax Secretion . . . . . . . . 9.3.2 Nectariferous Glands and Nectar Secretion . . 9.3.3 Terpenoid Secreting Structures . . . . . . . . . . 9.4 Secretory Processes in Insectivorous Plants . . . . . . . 9.5 Water Elimination in Plants . . . . . . . . . . . . . . . . . . 9.6 Ecological Role of Substance Elimination . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Temperature . . . . . . . . . . . . . . . . . . . . . Light . . . . . . . . . . . . . . . . . . . . . . . . . . The Molecular Targets of Light Signaling Rhythmic Regulation of Light Signaling . ...............................

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10 Physiology of Plant Resistance to Unfavorable Environmental Factors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1 The Concepts of Resistance and Adaptation . . . . . . . . . . . 10.2 Unfavorable Factors of the Winter-Spring Period . . . . . . . 10.3 Plant Resistance to Cold and Frost . . . . . . . . . . . . . . . . . 10.4 Plant Resistance to Drought . . . . . . . . . . . . . . . . . . . . . . 10.4.1 Physiological Basis of Irrigation . . . . . . . . . . . . . 10.5 Plant Resistance to Saltiness. . . . . . . . . . . . . . . . . . . . . . 10.6 Regulation of Physiological Processes in Halophyte Plants. 10.7 Plant Resistance to Environmental Pollution . . . . . . . . . . . 10.7.1 Resistance to Heavy Metals . . . . . . . . . . . . . . . . 10.7.2 Resistance to Radiation . . . . . . . . . . . . . . . . . . . 10.7.3 Resistance to Gases. . . . . . . . . . . . . . . . . . . . . . 10.8 Metabolism of Pollutants in Plants . . . . . . . . . . . . . . . . . 10.9 Biochemical Mechanism of Pollutant Transformation in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.10 Self-regulation of Plant Growth and Development in Unfavorable Environmental Conditions . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 1

Introduction to the Educational Course of Plant Physiology

Abstract Plant physiology is a science that studies vegetal organisms in ontogenetic dynamics—the diversity, the laws and the mechanisms of physiological and biochemical processes, their biological significance, their dependence on environmental factors. Traditionally, it was based on two directions: anatomical/morphological and physiological, but this division is somewhat relative, because structure and function have evolved in parallel and cannot be studied separately. This interdisciplinary research field focuses on a series of compartments like: plant cell physiology; water regime; photosynthesis; mineral nutrition; respiration; growth and development; resistance to unfavorable factors; phenomena of self-regulation at all the levels of organization (including at the organism level by means of interacting centers of dominance). While as a theoretical science plant physiology tries to obtain an integrated, detailed picture of the molecular, biochemical, physiological, morphogenetic processes going on in the living plant and the interconnection between these, at the applicative level its aim is to be able to direct vital processes in the life cycle of a plant like growth, development, metabolism, photosynthesis, nutrition, resistance, fructification in order to control the vitality or yield of the crop species and to maximize economic benefits. Classical research in plant physiology is carried out in the field, in vegetation pots, solariums, greenhouses, phytotrons, laboratories. Experiments make use of a diverse range of methods like: imaging technologies (optical and electronic microscopy), centrifugation, chemical analysis, chromatography, radioactive labeling, gel filtration, electrophoresis, X-ray analysis, in vitro culture, but also in silico mathematical modeling to predict the behavior of various systems and the output parameters.

© Springer International Publishing Switzerland 2015 M. Duca, Plant Physiology, Biological and Medical Physics, Biomedical Engineering, DOI 10.1007/978-3-319-17909-4_1

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Historical Background 1727—St. Hales identifies the pathways of water, mineral salts and organic substances circulation. 1771—J. Priestley discovers photosynthesis. 1775—M. Malphigi describes the cycle of substances in plants—the ascending and descending currents. 1800—J. Senebier edits “Plant Physiology” in 5 volumes. 1804—J. Senebier and Th. Saussure argue that photosynthesis represents the nutrition of plants with carbon. Brief Updates During the last decades, by using gene engineering methods, plants with recombinant DNA have been created, also called genetically modified plants (GMPs), this fact favoring the emergence of a new direction in plant physiology—the physiology of transgenic plants which aims to determine the physiological and biochemical changes of transgenic plants as a result of the inclusion of new genes into their genome. Thus, the use of GMPs has allowed the elucidation of the genetic and physiologic mechanisms of the activity of genes artificially included in the plant

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genome, among which are also those that are normally found in animal organisms, such as the Green Fluorescence Protein gene (GFP) from certain jellyfish species. The GFP emits a green fluorescence under UV light, and its fusion with any other protein allows the positional analysis of the last within the cell, the mechanism being similar to that of radio-labeling. Inserting auxine phytohormone biosynthesis genes (iaaM and iaaH) into the tobacco genome resulted in more viable transgenic plants with a more active vegetative morphogenesis and reproductive development and with both a higher amount of water stored in tissues and a higher resistance to drought. Another example is represented by the ferric superoxide dismutase gene (FeSOD) from Arabidopsis thaliana (one of the genes involved in antioxidative protection) which was included into the genomes of tobacco and wheat. The genetically modified plants proved more resistant to the oxidative stress than the control, confirming that the gene is expressed. Lately, to study a particular gene function the antisense strategies are often applied. The best known example is given by the gene that encodes the synthesis of the polygalacturonase enzyme, involved in cell wall degradation in ripening tomato fruits. After including this gene in the tomato genome, in reverse orientation, sense and antisense RNA will bind on the basis of complementarity, thus obstructing translation and leading to longer fruit preservation.

1.1 The Definition and Scope of Plant Physiology Plant physiology is a very important branch of biological sciences that studies the life of plants—the laws and mechanisms of physiological and biochemical processes, their significance, their interdependence with environmental factors in ontogenetic dynamics. The notion of physiology originated from Greek by joining the words physis, which means “function” and logos—“science”. Plant physiology has appeared in 1800, when the Frenchman J. Senebier edited his first monograph in five volumes “Plant Physiology”, which included not only his own experimental results, but also those obtained in this scientific field by: M. Malpighi, who has described the flow of substances in the plant (1775); St. Hales, who demonstrated that water and mineral salts flow through the xylem, while organic substances—through the phloem (1727); J. Pristley, who has discovered photosynthesis (1771), etc. During the development of plant physiology as a science, it has been based on two directions: anatomical/morphological (descriptive) and physiological (experimental), which, in principle, can be considered two basic research methods. This division is relative, because vegetal organs can’t be studied without taking into account their function, just as any processes cannot be studied without knowing the

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structures they are localized in. Any physiological process should be regarded as a product of long evolution, which forms the plant ability to adapt to variable environmental conditions. The function has evolved in relationship with the structure of the organism and the structure has stabilized under the action of environmental factors and according to the function. Thus, to study respiration, it is necessary to know the structure and ultrastructure of mitochondria, and to reveal the mysteries of photosynthesis, a unique and specific process happening only in green plants, it is important to know the structure and ultrastructure of the assimilatory apparatus. Most of the compartments of plant physiology have been delimited in the nineteenth century and are valid even nowadays. These are: 1. 2. 3. 4. 5. 6. 7. 8. 9.

Cytophysiology (plant cell physiology); Water regime of plants (H. Dutrochet, H. de Friz, J. Sachs); Photosynthesis (G. Busengo, M. Ţsvet, J. Pristley, K.A. Timireazev); Mineral nutrition (I. Leibih, G.B. Busengo, D.N. Preanishnikov); Respiration (A.S. Famiţsin, I.P. Borodin, L. Paster); Growth and development (J. Sachs, A.S. Famiţsin); Plant movements (T. Nait, J. Sachs, Ch. Darwin); Irritation (B. Sanderson, Ch. Darwin); Resistance to unfavorable factors (D.I. Ivanovski).

Thus, plant physiology as a distinct branch of biology, aims to study successively all vital processes that occur in vegetal organisms. In the second half of the twentieth century the basics of a new branch of plant physiology named self-regulation were laid. The phenomena of self-regulation and coordination of physiological processes, as well as other processes, are studied at all the levels of organization of living matter (molecular, intracellular, at the levels of tissue, organ, organism, biocoenosis) the mechanisms of implementation being diverse and specific. Self-regulation (autoregulation) is the property of biological systems to maintain the stability of the physical and chemical conditions of the internal environment, of the structure and properties of the organism in their elementary form, all these in conditions of a dynamic equilibrium. Autoregulation represents the process, which minimizes various deviations in the biological systems (pH, viscosity, redoxpotential, etc.), resulting from the influence of causative agents. Therefore, the capacity of the vegetal organism of carrying out vital functions amidst changing and unfavorable environmental conditions is implemented. Such a stability has a dynamic and active character. It is maintained by complex mechanisms, which determine the coordinated physiological activity of different organs, thus allowing autoregulation of plant growth and development, organism temperature, raw sap composition, regeneration of damaged tissues, adaptation to stress conditions, etc. (Figure 1.1). Self-regulation ensures integrity and homeostasis of plant organisms, allows harmonious growth and development and helps react adequately to the alternating

1.1 The Definition and Scope of Plant Physiology

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Fig. 1.1 Scheme representing regulative and directive processes in living organisms (Polevoy 1989)

factors of the environment. Autoregulation mechanisms are turned on automatically in the appropriate place and time, according to the needs of the organism. The notion of self-regulation is characteristic both for the whole vegetal organism and for the individual cells. In fact, it is at the cellular level that the integration of plant physiology with genetics, cytology, molecular biology, biochemistry, biophysics, etc. happens. Study of the autoregulation phenomena may contribute to the transition from describing the processes happening in plant organisms to their direct manipulation by acting upon the corresponding regulatory systems. In the last decades, the number of theoretical and experimental studies dealing with regulation and autoregulation of gene and enzyme functional activity, with membrane, electro-physiological, phytohormonal control (particularly related to the development of gene engineering and biotechnology) has considerably increased. Self-regulation determines plant homeostasis and creates conditions for the epigenesis of functions, which implies a strong collaboration between the factors of the environment and the plant genome. Consequently, it leads to the appropriate phenotypic expression. Regulatory systems (Fig. 1.2) at the cellular level include: • the mechanisms that determine qualitatively the enzymatic equipment of the cells and which consequently determine the metabolic profile of the cells, tissues and organisms; • the mechanisms maintaining a relative constant of the cellular metabolism (quantitative regulation of enzyme activity, of membrane transport etc.). All these regulatory systems are interdependent. For example, gene activity determines the properties of the cell membrane, and biological membranes also influence the differential activity of the genes. With the advent of multicellular organisms, intercellular regulatory systems have emerged including trophic, hormonal, electrophysiological regulation, contributing

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1 Introduction to the Educational Course of Plant Physiology Dominant centers

Polarity

Biorhythms

Channel connections

Regulatory contours

Phyto hormonal regulation

Electrophysiological regulation

Trophic regulation

Genetic regulation

Membrane regulation

Enzymatic activity regulation

Fig. 1.2 Interaction of regulatory systems (Polevoy 1989). Regulatory level: I intracellular, II intercellular, III organismal

to the interaction between plant organs. Such an interaction can be observed during cultivation of different vegetative explants in vitro. However, the existence of trophic, hormonal and electrophysiological interactions between cells, tissues and organs does not fully explain the behavior of a plant as a whole living organism. There are higher level regulatory systems and mechanisms connecting organs and functional systems of the plant during the life cycle of the organism and its ontogenetic transitions. The basic autoregulation mechanism at the organism level relies on the presence of a few centers of dominance (the stem and the root apexes), which receive information both from the external and internal medium and influence the living organism by driving tissue morphogenesis, by creating physiological gradients, polarity, channel connections (conductive fascicules), physiological oscillations.

1.1 The Definition and Scope of Plant Physiology

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Polarity and channel connections coordinate the space orientation of morphological processes, while oscillations help achieve time coordination. At the organism level these regulatory centers unite in regulatory contours with reversible relationships—positive or negative, which constitute the irritability effect. Thus, the integrity of the plant organism is determined by the interaction of control systems with central directing elements represented by the dominant centers. All the physiological processes occurring in plants are studied from various aspects. From the biochemical aspect, plant physiology studies the biosynthesis of organic compounds from inorganic ones, the functional importance of the diversity of organic substances formed as a result of the primary and secondary metabolism of the plant. It also researches the pathways of photosynthesis, reveals the laws of mineral nutrition, the importance of mineral substances as regulators of metabolic processes, their role in electrical phenomena occurring inside the cell or in the synthesis of organic compounds, etc. From the biophysical aspect the following problems are studied: cell energetics, electrophysiology of vegetal organs, physical and chemical laws of the water regime, those of nutrition via the root system, of growth, photosynthesis, respiration, the electrical aspects of irritability. From the aspect of evolution, researchers study the physiology of the genus, species, individual, as well as ontogenesis as a function of the genotype, which has transformed during phylogenesis. The ontogenetic aspect implies the analysis of the age-related laws governing plant growth and development based on the physiological and biochemical processes occurring in cells, tissues, organs as well as the study of morphogenesis and possible ways of acting on plant development (interfering with the photoperiodism, hardening plants, manipulating phytohormone signaling pathways to control plant stature, etc.). The ecological aspect consists of studying the dependence of the internal processes and of the particularities of individual development of the vegetal organism on the multitude of environmental conditions. Plant physiology is an experimental biological science that summarizes the ensemble of theoretical and practical knowledge, based on which, by using the principles of the scientific method, it is possible to intervene in the most important processes in the life cycle of plants: growth, development, metabolism, photosynthesis, nutrition, resistance, fructification. In describing the studied phenomena, plant physiology integrates knowledge from different areas of biology and life sciences such as: botany and plant morphology (studying the structure and components of the vegetal organism), cytology (studies the cells), biochemistry (investigates chemical substances and reactions occurring in living organisms), biophysics (focuses on the description of physical phenomena related to living organisms for instance energy exchange between plants and the environment, etc.), ecology (provides data on the effect of environmental factors on plants), chemistry, physics, mathematics, etc. At the same time, plant physiology represents a theoretical basis for plant cultivation, phytopathology, plant breeding, agriculture, agro-chemistry, genetics and pedology.

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1 Introduction to the Educational Course of Plant Physiology

1.2 Purposes of Plant Physiology as a Science The possibility to constantly build up on available research knowledge and the potential for implementing the final results make plant physiology a fundamental science with practical applicability. The purpose of plant physiology as a science is to investigate the peculiarities of the life of different species of vegetal organisms both cultivated ones and those from the spontaneous flora, in order to direct key processes like growth, development, nutrition, metabolism and others. Being a fundamental science, plant physiology aims to examine the molecular, physiological, biochemical and morphogenetic mechanisms of the vital processes in their dynamical succession and as a function of alternating environmental conditions, including: • discovering the essence of the organism’s individual development and studying the interaction of genetic, physiological, enzymatic mechanisms during growth and development; • elucidating regulatory and autoregulatory mechanisms under the action of external factors; • detailing the biochemical theory concerning mineral nutrition of plants; • elucidating the ways used by plants to improve the efficiency of solar energy utilization; • investigating the mechanism of atmospheric nitrogen fixation and its utilization by superior plants; • developing and detailing the theoretical bases of the use of biologically active substances; • elucidating the laws of plant viability (mechanisms of nutrition, growth, movement, reproduction); • improving the theoretical knowledge on maximizing crop yields; • researching endogenous mechanisms of regulating physiological functions, including basic mechanisms of enzyme biosynthesis, transport of substances and regulatory action of biomembranes; • decoding mechanisms that control the chronological sequence of genetic program implementation during plant ontogenesis, including intracellular interdependence, interaction between vegetal organs during growth, reproduction and, finally, during crop formation; • studying the regulation of secondary metabolite biosynthesis (alkaloids, rubber, phenolic compounds, etc.) which are often of great economical importance. As an applicative science, plant physiology aims to increase plant productivity. It is known that in an ear of wheat there are 3–5 flowers, of which only 1, 2 or 3 are fructifying. To make all these flowers fructify is the kind of challenges put in front of plant physiology. To achieve this goal, it is necessary to know the causes preventing metabolite formation and grain filling. At the core of such phenomena lies, for instance, the poor activity of the photosynthetic apparatus, caused by the

1.2 Purposes of Plant Physiology as a Science

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depletion of chloroplast enzymes or the exhaustion of cell energy resources in the form of adenosine triphosphate. In order to solve tasks like this, plant physiology investigates: • • • • • • •

photosynthetic apparatus activity and efficiency of solar energy use; plant requirements for mineral nutrition; water regime and efficiency of water utilization; plant resistance to various unfavorable factors; the possibility of using growth regulators; critical phases of ontogenesis; physiological bases of implementing the morphogenetic program.

A problem of the physiology of mineral nutrition, with promising prospects in plant breeding, is to study the absorption of nutrients by the root system. Knowing the rhythm and the rate of nutrient absorption in the multitude of varieties resulting from plant breeding, we can choose the biological material with a maximum capacity of fertilizer intake, this being a prerequisite for big crops. Discovering the functions of growth regulators may have multiple practical applications. Thus, gibberellins can be used to spray tree seedlings in greenhouses to force their growth in the first year and to reduce the overall time spent in a greenhouse, while auxins can be used to stimulate seedling rooting. It is again the task of plant physiology to determine experimentally for various species the duration of seedling exposure to these phytohormones, their optimal concentrations and the optimum age of the treated sprouts in order to achieve the best results. Another important task of plant physiology is to find out plant requirements with regard to nutrients and water during different vegetation periods. In autumn cereals, regrowth of the foliar system that has been destroyed during the winter by the frost involves the consumption of large amounts of nitrogen in the early spring. In vegetable production, the practical purpose is to obtain seedlings in greenhouses during winter, when the light intensity is low because of permanent cloudiness. The etiolation phenomenon (plants have long, weak stems; smaller, sparser leaves due to longer internodes; and a pale yellow color) can be prevented by illuminating plants with artificial light or spraying them with diluted solutions of retardant substances that hamper seedling elongation.

1.3 Research Methods Used by Plant Physiology As mentioned above, plant physiology is a pronouncedly experimental science, the experiment representing the main research method. The experiment is preceded by the hypothesis. Studies and experiments on plants are performed under three basic aspects: (1) Passing from a higher level to a more elementary one, from analyzing complex biological laws to studying simpler ones—physical and chemical. This

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research direction led to the advent of molecular biology, to the discovery of the hereditary code, the protein biosynthesis mechanism, the main laws of absorption and utilization of light quanta in photosynthesis. This, however, is not sufficient to understand the laws of physiological processes occurring in vegetal organisms. (2) Currently another approach is used—from simple to complex, called by V.A. Engelhardt integral. Generally, this path allows pursuing the evolution of certain processes at the level of DNA–RNA–protein–enzyme–biochemical reaction–physiological process–property of the cell. At any level of this path, regulation is possible and there are also internal mechanisms of autoregulation (targeting DNA replication, RNA and protein biosynthesis, enzymatic activity, but also cell, tissue and organ differentiation). (3) Physiological processes are studied in ontogenetic dynamics and in relationship with environmental factors. Plant physiology research is carried out in the field, in vegetation pots, solariums, greenhouses and laboratories. The most modern investigations are carried nowadays in phytotrons (the name was given by R.A. Millikan in 1949). Phytotrons are constructions with special rooms with either natural or artificial illumination, heated or cooled artificially, with adjustable air temperature and humidity. With the help of automatic electrical installations, vegetation parameters can be maintained at certain established levels. Investigations in the field of plant physiology imply carrying out experiments and exploring processes and phenomena at different levels of organization of the living matter by means of biochemical, biophysical, physicalchemical and biological methods. At the molecular level the physical-chemical processes occurring in living organisms are studied: the synthesis, assembly and restructuring of the proteins, nucleic acids, polysaccharides, lipids and other substances, the energetic and informational metabolism that regulate these processes. At the cellular level the structure and properties of the cell and its components is studied, the relationship between organelles etc. At the intercellular level knowledge from multiple disciplines is integrated and the principles of photosynthesis, respiration and interaction between tissues and organs are provided. At the organismal level the processes and phenomena taking place in an individual organism are studied as well as the coordinated functioning of its organs and systems, interactions between different organs and their individual roles, changes caused by accommodation. At the population level research focuses on the basic unit of the evolutionary process—the population. It means that the interactions between individuals that inhabit a certain territory (more or less isolated) is investigated. The composition and dynamics of the population is strongly correlated with the molecular, cellular, intercellular and organismal levels of organization. At the level of the biosphere the processes taking place in the biogeocenoses are studied including the interactions of biotic and abiotic components of the ecosystems. Each of the mentioned levels of organization has its own specific research methods. The observation of various phenomena is carried out with the naked eye

1.3 Research Methods Used by Plant Physiology

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(macroscopically) or using a microscope (microscopically). The discovery and constant improvement of imaging technologies with the electronic microscope had marked a significant stage in plant physiology development—the era of cellular organelle physiology. The electronic microscope together with the range of methods for cellular homogenate ultracentrifugation allowed the study of the submicroscopic structure of cellular organelles, while chemical micro-analysis allowed elucidation of their chemical composition. Based on knowledge about the ultrastructure and chemical composition it has become possible to decode physiological functions of various cell organelles. The scientific works in the field of plant physiology often make use of: ordinary microscopy, electronic microscopy, centrifugation, chemical analysis, chromatography, radioactive labeling, gel filtration, electrophoresis, roentgen analysis, artificial modeling of systems, autoradiography, in vitro culture. Lately, side by side with physiological and biochemical methods, mathematical modeling of life processes and plant productivity in defined conditions of growth and development is used by utilizing model-algorithm-program triads.

Glossary Adaptation The evolutionary process by which the organism or species survives and reproduces in new environmental conditions. Self-regulation (autoregulation) The general feature of biological systems that assures the control and autonomous coordination of the functioning of system elements and the maintenance of a dynamic equilibrium in the system. Evolution The progressive development of living organisms during successive generations by means of accumulating favorable hereditary variations enforced by natural selection. Enzyme A protein produced by the cell which controls the reactions of synthesis and degradation via its catalytic activity, playing a fundamental role in metabolic processes regulation. Phylogenesis The history of the development of a species or other taxonomical unit during the evolutionary process. Phytohormone A substance secreted by the plant cell in small quantities, which controls various aspects of growth, developmental transitions, organ morphogenesis, response to various stress factors etc. Photosynthesis The fundamental process of synthesis of organic compounds from inorganic ones (CO2 and H2O) in the presence of light, carried out by green plants and photosynthesizing microorganisms. During the process of photosynthesis the solar energy is transformed into the energy of chemical bonds in organic molecules.

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Metabolism The totality of all the complex processes of synthesis (energy storage) and degradation (energy release) undergone by the substances in a living organism. Morphogenesis Cyto-differentiation and development of visible structures (organs or parts) in an organism during ontogenesis. Levels of organization Systems with a specific organization (characteristic of biological systems only) and with a character of universality. Ontogenesis The series of transformations undergone by the organism, from egg fecundation to death, according to the scenario for the respective species. Respiration The process of oxidative degradation of complex organic substances into inorganic ones accompanied by energy release.

References Acatrinei Gh (1991) Reglarea proceselor ecofiziologice la plante. Editura Junimea, Iaşi, p 280 Burzo I, Toma S, Crăciun C ş. a (1994) Fiziologia plantelor de cultură, vol 1–4. Chişinău, Ştiinţa Crăciun T, Crăciun L (1989) Dicţionar de biologie. Editura Albatros, Bucureşti, p 285 Derfling K (1985) Gormony rasteniy. Mir, 304 p Duca M (1996) Sisteme şi mecanisme de autoreglare la plante. Chişinău, USM, 199 p Duca Gh, Zănoagă C, Duca M, Gladchii V (2001) Procese redox în mediul ambiant. Chişinău, 381 p Lebedev SI (1982) Fiziologiya rasteniy. M. Kolos, 544 p Milică C, Dorobanţiu N. ş. a (1982) Fiziologia vegetală. Bucureşti, Ed. Didactică şi Pedagogică, 375 p Polevoy VV (1989) Fiziologiya rasteniy. M. Vysshaya shkola, 464 p Polevoy VV (1982) Fitogormony. L. Izd. Leningradskogo universiteta, 248 p Tarhon T (1992) Fiziologia plantelor, vol I, II. Chişinău, Lumina Udovenko GV, Sheveluha VS (1995) Fiziologicheskie osnovy selektsii rasteniy, vol 2. VIR Yakushina NI (1980) Fiziologiya rasteniy. M. Prosveshchenie, 303 p

Chapter 2

Plant Cell Physiology

Abstract The cell is the smallest structural and functional unit of all living organisms, at the level of which all the fundamental characteristics of life are manifested. The cell is composed of several structures which have evolved to perform unique functions: • the cell wall contains a middle lamella, a primary cell wall and a secondary cell wall (in order of their formation as a result of cell division) and is composed of cellulose micro and macrofibrils immersed in an amorphous matrix consisting of hemicellulose, pectic substances, proteins, but also of optional substances like suberin and lignin that add up to its rigidity. The primary function of the cell wall is that of a mechanical exoskeleton and delimiting barrier. The system of interconnected gaps in the cell wall forms the apoplast which is a transport path for liquids in the vegetal organism along with the symplast formed by the plasmodesmata which connect the cytoplasm of the cells. • the protoplasm is made of a viscous liquid matrix—the hyaloplasma which serves as a medium for metabolic and energy exchange reactions, deposition of substances, etc. and is also the place where the cellular organelles reside: the nucleus, mitochondria, plastids, the endoplasmic reticulum, the Golgi body, ribosomes, the vacuole. • biological membranes (plasmalemma, tonoplast, membranes of the organelles) represent fluid amphiphilic phospholipid bi-layers with various amounts of embedded proteins which perform a diversity of functions and determine the unique properties of the membranes. Biological membranes perform a variety of functions like: compartmentalization, mechanical barrier, transport of various substances including water (by osmosis), ATP synthesis (in chloroplasts, mitochondria), receptor function, etc. Cells constantly exchange substances with the external environment via active or passive transport of the ions into or out of the cell. Active transport requires energy and happens via vesicles, ion pumps and some carrier proteins, while passive transport (no energy requirement)—via simple diffusion through the selectively permeable membrane, or via facilitated diffusion through some carrier proteins or protein channels. Water can also enter or exit the cell by means of osmosis, © Springer International Publishing Switzerland 2015 M. Duca, Plant Physiology, Biological and Medical Physics, Biomedical Engineering, DOI 10.1007/978-3-319-17909-4_2

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electroosmosis, imbibition, water currents, diffusion. A high osmotic pressure is essential for maintaining the normal physiological state of cell turgidity, while a low content is detrimental and can cause the phenomenon of plasmolysis. The totality of forces that contribute to water absorption by the cell form the suction force.

Historical Background 1667—The cell was discovered by R. Hooke. 1838–1839—T. Schwann and M.J. Schleiden formulated the “Cell theory”. 1877—W. Pfeffer studied the osmosis phenomenon in vegetal cells. 1880—G.D. Thuret and J.B. Bornet discovered plasmodesmata. 1890—S. Altman discovered mitochondria. 1895—Ch. Owerton formulated the theory of protoplasm permeability. 1897—A. Garnier discovered the endoplasmic reticulum. 1898—C. Golgi discovered the dictyosomes. 1955—G.E. Palade discovered the ribosomes. 1958—R. Buvat launched the theory of vacuole emergence from the endoplasmic reticulum. 1959—J. Robertson demonstrated the structural uniformity of all biological membranes.

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1960—K. Porter studied the endoplasmic reticulum in plant cells. 1972—S.J. Singer and G.L. Nicholson proposed the fluid mosaic model of lipoprotein organization in biological membranes. Brief Updates Cell death (apoptosis) represents the last stage in the life of a cell and is genetically programmed, but can also be triggered by the action of various unfavorable factors. The apoptosis process takes place when the metabolism ceases and is accompanied by some morphological and structural modifications—the destruction of the genetic nuclear apparatus, of the mitochondria and chloroplasts, an increase in the amount of Ca2+ ions, etc. The gravest changes occur at the DNA level, where endonucleases cleave the macromolecule in small fragments of 50–300 kb. A crucial role in the control of apoptosis is played by phytohormones (like ethylene) and by DNA methylation. Serine and cysteine proteases are also involved in this process. There are very few data relative to how the apoptosis signal is passed from one cell to another one. The H52-gene was discovered in tomatoes, which encodes an HD-Zip transcription factor involved in this process. The artificial inhibition of this gene’s expression leads to an imbalance in the control of apoptosis and to an increase in the amount of ethylene and salicylic acid. The induction of apoptosis in plants and animals share many common features. One of them is the presence of the BI-1 gene homologues both in plants and animals, for instance the Arabidopsis AtBI-1, which manifests itself under the action of biotic (infections, pathogens) and abiotic factors (oxidative stress). In some plant species (Arabidopsis thaliana, peas and rice) the dad1-gene was detected which prevents apoptosis onset just like its analogue in the animal cell. The activity of this gene decreases abruptly in senile plants and during seeds formation. The inclusion of this gene in guinea pig cells delayed the apoptosis onset. This fact demonstrates that the mechanism of apoptosis, especially its suppression is similar in both plants and animals.

2.1 The Cell as a Structural, Morphological, Functional Unit of Living Organisms Living organisms that inhabit the planet consist of cells. The cell (from lat. cellula or gr. cytos—“room”) is the smallest structural and functional unit of all living organisms. Only viruses are recognized as non-cellular forms of life. Cells vary in shape, size and color, but, whatever the cell type, it fulfills all the criteria of living organisms, this phenomenon being demonstrated by cell cultivation on artificial media, their reproduction in vitro and even their possibility to regenerate the entire plant.

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Fig. 2.1 Cellular theory founders

The cell was discovered in 1665 by an English physicist, Robert Hooke, who has perfected the microscope and studied samples of cork. The cell theory has crystallized during 1838–1839 owing to a number of German researchers: the botanist M.J. Schleiden (1839) and the zoologist T. Schwann (1839), who recognized the cell as the basic structural unit of living matter. Later R. Virchow (1855) confirmed that cells could originate only from other cells—omnis cellula e cellula (Fig. 2.1). The principles of cell theory are as follows: The cell is a structural, morphological, functional unit of all living beings. The cell is an open thermodynamic system, which constantly exchanges matter with its environment and constantly transforms matter within the cell. All the eukaryotic cells have a special complex of organelles that regulate metabolism, accumulate and consume energy. The cell can exist as a separate organism (bacteria, protozoa, certain algae and fungus species) or as a component of multicellular organism tissues. Plants and animals consist of billions of cells, each of them specialized for different functions (contraction, excretion, transport of substances and their deposition, photosynthesis, etc.). More than 60 types of cells with various specialization have been described in vegetal organisms. Vegetal and animal cells have similar structure, functions and chemical composition. Although the cell is the simplest form of life with a few microns in diameter, its structure is very complex. In spite of this, the cell types share similar functions and structure of cellular components. The vegetal cell, unlike the animal one, has the ability to accumulate solar energy, turning it into chemical or mechanical energy. Unlike other eukaryotic cells, the vegetal cell is characterized by (Table 2.1): • a system of plastids, present due to autotrophic nutrition; • a central vacuole in the mature vegetal cell playing an essential role in osmosis regulation and maintenance of the turgor pressure; • a cell wall (cell envelope) which confers rigidity to the tissues.

2.1 The Cell as a Structural, Morphological, Functional Unit …

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Table 2.1 Chemical composition of different plant cell organelles (% of dry matter) Structural elements of the cell

Proteins

Lipids

RNA

Cytoplasm Plastids Mitochondria Ribosomes

80–95 30–45 30–40 50–57

2–3 20–40 25–38 3–4

– 0.5–3.5 1–6 35

Fig. 2.2 Chemical composition of the cell

Qualitatively, the chemical composition of the vegetal and animal cells is similar (Fig. 2.2, Table 2.2). Inorganic substances (water and inorganic ions) play the role of structural elements of various organic substances, as well as that of a reaction medium for cellular metabolism. Traditionally, organic substances in vegetal cells can be divided in the following groups: • structural molecules, normally not involved in cellular metabolism and forming instead the plant skeleton (cellulose, pectin, myosin, certain lipids, carbohydrates, proteins); • enzymes—macromolecules catalyzing cellular metabolism (ribulose-1,5biphosphate carboxilase, phosphofructokinase, phospholipases, chlorophyllases, amylases, ribonucleases, catalase, ascorbate oxidase, superoxide dismutase); • micromolecular active substances (pigments, vitamins and phytohormones); • micromolecular metabolites (resulting from specific catabolic reactions, serve for the transport of electrons and protons, usually CoF); • excretions deposited in the cell wall and vacuole (tannin, anthocyanin, lignin); • reserve substances (starch).

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Table 2.2 Comparing the structure and composition of plant and animal cells (Pickering 1998) Characteristics of an animal cell (the result of heterotrophic nutrition)

Common features of animal and vegetal cells (with regard to the processes of life maintenance)

Characteristics of a vegetal cell (the result of autotrophic nutrition)

In animal cells secretory vesicles containing cellular products like hormones or enzymes can be often found

The cell membrane surrounding the cytoplasm controls the entrance and exit of soluble substances being, thus, responsible for cell content separation from the environment The cytoplasm contains water, soluble substances like amino acids and carbohydrates and supports cellular organelles. Various metabolic reactions occur in both the cytoplasm and cellular organelles The nucleus contains the genetic material (which makes up the genes and chromosomes and encodes genetic information). Chromosomes become visible only during cell division

The cellulosic cell wall offers support and protection against potential lesions caused by water flowing into cells due to the osmotic force

The cytoplasm of animal cells is more dense and contains more cellular organelles

Vacuoles are small and temporary. They may be involved in digestion (e.g. phagocytosis) or excretion processes (contractile vacuoles can remove water excess from the cell) Carbohydrates are stored in the form of glycogen

Chloroplasts contain a special pigment called chlorophyll (which absorbs light) and the enzymes necessary for glucose production via photosynthesis

A big, permanent vacuole contains the water needed to generate the turgor pressure and is also a repository for ions and molecules

Carbohydrates are stored in the form of starch (found in the cytoplasm and in chloroplasts)

Each daughter cell originates from the mother cell after division. The cellular theory is considered to be one of the three greatest discoveries of the 19th century, along with the law of mass and energy conservation and transformation (A.L. Lavoisier, M.N. Lomonosov) and the theory of evolution (Ch. Darwin).

2.2 Structural Organization, Chemical Composition and Function of the Cell Wall The cell wall (cellular envelope) is a complex formation with 3 basic layers: • the middle lamella; • the primary cell wall; • the secondary cell wall.

2.2 Structural Organization, Chemical Composition ...

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Fig. 2.3 Cell wall structure

The middle lamella appears during the telophase of cell division (mitosis) between two daughter cells as a product of the Golgi apparatus activity. For young growing cells from meristematic tissues a primary cell wall is characteristic, and while aging, the secondary structure of the cell wall emerges, both being produced by the protoplast (Fig. 2.3). Functions of the cell wall: • represents a mechanical exoskeleton for the cell; • confers shape to the cell and rigidity to vegetal tissues; • prevents the rupture of the cytoplasmic membrane due to hydrostatic forces acting from the cell interior; • represents a barrier for various infections; • participates in the absorption and transport of water and mineral salts; • represents a specific ion exchanger; • participates in the exchange of substances; • lectins contained in the cell wall recognize symbiotic bacteria, which cause the formation of root nodules in plants. Properties. The cell wall has a high rigidity but can also support elastic deformations. The thickness of the cell wall in different plant species are ranging from 0.1 to 10 µm. Chemical composition. The main components of the cell wall are: cellulose, hemicellulose, pectic substances, proteins, etc. Cellulose molecules (C6H10O5)n represent long unbranched chains, consisting of 3–14 thousand glucose residues.

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Structure. The cell wall consists of two basic components: • the micro-and macrofibril complex; • the amorphous complex (the matrix). Cellulose macromolecules aren’t in a free state, they are linked by hydrogen bonds and form microfibrils, which are woven into larger scale rope-like macrofibrils. The structure of macro- and microfibrils is not uniform. Well-organized as well as paracrystalized or amorphous sectors can be found. Cellulose macro- and microfibrils contained in the cell wall are immersed in an amorphous mass (a gel matrix) which consists of hemicellulose, pectic substances and proteins. Hemicellulose is a soluble polymer made of a hexose and a pentose. The polymerization degree of these substances is lower when compared to cellulose (150–130 monomers). Pectic substances are also polymeric compounds from the class of carbohydrates. The proteins of the cell wall confer elasticity (extensibility) or perform enzymatic functions. The middle lamella is based on the amorphous complex formed by protopectin and hemicellulose. These molecules intertwine with each other to form a mesh with a particular degree of rigidity. In mature cells the middle lamella contains calcium pectate, which cements cells. If there is a calcium or pectic substance insufficiency a mucous-like state of the cells can be noticed and tissue maceration occurs. During fruit ripening pectic substances in the middle lamella become soluble and fruits soften. During the growth and development process, cellulose, hemicellulose and pectin molecules are deposited on both sides of the middle lamella forming the primary cell wall, which consists of the amorphous complex (*70 %) and the microfibrillar complex (*30 %). Cellulose molecules aren’t too large (*2,000 glucose residues) and are linked by hydrogen bonds or are glued together by the matrix. Due to the chaotic arrangement of cellulose molecules, the primary cell wall is not too hard and allows the cell to grow and extend. The primary cell wall is characteristic for cells which are in the second stage of growth—the cell elongation stage. The secondary cell wall is localized on the inner side of the primary cell wall. It contains up to 80 % cellulose, forming micro- and macrofibrils. Cellulose molecules are long and contain up to 12,000 glucose residues. Their arrangement is clearly defined, forming a network of maximum strength. The gaps in the mesh of the cell wall can be impregnated with various substances insoluble in water—lignin, suberin. In this case cell wall rigidity increases at the expense of its elasticity. Via the gaps in the mesh system the cell walls of many cells in the vegetal organism are connected forming what is called the apoplast which also represents a transport path and contributes to the integration of all components of the plants. The apoplast occupies 5 % of the volume of aerial plant organs. The cell wall also contains pores which allow the cytoplasm of neighboring cells to be interlinked, thus establishing a continuity on the level of the entire organism. These fibers are called plasmodesmata. Every surface unit of 100 µm2 contains 10– 30 plasmodesmata with a diameter of 0.2 µm. Due to these cell structures the direct transport of water and other substances from one cell to another is possible forming a permanent flow—through what is called symplast. The apoplast and the symplast carry the transport function and contribute to the integrity of the vegetal organism.

2.3 Structure and Ultrastructure of Cell Protoplasm

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2.3 Structure and Ultrastructure of Cell Protoplasm The cellular protoplasm, or cytoplasm (from gr. cytos “cavity” and plasma “structure, substance”) represents the fundamental mass of the cell. Optical microscopy reveals cytoplasm as a viscous, transparent, colorless, homogeneous liquid, immiscible with water, characterized by surface tension, constant pH and rH values, semipermeability and selectivity, excitability, viscosity and movement, in which a series of organelles with varying size, shape, structure, chemical composition and functions. Electron microscopy, reveals cytoplasm as a heterogeneous and complex structure. It consists of a cytoplasmic matrix—the hyaloplasma, in which the cellular organelles are suspended (Figs. 2.4 and 2.5). Hyaloplasm (from gr. hyalos “transparent, glass”) represents the soluble phase, which performs a series of functions: • • • •

a matrix for metabolic and energy exchange reactions (glycolysis); deposition of organic (glycogen, starch) and inorganic substances; cell movement; cell adaptation to environmental conditions.

The hyaloplasm includes: • a structural part, representing several types of fibrillar and globular proteins, arranged in microfilaments (with the diameter of 6–10 nm) or in microtubules

Fig. 2.4 The components of the vegetal cell (Yakushina N.I., 1980)

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Fig. 2.5 Structure of the vegetal cell (Johnson et al. 2002)

(with 25–30 nm in diameter). This is a three-dimensional structure and facilitates vesicular and organelle transport within the cell; • the liquid contained in the gaps of the fibrillar network, which consists of approximately 70 % water and 30 % of organic compounds—carbohydrates (usually, in small amount, in particular carbohydrate monomers or oligomers), lipids (especially phospholipids), proteins, nucleotides, RNA, phytohormones, vitamins and mineral compounds—either as dissociated ions (K+, Ca2+, Mg2+,

2.3 Structure and Ultrastructure of Cell Protoplasm

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Fe2+, Mn2+, Cu2+, Zn2+, NH4+, CI−, PO43−, SO42−) or ions bound by organic molecules and, finally, inclusions (reserve substances). The ratio between these electrolyte and non-electrolyte substances contribute to the formation of a particular physical-chemical state of the hyaloplasm, leading to autoregulation of metabolic processes in the cell and maintaining the physiological and biochemical homeostasis, characterized by perfect integrity. As the hyaloplasm contains filamentous protein macromolecules it is more fluid when globular proteins predominate and more viscous if fibrillar proteins are present in big quantities, in the last case a transition from a sol to a gel state being possible. The intensity of metabolic processes is inversely proportional to viscosity and is often indicative of adaptation phenomena (to cold, drought, humidity). Protoplasm viscosity. is 16–20 times higher than water viscosity. It also changes during ontogenesis The lowest values of viscosity were detected during flowering while the highest ones—in seeds during maturation and anabiosis. The hyaloplasm is in constant motion, which can be circular or sliding. It is characterized by a specific isoelectric point, because the major part of its content is represented by proteins. Several cellular organelles are incorporated in the hyaloplasm. They have different shapes and sizes, perform specific functions and provide the vital activity of the vegetal organism. These organelles can be divided into two major groups: (a) membranous organelles (bi-membranous and uni-membranous); (b) non-membranous organelles, which have no phospholipid membrane on their surface.

2.4 Structure and Function of Biological Membranes Biological membranes (from lat. membrane—“parchment”) are two-dimensional structures composed of lipids and proteins, providing compartmentalization of living matter. There are several types of membranes, which differ in origin, structure and function. Cytoplasmic membrane (plasmalemma) is a thin, semipermeable film (6– 10 nm in thickness) on the surface of the protoplasm, which is located under the cellulose envelope and which separates the cell contents from the outside and controls the exchange of substances between the cell and the environment by assuring selective transport of nutrients. Plasmalemma is not separable from the cytoplasm unlike the cell wall, a fact that can be noticed during plasmolysis and deplasmolysis. It is flexible and, depending on the changes that occur in the water content of the cytoplasm, it can either contact with the cell wall or detach from it. This membrane is also responsible for the synthesis and assembly of the cell wall. Tonoplast (from gr. tonos—“tension”, plastos—“formation, structure”) is a membrane (structurally similar to plasmalemma) that embeds a vacuole. The term of “tonoplast” was introduced by Hugo de Vries in 1885.

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The membranes of the cellular organelles have a thickness of 6–7 nm and can be classified into: • double (internal and external); • simple. Double membranes are characteristic for the nucleus, mitochondria and chloroplasts, while the simple membranes can be found in the endoplasmic reticulum, the Golgi apparatus, in lysosomes and vacuoles. Properties of biological membranes. Biological membranes represent a homogeneous and transparent film that forms a system of folds, which increases the surface area. The thickness of the membranes varies from one organelle to another (from 5.5 to 20 nm). The fine structure of biological membranes reveals the property of selective permeability, this fact contributing to a strict control of matter inflow and outflow. For example, the plasmalemma is slightly permeable for glucose, while the tonoplast is impermeable. Thus, the products of photosynthesis are transported from the cell and do not accumulate in the vacuole. After cell death, biological membranes lose their property of semipermeability which proves that their structure and chemical composition are maintained by energy consumption. Chemical composition. Biological membranes are composed of lipids (1/2), proteins (1/3), polysaccharides (in small amounts), enzymes and various ions. The protein/lipid ratio of a membrane reflects the intensity of its functional activity. The most abundant lipids are phospholipids, followed by glycolipids and cholesterol. Sterols and unsaturated fatty acids confer porosity to the phospholipid double-layer. Phospholipids are amphiphilic which means that the molecules have one hydrophilic (polar) end and another highly hydrophobic end. This property allows lipids to self assemble in a two-layered structure in the presence of water. Lipids are not fixed stably in the membranes. They change permanently their location by lateral diffusion (within the monolayer) or by transverse diffusion (“flipflop”) occurring between two distinct lipid monolayers (this occurs much less frequently). Plasmalemma contains 35–40 % of lipids, mitochondrial membranes— up to 28 %, while the myelin membranes—up to 80 %. The proteins of the biomembranes (enzymes, receptors, pumps, channels, regulatory and structural proteins) consist mainly of hydrophilic (polar) amino acids, which are responsible for some specific properties. Membrane proteins are characterized by the ability to move within the fluid double lipid layer and can be divided into 3 groups: • Integral—which form hydrophobic links with the lipids and pierce the double lipid layer; • semi-integral; • peripheric—proteins are on the surface of the biological membranes. Biological membranes may also contain heterogeneous macromolecules (glycoproteins, glycolipids) and several minor components (coenzymes, nucleic acids, antioxidants, carotenoids, pigments, etc.) depending on the role they perform.

2.4 Structure and Function of Biological Membranes

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Fig. 2.6 Ultrastructure of the cellular membrane (Johnson and Raven 2002)

Glycoproteins usually consisting of up to 15 monomers, function as receptors. Membrane components are formed in the endoplasmic reticulum and then modified in the Golgi body. Structure. According to the model developed by Singer-Nicholson, biological membranes are composed of a double lipid layer, impermeable to any polar molecules, particularly to ions (Fig. 2.6). This layer is fluid, thus, phospholipid molecules are capable of very fast free lateral movements (rotation, diffusion, etc.). On both sides of the double lipid layer there are proteins integrated or partially integrated into it, these proteins being mostly globular and performing the function of trans-membranous transport. Biological membranes are asymmetric, they have internal and external foils that contain different compounds, corresponding to specific biochemical activities. All biological membranes originate from preexisting ones. Functions of the biological membranes. The primary function of biological membranes was to separate the internal environment of the cell from the external one, representing a barrier in the circulation of substances. Along the process of complication of the cell structure during evolution, several additional functions have emerged, including: • Protection. Membranes serve as mechanical barriers to harmful factors of the environment, protecting the internal content of the cell, nucleus, cellular organelles, etc.; • Transport. Biological membranes contain structural and functional systems necessary for passive and active transport of substances (Fig. 2.7); • Energetic function. The internal membranes of the chloroplasts and mitochondria transform solar energy and the energy of redox processes into ATP macroergic bonds; • Osmosis regulation. Due to semipermeability, biological membranes contribute to the emergence of a chemical gradient causing water absorption while in some aquatic plants they represent a barrier for water passive diffusion, thus protecting them from destruction;

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Fig. 2.7 Functions of the components of biological membranes (Johnson and Raven 2002)

• Enzymatic activity regulation. Different biochemical reactions take place at the level of biological membranes, which are catalyzed by specific enzymes, so that they represent themselves specific matrices on which enzymatic systems are assembled. Most of the enzymes that catalyze redox reactions, those of hydrolysis and biosynthesis are linked with biological membranes; • Compartmentalization. Multiple reactions of biosynthesis, degradation, transport, etc., with the participation of tens and hundreds of specific enzymes take place simultaneously in the living cell. Thus, the cell is a complex structure where different physiological and biochemical processes constantly interfere with each other. Biological membranes contribute to the formation of compartment systems specific for certain types of chemical reactions (ATP biosynthesis in separate compartments—mitochondria and chloroplasts, etc.), thus assuring and facilitating autoregulatory processes of cellular metabolism. The most important regulatory factors are cellular metabolites which control gene expression levels and, thus, affect enzyme concentrations or alter their activity. • Receptor function. By means of membrane embedded receptors the information related to fluctuations of the environmental factors is perceived and, based on this, the adjustment of the metabolism is carried; • Structural role. The cytoplasmic membrane forms a lot of folds, which together with the nuclear membrane and the endoplasmic reticulum forms a complex system of structures (Fig. 2.8).

2.5 Exchange of Substances Between the Cell and the Medium

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Fig. 2.8 Complex membrane structures in the cell

Table 2.3 Content of several ions in sea water and vacuole sap

Chemical element

Ion content (in milliequivalents) Sea water Vacuole sap

Chlorine Sodium Potassium

0.580 0.498 0.012

0.597 0.090 0.500

2.5 Exchange of Substances Between the Cell and the Medium The vital activity of each living organism is determined by its provision with nutrients and water. A constant exchange of substances between the exterior and interior environments occurs on the cellular level during its entire life cycle. Flow of substances inside the cell is called absorption. The transport of substances from the cytoplasm to the vacuole or to the lumen of xylem vessels is called secretion. If ions or other substances are leaving the cell, this process is called excretion or desorption.

2.5.1 Ion Flow into the Cell The living cell is capable of selective absorption and accumulation of mineral elements (see Table 2.3). For instance, water flows freely through the membranes, while macromolecular substances don’t. Semipermeability is one of the basic mechanisms of selective accumulation of ions in the cell and is owed to a large extent to the hydrophobic layer of the

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Fig. 2.9 Transport of substances in the cell

membrane (which prevents charged ions from passing through with varying efficiency depending on ion charge, diameter). With the advent of phospholipid membranes, transmembrane ion transfer mechanisms have emerged (Fig. 2.9). When lipids predominate in the membranes, permeability is higher for organic substances, while, if they are composed mainly of proteins, permeability is higher for water and mineral ions. Ion transport across biological membranes, may be passive or active. Passive transport of ions occurs according to the chemical and electrochemical gradients, without metabolic energy consumption. This phenomenon is based on simple and facilitated diffusion. Simple diffusion is the movement of ions and molecules along the concentration gradient which is from a region of higher concentration to one of lower based on the kinetic energy of the molecules, which increases with temperature and concentration. From the thermodynamic point of view, the diffusion vector is determined by the chemical potential of the substance. The higher the concentration of the substance, the higher its chemical potential. Passive transport across phospholipid membranes can occur with substances which are soluble in lipids or if the molecular diameter of the molecules is smaller than the pore diameter. The transport rate is inversely proportional to the diameter and mass of the transported molecules (the ultrafilter concept elaborated by W. Ruhland) and directly proportional to their solubility in lipids (the liposolubility concept developed by Overton). An example of passive transport is the movement of carbon dioxide from the air into leaf tissues.

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The transport of polar molecules and ions along the concentration gradient takes place with the help of transport proteins via facilitated diffusion. These proteins can be integral to the membrane or they can reversibly bind the transported molecules, cross the biological membranes and release the cargo, for instance, within the cell, and the protein travels back to reinitiate the cycle. Active transport is performed contrary to the concentration gradient with metabolic energy consumption (ATP, NADH, NADPH) derived from the respiration process. Active transport can occur via specific transport proteins included in the biological membranes and forming protein carriers or ion pumps. Transporters in this case have functions similar to those of enzymes. Absorption on one side of the membrane and desorption on the other side, are very selective due to the high specific affinity of the transporters for the cargo (e.g. ions). This transport type is characteristic for carbohydrates, amino acids, nucleotides, etc. and may be simple, in the case of one substance being translocated in a particular direction (Fig. 2.11), or coupled (cotransport)—when the simultaneous transport of two substances takes place (Figs. 2.10 and 2.12). When two molecules are transferred in the same

Fig. 2.10 Sodium and potassium ion transport carried out by a single pump (the K+/Na+-ATP-ase) (Johnson and Raven 2002)

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Fig. 2.11 Active transport of a single substance by the H+-pump through a series of conformational modifications

Fig. 2.12 Transport of two substances in different directions (Na+ and K+) and in the same direction (Na+ and carbohydrates) (Johnson and Raven 2002)

direction this kind of cotransport is called symport whereas if in opposite directions —antiport. As an energy source for the cycle consisting of: transporter activation, cargotransporter complex formation, conformational change that helps translocate the cargo, cargo release and the return of the transporter to the initial state (note that usually just some of these events require energy) the following reactions are used: • redox-reactions; • ATP hydrolysis—K+/Na+-ATPase (Fig. 2.10); Ca2+/H-ATPase; • the electrochemical gradient of an ion used for cotransport. Proton pumps use ATP energy, hydrolyzed by the H+-ATPase located in the membranes. The enzyme generates a high electrochemical potential and a pH gradient, which is the driving force for the absorption of substances that are cotransported with H+. An example of active transport is the intake of mineral ions from soil via the root hairs. Amino acids, glucose, K+ and Na+ ions are transported

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contrary to the concentration gradient and with energy consumption, usually in symport with H+ ions. The direction of ion movement is determined not only by the concentration gradient of the chemical species but also by the electrostatic potential across the membrane. These two components (chemical and electrical) form what is called the electrochemical gradient. Thus the symport system of Na+ ions with amino acids or carbohydrates, is paralleled by a dual effect of the Na+/K+ pumps: first—that of a chemical gradient creation (as a result, Na+ is in large excess outside of the cell compared to the interior) and, second—that of a charge gradient creation (for every 2 positively charged K+ ions that are transported inside of the cell, 3 positively charged Na+ ions are transported outside of the cell) (Fig. 2.12) Pinocytosis is a special type of active transport based on membrane modification by forming evaginations which latter give rise to special vesicles: these vesicles embed various substances that may be released inside or outside the plasmalemma or tonoplast. In this manner, anthocyanins are transported from the cytoplasm to the vacuole through the tonoplast and Golgi vesicles through the plasmalemma to the cell wall.

2.5.2 Water Flow into the Cell Water exchange between cells and the environment is achieved through the intervention of the following physical phenomena, characteristic of both the biological systems and the non-living matter: • • • • • •

water currents diffusion osmosis electroosmosis imbibition suction force of the cell.

Fig. 2.13 Diffusion of ions in water solutions through a semipermeable membrane (based on the example of urea)

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Fig. 2.14 Comparison of the diffusion and osmosis processes

Water currents represent the total movement of water, based on its free energy (the water potential, which alternates depending on several physical factors). Water moves from the region with higher potential of water to the region with a lower one. Diffusion is the random movement of molecules from a region of higher concentration to one of lower concentration until the balance is achieved. The rate of diffusion is determined by the temperature, the number and size of the particles, the fluidity of the medium, etc. This is a slow process that occurs at small distances in solutions with high difference in the potential of a chemical species (Fig. 2.13). Osmosis (from lat. osmos—“impulse, urge”) represents the movement of water or solvent molecules through a selectively permeable membrane. The direction of this movement is from a lower concentration to a higher one, i.e. from a higher water potential to a lower one (Fig. 2.14). The phenomenon of osmosis is the fundamental way of water flow into the cell, being very important for directing and distribution water in living organisms. The resistance force that opposes water entrance into the cell is directly proportional to the water concentration of the cellular juice and is called osmotic pressure (P). Osmosis is responsible for water flow from the tissular fluids into the cells, from the the soil (the accessible water) to the tiny root hairs, from the xylem into the cells that form the leaf mesophyll. The lowest osmotic pressure (0.1 atm) can be observed in aquatic plants and the highest pressures—in halophytes containing a high percentage of mineral salts in the cellular juice. In most crop plants the osmotic

2.5 Exchange of Substances Between the Cell and the Medium

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Fig. 2.15 Types of solution depending on the osmotic pressure. a Isotonic solution b Hypertonic solution c Hypotonic solution

Fig. 2.16 Plasmolysis

pressure values range from 0.5 to 3.0. Usually, the osmotic pressure of terrestrial plants is in the 506–1010 kPa limit, while for aquatic plants—in the 101–304 kPa limit. A high osmotic pressure—2,026–4,052 kPa—is identified in fruits, vegetables, berries, sugar beet. The osmotic character of water exchange between the cell and its environment is manifested in the phenomena of turgidity and plasmolysis, which depend directly on the tonicity of the internal environment of the cell. Depending on the chemical potential value, different situations may occur in the process of water exchange between cells and the environment, resulting in different types of solutions—isotonic, hypertonic and hypotonic (Fig. 2.15). In isotonic solutions ion concentration inside the cell is equal to their concentration outside the cell, i.e. the cells have the same osmotic pressure as the environment. In such solutions there is no active water movement except for diffusion and occasional short-distance water travel. In hypertonic solutions ion concentration in the cell is lower than in the external environment. Thus, water is eliminated from the cell by exosmosis causing detachment of the plasmalemma from the cell wall (a phenomenon called plasmolysis) and, consequently, plant wilting.

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The plasmolysis can be concave (early) and convex (final). In convex plasmolysis the protoplasm is contracted around the vacuole and its contact with the cell wall is maintained only via fine threads (Hecht’s filaments). Plasmolysis forms and types are determined by the degree of protoplasm hydration and by the microstructural features of the plasma membrane (Fig. 2.16). Hypotonic solutions are characterized by a higher concentration of ions in the cell as compared to the external environment. This phenomenon contributes to endosmosis, i.e. water molecule active movement from outside into the cell, causing deplasmolysis, leading to an increase in cell turgidity. If the plant cell is in a hypotonic medium, water enters it through the endosmotic flow and the cell expands. As this happens, the cell wall exerts an increasing counter-pressure on the cellular content. This is the turgor force (T), that gives the cell the state of rigidity and tension. T represents the hydrostatic force of the intracellular solution exerted on the plasmalemma, which shapes and stiffens the vegetal cell. The turgor force reaches its maximum value in the morning and its minimum—in the afternoon ranging from 5–10 atm in thallophytes to 100 atm in fungi. If a drought or intensive transpiration takes place, the turgor force value reduces to 0 and the plant wilts. Plasmolysis and deplasmolysis are phenomena characteristic only of living cells, which have maintained semipermeability. The speed and form of plasmolysis characterize the viscosity of the protoplasm. Plasmolysis allows to determine osmotic pressure values, this fact being important in ecological research and explains the ability of plants to absorb water from soil and retain it. Osmotic processes in plants are of primary importance in ensuring water delivery through absorption, efficient water circulation and the exchange of substances. Electroosmosis is the movement of liquid through the pores of a membrane under the influence of an electric field. This movement is possible because an electric potential can install in the vegetal cell at the cell wall level due to pectin and other substances containing carboxyl groups that dissociate in COO− and H+. COO− ions give a negative charge to the cell wall. In order to reach an electrostatic balance, ions of opposite charge are attracted and arranged parallel to the membrane, thus, an electrical gradient being established. Biological membranes are characterized by a certain electrochemical potential that contributes to water circulation. Imbibition forces are those which contribute to water penetration through macromolecules of protein colloids and cellulose-pectin microfibrils. Imbibition is based on colloidal and capillary effects and causes an irreversible increase in volume and mass. Imbibition based on colloidal effects predominate in the protoplasm, but both types are characteristic for the cell wall. This process is very important during seed germination, as the organic reserve matter, being hydrophilic, has the ability to bind a large number of water molecules, which helps develop in seeds an inner force of up to 1,000 kPa. Cellular proteins have a higher imbibition capacity while cellulose has a weaker one. Each OH− radical of the β-glucose residues that make up cellulose, can fix 3 water molecules while the acidic COOH group and the NH2 amino group can retain 4 water molecules. Imbibition may be limited, when

2.5 Exchange of Substances Between the Cell and the Medium

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Fig. 2.17 K. Hoffler’s diagram representing the value of cellular suction force as a function of cell volume (starting with a state of plasmolysis and ending with a state of maximum turgor) (Milica 1982)

the imbibed mass remains in the gel state and unlimited when the colloid transitions entirely into the sol state. The totality of forces that contribute to water absorption in the cell form the suction force, marked with S. It is determined by the difference between the osmotic and turgor pressures. This force depends on internal and external factors, on the plant species as well as on environmental conditions. As the turgor tends to maximum values, the rate of water entrance into the cell due to the osmotic force reduces gradually (S = P−T), until an equilibrium state is reached (P = T). In this case the turgor pressure is equal to the osmotic pressure and the suction force value is 0. During endosmoses the cell manifests itself as a self-regulating osmotic system, because the relationship between the turgor force and the osmotic pressure determines the absorption intensity and the volume of water that flows in it. This relationship was portrayed in a diagram by the German scientist K. Hoffler (Fig. 2.17).

Glossary Apoplast The totality of interfibrillar gaps, the so-called free space of the cellular envelope. Water and various substances circulate through the apoplast from cell to cell throughout the plant body. Cellulose (C6H10O5)n A polysaccharide that represents the basic component of the plant cell wall, and is usually in the form of fibrils. Its molecules are grouped into micelles, microfibrils and macrofibrils, forming the housing for the membrane

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that is embedded in an amorphous matrix of hemicellulose, pectins, lipids and proteins. It is the most common organic compound on earth. Hemicellulose A group of polysaccharides that don’t dissolve in water, but are soluble in alkaline bases. They are located in woody parts of the plants: straw, cobs, seeds, nuts, wood, bran. At acids hydrolysis form mannose, galactose, arabinose or xylose. Biological membranes Lipoprotein structures, with a thickness greater than 100 Å (there are internal and external membranes). Membranes delimit the protoplasm from the cell wall and the vacuole as well as from the cellular organelles. (network endoplasmic membranes). Membranes perform barrier, transport, osmotic, electrical, structural, secretory, assimilatory, receptory functions. Cellulose membrane (cell envelope cell wall) A constituent specific only for plant cells, the product of the metabolic activity of the protoplast. Protects the cell contents from injuries, confers a defined shape to the cell and is the basis of plant tissue mechanical strength and rigidity, participates in absorption and transport, accumulation and secretion processes. Osmosis Movement of water or solvent molecules through a selectively permeable membrane. Selective permeability The ability of biological membranes (plasmalemma, tonoplast) to allow the entrance of some substances and to prevent the elimination of others due to the particular structure of the membranes and their ability to perform active transport with the participation of membrane translocators and energy consumption. Plasmalemma The semipermeable external membrane that separates the cytoplasm from the cell envelope. Has a central role in the transport of ions and other substances in and out of the cell. Participates in cell wall formation. Plasmodesmata Microscopic channels which traverse the cell walls of plant cells connecting their cytoplasm. Due to plasmodesmata the protoplasm of all cells joins into a single whole called symplast. Proteins Macromolecular organic substances composed of amino acids bound by peptide bonds with a fundamental role in the lives of all living organisms. Perform most of the functions in living matter. Tonoplast A semipermeable membrane surrounding the vacuole and participating in the active transport of ions (identical in structure to other biological membranes).

References

References Acatrinei Gh (1975) Biologia celulei vegetale. București, Bolsover SR et al (2004) Cell Biol A short Course 2:531 Holl J et al Plant cell structure and metabolism. Ed 2. London, 1981, p 543 Burzo I (1999) Fiziologia plantelor de cultură. Știința 1:462 Johnson GB, Raven PH et al Biology. Ed. 6, McGraw-Hill, NY, 2002 Milică CI et al Fiziologie vegetală. București, 1982, p 376 Pickering WR (1998) Biologie, recapitulări prin Diagrame 1(2):71 Tarhon P (1992) Fiziologia plantelor, vol 1. Lumina, Chișinău, p 230

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Chapter 3

Water Regime

Abstract Water is a most important compound of living matter which is crucial for maintaining the normal physiological state of the cell. Due to its unique physical and chemical properties it serves as a basic solvent for mineral salts and organic compounds and is a medium for biochemical reaction progression. It has also a structural role ensuring the state and conformation of organic and inorganic molecules and macrostructures (like organelles), it is an important factor in plant temperature control through transpiration, ensures the turgid state of the cells and, thus, the rigidity of plant tissues, the paths of water flow in plants represent a means of transporting nutrients and regulators within the organism. The types of water in soil are distinguished by the force with which they are retained: constitutional water (enters in the composition of organic or inorganic molecules), hygroscopic water (retained by very strong forces as hydration layers of soil particles), pellicular water (retained as hydration layers around the hygroscopic water), capillary water (circulates freely and is weakly retained), and gravitational water (the excess water associated with precipitations, etc.). Plants use mostly capillary water but can also partially use gravitational and pellicular water. Water absorption (along with that of mineral elements) is the primary function of the root system and happens primarily at the level of root hairs which, due to their density, form a big absorption surface. The absorbed water is eliminated by transpiration and the balance between the two processes determines the amount of available water and the physiological state of the cells. The ascendant water flow (1.0–2.5 atm) generated by the root (at the level of the Kaspari strips) is called root pressure and is based on the use of ATP. During photosynthetic periods of vegetation, transpiration at the level of parenchymal cells of the leaves is generating a continuous ascendant and passive water flow (30–35 atm) called transpiration pull. Plant leaves have acquired a certain morphology and structure during evolution to balance the need to capture the scarce CO2 from the atmosphere and to minimize water loss. Thus leaves are covered with a cuticle made of waxes or with hairs that diminish transpiration intensity to only 10 % of the total. The other 90 % of the transpiration happen through stomata which are special pores whose opening or closure is controlled by the turgid or flaccid state of the two guard cells surrounding the pore. The turgor pressure of these cells © Springer International Publishing Switzerland 2015 M. Duca, Plant Physiology, Biological and Medical Physics, Biomedical Engineering, DOI 10.1007/978-3-319-17909-4_3

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depends on their ionic balance, on the humidity of the environment, CO2 concentration, exposure to sunlight, phytohormone action. According to their water regime plants have been classified in hydatophytes, homeohydrophytes and poichilohydrophytes.

Historical Background 1634—JB. Van Helmont concluded that water is a constituent of the organic mass of the cell. 1664—J. von Sachs demonstrated the role of temperature in water absorption by the root. 1671—M. Malpighi discovered stomatal cells and their function. 1727—S. Hales measured for the first time the intensity of plant transpiration. 1837—R.J.H. Dutrochet discovered the osmosis phenomenon. 1901—H.H. Dixon worked out the theory of the cohesion force. 1926—N.A. Maximov formulated his theory of plant resistance to drought. 1976—L.N. Babuşkin discovered the absorption mechanism of water vapor from the intercellular spaces in plants.

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Brief Updates The most important data that allow to understand the mechanism of water movement in living organisms were revealed with the discovery of the membrane proteins called aquaporins. Aquaporins belong to a large family of proteins homologous to the major intrinsic bovine protein (MIP) and are characterized by a structure containing integral membranous domains and by two repeats of a highly conserved amino acid sequences Asn-Pro-Ala. These proteins were discovered in plants in the late 80s of the twentieth century, concluding that some of them can act as water channels and can facilitate the diffusion of an enormous water amount along the transmembranous gradients. The research that had followed the first discovery of aquaporins found that they are very different in plants. Thus, the aquaporins of Arabidopsis are encoded by more than 30 homologous genes. Based on the homology between sequences, aquaporins biosynthesis genes can be classified into three subfamilies specific to plants, two of which correspond to a specific subcellular localization (for instance intrinsic to the plasma membrane or to the tonoplast). It is quite surprising that some aquaporins are multifunctional proteins and, besides water transport, may be also involved in the transport of osmosis-compatible solutions, like glycerol. Involvement of aquaporins in the transportation of gases like CO2 or NH3 is also possible, this process taking place in leaves or in symbiotic root nodules.

3.1 Role of Water in Plants Water is the main mineral compound of the living matter, on which practically all the vital processes depend and which maintains the normal physical state of the cell. The physiological function of the plant organs are only possible when the cells are saturated with water. Water is a most important constituent of plant life due to the physical and chemical properties it possesses. In the process of homeostasis maintenance and cell composition formation, water (Fig. 3.1) fulfills multiple roles: • a basic solvent for mineral salts and organic compounds and, at the same time, a dispersion medium for colloidal macromolecules and a medium for biochemical reaction progression; • an important factor in maintaining the stability of plant temperature helping to avoid tissue overheating, which could arise due to the heat released during the metabolic processes or under direct sunlight (summer); • an element of protoplasm structure, which is fixed electrostatically among the long catenae of polypeptides, allowing the physical and chemical properties of the protoplasm, favoring the formation of colloidal systems and determining the conformational structure of the proteins crucial for their functioning and also necessary to ensure the maintenance of the ultrastructure and the functional

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Fig. 3.1 a A water molecule, b hydration of polar molecules

• • • • • • •

activity of cell organelles. Protein dehydration leads to coagulation and sediment deposition; ensures the phenomenon of osmosis and allows turgidity, contributing to stomata movement, to plant orientation in space and to sprout, leave and other organ positioning and orientation; serves as a donor of protons and electrons for CO2 reduction in the dark phase of photosynthesis; is a component of the redox reactions of the Krebs cycle; participates in the reactions of hydrolysis, oxidation and reduction, assimilation and dissimilation; structural water in biological membranes ensures the assembly of the phospholipid bilayer, and thus, influences on the permeability of these membranes to electrons and protons; represents a universal carrier, ensuring the transport of dissolved substances through the xylem and phloem vessels, as well as the radial transport though the symplast and apoplast; ensures the integrity of plant organisms, forming a continuous flow from the root to the leaves, via which mineral salts and organic substances are transported.

3.2 Water Content and State in Plants

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3.2 Water Content and State in Plants Total water content in plants is highly variable and depends on plant species and, within the same species,—it depends on the organ, tissue, ontogenetic phase, etc. Thus, algae contain 94–98 % of water, succulent leaves—95 %, reserve organs— 85 %, leaves—80 %, dry seeds—12–14 %. Environmental factors and the organ type may influence the hereditarily expected values for this index. The variability of water content in this case is determined by the water retention capacity of the plant. Water retention is caused by osmotic forces, colloidal and capillary imbibition forces, etc. Protoplasmic colloids have a higher capacity to retain water in young leaves compared to older ones. Water in plant cells is retained in the cell wall, the protoplasm (up to 90–95 % of water), the vacuole sap (98 %). The amount of water retained by cellular envelopes depends on its thickness, structure and chemical composition. Approximately 7–8 % water is bound to cellulose polymeric chains and is retained by superficial bonds. The vacuolar sap contains up to 98 % water, which is retained by osmotic, electroosmotic and imbibition forces. Water is also a structural component of biological membranes—water interacting with the membrane surface, water located in the space between the internal and external chondriosome (mitochondria) membranes. There are 3 water aggregation states that can be found in plants. Water in liquid state is the basic component of all cells, because it is a component of the membranes (30–35 % of the membrane weight), of the protoplasm and vacuole. In its gaseous state (vapors), water can be found in intercellular spaces and in all the aeriferous tissues. Water in the solid state of aggregation represents ice crystals, formed during severe frost in intracellular and especially in intercellular spaces. Intracellular crystals break cytoplasmic membranes deteriorating the cells. Liquid water in the vegetal organism can be free (95 %), representing the basic solvent for mineral and organic substances, ensuring colloid micelle dispersion in the cytoplasm, or bound (4–5 %), retained by hydrogen bonds or by other types of chemical bonds or immobilized in fibrillar structures of macromolecules (Fig. 3.2). Free water is retained weakly in the plant organism. It circulates very easily in vacuoles, cytoplasm and conducting vessels either inside the cell, or from cell to cell, enabling, at the same time, turgidity. Free water represents the medium where the biochemical processes take place and it often directly participates in these reactions. Free water freezes at temperatures down to minus 10 °C, so plants with high content of free water are less resistant to low temperatures. Bound water is retained in plants very strongly. This water type is made up of immobile molecules with no possibility for diffusion or evaporation, it is hardly released by the cell. Bound water freezes at temperatures lower than −10 °C. It does not circulate in the cell or in the entire plant, doesn’t take part in biochemical processes and in dissolving organic or inorganic substances. Due to inability to act as solvent, bound water doesn’t participate in the transfer and circulation of substances.

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Fig. 3.2 Hydration of NaCl molecules

Bound water is held by: • osmotic forces, caused by dissolved substances whose dispersed particles retain water. Water retained by osmotic forces is called osmotic water (dissolving water). The elimination of this water form from the tissues (by transpiration) is the more difficult, the more concentrated the vacuolar sap is. • imbibition forces, caused by hydrophilic colloid soaking. Water retained by these forces is called imbibition water. There can be found numerous hydrophilic colloids in the cell (proteins, mucilage, cellulose), which retain water very strongly. Each mole of protein amino-groups is able to bind 2.6 mol of water and each mole of protein molecules (they can vary greatly in size)—tens of thousands of moles of water. Water can be fixed on the surface of various particles (ions or molecules) by absorption forces, which are mostly electrostatic forces, due to the bipolarity of water molecules. This fraction of water (adsorption or hydration water) determines the dissolution of colloidal particles by forming around them a dense shell of water molecules. Many organic substances from vegetal cells offer enormously large surfaces for water adsorption. For example, 1 g of cellulose provides an area of 1 million cm2, 100 average protein molecules bind 4–5 thousand water molecules. Regarding ions, usually the hydration layer is the thicker, the smaller the ion radius and the bigger its electric charge are. The imbibition of different proteins and mucilaginous substances in water is caused by electrostatic phenomena. Proteins are imbibed stronger in electrolyte solutions than in water, because they can fix ions strongly and the latter bring with them their hydration layers resulting in a significant combined effect. Adsorption of ions is inversely proportional to the size of their aqueous envelopes. Mucilaginous substances, by contrast, are imbibed more in water than in electrolyte solutions, because, fixing ions weakly, they have to compete with the ions in the solution for the solving water.

3.2 Water Content and State in Plants

45

There are numerous capillary spaces in plant cells (in vacuoles, in the spaces between the colloidal micelles of the membrane and the protoplasm (while at the tissue level—especially in the conducting vessels) in which the capillary forces retain water molecules. This fraction is called capillary water. There is also the constitutional water, chemically bound by certain molecules. Release of this water by molecules implies their destruction. The notions of free and bound water are relative, because these two forms of water can transition one into another. In unfavorable environmental conditions, when the vital activity of the plants is essentially reduced, the amount of free water decreases, while the amount of bound water increases, resulting in a higher stress resistance in plants. Quantitatively, free water always prevails when compared to bound water, however in drought conditions this difference is smaller. The critical limit of cell dehydration is 35 %, when vital processes are reduced to a minimum. According to its origin, water can be exogenous and endogenous. Most of it is of exogenous origin, absorbed by plants from the soil through the root system or, in a small amount, from the atmosphere in the form of vapors. Plants can obtain water through its aerial organs—leaves (from dew, rain water). During the drought period the dew has a marked impact on young leave hydration—50–70 % H2O, but less so in the case of old leaves—5–7 % H2O. Endogenous water is synthesized during the process of respiration in mitochondria.

3.3 Forms of Water in the Soil. Accessible and Inaccessible Water The amount of water absorbed by plants depends not only on the root system size, but also on the amount of water available in the soil and on the forces with which the last retains water. Water retention forces depend on the osmotic pressure of the solution present in the soil. Water can be found in many forms in the soil: • Constitutional water (crystallization water) enters in the composition of organic or inorganic molecules from the soil as crystallization water: for instance CuSO4·5H2O, Na2CO3·10H2O etc. This water form can’t be used by plants due to its huge retention force. • Hygroscopic water forms a very thin hydration layer on the surface of soil particles and is retained by very high forces (approximately 10,000–31 atm.). This water form can’t be used by plants, its removal from the particle’s surface being possible only by drying at 105 °C. • Pellicular water from the surface of soil particles is retained by the hygroscopic water film with forces higher than 30 atm, while the external layers of the film— with 0.5–30 atm. Plants can absorb only a certain part of pellicular water (that from the peripheral layers). Exceptions are some halophyte species that absorb water from the deeper layers as well.

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• Capillary water is mobile, moving ascendantly, being retained in the soil by forces smaller than 1 atm. It is easily absorbed by plants, contains dissolved minerals and represents their basic water supply. • Gravitational water is very mobile, moves descendantly, is located in big amounts in the large gaps between soil particles, accumulating after heavy rains or irrigation. Water contained in the soil, which is inaccessible for plants, is called physiologically dead water or dead water reserve in the soil. The soil dried up to the limit when it can’t release water contains inaccessible pellicular, hygroscopic and constitutional water. The amount of unused water in the soil during plant wilting got the name of wilting point (wilting coefficient). The weaker water is retained by the soil particles and solution the more plants are able to absorb it. Water mobility decreases, retention forces increase and the absorption process complicates as the soil is drying. The wilting point (θwp) can be calculated according to the formulae proposed by Brigs (θwp = hygroscopic water/0.67) and Bogdanov (θwp = 2·hygroscopic water). Wilting coefficients in different soil types are as follows: θwp = 1.0–1.1 for sandy soil, 6.5–6.9 for clayey-sandy soil and 16.6 for loam-clay soil. The useful water reserve in the soil is the amount of water available for plant growth and development.

3.4 The Root System as a Specialized Organ for Water Absorption During the evolutionary process, vegetal organism saturation with water has been possible permanently and sufficiently only for submerged plants (algae). Terrestrial plants, by contrast, are living in an aerial environment and loose a substantial amount of water through transpiration. Angiosperm plants have attained a continuous flow of water in their organisms, in order to keep the protoplasm of the assimilatory and other tissues saturated and to counter water loss through transpiration. This has been possible due to the development of a strong root system, of a dense network of conducting vessels, capable to deliver water in the most remote parts of the plant and due to the development of specialized protective tissues that protect the aerial parts against water loss. Plants absorb water from the substrate continuously during the entire life cycle through the root system, but can retrieve it also from precipitations, from fog or from dew with its aerial organs. Water is removed in a proportion of 99 % through transpiration and guttation processes. Continuous water currents between the root system and aerial organs represents an essential requirement for the metabolic activity and therefore for plant survival. In this case the root system plays the most important role in plant water supply.

3.4 The Root System as a Specialized Organ for Water Absorption

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Fig. 3.3 The root system of various species of plants (http://www.puc.edu/ Faculty/Gilbert_Muth/ botglosr.htm)

Root system functions: fixation in the substrate (laminaria rhysomes fulfill only the function of mechanical fixation), delivery of water and minerals, respiration, assimilation, substance storage, tissue regeneration. For an efficient execution of these functions, plants have developed the ability to orient in space, to respond to gradients of vital factors, to form a maximum area of contact with the soil. Morphological features: root system growth and development into the depth of the soil and close to the surface. The root system of plants varies in size and shape. The total length of the roots with their branches reaches some dozens of meters even in small plants. The total surface of the root is hundreds of times bigger than the surface of the stem and the leaves. The contact surface of the root system with soil particles, as well as the depth of root penetration into the soil vary in different species (alfalfa (lucerne)— 0.3 m, grapevine—18 m, spring wheat—2 m, potato—0.5 m) (Fig. 3.3). Those plants that have adapted to intense sunlight (heliophytes) lose more water through their aerial organs and have a much more developed root system than plants adapted for diffused light (sciophyts), which always grow in wet places and have a weakly developed root system. V.G. Rotmistrov, V.A. Kolesnik, A.L. Modestov had investigated the actual size of the root system. The length of all roots in cereals is 10,000 m, with 14 billion absorbing hairs. An apple tree, which has 10 branches on the surface, develops 45,000 branches at the root system level. The root system is developing better in a structured soil with sufficient humidity and adequate aeration. Root system formation can be stimulated through a set of actions like soil irrigation and fertilization and a series of agricultural techniques.

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Fig. 3.4 Anatomical structure of the root system: (a, b) (http://www.emc.maricopa.edu/faculty/ farabee/BIOBK/BioBookPLANTHORM.html)

Anatomical features: the absorption zone, Kaspari stripes and passage cells (Fig. 3.4). Water absorption in superior terrestrial plants is carried out by roots, and namely by the fine and always young root endings—the root hairs. Root hairs have a length of 0.15–8.0 mm and a thickness of 0.1 mm. They are formed at a short distance from the root tip in the area, where root growth in length ceases and they originate from the elongated external cells of the rhyzoderm. To the internal wall of the root hairs the protoplasm and the nucleus are localized, the interior is occupied by a large vacuole and a vacuolar sap that is more concentrated than the electrolyte solution in the external environment; thus a relatively high water permeability is conditioned. Root hairs are regenerating every 2–3 days, determining the continuous activity of the absorbing area. In Pinus silvestris up to 220 hairs per 1 mm2 can be found, in Secala cereale—about 2,500. The apical meristem can produce 200– 400 new cells per day (Vicia faba) or up to 2,100 (Zea mays), in conditions of sufficient water and nutrients. The membranes of root hairs lack cellulose, but contain callose, which is a product of the Golgi body, transported to the plasmalemma and cell wall through pinocytosis. Callose represents a β-glucane, with increased permeability compared to cellulose. Physiological features: as a result of evolution, the root system in superior plants has acquired the ability to perform growth movements towards water sources, called hydrotropism. Root orientation in space, its hydro-, chemo-, geotropic and branching behavior is regulated by endogenous phytohormones (AIA and ABA). Absorption of water and mineral salts is facilitated by the presence of the rhyzosphere and symbiotic bacteria and fungi.

3.5 The Influence of External Factors on Water Absorption Intensity

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3.5 The Influence of External Factors on Water Absorption Intensity The intensity of water absorption is conditioned by several environmental factors— soil temperature, aeration, pH value, the presence of toxic substances etc. During soil cooling, water absorption by plants ceases to the extent that it cannot compensate the water deficit in leaves. Plants from warmer regions stop water absorption at +5 °C. During sudden temperature decreases the absorption intensity reduces stronger than in the case of its gradual decrease (characteristics of protoplasm are being changed—its viscosity increases). Although the cold ground contains enough water it is physiologically dry to plants. For normal functioning and growth of the root system a good supply of oxygen (O2) is needed. In the absence of O2, root respiration stops and the alcoholic fermentation intensifies, resulting in alcohol as the final product. Its accumulation may cause root intoxication and therefore a decrease in water absorption. H2O absorption by the roots is reduced also when the soil accumulates large amounts of CO2 (4–5 % of CO2 in the soil has damaging action on roots). Hydrogen ion concentration in the soil solution changes protoplasm permeability, affecting the intensity of root growth. As a result of root activity and of various reactions taking place in the soil, different organic and inorganic substances are being formed that, by being adsorbed to the root surface prevent water absorption, a phenomenon that occurs especially in poorly aerated soils. The accumulation of high salt concentrations in the soil also negatively affects water absorption.

3.6 Water Elimination. Physiological Importance of Plant Transpiration Transpiration is the physiological phenomenon of water elimination through leaves. The transpiration mechanism in vascular plants consists of three stages: • water elimination on the surface of leaf cells and transformation of liquid water into vapor (liquid water is transported to the evaporation surface through the cell walls, where there is a lower resistance in comparison to the protoplasm and vacuoles); • water vapor transport through the lacunes and their accumulation in the subostiolar chamber; • water vapor diffusion and elimination through the open stoma. For evaporation of one gram of water from the leaf surface 10.5 calories are required, the major part of this energy being used for breaking hydrogen bonds. This process is very important in the life of a plant.

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The importance of transpiration • Transpiration is a means to achieve thermoregulation, because a transpiring plant has a temperature 6–7 °C lower than the temperature of a wilting leaf. Overheating could result in chloroplast damaging and to photosynthesis arrest • Due to transpiration, continuous water absorption and transport takes place together with the ions of mineral salts dissolved in it • Owing to transpiration, a continuous flow from the roots to the tip of the plant is established, thus contributing to the formation of an integrated communication system between all plant organs

3.6.1 Indices of Transpiration Transpiration intensity represents the index reflecting the amount of water evaporated from a surface unit per one unit of time. This depends on the species, light intensity, temperature, wind speed (15–250 g/m2/h during the day and 1–20 g/m2/h during the night). During the 24 h cycle the intensity of the transpiration forms a characteristic curve in which the minimum values are registered in the morning, then they gradually increase at 1–2 PM (when they reach the maximum values), and later decrease until 6 PM (when the intensity is very weak). Transpiration productivity is the index reflecting the amount of organic mass accumulated in plants during the elimination of a kilogram of water through transpiration (1–8 g of dry matter per 1 kg of water). The transpiration coefficient is the amount of water needed for the synthesis of a gram of dry mass, it varies from 250–300 to 700–800 l/kg, depending on plant species. Relative transpiration is the index showing the water removal rate per unit of leaf surface divided by the water evaporation rate from a free surface and has values ranging from 0.1 to 0.5. During the summer a corn plant eliminates through transpiration 150 g of water, a pea plant −5 kg, 1 ha of oats—3,000 kg while cacti that grow in deserts eliminate only 2,700 kg per ha annually.

3.7 Structure of the Leaf as an Organ of Transpiration The leaf is the basic organ where the process of photosynthesis takes place and is the main body for water removal via transpiration. For creating a larger surface area contacting with the atmospheric air in order to absorb more carbon dioxide (0.03 %) and sunlight, plants have to develop a foliar system as large as possible, but this creates a large surface of water evaporation as well. This is why, during evolution plants have developed a morphological-anatomical structure to balance and optimize both of the functions in order to survive (Fig. 3.5).

3.7 Structure of the Leaf as an Organ of Transpiration

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Fig. 3.5 Anatomical structure of the leaf

Leafs have a thickness of about 100–200 µm. Parenchymal cells of the leaf are arranged to form a sponge-like tissue. Between them there is a system of lacunae (gaps) constituting 15–25 % of the leaf volume. Carbon dioxide diffuses through assimilatory tissues, especially through the lacunar one (15–20 %). The leaf is covered with a protective tissue—the unicellular epidermis, consisting of compact cells. Each second cell in the leaf mesophyll is surrounded by a xylem vessel. Both in the petiole and mesophyll water moves through the veins, whose number decreases in the direction of the petiole. The smallest ribs consist of unique tracheids. In the leaves of some plants, especially those with C4 photosynthesis type, the conducting vessels are covered with a compact layer of parenchymal cells surrounding the vessels and serving as a mechanical support. The leaves of certain species are covered with a cuticle containing oxymonocarbonic acids impermeable to water. The cuticle contains waxes or hairs that diminish water loss, reducing the speed of air movement and scattering the light.

3.8 Stomatal and Cuticular Transpiration Throughout the period of growth, plant transpiration at the leaf level happens in a proportion of about 90 % through the stomata and 10 % through the cuticle. Stomata are found on the epidermis of unsuberized leafs and stems (Fig. 3.6).

3.8.1 Stomatal Transpiration Stoma represent special pores in the epidermis of leaves and other organs which are surrounded by two guard cells with a special shape (specialized parenchymal cells)

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Fig. 3.6 A stoma (photon microscope image)

and are the basic structures participating in water removal. Guard cells are characterized by: • a prolonged shape in monocotyledonous plants and a semioval shape in dicotyledonous plants; • a thickened inner cell wall; • chloroplasts containing chlorophyll. Mature leaves can contain between 50 and 500 stomata. The majority of cultivated plants with the horizontal positioning of the leaves contain stomata only on the bottom side of the leaf and are called hipostomatic. Plants with leaves positioned more or less vertically contain stomata on both sides of the leaf and are called amphystomatic. Aquatic plants containing stomata on their top parts, are called hyperstomatic. In most of the plants cultivated in the temperate zones, guard cells are located in the leaf epidermis. Plants growing in wet zones have the stomata exposed on cells located outside of the epidermis to stimulate transpiration. In drought-resistant plants stomata are buried in the leaf mesophyll. On average 10,000 stomata can be found on 1 cm2, making up 1–2 % of the foliar surface. Transpiration of branches and stems is very reduced compared to that of the leaves and happens at the level of lenticels. Lenticels correspond to a lack of cohesion between the suberized cells or to an absence of suberization. They make only up to 2 % of the suberized surface (see Chap. 9—Elimination of substances in plants). Plants can also regulate transpiration intensity and the volume of eliminated water. This regulation is carried out by the alteration of guard cell shape that contributes to opening and closing of the stomatal pore (the osteole). The mechanism of such changes in shape is based on cell turgidity variation. The following types of deformations can be distinguished in stomata (Figs. 3.7 and 3.8):

3.8 Stomatal and Cuticular Transpiration

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Fig. 3.7 Physiological deformations of guard cells

Fig. 3.8 Different states of the stoma

• Passive, conditioned by changes in the turgidity state occurring in neighboring cells surrounding the guard cells; • Active, caused by turgidity state changes taking place directly in the guard cells. Passive movements are hydropassive (those of opening or closing stomatal pores), they are determined by changes in water content. Active movements can be both hydroactive (those of opening and closing), dependent on water content and photoactive (also of opening and closing) caused by light. Hydropassive closure of stomata is related to the mechanical pressure exerted by the neighboring cells of the epidermis under full turgidity. Hydropassive opening occurs when this pressure is released during weak water deficit conditions. Hydroactive opening and closure of the stomata occur when transpiration levels become greater than water absorption by the roots and when the decrease in guard cell turgidity reaches critical levels. The photoactive opening and closure is caused mainly by the photosynthesis process occurring in guard cells. Internal and external environmental factors influence directly or indirectly the process of transpiration by affecting the turgidity of guard cells, which, therefore, lead to closing or opening osteoles. Osteole opening and closure depend on humidity, CO2 concentration in the atmosphere and in the intercellular space, on the ionic balance in guard cells, on the phytohormone amount (cytokinin stimulates

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osteole opening and abscisic acid—its closure). The degree of water supply, the deficit in relative humidity of the atmosphere, the wind speed etc. are important factors as well.

3.8.2 Cuticular Transpiration The cuticle is a barrier to water loss, because it is rich in hydrophobic substances, derived from the oxidation of fatty acids, and is less permeable for water. Its permeability varies considerably depending on the age and thickness of the leaf. The cuticle may also contain microscopic perforations or less hydrophobic areas, where water vapor diffusion is possible. Slight transpiration can also take place through thin cuticles. This type of transpiration is prevalent in young plants (50 %), which have not developed yet a thick cuticle, but it can also happen in mature plants during senescence (40 %), when the epidermis and the cuticle of the leaf begin to deteriorate. During vegetation the rate of cuticular transpiration does not exceed 10 % of the total transpiration. During winter, transpiration and water elimination from reproductive organs and buds happens through the cuticle. The intensity of cuticular transpiration is determined by the structure and the thickness of the cuticle and is 10–20 times lower than the intensity of transpiration through stomatal pores. For example, it is very insignificant in conifers and magnolia, which have a very thick and rigid cuticle. It is also less important in the life of cultivated plants.

3.9 Water Absorption Mechanism and Ways of Its Circulation in Plants Water absorption happens on the entire root system surface (total absorption), but the process is much more active through the specialized root hairs (active absorption). Enabling absorption are: imbibitions forces, the osmotic pressure, the suction force of the cells, cohesion forces, etc. The imbibition force of protoplasmic colloids is ubiquitous but has a most important role in seed germination. The seedlings formed during embryo germination contain cells with no vacuoles, which do not develop a strong osmotic force that would allow water absorption. Water penetration is conditioned by the hydrophilic colloids from the protoplasm and from the seed storage tissue. Water entering by means of imbibition is sufficient to determine rupture of the seed tegument, seed germination and certain levels of seedling growth. The osmotic force is developed by the osmotic pressure of the vacuolar sap in root hairs and cortical parenchyma. Water is absorbed when the osmotic force of the plants exceeds the retention of the water by the soil. The osmotic force value is

3.9 Water Absorption Mechanism and Ways of Its Circulation in Plants

55

determined by the concentration of osmotically active substances (sugars, organic acids, mineral substances) in the vacuolar sap. Water entrance into the cell does not depend on the osmotic pressure value, but rather on the difference between this value (P) and that of turgidity (T). This pressure difference, called the suction force (S), is the true active force, participating in water absorption (S = P – T). The suction force will increase when the value of the P – T difference will increase. For water absorption initiation the plant must have a suction force bigger than the osmotic pressure of the external solution from the soil. If the suction force of the hairs is lower than the osmotic pressure of the external solution, the hairs will lose water, will lose turgidity and will perish. Such situations occur very rarely, only when a large amount of fertilizers is negligently introduced into the soil. A similar situation can be created during a prolonged drought. Leaves are able to absorb part of the water fallen on them during rain, part of the condensation water (dew), fog water and even water vapors straight from the atmosphere. Absorption takes place mainly through the cuticle due to the suction force of epidermal cells. This absorption type is rapid, when the cuticle is thin and slow, if the cuticle is thick.

3.9.1 Water Transport in Plants In nonseptate thalophytes water flow takes place only intraprotoplasmically while in septate talophytes and in cormophytes—intra-and extraprotoplasmic. The intraprotoplasmic path allows only a slow water flow, but allows instead a physiological control of the water absorption speed and the nature of substances dissolved in it. The extraprotoplasmic path consists in water flow assisted by forces of colloidal or capillary imbibition and through diffusion in free aqueous spaces. This flow is faster and more economical, because it is passive and does not require energy consumption. Water can circulate in superior plants by: • transport through living cells and tissues; • transport trough dead cells and tissues (Fig. 3.9). Thus, from the root hairs the absorbed water is transported by tissues to the xylem vessels through the symplastic path (based on colloidal imbibition forces, on osmosis, electroosmosis, suction force, diffusion, etc.) and through the apoplastic path (based on capillary and colloidal imbibition forces, diffusion, etc.). This transport type is called radial transport. The rate of radial transport of water is 1– 2 mm/h and the distance does not exceed 1 cm (Fig. 3.10). Water is transported from the periphery to the center due to a higher osmotic pressure in the tissue neighboring xylemic vessels. This gradient is present even at the individual cell level. These examples show the phenomenon of polarity, characteristic for living organisms. The inner layer of the primary cortex—the endoderm, has a particular anatomical structure—the passage cells with thin cell walls,

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Fig. 3.9 Water transport in plants

Fig. 3.10 Pathways of water penetration into the cell

exposed opposite to the xylemic vessels, and to the cells with suberized cell walls. At the level of the Kaspari strips the flow of water reaches the xylem vessels only trough the passage cells, forming a pressure pushing water into xylem vessels with a force of 1.0–2.5 atm due to the free water potential. This force is called root pressure. The action of the pressure contributes to water flow up the xylem vessels when the foliar system is not present. This force is active and is based on the use of metabolic energy (ATP). An eloquent demonstration of the root pressure is the process of elimination of crude sap from stems or sprouts when cut. (After the leaves unfold, stem injuries do not cause sap elimination due to the suction force generated by the leaves.) In perennial plants this phenomenon can be noticed mostly during the spring period (birch juice) while for herbaceous—during the entire vegetation period. Crude sap, released through this process, contains minerals, organic substances, sugars, etc.

3.9 Water Absorption Mechanism and Ways of Its Circulation in Plants

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Summer crude sap in pumpkin plants contains ash particles, organic acids but no sugar. With the formation of the foliar system the importance of root pressure diminishes. During the warm summer period the essential role in water absorption is played by transpiration. During water elimination the osmotic potential in mesophyll cells decreases, aspiring water from xylem vessels. A suction force of up to 30–35 atm forms in this case, contributing to water flow from conductive vessels to the leaves and from roots—to xylem vessels. This force is called transpiration pull, and is generated in the parenchymal cells of the leaf and has self-regulating mechanisms. It is a passive force owed entirely to solar energy, which determines the intensity of transpiration by heating the leaves. Due to this force, a continuous flow is developing from the level of leaves to the root system level and during the vegetation period plant water supply is ensured. Transpiration pull values are 15–20 times higher than the values of the root pressure (Fig. 3.11). The distance water travels through leaf or root tissues is the fraction of a millimeter, but it faces more resistance here, compared to when travelling through the stem. Therefore, water flows through cortical and foliar parenchyma with a speed 10,000 times lower than the flow speed through xylem vessels. Specialized elements for water transportation appeared in cormophytes—the xylemic vessels, which supplied all plant organs and tissues. Xylem elements do not contain cytoplasm and are formed from the procambial cells of the root and stem. Cell walls rigidify and cells become dead to serve their role as conducting tissue. Tracheids appear in pteridophytes and are composed of solitary cells with perforated side walls. Tracheid appearance was an important step in the evolution of the plant kingdom. Tracheids appear in gymnosperms and angiosperms and consist of a large number of cells exposed in spirals, which have lost their lateral cell walls, forming xylemic vessels. Xylemic transport is obeying the laws of hydrodynamics by travelling along the gradient of water potential from the root to the leafs. The active forces that determine water circulation are created by the living cells at the upper and lower

Fig. 3.11 Water and nutrient circulation through the plant (John Johnson et al. 2002)

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ends of the vascular system but a special role is also played by cohesion forces. According to the theory of cohesion, water rises through the capillaries of the conducting vessels owing to the transpiration pull and also due to the cohesion forces between water molecules and the forces of cohesion of water molecules with hydrophilic capillary walls (adhesion forces). The speed of water transport by xylem vessels is 0.7–1.5 m/h in herbaceous plants and much higher in woody plants. Cohesion force values can reach 300–350 atm. When the stomata are closed and plants are supplied with water abundantly, water elimination takes place due to the phenomenon of guttation, i.e. removal of water drops by special structures called hydathodes. Guttation was observed in more than 300 types of plants and is characteristic for tropical and equatorial plants. Guttation is also an eloquent evidence of the root pressure.

3.10 Ecology of the Water Regime in Plants The water regime is the totality of the processes of absorption, transport and elimination of water, determining the ratio between the amount of water the plant receives and the amount which is used by it in a time unit. The water regime reflects the state of the water in the soil, the plant, the atmosphere and is in a dynamical equilibrium. Water regime regulation contributes to the maintenance of the hydric homeostasis. Life had appeared in water and remained enclosed in the cells of living organisms in a watery environment. During evolution, plants became more and more independent from it, this fact allowing their expansion on the globe. Sporophyte plants have retained their dependence on water in the reproductive process—their gametes move through water drops and by using flagella. Gymnosperm and angiosperm plants, by contrast, don’t need water for reproduction which is an evolutionary advantage. The ratio between the amount of absorbed and transpiration water characterizes the water regime of different groups of plants and represents the water balance. In gymnosperms and angiosperms this ratio is ≈1, due to: • • • •

a perfectly developed root system, necessary for absorption; the conducting vessels, necessary for transport; the protective tissues, necessary to minimize evaporation; stomata, which regulate transpiration. WB ¼ A=T  1ðMaximov 1926Þ;

3.10

Ecology of the Water Regime in Plants

59

where WB water balance, A absorption, T transpiration. The water balance (WB) depends on the environmental conditions and determines the optimal functional activity of the plant. As a result of evolution, several changes in organ structure and functions have emerged determining the water balance and contributing to the formation of certain ecological groups of plants characterized by specific water regimes. Terrestrial plants show a deficit of water (5–10 %), which doesn’t lead to functional disorders. But a higher deficit causes plants to lose their turgidity and wilt. Wilting may be temporary and permanent. Plants have adapted to survive in conditions of water deficit, developing various mechanisms to regulate water balance. Accordance to this, in 1973 Antipov proposes a classification of plants (Fig. 3.12):

Fig. 3.12 The particularities of the water regime in different groups of plants

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(1) aquatic plants (hydatophytes): • primary aquatic plants; • secondary aquatic plants; terrestrial plants (poikilohydric, homeohydric, hydrophytes). Hydatophyte plants include algae and secondary aquatic plants, which contain 90–95 % water, spend all their vegetation period in water, absorb it with the entire surface of the organism. Water regime control is reduced to mechanisms designed to remove water from the organism (contractile vacuoles). If the living environment dries up and dehydration happens the plant dies. Water regime is not regulated by the vital processes of the plant. Phylogenetically it is the most primitive mechanism of the water regime. In these plants there is no transpiration and no protoplasm imbibition. Poichilohydrophytes are more advanced plants (for example some algae, mosses and others). These plants contain 70–75 % of water and are also able to absorb water through the entire body surface. They do not have some specific mechanisms for water regime regulation, but they pass into the state of anabiosis in the case of dehydration, forming spores resistant to a very strong water deficit (2–3 %). Protoplasm transition into a gel-like state takes place. These phenomena are based on such physical processes as hygroscopicity, capillarity, imbibition, evaporation. In this case the water regime is more advanced and is characteristic for the first terrestrial plants, for which the intensity of metabolic processes depends on the amount of water available. But even this type of water regime is passive and simple. Homeohydrophytes are represented by some pteridophytes, angiosperms and gymnosperms. They differ by their water content in different organs (for example, 35–50 % in coniferous leaves, 90–95 % in cabbage leaves). This group of plants is characterized by specific mechanisms of self-regulation of cuticular and stomatal transpiration, opening and closing of the osteole, root system volume, hydrotropisms, osmotic pressure. These plants actively regulate water exchange. Water absorption and elimination is ensured by complex and active physiological mechanisms. They are less resistant to drought and die if there is a strong dehydration. The homeohydric type of water regime has appeared later than the hydatophytic and poichilohydrophytic as a result of phylogenetic adaptations to terrestrial life. Homeohydrophytes are xerophytes, hygrophytes and mesophytes. Xerophytes populate the steppe, dry zones, with a strong water deficit in soil and very hot and dry air. They are characterized by certain structural and anatomical features (a large number of cells, a greater number of stomata per surface unit, high chemical potential, high osmotic pressure), which allow them to adapt easily to drought. These peculiarities are called xeromorphic. The leaves of these plants can be covered with a brush of hairs or the leaf blades have a filiform shape to reduce

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the transpiration area. The trees and shrubs from this group of plants have an underground part often largely exceeding the size of the aerial one. Hygrophytes are plants that grow in the regions with constant humidity. These plants do not suffer from water deficit, so they have no special mechanisms that would limit transpiration. They possess a specific type of parenchymal tissue— aerenchyma, which stores oxygen needed for photosynthesis. The leaves are hyperstomatic. Mesophytes include most of the crop plants. The water regime in mesophytes was studied by the Russian scientist B.D. Zalenski. He elaborated a law that bears his name and states that leaves positioned higher on the stem possess better expressed xeromorphic features—the cells are smaller, have a larger number of stomata per surface unit of the leaf, the network of conducting vessels is denser, the number of root hairs per surface unit is larger, the palisade tissue is better developed. Upper leaves are distinguished by a stronger assimilation and more intense transpiration, the concentration of the cellular sap is higher and during wilting, they remove the water from inferior leaves, leading to their drying and death. One of the most important problems in biology is the problem of the evolution of the organic world, i.e. the investigation of the ways and mechanisms that have contributed to the formation of more complex structures and functions during phylogenesis. Any function has evolved in parallel to its corresponding structure and any structure—in parallel with its function—the evolution of photosynthesis, transpiration, protein synthesis, etc. It is possible that the development of the water regime has evolved with the emergence of different ecological groups of plants and biological phenomena related to the general level of organization, for example, plant adaptation to terrestrial life, the appearance of transpiration, the improvement of the photosynthesis process, respiration, phytohormonal regulation, etc.; special adaptive responses to one of the environmental factors, such as adaptation to drought, to water excess, etc. Based on this, there are hydrophytes (water-loving plants) xerophytes (resistant to draught) and mesophytes. It is considered that the evolution of superior plants has taken place at the mesophyte level, characterized by the highest adaptability range to different environmental factors. In homeohydrophytes, during the ontogeny process, all types of water exchange from the species phylogenesis appear, proving once more the universal law of biology, the biogenetic law of Müller and Hegel—onthogenesis repeats the phylogenesis. From the zygote to the embryo stage the plant’s water regime is hydatophyte. Then, from the seed formation to germination—the regime is poichilohydric. During vegetative growth plants develop certain features that allow strict regulation of the water balance and adopt the homeohydric water regime.

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Glossary Bound water Water linked with hydrophilic colloids of the protoplasm after their hydration, being retained with big forces, doesn’t diffuse, freezes at temperatures lower than −10 °C, doesn’t take part in the transformation and circulation of substances. Free water Water that preserves all the properties of pure water, moves freely, is retained by relatively small forces, has solvent properties, evaporates via transpiration and freezes at temperatures higher than −10 °C. Water balance of plants The ratio between the amount of absorbed water and the amount of water eliminated through transpiration. The value of this ratio depends on environmental factors, A/T > 1 is characteristic for humidity excess, and A/T < 1—for drought conditions. The volumes of transpired and absorbed water should be equal for normal growth and development. Wilting coefficient The amount of water in the soil expressed as a percentage, that has remained unused by plants during their wilting. For sandy soils wilting coefficient is 0.9 % and for the clayey ones—9.7 %. Transpiration coefficient The amount of water (g) eliminated by plants through transpiration, necessary for accumulation of 1 g of dry matter. Usually varies from species to species within the limits 300–1,000. Cohesion Property of water molecules to remain united due to attraction forces (hydrogen bonds). This phenomena can be observed in xylem tissues enabling water circulation in plants. Hydropassive stomata movement Osteole closure in conditions of high humidity. Takes place when guard cells are being pressed by the surrounding tissue at high water concentration in leaves (for example, during the long-periods of rain). Stomata open passively when weather is stabilized again. Osmosis Diffusion of water from a higher water potential to a lower one through semipermeable membranes. Root pressure The force that causes unilateral (upward) water movement through the root vessels. Transpiration productivity The value, which indicates the amount of dry matter (g) accumulated by the plant during the evaporation of 1 kg of water through transpiration. The average value of this index is 2–8 g. Dead water reserve of the soil The amount of water absolutely inaccessible to plants consisting of pellicular, hygroscopic and chemically bound water. The dead water amount depends on the soil type and its mechanical composition. The dead water content of fine sand is 1.3 %, of the sandy-clayey soil—10.2 %, of the silty-clayey soil—14.5 %.

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Crude sap Liquid eliminated by plants from the injured tissues of the stem or the root under the action of the root pressure. Chemically, the sap is an aqueous solution containing minerals and organic substances. Turgidity Water saturation state of the cells. Such a state provides the mechanical rigidity and strength of the tissues, contributing to plant shape maintenance and orientation of plant organs in space.

References Burzo I et al (1999) The physiology of crop plants, vol 1. Ştiinţa, 462 p Duca G, Zănoagă C, Duca M, Gladchii V (2001) Procese redox în mediul ambiant. Chişinău, 381 p Gusev NA (1979) Sostoyanie vody v rastenii. M. 1979, 130 p Johnson GB, Raven PH et al (2002) Biology, 6th edn. McGraw-Hill, NY Malinovskiy VV (2004) Fiziologiya rasteniy. DVGU Publishing, 103 p Polevoy VV (1989) Fiziologiya rasteniy. M. 463 p Sleycher R (1970) Vodnyy rezhim rasteniy. M. 265 p Zholkevich VN i dr (1989) Vodnyy obmen rasteniy. M. Nauka, 256 p http://www.puc.edu/Faculty/Gilbert_Muth/botglosr.htm http://www.emc.maricopa.edu/faculty/farabee/BIOBK/BioBookPLANTHORM.html

Chapter 4

Photosynthesis

Abstract Photosynthesis is the process of solar energy absorption by chlorophyll molecules and its conversion to the energy of chemical bonds by synthesis of organic substances from carbon dioxide and water. The advent of photosynthetic organisms during evolution means that food chains gained access to a limitless source of energy while the atmosphere started accumulating oxygen, which made possible the appearance of other species. The main photosynthetic organ is the leaf. It has acquired several adaptations during evolution, for instance a large surface area for absorbing CO2, a multitude of stomata for water but also for gas exchange, the presence of photosynthesizing cells organized in a bilayer structure to form a palisade parenchyma and a spongy parenchyma adapted for gas exchange, the synthesis of photoprotective pigments such as anthocyanins and carotenoids. Chloroplasts are the main photosynthesizing organelles, with a double membrane of which the internal one forms folds rich in chlorophyll and which either have the form of overlaid disks (granal thylakoids) or traverse the chloroplast from one edge to another (thylakoids of the stroma). The synthesis of the chloroplast components is regulated by both the chloroplast and the nuclear genome. The process of photosynthesis is possible due to a series of pigments for which the presence of a chromophore and a system of conjugated bonds are characteristic. These are represented by chlorophylls (a, b, c, d characteristic for different taxonomic units), carotenoids (carotenes, xantophylls, carotenoidic acid) and phycobilins (phycoerythrin, phycocyanin) all of these having specific light absorption patterns along the radiation spectrum. Consequently some of these molecules act as auxiliary pigments absorbing and transferring energy to the “main” pigments that carry the most important reactions of photosynthesis. Photosynthesis occurs in two stages: the light phase (Hill phase), which happens in the granal thylakoids and the dark phase (Blackman phase), progressing in the chloroplast stroma. The light phase is characterized by energy absorption by the light-harvesting complexes containing chlorophyll molecules “a” and auxiliary pigments (chlorophyll “b”, carotenoids, phycobilins), the transfer of the electrons from the reaction center by the Electron Transport Chain (ETC) coupled with the transport of protons, transformation of the

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proton gradient into ATP and NADPH+H+, water photolysis and O2 release. In the dark phase, fixation of CO2 by ribulose-1,5-diphosphate happens mediated by the enzyme RUBISCO, carbohydrate synthesis, with consumption of the ATP and NADPH+H+ formed during the light phase. In parallel with photosynthesis a process called photorespiration occurs characterized by CO2 elimination and O2 absorption. It is known to intensify during intense illumination, high temperatures or low CO2 concentration.

Historical Background 1771—J. Priestley has demonstrated that O2 is consumed by animals and is produced by plants. 1779—J. Ingenhousz has shown that light is necessary for green plants to produce oxygen. 1818—J. Pelletier and J. Caventou extracted a pigment from green leaves and called it chlorophyll. 1840—J.B. Bousingault proposed a global reaction for photosynthesis. 1845—J.R. Mayer showed that solar energy is transformed in the energy of chemical bonds. 1875—K.A. Timireazev formulated the idea of the global role of green plants.

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1877—W. Pfeffer introduced the notion of photosynthesis, from gr. photos —“light” and synthesis—“to put together”. 1922—O. Warburg realized the first measurements of the quantum outcome of photosynthesis. 1936—H. Gaffron and K. Woll formulated the concept of the presence of the reaction’s photochemical centers. 1938—R. Hill demonstrated elimination of oxygen during the light phase of photosynthesis. 1943—R. Emerson and collaborators investigated the phenomenon of photosynthesis relaunching—“the Emerson effect”. 1951—L. Duysens investigated the absorption and migration of the excitation energy in auxiliary pigment complexes. 1954—D. Arnon and collaborators discovered photosynthetic phosphorylation. 1956—M. Calvin and collaborators identified the reactions of CO2 reduction to carbohydrates. 1960—R. Hill and F. Bendall proposed the Z-scheme of electron transport during the light phase. 1960—S.E. Karpilov, M.D. Hatch and C.R. Slack (1966) discovered the C4 cycle of carbon assimilation. 1961—P. Mithcell elaborated a chemiosmotic hypothesis of the oxidative and photosynthetic phosphorylation mechanism.

Brief Updates Solar energy accumulation during photosynthesis depends to a big extent on the level of cell protection against oxidative degradation. Multiple antioxidant components (omega-3 fatty acids, vitamin E, carotenoids, etc.)—substances, which act to neutralize free radicals—protect both vegetal and animal cells preventing apoptosis, because the fundamental cellular signaling processes and the mechanisms of protection and adaptation are very conservative throughout the entire living world. Multiple extracts of algae and higher plants, products of photosynthesis, are capable to manipulate signaling processes in human cells and as a result, gene expression. For example, phytoestrogens (a group of flavones that play the role of messengers in plant—microbe interaction) mimic the activity of the human hormone estrogen. Carotenoids, such as zeaxanthin and lutein protect the photosynthetic systems from the destructive action of ultraviolet light, but can also be found in the human retina (Lutein is apparently employed by animals as an antioxidant and for blue light absorption while zeaxanthin may serve as a photoprotectant for retina from the damaging effects of free radicals produced by blue light). An important role in the regulation of plant life is played by the solar light spectrum, which, besides photosynthesis, participates in the regulation of certain physiological processes (like growth and development).

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4.1 Importance of Photosynthesis and the Global Role of Green Plants Photosynthesis is the process through which the energy of light is absorbed by chlorophyll molecules and converted to potential chemical energy of organic substances, composed of carbon dioxide and water. This process includes a large number of reactions, however, for autotrophic organisms producing oxygen, it can be summarized by the following equation: 6CO2 þ 6H2 O þ 673:8 kcal ¼ C6 H12 O6 þ 6O2 "

According to this equation, the outcome of photosynthesis is light-dependent CO2 fixation with its reduction to carbohydrates and the oxidation of H2O to O2. However, oxygen formation is not characteristic for all photosynthetic organisms. Some photosynthetic bacteria use as a hydrogen donor other inorganic substances (hydrogen sulfide, thiosulfite, etc.) or organic compounds (lactic acid, isopropanol). For instance, green sulfur bacteria use hydrogen sulfide. In this case the summary equation of photosynthesis is as follows: hv

CO2 þ 2H2 S !½CH2 O þ H2 O þ S2 In the process of photosynthesis, besides carbon dioxide that is the main acceptor of hydrogen ions in photosynthetic autotrophic organisms, sulfate or nitrogen can be reduced, forming hydrogen. Thus, the process of photosynthesis in different photosynthetic organisms can take place with the participation of different donors and acceptors of hydrogen ions.

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The appearance of green plants capable of photosynthesis (2 billion years ago) marked a very important step in the evolution of life on Earth. Photosynthesizing organisms and, consequently, all living organisms have gained access to a limitless and renewable source of electrons, participating in all bioenergetic processes— water. This fact has determined the extent of photosynthesis, assuring energy flow and transformation in the biosphere. Eukaryotic organisms (superior green plants and eukaryotic algae) and prokaryotes (photosynthetic bacteria and cyanophytic algae) capture solar energy, and convert it into potential chemical energy. This is why photosynthesis can be considered a phenomenon of cosmic nature, and the only means by which energy from a celestial body is fixed and stored on Earth in the form of biomass used later in all life processes of vegetable and animal organisms. The cosmic role of green plants has been widely described and argued by the Russian scientist K.A. Timireazev. Photosynthesis ensures the continuous existence of life by purifying the atmosphere from carbon dioxide. The oxygen released within this physiological process, replenishes its amounts in atmosphere, keeping it within limits optimal for respiration. Annually, green plants release 460 billion tons of oxygen in the external environment, representing the only natural source of oxygen. Photosynthesis represents also the primary source of all organic substances as well: carbohydrates, lipids, vitamins, proteins, hormones, glycosides, tannins, etc. These compounds synthesized by plants for their own needs serve later as the main nutrition source for the multitude of trophic chains. Photosynthesis constitutes a most crucial step for energy and matter circulation in nature (Fig. 4.1).

Fig. 4.1 The cycle of photosynthesis products in nature

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The unique physiological importance of this process can be generalized by the following contribution of photosynthesis in: • transforming the nature of the atmosphere from a reducing one into an oxidant one which lead to the advent and spread of aerobic organisms; • conversion of solar radiation into metabolic energy; • purifying the atmosphere through gas exchange; • formation of organic substances from inorganic ones; • formation of the reserves of mineral resources; • ensuring the circuit of carbon in nature. Photosynthesis as the most complicated fundamental biological process, is a research object for biologists, physicists, chemists, mathematicians, etc. Knowledge of the molecular mechanisms of photosynthesis is very important in solving many industrial-economic problems related to the use of ecologically pure unlimited energy sources (for example, obtaining oxygen and molecular hydrogen through water photooxidation), in increasing the photosynthetic productivity of plants, ensuring long-term cosmic expeditions with organic matter and molecular oxygen.

4.2 The Leaf as a Specialized Photosynthesis Organ Photosynthesis takes place in all plant cells that contain green pigments (leaves, branches, young stems, sepals, unripe fruits), but the organ specialized in fulfilling this function is the leaf, which shows some features formed during a long process of adaptation and improvement: • a large, flat area, adapted for absorption of large amounts of solar energy and CO2 from the atmosphere; • an epidermis provided with stomata through which gas exchange and transpiration occurs. Depending on the positioning of stomata in plants, they can be divided in 2 groups: amphistomatic (with stomata present on both sides of the leaf) and epistomatic (with stomata located only on one side of the leaf); • the presence of organelles specialized for photosynthesis—chloroplasts; • a bilayer structure, the assimilatory parenchyma being differentiated in palisade parenchyma that plays the main photosynthetic role and spongy parenchyma with a pronounced role in gas exchange. In young branches, seeds and unripe fruits, assimilatory cells with chloroplasts are located in the parenchymal layers under the epidermis. Intercellular spaces are very small which causes reduced CO2 absorption from the external environment (in comparison with green leaves); • the presence of conducting channels (phloem and xylem) which deliver mineral compounds and water to mesophyll cells and transport the elaborated sap with synthesized organic compounds to all plant organs. During evolution leaves have changed generating a great diversity, determined by the structural changes adopted for carbon assimilation (Fig. 4.2). Some plants

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Fig. 4.2 Structure of the leaf blade in plants with C3 and C4 types of photosynthesis

originating from tropical and subtropical zones (corn, sugar cane, etc.) have leaves with a particular anatomical structure that differs from the leaves of plants growing in temperate climate (300.000 species of plants), adapted to carry out photosynthesis in certain environmental conditions. The leaves of these species are well vascularized, the mesophyll is homogenous containing granal chloroplasts while conducting vessels are surrounded by a compact layer of parenchymal cells, forming a sheath of perivascular assimilatory tissue with big agranal chloroplasts (Fig. 4.2). Perivascular sheath cells are separated from the mesophyll and from the air of intercellular spaces by a film, resistant to carbon dioxide diffusion. Adaptive changes developed for the fulfillment of photosynthetic functions have been directed both towards ensuring the optimal conditions for intense absorption of solar radiation and to protect cells from photooxidation caused by visible spectrum radiation and UV rays. Depending on environmental conditions, the cell size, the morphology of the assimilatory tissue, the content and ratio of basic pigments (chlorophylls and carotenoids) change, allowing photosynthesis to proceed in conditions of both strong radiation (for some desert species), and low light (for tropical species living in the shade). In some species, the cells of the superior epidermis of the leaves, can focus light due to their shape increasing its intensity by 15–20 times. Leaves of the plants from sunny zones, have a small area, are thick, have a larger number of stomata and long palisade cells with chloroplasts containing less chlorophyll, but assimilating carbon more efficiently. Another measure to protect the cellular structures from optical radiation consists in the synthesis of auxiliary pigments with photoprotective properties. Such substances are anthocyanins, present in higher concentrations in young and senile plants; they are often formed as a result of plant response to a high intensity of the visible light, to ultraviolet radiation, to low or high temperatures and to other stress factors. These red pigments are located in the cells of the superior epidermis and provide an effective screening in the green region of the spectrum in which the leaves are mostly “transparent”. On the action of UV radiation, the synthesis of several phenolic compounds is induced. They are accumulating in the cuticle and epidermal cells, ensuring UV absorption and tissue protection from its damaging effect. Other compounds playing the role of photoprotection in foliar tissues are carotenoids, which ensure, at low concentrations, a strong absorption in the

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indigo-blue region of the spectrum blocking photo-destructive processes. Their synthesis is activated before the period of vegetative pause in deciduous trees and before drought in tropical species—a period associated with the destruction of the photosynthetic apparatus and exposure to photooxidative stress determined mostly by the high intensity of the solar light.

4.3 The Structure, Chemical Composition, Function and Origin of Chloroplasts Chloroplasts are organelles specialized for fulfilling the photosynthetic function and represent microstructures with the length of 5–10 µm and a diameter of 2–3 µm, with spherical, oval, discoid or ellipsoid shape. In the majority of green plants ellipsoid chloroplasts predominate; this shape proved to be the most rational, developing during the evolution of the vegetal world. The number of chloroplasts varies from 20 to 100 per cell, depending on the species, environmental conditions, foliar tissue. The plastids of the cell are constantly moving, either passively with the cytoplasmic flow or actively, requiring energy consumption and being determined by light intensity and by other factors. The structure of chloroplasts. The fundamental substance of the chloroplasts, called stroma, is limited to the exterior by a double lipoprotein membrane (with the thickness of 10–30 nm) containing a large number of pores with a surface area of 30–40 nm2. The internal membrane, that has no pores, is less permeable in comparison to the external one, but it can be passed by molecules of trioses and amino acids. It forms folds called thylakoids (from thylacoides—“bag-shaped”) along the longitudinal axis of the chloroplast and which either have the form of overlayed disks (these are called granal thylacoids) forming the structures called grana (from granum—“granule”) or traverse the chloroplast from one edge to another—thylakoids of the stroma (Fig. 4.3). Fig. 4.3 Structure of a chloroplast (Tihonov 1996 )

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The number of grana in chloroplasts and the number of thylakoids in a granum vary within large limits. It is considered that a chloroplast contains about 40–100 grana and the total area of the thylakoids is 500 times bigger than the external membrane surface—a peculiarity that is considered an adaptation for carbon assimilation by chlorophyll given the low concentration (<0.03 %) of carbon dioxide in the atmosphere. The chloroplasts of some superior plants, just like those of algae, have no grana, each 2–8 lamellas being united in “packages”. Chloroplasts can be of several types: • granal (containing granal and stromal thylakoids), characteristic for all superior plants; • agranal, containing only stromal thylakoids—characteristic for algae and for the perivascular sheath cells from plants with C4 photosynthesis type. According to the results of electronic microscopy, granal and stromal thylakoids contain a large number of lipoproteic spherical structures, called quantasomes or photosynthetic units consisting of pigments and components of the system of photosynthetic electron transfer (ETC) and components of the system of ADP phosphorylation that is coupled with this transfer (Fig. 4.4). The synthesis of the proteins and lipids (glycosyl-glycerides) of the thylakoidal membranes is regulated by both the nuclear and chloroplast genomes. Approximately 50 % of the proteins involved in the formation of ETC complexes (40 proteins) are encoded by chloroplast DNA, the rest—by nuclear DNA. The latter are synthesized in the cytoplasm then enter the chloroplasts, where, by binding specific proteins, they form functionally active protein complexes assembled and distributed in an oriented manner in the thylakoidal membrane. There are several types of such macromolecular complexes which ensure the functions of absorption and transformation of the solar energy into that of chemical bonds (light phase of photosynthesis): • • • •

photosystems I (PSI) and II (PSII); cytochrome complex b/f; light-harvesting complex; ATPase complex.

Fig. 4.4 Schematic representation of quantasome localization in the thylakoid membrane

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The coordinated activity of the nuclear and chloroplast genomes in the synthesis of these molecular complexes is represented schematically in Fig. 4.5. Another example of regulation of the photosynthetic apparatus by the nuclear genome is the synthesis of the key enzyme of the dark phase (the Calvin-Benson cycle)—ribulose-1,5-bisphosphate carboxylase (RUBISCO). The functionally active enzyme is composed of eight small and eight large subunits (Fig. 4.6). The bigger subunit (54 kDa) is encoded by the chloroplast DNA, and the smallest (14 kDa)—by the nuclear DNA, which, after being synthesized (translated) in the cytoplasm, is transported into chloroplasts, where the correct assembly of the

Fig. 4.5 The assembly of the basic ETC complexes, formed by proteins encoded by the nucleus (yellow) and the chloroplast (green) (Hermann 1992). Violet color shows unknown origin. 1 PS II; 2 cytochrome complex b/f; 3 PS I; 4 ATPase complex

Fig. 4.6 Subunits of the enzyme ribulose-1, 5-biphosphate carboxilase (Kulaeva 1997)

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enzyme takes place in the presence of another chaperone protein of 60 kDa also encoded by the nucleus. Proteins encoded by the nucleus with chloroplast destination enter the organelle using a localization signal (a peptide of about 40 aminoacids for stromal proteins and more than 80 for thylacoidal proteins localized at the N-terminus of the protein molecule. This region recognizes the receptors of the external membrane of the chloroplast, triggering the translocation of the entire protein into the stroma, where, under the action of a specific peptidase (signal peptidase) cuts the localization signal from the protein (by hydrolyzing a peptide bond). The protein can then be included in the corresponding poly-enzymatic complex. The biosynthesis of proteins encoded by genes of the chloroplast genome occurs in the stroma—the fundamental substance of the organelle, which contains ribosomes, RNA, DNA and other components of the proteosynthetic system. Proteins, specific for thylacoidal membranes are distributed according to certain physicalchemical factors, including the amount of charge on the membrane surface. Big differences have been noticed between the localization and the ratio of protein complexes within the thylakoids of the grana and that of the stroma. Photosystem I and the H+-ATPase complex are located in stromal thylakoids, while photosystem II and the proton pumps—in granal thylakoids. The physiological activity of the chloroplast is ensured by a certain level of hydration (on average 58–75 %) and by a whole range of organic and mineral substances present in the stroma, in which the enzymatic reactions of photosynthesis take place (the dark phase). Chloroplasts contain enzymes involved in the process of photosynthesis, carbohydrate phospholipid and chlorophyll biosynthesis, in reactions of chlorophyll and starch degradation etc. Some enzyme molecules are absorbed by the chloroplast lamellas, others can be found in free state. The largest amounts of chlorophyll and carotenoids are concentrated in thylakoids (Table 4.1). The formation of all molecular and structural components which enable the physiological role of the chloroplasts (Fig. 4.7) is ensured by the nucleus-cytoplasm-chloroplast interaction which explains the failure to obtain isolated cultures of chloroplasts on a nutrient substrate. The origin of chloroplasts. Chloroplasts represent a variety of the organelles specific for plant cells—the plastids, formed from the so-called proplastids, found in meristematic cells. Proplastids are colorless vesicles with a diameter of 0.5–1.5 µm, delimited by a double lipoproteic membrane containing a double stranded circular

Table 4.1 The chemical composition of the chloroplast (% of dry mass)

Proteins (enzymes, membranes, stroma) Lipids (membranes, stroma) Mineral elements (ionic pumps, pigments, stroma) Carbohydrates (membranes, stroma) DNA/RNA (stroma) Chlorophyll (membranes) Carotenoids (membranes)

35–55 20–30 6–16 8–10 ≤0.5/2– 3 5–9 4–5

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Fig. 4.7 The schematic representation of the physiological role of the chloroplast

coiled DNA molecule with a size of 130–160 thousand base pairs (bp) encoding about 130 genes. Proplastids have no developed internal membrane system. The cytoplasm of the mother cell contains some proplastids, which pass into the daughter cell during division, these proplastids in turn divide to increase their number. Thus, genetic characteristics determined by the chloroplast DNA are transmitted only through the maternal line (cytoplasmic inheritance). Other types of plastids from plant cells, which may be colorless (leucoplasts, etioplasts) or colored (chloroplasts, chromoplasts) can be also formed from proplastids. With cell growth proplastids increase their size as well, the internal membrane is growing more intense, forming vesicles (thylakoids), which lie parallel along the stroma. In the presence of light, these discs arrange in a certain position relative to the plastid axis, suffering a differentiation into stromal and granal thylacoids. This process is accompanied by the biosynthesis of lipids, proteins, chlorophyll and their inclusion in the structure of the membranes. In the dark, etioplasts form, which have an internal structure similar to a crystalline network of protochlorophyllids (chlorophyll a precursors lacking the phytol side chain, which can be added only in the presence of light in angiosperms). Ethyolated tissue exposure to light causes the reorganization of the etioplast internal structure into a membranous structure, characteristic for chloroplasts (Fig. 4.8). Under the influence of certain environmental factors as well as according to a specific genetic program, chloroplasts can transform into etioplasts, amyloplasts (plastids that deposit starch) and chromoplasts (that are forming during the autumnal period in leaves and some fruits and flowers during their maturation, giving them the characteristic yellow-orange color, determined by the carotenoid pigments). Chloroplast transformation into chromoplasts is caused by the destruction of the granal thylakoide structure and the formation of a new internal structure

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Fig. 4.8 Development and transformation of chloro-chromo-amyloplasts

under the control of the nucleus through specific proteins encoded by it and synthesized in the cytoplasm. For example, the 58 kDa polypeptide that forms complexes with carotenoids represents half of all the proteins contained in the membrane structures of the chromoplast. The differentiation of plastids from proplastids into green photosynthesizing chloroplasts, into white amyloplasts containing starch or into yellow-orange chromoplasts, full of carotenoids and their inter-conversions are controlled by their own DNA and the nucleus-cytoplasm interaction. The study of molecular mechanisms of plastid differentiation and transformation represents a big interest to modern molecular biology, because this knowledge allows to understand better the signaling systems, which ensure the coordinated functioning of the nuclear and chloroplast genome in the plant cell.

4.4 Photosynthesis Pigments The selective absorption of solar radiation in the visible spectrum region of 400– 700 nm at levels sufficient for photosynthesis is a particular feature of several organic compounds (pigments), which have in their structure chromophore groups and systems of conjugated bonds. Plastid pigments may have a different chemical composition and are classified into three groups: chlorophylls, carotenoids and phycobilins.

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Chlorophyll pigments are present in superior plants and in algae in four forms: chlorophyll a”, “b”, “c” and “d”. All photosynthesizing plants and all groups of algae and cyanobacteria contain chlorophyll “a”. Chlorophyll “b” is typical for superior plants and green algae. Brown algae contain chlorophyll “c” and red algae —chlorophyll “d”. Photosynthesizing bacteria contain various types of bacteriochlorophyll (Fig. 4.9). In the seeds of certain plants (pumpkin, hemp) protochlorophyll was detected. In superior plants this chlorophyll type is synthesized during the dark phase and on light exposure transforms into chlorophyll. Chlorophyll is one of the most complex organic substances, representing a double ester of chlorophyllin with phytol (the alcohol of a higher carbohydrate with 20 carbon atoms and a double bond) and methyl alcohol. The most common are chlorophyll “a” and “b” (Fig. 4.10).

Fig. 4.9 The diversity of chlorophyll pigments

Fig. 4.10 The structure of a chlorophyll molecule

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Four pyrrol rings (pyrrol—heterocycle of 4 carbon atoms and a nitrogen atom), interlinked by methyl bridges, form the porphyrin core with one atom of Mg in the center, to which the phytol group (C20H39OH)is linked and a methyl group (–CH3) for chlorophyll “a” or aldehyde group (–CHO)—for chlorophyll “b”. Chlorophyll molecules also contain a pentanonic cycle (the fifth) consisting only of carbon atoms. Chlorophyll manifests hydrophilic properties due especially to the pentanonic homocycle but also hydrophobic and lipophilic properties—due to the phytol group. The presence of magnesium and of the conjugated system of double bonds in the porphyrin core confers the green color characteristic for chlorophyll. The spatial organization of chlorophyll is very important for its activity, during the process of photosynthesis. The porphyrin core—the hydrophilic part of the chlorophyll molecule, with a surface of 1 nm2 and a thickness of 0.42 nm—is located in the space between the contacting membranes of the granal thylakoids. The phytol radical (2 nm) forms an angle with the porphyrin core and is oriented to the interior of the spherical photosynthetic unit, being in contact with the hydrophobic groups of membranous proteins and lipids (Fig. 4.10). In plants, chlorophyll represents a lipoprotein complex in monomeric form (active) or aggregate (storage form) one only 3–4 % of its total amount can be found in free state. The quantity of these pigments in leaves varies within the limits of 0.2–0.8 % of the total dry mass, a high content is characteristic for sciophytes. In green algae, the amount of chlorophyll can reach 3 % of the dry mass. The ratio between chlorophyll “a” and “b” in plant tissues is 3:1, but may increase in heliophytes. Chlorophyll is in a continuous process of biosynthesis and degradation. It is constantly renewed (40 % in 2–3 days). Carotenoid pigments are liposoluble compounds present in chloroplasts and chromoplasts. They can’t be observed in green leaves, because of the presence of chlorophyll. In autumn, when the latter is destroyed, the foliage becomes yelloworange due to these pigments. Bacteria and fungi also contain carotenoids. Approximately 400 pigments from this group were studied (Fig. 4.11). Carotenoids are unsaturated carbohydrates with conjugated double bonds, derived from isoprene CH2 = C(CH3)–CH–CH2. They can be acyclic (lycopenes), mono and bicyclical (Figs. 4.12 and 4.13). Due to conjugated double bonds carotenoids are able to perform redox reactions. Carotenoids, like chlorophylls, are non-covalently linked with proteins and lipids of photosynthetic membranes. Due to the physical-chemical properties, this group of pigments fulfills a double role: that of solar energy absorption and transfer to the chlorophyll pigments and, at the same time, it plays a photoprotective role. Carotenoids are synthesized in chloro-and chromoplasts by the mevalonate cycle starting with acetyl-CoA through a series of intermediates—mevalonate, geranylpyrophosphate, lycopene, which serve as precursors for other carotenoids. Phycobilin pigments enter the group of biliary pigments (in animals, a representative of this group is bilirubin). They can be found in some red, green, blue and pyrophyte algae and in a very small amount in chloroplasts of green plants (Fig. 4.14). The following pigments from this group are known:

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Fig. 4.11 Principal groups of carotenoids

Fig. 4.12 Molecular model of carotene

• Phycoerythrin—C34H47N4O8, present in red, green, blue and pyrophyte algae; • Phycocyanin—C34H42N9O4, present in blue-green algae. Phycobilins are tetrapyrroles in which the pyrrol rings are linked by methyl and methine groups as an open chain (Fig. 4.14). Phycobilins are chromophore groups of phycobiliproteins (globulins which connect via covalent bonds, unlike chlorophyll). Phycobilichromoproteids are located in specific granules, called phycobilisomes, situated on the external surface of photosynthetic lamella of algal cells. These hydrosoluble chromoproteids serve as auxiliary pigments for light absorption (analogically to chlorophyll “b” in higher plants). About 90 % of the light energy is transmitted to chlorophyll “a”. All plants contain a special pigment—the phytochrome, whose prosthetic group, similar to phycobilins, is the bilinic pigment. The phytochrome is a photoreceptor of the red spectrum, performing the function of physiological regulation in many processes.

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Fig. 4.13 Structural formulae of some carotenoids (I β-Carotene, II Astaxanthine, III Cantaxanthine, IV Zeaxanthine, V Lutein, VI β-Criptoxanthine)

4.5 Photosynthesis Energetics The most important peculiarity of photosynthesis is the use of solar energy, that is the energy of electromagnetic oscillations, characterized by a certain wavelength (distance between two consecutive maximum points of a cycle), an oscillation frequency and a speed of dispersion: k¼

c m

where λ—wavelength, nm; c—speed of light, 2.997 × 108 m/s; ν—oscillation frequency, Hz. Solar light includes radiation from: The visible spectrum: violet (400–450 nm), indigo (400–450 nm), blue (450– 500 nm), green (500–570 nm), yellow (570–590 nm), orange (590–610 nm), red (610–700 nm) as well as from the invisible one, which includes:

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Fig. 4.14 Molecular model of phycobilin

ultraviolet: (λ < 400 nm) and infrared: (λ > 700 mm) (Fig. 4.15). Radiant energy is distributed in the form of quanta or photons. A light quantum contains a quantity of energy directly proportional to the oscillation frequency and inversely proportional to λ: E ¼ hm ¼

hc k

where h—Planck’s constant (6.62 × 10−27 erg/s, 1.584 × 10−34 cal × s or 6.626 × 10−34 J × s). The energy value for quanta of each radiation type is constant and depends on the wavelength. Thus, the bigger the wavelength, the lower the quantum energy. At the molecular level quantum energy values are significant. For example, quantum energy for 500 nm radiation is equal to 2.48 eV, which is 10 times bigger, than the energy derived from ATP hydrolysis. Quantum energy can be expressed in different measure units: ergs, calories, electron volts (eV), but the international measure unit for energy is Joule (J). The Einstein unit (Es) which represents the energy contained in one mole of photons can also be used. Photochemical reactions are possible in the case of visible rays of solar light with quantum energy of 1–3 eV or 150–587 kJ/mol. Plants absorb this radiation at high speed, but depending on the wavelength of the corresponding radiation. Infrared rays (0.01–0.1 eV) and ultraviolet ones (6–10 eV) are not used in photosynthesis.

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Fig. 4.15 Spectral composition of the solar light

Ultraviolet radiation absorbed by leaves in large quantities induces mutagenic effects, caused by the very high energy of the quanta, which exceeds the energy value of many chemical bonds in proteins, nucleic acids and other components of the cell. According to the energy outcome of photosynthesis, for reducing one molecule of CO2 to carbohydrates 480–528 kJ (3–4 quanta of light) are needed. But part of the energy absorbed by the leaf is eliminated as heat and fluorescence. That is why it is estimated that 8–12 quanta per molecule of carbon dioxide are needed. Light utilization coefficient (amount of light energy used in photosynthesis from the total amount of energy absorbed by the leaf) is very low. Only 2–5 % of the total quantity of solar energy that reaches the leaf surface, are used in the process of photosynthesis; the rest remains unused (Fig. 4.16): • 10–15 % is reflected from the leaf surface depending on the properties of the cuticle (smooth/rough, glossy/matte); • 10–15 % of the energy passes through the leaf without being absorbed, depending on the thickness of the leaf blade; • more than 70 % of the light energy that reaches the leaf surface is absorbed, 20 % dissipates as heat, about 45 % is used in the transpiration process as latent heat of vaporization. Green leaves strongly absorb light radiation (a low value of light reflection and passage through the leaf) in the violet, indigo, blue and red spectral regions. In this region of the solar spectrum, light is practically totally absorbed by the leaf surface,

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Fig. 4.16 Schematic representation of light interaction with the leaf

varying slightly depending on the concentration of chlorophyll. Rays with a wavelength of 520–580 nm to a great extent pass through the leaf without being absorbed, therefore leafs looks green (Fig. 4.16). During autumn chlorophyll degrades and leaves become more “transparent”, allowing not only green, but also orange and red rays to pass through. The light energy utilization coefficient varies from species to species, depending on the physiological, morphological and anatomical particularities of the plants (see Table 4.2). The reduced value of the light energy utilization coefficient is determined by the high percentage of the radiation unused by chloroplasts: both from the visible spectrum (green, yellow) and the invisible one (infrared, ultraviolet). Infrared radiation (λ > 750 nm) is absorbed less compared to the radiation of the visible spectrum, because it is reflected to a big extent (50 %), especially in the case of direct sunlight and less so in the case of diffused light. Diffused light is used in photosynthesis at a higher rate compared to direct light, this fact explaining high light utilization coefficients in submerged aquatic plants (in seas and oceans). Capturing the entire spectrum of solar light in its visible region is ensured by the presence of a wide range of pigments that selectively absorb light energy. There are two absorption maxima in chlorophyll types “a”, “b”, “c” and bacterioviridin: one in the red and another in the violet-blue region of the spectrum. Absorption maxima for chlorophyll “a” (in organic solvents) are within the limits of 660–663 nm in the red region and 428–430 nm in the blue region. For chlorophyll “b” these limits are respectively 642–644 and 452–455 nm. Absorption maxima of chlorophyll “b” are situated between the absorption maxima of chlorophyll “a” (Fig. 4.17). Absorption in the blue-violet region is determined by the system of conjugated double and simple bonds and the porphyrin ring while in the red region—by the presence of

4.5 Photosynthesis Energetics Table 4.2 Light energy utilization coefficient

85 Plant species

Light utilization coefficient (%)

Corn Barley Oats Spring wheat Potato Fall rye Beet

4.5 2.9–3.5 3.3 3.3 3.0 2.6 2.1

Fig. 4.17 The absorption maxima of some pigments

magnesium in the porphyrin core. Chlorophylls absorb orange and yellow light very weakly and do not absorb green and infrared light at all. The position of the absorption maxima in the spectrum is influenced by the nature of solvents, the extent of interaction between chlorophyll molecules with themselves and with other pigments, lipids and proteins. The absorption maxima for carotenoids lay in the violet-blue region of the spectrum—400–500 nm (Fig. 4.17). Energy absorption in the blue-green region of the spectrum, 70 % of the absorption is due to these pigments and only 30 %—due to chlorophyll. This feature of carotenoid absorption is important in carrying the process of photosynthesis during cloudy weather when blue-violet rays dominate, because the molecules of carotenoids (in contrast to xanthophylls) function as auxiliary pigments in transferring energy to chlorophyll molecules. Chlorophylls absorb violet-blue light more intensively, than red light (Fig. 4.18) but carbon assimilation in the red region of the spectrum is more efficient. The intensity of the photosynthesis process in different regions of the spectrum is called action spectrum. The action spectrum is similar to the absorption spectrum by the position of the light absorption regions and photosynthesis but it reflects photosynthesis intensity values (rather than absorption values) which indicate the varying efficiency of using energy of different wavelength absorbed during photosynthesis.

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Fig. 4.18 Dependence of the reflection coefficient (R) at 550 and 700 nm on the species

The intensity of photosynthesis in the violet-blue spectrum zone makes up only about 14 % of the photosynthesis happening under red light, indicating the inefficient use of energy in this spectral region. This phenomenon can be explained by the photoelectric effect theory, formulated by Einstein, which also states that the number of molecules involved in photochemical reactions is determined by the number of absorbed photons and no by the amount of energy contained in the photon (within energy limits which make possible a photochemical reaction). Red radiation quanta have the smallest amount of energy −159 kJ from the entire photosynthetically active radiation zone (A = 400–700 nm), but it is used efficiently by the molecules of chlorophyll in most of the plants. Blue radiation quanta have big energy value (297 kJ), this fact determining the achievement of a photosynthetic maximum, but quantum absorption in this spectral region is accompanied by the dispersion of an important amount of energy as caloric rays which is the reason why photosynthesis efficiency is less than in the case of red rays. Therefore the green color of the plants is not accidental. During evolution, plants have adapted to the composition of solar light, absorbing those rays of light (red) that are used more efficiently in the process of photosynthesis—the latter being a color complementary to green.

4.6 Photosynthesis Mechanism According to the modern theory regarding the molecular mechanism of photosynthesis, this process is a chain of successive redox-reactions, which requires sunlight at early stages (Robin Hill phase), while subsequent steps can occur in the dark (F.F. Blackman phase) (Table 4.3).

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Table 4.3 Comparison of the light and dark phases of photosynthesis Light phase

Dark phase

• Different photochemical and photophysical processes, including water photolysis take place • H2O, chlorophyll and solar energy are used • O2 is formed • Solar energy is included in ATP and NADPH+H+ • Temperature has no influence on light reactions

• Enzymatic reactions take place

• Reactions depend on the amount and intensity of light (Emerson effect) • Reactions take place in granal thylakoids • Is common for all species

• CO2, ATP and NADPH+H+ are used • Carbohydrates (CH2O)n are formed • The energy of ATP and NADPH+H+ is included in organic substances • Temperature influences enzymatic reactions. A 10 °C increase in temperature leads to a 2–3 times increase in reaction rates • Enzymatic reactions don’t depend on light intensity • Reactions take place in the chloroplast stroma • The mechanism differs from one plant species to another (C3, C4, CAMphotosynthesis)

In the light phase of photosynthesis absorption of light occurs by chlorophyll molecules “a” with the participation of auxiliary pigments (chlorophyll “b”, carotenoids, phycobilins) and transformation of solar energy into ATP and NADPH +H+. All these processes are carried out in photochemically active chloroplasts membranes, and represent a complex system of photophysical, photochemical and chemical reactions. In the dark phase of photosynthesis carbon fixation by the primary acceptor (ribulose-1,5-diphosphate) happens, involving enzymes located in the chloroplast stroma and with energy consumption in the form of ATP and NADPH+H+ which are the final products of the light phase.

4.6.1 Light Phase of Photosynthesis The processes occurring during the light phase of photosynthesis can be related to: (1) Absorption of carbon dioxide; (2) Absorption of solar energy and its transformation into chemical energy. (1) Absorption of carbon dioxide from the external environment happens through the open osteole (photoactive physiological reaction). Carbon dioxide enters the sub substomatal cavity, from where it diffuses through the free intercellular spaces to directly contact the cellulose membranes of palisade assimilatory parenchyma, situated on the upper side of the leaf blade, or the cells of the spongy parenchyma from the inferior side (Fig. 4.19).

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Fig. 4.19 Carbon dioxide penetration mechanism

In the envelopes of assimilatory cells are continuously irrigated with water absorbed from the soil, the CO2 from the air that circulates in the intercellular spaces, possessing a high hydrosolubility, dissolves and forms carbonic acid (H2CO3), which dissociates in H+, HCO3  , CO3 2 . In the ionic form carbon dioxide enters the cytoplasm and reaches chloroplasts. Consequently, it results that the first condition of photosynthesis is the degree of osteole opening and the presence of a sufficient amount of water in foliar tissues. At night, when stomata are closed (photoactive closure) as well as in drought conditions (hydroactive closure), when the cellular membranes of the leaf mesophyll cells are dry, photosynthesis is blocked and plant growth stagnates. 1. Absorption of solar energy and its transformation into chemical energy happens via several successive stages: • solar energy absorption and excitation energy migration to the system of pigments; • oxidation of the reaction centre and stabilization of the separated charges; • electron transfer through the electron transport chain (ETC); • water photooxidation and molecular oxygen elimination; • conjugation of electron transport with proton transfer and the synthesis of ATP. These processes are carried out in granal and stromal thylakoids with the participation of different molecules that make up two specific structures in superior plants—photosystem I (PS I) and photosystem II (PS II), which differ in their protein components, pigments and optical properties. Each photosystem is formed of a reaction center conjugated with electron donors and acceptors together with the “antenna” pigments (Fig. 4.20). Chromoproteids of the antenna-complexes have no photochemical and enzymatic activity. Their role is reduced to the accumulation and transmission of energy quanta to a limited number of molecules, which carry out photochemical reactions.

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Fig. 4.20 Energetic levels of the molecules and transitions between electronic states

These chromoproteins, which absorb sunlight with λ = 700 nm and λ = 680 nm found in a ratio of one molecule to 200–400 molecules of chlorophyll and other auxiliary pigments, were called reaction centers. In the reaction centers, the energy of the excited state of chlorophyll is transformed into the energy of separate charges —chemical energy. According to their absorption characteristics, reaction centers are denoted as P680 (PS II) and P700 (PS I). The pigment molecule in the reaction center is in a complex with the electron donor and acceptor; due to which the coordination of photophysical reactions of electronic excitation with enzymatic reactions of electron transfer from donor to acceptor is possible. A small part of protein molecules fulfill the role of reaction centers, while the majority (more than 90 %) form the antenna-complexes, consisting of hundreds of chlorophyll molecules and a smaller number of auxiliary pigments—all these, in complex with proteins, form the so-called antenna complexes (light-harvesting complexes). The antenna pigments of PS I absorb light with wavelength λ = 700–730 nm, and those of PS II—with wavelength of 680–700 nm. Auxiliary pigments are represented by carotenoids, with an absorption maximum at 450–480 nm (plants), phycoerythrins, with λ = 495–565 nm (red algae) and phycocyanins, with λ = 550–615 nm (bluegreen algae). The light harvesting antenna of PS I contains: 200 chlorophyll “a” molecules, 50 chlorophyll “b” molecules and 50 carotene molecules. The PS II antenna includes 200 chlorophyll “a” molecules, 200 chlorophyll “b” molecules and 50 molecules of xanthophyll and carotene. Absorption of solar energy and excitation energy migration in system of pigments. The primary processes of the light phase consist in light capturing in the form of photons by antenna-pigments. The intensity of this photophysical process is proportional to the number of absorbed photons, that’s why the light necessary for photosynthesis can be expressed by the number of quanta per molecule (Einstein). Pigment molecules, absorbing the energy of light quanta, enter an electron excitation phase (the electron jumps to a higher energetic level). Electrons from the molecule have certain energy values, which can be associated with energetic levels (Fig. 4.21).

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Fig. 4.21 Organization of the light-harvesting antenna and migration of excitation energy to the reaction center (Tihonov 1996)

Electrons rotate around the nucleus, but also around their own axis, creating mechanical and magnetic states—the so-called electron spins. Spins of two electrons occupying the same orbital are oriented anti-parallel. The basic energy state of the electrons, called singlet S0 (their total spin equals zero) is established, when all the electrons are coupled (in pairs of 2) and occupy the lowest energy orbitals. During photon absorption, electrons jump to higher energy levels, determining the appearance of two singlet states S1 and S2, if the electrons don’t change their spin and triplet T1, if one of the electrons changes its spin. The highest energy level is level two singlet (S2). At this level the electron comes under the influence of violet-blue rays, whose quanta carry a big amount of energy. In the first excitation state (S1), electrons can continue absorbing quanta of lower energy. The duration of the S2 excitation state is very short (10−12 s), and is followed by energy loss as heat and transfer of the electron without changing spin direction, to an inferior energetic level of excitation—the level one singlet (S1). The electron can stay in the S1-state for a longer period of time (10−9–10−8 s). The duration of the excitation state is a thousand times longer at the triplet level (10−5–10−4 s), which appears on energy level transformations accompanied by electron spin changing. Only electrons in S1 and possibly T1 states participate in photochemical reactions. From the excitation state, electrons go back to the fundamental state S0 through: • transfer of electronic excitation energy to a neighboring molecule of pigment (photochemical reaction); • its elimination as heat, fluorescence or phosphorescence (when transitioning from T1 to S0). The wavelength of fluorescent light is higher than that of absorbed light, because the amount of energy is lower compared to the absorbed energy. In the chlorophyll molecule there are two excitation levels, which determine the presence of two absorption peaks. The first excitation level is related to the electron transfer to a superior energetic level in the system of conjugated double bonds

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(18 delocalized π-electrons); the second excitation level is related to the paired electrons of the nitrogen and oxygen atoms from the porphyrin core. As mentioned earlier not all chlorophyll molecules fulfill the function of photochemical energy transformation and those who do, absorb only one quantum of light in 0.1 s and have a very short excitation period. Hence, the full use of energy from light quanta is possible only with the participation of additional pigments that absorb and transmit solar energy to the photochemically active molecules, thus ensuring an efficient functioning of photosynthetic system even at low values of light intensity. When a light quantum is absorbed, chlorophyll or auxiliary pigments from the antenna transit into the state of electronic excitation: Chl þ hm ¼ Chl The energy of the excited molecule Chl* is transferred to the neighboring pigment, which transfers it, in turn, to other pigment molecules with higher and higher absorption wavelengths characterized by a lower level of singlet excitation down to P680 or P700: Chl þ Chl ! Chl þ Chl ! Chl þ P ! Chl þ P The migration of energy among different types of antenna pigments occurs according to the principle of inductive resonance (which involves transfer of the electron excitation energy from a pigment molecule to another one without charge transfer and without its prior emission as fluorescent radiation (Fig. 4.22). Around the excited molecule an alternating electric field with a certain oscillation frequency is formed, which induces the nucleus-electron oscillation of the neighboring molecule. The acceptor molecule passes into the excitation state and donor molecule—in its fundamental state. Formation of a resonance system, in which electron excitation energy can be transferred to another molecule, is possible only at small distances (up to 10 nm) between two molecules with the same oscillation frequency. The period of time needed for the excitation energy to migrate from one pigment to another until it reaches the reaction center, does not exceed 10−10–10−9 s.

Fig. 4.22 Absorption and fluorescence spectra in light-harvesting systems

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Oxidation of the reaction center and stabilization of separated charges. The first reactions following light absorption and chlorophyll excitation in the reaction centers are processes involving electron transfer between different macromolecular entities. The sequence of events occurring in the reaction center is similar in all photosynthesizing systems. The first photochemical reaction in the reaction center is the fast transfer of electrons (τ ≈ 10−12 s) from the photoactive pigment (P*) to the primary acceptor (I), for instance bacteriopheophytin (BPP) in case of bacterial photosynthesis, the monomeric form of chlorophyll (A0) or pheophytin (Phe) for photosystem I and photosystem II, respectively (Table 4.4). Finally, this process yields a reductant I− (donor of electrons) and a strong oxidant P+ (acceptor of electrons): hc þ PI ! P I ! Pþ I This marks the second important stage of solar energy transformation within the process of photosynthesis (charge separation in the reaction center). The subsequent electron transfer happens outside the center of reaction, through an electron transport chain (ETC), which unites both reaction centers by means of cytochrome b6-f. The positive charge of the reaction center, formed during its oxidation, is neutralized within 2 μs by acceptance of an electron, returning to its fundamental reduced state. The function of the electron donor is performed by cytochrome “c” in bacteria, by plastocyanine in PSI and by the tyrosine radical—a component of the water dissociation system in the PS II. Electron transfer mechanism. The electron is transferred from the reaction center at long intermolecular distances from one side of the membrane to another at high speed. For example, the electron is transferred from P* to QA, at a distance of about 50 Å in 150 ps (Fig. 4.23). Table 4.4 The chemical nature of transporters in reaction centers Electron transporter

Bacterial photosynthesis

Photosystem I

Photosystem II

P (reaction center) I (primary acceptor) QA (secondary acceptor) QB (secondary acceptor) D (electron donor)

P870 Bacteriopheophytin Ubiquinone Ubiquinone Cytochrome C

P700 Chlorophyll “a” 695 (A0) Phyloquinone (A1) Fe-S proteins Plastocyanin (Pc)

P680 Pheophytin (Phe) Plastoquinone Plastoquinone Tyrosine (TyrS)

Fig. 4.23 Energetic levels of the photosystem at the different states of the: reaction center (P), electron donor (D), primary acceptor (I) and secondary acceptors (QA and QB)

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An important role in electron transfer is played by the protein medium between the redox cofactors (with transport function), which represents not only a physical support for the carriers, but also actively influences this process. Two theories are currently available explaining the mechanism of electron transport through ETC. (1) Electron transfer mediated by proteins is based on the so-called electron tunneling effect—a quantum mechanics phenomenon. Due to its oscillatory nature, the electron literally “slips” under energetic barriers, tunneling from one transporter to another (with a probability which decreases exponentially with the increase of the height of the barrier). It is considered that during tunneling, the electron loses part of the energy, which passes into oscillations of the light atomic groups of the proteins. (2) Electron transfer through the ETC in photosynthesis or in respiration is sometimes presented as a ball that moves downwards on a ladder where each step represents the energy level of the transporter. While moving to a new step, part of the electron energy can be converted into heat or stored in the form of ATP. Every time when the ball falls down on the corresponding step it rotates it in the direction that will facilitate ball movement to the next step. This process is so fast that the possibility of backward movement is much smaller than that of forward transport, which provides for the efficiency and irreversibility of the transport. Transfer of electrons through the electron transport chain (ETC). Electron transport chains contain a wide variety of carriers. Some carriers transport a single electron, while others transport an electron and a proton, or even more electrons and/or protons. But, an important feature of theirs is the fact that they are located in membranous structures of the cell—in chloroplast and mitochondrial membranes. Among the compounds that participate in processes of electron transfer one could distinguish: • cytochromes (proteins whose prosthetic groups are represented by the heme group). The active redox component is represented by the iron atom, that can exist as both Fe(II) and Fe(III) (Fe3++ē → Fe2+); • Proteins with iron and sulfur. The iron atoms in these proteins are in tight association with groups of sulfur containing atoms, which are normally found in the cysteine residues of the protein chain. Iron and sulfur clusters contain an equal number of sulfur and iron atoms and their common combinations are 4Fe+4S, 3Fe3+S → and 2Fe+2S; • Flavoproteins, which are widely spread among the redox enzymes, are characterized by the presence of some strong non-covalent flavin-nucleotide bonds. The structure of the isoalloxazine ring undergoes the transfer of two ions of hydrogen (2ē + 2H+); • Copper proteids, the paramagnetic ion of copper (Cu2+), is part of the structure of the active center.

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• Chinones, which have methyl, methoxy, amino—or hidroxyl substituents, that significantly affect their electrochemical properties (especially their potential for oxidoreduction). Chinones in water solutions undergo a reduction reaction (2ē + 2H+). These compounds form macromolecular protein complexes located in the thylacoid membrane (cytochrome b6-f, of water photo-oxidation; electron transporter ferredoxin—NADP—reductase), which, together with the mobile transporters (plastoquinone, plastocyanin and ferredoxin molecules), transfer electrons, ensuring conjugated functioning of the reaction centers (Fig. 4.24). Based on the quantum yield of photosynthesis, on the chemical composition of the transporters and their redox potential values, the consecutive scheme of electron transfer reactions in the light phase of photosynthesis was developed. Due to its similarity to the Z letter, this scheme is called the Z-scheme (the acyclic transport of electrons). The light induced transfer of charge in the reaction centers of PS I and PS II assures the transport of an electron from the water dissociated in photosystem II (the source of electrons for PS I) to the NADP + molecule, a transfer which is carried by the transporters distributed in a crescent order according to their redox capacity. In the excited PS II reaction center, the electron is consecutively transported from one side of the membrane to another by the primary acceptor (pheophytin) and the secondary acceptors—plastoquinone QA, which is strongly connected to one of the proteins of the photosystem II and the second molecule of plastoquinone—QB, which can move easily through the phospholipid layer and can receive two electrons. After two actions of the reaction center of this photosystem, the plastoquinone QB molecule receives two electrons: QB þ 2e ¼ QB 2 : The positively charged molecule QB 2 has a high affinity for hydrogen ions which it accepts from the stroma: QB 2 þ 2Hþ ¼ QH2 , building the electro-neutral form of this molecule—QH2 (Fig. 4.25). Fig. 4.24 Schematic representation of electron transfer in the reaction center (Rubin 1997)

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Fig. 4.25 Location of electron transporting complexes (PS I, PS II and b/f) and their interaction in thylakoid membranes

QH2 leaves the photosystem II and can easily move inside the thylacoid membrane ensuring the connection of PSII with other transporters of the ETC. Thus, the plastoquinone play the role of a mobile carrier of two electrons. The plastoquinone molecule diffuses into the galactolipid layer towards the complex of cytochromes b/f with which it connects by yielding 2ē to it. For each molecule of plastoquinone oxidized by the cytochrome complex, two hydrogen ions are eliminated inside the thylacoid (QH2↔Q + 2ē + 2H+). The oxidized plastoquinone returns back to its location in order to make a new transport of protons and electrons. The b/f complex serves as an electron donor for plastocyanin—a relatively small hydrosoluble protein (the redox reactions of the plastocyanin are accompanied by a change in the valence of copper ions, Cu2++ē ↔ Cu+). The molecules of plastocyanin move easily along the lumen of the thylakoid and transport one electron from the b/f/complex to PS I: Cuþ þ Pþ 700 ¼ Cu2þ þ P700 The oxidized form of the reaction center of photosystem II Pþ 680 is reduced, returning to its initial state, after accepting an electron from the water dissociated through the tyrosine radical of the manganese complex for water photodissociation. The molecules of chlorophyll from the reaction centers of PSI and PSII, being excited simultaneously by the energy transmitted via resonance from the light harvesting antenna, loose the electron. The electron is transported successively from the primary acceptor (monomeric form of chlorophyll—A0) to the secondary acceptors (phylloquinone—A1) and the proteins that contain iron and sulfur (Fe–S) reaching, ultimately, the ferredoxin molecules (Fd)—a hydrosoluble protein, located in stroma. The last phase of this transport consists in releasing electrons by the reduced ferredoxin to the complex of ferredoxin—NADP—oxidoreductase (FNR) with FAD as a cofactor. This complex functions on the external side of the

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thylakoid membrane and when it receives two electrons it reduces the molecule of NADP+ to NADPH+H+ in the presence of protons from the stroma. Chlorophyll molecules in oxidized form Pþ 700 from the PS I reaction center are reduced and return to their initial state by receiving an electron from PS II through the reduced plastocyanin, which is being oxidized after releasing the electron. Thus, as a result of simultaneous functioning of both photosystems, two electrons from the molecule of water dissociated by the PS II are transported through the ETC towards NADP, forming a strong reducing agent (NADPH2). Photo-oxidation of water and elimination of molecular oxygen. The photosynthetic oxidation of water is performed by the macromolecular complex PS II, which includes three basic structural and functional parts: • the complex of antenna-pigments, located on proteins with a molecular mass ranging from 25,000 to 47,000; • the reaction center with all its basic components located on a complex formed by 2 proteins with a molecular mass of about 32,000, called D1 and D2 which are located across the photosynthetic membrane (Fig. 4.26);

Fig. 4.26 Redox transformations and diagram of probable location of plastoquinone molecules in the membrane (Tihonov 1996)

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• the enzymatic center for oxygen elimination, which, undergoing multiple oxidations determined by the oxidized form of the chlorophyll from the reaction center Pþ 680 , oxidizes water, accepting the missing electrons and transforms it into molecular oxygen. The strongest biologic oxidant—Pþ 680 is formed as a result of the primary photosynthetic reaction and its redox potential is sufficient for water oxidation (+1, 1 V in comparison with the ordinary hydrogen electrode). In order to form a molecule of O2 from water, four electrons and four protons are required according to reaction: 2H2 O þ 4e þ 4Hþ ¼ O2 þ 4H2 It is known that the reaction center of PS II forms a single oxidative equivalent Pþ 680 when absorbing a single light quantum. Thus, four photochemical reactions of the reaction center are necessary in order to form a molecule of O2 out of two molecules of water. This is only possible in the presence of a system, which can accumulate four oxidative equivalents resulting from four successive photochemical reactions. Accumulation of these equivalents takes place in the enzymatic center, which contains four atoms of manganese (a metal with variable valence) located close to the reaction center. It is supposed that both, the 33,000 Da protein and the 47,000 Da pigment-protein complex play a significant role in stabilizing this complex, which has its binding groups located on the D1 and D2 proteins of the reaction center (Fig. 4.26). The structural model of the water oxidation center includes 4 atoms of Mn, which form two Mn–Mn dimers with the distance between them of approximately 2.7 and 3.3 Å (Fig. 4.27). Accumulation of oxidative equivalents (as a result of photoexcitation of the reaction center) determines a change in the valence of Mn ions from +2 (or +3) to +4: Mn2þ  e ! Mn3þ  e ! Mn4þ The ions of manganese, when reaching a high level of oxidation, determine the oxidation of water molecules: 2Mn4þ þ 2H2 O ¼ 2Mn2þ þ 4Hþ þ O2 The resulting electrons are taken by the manganese atoms which, thus, reduce. The protons are accumulating in the lumen of the thylakoides while oxygen diffuses in the cytoplasm, where it is dissolved in water, passes into the intercellular spaces in the form of gas and is eliminated into the external environment through the open stomatal pores. The tyrosine radical (Tyr Z)—the aromatic amino acid from the D1 protein (161st from the N-terminus) which plays the role of intermediary transporter

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Fig. 4.27 Location of the basic components of the water photo-oxidation complex PS II (Klimov 1996). P680 Primary electron donor; Phe, QA, QB Electron acceptor; TyrZ Secondary electron donor; D1 and D2 Proteins of PS II; cyt b559 Cytochrome with protective function by neutralizing separate charges in cas of photoinactivation of PS II; 17, 24, 33, 43, 47 proteins with Mn (17,000, 24,000, 33,000, 43,000, 47,000 Da)

of electrons between Pþ 680 and the Mn complex or the radical of another aromatic amino acid—histidine, also participate in the process of accumulating oxidative equivalents. Also, it has been found that the ions of Ca2+, Cl− and possibly HCO3− have a significant role in the functioning of the water oxidation system. Conjugation of electron transport with proton transport and ATP synthesis. The transfer of electrons at the level of mobile plastoquinone conjugated with transmembrane crossing of the H+ ions from the stroma to the inner space of the thylakoids (against the concentration gradient) and the continuous influx of protons generated by the enzymatic reactions of water photo-oxidation determine their accumulation in the lumen of thylakoids. Due to the fact that the membrane of the thylakoids is impermeable to protons, their concentration inside the thylakoids increases a 100–1000 times in comparison with the stroma, causing the appearance of a gradient of protons ΔpH and of a membrane potential Δφ conditioned by the appearance of a positive charge inside and of a negative charge outside it. The electro-chemical potential of hydrogen ions ΔµH+ (electrical potential—Δφ and chemical potential ΔpH) is the driving force for the process of phosphorylation. In

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Fig. 4.28 Scheme of the enzymatic system of water photo-oxidation (Klimov 1996)

accordance with the chemiosmotic hypothesis, the trans-membrane transfer of hydrogen ions from the cavity of thylakoids into the stroma is going on passively (according to the concentration gradient) through the proton channel of the H+ATPase, combined with the synthesis of ATP from ADP and inorganic phosphorus. It is considered that the synthesis of ATP is accompanied by the transfer through the macromolecular complex CF0–CF1 of three protons (Fig. 4.28). Ions are marked with: red—manganese ions, green—carbon ions, blue—oxygen ions ADP þ Pi þ 3Hþ ¼ ATP þ H2 O þ 3Hþ This phenomenon of solar energy conversion into chemical energy, accumulated in the macroergic bonds of the ATP is called photophosphorylation. The synthesis of ATP by the oligo-enzymatic complex of reversible H+-ATPase, which performs in this case the function of ATP-synthesis, in the presence of ΔµH+ on the thylakoidal membrane generated by the acyclic transfer of electrons, from water to NADP+ (functioning of both the photosystems), is called acyclic photophosphorylation. In this case, the energy of light is transformed into macroergic ATP bonds, into the chemical potential of the reduced form of NADP and into molecular oxygen according to the final equation: 2ADP þ 2Pi þ 2NADPþ þ H2 O ¼ 2ATP þ 2NADPH þ Hþ þ O2 þ 2Hþ Acyclic photophosphorylation is specific only for green plants and for algae. It has been noticed that during the light phase of the photosynthesis the quantity of the synthesized ATP is higher than that of NADPH+H+. This surplus of ATP is

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formed only in one photosystem—PS I. In this case, the transfer of electrons by the transporters is cyclic (closed) and only includes the acceptors from the reaction center (A0, A1, Fe-S proteins, Fd), the mobile plastoquinone, the complex of cytochromes b6/f and the plastocyanin. The electron of the excited molecule from the reaction center is successively accepted by both the primary and the secondary acceptor until it reaches Fd. From there, it is accepted by PQ, which performs the transfer of the electrons conjugated with the transmembrane transfer of protons (from the stroma into the thylacoid cavity), on the cytochrome complex b6/f from where it is accepted by Pc, which is passing it to the oxidized reaction center Pþ 700 reducing it. In this case NADP+ is not reduced, and the electro-chemical potential generated by this electron transfer provides the phosphorylation of ADP (Fig. 4.29). The summary equation of the cyclic phosphorylation is: ADP þ Pi þ hm ¼ ATP þ H2 O The final products of the light phase are O2, ATP and NADPH+H+. The later are used, at a rate of 3/2, in the second phase of photosynthesis—the enzymatic phase of carbon assimilation by reducing it to primary sugars (CH2O). This phase of the photosynthesis takes place in the stroma of the chloroplasts. The surplus of ATP produced during photosynthesis is used in other processes occurring in chloroplasts (synthesis of fatty acids, of certain amino acids, reduction of nitrites etc.). Fig. 4.29 Phosphorylation process diagram

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4.6.2 The Dark Phase of Photosynthesis There are different methods of reducing carbon dioxide: the Benson-Calvin cycle (C3), the Hatch-Slack-Karpilov cycle (C4), the metabolism of organic acids in Crassulaceae (CAM—“crassulacean acid metabolism”) and photorespiration (Fig. 4.30). The Benson-Calvin cycle (pentose-phosphate reduction pathway, photosynthetic type C3) is specific for a group of superior plants and includes a cycle of enzymatic reactions that can be grouped in 3 main stages: carboxylation, reduction and regeneration. The carboxylation phase, the primary CO2 acceptor is a compound with 5 carbon atoms, ribulose-1,5-diphosphate, which forms as a result of secondary phosphorylation of ribulose-5-phosphate with the participation of ATP and of ribulose phosphate kinase. Under the action of ribulose phosphate carboxylase/oxygenase (RUBISCO), ribulose-1,5-diphosphate attaches a molecule of CO2 to the second carbon atom and one molecule of water forming an instable compound with 6 atoms of carbon, which splits into 2 molecules of 3-phosphoglyceric acid. RUBISCO, the keyenzyme of the photosynthesis processes is the most widely spread enzyme on earth. It is considered that the general quantity of this enzyme is 10 million tones or approximately 20 kg per human being.

Fig. 4.30 General scheme of photosynthetic assimilation of carbon dioxide

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It is a hydro-soluble, complex enzyme, with a general molecular mass of 500,000 Da, which is formed of eight big and eight small protein subunits (Fig. 4.31). Due to the fact that the outcome of this reaction is a molecule with 3 carbon atoms, the cycle is also called C3. The reduction phase. The 3-phosphoglyceric acid has an energetic level lower than that of carbohydrates and the reduction of this compound to the level of triosephosphates (carbohydrates with 3 atoms of carbon) can happen only when using the energy of ATP and NADPH+H+ (energy that is called assimilation factor). The phosphoglyceric acid is reduced to phosphoglyceric aldehyde by two reactions. Phosphorylation of 3-phosphoglyceric acid to obtain 1,3 phosphoglyceric acid is performed in the presence of ATP and is carried by phosphoglycerate kinase. In the second reaction, the latter is reduced to phosphoglyceric aldehyde in the presence of NADP+H+ and with participation of phosphoglyceraldehyde dehydrogenase.

Fig. 4.31 Identification of the large (a) and small (b) subunits of the enzyme ribulose-1,5diphosphate carboxylase with the indication of the amino acid sequence. M Protein markers with known molecular weights; [1–3] proteins extracted from leaves of different genotypes of sunflower; [4, 5] proteins extracted from calathidia of different genotypes of sunflower

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The phosphoglyceric aldehyde, under the action of triosephosphate isomerase is transformed into phosphodioxiacetone by isomerization. This enzymatic phase is the only Calvin cycle reduction step in which the NADP +H+ accumulated in photochemical reactions is used while the ATP was used as an additional energy for producing these reactions. The regeneration phase. During this phase the acceptor-ribulose-1,5-diphosphate is regenerated in a cycle of reactions of reciprocal transformation of the carbohydrates with a different number of carbon atom: trioses, tetroses, hexoses, sedoheptuloses. Transketolases and transaldolases participate in such reactions by catalyzing the transfer of fragments made of two carbon atoms (–CO–CH2–OH) and three carbon atoms (–CHOH–CO–CH2–OH) respectively from ketoses to aldoses and isomerases. When three molecules of CO2 are absorbed, six molecules of reduced phopsphotrioses are formed. Five of these molecules are used in the regeneration of ribulose 5-phosphate and one molecule is set free. During the reaction, three molecules of pentose phosphate are formed out of 5 molecules of triose phosphate. After a second phosphorylation, in the presence of ATP, these molecules are transformed into ribulose diphosphate and can again perform the function of primary acceptor of carbon dioxide. In order to perform a cycle of reactions the presence of 3 molecules of ATP and 2 molecules of NADPH+H+ is necessary. The additional phosphoglyceric aldehyde molecule which was not consumed can be used in chloroplasts for the biosynthesis of glucose, fructose, starch, of some amino acids etc. Molecules of phosphoglyceric aldehyde can pass through the membranes of the chloroplast and reach the cytoplasm where hexoses are synthesized (Fig. 4.32). Fructose-1,6-diphosphate is formed by means of aldolic condensation of phosphodioxyacetone and phosphoglyceric aldehyde. When two molecules of phosphoglyceric aldehyde are condensed glucose-1,6-diphosphate is formed. Saccharose, which is the main form of carbohydrate transported in plants, consists of a molecule of glucose and one of fructose. Glucose and fructose represent substrates for the process of respiration, while the resulting intermediary compounds are used to synthesize different organic substances. The Hatch-Slack-Karpilov cycle. At the beginning of the 1960s it has been found out that in certain plants of tropical or subtropical origin (maize, sugar cane, sorghum, millet etc.) photosynthesis deviates from the basic cycle. In this species, phosphoenolpyruvic acid serves as a CO2 acceptor. This acid contains a macroergic bond, due to which it has a high reactive capacity. The primary products that are

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Fig. 4.32 General scheme of type C3 photosynthesis

Fig. 4.33 C4 leaf anatomy and C4 photosynthetic mechanism

formed as a result of CO2 reduction, consist of 4 carbon atoms. Due to this fact, this type of carbon assimilation is called the photosynthetic type C4. This photosynthetic type, is common for more than 1000 species originating from the tropical areas, which are adapted to conditions of intense illumination and high temperature. Plants in which photosynthesis takes place according to the C4 cycle, have leaves with a particular anatomic structure (Fig. 4.33). The cells of the palisade mesophyll have a small number of chloroplasts with CO2 fixation role, while the cells of the perivascular sheath are reach in big chloroplasts and have the function of performing photosynthesis of the C4 type. Fixation of the carbon dioxide takes place in the cytoplasm of mesophyll cells of, through a carboxylation reaction of the phosphoenolpyruvate in the presence of phosphoenolpyruvate carboxylase, resulting in a compound with 4 atoms of carbon—oxaloacetic acid. In chloroplasts, oxaloacetic acid in the presence of NADP+H+ formed during

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the light phase and of NADP-malate dehydrogenase is reduced to malic acid. In the presence of NH4+ ions the oxaloacetic acid can be aminated resulting in aspartic acid. The malate (or aspartate) is transported through the plasmodesms from the mesophyll cells to the cells of the perivascular sheath, which are permeable to organic acids and impermeable to CO2. Here, it is decarboxylated, with the formation of pyruvate and CO2. The pyruvate from the perivascular sheath is transported back to the chloroplasts of mesophyll cells where it undergoes phosphorylation in the presence of ATP and phosphopyruvate synthase, thus regenerating the primary acceptor—the phosphoenolpyruvic acid. In the chloroplasts of the perivascular sheath, PS II is weakly developed, in comparison to PS I. The ATP necessary to fix CO2 is synthesized as a result of the cyclic transport of electrons while NADP + H+ is formed as a product of the oxidative decarboxylation of the malate concomitantly with CO2. Due to the fact that two types of cells with two types of chloroplasts participate in this mechanism of photosynthesis, this particular type of photosynthesis is seen as “cooperative photosynthesis” (Karpilov 1970). Fixation of CO2 via C4 has some advantages: • The phosphoenolpyruvate carboxylase enzyme has a reaction speed higher than that of ribulose 1,5-diphosphate carboxylase and this fact determines the accumulation of carbon dioxide in the cells of the perivascular sheath. • Some species can perform the first stages in which the organic acids are formed during the night while, during the day, CO2 is released with subsequent reassimilation in the Calvin cycle. This fact allows plants to carry some carbon assimilation reactions during the day, even when the stomatal pores are closed (in the absence of exogenous CO2), thus, avoiding strong water elimination. It is considered that such peculiarities form the basis of a higher drought resistance of this group of plants; • Carbon dioxide accumulation in the cells of the perivascular sheath, where the C4 type photosynthetic reaction takes place determines the stimulation of this process and concomitantly blocks the oxidase activity of ribulose 1,5-diphosphate carboxylase and, respectively, photorespiration. Thus, the unnecessary consumption of organic substances is reduced and the productivity of the plants is increased. Crassulacean acids metabolism (CAM). Plants from the Crassulaceae, Liliaceae, Cactaceae families and some of the Compositae, Osteraceae species etc. have adapted to carry photosynthesis even in regions with drought and in deserts. These plants fixate carbon dioxide in the same manner in which C4 plants do. But for them two stage photosynthesis is specific: one during the night and another during the day. The stomata of these plants are open particularly during the night when most of the gas exchange processes happen. During daylight, they close reducing thus the loss of water through stomatal transpiration. The reaction of CO2 fixation by the phosphoenolpyruvic acid (first carboxylation reaction) takes place in the cytoplasm in the presence of phosphoenolpyruvate carboxylase. As a result of this reaction oxaloacetate forms which, under the action of

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NADP+H+ dependent malate-dehydrogenase, is reduced with the formation of malic acid which accumulates in the vacuoles. The transport of malate through the tonoplast is passive, according to the electrochemical gradient generated by the accumulation of protons in the vacuoles by means of active transport by proton pumps. Accumulation of malate in cells vacuoles determines an increase in their acidity. During the day time, when stomata are closed, malic acid diffuses through the tonoplast into the cytoplasm, where it is decarboxylated, resulting in pyruvic acid and carbon dioxide. CO2 is transported in chloroplasts and used in phosphoglyceric acid synthesis reactions by means of the C3 photosynthetic path, the second reaction of carboxylation being performed in this manner. Thus, the biochemical mechanism of performing C4 and CAM photosynthesis is identical; it only differs by the isolation in time (C4) and in space (CAM) of the two carboxylation reactions. In some species with CAM or C4 photosynthesis, under favorable life conditions (CO2 and water), photosynthesis takes place according to the C3 photosynthetic type and vice versa (Figs. 4.32, 4.33 and 4.34; Table 4.5). It is considered that these spatial and time variations in photosynthesis emerged as an adaptation to environmental conditions.

Fig. 4.34 Comparison between C4 and CAM photosynthesis. a Spatial separation of the stages, b temporal separation of the stages

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Table 4.5 Comparison of the different types of CO2 fixation in plants Features

CO2 fixation type C3

C4

CAM

Most common groups, families or genera

Most plants in the temperate region including wheat, barley, rice, Fabaceae species, sugar beet 15–35

Plants of tropical and subtropical origin, corn, sugar cane, sorghum, Amaranthus, Atriplex 40–48

Herbaceous plants from warmer regions and deserts: Euphorbiaceae, Cactaceae, Compositae, Crassulaceae 5–7

Phosphoglyceric acid

Oxaloacetic acid

Present (20–50 % of actual photosynthesis, 3–5 times higher than dark respiration) Big

Malic or oxaloacetic acids Difficult to determine (10 times weaker than dark respiration)

Very low

Low

Low or absent

450–490

3–5

50–55

22 ± 3.3

22 ± 3.3

Weaker in comparison to C3 plants

Maximum intensity of photosynthesis (mg CO2/dm2/h) First product of CO2 fixation Photorespiration

Extent of stomata opening in daylight Transpiration intensity (g water/g dry matter) Dry matter production (t/ha/year)

4.7 Photorespiration Photorespiration is a process of CO2 elimination and O2 absorption in the presence of light which takes place simultaneously with the photosynthesis. As for the biochemical mechanism, this process differs substantially from photosynthesis as well as from respiration. Photorespiration is more prominent in C3 photosynthetic plants under natural conditions, at an oxygen concentration of 21 % and a carbon dioxide concentration of 0.03 % (photorespiration constitutes 25–30 % of the photosynthetic gas exchange in leaves). Under intense illumination and lower carbon dioxide concentration and higher oxygen concentration, the photorespiration process intensifies. High temperature has a similar effect on the intensity of photorespiration. The essence of this process is that the basic enzyme in photosynthesis, RUBISCO, can act as both a carboxylase and an oxidase (Fig. 4.35), catalyzing the decomposition of ribulose-1,5-diphosphate into phosphoglyceric acid and phosphoglycolic acid, which subsequently dephosphorylases into glycolic acid. Since the primary product of the oxidation reaction is the glycolic acid, this path has taken the name of the glycolic pathway of carbon transformation. Originally the acceptor of carbon dioxide—ribulose-1,5-diphosphate forms a complex with the active center of the enzyme and, only after this, it fixates a

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Fig. 4.35 The carboxylation and oxygenation reaction mechanisms performed by ribulose disphosphate carboxylase/oxygenase RBF/ O, RBFC—enzyme of the complex with RBF and CO2, RBFO—enzyme of the complex with RBF and O2

molecule of CO2 or O2, performing the function of carboxylation or oxygenation (Fig. 4.35). The molecules of oxygen and carbon dioxide start competing for the active center. However, the ability of the enzyme to react with carbon dioxide is higher than its ability to react with oxygen. It is considered that for 3 molecules of carbon dioxide fixed as phosphoglycerate, approximately one molecule of oxygen is fixed together with the formation of a molecule of 3-phosphoglycerate and 3-phosphoglycolate. Photorespiration takes place during the interaction of three organelles: chloroplasts, mitochondria and peroxisomes (Fig. 4.36). The first reaction of oxidation with the formation of 3-phosphoglycerate and 3-phosphoglycolate takes place in the chloroplasts. The phosphoglyceric acid formed is reduced in the Calvin cycle and the dephosphorylated glycolic acid, in the presence of a phosphatase, is transported into the peroxisomes. In the presence of glycolic acid oxidase, it is oxidized to form glyoxylic acid and hydrogen peroxide, which is processed by the catalase from these organelles (into oxygen and water). The glyoxylic acid is converted to glycine by transamination with glutamic acid. The glycine is then transported to mitochondria. By means of an oxidative decarboxylation reaction related to NADP+ reduction, from two molecules of glycine, serine is formed and CO2 and NH3 are released. Serine can be used in protein biosynthesis or it can be deaminated in peroxisomes with the formation of glyceric acid, which is transported in chloroplasts, where after phosphorylation it is included in the Calvin cycle. This sequence of reactions does not always form a cycle. It can stop in mitochondria at the final products—serine and CO2. Release of CO2 is the reason of low photosynthetic productivity when photorespiration has a high intensity.

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Fig. 4.36 Photorespiration. (1) Ribulose-1,5-biphosphate carboxylase/oxygenase; (2) Phosphoglycolate phosphatase; (3) Glycolate oxidase; (4) Glutamate: glyoxylate aminostransferase; (5) Glycine decarboxylase and serine hydroxymethyl transferase; (6) Serine: glyoxylate aminotransferase; (7) Hydroxypyruvate reductase; (8) Glycerate kinase; (9) Catalase

In C4 plants, the CO2 released in the process of photorespiration is used in the carboxylation reaction of phosphoenolpyruvate from the mesophyll cells to form oxaloacetate and, then, by a reduction reaction—in malate, which is decarboxylated in the perivascular sheath cells, releasing CO2 in chloroplasts and then the latter is involved in the Calvin cycle again. This peculiarity explains the high net photosynthesis. The physiological relevance of glycolic acid formation reactions in chloroplasts can be seen in several ways: • Glycolic acid represents a mobile form of carbon transport from chloroplasts into the cytoplasm; • Amino acids glycine and serine are formed; • NADPH+H+ is formed in peroxisomes; • It is considered that photorespiration serves to protect the photosynthetic apparatus. Factors determining the damaging of the photosynthetic apparatus are intense light at a relatively low concentration of carbon dioxide and oxygen. If in such conditions oxygen concentration increases, the glycolate formation mechanism is induced. The glycolate carbon, undergoing a cycle of transformations until the formation phosphoglycolic acid, is removed partially as CO2, which is included in the Calvin cycle where it is reduced by the use of the “assimilation factor” of the chloroplasts—NADPH+H+ and ATP. Consequently, the photosynthetic apparatus works in vain without absorption of external CO2, but photosynthetic structures are

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protected in this manner by keeping the chloroplasts active. Such situations occur during drought conditions, which make the stomata close. According to this hypothesis, photorespiration which is useful in drought conditions becomes a parasitic process under optimal conditions of life.

4.8 Endogenous Regulatory Elements of Photosynthesis Photosynthetic structures are highly organized self-regulating systems characterized by: • highly efficient conversion of solar energy; • development of mechanisms for fine tuning to the varying conditions of the environment; • development of mechanisms for protection against various unfavorable factors. Cellular complexes involved in photosynthesis work coordinately and adaptively when environmental factors change: light, temperature, humidity, radiation, chemical agents and toxic substances. This coordination is ensured by fine adjustment mechanisms by which adaptation of organisms and optimization of the photosynthetic apparatus under varying conditions occur. Genetic regulation. Regulation to the process of photosynthesis and of the photosynthetic systems is demonstrated at the cellular transcription level as well as at the levels of translation, processing, assembly of macromolecular complexes, of protein activity and biochemical pathways. Chloroplasts are the most complex organelles in the cell. They are semiautonomous in their structure and activity. The ontogenesis of chloroplasts, their function and molecular mechanisms of endogenous regulation are under a dual control by the chloroplast and nuclear genomes. The synchronized activity of the chloroplast and nuclear genes ensures the implementation of the genetic program in forming and developing the photosynthetic apparatus (Fig. 4.37). According to this scheme of execution of the main events in the development of the photosynthetic apparatus, the first step begins with chloroplast genome replication in proplastids of meristematic cells. Carrying out of this process is entirely ensured by nuclear encoded genes. When the cells grow bigger and the number of copies of the chloroplast genomes increases (stage II in the scheme), the process of decoding the genome starts, initially with the synthesis of genes responsible for ribosomal and transport RNA synthesis. Chloroplast gene transcription is carried out by the nuclear encoded DNA polymerase. Also, part of the ribosomal proteins and aminoacyl-tRNA-synthetase, necessary for translation, enter the chloroplasts from the cytoplasm. Assembly of ribosomes in the chloroplasts enables the synthesis of another RNA polymerase, whose core subunits are encoded by chloroplast genes and only one subunit involved in promoter recognition is encoded by a nuclear gene. It is this chloroplast-specific RNA polymerase which transcribes most of the genes whose expression products are involved in the formation of both the thylakoid

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Fig. 4.37 Chloroplast development and interaction of chloroplast genes with nuclear genes (Shestakov 1998)

membranes and the photosynthetic apparatus. Also, during the same period, following certain signals received from the chloroplasts, several genes that determine synthesis of proteins destined for these organelles are activated in the nucleus. For example, the proteins of the light-harvesting complexes which are involved in the absorption and transfer of energy towards the reaction centre and those which participate in the transfer of electrons from PS II to PS I are encoded only by nuclear genes, synthesized in the cytoplasm and then transported to the chloroplast. In the last stages of chloroplast development, self-assembly of the basic macromolecular protein complexes of the photosynthetic apparatus takes place with a maximum activity photosynthesis-related genes (Table 4.6). Most of the genes from the chloroplast genome that encode photosynthetic proteins become inactive in the mature phase of the organelle, except for those that encode PS II proteins (e.g. th epsbA gene with D1 as its protein expression product) (Table 4.7). At the same time, the level of expression of many nuclear genes remains very high. By applying molecular biology techniques, important information was obtained regarding the structural organization of the photosystems, the cytochrome complex b6/f, the ATP-synthase complex (Fig. 4.37; Table 4.7).

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Table 4.6 Genetic control of the photosynthetic apparatus System

Chloroplast genes

Nuclear genes

Gene

Photosystem I Photosystem II Cyt b6/f complex Electron transport (plastocyanine etc.) ATP-synthase Light harvesting complexes

10 5 4 0 6 0

5 8 1 8 3 (>) 10

psb psa pet pet atp cab

Table 4.7 Chloroplast genes and their function Gene

Localization

Protein dimension (kD)

Function

psbA psbD psbB psbC psbE psbF psbI psbH psbJ psbK psbL psbO psbP psbQ psbS psbW psbT

CL CL CL CL CL CL CL CL CL CL CL N N N N N CL

D1(32) D2(32) CP47(47) CP43(43) Cyt.b559(9) Cyt.b559(5) PI(4.5) PPH(10) PJ(4) PK(3.5) PL(4) MSP(33) PP(23) PQ(16) PS(10) PW(6) PT(4)

Reaction center QB Reaction center QA Chlorophyll binding Heme binding Heme binding Unknown Phosphoprotein, QB stabilization PS II formation Unknown QA stabilization Stabilization of the Mn cluster Ca2+ and Cl− binding Ca2+ and Cl− binding Chlorophyll binding Unknown Protection from photoinactivation

The synchronized activity of nuclear and chloroplast genes ensure the formation of the structures and components of the photosystems, as well as of a number of proteins that are not directly involved in electron transfer reactions, but still have an important role in photosynthesis. These are the enzymes of chlorophyll and carotenoid pigment biosynthesis as well as proteases, kinases, phosphatases, translocases with the function of transferring certain proteins from the cytoplasm to the chloroplast, metal-carrying proteins, ions, cofactors and other auxiliary proteins involved in the assembly and renewal of the photosynthetic complexes, in metabolizing degraded proteins etc. Mutations produced in genes that encode these proteins can cause a decrease in photosynthesis efficiency or total inhibition of the process, similarly to the effect of inactivating many genes that encode proteins of the photosystems. The molecular mechanisms of photosynthetic gene regulation in chloroplasts are different from those in the nucleus. Chloroplasts genes and ribosomes are similar in

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structure to the prokaryotic genes and translation apparatus. Most of the chloroplast genes lack introns, which are ubiquitous in eukaryotic genes. Also, many genes are grouped in clusters, forming transcription units with promoters recognized by an RNA polymerase of a prokaryotic type. In some photosynthetic genes, in the promoter region, regulatory sequences with functions of transcription stimulation and inhibition were identified. At the same time, it is known that the expression of most chloroplast genes is subject to control after mRNA formation (post transcriptionally) but not at the transcription level. Differential regulation of protein synthesis in the chloroplasts is determined by mRNA transcribed with approximately the same efficiency from different genes, but which are then subject to different levels of processing or degradation, depending on the requirements for certain proteins (Fig. 4.38). The stability of mRNA molecules also represents an important target for the regulation process. Some mRNAs are subjected to very fast degradation, others are stable for a long time, participating in the synthesis of necessary proteins. mRNA stability is determined by mRNA-specific proteins encoded by nuclear genes, which are located in the 5′ untranslated regions. It is considered that for each mRNA there are specific control proteins which determine their life span, processing and translation rates. Fig. 4.38 Regulation of photosynthetic apparatus formation. 1 Nuclear gene expression during daylight; 2 daylight regulation of nuclear and chloroplastic mRNA translation; 3 regulation of the stability and translation rate of chloroplastic mRNA by nuclear gene products (Shestakov 1998)

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The expression of many nuclear genes, unlike those from the chloroplasts, is effectively regulated at the level of transcription in the presence of specific regulatory proteins—photoreceptors. Both the phytochrome, which absorbs red light with short and long wavelengths and some proteins which receives blue and ultraviolet light have photoreceptor function. Phytochromes carry the specific regulation of different groups of genes at different ontogenetic stages. The number of nuclear genes whose expression is regulated by light by means of photoreceptors is high, including the cab genes which encodes proteins of the light-harvesting complexes. The activity of the cab genes is regulated not only by phytochromes but also by phytohormones, by the redox-dependent systems of the cell. It is considered that phytochromes act through other regulatory proteins—intermediate mediators which are involved in the transmission of the light signal to genes controlling the processes of photo morphogenesis and adaptation of the photosynthetic apparatus. Membrane regulation of photosynthesis. Photosynthesis, like most of the natural processes, is subject to feedback control, which implies that the speed of a process which undergoes adjustment depends on its outcome. The electron transfer speed in the ETC of a chloroplast or a mitochondria depends on the ratio between the amount of substrate and the products of the ATP synthesis reaction (ADP + Pi → ATP + H2O). Proton transfer processes conjugated with ATP synthesis reactions play the key role in this regulation phenomenon. ETC functioning determines the accumulation of hydrogen ions in the internal space of the thylakoids, resulting in pH decrease. It has been proven experimentally that illumination determines the decrease of chloroplast pH by 2.5 units. This causes the speed of electron transfer to decrease. This reaction takes place at the level of plastoquinone—an ETC step where electron transfer is accomplished the slowest. Plastoquinone oxidation rates depend on the concentration of hydrogen ions inside the thylakoids: the higher their concentration, the slower the oxidation of QH2 occurs. This phenomenon is explained by the fact that electron transfer from a reduced plastoquinone QH2 and its semi reduced form—plastosemiquinone QH∙ to the b/f complex (reactions 2 and 4) is preceded by proton dissociation states in the internal space of the thylakoids—reactions 1 and 3 (Fig. 4.39). The QH∙ and Q∙ forms are electron donors for the b/f complex. Dissociation reactions 1 and 3 during which these active forms are produced depend on the pH inside of the thylakoids. The lower the pH, the lower the probability of proton dissociation, because the high proton pressure shifts the equilibrium of reactions 1 and 3 to the left in the direction of the synthesis of the inactive protonated forms QH2 and of the plastosemiquinone QH∙. This is why accumulation of hydrogen ions inside the thylakoids causes the decrease of the electron transfer rate. The amount of ATP and ADP affects indirectly the electron transfer speed through the ETC, at the level of proton transfer from thylakoids outwards through the ATPase complex. The electron transfer speed stays at the highest rate as long as substrate surplus exists: ADP and Pa (Fig. 4.39). Under these conditions, proton transport through the ATP-synthase coupled with ATP synthesis occurs. When ADP deficiency appears ATP synthesis literally stops. At the same time, the transport of protons from the thylakoids into the stroma stops as well, but the

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Fig. 4.39 Photosynthesis control in chloroplasts (Shestakov 1998). The chloroplast under conditions of active ATP synthesis is shown at the top, while at the bottom—in conditions of photosynthetic control where ATP synthesis is impaired. On the left the dependence of the rate of plastoquinone oxidation by the b/f complex on illumination duration is shown

ETC continues to function (Fig. 4.39). Thus, proton transfer, combined with that of the electrons determines the pH decrease, causing a slowdown in the oxidation reactions of the reduced plastoquinone. Subsequently, these events slow down the transfer of electrons between PS II and PS I. When chloroplast ATP reserves are exhausted through their use in the Calvin cycle and other processes in the chloroplast, ADP accumulation occurs and the same sequence of events repeats: excess of ADP, ATP synthase stimulation, increase of the internal pH, intensification of electron transfer. Regulation of enzymatic activity through phosphorylation. To adjust the distribution of solar energy in chloroplasts, besides the light-harvesting complexes linked with PS I and PS II, there exists another complex of mobile pigments, which acts as an additional antenna. PS I and PS II are not uniformly distributed in the membranes of the granal and stromal thylacoids. Most of the PS I components are located on stromal thylakoids, while PS II complexes—on granal thylakoids (Fig. 4.40). It is assumed that under low light conditions, the mobile antenna is localized mainly in granal thylakoids near the PS II, due to which the size of the PS II antenna increases. And under certain specific conditions, when the need to increase the efficiency of PS I emerges, this mobile antenna complex leaves PS II, moving through the membrane towards stromal thylakoids, where it contacts PS I. The

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Fig. 4.40 Structure of the chloroplast ETC and the ATP-synthase complex (PQ Plastoquinone, PC Plastocyanine, Fd Ferredoxin, Cyt Cytochrome) (Purves et al. 2005)

signal that determines the translocation of the mobile antenna is the surplus of reduced transporters in the ETC chain between photosystems I and II. This surplus may appear when PS II operates more intensely than PS I. Movement of the antenna towards PS I determines the “unloading” of the ETC between the photosystems based on more intense PS I functioning. This mechanism, which regulates the movement of the mobile light collecting complex is determined by the phosphorylation of a subunit of the protein complex through the attachment of a phosphoric radical by a protein kinase. The phosphorylated, negatively charged complexes, situated close to each other, reject reciprocally (rejection forces are of electrostatic nature). Following such reorganization, of the light-harvesting complexes of PS II decreases in size and that of the PS I increases, thus, ensuring a coordinated operation of both the photosystems. This regulating mechanism allows the photosynthetic apparatus to react adequately to changes in lighting conditions. When the need to expand the PS II antenna arises (in response to varying light intensity or spectral composition), a protein phosphatase is activated which eliminates the phosphate group by hydrolysis. The mobile dephosphorylated complexes move again to the granal thylakoids in order to contact PS II. Thus, by means of protein kinase and protein phosphatase action the chloroplast can optimize its power distribution between the light harvesting antennae of PS I and PS II. Redox regulation of photosynthetic enzymes with thiol groups of protein derived amino acids. Another mechanism of photosynthesis regulation includes the redox transformations of the photosynthetic proteins. The carboxylation

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reaction catalyzed by the RUBISCO enzyme is the slowest in the carbon fixation cycle. The activity of this enzyme is controlled at the level of thiol groups, which if in an oxidized state (S-S bridges) keep the enzyme idle and when reduced (–SH), activate the enzyme. An intermediate between this enzyme and the ETC, which is the source of electrons to activate RUBISCO is a specific protein thioredoxin, which is widespread not only in plants but also in the animal kingdom and bacteria. Thioredoxin is subject to redox transformations in cells by reduction of the thiol groups (-S-S + 2 ē + 2H+ → 2-SH). In chloroplasts, the thioredoxin is reduced by receiving two electrons from two molecules of reduced Fd in the presence of a specific enzyme ferredoxin-thioredoxin reductase. The reduced thioredoxin oxidizes yielding the electrons to RUBISCO. Thus, transition from darkness to light induces ETC activity, formation of reduced ferredoxin molecules and activation of the RUBISCO enzyme (ferredoxin-thioredoxin-RUBISCO). Due to ribulose diphosphate carboxylase activity, the speed of carbon dioxide utilization in the Calvin cycle increases. Activation of this enzyme also depends on other factors: changes in the pH and Mg2 ion content in the chloroplast stroma, that occur when chloroplasts are exposed to light.

4.9 Ecology of Photosynthesis By exchanging substances and energy plants are in constant contact with the environment. The ecology of photosynthesis involves the study of photosynthesis productivity dependence on environmental factors—light, O2 and CO2 concentration, temperature, water, humidity, minerals (Mg, N, P, K), presence of toxic substances which may inhibit photosynthesis etc. The dependence of photosynthesis on light intensity and spectral composition. Plant adaptation to different light intensities happened by means of different morphological and physiological changes. If seedlings are exposed to darkness, chlorophyll biosynthesis stops which determines the formation and accumulation of colorless protochlorophyllides, thus resulting in etioplasts in which the process of photosynthesis cannot take place. Exposure of etioplasts to light is accompanied by structural and functional changes, which cause the photosynthesis process to resume. Also, it induces differentiation of the internal membranes system as well as synthesis of proteins and lipids. On average, leaves absorb 80–95 % of the photosynthetically active solar spectrum (at a wavelength of 400–700 nm) and 25 % of the infrared energy, which constitutes about 55 % of the total radiation energy. Photosynthetic efficiency and the direction of organic biosynthesis depend on the quality of light absorbed by the leaf. The dependence curve of the photosynthesis process on the intensity of light has a logarithmic shape. A directly proportional dependence is observed only at low light intensities. Red light is always present in direct sunlight. Plants that have been grown in blue and red light differ essentially by the composition of photosynthesis products. In blue light other compounds are

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also formed besides carbohydrates. Photosynthesis rates increase by 20 % when blue light complements red light. The dependence of photosynthesis on the amount of CO2 in the air. CO2 concentration in the air is roughly 0.03 %. Photosynthesis can proceed at concentrations of at least 0.08 %. An increase in the concentration of this gas up to 1.5 % causes a proportional growth in photosynthesis intensity. At concentrations of 15– 20 % photosynthesis intensity doesn’t grow anymore, while at 70 % the process is inhibited. The effect of this growth is in close relationship with the manner in which other factors manifest themselves especially light intensity and temperature. There are plant species which are very sensitive to variations in CO2 concentration with significant slow-down of the process at only 5 %. Growing CO2 concentrations cause stomatal closure. During the day carbon dioxide concentrations in the immediate vicinity of the plant fall down and to increase their levels organic fertilizers are applied. CO2 absorption by the leafs is in a direct relationship with the intensity of air currents and atmospheric pressure values. According to J.P. Decker, the relationship between atmospheric pressure and CO2 diffusion rates is one of the fundamental factors determining the vertical (altitudinal) zonation of the vegetation. The dependence of photosynthesis on temperature. In the majority of plant species, photosynthesis starts at approximately 0 °C and intensifies as the temperature goes up. Every time, when the temperature increases by 10 °C there is a 1.5 to 1.6 times increase in the intensity of photosynthesis, which is directly related to the increase in chemical reaction speed. The influence of temperature on photosynthesis depends directly on illumination. In low light, photosynthesis does not depend on temperature (Q10 = 1) because the photosynthesis rates are limited by the speed of photochemical reactions. And vice versa, under intense light the overall rate of photosynthesis is limited by reactions of the dark phase and, in this case, the temperature begins to exhibit its influence. The optimal temperature for photosynthesis is 20–37 °C, with some variations depending on species. When temperature exceeds 37 °C the intensity of the process slows down quickly, due to harmful action on chloroplasts. When the temperature exceeds 37–40 °C, photosynthesis decreases quickly until its final interruption due to hampered enzymatic activity and chloroplast photoinactivation. The increase of temperature can also make the stomata close or can influence the viscosity of assimilatory cells. The dependence of photosynthesis on the quantity of water. Photosynthesis depends to a large extent on water supplies to leaf tissues, which assures guard cell turgidity involved in opening and closure of the stomata. A relatively small water deficit in cells (5–15 %) can influence positively the photosynthesis process because, when fully saturated, stomata cannot open due to the pressure from the neighboring mesophyll cells. If the water deficit exceeds 15 %, photosynthesis intensity slows down as stomata close which prevents CO2 from entering the leaves. This causes transpiration rates to decrease and, as a consequence, the temperature of the leaves grows up. Dehydration influences the configuration and implicitly, the activity of the enzymes that are involved in the dark phase. Research carried on sunflower has proven the mutual dependence of the degree of cell hydration and photosynthesis intensity, which is known as the V.A. Brilliant

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phenomenon. This relationship depends on the biological peculiarities of the species (hydrophytes, hygrophytes, mesophytes, xerophytes). At high water deficit in leaves, xerophyte species continue assimilation of CO2 while mesophytes and hydrophytes feel its absence more acutely. The influence of minerals on photosynthesis intensity is explained by the fact that they are part of some organic compounds involved in this process or serve as catalysts in some chemical reaction. The role of mineral nutrition in photosynthesis is multiple. Potassium acts indirectly by increasing the hydration degree of the cytoplasm, speeding up the transport of metabolites in leaves and opening stomata by being used to regulate the turgidity of guard cells. Potassium ions act directly activating the phosphorylation process. The importance of phosphorus in photosynthesis is also very important. Phosphorylated compounds are present in all the phases of photosynthesis and energy is accumulated in the form of phosphoric bonds. Many compounds of the ETC contain iron (cytochromes, ferredoxin) or copper, this is why in the absence of these elements the intensity of photosynthesis decreases. When the quantity of iron is insufficient, leaves lose their green color and turn yellow (chlorosis). Nitrogen and magnesium are parts of chlorophyll, with nitrogen being also part of amino acids and proteins. Photosynthesis is very sensitive to toxic substances (SO2, As2O3, H2S, herbicides, insecticides, chloroform etc.). In their presence, assimilation intensity decreases due to a harmful effect on cytoplasm, chloroplasts and the oxidation-reduction enzymes involved in photosynthesis.

• Autotrophic plants (from Greek autos “self” and trophe “nutrition”) produce by themselves all the necessary organic material using mineral substances absorbed from the external environment. Depending on the source of energy they use, autotrophic plants are grouped in phototrophic plans, which use solar energy, chemoautotrophic plants, which use chemical energy produced by oxidation of certain mineral substances (H2S, NH3 etc.) in the process of chemosynthesis. • The total amount of carbon, fixed by means of photosynthesis during the entire year, is about 7.8 × 1010 tones. This quantity is compensated by the same amount of CO2 which is eliminated through respiration by heterotrophic organisms. Assimilated carbon amounts to about ¼ of the total CO2 reserves in the atmosphere. Every year up to 0.3–0.4 % of the total reserve of carbon from the hydrosphere and troposphere is assimilated. • The total production of organic substance, synthesized by the vegetation of the planet, calculated in glucose, sums up to 4.5 × 1011 tons per year. • The world’s annual energy consumption (5 billion people) is 3 × 1020 J— 10 % of the energy accumulated annually in the process of photosynthesis. About 1 billion tons is used in the form of food products solely. It equals to

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an energy of 15 × 1018 J, which sums up to 0.5 % of the total energy accumulated during 1 year. Photosynthesis in some inferior organisms takes place in chromatophores of various forms and sizes: spiral ribbon (spirogyra), stars (zignems), parietal plates (ulotrix). The hydrosphere contains 60 times more carbon than the atmosphere (3.5 × 1013 tons), which is determined by the high solubility of CO2 in water. Therefore, it can be assumed that the insignificant accumulation of CO2 as a result of oil burning (less than 1 % of the total annual amount of CO2 in the atmosphere) should not induce an essential increase of carbon dioxide amounts in the atmosphere. But in fact, the exchange of carbon dioxide with the atmosphere is carried out relatively quickly, during 6– 7 years, only in the outer layers of the world ocean which concentrate 1.5 % of the total amount of carbon dioxide dissolved in water, while the establishment of such an equilibrium at a deeper level will take several thousands of years. As a consequence, industrial burning of natural deposits caused the increase of CO2 in the atmosphere from 0.027 % (the preindustrial period) up to 0.034 %—current level. It is estimated that by 2035 the quantity of CO2 in the atmosphere will double (0.06 %), thus causing global climate warming (the greenhouse effect). Increasing the surface of green areas could be one of the solutions to the big ecologic problem of global warming. The combined area of chloroplasts is 200 times bigger than the surface of the organ in which they reside. On 1 mm2 one can find from 100 to 300 stomata. The amount of CO2 diffusion through stomata is 50 times higher than its diffusion through the cuticle. The number of thylakoids in cells of different species differs essentially. In cells of photosynthesizing bacteria, for example, there can be several thousands of them. In others, only a chromatophore, with a big number of thylakoids can be observed. For example, in different species of spirogyra one can find from 1 up to 12 chromatophores in the shape of a spiral ribbon. The external membrane of the plastid is found in most of the thallophytes and in all cormophytes. It is not present, however, in blue algae and in some bacteria. In blue algae, the lamellae of the plastids are embedded directly in the cytoplasm, forming with it a complex called chromatoplasma. This findings show that the external membrane appeared later than the stroma and the lamellae during phylogenesis of the chloroplasts, while in ontogenesis the external membrane develops first, followed by the stroma and the system of lamellae. Every second, the Sun emits an amount of energy equal to 9 × 1022 kcal, which means 3 × 1033 kcal per year. Out of this energy, about 5 × 1020 kcal (1 over 6 billion of the total) reaches the Earth. The land gets about 40 %

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(2 × 1020 kcal) or 1 over 15 billion of the total energy issued by the Sun. Thus, out of the 40 % of the Solar energy that reaches the surface of the Earth, 2–5 % is absorbed by plants, out of which only 0.1–0.22 % is used in the production of organic matter.

Glossary Carotenoids Yellow and orange pigments that are found in chloroplasts and chromoplasts that participate in light absorption as supplementary pigments and protect the molecules of chlorophyll and other active substances from irreversible photo-destruction. One can distinguish oxygen free carotenoids (C40H56lycopeneα-β-γ-carotenoids) and oxidized carotenoids (C40H56O2C40H56O4xanthophyllsluteinzeaxanthin). Chloroplasts Specialized organelles of the vegetal cells in which photosynthesis takes place delimited to the exterior by two membranes—internal and external with the second being incorporated in the homogeneous environment (stroma). The internal membrane form the folds called stromal thylacoids and granal thylakoids, in which all the photochemical reactions of the light phase are carried. The dark phase of photosynthesis A complex process that includes the sequence of enzymatic reactions that lead to the formation of photosynthesis products and of the organic acceptor of carbon dioxide. The light phase of photosynthesis A phase of photosynthesis during which light absorption and transformation of solar energy into the chemical energy of ATP and NADPH+H+ happens. This process occurs in the active photochemical membranes of the chloroplast and represents a system of photophysicalphotochemical and chemical reactions. Acyclic phosphorylation A process during which light energy is transformed into the macroergic bonds of ATP and NADPH+H+. It is then followed by water photolysis and oxygen elimination. Cyclic phosphorylation A process during which the electron emitted by chlorophyll through a series of transformations returns back to the pigment. The absorbed energy is fixed in the macroergic ATP bonds. Photosynthetic phosphorylation A process of converting light energy quanta into ATP.

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Photodissociation of the water The light induced decomposition of water molecules that occurs during the light phase of photosynthesis. As a result of water photodissociation free oxygen which is eliminated and hydrogen which is used to reduce CO2 in the dark phase are produced. Photosynthesis (Carbon nutrition)—a fundamental process during which organic compounds are synthesized by green plants and photosynthesizing microorganisms out of simple inorganic substances (CO2 and H2O) in the presence of light and during which the solar energy is transformed into the energy of chemical bonds of organic substances. Photosystem I The assimilation unit which has as reaction center a molecule of chlorophyll “a” capable to absorb light with a wave length of 700 nm (noted as P700)as well as 200 molecules of chlorophyll “a” (sometimes chlorophyll “b”) and 50 molecules of carotene in the composition of the light harvesting complex. Photosystem II The assimilation unit which has as reaction center a molecule of chlorophyll “a” (P600) and auxiliary light sensitive pigments: 200 molecules of chlorophyll “a” 200 molecules of chlorophyll “b” and xanthophylls. Quantum efficiency of photosynthesis The number of CO2 molecules subjected to photochemical transformation per each absorbed light quantum. It equals roughly 0.25 which means that 4 photons of red light are consumed to reduce a CO2 molecule. The Robin Hill reaction The elimination of oxygen from the water molecule by isolated chloroplasts under the action of light and in the presence of artificial acceptors of electrons. It explains the essence of the 2 phases in the chemistry of photosynthesis.

References Gavrilenco VF et al (1986) Selected paragraphs from plant physiology. M., 440 Govindji M (ed) (1987) Photosynthesis, vol 2, pp 728 and 460 Gudwin T, Merser E (1986) Introduction into plant biochemistry. T. 1. M., 392 (1986) Kleiton R (1984) The photosynthesis. Physical mechanisms and chemical models. M. p. 350 Klimov VV (1996) Photosynthesis and the Biosphere. Soros Educ J 8:6–13 Kulaeva ON (1997) The chloroplast. Soros Educ J 7:2–9 Makronosov AG (1981) The photosynthetic function and the integrity of the plant organism. In: 42nd, Timireazev’s reading. M. p. 64 Purves WK, Sadava D, Orians GH, Heller HC (2005) Life Science of Biology, 7th edn. W.H. Freeman & company, USA, p. 1121 Rubin BA, Gavrilenco PF (1977) Biochemistry and the physiology of photosynthesis. M. p. 328 Rubin BA (1997) The primary processes of photosynthesis. Soros Educ J 10:79–84 Shestakov SV (1998) The molecular genetics of photosynthesis. Soros Educ J 9:22–27 Tihonov AN (1996) Energy transformation in chloroplasts—the energy converting organelles of the plant cell. Soros Educ J 4:24–32

Chapter 5

Plant Respiration

Abstract Respiration is a process in living organisms involving the production of energy, typically with the intake of oxygen and the release of carbon dioxide from the oxidation of complex organic substances. The energy gained during respiration is used to fuel all endergonic reactions of the organism. The catabolism of the substances can be either complete (aerobic respiration) with water and CO2 as final products and complete energy release or partial (anaerobic respiration or fermentation). For instance, in plants, under specific anaerobic conditions dictated by the environment (e.g. during flood or under thick layers of snow) alcoholic fermentation takes over which is energy inefficient and can be detrimental for prolonged periods. The first stage of respiration, glycolysis is represented by the reactions that lead to glucose break down into pyruvate yielding four molecules of ATP derived from substrate-level phosphorylation (of which two are reused for glucose activation), two molecules of NADH+H+, but also metabolically important intermediates. Next, pyruvate molecules undergo oxidative decarboxylation which results in the synthesis of an acetyl-CoA and another NADH+H+ molecule per molecule of pyruvate. Acetyl-CoA is utilized in the Krebs cycle (tricarboxylic acid cycle) in a stepwise release of metabolic energy accompanied by the synthesis of a GTP molecule, 3 molecules of NADH+H+, 1 of FADH+H+. The Krebs cycle also results in a series of keto acids used for the synthesis of amino acids and other valuable organic substances including acetyl coenzyme A, involved in fatty acid synthesis. Inside mitochondria, the energetic function is located on the internal membrane which forms the crystae. Here, the NADH+H generated during all of the above mentioned stages as well as the FADH+H+ are used by the components of the Electron Transport Chain in the process of oxidative phosphorylation generating respectively up to 3 and up to 2 molecules of ATP each. Electron transport is coupled with proton transport and the formation of a proton gradient across the inner membrane which drives the action of the ATP synthase. Consequently, the break down of one molecule of glucose leads to the synthesis of the energy equivalent of up to 38 molecules of ATP. There are also variations of the classical biochemical reactions in the Krebs cycle represented by the pentose phosphate cycle, the glyoxylate cycle, the direct sugar oxidation path.

© Springer International Publishing Switzerland 2015 M. Duca, Plant Physiology, Biological and Medical Physics, Biomedical Engineering, DOI 10.1007/978-3-319-17909-4_5

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Historical Background 1770—A.L. Lavoisier for the first time emphasizes the role of oxygen in living organisms. 1897—A.N. Bach formulated the peroxide theory of biological oxidation. 1912—V.I. Palladin proposed the idea of two stages in respiration: anaerobic and aerobic. 1912—H. Wieland demonstrated the role of oxygen in oxidation processes. 1921—O.G. Varburg showed that oxygen assimilation is inhibited by carbon dioxide. 1925—D. Keilin discovered cytochrome oxidase. 1935—G. Embden, O. Meyerhov and I. Parnas determined the most important products of glycolysis. 1937—H.A. Krebs described the citric acid cycle (the Krebs cycle) in animals. 1939—A.C. Ibnell discovered the presence of the Krebs cycle in plants. 1939—H.M. Kalcar and B.A. Belitzer discovered oxidative phosphorylation. 1961—V.A. Engelhardt submitted the idea of oxidative phosphorylation and aerobic respiration. Brief Updates According to proteomic studies, each cellular body contains 1,000–2,000 different polypeptides. Approximately 15 % of the cellular proteins are located in

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mitochondria, 30 % of which have a role in respiration and 25 %—in the primary metabolism. Protein-synthesis in the plant cell is achieved by the interaction of three genetic systems—nucleus, chloroplasts, mitochondria. Most of the mitochondrial proteins are synthesized in the cytosol as precursors, which are subsequently imported into organelles. All mitochondrial proteins are classified as: • Proteins encoded by the mitochondrial DNA; • Proteins encoded by the nuclear DNA (the majority); There are also protein complexes encoded by both mitochondrial and nuclear genes. The nuclear genome encodes structural proteins from the mitochondrial membranes, the soluble polypeptides from its matrix and the enzymes involved in respiration. The circular chloroplast genome has 120–160 kb and encodes only 120 proteins. Chloroplast proteome analysis showed that about 21 % of proteins are involved in energy processes, 2 % in transcription and translation, 10 % in growth and division, 3 % in defensive processes and 2 % in transport. About 17 % of the proteins have an unidentified function while for 31 % of them the encoding genes are not known. The mitochondrial genome, which can be present in plants in both linear and circular forms, can encode about 100 polypeptides, while the mtDNA in animal cell can only encode 10/15 different polypeptides. Most of the mitochondrial proteins have their molecular weight between 30 and 60 kDa. Researchers have demonstrated the role of mitochondria in programmed cell death (apoptosis) and synthesis of shock proteins (HSP10, HSP60, OZ11, MnSOD), involved in the stress response to ozone and bacteria. Studying mtDNA with the help of restrictases (restriction analysis) and hybridization techniques has proved the presence of several types of mitochondrial DNA, each of them containing specific genetic information.

5.1 General Notions of Respiration Respiration is one of the basic characteristics of life and represents the physiological process of oxidative degradation of complex organic substances to intermediary products (fermentation) or to final products (aerobic respiration). Aerobic respiration occurs in the presence of O2 and ends up with total substrate degradation (catabolism) and energy release. Aerobic respiration happens in all the cells at the mitochondrial level (Fig. 5.1). C6 H12 O6 + 6O2 = 6CO2 + 6H2 O + 686kcal=mol 2867kj=mol




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Fig. 5.1 The inner structure of a mitochondrion

Anaerobic respiration occurs in the absence of oxygen and ends up with partial energy release, which is characteristic of anaerobic organisms: C6 H12 O6 = 2CO2 + 2C2 H5 OH + 48kcal=mol The amount of released energy depends on the organic substances involved. Thus, biodegradation of 1 g of glucose yields 3.9 kcal, of 1 g of starch—4.2 kcal, of 1 g of protein—5.7 kcal, of 1 g of lignin—5.7 kcal, of 1 g of lipids—9.4 kcal. Importance of plant respiration • During respiration gas exchange takes place (discovered by A.L. Lavoiser 1773), which results in complete oxidation of the catabolites. • Biological oxidation contributes to the formation of certain intermediary products which are used in the synthesis of other cellular compounds. Thus, 11 organic acids are synthesized in the Krebs cycle, which can be used in different metabolic cycles (α-ketoglutarate and succinic acid participate in direct amination with amino acid formation; acetyl coenzyme A participates in fatty acid synthesis). • During respiration, the energy from organic substrate degradation accumulates in mitochondria. The biggest part of the released chemical energy (65 %) is stored in the macroergic bonds of ATP—like phosphate compounds, as physiologically usable chemical energy. This energy is used in all the vital processes which require metabolic energy—endergonic processes (nitrate reduction in plants, polymerization reactions and a variety of other synthesis reactions, water and mineral salt absorption, in plant movements—tropisms and nasties, for growth and development processes). A relatively small amount of the released energy is used to maintain the structure of the cell protoplasm—maintenance respiration. In growing cells, a bigger amount of energy is used in the process of structural organic substance synthesis—growth respiration. Another part of the energy released during respiration is transformed in mechanical energy, which is used in creating the cytoplasmic flow. In some cases, this

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energy causes the temperature of the tissues to be a few degrees higher than that of the environment. Release of chemical energy and ATP formation are the essence of the respiration process called energogenesis. The process of caloric energy release is known as thermogenesis.

Organic substances which are subject to degradation through catabolic reactions are called respiratory substrates. The fundamental and universal respiratory substrate is represented by reserve carbohydrates. Proteins and lipids are used in the germination of fat seeds and in olive plant fruit respiration. Citric acid is used in respiration in lemon fruits, tartaric acid—in young grapes, malic acid—in young apples, mannitol—in young ash trees, olives, bay leaves, coffee, cinnamon tree and sorbate—in apple, pear and rowan. One index that reflects the respiration type and the oxidized respiratory substrate as well as the nature of oxidation during is known as the respiratory coefficient (quotient) which is marked by QR or CR. QR represents the ratio between the volumes of eliminated CO2 and absorbed O2 during the respiration process. The respiratory index is equal to one in case of glucose oxidation: CR ¼

nðCO2 Þ 6ðCO2 Þ ¼ ¼1 nðO2 Þ 6ðO2 Þ

When lipids and oxygen-poor proteins are degraded CR < 1 and, with its values ranging between 0.3 and 0.5. For organic acids CR is ranging between 1 and 4. The less oxygen is contained in the molecules of the respiratory substrate the more free oxygen it will need during respiration, and the lower the CR value will be. 2C2 H2 O4 ¼ 4CO2 þ 2H2 O þ Q C18 H36 O2 þ 26O2 ¼ 8CO2 þ 18H2 O þ Q

CR ¼

nðCO2 Þ 4ðCO2 Þ ¼ ¼4 nðO2 Þ 1ðO2 Þ

CR ¼

nðCO2 Þ 18ðCO2 Þ ¼ ¼ 0:69 nðO2 Þ 26ðO2 Þ

The respiratory index also differs depending on the age of the organ and of the organism. Thus, in green fruits the respiratory index is 0.5–0.1, in mature fruits it varies between 2.5 and 3.0, in flax seeds it is 0.65 and in tangerines it is 2.65. CR is of crucial importance in the process of seed germination, as well as in regulating fruit maturation and preservation. A low CR in oleaginous plants means that much aeration is needed during germination. In tomatoes, blueberries, plums and melons, by the end of their growth when organic acids are still being synthesized, CR is 0.85, during the aromatic phase in the same fruits it is 1.25, and during the maturity phase CR is as high as 2.7.

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Table 5.1 The effect of different factors on the process of respiration Stimulatory effect

Inhibitory effect

Presence of elements with the role of coenzyme (Fe, Cu, Zn, Mo, Co) Presence of phosphorus, which amplifies the activity of dehydrogenases Attack of certain pathogens (fungi, bacteria, viruses etc.) Trauma (wounds, insect bites etc.)

Inhibitors which act on enzymes (pesticides, cyanides, CO2) Temperatures under and over optimal threshold Excess of water

Another specific index of respiration is the intensity of respiration, which depends on the substrate, organism age and environmental conditions, and varies in different groups of plants. The intensity of respiration also correlates with the biological activity of different organs and tissues. The intensity of respiration is very high in inferior plants, which have a short ontogenetic life cycle, in germinating seeds, in floral organs and in ripen fruits. The lowest values are found in woody roots, underground stems and in dry seeds. Due to growth respiration, there is an active consumption of carbohydrates in young organs, while in mature organs, which require less energy, there is only a maintenance respiration. Flowers breathe more intensely than leaves, and leaves breathe more intensely than other vegetative organs. Deciduous species breathe more intensely than those with persistent leaves. Species with C3 type of photosynthesis breathe more intensely than those with C4 type. The intensity of respiration is subject to positive or negative influence of various factors (Table 5.1).

5.2 Respiratory Enzymes Respiration represents a successive chain of oxidation-reduction reactions. It is an enzymatic process in which three groups of oxidoreductases participate: aerobic dehydrogenases, anaerobic dehydrogenases and oxidases Enzymes form enzyme-substrate complexes, in which the activation energy for the reaction they catalyze is reduced. The active center and the substrate are perfectly complementary (the principle of “lock and key”) (Fig. 5.2). Anaerobic dehydrogenases (alcohol dehydrogenases, lactate dehydrogenase, malate dehydrogenase etc.) transport the protons and the electrons to different intermediary acceptors, except for O2 and consist of two components: NAD cofactor and the prosthetic part. The NAD cofactor temporarily binds the active center and participates directly in the oxidation-reduction reaction. Aerobic dehydrogenases transfer electrons and protons to different intermediary acceptors, including O2. Aerobic dehydrogenases contain FAD (flavin-adeninedinucleotide) or FMN (flavin-mononucleotide) as a cofactor which is why they are also called flavoproteins. They transport electrons and protons to the oxidases. In

5.2 Respiratory Enzymes

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Fig. 5.2 Activation energy with and without catalyst and the mechanism of enzyme action

this group of enzymes the most important one is succinate dehydrogenase, which transfers electrons from aerobic dehydrogenases to quinones, cytochromes or O2. Oxidases are enzymes which transport electrons and protons to the final acceptor—O2. These enzymes are situated on the mitochondrial cristae and participate in the redox reactions of the electron transport chain (ETC). Given their action mechanism, it is considered that they participate in the process of activation of atmospheric oxygen. Depending on the number of electrons transported, oxygen can be turned into H2O, H2O2, or O2. The cytochrome oxidases represent a coupling between A and a3 cytochromes. Their structure looks like that of hemoglobin, because they are formed of a porphyrin core with four pyrrolic groups having iron as a cofactor in the center, which during electron transport is oxidized and reduced successively: Fe2þ 
e ! Fe3þ þ
e ! Fe2þ The transport of electrons in the electron transport chain is carried out according to the electric potential gradient. Electrons can be transferred to oxygen only by the cytochrome-oxidase (A+a3) (Fig. 5.3). Polyphenoloxidases contain copper atoms as cofactors. These enzymes participate in the oxidation of both polyphenols (respiratory chromogens) and quinones (respiratory pigments). Peroxidase and catalase, which contain the iron cation as a cofactor are part of the oxidase group too. Peroxidase oxygenizes the organic substrate by transferring protons to the peroxide oxygen resulting from oxygenated water decomposition: 2H2 O2 ! 2H2 O þ O2 ðin the presence of catalaseÞ Catalase is the main enzyme which characterizes the intensity of metabolic processes during ontogenesis. Frost resistant plants have a reduced catalase activity in winter, which reflects a deep hibernation state. The more productive forms have a higher catalase activity during generative organ formation.

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Fig. 5.3 The path travelled by electrons through the cytochrome system down their way to oxygen

5.3 A.N. Bach’s and V.I. Palladin’s Theories Efforts made by the physiologists and biochemists at the end of the nineteenth century contributed to the elucidation of the chemistry and mechanism of the respiration process. Two theories related to the primary processes of substrate degradation, are recognized: • the theory of slow oxidation, defined in 1897 by A.N. Bach; • the theory of respiration, defined in 1915 by V.I. Palladin. According to the theory of slow oxidation, in aerobic respiration the main role goes to oxygen which is activated by a easily oxidizable substance (A) from the plant body. As a result of breaking the double bond between oxygen atoms and the coordinative bond of –O–O type, peroxides are formed. This kind of reaction is carried out with the help of peroxidase and cytochrome oxidase enzymes, which together with O2 create peroxide combinations. This type of reactions is assisted by enzymes of the peroxidase and cytochrome oxidase type which form peroxidic combinations with O2. The oxidation cycle is repeated until the complete oxidation of the substrate:

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The respiration theory, formulated by V.I. Palladin, is based not on the activation of atmospheric oxygen, but on hydrogen extraction from the respiratory substrate, a process occurs with the participation of dehydrogenases. In plants, there are certain specific substances—respiratory pigments (R), which are capable of sequestrating the hydrogen from the respiratory substrate and forming intermediary compounds—respiratory chromogens (RH)-, which, during the final stage, are decomposed under the influence of O2: 1: C6 H12 O6 þ 6H2 O þ 12R ! 6CO2 þ 12RH2 2: 12RH2 þ 6O2 ! 12R þ 12H2 O C6 H12 O6 þ 6O2 ! 6CO2 þ 6H2 O Palladin’s theory explained the following: • the biphasic nature of the respiratory process (the anaerobic and aerobic phases); • atmospheric O2 does not participate directly in glucose oxidation but rather oxidizes certain intermediary products; • CO2 has anaerobic origin; • the role of water in the respiration process. Recent researches have proved that the respiration mechanism is based on double activation and consists of a successive chain of specific reactions, during which activation of oxygen and hydrogen represents coupled phases of the same process.

5.4 Respiration Mechanism The modern concept concerning the chemistry of the respiration process has been elaborated based on the works of the following scholars: A.N. Bach, V.I. Palladin, O. Varburg, V.A. Enghelhardt, A.I. Oparin and others. It implies carrying out a chain of redox reactions, catalyzed by specific enzymes. In case the respiratory substrate is represented by soluble carbohydrates, the degradation reactions comprise two phases: • anaerobic degradation; • aerobic degradation.

5.4.1 Genetic Link Between Respiration and Fermentation Between 1912 and 1928 the physiologist S.P. Kosticev, continued to research the mechanism of respiration in different living organisms and concluded that the initial chemistry of the respiration process in plants, animals, human beings and microorganisms is identical. In other words the first stage of respiration, during which

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Intermediate products

Pyruvic acid (PA)

Complete degradation of the substrate occurs in aerobic conditions

In conditions of saturation with metabolic energy, AP takes part in carbohydrate resynthesis

Various types of fermentation take place in anaerobic conditions

Fig. 5.4 The link between aerobic and anaerobic respiration

hexoses are decomposed anaerobically until pyruvic acid is formed, are based on reactions identical for all living organisms. The subsequent metabolic paths are different. In anaerobic conditions the pyruvic acid is subjected to degradation through fermentation, while in aerobic conditions—through the Krebs cycle. In cases when the cell does not require energy, resynthesis of hexoses occurs. Thus, Kosticev proved the genetic link between respiration and fermentation, at the level of pyruvic acid. He also determined that this acid represents half of the glucose molecule and is the fundamental plate of the cellular metabolism for both catabolic and anabolic processes (Fig. 5.4). Under anaerobic conditions, plants (germinated seeds, fruits and vegetables stored in poorly ventilated warehouses, vegetal organs that are submerged in water during floods or are under thick layers of snow) breathe through alcoholic fermentation, but this process is inefficient from the energetic point of view and accumulation of ethylic alcohol in plants can be toxic for the functioning of living cells.

5.4.2 Glycolysis—The Anaerobic Phase of Respiration Glycolysis, the first stage of the respiration process was discovered by G. Embden, O.F. Meyerhof and I.O. Parnas (it is also called the EMP path, dichotomyic path, anaerobic phase of respiration). It happens in the cellular hyaloplasm and in chloroplasts. This stage involves endogenous oxygen and consists of three

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Fig. 5.5 The cleavage of the molecule of fructosebisphosphate by an aldolase results in the formation of two isomers—glyceraldehyde-3phosphate and dihydroxyacetone phosphate

successive phases during which the molecule of glucose is divided into two molecules of pyruvic acid: • activation of the hexoses with the consumption of two molecules of ATP and their dichotomic decomposition into two phosphotrioses—unstable intermediary products (Fig. 5.5); • the first substrate phosphorylation, which starts with the transformation of phosphoglyceric aldehyde into phosphoglyceric acid with energy release in the form of reduced NADH+H+ and synthesis of one molecule of ATP; • the second substrate phosphorylation, during which the 3-phosphoglyceric acid, as a result of intramolecular oxidation yields the phosphate while a molecule of ATP is formed. Glycolysis is energetically poor compared to oxidative phosphorylation. During it, a macroergic bond is formed—the enolphosphoric bond (the highest-energy phosphate bond found in living organisms, −61.9 kJ/mol), the energy of which is used for synthesizing ATP:

Glycolysis (Fig. 5.6) depends on the availability of oxidized NAD (NAD+), which is a limiting factor for the entire process and can be produced by mitochondrial oxidative phosphorylation.

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Fig. 5.6 Scheme of glycolysis

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General reaction: C6 H12 O6 þ 2NAD þ 2ADP þ 2Pi ! 2CH3 COCOOH þ 2NADH þ 2ATP glucose

pyruvate

The process ends up with the production of two molecules of pyruvic acid from a molecule of glucose. Over time, two molecules of ATP are consumed and four molecules of ATP and two of NADH+H+ are produced. Importance of glycolysis • Glycolysis is the initial and common phase of aerobic respiration and fermentation. • It is the link between the respiratory substrate and the Krebs cycle. • It yields two molecules of ATP and two of NADH+H+ per each molecule of hexose. • A series of intermediary products which can be used in different metabolic cycles are formed. • In chloroplasts, it represents an independent way of ATP and NADH+H+ synthesis. • By means of glycolysis, in this cellular organelles starch breaks down into phosphotrioses—a compound that can be transported through the chloroplast membrane.

5.4.3 Krebs Cycle (Tricarboxylic Acid Cycle) The pyruvic acid that results from glycolysis is subjected to activation during which an acetyl-CoA molecule is formed which goes into the Krebs cycle (tricarboxylic acid cycle). These transformations, as well as the cycle itself occur in the mitochondrial matrix. The Krebs cycle is a succession of oxidative decarboxylation reactions, of dehydrogenation reactions, water fixation or elimination reactions which result in the formation of a series of intermediary products: citric acid → isocitric acid → ketoglutaric acid → succinic acid → fumaric acid → malic acid → oxaloacetic acid (the latter is converted into citric acid when pyruvate is provided and the cycle restarts). In the mitochondrial matrix different transformations occur that can be grouped in 5 important phases (Fig. 5.7): 1. Oxidative decarboxylation of the pyruvic acid, along with substrate dehydrogenation in the presence of the dehydrogenase, which transports the electrons and protons to O2 resulting in water formation. Through acetylation acetyl coenzyme A is formed (an important metabolic and energetic compound, which includes a macroergic bond of 11 kcal) with the participation of thiamine

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Fig. 5.7 Krebs cycle and its importance

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5.4 Respiration Mechanism

2. 3. 4.

5.

137

pyrophosphate which serves as a coenzyme for pyruvate dehydrogenase. Coenzyme A is a derivative of adenine, which consists of pantothenic acid, thioethanolamine amino acid and three phosphoric acid residues. The high energetic activity of acetyl coenzyme A is determined by the sulfhydryl group SH from the thio-ethanolamine, which is linked to the molecule through a macroergic bond. Coupling of acetyl coenzyme A with oxaloacetic acid in the presence of a synthetase, and formation of the citric acid (with coenzyme A release). Isocitric acid formation occurs through water loss when citric acid is converted to cis-aconitic acid, and isocitric acid is formed as a result of aconitase action. Formation of di- and tricarboxylic acids occurs via dehydration, decarboxylation and water fixation reactions, having the α-ketoglutaric, succinic, fumaric, abd malic acids as intermediary forms, with intercalation of a molecule of succinyl coenzyme A before the formation of the succinic acid. Reinitiation of the pyruvic acid cycle. By malic acid dehydrogenation, under the action of malate dehydrogenase enzyme and of the energetic group NAD+, oxaloacetic acid is formed, which, through decarboxylation, is converted into pyruvic acid or is coupled with acetyl coenzyme A, producing the citric acid, reactions which reenter the Krebs cycle.

Importance of the Krebs cycle • It is the universal pathway of substrate degradation. • Pairs of electrons are gradually released and stored in intermediates like NAD and FAD to be latter used in the ETC for ATP synthesis. • Plants are supplied with energy and metabolites, hence its role not only in catabolism but also in anabolism (keto acids are used in the synthesis of amino acids and other valuable organic substances, acetyl coenzyme A participates in the process of fatty acid synthesis).

This reaction cycle is the universal pathway of respiratory substrate degradation for carbohydrates as well as for other organic substances and represents the main link in cellular metabolism. Most of the intermediary products are used in other metabolic cycles (Fig. 5.5). During respiratory substrate oxidation a stepwise release of metabolic energy occurs. During each cycle one molecule of GTP (equivalent to 1 ATP) and 4 molecules of reduced coenzyme are generated, which, when transported to the ETC, are subjected to oxidative phosphorylation.

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5.4.4 The Electron Transport Chain and the Energetic Outcome of Aerobic Respiration Respiration is located in mitochondria, semiautonomous cellular organelles, with an external membrane which separates the mitochondria from the cellular cytoplasm and an internal one where the energetic function is predominantly located (the internal membrane forms folds called crystae). The matrix represents a homogeneous colloidal mass and contains ≈50 % protein. It contains all the enzymes of the Krebs cycle and enzymes for protein, DNA and ARN biosynthesis, all the components of proteosynthesis and the enzymes of fatty acid synthesis. The internal membrane contains 20–25 types of protein with structural and enzymatic role. The components of the electron transport chain (ETC) are located here (ferroprotein, coenzyme Q, cytochromes b, c, c1, a, a1). They are arranged in growing order according to their redox potential from “+” to “–”. The process of electron transport in the ETC is coupled with that of proton transport and creation of a proton gradient across the inner membrane of the mitochondrion which later fuels ATP synthesis by oxidative phosphorylation. Aerobic oxidation occurs at the level of oxysomes which are situated on the internal membrane of the mitochondria. The order of ETC components depends on their redox potential and can be illustrated by the following scheme: FAD # NADH ! FMN ! ubiquinone ! cytochrome b ! cytochrome c1 ! cytochrome c ! cytochrome a1 a3 þ 0:82V H2 O " 1=2O2 þ 2Hþ

ATP biosynthesis happens only on the internal membrane. At this stage, water forms from atmospheric oxygen and the hydrogen resulting from the oxidation of NADH+H+, FADH+H+. Electron transport is an exergonic process, which occurs with release of energy and is powered by the difference of redox potential between the initial and final systems. During transportation of a molecule of NADH+H+ and its oxidation in the ETC, biosynthesis of up to three molecules of ATP occurs, while in case of oxidation of one molecule of FADH+H+ or FMNH+H+, the biosynthesis of up to two ATP molecules happens. Thus, the energetic balance of respiration can be represented as follows: • glycolysis. 4 molecules of ATP result from the glycolysis process, out of which 2 are consumed for hexose activation through phosphorylation. In addition two molecules of NADH are produced, which are transported to mitochondria (the process of transport also consumes energy) and result in up to 6 molecules of ATP in the ETC.

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139

4ATP  2ATP ¼ 2ATP 2NADH þ Hþ  3ATP ¼ 6ATP 8ATP=glucose molecule • pyruate decarboxylation and The Krebs cycle. 15 molecules of ATP for each molecule of pyruvic acid and consequently, 30 molecules of ATP for each molecule of glucose are produced. Out of these, 24 are incorporated in NADH +H+ and, 6—in FADH+H+ while two molecules of GTP are obtained separately during the cycle. 4NADH þ Hþ  3ATP ¼ 12ATP 1FADH þ Hþ  2ATP ¼ 2ATP 1GTP ðequivalent to 1ATPÞ 15ATP=pyruvate molecule ¼ 30ATP=glucose molecule In sum 8ATP + 30ATP = 38ATP are produced per molecule of glucose. In reality, this number is around 30–32 or lower, due to the need to transport compounds (e.g. the pyruvate and the NADH produced during glycolysis, the phosphate, and the ADP) across the membrane into the mitochondrion which requires energy, but also due to the “leakiness” of the mitochondrial membrane for hydrogen protons, which means that not all of the hydrogen protons are used for ATP synthesis. By taking the ratio between the amount of energy released through respiration to the amount of energy expended on anabolic processes the following energy efficiency is obtained. 1.591 kJ:2.871 kJ 55.4 % 381 kcal:686 kcal There are three theories which explain the mechanism of oxidative phosphorylation: • Chemical • Mechanical • Chemiosmotic. According to P. Mitchel’s theory, there is a direct connection between chemical and transport processes. The transfer of protons across the internal membrane of the mitochondrion causes the appearance of a pH gradient and of a difference in electric potential— membrane potential (the inner side of the membrane is negatively charged, while the outer side—positively charged.) As a consequence, an electrochemical proton gradient emerges (ΔμH). This gradient drives the transfer of protons inside the mitochondrial matrix through the ATP synthase complexes embedded in the

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membrane. In this protein complexes, a transmembrane protonic channel exists. When protons flow from the outer side of the membrane into the matrix through this channel, ATP is synthesized from ADP and phosphate. Thus, the biological meaning of the ETC consists in harnessing the electrochemical potential and synthesizing ATP by means of a process called oxidative phosphorylation. To sum up the above mentioned, this process can be represented as follows: NADH + Hþ + 3ADP + 3Pi + 1=2O2 ! NADþ + 3ATP + 3H2 O

5.5 Different Types of Respiratory Substrate Oxidation The Krebs cycle represents a universal way of substrate degradation, which is common for the majority of living organisms. In addition, alternative pathways are known: • direct sugar oxidation; • the pentose phosphate cycle; • the glyoxylate cycle. Direct sugar oxidation, is also called the Entner-Doudoroff pathway. It was discovered in 1952 in Pseudomonas (a genus of Gram-negative, aerobic bacteria), although in 1904 N.A. Maximov mentioned about a distinct type of respiration specific for Aspergillus niger. This cycle is based on the activity of the glucose oxidase enzyme which oxidizes glucose until the formation of gluconic acid, which is later decomposed into pyruvic acid that goes into the Krebs cycle and into phosphoglyceric aldehyde. The oxidation process occurs without substrate phosphorylation. At the same time, gluconic acid is formed which serves as a substrate for other acid biosynthesis, including the ascorbic acid. The pentose phosphate cycle (the D.H. Dickens–B.L. Horecker path), discovered in 1935–1938 by O.G. Varburg, F. Dickens, and V.F. Lipmann practically represents the reverse side of the photosynthesis process (the Calvin cycle) and is also predominantly occurring in plastids. In terms of the quantity of synthesized energy (36 ATP molecules), it does not fall behind the Krebs cycle. According to the results of the research carried by Dickens (1938) and the data provided by O.G. Varburg, some monophosphorylated hexoses (the Robinson ester) are not degraded via anaerobic glycolysis. They follow another path which is carried out in the hyaloplasm. So, initially, glucose phosphorylation takes place which is converted to phosphogluconic acid, which through repeated decarboxylations is transformed into pentoses (5-phosphoribulose) or other simpler oses, which are subjected to degradation in the pentose phosphate cycle (Fig. 5.8). As a result of dephosphorylations, unphosphorylated pentoses (ribulose, ribose) or trioses (glyceric aldehyde) are formed. The oxidation process goes on until pyruvic acid forms, which is subjected to degradation in the Krebs cycle or enters

5.5 Different Types of Respiratory Substrate Oxidation

141

Fig. 5.8 Pentose phosphate pathway (F. Binet and J.P. Brunel)

other metabolic processes, producing polyphenols, tannins, nucleotides, lignin, and aromatic amino acids (phenylalanine, tyrosine and tryptophan). In these reactions, the hexoses from the cell are totally reduced down to carbon dioxide and water, while the remaining ones are resynthesized in hexoses by two paths: • Conversion of pentose phosphate into trioses, which can couple according to the reactions described in the Calvin cycle (C3 + C3 = C6); • Formation of such intermediaries like sedoheptulose (C7) and erythrose (C4) which in different combinations produce hexoses. In the pentose phosphate cycle, by breaking down a glucose molecule, 12 molecules of NADPH are formed. Each of them contributes to the synthesis of 3 ATP molecules during the oxidative phosphorylation process. This cycle mainly occurs in an unfavorable environment, especially during droughts. The importance of the cycle is the following: • A big number of carbohydrates and pentoses is formed which are used later in different metabolic pathways—in the biosynthesis of nucleotides, nucleic acids, coenzymes (NAD, FAD); • By means of this pathway, erythrose-4-phosphate is synthesized which serves as the basis for aromatic amino acid and auxin biosynthesis; • A limited number of enzymatic systems is used (dehydrogenases, transketolases, transaldolases, isomerases, epimerases);

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Fig. 5.9 Glyoxylic acid cycle. Enzymes involved: 1 citrate synthase, 2 aconitase, 3 isocitrate lyase, 4 malate synthase, 5 malate dehydrogenase

• NADPH+H+ is used in the synthesis reactions of fatty acid, steroids, isoprenoides and hexoses (NADP has an anabolic role in contrast to NAD which is primarily associated with catabolism); • The cycle is favorable from the energetic point of view. The glyoxylate cycle was discovered in 1957 by H.L. Cornberg and H.A. Krebs in bacteria. This cycle is produced in glyoxysomes as a shortened variant of the Krebs cycle. It can be observed in the process of seed germination in plants which have a high concentration of vegetal oils and allows for the utilization of lipid reserves as respiratory substrate. It lacks a cytochrome system and cannot be observed in animals. The reactions of this cycle stay at the basis of the transformation of lipids into carbohydrates (4 acetate molecules lead to the biosynthesis of one molecule of glucose). In the presence of the isocitrase enzyme, the isocitric acid is converted into succinic acid. The latter is transformed into glyoxylic acid, which captures succinilcoenzyme A in the presence of the malate synthethase enzyme and is transformed into malic acid (Fig. 5.9). Through dehydrogenation the oxalic acid (a more oxidized form) is formed and through decarboxylation it can return to the pyruvic acid, which undertakes the path of cellular oxidations. In the glyoxylate cycle both acetic acid CH3COOH and glyoxylic acid CHOCOOH are used as a carbon source. The cycle has a great importance for leaf respiration in daylight and allows the rapid and efficient synthesis of dicarboxylic acids. The glyoxylic acid contributes to the biosynthesis of glycine.

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143

5.6 Ecology of Respiration The internal factors that determine the intensity of plant respiration are: • Temperature. Changes in temperature can produce certain changes in the intensity of respiration, through the influence of temperature on the activity of enzymes which act in biological oxidation processes. Temperature influences the process of enzymatic glycolysis of glucose and the activity of enzymes that activate hydrogen in the respiratory chain. At low temperatures, the respiration process is catalyzed by flavoprotein enzymes, while at higher temperatures—by oxidases, which play a more significant role. The different roles of these enzymes in respiration depending on temperature demonstrates the adaptive nature of the respiration process in plants. • Light influences the formation of the organic respiratory substrates which are consumed during the bio oxidative processes, thus, indirectly, it influences the respiration process of green plants. The coenzymes involved in photosynthesis (NAD, ATP, ADP) have also an important role in respiration. Thus, ADP is essential for the terminal phases of oxidative phosphorylation, while the reduction of NAD during photosynthesis inhibits the pentose phosphate cycle and aerobic respiration. • The influence of mechanical trauma, such as cutting or biting increases the intensity of respiration, because some synthetic processes are intensified. • Cytoplasm hydration degree. The substances which are decomposed in plants during oxidation, are oxidized only if they are dissolved in water. This means that the degree of protoplasm hydration influences the intensity of respiration. In case of a prolonged water deficit, an essential change in the organic metabolism can occur which can lead to a considerable drop in the crop yield. • Presence of mineral salts in the soil (nitrates, nitrites, sulfates, ammonium and potassium salts) intensifies respiration in vegetal tissues (anionic respiration). In this case respiration intensity is directly proportional to the amount of absorbed ions. The NH4+ ions participate in glutamine synthesis—a process during which NADPH+H+ is formed. Potassium has a role in maintaining mitochondrial structures. Sulfur is part of certain respiration enzymes (acetyl-CoA), iron is an important element in the composition of cytochromes. • Certain chemical substances can have an inhibiting action on respiratory enzymes. Fluoride can stop glycolysis, while malonic acid can inhibit the Krebs cycle. Cyanides influence the oxidative phosphorylation process as well as electron transportation in mitochondria. Arsenates, 2,4-dinitrophenol break down macroergic intermediary precursors, which precede ATP formation by blocking oxidative phosphorylation, even though they stimulate electron transport in the ETC. • The chemical composition of the air (oxygen and carbon dioxide concentration) has a great influence on the respiration process. If the pressure of carbon dioxide in the environment is high, than the intensity of respiration will gradually decrease and the plant will absorb lower and lower quantities of oxygen.

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High carbon dioxide concentrations have metabolic effects on plants. Thus, treatment with CO2 can increase the storage duration for fruits during winter. • Vegetal organ infestations with parasitic fungi intensify respiration and alter enzymes activity. In cases of mycoses, intensification in the activity of phenoloxidases, cytochromoxidases and peroxidases occurs. During invasions, a decrease in the activity of certain enzymes is observed, the level of enzymatic activity in cells is increased and respiration in infected plants is mainly carried out through the pentose phosphate cycle.

5.7 Regulation and Self-regulation of the Respiration Process Even when environmental factors alternate, a certain equilibrium of the metabolic processes, among which respiration, is maintained in plants (Fig. 5.10) which ensures favorable conditions for the plants to grow and develop. Glycolysis regulation. Glycolysis intensity is controlled at several levels. Glucose recruitment into the process of glycolysis is regulated at the level of the hexochinase in a feedback manner: the surplus of the reaction product (glucose-6phosphate) inhibits allosterically enzyme activity. The second stage of glycolysis regulation is at the level of phosphofructokinase. The enzyme is allosterically inhibited at high ATP concentrations and is activated

Fig. 5.10 Carbohydrate degradation via respiration with CO2 release and ATP formation (Milica et al. 1982)

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145

in the presence of orthophosphoric acid and ADP. Inhibition by ATP prevents a reverse reaction at high concentrations of fructose-6-phosphate. Besides this, the enzyme is suppressed by a Krebs cycle product—citrate and is activated by its own product—fructose-1,6-diphosphate (inverse positive connection). High concentrations of ATP suppress the activity of pyruvatkinase, decreasing the affinity of the enzyme for phosphoenolpyruvate. Pyruvatkinase also suppresses acetyl-CoA. The pyruvate dehydrogenase complex, which participates in the formation of acetyl-CoA from pyruvate, is inhibited by its own product and at high concentrations of ATP and NADH+H+. Regulation of the Krebs cycle. Further use of acetyl-CoA, which is obtained from pyruvate depends on the energetic state of the cell. When the cell has a low demand for energy, respiratory control is slowed down by the activity of the respiratory chain and, as a result, the reactions of the tricarboxylic acid cycle and the formation of intermediary substances including oxaloacetate which involves acetyl-CoA into the Krebs cycle are also slowed down. This leads to an increased use of acetyl-CoA in synthesis processes, which also consumes energy. An important peculiarity of tricarboxylic acid cycle regulation is the dependence of the four dehydrogenases (isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinate dehydrogenase and malate dehydrogenase) on the [NADH]/[NAD+] ratio. Citrate synthase activity is hampered by the high concentration of ATP as

Fig. 5.11 The connection between respiration and photosynthesis

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Table 5.2 Interdependence between respiration and photosynthesis Characteristics

Respiration

Photosynthesis

Summary reaction Metabolism

nC6H12O6 + nO2 = nCO2↑ + nH2O

nCO2 + nO2 = nC6H12O6 + nO2↑

Catabolic reactions of oxidative decomposition of organic substances Carbohydrates, proteins, lipids, etc.

Anabolic reactions of organic substance synthesis from minerals CO2, H2O

CO2, H2O, ATP

O2, carbohydrates

Cytoplasm, mitochondria Glycolysis (hyaloplasma) Tricarboxylic acid cycle—the Krebs cycle (mitochondria) Mitochondria:

Chloroplasts Light phase (granal chloroplasts) Dark phase—Calvin cycle (chloroplast stroma) Chloroplasts:

• two: internal and external • cristae • matrix

• two: internal and external • thylakoids • stroma

• possess a genetic apparatus • oxidative decomposition of the substrate (AP); ATP and water synthesis

• possess a genetic apparatus • solar energy uptake and conversion into chemical energy; water photo-dissociation with O2 release Two electron transport chains: in the first one descendant movement of the electrons that determines the transport of protons into the intrathylakoid space occurs; the second ETC transfers electrons to NADP+ Photophosphorylation: • cyclic • acyclic Towards higher redox potential

Initial products of the process Final products of the process Location Stages

Particularities of the organelles • membranes • formations • internal environment • autonomy • function

ECT

An electron transport chain that carries out the oxidative phosphorylation of NADH (3 molecules of ATP), FADH, FMNH (2 molecules of ATP)

Formation of ATP

Phosphorylation: • substrate • oxidative Towards lower redox potential

Direction of electron movement inside the ETC Conversion of intermediates

Progression depends on the activity of:

In glycolysis: Dioxyacetone phosphate → 3 phosphoglyceric aldehyde → 3 phosphoglycerate Phosphofructokinase, which is inhibited by ATP molecules and is activated by AMP molecules

3-phosphoglycerate → 3phosphoglyceric aldehyde → dioxyacetone phosphate Ribulose bisphosphate carboxylase, which is activated allosterically by fructose-6-phosphate and inhibited by fructose-1,6-diphosphate

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well as by its own product—citrate. Isocitrate dehydrogenase is inhibited by NADH and activated by the citrate. α-ketoglutarate dehydrogenase is suppressed by the reaction product succinyl-CoA and activated by adenylates. Oxidation of succinate by succinate dehydrogenase is hampered by oxaloacetate and accelerated by ATP, ADP and reduced ubiquinone (QH2). Finally, malate dehydrogenase is inhibited by oxalacetate and, in some cases, by the high concentration of ATP, but the extent to which the electric potential or adenylic nucleotides are involved in regulation of the Krebs cycle in plants has not yet been described. Also, a regulatory role in plant mitochondria can be played by an alternative ETC. Under the conditions of a high ATP content, when the activity of the main respiratory chain is lower, substrate oxidation continues by means of an alternative oxidase (without ATP formation). This keeps the [NADH]/[NAD+] ratio low and decreases ATP levels. All these allow the functioning of the Krebs cycle. Respiration acts contrary to photosynthesis and is part of the catabolic side of metabolism (Fig. 5.11; Table 5.2).

Glossary ATP (adenosine triphosphoric acid) An organic compound that contains two macroergic bonds and serves as a source of energy for different transformations that occur in the cell. The energy of a phosphate macroergic bond is approximately 30.5 kJ. Catalase An anzyme from the oxidorecductase class which catalyzes decomposition of the oxygenated water into water and molecular oxygen. Catalases are ferroproteids which are widespread in the plant and animal kingdoms as well as in microorganisms. Cytochromes Compound proteins which contain a porphyrin group with an iron atom in the center. About 20 cytochromes are known which are divided into four classes: a, b, c, d and differ by the nature of the prosthetic group. The role of cytochromes consists in transporting electrons from flavinic enzymes to the atmospheric oxygen. The a3 cytochrome or the cytochromoxidase interact directly with oxygen. Due to electron transport, a change in the valence of the iron atom, as well as elimination of energy and its accumulation in ATP molecules occur. Dehydrogenase Enzymes of the oxidoreductase class which catalyze the transport of hydrogen originating from different substrates towards the donor, being localized in mitochondrial membranes. Depending on the nature of the donor they are divided into aerobic and anaerobic ones. Aerobic dehydrogenases A group of enzymes that catalyze the reaction in which the hydrogen is transported to intermediary acceptors or to molecular oxygen. Their coenzymes are products derived from B2 vitamins—flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN).

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Anaerobic dehydrogenases Enzymes which have NAD and NADP as coenzymes. These coenzymes are reduced by accepting two ions of hydrogen and two electrons from the substrate. Depending on the protein carrier to which the coenzyme is attached, one can distinguish more that 150 natural dehydrogenases with specific qualities with regard to the substrate. Dehydrogenases transport hydrogen and electrons to intermediary acceptors and are oxidized in the process. Oxidases Metalloprotein enzymes of the oxidoreductase class, which transport electrons directly to the atmospheric oxygen with simultaneous formation of water. They act in the final phase of respiration. Oxides are enzymes which contain copper (polyphenol oxidase, ascorbate oxidase) and iron (cytochrome, cytochrome oxidase, catalase, peroxidase). Plastoqhinones Methylated derivatives of the para-benzoquinones that have an isoprenic chain of varying length. It is the most important system for electron transfer in a phosphorylation reaction. Respiration A process of oxidative degradation of compound organic substances into inorganic ones during which energy is released. During respiration, O2 from the air serves as electron acceptor. The global equation is: C6 H12 O6 þ 6O2 ! 6CO2 þ6H2 O þ 2:824 kJ

References Burzo I et al (1999) Physiology of crop plants. Ştiinţa 1:462 Godwin T, Merser E (1986) Introduction into plant biochemistry. T.1.M, p 392 Kretovici VL (1986) Plant biochemistry, 2nd edn. p 504 Milica CI et al (1982) Plant physiology. Bucharest, p 368 Nicols DJ (1985) Bioenergetics. Introduction into the chemiosmotic theory. p 190 Polevoy VV (1989) Plant physiology. p 463 Rubin BA, Ladygina ME (1974) Physiology and biochemistry of plant respiration. p 512 Sculaciov VP (1972) Transformation of energy in the biomembranes. p 203 Tarhon P (1992) Plant physiology, vol 1. Lumina, Chişinău, p 230 Zemleanuhin A et al (1986) The glyoxylate cycle of plants. Voronej, p 148

Chapter 6

Mineral Nutrition of Plants

Abstract Mineral nutrition in plants is a series of biochemical, biophysical and physiological processes by means of which mineral ions are absorbed from the soil, transported and included in the metabolism. In total, 15 chemical elements are necessary to provide the normal growth and development of plants. Of these, four basic chemical elements: carbon and oxygen, which are obtained from the atmosphere, hydrogen which is dissociated from water and nitrogen, derived from mineral compounds are called organogenic elements, since they represent the basic constituents of organic substances. Depending on the quantity of the mineral element one can distinguish macroelements (O, C, H, S, P, Na, Ca, K, Mg) which represent at least 0.01 % of the dry weight of the plant, microelements (Fe, Co, Cu, B, I, Mn, Mo)—at least 0.001 % and ultra microelements. Chemical elements can have specific functions characteristic only for them (functioning for instance as constituents of enzymatic catalytic sites or prosthetic groups, as enzyme activators by modifying the conformational structure of the proteins, as regulators of the colloidal status of the cytoplasm) or they can have nonspecific functions in which case they can be substituted by other elements (e.g. osmotic pressure can be achieved by increasing the concentration of various molecular species). Absorption of mineral elements in plants happens at the level of root hairs via endosmosis and is often facilitated by mycorrhizae (symbiotic relationships between the root and microscopic fungi) and bacteriorrhizae, (symbiosis between the roots and bacteria). Nitrogen is absorbed from the soil as nitrites and nitrates which are later converted to ammonia and used for the synthesis of glutamine and asparagine through amination reactions. It is a ubiquitous component of most organic substances. Sulfur is absorbed as SO42− ions and is part of the highly catalytic proteinogenic aminoacids cysteine and methionine, it is part of coenzyme A and vitamins and also is crucial in controlling the redox state of the cell through glutathione levels. Phosphorus is in the composition of ATP, nucleic acids, phospholipids in biological membranes, it is at the core of signaling and other processes of controlling protein activity. It is assimilated in the form of salts of the orthophoshoric acid. Besides these, metal ions (Ca, K, Fe, Mg, Cu, Mb, Zn) represent key-components in the process of mineral nutrition and are absolutely necessary for plant growth and development.

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Historical Background 384–322 b.c.—Aristotle considered that plants utilize nutrients in a complex form directly from the soil. 1675—M. Malpigi discovered the phenomenon of ascendant and descendent circuit of substances in plants. 1772—D. Rutherford discovered nitrogen. 1840—J. von Leibig develops the “Mineral Nutrition Theory”. 1859—A.A. Knop and J. Sachs produced nutritive environments for growing plants. 1878—J.B. Boussingault discovered that Fabaceae species can assimilate nitrogen from the atmosphere. 1880—H. Hellriegel and H. Willfarth stated that nitrogen fixation occurs with the help of bacteria. 1937—A.E. Braunstein and M.G. Kritzmann discovered the transamination reactions. 1950—S. Ratner and E. Racker studied ion exchange between plants and the environment. 1952—A.B. Hope and P.G. Stevens introduced the term of “free space” of the cell. 1974—B.J. Miflin discovered the pathway of ammonium ion assimilation.

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Brief Updates Metabolism consists of a series of tightly coordinated enzyme-mediated chemical reactions, which occur in living organisms with the direct or indirect involvement of mineral ions and resulting in the synthesis and utilization of a big variety of molecules from the category of carbohydrates, amino acids, fatty acids, nucleotides and of the polymers which are derived from them (polysaccharides, proteins, lipids, DNA, RNA etc.). All these processes represent the primary metabolism and the respective compounds which are essential for plant survival, are defined as primary metabolites. Besides primary metabolites (proteins, carbohydrates and lipids), with an important role in life maintenance, a series of compounds which belong to the secondary metabolism are synthesized (terpenes, steroids, anthocyanins, anthraquinones, phenols and polyphenols). Both primary and secondary metabolites derived from plants are very interesting from the economic point of view. Most of them are nonproteic chemical compounds which can be extracted from the vegetal material by steam distillation with organic or water solvents. Every year, more than 1,500 new compounds are identified in different plant species. Except for natural rubber, condensed tannins and some polysaccharides like gums, pectins and starch, secondary metabolites are compounds with low molecular weight (under 2000 Da), which are widely used in industry, medicine etc. Almost one third of the pharmaceutical products contain substances of vegetal origin. Although there are relevant differences with regard to synthesis and storage of secondary metabolites in different plant tissues or at different stages of plant development, the genome of each cell contains the information for synthesizing all secondary metabolites which are characteristic for a certain species. The progress achieved in molecular biology and gene engineering offer some promising perspectives for intensifying controlled biosynthesis of specific secondary compounds, by influencing gene expression levels. By increasing the number of copies, or by attaching the respective genes to a strong promoter more transcription and translation can be induced and, as a consequence, specific biosynthetic enzymatic functions can be amplified.

6.1 Importance of Mineral Elements in Plant Nutrition Mineral nutrition in plants is a chain of biochemical, biophysical and physiological processes with direct and indirect connections during which absorption, transportation and metabolization of mineral ions occurs. As a result, plants are supplied with all the elements which are necessary for them to grow and develop. During the mineral nutrition process, they absorb mineral salts from soil and integrate them into complex organic substances or cell structures.

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In order to grow and develop, plants need a big number of chemical elements that come either directly from minerals, or from mineralization of the decaying organic substances in the environment. Mineral elements are required in different amounts by plants. The amounts required differ from species to species and even from variety to variety. In addition, plants have somewhat different specific requirements which end up determining the chemical composition of different species. Chemical analyses showed that the vegetal matter contains more than 60 chemical elements, but only 15 are necessary to provide the normal growth and development of plants. The basic chemical elements: carbon(IV) and oxygen, which are obtained from the atmosphere, hydrogen which is dissociated from water and nitrogen—derived from mineral compounds are called organogenic elements, since they represent the basic constituents of organic substances (amino acids, proteins, carbohydrates etc.) and are most widely spread, representing 95 % of the dry vegetal mass. The ions of nonmetals (N, P, S) and metal ions (Ca, K, Fe, Mg, Cu, Mb, Zn) represent the key-components in the process of mineral nutrition and are absolutely necessary for plant growth and development. They have different function in the vegetal organism. These functions can be grouped in: • specific functions, in case the mineral ion has a certain role and cannot be substituted by another element (a catalyzing and structural role, a role in ensuring the colloidal status of the protoplasm, in electron transport during a specific process etc.). Mineral ions can act like biocatalyzers, in which case they: • are part of the prosthetic groups of proteins (cytochrome enzymes, from the electron transport chain); • are part of the catalytic site of the enzymes (Cu in peroxidase, Fe in catalase) • are enzyme activators (K does not enter in the composition of any organic substance, but it activates about 50 enzymes by modifying the conformational structure of the proteins). Chemical elements (like Mg, Zn etc.) merge with enzymes and form compounds called chelates, which enable enzyme interaction with their substrates. They can also be found in the composition of specific amino acids (S), proteins (N, S), nucleic acids and ATP (P), chlorophyll (Mg), cellular wall (Ca) etc., where they have a structural function. The function of protoplasm colloidal status regulation is carried by K ions which act to decrease the viscosity of the protoplasm and by Ca ions that act to increase it thus influencing the hydration level of protoplasmic colloids; • nonspecific functions—when performing these functions an ion can be substituted by another one (osmotic regulation—inflow and elimination of water from the cell is determined by the quantity of ions and not by their type). The physiological significance of mineral elements has been studied extensively (Table 6.1). It is known that mineral elements influence the exchange of substances, alter cell turgidity and the permeability of biological membranes, enter in the

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Table 6.1 The role of different chemical elements in plant physiology Chemical element

Source

Functions

Carbon Oxygen Hydrogen Nitrogen

CO2 H2O, O2 H2O NO3−, NH4+

Chlorine, sodium

Cl−, Na+

Potassium

K+

Is part of the structure of all organic compounds Is part of the structure of all organic compounds Is part of the structure of all organic compounds Is part of proteins, amino acids, nucleic acids, chlorophyll, nucleotides, coenzymes important in maintaining ion equilibrium and in regulating osmotic pressure Ensures the structural conformation of proteins, important in stomata closure and opening, serves as activator for many enzymes Is part of the structure of nucleic acids, macroergic compounds, phospholipids, participates in phosphorylation reactions (e.g. in signal transduction) Part of coenzyme A and chlorophyll Part of proteins, amino acids and nucleic acids Important in the synthesis of chlorophyll, cytochromes and nitrogenase Enzyme activators

Phosphorus

H2PO4−, HPO42

−

Magnesium Sulfur Iron

Mg2+ SO42− Fe3+,2+

Copper, Magnesium Calcium

Cu2+, Mg2+ Ca2+

Part of cell wall structure, has a role in cellular permeability, enzymatic cofactor

composition of vitally crucial organic substances, participate in macromolecule and colloidal particle stabilization and are involved in different catalytic reactions. One and the same element can perform multiple functions in plants (Table 6.1).

6.2 Chemical Composition of the Ash Plants, depending on their age, species and organ contain 90–98 % water, the rest being represented by dry mass which contains the following average amounts of elements: C—45 %, O—42 %, H—6.5 %, N—1.5 %. At high temperatures, organic substances are burned and eliminated, while the remaining mineral elements form the ash, which makes up 0.2–20 % of the dry mass. The total amount of ash varies depending on the species, on the nature and age of the organs and is tightly connected to the composition of the soil and its humidity. The chemical composition of the plants reflects their need in mineral elements. Burning different organs and plant parts result in different amounts of ash. Wood, for instance, contains—1 %; seeds—3 %; leaves—5–15 %; bark—7 % of the dry weight. These figures demonstrate that mineral elements are concentrated in cells and organs with a high vital activity. All the elements of the Mendeleev table are

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part of the ash composition. This allowed the Russian scholar V.I. Vernadski to mention the role of plants in the circuit of elements in nature (especially of the rare ones). The content of mineral elements in ash varies within certain limits. Depending of the percentage of elements in the ash and in living plants, they can be classified as follows: • macroelements (O, C, H, S, P, Na, Ca, K, Mg), which represent at least 0.01 % of the plant’s dry weight; • microelements (Fe, Co, Cu, B, I, Mn, Mo) which make from 0.01 to 0.001 % of the dry mass of the plants; • ultra microelements (minor traces of elements).

6.3 Methods of Mineral Nutrition Research J. Sach, W. Pfeffer, J.B. Boussingault, H. Lundgardh, D.N. Preanishnikov carried out numerous, extensive studies on plant nutrition. In their research they made use of three basic research methods: • the chemical method (analytical); • the physiological method (mixed); • field experiments. The chemical method was used in researching the composition of the raw sap, of the dry mass, of the ash, of the leaves at different ontogenetic stages, as well as of fruits and vegetables. The physiological method includes research under controlled conditions (Fig. 6.1) of cultivation in soil, in sand, in water (hydroponics) and in air (aeroponics). This method is also called the mixed or vegetative method. It involves the use of different nutrition media, which represent physiological solutions

Fig. 6.1 The physiological research method applied on plants

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(A.A. Knop, H. Hellriegel, D.N. Preanishnikov and others) characterized by a specific pH and a balanced ionic equilibrium and contain all the necessary elements for plant growth and development. This method allows to study the effects of deprivation for various elements (when nitrogen is excluded, lower leaves turn yellow, lack of iron determines upper leaves to turn yellow; the insufficiency of phosphorus causes a reddish color around the veins etc.). The method of field experiments is used to determine the dosage of the mineral elements. It supposes cultivation on small land parcels and is used mostly in phytotechny to optimize the conditions for maximum crop yield for different agricultural species.

6.4 The Root System as an Organ for Absorption and Transport of Mineral Elements The root is the organ which grows into the soil having the function to fixate the plant and to absorb water and mineral salts (see: Chap. 3, The water regime.). Roots can form mycorrhizae (symbiotic relationships between the root and some microscopic fungi), bacteriorrhizae, (symbiosis between the roots and bacteria). If the tip of a young root is analyzed with a magnifier, five regions can be distinguished (Fig. 6.2): the root cap (calyptra), the apical meristem (division zone), the elongation region (smooth), the root hair region (differentiation zone) and the mature region (rough). The root cap region protects the tip of the root and is also called the calyptra. This region has a constant length, because new cells are constantly born to replace old ones that disappear as a result of friction with the soil. The apical meristem is the region where cell division promoting root growth occurs. It gives birth to the primordial meristem. The elongation region is a root region where cell do not divide but rather increase their longitudinal dimensions manyfold. The root hair region is a region where cell differentiation occurs. Here, histogenesis gives birth to the rhizoderm, to the epidermis and to the central cylinder of the root. Rhizoderm cells differentiate and form cylindrical projections perpendicular to the root axis, hence the name of this root zone. The function of the root hairs is to absorb, through endosmosis, water and mineral elements from the soil. They have a thin cellulosic membrane, a nucleus located close to the tip and surrounded by cytoplasm, while the center of the cell is filled by a big vacuole. Root hairs help create huge absorption surfaces that can reach tens or even hundreds of square meters. The maturation region has a rough dentate surface due the root hairs that have detached in the process of constant renewal.

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Fig. 6.2 Root structure. The main root regions and tissue types are represented

6.5 Physiological Role of Macroelements The macroelements are the mineral elements which constitute at least 0.01 % of the plant dry weight. They are part of the tissue composition and have a key-role in plant growth and development.

6.5.1 Absorption, Transport and Metabolism of Nitrogen Nitrogen (N) was discovered by D. Rutherford in 1772 in the form of a gas, which cannot maintain the processes of burning or life. It is, however, part of the most

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important vital components and is used in the biosynthesis of most organic substances. The importance of nitrogen in plants. Nitrogen makes up 1.5 % of the vegetal dry mass and plays a central role in plant metabolism: • • • • •

it is part of amino acids and proteins; is part of vitamins and other biologically active substances; enters in the composition of nucleic acids—DNA, RNA; is a component of ATP—the universal energy source; is an element found in the molecule of chlorophyll;

The forms of nitrogen in nature are diverse (Fig. 6.3). But the biggest amounts of nitrogen are found in the atmosphere (N2, vapors of NH3), where it constitutes 72–73 % of the weight. In the soil nitrogen is found in inorganic form (in nitrates, nitrites, ammonium salts) and organic form (the nitrogen of amino acids, amides, proteins and humus). The inorganic nitrogen in the soil is formed as a result of mineralization of the remnants of animals, plants, etc., and constitutes 1–2 %, while organic nitrogen constitutes 98–99 %. Microorganisms fix nitrogen from the atmosphere, while plants use the inorganic nitrogen from the soil in the form of NH4+, NO3−, NO2−. It is considered that plants can also absorb organic nitrogen in the form of urea, amides and simple amino acids. Recovery of nitrates in plants. Of all the absorbed forms of nitrogen, the ammonia form is included in the composition of organic substances. Nitrates and nitrites are reduces to this form at the level of the root and the foliar system (this process is called nitric photosynthesis). At the root level the process is located in mitochondria, while at the level of leaves—in chloroplasts. The root system utilizes efficiently nitrates and nitrites. After absorption, they undergo fermentative recovery up to the level of nitrites and, subsequently,

Fig. 6.3 Nitrogen sources for plants

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ammonium in a series of stages. Nitrates are converted to nitrites by nitrate reductase (a flavoprotein which contains Mo).

To reduce the nitrates the following are necessary: metabolic energy, the presence of proton and electron donors which are NADPH+H+ or NADH+H+ (reduced nicotinamide adenine dinucleotides). The source of these substances in the root system is represented by the process of respiration, while in leaves—by photosynthesis (nitric photosynthesis). Thus, nitrate reduction is mainly determined by the intensity of these processes. For proper respiration, a certain quantity of carbohydrates (respiratory substrate) is sufficient. If the quantity of carbohydrates is reduced artificially below this level, nitrites will not be reduced, and will accumulate instead in all the organs of the plant. An excessive accumulation of nitrites in plants may have a negative (toxic) impact on them. Nitrate recovery is also stimulated significantly by light. Presumably, for these reactions the products resulting from acyclic photophosphorylation (NADPH+H+, ATP) can be used directly. Blue light is stimulatory in this process due to the fact that flavin, which is an integral part of nitrate reductase, absorbs blue light and is activated by it. Another nitrogen source for plants is the ammonium cations, which enter plant tissues very easily (faster than nitrites) and are used directly. Amination reactions and amino acid biosynthesis. The ammonium cation is a basic component of plant metabolism. Its origin can be: • directly from the soil; • formed as a result of nitrite reduction; • formed as a result of protein catabolism in aging organs. Accumulation of ammonium in cells may have a negative impact. However, plants can sequestrate ammonium with the help of organic acids forming amides (glutamine, asparagine etc.). This process is similar to that of forming urea in animal organisms. When the intensity of respiration is low, or in case of carbohydrate insufficiency, amides are not formed and NH4+ accumulates, which causes plant intoxication. There is a whole group of plants which accumulate a big amount of organic acids that are used to neutralize ammonium. This caused the division of plants into: (a) those that form amides (for instance asparagine and glutamine) and (b) those that form ammonium salts.

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A change in the pH of the cytoplasm, may alter the nitrogen metabolism causing a switch between amide and ammonium salt formation. Amino acid and amide biosynthesis pathways in plants. During respiration, the α-ketoglutaric and oxalic organic acids are produced as intermediary products. These acids incorporate ammonia as a result of a direct amination reaction:

The reaction is catalyzed by glutamate dehydrogenase, which contains an active NAD group. This enzyme is located in mitochondria, since this is the place where organic acids and recovered nicotinamide coenzymes are formed. Aspartic acid is formed similarly after amination of the oxalic acid. Also, aspartic acid can be formed by direct amination of the fumaric acid, with the participation of the aspartase enzyme:

Aspartic acid synthesis is stimulated by light and is localized in chloroplasts. The glutamic and aspartic acids incorporate one more molecule of ammonia each and form glutamine and asparagine (amides):

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The quantity of formed asparagine and glutamine and their importance differ depending on the plant species and environmental conditions. Glutamine is formed in leaf and root cells. Formation of asparagine predominates during protein catabolism in seeds. Thus, asparagine is the neutralization form of the NH3 formed during protein degradation (the regressive branch of nitrogen exchange in plants):

The age of the plant plays an important role in amide formation. The younger the plants, the higher its ability to form amides. In younger organs and even in younger cells of the same organ, the intensity of amide formation is higher. Crude sap and guttation sap both contain amides which proves that the nitrogen absorbed from the soil is transformed into amides in the living cells of the root. Amides in plants represent: • the form of NH3 neutralization; • the form of transport for nitrogen compounds; • a building block for the synthesis of other amino acids in the process of transamination. Transamination and protein synthesis. Each of the amino acids which are formed by direct amination (the glutamic and aspartic acids) are predecessors for a whole group of amino acids. Out of the 20 aminoacids which enter in the composition of plant proteins (proteinogenic amino acids), only two can be formed in the process of direct amination. The others are formed as a result of transamination and retransformations. These reactions are catalyzed by special enzymes—aminotransferases and occur with

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participation of the pyridoxal phosphate coenzyme (the active form of the B6 vitamin). The role of this catalyzer consists in ligating the amino group and forming pyridoxamine phosphate and keto acids. The most common reaction is that by which the amino group is dissociated from the glutamic acid. Different amino acids synthesized by transamination produce other amino acids by transforming the carbon backbone. Thus amides serve as donors of amino groups. Plants, in comparison to animals, have the ability to synthesize all the necessary amino acids. They can be formed in different organs of the plant—leaves, roots, the apex of the stem: Transamination

Some amino acids are formed directly in the chloroplasts, where they are used to synthesize proteins. Protein synthesis occurs is more intense in meristematic tissues undergoing development. Protein biosynthesis stops in cut leaves, which proves the requirement of factors formed in plant roots (mot probably a phytohormone from the group of cytokines or a similar substance): Deamination

The following conditions are necessary to ensure proper protein biosynthesis: (1) a sufficient quantity of nitrogen (2) sufficient amounts of carbohydrates;

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(3) a high intensity of respiration and phosphorylation (ATP is necessary for all the stages of nitrogen compound transformation: recovery of nitrates, formation of amides, activation of amino acids in the process of protein synthesis); (4) presence of the basic components of the proteosynthetic apparatus: • DNA—as the molecule encoding the information about the sequence of amino acids in the protein chain, • mRNA—the agent transmitting information from DNA to ribosomes, • tRNA—associates the nucleotide triplets (codons) of the mRNA with their corresponding amino acids decoding the nucleotide sequence into the amino acid sequence; (5) presence of the ribosomes—the structural units of the cell which carry protein synthesis; (6) presence of enzymes catalyzing protein biosynthesis (aminoacyl-tRNA-synthetases etc.) and protein factors; (7) Mineral elements (Mg2+, Ca2+). The progressive branch of nitrogen exchange in plants, found mainly in young organs, ends up with protein synthesis (primary synthesis of proteins). However, a continuous process of protein decay also occurs in plants in parallel. Protein renewal takes place rather rapidly (about 60 % of the proteins are renewed during 48 h). Proteins are degraded to amino acids and further to NH3 (the regressive branch of nitrogen exchange), which is later again neutralized by the formation of asparagine and glutamine that serve for amino acid synthesis. This process allows plants to form a new set of amino acids, which are used in protein anabolism (secondary synthesis of proteins). Thus, both mineral and organic nitrogen can be found in vegetal organs. Organic nitrogen is represented by micromolecules (amides, organic acids, nitrogenous bases etc.) and by macromolecules (proteins, nucleic acids). 80–85 % of all the nitrogen in the vegetal mass is represented by enzymes, while in seeds, by reserve substances such as proteins. The symptoms of nitrogen deficiency in plants. Nitrogen deficiency causes delay in plant growth and development, plants turn green-yellow. The lower leaves turn yellow (chlorophyll molecules are destroyed and nitrogen compounds are transported to younger leaves), the vegetation period is reduced, the intensity of protein and enzyme biosynthesis is diminished, which has an impact on cellular metabolism. In higher plants anthocyanin biosynthesis intensifies. Atmospheric nitrogen fixation. Some plant species (leguminous plants), which live in symbiosis with microorganisms of the Rhizobium genus have the capacity to fix atmospheric nitrogen. About 500 species of microorganisms belonging to this genus are known, which coexist with higher plants by forming specific root nodules and recruiting free nitrogen from the atmosphere (Figs. 6.4 and 6.5). These microorganisms contribute to atmospheric nitrogen conversion into ammonium

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Fig. 6.4 Root nodules containing nitrogen fixing bacteria of the genus Rhizobium

Fig. 6.5 The mechanism of nodule formation by Rhizobium bacteria

ions by using the necessary energy and metabolites from higher plants (Figs. 6.6 and 6.7). N2 þ 8e þ 8Hþ þ ATP ! 2NH3 þ H2 þ ADP þ Pi The interaction between bacteria and the plant is triggered by certain flavonoids synthesized by the roots, which induce the expression of genes in microorganisms that are involved in nodule formation.

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Fig. 6.6 Nitrogenase structure

Fig. 6.7 Symbiotic nitrogen fixation by bacteria of the Rhizobium genus

Nitrogen fixing bacteria have the enzyme nitrogenase encoded by genes of the fix and nif type. This enzyme consists of two proteins which contain Mo and Fe and one which contains Fe. The genes are active in anaerobic conditions, while the level of oxygen in nodules is regulated by leghemoglobin, the protein part of which is encoded by the plant while the heme—by the bacteria.

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Fig. 6.8 Nitrogen fixation

Atmospheric nitrogen can also be fixed chemically in an industrial process (Fig. 6.8) according to the reaction: N2 + 3H2 → → 2NH3 however this requires extreme conditions (500 °C and 200 atm). Thus, nitrogen fixing bacteria are a perfect example of how enzyme evolution has made possible even the most extreme reactions (with big activation energies or big energy consumption). Soil bacteria, which belong to the Pseudomonas genus participate in plant growth by synthesizing different biologically active compounds—IAA and gibberellins, by fixating nitrogen and participating in the process of metabolization of the organic and inorganic phosphorus from the soil by eliminating phosphatases and dissolving inorganic phosphorus salts in acids. Nitrates can induce gene expression in plants. In Arabidopsis thaliana NO3− activates 15 different genes, the majority of which encode transcription factors, metabolic enzymes (transaldolases, transketolases, malate dehydrogenases) and participate in signal transduction in the root.

6.5.2 Absorption, Transport and Metabolism of Sulfur Sulfur belongs to the category of nutritive elements which are absolutely necessary for vital activity. This element enters the plant in the form of sulfate ions (SO42−). Its amount in plants is relatively low, making up 0.2–1.9 % of the dry mass. The importance of sulfur. This element is part of cysteine and methionine which are among the most important amino acids given their role in catalytic reactions mediated by enzymes. These amino acids can be found in a free state or as residues in protein sequences. Methionine is one of the essential amino acids and has some unique properties due to the sulfur and the methyl groups. It was identified in the active centers of many enzymes. Methionine confers hydrophobic properties to protein molecules, which plays an important role in stabilizing their structural conformation. One of the most important functions of sulfur in proteins and polypeptides is the participation of SH groups in the formation of covalent, hydrogen and mercaptide bonds which determine the three-dimensional structure of proteins. Disulfide bridges— covalent bonds between the thiol groups of, for instance, two cysteine residues

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(located on different or the same polypeptide chain) stabilize the 3D configuration of a protein or protein complex. Another important function of sulfur in the vegetal organism consists in maintaining a certain level of the oxidation/reduction potential within the cell, based on the reversible reactions: cysteine–cysteine and SH–glutathione or S–S glutathione. This redox systems can bind or release hydrogen cations depending on the state of cell environment. Sulfur is also in the composition of the most important biological compounds such as: coenzyme A and vitamins which are important in various enzymatic reactions. The forms of sulfur in soil and plants. In the soil, sulfur can be found in organic and inorganic forms. In most of the soils, organic sulfur, which originates from animal and vegetal remnants prevails. The basic inorganic form is the sulfate SO42− which can be found in the form of salts: CaSO4, MgSO4, Na2SO4 dissolved in the soil solution and in the form of ions absorbed into the soil colloids. Sulfate is relatively stable in soil. In saline soils, its content can reach up to 60 % of the dry weight. The average sulfur content can vary between 0.005 and 0.04 %. Sulfur, like all other biogenic elements, participates in the biological circuit of elements in nature. The organic sulfur from the soil and water is mineralized by saprofite microorganisms to H2S. A proportion of the H2S can be converted into insoluble compounds, while the rest is released into the atmosphere. Colorless sulfur bacteria (chemosynthetic) in the presence of O2 and purple and green sulfur bacteria (photosynthetic) in anaerobic conditions, oxidize H2S to free sulfur and sulfate: H2 SSOSO3 2 SO4 2 And vice versa, chemiosynthetic bacteria which recover sulfur, in anaerobic conditions, use sulfur as a source of O2: 4H2 þ SO4 2 ! S2 þ 4H2 O ðsulfate respiration) These transformations are depicted in Fig. 6.9. The microbiological oxidation of H2S (or FeS) to SO42− is accompanied by an increase in soil acidity (a decrease in pH). Forms of sulfur in plants. Autotrophic plants absorb sulfur in the form of sulfates—SO42−, which are ultimately converted into the SH groups of organic substances. Transmembrane sulfate transport is carried out in co-transport with H+ or in exchange with HCO3− ions. The less oxidized inorganic forms (SO2) and the more reduced ones (H2S) are toxic for plants. Plants can also use SO2 from the atmosphere—it is favorable in concentrations of 0.1–0.2 mg/m3. If the concentration of SO2 exceeds 0.5–0.7 mg/l, it becomes toxic, and causes necrosis of the leaves, which is explained by the fact that accumulation of SO2, HSO3− and SO32− in tissues unbalances photophosphorylation and destroys chloroplast membranes. Sulfur is found in plants in two essential forms: oxidized (in the form of inorganic sulfur) and reduced. The absolute amount and ratio of the oxidized/reduced

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Fig. 6.9 Sulfur circuit in nature

forms in plant organs depends on the intensity of sulphate reduction and assimilation, on the concentration of SO42− in the nutritive environment. A part of the sulfur which was absorbed by plants is retained in the roots. The biggest part of it, however, moves from the roots into the xylem vessels, from where it is transported to the fast growing young organs, which are involved in a dynamic metabolic process. From the leaves, sulfate and reduced sulfur forms (amino acids which contain sulfur and glutathione) enter the phloem and are deposited. In seeds, sulfur is present in the organic form, but when growing, it is partially converted to the oxidized form. At the same time, during seeds maturation, reduction of sulfur and synthesis of sulfur-containing amino acids takes place. A summary of the process of complete sulfur reduction can be expressed by the following formula: SO4 2 þ ATP þ 8Hþ þ Oacetylserine ! cysteine þ acetate þ 3H2 O þ AMP þ PPi

Recovery of SO42− in leaves is linked to photosynthetic processes (ATP, feredoxin) and is located in chloroplasts, but all sulfur reduction enzymes are also contained in mitochondria. Cysteine is the first stable product in which sulfur is present in a reduced form and represents the predecessor of many organic products which and first of all of methionine—the final product of sulfur assimilation: CysteineHomocysteineMethionine The symptoms of sulfur deficiency. Sulfur deficiency causes leaves to turn yellow, like in the case of nitrogen deficiency, but it first occurs in youngest leaves. Sulfur deficiency delays thio amino acid and protein synthesis and reduces photosynthesis intensity as well as plant growth rate. In case of a significant deficiency, chloroplast formation is affected and their degradation can occur.

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6.5.3 Absorption, Transport and Metabolism of Phosphorus Phosphorus (P) is part of the essential nonmetal group and forms 0.2 % of the total dry mass. This element enters the plant in its oxidezed PO43− form. After undergoing transformations it preserves its state and degree of oxidation. The importance of phosphorus. In plant tissues, phosphorus is present in organic (proteins, nucleotides, vitamins and other compounds) and inorganic form (orthophosphoric acid and its salts). The phosphate group determines the hydrophilicity of a phospholipid molecule while its lipid part remains hydrophobic. This is why, at the border of phase separation in biological membranes, the phospholipids are oriented with their phosphate ends outside, while lipophilic groups are retained stably inside the lipid bilayer, stabilizing the membrane. Another unique function of phosphorus is its participation in the process of phosphorylation of cellular proteins mediated by protein kinases. This mechanism is a means of regulating many metabolic processes, because inclusion of the phosphate in the protein molecule causes a redistribution of electric charges in it and, as a consequence, leads to changes in its conformation and, consequently, its function. Protein phosphorylation regulates such processes like ARN and protein synthesis, is important in signal transduction and many other processes etc. Phosphorus is the basic bioenergetic element which has an important limiting role in plant growth and development. Its action is also crucial for entering and exiting states of decreased metabolism (e.g. anabiosis). The symptoms of phosphorus deficiency. The deficiency of phosphorus has an impact on all vital processes—photosynthesis, respiration, plant growth and development. It produces a change in leaf color into blue-green with purple and golden shades, which is a result of the delay in protein synthesis and carbohydrate accumulation. Leaves become smaller and thinner. Plant growth as well as fruit ripening (maturation) is delayed. During phosphorus scarcity O2 absorption speed is reduced and the activity of enzymes which participate in respiration is altered. Plants are more sensitive to phosphorus deficiency during the primary stages of their growth and development. A proper phosphorus input, at later stages, causes fast maturation of plants and fruits. Forms of phosphorus in nature. Phosphorus reserves in soil are relatively small (2.3–4.4 t/ha—P2O5). Out of this quantity two thirds are represented by the mineral salts of the ortophosphoric acid (H3PO4) and one third—by organic compounds (from dead remnants, humus). The concentration of phosphorus in the soil solution is low (0.1–1 mg/l). Organic compound containing phosphorus are by large insoluble in this solution. But some agricultural species (buckwheat, peas) use poorly soluble forms as well. Phosphorus from organic remnants and from humus is mineralized by microorganisms and the biggest part of it is converted in poorly soluble salts which can be assimilated by plants and converted to mobile forms. This is possible due to the fact that roots release organic acids that acidify the rhizosphere contributing to the sequence of transformations:

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Fig. 6.10 Phosphorus circulation in the biosphere

PO4 3 ! HPO4 2 ! H2 PO4  The circuit of phosphorus in nature is depicted in Fig. 6.10. Transformations of phosphorus in plants. Phosphorus plays an important role in cellular energetics, since energy is accumulated and stored in the form of macroergic ester bonds (C–O–P) or pyrophosphate bonds. All the transformations of phosphorus in plants are reduced only to transferring the phosphoric acid residue to an organic substance by formation of ester bonds (phosphorylation). Transphosphorylation is a process in which the rest of the phosphoric acid which is contained in a organic substance is transferred to another organic substance. The main form phosphorus reserves in a cell is phytin (the Ca, Mg salt of the inosit phosphoric acid). A big quantity of phytin (0.5–2.0 % of the dry weight) is stored in seeds. The radial movement of phosphorus in the absorption region of the root occurs through the symplast. The concentration of phosphorus in root cells is tens or even hundreds of times higher than in the soil solution. Transportation through the xylem occurs in the form of inorganic phosphorus and in this form it reaches the stem and the leaves. From the cells of the leaves, the phosphorus enters the floem and is transported to all the organs, cells and tissues of the plant, especially to the growth cone and to the developing fruits. A series of compounds of phosphorus, which are important from the biological point of view, contain several phosphoric acid residues (polyphosphates).

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Phosphorus is part of a big list of organic compounds such as: nucleic acids (DNA and ARN), nucleotides, phospholipids, vitamins, and plays an important role in the exchange of substances in plants. Many vitamins which contain phosphorus and its derivatives are coenzymes and are directly involved in cell catabolism, accelerating the substance exchange processes. Phosphorus has the specific ability to form instable, macroergic bonds, this representing a convenient, controllable, recyclable means of energy utilization in different biochemical and physiological processes. When phosphoric acid reaches the root cells, it is rapidly included in the composition of nucleotides forming AMP (adenosine monophosphate) and ADP (adenosine diphosphate). Later, ATP is formed as a result of substrate and oxidative phosphorylation (the aerobic and anaerobic phases of respiration). After just 30 s from the moment of its absorption by the plant, phosphorus is included ATP molecules, which is later used to activate amino acids, in the process of nucleic acids synthesis etc.

6.5.4 The Physiological Role of Other Macroelements Potassium or kalium (K) is one of the most important and necessary nutritive elements. Its amount in plants varies between 0.5–1.2 % of the dry mass. The content of potassium in the cell is hundreds of times higher than in the environment, thus for quite a long time the ash was the only source to obtain this element. The reserves of kalium in soil are significant and can be found in the following forms: • in colloidal particles; • in the composition of organic remnants and microorganisms; • as mineral salts dissolved in the soil solution. The most accessible source of potassium are the 0.5–2.0 % of the soil reserves. Periodic drying and wetting of the soil, the activity of the root system favors potassium conversion into accessible forms. Fertilizers containing potassium are water soluble and, by entering the soil, they interact with its colloids from where it can be absorbed by plants. However potassium input displaces other ions (H+, Ca2+, Al3+, Mg2+) from the colloids into the soil solution. Kalium fertilizers are physiologically acid salts which stimulate HCI and H2SO4 accumulation in the soil That’s why, in acid soils the efficiency of potassium fertilizers decreases. In plants, higher amounts of potassium can be found in young, growing tissues, which are characterized by high rates of substance exchange—meristems, cambium, young leaves, buds, sprouts. In cells, potassium can be found in the ionic form. It is not part of organic substances, it has a high mobility and is easily recyclable. The rapid migration of kalium from mature cells to young ones happens due to sodium, which replaces it in cells that stopped growing.

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In vegetal cells, about 80 % of potassium is contained in vacuoles. It is the main cation in the vacuolar sap. This is why kalium is washed out from old leaves during rain. A small part of it (about 1 %) is tightly linked to mitochondrial and chloroplast proteins. Potassium stabilizes the structure of these organelles. In case of its scarcity, the lamellar structure of the chloroplasts is disrupted and the membranous structure of the mitochondria degrades. Up to 20 % of the cellular potassium is absorbed by the colloids of the cytoplasm. In the presence of light, potassium is bound tighter by the colloids compared to darkness. During the night, even the elimination of this element through the root system can occur. The importance of potassium is revealed by the following: • It provides counter ions to neutralize the negative charges of both organic and inorganic anions. It is the presence of kalium, that determines the main chemical and colloidal proprieties of the cytoplasm, which influence effectively all the processes in the cell. • It contributes to the maintenance of the hydrated state of cytoplasm colloids, by affecting their capacity to retain water. Increasing protein hydration and water retention by the cytoplasm positively affects plant resistance to drought and frost. • Potassium is necessary for the process of absorption and transportation of water through the plant. Calculations have revealed that root pressure, is determined in a proportion of ¾ by the presence of kalium in the cellular sap. • It has a great importance in opening and closing the stomata (Fig. 6.11). In daylight, the concentration of kalium ions in the vacuoles of guard cells increases dramatically (fourfold to fivefold), which causes water inflow, an increase in turgidity and the opening of the stomata. In the dark, kalium starts to be released from the cells, the turgor pressure inside them goes down and the stomata close.

Fig. 6.11 Role of potassium and chlorine in stomatal opening and closure

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• Potassium is absorbed by plants as cations and only forms weak bonds with different compounds of the cell. This is one of the reasons why it creates an ionic asymmetry and an electrochemical potential at the membrane separating the cell and the environment. • It is a cation which activates fermentation systems. Nowadays, more than 60 enzymes activated by potassium are known. It is needed for including phosphate in organic compounds, in the reactions of phosphate group transfer, for protein and polysaccharide synthesis. It participates in the biosynthesis of riboflavin—a component of all flavin dehydrogenases. • Potassium increases the accumulation of starch in potatoes, of sucrose in sugar beet, of monosaccharides in fruits and vegetables, of cellulose, hemicellulose and pectic substances in the cell wall. As a consequence, it increases the strength of the stems in cereals, the quality of hemp and flax fibers is improving. A sufficient supply of kalium in plants increases their resistance to diseases caused by fungi and bacteria. If the levels drop, the quantity of Na, Mg, Ca NH3, and of the H+ ions increases to compensate the osmotic effect, but they cannot compensate for its specific functions. The critical period for supplying plants with potassium is 1–2 weeks after their appearance. However, the biggest quantity is absorbed during the period of vegetal mass growth. In case of scarcity, leaves start turning yellow from plant bottom to its top (from old to young leaves). Leaves start changing color from the edges, later the edges and the tips turn brown with red spots (rust color), then their death and decay occurs. The scarcity is strongly felt in young and in actively growing organs, this is why the functionality of cambium is reduced, the development of conducting tissues is disturbed, the epidermis and the cuticle become thinner, the processes of cell multiplication and elongation are delayed. Shortening of the distance between internodes leads to dwarf phenotypes. Potassium deficit causes a drop in the dominating effect of the apical bud, lateral sprouts develop more intensely and plants get a shrub-like aspect. This deficit also causes the productivity of photosynthesis to drop. In this case, neither phosphorous nor nitrogen containing fertilizers can replace potassium. Calcium (Ca) is another chemical element which is important for plants metabolism. The general quantity of Ca2+ in different plant species is of 5–30 mg/g of the dry mass. Depending on their “attitude” towards Ca2+, plants can be divided into three groups: • calciphiles; • calciphobes; • neutral. Legumes, buckwheat, sunflower, potatoes, cabbage and hemp are reach in calcium ions. Grasses, flax and sugar beet are much poorer in calcium. The tissues of dicotyledonous plants usually contain much more calcium than those of the monocotyledonous ones. Ca2+ is accumulated and stored in older

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organs and tissues. This is due to the fact that transportation happens through the xylem which makes its reutilization difficult. When cells grow old or when their physiological activity drops down, Ca2+ moves from the cytoplasm into the vacuoles and is stored in the form of insoluble salts of the citric and oxalic acids etc. These crystals impede the reutilization of this cation. In most crop plants, Ca2+ accumulates in vegetative organs. In the root system Ca2+ can be found in the form of phytin and its quantity is lower than in aerial organs. In cells, a big quantity of Ca2+ is bound by pectic substances from the cell wall. It is also contained in chloroplasts, mitochondria and nucleus, complexed with biopolymers. Ca2+ performs different functions in the process of exchange of substances. These functions depend on calcium influence on: • the structure of membranes, determining ion circulation through them and the bioelectrical processes; • the processes of cytoskeleton transformation—actiniform proteins, which participate in the processes of cytoplasmic flow, in the reversible processes during which its viscosity is altered (conversion from sol into gel and vice versa), in the spatial organization of the enzymatic cytoplasmic systems (e.g. glycolysis). Calcium is necessary for processes of plants secretion. This element activates a series of fermentative systems in the cell—dehydrogenases, α-amilases, lipases, phosphatases etc. in this case, Ca2+ can determine the assembly of protein subunits, can serve as a bridge between the enzymes and the substrate, can modulate enzymatic action allosterically. Ca2+ surplus in ionic form depresses photophosphorylation and oxidative phosphorylation. The regulatory action of Ca2+ on metabolism depends on the interaction with the intracellular receptor of calcium—calmodulin protein (recruits 4 ions of Ca2+). The Ca2+—calmodulin complex activates several enzymatic systems. The ions of Ca2+ have the important role to stabilize membranes. By interacting with the negatively charged phospholipids groups they stabilize the membrane and decrease its passive permeability. In case of a deficit, membrane permeability increases, fragmentation and ruptures appear and the process of membranous transport is disturbed. By limiting the absorption of other ions into the organism, Ca2+ counters the toxicity determined by the surplus of ammonium ions, Fe, Al and Mn, increases plant tolerance to salts, decreases soil acidity. It is Ca2+ which performs the role of ionic balancer in creating equilibrated physiological solutions, since its quantity in soil is high. In case of calcium scarcity the first to suffer are young meristematic tissues and the root system. In multiplying cells, new cell envelopes are not formed and, as a consequence, multinucleate cells are produced. Formation of lateral roots and root hairs is ceased, root growth ceases. Ca2+ insufficiency causes pectic substances to swell which leads to the appearance of mucus on cell envelopes and cell destruction. As a consequence, the roots, leaves, some parts of the stem rot and die. The tips and the edges of the leaves first turn white and later, black, the leaf blade loses

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its shape and twists. On the fruits, in the conducting and reserve tissues necrotic regions appear. The structure of the plasmalemma and of the cellular organelle membranes is distorted. Most types of soil are rich in calcium and scarcity is very infrequent (in soils with increased acidity and in those with high salt content). Magnesium (Mg), in terms of its quantity in plants, occupies the fourth position after K, Na and Ca. In higher plants, its content in the dry mass is as high as 0.02–3.1 %. It is usually found in the so called short day plants—maize, millet, sorghum, potato, beet, and tobacco. A kilogram of fresh leaves contains 300–800 mg of magnesium. Of them, 30–80 mg are part of chlorophyll. The biggest part of magnesium is contained in young leaves and in the tissues in which reserve substances are stored. In seeds, magnesium is accumulated in embryos, where its levels are several times higher than in the endosperm. About 10–12 % of the magnesium which is part of the chlorophyll performs a unique function in the vegetal body. Magnesium is needed for the synthesis of protoporphyrin IX—the direct predecessor of chlorophyll. During daylight, Mg2+ ions are released from the thylakoids into the chloroplast stroma. When Mg2+ concentration in the stroma goes up, the RDF-carboxilase and other enzymes are activated. It is supposed that the increase of Mg2+ concentration (up to 5 mol/l) causes the activation of CO2 reduction. Magnesium influences directly the enzymatic conformation the enzyme and the proper conditions for its functioning, determining the pH of the cytoplasm as an anti-ion of the protons. Magnesium influences a series of reactions of electron transport during phosphorylation: (a) NADP+ recovery; (b) the speed of the Hill reaction; (c) Electron transfer from PS I to PSS II. In most of the cases, the influence of Mg2+ on other processes of substance exchange is connected to the ability of enzyme regulation, so its importance for a series of enzymes is unique. Mg2+ is the coenzyme of all the enzymes which catalyze phosphate groups transport (phosphokinases, phosphotransferases, ATPases, pyrophosphatases). This happens due to the ability of Mg2+ to form complexes. Mg2+ is also needed for many enzymes involved in glycolysis and for those of the Krebs cycle. In case of its scarcity, the number of mictochondrial cristae is reduced, their form is altered and consequently they disappear. In 9 out of the 12 reactions of glycolysis, participation of activating metals is necessary and magnesium participates in 6 of them. Mg2+ is also necessary for the activity of enzymes which participate in lactic and alcoholic fermentation; it accelerates the synthesis of ethereal oils, calcium, and A and C vitamins; it is necessary for the formation of ribosomes and polisomes, for the activation of amino acids and protein synthesis and is used in all the processes in a quantity of at least 0.5 mol/l. Magnesium activates ADN and ARN-polimerases. The process of Mg2+ absorption by the plant, depends on its supply with other cations. Thus, if a big quantity of K+ or NH4+ is contained in the soil, the level of

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Fig. 6.12 Symptoms of magnesium deficiency

magnesium is decreasing, especially in vegetative organs. Ca2+ and Mn2+ also act like competitors in the process of Mg2+ absorption by plants. The Ca/Mg ratio has a great importance in the vital activity of the plant and regulates many metabolic processes. When the soil pH value is decreasing, Mg2+ enters plants in lower amounts. The insufficiency of magnesium in plants is felt at the level of 2 mg per 100 g of soil. Mg2+ deficit manifests by the formation of yellow-green spots in the vicinity of the veins. The edges of the leaf blade become yellow, red, orange (Fig. 6.12).

6.6 Physiological Role of Microelements Microelements represent an irreplaceable group of mineral elements, which perform an important function in the vital activity of plants. Their content in plants is of 0.001–0.01 % of the dry mass. Microelements participate in the processes of oxidation/reduction, photosynthesis, and nitrogen and carbon exchange. They are part of the active centers of the enzymes and vitamins and increase plant immunity and resistance to unfavorable conditions. The scarcity of microelements causes a series of illnesses and it often leads to the death of the cell. Mn (manganese) is required by all plants. 1 kg of dry mass contains 1 mg of the element. It enters the cell in the form of Mn2+. It is stored in leaves, participates in O2 generation (via water photodissociation) and in CO2 reduction during photosynthesis. Manganese stimulates an increase in sugar quantity in leaves. Two dehydrogenases of the Krebs respiratory cycle (malate- and isocitrate- dehydrogenases) are activated by the ions of Mn2+. Mn2+ is required in the metabolism of nitrogen as part of the nitrate reductase complex in nitrate reduction. Manganese is also important for cell growth. It is:

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(1) a cofactor of the ARN-polymerase II enzyme, responsible for mARN synthesis; (2) Cofactor of the auxine oxidase—an enzymatic complex which decomposes IAA (indole acetic acid or auxin). If Mn2+ is excluded from the nutritive solution, the levels of the main mineral elements in plant tissues rise and their ratio is disturbed. Although the quantity of manganese in the soil is high enough, this element can be difficult to access by plants, especially in soils with a neutral pH. Grasses and the potato plant are very sensitive to Mn2 scarcity in the soil. A phenotype marked by dotted chlorosis is peculiar for Mn2+ scarcity. Yellow spots appear in between leaf veins and the tissue in this areas die. Mo (molybdenum)—the biggest quantity is characteristic of the family of Fabaceae—legumes (0.5–20 mg per 1 kg of dry mass). Grasses contain 0.2–2.9 mg/1 kg molybdenum. It enters plants in the form of anion—MoO42− and is concentrated in young growing organs. It is present more in leaves (especially concentrated in chloroplasts) rather then in roots and stems. The molybdenum participates in nitrate recovery, entering into the composition of the active center of bacterial nitrogenases which fix atmospheric nitrogen in the nodules of the legumes. The molybdenum like the iron is required for the synthesis of leghemoglobin—the protein which transports O2 in the nodules. As an activating metal, molybdenum is required for amination and transamination reactions, for the inclusion of amino acids in the peptide chain, for other enzyme activity. In case of molybdenum insufficiency, a big quantity of nitrates accumulate in the tissues, root nodules are do not develop (the nodules become grayish or yellow, while their ordinary color is red), plant growth is delayed, the leaf blade is deformed. Iron (Fe) in plants makes up about 0.02–0.08 % (20–80 mg/1 kg of dry mass). Fe3+ from the soil solution is converted by the redox system of the plasmalemma of the rhizodermis cells to Fe2+ and, in this form, it enters the plant (the root). By means of compounds which contain heme (cytochromes, catalase, peroxidase), iron participates in the functioning of the main redox systems of photosynthesis and respiration. Together with the molybdenum, iron is required for nitrate reduction and for nitrogen fixation by bacteria in the nodules, being part of the nitrate reductase and nitrogenase. It also catalyzes the initial phases of chlorophyll synthesis (formation of the β-aminolevulinic acid and of the protoporphyrin). This is why iron deficiency in plants in high humidity conditions in carbonate soils leads to a decrease in respiration and photosynthesis intensity and is expressed by a change in the color of the leaves to yellow (chlorosis) and their rapid falling. Iron can be stored not only in catalytically active systems but also in association with proteins like ferritin. Si (silicon) is identified in all plants. A lot of silicon is present in cellular envelopes. Plants that store silicon have solid stems. The insufficiency of silicon can retard the growth of grasses (like oat, barley) and of dicotyledonous plants

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(cucumbers, tomatoes, tobacco). Exclusion of silicon during the reproduction period causes a decrease in the number of mature seeds. When silicon is lacking from the list of nutrients, the ultrastructure of cellular organelles is disturbed. Al (aluminum) has a high importance for the exchange of substances in hydrophytes. This cation is accumulated by tea and sugar cane. In case of aluminum insufficiency in tea, chlorosis can be observed, but at high concentrations it becomes toxic for plants, because it immobilizes phosphorus, which, as a consequence, leads to a phenomenon called “phosphorus hunger”. B (boron) increases the content of carbohydrates, favors flowering, increases pollen viability, the active absorption of salts, influences the absorption of the nitrogen, increases the content of the later in water, facilitates the transportation of the phytohormones in plants. Boron, has an important role in the processes of cell division and elongation. The first symptoms of boron scarcity appear in roots and embryos causing meristem degradation. In legumes, the lack of boron determines reduced root growth, while at high deficit they become brown and mucilaginous. Excess of boron has a toxic effect, the leaves become twisted and necrosis appears on their edges. The activity of Cu (copper) in plant life is quite important. This element is also called a “life giving” element. It is absorbed by the plant from the soil particles in the form of ions. It is an easily oxidizable element. It has the ability to convert Cu+ into Cu2+ and vice versa. Copper is part of plastocyanin composition, being a component of the electron transport chain in the process of photosynthesis. Usually, transportation of Cu2+ occurs in the direction of dominating centers—organs which are in the process of formation, germinated seeds, etc. The amounts of ascorbic acid in plants grow under the influence of copper. Copper intensifies the formation of substances with energy rich phosphate bonds, facilitates the energetic metabolism of carbohydrates. Copper insufficiency can be noticed best in terminal buds and in young leaves which have a small size, a blue-green color and are wilted, but don’t have stains or signs of chlorosis. In grasses, the color is very bright, up to white; leaves are twisting and folding. Copper is scarce in swamp lands, where salts of copper are converted to an insoluble form, inaccessible to plants. Copper scarcity in citric plants, in apples, pears and plums causes the terminal buds and later on the entire sprout to die. Zn (zinc) is part of certain enzymes, such as: carbohydrase, phosphatase, aldolase, carboxypeptidase. It participates in the activation of numerous enzymes, such as: dehydrogenases, enolases, lecithinases. It plays an important role in photosynthesis, due to its presence in carbohydrase, which participates in CO2 fixation. Participates in the biosynthesis of proteins, nucleic acids, certain vitamins, chlorophyll and triptofan. It facilitates an increase in the amounts of solvable carbohydrates in leaves and reduces the intensity of respiration. It determines growth of protoplasm viscosity, by reducing the quantities of free water in the cell. It is absorbed from the soil solution into plants in the form of Zn2+. Plant sensitivity to zinc scarcity differs depending on the species, the age and a series of other external factors. Plants which are sensitive to the lack of this element are: the

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flax, the hop, the ricin, the maize and the grapevine. The symptoms of scarcity differ from species to species and are normally exhibited by a reduction in plant growth: short internodes in grasses, a “rosette” arrangement of the terminal leaves and brunches, emergence of yellow spots on the leaves. In apples, apricots, plums, cherries and grapevine zinc insufficiency causes the formation of small leaves with chlorotic spots.

6.7 Mechanism of Absorption and Transport of Ions in Plants Mineral salts are found in the soil in the form of: • Salts soluble in water; • Salts with low solubility; • Insoluble salts. For their mineral nutrition, plants mainly use the soluble salts from the soil solution as anions and cations. Ion absorption by plants is a complex, selective and self-regulating process, which allows plants to undertake mineral salts from the environment, in a selective manner. As a result, the degree of ion absorption is not proportional to their concentration in the soil solution, for example, in spite of the fact that the ions of NO3- and of H2PO4− are found in small concentrations, they are absorbed by plants in high quantities. Other ions (Cl−, Na+, Al3+ etc.) are found in big quantities, but are only used in small amounts. Plants can also use the ions of insoluble salts, by dissolving the later.

6.7.1 Mineral Element Absorption Absorption (through the differentiation region of the root) and transportation (radial and xylemic transport) of the ions is carried out by the same pathways as water but at a lower speed. In the process of mineral element absorption two phases have been delimited: • the rapid phase of absorption and desorption; • the slow phase of absorption and desorption. The rapid phase is linked to the absorption and desorption of the ions at the level of the cell wall, while the slow phase—at the level of plasmalemma. Absorption takes place through the entire root system (the total volume of absorption), but in an active manner it occurs only at the level of the root hair region (the active volume). The first barrier in the way of mineral elements entrance into

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the cells is the cell wall. Since it has a fibrillar structure, the ions cross the cell wall through: (1) diffusion; (2) exchange of ions; (3) adsorption. Diffusion is a physical phenomenon through which the atoms or molecules of a substance slowly mix with the molecules of the other substances which they contact. At the cell wall level it happens through the free spaces of the macro- and microfibrils. Absorption is obtained through mutual ion exchange between the cells of the plant and the soil solution, in which the cell wall has the role of a cation exchanger. This is possible due to the carboxylic groups (-COOH) of the pectic acids, which participate in the exchange of hydrogen ions with monovalent cations of potassium (K+), sodium (Na+), ammonium (NH4+) as well as due to proteins, which contain both carboxylic (-COOH) and amine (-NH2) groups. These functional groups have the property to accumulate cations and anions from the soil solution. The exchange of ions also happens due to the respiration process. The carbon dioxide which is released as a result of this process, being very soluble in water, forms carbonic acid, which dissociates to generates anions of HCO3−, CO3− and cations of H+ able to participate in ion exchange with the soil solution. Thus, in the cell wall and the plasmalemma one can find ions of H+, HCO3− and OH−, which are released by the roots back into the soil, by absorbing in turn ions of NO3−, PO43−, K+, Ca2+, Mg2+, etc. Another mechanism of ion uptake by the cell wall is the adsorption phenomenon. There are two types of adsorption: mechanical and by means of chelates. The mechanical adsorption is performed via very labile, transient bonds between the adsorbed substance and the cell wall. Adsorption by means of chelates is stronger and is carried out with the formation of stronger bonds between mineral ions (cations or anions) and organic substances. Ion penetration through biological membranes, which is the next obstacle, can be either active or passive. Passive transport through biological membranes is carried out according to the concentration gradient, without metabolic energy consumption and the process is connected with diffusion. From the thermodynamic point of view, the direction of the diffusion process is determined by the chemical potential of the substance. The higher the concentration of the substance, the bigger the chemical potential will be. Diffusion is oriented towards a lower chemical potential. Importantly, the direction of ion movement is also determined by the electrostatic potential. In ion diffusion, the values of the kinetic molecular energy count. This energy grows with temperature increase and concentration increase. Ions with different charges have different diffusion rates through the semipermeable membranes. This fact leads to a difference in the electric potential, which can serve as a driving force for transferring other ions. The electric potential can also appear as a result of irregular

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distribution of charges inside the membrane. Thus, passive movement happens according to the electrochemical potential. The following substances can be transported through diffusion: • liposoluble substances; this phenomenon has been described by Overton in 1895, who proved that the speed of transportation is directly proportional with the liposolubility of the substances. The permeability of plasmatic membranes for organic and mineral substances depends on their dynamic composition (the ratio of lipids and proteins). When lipids are predominant in the plasmatic membrane, its permeability is much higher for organic substances; while if proteins are predominant, its permeability is higher for water and mineral ions. • molecules whose diameter is smaller than that of the pores from lipoprotein membranes. This phenomenon was described in 1867 by Traube, who proved that the speed of transport is higher when the diameter of the molecule is smaller and vice versa. Facilitated diffusion is another mechanism of passive transport which is carried out without energy expenditure. The transporters are proteins which act selectively on certain ions. There are several types of transporters according to the functioning mechanism: (1) diffusing transporters; (2) sliding transporters (along the walls of the membrane pores); (3) transporters that rotate inside the membrane. Facilitated diffusion occurs much faster than simple diffusion. At the same time, active transport of ions occurs inside the cell. This happens with metabolic energy consumption. Active transport happens against the degree of concentration. This process has a great significance for the normal functioning of the cell. The energy which is necessary for active intake of the ions by the cell is produced during respiration and is stored as ATP. It has been proved that in order to transport ions against the concentration gradient, it is necessary to spend an energy amount of up to 4600 J/mol. To access the ATP energy, ATP-ase domains are included as part of the sequence of the transmembrane transporter proteins (Na+, K+-ATP-ase etc.). A directly proportional dependence exists between ATP-ase activity and ion transport. The activity of ATP-ases is also specific for the genotype and the studied organ (Fig. 6.13). Higher ATP concentrations accelerates penetration of the ions into the cell, which proves the link between absorption of mineral salts and respiration. After passing through the membranes, ions enter the protoplasm where they are included in the cell metabolism. Intracellular transport is carried out through the channels of the endoplasmic reticulum. Cytoplasmic organelles play an important role in nutrient acquisition. The nucleus, the mitochondria and the chloroplasts are competing in the absorption of cations and ions from the cytoplasm. The surplus of ions enters the vacuoles, contributing to the formation of the chemical potential of the cell and represents a reserve form of it. In order to accede to the vacuoles, the

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Fig. 6.13 The activity of Mg2+, Na+, K+—ATPase in the calathides and leaves of different sunflower genotypes in the flowering phase (μmol ATP/min g fresh weight) (Glijin 2002)

ions have to pass yet another barrier—the tonoplast. The permeability of the tonoplast is lower than that of the plasmalemma. It is known that there are two active systems of ion transport through the cell: • located in the plasmalemma and which functions in conditions of low intracellular concentrations; • located in the tonoplast and functioning only at high intracellular concentrations, when the cytoplasm, is saturated with these ions. It is the second mechanism which is responsible for ion entry into the vacuoles. Thus, ions which enter through the plasmalemma are used for cellular needs or are transported into the neighboring cells and only their surplus enters the vacuoles.

6.7.2 Mineral Element Transport Transportation of mineral salts in plants are carried out by means of 2 paths (radial and xylem). The mineral substances which are accumulated in the cytoplasm of root hair cells and in cortical cells are transported towards the xylem vessels (radial transport) through the symplast and apoplast (Fig. 6.14). Transport of ions through the apoplast is based on the process of diffusion of ions at the cell wall level. The symplastic path is permanently active and occurs from cell to cell through the plasmodesmata. Ions from the soil solution are transported through the apoplast to the plasmalemma simultaneously in all cortical cells. The ion pumps from the plasmalemma of root hair cells and cortical cells function in the same direction, transporting ions from the apoplast into the symplast and vacuoles, where they generate the turgor pressure, or from cell to cell towards the conducting vessels.

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Fig. 6.14 Transport of mineral elements in plants

Transport of the crude sap from the roots towards the leaves is performed through the xylem. The crude sap is a diluted solution which contains ions and organic compounds which originate from the soil. The delivery of mineral substances and water to the leaves is carried out through their branching veins (xylem). The xylem transport of minerals follows the water stream up to the level of the foliar system, but at a lower speed. Movement of the nutritive substances in an ascendant manner through the xylem is a passive process. But the distribution of nutritive substances is not determined by the intensity of respiration, but by the exchange of substances in the respective organ and by the presence of auxins in the growing apex which has the role of a dominant center. Its removal leads to a uniform distribution of mineral ions in all plant organs. The distribution of ions is determined by the functional activity of the tissue. The biggest quantities of ions are provided to the young growing tissues.

6.8 Soil as a Substrate for Plant Nutrition The soil represents the main substrate for mineral nutrition and is formed by three types of substances (solid, liquid and gaseous). It is characterized by a certain chemical composition, pH, structure etc. The soil solution is a physiologically equilibrated solution, in which ion antagonism is present—reducing the negative effects of certain ions by others. The concentration of the soil solution is 0.05–0.15 %. Depending on the pH of the soil, selective absorption of certain ions occurs. Anions are absorbed at a weak acidic pH, while cations—at a weak basic pH. At the

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optimal pH the monovalent anions and cations are absorbed at the same rates. The salts that do not affect the soil pH are called neutral: NH4 NO3  NH4 þ þ NO3  The speed of cation absorption is higher than that of anion absorption. If the soil contains many anions it is called physiologically acid soil: ðNH4 ÞSO4  2 NH4 þ þ SO4 2 If cations accumulate in the soil solution, it will become alkaline (physiologically basic soil): CaðNO3 Þ2  Ca2þ þ 2NO3 

6.9 Influence of Various Environmental Factors on Mineral Nutrition in Plants The process of mineral element absorption can be influenced by a series of internal and external factors. Among the internal factors an important role is played by the species and the age of the plant, its metabolism etc. The highest intensity of mineral element absorption can be observed during the phase of active growth and fructification. The main external factors which influence mineral nutrition are soil humidity, temperature, pH, light etc. At a soil humidity of 75–80 % intense water absorption of mineral compounds and water from the soil occurs. At low values of humidity the absorption rate is low. At temperatures around zero degrees Celsius, the rate of absorption decreases dramatically or ceases altogether, the temperature range at which absorption is done with a maximum intensity is between 20 and 35 °C. At higher temperatures, the intensity of absorption decreases. When temperature reaches 50 °C absorption also stops. Lack of oxygen can influence the process of absorption because in this case insufficient energy will be released during respiration. Big concentrations of mineral salts in the soil solution are toxic for plants. Absorption of ions with a high intensity is performed at a concentration of 0.2–1 %. A normal concentration of potassium ions influences favorably the absorption NO3− and Fe2+ ions but decreases the absorption of Ca2+, Mg2+ and PO43− ions. It results that the antagonism between ions and their mutual influence play an important role in plant mineral nutrition.

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Glossary Absorption of nutritive elements Selective entry of mineral and organic substances into the plant together with water. It can happen through active or passive transport. Passive transport is carried out without energy consumption, being determined by diffusion processes according to electrical and chemical gradients. Active transportation of the ions are performed with metabolic energy consumption and are carried by specialized transporters (e.g. ionic pumps) etc. These two processes take place simultaneously and are interdependent. Aeroponics Cultivation of plants without a substrate (in the air). During this process their roots are fed periodically with nutritive solutions. Deficiency Lack of nutritive elements that causes structural and morphological changes in plant organs. A deficiency of macroelements (N, K, P and Mg) manifests itself in the lower parts of the plant, while microelements deficiency (Fe, Mn, B, Mo, Zn) manifests itself in young leaves at the top of the stem. Chlorosis The appearance of yellow-colored regions on the leaf blades, caused by sickness and by nutrition deficit (lack of accessible forms of iron, magnesium and other elements) or by a prolonged surplus of humidity, by lack of light as well as by viruses, bacteria and fungi. Mineral nutrition Intake and assimilation by plants of the inorganic compounds (macro- and microelements), which include the processes of absorption, transportation and metabolization. Plant nutrition The process of supplying the plant with nutritive substances. Depending on the nature of their nutrition, plants are divided into: heterotrophic, which feed on available organic substances (higher plants without chlorophyll), autotrophic, which implies synthesis of organic compounds from inorganic substances (higher green plants, algae) and mixotrophic, which can have both autotrophic and heterotrophic nutrition (some green algae and some carnivore plants).

References Brei S (1986) Nitrogen exchange in plants M., p 240 Burzo I et al (1992) Physiology of crop plants, vol 1 Ştiinţa, p 462 Glijin A (2002) The exogenic action of gibberellins on protein synthesis in sunflower. Ph.D. thesis in biology. Chişinău, p 22 Izmailov SF (1986) Nitrogen exchange in plants M., p 320 Klarkson D (1978) Ion transport and the structure of plant cells M., p 368 Liutghe U, Highinbotam H (1984) Transportation of substances in plants M., p 408 Molecular mechanisms of nitrogen absorption in plants, M. 1983, p 263

References

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Rubina AB (ed) (1980) Results of science and techniques. Plant physiology T. 4. Ion Transportation in plants, p. 176 Şcolinik MIa (1974) Microelements in the life of a plant L. p 324 Şeveakova NI (1979) The metabolism sulfur in plants M. p 166 Sîtnic KM, Kniga NM, Musatenko LM (1972) Root physiology Kiev. p 356

Chapter 7

Plant Growth and Development

Abstract Growth and development are two interrelated processes occurring simultaneously throughout the ontogeny of any living organism. Growth represents a combination of physiological and biochemical processes through which the irreversible increase in plant volume, mass happens due to cell, tissue and organ expansion. Plants, unlike animals can grow throughout their life due to the maintenance of active meristematic tissues (apical, lateral, intercalary etc.) in the centers of growth. However, it can be interrupted, especially in a seasonal manner (dictated often by photoperiodism, temperature drop), by periods of genetically programmed latency or dormancy. This dormancy can be biological (deep), characteristic for all plants and genetically programmed or forced, resulting from the immediate environmental conditions. Growth is accompanied by development characterized by the formation of new organs which simultaneously suffer significant changes in size, shape and structure, often acquiring new functions. These changes are reflected down to the cell and molecular levels determined by the transition between different genetic programs (e.g. induction of flower development after vegetative growth). Individual development in plants is divided into four periods: embryonic (from the zygote to mature seeds), juvenile (begins and ends with the formation of vegetative organs), reproductive (period of seed and fruit formation) and senescent (starts when fruit formation ceases and ends up with death). Plants control their physiological programs, developmental transitions and responses to the environment with the help of phytohormones. These are organic compounds of varied chemical structure synthesized by plants in low concentrations in specialized tissues and transported throughout the plant body where they alter essential physiological processes qualitatively and quantitatively. The main groups of plant hormones include auxins, cytokinins, gibberellins, abscisic acid, ethylene, brassinosteroids and jasmonates. Phytohormones have multiple functions and various combinations of them can act either synergistically (auxins and gibberellins) or antagonistically (abscisic acid and auxins) to promote very specific responses.

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Historical Background 1901—D.N. Neliubov described the morphogenetic action of ethylene. 1903—G. Krebs demonstrated the role of external environmental factors on plant growth and development. 1920—U.U. Garner and G.A. Allard discovered the phenomenon of photoperiodism. 1926—N.G. Holodnâi and F. Vent developed the hormonal theory of tropisms. 1934—F. Kogl and others determined the chemical nature of heteroauxin. 1935—N.I. Vavilov layed the foundations of plant growth and development in different ecological conditions. 1937—L.M. Chailakhian forwarded the hormonal theory of plant development. 1946—H. Bortwick and others demonstrated floral induction by red light. 1956—A. Lang shows the significance of gibberellins in flowering. 1958—L.M. Chailakhian launches the hypothesis regarding the dual nature of florigen. 1959—V.O. Kazarian has studied the role of functional correlation between the processes of root and leaf senescence. 1965—J. Bonner develops morphogenetic tests.

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Brief Updates Parasitic bacteria Agrobacterium tumefaciens and A. rhisogenes use phytohormones to induce tumors in plants. A. tumefaciens contains the Ti plasmid, harboring the iaaM1 and iaaH2 genes involved in the synthesis of auxin (IAA) while the ipt gene encodes cytokinin biosynthesis. Because these genes lead to uncontrolled cell proliferation, they were assigned to oncogenic agents. The Arabidopsis thaliana gene Leafy (LFY) is involved in plant transition from the vegetative to the reproductive state, contributing to the initiation of flower formation. In addition to this gene the Apetala (AP1) and cauliflower (CAL) genes are involved in flower formation. An opposite effect is exerted by the terminal flower (TFL1) gene, which retains the formation of flowers, inhibiting gene expression of LFY and AP1. Genes with similar effect were detected in rice (OsRCN1) and rye (LpTFL). Jasmonic acid (JA), which is synthesized from linolenic acid, by the octadecanoic pathway has been found in higher plants and is one of the key phytohormones involved in stress signaling. This phytohormone manifests an inhibitory effect on photosynthesis, callus, growth, cell division and DNA replication and induces senescence. Exogenous treatment of the Quercus ilex plant with jasmonate decreased the rate of photosynthesis, induced an alteration in the stomatal conductance levels and determined the elimination of volatile monoterpenes and methyl salicylate.

7.1 The Concept of Plant Growth and Development Vegetable bodies absorb water and mineral salts, accumulate solar energy, make countless reactions of substance exchange. As a result of these activities plants grow and develop. Growth and development are two specific integrated processes of any living organism occurring simultaneously. Growth and development occur throughout ontogeny (from gr. ontos “being” and genesis, “origin”) from zygote until death. During ontogeny the hereditary information (genotype) is implemented in strict relationship with the conditions of the environment which ultimately results in a phenotype characteristic for the species. Growth represents a combination of physiological and biochemical processes through which the irreversible increase in volume, mass, size of the plants happen due to tissue and organ expansion (Fig. 7.1). Unlike animals, plant growth can occur throughout the life, because the centers of growth (stem and root tips, shoots, cambium, phellogen) have active meristematic tissues that work continuously. The process of growth in plants is rhythmically interrupted by periods of rest (latency). Latency in plants is a normal periodical phenomenon which is genetically programmed and during which a decrease in the intensity of life processes occurs. Growth intensity varies depending on the species, organ, environmental conditions etc. The highest rates of growth were found in mushrooms (5 mm/min) and in

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Fig. 7.1 Seed germination

plants—the bamboo. Growth intensity decreases with age. Growth is determined by a large number of genes the individual effects of which can be traced in the phenotype even by simple measurements (with a ruler or auxanometer). During growth new organs form which simultaneously suffer significant changes in size, shape and structure or, in other words, growth is accompanied by development. Intensive growth occurs rather in the dark than in daylight. Ontogenetic development is the evolution of the individual specimen from undifferentiated cells to a mature organism and includes quantitative changes, structural, functional changes in the properties of cells, tissues and organs (Fig. 7.2). Development is manifested by the emergence of differentiated specialized cells from undifferentiated embryonic cells (cytogenesis), of tissues (hystogenesis) and organs (organogenesis) based on morphological, anatomical, physiological, biochemical changes that happen in a predefined sequence encoded by the genetic program. Translation of the genetic information during ontogeny is performed by differential gene expression in space and time. Development is determined by the activity of a variety of specific proteins, whose biosynthesis and action varies depending primarily on the gene expression levels (DNA–RNA–protein). We can distinguish vegetative development (Fig. 7.3) and reproductive development (Fig. 7.4), which includes all fundamental processes of forming: reproductive organs—flowers, seeds and fruits (for generative propagation), as well as specialized bodies—tubers, bulbs (for vegetative propagation). In plant morphogenesis, light is influencing growth and development through the phytochromes.

7.1.1 Dormancy in Plants (Repose) Dormancy in plants is a physiological state during which the intensity of metabolism and growth rates decrease sharply, but morphogenetic processes may be progressing slowly. It manifests by a delay in seed, tuber, bulb germination, or a delay in bud opening and represents a form of adaptation in order to survive adverse

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Fig. 7.2 Plant development

Fig. 7.3 Tissue differentiation in different organs of the plant (Gilbert 2000)

environmental conditions during certain periods of the life cycle or an unfavorable season. During the dormancy period plant ability to withstand drought as well as high or low temperatures increases. But dormancy is not just a protective reaction of the organism to unfavorable environmental conditions. Plants enter this state even with all the necessary conditions for growth. After it growth is resumed with increased intensity which further highlights its importance in the ontogeny of plants. Dormancy may be: • biological or deep—characteristic for all plants and genetically determined; • forced—determined by environmental conditions.

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Fig. 7.4 The transition from vegetative to reproductive development (Gilbert 2000)

Plants or organs that are in biological repose are characterized by the accumulation of growth inhibitors and by certain physiological and biochemical processes that prepare future growth. This is part of the reason why growth is not restored even when all the necessary conditions exist. Usually, plants enter the forced dormant state in the absence of one of the factors necessary for growth and as soon as these factors become available, growth processes are resumed. Plants exit forced dormancy only when the biological repose is due to begin. Perennials fall in deep dormancy in autumn and in late winter they long before budding they enter forced dormancy. Either the whole organism or pats of it (seeds, tubers, roots) can be dormant. Under certain conditions some plant organs can grow while others (buds) are dormant. Transition to the repose state is often accompanied by loss of organs (falling leaves or even entire shoots). It is in such a state that perennials survive winter. While dormancy can be different and can affect different organs differently there are also common features that characterize the phenomenon: (1) lack of active growth (there may be a latent growth); (2) decrease in the intensity of metabolic processes; (3) reduction in the amount of growth promoters. In most of the crop plants the state of repose is controlled by photoperiodism. Long days accelerate vegetative growth and short days lead to growth inhibition and formation of dormant buds. There are different ways to stop this phenomenon (etherization, hot baths, treatment with volatile substances or low temperatures, growth stimulating substances) which are used in greenhouses for growing winter flowers. Bud and other organ dormancy may be interrupted with gibberellins, cytokinines, ethylene and triggered by abscisic acid.

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7.2 Types of Plant Growth Plant growth occurs in areas of embryonic tissue—meristems, where cells are dividing. Apical meristems are located at the tip of the shoots and roots. Lateral meristems in dicotyledonous plants form the cell layers around all the shoots and roots and include primary meristems—procambium and pericycle and secondary— cambium and phellogen (Fig. 7.5). Intercalary meristems are localized at the leaf or node bases. Based on these considerations, we distinguish the following types of plant growth: • • • • •

apical—characteristic for root or stem growth; lateral—for plants with a secondary anatomical structure; intercalary—for grasses; basal—specific for leaves and fruits; traumatic—specific to all organs able to regenerate tissues.

Apical meristems are not only the tissues that give birth to the plant but also the dominant centers of coordination that affects morphogenetic processes throughout the plant organism.

7.3 Phases of Cell Growth and Development The basis for multicellular organism growth consists in increasing the number and size of the cells, accompanied by differentiation. Growth and development start from a single cell and follow three phases: Embryonic stage covers the period of cell preparation for the replication process and the process itself which usually happens 2–6 times. At this stage, growth intensity is not too high, because it is based on the increase in the number of cells and less on their volume increase. During this phase accumulation of organic matter Fig. 7.5 Areas of growth and development of the root system cells

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occurs in the protoplasm—enzymes, structural proteins, starch, lipids, phosphatides, nucleic acids. At the same time cell organelles form. Expansion (elongation) phase is characterized by structural and physiological changes such as the formation of the central vacuole, protoplasmic colloid hydration, increased protein biosynthesis and high metabolic rates. The suction force (S) of the cells increases, leading to increased water retention. As a result their volume and mass increase considerably and cells extend. In the process of cell elongation a special role is played by growth regulators, including auxins (IAA). IAA, in the presence of Ca ions, acts on hydrogen pumps, thus contributing to the acidification of the primary cell wall and the activation of hydrolases, including glycosidases, which, in conditions of low pH, break the glycosidic bonds. In turn, glycosidases contribute to the synthesis of specific cell wall polysaccharides that enhance cellular envelope plasticity. The accumulation of hydrogen ions in the cytoplasm as a result of hydrogen pump activity and various osmotically active metabolites contribute to an increase in the osmotic pressure and in the suction force of the cells. Thus, on one hand, breaking the hydrogen bonds that link cellulose microfibrils with hemicellulosic xyloglucans and the glycosidic bonds that links xyloglucans with rhamnogalacturonan and, on the other hand, the increased turgidity as a result of intensive endosmosis, contributes to cell wall expansion and cell elongation (Fig. 7.6). Cells are covered with a primary cell wall, which is elastic and is capable of unlimited expansion, so that size adjustment can happen. Growth intensity is maximal since it is based on the extension of each individual cell. This phase can occur immediately after cell division or after a certain period of time. The phase of differentiation is the phase when stabilization of the structure and the outer shape of the cell happen and is characterized by reduced growth. Cell hairs can form, differentiation of cell organelles completes and specific structures are formed according to the location of the cell. Differentiation is determined both

Fig. 7.6 Mechanism of cell elongation

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hereditary and by the ratio of endogenous phyhormones. Cells can be totipotent (omnipotent). Each cell contains hereditary information necessary for growth and development of the entire body. Omnipotence has been demonstrated by in vitro culture. Functional differentiation of the cells takes place throughout the duration of cell growth. Even in daughter cells, which emerge immediately after division, there are some differences, manifested in the chemical composition, morphological features of the nucleus and organelles. Differentiation culminates with the establishment of the different tissues of the living organism that perform a wide variety of functions.

7.4 Phases of Plant Growth and Development Plant morphogenesis includes processes of formation growth and development of cells, tissues and organs which are genetically programmed and mutually coordinated. Individual development in plants is divided into four periods: embryonic, juvenile, reproductive and senescent (Figs. 7.7 and 7.8). The embryonic stage in angiosperm plants includes embryo development from the zygote to mature seeds and happens on the parent organism.

Fig. 7.7 Individual development in gymnosperms

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Fig. 7.8 Individual development in angiosperms

The juvenile stage is characterized by an intensive increase in vegetative development. In seed plants, the seed germination stage begins and ends with the formation of vegetative organs. During this period, plants do not have the ability to reproduce sexually. Reproductive stage is the stage of maturity and multiplication with a reduced growth. It represents period of seed and fruit formation—the plant is more active in this respect, because it possesses a vegetative mass sufficient to supply the growth and development of flowers, seeds and fruits, which are heterotrophic organs of the plant. Senescence phase is the phase of aging and starts when fruit formation ceases and ends up with plant death. It is a period of gradual weakening of the vital activity of the organism. It was showed that the growth rate obeys a common law and represents a curve, which is called “growth curve” or “S-curve”. At first growth usually is reduced, then, it intensifies and, in the end, decreases again. This regularity is manifested at all levels of organization of the living matter. In the process of plant growth and development a succession of dominant centers occurs (Fig. 7.9), characterized by a high functional activity, high concentrations of auxins, which retain the growth of shoots. These phenomena regulate and determines plant metabolites distribution.

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Fig. 7.9 Dominant centers. Apical dominance

7.5 Genetic Aspects of Plant Morphogenesis The biology of plant growth and development includes the fundamental analysis of the mechanisms and phenomena that underlie cellular differentiation, intra- and intercellular interaction within the entire organism to form cells, tissues and specialized bodies. Plants, unlike animals, have a reversible plastic morphogenetic process. Although the number of structural genes in plants and animals is almost the same, these kingdoms differ substantially by the number of specialized cell types and tissues. Thus, in plants were identified only a few tens of types of tissues and 60 different types of specialized cells, while in vertebrates the number of the latter is several hundreds. Structural and morphological differences between plants and animals are determined by morphogenesis and development mechanisms, which were formed during the evolution and ensured the phylogenetic separation of these kingdoms. The primary role in the occurrence of certain features of morphogenesis is owed to the fact that plants are fixed to the substrate, so that growth and development is dependent on and is in direct connection with varying environmental factors. Development in plants and animals has a cyclic character. In plant organisms the succession of generations occurs—sporophyte–gametophyte (haploid-diploid or sexual-sexual). During individual development highly specialized complex structures like the flowers and the fruits form from a practically undifferentiated embryo. At the cellular level, the formation of different morphological structures occurs through continuous functional activity of undifferentiated meristems. The rigid cellulose-pectin cell walls exclude cell migration during morphogenesis. Plant cells are omnipotent—a unique feature, determined by the plasticity of the plant genome, which allows in certain circumstances, even partially differentiated cells to switch to another program of morphogenetic development and to even ensure regeneration of the entire organism. In animals stem cells etc. are similar to meristematic tissues, but only embryonic stem cells have a comparable plasticity. In terms of molecular biology, the development process represents differential gene activation and suppression. Inclusion of each new developmental program is reflected in the modified spectrum of gene expression products—mRNAs and proteins. Development of modern research methods of nucleic acids and proteins

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allowed to determine the mechanisms of temporal and spatial gene regulation which contributes to the elucidation of plant growth and development. It was found that only about 4 % of the polypeptides are organ-specific, while the mRNA level of specificity is much higher—25 %. An important issue is finding the nature of these differences and the role of specific proteins in inducing morphogenetic programs and organ differentiation. It’s possible that these key proteins are synthesized in small quantities during short time periods. A key direction in the study of plant growth and development is the isolation and functional analysis of specific genes with an important role in morphogenesis. In this regard the creation of cDNA libraries (Fig. 7.10) and their subsequent hybridization with DNA or RNA enables the identification of specific active genes for different tissues or organs at different stages of vegetation.

Fig. 7.10 Creating cDNA libraries

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Investigating promoters with the help of reporter genes is another way to determine the mechanisms of differential gene expression. The most common reporter genes—genes that synthesize enzymes whose activity can be easily identified in plant tissues in vivo and in vitro are—β-glucuronidase, luciferase, β-galactosidase, octopine- and nopaline synthase, chloramphenicol acetyltransferase, neomycin phospho transferase. The role of specific genes in morphogenesis can be studied by genetic transformation, particularly by antisense technologies. In this case, the obtained genetically modified plant contains a construct comprising cDNA, in which the promoter reads and transcribes mRNA(−) from short regions of antisense DNA. This mRNA (−) later interacts with the target complementary mRNA(+) available in the cell, thus blocking the translation process. As a result, protein synthesis is greatly reduced, which leads to a detectable phenotypic effect, so that gene and protein functions can be deduced during morphogenesis. Each stage of growth and development is characterized by the implementation of a specific genetic program, the functional activity of certain genes and a complex of morphological and biochemical indices. The transition to flowering is one of the most studied morphogenetic phases in plants. Anthesis and floral induction are determined by photoperiod, vernalization, phytohormones and certain gene families that control these factors. For example, in peas seven basic genes Veg, Lf, Sn, Dne, E, Hr and Gi were found, which are responsible for the transition from vegetative growth to floral primordia differentiation. The Veg gene determines the ability to switch to flowering plants. In Veg mutants flowering does not happen. Flowering is determined by the balance between inducers (locus Gi) and inhibitors (dominant alleles Sn and Dne loci). Sn and Dne also control plant photoperiodism. The Hr gene is active in leaves and amplifies Dne-Sn gene function while gene E is active in cotyledons and act to diminish the functional activity of these genes. The Lf gene is active in the shoot apex and is a key element in recognizing the ratio of inducers to inhibitors required for the transition of the apical meristem into flower formation. Different alleles of this gene determine the sensitivity threshold of this ratio, which corresponds to appearance of the first flower at the internode. Flower development is a classic example of cell differentiation, the transition of the morphogenetic program from vegetative growth, characterized by endless divisions, to the reproductive stage, characterized by flower formation.

7.6 Endogenous Factors of Plant Growth and Development Plant growth and development are complex processes conditioned by three key types of factors—nutritional, genetic and hormonal. In 1675 the Italian scientist Marcello Malpighi predicted that plants contain substances with regulatory effect. First experiences that have shown the presence of stimuli acting on the movement of plants were exposed by Francis Darwin and his

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father, Charles Darwin (1880) in his famous On the Movements and Habits of Climbing Plants. The first scientist who worked with hormones and active extracts from plants and introduced the concept of “hormone” in plant physiology was Fitting (1909– 1910). Phytohormones (from gr. phyton “plant” and hormaein “to stimulate, to excite”) are natural organic compounds with relatively low molarity and varied chemical structure, participating in the interdependent activation of cells, tissues and organs and required in small quantities (10−6–10−11) for activation and implementation of physiological programs. They are synthesized in specialized tissues of higher plants and transported throughout the plant body coordinating ontogenesis, stimulating or inhibiting the morphogenetic pathways or altering the quantity and quality of the essential processes in the organism. In contrast with animals that have special glands that synthesize hormones, their biosynthesis in plants takes place mainly in the meristematic tissues of growth centers, phytohormones are multivalent and polyfunctional (Fig. 7.11). Traditionally phytohormones are divided into 5 groups (Table 7.1): • • • • •

auxins (derivatives of the indole-3-acetic acid); cytokinins (derivatives of 6-aminopurine-zeatin); gibberellins (tetracyclic carbonic acids of the diterpenoid class-GA3); abscisins (abscisic acid ABA—a sesquiterpenoid with optical activity); ethylene (colorless gas, unsaturated hydrocarbon with a double bond).

Similar actions have some oligosaccharides—salicylic acid, polyamines. Brassinosteroids are also considered hormones (with stimulatory action), as well as fusicoccins and anthesins. Novel compounds similar to cytokinins—4-phenilthio redo salicylic acid and 1-(3-chlorophenyl)-3-(2-pyridyl) urea are also being tested. Between different groups of hormones there are synergistic relationships (auxins stimulate gibberellins) and antagonistic ones (between abscisic acid and auxins).

Fig. 7.11 The diagram shows gradual changes in the location of certain regions of the leaf (blue dots) and the concentration (the size of blue dots) of free IAA production during the development of the leaf primordium in Arabidopsis. Arrows indicate locations with the highest level of primary production of free auxin located on the margins of the leaf blade at each stage of development (a–d) while short arrows indicate the location of reduced auxin synthesis rates (d, e)

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Table 7.1 Principal groups of phytohormones Hormone

Synthesis location

Target tissue

Auxine Cytokinin Giberelline Abscisic acid Ethylene

Stem apex, developing fruits Actively growing regions Immature seeds Leaves Fruits, flowers, leaves, roots

Primary cell wall Roots, stem, phloem, xylem Internodes, seeds, fruits Stomata Buds, seeds, fruits

Fig. 7.12 Differential effects of phytohormones on life processes in plants

Natural and synthetic substances that act as growth regulators, according to the mode of action, are divided into (Fig. 7.12): • growth promoters; • growth inhibitors; • retardants (only synthetic). Auxins, gibberellins and cytokinins are considered stimulants and abscisins and ethylene—inhibitory hormones. There are multiple links between them, they have a versatile action, which depends, on one hand, on the concentration of the hormone that has reached the target cells, on the other hand, on the tissue competence (ability to respond, type and intensity of the response) (Fig. 7.13). These three groups of

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Fig. 7.13 Induction of the hormonal response by hormonal or environmental factors

substances are not acting separately. They interact in such a manner that all stages of growth and development are the result of an equilibrium between stimulators and inhibitors that manifest mainly in seasonal processes. Growth stimulants (auxins, gibberellins, cytokinins, substances of the vitamin B group, ethylene chlorohydrin, thiourea, etc.) are organic substances that stimulate endogenous plant morphogenesis and regulate physiological correlations between different organs of the plant. If a paste containing vitamins is applied on the growth cone or at the basis of a leaf, we would see increased growth, the formation of shoots and fruit components. Stimulants are used in agriculture to facilitate the rooting of certain cuttings, in order to increase plant productivity. Growth inhibitors (unsaturated lactones, phenolic compounds, organic acids and flavonoids) are endogenous organic substances, which inhibit the plant physiological activity and the development of organs. Growth inhibitors are present in different organs of the plant—seeds, bulbs, tubers, shoots and reduce or cancel the activity of stimulants and inhibit plant growth, seed germination, alter the activity of enzymes, inducing deep dormancy. Their content increases maximally in autumn during the transition to the dormant state, which is related to interruption of meristematic tissue growth. Among natural inhibitors are: the β inhibitor, derived from stems and roots, which inhibits seed germination inside the fruit, the abscisic acid, derived from dormant buds that has multiple properties (anti-stimulating action, maintenance of the repose state, young fruit detachment etc.); phlorizin is synthesized only in leaves only during short days and inhibits the respiration process, synthesis of nucleic acids and proteins; coumarin inhibits seed germination etc. Fungicides, nematicides, insecticides and other groups of toxic substances used in agriculture, also act by inhibiting important vital processes.

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The inhibitory effect can be noticed also during the action of physical factors (cold or extreme weather, light excess or shortage, deficit or excess of moisture) and chemical factors (chloropropane, butylate and alachlor impede germination, atrazine, simazine, propazine block photosynthesis). Retardants are artificial substances retaining plant growth and development.

7.6.1 Auxins Natural auxins (indole-3-acetic acid = IAA) were detected in different organs of actively growing plants—buds, young leaves, roots and stem apices, cotyledons, etc. and are the only phytohormones that have analogues in the animal world.

Biosynthesis. The auxin precursor in plants is tryptophan or substances derived from its degradation. It is formed by following three steps involving three enzymes: transaminase, which catalyzes the conversion of tryptophan into tryptamine, decarboxylase—from tryptamine to indole pyruvic acid, which transforms into βindole acetaldehyde and aldehyde dehydrogenase, which catalyzes the formation of β-indole acetic acid (Fig. 7.14). The presence of two active groups—carboxyl and amine and the properties conferred by the nucleus of the indole molecule, transform IAA in a very active substrate for biochemical reactions which leads to its rapid inactivation, both in vivo and in vitro. The quantity of auxins in plants is determined by the action of the enzyme auxin oxydase that interferes in controlling the levels of endogenous auxins in the root tip and can interfere with auxin metabolism. Indole acetic acid is found in plants in two forms: • bound (70 %) to other macromolecules, less mobile, which in most cases, don’t have phytohormone activity and lack toxicity. Auxins linked with proteins bind to the active cell centers and suppress their activity. Under the action of proteolytic hydrolases auxins are released from their protein substrates and exert again their phytohormone activity; • free (30 %)—available auxins are mobile and are easily transported to different organs of growth.

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Fig. 7.14 Indole acetic acid biosynthesis

Transport. Chromatography investigations and the use of labeled atoms showed that auxins are circulating in a polar manner at a speed of 10–20 mm/h, basipetally through the phloem and parenchyma. Transport requires energy and can be blocked by the presence of alkaloids and lack of oxygen. Acropetal movement is less intense. Auxins circulate in plants usually during the period of activation of morphogenetic processes of growth and development, when some regions are involved in the synthesis of phytohormones while others are involved in various growth processes. Migration of auxin molecules are based on the electrical charge of its carboxylic groups. The mechanism of polar auxin transport consist in the fact that, in apical cone cells, IAA enters passively with hydrogen ions while on the basal part it is actively secreted through the cell membrane and is based on the difference in the electrical potential between the top of the plant, with negative charge, and its base with a positive electric charge (Fig. 7.15). Mechanism of action. Auxins act on gene expression by activating the cytosolic protein ARF (auxin response factor).

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Fig. 7.15 Polar auxin transport

ARF is a transcription factor that can enter the nucleus, where it binds the promoter sequences of various genes and alters gene expression levels. IAA influences polyribosomes and the activity of the nuclear apparatus including: • RNA polymerase (RNA polymerase-1) due to the increasing content of the transcription initiation factor ϒ;

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Fig. 7.16 Auxin location revealed by immunostaining and viewed with a confocal microscope (a) and expression of the genes DR5:GUS in transgenic Arabidopsis thaliana, demonstrating histochemical localization of GUS activity during morphogenesis of leaf primordia (b–f). a Lamina paradermal section with IAA strong marking (characteristic) (green-fluorescent staining by secondary antibody conjugation) in chloroplasts (indicated by arrows) and a lower concentration inside the cytoplasm of elongated cells near the fibro-vascular bundle. b Strong expression of the GUS gene in stipules (marked with arrows), the promeristem without the GUS gene (marked with a large arrow), foliar primordium without the GUS gene (marked with a short arrow) and early production (low expression of the GUS gene) without IAA production (marked with a large short arrow) in the tip of a leaf primordium. c Gene expression in all active hydathodes (two are marked by arrows) of the leaf primordia. d Strong expression of the GUS gene in a marginal hydathode during development (marked with arrows) and a fibro-vascular bundle in differentiation (indicated by short arrows) with low activity of the GUS gene. e GUS gene expression at the base of trichomes (arrows) and two venules ending free (short arrows) associated with trichomes. f Reporter gene expression (marked with a short arrow) at the top of the venule that ends in the primordium of the developing lamina

• cellulose synthases by inducing de novo synthesis of certain types of proteins like citrate synthase, invertase, peroxidase, of co-enzymes, vitamins, etc. (Fig. 7.16). In the membrane, auxins interact with specific receptors ABP1 (auxin-binding protein 1) (low concentrations of the phytohormone triggers cell division). Biological significance. The functional range of auxin is very broad. Low doses accelerate growth having a catalytic role while larger doses halts the growth of roots and shoots. IAA is the main hormone of cell division and cambium activity, one of the hystogenetic factors and, in particular, of root formation by inducing true secondary meristems. It contributes to root differentiation, stimulating the formation of lateral roots and determines cell elongation. Physiologically, auxins trigger particular metabolic reactions, stimulating plant growth, seed germination and inhibiting aging processes in tissues. Tissues rich in auxin attract nutrients conditioning apical dominance and inhibiting lateral bud growth, adjust leaf falling, callus formation in tissue culture, fruit growth, slows down the aging process. IAA plays an important role in the phenomenon of tropisms and nasties. Ensures integration of plant organ activity. Auxins stimulate photosynthesis by increasing CO2 conversion into photosynthesis products and their subsequent translocation. Practical applications. Synthetic auxins are used as herbicides, they function as inhibitors at high concentrations. They are used for rooting, to obtain parthenocarpy, for switching sex in plants etc.

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7.6.2 Gibberellins Gibberellins are named after the fungus Gibberella fujikuroi, in which they have been identified for the first time (1926). From the chemical point of view, they are tetracycle diterpenoids. The symbol used for gibberellin notation is GA, equipped with a numeric index starting with 1 (GA1, GA2, GA3, …). There have been identified over 70 types of gibberellins, gibberellic acid 3 (GA3) is considered more active. Gibberellins are found in the free state and bound with glycosides.

Biosynthesis. Gibberellin predecessor is kaurene. In the chemical structure of both of them there is a common backbone—gibban, to which certain side groups are attached that determine their specificity. Thus each plant species has its own set of gibberellins (Fig. 7.17). Gibberellins precursors ent-kaurene and ent-gibberillan are synthesized in young leaves, in germinated seeds, apical buds, stem apices, etc. under the action of entkaurensynthase, an enzyme encoded by a nuclear gene (Le) and localized in plastids. In young leaves light stimulates gibberellins. The passive transport of gibberellins happens with the phloemic and xylemic flow (5–20 mm/h). Some authors claim that they migrate like organic metabolites and are accumulated in areas of growth. Mechanism of action. The mechanism of gibberellin action is interpreted as an intervention at the level of genes, inducing the de novo synthesis of α-amylase and protease. In cotton, the Ltp3 gene encoding the LTP3 protein has a maximum concentration during fiber elongation and maturation and occurs under the action of plant hormones. GA3 induces the expression of the mitochondrial gene analogous to orfH522 (Fig. 7.18) and the synthesis of the 16kD protein (Fig. 7.20) associated with cytoplasmic male sterility in sunflower. Exogenous treatment with GA3 does not alter the transcriptional profile in the cytoplasmic male sterility line (S+), but determines the appearance of a discrete fragment of 321 bp specific to the nucleotide sequence analyzed in the fertile line (F+). The PCR product (317 pb) obtained with specific primers for the actin gene sequence, used as positive control for reverse transcription reactions showed that cDNA synthesis and subsequent PCR amplifications occurred in all studied genotypes (Fig. 7.18). So the absence of amplification products of 321 bp in the

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Fig. 7.17 Gibberellin biosynthesis

fertile line and its appearance after application of gibberellins GA3 tells about the induced expression in the fertile line of a gene similar to orfH522 associated with cytoplasmic male sterility (CMS) in sunflower. Sequencing and comparison with the nucleotide sequence of orfH522 present in the EMBL database (EMBL/GenBank Accession X55963) showed 99–100 % homology with the mitochondrial gene sequence orfH522 selected for amplification (Fig. 7.19). Consequently, the exogenous application of GA3 during budding induces the de novo synthesis of a protein compound with the relative molecular weight of 16 kDa,

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Fig. 7.18 RT-PCR products obtained with primers specific for orfH522 (a) and actin (b). Total RNA was extracted from calathidium of fertile SW501 (F) and sterile SW501CMS (S) plants treated and untreated with GA3 (“+” “–”), M—marker 100 bp and 1 kb. (Duca et al. 2006)

Fig. 7.19 The nucleotide sequence of the sunflower mitochondrial orfH522 and the amplified sequence (I)

similar to that identified in CSM lines (Fig. 7.20). These results support the idea that GA3, causes male sterility by inducing the expression of a similar open reading frame, because the translation products are also identical. Biological significance. Physiological effects of gibberellins on plants are multilateral. Among the most important functions can be mentioned: • stimulates stem elongation in dwarf plants, so many dwarfism genes are gibberellin deficiency genes; • accelerates flowering in long day plants; • stimulates caryopsis germination in cereals, stimulates fruit growth; • determines changes in the photoperiod; • intervenes in ceasing bud dormancy. Brings seeds out of the dormant state and influences their germination by intensifying the formation of ribosomes and nucleic acids, but also by permeating membranes; • in the endosperm gibberellins are participating in endoplasmic reticulum development, cell wall degradation and synthesis of a large number of hydrolytic enzymes that catabolize seed reserves and the formed metabolites ensure embryo and seedling development (Fig. 7.21);

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Fig. 7.20 Electrophoresis of total protein pools from the leaves (a) and inflorescences (b) of fertile sunflower plants SW501 (F) and sterile SWS01ASC (S) treated and untreated with GA3; M—marker (10–100 kDa); “+”—variants treated with GA3. (Duca et al. 2006)

• among the enzymes induced by gibberellins are α-amylase, some proteases, acid phosphatase, β-gluconase, α-glucosidase and ribonuclease; • determines the sex switch in plants, causes parthenocarpy; • intensifies transpiration, photosynthesis and respiration; • shows synergism with auxins, due to its action on auxinoxidases; • by stimulating cell division, gibberellins control mitotic activity, activate enzymes responsible for phospholipid biosynthesis. Practical applications. Based on these properties, gibberellins have wide application in practice—they are used to stimulate tomato fruit formation, to stop dormancy in tubers, buds, seeds, etc.

7.6.3 Cytokinins This group of hormones include kinetin, benzylaminopurine, zeatin and isopentenyladenine—compounds ten times more powerful than kinetin and other natural or synthetic compounds with morphogenetic roles used in tissue cultures. Cytokinins are derivatives of adenine. Functional activity of cytokinins occurs in the presence of auxins.

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Fig. 7.21 The role of gibberellins in seed germination. 1 GA induces water imbibition of the embryo; 2 GA stimulates the production of α-amylase by aleurone cells; 3 Amylase cleaves starch reserves from the endosperm; 4 Carbohydrates fuel the growth processes of the embryo

Biosynthesis. Zeatin is synthesized from mevalonic acid and adenine. It was found that cytokinins are the result of degradation of nucleic acids and therefore, could serve as an indicator of the rate of DNA replication. In plants cytokinins exist in free and bound form. Bound cytokinins are synthesized in the cytoplasm and chloroplasts. It is assumed that they may be synthesized also in mitochondria based on their own DNA. This confirms the endosymbiotic theory organelle genesis. Transport. Very rich in cytokinins are green seeds, meristematic tissues, but the main site of cytokinin synthesis is the apical meristem, especially the root (Fig. 7.22), from where they are transported ascendantly (acropetal), passive with the flux of other metabolites. Mechanism of action. Cytokinins may impact the structural and functional status of the cell. Acting artificially with a synthetic analogue of cytokinin the following effects were observed: cell rejuvenation and restoration of damaged structures, an increase in the size of the nucleus, chromatin structure becomes more diffuse, mRNA biosynthesis is enabled, more ribosomes are formed as well as new grana and lamellae in the chloroplasts stroma, more cristae in mitochondria. All these changes demonstrate an increased functional activity of the cell by increasing the biosynthesis of RNA and protein, an increase in the amount of chlorophyll and also an increase in the intensity of photosynthesis (Fig. 7.23). Under cytokinin action activation of all types RNA in cut leaves (mRNA, tRNA, rRNA) occurs. Biological significance. Cytokinins exhibit the following physiological features: • stimulate cell division and germination of the seeds; • determine bud and shoot formation from undifferentiated callus in cultures in vitro; • delay senescence, increasing proteosynthesis and metabolic activity;

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Fig. 7.22 The gene for cytokinin biosynthesis. a Chloroplasts from seeds grown in the dark without cytokinins; b Chloroplasts from seeds grown in the dark with cytokinins, notice the formation of thylakoids Fig. 7.23 Cytokinin effect on germination of wild-type Arabidopsis grown in the dark

• • • • • •

mobilize and attract metabolic substances and minerals to the dominant centers; stimulate the exit from dormancy and neutralize apical dominance; activate callogenesis as a result of active cell division; activate differentiation of adventitious buds on stems and roots; involved in apical dominance, stimulate seed germination and induce anthesis; increase resistance to cold and toxic substances;

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• • • • • • • •

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together with auxins, activate formation of generative organs; activate enzymatic systems (nitrate reductase, isocitrate lyase, protease etc.); enhances RNA polymerase and chromatin activity; cytokinins increase the amount of polyribosomes; stimulate cell division, germination of seeds; stimulates the synthesis of RNA, DNA and proteins; delay aging; have a correlating action on lateral and apical buds.

Practical applications. Cytokinins stimulate opening of the buds, enhance leaves and cotyledon growth, have a revitalizing effect on aging leaves, have an influence on nutrients. Also they stimulate rooting of the cuttings and cause the transition of a greater number of vegetative buds into floral buds.

7.6.4 Abscisic Acid Is an inhibitor of growth with retardant action. It is present in all plant tissues. Abscisic acid is an antagonist of auxin, gibberellins and cytokinins. It accumulates in large quantities during autumn, in the period of transition to a state of latency, particularly in reaction to stress.

Has the ability to form glycoside by interacting with glucose, thus representing an inactive non-toxic form. ABA is also inactivated by hydroxylation in the endoplasmic reticulum. Biosynthesis. Is synthesized in mature leaves and fruits. ABA biosynthesis occurs via two pathways (Fig. 7.24): • from mevalonic acid → isopentenyl pyrophosphate → heranylpirophosphate; • by carotenoid and violaxanthine decomposition → xanthoxin → abscisic acid. It was found that induction of ABA synthesis occurs during genome reprogramming and synthesis of increased amounts of ABA-inducing polypeptides, of which lectins are more significant (especially agglutinins in wheat). Transport. ABA is transported from its synthesis location through the phloem and xylem to the parenchyma, the descendent flow being three times greater than the ascendant. Mechanism of action. The primary action is carried in the membranes of target cells which contain protein receptors specific for ABA, influencing the lipid phase

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Fig. 7.24 ABA biosynthesis

of the membranes, causing degradation of hydrocarbon bonds and of membrane hydrophilic groups. ABA influences the level of calcium in the cytosol by opening calcium channels (calcium is a secondary messenger in phytohormone signaling). ABA mechanisms of action determine plant response reactions: • immediate or • late. The most studied immediate response is stomatal closure induced by ABA, by altering the concentration of CO2 in intercellular spaces and the turgor pressure. ABA slows potassium ion accumulation by inhibiting the corresponding proton pumps in the plasmalemma (Fig. 7.25). It has an important influence on water transport, increasing membrane permeability. The late response is represented by the influence of endogenous ABA on transcription of genes into mRNA and cytoplasmic protein synthesis. Among the reactions of late response to ABA content increase under stress conditions are phenomena like RNA and protein synthesis inhibition. In a concentration of 1–10 μM ABA reduces RNA polymerase activity by 22–38 % in active chromatin, blocks the synthesis of α-amylase and protease, alters the activity of nuclear and

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Fig. 7.25 ABA action on stomata opening and closure

cytoplasmic protein kinase, chloroplast differentiation, synthesis of chlorophyll and enzymes photosynthetic apparatus (Fig. 7.26). In isolated chloroplasts ABA and cytokinins are inactive therefore we can say that their action is determined by the nuclear genome. Biological significance. ABA has the following properties: • contributes to the distribution of metabolites in plants, in case of repeating stress; • in wheat and soybean, stimulates the movement of photosynthesis products from the leaves into the ear; • accumulates sucrose in seeds, sweet fruits, reserve tissues of the roots; • promotes leaf detachment by forming the separation layer between the petiole and the peduncle; • induces the synthesis of shock proteins and of certain osmotins and dehydrins; • inhibits seed germination and stem growth; • contributes to oriented movements of the root in the soil; • has anti-gibberellin, anti-auxin, anti-quinine action; • maintains plant dormancy (latency); • increases plant resistance to stress factors; • prevents seed germination in the fruit. Practical applications. It is used to control the number of flowers and fruits per unit of surface to achieve the optimum yield in terms of quality and quantity.

7.6.5 Ethylene Ethylene (H2C = CH2)—a gas that is formed in plant organs, being an inhibiting hormone and an auxin antagonist. It was found in plants and fungi and does not

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Fig. 7.26 The axes of incubation depending on ABA concentration (Ried et al. 1990). The axes of hibernating and non-hibernating wheat seeds incubated for 28 h at 20 °C on agar-agar containing varying concentrations of ABA

occur in bacteria, algae, and animals. It is considered as the maturation and aging hormone, since it stimulates apoptosis. At low concentrations (0.04–1.0 μl) it shows strong morphogenetic effects. It was described in 1901 by Neliubov, which demonstrated the role of ethylene in inhibiting stem elongation, thickening and horizontal orientation. In the 20s of XXth century it was found that ethylene accelerates fruit ripening. Biosynthesis. Ethylene in higher plants is synthesized in the presence of light, from methionine, with 1 amino cyclopropan-1-carbonate (ACC) as precursor, which can serve as a transportation form. Transport. Ethylene diffuses freely through the intercellular spaces. Mechanism of action. Ethylene action on biological processes is very fast, which leads to the idea that like other phytohormones, ethylene has a signaling role. Biological significance (Fig. 7.27). • • • • • • • •

causes epinasties; delays growth and development; stimulates organ aging; accelerates fruit ripening and their detachment (Fig. 7.28); contributes to flower, bud and leaf detachment; blocks the growth of leaves, plant elongation, mitosis; slows down polar auxin transport and removes apical dominance; switches the direction of growth form the longitudinal one to the transversal one, contributing to thickening of the stems;

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Fig. 7.27 The role of ethylene in various physiological processes. (I) Germinating seed of Arobidopsis at 4 °C for 4 days and then transferred to dark for 72 h; a Wild-type genotype grown in the presence of ethylene at 10 μl/l; b Wild-type genotype grown without ethylene (Guzman et al. 1990); (II) Leaf detachment

Fig. 7.28 Involvement of ethylene in fruit ripening. (I) Model of ethylene receptor action. In the absence of ethylene, receptors (AR) actively suppress ethylene responses and fruit ripening. Before binding ethylene receptors become inactive (IR) and ethylene responses can be initiated. Mutated receptors (M) cannot bind ethylene and continue to actively suppress ethylene responses (Klee 2004). (II) Induction of fruit ripening in the presence of ethylene. (III) Lack of the ethylene receptor gene in tomatoes on the left

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• increases membrane permeability, stimulating respiratory contact between enzymes and substrates; • these effects are removed when the concentration of CO2 increases. Practical applications It is used to preserve fruits during winter. Other natural inhibitors such as the β inhibitor, phlorizin, coumarin, cinnamic acid, scopoletin etc., are poorly studied.

7.7 Photoperiodism and Yarovization Plant growth and development is determined by the interaction of the hereditary system and phytohormones, which is happens in certain environmental conditions in which the organism develops. An important role in plant development and in the transition from vegetative to generative stage have temperature and the duration of the day and night. Dependence on temperature is called vernalization (yarovization) and dependence on plant transition from flowering to reproduction is called photoperiodism. Vernalization and photoperiodism are formed in the process of evolution as specific adaptations that lead to flowering in the most favorable conditions. With these adaptations, the organism determines the flowering time. Photoperiodism is a specific plant reaction to the ratio between the duration of the day and the night (Fig. 7.29). After their response to photoperiodism plants are divided into 3 groups: (1) Long day plants—which are flowering faster if during early vegetation have long days (12 h)—barley, wheat, sugar beet. (2) Short day plants—which are flowering more quickly if at the beginning of the vegetation period have short days and long nights—beans, tobacco, cotton. (3) Neutral plants—sunflower. The reaction of plants to day length is called photoperiodic sensitivity. The higher the index is, the more difficult plants bloom or they do not bloom at all. Short day or long day plants bloom if they have a certain number of days with favorable light conditions—a continuous optimum photoperiod isn’t necessary. Vernalization (yarovizarea) is a complex process that occurs at low positive temperatures, needed for the buds and seeds to initiate reproductive processes necessary for flowering. Vernalization is a biological process specific to each species. For a large number of plants transition to the stage of maturity occurs only after exposure at low temperatures for defined periods of time (Fig. 7.30).

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Fig. 7.29 Photoperiodism and plant flowering

According to this factor, plants were divided into three groups: (1) Annual autumn plants starting their vegetation in late autumn, resist the winter as seedlings and the germinated seeds are vernalizated (wheat, barley); (2) Biennial plants, which enter the winter as large plants, are flowering in the next years and are vernalizated at an advanced stage of growth and development—rhizocarps, tubers (carrot, sugar beet); (3) Perennials—every season produce shoots or branches, which require low temperatures to move from the vegetative to the flowering stage. Development dependence on temperature is very obvious in biennial plants. It is well known that if you sow autumn wheat, rye, oats during spring, they grow better but do not pass the reproductive stage and do not form the ear. If seeds are exposed to low temperatures, being sown in the spring, they form the ear, blossom and form wheat grains.

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Fig. 7.30 Examples of plants that require vernalization (Amasmo 2004). a Brassica oleracea, b Hyoscyamus niger, c Arabidopsis

Yarovization does not cause a direct transition to reproduction, it only prepares it, causing certain physiological and biochemical changes in the centers of growth. There are plants that pass the yarovization stage only in the presence of leaves. The best temperature for winter wheat is 4–0 °C. Vernalization duration varies (35–65 days) and depends on the plant species. A prerequisite for vernalization is moderate humidity (40–45 % of seed mass), normal aeration and the presence of cells able to divide. Vernalization occurs in the embryo or the stem apical meristems, in young leaves, buds and shoots. Under the action of low temperatures in meristematic cells the isoelectric point of the proteins changes towards a more acidic one, increasing the amount of RNA. Yarovization processes are reversible. Under the action of high temperatures deyarovization occurs and meristematic cells lose their reproductive ability. After yarovization synthetic activity in plants reduces and the activity of hydrolytic enzymes increases. In some species of plants, during vernalization the amount of gibberellins in tissues increases. But the physiological and biochemical essence of vernalization are not established.

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7.8 The Influence of Exogenous Factors on Plant Growth and Development Other external factors affect plant growth and development and the speed of growth depends on the intensity of all physiological processes—nutrition (carbon dioxide and nutrients), water supply, energy and substance exchange. Temperature. Plant growth is possible in different temperature amplitudes. Early spring plants grow even at temperatures below 0 °C (wheat, peas at −20 °C). There are plants which start their growth at the limit of +50 °C. For each species, according to its characteristics and in particular, the geographical origin there are specific temperature limits within which they can grow and develop. For each species there are three important temperatures: (1) The minimum—which starts growth; (2) The optimal—providing the most favorable conditions; (3) The maximal—which stops growth. It was established that plants grow very intense during night. For many plants, alternating temperatures are favorable: very high during the day and low at night. This process was named by F. Vent, thermoperiodism. Mineral nutrition. The increase is significantly influenced by the amount of nutrients in the soil and, in particular, by the concentration of nitrogen. A high concentration of nitrogen, however, stimulates plant rapid growth, inhibits differentiation of organs, such as flower formation and possibly, blooming. A rich background of minerals contributes to the formation of abundant green mass, which is favorable for forage cultivation (so, fertilizers lead to a decrease in the abundance of fruit and seeds). The amount of water. During the process of growth an invaluable importance has the water supply. Lowering the quantity of ground water leads to lower concentration of water in plants, and this in turn leads to slow growth—reduced rate of cell division and, in particular, elongation. Light influences intensity and character of growth. Photosynthesis and organic mass accumulation occurs more intense in daylight and cell elongation is more intense in the dark. Light has a great influence on the process of organ formation. In plants grown in the dark chlorophyll “b” is not synthesized, so they have a yellowish tint and are called etiolated. Etiolated plantlets are characterized by a number of anatomical and morphological features—a simplification of the anatomy of the stem —there is a weak development of central cylinder tissues and mechanical tissues, therefore long thin stems form; leaves are reduced in size. Stem and root elongation in etiolated seedlings appeared during the evolution process because, in most cases, seed growth occurs in the soil, in absence of light and the lack of leaves and other mentioned peculiarities facilitate seedling passage through the soil. It is possible that stem elongation in the absence of light is due to the lack of growth inhibitors. During the dark phase many hormones, auxins. Disturbance of the auxins/growth inhibitors ratio causes irregular growth.

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Once the seedlings grow through the soil surface, internal and external changes occur. In the dark, in dicotyledonous plants, hypocotyl is upside down and protects the growing point from damage. Under the action of light it is straightening. In light stem growth slows down, leaf growth accelerates and they acquire their regular shape. Under the action of light the stem epidermis forms. These changes are caused by red light with a wavelength of 660 nm absorbed by the phytochrome pigment, which is a chromoprotein with molecular weight of 120,000 kDa. Phytochromes which absorb red rays are called red phytochromes (Pr), and those that absorb red rays with the wavelength of 730 nm, are called longwave red phytochroms (Prf). Phytocromes can be found in different plant organs, being involved in photomorphogenetic programs, including photoperiodic response in plants. All the processes controlled by the phytochrome system can be classified into two groups: • processes that under the influence of red light intensify—differentiation of the epidermis, anthocyan biosynthesis, seed germination; • processes that are inhibited—stem growth, hypocotyl elongation. The phytochrome system is evolutionary very old, and is also encountered in blue-green algae and heterotrophic organisms. Oxygen nutrition. Growing process require a lot of energy resulting from the process of respiration. Therefore the influence of O2 reflects on growth. Reducing the amount of O2 under 5 % retains growth. This is the result not only of disturbed energy balance, but also of the accumulation of products of anaerobic exchange (alcohol, lactic acid).

7.9 Plant Growth Movements—Tropism and Nasties Excitability and movement, which are essential qualities of living matter, are also present in plants. Various movements can be found in plants and they are based on the changes in the turgidity of different groups of cells and tissues. Plant growth movements are known from ancient times, being described by Teofrast that detected movements in Mimosa pudica and clover plants. Physiological mechanisms of growth movements have been explained by the 1960–1970 years, when Holodnâi and Vent developed the hormone theory of growth movements. At the basis of these mechanisms stay changes in phytohormone content. Plant movements are divided into: • passive; • active.

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Passive movements are favored by the presence of adaptations that allow entire plants or parts of their bodies to be moved by physical or biological agents from one place to another. They are produced by metabolic energy expenditure, as determined by physical and chemical factors (movement of pollen, seeds, etc.). Active movements are made by plants with the use of their own energy. Regardless of the structure in which they occur (inside plant cells or organs), active movements can be autonomous or induced. Autonomous movements are executed under the influence of internal factors, specific to the organism. These movements in higher plants are known as nutations. Induced movements are determined by the direction of action and intensity variations of some external environmental factors. Induced movements performed by the organs of fixed plants are called tropisms while the ones executed by plant organs under the influence of intensity variation of environmental factors are called nastic movements. At the cellular level both autonomous and induced movements are present. Among these movements ciclose movements, cytoplasmic currents and chloroplast movements can be highlighted. Active movements are accompanied by ATP and metabolic energy expenditure. Based on the above mentioned, we can deduce: • intracellular movements—movements of fixed plants (tropisms, nastic movements); • intercellular movements—movements of cytoplasm, chloroplasts, the nucleus. Tropisms are movements related to the unilateral direct action of a factor (light, gravitation). These are directed growth movements towards the stimulant. Movements oriented to excitation source are called positive movements, while those away from this source—negative movements. Depending on the stimulating factors that influence the physiological and biochemical processes from the protoplasm, tropisms are divided into: • geotropisms—caused by unilateral and directed action of the earth gravitational force. Stem growth via its apex is negative geotropism, root growth—positive geotropism; • phototropisms—caused by the unilateral action of light. Stem growth is positive phototropism and root growth—negative phototropism; • seismotropisms—the movement of plants towards an object (vines that cling to other plants, for example, beans) (Fig. 7.31); • chemotropisms—root growth towards the source of granular fertilizers, pollen tube growth through the stigma to the ovary (Fig. 7.32); • hydrotropisms—growth towards the source of water in the soil; • electrotropisms—passage of electric current through the organism of a plant induces bending towards the positive pole.

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Fig. 7.31 Examples of tropisms

Fig. 7.32 Pollen movement. Cytoarhitecture is maintained after treatment with incompatible protein-S. Images were recorded in the apical pollen tube after 1 min. (a) after 4 min. (b) after 6 min. (c) after 8 min. (d) of -S protein induction

Nastic movements represent growth movements that are determined by the diffuse action of excitatory factors. They are observed in plants with dorsoventral symmetry and cause two types of movements: epinasties—bending downwards and hyponasties—bending upwards (Fig. 7.33).

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Fig. 7.33 Nyctinastic movements in Mimosa pudica caused by touch. The yellow arrow indicates the place where the response is triggered

If growth is faster at the top of the organs, then movements are called epinasties. Such kind of movements cause floral buds to open. If the increase is more intense in the organs, the movements are called hyponastic movements. Depending on the factor causing nastic movements, we distinguish: • nyctinastic movements; • seismonastic movements; • termonastic movements. Nyctinastic movements are determined by the alternation of day and night. These are movements of flowers and leaves caused by changes in temperature and light. Seismonastic movement are the phenomenon of closure and opening of the petals in Mimosa pudica induced by touch. Termonastic movements represent growth movement caused by the diffuse action of temperature, opening and closure of tulip flowers depending on temperature.

7.10 Self-Regulation of Plant Growth and Development Plants are integral organisms and consist of 50–60 cell types, 12–15 types of tissues and 5–6 organs and specialized organ systems. Although they differ by their structure and functions during growth and development, they interact through conducting vessels forming a whole. In the growth and development processes regulation and self-regulation of all physiological processes occurs which ensures homeostasis (morphological, biochemical, genetic). Self-regulation represents all the systems and mechanisms that help maintain homeostasis in the fluctuating environmental conditions at the cellular level, based on the interaction of the: • genetic system; • enzymatic system; • membrane system.

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The interaction between organs is based on different systems and mechanisms, including: • phytohormone regulation; • trophic regulation; • electrophysiological control. At the organism lever autoregulation of growth and development is determined by: • dominant centers—points with high functional activity where the highest concentrations of plant hormones are found; • the phenomenon of polarity—through the anatomical structure, stem and root apexes are absolutely polar; • regulatory contours; • oscillations (biorhythms); • conducting system, which is achieved through conducting vessels. Growing body integrity as a universal biological property is determined by the polarity of biological structures, i.e. the gradients of chemical, physical nature and the anatomical morphological and physiological gradients, reflecting the specific orientation of structures and processes in space. All types of gradients are interdependent and interconnected. Polarity underlies mutual relationships between organs and various parts of the plant, contributing to self-regulation of physiological processes and is a result of internal correlative links based on the currents of substances—acropetal and basipetal. Polarity is a known phenomenon in the plant kingdom and is expressed by increasing or decreasing (gradual) ion concentration in the plant axis, metabolites, phytohormones, Rh, pH, osmotic pressure, enzyme activity, intensity of respiration etc. In radial stem and root tissues there are varying amounts of O2, CO2. Polarity is manifested both in morphological/anatomical structures and in the diversity of plant physiological properties. It is characteristic of all forms starting with the cell. The polarity of the cells is determined by the polarity of the macromolecules (proteins, nucleic acids) in the cytoplasm, which occurs as a result of different tasks, ionic composition, pH of the medium, the influence of the electrostatic field, etc. In higher plants polarization conditions, i.e. to form the main axis (shoot root) is created already in the ovary during ovule formation. Ovary polarization in the direction of micropyle is observed with the microscope. The ovary is oval, pear-shaped. The elongated face is directed towards the micropyle pole. In this part of the ovary there is a large vacuole that moves the nucleus towards the apex. The zygote is also polar and its division leads to the formation of two physiologically different cells—one of them (the apex) will form the stems and cotyledons, and the second (basal) will form the radicle. It is assumed that after the electric polarization in plasmalemma electrophoretic transport of lipoprotein components with positive and negative charges occurs. These components (ion channels, pumps, enzymes) are fixed at the cell poles with cytoskeleton microfilaments and microtubules, setting a primary polarization and generating the main axis of the plant body.

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After further division of the egg (the division plane is perpendicular to the polarization axis), daughter nuclei have different conditions which create the polarized cytoplasm. As a consequence, different genetic programs and differentiation paths may be established. The direction of cell polarization changes continuously along embryogenesis, during the formation of primordia in the apex, during the process of hystogenesis in the leaves, root formation in the pericycle. Each meristematic polarized cell can divide into two different or identical cells, in the first case there is division of differentiation, and in the second—one of multiplication. An important role in creating polarity in higher plants have phytohormones which create concentration gradients in various plant organs. Heteroauxin and its physiological analogues β-indolyl acetic acid and α-naphthylacetic acid, substances which actively influence growth and development of the root. Acropetal gibberellic acid acts on the acropetal current leading to the stimulation of sprouting and development of shoots. Auxins stimulate cell growth in areas of the basal shoots and gibberellins stimulate apical bud areas. IAA, by circulating in a polar manner, accumulates at the bottom end which induces morphological and genetic programs of root formation. Thus, in plants, opposite ends of the axis differ essentially, for instance in grapevine cuttings by the formation of roots on the morphological bottom and of shoots—from buds exposed on the morphological top. Phytohormones do not act directly on basipetal and acropetal currents of substances. They lead to the intensification of growth processes in the locations where they are applied and in this manner the ability forms in these centers to attract substances that contribute to the polarization of the plant body. Biopotentials in higher plants are polar and are the expression of metabolic gradients between dominant centers—apical and root portions corresponding to the transport of water and mineral salts. In a leaf, the apical electrode is usually positive compared to the basal electrode (≈100 mV), and the difference can also show variations between day and night. The inner side of the leaf is negative compared to the top surface; between the illuminated zone of a leaf and a zone which stays in shadow, a potential difference of 50–100 mV appears in a few minutes (the shaded portion is negative). Periods of light and dark can be alternated several times with the same result. Along onion root cells there are dipoles placed in series. The root tip is positive compared to the middle part and they are negative in comparison to the core. The magnitude is of the order of tens of millivolts. Probably in the middle zone of the root cell dipoles change orientation (along the roots of beans spontaneous potential oscillations were revealed with small amplitude (<2.5 mV) and with a period of a few minutes). They are due to fluctuations in time of the currents of Na+ and K+, as a result of self-regulation of the cellular osmolarity.

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Glossary Chemotropism Plants growth movements, oriented, caused by the action of chemical stimulants (positive or negative chemotropism). Cytokinins Products of purines with an action similar to auxin, activates meristematic cell multiplication and seed germination, resuming dormancy, causing bud differentiation. Predominant in flowers, fruits and seeds. Geotropism Stem, root and leaf orientation, caused by unilateral action of the terrestrial attraction force. Root positive geotropism is conditioning growth towards the center of the earth, and stem negative geotropism—from the center of the earth. Growth curve An S-shaped graphical curve, describing growth: length increase, volume and mass of the cells, tissues, organs or entire body or even population. We can distinguish the initial, acceleration, stagnation and the stationary phase. Photoperiodism Plant specific reaction to day and night duration (photoperiod). It manifestes via morphological and physiological peculiarities of growth and development in connection with the ontogenesis adjustment to seasonal changes in climatic conditions. Phototropism Organ unilateral orientation expressed by unidirectional growth and bending towards light (stem positive phototropism) or from light (root negative phototropism). Dormancy The state of the seed bud or organ of a plant when growth is interrupted, but morphogenetic processes may be ongoing. Biological dormancy is caused by the accumulation of growth inhibitors and forced dormancy is caused by unfavorable environmental factors. Retardants Organic compounds with different chemical structure that slow down stem and shoot growth. Among retardants are: CCC, B-995, which inhibits foliar crown growth in fruit trees and stimulates fruiting.

References Amasmo R (2004) Vernalisation, competence, and the epigenetic memory of winter. Plant Cell 16:2553–2559 Chaylahyan MX et al (1962) Terminologiya rosta i razvitiya vysshih rasteniy, 96 pp Duca M, Port A, Orozco-Cardenas ML, Lovatt C (2006) Mecanisme moleculare ale androsterilității ereditare şi induse la floarea-soarelui. Buletinul AŞM 298(1):86–93 Gilbert SF (2000) Developmental Biology, 6th edn. 709 pp Guzman P, Ecker JR (1990) Exploiting the triple response of Arabidopsis to identify ethylenerelated mutants. Plant Cell 2:513–523 Ivanov BB (1974) Kletochnye osnovy rosta rasteniy, 223 pp

References

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Kefeli VI (1984) Rost rasteniy, 175 pp Klee HJ (2004) Ethylene signal transduction. Moving beyond Arabidopsis. Plant Physiol 135: 660–667 Molotovskii GX (1961) Polyarnost’ razvitiya rastenii. L’vov, 262 pp Nikell Dzh (1984) Regulyatory rosta rasteniy. Primenenie v sel’skom hozyaystve, 192 pp Nikolaeva M (1967) Fiziologiya glubokogo pokoya semyan, 207 pp Ried JL, Walker-Simmons MK (1990) Synthesis of Absisic acid-responsive, heat-stable proteins in embryonic axes of dormant wheat grain. Plant Physiol 93:662–667 Sheveluha VS (1980) Periodichnost’ rosta sel’skohozyaystvennyh rasteniy i puti ee regulyatsii, 456 pp Sinnot Je (1963) Morfogenez rasteniy, 603 pp Uoring F, Fillips I (1984) Rost rasteniy i differentsirovka, 512 pp

Chapter 8

Plant Biorhythms

Abstract Biorhythms represent periodic fluctuations within a range of certain biological processes or phenomena which are endogenous in nature, being determined and regulated by genes but are also closely related, adjusted and responsive to external environmental factors. The internal timing mechanism is called biological clock. Oscillation periods can vary in duration in which case biorhythms can be diurnal (succession of light/dark cycles), monthly, seasonal, annual, etc. Various biological traits can be subjected to rhythmic fluctuations for instance metabolism intensity during the year as well as during day and night, the quantity of photosynthesizing pigments in the leaves during the year, the spatial orientation of the leaves during day and night. The biological response of the plants to day/night fluctuations that take place in the 24 h is called “photoperiodism” and has a crucial importance in controlling plant genetic programs. It is the relative duration of day and night that is the main factor that controls the transition from vegetative growth to flowering in many plants. According to this, plants are classified into short-day, long-day, neutral plants, etc. The circadian rhythm is characterized by the following parameters: an endogenous duration of approximately 24 h (the fluctuation period) “trained” in order to fit the duration of an astronomical day, a certain amplitude of the fluctuating parameters, the dependence of the period on light and temperature, the maintenance of the rhythm in the absence of the input signal. The molecular mechanism of the circadian clock in mammals, insects, fungi, cyanobacteria and plants involves a genetic circuit with negative feedback characterized by a 24h rhythm and by positive and negative transcriptional regulation. These “clock associated genes” are maintaining the molecular oscillation that controls all other traits of the circadian rhythm. To sense fluctuations in the quantity and quality of light plants use specific photoreceptors like phytochromes, which respond to light from the red and infrared spectrum and cryptochromes, sensitive to UV/blue rays.

© Springer International Publishing Switzerland 2015 M. Duca, Plant Physiology, Biological and Medical Physics, Biomedical Engineering, DOI 10.1007/978-3-319-17909-4_8

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Historical Background 1814—J. Vire first used the term “biological clock—horloge vivante”. 1926—U. Garner and G. Allard introduced the notion of photoperiodism. 1932—E. Bunning described a hybrid of beans which differed in the duration of the circadian rhythm. 1958—E. Bunning published the first monograph in the field of biological rhythms. Brief Updates An important role in the regulation and coordination of the plant biorhythms is played by the phytochrome pigment. The phytochrome is a chromoprotein, formed by an apoprotein with a relative molecular weight (Mr) of 250 kDa, which consists of two similar subunits of 125 kDa each, linked by disulfide bonds. The protein is linked to the nonprotein part, a tetrapyrrole pigment containing molybdenum. Genes that are involved in the synthesis of phytochromes in plants have been isolated recently. In Arabidopsis thaliana, characterized by a relatively small genome (only 120 mln. bp) a family of five genes was detected (PHYA, PHYB, PHYC, PHYD and PHYE) while in tomato plants—seven genes involved in the biosynthesis of these compounds. The phytochrome A gene is expressed more intensely in the dark compared to genes encoding other phytochromes, but transcripts and proteins originating from the later are more stable.

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A spectacular breakthrough in identifying the expression pattern of these genes was obtained by using transgenesis with the GUS reporter gene (encoding enzyme β-glucuronidase), which determines the blue color of the tissue in which the gene is expressed. Thus, the phytochrome encoding genes were fused with the GUS gene under the control of a single promoter (which leads to the expression of these genes at the same time). The results have shown that the gene is very active in young, undifferentiated tissues of the stem and roots and participates in gene activation, chloroplast division, flower formation etc. Phytochromes also play an important role in regulating the synthesis of proteins involved in photosynthesis.

8.1 Classification and Mechanisms of Biological Rhythms Biorhythms are one of the ways in which organism integrity is manifested, representing a periodic fluctuation within a range of certain biological processes or phenomena, a controlled chronobiological variation. The notion of rhythm includes the entire set of processes and transformations based on self-regulating mechanisms and systems that contribute to a harmony, a strict organization of events that occur in nature. Biological rhythms are characteristic of all levels of organization of the living matter—from the molecular and submolecular to the biospheric, which shows that biorhythmicity is a universal property of living systems. Biorhythms are recognized as adjustment mechanisms that maintain body homeostasis, dynamic equilibrium and adaptive processes in living systems. Biological rhythms are endogenous, being determined and regulated by genes, but are also closely related to external environmental factors. The temporal organization of living systems happened during the evolution of the organic world in a certain manner. Biological rhythms are found in all homeostatic control systems, allowing organisms to behave as complex entities, integrated in the multitude of influences exerted by the external environment: diurnal, monthly, seasonal. Particular attention should be paid to the fact that the very existence of rhythmic changes in nature contributed to the evolution of organisms, being reflected in certain aspects of phylogenesis. One of the most important factors that dictates periodicity phenomena is the rotation of the Earth around its axis and around the sun which reflects on the succession of light/dark cycles, weather, droughts, floods etc. Besides this contributing to the rhythmicity of life in ecosystems can be the seismic and volcanic activity, or other local phenomena. Altogether, these factors create a rhythm which is strongly intertwined with the life cycle of an organism or species. Over the years scientists have been concerned with the idea of classifying biological rhythms.

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For example, according to Haliberg one can distinguish: • • • •

circadian rhythms (from lat. circa “approximately”; dies “day”); circaseptal rhythms (weekly); circalunar rhythms (monthly); circannual rhythms (annual). According to other classifications there are:

• ecological rhythms—monthly, seasonal, solar rhythms (11 years), diurnal; • physiological rhythms—the bioelectric activity of various organs. According to their duration, biorhythms can have: • a short period, on the order of seconds or minutes, such as the exchange of ions and water across cell membranes. The movement of the cytoplasm is characterized by oscillations lasting a few minutes. • a long period, on the order of days, months, which represents the sequence of events or quantitative changes regulated at the cell, tissue, organ level. For instance a dependence of the circadian rhythm on the phase of plant development was demonstrated. The circadian rhythm behaves as an oscillator capable of self-regulation and the external environment acts as a synchronizing agent. Recent data lead to the idea that biological rhythms are controlled by cellular membranes, based on such processes as: • regulation of ion transport in the cell; • subcellular compartmentalization; • regulation of energetic metabolism at the level of plastidial and mitochondrial membranes. Over many years biologists have been concerned about the origin of biological rhythms, trying to understand if biological rhythms occur only under the action of environmental factors, cosmic rays, Earth’s magnetic field. After a series of experiences, involving transportation of organisms into different regions of the globe and environments (the South Pole, the cosmic orbit etc.) it has been found that, in addition to external factors, biological rhythms are controlled by endogenous factors (genetic), which have formed under cyclic fluctuations of the environment (Fig. 8.1). The internal timing mechanism is called “biological clock”. Most of the processes of growth and development of living organisms, including plants, have a rhythmic character. Different biological traits may be subjected to rhythmic changes: orientation of molecules, ion concentration, growth shape etc. There are several hypotheses on the endogenous mechanism of the biorhythms: (1) The “chronon” hypothesis (Epe and Tracco), which states that there is a “chronon”, i.e. a DNA sequence considered as the material substrate that controls biorhythms. In support of this hypothesis comes a series of studies

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Fig. 8.1 The circadian system in Arabidopsis and its relationship with the CO flowering gene (Hayama and Coupland 2004). The model of the circadian clock is as follows. Phytochromes and cryptochromes receive light and are involved in resetting the circadian horologe. ELF3 and ZTL are intermediaries between the photoreceptors and the circadian clock. TOC1/ELF4 and LHY/ CCA1 form a negative inverse relationship with circadian oscillators. LHY/CCA1 put in action negative regulators for TOC1 and ELF4 which positively regulate LHY/CCA1 transcription. Oscillating functions determine the transcription stage of CO, which is a key intermediary gene between the circadian clock and flowering. CO transcription is regulated by FKF1 and GI, whose transcription is controlled by the circadian clock. The FKF1 protein is controlled directly by light, and this fact allows to increase CO transcription during long day conditions. The CO protein is also directly activated by light, and this allows CO to generate a long day signal and to activate the flowering time gene—FT to promote flowering especially in conditions of long days. Circadian Clock Associated 1 (CCA1), Late elongated hypocotyl (LHY), Timing of Cab Expression 1 (TOC1), Early Flowering 4 (ELF4), Early Flowering 3 (ELF3), Zeitlupe (ZTL), Flowering-time genes Constants (CO), Flowering Locus T (FT), Flavin binding, Kelch Repeat, F-Box (FKF1), Gigantea gene (Gl)

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showing that the use of actinomycin D, which blocks polypeptide synthesis by ribosomes and controls the status of the neural membrane of the abdominal ganglion in Aplysia, leads to the disruption of the rhythmic activity of the nerve cells linked to day and night cycle. RNA synthesis coincides with a certain time of the day and reflects the lifestyle of the animal. Thus, in nocturnal rodents, RNA synthesis in liver cells is more intense in the morning while protein synthesis—during the first half of the night. (2) The membrane hypothesis according to which the cyclicity of processes is related to the state of lipoprotein membranes and their permeability to K+ ions. The membranous structures of the cell possessing receptor properties, are in control of biorhythms dictated by temperature and photoperiodism. (3) The hypothesis of the multioscillatory model of biorhythms. According to this hypothesis, a central “pacemaker” may operate in the multicellular organism which imposes its own rhythm to all the other systems that are unable to generate their own oscillatory processes. For example, the epiphysis is considered as one of the central controllers of rhythm, by releasing melatonin, which is connected to photoperiodism. According to several authors, the epiphysis has a proper biological clock whose timing is in correlation with external factors.

8.2 Biological Rhythms in Plants A characteristic property for plants besides the bipolar axes system is the typical alternation of nodes with internodes, of leafs with lateral buds etc. These metamers develop consecutively and determine a structure that optimizes the photosynthetic activity of leaves. It should be mentioned that plants are characterized by a certain rate by which they generate new leaves, nodes and internodes and which has been called the plastochron, so that the physiological rhythm in the apical bud of the plant (where the meristematic tissue is located) turns into a spatial pattern. For instance, the presence of oscillatory processes can be observed in the structure of the plant corm. Presence of regularity can be noticed in the layered arrangement of the starch grains, cell walls and annual rings, in the very complicated configuration of leafs, flowers, in the complex structure of the inflorescences etc. (Fig. 8.2). It is assumed that, in sunflower, oscillations with a certain periodicity are the phenomenon responsible for the helical arrangement of leaves on the stem and the helical arrangement of flowers on the calathidium. After the periodical activity of the cambium, which in temperate zones is a seasonal phenomenon, concentric layers are forming in the secondary phloem and xylem, which correspond to a year and are called annual rings. The width of these layers varies depending on the climatic conditions for that particular year, namely, the amount of light, temperature, rainfall, soil moisture, the duration of the growth season etc.

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Fig. 8.2 The temporal pattern of sunflower flowering

In the reserve tissues of different organs, especially in tubers, bulbs, rhizomes, in the amyloplasts carbohydrates are deposited in the form of starch. Stratification has a certain periodic pattern and is a result of intermittent deposition of starch within the hilum. All organisms have a biological clock, starting from unicellular organisms and ending with the humans. This is also true for plants. Periodic movements of the leaves towards the sun, opening and closing of the flowers, their movement, the pattern of photosynthesis shows the presence of a biological clock. Each species of flower wakes up and opens its petals at a strict time. Some species of plants open their petals at night (Fig. 8.3). The swedish botanist Carl von Linne, as early as in the eighteenth century has “built” a “floral clock” for several plant species. The rose for instance opens its flowers at 4–5 in the morning and closes them at 19–20; the chicory, respectively at 4–5 and 14–15; the poppy at 5 and 14–15; the dandelion at 5–6 and 14–15; the potato at 6–7 and 14–15; the flax at 6–7 and 16–17; the white lily at 7–8 and 18–19; the marigold at 9 and 15–16; the surelle opens at 9–10 and closes at 17–18; the coltsfoot at 9–10 and 17–18. The Intensity of many processes in plants change within the 24 h period— photosynthesis, respiration, transpiration, transport of metabolites. In the bark of young shoots of blackberry a circadian regime for florigen content (a biologically active substance) was detected, whose concentrations vary according to the phases of intensive shoot growth and flowering. There are rhythmic changes also in the sensitivity of the organism to the destructive factors of the environment. Study of the circadian and circannual rhythms of plant cell resistance to the action of various kinds of irradiation revealed that the lethal doses of ultraviolet rays for the cells of the upper and lower epidermis

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Fig. 8.3 Flower clock by Carl von Linne (Moore-Ede et al. 1982)

in onion leaves or in certain moss species changes 1.5–2-fold over a period of 24 h. Onion epidermal cells showed a maximum resistance to irradiation at 12 in the noon and a minimum—at 8 am, which correlates with changing nucleus size during the 24 h cycle. It was also found, that plant leaves also change spatial orientation several times a day (as, for example, Oxalis leaves which close during the night). One hypothesis says that this protects them from moonlight and thus prevents photoperiodic reactions. Another hypothesis, proposed by Darwin 100 years ago, assumed that leaf bending protects them from overcooling during the night. In many plants the leaves are positioned perpendicular to the stem and to the sun rays while at night—parallel to the stem. These “falling asleep” movements may be recorded on a cylinder which is rotating by using a well adjusted system consisting of a lever attached to the leaf by a string, a brush. In many plants, such as the bean (Phaseolus vulgaris), leaves continue the movement for a few days, even at lower illumination. The persistence of this circadian rhythm demonstrates its stability and manifestation even in poor lighting conditions. The biological response of the plants to day/night changes that take place in the 24 h is called “photoperiodism”. Experiments clearly demonstrate that a decisive regulating factor of the transition from vegetative growth and development to the reproductive phase for many species of plants, is given by the relative length of day and night. There are several types of photoperiodic flowering reactions and, depending on the reactions, there are several groups of plants: • neutral, which do not exhibit photoperiodic sensitivity; • long day plants, that bloom at longer day durations than the critical one. These species bloom in conditions of continuous illumination;

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Fig. 8.4 The photoperiodic characteristic of flowering (Eriksson and Millar 2003)

• short-day plants, which flower also in the presence of a dark period longer than the critical period of day/night alternation (Fig. 8.4); • plants, which, in order to reach the reproduction stage, need a long day duration, then a short one; • plants that are flowering faster if they first pass through a period of short days, followed by long days. There is also the qualitative photoperiodic response of plants with photoperiodic control, which determines the flowering of plants (short-day or long-day) in unfavorable lighting conditions. The photoperiodic response can be suddenly altered under the influence of other environmental factors. It should be noted that photoperiodism which influences plant growth, ultimately regulates also the intensity of photosynthesis.

8.3 Circadian Rhythms in Plants Eukaryotic organisms have adapted to the day/night cycle of 24 h by developing the circadian clock that controls many aspects of metabolism and physiology. The plant circadian clock is maintained by the light and temperature signals of the environment which is reflected in a complex relationship of rhythmic control elements and environmental signals.

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Thus, the role of the circadian system in the signaling network is that of an interface between the environmental factors and the internal programs. The circadian clock evolved as an adaptation to the 24h rotation of the Earth around its axis and the fluctuations of light intensity and temperature that accompany it. Photoautotrophic organisms must be exposed to sunlight in order to carry photosynthesis. Thus, all plants are subjected to the day/night cycle, except for the seedlings that germinate in the soil or the seedlings that grow and develop in the subpolar regions of the globe. The circadian system allows organisms to anticipate these cycles by detecting light or heat levels. The 24h biological rhythm characterizing the circadian clock is not a direct response to external factors and persists even in constant environmental conditions, with a periodicity that often differs from 24 h. The circadian system retains information from previous days. Rhythmic processes are similar for all organisms. Molecular research on circadian rhythms in plants have advanced mostly in Arabidopsis thaliana, although this physiological phenomenon was also investigated in other species, among which could be mentioned Ipomoea nil(Pharbitis nil), Kolanchoe and Phaseolus. The circadian rhythm is characterized by the following parameters: an endogenous duration of approximately 24 h (the fluctuation period), a quantitative extent in the fluctuation of the parameters (amplitude) which is also dependent on external stimuli, the dependence of the period on temperature, the presence of rhythmicity in the absence of the input signal (Fig. 8.5). Microarray analyzes showed that at least 6 % of Arabidopsis genes are expressed rhythmically, with expression peaks at all stages throughout the day and night. Gene expression patterns produce an impact on the physiological processes in plants, some of which are obvious (like “sleep movements” of leaves in legumes), while others less so. In many cases, the genes that affect a particular pathway or process are expressed during the same phase. Multiple genes encoding the biosynthesis of phenylpropanoid enzymes have maximum RNA levels before dawn,

Fig. 8.5 Characteristic of the circadian rhythm (Somers 1999)

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probably in order to allow accumulation of photoprotective flavonoids before sunrise. A large amount of rhythmically regulated genes may be indicative of a response to environmental stress. Rhythmic expression of these genes in anticipation of expected environmental changes act to prepare the plant to withstand stressful environmental conditions. Photoperiodism is a special case in which the circadian rhythm is combined with the transmission of light signals. The photoperiod sensor allows plants to respond to the annual cycle of day length by developing flowers, tubers or buds able to resist in frost in the appropriate season. Correct “training” is crucial for photoperiodism. General physiology indicates that the essential difference between plants which bloom during long days and those that bloom during short days lays in different training of their photoperiodic rhythm.

8.4 The Molecular Mechanism of the Circadian Clock The molecular mechanism of the circadian clock in mammals, insects, fungi, cyanobacteria and plants includes a genetic circuit with negative feedback involving a 24h rhythm acting at the level of positive and negative transcriptional regulation. These “clock associated genes” are maintaining the molecular oscillation that controls all other circadian rhythms. Any factor that involves the circadian clock has to affect the expression of at least one of these components. Molecular components of the biological clock are specific to each taxon, so common architecture appeared at least four times during evolution, which ensures a rich basis for comparative research. Oscillatory components from Arabidopsis are represented by two small gene families, which are based on the LHY protein (late elongated hypocotyl) and CCA1 (Circadian Clock Associated 1) that bind to DNA and proteins like TOC1 (timing of cab expression 1) that act as regulators of the pseudo response. It is believed that genes LHY and CCA1 are rhythmically expressed reaching the circadian maximum approximately at sunrise and are rapidly induced by sunlight. Related proteins are produced during the course of the next 2–3 h, inhibiting transcription from promoters starting with the promoter of TOC1. As the LHY and CCA1 protein levels decrease towards the end of the day, the abundance of gene TOC1 RNA increases and is maintained until midnight, contributing to TOC1 protein synthesis, which may be involved in the transcriptional activation of the CCA1 and LHY genes (Fig. 8.6). Overexpression of the CCA1 or LHY genes disturbs all tested rhythms, with a possible exception represented by the rhythmic expression of the early flowering gene ELF3 (Early-flowering 3). The LHY and CCA1 activation mechanisms are still unknown, but require the presence of at least three genes that are expressed approximately during the same phase, like TOC1. For example, ELF4 encodes a polypeptide that consists of 111 amino acids with no apparent homology to other known genes. Other genes like TIME FOR COFFEE (TIC), regulate the expression

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Fig. 8.6 The scheme of the circadian rhythm in Arabidopsis (Onai and Ishiura 2005)

of LHY, CCA1 and TOC1 and profoundly affect the circadian system, but they have not yet been cloned and sequenced.

8.4.1 Environmental Signals Involved Nature provides a complex set of signals during the day/night cycle, including changes in temperature, light quantity and quality. Thus, the combination of signals during the day/night cycle reinforces the precision of biological clock functioning and maintain its correct period. If the circadian clock was lagging behind, the biological processes would occur later than during certain optimal environmental conditions. By contrast, a faster circadian clock would be initiating the rhythmic processes too early. A constant signal, for instance a short light pulse transiently affects oscillatory components and, subsequently, the length of the period resulting in either clock acceleration or delay.

8.4.2 Temperature Circadian rhythms are also reflected in varying temperatures during the day/night cycles. The role of temperature in manifestation of the biological rhythms is eloquently represented in Arabidopsis, where, depending on the day or night, temperature values have a difference of only 4 °C or even less.

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8.4.3 Light Cellular components involved in signal perception and transduction in plants, including phytochrome and cryptochrome photoreceptors, contribute to the induction of biological rhythms. Phytochromes (phy) respond to light from the red and infrared spectrum, while cryptochromes absorb UV/blue rays. Arabidopsis has five phytochrome genes: PHYA-PHYE (phytochromes A, B, C, D, E) and two cryptochrome genes: CRY1 and CRY2 (cryptochrome 1 and 2). Using mutants of these genes allowed the in vivo modeling of different types of circadian rhythms. Arabidopsis plants in constant light (“standard” laboratory conditions with white fluorescent light of about 100 mmol/m2s) have a period of about 24 h, while after several days spent in the dark, the period may exceed 30 h. All major photoreceptors conduct signals to the clock system, causing a shortening of the period under the influence of red light (phyA and phyB) and under the influence of blue light (cry1 and cry2). It was noticed, that the curve of the response phase is similar in various plants. A pulse of light during the afternoon causes reduced alteration of the period. In the evening, the presence of light contributes to the delay of the timing while, in the morning, it accelerates it. In this case there is a well noticeable transition from major delays to big leaps in the middle of the night. Many of the biological rhythms revealed in Arabidopsis (for example, the expression of the CAB chlorophyll a/b binding gene) are associated with photosynthesis and rapidly lose the extent and intensity in the dark.

8.4.4 The Molecular Targets of Light Signaling Exposure of the plants to light affects one of the oscillatory components, which is to reactivate the clock. Several target-mechanisms involved in the initiation of biological rhythms have been described in plants. The first mechanism can be determined by the involvement of PIF3 (phytochrome-interacting factor 3). PIF3 is a protein that binds to DNA, including to the G-box sequence of the CCA1 and LHY promoters, which is present in several genes activated by light. In enacting biological rhythms in plants proteins from the ZEITLUPE (ZTL), Flavin-binding-Kelch-F-box (FKF) and LOV-Kelch 2 (LKP2) families might also be involved. They contain a domain related to Period-ARNT-Sim (PAS), which binds with the flavin chromophore in phototropin photoreceptors. ZTL mutant phenotypes are light dependent, suggesting a possible photoreceptor role. The ZTL protein has the potential to interact both with phyB, and cry1, which may cause indirect dependence on light.

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Fig. 8.7 Induction of flowering in Arabidopsis depending on day length (long day and short day) (Hayama and Coupland 2004)

8.4.5 Rhythmic Regulation of Light Signaling Four genes of the pseudo-response regulator in Arabidopsis (called PRR) homologous with TOC1 are rhythmically expressed, in a single sequence every 2–3 h from sunrise until sunset, PRR9-PRR7-PRR5-PRR3-TOC1. PRR9, PRR5 and TOC1 are characterized by light-dependent effects during the circadian period. The three PRRs were linked with light signaling. Thus PRR9 expression is activated by light and inhibited by TOC1 overexpression. PRR7 was

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identified as a change in the phytochrome signal during hypocotyl elongation. PRRS can interfere with the phytochromes, altering their signal either via PIF3, either by other parallel paths. Photoreceptor genes PHY and CRY are targets of circadian regulation at the level of RNA (transcript) abundance. phy and cry photoreceptors are modified by posttranslational phosphorylation followed by nuclear translocation. Thus, plant growth and development are based on periodic, oscillatory processes with circadian rhythms caused by external factors such as thermoperiodicity, light intensity variation etc. The character of circadian periodicity for various species of plants depends largely on the morphological and physiological structure, stage of development, the level of ecological adaptability to the conditions of growth (Fig. 8.7).

Glossary Biorhythm Biological phenomena that occur repeatedly at regular, genetically conditioned intervals characterized by a certain period of time; “biological clock”. Florigen The flowering hormone that accumulates in the apical meristemes of the stem and which alters the physiology of the growth centers leading to their transition into a floral meristeme. Photoperiodism The ensemble of plant responses to variations in the duration of the day. Hilum The scar on the outer surface of the seed which marks the place of its former attachment to the ovary wall. Metamer Each of the elements similar in structure that form the body of an organism. Plastochron The phenomenon of rhythmic emergence of leaves, nodes and internodes along the stem.

References Agazhdyan N (1987) Nauka o fiziologicheskih i biologicheskih ritmah/Uspehi fiziologicheskih nauk, vol 18. pp 80–104 Ashoffa J (1984) Biologicheskie ritmy, vol 1. Mir, 414 p Duca M (1997) Sisteme şi Mecanisme de Autoreglare la Plante. Chişinău, U.S.M., p 99 Enciclopedia tânărului naturalist (1988) Chişinău, Ed. Pedagogică, p 43 Eriksson ME, Millar AJ (2003) The circadian clock. A plant’s best friend in a spinning world. Plant Physiol 132:732–738

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Hayama R, Coupland G (2004) The molecular basis of diversity in the photoperiodic flowering responses of arabidopsis and rice. Plant Physiol 135:677–684 Makarov V, Poznyakova V (1989) Nauka o biologicheskih ritmah, sostoyanie, problemy, perspektivy. Biologiya v shkole, nr. 6, pp 5–10 Melnic (1985) Metronomul biologic. Chişinău Moore-Ede MC, Sulzman PM, Fuller CA (1982) The clocks that time us. Harvard University Press, Cambridge Naumov SP (1989) Zoologia vertebratelor. Chişinău, Lumina Onai К, Ishiura M (2005) PHYTOCLOCK 1 encoding a novel GARP protein essential for the Arabidopsis circadian clock. Genes Cells 10:963–972 Polevoy VV (1982) Fitogormony. Leningrad, pp 69–74 Polevoy VV, Salamatova TS (1991) Fiziologiya rosta i razvitiya rasteniy. Leningrad, pp 158–161 Reyvn P, Jevert R, Aykhorn S (1990) Sovremennaya botanika, vol 2. M. Mir, pp 119–123 Somers DE (1999) The physiology and molecular bases of the plant circadian clock. Plant Physiol 121:9–19

Chapter 9

Elimination of Substances in Plants

Abstract Plants lack a well-differentiated excretory system and the elimination of substances is performed by morphologically identical cells and tissues. Compared to animals, they do not feed on protein and as a result, the small amounts of ammonia formed in the metabolic processes are eliminated by simple diffusion. Plants remove both inorganic substances (mineral ions) and a big number of primary and secondary organic metabolites (monosaccharides, polysaccharides, amino acids, proteins, waxes, terpenes, alkaloids, volatile organic compounds—propanol, acetaldehyde and so on). Excess water can also be eliminated from plants by means of phenomena like tearing and guttation (in addition to transpiration). The eliminated substances can be final products of metabolism—this process is called excretion, or substances that participate in metabolism, in which case it is considered secretion. In contrast to the former the last is carried with metabolic energy expenditure. The secretion of the components is carried out at the level of: • the cell wall (deposition of lignin, suber, salts of calcium, silicon dioxide, carbonates, sulfates but also of the cutin and waxes by leaf epidermal cells in the process of cuticle formation, representing at the same time an important aspect of cell and plant organ morphogenesis); • specialized secretory structures (nectary glands, idioblasts, glandular hairs, osmophores); • collecting channels that store substances inside the plants which can only be released by mechanical lesions (resins, arabic gum); • many organic substances and metabolic residues are deposited in dead or detachable plant tissues. The variety of substances secreted by plants is great. These can be substances involved in trophic relationships within the ecosystem, substances with signaling or regulatory functions, etc. Root exudates consisting of various biologically active substances, solubilizers and nutrients are important for promoting growth of soil bacteria in the rhizosphere or directly on root nodules.

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Historical Background 1735—C. von Linne proposed the term “nectar”—nectarium florum. 1848—G. Kaspar classified the nectariferous glands. 1949—A. Frei-Vissling has classified physiological eliminations. 1971—U. Luttghe developed the classification of substances eliminated by plants. 1985—O. Zauralov proposed a more complete classification of eliminated substances. 1989—G. Denisova proposed a classification of terpenoid compounds. Brief Updates Today several hundred species of insectivorous plants (carnivorous) are known. Similar to other typical green plants, carnivorous plants have organs required for normal nutrition. Thus, they possess green leaves that can absorb CO2 from the air and the process of photosynthesis takes place mostly according to the C3 type. This type of nutrition in carnivorous plants was caused by low nitrogen content in the soil. It is considered that nitrogen compounds extracted by insectivorous plants from captured prey significantly enhance photosynthesis. In some representatives, the leaves have a completely altered shape while in others they have only partially transformed in specialized organs designed to capture insects and possess secreting glands or other aromatic substances. Carnivorous plants are divided into three

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groups, based on the adaptations they have developed during evolution that allow capturing the prey. The first group includes plants that capture prey with sticky leaves. The second group is comprised of plants that have traps in the form of vessels. Plants of the third group make quick and active movements to capture the prey. The quick closing mechanism of the metamorphosed leaves (trap) occurs by the rapid change in the turgidity of the mesophyll cells, with ATP consumption. Traps belonging to the plants of the genus Pinguicula and Byblis are functioning due to sticky substances with a high content of carbohydrates, which are secreted by specialized tentacular glands located on the leaves and which are directly used to capture insects. Plants belonging to the genus Utricularia and Polypompholys live in swamps. The pyriform vesicles which grow on the leaves can be closed with a lid. Special glands will remove water from inside the vesicle, so that the lid will be kept closed by the external water pressure. These plants secrete a substance with a high content of carbohydrates that will both attract prey and help fixate the closed lid. As soon as the victim touches the hairs of the lid the later opens and the difference in pressure pushes the lid inwards while the victim is sucked together with water. The lid closes, water is removed from the vesicle by glands and digestion of the prey occurs by hydrolytic decomposition of complex organic substances, in particular proteins, followed by their absorption.

9.1 Classification of the Types of Substance Elimination Plants play a special role in the biogeochemical circuit of substances in nature, as they are open systems continuously participating in the processes of matter and energy exchange with the environment. One of the main aspects of this exchange is the elimination of substances which is a phenomenon specific to all living organisms and a necessary prerequisite for homeostasis. Plants lack a well-differentiated excretory system and the elimination of substances is performed by morphologically identical cells and tissues. The process of elimination of substances is achieved at the level of cells, tissues, organs and organisms. Plants remove both inorganic substances (water, mineral ions) and a big number of primary and secondary organic metabolites (monosaccharides, polysaccharides, amino acids, proteins, terpenes, alkaloids, volatile organic compounds—propanol, acetaldehyde and so on). Eliminated substances can perform functions that enable chemical interaction between plants within the biocenosis, representing one of the mechanisms of adaptation of the organisms to external environmental conditions, ensuring the maintenance and perpetuation of the species. As plant growth and development take place in the soil, their secretions end up in this environment, where they are

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absorbed and utilized by the rhizosphere microflora. Secretions from algae and higher plants form a chemical environment that functions as an aquatic biocenosis. Plants can eliminate final products of metabolism—this process is called excretion, or substances that participate in metabolism, in which case it is considered secretion. Thus, secretion is the active elimination of specific products of the metabolism by first transforming active components of the cellular metabolism into less active components. The process of secretion also requires the participation of active mechanisms of substance transport, accompanied by consumption of metabolic energy. Excretion is the removal of remnants, waste and final products of metabolism, unnecessary for further growth and development of living organisms and is performed along the concentration gradient without the expense of metabolic energy (ATP). A separate phenomenon is represented by water removal from plants through various mechanisms—transpiration, guttation (Fig. 9.1) and plant tearing. Eliminations are represented by plant proteolytic enzymes, nectar sugars, waxes and terpenes, water and mineral salts (Fig. 9.2) and can be grouped into: (1) internal eliminations that remain within plants; (2) external eliminations which are excreted in the environment (Table 9.1). In the 1970s, taking into account the ecological role assigned to these eliminations for plant interaction within the biocenosis, the term “exometabolites” was proposed for substances eliminated by plants under normal conditions and “pathological eliminations” or “stress metabolites” for substances that are secreted under stress.

Fig. 9.1 Guttation in plants

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Fig. 9.2 Metabolic substances eliminated by plants

Table 9.1 Classification of eliminations according to O.A. Zauralov Group

Subgroup

The eliminated substances

Internal eliminations

That accumulate outside the cells That accumulate in tissues Located in glands Not located in glands

Cutin, suber, waxes, polysaccharides, vacuolar substances, essential oils (ethereal oils), terpenes Essential oils, mucus; oils and resins, rubber, latex, arabic gum Nectar, essential oils; mineral salts Eliminations from roots; volatile eliminations from leaves

External eliminations

9.2 Excretion Excretion is the removal of the surplus of substances from plants (waste, toxic waste), whose accumulation would disturb homeostasis of the internal environment. Excretion in plants differs greatly from that seen in the animal world. These fundamental differences are a direct consequence of the physiology and lifestyle of the representatives of the two kingdoms—animal and vegetable. Plants are autotrophic organisms, which are capable of synthesizing organic matter, forming small

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amounts of waste and therefore it was not necessary during development to develop a well-differentiated excretory system. Representatives of the animal kingdom, being heterotrophic and using organic substances in their nutrition, evolved to eliminate nitrogen waste as urea, uric acid and ammonia. Whereas plants do not feed on protein and don’t possess muscle activity (key factors of nitrogenous waste formation), they don’t form this type of waste and small amounts of ammonia formed in the metabolic processes are eliminated by diffusion. Plants are primary producers and synthesize sufficient amounts of all organic compounds required for growth and development. Only the amounts of substances immediately required for the biological processes are synthesized at any moment in plants, which explains why the plant body does not contain surpluses of protein, respectively, does not eliminate nitrogenous waste. But if the proteins are decomposed to amino acids, they are reused in the biosynthesis of other proteins. The three final products produced as a result of cellular metabolism in plants— O2, CO2 and H2O—can be used again by plants as initial material for other reactions. In the presence of sunlight, plants form as a result of photosynthesis much larger quantities of molecular oxygen than are necessary for respiration. Thus, the excess of O2 is released into the atmosphere by diffusion. Many organic substances and metabolic residues are deposited in plant dead tissues, in bark or in leaves that fall periodically. The substances destined for excretion are removed also through sepals, petals, fruits and seeds, although at a smaller extent. Mineral salt ions absorbed by the plant root system are deposited together with other cations in the form of insoluble crystals which stay in the cell, but are neutral for their functional activity. For example, plants absorb both Ca2+ and SO42− ions, but sulfates are included immediately in the process of amino acid (cysteine, cystine and methionine) synthesis, while the divalent calcium cations keep accumulating. Neutralization takes place due to interaction with the oxalacetic and pectic acids, forming neutral substances insoluble in water such as calcium oxalate and pectate. The later “cements” the medial lamella of plant cells. Lack or deficiency of Ca2+ ions leads to maceration of plant tissues. Other ions (Fe2+ and Mn2+) and some organic acids (nicotinic acid, and tannins) are transported in leaves where they accumulate, conferring a specific color before detaching. In the majority of aquatic plants, most of the waste products pass directly into the environment through diffusion. Excretion of substances in higher plants differs according to the stage of vegetation, the capacity of excretion substantially increasing after flowering. Towards the end of the vegetating season a series of substances is eliminated: previously absorbed minerals, as well as organic substances such as acids, carbohydrates, proteins with simple structure, vitamins, hormones, enzymes. Ageing of protoplasmic colloids accompanied by a decrease in their ability to retain ions, makes the precipitations from the end of the vegetating season to “wash” plants, sequestering significant quantities of ions, minerals, substantially reducing forage and fruit quality. For instance, wheat during maturity eliminates K+, Ca2+ and Mg2+ ions in considerable amounts while vine releases K+ and Ca2+ ions.

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Fig. 9.3 Effect of root system exudates on nutrient availability (Dakor and Phillips 2002). AO Organic acids; AA amino acids, including phytosiderophores; CF phenolic compounds

Around the plant roots an ecological environment is created that is different from that of the surrounding soil and which is called rhizosphere, formed by plant exudates that attract microorganisms through chemoattraction (Fig. 9.3). The microflora of the rhizosphere, especially that of legumes, produces organic acids (e.g. Ketoglutarate) used as solubilizers for phosphoric compounds and silicates. It was shown that the wheat roots excrete glucose, flavones and nucleotides, while pea plants eliminate only glucose and nucleotides. Through their roots, plants also excrete other substances: amino acids, biologically active substances. Pea plants eliminate much larger quantities of amino acids compared to oat. In general, legumes often excrete biotin, niacin and pantothenic acid and less frequently riboflavin and thiamine.

9.3 Secretion A particular feature of plant cells is their ability to perform secreting functions throughout the life cycle. The secretion means a controlled cellular activity, as opposed to simple diffusion. As mentioned above, the secretory processes in plants

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Fig. 9.4 Synthesis and secretion pathways in the living cell

are achieved by means of metabolic energy consumption and comprise a number of steps (Fig. 9.4): • incorporation and storage inside the cell of the substrates which will be transformed into secretions; • synthesis and concentration of the secretions; • elimination of the secretions; • restauration of the cellular structures. Secretions can be of three types: • merocrine—the product is eliminated through the membrane; • apocrine—when the product is accumulated in the apical part of the cell, which is “melting”; • holocrine—when the entire cell is destroyed and converted into secretion products. An example is given by mucus eliminations on the surface of the root cap. The secretion of the components is carried out at the: • cell wall, during the process of phragmoplast formation, formation of the first and the second layers of the cell wall, during the deposition process of lignin, suber and waxes, representing at the same time an important aspect of cell and plant organ morphogenesis;

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• specialized secretory structures—glandular papillae and hairs, nectary glands (nectaries), resin channels localized on different plant organs; • structures on the surface of plant organs—osmophores, nectary glands, digestive glands of carnivorous plants—all of these eliminate secretions on the surface, that can evaporate, can be washed by rain, collected by insects; • collecting channels, the niches in the tissues through which secretions that are not eliminated on the surface are collected inside the plant, these can only be released by mechanical lesions (resins, arabic gum). In the process of secretion substances with a very variable composition are eliminated which may be of use for plants (such as the nectar in flowers for attracting insects), which may have an undefined physiological role, like the latex, or which represent waste, such as ions in halophytic plants.

9.3.1 Lignin, Cutin and Wax Secretion Conquering the terrestrial environment by plants and increasing their body size have dictated the development of the central conducting system in order to supply the cells with water. It is precisely these phenomena that have contributed to the appearance of lignin in cell walls (Fig. 9.5). Lignin has not been detected in current and ancient aquatic plants; in the cell walls of a number of mosses there are aromatic compounds, while true lignin appears only in ferns. In contemporary higher plants, lignification represents the process of cell wall impregnation with lignin, which is deposited between the cellulose microfibrils. In this manner lignification occurs in hazelnut walls, nut mesocarps, wooden vessel walls etc. Lignins are aromatic polymers contained in the tertiary cell walls and which confer them their specific rigidity. Lignification is a process that occurs throughout the life cycle of plant cells and increases at the end of the differentiation process. More intensely, cell wall lignification occurs after completion of cell growth.

Fig. 9.5 Lignification of the cell wall

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Fig. 9.6 Cross section through the leaf blade

On the surface of the epidermis an outer skin called the cuticle forms (Fig. 9.6). The cuticle consists of cutin and waxes and has a thickness of 1–15 nm, having a low permeability for water and gas. The cuticle appeared in archaic terrestrial plants, which resemble contemporary mosses and can be detected in sediments of the silurian period. It is assumed that, with the advent of the cuticle, terrestrial plants have adapted to water scarcity and survived under unfavorable conditions of life. At the same time, the cuticle protects plants from short-wave radiation and has implications on the rates of water and CO2 exchange. The cuticle is formed by depositing cutin produced by the cytoplasm of epidermal cells on the external cell walls and contains lipid polymers with 16 and 18 carbon atoms and minor amounts of phenolic compounds. The fatty acids of these polymers contain two or more hydroxyl groups. Kolattuduy supported the idea that cutin biosynthesis takes place on the outside of the cell wall, using monomers formed in the smooth endoplasmic reticulum that are transported to the surface of the cells in the form of vesicles or droplets. The cutin layer is coated with polysaccharide components of the cell wall (e.g. cellulose) and forms the cuticle, a structure containing numerous molecular pores. The cuticle surface in many plants has a polymer layer more hydrophobic than cutin, consisting of waxes. Waxes deposited on the external surface of the cell walls are a mixture of cerides: esters of fatty acids and hydroxyacids (with 15–35 carbon atoms) with alcohols (having 28–34 carbon atoms). These substances also contain free molecules of fatty acids, alcohols, ketones, hydrocarbons, aldehydes etc. The waxes are synthesized by epidermal cells and transported outside through the plasmodesmata and cuticular pores. Wax components are transported through the cell wall embedded in a lipo- or glycoprotein “capsule”, which has a hydrophilic outer surface and can, thus, be transported through the hydrophilic cell wall. It was indicated that transport of the precursors of epicuticular waxes happens in a dissolved state. These substances pass through the ectodesmata and the cuticular pores onto the surface of the cuticle where solvents evaporate and waxes crystallize. Wax

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Fig. 9.7 Longitudinal section through a salt secreting gland of Tamarix aphylla. The surface transfusion can be seen on the left side of the main wall. Small vacuole proliferation was detected near the secreting cells and their apparent association with the plasmalemma in the region of wall extinction (Echeverria 2000)

deposition on the outer cell walls reduces water loss from tissues through the process of transpiration. Each type of wax is deposited in a specific pattern (we can distinguish 14 types)—from amorphous layers (the leaves of beet, the common bean) to various forms of filaments and tubules. Another process, suberization represents the deposition of suberin layers on the inside of the epidermis and endoderm cell walls. This process also occurs in mechanically damaged cells, contributing to the isolation of the tissues that were attacked by parasitic organisms from the healthy ones. Gelation represents the process of excessive production of pectic substances, which in contact with water forms mucilage. This process is found frequently in peach fruits. Mineralization is the impregnation of the cell wall with silicon dioxide in grasses, sedges, diatoms etc., with salts of calcium, carbonates and, less frequently, sulfates in some woody plants. Trough mineralization of the cell walls the latter acquire a greater mechanical strength. Salt-secreting glands are located in the epidermis of the leafs or stems and have a similar structure to that of hydathodes. These glands are characteristic for some species of halophytes and are designed to collect and remove excessive amount of mineral salts (Fig. 9.7). Such secreting glands are found in statice, which actively eliminates chlorine and passively eliminates sodium. Nectary glands are made of a special type of epidermal cells and are localized on some floral or even extrafloral components on stipels, receptacles, stamen filaments, on the ovary or on the style (stilus) (Fig. 9.8).

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Fig. 9.8 Nectariferous glands in Dionea muscipula (a), Drosera intermedia (b) and Phyostegia virginiana (c)

Fig. 9.9 The nectary gland in Croton glandulosus. Nectar secretion may cause the separation of the cuticle from the outer periclinal walls of palisade cells that form the secretory epidermis

9.3.2 Nectariferous Glands and Nectar Secretion Nectariferous glands (nectaries) (Fig. 9.9) secrete substances used in pollen germination and fecundation of the ovum. Unused nectar can be absorbed by the ovary for additional nutrition of the generative parts of the plant. Intrafloral nectaries have the role to attract insects that carry out cross-pollination, while the extrafloral nectaries serve to remove the excess of glucose in the periods of high photosynthetic activity. The nectar has phytoncide and bactericidal properties, protecting the ovary from various microorganisms. Extrafloral nectaries defend the plants from the insects feed on them. There are two possible ways of transporting the nectar in the cells of the nectary glands. The transport of pre-nectar from cells to the nectaries can occur by passive transport through the apoplast due to the proton pump localized in parenchymal cells of the passage elements, which eliminates the pre-nectar from the passage elements under pressure. Pre-nectar moves from cell to cell and through the symplast, the plasmodesmata, turning into a type of nectar specific for the plant species in the cytoplasm of the nectary gland cells, followed by its elimination. This transport is carried with consumption of metabolic energy, as confirmed by increasing oxygen intake during secretion and an increase in the number of mitochondria in the cells of the nectaries. Both the symplastic and apoplastic transport

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can be identified at different stages of the traveled path. At the same time, in nectary glands, with lignified and suberized cell walls, in which the transport through the apoplast is excluded, the transport happens exclusively through the symplast. Elimination of the nectar from nectariferous epithelium occurs in the form of droplets and is similar to guttation.

9.3.3 Terpenoid Secreting Structures The morphology of the structures that eliminate terpenes are of a great diversity— solitary cells or groups of secretory cells. Isolated secretory cells called idioblasts can be found in different plant tissues: leaves, stems, etc. Glandular hairs represent outgrowths consisting of one or more cells derived from the epidermis and, sometimes, from the hypodermis. They are located on the leaves and stems of the plants and have a terminal part formed of one or more secretory cells. Glandular hairs from Primula, Alnus and Populus producing flavonoids are characterized by the presence in the terminal cell of a large number of ducts of the endoplasmic reticulum, while the hairs producing carbohydrates are characterized by welldeveloped Golgi complexes. The essential oils synthesized by Mentha piperita accumulate in the vacuoles of the secretory hairs, while stinging hairs of Urtica dioica accumulate histamines and acetylcholine. The secretions are released through pinocytosys in the space between the plasmalemma and the cell wall, from where it diffuses towards the exterior, accumulating in the subcuticular space. Glandular papillae are composed of epidermal cells, elongated outwards. They can be found in species of the genus Rosa, Syringa, Lilium, Hyacinthus and the secretions consist of volatile oils. The osmophores are specialized glandular cells which secrete essential oils that determine floral aroma in a group of plants. Various parts of the flower can morph into osmophores accepting a hair-like, wing-like form etc. From the essential oils almost 1,000 organic components are extracted—terpenes, carbohydrates, aldehydes, alcohols, ketones, phenols and others. The linalool is characteristic for oils derived from the lily of the valley, oranges, coriander, eucalyptus, roses, geraniums. Several phytohormones also belong to the class of terpenes—the abscisic acid (C15H20O4), gibberellins (C19–20), steroids (C28–30). The composition of essential oils specific for each plant species is characterized by the presence of tens of terpenoids (Fig. 9.10). Lactiferous cells consist of long secreting cells, branched or unbranched, arranged in rows (unbranched lactiferous cells) or made of cells placed end to end, which have their separation walls reabsorbed (articulated lactiferous cells). During the growth of the lactiferous cells, a single large vacuole is formed for the accumulation of latex— an emulsion containing 50–80 % water, as well a series of substances in the form of a suspension: carbohydrates, tannins, glycosides, calcium malate, essential oils, waxes, gums, resins, aromatic acids, polyisoprenic carbohydrates and sometimes toxic alkaloids. The color of latex is white, in contact with air, it strengthens.

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Fig. 9.10 Terpenophenols in hops (Humulus lupulus). The paternal genotype produces cones (a) used in beer fermentation. Glandular trichomes (lupulin glands), covering the cone bracts (b) represent the main place of terpenophenol biosynthesis in hops

The biosynthesis of latex is achieved with the participation of the cytoplasm, endoplasmic reticulum and Golgi complex. Substances with a high molecular weight accumulated in transport vesicles, are transferred into vacuoles by a process of pinocytosis while low molecular weight substances cross the tonoplast through an active or passive transport process. Some of the substances accumulated in lactiferous cells can be re-used in metabolic processes, while others stay accumulated in the cells. Internal glandular tissues are composed of a massive group of secreting cells, that delimitate a common space in the form of a bag, a pocket or a canal, which accumulates secreted substances. In the species of the Citrus genus schizolysigenous pockets can be found, which accumulate monoterpenes and sesquiterpenes. Essential oils are synthesized and accumulated in plastids in the form of droplets. When reaching maturity, they form a cavity in which volatile oils accumulate from lysed plastids. Volatile essential oils contain terpenes with a varying numbers of carbon atoms (C5, C10, C15 and C20) aliphatic and aromatic esters, phenols, etc.

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9.4 Secretory Processes in Insectivorous Plants Among higher plants more than 400 species of carnivorous species are known that have various mechanisms of hunting and different secretory glands. These plants, in addition to autotrophic nutrition processes, are able to use small animals (insects, juvenile fish) as a source of food. The leaves of insectivorous plants turn into special traps, capable both to photosynthesize and capture prey. Usually, the latter sticks to the leaves, whose glands eliminate a sticky mucus (as is the case of sundew—Drosera rotundifolia) or falls into traps in the form of jugs, tubes, bags that are brightly colored and eliminate sweet, aromatic secretions (Sarracenia, Heliamphora). The principle of the trap is based on catching the victim with the help of quickly closing leaves or by sucking the prey with the help of a sudden water current, created with the help of a vacuum formed in the trap. Common to all the types of traps is the need to attract insects with nectar and polysaccharides (in the mucus), which are either secreted by the specialized trapping organ or by glands localized near the trap. Responsiveness of the trap mechanisms is triggered by the irritation of sensitive hairs that is caused by prey movement (Figs. 9.11 and 9.12). Secretions produced by digestive glands contain proteolytic enzymes and mucilaginous, sticky substances, ions of sodium, potassium, magnesium, calcium and chloride in a concentration of 20 mmol. Secretions also contain amylases, glucuronases and cell wall hydrolytic enzymes, which are produced by the endoplasmic reticulum and ribonucleases, proteases, phosphatases generated by the Golgi complex. Insects stick to the polysaccharide containing mucus, which fixes them in place. The biological importance of the secretory processes in insectivorous plants consists in achieving heterotrophic digestion, in addition to the process of

Fig. 9.11 Drosera intermedia. General aspect (left) and nectary glands (right)

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Fig. 9.12 The stages of prey capture by Drosera intermedia

photosynthesis. This property is very useful in conditions of nitrogen deficit (for epiphytic plants, growing on trees which have a poorly developed root system). Compared with aquatic plants, there is no mycorrhiza in insectivorous plants, therefore nutrients provided by the captured prey are of significant importance. Plants receive nitrogen, phosphorus, potassium, sulfur from the captured insects. The carbon, which is contained in the hydrolysate, is also used in the plant metabolism.

9.5 Water Elimination in Plants Water is so important to living organisms, that life on Earth, would be impossible without it. Life is present only where there is enough water. A normal water regime provides a high degree of water saturation even under conditions of relatively dry air so that the cells remain in a state of turgidity. In conditions of drought, plants lose significant amounts of water that they can’t compensate for. This poor water regime has unfavorable consequences on the vital activity and if it last too long it leads to death of the organism. Plant tissues contain greater amounts of water than animal tissues and the normal functioning of cells, tissues, organs and of the entire organism depends on the

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Table 9.2 Methods of maintaining water homeostasis in plants The basic principle

Adaptation

Examples

Reduction in the rate of transpiration

Cuticle waxes Fewer stomata Leaves covered with hairs

Opuntia

Water storage

Water absorption

Burying the stomata in the leaf mesophyll Osteoles are open during the night and closed during the day Fleshy succulent leaves Fleshy succulent stems Fleshy underground parts A root system well developed in depth A root system well developed at the surface

Mesembryan theum Pinus

Bryophyllum Kleinia Raphionacme Medicago sativa Medicago falcata

Fig. 9.13 Forms of water elimination from plants

stability of the water content. The water regime of plants is one of the determining factors for a normal metabolism of the organism, hence its role in homeostasis (Table 9.2). The main processes by which the plants are eliminating the water are transpiration, tearing and guttation (Fig. 9.13). Removing water in the form of vapors represents transpiration—the process that is characteristic for all terrestrial plants and which occurs with varying intensity in various plant organs (except for the roots) and in different groups of plants (Table 9.3). The process of transpiration does not require metabolic energy, it uses light energy that by being absorbed is converted into heat which fuels the process. From this point of view, the mechanism is called passive. But in terms of the suction force it generates it exceeds the active mechanisms of absorption and movement of water through the plant, which is carried by the root system, based on root pressure. Removing water from the plant is essential because it ensures: crude sap ascension

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Table 9.3 Intensity of stomatal and cuticular transpiration in different groups of plants Group of plants

mg H2O (dm2/h) Stomatal transpiration

Cuticular transpiration

% from stomatal transpiration

Hydrophytes Helyophytes Ombrophytes Pomoideae Prunoideae Vitaceae Cactaceae

1,800–1,400 1,700–2,500 500–1,000 700–1,000 400–700 400–500 200–600

– 200–300 50–250 120–160 80–110 80–90 3–10

60–70 up to 20 up to 25 10–20 15–20 17–24 1–2

through the xylem vessels, avoidance of cell saturation with water and plant overheating (vaporization of 1 g of water is accompanied by absorption of 2.2 kJ of energy). Due to these reasons, transpiration is considered a necessary physiological evil for plants. Transpiration occurs through stomata and lenticels but also through the external protective layers: cuticle, suber, etc. Osteolar transpiration is correlated with the rate of water provision and their degree of openness. Stomata have a wide distribution on the leaf surface, depending on the species. Approximately 71 % of the woody species are hypostomatic and almost all of the crop plants are amphystomatic. Water from the xylemic vessels is transported through the apoplast into the lacunar parenchyma cells and the process of evaporation takes place in the substomatal cavity. Water vapor transport through the lacunar spaces, has a lower extent. Air from the substomatal cavity is saturated with water vapor, while the ambient air has a low relative humidity (40–60 %). As a result, water vapor diffuses into the environment from the substomatal cavity. After harvesting vegetables, the stomata close in about 10 min which helps to maintain turgidity during further processing. Cuticular transpiration has a reduced extent due to the low permeability of the lipid components of the cuticle. This type of transpiration makes up 1–2 % of the total transpiration in Cactaceae plants, between 10 and 25 % in mesophilic plants and reaches up to 60–70 % of the total transpiration in hydrophytes. In plants with very thick cuticle, like the Hedera helix, cuticular transpiration does not occur at all. Lenticular transpiration is evident in stems, fruits and leaves of conifers, containing lenticels which are gaps in the epidermis, periderm or modified stomata. By means of the lenticels, living cortical cells are connected to the ambient atmosphere. When provided with abundant amounts of water and in the presence of a high content of water in the atmosphere, several plants, especially young ones, eliminate the raw sap through the upper leaves in the form of droplets. This phenomenon is called guttation. In dicotyledonous plants, guttation occurs through special openings called hydathodes, localized on the tips of the leaves or the lobes and can be

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noticed at night or in the morning, during spring and summer. Among cultivated plants, considerable levels of guttation can be noticed in potatoes, cereals, primula. Some tropical plants such as Caesalpinia gilliesii have an abundant guttation such that under the tree water is dripping. Guttation was observed in more than 300 genera of plants and it is characteristic for tropical and equatorial plants. Like in the case of plant tearing guttation is an eloquent proof of the root pressure. At the basis of this phenomenon lies the disparity that is created between water absorption by the roots and its elimination in the form of vapors. In grasses, guttation is observed at a young age—elimination of water takes place not through the hydathodes, but trough the tips of the leaves, after the layers of young cells rupture under the internal pressure. In the guttation water of the cereals, along with other substances (salts of calcium, potassium, phosphorus), malic acid is almost always present. Guttation in this group of plants can be easily induced by covering the pot in which the plant (wheat, barley and maize) grows with a glass bell for a certain period of time. From the biological point of view, guttation can be regarded as an adaptation of the plant in order to remove the water excess from the body. Due to this phenomenon, the equilibrium between the absorbed and eliminated water is maintained. If plants couldn’t remove water excess, the later would penetrate the intercellular spaces and would cause tissue death by suffocation. Some physiologists believe guttation is important because, along with water, plants eliminate many toxic salts. Besides guttation the phenomenon of plant tearing can be noticed which is happening due to the root pressure demonstrating an active, purely physiological water absorption and conductance mechanism. The root pressure is the force that pumps water into the symplast of the endoderm through the conducting vessels of the central cylinder. During the spring, when many plants don’t have leaves, the only force that moves the water through the plants is the root pressure. The value of the generated force depends on the accumulation of plastic substances in the cortical parenchyma cells and on the energy metabolism of the cells. In some circumstances, depending on the root pressure values, the crude sap can elevate to a certain height of the plant. This effect can be easily noticed in vines during early spring, which when cut releases large amounts of sap. This phenomenon was called “tearing”. It can be observed in many plants also during the summer, when the aerial part of the plant is cut. Moreover, crude sap can be collected, and its chemical composition–determined. Also the value of the root pressure and the osmotic pressure of the sap can be measured. The value of the root pressure is relatively low. In some trees and vines values of 2–3 atm, where detected which can lift water up to 15–20 m high. Once the leaves come into action the leaf suction force is generating a counterforce in the conducting vessels and often prevent sap eliminations after mechanical lesions. In perennial plants, this phenomenon manifests itself especially during the spring (birch juice) while in herbaceous species—throughout the vegetation period. The crude sap eliminated by the process of tearing contains mineral salts, organic substances, sugars, etc. Summer raw sap contains ash—derived elements, organic acids, but no sugar.

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Plant tearing lasts from a few days up to 5–6 months, and the eliminated water amount varies from one species to another. A palm tree can eliminate by tearing about 10 L of sap, a birch tree—5 L, and a vine plant—almost 1 L, with a force of 1.6 atm, elevating water to about 16–21 m. The crude sap elimination mechanism was explained by A. Sabinin by the different nature of the metabolism occurring in the cells surrounding the xylem vessels, which leads to the creation of a difference in the suction force, as result of which, at one end of the cell water is being absorbed while at the other—eliminated. If a plant is exposed in a solution with the osmotic pressure equal to the osmotic pressure of the raw sap, tearing ceases immediately. Tearing is closely related to the vital activity of the plant. If soil aeration is low, the rate of water elimination by tearing is reduced significantly.

9.6 Ecological Role of Substance Elimination In the process of ontogeny, plants eliminate different chemical compounds. These can be both trophic substances or intermediates, having signaling or regulatory functions. The later group has a less important role in comparison with the group of trophic substances, which are involved in the chemical interaction of the organisms that are part of any ecosystem. Between each living cell and its surrounding environment there is a permanent exchange of substances. Eliminations from one species can exert an influence of the growth, survival and reproduction of other species. The phenomenon is called allelopathy and it can lead to a reduction in crop yields. Both the quantity and quality of the harvest can decrease in this case despite the fact that the soil contains sufficient nutrients. The above is also true for monocultures. This phenomenon is explained by the accumulation of large quantities of certain microorganisms, pests and toxic root eliminations, which negatively act on the plant and the microflora of the rhizosphere, inhibiting plant growth and development and in some cases, leading to their death. In this case application of fertilizers containing micronutrients and cultivation of different species on the same territory are recommended. Investigation of the mechanisms and characteristics of these interactions between organisms represents the object of biochemical ecology. Obviously, this kind of research requires knowledge about the stages, mechanisms and regulation of secretory process in plants as the main components of the biocenosis. In natural biocenoses, the separation of the effects caused by regulatory and trophic substances is a difficult task. But for microorganisms that grow in conditions of organic food deficiency, it is possible to identify the role of eliminated regulatory substances. Investigation of the interaction processes in 100 microorganism cultures showed that they all produce biologically active volatile substances: acetone, volatile fatty acids, amines, CO2, N2, O2, ethane, butane and others. The chemical composition of the volatile biologically active substances in various cultures, depend on their age and the metabolites produced by other microorganisms.

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• After elimination of waxes from the surface of the plants, their permeability for water increase over 500 times. The osteoles of many angiosperms and gymnosperms are also coated with wax containing structures that are designed to minimize water loss from the aerial niches of the osteoles. The waxy pellicle on the osteole in some xerophytes intensifies, during the night, water condensation from the air. • The suberin is formed during secondary growth, causing Kaspari thickenings in endodermal cells or impregnates the outer cells of the potato tuber, during the mature stage. • The leaves of Laurus nobilis contain large cells that secrete volatile oils. In the bark of Quercus species and in the medular tissue of Rosa species cells can be found in which substances like tannins, alkaloids and heterosides are synthesized and accumulated. • Plants from the Tiliaceae family have cells containing mucilages derived from cytoplasm degeneration or from the cell wall gelling. • In the petiole of Begonia, Prunus, Allium species cells that accumulate crystals of calcium oxalate can be found. • In cells of Liriodendron and Magnolia volatile oils are accumulating in vesicles formed by invagination of the plasmalemma. The accumulation of these substances takes place in cells of the leaf mesophyll, of the fruit mesocarp and, in some cases, in roots and stems. • Secretory glands in plants of the Fabaceae family are rounded, consisting of 6–12 cells located radially, while in species of the Compozitae family they are elongated, bicellular. • The latex of the Papaver species contains alkaloids, that of the Ficus—protein substances, that of the Musa—tannins while Carica papaya contains papain. • On a 1 year old branch of Gledistria triacanthosa with a length of 20 cm 72–210 lenticels can be found, while tubers of Solanum tuberosum have a lenticel per 1 cm2. • A plant of Zea mays absorbs during the vegetating season about 250 L of water, of which about 98 % is eliminated by transpiration.

Glossary Allelopathy Mutual biochemical influence of plants, consisting of inhibition, rarely—growth stimulation of a plant by the metabolic products of other plants, eliminated in the environment. By extrapolation, the term allelopathy now applies to all biochemical processes of mutual influence between organisms in the biocenosis. Allelopathy acts as an instrument of the self-regulation mechanisms of the biocenosis, based on which a “biochemical metabolism” of the biocenosis is established.

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Biocoenosis The term designating a living community including also the atmosphere and the soil. The biocoenosis comprises the phytocoenosis, zoocenosis and microbiocoenosis. Halophytes Plants that can grow on soils with high salt content due to the presence of a number of characters and traits that have emerged under the action of specific environmental conditions and natural selection during evolution. Homeostasis An autoregulatory process by means of which the organism maintains a certain chemical composition a set of physical and morphological parameters and can re-establish a disrupted balance, either by returning to its original state, either by returning to an equivalent state. Median lamella The cellular structure that appears in the telophase of the mitotic process between two daughter cells and is composed of insoluble pectic substances (protopectin), which serve as a ligand between cells. Tissue softening in fruits is caused by the enzymatic decomposition of pectic substances from the middle lamella resulting in easily separating cells, a process which characterizes the stages of maturity and senescence. Terpenes Organic substances widely spread in plants as constituents of essential oils, also some insects have terpenes as a component of protective substances and pheromones. Sometimes the term designates only the corresponding carbohydrates while their oxidized derivatives are called monoterpenes.

References Burzo I. ş. a (1999) Fiziologia plantelor de cultură, vol I. Chişinău Dakora FD, Phillips DA (2002) Root exudates as mediators of mineral acquisition in low-nutrient environments. Plant Soil 245:35–47 Denisova GA (1989) Terpenoidosoderzhashhie struktury rasteniy. L., 1989 Echeverria E (2000) Vesicle-mediated solute transport between the vacuole and the plasma membrane. Plant Physiol 123(4):1217–1226 Fahn A. Plant. Anatomy. Oxford, 1995 Ostroumov SA (1986) Vvedenie v biohimicheskuyu ekologiyu. M., 176 pp Polevoy VV (1989) Fiziologiya rasteniy. M., 502 pp Popovici L, Moruzi C, Toma I (1994) Atlas botanic. Bucureşti Roshhina VD, Roshhina VV (1989) Vydelitel’naya funktsiya vysshih rasteniy. M., 214 pp Salamatova TS (1983) Fiziologiya rastitel’noj kletki. P., 232 pp Salamatova TS, Zauralov OA (1991) Fiziologiya vydeleniya veshchestv rasteniyami. P., 1991 Sebanec J (1992) Plant physiology. Amsterdam, Oxford Shubnikova EA, Korot’ko GF (1986) Sekretsiya zhelez. M., 131 s Tarhon P (1992) Fiziologia plantelor, vol I. Chişinău Vasil’eva AE (1997) Funktsional’naya morfologiya sekretornyh kletok rasteniy. P., 1977

References Zauralov OA (1985) Rastenie i nektar. Saratov, 177 pp Zeiger E (1987) Stomatal function. Standford, California http://ridge.icu.ac.jp http://www.cactus-art.biz

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Chapter 10

Physiology of Plant Resistance to Unfavorable Environmental Factors

Abstract Resistance is defined as the ability of organisms to oppose the alteration of the metabolism under the action of unfavorable factors of the environment, to survive and to ensure the normal flow of physiological processes. This is most often a polygenic trait. Plants can survive in unfavorable environmental conditions: by using special adaptive structures; due to physiological properties that determine resistance to adverse conditions; due to various physiological mechanisms involved in avoiding the adverse effect. Multiple negative phenomena can have a detrimental and even lethal effect on plants in natural conditions for instance asphyxiation under snow or water, uprooting, spring frosts, low temperatures, drought, etc. According to their resistance to cold there are cold resistant plants (which can withstand low positive temperatures) and frost resistant plants (which can withstand temperatures below 0 °C). The main impact of low temperatures on plants consist in disturbing the water balance by altering the fluid state of biological membranes, by formation of intercellular and intracellular ice crystals which can cause dehydration and mechanical damage. In adaptation to these, plants often increase the cellular concentration of dissolved substances which results in more water stored in the hydration layers of the molecules and in a lower freezing temperature. The resistance of crop species to cold can be increased by hardening seeds with low temperatures. Periods of prolonged drought result in plant wilting by affecting the water regime. Water loss leads to an alteration of the metabolic processes at various levels: protein hydrolysis increases, the processes of carbohydrate biosynthesis and phosphorylation are destabilized; photosynthesis halts, enzymes are blocked all these leading to an impact on the ongoing morphogenetic processes. To counter drought effects plants increase respiration rates which leads to the synthesis of more endogenous water and ensures energy supplies in conditions of hampered photosynthesis. Salinization can be determined by the presence of: sulphate (Na2SO4), chloride (NaCl), carbonate (Na2CO3) or it can be combined. Depending on their resistance to saltiness plants can be either glycophytes or halophytes. The last group can be further divided based on whether they absorb or not salts through their roots and whether they accumulate them or not in the cell sap. Besides stress factors naturally encountered in the environment a series of pollutants such as toxic gases, radiation, heavy metals, dust, etc. can also have a devastating impact on the vegetal © Springer International Publishing Switzerland 2015 M. Duca, Plant Physiology, Biological and Medical Physics, Biomedical Engineering, DOI 10.1007/978-3-319-17909-4_10

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organism. Plants try to prevent toxic gases from entering the leaves or to integrate them in the metabolism, to immobilize the ions of heavy metals in the cell wall reducing their toxic effect.

Historical Background 1896—B. Lindforss demonstrates the role of carbohydrates as cryoprotectants in freezing cells. 1904—B.D. Zalenski studies the role of the anatomy of leaves in drought resistance. 1931—I.I. Tumanov launches the idea of plant hardening in frost. 1958—Rudolf W. and H. Kapert underline the role of unfavorable factors in the development of functional and structural disorders in plants. 1962—B.P. Stroganov demonstrates that plant adaptation to different kinds of salinization is different. 1962—F. Ritossa reveals heat shock proteins. 1971—M.N. Christiansen finds that low temperatures cause metabolic aberrations in plants.

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Brief Updates A large group of proteins that are involved in stress responses are heat shock proteins (HSP) or chaperons, which are synthesized in cells following exposure to high temperatures. Such proteins are found in microorganisms, animals and plants, presenting high structural similarity, which is indicative of their evolutionary conservation. For example, the structure of the HSP with the Mr 70 kDa is similar in insects, birds, mammals, fungi and plants. In plants, HSPs can have a big mass (Mr between 60 and 110 kDa) and a small mass (with Mr of 15–35 kDa). Their function is to bind normal proteins and prevent their aggregation induced by high temperatures, the energy process is ATP-dependent. HSP synthesis is initiated when temperature changes with 8–10 °C compared to the normal. In such stressful conditions, the genetic program stops normal protein synthesis and replaces them with stress proteins. Synthesis of mRNA encoding HSP starts already after 5 min and the maximum is recorded after 2–4 h and followed by decrease. It has been demonstrated that HSP are synthesized also during other stress situations, such as, for example, the action of heavy metals. Induction of HSP synthesis serves as a model for the study of the molecular mechanisms regulating gene expression in the process of adaptation to stress factors. In the future it is expected to obtain transgenic plants containing genes encoding HSP, which would contribute to an increased resistance to various extreme conditions. Dehydrins are proteins similar to the HSPs that are synthesized in the late stages of embryogenesis under the influence of abscisic acid (ABA). Their elevated content correlates with cell dehydration that occurs during this developmental stage. In cotton there were found 18 molecules of mRNA, 13 of which can be induced by exogenous treatment with ABA. It is assumed, therefore, that these proteins may be involved in the protection of plants during water deficit. The expression of the genes encoding such proteins may be induced by ABA, by low temperatures, by drought and salinity.

10.1 The Concepts of Resistance and Adaptation On vast territories of the globe in different climatic zones plants periodically undergo unfavorable conditions of life, such as, pedological drought, atmospheric drought, high or low temperatures, pesticides etc. All these lead to lower productivity or total loss of the crop, hence the need to counter these effects by boosting the resistance of the cultivated species. Resistance is the ability of organisms to oppose the alteration of metabolic processes under the action of unfavorable factors of the environment, to survive and to ensure normal life processes.

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Plant resistance to unfavorable factors is a complex biological trait. In terms of genetics, resistance is a polygenic index (it is under the control of a set of genes). Quantitatively, there are two types of crop resistance: biological resistance and agronomic resistance. Biological resistance is defined by the limits of the stress levels at which plants are still able to form viable seeds (the function of the maintenance of the species as a biological unit). Quantitatively, biological resistance is estimated in units which are used to measure the extreme factor which is acting on the plant (temperature, concentration of substances in the environment, water potential, etc.). Agronomic resistance reflects the degree of decrease in the crop yield caused by the action of stress factors and is expressed in units of plant productivity per action of the stressor or in percents that reflect the ratio of plant productivity under stress compared to their productivity in the absence of the stress agents. Usually, plants are characterized by high, medium and low (weak) resistance to particular types of abiotic stress (heavy metals, radiation, gas, drought, salinity etc.). Plants can survive in unfavorable environmental conditions: • by using special adaptive structures; • due to physiological properties that determine resistance to adverse conditions; • due to various physiological mechanisms involved in avoiding the adverse effect. There are three types of mechanisms involved in the manifestation of plant resistance: genetical, morpho-anatomical and physiological involving adaptation. For example, many desert plants typically have specific mechanisms of photosynthesis that makes it possible to avoid water loss (CAM and C4 photosynthetic cycles). Adaptation represents the totality of processes and adaptive responses of the organisms (individuals, species, populations) or organs that maintain their resistance to various environmental factors (Figs. 10.1, 10.2 and 10.3) during ontogeny. The essence of each adaptation process is reduced to the maintenance of the anatomical and functional resistance of the living systems that ensures progression through ontogeny and reproductive organ formation under variable environmental conditions. Adaptation is a prerequisite of species survival and determines the boundaries of life for that species in certain environmental conditions. Adaptation occurs and develops under the action of three main factors of the evolution— heredity, variability and selection (natural and artificial). The basis of the adaptation process of the plant organism is formed by a set of constitutive and inductive systems. Constitutive resistance systems are formed during evolution and are expressed continuously, while inductive systems are manifested only as a response to the action of the unfavorable factor (Fig. 10.3). Plant adaptation to the conditions of existence occurs due to genetic and phenotypic variability by restructuring a complex set of physiological, biochemical, morphological and anatomical parameters in ontogeny and the formation of new reaction patterns during phylogenesis.

10.1

The Concepts of Resistance and Adaptation

Fig. 10.1 Ecological factors

Fig. 10.2 Properties of environmental factors

Fig. 10.3 The main types of adaption of living organisms to the environmental conditions

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The ontogenetic adaptation can be genotypic, if there is a selection of genetically determined characteristics or phenotypic when variability is determined by the norm of reaction, the stability of the genotype. The strategy for biological adaptation is implemented at different levels of development of the living matter: molecular, subcellular, cellular, organ, organism, population, species, biocoenosis, biosphere and is reflected on different levels: genetic, biochemical, physiological, morphological and anatomical. According to current knowledge, three types of adaptations can be distinguished: • genetic adaptation; • phenotypic adaptation; • instant adaptation. Genetic adaptation is implemented based on the entire complex of “adaptive strategies”, formed during the evolution of the species. Phenotypic adaptation happens within the limits of the genetic information stored and executed by the genome and occurs only at the level of the phenotype. Instantaneous adaptation is related to variations in the conformations of the macromolecules, in enzyme activity, in pH, ion concentration, chemical composition, intracellular microenvironment etc. These processes are targeted towards homeostasis and vital function maintenance. Instant adaptation is being examined as the primary protective response of the body to the action of unfavorable environmental factors. Thus, plant adaptation to extreme environmental factors reflects the variety of relationships between plants/phytocenoses and the environment and represents a complex of complicated processes, driven by the self-regulating systems of the organism.

10.2 Unfavorable Factors of the Winter-Spring Period During winter, plants, especially perennial and multi-annual grasses are influenced by unfavorable conditions caused by low temperatures. In addition, perennial plants may suffer from other damaging phenomena such as asphyxiation under snow (e.g. winter wheat may perish due to the thick snow layer), asphyxiation under water, uprooting, alternating low and high temperatures and winter drought. Asphyxiation under snow represents the death of plants trapped under a thick layer of snow for 2–3 months under moderate winter conditions at a temperature of about 0 °C in unfrozen ground. Snow is a bad conductor of heat, so the temperature rises under the snow sheath to about 0 °C, causing an intensification of the respiratory process. Plants start utilizing carbohydrate reserves, whose amount decrease from 20 to 2–4 % and plants may die due to exhaustion of energy resources, a phenomenon called inanition. It was noticed that in thermophilic plants, a decrease in temperature is accompanied by a sharp increase in cytoplasm viscosity and the oxidation-phosphorylation correlation is disturbed. Thus, plants lose resistance to adverse conditions and

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during spring are easily attacked by Fusarium nivale and perish. Consequently, in areas with heavy snow crop varieties containing increased amounts of carbohydrates are needed. Plants often perish during the winter due to the formation of a thin crust of ice on the surface of the soil, which causes plant uprooting. Uprooting is the process through which the ice crust detaches the top layer of the soil together with the plant, leading to rupture of the root and organism death. Uprooting is conditioned by the attraction of water from the deeper layers of the soil to the ice layer formed on the surface. If, as a result of the frost, the layer at 2.5–5.0 cm from the soil surface freezes and if there is no further cooling, this layer begins to absorb water from deeper soil layers. As a result, the soil crust forms a frozen ice crust which elevates and detaches the frozen soil layer from the unfrozen one damaging plant roots. Frost resistant varieties which are characterized by a greater extensibility of the roots support these conditions easier, while those with poorly developed root systems perish. In order to prevent this phenomenon, it is necessary to create varieties with high extensibility of the root system and to use methods which reduce the amount of water on the surface of the soil. Another common phenomenon in certain regions of the globe are spring frosts. During the spring time plants die from an alternation of high and low temperatures. Thus, plants that survived the winter at −30 °C can die in the spring at less extreme temperatures, because they have completed the processes of hardening and began the processes of growth. In early spring, water accumulation from melting snow can cause plant asphyxiation under water. Typically, these phenomena occur in the spring, but can also occur during the winter when temperatures stay for a long time around 0 °C and plants remain for a long period under water. Under excessive humidity conditions, in these plants due to oxygen shortage, aerobic respiration decreases in intensity while anaerobic respiration intensifies. Accumulation of toxic substances (alcohol) as a result of anaerobic respiration leads to plant poisoning. In addition, excessive consumption of organic substances, weakens plant fitness (Fig. 10.4).

10.3 Plant Resistance to Cold and Frost Plants in different climatic zones are characterized by their different potential to tolerate a range of low temperatures. Therefore, according to their resistance to lowtemperatures, plants are divided into: • cold-resistant plants, which can withstand low positive temperatures; • frost-resistant plants, that can withstand temperatures below 0 °C. Heat-loving plants, after being exposed to low positive temperatures, alter their water supply processes, which causes a loss in cell turgidity (for example, cucumber leaves lose turgidity at 3 °C on the third day). So, at low positive

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Fig. 10.4 Influence of oxygen deficiency on various metabolic processes (Igamberdiev et al. 2004)

Fig. 10.5 Testing plant frost resistance

temperature the water regime of the plants is disturbed. The main cause of the harmful action of low temperatures on heat-loving plants is the perturbation of the functional activity of the membranes, as a result of the transition of the saturated fatty acids from the liquid crystalline state to the gel state. This alters the normal balance in substance exchange, while a longer action of this negative factor can lead to death (Fig. 10.5). Agricultural plant resistance to cold can be increased by hardening seeds with low temperatures (with the periodicity of 12 h—1–5 °C, 10–20 °C). The same

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Plant Resistance to Cold and Frost

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Fig. 10.6 Acclimatization to frost in different Arabidopsis lines. a Three week plants were acclimatized at 1 °C with a photoperiod of 16 h for 0 (NA), 1, 2 or 3 days or acclimatized for 3 days followed by deacclimatization at 21 °C for 1 day. These plants were frozen at −4 °C (left), −7 °C (center) or −10 °C (right); b three week plants were not acclimatized (NA) or kept for 3 days at 1 °C in darkness or in conditions of a 16 h photoperiod; c Three week plants were acclimated to a 12 h photoperiod at a high temperature (1 °C) during the day and a low temperature (−1 °C) during the night (1 °L/−1 °D), at a low temperature during the day and a high temperature during the night (−1 °L/1 °D), or at 1 °C (1 °L/1 °D) for 2, 3 or 5 days before freezing at −8 °C; d three week plants were acclimatizated at 1 °C and 16 h of daily light for 2 or 3 days in the presence or absence of 50 mM DCMU before freezing at −8 °C

method is also used for treating seedlings, except that in this case a 25 % solution of microelements or ammonium nitrate is added for 20 h. The frost resistance of plants is their ability to survive under the influence of low temperatures well below 0 °C (Fig. 10.6). It was established that freezing temperatures below −20 °C are specific to about 42 % of the globe. Low temperatures have different effects on plants. The main causes of cell death at low temperatures are cell dehydration and mechanical trauma caused by ice crystal formation. A rapid decrease in temperature in extreme conditions leads to intracellular ice formation and cell death. A gradual decrease in temperature, which is more likely to happen in natural conditions, leads to ice formation in intercellular spaces. The formed ice crystals remove the air and frozen tissues may seem transparent. Specific physiological adaptations allow plants to partially avoid wounds that occur after frost. It is known that in case of tissue freezing the biggest damage is produced by the crystals of ice formed within the cell. These crystals grow, break cell membranes and the cell dies. Increasing the concentration of dissolved substances in the vacuolar sap, mainly carbohydrates, protects plants from frost damage, because it increases the probability of formation of large ice crystals. Another method of protecting plants from damage caused by frost is by increasing the quantities of proteins, which are able to recruit water into their

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hydration layers. Hydration water practically does not freeze. It is held around protein molecules with larger forces which prevents formation of ice crystals. Therefore, the more proteins of this type are synthesized in a cell, the more resistant is the cell to frost. It was found that many plants synthesize such proteins in autumn while in spring they are utilized in metabolism.

10.4 Plant Resistance to Drought Drought is a reflection of weather conditions, and is determined by the lack of water in the soil, by high temperatures and a decrease in the relative humidity of the air. Two types of drought can be distinguished: pedological and atmospheric. Drought in the majority of the cases starts in the form of atmospheric drought and then, as the ground water is consumed, it turns into pedological drought. Depending on the season, three types of drought are distinguished: spring, summer and autumn. Drought may be temporary or long-term. Temporary drought, although acting for a short period, can be very destructive for plants, especially when there is water deficit in the soil. Plant transpiration in such conditions is much more intense than the absorption of water by roots and, as a result, an imbalance is created in the hydrological regime. When transpiration exceeds water absorption an acute shortage of water forms very soon causing plants to wither, thus, tending to reduce transpiration rates. Therefore, the first symptoms of drought are wilting, loss of cell and tissue turgidity, yellowing of the leaves (Fig. 10.7). Plant wilting may be temporary or permanent. Temporary wilting occurs during hot periods, during atmospheric drought. Permanent wilting occurs when pedological and atmospheric drought are simultaneous. If the water deficit is not compensated during the night and the turgor pressure of the cells is not restored competition for water begins between different organs of the plant. It was found that drought affects physiological and biochemical processes in plants, determining the physical and chemical properties of the protoplasm colloids. As a result, the permeability of biological membranes decrease and metabolic processes reduce in intensity. During drought plants first lose the free water, then the bound water. Water loss leads to more intense respiration and blocking of the photosynthesis process (Fig. 10.8). Numerous studies have shown that in drought conditions the rates of protein hydrolysis increase, the processes of carbohydrate biosynthesis and phosphorylation are destabilized; enzyme activity is blocked all these leading to an impact on the ongoing morphogenetic processes. Slowing down the processes of carbohydrate biosynthesis in leaves has a negative impact on certain stages of photosynthesis (in some plants photosynthesis intensity may drop by 40–70 %).

10.4

Plant Resistance to Drought

Fig. 10.7 Drought resistance for the 35S: STZ phenotypes and for the wild lines of Arabidopsis a transgenic and control plants grown in pots on agar medium for 21 days. Plants transformed with the pBI121 vector were used as control; b plants grown in soil for 36 days; c STZ gene expression in control and transgenic plants. 20 µg of RNA extracted from Arabidopsis plants of 3 weeks age were introduced in each of them, d resistance of transgenic and control plants to pesticides. The 35S:STZa and 35S:STZb phenotypes showed an increased resistance to pesticide stress; e the difference in recovery after rehydration between control plants, 35S:STZa and 35S:STZb; f electrolyte leakage was assessed after the dehydration event. 17 day old plants were used in each experiment. Plants were removed from the agar and dehydrated in plastic dishes for 4 h

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Fig. 10.8 Plant organism response to water deficit (Bohnert et al. 1995). Under the influence of drought various cellular processes can be affected. These changes allow the plant to maintain metabolism and restore the conditions that allow continued growth under stress

The increase in respiration intensity is one of the mechanisms of adaptation to drought. During respiration, metabolic water is synthesized, which in drought conditions can constitute up to 13 % of the total water content. Different plant species are characterized by different resistance to drought (Figs. 10.7, 10.9 and 10.10) at certain critical stages. For most crop species (e.g. cereals) the critical stage is considered the reproductive phase, which begins with the formation of the reproductive organs and ends with fertilization. The sensitivity of the various organs of the plants to drought is also different. More susceptible to dehydration are the flowers and leaves from the lower region of the plant. The upper leaves can extract water from the bottom ones and from the fructification organs. Therefore drought causes floral primordia extinction, whereas if it acts at later stages of ontogeny it leads to the formation of small, wrinkled

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Fig. 10.9 Transgenic tomato plants resistant to water shortage (Tsai-Hung et al. Tsai-Hung et al. 2002). The wild-type control and three T1 transgenic plants (M, C5, C15 and C21) were grown at 24 °C without being watered for 21 days. The leaves of the wild forms wilted and have twisted considerably. To test survival, the control (M) and the three transgenic plants T1 (C5, C15 and C21) were grown at 24 °C without watering for 28 days. The number of surviving plants to the total number of plants tested is indicated in the middle of the figure

seeds, small fruits etc. Long-term drought causes cytorrhysis (strong dehydration of the cytoplasm) which destroys the integrity of the cell. For many years it was thought that water is available to plants, as long as, the soil moisture does not reach the wilting coefficient beyond which water remains inaccessible. According to this conception, physiological processes, plant growth and development proceed normally until reaching the wilting coefficient of the soil. But recent scientific research has shown that the exchange of substances is influenced even by a small shortage of water. Such a deficit occurs inside tissues and cells long before soil moisture reaches the wilting coefficient. Water scarcity in plants acts on a range of physiological and biochemical processes such as water uptake, root pressure, seed germination, stomata movements, transpiration, photosynthesis, respiration, general enzymatic activity, etc. Plants that had a strong water deficit even for a short period of time do not return to a normal exchange of substances. The water balance and the degree of affection depend on a number of internal and external factors related to the type of plant (drought resistance, the depth of the root system penetration into the soil and its branching, the stage of development), the number of plants per unit of area, weather conditions (temperature and air humidity, wind, light, amount of atmospheric

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Fig. 10.10 Northern-blot analysis of transgenic tomato plants (Tsai-Hung et al. 2002) Total RNA (10 µg) was extracted from control plants (M, track 1) and from T1 transgenic plants which are characterized by an exaggerated expression of CBF1 (C repeat/dehydration-responsive element binding factor 1) (tracks 2–9). The used cDNA samples (Arabidopsis CBF1, the GUS reporter gene from pCAMBIA 2301, CAT1 from tomato and β-TUBULIN, rRNA) were labeled with P32

precipitation), soil-related factors (the quantity of water in the soil, the osmotic pressure of the soil solution, the physical properties and composition of the soil). The influence of the water deficit on metabolic processes depends largely on the duration of drought action. Thus, in cereals a long withering period increases the rate of RNA and protein degradation, which is leading to an increased amount of nitrogen-containing non-protein organic compounds and their deposition in the stem and the ear. As a result, during drought there is an increase in the quantity of protein in seeds and a decrease of their content in leaves. Water scarcity also affects substantially the exchange of carbon, lowering the intensity of photosynthesis and reducing the amount of synthesized ATP during photophosphorylation. Under the action of pedological and atmospheric drought, the processes of translocation of photosynthesis products in other organs are slowed down. Drought, be it long or short, ultimately leads to lower plant productivity. In the process of evolution, a group of plants has formed with a number of adaptations to drought represented by false xerophytes (ephemeral and ephemeroids). To combat the effects of drought it is required to plant forest belts in dry areas, to create resistant varieties, able to adapt to drought conditions and to practice irrigation. In order to increase plant resistance to drought, the method proposed by P.A. Henkel is used, which consists in drying the swollen seeds due to which the emerging plants will acquire xeromorphic qualities. An important role in increasing plant resistance to drought, as shown by K.A. Timireazev, is played by mineral fertilizers. Administration of phosphorus during sowing, of nitrogen during tillering, and of K, B, and Cu during the critical period significantly increases plant resistance to water scarcity. Under the influence

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of B, Mn, Cu, Co, Mo, Zn the hydrophilic protein colloid content increases, as well as their hydration degree, the viscosity of the protoplasm becomes higher and the amount of “bound water”. It was demonstrated that Zn and B may favor the formation of organic acids, which help fixate the ammonia produced during drought, converting it into a harmless form. Microelements contribute to the formation of phosphorylated compounds, which is of great importance, because the phosphorylation processes in plants slow down during drought.

10.4.1 Physiological Basis of Irrigation Plant growth depends largely on the amount of water available for the plants in the soil. Approximately one third of the land area is occupied by regions with essential moisture deficit, half of which, or about 12 % of Earth’s land is very dry. Regions with moisture excess occupy about 9 % of the surface of the land. If the amount of annual rainfall is greater than the amount of evaporated water then the area is a humid area and if intense water evaporation takes place and the rainfall is low, the area is called arid. Many agricultural regions are located in arid zones and farming here is possible only by practicing artificial irrigation. At the moment there is a system of measures to combat the drought, one of the most important being artificial irrigation, which involves also administration of mineral elements in different climatic zones. However, for an efficient use of water resources it is important to know the physiological parameters of the crop species that characterize their water regime. One of the problems of irrigation is to determine the maximum and minimum humidity allowable levels. The upper limit of allowable humidity is called soil humidity. Exceeding the allowable upper limit of soil humidity is inadmissible because excess water is not useful for plants, but on the contrary, is harmful because it creates a lack of oxygen. As the soil dries up until it reaches the humidity at which permanent wilting occurs plants suffer increasingly from water scarcity. The humidity of constant wilting cannot be used as an index for starting irrigation, as permanent wilting leads to a water deficit in tissues and therefore decreases the productivity of plants. Determination of the optimum levels of soil moisture, which do not cause physiological disorders—the upper limit of optimum humidity, is important for controlling the processes of plant growth and development. In practical terms, however, it is very difficult to know these parameters. For this purpose, various methods for their determination have been developed starting with measurements of leaf saturation with water, of leaf osmotic pressure, of stomata opening, of the generated suction force. Nowadays, for different crops the values are known for the critical suction force of the leaf, which correlates with the state of the plant that still does not cause crop yield reduction. Identification and assessment of physiological indices for determining the forms and limits of more accurate and economical irrigation is an

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element of modern agriculture, however, although irrigation ensures stable harvest of the crop, currently only 16 % of the world farmland surface are irrigated.

10.5 Plant Resistance to Saltiness Currently, about 20 % of the cultivated areas and about half of the world’s arable land are marked by salinization. According to data published by the International Society for Soil Science, saline soils occupy an area of 3 million km2 only in Europe and Australia. Soils characterized by an excessive concentration of salt, may adversely affect or even destroy the flora. In addition, improper irrigation is leading to salinization of the soils. A harmful effect characterized by high concentrations of salt occurs after the application of excessive doses of mineral fertilizers. Salts accumulate in the ground, as in dry regions the soil is not washed enough by water to carry the salts in the deeper layers of the ground. On the other hand, salts can be brought to the surface by the upward flow of water in the process of evaporation. These soils have a low fertility potential and the salt excess has many negative effects, including: • increases the osmotic pressure of the soil solution making water absorption more difficult; • inhibits imbibition and germination of the seeds; • inhibits the growth of the root system; • has a toxic influence on the protoplasm; • inhibits anabolic processes—photosynthesis and protein synthesis; • prevents starch formation in somatic cells; • causes oversaturation of the root system cells with salts, which leads to loss of selectivity in the process of ion absorption, the later entering passively into the cell in order to be accumulated in abundance in the vacuole. In 1898 it was developed a theory according to which the action of salts on plants consists in creating a “physiological drought” because the osmotic pressure in plant cells is 15–20 atm compared to 30–50 atm in the soil. Plants can’t absorb water, so these soils are called dry. Salinization can be determined by the presence of: sulphate (Na2SO4), chloride (NaCl), carbonate (Na2CO3) or it can be combined (Fig. 10.11). A high saline content in the soil leads to the perturbation of the metabolic processes. For example, it has shown that, under the action of salt excess, in plants, the exchange of nitrogen is disturbed, leading to the accumulation of ammonia and other toxic products. The most harmful action on plants is played by the excess of carbonates as these degrade in the soil forming strong bases (NaOH). An abundance of sulphates in the soil leads to the accumulation of sulfur oxidation products that can be toxic to plants. A high concentration of salts, in particular chlorides, can disrupt the process of oxidation and phosphorylation i.e. plant supply with energy.

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Fig. 10.11 Types of salinization

Fig. 10.12 ST overexpression (salinity resistance gene) increases resistance to salinity. The picture shows germination of the control and transgenic ST genotypes. Four-day seedlings were transferred to a fresh medium supplemented with 0, 50 and 100 mM NaCl and grown for 10 days (a). Seedling root growth in the basic medium supplemented with NaCl has been established in control samples and transgenic ST samples after 10 days (b). The white bar control samples; black bar transgenic ST plants

Resistance to salinity is determined by a whole family of genes, which, in case of incorporation by genetic engineering techniques into different genotypes, may significantly increase this feature (Fig. 10.12). Morpho-physiological changes resulting from excess salinity are: • disruption of chloroplast submicroscopic structure, leading to etiolated leaves; • reduction of the leaf area; • thickening of the cuticle and palisade tissue in leaves under the action of chlorides and sulphates; • increased succulence of the tissues; • a reduction in the diameter and height of the stem and its lignification. According to their reaction to salts, plants are classified into: • glycophytes, plants that can’t withstand saline soils; • halophytes plants that during phylogenesis have developed various adaptations that allow them to withstand high concentrations of salts.

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Depending on the mechanisms of detoxification and elimination of salts by plants, halophytes are divided into three groups: (1) Euhalophytes (Suaeda, Salsola, Salicornia). Succulent plants with increased resistance to salinity. The cells of these plants have the ability to withstand high salt concentrations due to the fact that the cells have a high osmotic pressure (about 100 atm), allowing them to absorb water from the saline soil. (2) Crinohalophytes (Statice gmelini, Tamarix gallica). These plants absorb salts from the soil, but do not accumulate them in the cell sap but eliminate them through the pores. An increased intensity of photosynthesis characteristic for these plants leads to an increased salt concentration in the cell sap and enables water absorption in soils with a high water retention capacity. (3) Glycohalophytes (Artemisia maritima, Artemisia salina). The cytoplasm of the root cells is impermeable to salts and they do not penetrate the cell. The high osmotic pressure in cells allows water absorption due to the accumulation of soluble sugars and organic acids. All these features of halophytes are genetically determined and occur only in saline soils. Among the cultivated species cotton and partially sugar beet have such properties. According to the degree of salinity resistance crop species are classified as follows: • salt-sensitive plants: peas, beans, rice, corn, flax, potato, buckwheat, oats, cucumber, radish, carrot, garlic, clover, alfalfa, timothy, fruit trees; • plants with average resistance: rye, wheat, barley, millet, cotton, sunflower, cabbage, soybean, tomato plant, couch grass, grass-of-Sudan; • salt resistant plants: some varieties of barley, sorghum, sugar beet, fodder beet, pumpkin, eggplant, kale. Plant resistance to salt is not a static trait, but an entire organism reaction to the unfavorable conditions of life on saline soils. In order to improve crop plant resistance to high concentrations of salts in the soil various methods may be used which improve both the chemical and colloidal characteristics of the cell protoplasm and the physicochemical properties of the soil. Depending on the type and amount of salt the following are used: • gypsum and phosphogypsum on saline and alkaline soils with strong alkaline reaction; • finely ground limestone (calcium carbonate) and quicklime (calcium oxide) in pulverized state on saline soils; • organic fertilizers—vegetal remains, manure and other to improve the physicochemical, mechanical, chemical and biological properties of the soils; • physiologically acidic fertilizers: (NH4)2SO4, ammonia water (NH4OH), urea; • the administration of microelements (Al, Mn, Cu, B, Zn) in order to modify the permeability of the plasma membrane of absorbent cells; • treatment of the seeds with salt solutions of increasing concentration (method developed by Henkel).

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Salt resistance can be increased by using of specific hardening methods: germinated seeds are kept for an hour in 3 % NaCl solution and then washed. Such seeds have a lower exchange of substances, but possess a higher resistance to salt.

10.6 Regulation of Physiological Processes in Halophyte Plants About 25 % of terrestrial plants and 95 % of aquatic plants are living in saline environments. Saline soils create unfavorable conditions of life for most plants, leading to the formation of specific communities composed of halophyte species. Plant evolution with regard to salinity resistance followed the path of diversification of mechanisms and specialization of ionic regulation, some halophytes having the ability to accumulate up to 50 % of salts in their dry mass without toxic effects. Halophytic microorganisms adapted to the excess of salts by adjusting the entrance of the ions into the cells. Ion adaptation of the microorganisms is characterized by the fact that the cell osmotic potential is almost indistinguishable from that of the external environment, allowing them to live in conditions of salinity of 3–5 M. Synthesis of DNA, RNA, ATP, proteins etc. in halophytes and glycophytes happens in the same manner, however, halophyte enzymes work better in the presence of salts, whereas glycophyte enzymatic activity is inhibited. Halobacteria enzymes are different from those of glycobacteria by the amino acid content. For example, glycobacteria have enzymes with a high content of basic amino acids, while in halobacteria acidic amino acids are dominating. Terrestrial higher plants come in contact with the external saline environment just through the root system. But during the evolution genetic changes of the organism, organs, tissues and cells involved in regulating the speed of ion transport have occurred. An internal environment with a higher ion concentration in comparison with the external environment has formed. The adaptation of plants to salt is related to the phenomenon of intracellular and intercellular ionic regulation, through the conducting vessels and intercellular spaces. During development two groups of halophyte plants appeared with specific adaptations to salinity: • obligatory halophytes, with a reduced leaf blade, with succulent stems, which accumulate large amounts of salts; • facultative halophytes, which differ from the first by the amount of salt accumulated (5–20 times lower). Higher plants differ from halobacteria by specific physiological mechanisms involved in maintaining the homeostasis. Avoiding salt toxicity due to absorption and concentration of ions in the vacuoles. Plants exposed to high salt concentrations in the soil at some point suffer

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from physiological drought. Under these conditions the absorption of ions from the external environment is regulated by active transport mechanisms localized in the cell membranes, but the control is relative. Thus, if in the external environment toxic ions are present, they will be partially absorbed. It was established that with an increasing soil salinity the absorption of Na+ ions and Cl− increased and of K+ and Ca2+ decreased. Increasing the osmotic pressure due to the accumulation of ions through pinocytosis. An adaptive process in plants is the formation of pinocytotic vesicles in root and cotyledonary cells of the halophytes facilitating an increase in the osmotic pressure. It was proved experimentally in barley seedlings that, under the influence of NaCl, pinocitotic vesicles appear that stimulate Na+ accumulation and K+ efflux, increasing the Na/K ratio 100 times, while the Na/K ratio in halophytes was 5 times higher. By invagination of the plasmalemma and vesicle formation in the cytoplasm the compartmentalization of the high salt concentrations occurs, thus protecting the enzymes from toxicity. Increasing the resistance of the enzymatic system. Homeostasis maintenance in halophytes is determined by increasing the resistance of the enzymatic system that preserves its high activity under the presence of relatively high concentration of toxic salts in the cell. It was found that enzymes isolated from halophytic plants slow their work under the action of concentrated salt solutions, while enzymes isolated from glycophytic plants completely lose enzymatic activity. Thus, ATPases and malate dehydrogenases of halophyte species under high salt conditions had higher levels of activity and varieties of the haloresistant Pennisetum americanum had an elevated activity of their NAD-nitrate reductase, NADP-glutamate dehydrogenase, of the enzymes of glutamine and glutamate synthetase in comparison with the same enzymes from salt sensitive species. (The high enzymatic activity of malate dehydrogenase at high concentrations of NaCl reflects some structural adaptations of the enzyme molecule.) Increasing succulence represents a way to adapt to salinity. It was established that succulent halophytes have high selectivity for chlorine absorption, which accumulates in the intercellular space (30–35 %). Experimental work has shown that NaCl slows down pectinesterase activity in Aster tripolium and salt is retained by methylated pectic substances, increasing the plasticity of the cell wall and facilitating cell elongation. Increasing the osmotic pressure by accumulating organic substances. Halophytes for which the accumulation of salts is toxic and whose roots have a low permeability for salts adapted to high salinity by increasing the concentration of the vacuolar sap, accumulating active osmotic organic compounds—carbohydrates and organic acids, organic nitrogen in the form of glycine, beatin and proline which are involved in osmotic regulation and act as protective substances. Modification of protein biosynthesis. Under the influence of high salt concentrations protein synthesis is altered and accumulation of high molecular weight proteins is enhanced as a measure of adaptation. The content of alkaline soluble proteins and water increases in plants under high salinity conditions, which plays a protective role. Metabolism rates decrease.

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Plasmodesmata disorganization. Under saline conditions, in halophytes, plasmodesmata disorganization and therefore cell communication disruption can be observed. At the same time, the passive permeability of the tissue increases as well as the volume of intercellular spaces as a result of the detachment of the protoplast from the cell envelope, while cells accumulate a large amount of free ions. Salt secretion. Most halophyte adaptation mechanisms are based on avoiding ion toxicity by removing them through salt glands and vesicular hairs. Secretion intensity is very high and secretions from vesicular hairs contain Na+, K+, Mg2+, Ca2+ ions, CI−, NO3−, HCO3−, SO42− anions and some cellular metabolites. Highly selective absorption and cellular tolerance to high ionic concentrations. Halophytes are characterized by an ascending transport of Na+ ions and their more intense absorption compared to K+ ions whereas glycophytes—on the contrary. In halophytic plants the process of adaptation to the high concentration of salts is carried out by their entrance and compartmentalization in certain regions of the cell (vacuoles, plastids), thereby protecting the areas with high metabolic activity. It has been found that the speed of osmotic adjustment is 4–20 times higher in halophytes than in glycophytes while the concentration of ions in the “living” cytoplasm is 1/3 compared to the vacuolar ion concentration. Halophytes accumulate salt ions in tissues that do not have a great metabolic load while in tissues with highly important metabolic functions they have a low salt content. Active transport of substances due to the energy supplied by the proton pumps. Selective adjustment of ions in the cellular sap is determined by the energetic processes involved in active transport, which are carried out against the concentration gradient. All active processes of substance accumulation in living systems occur with the involvement of ionic pumps and the membrane electrochemical gradient. The high activity of ATP-ases from halophyte cell membranes is considered as a protective mechanism. Glycophyte plants suffer some anatomical, morphological and physiological changes that occur as an adaptation reaction to the living conditions created by the saline environment such as foliar surface reduction, cuticle and palisade tissue thickening, changing the size and distribution of stomatal cells, alteration of the chloroplast structure (appearance of ethyolation), increased succulence of the tissues (on soils with chloride), increased nuclear volume (which acquires an amoeboid form), reduced height and stem diameter, a decreased number of cell layers of the root cortical parenchyma. Under the influence of salinity the leaf blade is thickening, the degree of stomatal opening increases as well as the amount of wax on the surface of the leaves. Auxins and the abscisic acid inhibits the flow of sodium at the level of the plasmalemma by blocking parts of the system of Na+/K+ exchange. This causes a slowdown of the secondary protein synthesis, modifying the degree of colloid hydration, the viscosity, elasticity of the protoplasm, reducing the percentage of free water, increasing respiration intensity, modifying the content of pigments, reducing the intensity of photosynthesis, retaining leaf growth. Obtaining plants resistant to high concentrations of salt would allow to both increase plant productivity and to expand the cultivated area. Recently, it was

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shown that the genes encoding vacuolar Na+/H+ antiporters increased salinity resistance of Arabidopsis plants, cabbage, tomato, rice. Currently investigations are carried out in order to obtain transgenic wheat plants resistant to salinity by transferring the Na+/H+ gene to the vacuolar antiporter HNHX cloned from the barley genome.

10.7 Plant Resistance to Environmental Pollution Currently the global public attention is drawn to the rate of ecological situation worsening. Various issues such as continuing degradation of the environment, reduction of plant and animal species number, decreased soil fertility, increased content of greenhouse gases in the atmosphere, the occurrence of acid rains, climate change, contamination of water basins, food contamination, loss of food security requires development of a penalty system and urgent action, integrated and efficient to solve global problems of the mankind, reducing and eliminating the negative consequences of human impact on ecological systems. This requires the creation of efficient mechanisms to minimize the negative impact of the economic activity on ecological systems, the development of measures related to the implementation of environmental management to restore ecological balance. Pollution is characteristic to the modern, chemically intensive, mechanized agriculture while air pollution is a common phenomenon in large cities and surrounding areas. It has been found that in countries with a highly developed industry air is polluted by transport (up to 50 %), by heating systems (18 %), by industrial production (18 %). Considerable damages are caused by chemicals used in modern agriculture, that don’t have analogy in nature and affect large areas. Environmental disturbances caused by pollutants are manifested by decreasing the number and diversity of plant and animal species by ecosystem reduction in size and degradation, disruption of food chains, respectively, of the biocoenosis. On the other hand, there is an uncontrolled multiplication of organisms (some insects, microorganisms) that adapt easily to these conditions due to the mechanisms of resistance they possess. Living organisms suffer directly or indirectly due to enhanced pollution of the living environment. Thus the problem of environmental protection requires a thorough study of the influence of different types of pollution on living organisms. In this context it becomes particularly important to study the properties and directions of the ongoing changes in natural systems, to unveil the adaptation reactions and research the resistance mechanisms to unfavorable factors exhibited by living organisms. Plants represent open systems that are in constant exchange of matter and energy with the environment, they respond appropriately to adverse conditions while the physiological and biochemical processes are perfectly coordinated with the ambient factors. At the same time, pollutants generate in plants the so-called stress conditions that represent the alteration of the processes of growth

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and development, of photosynthesis, respiration, hormonal activity and other processes regulated at the molecular level. The higher the degree of environment pollution the more pronounced are the disorders in cellular metabolism. So, the existence of living organisms in unfavorable environmental conditions is determined by the resistance and ability to adapt. It is known that plants, through specific biochemical reactions, are incorporating, metabolizing and partially detoxify pollutants, thereby helping to reduce the effects of environmental pollution. However, food contaminated with pollutants is a major risk factor and the continuous negative influence of the polluted environment on human health and the biosphere as a whole imposes a monitoring of the environmental conditions to foresee and prevent the consequences caused by anthropogenic changes, to solve problems of waste recycling, residual water treatment, to reduce the amount of toxic substances in food, to minimize the risk posed by the polluted environment on human health and the ecosystems. Solving these problems is possible only on the basis of deep theoretical studies and is indissolubly linked to the formation of a new green mentality. Pollutants affect all three components of the environment—water, air and soil. The composition of the atmospheric air is not constant. Currently this composition has altered a lot due to the industrial development. As a result of human activities over 200 chemical compounds are eliminated into the atmosphere, including SO2, NO, NO2, CO, vapors of acids, phenols, ash particles, dust, smoke, oxides containing toxic heavy metals, etc. Depending on the chemical composition, the pollutants can be divided into: • acid gases very toxic for plants (fluorine, chlorine, oxides of sulfur, oxides of nitrogen, carbon monoxide, phosphorus oxide, hydrogen sulfide, etc.); • vapors of acids (hydrochloric acid, sulfuric acid, nitric acid, organic acids); • metal oxides (lead, zinc, selenium, manganese oxides etc.); • alkaline bases (ammonia); • mercury vapors; • various organic gases and carcinogens (saturated and unsaturated hydrocarbons, phenols, etc.). According to particle size, sedimentation rate and electromagnetic spectrum, chemical compounds that pollute the atmosphere are classified as: dust, fumes, fog and smoke. Vapors and gases enter the plant easily through the ostioles and directly affect cell metabolism, chemically interacting at the level of the cell wall and cell membrane. Very toxic are the acid gases which acidify the cell cytoplasm permanently, alter the activity of the membrane transport systems and contribute to the accumulation of different elements (Ca, Zn, Pb, Cu, etc.). Plants resistant to toxic gases after their penetration through ostioles, integrate them in the exchange of substances, thereby detoxifying them. However other plants close their ostioles, thus regulating leaf gas penetration. Dust, penetrates the leaf surface, clogs the ostioles and thus disturbs gas exchange, transpiration and light absorption. Air

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pollution leads to a decrease in respiration intensity, has a negative influence on the soil microflora and other negative effects. The soil is the main source of mineral nutrition for plants and its pollution (physical, chemical, biological, etc.) leads to the alteration of its functions. The constantly increasing use of pesticides and nitrogen fertilizers causes soil pollution with heavy metals and nitrates, increasing their content in plants and vegetal food products. Groundwater is also subjected to pollution with chemical fertilizers and pesticides, applied improperly in agriculture and are washed into the deeper layers of the soil by rainfall.

10.7.1 Resistance to Heavy Metals Resistance of plants to heavy metals is provided by the molecular and physiological mechanisms that depend on the nature of the substances and on the peculiarities of the species. Tolerance to heavy metals is ensured by preventing the penetration of the metals into the cell and by intracellular mechanisms for heavy metal detoxification (Fig. 10.13).

Fig. 10.13 Influence of heavy metals (Cd) on the cellular redox control (Schutzendubel and Polle 2002)

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Avoidance of heavy metal ion penetration into the plant cell is based on the immobilization of heavy metal ions by the cell wall and removing the cell ligands that act like chelating agents for metals. As a result, the plant avoids the toxic action of heavy metals on intracellular processes. Immobilization and significant accumulation of heavy metals happens in the free space of the cell wall or by binding metals to specific sites of the extracellular matrix. Thus, in Agrostis tenuis, a plant tolerant to Zn, there is a direct correlation between the ability of the cell wall to bind metal, and the degree of resistance to the metal. Cultivation of Oryza sativa on media with toxic concentrations of Cd and Zn and of Lupinus luteus on media with high concentration of Cu and Pb led to the accumulation of these metals namely in the cell wall. The accumulation of metal ions in the free space of the cell wall is determined by the ion exchange coefficient, which depends largely on the number of histidine groups in proteins and the number of carboxyl groups on the surface of pectic substances from the cell wall. It is known that pectins play an important role in the regulation of nutrient entry into the cell, including microelements. Carboxyl groups create charges on the surface of pectins, which allows the retention of metal ions. The higher the ion exchange coefficient, the more likely are toxic ions to be retained in the gap of the cell wall, and vice versa. The efficiency of heavy metal immobilization by the cell wall is also dependent on the decrease of their activity after formation of stable chemical bonds with some substances in the cell. It is assumed that heavy metals are bound by specific proteins of the cell wall. The plasma membrane serves as a selective barrier for heavy metal ions to enter the cell. It can play an important role in the formation of plant resistance to heavy metals, completely blocking the penetration of toxic ions into the cell. The mechanisms involved in heavy metal detoxification and removal of these ions from the cytoplasm is based on the property of organic compounds to bind these metals. Plant tolerance to heavy metals may be provided by mechanisms that participate in heavy metal detoxification and elimination from the cytoplasm, in allowing plant cells to function normally in the presence of heavy metals or in ensuring rapid restoration of damage caused by heavy metals. Metal detoxification may be carried out by using organic acids, metallothioneins, phytochelatins, glutathione and ferritins. Also to this type of mechanism is related the active removal of heavy metal ions from the cell and appearance of tonoplast transferases, capable to transport quickly metal ions from the cytoplasm into the vacuole. All these mechanisms participate directly in binding or removing metal ions from the cytoplasm.

10.7.2 Resistance to Radiation Cosmic and terrestrial radiation on Earth existed long before the appearance of life. The sum of all such cosmic and terrestrial irradiation form the global radiation background.

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In nature two types of radioactive pollution occur: • natural, represented by the radiation emitted by radioactive elements (Ur, Ra, Po, Tr, Ac), components of the earth’s crust; • artificial, resulting from nuclear fuel production, chemical processing and metallurgical materials in reactors, radioactive element use in industry, medicine, research and nuclear explosions. Moreover, the use of nuclear energy, of nuclear power brings with it increased pollution through improper handling and storage of radioactive waste. It was found that radioactive residues accumulate in the stratosphere, concentrating due to air currents in the northern temperate zone, where 80 % of the world population live. The amount of natural radiation to which an individual is exposed is typically 70–200 mrem/year ≈ 3 rem/generation the ranging from minimum values at the sea level and maximum values at high altitude. There are, however, areas with soil which is richer in radioactive elements. A resident of the volcanic regions of Brazil receives on average 1,600 mrem/year, Hindus inhabitants of some coastal regions of India ≈ 1,300 mrem/year. Radioactivity is the quality of radioactive isotopes to emit radiation (particles and energy) spontaneously from the nucleus, which leads to the formation of atoms of other elements or isotopes of the original element. The phenomenon of radioactivity was discovered by Becquerel, who noticed that the substances containing uranium emit invisible rays. Later Marie and Pierre Curie established that similar rays are emitted by all the substances containing uranium and thorium. In the uranium ore they discovered two new radioactive elements Po and Ra. The effects of radiation pollution are felt in the air, water, soil, while radioactive particles can accumulate in food chains, influencing living organisms. Pollution can be direct, as a result of the interaction of the radiation with biological tissues, by modifying the composition and structure of the matter, sometimes associated with genetic mutations in the DNA, or indirect, when the biological structure is not affected, only the habitat. According to their sensitivity to treatment with ionizing radiation, the representatives of gymnosperms are radiosensitive plants while angiosperms contain both sensitive and less sensitive groups. In monocots, all orders, families, genera and species studied were found to be moderately radiosensitive and resistant. In general, it was found that phanerophytes are most susceptible, the terrophytes are the most radioresistant, talophytes and bryophytes are less sensitive to radiation than woody species. Blue algae are the most radioresistant plants on earth, with LD50 = 1200 kRad. It was found that only lower plants (Cladonia, Parmelia) survive a daily level of irradiation of 1000 R, while among higher plants only isolated specimens of herbaceous or shrub plants survive after 1 year of exposure to 200 R/day. Differences in sensitivity to radiation occur not only between different species but also within the same species, with different sensitivity levels attested in different varieties and even individuals. There is also different radiosensitivity depending on gender. For example, a high radiosensitivity characterizes in particular the uninucleate stage of the male gametophyte. The root system is less sensitive to radiation,

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as compared to the aerial parts of the plant and the lateral roots are more sensitive than the main, old leaves are more resistant than younger and so on, while the pollen of the plants is more resistant than the oosphere, withstanding very high doses (125–800 kR) of irradiation without losing germination capacity. At the tissue level, meristematic cells and tissues, including those that divide intensely are more sensitive than differentiated cells and tissues that divide slowly. Specialized cells, such as stomata, are more resistant than the epidermis and the latter, in turn, has a higher radioresistance than the meristematic tissue. A different response to irradiation is recorded even in cellular organelles and structures. The nucleus is more sensitive to radiation than the cytoplasm and mitochondria. From the cell organelles, the strongest are the chloroplasts, which undergo reduced changes even at lethal doses. Radiosensitivity depends of the specificity of the irradiated biological material and is determined by the seed size, the volume of the nucleus, the genetic nature of the organism, size and number of chromosomes, DNA content and functional condition, metabolic peculiarities of the species, the efficiency of the reparation systems etc. The morphological and anatomical peculiarities mentioned above as well as the physiological and biochemical particularities of the vegetal organisms are causing the differences in the radiosensitivity among different species. Plant radiosensitivity depends largely on the chemical composition of the cell, the metabolic particularities of certain substances. Among the biochemical components that provide increased plant resistance to radiation are: fats, ascorbic acid, compounds with sulfide groups, growth substances, pigments “a”, oxidoreductases (peroxidase and catalase), etc. Radioresistance is a genetically determined character, whose level of expression depends on the internal conditions of the organism as well as on the environmental conditions before and after irradiation (Fig. 10.14).

Fig. 10.14 Specific staining of lignin from the leaf of a mutant line of duckweed resistant to UV radiation-mTR and sensitive to UV-PL 3FL7-11 (Jansen et al. 2001) phloroglucinol/HCI was infiltrated in the branch with the leaves and it was studied under a light microscope. Mesophyll cell walls are colored in red-orange.

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Enzymatic systems of restoration and DNA repair find the region damaged by radiation, destroy it and restore the integrity of the DNA molecule. At the cellular level there are some radioprotective substances: glutathione, cysteine, ascorbic acid, metal ions and a number of enzymes and cofactors (catalase, peroxidase, polyphenol oxidase, cytochromes C, NAD) which protect cells from the damaging action of the radiation. The function of radioprotective substances is to neutralize free radicals arising from the action of the radiation, to create local O2 insufficiency or to block reactions involving derivatives of radiochemical processes. At the level of the organism restoration is provided by: (a) the heterogeneity of the population of meristematic cells; (b) asynchronous division in meristems, so that at any time cells are involved in different mitotic stages characterized by different radioresistance; (c) the presence in apical meristems of the mitotic background similar to the latent center, which starts to divide very actively when division of the basic meristem stops which leads to the regeneration of both the original cells and the meristem (d) presence of latent meristems of the type of dormant buds which in the case of apical meristem destruction begin to function actively and repair lesions. Protection and repair mechanisms are not specific for plants only and hence, the importance of studying them for solving the problem of radioresistance in both plants and other living organisms.

10.7.3 Resistance to Gases Among the other types of resistance an important place has the resistance to high concentrations of gases, which is the ability to maintain vital processes of plants under the action of harmful gases (Polevoy 1982). The degree of plant resistance to gases varies depending on the physical, geographical and meteorological conditions. It is known that the atmosphere composition varied over time along the evolution of the organic world on earth. During abiogenesis Earth’s atmosphere contained a number of compounds toxic for contemporary organisms (methane, ammonia, hydrogen sulfide, carbon monoxide, etc.) which gradually changed with the advent of life and due to other reasons as well. Nowadays, however, with the rapid development of industry, atmospheric composition changes substantially in record time. Presumably, the clearance of the primary earth atmosphere from these gases was performed by early autotrophic plants, which are equipped with the mechanisms of resistance to gas. Possibly, at that time, plants developed specific mechanisms of resistance to toxic gases. Contemporary flora formed in conditions when air pollution has occurred on account of geological and chemical processes. As a result of the decrease of the content of toxic gases, of the increase in the oxygen content of the atmosphere (determining its oxidative character), organisms have partially lost their resistance

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to toxic gases. In the process of development they have adapted to extreme temperatures (heat, cold), lack of water, soil salinity and probably lost resistance to toxic gases. Therefore, contemporary species do not have a specific adaptation to toxic gases. In the process of evolution hereditary physiological and biochemical features have appeared in plants, which indirectly may enhance or attenuate gas resistance. Based on what was said, three types of gas resistance can be highlighted: biological, anatomical-morphological and physiological-biochemical. Biological resistance is the ability of the plants to restore damaged organs after removal of the pollutant. It is determined by the biological characteristics of the species (intensity of morphological, physiological processes of growth and development, ecological plasticity, age, developmental stage, etc.). Morphological and anatomical resistance is determined by structural features of the plants, by presence of the cuticle, the mechanical tissues, etc. Physiological and biochemical resistance is determined by the physical and biochemical properties of the plants (total water content, water retention capacity, vacuolar sap concentration, redox potential, the ability of plants to recruit toxic gases in the exchange substances, detoxifying them). The degree of plant contamination with gases depends on their sensitivity to external environmental factors and the duration of action of the factor and, depending on the nature of the response, plants can be either sensitive or resistant to gases. As mentioned earlier, gas resistant plants have the ability to adjust the penetration rate of toxic gases and to detoxify them. Some resistant plants close their ostioles, thus preventing the penetration and accumulation of various toxic gases in their tissues. As a result, in conditions of air pollution, photosynthesis and the synthetic processes can be maintained at a high level. Resistance to toxic gases can be linked to the amount of cations in the cell (K+, + Na , Ca2+) that are able to neutralize acids. Usually, plants resistant to drought, salts and other stress factors, have a high resistance to gas, possibly due to their ability to regulate the water regime and the ionic composition. Resistance to gases can be increased by optimizing mineral nutrition and hardening the plant material. The hardening is carried out by treating the seeds with weak a solution of sulfuric acid or hydrochloric acid.

10.8 Metabolism of Pollutants in Plants Phytotoxicity of pollutants in the environment is determined by their ability to be metabolized by plants. Therefore, despite the fact that pollutants adversely affect growth, plants continue to grow and develop even in rather toxic environments. This is due to the presence in plant tissues of highly active enzymes (esterases, oxidases, amidases, hydrolases, phosphatases, etc.) and of efficient mechanisms for

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metabolizing, detoxifying, inactivating and eliminating various xenobiotics that have made their way into the cells. Plants play an important role in the process of rehabilitation of the environment. As a result of their vital activities they clean the atmosphere, the water and the soil. The purifying role is due to: • mechanical cleaning of the air, which is achieved by retaining dust by trees, bushes and grass. According to the literature, coniferous species precipitate about 80 % of the polluting particles in the atmosphere. A tree of Populus nigra of an average age precipitates during the growth 44 kg of dust, Salixalba— 34 kg, Acer traxinifolia 30 kg of cement particles and about the same amount of dust. During the vegetation Picea abies absorbs 94 mg/h of SO, per 1 kg of green mass of leaves, Green plants also absorb about 26 % of the sound. Thus, the amount of SO2 absorbed by the plant body is proportional to the amount of sulfur found in the plant. Immediately after absorption the sulfur is found as sulfate, then, after more than 15 days it may be found at a proportion of 2.2 % in thio-amino acids, at 5 % in proteins, at 92 % in sulfates. Thus, plants are able to turn into sulfates about 92 % of the SO2 accumulated in the atmosphere, thus annihilating some of its harmful effects. The amount of sulfur contained in plants can be used to determine the degree of atmospheric pollution with SO2. It is known that the SO42− ion is 30 times less toxic than the SO32− ion; • bacteriological purification of the air that occurs after releasing bactericidal substances. Thus, the microflora deposited on leaves is subjected to a natural sterilization by the simultaneous action of ultraviolet radiation and ozone released by plants, particularly conifers. Ozone has an oxidizing action on organic matter deposited on the leaves, destroying that nutrient substrate material that would facilitate the conservation and development of germs. The leaves and flowers of many plants eliminate volatile phytoncides that destroy some bacteria and pathogenic fungi. Tree species like Betula, Quercus, Tilia, Pinus etc. eliminate substances with bactericidal character for the diphtheria bacillus, bacillus tuberculosis and other pathogenic bacteria; • chemical cleaning of the air, which is mainly related to the activity of plant chlorophyll. Photosynthetic processes create an alternating rhythm of release and absorption of O2 and CO2. In addition, it is known that plants have the property to fix harmful gases from atmosphere. However plants purifying air by various mechanisms may suffer themselves when atmospheric pollutants are in excess. The large amounts of fungicides, gases, acids, etc. act have a harmful effect on plants, causing illness and often death. Especially harmful for plants are sulfur oxides and nitrogen oxides, fluorine, chlorine, ash dust. The toxic SO2 acts on cell protoplasm, harming and destroying it at a concentration of about 1 mg/m3. Ash particles deposited on leaves, obstructs breathing through stomata and also impedes light passing into the tissues of the leaves affecting plant energetic metabolism. On the outside this is manifested by color loss in leaves, which become brown, yellow or red, dry out and fall prematurely.

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Air pollution affects differently the various species of trees and lower plants. Lichens, trees like the elm, the ash tree and the pine are so sensitive to SO2 that may serve as biological indicators of air pollution with this substance. Walnut and apple trees are very sensitive to the harmful substances released into the atmosphere. In these species drying of the tips of the branches and early flower detachment can happen. In Quercus petraea and the black poplar Populus nigro significant defoliation and massive drying of the branches occur. Pelargonium zonale because of its increased sensitivity to pollutants, can serve as an indicator of SO2 in the air by presenting a yellowing of leaves, defoliation, pronounced chlorosis of the leaf blade (at first on the edges, then progressing inwards along the leaf nerve). In Iris germanica, Vitis vinifera, Lotus corniculatus burns on the leaves and leaf necrosis caused by SO2 can be noticed which by reacting with water, produce H2SO3 and H2SO4. Plant species such as tobacco are sensitive to oxidants and leaves of cabbage (Brassica)—to aromatic polycyclic hydrocarbons, as well as lead, iron, chlorine. Plants have an important role in cleaning the soil. Due to the absorption capacity of the root system, a decrease in the content of all types of pollutants in the soil happens. The plants are able to absorb from the soil nitroso compounds, oxides, pesticides, heavy metals, etc. Uptake of pollutants by plants occurs also in the aquatic environment. Aquatic plants are involved in water purification and absorption of biogenic elements, of hydrocarbons, phenols. In the Netherlands Scirpus is widely used for water purification. An active water purifier is Elchornia crassipes, which absorbs salts of heavy metals, insecticides and detergents. Lemna absorbs heavy metals, nitrogen, phosphorus, limiting their spread.

10.9 Biochemical Mechanism of Pollutant Transformation in Plants Transformation of pollutants in glycosides and elimination of xenobiotics is performed by compartmentalization of the metabolites in certain cellular structures (organelles). In the process of metabolizing xenobiotics degradation occurs by means of oxidation, reduction, decarboxylation, hydrolysis, etc. reactions by means of conjugation or biosynthesis reactions. By conjugation reactions some substances (amino acids, glucuronic acids) or some radicals (methyl, acetyl, sulfate, etc.) are introduced in the initial compound. Enzymes that catalyze the metabolism of xenobiotics are localized in the cytoplasm, mitochondria and predominantly in the endoplasmic reticulum (microsomes). Thus, selectivity of herbicide action on different plants is owing to the specific enzyme system found in a particular species. 2,4-dichlorophenoxyacetic acid, also known as the 2,4-D herbicide is absorbed by all sprayed plants. However crops species, especially cereals have the ability to

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Fig. 10.15 The influence of 2,4-dichlorophenoxyacetic acid on cotton seedlings

easily break down the side chain of the herbicide 2,4-D, which leads to its rapid metabolization, whereas weeds are perishing as a result of accumulating the 2,4-D herbicide, not due to its too high toxicity, but rather due to the excessive growth of plants in a short time. Crop species can detoxify from the 2,4-D herbicide either by esterification of its carboxyl group with a monosaccharide either by forming a peptide bond with glutamic acid. Detoxification is achieved also by hydroxylation of the aromatic nucleus, followed by a reaction of glycosidation, or by oxidation of the side chain. If the side chain of the herbicide is removed, it loses its hormonal activity and 2,4dichlorophenol is formed as an intermediate product, which by means of natural glycosidation turns into the corresponding glycoside, which is not toxic for plants (Fig. 10.15). The xenobiotic transformation mechanism includes three main stages in the detoxification of herbicides, insecticides and fungicides. Phase I—at this stage reactions of degradation occur and of inclusion of new functional groups in the initial structures, as a result of which the physical activity changes as well as the hydrophilic or hydrophobic properties, the mobility of substances, which reflects their ability to pass through the membrane and the localization of the xenobiotics. Primary reactions are carried out with a very high speed. As a result of these reactions modified molecules have one or more groups with high reactivity (−OH, −SH, −COOH, −sNH). Inclusion of new functional groups allows further conjugation of the formed metabolites with other endogenous substances from plants. Phase II (conjugation)—primary products are subjected to glycosidation, esterification, conjugation with amino acids etc. The conjugation reactions most frequently involve glucose. Reactions are produced by energy consumption in the presence of uridine diphosphoglucose (UDPglucose), under the action of glycosyltransferases. Depending on the nature of the groups which reacted, O-glycosides, N-glycosides, and S-glycosides result. Typically, glycosides and in particular the O-glycosides are highly labile substances in vitro. In this context, their prolonged storage in plants would be impossible without localizing them in cell compartments, where they are protected from the action of hydrolytic enzymes. At the same time, by compartmentalizing the conjugates of the xenobiotics, their action on the metabolic processes of the cell is

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blocked. For example: through glycosidation, water solubility of conjugated substances increases, which allows vacuolar storage of the formed glycosides in the vacuolar sap. Phase III—in some cases polymerization or other metabolite alterations that lead to toxin inactivation by formation of insoluble compounds. The result of xenobiotic metabolization may be the detoxification (inactivation) or, on the contrary, an increase in the toxicity (activation). Therefore, while some components are detoxified, others, such as parathion, malathion, dypterex, are converted during metabolism, into even more toxic compounds (paraoxon, malaoxon, dichlorophosphonomethyl). Activation and deactivation can alternate for the same substance resulting in parallel in highly toxic compounds and in products with low toxicity. Based on the above mentioned, we can generalize the biohygienic function of the plant consists not only in absorbing chemical compounds, but also in including them in the metabolism and in inactivating them. Thus, plants oxidize carbohydrates forming organic acids and amino acids. One of the end-products of alkane oxidation, alcohols, aldehydes, phenols, ketones is CO2, which can be used later in the process of photosynthesis by plants.

10.10 Self-regulation of Plant Growth and Development in Unfavorable Environmental Conditions The reaction of various species, varieties and hybrids to unfavorable environmental conditions, quantitatively, depends on the norm of reaction, on the genotype and the capacity of self control that underlies the adaptation mechanisms of the body. Comparative studies of the same genotype in different environmental conditions demonstrated that qualitatively, functional changes in the plant metabolism in different species and various stress factors are similar. Thus, elevated ion concentrations have been found at high concentrations of salt, at dehydration, at temperatures below 0 °C, under conditions of hyperthermia. In all these conditions a decrease in the water content and an increase in the fluid and osmotic potential of the cell have been noticed but also changes in the functional activity of the DNA as a key element in synthetic reactions. Under stress conditions there were stated disorders in the bioenergetic processes as well as in the active centers of the photosynthetic systems responsible for the primary process of transformation of the solar energy into chemical energy, for reducing the amount of free radicals in the cell and for partial blocking of the ETC. This leads to a reduction of energy production efficiency as a result of photophosphorylation and oxidative phosphorylation. Thus, under stress conditions, considerable amounts of energy are spent to restore the functionality and repair the damaged cell structures. Simultaneously, structural changes and loss of cellular membrane integrity occurs, as a result of lipid complex oxidation as well as the deregulation of the

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ability of metabolite compartmentalization within the cell and deregulation of the entire metabolism. Analysis of the physiological dynamics of the biological parameters during stress and the nature of the relationship in each metabolic chain allows distinguishing (Fig. 10.16): • primary disorders caused by direct action on the cell (disruption of osmotic processes, modification of the bioenergetics, impaired structural integrity of the membranes, reduced functional activity of the nuclear DNA). All these parameters alter significantly immediately after the onset of stress factor and, if this stress acts constantly, disorders persist throughout the entire period of the action of the factor; • secondary disorders caused by a primary disruption of the metabolic functions (changes in the processes of biosynthesis—protein biosynthesis retention, an increase in the content of inhibiting phytohormones, retention of cell multiplication and elongation. Primary and secondary physiological disorders lead to the modification of important physiological functions of the vegetal organism such as the intensity of nutrient absorption and utilization, the increase in the biomass, the productivity of seeds, fruits and vegetables. Damaging processes and the autoregulation mechanism that leads to adaptation under stress conditions differ by their physiological essence and are separate by the time of onset and manifestation, while the whole complex of metabolic changes under extreme conditions in vegetal cells has a phase character. For the first stress phase, called the irritation phase a rapid and sudden deviation is characteristic with a rapid return to the norm of several biochemical and physiological parameters. This kind of effect, which appears after a few minutes of stress, was identified, for instance, in the action of a high salt concentration on the quantity of water in chloroplasts and mitochondria, a correlation being established between the amplitude, the speed of the effect and the intensity of the stress. From the physiological point of view, these changes during the irritation phase are not yet disorders of the metabolic functions, but rather specific signals of the organism about the environmental deviations from the norm. This phase lasts only a few tens of minutes. The next phase, called the injury phase, lasts a few days. During this phase there is an inhibition of the anabolic reactions that are energy dependent and a considerable increase in the catabolic and hydrolytic reactions. A strong increase in the intensity of the stress factor, which surpasses by much the maximum resistance of plants creates an imbalance between the two aspects of cellular metabolism in a manner similar to a chain reaction and, eventually, the plant dies. During the irritation phase both specific and non-specific changes to various stress factors can be attested, but immediately after these changes nonspecific metabolic disorders appear that have actually a primary importance and are characteristic for the adaptation phase.

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Self-regulation of Plant Growth and Development …

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Fig. 10.16 Adaptation to abiotic stress (Arnholdt-Schmitt 2004). Typical phenotypic and epigenetic changes at the level of the entire vegetal organism and at the cellular level in response to phosphorus deficiency (a) and at the level of the factors involved in the global regulation of the genome (b)

If the intensity of the factor does not exceed the lethal threshold, over a certain time, the ratio of anabolic and catabolic products gradually returns to normal, and initiation of the process of regeneration of the disturbed intercellular structures

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(membranes, organelles) happens, which demonstrates the connection of the autoregulation mechanisms. At the basis of the adaptation process lies the reduction of catabolic and hydrolytic reactions, since under extreme conditions, the intensity of synthetic and energy forming processes maintains the same low level as in the phase of injury. It is possible that namely the weakening intensity of hydrolytic and catabolic processes is the basis and essence of the adaptation phase and reflects metabolic autoregulation in extreme conditions. If the action of extreme factors ends, and optimal conditions follow (rain after drought, etc.) the repair mechanisms kick in. In general, they all consist in increasing the synthesis processes previously blocked by stress, in the process of accelerated renewal of cell structures and in the adjustment of various functions to the optimum level. After the action of the unfavorable factor ceases, which causes partial or total damage to the organs, their rapid regeneration begins. According to the depth of physiological disorders during stress, the repair can be partial or total. Plant adaptability and autoregulation processes change during ontogeny. The lowest degree of adaptation is seen in young plants in the juvenile stage of growth and development and then increases slowly towards the end of the vegetation period, but is reduced during the formation of the reproductive organs, changes that correlate with the nature of the modifications during ontogenesis of the physicochemical properties of the cytoplasm. All the mechanisms of self-regulation and adaptation to unfavorable conditions are realized at the cellular and intercellular level. At the level of the organism they are complemented by additional mechanisms that reflect the interaction between organs. During stress, competition reactions between generative and vegetative organs for water and nutritive substances are increasing. If the adverse circumstances act until the differentiation of the generative organs a decrease in the number of floral primordia occurs. Receiving external signals about extreme conditions, the plant forms that minimum of generative organs that it is able to supply with nutrients up to ripening. If the stress factors are acting after the formation of the fructification organs, the competitive relationships begin between the seed forming organs, which constrains the development of some of them. At the population level another mechanism that works effectively in adverse conditions comes into play—selection. The variability of the level of resistance in a population serves as a motivation for the manifestation of this mechanism. Thus, only those individuals survive who have a greater genetically determined capacity for resistance. Plant adaptation to extreme environmental conditions is a complex process, coordinated by the body autoregulatory system. The higher the level of biological organization (cell, organism, population), the greater the number of mechanisms involved simultaneously in the adaptation process. The character of adaptive-protective reactions is unique and universal for different stress factors—salts, high temperatures, low temperatures, drought etc.

Glossary

307

Glossary Asphyxiation under water Plant death under conditions of excessive humidity, especially during the spring. Due to the high humidity and insufficiency of oxygen plants switch to anaerobic respiration. Accumulation of toxic substances like ethylic alcohol leads to cell intoxication. Asphyxiation under snow Plants are perishing under a thick layer of snow (typically in winter crops), in conditions of moderate winter, at temperature near 0 °C. The cause of asphyxiation are a high rate of respiration followed by the exhaustion of the organic matter. The amount of sugars in the tissues decreases from 20 to 2–4 %. Uprooting Movement of the tillering node to the surface of the soil caused by the pressure exerted by the ice crust on the plant organs, which results in rupturing of the root. Halophytes Plants resistant to salts which either accumulate, remove, localize salts in cell compartments or which don’t accumulate salts at all. Law of B. D. Zalenski The leaves and other plant organs located at the top of the plant are different from the leaves and organs at the bottom by a xeromorph structure and increased resistance to drought. Frost resistance The ability of plants to survive low negative temperatures. Frost resistance is hereditary. Salt resistance The property of plants to grow on saline soils. The category of agricultural plants, resistant to salinity includes sorghum, some varieties of barley and millet and sensitive to salinity—oats and corn. Heat resistance The ability of plants to support dehydration and overheating. Among the drought resistant plants are the succulents, among the crop species— rice, cotton, sorghum. Plant resistance The ability of plants to withstand extreme action of unfavorable factors of the environment without suffering damage. This property formed during evolution and is genetic determined. Winter drought The exposure of trees and shrubs to the winter winds and the sun which have a drying effect. Plant tissues experience a water deficit caused by water retention by the cold ground.

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References Aleksandrov VJ (1975) Kletki, makromolekuly i temperatura. L., 329 p Arnholdt-Schmitt B (2004) Stress-induced cell reprogramming. A role for global genome regulation? Plant Physiol 136:2579–2586. www.plantphysiol.Oig Bohnert HJ, Nelson E, Jensenay RG (1995) Adaptations to environmental stresses. Plant Cell 7:1099 Deveroll BD (1980) Zashchitnye mehanizmy rasteniy. M., 126 pp Genkel’ PA (1982) Fiziologiya zharo- i zasuhoustoychivosti rasteniy. M., 280 pp Grodzinskij DM (1983) Nadezhnost’ rastitel’nyh sistem. Kiev, 366 pp Igamberdiev AU, Hill RD (2004) Nitrate, NO and haemoglobin in plant adaptation to hypoxia on alternative to classic fermentation pathways. J Exp Bot 55(408):2473–2482 Jansen MAK, van den Noort RE, Tan MYA, Prinsen E, Lagrimini LM, Thorneley RNF (2001) Phenol-oxidizing peroxidases contribute to the protection of plants from ultraviolet radiation stress. Plant Physiol 126:1012–1023 Meglickij LV, Ozeretskovskaya OL (1985) Kak rasteniya zashchishchayutsya ot bolezney. M., 190 pp Nikolaevskiy VS (1979) Biologicheskie svoystva gazoustoychivosti rasteniy. Novosibirsk, 278 pp Polevoy VV (1982) Fitogormony. L. Izd. Leningradskogo universiteta, 248 pp Rubin BA, Arcihovskaya EV, Aksenova VA (1975) Biohimiya i fiziologiya immuniteta rasteniy. M., 320 pp Schutzendubel A, Polle A (2002) Plant responses to abiotic stresses: heavy metal-induced oxidative stress and protection by mycorrhization. J Exp Bot 53(372):1351–1365 Strogonov BI (1973) Metabolizm rasteniy v usloviyah zasoleniya. M., 51 pp Tsai-Hung H et al (2002) Tomato plants ectopically expressing arabidopsis CBFL show enhanced resistance to water deficit stress. Plant Physiol 130:618–626 Tumanov IM (1979) Fiziologiya zakalivaniya i morozostoykosti rasteniy. M., 352 pp

Index

A Abscisic acid (ABA), 200, 273 Absorption, 27 Absorption maxima, 84 Absorption zone, 48 Acceptor, 94 Acetyl coenzyme A, 135 Acid gases, 293 Acropetal, 226 Action spectrum, 85 Active transport, 29, 180 Acyclic photophosphorylation, 99 Adaptation, 274 Adaptation process, 306 Adhesion forces, 58 Adsorption, 179 Aerobic dehydrogenases, 128 Aerobic respiration, 125 Aeroponics, 154 Aggregation states, 43 Agro-chemistry, 7 Agronomic resistance, 274 Alcoholic fermentation, 132 Allelopathy, 266 Aluminum, 177 Amides, 158 Amino acid, 162 Aminotransferases, 161 Ammonia, 298 Ammonium, 157 Amorphous complex, 20 Amphiphilic, 24 Amphystomatic, 52 Amplitude, 240 Amyloplasts, 76 Anabolic reactions, 304 Anaerobic dehydrogenases, 128 Anaerobic respiration, 126 Anatomical/morphological, 3

Annual autumn plants, 219 Annual rings, 236 Anthesis, 199 Anthocyanins, 71 Antioxidant, 67 Antiport, 30 Antisense strategies, 3 Apical meristems, 193 Apocrine, 254 Apoplast, 20, 181, 258 Apoptosis, 125, 216 Aquaporins, 41 Arabic gum, 255 Ash, 153, 300 Asparagine, 158 Asphyxiation under snow, 276 Asphyxiation under water, 277 Assimilation factor, 102 Assimilatory cells, 70 ATP, 180 ATPase complex, 73 Autoregulation processes, 306 Auxiliary pigments, 85 Auxins, 200 B Bach, 130 Bacteriological purification, 300 Bacteriorrhizae, 155 Basipetal, 226 Benson-Calvin cycle (C3), the, 101 Biennial plants, 219 Biocenosis, 249 Biochemistry, 7 Biological clock, 234 Biological indicators, 301 Biological repose, 192 Biological resistance, 274 Biophysics, 7

© Springer International Publishing Switzerland 2015 M. Duca, Plant Physiology, Biological and Medical Physics, Biomedical Engineering, DOI 10.1007/978-3-319-17909-4

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310 Biopotentials, 227 Biorhythms, 226, 233 Biosphere, 10 Birch juice, 265 Blackman phase, 86 Boron, 177 Botany, 7 Bound water, 43 Brassinosteroids, 200 Brilliant phenomenon, 119 C Cab genes, 114 Calciphiles, 172 Calciphobes, 172 Calcium, 172 Calmodulin, 173 Cambium, 189, 193 Capillary water, 46 Carbohydrates, 68, 127 Carbonate, 286 Carbon monoxide, 298 Carboxylation, 108 Carotenoids, 71 Catabolic and hydrolytic reactions, 304 Catabolism, 125 Catalase, 129 Cell death (apoptosis), 15 Cell theory, 16 Cellular, 10 Cellulose, 19 Cell wall, 16 Cerides, 256 Chelates, 152 Chelating agents, 295 Chemical and electrochemical gradients, 28 Chemical cleaning, 300 Chemical method, 154 Chemoautotrophic, 119 Chemotropisms, 223 Chinones, 94 Chloride, 286 Chlorine, 300 Chlorophyll, 68 Chlorophyll “a”, “b”, “c” and “d”, 78 Chlorophyllin, 78 Chloroplast and nuclear genomes, 110 Chloroplasts, 72 Chlorosis, 176, 301 Cholesterol, 24 Chromatoplasma, 119 Chromoplasts, 76 Chronon hypothesis, 234 Circadian rhythms, 234

Index Circalunar rhythms, 234 Circannual rhythms, 234 Circaseptal rhythms, 234 Citric acid, 135 Coenzyme Q, 138 Cohesion forces, 58 Colloidal and capillary effects, 34 Colloid hydration, 291 Colloids, 44 Compartmentalization, 291 Conducting vessels, 181 Conjugated double bonds, 90 Conjugation reactions, 301 Constitutional water, 45 Copper, 177 Copper proteids, 93 Cotransport, 29 Crassulacean acid metabolism, 101 Crinohalophytes, 288 Critical stage, 282 Crude sap, 266 Cryptochromes, 243 Crystallization water, 45 Cuticle, 51, 256 Cuticular pores, 256 Cuticular transpiration, 264 Cutin, 256 Cysteine, 165 Cytochrome, 73 Cytochrome-oxidase, 129 Cytogenesis, 190 Cytokinins, 200 Cytology, 7 Cytophysiology, 4 Cytoplasmic male sterility, 207 Cytoplasmic membrane (plasmalemma), 23 Cytorrhysis, 283 D Dark phase of photosynthesis, 87 Dehydrogenases, 131 Deplasmolysis, 34 Detoxification, 302 Development, 190 Dichotomyic path, 132 Diffused light, 84 Diffusion, 179, 252 Digestive glands, 255 Direct sugar oxidation, 140 Disulfide bridges, 165 Division zone, 155 Dominant centers, 196 Donor, 95 Dust, 293

Index Dwarfism, 209 Dynamic equilibrium, 4 E Ecology, 7 Ectodesmata, 256 Electrochemical potential, 98, 172, 180 Electron, 95 Electronic microscope, 11 Electron spins, 90 Electron transport chain (ETC), 88, 129 Electron tunneling effect, 93 Electroosmosis, 31 Electrophysiological control, 226 Electrotropisms, 223 Elongation region, 155 Embryonic stage, 195 Emerson effect, 87 Endogenous, 45 Endogenous duration, 240 Endoplasmic reticulum, 24 Endosmosis, 155 Energetic level, 89 Energogenesis, 126 Energy and matter circulation, 69 Environmental factors, 10 Enzymes, 17 Epinasties, 224 Erythrose, 141 Esterification, 302 Ethylene, 200 Etiolation, 9 Etioplasts, 76 Euhalophytes, 288 Exchange of ions, 179 Excitation state, 90 Excretion, 27, 250 Exergonic, 138 Exogenous, 45 Exometabolites, 250 Experimental science, 9 Extrafloral nectaries, 258 Exudates, 253 F Facilitated diffusion, 29, 180 Facultative halophytes, 289 Fatty acid synthesis, 138 Fermentation, 125 Ferredoxin, 94 Ferritin, 176 Ferroprotein, 138 Fe-S proteins, 92 Field experiments, 154

311 Flavoproteins, 93, 128 Floral clock, 237 Fluctuation period, the, 240 Fluorescence, 90 Fluorine, 300 Forced dormancy, 192 Free water, 43 Frost resistance, 279 Fructose-6-phosphate, 145 Fumaric acid, 135 G Gametophyte, 197 Gas exchange, 126 Gelation, 257 Genetically modified plants (GMPs), 2 Genetic program, 199 Genetics, 7 Geotropisms, 223 Gibberellins, 200 Glandular hairs, 259 Global warming, 119 Glucose, 135 Glucose-6-phosphate, 144 β-glucuronidase, 233 Glutamate dehydrogenase, 159 Glutamine, 158 Glutathione, 166, 298 Glycohalophytes, 288 Glycolic acid, 109 Glycolic pathway of carbon transformation., 107 Glycolipids, 24 Glycolysis, 132 Glycophytes, 287 Glycosidation, 302 Glyoxylate cycle, 140 Golgi apparatus, 24 Granal and stromal, 73 Gravitational water, 46 Growth, 189 Growth and development, 4 Growth inhibitors, 201 Growth promoters, 201 Growth regulators, 9 Growth respiration, 126 Guard cells, 51, 171 Guttation, 46, 58, 263 H Halophytes, 287 Hardening, 278 Hatch-Slack-Karpilov cycle (C4), 101 Heat-loving plants, 277

312 Heat shock proteins, 273 Heavy metals, 294 Hecht’s filaments, 34 Heliophytes, 47, 79 Hemicellulose, 19 Herbicide, 302 Hexochinase, 144 Hexoses, 132 Hill phase, 86 Hipostomatic, 52 Holocrine, 254 Homeohydrophytes, 60 Homeostasis, 267 Hyaloplasma, 21 Hydathodes, 257 Hydatophytes, 60 Hydration water, 280 Hydroactive, 53 Hydrogen sulfide, 298 Hydropassive, 53 Hydrophilic, 24 Hydrophobic, 24 Hydroponics, 154 Hydrotropism, 48, 223 Hygrophytes, 60 Hygroscopic water, 45 Hyperstomatic, 52 Hypertonic, 33 Hyponasties, 224 Hypothesis of the multioscillatory model of biorhythms, 236 Hypotonic, 34 Hystogenesis, 190 I Idioblasts, 259 Imbibition, 31 Imbibition force, 44, 54 Inanition, 276 Inductive resonance, 91 Infrared, 82 Injury phase, 304 Inorganic ions, 17 Insecticides, 301 Insectivorous plants, 248 Insoluble compounds, 303 Intensity of respiration, 128 Intercalary meristems, 193 Intercellular, 10 Intrafloral nectaries, 258 Ionic asymmetry, 172 Ion pumps, 29 Irrigation, 285 Irritation phase, 304

Index Isocitrate dehydrogenase, 145 Isocitric acid, 135 Isotonic, 33 J Juvenile stage, 196 K Kaspari stripes, 48 α-ketoglutarate dehydrogenase, 145 Ketoglutaric acid, 135 Kinases, 112, 168 Krebs cycle, 126 L Lactiferous cells, 259 Latency, 189 Lateral meristems, 193 Latex, 255 Law of Müller and Hegel, 61 Leghemoglobin, 164 Leguminous plants, 162 Lenticels, 52 Lenticular transpiration, 264 Leucoplasts, 76 Light-harvesting complex, 73 Light phase of photosynthesis, 87 Light quantum, 82 Light utilization coefficient, 83 Lignin, 254 Liposolubility, 180 Localization signal, 75 Long day plants, 218, 238 Lysosomes, 24 M Macroelements, 154 Macroergic bonds, 126 Macroergic ester bonds, 169 Magnesium, 174 Maintenance respiration, 126 Malate dehydrogenase, 145 Malic or oxaloacetic acids, 107, 135 Manganese, 175 Mathematical modeling, 11 Maturation, 216 Mature region, 155 Mechanical cleaning, 300 Median lamella, 267 Membrane hypothesis, 236 Meristematic tissues, 189 Merocrine, 254 Mesophyll cells, 118 Mesophytes, 60

Index Metabolic disorders, 304 Metabolic water, 282 Metabolite compartmentalization, 304 Metal oxides, 293 Metamers, 236 Methane, 298 Methionine, 165 Micro-and macrofibril complex, 20 Microelements, 154 Middle lamella, 18 Mineral elements, 152 Mineralization, 152, 257 Mineral nutrition, 4 Mitochondria, 125 Mitochondrial cristae, 129 Mn-Mn dimers, 97 Molecular, 10 Molecular oscillation, 241 Molybdenum, 176 Morphogenesis, 6, 190 Mucilage, 257 Multicellular, 16 Mycorrhizae, 155 N Na+/K+pumps, 31 Na/K ratio, 290 NADPH+H+, 87 Nasties, 222 Necrosis, 166, 301 Nectar, 250 Nectary glands (nectaries), 255 Neutral plants, 218 Nitrate reductase, 158 Nitrates, 157 Nitrites, 157 Nitrogen, 156 Nitrogenase, 164 Nitrogen oxides, 300 Nitrogenous bases, 162 Nutations, 223 Nyctinastic movements, 225 O Obligatory halophytes, 289 Ontogenesis, 8 Ontogenetic dynamics, 3 Organelles, 21 Organic acids, 288 Organismal, 10 Organogenesis, 190 Organogenic elements, 152 Orthophosphoric acid, 168 Osmophores, 255

313 Osmosis, 31, 42 Osmotic force, 44, 54 Osmotic pressure, 32, 286 Osteole, 52 Oxidases, 129 Oxidation cycle, 130 Oxidative degradation, 67, 125 Oxysomes, 138 P P680, 89 P700, 89 Pacemaker, 236 Palisade parenchyma, 70 Palladin, 130 Passage cells, 48 Passive transport, 28, 179 Pectic substances, 19, 173, 257 Pedological and atmospheric drought, 284 Pedology, 7 Pellicular water, 45 Pentanonic homocycle, 79 Pentose phosphate cycle, 140 Perennials, 219 Pericycle, 193 Perivascular sheath cells, 109 Permanent wilting, 280 Peroxidase, 129 Peroxisomes, 108 Pesticides, 301 Phellogen, 189, 193 Phenolic compounds, 253 Pheophytin, 92 Phosphatases, 112 Phosphoenolpyruvate, 145 Phosphoenolpyruvic acid, 103 Phosphofructokinase, 144 3-phosphoglyceric acid, 102 Phosphoglyceric acid, 133 Phosphoglyceric aldehyde, 103, 133 Phospholipid molecule, 168 Phospholipids, 24 Phosphorescence, 90 Phosphorylation, 168 Photoactive, 53 Photoelectric effect theory, 86 Photoinactivation, 118 Photons, 82 Photooxidation, 70 Photooxidative stress, 72 Photoperiodism, 218, 236 Photoprotection, 71 Photorespiration, 101 Photosynthesis, 4

314 Photosynthetic apparatus, 111 Photosystems I (PSI) and II (PSII), 73 Phototrophic, 119 Phototropisms, 223 Phragmoplast, 254 Phycobilins, 77 Phycocyanin, 80 Phycoerythrin, 80 Phyloquinone, 92 Physiological, 3 Physiological drought, 286 Physiologically acid soil, 183 Physiologically basic soil, 183 Physiological method, 154 Phytin, 169 Phytochrome, 80, 222, 232 Phytohormone regulation, 226 Phytohormones, 9, 17, 200 Phytol, 78 Phytoncides, 300 Phytopathology, 7 Phytotoxicity, 299 Phytotrons, 10 PIF3, 243 Pinocytosis, 31 Plant breeding, 7 Plant cultivation, 7 Plant morphology, 7 Plant productivity, 8 Plasmodesmata, 20, 256 Plasmolysis, 33 Plastids, 16 Plastochron, 236 Plastocyanin, 92 Plastoquinone, 92 Poichilohydrophytes, 60 Polarity, 226 Polyphenols, 129 Population, 10 Porphyrin core, 79, 129 Potassium or kalium, 170 Primary acceptor, 92, 94 Primary and secondary metabolites, 151 Primary cell wall, 18 Primary disorders, 304 Procambium, 193 Processing, 113 Progressive branch of nitrogen exchange, 162 Proplastids, 75 Protein carriers, 29 Protochlorophyllides, 117 Protoplasm, 23 Protoplasm colloidal status, 152 Protoplast, 19

Index PRR, 244 Pyrophosphate bonds, 169 Pyrrol, 79, 129 Pyruvic acid, 132 Q Quantasomes, 73 Quinones, 129 R Radial and xylemic transport, 178 Radial transport, 55 Radiation, 295 Radicals, 301 Radioprotective substances, 298 Radiosensitivity, 296 Reaction centre, 88 Redox reactions, 79, 131 Redox system, 176 Regressive branch of nitrogen exchange, 160 Regulatory systems, 5 Relative transpiration, 50 Reproductive development, 190 Reproductive stage, 196 Resistance, 273 Resistance to drought, 280 Respiration, 4 Respiratory coefficient, 127 Respiratory pigments, 131 Respiratory substrate, 127 Retardants, 201 Rhizobium genus, 162 Rhizosphere, 48, 250 Ribulose-1,5-bisphosphate carboxylase (RUBISCO), 74 Root cap (calyptra), 155 Root hair region (differentiation zone), 155 Root hairs, 48 Rooting, 206 Root nodules, 162 Root pressure, 56 S Saline soils, 166 Salinization, 286 Salt glands, 291 Sciophyts, 47, 79 Secondary acceptor, 95 Secondary cell wall, 18 Secondary disorders, 304 Secretion, 27, 250 Sedoheptulose, 141 Seismonastic movements, 225 Seismotropisms, 223

Index Self-regulation (autoregulation), 4 Semipermeability, 21 Senescence phase, 196 Short-day plants, 218, 239 Silicon, 176 Simple diffusion, 28 Singlet, 90 Soil colloids, 166 Soil solution, 182 Spectral composition, 116 Spongy parenchyma, 70 Sporophyte, 197 Stomata, 51 Stress conditions, 292 Stress metabolites, 250 Stroma, 75 Structural molecules, 17 Suber, 254 Suberization, 257 Succinate dehydrogenase, 145 Succinic acid, 135 Succulence, 290 Suction force, 31, 55 Sugars, 288 Sulfate, 165 Sulfur, 165 Sulphate, 286 Symplast, 20, 181, 258 Symport, 30 T Tearing, 263 Temporary wilting, 280 Termonastic movements, 225 Terpenes, 250, 259, 267 Thermoperiodicity, 245 Thioredoxin, 117 Thylakoids, 73 Tonoplast, 23 Tonoplast transferases, 295 Toxic gases, 298 Tracheids, 51 Transamination, 160 Transcription, 114 Transition to flowering, 199 Translation, 113 Transphosphorylation, 169 Transpiration, 263 Transpiration coefficient, the, 50 Transpiration intensity, 50

315 Transpiration productivity, 50 Transpiration pull, 57 Tricarboxylic acid cycle, 135 Triplet, 90 Trophic regulation, 226 Trophic substances, 266 Tropism, 222 Turgidity, 33, 43, 171 Turgor pressure, 35 U Ubiquinone, 92 Ultracentrifugation, 11 Ultramicroelements, 154 Ultrastructure, 11 Ultraviolet, 82 Uprooting, 277 Upward flow of water, 286 V Vacuolar Na +/H + antiporters, 292 Vacuole, 16 Vegetative development, 190 Vapors of acids, 293 Vernalization (yarovization), 218 Visible spectrum, 81 W Water aggregation states, 43 Water balance, 58 Water oxidation, 97 Water regime, 4 Waxes, 250 Wilting coefficient, 283 Wilting point, 46 Winter drought, 307 X Xanthophyll, 89 Xenobiotics, 300 Xeromorphic features, 61 Xeromorphic qualities, 284 Xerophytes, 60 Xylem vessels, 57 Z Z-scheme, 94 Zeatin, 211 Zinc, 177