Chlorophyll a Fluorescence Advances in Photosynthesis and Respiration
VOLUME 19
Series Editor : GOVINDJEE University of Illinois, Urbana, Illinois, U.S.A.
Consulting Editors: Christine FOYER, Harpenden, U.K. Elisabeth GANTT, College Park, Maryland, U.S.A. John H.GOLBECK, University Park, Pennsylvania, U.S.A. Susan S. GOLDEN, College Station, Texas, U.S.A. Wolfgang JUNGE, Osnabrück, Germany Hartmut MICHEL, Frankfur t am Main, Germany Kirmiyuki SATOH, Okayama, Japan James Siedow, Durham, Nor th Carolina, U.S.A.
The scope of our series, beginning with volume 11, reflects the concept that photosynthesis and respiration are intertwined with respect to both the protein complexes involved and to the entire bioenergetic machinery of all life. Advances in Photosynthesis and Respirationis a book series that provides a comprehensive and state-of-the-art account of research in photo- synthesis and respiration. Photosynthesis is the process by which higher plants, algae, and certain species of bacteria transform and store solar energy in the form of energy-rich organic molecules. These compounds are in turn used as the energy source for all growth and reproduction in these and almost all other organisms. As such, virtually all life on the planet ultimately depends on photosynthetic energy conversion. Respiration, which occurs in mitochondrial and bacterial membranes, utilizes energy present in organic molecules to fuel a wide range of metabolic reactions critical for cell growth and development. In addition, many photosynthetic organisms engage in energetically wasteful photorespiration that begins in the chloroplast with an oxygenation reaction catalyzed by the same enzyme responsible for capturing carbon dioxide in photosynthesis. This series of books spans topics from physics to agronomy and medicine, from femtosecond processes to season long production, from the photophysics of reaction centers, through the electrochemistry of intermediate electron transfer, to the physiology of whole orgamisms, and from X-ray christallography of proteins to the morphology or organelles and intact organisms. The goal of the series is to offer beginning researchers, advanced undergraduate students, graduate students, and even research specialists, a comprehensive, up-to-date picture of the remarkable advances across the full scope of research on photosynthesis, respiration and related processes.
The titles published in this series are listed at the end of this volume and those of forthcoming volumes on the back cover. Chlorophyll a Fluorescence A Signature of Photosynthesis
Edited by George C. Papageorgiou National Center for Scientific Research Demokritos, Athens, Greece
and Govindjee University of Illinois, Urbana, Illinois, U.S.A.
4y Springer A C.I.P. Catalogue record for this book is available from the Library of Congress.
ISBN 1-4020-3217-X (HB) ISBN 1-4020-3218-8 (e-book)
Published by Springer, P.O. Box 17, 3300 AA Dordrecht, The Netherlands.
Sold and distributed in North, Central and South America by Springer, 101 Philip Drive, Norwell, MA 02061, U.S.A.
In all other countries, sold and distributed by Springer, P.O. Box 322, 3300 AH Dordrecht, The Netherlands.
The camera ready text was prepared by Lawrence A. Orr, Center for the Study of Early Events in Photosynthesis, Arizona State University, Tempe, Arizona 85287-1604, U.S.A.
Legend for solar photon flux densities and pigments spectra to 1000 nm
Solar photon flux densities at the top of the atmosphere and at the Earth’s surface, with estimated in vivo absorbance spectra of selected photosynthetic pigments from plants, algae and cyanobacteria, and fluorescence spectrum of chlorophyll a. The solar spectra were truncated beyond 1000nm, and the pigment absorption spectra below 400 nm. Sources: Top-of-the-atmosphere irradiance: 150-200 nm, Andrew Lacis, NASA Goddard Institute for Space Studies (GISS); 200-400 nm, Judith Lean (Naval Research Laboratory); 400-2500 nm, Brian Cairns, NASA GISS. Surface irradiance: 200-400 nm, J. Lean (Lean and Rind, 1998); 400-2500 nm, B. Cairns. Atmospheric gases, David Crisp (personal communication, NASA Jet Propulsion Laboratory). Chlorophyll a and Chlorophyll b absorbance measurements, made by Junzhong Li (H. Du and coworkers, 1998), in vitro, were shifted in wavelengths to match in vivo peaks, and absorbances were normalized to between 0 and 1. Carotenoid absorption spectra are estimated in vivo absorption spectra in green algae (Govindjee, 1960). Phycoerythrin and phycocyanin absorption spectra are unpublished absorption spectra from Govindjee’ s laboratory, and from Ke (2001). Chlorophyll a fluorescence spectrum, from spinach chloroplasts, is from Govindjee and Yang (1966). The color bar is from Dan Burton (Color Science web page: http://www.physics.sfasu.edu/astro/color.html). See chapter 1 by Govindjee for references cited above.
The figure for the cover is copyrighted and prepared by Nancy Kiang (NASA Goddard Institute for Space Studies) and Govindjee (University of Illinois at Urbana-Champaign). Printed on acid-free paper
All Rights Reserved © 2004 Springer No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work.
Printed in the Netherlands. This book is dedicated to L. N. M. Duysens, a pioneer of Photosynthesis
During his scientific life, Louis (Lou) Nico Marie ideas of Lou Duysens. Fundamental concepts that fol- Duysens quietly contributed some of the most lowed from these experiments include the carotenoid seminal ideas in photosynthesis, with still central to (bacterio-)chlorophyll energy transfer in plant influence over our thinking today. His approach was and bacterial light harvesting systems, the efficient a perfect blend of deep theoretical understanding energy transfer among a large number of quasi-iden- and experimental innovation. Typical of Duysens' tical chlorophylls to reach the small population of early work was his insightful application of Forster reaction centers, the Take model' of photosynthetic theory to light harvesting by the accessory pigment unit function, the control of the fluorescence yield beds, which firmly established the physics underly- of Photosystem II by the redox state of the quencher ing the function of the photosynthetic unit. Using his Q (now known as QA), and chlorophyll excitation own state-of-the-art absorption spectrophotometer, quenching by carotenoids. These concepts underlie he discovered the spectroscopic signature of oxida- the veritable industry of fluorescence-based, non- tion and reduction of the photochemical reaction invasive methods now used to study photosynthesis center in bacteria. Later, adapting this technique to in vivo and to analyze plant productivity, from the plant (algal) systems, he discovered the alternating lab bench to satellite monitoring. effects of green and red light on the redox state of Lou's understanding of physical principles is im- cytochrome fin red algae, to firmly establish the now mense and so facile that his colleagues and competi- familiar model of Photosystem I and Photosystem tors struggled to keep up with him. His seemingly II acting in series. These major contributions exem- simple analysis of the thermodynamic limits of pho- plified Lou Duysens' philosophy towards technical tosynthetic energy conversion sparked a literature developments. Technology never drove nor limited that lasted several decades, with each incremental his research, but was harnessed to address specific advance simply rediscovering what he had said at scientific questions and hypotheses. the outset. Indeed, this is still an area that few fully With his easy understanding of photochemical comprehend. and photophysical principles, Duysens pioneered Since Lou's retirement, many ultrafast spectro- the use of Chlorophyll fluorescence as a powerful scopic techniques have emerged, revealing intricate probe of photosynthetic function, in all classes of details of function in the photosynthetic apparatus, photosynthetic organisms, to discover many of the which can be well understood in terms of the fan- essential events of primary energy conversion in tastic atomic resolution structures of photosynthetic photosynthesis: excitation energy transfer in the pigment-protein structures that are now available. In light-harvesting antenna and charge separation in the this new era of understanding, it is amazing to see reaction center. Over many years, in his laboratory in how the concepts, developed by Duysens even 50 Leiden (The Netherlands), a multitude of state-of-the years ago, have survived as solid foundations of our art fluorescence methods was created, driven by the current models of photosynthetic activity.
v Editorial
Advances in Photosynthesis and Respiration Volume 19: Chlorophyll a Fluorescence: A Signature of Photosynthesis
I am delighted to announce the publication, in the 10. Photosynthesis: Photobiochemistry and Photo- Advances and Photosynthesis and Respiration biophysics (Bacon Ke, author, 2001); (AIPH) Series, the first book that focuses on the red 11. Regulation ofPhotosynthesis (Eva-Mari Aro and light that plants, algae and cyanobacteria emit when Bertil Andersson, editors, 2001); exposed to UV and visible light. This new volume, 12. Photosynthetic Nitrogen Assimilation and As- Chlorophyll a Fluorescence: A Signature of Photo- sociated Carbon and Respiratory Metabolism synthesis, is edited by George C. Papageorgiou and (Christine Foyer and Graham Noctor, editors, Govindjee. The other two recent Volumes 17 and 18 2002); (edited by David Day et al. and Hans Lambers and 13. Light Harvesting Antennas (Beverley Green and Miquel Ribas-Carbo, respectively) deal with several William Parson, editors, 2003); aspects of Plant Respiration.'Volume 19 is a sequel 14. Photosynthesis in Algae (Anthony Larkum, Su- to the eighteen volumes in the AIPH series. san Douglas and John Raven, editors, 2003); 15. Respiration inArchaea and Bacteria: Diversity of Prokaryotic Electron Transport Carriers (Davide Published Volumes Zannoni, editor, 2004); and 16. Respiration inArchaea and Bacteria 2 : Diver- 1. Molecular Biology of Cyanobacteria (Donald sity of Prokaryotic Respiratory System (Davide A. Bryant, editor, 1994); Zannoni, editor, 2004) 2. Anoxygenic Photo synthetic Bacteria (Robert E. 17. Plant Mitochondria: From Genome to Function Blankenship, Michael T. Madigan and Carl E. (David A. Day, Harvey Millar and James Whelan, Bauer, editors, 1995); editors, 2004) 3. Biophysical Techniques in Photosynthesis (Jan 18. Plant Respiration. From Cell to Ecosystem (Hans Amesz and Arnold J. Hoff, editors, 1996); Lambers and Miquel Ribas-Carbo, editors, 4. Oxygenic Photosynthesis: The Light Reactions 2005) (Donald R. Ort and Charles F. Yocum, editors, See
vi in ATP synthesis. Partial processes include the split- (Greece) presents information on fluorescence of ting of water to molecular 02 (which escapes to the photosynthetic pigments (Chapter 2); N. R. Baker atmosphere) and to electrons and protons, which and K. Oxborough (UK) show how one can exploit participate directly in the electrochemical reactions Chi a fluorescence to probe photosynthetic produc- through which redox and proton gradients are coupled tivity (Chapter 3); and R. M. Clegg (USA) provides to phosphorylation and to fixationo f C02 to sugars. the basics of excitation energy migration and transfer Chlorophyll, as the major chromophore of pigment- (Chapter 4). This is followed by discussions by R. protein systems, is engaged in photon harvesting, in van Grondelle and B. Gobets (The Netherlands) on the regulated distribution of excitation energy, and in transfer and trapping of energy in plant photosystems its primary conversion to redox potential and proton (Chapter 5); by W. J. Vredenberg (The Netherlands) on gradient. Although it performs these tasks with high a three-state model of Photosystem II (PS II) where quantum efficiency, a small fraction of absorbed pheophytin plays an important role in controlling photons is re-emitted as red fluorescence. This frac- Chi fluorescence (Chapter 6); and by M. Mimuro tion varies with metabolic state, and provides the (Japan) on energy migration and trapping in cyano- basis for the measurement of photosynthesis through bacteria (Chapter 7). V Shinkarev (USA) discusses fluorescence. the relationship between PS II reactions and Chi The goal of this book is to equip readers with fluorescence in multiple flashes of light (Chapter sufficient theory to enable them to interpret the in- 8); and S. Itoh and K. Sugiura (Japan) summarize formation from measurements of Chi a fluorescence, information on fluorescence of PS I (Chapter 9). D. and to facilitate the use of relatively inexpensive, M. Kramer, T. J. Avenson, A. Kanazawa, J. A. Cruz, portable fluorometers that permit field applications B. Ivanov and G. Edwards (USA and Russia) discuss in agriculture, and applications in biochemistry, the regulation of the photosynthetic electron transfer biophysics, physiology and ecology. It deals with (Chapter 10). This is followed by a presentation by successful applications of the use of Chi fluorescence U Schreiber (Germany) of the Pulse Amplitude as a convenient, non-invasive, highly sensitive, rapid Modulation (PAM) Fluorometry and its application and quantitative probe of some of the partial pro- to photosynthesis (Chapter 11); R. J. Strasser, M. cesses of photosynthesis. The detailed studies in the Tsimilli-Michael and A. Srivastava (Switzerland, laboratory have been extended to measurements of Cyprus and USA) present a detailed analysis of total photosynthesis of cells, leaves, plants and plant the Kautsky curve (Chi a fluorescence transient or ecosystems. The book also explains mechanisms for induction) (Chapter 12); E. Tyystjarvi and I. Vass 'photoprotection' of plants (against excessive light), (Finland and Hungary) discuss the relationship and regulation of the photosynthetic machinery in of fluorescence with delayed light emission and response to temperature extremes, drought, heavy thermoluminescence (Chapter 13). L. Nedbal and metal stress and UV stress. Further, it discusses J. Whitmarsh (The Czech Republic and USA) show newer findings using modern technologies such as and discuss fluorescence imaging of leaves and fruits fluorescence imaging of leaves and cells and remotely (Chapter 14), whereas K. Oxborough (UK) presents sensed fluorescence (from terrestrial, airborne, and the use of fluorescence imaging to monitor photo- satellite bases). The book provides a solid founda- synthetic performance (Chapter 15). This is followed tion of the basic theory, and applications of the rich by the chapter of I. Moya and Z. Cerovic (France) information contained in Chi fluorescence signal as on the remote sensing of Chi fluorescence; of J.F it relates to photosynthesis and plant productivity. Allen and C. Mullineaux (Sweden and UK) on the Research scientists, graduate students, and advanced state-transitions in plants, algae and cyanobacteria undergraduates in integrative biology, cellular and (Chapter 17). Non-photochemical quenching (NPQ) molecular biology, plant biology, biochemistry, of chlorophyll fluorescence and its role in photo- biophysics, plant physiology, global ecology and ag- protection is a theme covered in several chapters: riculture, will find this book invaluable in advancing G. H. Krause and P. Jahns (Germany) characterize and consolidating their knowledge of photosynthesis it (Chapter 18); D. Bruce and S. Vasil'ev (Canada) and Chi a fluorescence. discuss the multiple dissipation processes (Chapter The book has 31 chapters, written by 59 authors 19); T. Golan, X-R Li, P. Muller-Moule and K. K. from 18 countries. Govindjee (USA) discusses a bit Niyogi (USA) show how one can exploit mutants to of basics andhistory (Chapter 1); G. C. Papageorgiou study NPQ (Chapter 20). A. M. Gilmore (Australia
vii and USA) summarizes the use of global analysis of Goethe about light emission from minerals. time and wavelength-resolved fluorescence in probing energy dissipation mechanisms (Chapter 21); and W. 'The 'Stokes shift' of the luminescent emissions W. Adams III and B. Demmig-Adams (USA) empha- from mineral samples is qualitatively, but clearly size the use of Chi fluorescence in monitoring plant discussed by Goethe, and this shows that even at responses to the environment (Chapter 22); M. Tevini that time, before the phenomenon was understood, (Germany) focuses on the ultraviolet light effects on it was known that the color of the emitted light plant responses (Chapter 23); N. G. Bukhov and R. was different from the color to which the samples Carpentier (Russia and Canada) discuss the effects were exposed, and that the radiance of mineral of water stress on plant responses (Chapter 24), samples was shifted to the red. In his 1810 book whereas M. K. Joshi and P. Mohanty (India) focus on (Zur Farbenlehre, Historischer Teil: Wirkung the heavy metal stress on plants (Chapter 25). G. C. farbiger Beleuchtung auf verschiedene Arten von PapagergiouandK. Stamatakis (Greece) summarize Leuchtsteinen), Goethe discusses the work of the use of Chi fluorescence in monitoring water and 'Herrn Doktor Seebeck zu Jena,' who carried out solute transport in cyanobacterial cells (Chapter 26); experiments on the 'effect of colored illumination J. K. Hoober and J. Argyroudi-Akoyunoglou (USA on glowing stones, metal oxides and plants'. See- and Greece) address the assembly of the light har- beck had used: '(1) Barytphosphors, ... radiated vesting complexes of Photosystem II and discuss the yellow-red, as weakly glowing coals, after they had role of Chi b (Chapter 27); H. K. Lichtenthaler and been exposed to [author: direct] sunlight, or just F. Babani (Germany and Albania) discuss the use of to daylight. (2) Phosphors from artificial sulfuric Chi fluorescence in monitoring light adaptation and acid [author: treated] Strontian,... These radiated senescence in plants (Chapter 28). This is followed sea-green (3) Quicklime (CaO) phosphors, pre- by chapters on productivity of ecosystems: terrestrial pared from burned Oyster shells according to the (Chapter 29 by J. Cavender-Bares and F. Bazzaz, instructions of Canton, which radiate mostly bright USA); marine (Chapter 30 by P. G. Falkowski, M. yellow. These phosphors were successively exposed Koblizek, M. Gurbunov and Z. Kolber, USA); and to different prismatic colors [author: light that has inland waters (Chapter 31 by J. Raven and S. C, passed through a prism]. In blue and violet light Maberly, Scotland, UK). they all became equally luminous; however, the The book is dedicated to apioneer of Chlorophyll a radiated light was not changed in any way [author: Fluorescence: Louis N. M. Duysens (of Oegstgeest, from that produced by exposure to strong sunlight]: The Netherlands). in the dark, the Barytphosphors glowed yellow- red, the new Strotianphosphors sea-green, and so forth, exactly as though they had been exposed to Just for Fun: An Historical Note on Goethe sunlight. In the blue they were only slightly less radiant than when they were exposed to violet light. In the recent past, I have been fascinated by the Beyond violet [author: this would be nowadays in history of science, particularly of photosynthesis the near ultraviolet] where hardly a color can be research (see Photosynthesis Research, Volume 73, discerned, they took on a lively radiance exactly 2002; Volume 76, 2003, and Volume 80, 2004). In as though they had been exposed to violet light. Chapter 1 of the present book, I have included a bit With green they glowed markedly weaker than with of history of'Chlorophyll a fluorescence.' Professor blue, with yellow much weaker, and with red they Robert Clegg (Physics Department, University of Il- radiated the weakest,...' linois, Urbana, IL) recently told me about Goethe's contributions to science. Johann Wolfgang von Goethe (1749-1832), Germany's greatest poet and dramatist, famous for Faust, was also a distinguished Chlorophyll a Fluorescence Books scientist, who carried out careful observations in physics. Although his interpretations of color and There are only three other books that deal solely disagreement with present physical theory is well with 'chlorophyll a fluorescence:' (1) 1986: Light known, many of his other contributions are not well Emission by Plants and Bacteria (638 pages; Govin- known. Clegg sent me an interesting story from djee, J. Amesz andD. C. Fork, eds), Academic Press,
viii New York; (2) 1988: Applications of Chlorophyll able advances across the full scope of research on Fluorescence in Photosynthesis Research, Stress bioenergetics and carbon metabolism. Physiology, Hydrobiology and Remote Sensing (384 pages; H. K. Lichtenthaler, ed), Kluwer Academic Publishers, Dordrecht; (3) 2003: Practical Applica- Future Books tions of Chlorophyll Fluorescence in Plant Biology (280 pages; J. R. DeEll and P. M. A. Toivonen, eds), The readers of the current series are encouraged to Kluwer Academic Publishers, Dordrecht. The three watch for the publication of these forthcoming books books are multiauthored: Govindjee et al. (1986) (not necessarily arranged in the order of future ap- deals with both basics and applications. The focus pearance): of books edited by Lichtenthaler (1988) and of DeEll (1) Discoveries in Photosynthesis Research (Editors: and Toivonen (2003), however, is mainly applications, Govindjee, Howard Gest, J. Thomas Beatty and although some basics are also provided. The current John F. Allen); book (~ 820 pages) is the most extensive and com- (2) Chlorophylls and Bacteriochlorophylls: Bio- prehensive book on the basics and the applications chemistry, Biophysics and Biological Function of Chi a fluorescence in plant biology. (Editors: Bernhard Grimm, Robert J. Porra, Wolfhart Rudiger and Hugo Scheer); (3) Photosystem II: The Water/Plastoquinone Oxido- The Scope of the Series reductase in Photosynthesis (Editors: Thomas J. Wydrzynski and Kimiyuki Satoh); Advances in Photosynthesis and Respiration is a (4) Photosystem I: The Plastocyanin/Ferredoxin book series that provides, at regular intervals, a com- Oxidoreductase in Oxygenic Photosynthesis prehensive and state-of-the-art account of research (Editor: John Golbeck); in various areas of photosynthesis and respiration. (5) Photoprotection, Photoinhibition, GeneRegula- Photosynthesis is the process by which higher plants, tion and Environment (Editors: Barbara Dem- algae, and certain species of bacteria transform and mig-Adams, William W. Adams III and Autar store solar energy in the form of energy-rich organic Mattoo); molecules. These compounds are in turn used as (6) Photosynthesis: A Comprehensive Treatise: Bio- the energy source for all growth and reproduction chemistry, Biophysics and Molecular Biology, in these and almost all other organisms. As such, Part 1 (Editors: Julian Eaton-Rye and Baishnab virtually all life on this planet ultimately depends on Tripathy) photosynthetic energy conversion. Respiration, which (7) Photosynthesis: A Comprehensive Treatise: Bio- occurs in mitochondria and in bacterial membranes, chemistry, Biophysics and Molecular Biology, utilizes energy present in organic molecules to fuel Part 2 (Editors: Baishnab Tripathy and Julian a wide range of metabolic reactions critical for cell Eaton-Rye); and growth and development. In addition, many photo- (8) The Structure and Function ofPlastids (Editors: synthetic organisms engage in energetically wasteful Kenneth Hoober and Robert Wise) photorespiration that begins in the chloroplast with an In addition to these contracted books, we are oxygenation reaction catalyzed by the same enzyme interested in publishing several more books. Topics responsible for capturing C02 in photosynthesis. This under consideration are: Molecular Biology of Stress series of books spans topics from physics to agronomy in Plants; Global Aspects of Photosynthesis and and medicine, from femtosecond (10-15 s) processes Respiration; Protein Complexes of Photosynthesis to season long production, from the photophysics and Respiration; Protonation and ATP Synthesis; of reaction centers, through the electrochemistry of Functional Genomics; The Cytochromes; Laboratory intermediate electron transfer, to the physiology of Methods for Studying Leaves and Whole Plants; and whole organisms, and from X-ray crystallography of C-3 and C-4 Plants. proteins to the morphology of organelles and intact Readers are requested to send their suggestions organisms. The intent of the series is to offer begin- for these and future volumes (topics, names of fu- ning researchers, advanced undergraduate students, ture editors, and of future authors) to me by E-mail graduate students, and even research specialists, a ([email protected]) or fax (1-217-244-7246). comprehensive, up-to-date picture of the remark- In view of the interdisciplinary character of re-
ix search in photosynthesis and respiration, it is my Noeline Gibson (of Kluwer Academic Publishers) earnest hope that this series of books will be used in special thanks for her friendly and wonderful work- educating students and researchers not only in Plant ing connection with the production of this book. Sciences, Molecular and Cell Biology, Integrative Thanks are also due to Jacco Flipsen (also of Kluwer Biology, Biotechnology, Agricultural Sciences, Mi- Academic Publishers), and Jeff Haas (Director of crobiology, Biochemistry, and Biophysics, but also Information Technology, Life Sciences, University in Bioengineering, Chemistry, and Physics. of Illinois) for their support. I am also thankful to I take this opportunity to express my heartfelt thanks Nancy Kiang (of NASA, Goddard Space Center) for and appreciation to George C. Papageorgiou (co- the cover of this book, and Robert Clegg (of UIUC) editor of the current volume) for the highest quality for the story on Goethe. My wife Rajni Govindjee and friendliness of his editorial work. Both of us are deserves my special thanks for tolerating my work grateful to Larry Orr for typesetting this book and for habits and for her help when I needed it most. Our the preparation of the Index; we are indebted to him daughter Anita Govindjee and her husband Morten for his friendly reminders on the rules of the Series. Christiansen; our son Sanjay Govindjee and his wife We thank all the authors of Volume 19: without their Marilyn Govindjee provided facilities at different authoritative chapters, there will be no book. We owe times during the preparation of this book.
August 15,2004
Govindjee Series Editor, Advances in Photosynthesis and Respiration University of Illinois at Urbana-Champaign Department of Plant Biology 505 South Goodwin Avenue Urbana, IL 61801-3707, USA E-mail: [email protected]; URL: http://www.life.uiuc.edu/govindjee Contents
Editorial v
Contents xi
Preface xxi
Color Plates CP-1
1 Chlorophyll a Fluorescence: A Bit of Basics and History 1-42 Govindjee Summary 2 I. Introduction 2 II. The Two-Light Reaction and Two-Pigment System Concept 12 III. Photosynthetic Unit and Excitation Energy Transfer 18 IV. The Fluorescence Transient 22 V. The Photosystem II Reactions and Chlorophyll Fluorescence 24 VI. Non-photochemical Quenching of Chi Fluorescence 28 VII. Concluding Remarks 31 Acknowledgments 32 References 32
2 Fluorescence of Photosynthetic Pigments in Vitro and in Vivo 43-63 George Christos Papageorgiou Summary 43 I. Introduction 44 II. Origin and Evolution of Oxyphototrophic Organisms 44 III. Chromophores for Light Harvesting and Excitation Handling 46 IV Intramembranous Pigment Holochromes 50 V Extramembranous Light Harvesting Antennae—Phycobiliproteins and Phycobilisomes 57 VI. Concluding Remarks 58 Acknowledgments 58 References 58
3 Chlorophyll Fluorescence as a Probe of Photosynthetic Productivity 65-82 Neil R. Baker and Kevin Oxborough Summary 66 I. Introduction 66 II. Fluorescence Terminology 67 III. Fluorescence Parameters 68 IV Relationship between the Operating Efficiencies of PS I and PS II Electron Transport 71
xi V. Factors Associated with Changes in PS II Operating Efficiency 71 VI. The Relationship between PS II Operating Efficiency and the Quantum Yield of C02 Assimilation 75 VII. Can Rates of Electron Transport and C02 Assimilation be Calculated Accurately from PS II Operating Efficiencies? 77 VIII. Concluding Remarks 78 Acknowledgments 79 References 79
4 Nuts and Bolts of Excitation Energy Migration and Energy Transfer 83-105 Robert M. Clegg Summary 84 I. Introduction 84 II. Historical Background 84 III. Why Fluorescence Resonance Energy Transfer (FRET) Is Such a Popular Method of Measurement 87 IV FRET Basics: A Short Description 88 V What Can We Learn from Energy Transfer? 90 VI. Simple Portrayal of the FRET Process that Explicates the Different Ways of Measuring Energy Transfer Efficiency 91 VIII. Transfer between Identical Molecules Detected by Fluorescence Anisotropy 100 IX. Models of Energy Transfer through Photosynthesis Antennae Systems 100 X. Energy Transfer by Electron Exchange 101 XI. Assumption of Non-coherent Mechanisms. Cooling Off to the Equilibrium Position of the Nuclei Positions 102 XII. Cascade Mechanism of Transfer—Emission and Reabsorption of a Photon 102 Acknowledgments 102 References 102
5 Transfer and Trapping of Excitations in Plant Photosystems 107-132 Rienk van Grondelle and Bas Gobets Summary 107 I. Introduction 108 II. Transfer and Trapping of Excitations in Photosystem (PS) I 112 III. Transfer and Trapping of Excitations in PS II 118 IV Concluding Remarks 127 Note Added in Proof 127 References 128
6 System Analysis and Photoelectrochemical Control of Chlorophyll Fluorescence in Terms of Trapping Models of Photosystem II: A Challenging View 133-172 Wim J. Vredenberg Summary 134 I. Introduction 134 II. The 'Classic' Two-state Trapping Model Of Photosystem II 137 III. Photoelectrochemical Control of PS II Chlorophyll Fluorescence 148
xii IV. A Three-state Energy Trapping Model of Photosystem II 153 V. Concluding Remarks, Controversies and Perspectives 163 Acknowledgments 168 References 168
7 Photon Capture, Exciton Migration and Trapping and Fluorescence Emission in Cyanobacteria and Red Algae 173-195 Mamoru Mimuro Summary 174 I. Introduction 174 II. Antenna Systems in Cyanobacteria and Red Algae 175 III. Excitation Energy Transfer and Trapping 183 IV. Energy Transfer Mechanisms 188 V. Diversity of Pigments and Antenna Systems in Cyanobacteria 189 VI. Concluding Remarks 191 Acknowledgments 191 References 192
8 Photosystem II: Oxygen Evolution and Chlorophyll a Fluorescence Induced by Multiple Flashes 197-229 Vladimir Shinkarev Summary 198 I. Introduction to Photosystem II 199 II. Biochemical Organization of Photosystem II 199 III. Electron Transport 204 IV The Kok Model for the Flash-induced Oxygen Evolution 208 V. Binary Oscillations of the Plastosemiquinone on the Acceptor Side of Photosystem II 213 VI. Chlorophyll a Fluorescence 214 VII. Conclusions 223 Acknowledgments 224 References 225
9 Fluorescence of Photosystem I 231-250 Shigeru Itoh and Kana Sugiura Summary 231 I. Introduction 232 II. Fluorescence of Photosystem I in Vivo 234 III. Fluorescence in Isolated Photosystem I Reaction Centers 238 IV Fluorescence in the Chlorophyll-depleted Reaction Center of Photosystem I 241 V Photosystem I with Chlorophylls Other than Chlorophyll a 244 VI. Concluding Remarks 246 Acknowledgments 247 References 247
xiii 10 The Relationship between Photosynthetic Electron Transfer and its Regulation 251-278 David M. Kramer, Thomas J.Avenson,Atsuko Kanazawa, Jeffrey A. Cruz, Boris Ivanov and Gerald E. Edwards Summary 252 I. Introduction 252 II. A'Static'Model for Photosynthesis and Down-regulation 253 III. Possible Mechanisms of Short-term Variation in Down-regulatory Sensitivity 255 IV. Conclusions and Working Model 270 Note Added in Proof 270 Acknowledgments 271 References 271
11 Pulse-Amplitude-Modulation (PAM) Fluorometry and Saturation Pulse Method: An Overview 279-319 Ulrich Schreiber Summary 280 I. Introduction 280 II. Principle of Pulse-Amplitude-Modulation 282 III. Information Carried by Chlorophyll Fluorescence Yield 284 IV Saturation Pulse Method of Quenching Analysis 287 V Assessment of Quantum Yield and Relative Electron Transport Rate 294 VI. Intrinsic Heterogeneity of Variable Chlorophyll Fluorescence 297 VII. Pulse Amplitude Modulation (PAM) Fluorometry for Special Applications 306 Acknowledgments 312 References 312
12 Analysis of the Chlorophyll a Fluorescence Transient 321-362 Reto J. Strasser, Merope Tsimilli-Michael and Alaka Srivastava Summary 322 I. Introduction 323 II. Theoretical Background 325 III. Fluorescence Transients in the Presence of Diuron at Room Temperature 335 IV Fluorescence Transients at Low Temperature (77K) 337 V Polyphasic Fluorescence Transients in Vivo 339 VI. Concluding Remarks and Future Perspectives 356 Acknowledgments 358 References 358
13 Light Emission as a Probe of Charge Separation and Recombination in the Photosynthetic Apparatus: Relation of Prompt Fluorescence to Delayed Light Emission and Thermoluminescence 363-388 Esa Tyystjarvi and Imre Vass Summary 364 I. Introduction 364 II. Thermodynamics of Reaction Kinetics 364
xiv III. Variable Chlorophyll Fluorescence 372 IV. Delayed Light Emission (DLE) from Photosynthetic Systems 374 V. Thermoluminescence (TL) 379 VI. Concluding Remarks 381 Acknowledgments 382 References 382
14 Chlorophyll Fluorescence Imaging of Leaves and Fruits 389-407 Ladislav Nedbal and John Whitmarsh Summary 390 I. Introduction 390 II. Imaging Technology and Techniques 394 III. Sources of Heterogeneity in Fluorescence Images 401 IV Future Applications 403 Acknowledgments 404 References 404
15 Using Chlorophyll a Fluorescence Imaging to Monitor Photosynthetic Performance 409-428 Kevin Oxborough Summary 410 I. Introduction 410 II. Theoretical Background 412 III. Technical issues 412 IV High Resolution Examples 419 V. Low Resolution Examples 422 VI. The Immediate Future and Concluding Remarks 425 Acknowledgments 427 References 427
16 Remote Sensing of Chlorophyll Fluorescence: Instrumentation and Analysis 429-445 Ismael Moya and Zoran G. Cerovic Summary 429 I. Introduction 430 II. Ground Based Measurements 431 III. Long Distance Fluorosensing 440 IV Concluding Remarks 443 Acknowledgments 443 References 443
17 Probing the Mechanism of State Transitions in Oxygenic Photosynthesis by Chlorophyll Fluorescence Spectroscopy, Kinetics and Imaging 447-461 John F.Allen and Conrad W. Mullineaux Summary 447 I. Introduction to State Transitions 448
xv II. Studying State Transitions using Continuous Measurements of Fluorescence 451 III. Studying State Transitions using Picosecond Fluorescence Kinetics 452 IV. Using Fluorescence Recovery after Photobleaching (FRAP) to Study Protein Mobility 455 V. Screening for State Transition Mutants 457 VI. Concluding Remarks 458 Acknowledgments 460 References 460
18 Non-photochemical Energy Dissipation Determined by Chlorophyll Fluorescence Quenching: Characterization and Function 463-495 G. Heinrich Krause and Peter Jahns Summary 464 I. Introduction 464 II. Definition of Quenching Parameters 465 III. Characterization and Mechanisms of Non-photochemical Quenching 467 IV Function of Thermal Energy Dissipation 481 V Conclusions 485 Acknowledgments 485 References 485
19 Excess Light Stress: Multiple Dissipative Processes of Excess Excitation 497-523 Doug Bruce and Sergej Vasil'ev Summary 498 I. Introduction 498 II. Origins, Measurements and Interpretations of Variable Chlorophyll Fluorescence 500 III. Fluorescence Quenching, Multiple Mechanisms for the Dissipation of Energy 509 IV Concluding Remarks 518 Acknowledgments 519 References 519
20 Using Mutants to Understand Light Stress Acclimation in Plants 525-554 Talila Golan, Xiao-Ping Li, Patricia Muller-Moule, and Krishna K. Niyogi Summary 526 I. Introduction 526 II. Biochemical and Physiological Aspects of Light Stress 527 III. Genetic Methods to Study Abiotic Stress 531 IV Insights into Light Stress Acclimation 538 V Genomics and the Future 546 Acknowledgments 547 References 547
xvi 21 Excess Light Stress: Probing Excitation Dissipation Mechanisms through Global Analysis of Time- and Wavelength-Resolved Chlorophyll a Fluorescence 555-581 Adam M. Gilmore Summary 556 I. Introduction 556 II. Time- and Wavelength-Resolved Fluorescence Instrumentation 560 III. Overview of Global Analysis 564 IV. Applications of Global Statistical Analysis 569 V. Conclusions and Future Research 577 Acknowledgments 578 References 578
22 Chlorophyll Fluorescence as a Tool to Monitor Plant Response to the Environment 583-604 William W.Adams III and Barbara Demmig-Adams Summary 584 I. Introduction 584 II. Regulation of Excitation Energy Transfer within Photosystem II Complexes 585 III. Photoinhibition, Zeaxanthin Retention, and Sustained Decreases in Fv/Fm 592 IV. Using Chlorophyll Fluorescence to Assess Photosynthetic Performance 595 V Strategies of Adjustment to Excess Light: Light Harvesting Capacity, Photosynthetic Electron Flow, and Excitation Energy Transfer Efficiency 597 VI. Concluding Remarks: What Chlorophyll Fluorescence Can and Cannot Reveal about Stress in Plants 598 Acknowledgments 599 References 599
23 Plant Responses to Ultraviolet Radiation Stress 605-621 Manfred Tevini Summary 605 I. Introduction: Ozone Reduction and UV Radiation Stress 606 II. General Responses to UV Radiation 607 III. Responses in Photosynthesis 609 IV Photosynthesis Under Ecological Conditions 614 V Conclusions 615 Acknowledgments 615 References 615
24 Effects of Water Stress on the Photosynthetic Efficiency of Plants 623-635 Nikolai G Bukhov and Robert Carpentier Summary 623 I. Introduction 624 II. Water Deficit in Desiccation-tolerant or Poikilohydric Lower Plants 624 III. Water Deficit in Desiccation-tolerant Vascular Plants 626 IV Water Deficit in Desiccation-sensitive Higher Plants 627
xvii V. Photosystem II Function in Crassulacean Acid Metabolism Species under Drought Conditions 631 VI. Conclusions 632 References 632
25 Chlorophyll a Fluorescence as a Probe of Heavy Metal Ion Toxicity in Plants 637-661 Manoj K. Joshi and Prasanna Mohanty Summary 637 I. Introduction 638 II. Dynamics of Chlorophyll a Fluorescence Changes and Their Relationship to the Structure-Function of Photosynthetic Membranes 639 III. Role of Chlorophyll a Fluorescence Imaging in Detection/Understanding of Metal Ion Stress 641 IV. Commonality in Metal Ion Action 642 V Amelioration of Metal Ion Action by Other Metal Ions 644 VI. Action of Selected Heavy Metal Ions on Plants 645 VII. Conclusions and Perspectives 652 Acknowledgments 652 References 652
26 Water and Solute Transport in Cyanobacteria as Probed by Chlorophyll Fluorescence 663-678 George C. Papageorgiou and Kostas Stamatakis Summary 663 I. Introduction 664 II. Light-induced and Osmotically-induced Changes of Chlorophyll a Fluorescence in Cyanobacteria 664 III. Applications 669 IV Do Osmotically-induced Changes in Chlorophyll a Fluorescence and State Transitions Share a Common Mechanism in Cyanobacteria? 674 V Conclusions 675 Acknowledgments 675 References 675
27 Assembly of Light-Harvesting Complexes of Photosystem II and the Role of Chlorophyll Jb 679-712 J. Kenneth Hoober and Joan H.Argyroudi-Akoyunoglou Summary 680 I. Introduction 680 II. Biological Context for Considering LHC II Assembly 683 III. The Role of Chlorophyll (Chi) b 690 IV Identification of Chls within Native and Reconstituted LHC II 698 Note Added in Proof 703 Acknowledgment 704 References 704
xviii 28 Light Adaptation and Senescence of the Photosynthetic Apparatus. Changes in Pigment Composition, Chlorophyll Fluorescence Parameters and Photosynthetic Activity 713-736 Hartmut K. Lichtenthaler and Fatbardha Babani Summary 713 I. Introduction: Occurrence and Function of Photosynthetic Pigments 714 II. Light Adaptation of Pigment Composition and Chloroplast Function 716 III. Chlorophyll Fluorescence Parameters as Indicators of Photosynthetic Function 721 IV. Chlorophyll Fluorescence and Pigment Changes During Autumnal Senescence 724 V. Chlorophyll Fluorescence Imaging of Photosynthetic Activity 730 VI. Conclusions 733 Acknowledgments 733 References 734
29 From Leaves to Ecosystems: Using Chlorophyll Fluorescence to Assess Photosynthesis and Plant Function in Ecological Studies 737-755 Jeannine Cavender-Bares and FakhriA. Bazzaz Summary 737 I. The Role of Photosynthesis in Ecological Research 738 II. Definition and Explanation of Fluorescence Parameters 739 III. Detecting Stress in Plants at the Leaf and Whole Plant Level 741 IV Measuring Productivity at the Ecosystem Level 747 V Scaling from the Bottom Up — The Role of Species Composition in Ecosystem Dynamics 750 VI. Concluding Remarks 752 Acknowledgments 752 References 752
30 Development and Application of Variable Chlorophyll Fluorescence Techniques in Marine Ecosystems 757-778 Paul G. Falkowski, Michal Koblfzek, Maxim Gorbunov and Zbigniew Kolber Summary 757 I. Introduction 758 II. Fluorescence-based Estimation of Photosynthetic Electron Transport 762 III. The Functional Absorption Cross Section of Photosystem (PS) II 764 IV Measuring Variable Chlorophyll Fluorescence in Marine Environment 766 V Variations in the Maximum Quantum Yield of Fluorescence in Marine Environments 768 VI. Fluorescence-based Estimates of Primary Production 771 VII. Applications of Variable Fluorescence in Benthic Ecosystems 773 VIII. Aerobic Anoxygenic Phototrophs 774 IX. Concluding Remarks 776 Acknowledgments 776 References 776
xix 31 Plant Productivity of Inland Waters 779-793 John A. Raven and Stephen C. Maberly Summary 780 I. Introduction 780 II. The Habitat 781 III. The Organisms 785 IV. Primary Production and Biomass 787 V. Conclusions and Future Prospects 791 Acknowledgments 791 References 791
Index 795
XX Preface
If we were to single out one line of technological AIPH series, edited by H. A. Frank, A. J. Young, G. inventions that provided the greatest stimulus to Britton and R. J. Cogdell biological research, and was really behind the un- Most of the light which photosynthetic organisms precedented advances that biological sciences made absorb is invested in the photoinduced transport of since World War II, then we would choose technolo- electrons and protons from water to nicotinamide gies to detect weak light signals by transducing them adenine dinucleotide phosphate and in the phos- to electrical signals, be it by the photomultiplier phorylation of ADP to ATP (see Photosynthesis: tube of the late 1940s, or the transistor photodiode Photobiochemistry and Photobiophysics by B. Ke, and the charge coupled device detector of the late AIPH series, Volume 10). Both products are needed 1960s and early 1970s. And if we were to name the to convert carbon dioxide to carbohydrate via the one scientific discipline these technologies and their enzymes of the Calvin-Benson-Bassham Cycle. A variants benefited the most, then we would place minor part of the electronic excitation energy, that our bet on photosynthesis. And for good reason. In is acquired by light absorption, escapes back as photosynthesis, light is a reactant, therefore it is used Chi a fluorescence, the focus of the present volume. quantitatively by all kinds of photosynthetic organ- Decades of imaginative research, technological in- isms which employ pigment assemblies to capture it novations, and advances in many scientific fronts and utilize its energy. Photosynthesis is of course the combined in elevating Chi a fluorescence to a major ubiquitous, fundamental and characteristic process diagnostic tool of both static and dynamic aspects of of our planet, the very foundation of all food chains. photosynthesis. For a description of the structure and function of Chlorophyll a fluorescence has always been a fasci- pigment-protein complexes, that contain these pig- nating area of intellectual adventure, as well as aprac- ment assemblies, see Volume 13 of the Advances tical tool, to scientists of diverse origins and tastes. in Photosynthesis and Respiration (AIPH) series, Together with other spectroscopic and biochemical 'Light-harvesting Antennas in Photosynthesis', edited methods it helped elucidate many important features by B. R. Green and W A. Parson. of the mechanism of photosynthesis: the modes and Actually, photosynthesis is a mosaic of partial dynamics of exciton migration in pigment assemblies; processes, some of which may trace their origin back the trapping of excitonic energy by special reaction to the late Archaean era (about 3000 million years center chromophores and its stabilization as redox ago). In spite of its long evolutionary course, and the potential difference; and the adaptive mechanisms great diversity of present day photosynthetic organ- which oxygenic photosynthesizers mobilize in order isms, the biological machinery of photosynthesis is to withstand successfully environmental stresses, such remarkably conservative. Its key pigment, chlorophyll as light, heat, salinity, dehydration, and pollution. (Chi) a, is present in all oxygenic photosynthetic More importantly, basic research in the 1960s organisms, and with one recently identified excep- and 1970s led to the realization that Chi a fluores- tion, it is the exclusive photoactive chromophore of cence, which measures the fraction of the absorbed all photosynthetic reaction centers, which convert photosynthetically active radiation that is not used and store electronic excitation energy in the form for photosynthesis, is in a way an inverse measure of oxidoreduction potential difference. In addition, of the productivity of green plants. Thus, the more only a few other kinds of colored molecules (e.g. Chi a fluorescence a plant emits, the less productive Chi b, Chi c, Chi d, carotenes, xanthophylls and it is. However, a major caveat to remember in this phycobiliproteins) assist Chi a in photon harvesting. inverse relationship is that the heat loss must remain Indeed, the disparity between the great multitude of constant. However, during exposure to excess light, photosynthetic organisms and the small number of both fluorescencean d photochemistry decrease and photosynthetic chromophores is so enormous, that heat loss increases. And, this has been exploited to the latter are used to characterize broad taxonomic understand the mechanism of photoprotection by divisions of the former. 'The Photochemistry of plants and algae. Carotenoids' has been discussed in Volume 8 of the Light-induced transients of Chi a fluorescence
xxi have been particularly useful in assessing plant and biophysicists. It has been designed so that not productivity, since photosynthetic efficiency could only the beginning and advanced researchers, but the be quantified in terms of the transient amplitudes. advanced undergraduate and graduate students will Subsequent developments in Chi a fluorescence find it invaluable. imaging enabled the visualization of the metabolic We take this opportunity to thank first the one status of whole leaves. These discoveries, combined person who was most crucial in bringing this with technical advances that increase the signal/noise book to publication stage: our good friend Larry ratio and allow the registration of reliable fluorescence Orr (of Arizona State University). A book of this signals even in strong light backgrounds, took Chi a magnitude and depth on 'Chlorophyll a Fluo- fluorometry out of the dark room and into the broad rescence' was possible only because our authors daylight. Equipped with portable and reasonably are in the forefront of their fields. We thank all of priced Chi a fluorometers, field scientists are now them for their cooperation, for providing us with able to monitor the photosynthetic productivity of authoritative chapters, and above all for their patience individual leaves and plants rapidly, conveniently, with our regular and sometimes annoying e-mails sent repeatedly, non-destructively, and inexpensively. past midnight. Noeline Gibson and Jacco Flipsen Moreover, they can enter masses of numerical data (both at Kluwer Academic Publishers) deserve our into suitable mathematical models which return time gratitude for their spirit of cooperation and their help and space snapshots of productivity of oxygenic pho- in producing the book. Without the advice of several tosynthesizers. Still another technological advance anonymous reviewers, a book of this scope would not is the ability for the sensing of Chi a fluorescence have been possible. Nancy Kiang (at NASA Goddard from terrestrial, airborne and satellite platforms. In Institute of Space Studies) is thanked for the striking principle, the detected fluorescence bears informa- diagram on the book's cover. We also acknowledge tion not only on the ecosystem productivity, but also the support of our respective institutes (Institute of on the effects of environmental stresses and on the Biology, NCSR, Demokritos, Athens, Greece; and diversity ofplant species. All these applications, more Department of Plant Biology, University of Illinois, than justify Chlorophyll Fluorescence: A Signature Urbana, IL, USA). Finally, we owe special gratitude of Photosynthesis, as the title of this book to our wives (Sophie Papageorgiou and Rajni Gov- The book comprises 31 chapters, authored by 59 indjee) for tolerating us and supporting us when we experts in Chi a fluorescence in vivo. The outstand- most needed their help. ing contributions of many more distinguished pho- tosynthesis scientists are paraded through its pages. We mourn the loss of our dear friend and photobi- Chapters are arranged in seven sections. The opening ologist, Professor Gauri Shankar Singhal (Jawaharlal section (1) comprises introductory overviews on Chi Nehru University, New Delhi), who passed away on a fluorescence. It is followed by sections in photobio- July 3, 2004. We knew him as a wonderful human physics (2), photobiochemistry (3), instrumentation beingand an outstanding young photo chemist when and analysis (4), the use of Chi a fluorescence as a he was a postdoctoral associate with Eugene Rabi- sensor of environmental stresses (5), and chloro- nowich, at Urbana, Illinois, in the 1960s, and when phyll biosynthesis and degradation (6); and the last he hosted several international conferences in New section (7) includes chapters on the productivity of Delhi, in the 1980s. ecosystems (terrestrial, freshwater and marine) as detected and quantified by measurements of Chi a George C. Papageorgiou fluorescence. Athens, Greece The present volume covers both the basics as well E-mail: [email protected] as the applications of Chi a fluorescence in one vol- ume. It brings to laboratory scientists in touch with Govindjee the realities of photosynthetic productivity, and open- Urbana, Illinois,USA field scientists in solid awareness of the principles, the E-mail: [email protected] physical basis, the potential and the instrumentation of Chi a fluorometry. It is an indispensable book not only for plant physiologists, agronomists and ecolo- gists, but equally for plant biologists, biochemists,
xxii Res 80: 427-437). George was Govindjee's first Ph.D. student; other students being John C. Mun- day, Jr., Frederick Cho and Ted Mar; George did his graduate research using the homemade fluorometer of Govindjee, a rare instrument in early 1960s when all existing fluorometerswer e homemade. In 1968, he obtained a Ph. D. in Biophysics , with a thesis on 'Fluorescence induction in Chlorellapyrenoidosa and Anacystis nidulans and its relation to photohosphory- lation.' After graduation, and some post-graduate work at the University of Illinois, he was appointed as research scientist in the Photosynthesis Laboratory of NCSR Demokritos (head George Akoyunoglou; see G. C.Papageorgiou (2003) Photosynth Res 76 :427-433) and few years later he was named head of the Laboratory of Membrane Biophysics and Biotech- nology at the Department of Biology (now Institute of Biology). George has held several administrative positions in Demokritos, served in national and inter- national committees, organized or participated in the organization of a number international conferences and symposia (including the organization in 1980 of The above photograph of George Papageorgiou, the 5th International Congress of Photosynthesis in standing on the volcanic ash beach, was taken by Greece, whose chief organizer was George Akoyu- Sophie Papageorgiou in 2004 on the Island of Melos, noglou). George is one of the founding members of Greece. the Hellenic Biochemical and Biophysical Society (now Hellenic Society of Biochemistry and Molecular George Papageorgiou Biology), and has served this Society as its secretary for several years His scientific interests centered on membrane bioenergetics and biophysics, withparticu- George C. Papageorgiou, one of the editors of this lar emphasis onphotosynthetic membranes of higher volume, retired in 2001 as Research Director, after plants, algae and cyanobacteria. During his scientific 32 years of service, at the Institute of Biology of the career, he has collaborated with many distinguished National Center of Scientific Research (NCSR) De- colleagues in the fields of photosynthesis and mem- mokritos in Athens, Greece. He received a diploma brane bioenergetics. Special mention is made here in chemistry in 1958 from his hometown university of Govindjee (UIUC), the late Eugene Rabninowitch (University of Thessaloniki, Greece), then served in (UIUC), the late George Akoyunoglou (NCSR, De- the Greek Army as an artillery reserve officer. After a mokritos), Joan Argyroudi-Akoyunoglou (NCSR, number of teaching jobs in Greece, he was admitted Demokritos), Lester Packer (University of California, in 1963 for graduate studies in the Department of Berkeley, CA), the late David Hall (University of Physiology and Biophysics of the University of Illi- London, King's College), Sergio Papa (University of nois at Urbana-Champaign (UIUC). He did his thesis Bari) and Norio Murata (National Institute of Basic research in the laboratories of Govindjee and Eugene Biology, Okazaki). After retirement, George has been Rabinowitch, where he was introduced to 'Photosyn- a collaborating research scientist at the Institute of thesis.' There, he met and befriended several young, Biology of NCSR Demokritos. His current research at that time, scientists who were to become later well (with his successor, Dr. Kostas Stamatakis) centers known photosynthesis personalities in the subsequent on osmotic volume changes of cyanobacterial cells, years (e.g., Prasanna Mohanty, Alan Stemler, Gauri as detected by changes in the intensity of phycobili- Singhal, W. Patrick Williams, Laszlo Szalay, Danuta some sensitized chlorophyll a fluorescence. Fr^ckowiak; also see S. S.Brody (2002) Photosynth Res 73:127-132; andA. K. Ghosh (2004) Photosynth
xxiii A 1954 group photograph ofGovindjee (see arrow) with his fellow students at the University ofAllhabad, Allahabad, India. Photo, taken at 14B Bank Road, courtesy ofDurga Prasad Tiwari.
Govindjee
Govindjee is Professor Emeritus of Biochemistry, tinguished Lecturer of the School of Life Sciences, Biophysics and Plant Biology at the University of UIUC (1978); President of the American Society of Illinois at Urbana-Champaign (UIUC), Illinois, Photobiology (1980-1981); Fulbright Senior Lecturer USA, since August 1, 1999. He received his B.Sc. (1996-1997); and honorary President of the 2004 (Chemistry,BotanyandZoology)andM.Sc. (Botany) International Photosynthesis Congress (Montreal, from the University of Allahabad, Allahabad India, in Canada). Govindjee's research has focused on the 1952 and 1954, respectively. He specialized in Plant function of 'Photosystem IF (water-plastoquinone Physiology under Shri Ranjan (a former student of oxido-reductase), particularly on the primary pho- Felix Frost Blackman); he was a Lecturer in Botany tochemistry; role of bicarbonate in the electron and at Allahabad University from 1954-1956. He came proton transport; thermoluminescence, delayed and to the University of Illinois at Urbana-Champaign prompt fluorescence (particularly lifetimes), and their (UIUC), Illinois, USA in 1956, as a Fulbright scholar, use in understanding electron transport and photo- to work for his Ph.D. in physico-chemical biology protection against excess light. He has coauthored under Robert Emerson. After Emerson's untimely Photosynthesis (1969); and has edited (or co-edited) death in a plane crash in February, 1959, he worked Bioenergetics of Photosynthesis (1975); Photosyn- with Eugene Rabinowitch, and obtained his Ph.D. thesis (in2 volumes, 1982); Light Emission by Plants in Biophysics from UIUC, in 1960, with a thesis on and Bacteria (1986), among several other books. the Action Spectra of the Emerson Enhancement He is a member of the American Society of Plant Effect in Algae'. From 1960-1961, he served as a Biology (formerly Physiology), American Society United States Public Health (USPH) Postdoctoral for Photobiology, Biophysical Society of America, Fellow; from 1961-1965, as Assistant Professor of and the International Society of Photosynthesis Re- Botany; from 1965-1969 as Associate Professor of search (ISPR). Govindjee is interested in 'History Biophysics and Botany; and from 1969-1999 as of Photosynthesis Research,' and in 'Photosynthesis Professor of Biophysics and Plant Biology, all at the Education.' For further information, see his web page UIUC. His honors include: Fellow of the American at: http://www.life.uiuc.edu/govindjee. He can always Association of Advancement of Science (1976); Dis- be reached by e-mail [email protected].
xxiv Color Plates
Color Plate 1
Fig. 1, left: Structural model of monomeric LHCII. Red, protein backbone; blue, Chi a; green, Chi b; blue/green, mixed Ch\a/Ch\b. Right: Constructed image of a 'complete' PSII-LHCII supercomplex, the hypothetical C2S2M2L2 supercomplex (adapted from Boekema et al., 1999b). The PSII core complexes are depicted in blue, the 'minor' peripheral antenna complexes CP29, CP26 and CP24 are de- picted in red and the trimeric LHCII complexes at the so-called S, M and L positions are depicted in green. S, M and L refer to strongly, moderately and loosely bound LHCII, respectively. See Chapter 5, van Grondelle and Gobets.
Fig. 2. The Photosystem II super-complex viewed from the side (a) and from the lumen (b). In (c) the docking sites of the 17 kD, 23 kD and 33 kD proteins of the extrinsic oxygen-evolving complex and the trans-membrane a-helices of the protein subunits in the core complex are shown at higher magnification. The structures of LHCII, CP24 and CP26 were derived from Kiihlbrandt et al. (1994) and their positions are based on internal density distribution in the super-complex, determined by electron microscopy, and chemical cross- linking (From Nield et al., 2000; used with permission). See Chapter 27, Hoober and Akoyunoglou.
George C. Papageorgiou and Govindjee (eds): Chlorophyll a Fluorescence: A Signature of Photosynthesis, pp. CP-l-CP-8. © 2004 Springer. Printed in The Netherlands. Color Plate 2
Fig. 1. A leaf'of Nerium oleander. Heterogeneity was artificially Fig. 2. Coefficient of nonphotochemical quenching, qN, imaged induced by dipping the tip of the leaf in 45 °C water for 5 min; at the upper surface of a dandelion leaf (Taraxacum officinale) continuous illumination at 400 jimol photons nr2 s"1. (a) Coef- showing spatiotemporal variations after spot heating by a pulse ficient of absorbed PAR; (b) Effective quantum yield (AF/Fm'); of near-infrared laser light. The leaf was illuminated by blue light (c) Relative rate of photosynthesis; (d) Nonphotochemical (470 nm) at 80 jimol photons nr2 s-1 . Saturation pulses for the quenching (NPQ). Relative values ranging from 0 to 1 are dis- assessment of quenching parameters were applied every 10 s. The played using the indicated false color scale (black corresponds displayed images were recorded: (a) 10s before the infrared laser to 0). Measurements with the IMAGING-PAM fluorometer. See pulse; (b) 20 s after the infrared laser pulse; (c) After additional Chapter 11, Schreiber. 60 s; (d) After additional 60 s. qN values between 0 and 1 are coded by a scale of gray tones ranging from black to white. The green color dominating before the laser pulse corresponds to qN a 0.4, whereas the blue color spreading via the vein system after the laser pulse corresponds to qN a 0.7. Measurements with the IMAGING-PAM fluorometer. See Chapter 11, Schreiber.
Fig. 3. An attached Nicotiana tabacum leaf before and after injecting 50 mM CuS04 into the main vein. The image which represents the relative fluorescence decrease (Rfd = Fp - Fs/Fs) was created by pixel point processing. Fp and Fs are the fluorescence images after 1 s of illumination and at the end of the fluorescence induction (steady state), respectively, (a) False color scale, with blue indicating high and red low Rfd; (b) before the CuS04 treatment (high Rfd values); (c) 30 min after the CuS04 treatment (a decline in Rfd); (d) 60 min after the CuS04 treatment (further decline in Rfd). Adapted from Ciscato and Valcke, 1998. See Chapter 25, Joshi and Mohanty.
CP-2 Color Plate 3
Fig. 1, Left. Solar induced fluorescence yields as retrieved by MODIS, an Earth orbiting satellite based sensor of moderate resolution. The fluorescence retrievals are calculated from the water leaving radiances at 685 nm, which are subsequently corrected for atmospheric scattering, and then normalized to chlorophyll a and total spectral irradiance. Right. Chlorophyll distribution, variable fluorescence, photosynthetic yields, functional absorption cross section, and primary production calculated along the transect across the Middle Atlantic Bight off the New Jersey coast. See Chapter 30, Falkowski et al.
CP-3 Fig. 1. Images of chlorophyll fluorescence showing F0, FM, Fs, and FM' for an African violet leaf (Saintpaulia) infiltrated by the photosys- tem II inhibitor DCMU (top row). The bottom row shows the ratios FV/FM (= (FM- F0)/FM,), 1^,= (FM- FS)/FM), PSII (= (FM' - FS)/FM'), and NPQ (= (FM- FM')/FM') calculated pixel-by-pixel using the data in the top row. The numerical values shown to the right of the rows are color-coded using a red (high level) to blue (low level) color scale. See Chapter 14, Nedbal and Whitmarsh.
Fig. 2. Three-dimensional visualization of signals imaged by a CCD camera. The two-dimensional image is represented by the x-y axes and the signal amplitude is represented by the z-axis. A red-blue color scale is used to enhance visual perception, with high signals shown in red (maximum 255) and low signals in blue (minimum 0). (A) Fluorescence signal of a plastic sheet containing a uniformly distributed fluorescent dye (Nile Blue) illuminated by diffuse homogenous light. The non-uniformity of the image is due to the proximity of the camera objective lens (9 cm above the Nile Blue sheet). (B) Fluorescence signal emitted by the Nile Blue sheet excited by measuring flashes from an array of orange light emitting diodes (l^ = 635 nm). The non-uniformity of the signal is due to the camera optics and the non-uniformity of the measuring light field. (C) Fluorescence signal emitted by the Nile Blue sheet excited by an intense pulse of light from a 250 W halogen lamp (3000 (xmol photons rrr2 s_1) The non-uniformity of the signal is due to the combined effect of the camera optics and inhomogeneity of the actinic light field. (D) Scattered light signal from a leaf (Hedera) placed on white paper that was illuminated by the orange LED array. Scattered light was selected by placing an orange filter in front of the camera. The low signal in the center of the image is due to light absorption by the leaf. (E) Image of the fluorescence signal emitted by the leaf excited by the orange LED array. Fluorescent light was selected by placing a red filter in front of the camera that passed 695-750 nm light.The camera sensitivity was adjusted to provide similar signal amplitude in each experiment. See Chapter 14, Nedbal and Whitmarsh.
Fig. 3. Scattered light and fluorescence images of a Hedera leaf. (A) Color photograph of the leaf illuminated by white light (24 bit, 2048 x 1536 pixels). (B) Color photograph of the leaf illuminated by the orange LED measuring flashes (635 nm). (C) Map of scattered measuring light (635 nm) from white paper without the leaf. Note that the scattered light signal is shown only for the area previously occupied by the leaf. The image was captured without a red filter in front of the camera. (D) Map of scattered measuring light from the leaf (same as C except with the leaf). (E) The difference between images (C) and (D), showing the decrease in scattering due to light absorption by the leaf. (F) Map of the fraction of measuring light absorbed by the leaf (calculated by dividing the pixel data in image (D) by the pixel data in image (C). Images (C-F) were captured using a monochrome CCD camera (8-bit, 400 x 300 pixels). See Chapter 14, Nedbal and Whitmarsh.
Fig. 4. Fluorescence images of F0, FM, Fv, and FM' emitted by a Hedera leaf (top row). The bottom row shows the same fluorescence im- ages after normalization by dividing each pixel by the value of the corresponding pixel in the image mapping the absorbed light shown in Fig. 3E. Normalization serves to correct for heterogeneity in the distribution of chlorophyll in the leaf and heterogeneity in the measuring light field, which yields a more accurate map of the fluorescence yield parameters. See Chapter 14, Nedbal and Whitmarsh.
Fig. 5. Images of the red Chi fluorescence of a sun (A) and a shade (B) leaf of beech at maximum fluorescence Fp as well as at steady- state fluorescence Fs (C,D) after 5 min of illumination at the end of the Chi fluorescence induction kinetics (Kautsky effect). The scale in (A) to (D) indicates the intensity of the Chi fluorescence of the leaf pixels given in false colors from high (red) to low fluorescence (blue). Note the different scales for sun and shade leaves. The images of the Chi fluorescence decrease ratio (RFd = Fd/Fs) of the sun (E) and shade (F) leaf calculated from the induction kinetics are in the same scale. Chi fluorescence images were measured with the Karslruhe flash-lamp fluorescence imaging system (FL-FIS) using blue excitation light. See Chapter 28, Lichtenthaler and Babani.
Fig. 6. Images of the red chlorophyll fluorescence (F690) of a bean leaf at maximum fluorescence Fm (A) and at steady-state fluores- cence Fs (B) of the Chi fluorescence induction kinetics (Kautsky effect). (C), Image of the Chi fluorescence decrease ratio (RFd = Fd/Fs) which is an indicator of the photosynthetic quantum conversion and the C02 fixation rates of a normal bean leaf and (D), image of the Chi fluorescence decrease ratio of a water-stressed bean leaf. The scale in A and B indicates the intensity of the Chi fluorescence. In (C) and (D) the scales are different and indicate the values of the fluorescence decrease ratio RFd. Excitation with pulsed blue light (Xenon flash lamp with blue filter Corning No. 9782, X = 465 nm ± 50 nm). See Chapter 28, Lichtenthaler and Babani.
CP-4 Color Plate 4
1 2
CP-5 Fig. 1. Images from guard cells and surrounding mesophyll in a tradescantia leaf. (A) reflected light image, showing the stomatal opening. (B) F' image taken with a 680 nm bandpass filter. (C) F' image taken with a Schott RG695 (695 nm longpass) filter. (D) and (E) Images of Fq 7Fm' constructed using (B) and (C), respectively, plus Fm' images taken immediately after (B) and (C). (F) and (G) Fq '/Fm' images of the chloroplasts within guard cells only, isolated from (D) and (E), respectively. The incident actinic PAR was 93 /anol photons nr2 1 2 1 s" . The incident PAR during the saturating pulses used to measure Fm' was 6500 ,umol photons nr s" . Mean values of Fq 7Fm' in (D), (E), (F) and (G) are 0.60, 0.55, 0.56 and 0.55, respectively. The scale at the bottom of G represents 50 fim. The palette scale (which is a monochrome version of a false color palette) applies to (D), (E), (F) and (G). See Chapter 15, Oxborough.
Fig. 2. High resolution images taken from an intact biofilm. (A) F' image taken over 200 ms at an incident PAR of 200 ,umol photons 2 _1 2 -1 nr s . (B) Fm' image taken during the last 40 ms of a 400 ms saturating pulse at an incident PAR of 6500 /anol photons nr s . (C) Image of Fq 7Fm' constructed from (A) and (B), which illustrates the movement of cells between the F' and Fm' images being taken. (D) Image of Fq 7Fm' constructed from cells that were isolated within (A) and (B) and then nudged together to gain the best possible overlap. Cells can usually be identified to the genus level within raw or parameter images. Within (D), (i) Euglena sp.; (ii) Plagiotropis sp.; (iii) Pleurosigma sp.; (iv) Cylindrotheca sp.; (v) Gyrosigma sp. The white scale at the bottom of (D) represents 200 fjm. See Chapter 15, Oxborough.
Fig. 3. Images of two unidentified pelagic algae, which were isolated from a water sample taken from the Odense Fjord Denmark st on 21 August, 2002. Cells were concentrated onto a black Nucleopore filter. (A) Fo image taken over an integration period of 48 ms, 2 _1 during which 1 /is pulses of approximately 5000 fimol photons nr s were provided at 500 /is intervals. (B) Fm image taken during an integration period of 12 ms at the end of an 800 ms saturation pulse. (C) Fv/Fm image constructed from (A) and (B) The numbers on the images are mean values for each cell. In (C), the number in brackets at the top of the image is an integrated mean for the cell at upper left in (A) and (B), while the lower number is a pixel mean for the adjacent cell. A pixel mean could not be calculated for the upper cell in (C) because it had moved several /an in the 800 ms interval between the taking of the Fo and Fm images. The scale in (C) represents 20 fim. Images were taken using the LED based system described in Section III.A and by Fig. l.B in Chapter 15, Oxborough.
Fig. 4. Fluorescence images from a detached leaf of Cornus sp., which had it's petiole submerged in a 10 mM solution of DCMU for the entire experiment. The incident PPFD was 300 jimol nr2 s"1. Images of F' were generated by synchronizing the camera shutter to a 1 ms 2 _1 measuring pulse at a PPFD of 4600 jimol nr s . To produce images of Fm and Fm', a sequence of images was taken at 20 fps over the last 600 ms of an 800 ms saturating pulse. The image with the highest mean value was taken to be the Fm or Fm' value. The remaining images 2 1 were discarded. The PPFD of the saturating pulse and measuring pulses during this procedure was 4600 jimol nr s" . Images of Fo were generated by applying 2 (xs pulses, at a PPFD 4600 jimol m"2 s"1, at 400 (xs intervals during a 16.7 ms exposure. In (A-C), the false color images are of Fq 7Fm'. The small monochrome images to the left of the false color images are the F' image (leftmost) andFm' image that were used in construction of the Fq 7Fm' images. These pairs of images were taken after 60 s (A), 1 h (B) and 2 h (C) illumination. In (D), the false color image is olFJFm. The small monochrome images to the left are the Fo (leftmost) andFm images used in construction of the F/Fm image. This pair of images was taken after 2 h illumination, followed by 30 min dark-adaptation. The histograms to the right of the false color images (A- D) show how data values are mapped to the palette. The numbers below the histograms reflect the range of values represented by the x-axis in each case. The scale in (D) represents 20 mm. See Chapter 15, Oxborough.
Fig. 5. Methods for the isolation of multiple plants within an image. (A), (B) and (C) are all from the same Arabidopsis plants grow- ing within a 96 well plate. The 12 columns of plants were treated with different amounts of Imazapyr, 48 h before measurements were made. Each well received 15 jiL of Imazapyr in 50% acetone. The concentrations applied were (left to right): 0, 8 mM, 4 mM, 0.8 mM, 0.4 mM, 0, 0, 8 mM, 4 mM, 0.8 mM, 0.4 mM, 0. Columns 6 and 12 are controls (no acetone). Details of the imaging system and all lighting conditions (actinic light, saturating pulses and measuring pulses) are the same as described for Fig. 7 of Chapter 15. (A) F' image after non-fluorescent pixels have been removed using a low-cut filter (Section III.E.2 of Chapter 15). Circles highlight wells that contain more than one plant. (B) Individual plants have been identified in (A), using an algorithm which searches for blocks of connected pixels.
(C) Fv/Fm image from the same sample of Arabidopsis plants and in (A) and (B), which has been divided into 96 zones, using vertical and horizontal lines. Mean values for all plants within each column are, from left to right: 0.81, 0.35, 0.31, 0.45, 0.71, 0.81, 0.81, 0.36, 0.32, 0.51, 0.65, 0.78. (D) Histogram showing the distribution oiFy/Fm values in (C). See Chapter 15, Oxborough.
CP-6 Color Plate 5
1
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CP-7 Color Plate 6
Fig. 1. Algal and plant life forms in standing water. Top: Planktophytes (phytoplankton community from North Basin of Windermere) showing unicellular, colonial and filamentous algae from arange of taxonomic groups (photo courtesy of Dr Hilda Canter-Lund). Middle: Haptophytic red macroalga Lemanea mamillosa; note another haptophyte, Fontinalis and an epiphytic red alga on some of the filaments (photo courtesy Mr Trevor Furnass). Bottom: Submerged rhizophytic angiosperms (from about 3 m in Loch Borralie, Scotland; photo by the late Professor D. H. N. Spence). See Chapter 31, Raven and Maberly
CP-8 Color Plates
Color Plate 1
Fig. 1, left: Structural model of monomeric LHCII. Red, protein backbone; blue, Chi a; green, Chi b; blue/green, mixed Ch\a/Ch\b. Right: Constructed image of a 'complete' PSII-LHCII supercomplex, the hypothetical C2S2M2L2 supercomplex (adapted from Boekema et al., 1999b). The PSII core complexes are depicted in blue, the 'minor' peripheral antenna complexes CP29, CP26 and CP24 are de- picted in red and the trimeric LHCII complexes at the so-called S, M and L positions are depicted in green. S, M and L refer to strongly, moderately and loosely bound LHCII, respectively. See Chapter 5, van Grondelle and Gobets.
Fig. 2. The Photosystem II super-complex viewed from the side (a) and from the lumen (b). In (c) the docking sites of the 17 kD, 23 kD and 33 kD proteins of the extrinsic oxygen-evolving complex and the trans-membrane a-helices of the protein subunits in the core complex are shown at higher magnification. The structures of LHCII, CP24 and CP26 were derived from Kiihlbrandt et al. (1994) and their positions are based on internal density distribution in the super-complex, determined by electron microscopy, and chemical cross- linking (From Nield et al., 2000; used with permission). See Chapter 27, Hoober and Akoyunoglou.
George C. Papageorgiou and Govindjee (eds): Chlorophyll a Fluorescence: A Signature of Photosynthesis, pp. CP-l-CP-8. © 2004 Springer. Printed in The Netherlands. Color Plate 2
Fig. 1. A leaf'of Nerium oleander. Heterogeneity was artificially Fig. 2. Coefficient of nonphotochemical quenching, qN, imaged induced by dipping the tip of the leaf in 45 °C water for 5 min; at the upper surface of a dandelion leaf (Taraxacum officinale) continuous illumination at 400 jimol photons nr2 s"1. (a) Coef- showing spatiotemporal variations after spot heating by a pulse ficient of absorbed PAR; (b) Effective quantum yield (AF/Fm'); of near-infrared laser light. The leaf was illuminated by blue light (c) Relative rate of photosynthesis; (d) Nonphotochemical (470 nm) at 80 jimol photons nr2 s-1 . Saturation pulses for the quenching (NPQ). Relative values ranging from 0 to 1 are dis- assessment of quenching parameters were applied every 10 s. The played using the indicated false color scale (black corresponds displayed images were recorded: (a) 10s before the infrared laser to 0). Measurements with the IMAGING-PAM fluorometer. See pulse; (b) 20 s after the infrared laser pulse; (c) After additional Chapter 11, Schreiber. 60 s; (d) After additional 60 s. qN values between 0 and 1 are coded by a scale of gray tones ranging from black to white. The green color dominating before the laser pulse corresponds to qN a 0.4, whereas the blue color spreading via the vein system after the laser pulse corresponds to qN a 0.7. Measurements with the IMAGING-PAM fluorometer. See Chapter 11, Schreiber.
Fig. 3. An attached Nicotiana tabacum leaf before and after injecting 50 mM CuS04 into the main vein. The image which represents the relative fluorescence decrease (Rfd = Fp - Fs/Fs) was created by pixel point processing. Fp and Fs are the fluorescence images after 1 s of illumination and at the end of the fluorescence induction (steady state), respectively, (a) False color scale, with blue indicating high and red low Rfd; (b) before the CuS04 treatment (high Rfd values); (c) 30 min after the CuS04 treatment (a decline in Rfd); (d) 60 min after the CuS04 treatment (further decline in Rfd). Adapted from Ciscato and Valcke, 1998. See Chapter 25, Joshi and Mohanty.
CP-2 Color Plate 3
Fig. 1, Left. Solar induced fluorescence yields as retrieved by MODIS, an Earth orbiting satellite based sensor of moderate resolution. The fluorescence retrievals are calculated from the water leaving radiances at 685 nm, which are subsequently corrected for atmospheric scattering, and then normalized to chlorophyll a and total spectral irradiance. Right. Chlorophyll distribution, variable fluorescence, photosynthetic yields, functional absorption cross section, and primary production calculated along the transect across the Middle Atlantic Bight off the New Jersey coast. See Chapter 30, Falkowski et al.
CP-3 Fig. 1. Images of chlorophyll fluorescence showing F0, FM, Fs, and FM' for an African violet leaf (Saintpaulia) infiltrated by the photosys- tem II inhibitor DCMU (top row). The bottom row shows the ratios FV/FM (= (FM- F0)/FM,), 1^,= (FM- FS)/FM), PSII (= (FM' - FS)/FM'), and NPQ (= (FM- FM')/FM') calculated pixel-by-pixel using the data in the top row. The numerical values shown to the right of the rows are color-coded using a red (high level) to blue (low level) color scale. See Chapter 14, Nedbal and Whitmarsh.
Fig. 2. Three-dimensional visualization of signals imaged by a CCD camera. The two-dimensional image is represented by the x-y axes and the signal amplitude is represented by the z-axis. A red-blue color scale is used to enhance visual perception, with high signals shown in red (maximum 255) and low signals in blue (minimum 0). (A) Fluorescence signal of a plastic sheet containing a uniformly distributed fluorescent dye (Nile Blue) illuminated by diffuse homogenous light. The non-uniformity of the image is due to the proximity of the camera objective lens (9 cm above the Nile Blue sheet). (B) Fluorescence signal emitted by the Nile Blue sheet excited by measuring flashes from an array of orange light emitting diodes (l^ = 635 nm). The non-uniformity of the signal is due to the camera optics and the non-uniformity of the measuring light field. (C) Fluorescence signal emitted by the Nile Blue sheet excited by an intense pulse of light from a 250 W halogen lamp (3000 (xmol photons rrr2 s_1) The non-uniformity of the signal is due to the combined effect of the camera optics and inhomogeneity of the actinic light field. (D) Scattered light signal from a leaf (Hedera) placed on white paper that was illuminated by the orange LED array. Scattered light was selected by placing an orange filter in front of the camera. The low signal in the center of the image is due to light absorption by the leaf. (E) Image of the fluorescence signal emitted by the leaf excited by the orange LED array. Fluorescent light was selected by placing a red filter in front of the camera that passed 695-750 nm light.The camera sensitivity was adjusted to provide similar signal amplitude in each experiment. See Chapter 14, Nedbal and Whitmarsh.
Fig. 3. Scattered light and fluorescence images of a Hedera leaf. (A) Color photograph of the leaf illuminated by white light (24 bit, 2048 x 1536 pixels). (B) Color photograph of the leaf illuminated by the orange LED measuring flashes (635 nm). (C) Map of scattered measuring light (635 nm) from white paper without the leaf. Note that the scattered light signal is shown only for the area previously occupied by the leaf. The image was captured without a red filter in front of the camera. (D) Map of scattered measuring light from the leaf (same as C except with the leaf). (E) The difference between images (C) and (D), showing the decrease in scattering due to light absorption by the leaf. (F) Map of the fraction of measuring light absorbed by the leaf (calculated by dividing the pixel data in image (D) by the pixel data in image (C). Images (C-F) were captured using a monochrome CCD camera (8-bit, 400 x 300 pixels). See Chapter 14, Nedbal and Whitmarsh.
Fig. 4. Fluorescence images of F0, FM, Fv, and FM' emitted by a Hedera leaf (top row). The bottom row shows the same fluorescence im- ages after normalization by dividing each pixel by the value of the corresponding pixel in the image mapping the absorbed light shown in Fig. 3E. Normalization serves to correct for heterogeneity in the distribution of chlorophyll in the leaf and heterogeneity in the measuring light field, which yields a more accurate map of the fluorescence yield parameters. See Chapter 14, Nedbal and Whitmarsh.
Fig. 5. Images of the red Chi fluorescence of a sun (A) and a shade (B) leaf of beech at maximum fluorescence Fp as well as at steady- state fluorescence Fs (C,D) after 5 min of illumination at the end of the Chi fluorescence induction kinetics (Kautsky effect). The scale in (A) to (D) indicates the intensity of the Chi fluorescence of the leaf pixels given in false colors from high (red) to low fluorescence (blue). Note the different scales for sun and shade leaves. The images of the Chi fluorescence decrease ratio (RFd = Fd/Fs) of the sun (E) and shade (F) leaf calculated from the induction kinetics are in the same scale. Chi fluorescence images were measured with the Karslruhe flash-lamp fluorescence imaging system (FL-FIS) using blue excitation light. See Chapter 28, Lichtenthaler and Babani.
Fig. 6. Images of the red chlorophyll fluorescence (F690) of a bean leaf at maximum fluorescence Fm (A) and at steady-state fluores- cence Fs (B) of the Chi fluorescence induction kinetics (Kautsky effect). (C), Image of the Chi fluorescence decrease ratio (RFd = Fd/Fs) which is an indicator of the photosynthetic quantum conversion and the C02 fixation rates of a normal bean leaf and (D), image of the Chi fluorescence decrease ratio of a water-stressed bean leaf. The scale in A and B indicates the intensity of the Chi fluorescence. In (C) and (D) the scales are different and indicate the values of the fluorescence decrease ratio RFd. Excitation with pulsed blue light (Xenon flash lamp with blue filter Corning No. 9782, X = 465 nm ± 50 nm). See Chapter 28, Lichtenthaler and Babani.
CP-4 Color Plate 4
1 2
CP-5 Fig. 1. Images from guard cells and surrounding mesophyll in a tradescantia leaf. (A) reflected light image, showing the stomatal opening. (B) F' image taken with a 680 nm bandpass filter. (C) F' image taken with a Schott RG695 (695 nm longpass) filter. (D) and (E) Images of Fq 7Fm' constructed using (B) and (C), respectively, plus Fm' images taken immediately after (B) and (C). (F) and (G) Fq '/Fm' images of the chloroplasts within guard cells only, isolated from (D) and (E), respectively. The incident actinic PAR was 93 /anol photons nr2 1 2 1 s" . The incident PAR during the saturating pulses used to measure Fm' was 6500 ,umol photons nr s" . Mean values of Fq 7Fm' in (D), (E), (F) and (G) are 0.60, 0.55, 0.56 and 0.55, respectively. The scale at the bottom of G represents 50 fim. The palette scale (which is a monochrome version of a false color palette) applies to (D), (E), (F) and (G). See Chapter 15, Oxborough.
Fig. 2. High resolution images taken from an intact biofilm. (A) F' image taken over 200 ms at an incident PAR of 200 ,umol photons 2 _1 2 -1 nr s . (B) Fm' image taken during the last 40 ms of a 400 ms saturating pulse at an incident PAR of 6500 /anol photons nr s . (C) Image of Fq 7Fm' constructed from (A) and (B), which illustrates the movement of cells between the F' and Fm' images being taken. (D) Image of Fq 7Fm' constructed from cells that were isolated within (A) and (B) and then nudged together to gain the best possible overlap. Cells can usually be identified to the genus level within raw or parameter images. Within (D), (i) Euglena sp.; (ii) Plagiotropis sp.; (iii) Pleurosigma sp.; (iv) Cylindrotheca sp.; (v) Gyrosigma sp. The white scale at the bottom of (D) represents 200 fjm. See Chapter 15, Oxborough.
Fig. 3. Images of two unidentified pelagic algae, which were isolated from a water sample taken from the Odense Fjord Denmark st on 21 August, 2002. Cells were concentrated onto a black Nucleopore filter. (A) Fo image taken over an integration period of 48 ms, 2 _1 during which 1 /is pulses of approximately 5000 fimol photons nr s were provided at 500 /is intervals. (B) Fm image taken during an integration period of 12 ms at the end of an 800 ms saturation pulse. (C) Fv/Fm image constructed from (A) and (B) The numbers on the images are mean values for each cell. In (C), the number in brackets at the top of the image is an integrated mean for the cell at upper left in (A) and (B), while the lower number is a pixel mean for the adjacent cell. A pixel mean could not be calculated for the upper cell in (C) because it had moved several /an in the 800 ms interval between the taking of the Fo and Fm images. The scale in (C) represents 20 fim. Images were taken using the LED based system described in Section III.A and by Fig. l.B in Chapter 15, Oxborough.
Fig. 4. Fluorescence images from a detached leaf of Cornus sp., which had it's petiole submerged in a 10 mM solution of DCMU for the entire experiment. The incident PPFD was 300 jimol nr2 s"1. Images of F' were generated by synchronizing the camera shutter to a 1 ms 2 _1 measuring pulse at a PPFD of 4600 jimol nr s . To produce images of Fm and Fm', a sequence of images was taken at 20 fps over the last 600 ms of an 800 ms saturating pulse. The image with the highest mean value was taken to be the Fm or Fm' value. The remaining images 2 1 were discarded. The PPFD of the saturating pulse and measuring pulses during this procedure was 4600 jimol nr s" . Images of Fo were generated by applying 2 (xs pulses, at a PPFD 4600 jimol m"2 s"1, at 400 (xs intervals during a 16.7 ms exposure. In (A-C), the false color images are of Fq 7Fm'. The small monochrome images to the left of the false color images are the F' image (leftmost) andFm' image that were used in construction of the Fq 7Fm' images. These pairs of images were taken after 60 s (A), 1 h (B) and 2 h (C) illumination. In (D), the false color image is olFJFm. The small monochrome images to the left are the Fo (leftmost) andFm images used in construction of the F/Fm image. This pair of images was taken after 2 h illumination, followed by 30 min dark-adaptation. The histograms to the right of the false color images (A- D) show how data values are mapped to the palette. The numbers below the histograms reflect the range of values represented by the x-axis in each case. The scale in (D) represents 20 mm. See Chapter 15, Oxborough.
Fig. 5. Methods for the isolation of multiple plants within an image. (A), (B) and (C) are all from the same Arabidopsis plants grow- ing within a 96 well plate. The 12 columns of plants were treated with different amounts of Imazapyr, 48 h before measurements were made. Each well received 15 jiL of Imazapyr in 50% acetone. The concentrations applied were (left to right): 0, 8 mM, 4 mM, 0.8 mM, 0.4 mM, 0, 0, 8 mM, 4 mM, 0.8 mM, 0.4 mM, 0. Columns 6 and 12 are controls (no acetone). Details of the imaging system and all lighting conditions (actinic light, saturating pulses and measuring pulses) are the same as described for Fig. 7 of Chapter 15. (A) F' image after non-fluorescent pixels have been removed using a low-cut filter (Section III.E.2 of Chapter 15). Circles highlight wells that contain more than one plant. (B) Individual plants have been identified in (A), using an algorithm which searches for blocks of connected pixels.
(C) Fv/Fm image from the same sample of Arabidopsis plants and in (A) and (B), which has been divided into 96 zones, using vertical and horizontal lines. Mean values for all plants within each column are, from left to right: 0.81, 0.35, 0.31, 0.45, 0.71, 0.81, 0.81, 0.36, 0.32, 0.51, 0.65, 0.78. (D) Histogram showing the distribution oiFy/Fm values in (C). See Chapter 15, Oxborough.
CP-6 Color Plate 5
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CP-7 Color Plate 6
Fig. 1. Algal and plant life forms in standing water. Top: Planktophytes (phytoplankton community from North Basin of Windermere) showing unicellular, colonial and filamentous algae from arange of taxonomic groups (photo courtesy of Dr Hilda Canter-Lund). Middle: Haptophytic red macroalga Lemanea mamillosa; note another haptophyte, Fontinalis and an epiphytic red alga on some of the filaments (photo courtesy Mr Trevor Furnass). Bottom: Submerged rhizophytic angiosperms (from about 3 m in Loch Borralie, Scotland; photo by the late Professor D. H. N. Spence). See Chapter 31, Raven and Maberly
CP-8