Table Of ContentCELL BIOLOGY: A Series of Monographs
EDITORS
D. E. BUETOW I. L. CAMERON
Department of Physiology Department of Cellular and
and Biophysics Structural Biology
University of Illinois The University of Texas
Urbana, Illinois Health Science Center at San Antonio
San Antonio, Texas
G. M. PADILLA A. M. ZIMMERMAN
Department of Physiology Department of Zoology
Duke University Medical Center University of Toronto
Durham, North Carolina Toronto, Ontario, Canada
Recently published volumes
Gary L. Whitson (editor). NUCLEAR-CYTOPLASMIC INTERACTIONS IN THE CELL CYCLE, 1980
Danton H. O'Day and Paul A. Horgen (editors). SEXUAL INTERACTIONS IN EUKARYOTIC MICROBES,
1981
Ivan L. Cameron and Thomas B. Pool (editors). THE TRANSFORMED CELL, 1981
Arthur M. Zimmerman and Arthur Forer (editors). MITOSIS/CYTOKINESIS, 1981
Ian R. Brown (editor). MOLECULAR APPROACHES TO NEUROBIOLOGY, 1982
Henry C. Aldrich and John W. Daniel (editors). CELL BIOLOGY OF PHYSARUM AND DIDYMIUM. Volume
I: Organisms, Nucleus, and Cell Cycle, 1982; Volume II: Differentiation, Metabolism, and Methodology, 1982
John A. Heddle (editor). MUTAGENICITY: New Horizons in Genetic Toxicology, 1982
Potu N. Rao, Robert T. Johnson, and Karl Sperling (editors). PREMATURE CHROMOSOME CONDENSA
TION: Application in Basic, Clinical, and Mutation Research, 1982
George M. Padilla and Kenneth S. McCarty, Sr. (editors). GENETIC EXPRESSION IN THE CELL CYCLE,
1982
David S. McDevitt (editor). CELL BIOLOGY OF THE EYE, 1982
P. Michael Conn (editor). CELLULAR REGULATION OF SECRETION AND RELEASE, 1982
Govindjee (editor). PHOTOSYNTHESIS, Volume I: Energy Conversion by Plants and Bacteria, 1982; Volume II:
Development, Carbon Metabolism, and Plant Productivity, 1982
John Morrow. EUKARYOTIC CELL GENETICS, 1983
John F. Hartmann (editor). MECHANISM AND CONTROL OF ANIMAL FERTILIZATION, 1983
Gary S. Stein and Janet L. Stein (editors). RECOMBINANT DNA AND CELL PROLIFERATION, 1984
Prasad S. Sunkara (editor). NOVEL APPROACHES TO CANCER CHEMOTHERAPY, 1984
Burr G. Atkinson and David B. Walden (editors). CHANGES IN EUKARYOTIC GENE EXPRESSION IN
RESPONSE TO ENVIRONMENTAL STRESS, 1985
Reginald M. Gorczynski (editor). RECEPTORS IN CELLULAR RECOGNITION AND DEVELOPMENTAL
PROCESSES, 1986
Govindjee (editor). LIGHT EMISSION BY PLANTS AND BACTERIA, 1986
In preparation
Peter B. Moens (editor). MEIOSIS, 1986
Robert A. Schlegel, Margaret S. Halleck, and Potu N. Rao (editors). MOLECULAR REGULATION OF
NUCLEAR EVENTS IN MITOSIS AND MEIOSIS, 1987
Monique C. Braude and Arthur M. Zimmerman (editors). GENETIC AND PERINATAL EFFECTS OF
ABUSED SUBSTANCES, 1987
Light Emission
by Plants and Bacteria
Edited by
GOVINDJEE
Departments of Physiology and Biophysics and Plant Biology
University of Illinois at Urbana—Champaign
Urbana, Illinois
JAN AMESZ
Department of Biophysics
Huygens Laboratory of the State University
Leiden, The Netherlands
DAVID CHARLES FORK
Department of Plant Biology
Carnegie Institution of Washington
Stanford, California
1986
ACADEMIC PRESS, INC.
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COPYRIGHT © 1986 BY ACADEMIC PRESS, INC.
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United Kingdom Edition published by
ACADEMIC PRESS INC. (LONDON) LTD.
24-28 Oval Road, London NW1 7DX
Library o f Congres s Cataloging i n Publicatio n Dat a
Light emissio n b y plant s an d bacteria .
(Cell biology )
Includes index .
Ί. Plan t luminescence . 2 . Bacteria , Luminous .
I. Govindjee , Dat e . II . Amesz , Jan . III . Fork ,
David Charles . IV . Series .
QK844.L54 198 6 581.Ί9Ί2 5 86-792 0
ISBN 0-12-294310- 4 (alk . paper )
PRINTED IN THE UNITED STATES OF AMERICA
868 78 88 9 9 8 7 6 5 4 3 2 1
Foreword
Studies of the absorption and emission of ultraviolet and visible radia
tion occupy a central position among the methodologies employed in the
investigation of living systems at the molecular level. A detailed exami
nation would indeed show that this position is likely to be a permanent
one. On the one hand, we can count the elementary quanta in these
spectral regions, and are thus close to reaching the physical limits of
detection. On the other, quanta of visible and near-ultraviolet light cor
respond to the highest energies that can be absorbed without producing
irreversible changes in the absorbing molecules. The number of organic
chromophores that undergo photochemical reactions after absorption
of visible or ultraviolet photons is only a very small fraction of those
commonly found in organisms, while those that are rapidly and revers-
ibly deactivated after excitation are in the majority, a distinction of im
portance in the evolutionary selection of the photochemical mechanisms
that are at the basis of both vision and photosynthesis. As many of the
reversibly deactivated chromophores have detectable fluorescence, we
are not limited in our studies of light emission of plants and bacteria to
those cases in which photochemistry competes with radiative deactiva
tion. We can also analyze the far more common situations in which dark
chemistry of all kinds competes with the emission of light. This favorable
circumstance has been recognized and exploited for at least forty years.
In the 1920s and 1930s we began to understand the emission of "cold
light" as various forms of luminescence came to be explained by the
quantum theory. S. I. Wavilov showed that fluorescein solutions convert
almost all of the photons of excited light into fluorescence, and E. Ga-
viola was able to demonstrate, at about the same time, that the lifetimes
of photo-excited states in solution are a few nanoseconds, the length
predicted by the classical radiation theory. Also at this time Francis Per-
rin recognized the importance of the polarization of the radiation as a
quantitative measure of the molecular rotational motions in liquids, and
Gaviola and P. Pringsheim demonstrated the existence of long-range
electronic energy transfer among identical fluorophores in solution.
xiii
xiv Foreword
These observations and theoretical formulations are still the basis of
most of today's experiments. Although our views on each of these obser
vations have been considerably refined through the years, nothing of
similar fundamental importance has been added since then. What has
radically changed is our ability to observe these phenomena in the real
world. While the classical observations listed above were limited to some
of the strongest emitters, such as fluorescein, and only when studied
under exceptionally favorable circumstances, we can now make similar
measurements on virtually any system. In the past twenty years, develop
ments in instrumentation to detect and measure optical phenomena
have undergone a virtual revolution, exceeded only by the complemen
tary growth in the techniques for the digital manipulation and analysis
of the experimental data. Progress in the ability to make observations in
the shortest time domains has been particularly impressive. Light emis
sion is now studied by methods that exploit both the impulse response
and the harmonic analysis, with the result that the pico- and nanosecond
regions can be explored in such reliable detail that, paradoxically, we
now know the molecular properties that find expression within these
short times much better than those that emerge in micro- or milli
seconds.
Absorption, fluorescence, and Raman spectroscopy methods, which
involve the controlled interaction of radiation with systems of interest,
are being extended daily to new domains of interest in biology, but the
analysis of the spontaneous emission of light by plants and bacteria has a
special place beside them. It reveals the natural biological activities in a
fashion which requires little or no interference with the object under
study, and is thus often crucial in the interpretation of experiments
which involve the forced behavior of the systems.
This book presents a current view of the emission of light by plants
and bacteria discussing its physical basis as well as its biological signifi
cance. From such an approach, biophysics is emerging as a distinct sci
ence, an accomplishment similar to that of biochemistry a generation
ago. It may well be recognized in the future that in this emergence of
biophysics the study of light from organisms and their derived compo
nents played a major role. Indeed, "in the beginning there was light."
GREGORIO WEBER
Department of Biochemistry
Roger Adams Laboratory
University of Illinois
Urbana, Illinois 61801
Preface
Many plants and bacteria emit light as a result of numerous different
reactions. The light emitted by these organisms is of great importance in
biology since it provides a nondestructive, noninvasive, extremely sensi
tive, and versatile tool for measuring and studying various reactions of
the living system. This book deals with light emission from plants and
bacteria, with special emphasis on light emission from photosynthetic
organisms.
When a molecule absorbs a photon, it goes into an excited state. De-
excitation may occur by loss of energy as heat (internal conversion) or as
light (prompt fluorescence). Prompt fluorescence lasts from picoseconds to
hundreds of nanoseconds after the cessation of initial illumination. In
some instances, of which photosynthesis is by far the most prominent
example, excitation energy is stored in the form of chemical energy
(photochemistry). If the excited state is created by chemical reactions,
the emitted light is chemiluminescence {bioluminescence in living systems).
Delayed fluorescence (also called delayed light emission) is a special type of
chemiluminescence in which the substrates for the chemical reaction are
produced as a consequence of photochemistry, and may last from nano
seconds to hours after the cessation of initial illumination. If light is
emitted during the transition from a triplet state to a ground state, it is
called phosphorescence; this emission has a lower energy, i.e., it occurs at a
longer wavelength than the prompt fluorescence and lasts for a longer
period of time (e.g., microseconds) after the cessation of initial illumina
tion. Thermoluminescence is delayed fluorescence that is observed on heat
ing of a preilluminated and cooled sample. This book includes discus
sions of all these types of light emission from biological samples.
During the past fifty years, chlorophyll or bacteriochlorophyll fluores
cence has been extensively used to obtain information, among other
things, on (1) the composition, organization, and orientation of the
pigment systems; (2) the efficiency and the pathway of excitation energy
transfer from one pigment to another; and the regulation of excita
tion energy distribution and redistribution among and within the
XV
xvi Preface
photosystems; (3) photosynthetic models and photochemical efficien
cies; and (4) the nature of the early electron carriers, the times involved
in these early electron-transport steps, and the sites of action of inhibi
tors. Since almost all photosynthetic reactions from the time light is
absorbed until CO2 is fixed (see Govindjee: "Bioenergetics of Photosyn-
thesis,', Academic Press, 1975; and Govindjee: "Photosynthesis," Vol
umes 1 and 2, Academic Press, 1982, all in the Cell Biology series) could
potentially affect fluorescence, it is often a difficult task to interpret the
data without extreme caution and understanding of all the parameters
involved, especially when making practical applications. However, the
time scale of measurement usually defines the reactions being moni
tored, and in many cases fluorescence monitors a single reaction with
great precision and reliability. Examples of the above-mentioned uses
can be found in several chapters in this book.
This volume is divided into six parts. Part I provides an introduction
(Chapters 1-5), Part II (Chapters 6-12) deals with the relationship of
light emission to the various photosynthetic reactions in vivo, Part III
(Chapter 13) discusses bioluminescence, Part IV (Chapter 14) describes
light emission from bacteriorhodopsin and rhodopsin, Part V (Chapters
15-18) deals with the special light emission characteristics and their
relationship to specialized pigment systems in various groups of bacteria
and plants, and Part VI (Chapter 19) deals with the practical applications
of light emission from algae and higher plants.
L. Ν. M. Duysens (Chapter 1) discusses the historical development in
the area of chlorophyll a (and bacteriochlorophyll) fluorescence and
develops equations relating emission kinetics to the concentration of
photosynthetic components. W. A. Arnold (Chapter 2) relates how he
discovered delayed fluorescence, thermoluminescence, and the electric
field effect on delayed light in plants. J. R. Norris and G. van Brakel
(Chapter 3) summarize the energy trapping and the primary photo
chemistry in photosynthesis of purple bacteria, which is the best under
stood system to date. J. Lavorel, J. Breton, and M. Lutz (Chapter 4)
present the principles of the methods of measurement of light emitted
by photosynthetic systems. The authors have also included a discussion
of resonance Raman measurements. Part I ends with a chapter by G. R.
Seely and J. S. Connolly (Chapter 5) which deals with fluorescence of
photosynthetic pigments in vitro. This chapter also provides an introduc
tion to the physical processes of light absorption, singlet-state decay
processes, excitation energy transfer, fluorescence lifetimes, quantum
yields, and polarization of fluorescence, and helps the reader appreciate
the potential of light emission measurements both in vitro and in vivo.
N. Murata and Kimiyuki Satoh (Chapter 6) summarize absorption and
Preface xvii
fluorescence emission by intact cells, chloroplasts, and chlorophyll-pro
tein complexes. This chapter serves as an introduction to Chapters 16,
17, and 18. I. Moya, P. Sebban, and W. Haehnel (Chapter 7) discuss the
results and significance of the lifetime of excited states and quantum
yield of chlorophyll a (and bacteriochlorophyll) fluorescence in vivo.
R. van Grondelle and J. Amesz (Chapter 8) present an introduction to
excitation energy transfer in photosynthetic systems. This chapter pro
vides the basis for excitation energy-transfer measurements mentioned
in Chapters 15, 16, 17, and 18. A. J. Hoff (Chapter 9) provides a thor
ough discussion of triplets, phosphorescence, and magnetic resonance
measurements. H. van Gorkom (Chapter 10) presents a brief but lucid
discussion of the fluorescence measurements in the study of photosyn
thetic electron transfer. This chapter provides information on a topic
that is of paramount importance to current research in several laborato
ries. P. A. Jursinic (Chapter 11) discusses the current concepts and status
of delayed fluorescence. Part II ends with a chapter by P. V. Sane and
A. W. Rutherford (Chapter 12), who summarize the current concepts
and status of thermoluminescence from photosynthetic membranes.
J. W. Hastings (Chapter 13) presents the current status of biolumines-
cence in both bacteria and dinoflagellates. The author also explains the
similarities between bioluminescence and delayed fluorescence in this
sole chapter of Part III. R. Govindjee and T. Ebrey (Chapter 14) provide
a summary of light emission from bacteriorhodopsin and rhodopsin and
relate these studies to the photocycle of these pigment proteins in this
sole chapter of Part IV. J. Amesz and H. Vasmel (Chapter 15) discuss the
absorption and fluorescence properties of photosynthetic bacteria
(green as well as purple). D. C. Fork and P. Mohanty (Chapter 16) review
the absorption and fluorescence properties of cyanobacteria, red algae,
and cryptomonads and discuss the special features and unique proper
ties of these interesting organisms. Govindjee and Kazuhiko Satoh
(Chapter 17) discuss the absorption and fluorescence properties of all
greenish (chlorophyll b—containing) and brownish (chlorophyll c—con
taining) algae. A brief discussion of evolution is also included. Part V
ends with the chapter by J.-M. Briantais, C. Vernotte, G. H. Krause, and
E. Weiss (Chapter 18), who discuss chlorophyll a fluorescence of higher
plants, both from leaves as well as from chloroplasts. G. Renger and
U. Schreiber (Chapter 19, the sole chapter of Part VI) summarize the
practical applications of fluorimetric methods to algae and higher plant
research. We hope that many more future applications are to come, and
that this book will be used to obtain background knowledge in this grow
ing field of biology.
This volume emphasizes the physiological, biophysical, and biochem-
xviii Preface
ical relevance of light emission studies in plants and bacteria. The aim of
most of the authors was to include a review of the historical develop
ments of the major concepts, critical analysis of experimental ap
proaches, and an exposition of recent findings. It is hoped that the
individual chapters will serve as a reference source that integrates exper
imental results and theoretical considerations from a large number of
research publications as well as from the authors' laboratories. Since
both background and up-to-date information are included, this book
should serve not only as a reference source for researchers but also as an
introductory book for graduate and advanced undergraduate students
in general biology, cell biology, microbiology, plant physiology, bio
chemistry, biophysics, and agriculture. We hope the readers will benefit
from this book by absorbing the exciting information contained on light
emission from both plants and bacteria.
A major setback in the preparation of this book was the untimely
death of Professor Warren L. Butler, who was scheduled to write a
chapter on "Photosynthetic Models." We felt that no one could replace
Warren, so this chapter was omitted. We have, however, attempted to
include the important concepts involved in the various chapters of this
work. We are aware of Warren's pioneering research on several topics
relevant to this volume: photosynthetic models and excitation energy
transfer in normal and developing plants; absorption spectroscopy of
highly scattering systems; lifetime and polarization of fluorescence; two-
light effect on chlorophyll a fluorescence; inhibition and reconstitution
of electron transport in photosynthesis; low-temperature reactions; the
redox potential and function of primary electron acceptors; quenching
of chlorophyll a fluorescence; and the role of cytochromes in photosyn
thesis. In view of this, and of the personal loss we all feel, we dedicate
this book to the memory of our friend Warren L. Butler.
GOVINDJEE
JAN AMESZ
DAVID CHARLES FORK
Warren Butler and Photosynthesis
The Early La Jolla Years
Warren Butler in 1979. Photograph by Helga Ninnemann.
Warren Butler received a Bachelor of Science degree in physics from
Reed College in 1949 and his Ph.D. in biophysics from the University of
Chicago in 1955 as the last graduate student of James Franck. He joined
the United States Department of Agriculture laboratory in Beltsville,
xix
