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Chlorophyll Fluorescence
Understanding Crop
Performance— Basics and Applications
Chlorophyll Fluorescence
Understanding Crop
Performance— Basics and Applications

By
Mohamed H. Kalaji
Vasilij. N. Goltsev
Krystyna Z uk-Golaszewska
Marek Zivcak
Marian Brestic
CRC Press
Taylor & Francis Group
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Boca Raton, FL 33487-2742

© 2017 by Taylor & Francis Group, LLC

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Library of Congress Cataloging‑ in‑ Publication Data

Names: Kalaji, Mohamed H., author.

Title: Chlorophyll fluorescence : understanding crop performance : basics and
applications / authors: Mohamed H. Kalaji, Vasilij N. Goltsev, Krystyna Zuk-Golaszewska,
Marek Zivcak, Marian Brestic.
Description: Boca Raton, FL : CRC Press, 2017. | Includes bibliographical references
and index.
Identifiers: LCCN 2016049531 | ISBN 9781498764490 (hardback : alk. paper)
Subjects: LCSH: Plants--Effect of stress on. | Photosynthesis--Measurement.
Classification: LCC QK754 .K35 2017 | DDC 572/.46--dc23
LC record available at https://lccn.loc.gov/2016049531

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Contents
Acknowledgments......................................................................................................ix
Authors ......................................................................................................................xi
Introduction............................................................................................................. xiii

Chapter 1 Photosynthesis....................................................................................... 1
1.1 General Description of Photosynthesis...................................... 1
1.1.1 PSII Antenna Complex.................................................. 5
1.1.2 Photosynthetic Pigments of Higher Plants.................... 6
1.1.3 Photosystem II (PSII)....................................................9
1.1.4 Cytochrome b6  f Complex ........................................... 10
  

1.1.5 Photosystem I (PSI)..................................................... 10

1.1.6 ATP Synthase.............................................................. 11
1.2 Light Phase of Photosynthesis.................................................. 12
1.2.1 Absorption of Photosynthetically Active
Radiation (PAR).......................................................... 12
1.2.2 Transport of Electrons and Non-Cyclic
Phosphorylation........................................................... 13
1.2.3 Cyclic Transport of Electrons and Cyclic
Photophosphorylation.................................................. 17
1.2.4 Pseudocyclic Transport of Electrons and
Pseudocyclic Photophosphorylation............................ 18
References ........................................................................................... 19

Chapter 2 Fluorescence of Chlorophyll a ............................................................ 23

2.1 Chlorophyll a Fluorescence as an Indicator of PSII
Performance.............................................................................24
2.2 Chlorophyll a Fluorescence Induction Curve.......................... 27
2.3 Techniques for Measuring Chlorophyll a Fluorescence.......... 30
2.3.1 Direct Fluorescence of Chlorophyll a......................... 30
2.3.2 Modulated Chlorophyll a Fluorescence ...................... 31
References ........................................................................................... 33

Chapter 3 JIP (OJIP) Test..................................................................................... 37

3.1 Parameters in JIP (OJIP) Test................................................... 39
3.2 Data Derived Indirectly from JIP Test..................................... 39
3.3 Fluorescence Parameters Derived from Measured Data..........40
3.4 Specific Energy Fluxes per QA -Reduced PSII Reaction
Center....................................................................................... 41
3.5 Yields or Energy Flux Ratios................................................... 41

v
vi Contents

3.6  Phenomenological Energy Fluxes per Excited Cross-

Section (CS) of a Sample.......................................................... 42
3.7 Performance Indices at t   =  0 and Density of Active PSII
Reaction Centers....................................................................... 43
3.8 Driving Forces (Logarithms of Performance Indices
at t   =  0).................................................................................. 43
3.9 Overall Grouping Probability................................................... 43
3.10 Structure and Function Indices................................................44
3.11 Association of OJIP Fluorescence Kinetics with
Measurements of Light Scattering at 820 nm Wavelength......44
References ........................................................................................... 53

Chapter 4 Delayed Fluorescence in Photosynthesis............................................ 57

4.1 Basics of Delayed Light Emission by Plants............................ 57
4.1.1 Mechanism of DF Quanta Generation ........................ 58
4.1.2 Decay Kinetics of Delayed Fluorescence ................... 62
4.1.3 Induction Kinetics of Delayed Fluorescence ..............66
4.2 Analysis of Relationship between Delayed and Variable
Chlorophyll a Fluorescence...................................................... 70
4.3 Methods for Recording Delayed Fluorescence........................ 73
References ........................................................................................... 77

Chapter 5 Pulse-Amplitude Modulated (PAM) Fluorescence Measurements.... 83

5.1 Rationale for the Saturation Pulse Method..............................84
5.2 Interpretation of the Saturation Pulse Method with the
Use of Mathematical Models.................................................... 85
5.2.1 Processes Affecting Fluorescence Emission in
Terms of Their Rate Constants ................................... 86
5.2.2 Sensitivity Factor S ..................................................... 87
5.2.3 PSI Fluorescence......................................................... 89
5.3 Parameters Derived from Pulse-Amplitude Measurements..... 91
5.3.1 Directly Measured Basic Parameters.......................... 91
5.3.1.1 Basal Fluorescence F0 ..................................92
5.3.1.2 Maximum Fluorescence FM .........................94
5.3.1.3 Variable Fluorescence FV ............................ 95
5.3.1.4 Steady-State Fluorescence Fʹ.......................
s 95
5.3.1.5 Minimum Fluorescence in Light-
Exposed Samples, Fʹ.0...................................96
5.3.1.6 Maximum Fluorescence in Light-
Exposed Samples, FʹM................................... 98
5.3.2 Parameters of Quenching Analysis........................... 100
5.3.2.1 Photochemical Quenching of Variable
Fluorescence qP, qL................................... 100
Contents vii

5.3.2.2 Non-Photochemical Quenching of

Variable Fluorescence, qN......................... 102
5.3.2.3 Non-Photochemical Quenching of
Maximum Fluorescence, NPQ.................. 103
5.3.2.4 Fractions of Non-Photochemical
Quenching.................................................. 104
5.3.3 Parameters of Energy Partitioning............................ 106
5.3.3.1 Rationale of Energy Partitioning............... 106
5.3.3.2 Maximum Quantum Yield of PSII
Photochemistry, Φ Po , FV /FM ...................... 106
5.3.3.3 Maximum Quantum Yield of Basal
Non-Photochemical Energy Losses,
Φ No , F0 /FM ................................................. 108
5.3.3.4 Maximum PSII Quantum Yield in a
Light-Adapted Sample, Φ P , FVʹ /FMʹ .......... 109
5.3.3.5 Efficient Quantum Yield at a Given
Light Intensity............................................ 111
5.3.3.6 Quantum Yield of Non-Light-Induced
Energy Losses............................................ 111
5.3.3.7 Quantum Yield of Light-Induced
Energy Losses............................................ 113
5.3.4 Electron Transport Rate (ETR PSII) ............................ 114
5.3.4.1 Theoretical Definition and Calculation ..... 114
5.3.4.2 Physiological Meaning and Application ..... 115
5.3.4.3 Limitations and Possible Errors in
Estimations of Electron Transport Rate .... 119
5.3.5 Summary of the Parameters...................................... 122
5.4 Most Useful Measurement Protocols Based on PAM
Method.................................................................................... 122
5.4.1 Single Saturation Pulse Applied to a Dark-
Adapted Sample (F V/F M Measurement) .................... 122
5.4.2 Simple Dark-to-Light Slow Fluorescence
Induction.................................................................. 124
5.4.3 Measurements of Samples Not Adapted to Dark
( Ф PSII Measurement) ................................................. 124
5.4.4 Slow Induction Curve and Recovery......................... 125
5.4.5 Light Response Curves.............................................. 128
5.4.6 Relative Fluorescence Decrease................................ 130
5.5 Special Applications of PAM Method.................................... 132
5.5.1 Chlorophyll Fluorescence Imaging........................... 132
5.5.2 On-Site Long-Term Monitoring of Fluorescence
Parameters................................................................. 134
5.5.3 Measurement of Fluorescence Parameters under
Multicolor Excitations............................................... 136
5.5.4 Simultaneous Measurements of PAM and Other
Signals....................................................................... 137
viii Contents

5.5.4.1 Chlorophyll Fluorescence Measured

Simultaneously with Photosynthetic
Gas Exchange............................................ 137
5.5.4.2 Simultaneous Measurements of
PSI Transmittance and Chlorophyll
Fluorescence.............................................. 140
5.5.5 Risks Associated with Multiple Saturation Pulse
Methods..................................................................... 144
References ......................................................................................... 145

Chapter 6 Application of Chlorophyll a   Fluorescence in Plant Research......... 163

6.1 Light Stress............................................................................. 164
6.2 Temperature Stress................................................................. 171
6.3 Water Stress............................................................................ 181
6.3.1 Drought...................................................................... 181
6.3.2 Flooding Stress.......................................................... 191
6.4 Salinity Stress......................................................................... 192
6.5 Stress Caused by Heavy Metals............................................. 195
6.6 Nutrient Deficiency................................................................. 201
References .........................................................................................204
Index ....................................................................................................................... 219
Acknowledgments
The authors would like to thank the reviewers for their thorough reading of this book
and their thoughtful comments. The authors also are very thankful to Prof. Reto
Strasser and Prof. Govindjee for their scientific support and advice. Many thanks to
Hansatech Instruments (UK), PP Systems (USA), and bbe Moldaenke (Germany) for
supporting this book with figures and schemes related to the instruments used in the
field of chlorophyll fluorescence measurements.

ix
Authors
Hazem M. Kalajiis a Polish plant physiologist of Syrian
origin. He has more than 30 years of experiences in the field
of photosynthesis and stress physiology. He works as an
international scientific counselor and cooperates with more
than 100 well-known international institutes, organizations,
laboratories, and companies, and has been working as Vice
Chair of MSCA at the Scientific Committee of European
Union in Brussels, Belgium. He has published four books
related to chlorophyll fluorescence (in Polish, Italian, and
Russian) and more than 200 scientific papers (124 of which
are on JCR list). He has also worked as Associate Editor of the journal
Photosynthetica and belongs to the editorial boards of seven other international
scientific journals. He has organized many international conferences and partici-
pated in more than 150 meetings, symposiums, and seminars. Recently, with his
team at Warsaw University of Life Sciences– SGGW, he has been working on the
biological behavior of photosystems (PSI and PSII), and the application of chloro-
phyll fluorescence technique and artificial neural networks (ANN) to identify and
predict stress type in plants. Other practical work, where chlorophyll fluorescence
technique has been employed, relates to enabling plants to control their growth
conditions in any greenhouse automatically, based on the “ live” -sensing of their
photosynthetic efficiency.

Vasilij Goltsevis a professor at St. Kliment Ohridski

University in Sofia, Bulgaria. For 18 years he was Head of
the Department of Biophysics and Radiobiology, Faculty of
Biology at University of Sofia. Professor Goltsev dedicated
more than 35 years toward research on light emission from
plants and teaching biophysics, photobiology, the biophysics
of photosynthesis, and mathematical modeling. His publica-
tions are in the field of plant stress response and adaptation
to adverse environmental, physical, chemical, and biological
factors. Along with his team he has been working on the biophysical aspects of
photosynthetic machinery stress response and on the development of a luminescent
approach for multi-parametric estimation of the physiological state and stress reac-
tion in plants at in vivo and in situ condition. Professor Goltsev currently serves on
the editorial boards of Genetics and Plant Physiology , NanoPhotoBioSciences , and
Technology of Living Systems , and is the review editor of Agroecology and Land
Use Systems .

xi
xii Authors

Krystyna Żuk-Gołaszewska has a PhDin agronomy and is an

associate professor with over 20 years of research and teaching
experience at the University of Warmia and Mazury in Olsztyn,
Poland, in the Department of Agrotechnology, Agricultural
Production Management, and Agribusiness. Her research
focuses on determining the relationship between the agrotechni-
cal factors of crop production, and the physiological parameters
of plant growth and development associated with measurements
and indices of photosynthetic efficiency, transpiration, water-
use efficiency, LAI, chlorophyll content, and other factors.

Marek Zivcakis a plant physiologist working at the Slovak

University of Agriculture in Nitra, Slovakia. He has been an
assistant professor (since 2008) and associate professor (since
2015) at the Department of Plant Physiology. He is an expert in
plant physiology, the biophysics of photosynthesis, and various
spectrometric methods for analysis of the photosynthesis
in vivo , including different applications of chlorophyll fluores-
cence methods. The recent activities of his group focus mostly
on the development of methodologies of high-throughput phe-
notyping of the aboveground parts of plants, mostly the photosynthetic apparatus,
using optical sensors, including different technical ways to measure the chlorophyll
fluorescence signal.

Marian Brestic is a leader of a research group at the Slovak

University of Agriculture in Nitra, Slovakia, dealing with
photosynthesis, abiotic stresses, and the development of physi-
ological criteria for improving crop tolerance to abiotic stress.
His group studies climate change and drought, and their
impacts on and challenges for sustainable agriculture and the
assessment of physiological traits in crop genetic resources, as
well as on mechanistic research in plant stress tolerance and
responses at the level of photosynthetic apparatus. Recently
his group has focused on the development of methodologies of
high-throughput phenotyping of the aboveground parts of plants, mostly the photo-
synthetic apparatus, using optical sensors, including different technical ways to mea-
sure the chlorophyll fluorescence signal. The applied research is closely connected to
the mechanistic research aimed at abiotic stress responses.
Introduction
Living organisms are exposed to numerous adverse environmental factors that
disturb their functions (e.g., high or low temperature, frost, excessive sunlight radia-
tion). Crops and other plants growing in natural habitats are at a higher risk because
they are typically subjected to more than one stressor. Efforts are underway to
develop and implement methods which will enable us to determine accurately the
effects of adverse environmental factors on the growth and development of plants.
Studies on stress-induced processes in plants involve various methods. The most
reliable results are obtained with techniques that analyze the course of photosynthe-
sis, one of the principal processes in plants that is extremely sensitive to stressors.
Among the range of stressful conditions, such as excessive solar radiation within the
photosynthetically active range or higher/lower temperatures, the balance between
the supply of the so-called assimilatory power generated by photochemical reactions
involving adenosine triphosphate (ATP) and a reduced form of nicotinamide adenine
dinucleotide phosphate (NADPH) and the demand for these products in the enzy-
matic reactions within the Calvin– Benson cycle (the dark phase of photosynthesis),
becomes a key consideration in photosynthesis. This situation necessitates the acti-
vation of various processes to disperse the excess energy absorbed by chlorophyll,
including changes in fluorescence emission and increase in heat production.
Chlorophyll a fluorescence is strongly influenced by many factors disrupting the
course of primary photosynthetic reactions in PSII, especially excessive radiation
in the visible and ultraviolet range, as well as high temperatures. Measurements
of chlorophyll fluorescence induction (the Kautsky effect) and modulated chloro-
phyll fluorescence are very useful methods for monitoring various events during
photosynthesis.
Research methods and techniques which employ chlorophyll fluorescence are
easy to use, quick, non-invasive, and highly sensitive. These attributes are particu-
larly helpful in ecophysiological investigations for monitoring crops and ecosystems
threatened by phytotoxic factors, in studies on the efficiency of photosynthesis, in
assessments of plant tolerance to various stress factors, and in determinations of the
nutritional requirements of crops. In some circumstances, fluorescence methods can
replace more time-consuming gasometric methods used in plant physiology stud-
ies, and further developments and improvements in measuring equipment will most
certainly widen the range of possible applications of these techniques. This book is
addressed to a wide circle of readers interested in the use of chlorophyll a fluores-
cence in research on plants and other photosynthesizing organisms, such as algae
and cyanobacteria.
The first chapter provides a general review of photosynthesis, especially its light
phase, where absorption of light quanta (photons) by photosynthetic pigments occurs,
as well as chlorophyll fluorescence and basic photochemical reactions leading to the
transformation of energy of absorbed photons into chemical energy in the form of
ATP and NADPH.

xiii
xiv Introduction

The second chapter presents the theory of chlorophyll a fluorescence, measuring

methods and techniques, and their applications in ecophysiological and agricultural
research.
Chapter three is dedicated to the so-called JIP test and is addressed to readers
interested in early reactions in the light phase of photosynthesis and the information
obtained from the analysis of numerous parameters.
In the fourth chapter, we introduce the analysis of delayed fluorescence. This
signal, which can be measured in parallel with fast fluorescence kinetics, provides
valuable additional information on the status of photosystem II (PSII) photochemis-
try. Because this method deserves wider application, it is described in detail.
In contrast, the fifth chapter presents a popular technique for measuring chloro-
phyll fluorescence— the pulse-amplitude modulated (PAM) technique. In addition to
basic information available in many other publications, we focus on explaining the
physiological meaning of individual parameters, and on useful protocols and pos-
sible pitfalls and sources of error that are important in practical applications.
The sixth chapter contains model applications of chlorophyll fluorescence read-
ings for assessing the impact of selected unfavorable environmental conditions on
the efficiency of the photosynthetic apparatus in different plant species.
While working on this book, we drew on our long-term experience in teaching
about the fluorescence of chlorophyll in various university settings (lectures, classes,
tutorials, courses, training courses, promotional events) and for both part-time and
full-time undergraduate, postgraduate, and doctoral students majoring in agriculture,
biology, environmental conservation, and biotechnology (especially in the context of
plant physiology). Our experience with the chlorophyll fluorescence method, gained
through studies funded with grants or by research commissioning institutions, has
also been very helpful. We have investigated the photosynthetic apparatus in a wide
range of plants, from cereals to woody and even aquatic plants. Also noteworthy, is
the experience Mohamed H. Kalaji and Vasilij N. Goltsev gained at the Bioenergetics
Laboratory, University of Geneva, headed by Reto J. Strasser (a renowned authority
on chlorophyll a fluorescence).
The objective of this book is to characterize the phenomenon of chlorophyll a
fluorescence, to describe the methods for its measurement, and to demonstrate— using
selected examples— the applicability of these methods to research into the response
of the photosynthetic apparatus and plant tolerance to unfavorable environmental
conditions.
 1 Photosynthesis

1.1 GENERAL DESCRIPTION OF PHOTOSYNTHESIS

Photosynthesis is the most vital bioenergy-generating process, without which life
on earth and the existence of our biosphere would be impossible. Sunlight enables
photosynthesizing organisms to transform CO2 and water and produce sugars and
other organic compounds. Photosynthesis occurs in plants, algae, and some bacteria,
including cyanobacteria, formerly called blue-green algae (for background informa-
tion, see Rabinowitch and Govindjee 1969). In plants and algae, this process takes
place in chloroplasts, which are filled with thylakoids. Thylakoid membranes are
where the light phase of photosynthesis occurs, while the stroma contains enzymes
essential for the dark phase of photosynthesis (the Calvin– Benson cycle) (Figure 1.1).
This multi-stage and extremely complicated process leads to the conversion of the
energy of absorbed photons into stable chemical energy of organic compounds. In
the most general terms, photosynthesis can be described with the following simpli-
fied formula:

CO2 + H 2O + Solar energy → CH 2O + O2

The so-called “ light phase” of photosynthesis consists of three major subphases:

1. Absorption of light and transfer of excitation energy within the pigment

antenna, followed by its trapping at the reaction centers (excitation of the
reaction center chlorophyll).
2. Transport of electrons: The primary event in the reaction centers involves
the transfer of an excited electron in a chlorophyll molecule to an inter-
mediate acceptor, that is, pheophytin (Pheo) (in Photosystem II [PSII]) or
chlorophyll (in Photosystem I [PSI]).
3. Stabilization of the energy of electrons during oxidation– reduction reac-
tions (the photosynthetic transport of electrons) during the generation of
ATP and the formation of the reducing power in the form of NADPH.

The absorbed photosynthetically active radiation (PAR) is used for the synthesis
of adenosine triphosphate (ATP) and Nicotinamide adenine dinucleotide phosphate
(NADPH), as noted in Subphase 3. These two products, obtained in the light phase,
are used (for the synthesis of sugars from CO2 and H2 O) during the dark phase,
also known as the biochemical phase, which includes the Calvin– Benson cycle (see
Govindjee 2010 for Benson’ s contributions).
As implied above, during the biochemical phase, CO2 is bound and reduced to the
level of sugars with the involvement of ATP and NADPH generated during the previ-
ous light phase. The key reaction during CO2 binding to a five-carbon compound is
catalyzed by the Rubisco enzyme (ribulose-1,5-biphosphate carboxylase/oxygenase)

1
2 Chlorophyll Fluorescence: Understanding Crop Performance

H2O
CO2
PAR
400–700 nm
NADP+

ADP
+
Light Pi Calvin- Dark
phase Benson phase
cycle
ATP

NADPH + H+

Sugars
O2

FIGURE 1.1 Two phases of photosynthesis and their mutual dependence in a chloroplast.
The light-dependent phase (light phase) occurs in thylakoids and generates assimilatory
power (NADPH and ATP), which is used in the dark phase (Calvin– Benson cycle). The
dark phase occurs in the stroma, where CO 2   is bound and reduced to sugars (organic com-
pounds). (Adapted from Kalaji, M.H. and Ł oboda, T., Chlorophyll Fluorescence to Study the
Physiological Status of Plants [fluorescencja chlorofilu w badaniach stanu fizjologicznego
roś lin]  , Wydawnictwo SGGW [in Polish], Warsaw, 2009.)

located in the stroma. Rubisco acts very slowly; it takes almost 1 s for one molecule
of the enzyme to bind three substrate molecules, which is why plants need large
amounts of this enzyme. The total amount of Rubisco makes up around 50% of
all proteins present in the chloroplast (see Blankenship 2014). In the carboxylation
process, a CO2 molecule is bound to ribulose-1,5-biphosphate (a five-carbon mol-
ecule) to form a transient six-carbon compound, but this is then quickly hydrolyzed
to produce two three-carbon molecules of 3-phosphoglyceric acid (PGA). Next,
PGA is reduced to the level of three-carbon sugar(s), 3-phosphoglyceraldehyde and
dihydroxyacetone phosphate, and it is at this stage that NADPH and ATP are used.
An accompanying process involves the regeneration of ribulose-1,5-biphosphate (as
a preparatory stage for the binding of another CO2 molecule). During these three
steps (carboxylation, reduction, and regeneration), three carbon dioxide molecules
and three molecules of ribulose-1,5-biphosphate produce a net yield of one molecule
of 3-phosphoglyceraldehyde (a triose), while three molecules of ribulose-1,5-biphos-
phate are regenerated. The entire cycle is known as the Calvin– Benson cycle (named
after its discoverers, Melvin Calvin and Andrew A. Benson; see Govindjee 2010).
Sugars produced during this cycle are used in the synthesis of sucrose, starch, and
many other organic compounds. In plants, the first products of the CO2 incorporation
in the Calvin– Benson cycle are three-carbon compounds (PGA), which is why the
cycle is also called the C-3 pathway , and plants in which it takes place are called C3
plants (Berg et al. 2005). Most plants grown in the moderate climate zone belong to
this group.
Rubisco acts in two different ways in C3 plants: as carboxylase or as oxygenase,
the latter catalyzing the binding of an oxygen molecule to ribulose-1,5-biphosphate.
Photosynthesis 3

In the second case (oxygenation), molecules of phosphoglycolate and PGA are pro-
duced, and because one oxygen molecule is also bound during the process, the reac-
tion is known as photorespiration (see Ogren 2005). The reaction causes depletion
of net O2 and release of CO2 . At 25˚ C, and at an O2 concentration of 21% and a CO2
concentration of 0.037%, the Rubisco activity is nearly three times higher than its
oxygenase activity (Berg et al. 2005).
Some warm climate plants have developed mechanisms that enable them
to enhance CO2 concentration at the site where Rubisco acts, which markedly
decreases photorespiration. In such plants, carbon dioxide, with the participa-
tion of phosphoenolpyruvate carboxylase (PEPC), becomes fixed to molecules of
phosphoenol pyruvate (PEP) in mesophyll cells. This process yields a four-carbon
compound, oxaloacetate, which is reduced to malate or aminated to aspartate,
depending on the plant. The above process has been named the C4 pathway ,
and plants in which it occurs are called C4 plants (this pathway is also called
the Hatch– Slack pathway after its main proponents; see Hatch 2005). C4 plants
include maize, sorghum, sugarcane, and numerous weeds. Malate or aspartate
molecules in C4 plants are transported to bundle sheath cells, where they are sub-
sequently decarboxylated to three-carbon compounds (pyruvate or PEP), which
involves CO2 release. Depending on the decarboxylating enzyme, C4 plants
form malate or aspartate in bundle sheaths. Plants can be divided into three sub-
types: NADP-ME, NAD-ME, and PEPCK (possessing NADP-dependent malate
enzyme (ME), NAD-dependent malate enzyme (ME), or PEP carboxylase (CK),
respectively).
The carbon dioxide released in bundle sheath cells is immediately fixed in the
Calvin– Benson cycle inside these cells. In cell surroundings, CO2 concentration
is low, because mesophyll cells effectively protect bundle sheath cells against air
access. In this way, Rubisco of C4 plants attains high carboxylase activity with very
low oxygenase activity. The elimination of photorespiration enhances the efficiency
of the whole process of photosynthesis, which enables C4 plants to successfully com-
pete with C3 plants (Edwards and Walker 1983).
To avoid high water loss, some plants growing in warm and dry climates, known
as Crassulacean acid metabolism plants (CAM type; see Black and Osmond 2005),
close the stomata during the day and open them at night. They fix carbon dioxide
at night using PEP carboxylase and reduce the produced pyruvate to malate, which
is then transported to and accumulated in vacuoles. Similarly to C4 plants, during
the day, they decarboxylate malate and use the produced CO2 in the Calvin– Benson
cycle (Ferreyra et al. 2003).
Chloroplasts are bounded by a double lipoprotein membrane (outer and inner
membrane). Chloroplasts contain stroma, a protein matrix with embedded disc-
shaped sacs known as thylakoids . Thylakoids are gathered in stacks referred to as
grana . Chloroplasts also contain inter-grana membranes, known as stroma lamellae.
Thylakoid membranes have photosynthetic pigments that capture light, as well as
proteins that build the photosynthetic apparatus (chlorophyll– protein complexes of
PSI and PSII antenna arrays). The light phase of photosynthesis occurs in thylakoids,
whereas the dark phase takes place in the stroma. Chloroplasts also possess DNA
and RNA (Figure 1.2).
4 Chlorophyll Fluorescence: Understanding Crop Performance

Chloroplast envelope
External membrane
Internal membrane
Stroma
Stromal thylakoids
Granal thylakoids
DNA
Ribosome
Starch grain
Assimilation
plastoglobules

FIGURE 1.2   Structure of a chloroplast in higher plants. (Adapted from Kalaji, M.H. and
Ł oboda, T., Chlorophyll Fluorescence to Study the Physiological Status of Plants [fluo-
rescencja chlorofilu w badaniach stanu fizjologicznego roś lin] , Wydawnictwo SGGW [in
Polish], Warsaw, 2009.)

Thylakoid membranes contain integral proteins, which can be hydrophobic or

amphipathic (they possess hydrophilic or hydrophobic regions), and they have hydro-
philic proteins on the surface, such as plastocyanin (a copper-containing protein) and
ferredoxin (a protein with non-heme iron and sulfur). Lipids constitute 35%– 40% of
these membranes. The major lipids are galactolipids (monogalactosyldiacylglycerol
[MGDG] and digalactosyldiacylglycerol [DGDG]), which are crucial for the effi-
ciency of photosynthetic light reactions. The membranes also contain phospholipids,
including phosphatidylglycerol, phosphatidylcholine, phosphatidylinositol, and phos-
phatidylethanolamine, as well as sulfolipids, such as plastoquinone, α -tocopherol,
and vitamin K. Chloroplast membranes are rich in polyunsaturated fatty acids and
have trace amounts of sterols, which makes them the most fluid among all known
biological membranes and facilitates the translocation of proteins in the lateral plane
of membranes.
Duysens et al. (1961) demonstrated the involvement of two photosystems in the
light phase of photosynthesis: PSI and PSII, which contain pigments and numer-
ous proteins (for the evolution of the concept of two photosystems and two pigment
systems, see Govindjee and Bjö rn 2012). Together with the cytochrome complex,
the two photosystems are the major components needed for the electron transport
chain from water to NADP+ . Individual complexes in the photosynthetic apparatus
of higher plants are distributed in a variety of ways within thylakoid membranes
(Figure 1.3).
PSII, as well as its light-harvesting antenna system (LHCII), is located in the
membranes of the thylakoid grana and is not exposed to the stroma. PSI and the ATP
synthase complex are located in thylakoid membranes, which are in contact with the
stroma and the stroma lamellae, while the cytochrome b 6 f complex (cyt b 6 f ) is situ-
ated in both stroma-contacting and granal membranes. The main PSII components
include the crucial oxygen evolving complex (OEC) required for oxidation of water
to molecular oxygen. The structure of chloroplasts, and the organization and activity
Photosynthesis 5

PSI

PSII

LHCPII

Cyt. b6 f

ATP synthase

FIGURE 1.3   Scheme of the heterogeneous distribution of protein complexes in the

thylakoid membrane. PSI and ATP synthase are found in membranes of thylakoids that are
in contact with the stroma, PSII is in thylakoid membranes that are not in contact with the
stroma, while cyt b 6 f is in all regions of thylakoids. (Adapted from Kalaji, M.H. and Ł oboda,
T., Chlorophyll Fluorescence to Study the Physiological Status of Plants [fluorescencja chlo-
rofilu w badaniach stanu fizjologicznego roś lin] , Wydawnictwo SGGW [in Polish], Warsaw,
2009.)

of the discussed complexes, depend on a number of factors (Garstka 2007; Garstka

et al. 2007), but they are also affected by the type of photosynthesis (Romanowska
et al. 2006).

1.1.1 PSII Antenna Complex

The absorption of solar radiation within the PAR range (400– 700 nm) by photo-
synthetic pigments (chlorophyll and carotenoids) occurs mostly in light-harvesting
antenna complexes, which are located in thylakoid membranes (Blankenship 2014;
Hall and Rao 1999; Lawlor 2001; Strzał ka 2002). An antenna complex is composed
of numerous molecules of chlorophylls and carotenoids connected by proteins
(Figure 1.4).
According to Ghanotakis et al. (1999) as well as Hall and Rao (1999), PSII antenna
complexes can be divided into

1. Internal antennae composed of protein and chlorophyll complexes, such as

CP43 (43 kDa) and CP47 (47 kDa) proteins, which contain chlorophyll a
and β - carotene molecules
2. Trimeric external antennae composed of LHCII, which contain chlorophyll
a , chlorophyll b , and xanthophylls
3. Monomeric proteins CP29 (29 KDa), CP26 (26 kDa), and CP24 (24 kDa)
binding chlorophyll a , chlorophyll b , and xanthophylls
6 Chlorophyll Fluorescence: Understanding Crop Performance

hν

100 molecules
of chl b (650 nm) +
several molecules
of carotenoids
200 molecules
of chl a (650 nm) +
Absorption

several molecules
PSII
Energy

of xanthophylls
antenna
complex
100 molecules
of chl a (670 nm) +
several molecules
of b-carotene

chl. red. Dimer chl a chl. excit.

(P680)

Donor e– chl. e– Acceptor

oxid.
Electron transport

FIGURE 1.4   (See color insert.) Simplified diagram of the flux of electrons in an energy
antenna of PSII. (Adapted from Kalaji, M.H. and Ł oboda, T., Chlorophyll Fluorescence to
Study the Physiological Status of Plants [fluorescencja chlorofilu w badaniach stanu fizjo-
logicznego roś lin] , Wydawnictwo SGGW [in Polish], Warsaw, 2009.)

The LHCII pigment and protein complex is composed of trimers whose mono-
mers consist of a polypeptide chain containing 232 amino acid residues, 8 mole-
cules of chlorophyll a , 6 molecules of chlorophyll b , and 4 molecules of carotenoids
(Garstka 2007). These complexes are capable of lateral migration in thylakoid mem-
branes, depending on their phosphorylation state. In non-phosphorylated form, these
complexes can transfer energy for the excitation of the PSII reaction center. In this
way, the inflow of absorbed energy to the reaction centers of individual photosystems
is regulated (Hall and Rao 1999).

1.1.2 Photosynthetic Pigments of Higher Plants

The major pigments participating in photosynthesis are chlorophylls, which give
plants their green color. Green plants contain two types of chlorophyll: chlorophyll
a , which is present in all photosynthesizing organisms that produce oxygen, and
chlorophyll b , which is present in one-third the amount of chlorophyll a and is found
in plants and chlorophytes (Hall and Rao 1999). A molecule of chlorophyll a is
composed of a porphyrin ring, in which nitrogen atoms of four pyrrole rings have
Photosynthesis 7

H CH2 H CH2
C H CH3 C H CHO

H 3C I II C 2H 5 H 3C I II C 2H 5
N N N N Porphyrin
H Mg H H Mg H ring head
H 3C N N H 3C N N
IV III CH3 IV III CH3
H H
CH2H V CH2H V
H H
CH2 O CH2 O
O C COOCH3 O C COOCH3
O O
CH2 CH2
CH CH
C CH3 C CH3
CH2 CH2
CH2 CH2
CH2 CH2
HC CH3 HC CH3 Phytol tail
CH2 CH2
CH2 CH2
CH2 CH2
HC CH3 HC CH3
CH2 CH2
CH2 CH2
CH2 CH2
CH CH
CH3 CH3 CH3 CH3

Chlorophyll a Chlorophyll b

FIGURE 1.5   The structural pattern of chlorophyll a and b . (Adapted from Kalaji, M.H.
and Ł oboda, T., Chlorophyll Fluorescence to Study the Physiological Status of Plants
[fluorescencja chlorofilu w badaniach stanu fizjologicznego roś lin] , Wydawnictwo SGGW
[in Polish], Warsaw, 2009.)

coordination links with the centrally located magnesium ion, an additional fifth ring,
and a residue of 20-carbon alcohol known as phytol (Figure 1.5).
A sequence of alternating double and single bonds in rings, which form a sys-
tem of conjugated bonds, is responsible for light absorption. The phytol chain is not
involved in light absorption, but it anchors the chlorophyll molecule in the thylakoid
membrane and provides it with the right orientation. In algae, there are many types
of chlorophyll, which are marked with consecutive letters of the English alphabet
and possess different structures. For example, chlorophyll c is present in diatoms and
brown algae, whereas chlorophyll d is found only in cyanobacteria (see Blankenship
2014). The structure of chlorophyll e is not yet determined, and the chemical struc-
ture of chlorophyll f from cyanobacterial cultures was discovered only recently
(Chen et al. 2010, 2012).
8 Chlorophyll Fluorescence: Understanding Crop Performance

Differences in the structure of these pigments result in certain variations in their

absorption spectra. All chlorophylls absorb radiation within the wavelength region
of 400– 720 nm and have two main absorption bands: one in the blue range (higher
excitation level) and the other in the red range (lower excitation level). In solutions
of organic solvents, chlorophyll a shows maximum absorption at the wavelengths of
around 420 and 660 nm, while chlorophyll b has absorption peaks at 435 and 642 nm
(Figure 1.6). In pigment and protein complexes located in chloroplasts, absorption
maxima are shifted toward long waves. For a discussion on why chlorophyll a was
chosen for photosynthesis, see Bjö rn et al. (2009): specific binding to specific amino
acids provides different absorption and redox properties.
In addition to chlorophylls, thylakoid membranes also contain other auxiliary
pigments (e.g., carotenoids and phycobilins), which absorb photons of other wave-
lengths of light and transfer them to chlorophyll a molecules in the antennae and the
reaction centers. Carotenoids are present in plants and in most algae, whereas phy-
cobilins are found in red algae and cyanobacteria. Carotenoids are isoprene deriva-
tives comprising orange carotenes and yellow-orange xanthophylls. These pigments
absorb radiation at different wavelengths than chlorophylls (including the violet and
blue range of the spectrum), but in collaboration with chlorophylls, they enhance the
efficiency of light capture (Figure 1.6). The pigments have three absorption peaks,
which are found (depending on the type of carotenoid) at 420– 425, 440– 450, and
470– 480 nm. Diatoms and brown algae also contain the carotenoid fucoxanthol,
which captures much of the green light (500– 600 nm range) and transfers it very
efficiently to chlorophyll a . Carotenoids also play an important role by protecting
chloroplast lipids against photo-oxidation (light-induced oxidation). Carotenoids
can intercept the excess energy from excited chlorophyll molecules by thermally

Chlorophyll a
Chlorophyll b

Chlorophyll a
Extinction

Carotenoids

400 500 600 700

Wavelength (nm)

FIGURE 1.6   Absorption spectra of main photosynthetic pigments. (Adapted from Kalaji,
M.H. and Ł oboda, T., Chlorophyll Fluorescence to Study the Physiological Status of Plants
[fluorescencja chlorofilu w badaniach stanu fizjologicznego roś lin] , Wydawnictwo SGGW
[in Polish], Warsaw, 2009.)
Photosynthesis 9

deactivating them, which prevents the production of singlet oxygen, a highly reactive
form of this element.
Unlike chlorophylls and carotenoids, phycobilins are water soluble. Together with
special proteins, they are located in structures known as phycobilisomes , which sup-
ply energy for the excitation of both PSII and PSI (Strzał ka 2002). As mentioned,
they are found in red algae and cyanobacteria, they absorb different wavelengths of
visible light, depending on the type of phycobilins (phycoerythrins and phycocya-
nins), and their absorption peaks are in the range of 490– 670 nm. Phycoerythrins
absorb more green light, whereas phycocyanins absorb more orange light. In anox-
ygenic photosynthesizing bacteria, the functions of chlorophyll are performed by
various types of bacteriochlorophylls, from BChl a to BChl g. These organisms will
not be discussed in this book.

1.1.3 Photosystem II (PSII)

Typically, chloroplasts contain more PSII than PSI, and the stoichiometric ratio of
the amount of PSII to PSI is often around 1.5, but it can vary subject to the environ-
mental conditions (Taiz and Zeiger 1991). PSII is located mainly in thylakoid grana,
at sites where they do not connect with the stroma (Figure 1.7).
PSII contains internal antennae CP43 and CP47; external antennae (LHCII); the
reaction center composed of six chlorophylls, including two chlorophyll a mole-
cules with maximum absorption at 680 nm (P680); Pheo; protein D1 (the site to

2H+ ½O2

Stroma

H2QB QA
LHCII Fe2+ LHCII
PQH2 CP
Pheo 47
CP Pheo
43 P680 D1
PQ D2
Y YZ
D
Mn4Ca

Intrathylakoid 17 33
space (lumen) 2H+
23
H2O

FIGURE 1.7   Structure of PSII. LHCII: light-harvesting chlorophyll and protein complex;
CP43, CP47: chlorophyll and protein complexes, which are the PSII cortical antenna; Pheo:
pheophytin; 17, 23, and 33: polypeptides of the OEC; P680: chlorophyll dimer (the PSII reac-
tion center); D1 and D2: chlorophyll P680 and pheophytin-binding polypeptides; QA : plas-
toquinone; H2 QB : reduced plastoquinone bound at the QB site; YD , YZ : tyrosine residues on
D2 and D1 proteins, respectively. (Adapted from Kalaji, M.H. and Ł oboda, T., Chlorophyll
Fluorescence to Study the Physiological Status of Plants [fluorescencja chlorofilu w badani-
ach stanu fizjologicznego roś lin] , Wydawnictwo SGGW [in Polish], Warsaw, 2009.)
10 Chlorophyll Fluorescence: Understanding Crop Performance

O OH

H3C H3C
CH3 CH3

H3C (CH2–CH=C–CH2)nH H3C (CH2–CH=C–CH2)nH

O n=6–10 OH n=6–10

Oxidized form of Reduced form of

plastoquinone (PQ) plastoquinone (PQH2)
(a) (b)

FIGURE 1.8   Structure of oxidized (a) and reduced (b) form of a plastoquinone. (Adapted
from Kalaji, M.H. and Ł oboda, T., Chlorophyll Fluorescence to Study the Physiological
Status of Plants [fluorescencja chlorofilu w badaniach stanu fizjologicznego roś lin] ,
Wydawnictwo SGGW [in Polish], Warsaw, 2009.)

which plastoquinone QB is attached); and protein D2, to which plastoquinone QA is

attached (for the structure of plastoquinone and plastoquinol, see Figure 1.8).
PSII also contains the OEC, located on the outer surface of thylakoid membranes.
The complex includes an amino acid, tyrosine (YZ), on protein D1, four atoms of
manganese, and external proteins PsbO (stabilizing the manganese cluster), PsbP,
and PsbQ. During the light phase of photosynthesis, two water molecules in this
complex are oxidized to oxygen and release four protons, whereas four electrons
are transferred individually, through a specific tyrosine, to oxidized P680 (P680+ )
produced during light-induced charge separation in PSII (for detail, see a review by
Govindjee et al. 2010; and for a high-resolution structure of PSII, see Umena et al.
2011). For a complete description of PSII (the water-plastoquinone oxidoreductase),
refer to the relevant chapters in Wydrzynski and Satoh (2005).

1.1.4 Cytochrome b6f Complex

The cytochrome b 6 f complex contains the Rieske iron-sulfur center with a low-
potential molecule of cytochrome b 6 and a high-potential molecule of cytochrome
b  6 , cytochrome f , and subunit IV (PetD). It acts as plastoquinol (PQH2 )-plastocyanin
oxidoreductase and plays an important role in the proton pump by carrying hydrogen
ions from the stroma into a thylakoid.

1.1.5 Photosystem I (PSI)

PSI is located in thylakoids of the stroma and in thylakoid grana connected to the
stroma. PSI includes an LHCI antenna complex, which contains mostly chlorophyll
a (the Chl a to Chl b ratio is 4:1); carotenoids; Lhca polypeptides; several poly-
peptides (A to N); a reaction center complex, which contains many antenna chlo-
rophyll a molecules and two molecules of special chlorophyll a with absorption
maxima at 700 nm (P700); phylloquinone (vitamin K1 ); several iron-sulfur proteins;
and a ferredoxin (Fd). PSI oxidizes plastocyanin in the thylakoid lumen, known as
Photosynthesis 11

H+
NADP+

FNR NADPH
Fd
Stroma D
E
M G FA C
FB H J
L I K
Fx
B A
A1

Lhca A0 Lhca
1–4 1–4

P700

e–
Intrathylakoid
space (lumen) N PC F

FIGURE 1.9   Structure of PSI. LHCI: light-harvesting chlorophyll and protein complex
containing Lhca 1– 4 polypeptides; A, B, … N: polypeptides; P700: chlorophyll dimer (the
PSI reaction center); A0 : a chlorophyll molecule; A1 : phylloquinone (vitamin K1 ); iron-sulfur
clusters FX , FA , and FB ; PC: plastocyanin; Fd: ferredoxin; FNR: ferredoxin-NADP+ reduc-
tase. (Adapted from Kalaji, M.H. and Ł oboda, T., Chlorophyll Fluorescence to Study the
Physiological Status of Plants [fluorescencja chlorofilu w badaniach stanu fizjologicznego
roś lin] , Wydawnictwo SGGW [in Polish], Warsaw, 2009.)

plastocyanin-ferredoxin oxidoreductase. On the outer side of PSI, Fd is adjacent

to the ferredoxin-NADP+ reductase (FNR) enzyme, which catalyzes the transfer of
electrons from Fd to NADP+ (Figure 1.9). A strong reductant, NADPH, is produced
when two electrons are accepted and a proton binds to the NADP+ molecule. For
details of PSI, see the relevant chapters in Golbeck (2005).

1.1.6 ATP Synthase

ATP synthase is a complex composed of a hydrophobic part (CFO ) in the thyla-
koid membrane, which is connected to the hydrophilic part (CF1 ) that protrudes
on the outside of thylakoid membranes. ATP synthase has catalytic sites. The CF1
possesses three α subunits, three β subunits, and regulatory subunits γ , δ , and
ε  (Figure 1.10). ATP synthesis occurs on subunit β . The CF0 contains a protein with
two ion channels. The ATP synthase complex enables the conversion of ADP and
inorganic phosphate (Pi) to ATP with the involvement of the proton motive force
(pmf  =  ∆ pH  +  ∆ Ψ ). The process of synthesizing ATP from ADP and inorganic phos-
phate, known as phosphorylation , is one of the most important reactions in the light
12 Chlorophyll Fluorescence: Understanding Crop Performance

ADP Pi
ATP

a b a

b CF1

b a

H+
Stroma II
g e

CF0
I

III
Intrathylakoid
space (lumen)
H+

FIGURE 1.10   Structure of ATP synthase. CF0 : binding part; CF1 : catalytic center. Letters
and figures designate subunits of the enzyme. (Adapted from Boyer, P.D., Nature , 402, 6759,
247– 249, 1999.)

phase of photosynthesis. For details relating to the use of pmf and a rotary mecha-
nism whereby pmf is converted into rotation energy and rotation energy is converted
into chemical energy, see Junge et al. (2009) and Mukherjee and Warshel (2012).

1.2 LIGHT PHASE OF PHOTOSYNTHESIS

1.2.1 Absorption of Photosynthetically Active Radiation (PAR)
When a molecule of chlorophyll or any other photosynthetic pigment present in
the antennae of the LHCII complex absorbs a PAR photon, it becomes excited. A
valence electron in the molecule of this pigment moves from the lowest (ground)
state to a higher energy level. The excited molecule, such as chlorophyll (X1 ), trans-
fers the excitation energy by resonance to an adjacent chlorophyll molecule (X2 ) and
returns to its ground state. For such excitation energy transfer to occur, the absorption
Photosynthesis 13

spectrum of X2 has to lie within the range of long waves of the spectrum relative to
the maximum of X1 . Such energy transfer is invariably associated with a loss of
transferred energy (Hall and Rao 1999). The shorter the distance between pigment
molecules involved in the transfer of excitation energy, the greater the probability
that it will be transferred. The energy transfer efficiency between chlorophyll mol-
ecules is close to 100%, and it is lower when the transfer occurs from carotenoids to
chlorophyll (Hall and Rao 1999). When energy migrating along the antenna reaches
chlorophyll a in the P680 photosynthetic reaction center, the electron in the excited
P680 is transferred onto an intermediary acceptor, Pheo, in PSII. This step of pri-
mary charge separation results in the formation of a pair of radicals (P680+ Pheo – )
in picoseconds (see Mamedov et al. 2015).
The electron on the reduced Pheo is transferred very quickly (after about 0.4 ns)
onto plastoquinone QA (P680+ Pheo –   QA → P680+ Pheo QA–  ). Plastoquinone QA ,
located on protein D2, is the first stable acceptor of electrons in PSII (Hankamer
et al. 1997). At the same time, the oxidized molecule of chlorophyll P680+ is neutral-
ized by an electron taken from tyrosine (Tyrz) in the water-splitting complex on the
electron donor side of PSII (Tyrz P680+ → Tyrz+ P680). The localization of separated
charges on the stable acceptor and the stable donor prevents their recombination,
which ensures effective use of energy during the following stages of photosynthesis
(Jones and Fyfe 2001) discussed later (Figure 3.1). A similar reaction takes place
simultaneously in PSI (see below; also Mamedov et al. 2015).

1.2.2 Transport of Electrons and Non-Cyclic Phosphorylation

The initiation of a series of oxidation and reduction reactions described in the previ-
ous section enables the transfer of electrons between successive carriers. The trans-
fer of electrons via a chain of special carriers located inside and outside PSII and
PSI complexes involves non-cyclic transport, because electrons move from water to
NADP+ without cycling.
After QA is reduced, subsequent separation of charges in the PSII reaction center
is not possible. Thus, fluorescence of chlorophyll from this photosystem becomes
more probable, because the de-excitation of excited chlorophyll can occur by three
pathways: photochemistry, fluorescence, and heat loss. The center where QA is in
the reduced state is referred to as a closed center . When the center opens after the
transfer of an electron from reduced QA onto plastoquinone QB on the D1 protein
(QA–    QB → QA QB–   ), the probability of chlorophyll a fluorescence is decreased. After
another light reaction, a subsequent reduction of QB ‾ by an electron transferred
from QA‾ would produce QB2 –    (QA– QB–   → QA QB2 –   ). By taking up two protons from the
stroma via amino acids and a bicarbonate ion bound to a non-heme iron localized
between QA and QB , QB2– +2H+ becomes QB H2 , which is a molecule of plastoquinol
that can be referred to as Q H2 for the sake of simplicity. The plastoquinol molecule
leaves protein D1 and joins the plastoquinol pool (Hankamer et al. 1997; Ott et al.
1999).
Another plastoquinone molecule from the pool of available plastoquinones is then
bound to the free QB site on D1. Plastoquinol moves to the other (lumen) side of
the thylakoid membrane, where it is oxidized to plastoquinone by transferring two
14 Chlorophyll Fluorescence: Understanding Crop Performance

electrons to the Cyt b 6 f complex: one to Cyt b 6 and the other to the Rieske iron cen-
ter. This reaction releases two protons, which are transferred to the thylakoid lumen.
The Cyt b 6 f complex cooperating with the pool of plastoquinones acts like a proton
pump. As a result, the inner part (lumen) of the thylakoid becomes acidified. The
action of the pump produces a proton gradient across the thylakoid membrane, which
is the driving force behind the process of ATP production (Kramer et al. 2004).
The reduced iron-sulfur center passes its electrons to plastocyanin (PC), which is a
mobile, copper-containing protein. Copper ions change their oxidation state. Thus,
PC transfers electrons to the oxidized P700+ reaction center each time PSI is excited,
which produces P700+ . The reduction of P700+ by PC takes between 20 and 200 μ s
(Bottin and Mathis 1985; Haehnel et al. 1980).
The water-splitting complex, located on the inner (donor) side of the thylakoid
membranes, plays a very important role in PSII (Figure 1.11). This complex is com-
posed of four manganese atoms and YZ , which is tyrosine 161 of protein D1 , as
well as extrinsic proteins of 33 (PsbO), 23 (PsbP), and 17 kDa (PsbQ). During this
process, four electrons are split off (one by one) from the manganese complex and
are subsequently donated, one by one, to the oxidized P680, that is, P680+ , which

hn hn

S1
e–
e–

O2
S2
S0
H+ hn

H+
H+
H+

S4 S3
2H2O e–

e–

hn

FIGURE 1.11   Schematic model illustrating the oxidation of the manganese complex dur-
ing the water-splitting process (formation of the oxidation states of the manganese complex,
release of H+ , and generation of O2 . S0 , S1 , … S4 are subsequent oxidation states of the man-
ganese complex from 0 to +4). (Adapted from Kalaji, M.H. and Ł oboda, T., Chlorophyll
Fluorescence to Study the Physiological Status of Plants [fluorescencja chlorofilu w badani-
ach stanu fizjologicznego roś lin] , Wydawnictwo SGGW [in Polish], Warsaw, 2009.)
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on totta: hän on lahjottanut minulle henkeni.»
 »Ja…?»

»Ja lähettänyt minut vankilaan.»

»Kuinka pitkäksi aikaa?» kuiskasi hän.

»Sitä en tiedä», vastasin minä. »Niin pitkäksi kuin kuningasta

miellyttää, pelkään minä.»

Hän värähti.

»Olen kenties tehnyt enemmän vahinkoa kuin hyötyä», mutisi hän

itsekseen ja katseli minua surkuttelevasti. »Mutta tein sen hyvässä
tarkoituksessa. Kerroin hänelle kaikki, ja kuitenkin olen kenties
tuottanut vahinkoa.»

Mutta kuulla hänen soimaavan itseään noin, tehtyään tämän

pitkän ja yksinäisen matkan minun pelastuksekseni, voitettuaan
vastenmielisyytensä vihollistaan kohtaan ja — siitä olin varma —
nöyrryttyään hänen edessään, sitä minun oli mahdoton sietää.

»Vaietkaa, mademoiselle, vaietkaa!» huudahdin kiivaasti. »Te

haavoitatte sydäntäni. Te olette tehnyt minut onnelliseksi, ja kuitenkin
soisin, että te ette olisi täällä, vaan Cocheforêtissa. Te olette tehnyt
puolestani paljoa enemmän kuin olisin voinut toivoa ja sata kertaa
enemmän kuin olen ansainnut. Mutta tähän täytyy sen loppua. Olin
auttamattomasti mennyttä miestä, ennen kuin tämä tapahtui, ennen
kuin olin teitä koskaan nähnyt, ja niin olen vieläkin, en enempää
enkä vähempää, enkä minä tahdo, että teidän nimeänne mainitaan
minun nimeni yhteydessä täällä Pariisissa. Sentähden — hyvästi.
Jumala varjelkoon minua sanomasta enempää teille ja antamasta
teidän viipyä täällä, missä joutilaat kielet pian ahdistaisivat teitä.»
 Hän katseli minua hiukan ihmetellen; sitten hän sanoi hymyillen ja
lempeästi:

»Se on myöhäistä.»

»Myöhäistä?» huudahdin. »Kuinka niin?»

»Sentähden että… muistatteko, herra de Berault, mitä

tienristeyksessä Agenin ulkopuolella kerroitte minulle
rakkaustarinastanne? Ettei se voinut saada mitään onnellista
loppua? Samasta syystä en minäkään hävennyt kertoa kardinaalille
omaani. Nyt se on joka miehen suussa.»

Katselin häntä, hänen seistessään ihan vastapäätä minua. Hänen

silmänsä säteilivät kiharaisten hiusten alta, jotka melkein peittivät ne.
Hän painoi kasvonsa alas, ja kuitenkin väreili hymy hänen huulillaan.

»Mitä kerroitte hänelle, mademoiselle?» kuiskasin läähättäen.

»Että rakastan», vastasi hän uljaasti ja käänsi kirkkaat silmänsä

minuun. »Ja että sentähden en hävennyt kerjätä — vieläpä
polvillani.»

Lankesin hänen jalkoihinsa ja tartuin hänen käteensä, ennen kuin

viimeinen sana oli kirvonnut hänen huuliltaan. Silmänräpäykseksi
unohdin kuninkaan ja kardinaalin, vankilan ja tulevaisuuden, kaikki,
kaikki, paitsi että tämä nainen, niin puhdas ja kaunis, kaikessa niin
suuresti yläpuolelleni kohonnut, rakasti minua. Silmänräpäykseksi,
sanon. Sitten palasi muistini. Minä nousin pystyyn ja irtauduin
hänestä äkillisessä tunteitteni vaihdoksessa.

»Te ette tunne minua!» huudahdin. »Te ette tiedä, mitä kaikkea
olen tehnyt!»
 »Juuri sen tiedän», vastasi hän ja katseli minua ihmettelevästi
hymyillen.

»Ei, te ette tiedä!» vakuutin taas. »Ja sitäpaitsi… on tämä meidän

välillämme!» Otin kardinaalin kirjeen, joka oli pudonnut lattialle.

Hän kääntyi ja kalpeni hiukan. Sitten hän huusi nopeasti:

»Avatkaa se! Avatkaa! Se ei ole suljettu eikä sinetöity.»

Tottelin koneellisesti, kauheasti peloissani siitä, mitä saisin nähdä.

Vielä silloinkin kun pidin sitä avoinna edessäni, loin vain pelokkaan
silmäyksen kaunismuotoisiin kirjaimiin. Se kuului näin:

»Kuningas näkee hyväksi käskeä, että herra Gil de Berault, joka

on sekaantunut valtion asioihin, viipymättä vetäytyy takaisin
Cocheforêtin herrastilalle ja pysyy sen alueella, kunnes kuningas
armollisesti toisin määrää.

Kardinaali Richelieu.»

Me menimme naimisiin seuraavana päivänä, ja neljätoista päivää

myöhemmin olimme Cocheforêtin kellastuneissa metsissä korkeiden
vuorten suojassa, suuren kardinaalin, joka oli vielä kerran voittanut
vihollisensa, katsellessa kylmäkiskoisesti hymyillen koko maailman
tungeksimista vastaanottohuoneessaan. Hänen onnensa nousua
kesti kolmetoista vuotta siitä ajasta lukien, ja se päättyi vasta hänen
kuollessaan. Sillä maailma oli kokemuksesta viisastunut, ja vielä tällä
hetkelläkin on se päivä, jona minä olin yksinäni vastaanotossa,
nimeltään »narrien päivä.»
*** END OF THE PROJECT GUTENBERG EBOOK KARDINAALIN
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