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We provide here a general introduction on chlorophyll (Chl) a fluorescence, then we present our measurements on fast (< 1 s) induction curves (the so-called OJIP transients) on dark-adapted intact leaves of Arabidopsis thaliana, under five different light intensities [in the range of ~ 500 to ~ 3,000 µmol(photons) m‒2 s‒1] using two different instruments: Handy PEA (Hansatech Instruments, UK; excitation light, 650 nm) and FluorPen (model FP-110; Photon Systems Instruments, The Czech Republic; excitation light, 470 nm). We then discuss the observed differences in the OJIP curves, as well as in Fo (F20μs, F50μs, or the extrapolated Ft→0), FP (the peak), and the ratios FP/Fo, and Fv (= FP ‒ Fo)/FP in terms of differences in excitation light intensity and absorptance (or absorbance) of the excitation light by the leaves, and other factors, as well as the data available in the literature. We suggest that such measurements be accompanied, in the future, by parallel measurements on Chl a fluorescence imaging, an area pioneered by Hartmut K. Lichtenthaler.

Govindjee, Professor Emeritus of Biochemistry, Biophysics and Plant Biology at the University of Illinois at Urbana-Champaign since 1999, is renowned for his pioneering work in the light reactions of photosynthesis and important accomplishments as educator, editor, historian and advocate of photosynthesis. In his honour, we review his contributions over the last twenty years, which were often achieved in collaboration with scientists working in laboratories around the world. We start with a short presentation about Govindjee’s career and continue with a section highlighting his passion and dedication to teach the younger generations of students and researchers about photosynthesis. His research work is grouped under different areas, including primary photochemistry; the relationship of chlorophyll a fluorescence to photosynthesis; short time-scale regulation processes of photosynthesis; studies toward improving photosynthesis and biomass yields; and, mathematical modelling of photosynthesis and artificial photosynthesis. He is best known for the discovery of the key role of bicarbonate on the electron acceptor side of Photosystem II. We also present his active involvement in the recognition of scientists at conferences and beyond, as well as of those who are no longer with us. He is committed to keeping alive the pioneers and discoverers in the field of photosynthesis in the collective memory of those in the field.

Photosynthesis can be probed through Chlorophyll a fluorescence induction (FI), which provides detailed insight into the electron transfer process in Photosystem II, and beyond. Here, we have systematically studied the natural variation of the fast phase of the FI, i.e. the OJIP phase, in rice. The OJIP phase of the Chl a fluorescence induction curve is referred to as "fast transient" lasting for less than a second; it is obtained after a dark-adapted sample is exposed to saturating light. In the OJIP curve, "O" stands for "origin" (minimal fluorescence), "P" for "peak" (maximum fluorescence), and J and I for inflection points between the O and P levels. Further, Fo is the fluorescence intensity at the "O" level, whereas Fm is the intensity at the P level, and Fv (= Fm - Fo) is the variable fluorescence. We surveyed a set of quantitative parameters derived from the FI curves of 199 rice accessions, grown under both field condition (FC) and growth room condition (GC). Our results show a significant variation between Japonica (JAP) and Indica (IND) subgroups, under both the growth conditions, in almost all the parameters derived from the OJIP curves. The ratio of the variable to the maximum (Fv/Fm) and of the variable to the minimum (Fv/Fo) fluorescence, the performance index (PIabs), as well as the amplitude of the I-P phase (AI-P) show higher values in JAP compared to that in the IND subpopulation. In contrast, the amplitude of the O-J phase (AO-J) and the normalized area above the OJIP curve (Sm) show an opposite trend. The performed genetic analysis shows that plants grown under GC appear much more affected by environmental factors than those grown in the field. We further conducted a genome-wide association study (GWAS) using 11 parameters derived from plants grown in the field. In total, 596 non-unique significant loci based on these parameters were identified by GWAS. Several photosynthesis-related proteins were identified to be associated with different OJIP parameters. We found that traits with high correlation are usually associated with similar genomic regions. Specifically, the thermal phase of FI, which includes the amplitudes of the J-I and I-P subphases (AJ-I and AI-P) of the OJIP curve, is, in turn, associated with certain common genomic regions. Our study is the first one dealing with the natural variations in rice, with the aim to characterize potential candidate genes controlling the magnitude and half-time of each of the phases in the OJIP FI curve.

BackgroundWith limited agricultural land and increasing human population, it is essential to enhance overall photosynthesis and thus productivity. Oxygenic photosynthesis begins with light absorption, followed by excitation energy transfer to the reaction centres, primary photochemistry, electron and proton transport, NADPH and ATP synthesis, and then CO2 fixation (Calvin–Benson cycle, as well as Hatch–Slack cycle). Here we cover some of the discoveries related to this process, such as the existence of two light reactions and two photosystems connected by an electron transport ‘chain’ (the Z-scheme), chemiosmotic hypothesis for ATP synthesis, water oxidation clock for oxygen evolution, steps for carbon fixation, and finally the diverse mechanisms of regulatory processes, such as ‘state transitions’ and ‘non-photochemical quenching’ of the excited state of chlorophyll a.ScopeIn this review, we emphasize that mathematical modelling is a highly valuable tool in understanding and making predictions regarding photosynthesis. Different mathematical models have been used to examine current theories on diverse photosynthetic processes; these have been validated through simulation(s) of available experimental data, such as chlorophyll a fluorescence induction, measured with fluorometers using continuous (or modulated) exciting light, and absorbance changes at 820 nm (ΔA820) related to redox changes in P700, the reaction centre of photosystem I.ConclusionsWe highlight here the important role of modelling in deciphering and untangling complex photosynthesis processes taking place simultaneously, as well as in predicting possible ways to obtain higher biomass and productivity in plants, algae and cyanobacteria.

Chlorophyll (Chl) a fluorescence induction (transient), measured by exposing dark-adapted samples to high light, shows a polyphasic rise, which has been the subject of extensive research over several decades. Several Chl fluorescence parameters based on this transient have been defined, the most widely used being the FV [= (FM-F0)]/FM ratio as a proxy for the maximum quantum yield of PSII photochemistry. However, considerable additional information may be derived from analysis of the shape of the fluorescence transient. In fact, several performance indices (PIs) have been defined, which are suggested to provide information on the structure and function of PSII, as well as on the efficiencies of specific electron transport reactions in the thylakoid membrane. Further, these PIs have been proposed to quantify plant tolerance to stress, such as by high light, drought, high (or low) temperature, or N-deficiency. This is an interesting idea, since the speed of the Chl a fluorescence transient measurement (

In this chapter, we focus on the use of chlorophyll (Chl) a fluorescence to study various photosynthetic processes and their regulation in cyanobacteria. Compared to algae and plants, thylakoid membranes of cyanobacteria not only have photosynthetic, but also respiratory electron transport components. Further, they have phycobilisomes (containing phycobilins) as outer antenna complexes, instead of Chl-containing light-harvesting complexes. We shall present here the use of Chl a fluorescence in monitoring: (a) excitation energy transfer to and in both the photosystems; (b) quantum yield of photosystem II, as inferred from the ratio of variable (FV) to maximum (FM) Chl a fluorescence, and its relation to overall photosynthesis; (c) regulation of photosynthesis, especially that of excitation energy distribution between the photosystems (the so-called state transitions); and (d) nonphotochemical quenching of the excitation energy. We will end this chapter by presenting the challenges in using Chl a fluorescence to monitor various photosynthetic processes in cyanobacteria.

The green alga Chlamydomonas (C.) reinhardtii is a model organism for photosynthesis research. State transitions regulate redistribution of excitation energy between photosystem I (PS I) and photosystem II (PS II) to provide balanced photosynthesis. Chlorophyll (Chl) a fluorescence induction (the so-called OJIPSMT transient) is a signature of several photosynthetic reactions. Here, we show that the slow (seconds to minutes) S to M fluorescence rise is reduced or absent in the stt7 mutant (which is locked in state 1) in C. reinhardtii. This suggests that the SM rise in wild type C. reinhardtii may be due to state 2 (low fluorescence state; larger antenna in PS I) to state 1 (high fluorescence state; larger antenna in PS II) transition, and thus, it can be used as an efficient and quick method to monitor state transitions in algae, as has already been shown in cyanobacteria (Papageorgiou et al. 1999, 2007; Kaňa et al. 2012). We also discuss our results on the effects of (1) 3-(3,4-dichlorophenyl)-1,4-dimethyl urea, an inhibitor of electron transport; (2) n-propyl gallate, an inhibitor of alternative oxidase (AOX) in mitochondria and of plastid terminal oxidase in chloroplasts; (3) salicylhydroxamic acid, an inhibitor of AOX in mitochondria; and (4) carbonyl cyanide p-trifluoromethoxyphenylhydrazone, an uncoupler of phosphorylation, which dissipates proton gradient across membranes. Based on the data presented in this paper, we conclude that the slow PSMT fluorescence transient in C. reinhardtii is due to the superimposition of, at least, two phenomena: qE dependent non-photochemical quenching of the excited state of Chl, and state transitions.

Using chlorophyll (Chl) a fluorescence many aspects of the photosynthetic apparatus can be studied, both in vitro and, noninvasively, in vivo. Complementary techniques can help to interpret changes in the Chl a fluorescence kinetics. Kalaji et al. (Photosynth Res 122:121–158, 2014a) addressed several questions about instruments, methods and applications based on Chl a fluorescence. Here, additional Chl a fluorescence-related topics are discussed again in a question and answer format. Examples are the effect of connectivity on photochemical quenching, the correction of FV/FM values for PSI fluorescence, the energy partitioning concept, the interpretation of the complementary area, probing the donor side of PSII, the assignment of bands of 77 K fluorescence emission spectra to fluorescence emitters, the relationship between prompt and delayed fluorescence, potential problems when sampling tree canopies, the use of fluorescence parameters in QTL studies, the use of Chl a fluorescence in biosensor applications and the application of neural network approaches for the analysis of fluorescence measurements. The answers draw on knowledge from different Chl a fluorescence analysis domains, yielding in several cases new insights. Fil: Kalaji, Hazem M.. Warsaw University of Life Sciences; Polonia Fil: Schansker, Gert. No especifica; Fil: Brestic, Marian. Slovak Agricultural University; Eslovaquia Fil: Bussotti, Filippo. University of Florence; Italia Fil: Calatayud, Angeles. Instituto Valenciano de Investigaciones Agrarias; España Fil: Ferroni, Lorenzo. University of Ferrara; Italia Fil: Goltsev, Vasilij. University of Sofia; Bulgaria Fil: Guidi, Lucia. Department of AgricultureFood and Environment; Italia Fil: Jajoo, Anjana. Devi Ahilya University; India Fil: Li, Pengmin. Northwest A&F University; China Fil: Losciale, Pasquale. Research Unit for Agriculture in Dry Environments; Italia Fil: Mishra, Vinod K.. College of Agriculture Science; India Fil: Misra, Amarendra N.. Central University of Jharkhand; India Fil: Nebauer, Sergio G.. Universidad Politécnica de Valencia; España Fil: Pancaldi, Simonetta. University of Ferrara; Italia Fil: Penella, Consuelo. Instituto Valenciano de Investigaciones Agrarias; España Fil: Pollastrini, Martina. University of Florence; Italia Fil: Suresh, Kancherla. Indian Institute of Oil Palm Research; India Fil: Tambussi, Eduardo Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentina Fil: Yanniccari, Marcos Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentina Fil: Zivcak, Marek. Slovak Agricultural University; Eslovaquia Fil: Cetner, Magdalena D.. Warsaw University of Life Sciences; Polonia Fil: Samborska, Izabela A.. Warsaw University of Life Sciences; Polonia Fil: Stirbet, Alexandrina. Newport News; Estados Unidos Fil: Olsovska, Katarina. Slovak University of Agriculture; Eslovaquia Fil: Kunderlikova, Kristyna. Slovak University of Agriculture; Eslovaquia Fil: Shelonzek, Henry. University of Silesia; Polonia Fil: Rusinowski, Szymon. Institute for Ecology of Industrial Areas; Polonia Fil: Bąba, Wojciech. Jagiellonian University; Polonia

In photosynthetic organisms, light energy is absorbed by a complex network of chromophores embedded in light-harvesting antenna complexes. In photosystem II (PSII), the excitation energy from the antenna is transferred very efficiently to an active reaction center (RC) (i.e., with oxidized primary quinone acceptor Q A), where the photochemistry begins, leading to O2 evolution, and reduction of plastoquinones. A very small part of the excitation energy is dissipated as fluorescence and heat. Measurements on chlorophyll (Chl) fluorescence and oxygen have shown that a nonlinear (hyperbolic) relationship exists between the fluorescence yield (Φ F ) (or the oxygen emission yield, $$ \Phi _{{{\text{O}}_{2} }} $$ ) and the fraction of closed PSII RCs (i.e., with reduced Q A). This nonlinearity is assumed to be related to the transfer of the excitation energy from a closed PSII RC to an open (active) PSII RC, a process called PSII excitonic connectivity by Joliot and Joliot (CR Acad Sci Paris 258: 4622–4625, 1964). Different theoretical approaches of the PSII excitonic connectivity, and experimental methods used to measure it, are discussed in this review. In addition, we present alternative explanations of the observed sigmoidicity of the fluorescence induction and oxygen evolution curves.

In higher plants, algae, and cyanobacteria, chlorophyll (Chl) a fluorescence induction (ChlFI) has a fast (under a second) increasing OJIP phase and a slow (few minutes) PS(M)T phase, where O is for origin, the minimum fluorescence, J and I for intermediate levels, P for peak, S for a semi-steady state, M for a maximum (which is sometimes missing), and T for the terminal steady-state level. We have used a photosynthesis model of Ebenhoh et al. (Philos Trans R Soc B, 2014, doi: 10.1098/rstb.2013.0223 ) in an attempt to simulate the slow PS(M)T phase and to determine the origin of the S-M rise in Chlamydomonas (C.) reinhardtii cells. Our experiments in silico show that a slow fluorescence S-M rise (as that observed, e.g., by Kodru et al. (Photosynth Res 125:219-231, 2015) can be simulated only if the photosynthetic samples are initially in a so-called "state 2," when the absorption cross section (CS) of Photosystem II (PSII) is lower than that of PSI, and Chl a fluorescence is low (see, e.g., a review by Papageorgiou and Govindjee (J Photochem Photobiol B 104:258-270, 2011). In this case, simulations show that illumination induces a state 2 (s2) to state 1 (s1) transition (qT21), and a slow S-M rise in the simulated ChlFI curve, since the fluorescence yield is known to be higher in s1, when CS of PSII is larger than that of PSI. Additionally, we have analyzed how light intensity and several photosynthetic processes influence the degree of this qT21, and thus the relative amplitude of the simulated S-M phase. A refinement of the photosynthesis model is, however, necessary in order to obtain a better fit of the simulation data with the measured ChlFI curves.

Using chlorophyll (Chl) a fluorescence many aspects of the photosynthetic apparatus can be studied, both in vitro and, noninvasively, in vivo. Complementary techniques can help to interpret changes in the Chl a fluorescence kinetics. Kalaji et al. (Photosynth Res 122:121–158, 2014a) addressed several questions about instruments, methods and applications based on Chl a fluorescence. Here, additional Chl a fluorescence-related topics are discussed again in a question and answer format. Examples are the effect of connectivity on photochemical quenching, the correction of F V/F M values for PSI fluorescence, the energy partitioning concept, the interpretation of the complementary area, probing the donor side of PSII, the assignment of bands of 77 K fluorescence emission spectra to fluorescence emitters, the relationship between prompt and delayed fluorescence, potential problems when sampling tree canopies, the use of fluorescence parameters in QTL studies, the use of Chl a fluorescence in biosensor applications and the application of neural network approaches for the analysis of fluorescence measurements. The answers draw on knowledge from different Chl a fluorescence analysis domains, yielding in several cases new insights.

Chlorophyll a fluorescence is a highly sensitive, non-destructive, and reliable tool for measuring, rather quickly, photosynthetic efficiency, particularly of Photosystem II (PSII), the water-plastoquinone oxidoreductase. We briefly review here the connection between the fast (up to 2 s) chlorophyll fluorescence rise and PSII, as well as the empirical use of the fluorescence rise kinetics in understanding photosynthetic reactions, particularly of PSII. When dark-adapted photosynthetic samples are exposed to light, a fluorescence induction is observed, known as the Kautsky effect, after Hans Kautsky, the discoverer of the phenomenon showing the existence of variable fluorescence. The chlorophyll fluorescence intensity rises from a minimum level (the O level), in less than 1 s, to a maximum level (the P-level) via two intermediate steps labeled J and I. This is followed by a decline to a lower semi-steady state level, the S level, which is reached in about one minute. We provide here an educational review on how this phenomenon has been exploited through analysis of the fast OJIP fluorescence transient, by discussing basic assumptions, derivation of equations, as well as application to PSII-related questions.

To honor Academician Alexander Abramovitch Krasnovsky, we present here an educational review on the relation of chlorophyll a fluorescence transient to various processes in photosynthesis. The initial event in oxygenic photosynthesis is light absorption by chlorophylls (Chls), carotenoids, and, in some cases, phycobilins; these pigments form the antenna. Most of the energy is transferred to reaction centers where it is used for charge separation. The small part of energy that is not used in photochemistry is dissipated as heat or re-emitted as fluorescence. When a photosynthetic sample is transferred from dark to light, Chl a fluorescence (ChlF) intensity shows characteristic changes in time called fluorescence transient, the OJIPSMT transient, where O (the origin) is for the first measured minimum fluorescence level; J and I for intermediate inflections; P for peak; S for semi-steady state level; M for maximum; and T for terminal steady state level. This transient is a real signature of photosynthesis, since diverse events can be related to it, such as: changes in redox states of components of the linear electron transport flow, involvement of alternative electron routes, the build-up of a transmembrane pH gradient and membrane potential, activation of different nonphotochemical quenching processes, activation of the Calvin-Benson cycle, and other processes. In this review, we present our views on how different segments of the OJIPSMT transient are influenced by various photosynthetic processes, and discuss a number of studies involving mathematical modeling and simulation of the ChlF transient. A special emphasis is given to the slower PSMT phase, for which many studies have been recently published, but they are less known than on the faster OJIP phase.

The in vivo variable fluorescence of chlorophyll a is used as a non-intrusive probe of the physiological state of intact leaves. It was established that the origin of this signal is the antenna system of photosystem II (PSII). The PSII population is heterogeneous with respect to antenna size, connectivity, electron transport activity, and localization. In this paper we have simulated the effect of these heterogeneities on the fluorescence transient using a mathematical model based on the redox reactions from the acceptor side of PSII. We have concluded that the systems with heterogeneous antenna size and connectivity can be simulated acceptably with a homogeneous population of PSII centres having average values for antenna size and the connectivity constant C . However, this is not possible if there are inactive PSII centres in the system (the so-called non-Q B active centres). Finally, we have presented some possible applications of these types of simulations.

Chlorophyll a fluorescence induction is extensively used as a probe of photosynthesis, and thus, it has become necessary to quantitatively analyse it to extend its usefulness. We simulate the experimental data of fluorescence transients in strong light through numerical integration, both in dark- and light-adapted plants. In the mathematical model used here we have considered for the first time the redox reactions at both the acceptor and the donor sides of photosystem II, and the non-photochemical quenching by the oxidised plastoquinone molecules from the lipid matrix of the thylakoid membrane. The model is based on assumptions established in the literature and also the values of input parameters used in simulations. The simulated fluorescence induction curves show the characteristics O→J→I→P steps as in the experimental ones and, in specific conditions, the presence of a dip (D) between the I and P steps of the transient. Moreover, it has been shown here how typical patterns of fluorescence kinetics are influenced by the state of the sample by studying the basic effects of the influence of some parameters [i.e. the connectivity between different PS II units, initial Q B :Q B − ratio and the ratio of the starting states of the oxygen evolving complex (S 1 :S 2 ), number of plastoquinone molecules in the plastoquinone pool, initial redox state of the plastoquinone pool, and the rate of plastoquinol oxidation]. In this way the information can be drawn from the experimental curves relative to these parameters.

The variable fluorescence in the fast phase of chlorophyll a fluorescence induction phenomenon is closely related to the electron transport in photosystem II (PS II) of plants. We present here two theoretical models that simulate successfully the experimental data on the O - J - I - P fluorescence transient using PS II reactions. The “core” of both these proposed theoretical models are charge stabilization and the two-electron-gate process on the acceptor side of PS II. The data for dynamic analysis of the models were obtained through numerical integration with a specialized simulation software Gepasi. We have simulated the fluorescence transients under three different experimental conditions: (1) variation of the light intensity; (2) DCMU treatment; and (3) re-exposure of the samples to actinic light after a defined period of darkness. The simulations match well the “in vivo” experimental data that can be obtained directly in the greenhouse or in the field. Therefore, the simulations presented here can be used in the screening of agro-ecosystems, fertilization and nutrition of plants, greenhouse control and horticulture or generally, in the screening of photosynthesis and productivity.

The interactions of four nytroxyl spin labels with photosynthetic membranes (thylakoids and liposomes) have been investigated by the Electron Paramagnetic Resonance technique (EPR). The obtained data (shapes of EPR spectra and kinetics of light induced reactions) allow us to localize the interactions between the markers and photosynthetic membranes. The pH influence on the reaction kinetics has also been investigated. On the basis of these experimental data, a theoretical model of the interaction between spin labels and the photosynthetic electron transport chain is proposed.

The fast (up to 1 s) chlorophyll (Chl) a fluorescence induction (FI) curve, measured under saturating continuous light, has a photochemical phase, the O-J rise, related mainly to the reduction of Q(A), the primary electron acceptor plastoquinone of Photosystem II (PSII); here, the fluorescence rise depends strongly on the number of photons absorbed. This is followed by a thermal phase, the J-I-P rise, which disappears at subfreezing temperatures. According to the mainstream interpretation of the fast FI, the variable fluorescence originates from PSII antenna, and the oxidized Q(A) is the most important quencher influencing the O-J-I-P curve. As the reaction centers of PSII are gradually closed by the photochemical reduction of Q(A), Chl fluorescence, F, rises from the O level (the minimal level) to the P level (the peak); yet, the relationship between F and [Q(A) (-)] is not linear, due to the presence of other quenchers and modifiers. Several alternative theories have been proposed, which give different interpretations of the O-J-I-P transient. The main idea in these alternative theories is that in saturating light, Q(A) is almost completely reduced already at the end of the photochemical phase O-J, but the fluorescence yield is lower than its maximum value due to the presence of either a second quencher besides Q(A), or there is an another process quenching the fluorescence; in the second quencher hypothesis, this quencher is consumed (or the process of quenching the fluorescence is reversed) during the thermal phase J-I-P. In this review, we discuss these theories. Based on our critical examination, that includes pros and cons of each theory, as well mathematical modeling, we conclude that the mainstream interpretation of the O-J-I-P transient is the most credible one, as none of the alternative ideas provide adequate explanation or experimental proof for the almost complete reduction of Q(A) at the end of the O-J phase, and for the origin of the fluorescence rise during the thermal phase. However, we suggest that some of the factors influencing the fluorescence yield that have been proposed in these newer theories, as e.g., the membrane potential ΔΨ, as suggested by Vredenberg and his associates, can potentially contribute to modulate the O-J-I-P transient in parallel with the reduction of Q(A), through changes at the PSII antenna and/or at the reaction center, or, possibly, through the control of the oxidation-reduction of the PQ-pool, including proton transfer into the lumen, as suggested by Rubin and his associates. We present in this review our personal perspective mainly on our understanding of the thermal phase, the J-I-P rise during Chl a FI in plants and algae.