Comparative modeling of fluorescence and P700 induction kinetics for alga Scenedesmus sp. obliques and cyanobacterium Synechocystis sp. PCC 6803. Role of state 2-state 1 transitions and redox state of plastoquinone pool.

The model of thylakoid membrane system (T-M model) (Belyaeva et al. Photosynth Res 2019, 140:1-19) has been improved in order to analyze the induction data for dark-adapted samples of algal (Scenedesmus obliques) and cyanobacterial (Synechocystis sp. PCC 6803) cells. The fluorescence induction (FI) curves of Scenedesmus were measured at light exposures of 5 min, while FI and P700 redox transformations of Synechocystis were recorded in parallel for 100 s intervals. Kinetic data comprising the OJIP-SMT fluorescence induction and OABCDEF P700+ absorbance changes were used to study the processes underlying state transitions qT2→1 and qT1→2 associated with the increase/decrease in Chl fluorescence emission. A formula with the Hill kinetics (Ebenhöh et al. Philos Trans R Soc B 2014, 369:20130223) was introduced into the T-M model, with a new variable to imitate the flexible size of antenna AntM(t) associated with PSII. Simulations revealed that the light-harvesting capacity of PSII increases with a corresponding decrease for that of PSI upon the qT2→1 transition induced by plastoquinone (PQ) pool oxidation. The complete T-M model fittings were attained on Scenedesmus or Synechocystis fast waves OJIPS of FI, while SMT wave of FI was reproduced at intervals shorter than 5 min. Also the fast P700 redox transitions (OABC) for Synechocystis were fitted exactly. Reasonable sets of algal and cyanobacterial electron/proton transfer (ET/PT) parameters were found. In the case of Scenedesmus, ET/PT traits remained the same irrespective of modeling with or without qT2→1 transitions. Simulations indicated a high extent (20%) of the PQ pool reduction under dark conditions in Synechocystis compared to 2% in Scenedesmus.

Machine learning methods for assessing photosynthetic activity: environmental monitoring applications.

Monitoring of the photosynthetic activity of natural and artificial biocenoses is of crucial importance. Photosynthesis is the basis for the existence of life on Earth, and a decrease in primary photosynthetic production due to anthropogenic influences can have catastrophic consequences. Currently, great efforts are being made to create technologies that allow continuous monitoring of the state of the photosynthetic apparatus of terrestrial plants and microalgae. There are several sources of information suitable for assessing photosynthetic activity, including gas exchange and optical (reflectance and fluorescence) measurements. The advent of inexpensive optical sensors makes it possible to collect data locally (manually or using autonomous sea and land stations) and globally (using aircraft and satellite imaging). In this review, we consider machine learning methods proposed for determining the functional parameters of photosynthesis based on local and remote optical measurements (hyperspectral imaging, solar-induced chlorophyll fluorescence, local chlorophyll fluorescence imaging, and various techniques of fast and delayed chlorophyll fluorescence induction). These include classical and novel (such as Partial Least Squares) regression methods, unsupervised cluster analysis techniques, various classification methods (support vector machine, random forest, etc.) and artificial neural networks (multilayer perceptron, long short-term memory, etc.). Special aspects of time-series analysis are considered. Applicability of particular information sources and mathematical methods for assessment of water quality and prediction of algal blooms, for estimation of primary productivity of biocenoses, stress tolerance of agricultural plants, etc. is discussed.

Model quantification of the light-induced thylakoid membrane processes in Synechocystis sp. PCC 6803 in vivo and after exposure to radioactive irradiation.

Measurements of OJIP-SMT patterns of fluorescence induction (FI) in Synechocystis sp. PCC 6803 (Synechocystis) cells on a time scale up to several minutes were mathematically treated within the framework of thylakoid membrane (T-M) model (Belyaeva et al., Photosynth Res 140:1-19, 2019) that was renewed to account for the state transitions effects. Principles of describing electron transfer in reaction centers of photosystems II and I (PSII and PSI) and cytochrome b6f complex remained unchanged, whereas parameters for dissipative reactions of non-radiative charge recombination were altered depending on the oxidation state of QB-site (neutral, reduced by one electron, empty, reduced by two electrons). According to our calculations, the initial content of plastoquinol (PQH2) in the total quinone pool of Synechocystis cells adapted to darkness for 10 min ranged between 20 and 40%. The results imply that the PQ pool mediates photosynthetic and respiratory charge flows. The redistribution of PBS antenna units responsible for the increase of Chl fluorescence in cyanobacteria (qT2 → 1) upon state 2 → 1 transition or the fluorescence lowering (qT1 → 2) due to state 1 → 2 transition were described in the model by exponential functions. Parameters of dynamically changed effective cross section were found by means of simulations of OJIP-SMT patterns observed on Synechocystis cells upon strong (3000 µmol photons m-2s-1) and moderate (1000 µmol photons m-2s-1) actinic light intensities. The corresponding light constant values kLΣAnt = 1.2 ms-1 and 0.4 ms-1 define the excitation of total antenna pool dynamically redistributed between PSII and PSI reaction centers. Although the OCP-induced quenching of antenna excitation is not involved in the model, the main features of the induction signals have been satisfactorily explained. In the case of strong illumination, the effective cross section decreases by approximately 33% for irradiated Synechocystis cells as compared to untreated cells. Under moderate light, the irradiated Synechocystis cells showed in simulations the same cross section as the untreated cells. The thylakoid model renewed with state transitions description allowed simulation of fluorescence induction OJIP-SMT curves detected on time scale from microseconds to minutes.

Analyzing both the fast and the slow phases of chlorophyll a fluorescence and P700 absorbance changes in dark-adapted and preilluminated pea leaves using a Thylakoid Membrane model.

The dark-to-light transitions enable energization of the thylakoid membrane (TM), which is reflected in fast and slow (OJIPSMT or OABCDE) stages of fluorescence induction (FI) and P700 oxidoreduction changes (ΔA810). A Thylakoid Membrane model (T-M model), in which special emphasis has been placed on ferredoxin-NADP+-oxidoreductase (FNR) activation and energy-dependent qE quenching, was applied for quantifying the kinetics of FI and ΔA810. Pea leaves were kept in darkness for 15 min and then the FI and ΔA810 signals were measured upon actinic illumination, applied either directly or after a 10-s light pulse coupled with a subsequent 10-s dark interval. On the time scale from 40 µs to 30 s, the parallel T-M model fittings to both FI and ΔA810 signals were obtained. The parameters of FNR activation and the buildup of qE quenching were found to differ for dark-adapted and preilluminated leaves. At the onset of actinic light, photosystem II (PSII) acceptors were oxidized (neutral) after dark adaptation, while the redox states with closed and/or semiquinone QA(-)QB(-) forms were supposedly generated after preillumination, and did not relax within the 10 s dark interval. In qE simulations, a pH-dependent Hill relationship was used. The rate constant of heat losses in PSII antenna kD(t) was found to increase from the basic value kDconst, at the onset of illumination, to its maximal level kDvar due to lumenal acidification. In dark-adapted leaves, a low value of kDconst of ∼ 2 × 106 s-1 was found. Simulations on the microsecond to 30 s time scale revealed that the slow P-S-M-T phases of the fluorescence induction were sensitive to light-induced FNR activation and high-energy qE quenching. Thus, the corresponding time-dependent rate constants kD(t) and kFNR(t) change substantially upon the release of electron transport on the acceptor side of PSI and during the NPQ development. The transitions between the cyclic and linear electron transport modes have also been quantified in this paper.

Simulation of chlorophyll fluorescence rise and decay kinetics, and P700-related absorbance changes by using a rule-based kinetic Monte-Carlo method.

A model of primary photosynthetic reactions in the thylakoid membrane was developed and its validity was tested by simulating three types of experimental kinetic curves: (1) the light-induced chlorophyll a fluorescence rise (OJIP transients) reflecting the stepwise transition of the photosynthetic electron transport chain from the oxidized to the fully reduced state; (2) the dark relaxation of the flash-induced fluorescence yield attributed to the QA- oxidation kinetics in PSII; and (3) the light-induced absorbance changes near 820 or 705 nm assigned to the redox transitions of P700 in PSI. A model was implemented by using a rule-based kinetic Monte-Carlo method and verified by simulating experimental curves under different treatments including photosynthetic inhibitors, heat stress, anaerobic conditions, and very high light intensity.

Influence of pH and ionic strength on electrostatic properties of ferredoxin, FNR, and hydrogenase and the rate constants of their interaction.

Ferredoxin (Fd) protein transfers electrons from photosystem I (PSI) to ferredoxin:NADP+-reductase (FNR) in the photosynthetic electron transport chain, as well as other metabolic pathways. In some photosynthetic organisms including cyanobacteria and green unicellular algae under anaerobic conditions Fd transfers electrons not only to FNR but also to hydrogenase-an enzyme which catalyzes reduction of atomic hydrogen to H2. One of the questions posed by this competitive relationship between proteins is which characteristics of thylakoid stroma media allow switching of the electron flow between the linear path PSI-Fd-FNR-NADP+ and the path PSI-Fd-hydrogenase-H2. The study was conducted using direct multiparticle simulation approach. In this method protein molecules are considered as individual objects that experience Brownian motion and electrostatic interaction with the surrounding media and each other. Using the model we studied the effects of pH and ionic strength (I) upon complex formation between ferredoxin and FNR and ferredoxin and hydrogenase. We showed that the rate constant of Fd-FNR complex formation is constant in a wide range of physiologically significant pH values. Therefore it can be argued that regulation of FNR activity doesn't involve pH changes in stroma. On the other hand, in the model rate constant of Fd-hydrogenase interaction dramatically depends upon pH: in the range 7-9 it increases threefold. It may seem that because hydrogenase reduces protons it should be more active when pH is acidic. Apparently, regulation of hydrogenase's affinity to both her reaction partners (H+ and Fd) is carried out by changes in its electrostatic properties. In the dark, the protein is inactive and in the light it is activated and starts to interact with both Fd and H+. Therefore, we can conclude that in chloroplasts the rate of hydrogen production is regulated by pH through the changes in the affinity between hydrogenase and ferredoxin.

The role of electrostatic interactions in the process of diffusional encounter and docking of electron transport proteins.

Electrostatic interaction of plastocyanin and cytochrome f in the process of protein-protein complex formation was investigated by computer simulation methods. It was shown that long-range electrostatic interaction promotes energetically favorable mutual orientation of protein molecules at distances between their cofactors shorter than 5 nm. At distances shorter than 3 nm, these electrostatic interactions lead to a significantly detectable increase in the rate of convergence of the cofactors.

Thylakoid membrane model of the Chl a fluorescence transient and P700 induction kinetics in plant leaves.

A new Thylakoid model is presented, which describes in detail the electron/proton transfer reactions between membrane protein complexes including photosystems II and I (PSII, PSI), cytochrome (Cyt) b 6 f, mobile plastoquinone PQ pool in the thylakoid membrane, plastocyanin in lumen and ferredoxin in stroma, reduction of NADP via FNR and cyclic electron transfer. The Thylakoid model parameters were fitted both to Chl fluorescence induction data (FI) and oxido-reductions of P700 (ΔA 810) measured from 20 µs up to 20 s in pea leaves. The two-wave kinetics of FI and ΔA 810 (O(JI)PSM and OABCDE) were described quantitatively, provided that the values of membrane electrochemical potential components ΔΨ(t), pHL(t)/pHS(t) are in physiologically relevant ranges. The time courses on the time scale from nanoseconds to tens of seconds of oxido-reduction changes of ET components as well as concentrations of proton/ions (K+, Cl-) were calculated. We assume a low constant FNR activity over this period. Charge movements across the thylakoid membrane by passive leakage and active ATPase transport and proton buffer reactions are simulated. The dynamics of charge fluxes during photosynthetic induction under low light (PFD 200 µmol photons m-2 s-1) were analyzed. The initial wave of P700 oxidation within 20 ms during independent operation of PSI and PSII was followed after 50 ms by PSI donor-side reduction from reduced PQ pool via Cyt b 6 f site. The Cyt b 6 f reactions contribute to the stabilization of fluxes in the time range 1 s < t < 10 s. The detailed analysis of Chl a fluorescence at the PSM stage (t > 10 s) would need the investigation of FNR activation effect in order to explain the transitions between cyclic and linear electron transport.

[Analysis of Chlorophyll Fluorescence Transient by Spectral Multi-exponential Approximation].

The method for analysis of chlorophyll fluorescence transient using approximation of measured signal by multi-exponential series is described. Visualization of partial sums of this series allows us to find amplitudes and characteristic times of individual phases of fluorescence induction curve. This method gives more rigid criteria of phase identification instead of semi-empirical approach currently used. Applied to Chlamidomonas reinhardtii sulfur deprivation case, it shows efficiency in finding visually undistinguishable phases of fluorescence transient for early detection of stress.

[Identification of Intermediate States of Electron Transfer Proteins Plastocyanin and Cytochrome f in the Process of Diffusional Encounter].

The Brownian dynamics method is used for qualitative analysis of events leading to formation of a functionally active plastocyanin-cytochrome f complex. Intermediate states of this process are identified by density-based hierarchical clustering. Diffusive entrapment of plastocyanin by cytochrome f is a key point of the suggested putative scenario of protein-protein approaching. Mobility of plastocyanin is characterized for different values of protein-protein electrostatic interaction energy.

Modeling of the redox state dynamics in photosystem II of Chlorella pyrenoidosa Chick cells and leaves of spinach and Arabidopsis thaliana from single flash-induced fluorescence quantum yield changes on the 100 ns-10 s time scale.

The time courses of the photosystem II (PSII) redox states were analyzed with a model scheme supposing a fraction of 11-25 % semiquinone (with reduced [Formula: see text]) RCs in the dark. Patterns of single flash-induced transient fluorescence yield (SFITFY) measured for leaves (spinach and Arabidopsis (A.) thaliana) and the thermophilic alga Chlorella (C.) pyrenoidosa Chick (Steffen et al. Biochemistry 44:3123-3132, 2005; Belyaeva et al. Photosynth Res 98:105-119, 2008, Plant Physiol Biochem 77:49-59, 2014) were fitted with the PSII model. The simulations show that at high-light conditions the flash generated triplet carotenoid (3)Car(t) population is the main NPQ regulator decaying in the time interval of 6-8 µs. So the SFITFY increase up to the maximum level [Formula: see text]/F 0 (at ~50 µs) depends mainly on the flash energy. Transient electron redistributions on the RC redox cofactors were displayed to explain the SFITFY measured by weak light pulses during the PSII relaxation by electron transfer (ET) steps and coupled proton transfer on both the donor and the acceptor side of the PSII. The contribution of non-radiative charge recombination was taken into account. Analytical expressions for the laser flash, the (3)Car(t) decay and the work of the water-oxidizing complex (WOC) were used to improve the modeled P680(+) reduction by YZ in the state S 1 of the WOC. All parameter values were compared between spinach, A. thaliana leaves and C. pyrenoidosa alga cells and at different laser flash energies. ET from [Formula: see text] slower in alga as compared to leaf samples was elucidated by the dynamics of [Formula: see text] fractions to fit SFITFY data. Low membrane energization after the 10 ns single turnover flash was modeled: the ∆Ψ(t) amplitude (20 mV) is found to be about 5-fold smaller than under the continuous light induction; the time-independent lumen pHL, stroma pHS are fitted close to dark estimates. Depending on the flash energy used at 1.4, 4, 100 % the pHS in stroma is fitted to 7.3, 7.4, and 7.7, respectively. The biggest ∆pH difference between stroma and lumen was found to be 1.2, thus pH- dependent NPQ was not considered.

[Brownian dynamics simulations of protein-protein interactions in photosynthetic electron transport chain].

The application of Brownian dynamics for simulation of transient protein-protein interactions is reviewed. The review focuses on theoretical basics of Brownian dynamics method, its particular implementations, advantages and drawbacks of the method. The outlook for future development of Brownian dynamics-based simulation techniques is discussed. Special attention is given to analysis of Brownian dynamics trajectories. The second part of the review is dedicated to the role of Brownian dynamics simulations in studying photosynthetic electron transport. Interactions of mobile electron carriers (plastocyanin, cytochrome c6, and ferredoxin) with their reaction partners (cytochrome b6f complex, photosystem I, ferredoxin:NADP-reductase, and hydrogenase) are considered.

Modeling chlorophyll a fluorescence transient: relation to photosynthesis.

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.

Model based analysis of transient fluorescence yield induced by actinic laser flashes in spinach leaves and cells of green alga Chlorella pyrenoidosa Chick.

Measurements of Single Flash Induced Transient Fluorescence Yield (SFITFY) on spinach leaves and whole cells of green thermophilic alga Chlorella pyrenoidosa Chick were analyzed for electron transfer (ET) steps and coupled proton transfer (PT) on both the donor and the acceptor side of the reaction center (RC) of photosystem II (PS II). A specially developed PS II model (Belyaeva et al., 2008, 2011a) allowed the determination of ET steps that occur in a hierarchically ordered time scale from nanoseconds to several seconds. Our study demonstrates that our SFITFY data is consistent with the concept of the reduction of P680(+) by YZ in both leaves and algae (studied on spinach leaves and cells of Chlorella pyrenoidosa Chick). The multiphasic P680(+) reduction kinetics by YZ in PS II core complexes with high oxygen evolution capacity was seen in both algae and leaves. Model simulation to fit SFITFY curves for dark adapted species used here gives the rate constants to verify nanosecond kinetic stages of P680(+) reduction by YZ in the redox state S1 of the water oxidizing complex (WOC) shown in Kühn et al. (2004). Then a sequence of relaxation steps in the redox state S1, outlined by Renger (2012), occurs in both algae and leaves as a similar non-adiabatic ET reactions. Coupled PT is discussed briefly to understand a rearrangement of hydrogen bond protons in the protein matrix of the WOC (Umena et al., 2011). On the other hand, present studies showed a slower reoxidation of reduced QA by QB in algal cells as compared with that in a leaf that might be regarded as a consequence of differences of spatial domains at the QB-site in leaves compared to algae. Our comparative study helped to correlate theory with experimental data for molecular photosynthetic mechanisms in thylakoid membranes.

Study of the effect of reducing conditions on the initial chlorophyll fluorescence rise in the green microalgae Chlamydomonas reinhardtii.

Incubation of Chlamydomonas reinhardtii cells under nutrient deficiency results in the faster initial rise in the light-induced chlorophyll fluorescence kinetic curve. We showed that short-term anaerobic incubation of algal cells altered initial fluorescence in a way similar to nutrient starvation, suggesting an important role of the plastoquinones redox state in the observed effect. Bi-component analysis of highly resolved initial fluorescence rise kinetics in sulfur- or oxygen-depleted C. reinhardtii cells suggested that one of the mechanisms underlying the observed phenomenon involves primary closure (photochemical inactivation via Qa reduction) of ß-type PSII as compared to α-PSII. Moreover, results of modeling of the fluorescence curve brought us to the conclusion that accumulation of closed centers in α-PSII supercomplexes may also cause a faster initial fluorescence rise. The observed correlations between nutrient supply rate and initial fluorescence rise pattern in green algae can serve to characterize culture nutritional status in vivo.

PS II model based analysis of transient fluorescence yield measured on whole leaves of Arabidopsis thaliana after excitation with light flashes of different energies.

Our recently presented PS II model (Belyaeva et al., 2008) was improved in order to permit a consistent simulation of Single Flash Induced Transient Fluorescence Yield (SFITFY) traces that were earlier measured by Steffen et al. (2005) on whole leaves of Arabidopsis (A.) thaliana at four different energies of the actinic flash. As the essential modification, the shape of the actinic flash was explicitly taken into account assuming that an exponentially decaying rate simulates the time dependent excitation of PS II by the 10 ns actinic flash. The maximum amplitude of this excitation exceeds that of the measuring light by 9 orders of magnitude. A very good fit of the SFITFY data was achieved in the time domain from 100 ns to 10s for all actinic flash energies (the maximum energy of 7.5 × 10¹6 photons/(cm²flash) is set to 100%, the relative energies of weaker actinic flashes were of ∼8%, 4%, ∼1%). Our model allows the calculation and visualization of the transient PS II redox state populations ranging from the dark adapted state, via excitation energy and electron transfer steps induced by pulse excitation, followed by final relaxation into the stationary state eventually attained under the measuring light. It turned out that the rate constants of electron transfer steps are invariant to intensity of the actinic laser flash. In marked contrast, an increase of the actinic flash energy by more than two orders of magnitude from 5.4×10¹4 photons/(cm²flash) to 7.5×10¹6 photons/(cm²flash), leads to an increase of the extent of fluorescence quenching due to carotenoid triplet (³Car) formation by a factor of 14 and of the recombination reaction between reduced primary pheophytin (Phe(-)) and P680(+) by a factor of 3 while the heat dissipation in the antenna complex remains virtually constant. The modified PS II model offers new opportunities to compare electron transfer and dissipative parameters for different species (e.g. for the green algae and the higher plant) under varying illumination conditions.

Direct computer simulation of ferredoxin and FNR complex formation in solution.

Ferredoxin reduced by Photosystem I in light serves as an electron donor for the reduction of NADP(+) to NADPH, and this reaction is catalyzed by enzyme ferredoxin:NADP(+)-reductase (FNR). Kinetics and mechanisms of this reaction have been extensively studied experimentally by site-specific mutagenesis, laser flash photolysis and stopped-flow methods. We have applied a method of multiparticle computer simulation to study the effects of electrostatic interactions upon the reaction rate of Fd-FNR complex formation. Using the model we calculated rate constants of Fd-FNR complex formation for the wild-type proteins and some mutant forms of FNR at different values of ionic strength. Simulation revealed that electrostatic interactions play an important role in Fd-FNR complex formation and define its specificity.

PS II model-based simulations of single turnover flash-induced transients of fluorescence yield monitored within the time domain of 100 ns-10 s on dark-adapted Chlorella pyrenoidosa cells.

The set up described in Steffen et al. (Biochemistry 40:173-180, 2001) was used to monitor in the time domain from 100 ns to 10 s single turnover flash-induced transients of the normalized fluorescence yield (SFITFY) on dark-adapted cells of the thermophilic algae Chlorella pyrenoidosa Chick. Perfect data fit was achieved within the framework of a previously proposed model for the PS II reaction pattern (Lebedeva et al., Biophysics 47:968-980, 2002; Belyaeva et al., Biophysics 51:860-872, 2006) after its modification by taking into account nonradiative decay processes including nonphotochemical quenching due to time-dependent populations of P680(+*) and (3)Car. On the basis of data reported in the literature, a consistent set of rate constants was obtained for electron transfer at the donor and acceptor sides of PS II, pH in lumen and stroma, the initial redox state of plastoquinone pool and the rate of plastoquinone oxidation. The evaluation of the rate constant values of dissipative processes due to quenching by carotenoid triplets in antennae and P680(+*)Q(A)(-*) recombination as well as the initial state populations after excitation with a single laser flash are close to that outlined in (Steffen et al., Biochemistry 44:3123-3133, 2005a). The simulations based on the model of the PS II reaction pattern provide information on the time courses of population probabilities of different PS II states. We analyzed the maximum (F(m)(STF)) and minimum (F(0)) of the normalized FL yield dependence on the rate of the recombination processes (radiative and dissipative nonradiative) and of P680(+*) reduction. The developed PS II model provides a basis for theoretical comparative analyses of time-dependent fluorescence signals, observed at different photosynthetic samples under various conditions (e.g. presence of herbicides, other stress conditions, excitation with actinic pulses of different intensity, and duration).