Photosystem II (PSII) uses solar energy to oxidize water and delivers electrons for life on Earth. The photochemical reaction center of PSII is known to possess two stationary states. In the open state (PSIIO), the absorption of a single photon triggers electron-transfer steps, which convert PSII into the charge-separated closed state (PSIIC). Here, by using steady-state and time-resolved spectroscopic techniques on Spinacia oleracea and Thermosynechococcus vulcanus preparations, we show that additional illumination gradually transforms PSIIC into a light-adapted charge-separated state (PSIIL). The PSIIC-to-PSIIL transition, observed at all temperatures between 80 and 308 K, is responsible for a large part of the variable chlorophyll-a fluorescence (Fv) and is associated with subtle, dark-reversible reorganizations in the core complexes, protein conformational changes at noncryogenic temperatures, and marked variations in the rates of photochemical and photophysical reactions. The build-up of PSIIL requires a series of light-induced events generating rapidly recombining primary radical pairs, spaced by sufficient waiting times between these events-pointing to the roles of local electric-field transients and dielectric relaxation processes. We show that the maximum fluorescence level, Fm, is associated with PSIIL rather than with PSIIC, and thus the Fv/Fm parameter cannot be equated with the quantum efficiency of PSII photochemistry. Our findings resolve the controversies and explain the peculiar features of chlorophyll-a fluorescence kinetics, a tool to monitor the functional activity and the structural-functional plasticity of PSII in different wild-types and mutant organisms and under stress conditions.

ELI ALPS ELI HU Nonprofit Ltd Szeged Hungary

Faculty of Science University of Ostrava Ostrava Czech Republic

Institute of Plant Biology Biological Research Centre Szeged Hungary

Laboratoire de R�activit� de Surface UMR 7197 Sorbonne University Paris France

Photosynthesis Research Center Key Laboratory of Photobiology Institute of Botany Chinese Academy of Sciences Beijing China

Photosynthetic Research Unit Institute of Biophysics National Research Council of Italy Milano Italy

Research Institute for Interdisciplinary Science and Graduate School of Natural Science and Technology Okayama University Okayama Japan

Universit� Paris Saclay CEA CNRS Institute for Integrative Biology of the Cell 91191 Gif sur Yvette France

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Abgaryan GA, Christophorov LN, Goushcha AO, Holzwarth AR, Kharkyanen VN, Knox PP, Lukashev EA (1998) Effects of mutual influence of photoinduced electron transitions and slow structural rearrangements in bacterial photosynthetic reaction centers. J Biol Phys 24: 1–17 PubMed PMC

Akhtar P, Nowakowski PJ, Wang W, Do TN, Zhao S, Siligardi G, Garab G, Shen JR, Tan HS, Lambrev PH (2020) Spectral tuning of light-harvesting complex II in the siphonous alga Bryopsis corticulans and its effect on energy transfer dynamics. Biochim Biophys Acta Bioenerg 1861: 148191. PubMed

Andrizhiyevskaya EG, Chojnicka A, Bautista JA, Diner BA, van Grondelle R, Dekker JP (2005) Origin of the F685 and F695 fluorescence in photosystem II. Photosynth Res 84: 173–180 PubMed

�rsk�ld SP, Masters VM, Prince BJ, Smith PJ, Pace RJ, Krausz E (2003) Optical spectra of synechocystis and spinach photosystem II preparations at 1.7 K: identification of the D1-pheophytin energies and stark shifts. J Am Chem Soc 125: 13063–13074 PubMed

Barber J (2004) Engine of life and big bang of evolution: a personal perspective. Photosynth Res 80: 137. PubMed

Barber J, Melis A (1990) Quantum efficiency for the photoaccumulation of reduced pheophytin in Photosystem II. Biochim Biophys Acta 1020: 285–289

Berthomieu C, Nabedryk E, Mantele W, Breton J (1990) Characterization by FTIR spectroscopy of the photoreduction of the primary quinone acceptor QA in photosystem II. FEBS Lett 269: 363–367 PubMed

Butler WL (1978) Energy distribution in the photochemical apparatus of photosynthesis. Annu Rev Plant Physiol 29: 345–378

Butler WL, Okayama S (1971) The photoreduction of C550 in chloroplasts and its inhibition by lipase. Biochim Biophys Acta 245: 237–239 PubMed

B�chel C, Garab G (1995) Electrochromic absorbency changes in the chlorophyll-c-containing alga Pleurochloris-Meiringensis (Xanthophyceae). Photosynth Res 43: 49–56 PubMed

Caffarri S, Broess K, Croce R, van Amerongen H (2011) Excitation energy transfer and trapping in higher plant Photosystem II complexes with different antenna sizes. Biophys J 100: 2094–2103 PubMed PMC

Cardona T, Sedoud A, Cox N, Rutherford AW (2012) Charge separation in photosystem II: a comparative and evolutionary overview. Biochim Biophys Acta Bioenerg 1817: 26–43 PubMed

Ceppi MG, Oukarroum A, Cicek N, Strasser RJ, Schansker G (2012) The IP amplitude of the fluorescence rise OJIP is sensitive to changes in the photosystem I content of leaves: a study on plants exposed to magnesium and sulfate deficiencies, drought stress and salt stress. Physiol Plant 144: 277–288 PubMed

Chmeliov J, Trinkunas G, van Amerongen H, Valkunas L (2014) Light harvesting in a fluctuating antenna. J Am Chem Soc 136: 8963–8972 PubMed

Chylla RA, Garab G, Whitmarsh J (1987) Evidence for slow turnover in a fraction of photosystem II complexes in thylakoid membranes. Biochim Biophys Acta 894: 562–571

Connolly JS, Samuel EB, Janzen AF (1982) Effects of solvent on the fluorescence properties of bacteriochlorophyll a. Photochem Photobiol 36: 565–574

Cseh Z, Rajagopal S, Tsonev T, Busheva M, Papp E, Garab G (2000) Thermooptic effect in chloroplast thylakoid membranes. Thermal and light stability of pigment arrays with different levels of structural complexity. Biochemistry 39: 15250–15257 PubMed

Dau H, Sauer K (1992) Electric-field effect on the picosecond fluorescence of Photosystem-II and its relation to the energetics and kinetics of primary charge separation. Biochim Biophys Acta 1102: 91–106

Delosme R (1967) Study of the induction of fluorescence in green algae and chloroplasts at the onset of an intense illumination. Biochim Biophys Acta 143: 108–128 PubMed

Demmig-Adams B, Stewart JJ, Burch TA, Adams WW 3rd (2014) Insights from placing photosynthetic light harvesting into context. J Phys Chem Lett 5: 2880–2889 PubMed

Duysens LMN, Sweers HE (1963) Studies on Microalgae and Photosynthetic Bacteria. Japanese Society of Plant Physiologists, University of Tokyo Press, Tokyo

Duysens LN (1978) Transfer and trapping of excitation energy in photosystem II. Ciba Found Symp 61: 323–340 PubMed

Farooq S, Chmeliov J, Wientjes E, Koehorst R, Bader A, Valkunas L, Trinkunas G, van Amerongen H (2018) Dynamic feedback of the photosystem II reaction centre on photoprotection in plants. Nat Plants 4: 225–231 PubMed

Feyziyev Y, De�k Z, Styring S, Bern�t G (2013) Electron transfer from Cyt b559 and tyrosine-D to the S2 and S3 states of the water oxidizing complex in photosystem II at cryogenic temperatures. J Bioenerg Biomembr 45: 111–120 PubMed

France LL, Geacintov NE, Breton J, Valkunas L (1992) The dependence of the degrees of sigmoidicities of fluorescence induction curves in spinach-chloroplasts on the duration of actinic pulses in pump-probe experiments. Biochim Biophys Acta 1101: 105–119

Fried SD, Boxer SG (2017) Electric fields and enzyme catalysis. Annu Rev Biochem 86: 387–415 PubMed PMC

Genty B, Briantais JM, Baker NR (1989) The relationship between the quantum yield of photosynthetic electron-transport and quenching of chlorophyll fluorescence. Biochim Biophys Acta 990: 87–92

Hodges M, Moya I (1986) Time-resolved chlorophyll fluorescence studies of photosynthetic membranes - resolution and characterization of 4 kinetic components. Biochim Biophys Acta 849: 193–202

Holzwarth AR, Wendler J, Haehnel W (1985) Time-resolved picosecond fluorescence-spectra of the antenna chlorophylls in chlorella-vulgaris - resolution of Photosystem-I fluorescence. Biochim Biophys Acta 807: 155–167

Hou JM, Boichenko VA, Diner BA, Mauzerall D (2001) Thermodynamics of electron transfer in oxygenic photosynthetic reaction centers: volume change, enthalpy, and entropy of electron-transfer reactions in manganese-depleted photosystem II core complexes. Biochemistry 40: 7117–7125 PubMed

Ikegami I, Katoh S (1973) Studies on chlorophyll fluorescence in chloroplasts II. Effect of ferricyanide on the induction of fluorescence in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylure. Plant Cell Physiol 14: 829–836

Joliot A, Joliot P (1964) �tude cin�tique de la r�action photochimique lib�rant l’oxyg�ne au cours de la photosynth�se. CR Acad Sci Paris 258: 4622–4625 PubMed

Joliot P, Joliot A (1979) Comparative study of the fluorescence yield and of the C550 absorption change at room temperature. Biochim Biophys Acta 546: 93–105 PubMed

K�lm�n L, Mar�ti P (1997) Conformation-activated protonation in reaction centers of the photosynthetic bacterium Rhodobacter sphaeroides. Biochemistry 36: 15269–15276 PubMed

Kawakami K, Shen JR (2018) Purification of fully active and crystallizable photosystem II from thermophilic cyanobacteria. Methods Enzymol 613: 1–16 PubMed

Kern J, Alonso-Mori R, Tran R, Hattne J, Gildea RJ, Echols N, Glockner C, Hellmich J, Laksmono H, Sierra RG, et al. (2013) Simultaneous femtosecond X-ray spectroscopy and diffraction of Photosystem II at room temperature. Science 340: 491–495 PubMed PMC

Kleinfeld D, Okamura MY, Feher G (1984) Electron-transfer kinetics in photosynthetic reaction centers cooled to cryogenic temperatures in the charge-separated state: evidence for light-induced structural changes. Biochemistry 23: 5780–5786 PubMed

Klimov VV, Klevanik AV, Shuvalov VA, Kransnovsky AA (1977) Reduction of pheophytin in the primary light reaction of photosystem II. FEBS Lett 82: 183–186 PubMed

Knox PP, Venediktov PS, Kononenko AA, Garab GI, Faludidaniel A (1984) Role of electric polarization in the thermo-luminescence of chloroplasts. Photochem Photobiol 40: 119–125

Koike H, Inoue Y (1983) Preparation of oxygen-evolving photosystem II particles from a thermophilic blue-green alga. InInoue Y, Crofts AR, Govindjee, Murata N, Renger G, Satoh K, eds, The Oxygen Evolving System of Photosynthesis. San Diego, CA: Academic Press, pp 257–263

Kupitz C, Basu S, Grotjohann I, Fromme R, Zatsepin NA, Rendek KN, Hunter MS, Shoeman RL, White TA, Wang D, et al. (2014) Serial time-resolved crystallography of photosystem II using a femtosecond X-ray laser. Nature 513: 261–265 PubMed PMC

Laisk A, Oja V (2020) Variable fluorescence of closed photochemical reaction centers. Photosynth Res 143: 335–346 PubMed

Lavergne J, Trissl HW (1995) Theory of fluorescence induction in Photosystem-II - derivation of analytical expressions in a model including exciton-radical-pair equilibrium and restricted energy-transfer between photosynthetic units. Biophys J 68: 2474–2492 PubMed PMC

Magyar M, Sipka G, Kov�cs L, Ughy B, Zhu Q, Han G, Špunda V, Lambrev PH, Shen JR, Garab G (2018) Rate-limiting steps in the dark-to-light transition of Photosystem II - revealed by chlorophyll-a fluorescence induction. Sci Rep 8: 2755. PubMed PMC

Malferrari M, Mezzetti A, Francia F, Venturoli G (2013) Effects of dehydration on light-induced conformational changes in bacterial photosynthetic reaction centers probed by optical and differential FTIR spectroscopy. Biochim Biophys Acta Bioenerg 1827: 328–339 PubMed

Mezzetti A, Leibl W (2017) Time-resolved infrared spectroscopy in the study of photosynthetic systems. Photosynth Res 131: 121–144 PubMed

Mezzetti A, Nabedryk E, Breton J, Okamura MY, Paddock ML, Giacometti G, Leibl W (2002) Rapid-scan Fourier transform infrared spectroscopy shows coupling of GLu-L212 protonation and electron transfer to QB in Rhodobacter sphaeroides reaction centers. Biochim Biophys Acta 1553: 320–330 PubMed

Nabedryk E, Bagley KA, Thibodeau DL, Bauscher M, M�ntele W, Breton J (1990) A protein conformational change associated with the photoreduction of the primary and secondary quinones in the bacterial reaction center. FEBS Lett 266: 59–62 PubMed

Nagy L, Mar�ti P, Terazima M (2008) Spectrally silent light induced conformation change in photosynthetic reaction centers. FEBS Lett 582: 3657–3662 PubMed

Nakanishi M, Sokolov AP (2015) Dielectric spectroscopy of hydrated biomacromolecules. In V Raicu, Y Feldman, eds, Dielectric Relaxation in Biological Systems. Oxford University Press, Oxford

Nelson N, Yocum CF (2006) Structure and function of photosystems I and II. Annu Rev Plant Biol 57: 521–565 PubMed

Nematov S, Casazza AP, Remelli W, Khuvondikov V, Santabarbara S (2017) Spectral dependence of irreversible light-induced fluorescence quenching: chlorophyll forms with maximal emission at 700-702 and 705-710nm as spectroscopic markers of conformational changes in the core complex. Biochim Biophys Acta Bioenerg 1858: 529–543 PubMed

Noguchi T (2007) Light-induced FTIR difference spectroscopy as a powerful tool toward understanding the molecular mechanism of photosynthetic oxygen evolution. Photosynth Res 91: 59–69 PubMed

Onoda K, Mino H, Inoue Y, Noguchi T (2000) An FTIR study on the structure of the oxygen-evolving Mn-cluster of Photosystem II in different spin forms of the S2 state. Photosynth Res 63: 47–57 PubMed

Papageorgiou GC, Govindjee (2004) Chlorophyll a Fluorescence: A Signature of Photosynthesis. Springer, Dordrecht, The Netherlands

Remelli W, Santabarbara S (2018) Excitation and emission wavelength dependence of fluorescence spectra in whole cells of the cyanobacterium Synechocystis sp. PPC6803: influence on the estimation of Photosystem II maximal quantum efficiency. Biochim Biophys Acta Bioenerg 1859: 1207–1222 PubMed

Rizzo F, Zucchelli G, Jennings R, Santabarbara S (2014) Wavelength dependence of the fluorescence emission under conditions of open and closed Photosystem II reaction centres in the green alga Chlorella sorokiniana. Biochim Biophys Acta Bioenerg 1837: 726–733 PubMed

Roelofs TA, Lee CH, Holzwarth AR (1992) Global target analysis of picosecond chlorophyll fluorescence kinetics from pea-chloroplasts - a new approach to the characterization of the primary processes in Photosystem-II alpha-units and beta-units. Biophys J 61: 1147–1163 PubMed PMC

Romero E, Novoderezhkin VI, van Grondelle R (2017) Quantum design of photosynthesis for bio-inspired solar-energy conversion. Nature 543: 355–365 PubMed

Ruban AV (2016) Nonphotochemical chlorophyll fluorescence quenching: mechanism and effectiveness in protecting plants from photodamage. Plant Physiol 170: 1903–1916 PubMed PMC

Schansker G, Toth SZ, Strasser RJ (2005) Methylviologen and dibromothymoquinone treatments of pea leaves reveal the role of photosystem I in the Chl a fluorescence rise OJIP. Biochim Biophys Acta 1706: 250–261 PubMed

Schansker G, T�th SZ, Holzwarth AR, Garab G (2014) Chlorophyll a fluorescence: beyond the limits of the QA model. Photosynth Res 120: 43–58 PubMed

Schansker G, T�th SZ, Kov�cs L, Holzwarth AR, Garab G (2011) Evidence for a fluorescence yield change driven by a light-induced conformational change within photosystem II during the fast chlorophyll a fluorescence rise. Biochim Biophys Acta Bioenerg 1807: 1032–1043 PubMed

Shen J-R (1998) Possible functional differences between dimer and monomer of Photosystem II complex. InGarab G, ed, Photosynthesis: Mechanisms and Effects: Volume I–V: Proceedings of the XIth International Congress on Photosynthesis, Budapest, Hungary, 17–22 August 1998, pp. 941–944

Shen JR, Inoue Y (1993) Binding and functional-properties of two new extrinsic components, cytochrome c-550 and a 12-kDa Protein, in cyanobacterial photosystem II. Biochemistry 32: 1825–1832 PubMed

Shen JR, Kamiya N (2000) Crystallization and the crystal properties of the oxygen-evolving photosystem II from Synechococcus vulcanus. Biochemistry 39: 14739–14744 PubMed

Shen JR, Kawakami K, Koike H (2011) Purification and crystallization of oxygen-evolving photosystem II core complex from thermophilic cyanobacteria. Methods Mol Biol 684: 41–51 PubMed

Shibata Y, Nishi S, Kawakami K, Shen JR, Renger T (2013) Photosystem II does not possess a simple excitation energy funnel: time-resolved fluorescence spectroscopy meets theory. J Am Chem Soc 135: 6903–6914 PubMed PMC

Shlyk-Kerner O, Samish I, Kaftan D, Holland N, Sai PSM, Kless H, Scherz A (2006) Protein flexibility acclimatizes photosynthetic energy conversion to the ambient temperature. Nature 442: 827–830 PubMed

Sipka G, Kis M, Mar�ti P (2018) Characterization of mercury(II)-induced inhibition of photochemistry in the reaction center of photosynthetic bacteria. Photosynth Res 136: 379–392 PubMed

Sipka G, M�ller P, Brettel K, Magyar M, Kov�cs L, Zhu QJ, Xiao YA, Han GY, Lambrev PH, Shen JR, et al. (2019) Redox transients of P680 associated with the incremental chlorophyll-a fluorescence yield rises elicited by a series of saturating flashes in diuron-treated photosystem II core complex of Thermosynechococcus vulcanus. Physiol Plant 166: 22–32 PubMed

Stirbet A (2013) Excitonic connectivity between photosystem II units: what is it, and how to measure it? Photosynth Res 116: 189–214 PubMed

Stirbet A, Govindjee (2012) Chlorophyll a fluorescence induction: a personal perspective of the thermal phase, the J-I-P rise. Photosynth Res 113: 15–61 PubMed

Strasser RJ, Tsimilli-Michael M, Srivastava A (2004) Analysis of the chlorophyll a fluorescence transient. InPapageorgiou GC, Govindjee, eds, Chlorophyll a Fluorescence: A Signature of Photosynthesis. Springer, Dordrecht, The Netherlands, pp 463–495

Suga M, Akita F, Sugahara M, Kubo M, Nakajima Y, Nakane T, Yamashita K, Umena Y, Nakabayashi M, Yamane T, et al. (2017) Light-induced structural changes and the site of O=O bond formation in PSII caught by XFEL. Nature 543: 131–135 PubMed

Szczepaniak M, Sander J, Nowaczyk M, M�ller MG, R�gner M, Holzwarth AR (2009) Charge separation, stabilization, and protein relaxation in photosystem II core particles with closed reaction center. Biophys J 96: 621–631 PubMed PMC

Tracewell CA, Brudvig GW (2008) Multiple redox-active chlorophylls in the secondary electron-transfer pathways of oxygen-evolving Photosystem II. Biochemistry 47: 11559–11572 PubMed PMC

Treves H, Raanan H, Kedem I, Murik O, Keren N, Zer H, Berkowicz SM, Giordano M, Norici A, Shotland Y, et al. (2016) The mechanisms whereby the green alga Chlorella ohadii, isolated from desert soil crust, exhibits unparalleled photodamage resistance. New Phytol 210: 1229–1243 PubMed

Tyystjarvi E, Vass I (2004) Light emission as a probe of charge separation and recombination in the photosynthetic apparatus: relation of prompt fluorescence to delayed light emission and thermoluminescence. InPapageorgiou GC, Govindjee, eds, Chlorophyll a Fluorescence. Advances in Photosynthesis and Respiration. Springer, Dordrecht, The Netherlands, pp 363–388

van Amerongen H, Croce R (2013) Light harvesting in Photosystem II. Photosynth Res 116: 251–263 PubMed PMC

van der Weij-de Wit CD, Dekker JP, van Grondelle R, van Stokkum IHM (2011) Charge separation is virtually irreversible in Photosystem II core complexes with oxidized primary quinone acceptor. J Phys Chem A 115: 3947–3956 PubMed

Vavilin DV, Ermakova-Gerdes SY, Keilty AT, Vermaas WF (1999) Tryptophan at position 181 of the D2 protein of photosystem II confers quenching of variable fluorescence of chlorophyll: implications for the mechanism of energy-dependent quenching. Biochemistry 38: 14690–14696 PubMed

Vredenberg W (2011) Kinetic analyses and mathematical modeling of primary photochemical and photoelectrochemical processes in plant photosystems. Biosystems 103: 138–151 PubMed

Witt HT (1979) Energy-conversion in the functional membrane of photosynthesis - analysis by light-pulse and electric pulse methods - central role of the electric-field. Biochim Biophys Acta 505: 355–427 PubMed

Zim�nyi L, Garab G (1989) Configuration of the electric-field and distribution of ions in energy transducing biological membranes: model calculations in a vesicle containing discrete charges. J Theor Biol 138: 59–76

Letter to the Editor

Letter to the Editor

Role of isotropic lipid phase in the fusion of photosystem II membranes

Letter to the Editor

Revisiting the nonregulatory, constitutive nonphotochemical quenching of the absorbed light energy in oxygenic photosynthetic organisms

Effects of lipids on the rate-limiting steps in the dark-to-light transition of Photosystem II core complex of Thermostichus vulcanus

Editorial

Monitoring the photosynthetic activity at single-cell level in Haematococcus lacustris

In appreciation of an ingenious scientist and a great friend: Győző Garab

Characterization of the Rate-Limiting Steps in the Dark-To-Light Transitions of Closed Photosystem II: Temperature Dependence and Invariance of Waiting Times during Multiple Light Reactions

Differences in susceptibility to photoinhibition do not determine growth rate under moderate light in batch or turbidostat - a study with five green algae

Light-induced reversible reorganizations in closed Type II reaction centre complexes: physiological roles and physical mechanisms

Insights on the regulation of photosynthesis in pea leaves exposed to oscillating light

Structural Entities Associated with Different Lipid Phases of Plant Thylakoid Membranes-Selective Susceptibilities to Different Lipases and Proteases

Dependence of the rate-limiting steps in the dark-to-light transition of photosystem II on the lipidic environment of the reaction center

An open Internet of Things (IoT)-based framework for feedback control of photosynthetic activities

Light quality, oxygenic photosynthesis and more

Photosynthesis dynamics and regulation sensed in the frequency domain

Lipid Polymorphism of the Subchloroplast-Granum and Stroma Thylakoid Membrane-Particles. II. Structure and Functions