Photosynthesis Research

, Volume 140, Issue 1, pp 1–19 | Cite as

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

  • N. E. BelyaevaEmail author
  • A. A. Bulychev
  • G. Yu. Riznichenko
  • A. B. Rubin
Original Article


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.


Chlorophyll a fluorescence yield Dissipative energy losses Electron transfer Model simulation Photosynthetic induction Photosystems I and II Proton transfer Transmembrane charge fluxes Non-photochemical quenching 




Cyt b6f

Cytochrome b6f complex


Cyclic electron flow (around PSI)


Electron transfer


Electron transport chain

Fd, Fdr







Minimal chlorophyll a fluorescence yield


Maximal chlorophyll a fluorescence yield (induced by multiturnover light pulses)

HL+, HS+

Protons in lumen (L), protons in stroma (S)


Nicotinamide adenine dinucleotide phosphate, oxidized form


Non-photochemical quenching (of the excited state of Chl a)


Photon flux density

Phe, Ph

Primary PSII electron acceptor, pheophytin

pHL, pHS

pH in lumen, in stroma








Photosystem II, Photosystem I


Proton transfer

P680, P680

Chlorophyll a acting as electron donor in PSII


Chlorophyll a acting as electron donor in PSI

QA and QB

Primary and secondary plastoquinone electron acceptors of PSII


Energy-dependent quenching


Reaction center (of PS II, or of PS I)


Water-oxidizing complex


Tyrosine 161 of the PS II D1 polypeptide


Electrical potential across the thylakoid membrane



This work was supported by the RFBR, Project No. 16-04-00318. We wish to thank Professor Wim J. Vredenberg (Wageningen University, The Netherlands) for thorough consideration of the manuscript and stimulating discussion. We are grateful to Reviewer 2 for critical reading and careful editing the manuscript. We thank Professor V. Z. Paschenko and Ph.D. I. V. Konyukhov (Biophysics Department, Moscow State University) for fruitful discussions.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Baake E, Schlöder JP (1992) Modelling the fast fluorescence rise of photosynthesis. Bull Math Biol 54:999–1021Google Scholar
  2. Baker NR (2008) Chlorophyll fluorescence: a probe of photosynthesis in vivo. Annu Rev Plant Biol 59:659–668Google Scholar
  3. Baker NR, Harbinson J, Kramer DM (2007) Determining the limitations and regulation of photosynthetic energy transduction in leaves. Plant Cell Environ 30:1107–1125Google Scholar
  4. Belyaeva NE, Lebedeva GV, Riznichenko GY (2003) Kinetic model of primary photosynthetic processes in chloroplasts. Modeling of thylakoid membranes electric potential. In: Riznichenko GY (eds) Mathematics computer education, vol 10. Progress-Traditsiya, Moscow, pp 263–276 (in Russian) Google Scholar
  5. Belyaeva NE, Schmitt F-J, Steffen R, Paschenko VZ, Riznichenko GY Chemeris YK, Renger G, Rubin AB (2008) 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. Photosynth Res 98:105–119Google Scholar
  6. Belyaeva NE, Bulychev AA, Riznichenko GY, Rubin AB (2011a) A model of photosystem II for the analysis of fast fluorescence rise in plant leaves. Biophysics 56(3):464–477Google Scholar
  7. Belyaeva NE, Schmitt F-J, Paschenko VZ, Riznichenko GY, Rubin AB, Renger G (2011b) PS II model based analysis of transient fluorescence yield measured on whole leaves of Arabidopsis thaliana after excitation with light flashes of different energies. BioSystems 103(2):188–195Google Scholar
  8. Belyaeva NE, Schmitt F-J, Paschenko VZ, Riznichenko GY, Rubin AB, Renger G (2014) Model based analysis of transient fluorescence yield induced by actinic laser flashes in spinach leaves and cells of green alga Chlorella pyrenoidosa Chick. Plant Physiol Biochem 77:49–59Google Scholar
  9. Belyaeva NE, Schmitt F-J, Paschenko VZ, Riznichenko GY, Rubin AB (2015) Modelling 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. Photosynth Res 125:123–140Google Scholar
  10. Belyaeva NE, Bulychev AA, Riznichenko GY, Rubin AB (2016) Thylakoid membrane model of the Chl a fluorescence transient and P700 induction kinetics in plant leaves. Photosynth Res 130:491–515Google Scholar
  11. Bernát G, Steinbach G, Kaňa R, Govindjee, Misra AN, Prášil O (2017) On the origin of the slow M–T chlorophyll a fluorescence decline in cyanobacteria: interplay of short-term light-responses. Photosynth Res. Google Scholar
  12. Brettel K (1997) Electron transfer and arrangement of the redox cofactors in photosystem I. Biochim Biophys Acta 1318(3):322–373Google Scholar
  13. Bulychev AA (2011) Induction changes in photosystems I and II in plant leaves upon modulation of membrane ion transport. Biochem (Mosc) Suppl Ser A Membr Cell Biol 5:335–342Google Scholar
  14. Bulychev AA, Vredenberg WJ (1999) Light-triggered electrical events in the thylakoid membrane of plant chloroplast. Physiol Plantarum 105:577–584Google Scholar
  15. Bulychev AA, Vredenberg WJ (2010) Induction kinetics of photosystem I—activated P700 oxidation in plant leaves and their dependence on pre-energization. Russ J Plant Physiol 57(5):599–608Google Scholar
  16. Bulychev AA, Cherkashin AA, Rubin AB (2010) Dependence of chlorophyll P700 redox transients during the induction period on the transmembrane distribution of protons in chloroplasts of pea leaves. Russ J Plant Physiol 57(1):20–27Google Scholar
  17. Bulychev AA, Osipov VA, Matorin DN, Vredenberg WJ (2013) Effects of far-red light on fluorescence induction in infiltrated pea leaves under diminished ∆pH and ∆φ components of the proton motive force. J Bioenerg Biomembr 45:37–45Google Scholar
  18. Carrillo N, Ceccarelli EA (2003) Open questions in ferredoxin-NADP+ reductase catalytic mechanism. Eur J Biochem 270:1900–1915Google Scholar
  19. Carrillo N, Lucero H, Vallejos RH (1981) Light modulation of chloroplast membrane-bound ferredoxin-NADP+ oxidoreductase. J Biol Chem 256:1058–1059Google Scholar
  20. Cruz JA, Sacksteder CA, Kanazawa A, Kramer DM (2001) Contribution of electric field (∆ψ) to steady-state transthylakoid proton motive force (pmf) in vivo and in vitro. Control of pmf parsing into ∆ψ and ∆pH by ionic strength. Biochemistry 40:1226–1237Google Scholar
  21. Cruz JA, Kanazawa A, Treff N, Kramer DM (2005) Storage of light-driven transthylakoid proton motive force as an electric field (∆ψ) under steady-state conditions in intact cells of Chlamydomonas reinhardtii. Photosynth Res 85:221–233Google Scholar
  22. Dau H (1994) Molecular mechanism and quantitative models of variable photosystem II fluorescence. Photochem Photobiol 60:1–23Google Scholar
  23. Demin OV, Westerhoff HV, Kholodenko BN (1998) Mathematical modeling of superoxide generation with the bc 1 complex of mitochondria. Biochemistry (Moscow) (6):634–649Google Scholar
  24. Ebenhöh O, Houwaart T, Lokstein H, Schlede S, Tirok K (2011) A minimal mathematical model of nonphotochemical quenching of chlorophyll fluorescence. Biosystems 103(2):196–204Google Scholar
  25. Ebenhöh O, Fucile G, Finazzi G, Rochaix JD, Goldschmidt-Clermont M (2014) Short-term acclimation of the photosynthetic electron transfer chain to changing light: a mathematical model. Philos Trans R Soc B 369:20130223Google Scholar
  26. Foyer CH, Lelandais M, Harbinson J (1992) Control of the quantum efficiencies of photosystems I and II, electron flow, and enzyme activation following dark-to-light transitions in pea leaves. Plant Physiol 99:979–986Google Scholar
  27. Foyer CH, Neukermans J, Queval G, Noctor G, Harbinson J (2012) Photosynthetic control of electron transport and the regulation of gene expression. J Exp Bot 63:1637–1661Google Scholar
  28. Gizzatkulov N, Klimov A, Lebedeva G, Demin O (2004) DBsolve7: new update version to develop and analyze models of complex biological systems. In: ISMB/ECCB conference, Glasgow, Scotland, UK, 31 July–5 August 2004.
  29. Govindjee (ed) (1982) Photosynthesis, vol 2. Academic Press, New YorkGoogle Scholar
  30. Harbinson J, Hedley CL (1993) Changes in P-700 oxidation during the early stages of the induction of photosynthesis. Plant Physiol 103:649–660Google Scholar
  31. Heldt HW, Werdan K, Milovancev M, Geller G (1973) Alkalinization of the chloroplast stroma caused by light-dependent proton flux into the thylakoid lumen. Biochim Biophys Acta 314:224–241Google Scholar
  32. Hope AB (1993) The chloroplast cytochrome bf complex: a critical focus on function. Biochim Biophys Acta 1143:1–22Google Scholar
  33. Joliot P, Joliot A (2002) Cyclic electron transfer in plant leaf. PNAS 99:10209–10214Google Scholar
  34. Junge W, Auslander W, McGeer A, Runge T (1979) The buffering capacity of the internal phase of thylakoids and the magnitude of the pH changes inside under flashing light. Biochim Biophys Acta 546:121–141Google Scholar
  35. Kamali MJ, Lebedeva GV, Demin OV, Beljaeva NE, Riznichenko GY, Rubin AB (2004) A kinetic model of the cytochrome bf complex with fitted parameters. Biophysics 49:1061–1068Google Scholar
  36. Klughammer C, Schreiber U (2016) Deconvolution of ferredoxin, plastocyanin, and P700 transmittance changes in intact leaves with a new type of kinetic LED array spectrophotometer. Photosynth Res 128:195–214. Google Scholar
  37. Kodru S, Malavath T, Devadasu E, Nellaepalli S, Stirbet A, Subramanyam R, Govindjee (2015) The slow S to M rise of chlorophyll a fluorescence induction reflects transition from state 2 to state 1 in the green alga Chlamydomonas reinhardtii. Photosynth Res 125(1–2):219–231Google Scholar
  38. Krause GH, Jahns P (2004) Non-photochemical energy dissipation determined by chlorophyll fluorescence quenching: characterization and function. In: Papageorgiou GC, Govindjee (ed) Chlorophyll a fluorescence: a signature of photosynthesis. Springer, Dordrecht, pp 463–495Google Scholar
  39. Kuvykin IV, Ptushenko VV, Vershubskii AV, Tikhonov AN (2011) Regulation of electron transport in C(3) plant chloroplasts in situ and in silico: short-term effects of atmospheric CO(2) and O(2). Biochim Biophys Acta 1807(3):336–347Google Scholar
  40. Laisk A, Walker DA (1989) A mathematical model of electron transport. Thermodynamic necessity for photosystem II regulation. Proc R Soc Lond B 237:417–444Google Scholar
  41. Laisk A, Eichelmann H, Oja V (2006) C3 photosynthesis in silico. Photosynth Res 90:45–66Google Scholar
  42. Lazár D (2003) Chlorophyll a fluorescence rise induced by high light illumination of dark-adapted plant tissue studied by means of a model of Photosystem II and considering Photosystem II heterogeneity. J Theor Biol 220:469–503Google Scholar
  43. Lazár D (2009) Modelling of light-induced chlorophyll a fluorescence rise (O-J-I-P transient) and changes in 820 nm-transmittance signal of photosynthesis. Photosynthetica 47(4):483–498Google Scholar
  44. Lebedeva GV, Belyaeva NE, Riznichenko GY, Rubin AB, Demin OV (2000) Kinetic model of photosystem II of higher green plants. Russ J Phys Chem 74:1702–1710Google Scholar
  45. Lebedeva GV, Belyaeva NE, Demin OV, Riznichenko GY, Rubin AB (2002) Kinetic model of primary photosynthetic processes in chloroplasts. Description of the fast phase of chlorophyll fluorescence induction under different light intensities. Biophysics 47:968–980Google Scholar
  46. Lyu H, Lazár D (2017) Modeling the light-induced electric potential difference (∆Ψ), the pH difference (∆pH) and the proton motive force across the thylakoid membrane in C3 leaves. J Theor Biol 413:11–23Google Scholar
  47. McDonald AE, Ivanov AG, Bode R, Maxwell DP, Rodermel SR, Huner NPA (2011) Flexibility in photosynthetic electron transport: the physiological role of plastoquinol terminal oxidase (PTOX). Biochim Biophys Acta 1807:954–967Google Scholar
  48. Mulo P, Medina M (2017) Interaction and electron transfer between ferredoxin–NADP+ oxidoreductase and its partners: structural, functional, and physiological implications. Photosynth Res 134:265–280Google Scholar
  49. Papageorgiou GC, Govindjee (eds) (2004) Chlorophyll a fluorescence: a signature of photosynthesis. Advances in photosynthesis and respiration, vol 19. Springer, DordrechtGoogle Scholar
  50. Papageorgiou GC, Govindjee (2011) Photosystem II fluorescence: slow changes—scaling from the past. J Photochem Photobiol B 104:258–270Google Scholar
  51. Papageorgiou GC, Tsimilli-Michael M, Stamatakis K (2007) The fast and slow kinetics of chlorophyll a fluorescence induction in plants, algae and cyanobacteria: a viewpoint. Photosynth Res 94:275–290Google Scholar
  52. Ptushenko VV, Zhigalova TV, Avercheva OV, Tikhonov AN (2018) Three phases of energy-dependent induction of P700 + and Chl a fluorescence in Tradescantia fluminensis leaves. Photosynth Res. Google Scholar
  53. Renger G (2004) Coupling of electron and proton transfer in oxidative water cleavage in photosynthesis. Biochim Biophys Acta 1655:195–204Google Scholar
  54. Renger G (2012) Mechanism of light induced water splitting. In Photosystem II of oxygen evolving photosynthetic organisms. Biochim Biophys Acta 1817:1164–1176Google Scholar
  55. Renger G, Schulze A (1985) Quantitative analysis of fluorescence induction curves in isolated spinach chloroplasts. Photobiochem Photobiophys 9:79–87Google Scholar
  56. Renger G, Eckert HJ, Bergmann A, Bernarding J, Liu B, Napiwotzki A, Reifarth F, Eichler HJ (1995) Fluorescence and spectroscopic studies on exciton trapping and electron transfer in photosystem II of higher plants. Aust J Plant Physiol 22:167–181Google Scholar
  57. Reynolds IA, Johnson EA, Tanford C (1985) Incorporation of membrane potential into theoretical analysis of electrogenic ion pumps. Proc Natl Acad Sci USA 82:6869–6873Google Scholar
  58. Rich PR (1988) A critical examination of the supposed variable proton stoichiometry of the chloroplast cytochrome b/f complex. Biochim Biophys Acta 932:33–42Google Scholar
  59. Roelofs TA, Lee CH, Holzwarth AR (1992) Global target analysis of picosecond chlorophyll fluorescence kinetic from pea chloroplasts. Biophys J 61:1147–1163Google Scholar
  60. Rubin A, Riznichenko GY (2009) Modeling of the primary processes in a photosynthetic membrane. In: Laisk A, Nedbal L, Govindjee (eds) Photosynthesis in silico: understanding complexity from molecules to ecosystems, advances in photosynthesis and respiration, vol 29. Springer, Dordrecht, pp 151–176Google Scholar
  61. Rutherford AW, Govindjee, Inoue Y (1984) Charge accumulation and photochemistry in leaves studied by thermoluminescence and delayed light emission. Proc Natl Acad Sci USA 81:1107–1111Google Scholar
  62. Satoh K (1982) Mechanism of photoactivation of electron transport in intact Bryopsis chloroplasts. Plant Physiol 70:1413–1416Google Scholar
  63. Schansker G, Strasser RJ (2005) Quantification of non-Q B-reducing centers in leaves using a far-red pre-illumination. Photosynth Res 84:145–151Google Scholar
  64. Schansker G, Srivastava A, Govindjee, Strasser RJ (2003) Characterization of the 820-nm transmission signal paralleling the chlorophyll a fluorescence rise (OJIP) in pea leaves. Funct Plant Biol 30:785–796Google Scholar
  65. Schansker G, Tóth SZ, Strasser RJ (2006) Dark-recovery of the Chl a fluorescence transient (OJIP) after light adaptation: the qT component of non-photochemical quenching is related to an activated photosystem I acceptor side. Biochim Biophys Acta 1757:787–797Google Scholar
  66. Schatz GH, Brock H, Holzwarth AR (1988) Kinetic and energetic model for the primary processes in photosystem II. Biophys J 54:397–405Google Scholar
  67. Semenov AYu, Cherepanov DA, Mamedov MD (2008) Electrogenic reactions and dielectric properties of photosystem II. Photosynth Res 98:121–130Google Scholar
  68. Steffen R, Eckert H-J, Kelly AA, Dörmann PG, Renger G (2005) Investigations on the reaction pattern of photosystem II in leaves from Arabidopsis thaliana by time-resolved fluorometric analysis. Biochemistry 44:3123–3132Google Scholar
  69. Stirbet A, Govindjee (2012) Chlorophyll a fluorescence induction: a personal perspective of the thermal phase, the J-I-P rise. Photosynth Res 113:15–61Google Scholar
  70. Stirbet A, Govindjee (2016) The slow phase of chlorophyll a fluorescence induction in silico: origin of the S-M fluorescence rise. Photosynth Res 130:193–213. Google Scholar
  71. Stirbet A, Govindjee, Strasser BJ, Strasser RJ (1998) Chlorophyll a fluorescence induction in higher plants: modeling and numerical simulation. J Theor Biol 193:131–151Google Scholar
  72. Stirbet A, Riznichenko GY, Rubin AB, Govindjee (2014) Modeling chlorophyll a fluorescence transient: relation to photosynthesis. Biochemistry 79:291–323Google Scholar
  73. Stirbet A, Lazár D, Kromdijk J, Govindjee (2018) Chlorophyll a fluorescence induction: can just a one-second measurement be used to quantify abiotic stress responses? Photosynthetica 56(1):86–104Google Scholar
  74. Takizawa K, Cruz JA, Kanazawa A, Kramer DM (2007) The thylakoid proton motive force in vivo. Quantitative, non-invasive probes, energetics, and regulatory consequences of light-induced pmf. Biochem Biophys Acta 1767:1233–1244. Google Scholar
  75. Talts E, Oja V, Rämma H, Rasulov B, Laisk AA (2007) Dark inactivation of ferredoxin-NADP reductase and cyclic electron flow under far-red light in sunflower leaves. Photosynth Res 94:109–120Google Scholar
  76. Tikhonov AN (2014) The cytochrome b6f complex at the crossroad of photosynthetic electron transport pathways. Plant Physiol Biochem 81:163–183Google Scholar
  77. Tikhonov AN (2015) Induction events and short-term regulation of electron transport in chloroplasts: an overview. Photosynth Res 125:65–94Google Scholar
  78. Tikhonov AN, Vershubskii AV (2017) Connectivity between electron transport complexes and modulation of photosystem II activity in chloroplasts. Photosynth Res 133:103–114Google Scholar
  79. Trouillard M, Shahbazi M, Moyet L, Rappaport F, Joliot P, Kuntz M, Finazzi G (2012) Kinetic properties and physiological role of the plastoquinone terminal oxidase (PTOX) in a vascular plant. Biochim Biophys Acta 1817:2140–2148. Google Scholar
  80. Tsimilli-Michael M, Stamatakis K, Papageorgiou GC (2009) Dark-to-light transition in Synechococcus sp. PCC 7942 cells studied by fluorescence kinetics assesses plastoquinone redox poise in the dark and photosystem II fluorescence component and dynamics during state 2 to state 1 transition. Photosynth Res 99(3):243–255Google Scholar
  81. van Kooten O, Snel JFH, Vredenberg WJ (1986) Photosynthetic free energy transduction to the electric potential changes across the thylakoid membrane. Photosynth Res 9:211–227Google Scholar
  82. Vredenberg WJ (2000) A 3-state model for energy trapping and fluorescence in PS II incorporating radical pair recombination. Biophys J 79:26–38Google Scholar
  83. Vredenberg WJ (2011) Kinetic analysis and mathematical modeling of primary photochemical and photoelectrochemical processes in plant photosystems. BioSystems 103:139–151Google Scholar
  84. Vredenberg WJ, Bulychev AA (2010) Photoelectrochemical control of the balance between cyclic- and linear electron transport in photosystem I. Algorithm for P700+ induction kinetics, Biochim. Biophys Acta 1797:1521–1532Google Scholar
  85. Walz D, Goldstein L, Avron M (1974) Determination and analysis of the buffer capacity of isolated chloroplasts in the light and in the dark. Eur J Biochem 47:403–407Google Scholar
  86. Zaks J, Amarnath K, Kramer DM, Niyogi KK, Fleming GR (2012) A kinetic model of rapidly reversible nonphotochemical quenching. PNAS 109:15757–15762Google Scholar
  87. Zhu XG, Wang Y, Ort DR, Long SP (2013) e-photosynthesis: a comprehensive dynamic mechanistic model of C3 photosynthesis: from light capture to sucrose synthesis. Plant Cell Environ 36:1711–1727Google Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • N. E. Belyaeva
    • 1
    Email author
  • A. A. Bulychev
    • 1
  • G. Yu. Riznichenko
    • 1
  • A. B. Rubin
    • 1
  1. 1.Department of Biophysics, Biology Faculty of the M.V. LomonosovMoscow State UniversityMoscowRussia

Personalised recommendations