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

Abstract

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 Q_A⁽⁻⁾Q_B⁽⁻⁾ 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 k_D(t) was found to increase from the basic value k_Dconst, at the onset of illumination, to its maximal level k_Dvar due to lumenal acidification. In dark-adapted leaves, a low value of k_Dconst 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 k_D(t) and k_FNR(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.

Appendix

The Thylakoid model components

The interacting components of ETC comprise various redox states of electron carriers in a membrane protein complex (PSI, PSII, b₆f) or mobile electron carriers (Pc, Fd, PQ). The model dynamic variable X_i(t), calculated using the ODE system (1), describes (Belyaeva et al. 2016) the time-dependent relative fraction (population) of the ith component multiplied by the total concentration of the ETC complex/mobile carrier in the system. The lumenal and stromal concentrations of H⁺, K⁺ and Cl⁻ are also model dynamic variables. The stoichiometry of the system constituents in the thylakoid membrane (Fig. 2), for PSI, PSII, Cyt b₆f complexes and PQ, Pc, Fd electron carriers was assumed to be 1:1:1:10:3:4.

Modeling catalytic cycles of PSII, Cyt b₆f and PS I (see Fig. 10).

Fig. 10

Scheme of the catalytic cycle of the photosystem II. Each rectangle denotes a certain state of PSII, determined by the redox states of the PSII cofactors: $\left\langle {\begin{array}{*{20}{c}} {{\text{Chl}}} \\ {{\text{P680}}} \end{array}} \right\rangle$—chlorophyll pigments of antenna and RC P680 (singlet excited states ¹Chl* being delocalized on all pigments); Phe—pheophytin, the primary electron acceptor of PSII; Q_A and Q_B—primary and secondary quinone acceptors; PQ—plastoquinone; PQH₂—plastoquinol; H_L⁺ or H_S⁺—protons in lumen or stroma. The model variables (x_i, y_i, z_i, g_i, i = 1,…,7) are defined above the rectangles. Reaction numbers are denoted above the arrows. Dashed arcs show irreversible reactions of non-radiative recombination of Phe⁻ with P680^+· (42–45) and Q_A⁻ with P680^+· (46–49). States capable of emitting fluorescence quanta are shaded. The gray backward arrows (see details in Fig. 3) specify the sum of all deactivation processes of ¹Chl^* (except the photochemical quenching) given by Eqs. (2)–(5)

Fig. 11

Scheme of the measuring system. Induction curves of chlorophyll fluorescence and differential absorbance changes at 810–870 nm were measured simultaneously using a PEA fluorometer (Plant Efficiency Analyzer, Hansatech, UK) in combination with a PAM-101 measuring system. 1—multi-branch fiber-optic light guide 101-F5 (Walz); 2—ED-P700DW dual-wavelength emitter-detector unit (Walz); 3—dual-wavelength P700 control unit (ED-P700DW, Walz); 4—computer-controlled LED source of white light; 5—leaf sample placed on a mirror support; 6—output from PAM101 amplifier to an analog-digital converter (PCI-6024E A/D, National Instruments, USA); 7—a sensor unit of Plant Efficiency Analyzer with an array of six light-emitting diodes (providing red light with a peak wavelength of 650 nm); 8—control unit of Plant Efficiency Analyzer; 9—output from a two-channel AD converter to computer

PSII model description

The electron carriers of PSII are modeled in reduced/neutral states for the cofactors Phe⁻/Phe and Q_A⁻/Q_A. In addition, the reduced/excited/ oxidized states of the primary electron donor P680 coupled to antenna, Chl-P680/(Chl-P680)*/Chl-P680⁺, respectively, are taken into account. The Q_B -site can exist in four redox states (in Fig. 10: neutral, reduced, protonated, double reduced, empty). Hence, the PSII model network has 48 hypothetical states whereas the model scheme in Fig. 10 contains 28 redox states of the PSII RC. Together with two states of PQ in the PQ pool, a set of 30 X_i(t) components is described when forward/backward transfer steps are given with rate constants k_n/k_−n (n = 1–49). Reduction of model forms was explained (Lebedeva et al. 2000; Belyaeva et al. 2015, 2016) with respect to information about the primary photosynthetic reactions (Govindjee 1982; Renger and Schulze 1985; Baake and Schlöder 1992; Dau 1994; Renger et al. 1995; Stirbet et al. 1998; Lazár 2003).

Thus the concentrations ${{X_i}(t)}$ for the different PSII states:

$${\sum {{X_{{\text{PSII}} - i}}(t)} =1.6\,{\text{mM}} \cdot \sum {({x_i}(t)+{g_i}(t)+{y_i}(t)+{z_i}(t))} }$$

are related to the population probabilities with ${\sum {({x_i}(t)} +{g_i}(t)+{y_i}(t)+{z_i}(t))=1},\quad i=1, \ldots ,7.$

Figure 10 includes the light-induced reactions of the PSII model pattern: charge separation (2, 9, 16, 29), charge stabilization (3, 10, 17, 30), electron transfer from Q_A⁻Q_B⁽⁻⁾ to Q_AQ_B(H⁻) (7, 14), PQH₂ release (21–27) and refilling of the empty Q_B-site (34–40). The WOC operation (reactions 4, 11, 18, 31) was assumed with one proton released into the lumen for one electron, transferred from WOC via Y_Z to P680⁺⁺ averaging the S_i cycles stoichiometry (refs. in Renger 2004, 2012).

For reactions with numbers n = 1, 5, 8, 12, 15, 19, 28, 32 the text in “Updating the Thylakoid Membrane model” introduces Eqs. (2)–(5) explaining the entity of gray backward arrows in Figs. 3 and 10 that define the sum of deactivation processes of ¹Chl^* via FL emission and heat dissipation. Moreover, dissipative energy losses are defined for closed RCs. The irreversible reactions of non-radiative recombination are shown (Fig. 10) by dashed arcs as transitions 42–45 from the 7th (with Phe⁻P680⁺) to the 5th forms (x₇, g₇, y₇, z₇) → (x₅, g₅, y₅, z₅) and transitions 46–49 from the 4th (with Q_A⁻P680⁺) to the 1st forms (x₄, g₄, y₄, z₄) → (x₁, g₁, y₁, z₁).

The Cyt b₆f model was shown earlier in details (Lebedeva et al. 2002; Kamali et al. 2004; Rubin and Riznichenko 2009), including the set of redox reactions known as the Q cycle (Hope 1993; Tikhonov 2014). The reduced scheme in Fig. 4a shows transitions in two catalytic centers at the lumenal (p) Q_o or stromal (n) Q_i sites that provide proton coupled electron transfer (PCET) reactions that can oxidize/reduce PQ/PQH₂ molecules.

Light-induced electron flow via PQH₂ diffusion delivers electrons into the Q cycle (step 41, Figs. 4c, 10) while mobile Pc and b_L heme are reduced in parallel (Fig. 4a, steps 50, 51 for the transitions f₁ → f₂ and f₃ → f₄). Step 52, f₂ → f₃ across the membrane reduces the heme b_H and PQ reduction in the catalytic stromal center becomes possible (f₄ → f₂ and f₃ → f₁) to produce the semiquinone (steps 53, 54) and plastoquinone (steps 55, 56) species that are protonated by two protons consumed from the stroma.

The PSI model is shown in Fig. 4d. Because of the very rapid (10^− 12–10^− 9 s) ET along the A₀–A₁–F_X chain (Brettel 1997), only the final electron acceptor in PSI, shown as FeS (uniting clusters F_X, F_A, and F_B) in Fig. 4d, is considered. Under light, states with P700⁺FeS^r are generated while two ways are active via steps 61, 62 or vice versa by steps 64, 63 giving rise to P700⁺ reduction from Pc and ET from reduced FeS complex to Fd. Subsequently, at the PSI acceptor side, the ET outflow is supplied by Fd mobile carriers that participate in both LEF and CEF fluxes: step 65, and steps 58–59, respectively (Fig. 4b).