Mechanisms of the plastoquinone pool oxidation in the dark after illumination

doi:10.21203/rs.3.rs-3025337/v1

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Mechanisms of the plastoquinone pool oxidation in the dark after illumination

https://doi.org/10.21203/rs.3.rs-3025337/v1

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Oxidation of the PQ-pool in leaves, intact chloroplasts and isolated thylakoids after its reduction by flash of light intensity 3000 µmol photons m^− 2 s^− 1 for 1.5 s was studied as a time-dependence in the dark of area above curve of fluorescence, A_fl. The fluorescence was excited with the same flash applied after dark intervals from 0.1 s to 10 min. The samples were either dark-adapted or pre-illuminated with a continuous light. The results showed a two-phase kinetic of A_fl across all samples, consistent with prior findings in thylakoids without added electron acceptors and in leaves. A comprehensive analysis of the kinetics in isolated thylakoids conducted under both aerobic and anaerobic conditions, with or without Fd and NADP⁺, and in the presence of catalase, enabled the proposition of mechanisms responsible for the observed two phases. Our findings suggest that the “fast” phase is likely a combination of residual electron flow from the PQ-pool to downstream acceptors and the oxidation of PQH₂ by the superoxide radical, formed during illumination and preserved in thylakoid membrane. The "slow" phase involves the oxidation of the PQ-pool through slow autocatalytic reactions with molecular oxygen, and this oxidation is enhanced in the presence of hydrogen peroxide. The role of the above processes in intact chloroplasts and leaves is discussed.

The plastoquinone pool (PQ-pool) serves as a central link in the photosynthetic electron transport chain (PETC) of higher plant chloroplasts. Molecules within the pool act as mobile electron carriers between Photosystem II (PS II) and the cytochrome b₆f complex in thylakoid membranes during linear electron transport. They also participate in cyclic electron transport around Photosystem I (PS I) and in chlororespiration. In the course of electron transport, they simultaneously transport protons from the chloroplast stroma into the thylakoid lumen, contributing to the building of a proton gradient across the thylakoid membrane. In addition to electron transport, the plastoquinone pool performs an antioxidant function, preventing PETC damage by reactive oxygen species (ROS), such as the superoxide anion-radical (Mubarakshina et al. 2006; Vetoshkina et al. 2017; Borisova-Mubarakshina et al. 2018), and singlet oxygen (Kruk and Trebst 2008; Kruk and Szymańska 2012, 2021; Ferretti et al. 2018). It has been suggested that the PQ-pool may interact with hydrogen peroxide (Khorobrykh and Tyystjärvi 2018; Borisova‐Mubarakshina et al. 2018). A wealth of evidence suggests that PQ-pool’s redox state plays a role in activating signal pathways aimed at plant adaptation to environmental conditions (Escoubas et al. 1995; Yang et al. 2001; Maciejewska et al. 2002; Foyer et al. 2012; Borisova-Mubarakshina et al. 2015). Therefore, the redox state of the PQ-pool affects many key functions of chloroplasts and, more broadly, photosynthesizing plant cells. In this context, elucidating the mechanisms of oxidation of the reduced PQ-pool under various functioning conditions is of evident importance.

Information about the oxidation processes of a reduced PQ-pool in the light and their mechanisms can be derived from studying these processes after illumination cessation. Several studies (McCauley and Melis 1986; Nixon and Rich 2006; Ivanov et al. 2007) have demonstrated that the kinetics of PQ-pool oxidation in the dark is biphasic, implying the involvement of several processes in the oxidation. In the studies (McCauley and Melis 1986; Nixon and Rich 2006), the redox state of the PQ-pool was assessed using the area above the chlorophyll fluorescence induction curve, while in the study (Ivanov et al. 2007), the appearance of oxidized plastoquinone molecules was spectrophotometrically measured. The main conclusion of the study (Nixon and Rich 2006), conducted with wild-type Arabidopsis leaves and IMMUTANS mutants, was that PTOX is not responsible for the biphasic nature of PQ-pool reduction, but detailed investigation of the mechanisms underlying the biphasic nature was not conducted. The studies (McCauley and Melis 1986; Ivanov et al. 2007) were conducted with isolated thylakoids without addition any acceptors from PS I into their suspension, and the authors considered the oxidation of the PQ-pool after its reduction as a process involving oxygen. Only in the study (Ivanov et al. 2007) an attempt was made to describe the occurring processes, particularly to explain the origin of the biphasic kinetics of oxidation. However, due to several methodological limitations, the authors were unable to study the process of PQ-pool oxidation in detail. In particular, they didn’t identify all possible processes contributing to each kinetic phase and the conditions determining the magnitude of their contribution.

In present study, we measured the kinetics of dark oxidation of the PQ-pool in leaves, chloroplasts, and isolated thylakoids. In the latter case, we performed a detailed analysis of the two phases of reoxidation of the PQ-pool in the dark after illumination cessation. We found that each phase may reflect a combination of at least two processes. In particular, the "fast" phase may include residual electron flow from the PQ-pool to the downstream acceptors and the oxidation of plastohydroquinone (PQH₂) molecules by superoxide radicals (O₂^•−), as was proposed in the work (Ivanov et al. 2007); the contribution of each of these processes may depend on the rate of electron transport along the PETC. An important result of the present study was the discovery that hydrogen peroxide (H₂O₂) can accelerate the oxidation of the PQ-pool in thylakoid membranes, primarily the "slow" phase of the oxidation. The results obtained in experiments with isolated thylakoids allowed us to explain some features of pool oxidation in intact chloroplasts and leaves.

The experiments were conducted with fresh spinach (Spinacea oleracea) purchased at a local grocery store, and 1.5-2-week-old pea (Pisum sativum) grown in a greenhouse at 21–23°C.

Thylakoids and chloroplasts isolation

Thylakoids from leaves of spinach and pea were isolated according to (Kozuleva and Ivanov 2010). The first and second tier leaves were used for pea thylakoids extraction. Isolated thylakoids were resuspended in a medium containing 0.4 M sucrose, 20 mM NaCl, 5 mM MgCl₂, 50 mM HEPES-KOH (pH 7.5), kept in the dark on ice and used on the same day.

Intact chloroplasts from spinach were isolated according to (Mubarakshina et al. 2010). Isolated chloroplasts were resuspended in a medium containing 0.33 M sorbitol, 60 mM KCl, 2 mM EDTA, 1 mM MgCl₂, 0.5 mM KH₂PO₄, 25 mM HEPES-KOH (pH 7.6). The intactness of the chloroplasts was measured as the ratio of oxygen uptake rates in the presence of 10 mM NH₄Cl and 1 mM K₃[Fe(CN)₆] before and after disruption of the outer membrane of chloroplasts by osmotic shock, according to (Heber and Santarius 1970) and in all experiments it was not less than 90%.

Chlorophyll concentration was measured according to (Lichtenthaler 1987) after extraction in 96% ethanol.

Measurements of fluorescence induction curves

The relative area above the fluorescence induction curve (A_fl), obtained with a HandyPea fluorometer (Hansatech, UK), was measured to assess the redox state of the PQ-pool. The induction curve was recorded during a 1.5 s flash of red light (λ = 660 nm) with an intensity of 3000 µmol photons m^-2 s^-1. This duration of the flash was chosen instead of the commonly used 1 s flash for measuring OJIP kinetics, as the maximum fluorescence level was not achieved within 1 s under all conditions used in our experiments. The indicated flash was also used to convert the PQ-pool into a fully reduced state both in samples adapted to the dark and after pre-illuminating the samples with continuous light just before turning off the light; the latter was used to create the same redox state of the PQ-pool before tracking the course of its oxidation in the dark under different conditions. To assess the redox state of the PQ-pool in the dark after illumination, a new suspension or a new leaf was used for each darkness interval from 0.1 s to 600 s. Thus, each leaf or suspension was illuminated twice with a flash: once to fully reduce the PQ-pool and a second time after a set darkness period to assess the redox state of the PQ-pool. An average of at least 3 leaves or suspensions were used for each time point.

The reaction medium for experiments with isolated thylakoids contained: 0.1 M sucrose, 20 mM NaCl, 5 mM MgCl₂, 50 mM HEPES-KOH (pH 7.5) and 1 µM Gramicidin D (GrD). The reaction medium for experiments with isolated chloroplasts contained: 0.33 M sorbitol, 60 mM KCl, 2 mM EDTA, 1 mM MgCl₂, 0.5 mM KH₂PO₄, 25 mM HEPES-KOH (pH 7.6) and 10 mM NH₄Cl. To facilitate effective electron flow from PS I, 500 µM NADP⁺ and 10 µM recombinant ferredoxin (petF from Chlamydomonas reinhardtii) obtained by heterologous expression in Escherichia coli BL21 cells and purified according to (Marco et al. 2018) were used. For creating anaerobic conditions in thylakoid or chloroplast suspensions, 50 U/mL glucose oxidase, 20 mM glucose, and 250 U/mL catalase were used. For inhibiting the oxidation of plastohydroquinone in the cytochrome b₆f complex, 5 µM dinitrophenyl ether of iodonitrothymol (DNP-INT) (Cayman Chemicals) was used. To control for the nonspecific action of catalase as a protein, bovine serum albumin (BSA) was used in an amount equivalent to the amount of catalase added (0.07 mg/mL).

Plotting of A_fl dependencies on the duration of darkness and their fitting were performed using the Origin software package.

Measurements of the P700 redox state

The kinetics of P700 redox transformations were measured using Dual-PAM-100 (Walz, Germany) as the differential optical density at 870 − 830 nm in a vertical cuvette to mitigate the adverse effect of thylakoid sedimentation on the P700 signal. The content of thylakoids corresponded to a chlorophyll concentration of 200 µg/mL. Changes in the level of P700⁺ in samples, both dark-adapted and pre-illuminated with light at an intensity of 650 µmol quanta m^-2 s^-1 for 30 s, were registered immediately before turning off the light in response to a flash of intensity 3000 µmol quanta m^-2 s^-1 and duration 800 ms. To determine the maximum range of P700 signal changes, a flash of intensity 10,000 µmol quanta m^-2 s^-1 and duration 300 ms was applied against a background of far-red light, and the resulting amplitude was used for the normalization of the measured kinetics.

Dark transformation of OJIP-kinetics after illumination of leaves, chloroplasts and thylakoids

Typical OJIP kinetics of chlorophyll a fluorescence induction during a light flash (intensity 3000 µmol photons m^− 2 s^− 1, duration 1.5 s), exhibiting distinct O-J, J-I, and I-P phases, are a characteristic feature of dark-adapted leaves (Fig. 1a, black line). Deviations from this typical kinetics are visible in isolated chloroplasts (Fig. 1a, red line), and are even more pronounced in isolated thylakoids (Fig. 1a, blue line). The divergence in OJIP kinetics in chloroplasts from that seen in leaves is evidently due to the lack of metabolite exchange with the cytoplasm. In thylakoids, the deviations primarily arise from the absence of stromal components, including the immediate soluble electron acceptor from PS I, ferredoxin (Fd). OJIP kinetics, measured for all three objects in darkness after a light flash, show over time a trend of returning towards the dark-adapted form, with each object exhibiting a unique recovery pattern (Fig. 1).

For intact leaves (Fig. 1b), a “dip” in fluorescence first appears around the I point, then shifts towards the P point within 0.1–2 s of darkness, and finally emerges in the J-I region after 2–5 s. The evolution of OJIP kinetics, characteristic for chloroplasts (Fig. 1c), shares certain similarities with that in leaves. This is evidenced by the wide “dip” in fluorescence during the initial darkness following a light flash, extending from point J to P, and gradually shifting to the I-P region over time, as observed in leaves. This may be attributed to the presence of Fd and NADP⁺ in chloroplasts, as in leaves. In the case of thylakoids (Fig. 1d), a minor “dip” in fluorescence in the J-I region is noticeable only during the very initial period of darkness (up to 1 s), while the I-P region remains largely unchanged.

Previous studies (McCauley and Melis 1986; Nixon and Rich 2006) have employed the area above the fluorescence curve (A_fl) as an indicator of the redox state of the PQ-pool, where a higher A_fl value corresponds to a more oxidized state. To investigate the oxidation of the PQ-pool in the dark, we examined the dependence of A_fl on the duration of darkness post-illumination, using data from the same experiments as shown in Fig. 1. The resultant dependencies were fitted using the sum of two exponential decay functions

$${A}_{fl}\left(t\right)={A}_{total}-{A}_{1}\times {e}^{-{k}_{1}t}-{A}_{2}\times {e}^{-{k}_{2}t}$$

where t is the time in darkness; A_total – the total amplitude of A_fl; A₁ and A₂ – the amplitudes of the “fast” and “slow” phases of A_fl changes; and k₁ and k₂ – rate constants of these phases. The fraction of the “fast” phase, computed as A₁/(A₁ + A₂), is henceforth referred to as r. In our analysis, we considered the half-time of the “fast” (τ₁) and “slow” (τ₂) phases:

$${\tau }_{n}=\frac{1}{{k}_{n}}\times ln\left(2\right)$$

The approaches used in this study have some inherent experimental limitations. The measuring flash induces electron transport not only from PS II, but also from the reduced PQ-pool to PS I. Since the light-induced electron flow from the PQ-pool is not a part of the dark oxidation processes examined here, we needed to estimate its magnitude. In our prior research we discovered a light-dependent inhibitory activity of dinitrophenyl ether of iodonitrothymol (DNP-INT), a competitive inhibitor of PQH₂ oxidation in the cytochrome b₆f-complex (Vilyanen et al. 2022). In that study we observed that after turning off the actinic light, the kinetics of P700⁺ reduction in the presence of DNP-INT was the same as in control (refer to Fig. 2a in (Vilyanen et al. 2022)). Essentially, in the darkness DNP-INT quickly ceased to inhibit electron transport through the b₆f-complex. In other words, DNP-INT serves as an effective inhibitor of electron transport from the PQ-pool only in the light, with no impact on electron transport to P700⁺ after the light is turned off. As a physical quencher of fluorescence, DNP-INT alters the shape of OJIP kinetics and the specific A_fl values derived from them. By comparing the amplitudes of the “fast” and “slow” phases of A_fl changes in the absence and presence of DNP-INT (data not shown), we estimated that the measurement flash-induced flow of electrons from the PQ-pool has negligible effect on the ratio (r) of “fast” and “slow” phases.

Data presented in Fig. 2 and Table 1 reveal that in leaves and intact chloroplasts, over 50% of the PQ-pool is oxidized during the “fast” phase (lasting 1–3 s), while in isolated thylakoids, which lack an efficient terminal electron acceptor (with only molecular oxygen serving as the acceptor), about only 25% of the PQ-pool is oxidized during the “fast” phase. The half-time of the “fast” phase, τ₁, for leaves and chloroplasts was approximately 0.6 seconds, while for isolated thylakoids, it was roughly double, reaching 1.3 s. The half-time of the “slow” phase, τ₂, for both the intact leaves and isolated thylakoids was similar, roughly 80–90 seconds, yet it was significantly shorter in chloroplasts, approximately 30 s.

Table 1

Kinetic parameters calculated from typical experiments similar to those depicted in Fig. 2. r – fraction of the “fast” phase; τ₁ – half-time of the “fast” phase; τ₂ – half-time of the “slow” phase
Object	r	τ₁, s	τ₂, s
Leaves	0.53 ± 0.01	0.66 ± 0.03	86 ± 9.7
Chloroplasts	0.58 ± 0.02	0.60 ± 0.03	31 ± 3.7
Thylakoids	0.27 ± 0.03	1.36 ± 0.25	82.6 ± 13.1

Numerous studies (Haldimann and Tsimilli-Michael 2002; Schansker et al. 2005) suggest that the fluorescence level at point J reflects the effectiveness of Q_A oxidation and can be used as an indicator of the redox state of the PQ-pool. Vj' was defined as:

$${V}_{J}^{{\prime }}=\frac{{F}_{J}-{F}_{OD}}{{F}_{M}-{F}_{OD}},$$

where F_J is the fluorescence level at point J from the OJIP kinetics; F_M is the maximum fluorescence level from OJIP kinetics; and F_OD is the minimum fluorescence level from OJIP kinetics, measured with dark-adapted samples.

The evolution of Vj' in the dark observed in leaves markedly deviated from those seen in isolated chloroplasts and thylakoids. In leaves, Vj' remains unchanged until 0.5 seconds, following which it experiences a decrease. The maximal rate of Vj' decline is observed within the 1–3 second interval in darkness (Fig. 2d, solid black line, black squares). In contrast, chloroplasts exhibit a significant drop in Vj' only after 2 seconds in darkness, but the changes are considerably less abrupt (Fig. 2d, solid black line, white squares). In thylakoids, Vj' decline is gradual, lacking any distinct phases (Fig. 2d, black dashed line, black triangles).

The variations in Vj' decline in darkness following illumination across different objects may be associated with the fact that in whole leaves, the accumulated NADPH is utilized in the stromal metabolic pathways, primarily the Calvin-Benson cycle, with stroma metabolites simultaneously exchanging with the cell cytoplasm. In isolated intact chloroplasts, NADP⁺ is also reduced to NADPH, but the pathways for NADPH oxidation in the stroma might be disrupted, especially due to a deficiency in inorganic carbon intake. In the case of isolated thylakoids, where the stroma is absent, oxygen serves as the sole electron acceptor.

Fast phase of plastoquinone pool oxidation kinetics in the dark after illumination

As seen from Table 1, τ₁ in isolated thylakoids is approximately double that observed in chloroplasts and leaves. To imitate the conditions found in chloroplasts, Fd + NADP⁺ were added into the thylakoid suspension. After a 1.5-s flash illumination, τ₁ decreased nearly fivefold in the presence of Fd + NADP⁺, but the “fast” phase's contribution increased insignificantly (Fig. 3a, Table 2). Kinetics of A_fl changes similar to those in chloroplasts was achieved after illuminating the suspension for 30 s with a light intensity of 650 µmol photons m^− 2 s^− 1. Under these conditions, the “fast” phase's contribution nearly doubled and τ₁ additionally decreased twofold, becoming 25 times shorter compared to the thylakoids without additions (Table 2); at that r increased only slightly.

Table 2

Kinetic parameters of A_fl changes in isolated thylakoids after illumination of the suspension under specified conditions ± SE. r – fraction of the “fast” phase; τ₁ – half-time of the “fast” phase; τ₂ – half-time of the “slow” phase
Illumination conditions	Additions	r	τ₁, s	τ₂, s
1.5 s with 3000 µmol quanta m^− 2 s^− 1	-	0.17 ± 0.01	0.70 ± 0.1	56.4 ± 6.5
1.5 s with 3000 µmol quanta m^− 2 s^− 1	10 µM Fd + 500 µM NADP⁺	0.22 ± 0.02	0.13 ± 0.04	60.4 ± 10.1
30 s with 650 µmol quanta m^− 2 s^− 1	-	0.33 ± 0.04	1.53 ± 0.17	80.2 ± 24.2
30 s with 650 µmol quanta m^− 2 s^− 1	10 µM Fd	0.19 ± 0.02	0.33 ± 0.06	61.9 ± 8.8
30 s with 650 µmol quanta m^− 2 s^− 1	10 µM Fd + 500 µM NADP⁺	0.39 ± 0.01	0.06 ± 0.02	22.9 ± 2.8

As Fig. 3 inserts illustrate, the course of A_fl changes in the initial seconds of darkness varies significantly in the presence and absence of Fd + NADP⁺. Having an effective electron flow from PS I results in a sharp acceleration and decrease in the “fast” phase's duration. At the same time, the contribution of the “fast” phase to the PQ-pool's oxidation in the dark increases when an effective electron acceptor from PS I is present. This suggests that in the initial darkness period, the oxidation of the PQ-pool involves electron flow from the PQ-pool to downstream acceptors.

The use of only Fd as an electron acceptor from PS I during 30-s illumination resulted in a decrease in the value of τ₁ to 0.33 s, compared to the 1.53 s observed in thylakoids without additions (Table 2). However, this value was higher than the 0.06 s observed when Fd + NADP⁺ were present. This data aligns with the observation that the level of P700⁺ during such illumination is higher when Fd + NADP⁺ are present, as opposed to when only Fd is present (Fig. S1b). This condition allows for a more efficient flow of electrons to PS I after the cessation of light.

Fd was present in an amount that is not sufficient to significantly increase the electron flow along the PETC (Kozuleva and Ivanov 2010), and the P700⁺ level with Fd was slightly higher than when oxygen was the only electron acceptor from PS I (Fig. S1b). Interestingly, despite the lower τ₁ value, the fraction of the "fast" phase in the presence of Fd was lower than that seen in the absence of additions (Table 2).

The insets in Fig. 3 show that when oxygen was the only electron acceptor from PS I, the increase in A_fl in the initial seconds of darkness occurred in a more gradual and prolonged manner. The post-illumination electron flow from the PQ-pool to downstream acceptors is likely to be insignificant, considering that oxygen is an inefficient electron acceptor from PS I. The latter is well illustrated by the fact that P700⁺ levels during illumination under aerobic and anaerobic conditions are very similar (Fig. S1a, S1b). Possibly, other processes can be included in the “fast” phase of PQ-pool oxidation after light cessation, besides the flow of electrons from the PQ-pool to the downstream acceptors.

Possibly such processes conditioned a slight increase in the share of the “fast” phase from 0.26 ± 0.01 to 0.30 ± 0.02 without change in initial A_fl rise when the light intensity was increased from 10 to 1000 µmol photons m^− 2 s^− 1 during a 30-s illumination of thylakoid suspension (Fig. 4a). Under anaerobic conditions after a 1.5-s flash illumination, the increase of A_fl in the dark in thylakoids without added acceptors was significantly suppressed compared to aerobic conditions. However, there was a small A_fl increase in the initial period of darkness (Fig. 4b). Since O₂ as an electron acceptor from PS I, was absent, the potential influence of electron flow from the PQ-pool to downstream acceptors was minimal, allowing the involvement of other processes in the fast phase to be more clearly discernible.

Slow phase of plastoquinone pool oxidation kinetics in the dark after illumination

Previous studies (Sanchez-Cruz et al. 2014; Khorobrykh and Tyystjärvi 2018) have demonstrated that H₂O₂ can oxidize reduced quinones. Moreover, the study (Khorobrykh and Tyystjärvi 2018) thoroughly investigated the oxidation of PQH₂ by H₂O₂ in organic solvents. The study (Borisova-Mubarakshina et al. 2018) suggested the possibility of PQH₂ oxidation in thylakoids by H₂O₂ produced during electron transfer in PETC.

As shown in Fig. 5a, pre-illuminating the thylakoid suspension for 30 s in the presence of catalase led to suppression of A_fl increase, which resulted in a significantly longer half-time of the “slow” phase, τ₂ (Fig. 5a, Table 3). Catalase also influenced the course of this increase after flash illumination alone, although the effect on τ₂ was less pronounced (Fig. 5b, Table 3). The effect of catalase was not a consequence of its protein nature and possible interactions with the thylakoid membrane, as was demonstrated in a separate experiment where the suppression of PQ-pool oxidation (increase in A_fl) did not occur in the presence of BSA added in equivalent quantity to that of the added catalase, under the same conditions as in Fig. 5a (Fig. S2, Table 3).

The effect of catalase indicated that hydrogen peroxide is involved in the oxidation of the PQ-pool. The accumulation of H₂O₂ when thylakoids are illuminated, and when the only electron acceptor from PS I is oxygen, can increase with longer illumination times. As seen from Fig. 5c and Table 3, the “slow” phase, which occurred both when the thylakoids are illuminated by flash only and after pre-illumination for 30 s, was considerably faster in the latter case, i.e. under longer illumination conditions.

However, the “slow” phase following flash illumination of the thylakoids was also observed under anaerobic conditions, albeit less pronounced than under aerobic conditions (Fig. 4b). Considering the revealed influence of H₂O₂ on the “slow” phase, this could be presumably attributed to the characteristics of our system for creating anaerobic conditions: due to the high K_m value of catalase for hydrogen peroxide, a small amount of H₂O₂ might have remained in the close vicinity of the thylakoid membrane. This assumption was supported by the fact that the “slow” phase was not observed under anaerobic conditions in experiments with isolated chloroplasts (Fig. 5d). Its absence could be because H₂O₂ produced during the oxidation of glucose by glucose oxidase in the chloroplast suspension was not located near the thylakoid membrane, but outside the chloroplast. Another possibility was that H₂O₂ formed outside the chloroplast during the reduction of oxygen by glucose oxidase permeated into the chloroplast through aquaporins ((Mubarakshina) Borisova et al. 2012) but was utilized by stromal and/or thylakoid-bound ascorbate peroxidases.

Table 3

Kinetic parameters of A_fl changes in isolated thylakoids after illumination of the suspension under specified conditions ± SE. r – fraction of the “fast” phase; τ₁ – half-time of the “fast” phase; τ₂ – half-time of the “slow” phase
Illumination conditions	Additions	r	τ₁, s	τ₂, s
1.5 s with 3000 µmol quanta m^− 2 s^− 1	-	0.25 ± 0.01	0.95 ± 0.08	80.2 ± 10.2
30 s with 650 µmol quanta m^− 2 s^− 1	-	0.23 ± 0.01	0.896 ± 0.075	53.4 ± 5.7
1.5 s with 3000 µmol quanta m^− 2 s^− 1	-	0.26 ± 0.01	0.75 ± 0.06	111 ± 5.18
1.5 s with 3000 µmol quanta m^− 2 s^− 1	250 U/ml Catalase	0.29 ± 0.01	0.65 ± 0.05	89.6 ± 3.87
30 s with 650 µmol quanta m^− 2 s^− 1	-	0.3 ± 0.02	1.5 ± 0.2	65.8 ± 8.4
30 s with 650 µmol quanta m^− 2 s^− 1	250 U/ml Catalase	0.45 ± 0.02	1.5 ± 0.2	136 ± 27
30 s with 650 µmol quanta m^− 2 s^− 1	-	0.31 ± 0.03	1.57 ± 0.22	48.5 ± 7.1
30 s with 650 µmol quanta m^− 2 s^− 1	0.07 mg/mL BSA	0.24 ± 0.02	1.1 ± 0.2	48 ± 6

To estimate the potential involvement of H₂O₂ in the oxidation of the PQ-pool in vivo, electron acceptors from PS I, either Fd alone or Fd + NADP⁺, were added into the thylakoid suspension. The influence of catalase addition into the suspension on the development of the “slow” phase of A_fl changes manifested as a suppression of increase in A_fl and longer τ₂ that was observed only in the presence of Fd alone (Fig. 6a, Table 4). It is known that addition Fd into the thylakoid suspension can increase the rate of O₂ reduction and, correspondingly, the accumulation of H₂O₂. In the presence of Fd + NADP⁺, the reduction of O₂ is much less than in the presence of Fd alone (Kozuleva and Ivanov 2010). Under these conditions, the effect of catalase manifested only as a decrease in the contribution of the “slow” phase to the observed increase of A_fl (Table 4).

Table 4

Kinetic parameters of A_fl changes in isolated thylakoids after pre-illumination of the suspension with light at an intensity of 650 µmol photons m^− 2 s^− 1 for 30 s under specified conditions ± SE. r – fraction of the “fast” phase; τ₁ – half-time of the “fast” phase; τ₂ – half-time of the “slow” phase
Additions	r	τ₁, s	τ₂, s
10 µM Fd	0.19 ± 0.02	0.33 ± 0.06	61.9 ± 8.8
10 µM Fd + 250 U/mL catalase	0.19 ± 0.012	0.25 ± 0.04	93.2 ± 15.2
10 µM Fd + 500 µM NADP⁺	0.283 ± 0.005	0.077 ± 0.006	38.9 ± 1.6
10 µM Fd + 500 µM NADP + 250 U/mL catalase	0.398 ± 0.013	0.08 ± 0.01	40.5 ± 3.8

Two-phase kinetics of PQ-pool oxidation after illumination have been observed in previous studies using isolated thylakoids (McCauley and Melis 1986; Ivanov et al. 2007) with oxygen acting as the sole terminal electron acceptor from PETC. In the present research, we used electron acceptors, Fd + NADP⁺, and varied light conditions, while evaluating the redox state of the PQ-pool at broad range of intervals after illumination ranging from 0.1 seconds to 10 minutes. This approach permitted to elucidate the mechanisms conditioning the "fast" and "slow" phases of PQ-pool oxidation in the dark, and allowed to propose their potential contribution to PQ-pool oxidation in isolated intact chloroplasts and leaves.

The rapid increase in A_fl that is observed under certain conditions within a timescale from 0.5 to 7 seconds corresponds to the "fast" phase of PQ-pool oxidation in the dark after illumination of isolated thylakoids. As seen in the insets of Figs. 3a and 3b, in the presence of effective electron acceptors, the rapid change in A_fl is restricted to the initial 1 second of darkness. Conversely, in the absence of effective acceptors, with oxygen as the sole acceptor (Figs. 3a, 3b, 4a, 5a-5c), the initial increase in A_fl in the dark proceeds more gradually over 7 seconds. It is established that the reduction of P700⁺ after turning off the light takes approximately 100 to 200 ms (Schreiber 2017). These observed variations in the rate of A_fl changes in the dark, depending on the conditions suggest the possible contribution of residual electron flow from the PQ-pool to downstream acceptors to the "fast" phase of PQ-pool oxidation in the initial moments of darkness, the magnitude of which is influenced by the status of PETC established during thylakoid illumination.

As Table 2 demonstrates, the inclusion of Fd and Fd + NADP⁺ in the thylakoid suspension results in a decrease in τ₁, the half-time of the "fast" phase, compared to conditions where oxygen serves as the only acceptor, and this decline in τ₁ closely correlated with an increase in the P700⁺ level as the efficiency of PS I acceptors increased (Fig. S1a). This figure shows increase in P700⁺ level during 800 ms flash applied to dark-adapted thylakoids in the presence of Fd + NADP⁺ which apparently reflects light-depended FNR activation. The absence of such increase in P700⁺ level when such flash was applied after pre-illumination of thylakoids with light of 650 µmol photons m^− 2 s^− 1 indicated that the FNR was already activated (Fig. S1b). The lower value of τ₁ after pre-illumination as compared with that in dark-adapted thylakoids (0.06 sec vs. 0.13 sec) could be attributed to such full activation of FNR. This activation of FNR can determine the significant electron flow from the PQ-pool to downstream acceptors in the initial period of darkness in the presence of Fd + NADP⁺ after pre-illumination.

Thus, the "fast" phase of PQ-pool oxidation in the dark can be partially attributed to the electron flow from the PQ-pool to downstream acceptors. However, when its contribution is weakly expressed, such as in the presence of only O₂ as an acceptor, the "fast" phase of A_fl increase may be facilitated also by another mechanism.

It was shown that higher light intensity led to an increase in the amplitude of the "fast" phase (Ivanov et al. 2007). The authors interpreted this result as a consequence of the increased accumulation of superoxide radicals, O₂^•−, in the membrane under high light intensity. These radicals are capable of oxidizing PQH₂ molecules in the dark in thermodynamically advantageous reaction. They were generated inside the membrane upon the reduction of O₂ molecules by PETC components (Shuvalov, V. A. and Krasnovskii, A. A. 1975; Kozuleva et al. 2011, 2015). Similarly, in our experiments, we noticed an increase in the fraction of the "fast" phase as light intensity increased in the absence of an effective electron acceptors from PS I (Fig. 4a). Additional evidence of increased oxidation of the PQ-pool by O₂^•− could be inferred from a larger fraction of the "fast" phase of A_fl changes in the absence of acceptors compared to the presence of Fd, despite similar P700⁺ levels in both scenarios (Fig. S1). Furthermore, the existence of the "fast" phase under anaerobic conditions could potentially be linked to the production of superoxide radicals from the oxygen released during illumination in the water-oxidizing complex (Fig. 4b). These results can be considered indirect evidence supporting the hypothesis of the role of the O₂^•− in the "fast" phase of PQ-pool oxidation in the dark.

The "fast" phase of PQ-pool oxidation after its reduction in the light appears to be a combination of electron flow to downstream acceptors of PETC and potential oxidation of PQH₂ molecules by O₂^•− produced by PETC components during illumination and preserved in the membrane. The contribution of the electron flow to downstream acceptors is heavily dependent on the presence of an effective electron acceptor from PS I.

The "slow" phase of PQ-pool oxidation is attributed to the autocatalytic reaction of PQ-pool components with molecular oxygen. The comproportionation reaction between plastoquinone and plastohydroquinone molecules leads to the formation of plastosemiquinone, PQ^•−:

PQ + PQH₂ → 2PQ^•− + 2H⁺ (Reaction 1)

Plastosemiquinone, having a low enough redox potential, can interact with molecular oxygen, leading to the formation of a superoxide anion-radical. This radical can oxidize plastohydroquinone, forming a new plastosemiquinone and hydrogen peroxide:

O₂ + PQ⁻ → PQ + O₂^•− (Reaction 2)

O₂⁻ + PQH₂ → PQ^•− + H₂O₂ (Reaction 3)

This slow, autocatalytic process of redox transformations involving PQ-pool components, oxygen, and ROS in the thylakoid membrane enables the gradual oxidation of the PQ-pool in the dark.

Our findings suggest that hydrogen peroxide could modulate the "slow" phase of PQ-pool oxidation in the dark. Long-term illumination of thylakoids in the absence of an effective electron acceptor from PS I significantly accelerated the "slow" phase. This effect was neutralized by addition of catalase into the thylakoid suspension (Fig. 5a, 5b).

Potential oxidation of the thylakoid PQ-pool by hydrogen peroxide in biological systems has not been thoroughly explored to date. However, studies exist on the mechanism of oxidation of reduced quinones in solutions containing H₂O₂. In the study by Sanchez-Cruz et al. (2014), a mechanism of such oxidation was described. According to this, H₂O₂ molecules react with semiquinones, forming a hydroxyl radical and a hydroxide ion, a process the authors termed the metal-independent Fenton reaction:

Q^●− + H₂O₂ → Q + OH^● + OH⁻ (Reaction 4)

The study (Khorobrykh and Tyystjärvi 2018) conducted the reaction of PQH₂ with H₂O₂ in methanol, proposing a sequential deprotonation process for PQH₂ oxidation by hydrogen peroxide:

PQH₂ + H₂O₂ → PQH⁻ + H₃O₂⁺ (Reaction 5)

PQH⁻ + H₂O₂ → PQ₂⁻ + H₃O₂⁺ (Reaction 6)

The authors proposed that the resulting deprotonated products could react with oxygen to form plastosemiquinone, which could subsequently be oxidized by oxygen as per Reaction 2:

PQH⁻ + O₂ → PQ^●− + HO₂^● (Reaction 7)

PQ²⁻ + O₂ → PQ^●− + O₂^●− (Reaction 8)

However, the interactions of PQ-pool components with H₂O₂ within thylakoids cannot be entirely explained by one of these mechanisms. It's important to consider that, according to (Sanchez-Cruz et al. 2014), the rate constant for the reaction of semiquinones with hydrogen peroxide (reaction 4) is approximately 10⁴ M^− 1s^− 1, significantly lower than the rate constant for semiquinones reacting with oxygen, given redox potential (E_m) values close to the E_m for the PQ/PQ^●− pair, which is around 10⁸ M^− 1s^− 1 (Wardman 1990). Secondly, in experiments by (Khorobrykh and Tyystjärvi 2018), incubating 75 µM PQH₂ with 5 mM H₂O₂ did not result in the complete oxidation of PQH₂, irrespective of incubation time, suggesting the absence of direct redox reactions of plastoquinone forms with H₂O₂ and a more complex process. We propose that a small accumulation of H₂O₂ in the thylakoid suspension during illumination, which could only react with PQ-pool components at the phase boundary, might catalyze the oxidation of the PQ-pool by molecular oxygen (reactions 1–3) through the deprotonation of PQH₂ molecules.

Thus, the "slow" phase of PQ-pool oxidation, which is observable in isolated thylakoids, appears to reflect only its oxygen-dependent oxidation. The rate of this process increases in the presence of hydrogen peroxide, which is formed in the PETC during illumination.

The experiments with isolated thylakoids in the presence of Fd + NADP⁺ (Figs. 3a and 3b) indicate that after pre-illumination the steady-state A_fl level established after 100 sec in the dark with Fd + NADP⁺ was substantially lower than in the absence of effective acceptors. In contrast, after flash illumination this effect was less pronounced. It was concluded (see above) that a more complete activation of FNR was established under pre-illumination conditions due to longer illumination time. These conditions could result in a larger accumulation of NADPH in the suspension compared to flash illumination. Therefore, the lower steady-state A_fl level and smaller τ₂ value (23 sec vs. 60 sec, Table 2) imply that a larger concentration of NADPH in the reaction medium facilitates more active NADPH-dependent reduction of the PQ-pool, maybe through NDH complex. This reduction occurring concurrently with oxidation by oxygen led to a decrease in the steady-state A_fl level in the dark.

Such situation in intact chloroplasts and its absence in thylakoids, in the latter case both with and without Fd + NADP⁺, after exposure to only a 1.5-sec flash, may explain the difference in the kinetics of PQ-pool oxidation in these objects (Fig. 2b vs. Figure 3a). Even when Fd + NADP⁺ are present in the thylakoid suspension, a sufficient concentration of NADPH may not be generated during the flash. However, in chloroplasts, such a flash can provide the reduction of almost all NADP⁺ in the limited volume of stroma. The similar τ₂ values for intact chloroplasts after flash illumination (31 s) and thylakoids with Fd and NADP⁺ after pre-illumination (23 s) (Table 1, 2), as well as the similar pattern of A_fl changes in the dark (Fig. 2b and Fig. 3b) point out the role of the NAPDH in the oxidation of PQ-pool in chloroplasts after illumination.

In chloroplasts, due to substrate scarcity, the pathways consuming NADPH are significantly suppressed. This makes the post 1.5-second flash illumination state in chloroplasts similar to the situation after continuous illumination of thylakoids with added Fd + NADP⁺, where NADPH is not consumed at all. However, when considering whole leaves, the nature of PQ-pool oxidation after a 1.5-second flash closely resembles that in isolated thylakoids without additions, rather than in chloroplasts, yet with a larger "fast" phase amplitude (Fig. 2a and 2c). The half-times of the "slow" phase, τ₂, for whole leaves and thylakoids without additions are both within the 80–90 s range (Table 1). At that, as shown in Fig. 2d, the pattern of Vj' changes in the leaves in the dark markedly differs from that in intact chloroplasts and thylakoids. This pattern can be well explained by the decrease in NADPH content accumulated in the stroma when its oxidation pathways are functioning under conditions of metabolite exchange between the stroma and cytoplasm, and regeneration of NADP⁺. Specifically, the absence of notable NADPH accumulation during flash illumination impedes PQ-pool reduction. Conversely, when NADPH accumulates, it leads to the observed slowdown of its oxidation, as is the case in intact chloroplasts. Therefore, the acquired data suggest a link between the redox state of the PQ-pool in vivo in the dark after illumination and the capacity of stromal metabolism to utilize NADPH.

The demonstrated in this study interaction of the PQ-pool components with H₂O₂ serves as an additional aspect of the pool's antioxidant function, leading to the neutralization of H₂O₂. It is well-known that stress conditions amplify ROS production (Allan and Fluhr 1997; Apel and Hirt 2004; (Borisova-Mubarakshina et al. 2012). During the initial stages of stress conditions, the utilization of NADPH may slow down, resulting in an accumulation of reduced Fd. This leads to the reduction of molecular oxygen and the production of H₂O₂ in the chloroplast’s stroma (Kozuleva and Ivanov 2010). Furthermore, the production of hydrogen peroxide in the thylakoid membrane significantly increases as well (see review Borisova-Mubarakshina et al. 2019). Thus, H₂O₂ considerably accumulates during the initial stages of stress conditions when the photosynthetic apparatus is yet to acclimate to the new conditions (Borisova-Mubarakshina et al. 2014). Simultaneously, the activity of ascorbate peroxidases (APX) decreases during the first stages of stress conditions, primarily due to ascorbate being exhausted as a result of its oxidation to dehydroascorbic acid (Zechmann 2011). An increase in ascorbate content, along with a rise in the content and activity of APX in chloroplasts, is only observed after a certain period of stress exposure (Caverzan et al. 2012; Zechmann 2018). Therefore, managing an excessive amount of hydrogen peroxide, beyond what is needed for its signaling role, can be critical for plant sustainability at the onset of stress factors. The neutralization of H₂O₂ by the PQ-pool components during this period prevents not only the inactivation of Calvin-Benson cycle enzymes by H₂O₂ (Kaiser 1976, 1979), but also the generation of the hydroxyl radical, an extremely effective oxidant that damages the functionality of the photosynthetic machinery.

PQ-pool – plastoquinone pool

PQ, PQ^•⁻, PQH₂ – plastoquinone, plastosemiquinone, plastohydroquinone respectively

PETC – photosynthetic electron transport chain

PSI, PSII – photosystem I, photosystem II respectively

Chl – Chlorophyll a

Fd – ferredoxin

FNR – ferredoxin:NADP⁺ oxidoreductase

NDH – NAD(P)H dehydrogenase

DNP-INT – dinitrophenyl ether of 2-iodo-4-nitrothymol

AUTHOR CONTRIBUTION

D.V. and I.N. have made equal contributions. D.V., M.B.-M. and M.K. designed experiments; M.B.-M. supervised experiments; I.N., and D.V. performed experiments; D.V. and B.I. wrote the article with contributions from all the authors.

ACKNOWLEDGMENTS

The work was supported by Russian Science Foundation (grant № 22-24-01074). Authors are thankful to Anna Krupyanko for help with thylakoids isolation.

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FigS1.tif
Fig. S1 Kinetics of P700⁺ redox changes measured in a thylakoid suspension in the presence of 10 μM Fd and 500 μM NADP⁺ (Fd + NADP⁺), in the presence of 10 μM Fd, in the presence of oxygen as the only electron acceptor from PS I, and under anaerobic conditions on dark-adapted thylakoids (a) and on light-adapted thylakoids (illumination for 30 seconds with red light at an intensity of 650 μmol photons m^-2s^-1) (b)
FigS2.tif
Fig. S2 Changes in the area above the fluorescence induction curve (A_fl) in the dark after illumination of isolated thylakoids with a flash at an intensity of 3000 μmol photons m^-2 s^-1 at the end of 30-s pre-illumination with light at an intensity of 650 μmol photons m^-2 s^-1, in the absence (–■–) and presence of BSA (0.07 mg/mL) (--□--). Thylakoid contents were equivalent to 30 μg Chl/mL (1 μM GrD). The data presented are from typical experiments; bars represent the standard error (SE) of the mean of 3-4 measurements with different suspensions

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Mechanisms of the plastoquinone pool oxidation in the dark after illumination

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Abstract

Figures

Introduction

Materials and Methods

Thylakoids and chloroplasts isolation

Measurements of fluorescence induction curves

Measurements of the P700 redox state

Results

Dark transformation of OJIP-kinetics after illumination of leaves, chloroplasts and thylakoids

Fast phase of plastoquinone pool oxidation kinetics in the dark after illumination

Slow phase of plastoquinone pool oxidation kinetics in the dark after illumination

Discussion

Abbreviations

Declarations

AUTHOR CONTRIBUTION

ACKNOWLEDGMENTS

References

Additional Declarations

Supplementary Files

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