Transthylakoid Proton Gradient Research Articles

Commonalities and specialties in photosynthetic functions of PROTON GRADIENT REGULATION5 variants in Arabidopsis.

The PROTON GRADIENT REGULATION5 (PGR5) protein is required for trans-thylakoid proton gradient formation and acclimation to fluctuating light (FL). PGR5 functionally interacts with two other thylakoid proteins, PGR5-like 1 (PGRL1) and 2 (PGRL2); however, the molecular details of these interactions are largely unknown. In the Arabidopsis (Arabidopsis thaliana) pgr5-1 mutant, the PGR5G130S protein accumulates in only small amounts. In this work, we generated a knockout allele of PGR5 (pgr5-Cas) using CRISPR-Cas9 technology. Like pgr5-1, pgr5-Cas is seedling-lethal under FL, but photosynthesis and particularly cyclic electron flow, as well as chlorophyll content, are less severely affected in both pgr5-Cas and pgrl1ab (which lacks PGRL1 and PGR5) than in pgr5-1. These differences are associated with changes in the levels of 260 proteins, including components of the Calvin-Benson cycle, photosystems II and I, and the NDH complex, in pgr5-1 relative to the wild type (WT), pgr5-Cas, and pgrl1ab. Some of the differences between pgr5-1 and the other mutant lines could be tentatively assigned to second-site mutations in the pgr5-1 line, identified by whole-genome sequencing. However, others, particularly the more pronounced photosynthetic defects and PGRL1 depletion (compared to pgr5-Cas), are clearly due to specific negative effects of the amino-acid substitution in PGR5G130S, as demonstrated by complementation analysis. Moreover, pgr5-1 and pgr5-Cas plants are less tolerant to long-term exposure to high light than pgrl1ab plants. These results imply that, in addition to the previously reported necessity of PGRL1 for optimal PGR5 function, PGR5 is required alongside PGRL1 to avoid harmful effects on plant performance.

Effects of enhanced UV-B radiation on photosynthetic performance and non-photochemical quenching process of intertidal red macroalgae Neoporphyra haitanensis

The effects of enhanced ultraviolet-B (UV-B, 280–320 nm) radiation reaching the Earth’s surface has become an important issue in terrestrial and marine ecosystems. Living in the land–marine boundary, intertidal macroalgae have various adaptive strategies to resist drastic environmental changes. In this study, we investigated the effects of enhanced UV-B radiation on the photosynthetic performance and photoprotective mechanisms of the red macroalgae Neoporphyra haitanensis . Our results showed that increasing UV-B radiation inhibited growth, damaged chloroplast structures and photosynthetic pigments, and reduced PSII activity and recovery ability in N. haitanensis . UV-B radiation significantly increased non-photochemical quenching (NPQ), indicating that NPQ is an effective photoprotection strategy in N. haitanensis . The transthylakoid proton gradient (ΔpH) uncoupler ammonium chloride (NH 4 Cl) significantly inhibited NPQ induction, showing that the induction of NPQ in N. haitanensis was dependent on ΔpH. Furthermore, violaxanthin and antheraxanthin, essential components of the xanthophyll cycle for modulating NPQ, were not detected in N. haitanensis , indicating the absence of the xanthophyll cycle in N. haitanensis . However, the increased NPQ under UV-B radiation was still associated with the accumulation of zeaxanthin. These findings will be useful for revealing adaptive strategies of intertidal macroalgae to cope with increasing UV-B radiation in the future. • Enhanced UV-B radiation significantly reduces photosynthetic performance of N. haitanensis. • NPQ can be used as effective strategy for photoprotection of N. haitanensis to resist UV-B radiation. • Although xanthophyll cycle is absent in N. haitanensis , NPQ is still regulated by the accumulation of zeaxanthin.

Cooperation of chloroplast ascorbate peroxidases and proton gradient regulation 5 is critical for protecting Arabidopsis plants from photo-oxidative stress.

High-light (HL) stress enhances the production of H2 O2 from the photosynthetic electron transport chain in chloroplasts, potentially causing photo-oxidative damage. Although stromal and thylakoid membrane-bound ascorbate peroxidases (sAPX and tAPX, respectively) are major H2 O2 -scavenging enzymes in chloroplasts, their knockout mutants do not exhibit a visible phenotype under HL stress. Trans-thylakoid proton gradient (∆pH)-dependent mechanisms exist for controlling H2 O2 production from photosynthesis, such as thermal dissipation of light energy and downregulation of electron transfer between photosystems II and I, and these may compensate for the lack of APXs. To test this hypothesis, we focused on a proton gradient regulation 5 (pgr5) mutant, wherein both ∆pH-dependent mechanisms are impaired, and an Arabidopsis sapx tapx double mutant was crossed with the pgr5 single mutant. The sapx tapx pgr5 triple mutant exhibited extreme sensitivity to HL compared with its parental lines. This phenotype was consistent with cellular redox perturbations and enhanced expression of many oxidative stress-responsive genes. These findings demonstrate that the PGR5-dependent mechanisms compensate for chloroplast APXs, and vice versa. An intriguing finding was that the failure of induction of non-photochemical quenching in pgr5 (because of the limitation in ∆pH formation) was partially recovered in sapx tapx pgr5. Further genetic studies suggested that this recovery was dependent on the NADH dehydrogenase-like complex-dependent pathway for cyclic electron flow around photosystem I. Together with data from the sapx tapx npq4 mutant, we discuss the interrelationship between APXs and ∆pH-dependent mechanisms under HL stress.

NTRC Effects on Non-Photochemical Quenching Depends on PGR5.

Non-photochemical quenching (NPQ) protects plants from the detrimental effects of excess light. NPQ is rapidly induced by the trans-thylakoid proton gradient during photosynthesis, which in turn requires PGR5/PGRL1-dependent cyclic electron flow (CEF). Thus, Arabidopsis thaliana plants lacking either protein cannot induce transient NPQ and die under fluctuating light conditions. Conversely, the NADPH-dependent thioredoxin reductase C (NTRC) is required for efficient energy utilization and plant growth, and in its absence, transient and steady-state NPQ is drastically increased. How NTRC influences NPQ and functionally interacts with CEF is unclear. Therefore, we generated the A. thaliana line pgr5 ntrc, and found that the inactivation of PGR5 suppresses the high transient and steady-state NPQ and impaired growth phenotypes observed in the ntrc mutant under short-day conditions. This implies that NTRC negatively influences PGR5 activity and, accordingly, the lack of NTRC is associated with decreased levels of PGR5, possibly pointing to a mechanism to restrict upregulation of PGR5 activity in the absence of NTRC. When exposed to high light intensities, pgr5 ntrc plants display extremely impaired photosynthesis and growth, indicating additive effects of lack of both proteins. Taken together, these findings suggest that the interplay between NTRC and PGR5 is relevant for photoprotection and that NTRC might regulate PGR5 activity.

An underlying mechanism of qE deficiency in marine angiosperm Zostera marina.

Non-photochemical quenching (NPQ) of photosystem II (PSII) fluorescence is one of the most important protective mechanisms enabling the survival of phototropic organisms under high-light conditions. A low-efficiency NPQ, characterized by weak NPQ induction capacity and a low level of protective NPQ, was observed in the marine angiosperm Zostera marina, which inhabits the shallow water regions. Furthermore, chlorophyll fluorescence and Western blot analysis verified that the fast-inducted component of NPQ, i.e., the energy-dependent quenching (qE), was not present in this species. In contrast with the lack of PSII antenna quenching sites for qE induction in brown algae and the lack of functional XC in Ulvophyceae belonging to green algae, all the antenna proteins and the functional XC are present in Z. marina. A novel underlying mechanism was observed that the limited construction of the trans-thylakoid proton gradient (ΔpH) caused by photoinactivation of the oxygen evolving complex (OEC) did not induce protonation of PsbS, thus explaining the inability to form quenching sites for qE induction. Although the ΔpH established under light exposure activated violaxanthin (V) de-epoxidase enzyme to catalyze conversion of V via antheraxanthin (A) and then to zeaxanthin (Z), the quenching capacity of de-epoxidized pigment was weak in Z. marina. We suggest that the low-efficiency NPQ was conducive to efficiently utilize the limited electrons to perform photosynthesis, resisting the adverse effect of OEC photoinactivation on the photosynthetic rate.

Photosynthetic regulation under fluctuating light at chilling temperature in evergreen and deciduous tree species

Plants usually experience fluctuating light conditions at chilling temperatures during the autumn season. We hypothesized that photosystem I (PSI) and PSII are more susceptible to photoinhibition under fluctuating light at chilling temperatures in deciduous species relative to evergreen species. We measured the photosynthetic performances under fluctuating light at 6 °C in two evergreen and two deciduous broadleaf tree species. Within the first 10 s after light increased at 6 °C, none of these species could generate an enough trans-thylakoid proton gradient. Meanwhile, PSI was highly oxidised in evergreen species but was highly reduced in deciduous species. This transient over-reduction of PSI in deciduous species was mainly caused by the higher electron flow from PSII. Furthermore, the deciduous species showed a significantly smaller violaxanthin pool and lower non-photochemical quenching under high light conditions at 6 °C, leading to more excess light energy could not be dissipated in PSII. Hence, we propose that fluctuating light combined with chilling temperature cause the over-reduction of photosynthetic electron chain in deciduous species.

Coordination of Cyclic Electron Flow and Water-Water Cycle Facilitates Photoprotection under Fluctuating Light and Temperature Stress in the Epiphytic Orchid Dendrobium officinale.

Photosystem I (PSI) is the primary target of photoinhibition under fluctuating light (FL). Photosynthetic organisms employ alternative electron flows to protect PSI under FL. However, the understanding of the coordination of alternative electron flows under FL at temperature stresses is limited. To address this question, we measured the chlorophyll fluorescence, P700 redox state, and electrochromic shift signal in leaves of Dendrobium officinale exposed to FL at 42 °C, 25 °C, and 4 °C. Upon a sudden increase in illumination at 42 °C and 25 °C, the water–water cycle (WWC) consumed a significant fraction of the extra reducing power, and thus avoided an over-reduction of PSI. However, WWC was inactivated at 4 °C, leading to an over-reduction of PSI within the first seconds after light increased. Therefore, the role of WWC under FL is largely dependent on temperature conditions. After an abrupt increase in light intensity, cyclic electron flow (CEF) around PSI was stimulated at any temperature. Therefore, CEF and WWC showed different temperature responses under FL. Furthermore, the enhancement of CEF and WWC at 42 °C quickly generated a sufficient trans-thylakoid proton gradient (ΔpH). The inactivation of WWC at 4 °C was partially compensated for by an increased CEF activity. These findings indicate that CEF and WWC coordinate to protect PSI under FL at temperature stresses.

The different patterns of post-heat stress responses in wheat genotypes: the role of the transthylakoid proton gradient in efficient recovery of leaf photosynthetic capacity.

The frequency and severity of heat waves are expected to increase in the near future, with a significant impact on physiological functions and yield of crop plants. In this study, we assessed the residual post-heat stress effects on photosynthetic responses of six diverse winter wheat (Triticum sp.) genotypes, differing in country of origin, taxonomy and ploidy (tetraploids vs. hexaploids). After 5days of elevated temperatures (up to 38°C), the photosynthetic parameters recorded on the first day of recovery (R1) as well as after the next 4-5days of the recovery (R2) were compared to those of the control plants (C) grown under moderate temperatures. Based on the values of CO2 assimilation rate (A) and the maximum rates of carboxylation (VCmax) in R1, we identified that the hexaploid (HEX) and tetraploid (TET) species clearly differed in the strength of their response to heat stress. Next, the analyses of gas exchange, simultaneous measurements of PSI and PSII photochemistry and the measurements of electrochromic bandshift (ECS) have consistently shown that photosynthetic and photoprotective functions in leaves of TET genotypes were almost fully recovered in R2, whereas the recovery of photosynthetic and photoprotective functions in the HEX group in R2 was still rather low. A poor recovery was associated with an overly reduced acceptor side of photosystem I as well as high values of the electric membrane potential (Δψ component of the proton motive force, pmf) in the chloroplast. On the other hand, a good recovery of photosynthetic capacity and photoprotective functions was clearly associated with an enhanced ΔpH component of the pmf, thus demonstrating a key role of efficient regulation of proton transport to ensure buildup of the transthylakoid proton gradient needed for photosynthesis restoration after high-temperature episodes.

The water-water cycle is more effective in regulating redox state of photosystem I under fluctuating light than cyclic electron transport

Photosynthetic electron flux from water via photosystem II (PSII) and PSI to oxygen (water-water cycle) may act as an alternative electron sink under fluctuating light in angiosperms. We measured the P700 redox kinetics and electrochromic shift signal under fluctuating light in 11 Camellia species and tobacco leaves. Upon dark-to-light transition, these Camellia species showed rapid re-oxidation of P700. However, this rapid re-oxidation of P700 was not observed when measured under anaerobic conditions, as was in experiment with tobacco performed under aerobic conditions. Therefore, photo-reduction of O2 mediated by water-water cycle was functional in these Camellia species but not in tobacco. Within the first 10 s after transition from low to high light, PSI was highly oxidized in these Camellia species but was over-reduced in tobacco leaves. Furthermore, such rapid oxidation of PSI in these Camellia species was independent of the formation of trans-thylakoid proton gradient (ΔpH). These results indicated that in addition to ΔpH-dependent photosynthetic control, the water-water cycle can protect PSI against photoinhibition under fluctuating light in these Camellia species. We here propose that the water-water cycle is an overlooked strategy for photosynthetic regulation under fluctuating light in angiosperms.

The highly efficient NDH-dependent photosystem I cyclic electron flow pathway in the marine angiosperm Zostera marina.

Zostera marina, a fully submerged marine angiosperm with a unique evolutionary history associated with its terrestrial origin, has distinct photochemical characteristics caused by its oxygen-evolving complex (OEC) being prone to deactivation in visible light. Based on the present phylogenetic analysis, the chloroplast NADPH dehydrogenase-like (NDH) complex was found to be completed in of Z. marina, unlike other marine plants, suggesting its crucial role. Thus, the responses of electron transport to irradiation were investigated through multiple chlorophyll fluorescence techniques and Western blot analysis. Moreover, the respective contribution of the two photosystem I cyclic electron flow (PSI-CEF) pathways to the generation of trans-thylakoid proton gradient (∆pH) was also examined using inhibitors. The contributions of the two PSI-CEF pathways to ∆pH were similar; furthermore, there was a trade-off between the two pathways under excess irradiation: the PGR5/L1-dependent PSI-CEF decreased gradually following its activation during the initial illumination, while NDH-dependent PSI-CEF was activated gradually with exposure duration. OEC inactivation was continuously under excess irradiation, which exhibits a positive linear correlation with the activation of NDH-dependent PSI-CEF. We suggest that PGR5/L1-dependent PSI-CEF was preferentially activated to handle the excess electron caused by the operation of OEC during the initial illumination. Subsequently, the increasing OEC inactivation with exposure duration resulted in a deficit of electrons. Limited electrons from PSI might preferentially synthesize NADPH, which could support the function of NDH-dependent PSI-CEF to generate ∆pH and ATP via reducing ferredoxin, thereby maintaining OEC stability.

Responses of photosystem I compared with photosystem II to combination of heat stress and fluctuating light in tobacco leaves

Moderate heat stress is usually accompanied with fluctuating light in summer. Although either heat stress or fluctuating light can cause photoinhibition of photosystems I and II (PSI and PSII), it is unclear whether moderate heat stress accelerate photoinhibition under fluctuating light. Here, we measured chlorophyll fluorescence, P700 redox state and the electrochromic shift signal under fluctuating light at 25 °C and 42 °C for tobacco leaves. We found that (1) the thylakoid proton conductance was significantly enhanced at 42 °C, leading to a decline in trans-thylakoid proton gradient (ΔpH); (2) this low ΔpH at 42 °C did not decrease donor-side limitation of PSI and thermal energy dissipation in PSII; (3) the activation of cyclic electron flow (CEF) around PSI was elevated at 42 °C; and (4) the moderate heat stress did not accelerate photoinhibition of PSI and PSII under fluctuating light. These results strongly indicate that under moderate heat stress the stimulation of CEF protects PSI under fluctuating light in tobacco leaves.

Decreased photosystem II activity facilitates acclimation to fluctuating light in the understory plant Paris polyphylla

In forests, understory plants are usually exposed to sunflecks on timescales of seconds or minutes. However, it is unclear how understory plants acclimate to fluctuating light. In this study, we compared chlorophyll fluorescence, PSI redox state and the electrochromic shift signal under fluctuating light between an understory plant Paris polyphylla (Liliaceae) and a light-demanding plant Bletilla striata (Orchidaceae). Within the first seconds after transition from low to high light, PSI was highly oxidized in P. polyphylla but was highly reduced in B. striata, although both species could not generate a sufficient trans-thylakoid proton gradient (ΔpH). Furthermore, the outflow of electrons from PSI to O2 was not significant in P. polyphylla, as indicated by the P700 redox kinetics upon dark-to-light transition. Therefore, the different responses of PSI to fluctuating light between P. polyphylla and B. striata could not be explained by ΔpH formation or alternative electron transport. In contrast, upon a sudden transition from low to high light, electron flow from PSII was much lower in P. polyphylla than in B. striata, suggesting that the rapid oxidation of PSI in P. polyphylla was largely attributed to the lower PSII activity. We propose, for the first time, that down-regulation of PSII activity is an important strategy used by some understory angiosperms to cope with sunflecks.

Effect of oxygen on the non-photochemical quenching of vascular plants and potential oxygen deficiency in the stroma of PsbS-knock-out rice

The excessive and harmful light energy absorbed by the photosystem (PS) II of higher plants is dissipated as heat through a protective mechanism termed non-photochemical quenching (NPQ) of chlorophyll fluorescence. PsbS-knock-out (KO) mutants lack the trans-thylakoid proton gradient (ΔpH)-dependent part of NPQ. To elucidate the molecular mechanism of NPQ, we investigated its dependency on oxygen. The development of NPQ in wild-type (WT) rice under low-oxygen (LO) conditions was reduced to more than 50% of its original value. However, under high-oxygen (HO) conditions, the NPQ of both WT and PsbS-KO mutants recovered. Moreover, WT and PsbS-KO mutant leaves infiltrated with the ΔpH dissipating uncoupler nigericin showed increased NPQ values under HO conditions. The experiments using intact chloroplasts and protoplasts of Arabidopsis thaliana supported that the LO effects observed in rice leaves were not due to carbon dioxide deficiency. There was a noticeable 90% reduction in the half-time of P700 oxidation rate in LO-treated leaves compared with that of WT control leaves, but the HO treatment did not significantly change the half-time of P700 oxidation rate. Overall, the results obtained here indicate that the stroma of the PsbS-KO plants could be potentially under O2 deficiency. Because the functions of PsbS in rice leaves are likely to be similar to those in other higher plants, our findings offer novel insights into the role of oxygen in the development of NPQ.

A siphonous morphology affects light-harvesting modulation in the intertidal green macroalga Bryopsis corticulans (Ulvophyceae)

Main conclusionThe macroalga Bryopsis corticulans relies on a sustained protective NPQ and a peculiar body architecture to efficiently adapt to the extreme light changes of intertidal shores.During low tides, intertidal algae experience prolonged high light stress. Efficient dissipation of excess light energy, measured as non-photochemical quenching (NPQ) of chlorophyll fluorescence, is therefore required to avoid photodamage. Light-harvesting regulation was studied in the intertidal macroalga Bryopsis corticulans, during high light and air exposure. Photosynthetic capacity and NPQ kinetics were assessed in different filament layers of the algal tufts and in intact chloroplasts to unravel the nature of NPQ in this siphonous green alga. We found that the morphology and pigment composition of the B. corticulans body provides functional segregation between surface sunlit filaments (protective state) and those that are underneath and undergo severe light attenuation (light-harvesting state). In the surface filaments, very high and sustained NPQ gradually formed. NPQ induction was triggered by the formation of transthylakoid proton gradient and independent of the xanthophyll cycle. PsbS and LHCSR proteins seem not to be active in the NPQ mechanism activated by this alga. Our results show that B. corticulans endures excess light energy pressure through a sustained protective NPQ, not related to photodamage, as revealed by the unusually quick restoration of photosystem II (PSII) function in the dark. This might suggest either the occurrence of transient PSII photoinactivation or a fast rate of PSII repair cycle.

Changes in activities of both photosystems and the regulatory effect of cyclic electron flow in field-grown cotton (Gossypium hirsutum L) under water deficit

To clarify the influence of water deficit on the functionality of the photosynthetic apparatus of cotton plants, leaf gas exchange, chlorophyll a fluorescence, and P700 redox state were examined in field-grown cotton Gossypium hirsutum L. cv. Xinluzao 45. In addition, we measured changes in the P515 signal and analyzed the activity of ATP synthase and the trans-thylakoid proton gradient (ΔpH). With increasing water deficit, the net CO2 assimilation rate (AN) and stomatal conductance (gs) significantly decreased, but the maximum quantum efficiency of PSII photochemistry (Fv/Fm) did not change. The photochemical activity of photosystem II (PSII) was reflected by the photochemical quenching coefficient (qP), quantum efficiency of photosystem II [Y(II)], and electron transport rate through PSII [ETR(II)], while the activity of photosystem I (PSI) was reflected by the quantum efficiency of photosystem I [Y(I)] and the electron transport rate through PSI [ETR(I)]. Both activities were maintained under mild water deficit, but were slightly decreased under moderate water deficit. Under moderate water deficit, cyclic electron flow (CEF), the fraction of absorbed light dissipated thermally via the ΔpH- and xanthophyll-regulated process [Y(NPQ)], and the fraction of P700 oxidized under a given set of conditions [Y(ND)] increased. Our results suggest that the activities of both photosystems are stable under mild water deficit and decrease only slightly under moderate water deficit. Moderate water deficit stimulates CEF, and the stimulation of CEF is essential for protecting PSI and PSII against photoinhibition.

High temperature specifically affects the photoprotective responses of chlorophyllb-deficient wheat mutant lines.

The effects of high temperature on CO2 assimilation rate, processes associated with photosynthetic electron and proton transport, as well as photoprotective responses, were studied in chlorophyll b-deficient mutant lines (ANK-32A and ANK-32B) and wild type (WT) of wheat (Triticum aestivum L.). Despite the low chlorophyll content and chlorophyll a-to-b ratio, the non-stressed mutant plants had the similar level of CO2 assimilation and photosynthetic responses as WT. However, in ANK mutant plants exposed to prolonged high temperature episode (42°C for ~10h), we observed lower CO2 assimilation compared to WT, especially when a high CO2 supply was provided. In all heat-exposed plants, we found approximately the same level of PSII photoinhibition, but the decrease in content of photooxidizable PSI was higher in ANK mutant plants compared to WT. The PSI damage can be well explained by the level of overreduction of PSI acceptor side observed in plants exposed to high temperature, which was, in turn, the result of the insufficient transthylakoid proton gradient associated with low non-photochemical quenching and lack of ability to downregulate the linear electron transport to keep the reduction state of PSI acceptor side low enough. Compared to WT, the ANK mutant lines had lower capacity to drive the cyclic electron transport around PSI in moderate and high light; it confirms the protective role of cyclic electron transport for the protection of PSI against photoinhibition. Our results, however, also suggest that the inactivation of PSI in heat stress conditions can be the protective mechanism against photooxidative damage of chloroplast and cell structures.

Cyclic electron flow provides acclimatory plasticity for the photosynthetic machinery under various environmental conditions and developmental stages.

Photosynthetic electron flow operates in two modes, linear and cyclic. In cyclic electron flow (CEF), electrons are recycled around photosystem I. As a result, a transthylakoid proton gradient (ΔpH) is generated, leading to the production of ATP without concomitant production of NADPH, thus increasing the ATP/NADPH ratio within the chloroplast. At least two routes for CEF exist: a PROTON GRADIENT REGULATION5–PGRL1–and a chloroplast NDH-like complex mediated pathway. This review focuses on recent findings concerning the characteristics of both CEF routes in higher plants, with special emphasis paid on the crucial role of CEF in under challenging environmental conditions and developmental stages.

Electron flow from PSII to PSI under high light is controlled by PGR5 but not by PSBS.

Absence of the Proton Gradient Regulation 5 (PGR5) protein from plant chloroplasts prevents the induction of strong trans-thylakoid proton gradient (ΔpH) and consequently also the thermal dissipation of excess energy (NPQ). The absence of the PSBS protein likewise prevents the formation of ΔpH-dependent NPQ. This component of NPQ is called qE, which is nearly exclusively responsible for induction of NPQ upon increase in light intensity. On the other hand, the pgr5 mutant is not only deficient in induction of strong NPQ but it also lacks the capability to oxidize P700 upon increase in light intensity. This, in turn, results from uncontrolled electron flow toward photosystem I (PSI), which has been proposed to be caused by the lack of PSII down-regulation by NPQ and by a poor control of electron flow via the Cytochrome b6f (Cyt b6f) complex. Here we asked whether NPQ really is a component of such regulation of electron flow from PSII to PSI at high light. To this end, the two NPQ mutants pgr5 and npq4, the latter lacking the PSBS protein, were characterized. It is shown that the npq4 mutant, despite its highly reduced Plastoquinone pool, does not inhibit but rather enhances the oxidation of P700 in high light as compared to wild type. This clearly demonstrates that the control of electron flow from PSII to PSI cannot be assigned, even partially, to the down-regulation of PSII by NPQ but apparently takes place solely in Cyt b6f. Moreover, it is shown that the pgr5 mutant can induce NPQ in very high light, but still remains deficient in P700 oxidation. These results challenge the suggestion that NPQ, induced by PGR5-dependent cyclic electron transfer, would have a key role in regulation of electron transfer from PSII to PSI. Instead, the results presented here are in line with our recent suggestion that both PSII and PSI function under the same light harvesting machinery regulated by ΔpH and the PSBS protein (Tikkanen and Aro, 2014; Grieco et al., 2015).

Repetitive light pulse-induced photoinhibition of photosystem I severely affects CO2 assimilation and photoprotection in wheat leaves.

It was previously found that photosystem I (PSI) photoinhibition represents mostly irreversible damage with a slow recovery; however, its physiological significance has not been sufficiently characterized. The aim of the study was to assess the effect of PSI photoinhibition on photosynthesis in vivo. The inactivation of PSI was done by a series of short light saturation pulses applied by fluorimeter in darkness (every 10s for 15min), which led to decrease of both PSI (~60%) and photosystem II (PSII) (~15%) photochemical activity. No PSI recovery was observed within 2days, whereas the PSII was fully recovered. Strongly limited PSI electron transport led to an imbalance between PSII and PSI photochemistry, with a high excitation pressure on PSII acceptor side and low oxidation of the PSI donor side. Low and delayed light-induced NPQ and P700(+) rise in inactivated samples indicated a decrease in formation of transthylakoid proton gradient (ΔpH), which was confirmed also by analysis of electrochromic bandshift (ECSt) records. In parallel with photochemical parameters, the CO2 assimilation was also strongly inhibited, more in low light (~70%) than in high light (~45%); the decrease was not caused by stomatal closure. PSI electron transport limited the CO2 assimilation at low to moderate light intensities, but it seems not to be directly responsible for a low CO2 assimilation at high light. In this regard, the possible effects of PSI photoinhibition on the redox signaling in chloroplast and its role in downregulation of Calvin cycle activity are discussed.

Low PSI content limits the photoprotection of PSI and PSII in early growth stages of chlorophyll b-deficient wheat mutant lines.

In vivo analyses of electron and proton transport-related processes as well as photoprotective responses were carried out at different stages of growth in chlorophyll b (Chl b)-deficient mutant lines (ANK-32A and ANK-32B) and wild type (WT) of wheat (Triticum aestivum L.). In addition to a high Chl a-b ratio, ANK mutants had a lower content of photo-oxidizable photosystem I (PSI, P m), and several parameters indicated a low PSI/PSII ratio. Moreover, simultaneous measurements of Chl fluorescence and P700 indicated a shift of balance between redox poise of the PSII acceptor side and the PSII donor side, with preferential reduction of the plastoquinone pool, resulting in an over reduced PSI acceptor side (high Φ NA values). This was the probable reason for PSI inactivation observed in the ANK mutants, but not in WT. In later growth phases, we observed partial relief of "chlorina symptoms," toward WT. Measurements of ΔA 520 decay confirmed that, in early growth stages, the ANK mutants with low PSI content had a limited capacity to build up the transthylakoid proton gradient (ΔpH) needed to trigger non-photochemical quenching (NPQ) and to regulate the electron transport by cytochrome b 6/f. Later, the increase in the PSI/PSII ratio enabled ANK mutants to reach full NPQ, but neither over reduction of the PSI acceptor side nor PSI photoinactivation due to imbalance between the activity of PSII and PSI was mitigated. Thus, our results support the crucial role of proper regulation of linear electron transport in the protection of PSI against photoinhibition. Moreover, the ANK mutants of wheat showing the dynamic developmental changes in the PSI/PSII ratio are presented here as very useful models for further studies.