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Linear electron flow (LEF) and cyclic electron flow (CEF) compete for light-driven electrons transferred from the acceptor side of photosystem I (PSI). Under anoxic conditions, such highly reducing electrons also could be used for hydrogen (H2) production via electron transfer between ferredoxin and hydrogenase in the green alga Chlamydomonas reinhardtii. Partitioning between LEF and CEF is regulated through PROTON-GRADIENT REGULATION5 (PGR5). There is evidence that partitioning of electrons also could be mediated via PSI remodeling processes. This plasticity is linked to the dynamics of PSI-associated light-harvesting proteins (LHCAs) LHCA2 and LHCA9. These two unique light-harvesting proteins are distinct from all other LHCAs because they are loosely bound at the PSAL pole. Here, we investigated photosynthetic electron transfer and H2 production in single, double, and triple mutants deficient in PGR5, LHCA2, and LHCA9. Our data indicate that lhca2 and lhca9 mutants are efficient in photosynthetic electron transfer, that LHCA2 impacts the pgr5 phenotype, and that pgr5/lhca2 is a potent H2 photo-producer. In addition, pgr5/lhca2 and pgr5/lhca9 mutants displayed substantially different H2 photo-production kinetics. This indicates that the absence of LHCA2 or LHCA9 impacts H2 photo-production independently, despite both being attached at the PSAL pole, pointing to distinct regulatory capacities.

The cytochrome b6f complex (b6f) has been initially considered as the ferredoxin-plastoquinone reductase (FQR) during cyclic electron flow (CEF) with photosystem I that is inhibited by antimycin A (AA). The binding of AA to the b6f Qi-site is aggravated by heme-ci, which challenged the FQR function of b6f during CEF. Alternative models suggest that PROTON GRADIENT REGULATION5 (PGR5) is involved in a b6f-independent, AA-sensitive FQR. Here, we show in Chlamydomonas reinhardtii that the b6f is conditionally inhibited by AA in vivo and that the inhibition did not require PGR5. Instead, activation of the STT7 kinase upon anaerobic treatment induced the AA sensitivity of b6f which was absent from stt7-1. However, a lock in State 2 due to persisting phosphorylation in the phosphatase double mutant pph1;pbcp did not increase AA sensitivity of electron transfer. The latter required a redox poise, supporting the view that state transitions and CEF are not coercively coupled. This suggests that the b6f-interacting kinase is required for structure-function modulation of the Qi-site under CEF favoring conditions. We propose that PGR5 and STT7 independently sustain AA-sensitive FQR activity of the b6f. Accordingly, PGR5-mediated electron injection into an STT7-modulated Qi-site drives a Mitchellian Q cycle in CEF conditions.

To sustain CO2fixation in the Calvin Benson-Bassham cycle, the multi-level regulation of the chloroplast ATP synthase (CF1Fo) is crucial to balance the ATP output of photosynthesis with protection of the apparatus. A well-studied mechanism is thiol modulation which involves reversible cleavage of a disulfide in the γ-subunit of CF1Fo. The disulfide in the dark-adapted enzyme from land plants establishes an inactive CF1Fosince it requires a too high transmembrane electrochemical proton gradientto catalyze ATP synthesis and hydrolysis. Here, we show in the green algaChlamydomonas reinhardtiithat CF1Fois differently regulated. Our in vivo investigation monitors the electrochromic shift (ECS) of pigments and reveals that an unusual hairpin structure in the γ-subunit redox domain lowers the algal activation threshold in CF1Foby ~1.4 ECS units. Thereby, thiol modulation is short-circuited so that the algal enzyme remains active in darkness. Prevention of γ-disulfide formation in a redox cysteine mutant increased the dark activity slightly which had a mild effect on raising thein darkness. Compared to wild type, the enzymatic activity was diminished ~3-fold in cells which carry a plant-like CF1Fo. These mutants sustained athat was ~2.6 ECS units lower in the dark. Our findings point to a diversity in the regulatory integration of CF1Fofor photosynthesis in Viridiplantae.

Linear photosynthetic electron flow (LEF) produces NADPH and generates a proton electrochemical potential gradient across the thylakoid membrane to synthesize ATP, both of which are required for CO2 fixation. As cellular demand for ATP and NADPH varies, cyclic electron flow (CEF) between Photosystem I and the cytochrome b6f complex (b6f) produces extra ATP. b6f regulates LEF and CEF via photosynthetic control, which is a pH-dependent b6f slowdown of plastoquinol oxidation at the lumenal site. This protection mechanism is triggered at more alkaline lumen pH in the pgr1 (proton gradient regulation 1) mutant of the vascular plant Arabidopsis (Arabidopsis thaliana), which contains a Pro194Leu substitution in the b6f Rieske Iron-sulfur protein Photosynthetic Electron Transfer C (PETC) subunit. In this work, we introduced the equivalent pgr1 mutation in the green alga Chlamydomonas reinhardtii to generate PETC-P171L. Consistent with the pgr1 phenotype, PETC-P171L displayed impaired NPQ induction along with slower photoautotrophic growth under high light conditions. Our data provide evidence that the ΔpH component in PETC-P171L depends on oxygen availability. Only under low oxygen conditions was the ΔpH component sufficient to trigger a phenotype in algal PETC-P171L where the mutant b6f was more restricted to oxidize the plastoquinol pool and showed diminished electron flow through the b6f complex. These results demonstrate that photosynthetic control of different stringency are established in C. reinhardtii depending on the cellular metabolism, and the lumen pH-sensitive PETC-P171L was generated to read out various associated effects.

Linear photosynthetic electron flow (LEF) produces NADPH and generates a proton electrochemical potential gradient across the thylakoid membrane used to synthesize ATP, both of which are required for CO2 fixation. As cellular demand for ATP and NADPH are variable, cyclic electron flow (CEF) between PSI and cytochrome b6f complex (b6f) produces extra ATP. The b6f regulates LEF and CEF via photosynthetic control, which is a pH-dependent b6f slowdown of plastoquinol oxidation at the lumenal site. This protection mechanism is triggered at more alkaline lumen pH in the pgr1 mutant of the vascular plant Arabidopsis thaliana, carrying Pro194Leu in the b6f Rieske Iron-sulfur protein. In this work, we introduced pgr1 mutation in the green alga Chlamydomonas reinhardtii (PETC-P171L). Consistent with pgr1 phenotype, PETC-P171L displayed an impaired NPQ induction along with slower photoautotrophic growth under high light conditions. Our data provides evidence that the ΔpH component in PETC-P171L is dependent on oxygen availability. Only under low oxygen conditions the ΔpH component was sufficient to trigger a phenotype in algal PETC-P171L where the mutant b6f was more restricted to oxidize the PQ pool and showed a diminished electron flow through the b6f complex.One sentence summaryChange of PETC to P171L via site directed mutagenesis alters the pH dependency of the photosynthetic control mechanism

The chloroplast ATP synthase (CF1Fo) contains a specific feature to the green lineage: a γ-subunit redox domain that contains a cysteine couple which interacts with the torque-transmitting βDELSEED-loop. This thiol modulation equips CF1Fo with an important environmental fine-tuning mechanism. In vitro, disulfide formation in the γ-redox domain slows down the activity of the CF1Fo at low transmembrane electrochemical proton gradient ( [Formula: see text] ), which agrees with its proposed role as chock based on recently solved structure. The γ-dithiol formation at the onset of light is crucial to maximize photosynthetic efficiency since it lowers the [Formula: see text] activation level for ATP synthesis in vitro. Here, we validate these findings in vivo by utilizing absorption spectroscopy in Arabidopsis thaliana. To do so, we monitored the [Formula: see text] present in darkness and identified its mitochondrial sources. By following the fate and components of light-induced extra [Formula: see text] , we estimated the ATP lifetime that lasted up to tens of minutes after long illuminations. Based on the relationship between [Formula: see text] and CF1Fo activity, we conclude that the dithiol configuration in vivo facilitates photosynthesis by driving the same ATP synthesis rate at a significative lower [Formula: see text] than in the γ-disulfide state. The presented in vivo findings are an additional proof of the importance of CF1Fo thiol modulation, reconciling biochemical in vitro studies and structural insights.

Ferredoxin-plastoquinone reductase (FQR) activity during cyclic electron flow (CEF) was first ascribed to the cytochromeb6fcomplex (b6f). However, this was later dismissed sinceb6finhibition by antimycin-A (AA) could not be reproduced. AA presumably fails to ligate with haembh, at variance with cytochromebc1complex, owing to a specific Qi-site occupation inb6f. Currently, PROTON GRADIENT REGULATION5 (PGR5) and the associated PGR5-Like1 are considered as FQR in the AA-sensitive CEF pathway. Here, we show that theb6fis conditionally inhibited by AA in a PGR5-independent manner when CEF is promoted. AA inhibition, demonstrated by singleb6fturnover and electron transfer measurements, coincided with an altered Qi-site function which required Stt7 kinase activation by a strongly reduced plastoquinone pool. Thus, PGR5 and Stt7 were necessary forb6factivity and AA-sensitive electron transfer in CEF-favouring conditions. Extending previous findings, a new FQR activity model of theb6fis discussed.

The chloroplast ATP synthase (CF1Fo) contains a specific feature to the green lineage: a γ-subunit redox domain which contains a cysteine couple and interacts with the torque-generating βDELSEED-loop. Based on the recently solved structure of this domain, it was proposed to function as a chock.In vitro,γ-disulfide formation slows down the activity of the CF1Foat low transmembrane electrochemical proton gradient. Here, we utilizein vivoabsorption spectroscopy measurements for functional CF1Foactivity characterization in Arabidopsis leaves. The spectroscopic method allows us to measure thepresent in dark-adapted leaves, and to identify its mitochondrial sources. Furthermore, we follow the fate of the extragenerated by an illumination, including its osmotic and electric components, and from there we estimate the lifetime of the light-generated ATP. In contrast with a previous report [Joliot and Joliot, Biochim. Biophys. Acta, 1777 (2008) 676-683], the CF1Foγ-subunit exists mostly in an oxidized form in the dark-adapted state. To study the redox regulation of the CF1Fo, we used thiol agent infiltration in WT and a mutant that does not form the γ-disulfide. The obtained-dependent CF1Foactivity profile in the two γ-redox statesin vivoreconciles with previous biochemicalin vitrofindings [Junesch and Gräber, Biochim. Biophys. Acta, 893 (1987) 275-288]. The highest rates of ATP synthesis we measured in the two γ-redox state were similar at high. In the presence of the γ-dithiol, similar rates were obtained at a ~45 mV lowervalue compared to the oxidized state, which closely resembled the energetic gap of 0.7 ΔpH units reportedin vitro.

Whereas photosynthetic function under steady-state light conditions has been well characterized, little is known about its changes that occur in response to light fluctuations. Chlororespiration, a simplified respiratory chain, is widespread across all photosynthetic lineages, but its role remains elusive. Here, we show that chlororespiration plays a crucial role in intermittentlight conditions in the green alga Chlamydomonas reinhardtii. Chlororespiration, which is localized in thylakoid membranes together with the photosynthetic electron transfer chain, involves plastoquinone reduction and plastoquinol oxidation by a Plastid Terminal Oxidase (PTOX). We show that PTOX activity is critical for growth under intermittent light, with severe growth defects being observed in a mutant lacking PTOX2, the major plastoquinol oxidase. We demonstrate that the hampered growth results from a major change in the kinetics of redox relaxation of the photosynthetic electron transfer chain during the dark periods. This change, in turn, has a dramatic effect on the physiology of photosynthesis during the light periods, notably stimulating cyclic electron flow at the expense of the linear electron flow.

International audience; The chloroplast F ⁠ 1 F ⁠ o-ATP synthase (CF ⁠ 1 F ⁠ o) drives ATP synthesis and the reverse reaction of ATP hydrolysis. The enzyme evolved in a cellular environment where electron transfer processes and molecular oxygen are abundant , and thiol modulation in the γ-subunit via thioredoxin is important for its ATPase activity regulation. Especially under high light, oxygen can be reduced and forms reactive oxygen species (ROS) which can oxidize CF ⁠ 1 F ⁠ o among various other biomolecules. Mutation of the conserved ROS targets resulted in a tolerant enzyme, suggesting that ROS might play a regulatory role. The mutations had several side effects in vitro, including disturbance of the ATPase redox regulation [F. Buchert et al., Biochim. Biophys. Acta, 1817 (2012) 2038–2048]. This would prevent disentanglement of thiol-and ROS-specific modes of regulation. Here, we used the F ⁠ 1 catalytic core in vitro to identify a point mutant with a functional ATPase redox regulation and increased H ⁠ 2 O ⁠ 2 tolerance. In the next step, the mutation was introduced into Chlamydomonas reinhardtii CF ⁠ 1 F ⁠ o , thereby allowing us to study the physiological role of ROS regulation of the enzyme in vivo. We demonstrated in high light experiments that CF ⁠ 1 F ⁠ o ROS targets were involved in the significant inhibition of ATP synthesis rates. Molecular events upon modification of CF ⁠ 1 F ⁠ o by ROS will be considered.

The chloroplast ATP synthase (CFoCF1) is crucial for photosynthesis since it controls the transmembrane electrochemical proton potential gradient which is generated by light-driven electron and proton transfer processes. Thereby, CFoCF1 produces ATP and, together with the provided reducing equivalents, it powers CO2 fixation. Since CFoCF1 represents the major proton-conducting element in photosynthesis, it is important to understand its tight integration in a feedback-regulated cellular energetics framework. CFoCF1 from green microalgae resembles its counterpart from vascular plants by consisting of a membrane-embedded CFo proton channel and a catalytic CF1 portion. The algal enzyme has been subject of numerous functional studies owing to the feasible generation and maintenance of point mutants and knockout strains. Thereby, a wealth of information has been obtained in vitro and in vivo concerning the genetic control of complex assembly and regulatory mechanisms of the enzyme on various levels. While many findings are transferable to vascular plants, some may concern specific adaptations of the microalgal enzyme. This chapter will present a selection of previous outcomes and summarize our current understanding of algal CFoCF1 regulation.

Proton gradient regulation 5 (PGR5) is involved in the control of photosynthetic electron transfer, but its mechanistic role is not yet clear. Several models have been proposed to explain phenotypes such as a diminished steady-state proton motive force (pmf) and increased photodamage of photosystem I (PSI). Playing a regulatory role in cyclic electron flow (CEF) around PSI, PGR5 contributes indirectly to PSI protection by enhancing photosynthetic control, which is a pH-dependent down-regulation of electron transfer at the cytochrome b6f complex (b6f). Here, we re-evaluated the role of PGR5 in the green alga Chlamydomonas reinhardtii and conclude that pgr5 possesses a dysfunctional b6f. Our data indicate that the b6f low-potential chain redox activity likely operated in two distinct modes — via the canonical Q cycle during linear electron flow and via an alternative Q cycle during CEF, which allowed efficient oxidation of the low-potential chain in the WT b6f. A switch between the two Q cycle modes was dependent on PGR5 and relied on unknown stromal electron carrier(s), which were a general requirement for b6f activity. In CEF-favoring conditions, the electron transfer bottleneck in pgr5 was the b6f, in which insufficient low-potential chain redox tuning might account for the mutant pmf phenotype. By attributing a ferredoxin-plastoquinone reductase activity to the b6f and investigating a PGR5 cysteine mutant, a current model of CEF is challenged.

Thiol-based redox-regulation is vital for coordinating chloroplast functions depending on illumination and has been throroughly investigated for thioredoxin-dependent processes. In parallel, glutathione reductase (GR) maintains a highly reduced glutathione pool, enabling glutathione-mediated redox buffering. Yet, how the redox cascades of the thioredoxin and glutathione redox machineries integrate metabolic regulation and detoxification of reactive oxygen species remains largely unresolved because null mutants of plastid/mitochondrial GR are embryo-lethal in Arabidopsis thaliana. To investigate whether maintaining a highly reducing stromal glutathione redox potential (EGSH ) via GR is necessary for functional photosynthesis and plant growth, we created knockout lines of the homologous enzyme in the model moss Physcomitrella patens. In these viable mutant lines, we found decreasing photosynthetic performance and plant growth with increasing light intensities, whereas ascorbate and zeaxanthin/antheraxanthin levels were elevated. By in vivo monitoring stromal EGSH dynamics, we show that stromal EGSH is highly reducing in wild-type and clearly responsive to light, whereas an absence of GR leads to a partial glutathione oxidation, which is not rescued by light. By metabolic labelling, we reveal changing protein abundances in the GR knockout plants, pinpointing the adjustment of chloroplast proteostasis and the induction of plastid protein repair and degradation machineries. Our results indicate that the plastid thioredoxin system is not a functional backup for the plastid glutathione redox systems, whereas GR plays a critical role in maintaining efficient photosynthesis.

Photosynthesis needs to adjust to dynamically changing light intensities in order to maximize its efficiency, notably by the employment of alternative electron pathways. One of them is chlororespiration - initially described in Chlamydomonas reinhardtii. This electron transfer pathway, found in all photosynthetic lineages, consists of a reduction of plastoquinone (PQ) through an NAD(P)H:PQ oxidoreductase and quinol (PQH2) oxidation by Plastid Terminal Oxidase, PTOX. Hence, chlororespiration constitutes an electron pathway potentially antagonistic to the linear photosynthetic electron flow from H2O to CO2. However, the limited flow chlororespiratory enzymes can sustain suggests that their relative contribution, at least in the light and in steady-state conditions, is insubstantial. Here, we focused on the involvement of PTOX in Chlamydomonas reinhardtii during transitions from dark to light and vice versa. We show that the kinetics of redox relaxation of the chloroplast in the dark was greatly affected when PTOX2, the major plastoquinol oxidase in Chlamydomonas, is lacking. Importantly, we show that this has a direct physiological relevance, as the growth of a PTOX2-lacking mutant is markedly slower in intermittent light. The latter can be rationalized in terms of a decreased flux sustained by photosystem II due to a redox limitation at the acceptor side of the PSI during the illumination periods. We finally show that the long-term regulation of cyclic electron flow around PSI is strongly affected in the PTOX2 mutant, substantiating an important role of chlororespiration in the maintenance of chloroplast redox balance.

The chloroplast F

The vast majority of organisms produce ATP by a membrane-bound rotating protein complex, termed F-ATP synthase. In chloroplasts, the corresponding enzyme generates ATP by using a transmembrane proton gradient generated during photosynthesis, a process releasing high amounts of molecular oxygen as a natural byproduct. Due to its chemical properties, oxygen can be reduced incompletely which generates several highly reactive oxygen species (ROS) that are able to oxidize a broad range of biomolecules. In extension to previous studies it could be shown that ROS dramatically decreased ATP synthesis in situ and affected the CF1 portion in vitro. A conserved cluster of three methionines and a cysteine on the chloroplast γ subunit could be identified by mass spectrometry to be oxidized by ROS. Analysis of amino acid substitutions in a hybrid F1 assembly system indicated that these residues were exclusive catalytic targets for hydrogen peroxide and singlet oxygen, although it could be deduced that additional unknown amino acid targets might be involved in the latter reaction. The cluster was tightly integrated in catalytic turnover since mutants varied in MgATPase rates, stimulation by sulfite and chloroplast-specific γ subunit redox-modulation. Some partial disruptions of the cluster by mutagenesis were dominant over others regarding their effects on catalysis and response to ROS.

Singlet oxygen ((1)O(2)) produced in plants during photosynthesis has a strong damaging effect not only on both photosystems but also on the whole photosynthetic machinery. This is also applicable for the adenosine triphosphate (ATP) synthase. Here we describe the impact of (1)O(2) generated by the photosensitizer Rose Bengal on the ATP hydrolysis and ATP-driven proton translocation activity of CF1CFo. Both activities were reduced dramatically within 1min of exposure. Interestingly, it is shown that oxidized thylakoid ATP synthase is more susceptible to (1)O(2) than CF1CFo in its reduced state, a new insight on the mechanism of (1)O(2) interaction with the gamma subunit.