Biology subjects

Buchert, F.

Publications and source records attributed to Buchert, F..

9 recordsLinked to original sources

bioRxiv2026

ATP synthase deficiency promotes prolonged hydrogenproduction in Chlamydomonas

Photosynthetic electron transport is tightly regulated to balance ATP and NADPH production and prevent over-reduction of the photosynthetic machinery. How this balance is maintained under severe ATP limitation, particularly under anoxia, is not fully resolved. Here, we used several deletion mutants of chloroplast ATP synthase subunits from Chlamydomonas reinhardtii to examine how electron flow is reorganized under anoxic conditions through the interplay between linear electron flow, cyclic electron flow, and alternative electron sinks. Using a combination of optical spectroscopy methods and membrane inlet mass spectrometry, we characterized changes in photosynthetic electron transport, proton motive force relaxation and gas exchange. Under oxic conditions, the {Delta}atpH mutant (lacking the c-subunit) showed strong acceptor-side limitation at photosystem I (PSI), whereas atpC1-2 (lacking the {gamma}-subunit) resembled the wild type. Under anoxia, this limitation was partially relieved, coinciding with sustained electron transport and prolonged hydrogen production. In particular, the {Delta}atpH mutant exhibited increased cyclic electron flow under oxic conditions and maintained PSI activity under anoxia despite impaired ATP synthesis. Prolonged hydrogen production was also observed in atpC1-2, which did not share the increased respiration of {Delta}atpH. Hydrogenase activity under anoxia is consistent with a role as an electron sink that may help alleviate PSI acceptor-side limitation and rebalance the redox state of the electron transport chain. These findings show that ATP synthase deficiency reorganizes photosynthetic electron flow and promotes prolonged hydrogen production, and point to hydrogenase as a candidate electron sink under energy-constrained conditions.

plant biology↗

bioRxiv2026

Lateral organization of cytochrome b6f in thylakoid membranes controls photosynthetic electron transfer efficiency

Photosynthetic electron transfer relies on the coordinated function and spatial organization of large protein complexes within the thylakoid membrane. The cytochrome b6f complex (b6f) functionally interconnects photosystem (PS) II and PSI in photosynthetic electron transfer and is equally distributed between appressed and non-appressed thylakoid membranes. Here, we investigate the functional link between the lateral distribution of b6f and efficient photosynthetic electron flow in Chlamydomonas reinhardtii. We engineered strains with stromal fusions between PetA of b6f and two fluorescent proteins (FPs) of different molecular mass: Clover and ATeam. Under oxic conditions, these strains exhibited significantly slower electron transfer rates (ETR), lower PSII quantum yields, and increased donor-side limitation of PSI. State transitions were diminished in the fusion strains, accompanied by a strong impairment of STT7-dependent function, suggesting that the presence of fused FPs at b6f interfere with STT7 function. Yet, ETR phenotypes were STT7-independent and FP fusion did not impact intrinsic b6f function. In situ cryogenic electron tomography revealed a significant depletion of b6f from appressed thylakoid membranes in the ATeam strains, while the overall membrane protein concentration remained unchanged. Overall, our data indicate that a balanced distribution of b6f between appressed and non-appressed thylakoid membranes is essential for regulating photosynthetic electron transfer, highlighting the functional importance of thylakoid molecular architecture in vivo.

plant biology↗

bioRxiv2025

N-terminal region of PetD is essential for cytochrome b6f function and controls STT7 kinase activity via STT7-dependent feedback loop phosphorylation

The cytochrome b6f complex (b6f) links photosystem I (PSI) and photosystem II (PSII) in the photosynthetic electron transport chain and is distributed across appressed and non-appressed thylakoid membranes. The b6f also activates the state-transition 7 kinase (STT7), which phosphorylates light-harvesting complex proteins, triggering their migration to enable energy redistribution between PSII and PSI. STT7-dependent phosphorylation has also been observed at T4 in the N-terminal domain of the b6f subunit-IV (PetD), though its functional significance has remained unclear. To investigate its role, we generated several chloroplast mutants. The phosphomimic mutation PetD T4E - but not T4A - inhibited STT7 kinase activity, as indicated by the absence of STT7-dependent phosphorylation and a State 1-locked phenotype. This reveals a novel feedback mechanism regulating STT7-dependent phosphorylation. Deletion of the first five N-terminal amino acids similarly inhibited STT7 activity and additionally disrupted electron transfer, underscoring a crucial role of the PetD N-terminus in b6f function.

plant biology↗

bioRxiv2024

Tethering ferredoxin-NADP+ reductase to photosystem I promotes photosynthetic cyclic electron transfer

Fixing CO2 via photosynthesis requires ATP and NADPH. Linear electron transfer (LET) supplies both metabolites, yet depending on environmental conditions, additional ATP is required which can be generated by cyclic electron transfer (CET). How the balance between LET and CET is set remains largely unknown. Ferredoxin(FD)-NADP+ reductase (FNR) has been suggested to act as the switch, channelling photosynthetic electrons to LET when it is bound to photosystem I (PSI) or CET when bound to cytochrome b6f. The essential role of FNR in LET precludes the use of a direct gene knock-out to test this hypothesis. We circumvented this problem by using CRISPR-Cas9 gene editing in Chlamydomonas reinhardtii to create a chimeric form of FNR tethered to PSI via PSAF. Chimeric FNR mutants exhibited impaired photosynthetic growth relative to the wild type, and decreased LET and non-photochemical quenching (NPQ) under high CO2 conditions. However, higher rates of CET and increased NPQ were found in the mutants under low CO2 or low O2 conditions where CET is normally up-regulated. Thus, rather than PSI-FNR interactions promoting LET, tethering FNR to PSI promotes CET at the expense of LET and CO2 fixation.

biochemistry↗

bioRxiv2022

The unusual chloroplast ATP synthase redox domain ensures enzyme activity and elevates the electrochemical proton gradient in dark-adapted Chlamydomonas reinhardtii.

To maintain CO2 fixation in the Calvin Benson-Bassham cycle, multi-step 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; a light/dark regulation through reversible cleavage of a disulfide in the CF1Fo {gamma}-subunit. The disulfide hampers ATP synthesis and hydrolysis reactions in dark-adapted CF1Fo from land plants by increasing the required transmembrane electrochemical proton gradient [Formula]. Here, we show in Chlamydomonas reinhardtii that algal CF1Fo is differently regulated in vivo. A specific hairpin structure in the {gamma}-subunit redox domain disconnects activity regulation from disulfide formation in the dark. Electrochromic shift measurements suggested that the hairpin kept wild type CF1Fo active whereas the enzyme was switched off in algal mutant cells expressing a plant-like hairpin structure. The hairpin segment swap resulted in an elevated [Formula] threshold to activate plant-like CF1Fo, increased by [~]1.4 photosystem (PS) I charge separations. The resulting dark-equilibrated [Formula] dropped in the mutants by [~]2.7 PSI charge separation equivalents. Photobioreactor experiments showed no phenotypes in autotrophic aerated mutant cultures. In contrast, chlorophyll fluorescence measurements under heterotrophic dark conditions point to a reduced plastoquinone pool in cells with the plant-like CF1Fo as the result of bioenergetic bottlenecks. Our results suggest that the lifestyle of Chlamydomonas reinhardtii requires a specific CF1Fo dark regulation that partakes in metabolic coupling between the chloroplast and acetate-fueled mitochondria. Significance StatementThe microalga Chlamydomonas reinhardtii exhibits a non-classical thiol modulation of the chloroplast ATP synthase for the sake of metabolic flexibility. The redox switch, although established, was functionally disconnected in vivo thanks to a hairpin segment in the {gamma}-subunit redox domain. Dark enzymatic activity was prevented by replacing the algal hairpin segment with the one from land plants, restoring a classical thiol modulation pattern. Thereby, ATP was saved at the expense of thylakoid membrane energization levels in the dark. However, metabolism was impaired upon silencing dark ATPase activity, indicating that a functional disconnect from the redox switch represents an adaptation to different ecological niches.

plant biology↗

bioRxiv2020

Cytochrome b6f complex inhibition by antimycin-A requires Stt7 kinase activation but not PGR5.

Ferredoxin-plastoquinone reductase (FQR) activity during cyclic electron flow (CEF) was first ascribed to the cytochrome b6f complex (b6f). However, this was later dismissed since b6f inhibition by antimycin-A (AA) could not be reproduced. AA presumably fails to ligate with haem bh, at variance with cytochrome bc1 complex, owing to a specific Qi-site occupation in b6f. Currently, PROTON GRADIENT REGULATION5 (PGR5) and the associated PGR5-Like1 are considered as FQR in the AA-sensitive CEF pathway. Here, we show that the b6f is conditionally inhibited by AA in a PGR5-independent manner when CEF is promoted. AA inhibition, demonstrated by single b6f turnover 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 for b6f activity and AA-sensitive electron transfer in CEF-favouring conditions. Extending previous findings, a new FQR activity model of the b6f is discussed.

plant biology↗

bioRxiv2020

ECS-based investigation of chloroplast ATP synthase regulation

The chloroplast ATP synthase (CF1Fo) contains a specific feature to the green lineage: a {gamma}-subunit redox domain which contains a cysteine couple and interacts with the torque-generating {beta}DELSEED-loop. Based on the recently solved structure of this domain, it was proposed to function as a chock. In vitro, {gamma}-disulfide formation slows down the activity of the CF1Fo at low transmembrane electrochemical proton gradient [Formula]. Here, we utilize in vivo absorption spectroscopy measurements for functional CF1Fo activity characterization in Arabidopsis leaves. The spectroscopic method allows us to measure the [Formula] present in dark-adapted leaves, and to identify its mitochondrial sources. Furthermore, we follow the fate of the extra [Formula] generated 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 CF1Fo {gamma}-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 {gamma}-disulfide. The obtained [Formula] -dependent CF1Fo activity profile in the two {gamma}-redox states in vivo reconciles with previous biochemical in vitro findings [Junesch and Graber, Biochim. Biophys. Acta, 893 (1987) 275-288]. The highest rates of ATP synthesis we measured in the two {gamma}-redox state were similar at high [Formula]. In the presence of the {gamma}-dithiol, similar rates were obtained at a ~45 mV lower [Formula] value compared to the oxidized state, which closely resembled the energetic gap of 0.7 {Delta}pH units reported in vitro.

plant biology↗

bioRxiv2019

PGR5 is required for efficient Q cycle in the cytochrome b6f complex during cyclic electron flow

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 downregulation 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, attributing a ferredoxin-plastoquinone reductase activity to the b6f. The latter mode allowed efficient oxidation of the low-potential chain in the WT b6f. A switch between the two Q cycle modes was dependent of PGR5 and relied on unknown stromal electron carrier(s), which were a general requirement for b6f activity. In CEF-favouring conditions the electron transfer bottleneck in pgr5 was the b6f and insufficient flexibility in the low-potential chain redox tuning might account for the mutant pmf phenotype and the secondary consequences. Models of our findings are discussed.

plant biology↗

bioRxiv2017

Photosynthesis is heavily chlororespiration-sensitive under fluctuating light conditions

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.

plant biology↗