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Abstract Background Lutein is the most abundant xanthophyll in the photosynthetic apparatus of higher plants. It binds to site L1 of all Lhc proteins, whose occupancy is indispensable for protein folding and quenching chlorophyll triplets. Thus, the lack of a visible phenotype in mutants lacking lutein has been surprising. Results We have re-assessed the lut2.1 phenotypes through biochemical and spectroscopic methods. Lhc proteins from the lut2.1 mutant compensate the lack of lutein by binding violaxanthin in sites L1 and L2. This substitution reduces the capacity for regulatory mechanisms such as NPQ, reduces antenna size, induces the compensatory synthesis of Antheraxanthin + Zeaxanthin, and prevents the trimerization of LHCII complexes. In vitro reconstitution shows that the lack of lutein per se is sufficient to prevent trimerization. lut2.1 showed a reduced capacity for state I – state II transitions, a selective degradation of Lhcb1 and 2, and a higher level of photodamage in high light and/or low temperature, suggesting that violaxanthin cannot fully restore chlorophyll triplet quenching. In vitro photobleaching experiments and time-resolved spectroscopy of carotenoid triplet formation confirmed this hypothesis. The npq1lut2.1 double mutant, lacking both zeaxanthin and lutein, is highly susceptible to light stress. Conclusion Lutein has the specific property of quenching harmful 3Chl* by binding at site L1 of the major LHCII complex and of other Lhc proteins of plants, thus preventing ROS formation. Substitution of lutein by violaxanthin decreases the efficiency of 3Chl* quenching and causes higher ROS yield. The phenotype of lut2.1 mutant in low light is weak only because rescuing mechanisms of photoprotection, namely zeaxanthin synthesis, compensate for the ROS production. We conclude that zeaxanthin is effective in photoprotection of plants lacking lutein due to the multiple effects of zeaxanthin in photoprotection, including ROS scavenging and direct quenching of Chl fluorescence by binding to the L2 allosteric site of Lhc proteins.

Some cyanobacteria can do photosynthesis using not only visible but also far-red light that is unused by most other oxygenic photoautotrophs because of its lower energy content. These species have a modified photosynthetic apparatus containing red-shifted pigments. The incorporation of red-shifted pigments decreases the photochemical efficiency of photosystem I and, especially, photosystem II, and it might affect the distribution of excitation energy between the two photosystems with possible consequences on the activity of the entire electron transport chain. To investigate the in vivo effects on photosynthetic activity of these pigment changes, we present here the adaptation of a spectroscopic method, based on a physical phenomenon called ElectroChromic Shift (ECS), to the far-red absorbing cyanobacteria Acaryochloris marina and Chroococcidiopsis thermalis PCC7203. ECS measures the electric field component of the trans-thylakoid proton motive force generated by photosynthetic electron transfer. We show that ECS can be used in these cyanobacteria to investigate in vivo the stoichiometry of photosystem I and photosystem II and their absorption cross-section, as well as the overall efficiency of light energy conversion into electron transport. Our results indicate that both species use visible and far-red light with similar efficiency, despite significant differences in their light absorption characteristics. ECS thus represents a new non-invasive tool to study the performance of naturally occurring far-red photosynthesis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In oxygenic photosynthesis, state transitions distribute light energy between PSI and PSII. This regulation involves reduction of the plastoquinone pool, activation of the state transitions 7 (STT7) protein kinase by the cytochrome (cyt) b6f complex, and phosphorylation and migration of light harvesting complexes II (LHCII). In this study, we show that in Chlamydomonas reinhardtii, the C-terminus of the cyt b6 subunit PetB acts on phosphorylation of STT7 and state transitions. We used site-directed mutagenesis of the chloroplast petB gene to truncate (remove L215b6) or elongate (add G216b6) the cyt b6 subunit. Modified complexes are devoid of heme ci and degraded by FTSH protease, revealing that salt bridge formation between cyt b6 (PetB) and Subunit IV (PetD) is essential to the assembly of the complex. In double mutants where FTSH is inactivated, modified cyt b6f accumulated but the phosphorylation cascade was blocked. We also replaced the arginine interacting with heme ci propionate (R207Kb6). In this modified complex, heme ci is present but the kinetics of phosphorylation are slower. We show that highly phosphorylated forms of STT7 accumulated transiently after reduction of the PQ pool and represent the active forms of the protein kinase. The phosphorylation of the LHCII targets is favored at the expense of the protein kinase, and the migration of LHCII toward PSI is the limiting step for state transitions., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Plant thioredoxins (TRXs) are involved in numerous metabolic and signalling pathways, such as light-dependent regulation of photosynthesis. The atypical TRX CDSP32, chloroplastic drought-induced stress protein of 32 kDa, includes two TRX-fold domains and participates in responses to oxidative stress as an electron donor to other thiol reductases. Here, we further characterised potato lines modified for CDSP32 expression to clarify the physiological roles of the TRX. Upon high salt treatments, modified lines displayed changes in the abundance and redox status of CDSP32 antioxidant partners, and exhibited sensitivity to combined saline-alkaline stress. In non-stressed plants overexpressing CDSP32, a lower abundance of photosystem II subunits and ATP-synthase γ subunit was noticed. The CDSP32 co-suppressed line showed altered chlorophyll a fluorescence induction and impaired regulation of the transthylakoid membrane potential during dark/light and light/dark transitions. These data, in agreement with the previously reported interaction between CDSP32 and ATP-synthase γ subunit, suggest that CDSP32 affects the redox regulation of ATP-synthase activity. Consistently, modelling of protein complex 3-D structure indicates that CDSP32 could constitute a suitable partner of ATP-synthase γ subunit. We discuss the roles of the TRX in the regulation of both photosynthetic activity and enzymatic antioxidant network in relation with environmental conditions., (© 2024 John Wiley & Sons Ltd.)

The nucleotides guanosine tetraphosphate and pentaphosphate (or (p)ppGpp) are implicated in the regulation of chloroplast function in plants. (p)ppGpp signalling is best understood in the model vascular plant Arabidopsis thaliana in which it acts to regulate plastid gene expression to influence photosynthesis, plant development and immunity. However, little information is known about the conservation or diversity of (p)ppGpp signalling in other land plants. We studied the function of ppGpp in the moss Physcomitrium (previously Physcomitrella) patens using an inducible system for triggering ppGpp accumulation. We used this approach to investigate the effects of ppGpp on chloroplast function, photosynthesis and growth. We demonstrate that ppGpp accumulation causes a dramatic drop in photosynthetic capacity by inhibiting chloroplast gene expression. This was accompanied by the unexpected reorganisation of the thylakoid system into super grana. Surprisingly, these changes did not affect gametophore growth, suggesting that bryophytes and vascular plants may have different tolerances to defects in photosynthesis. Our findings point to the existence of both highly conserved and more specific targets of (p)ppGpp signalling in the land plants that may reflect different growth strategies., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

A Chlamydomonas reinhardtii RuBisCO-less mutant, Δ rbcL , was used to study carbohydrate metabolism without fixation of atmospheric carbon. The regulatory mechanism(s) that control linear electron flow, known as "photosynthetic control," are amplified in Δ rbcL at the onset of illumination. With the aim to understand the metabolites that control this regulatory response, we have correlated the kinetics of primary carbon metabolites to chlorophyll fluorescence induction curves. We identify that Δ rbcL in the absence of acetate generates adenosine triphosphate (ATP) via photosynthetic electron transfer reactions. Also, metabolites of the Calvin Benson Bassham (CBB) cycle are responsive to the light. Indeed, ribulose 1,5-bisphosphate (RuBP), the last intermediate before carboxylation by Ribulose-1,5-bisphosphate carboxylase-oxygenase, accumulates significantly with time, and CBB cycle intermediates for RuBP regeneration, dihydroxyacetone phosphate (DHAP), pentose phosphates and ribose-5-phosphate (R5P) are rapidly accumulated in the first seconds of illumination, then consumed, showing that although the CBB is blocked, these enzymes are still transiently active. In opposition, in the presence of acetate, consumption of CBB cycle intermediates is strongly diminished, suggesting that the link between light and primary carbon metabolism is almost lost. Phosphorylated hexoses and starch accumulate significantly. We show that acetate uptake results in heterotrophic metabolism dominating phototrophic metabolism, with glyoxylate and tricarboxylic acid (TCA) cycle intermediates being the most highly represented metabolites, specifically succinate and malate. These findings allow us to hypothesize which metabolites and metabolic pathways are relevant to the upregulation of processes like cyclic electron flow that are implicated in photosynthetic control mechanisms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Saint-Sorny, Brzezowski, Arrivault, Alric and Johnson.)

Arabidopsis plants were grown from seeds at different photon flux densities (PFDs) of white light ranging from 65 to 800 µmol photons m -2  s -1 . Increasing PFD brought about a marked accumulation of plastoquinone (PQ) in leaves. However, the thylakoid photoactive PQ pool, estimated to about 700 pmol mg -1 leaf dry weight, was independent of PFD; PQ accumulation in high light mostly occurred in the photochemically non-active pool (plastoglobules, chloroplast envelopes) which represented up to 75% of total PQ. The amounts of PSII reaction center (on a leaf dry weight basis) also were little affected by PFD during growth, leading to a constant PQ/PSII ratio at all PFDs. Boosting PQ biosynthesis by overexpression of a solanesyl diphosphate-synthesizing enzyme strongly enhanced the PQ levels, particularly at high PFDs. Again, this accumulation occurred exclusively in the non-photoactive PQ pool. Mutational suppression of the plastoglobular ABC1K1 kinase led to a selective reduction of the thylakoid PQ pool size to ca. 400 pmol mg -1 in a large range of PFDs, which was associated with a restriction of the photosynthetic electron flow. Our results show that photosynthetic acclimation to light intensity does not involve modulation of the thylakoid PQ pool size or the amounts of PSII reaction centers. There appears to be a fixed amount of PQ molecules for optimal interaction with PSII and efficient photosynthesis, with the extra PQ molecules being stored outside the thylakoid membranes, implying a tight regulation of PQ distribution within the chloroplasts., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

The absorbance shift of pigments is proportional to the membrane potential (Δψ) in plants, green algae, and many photosynthetic bacteria. It is currently denoted as ElectroChromic Shift (ECS) at 515-520 nm for plant carotenoids. It is increasingly being used for phenotyping plants for traits related to photosynthesis or chloroplast metabolism because it is a non-invasive technique and also because more instruments are now commercially available from various manufacturers. The ECS technique is currently used to monitor the post-illumination decay of the proton-motive force (pmf), but it has a more general use for quantitative studies on photosynthetic energy transduction. Here we briefly summarize the basic knowledge on ECS, emphasize the full potential of this technique, and propose a quantitative analysis of the photosynthetic performance with the definition of a transmission coefficient for electrons along the photosynthetic chain., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Spontaneous photosynthetic mutants of the aerobic anoxygenic phototrophic bacterium Roseicyclus mahoneyensis, strain ML6 have been identified based on phenotypic differences and spectrophotometric analysis. ML6 contains a reaction centre (RC) with absorption peaks at 755, 800, and 870 nm, light harvesting (LH) complex 1 at 870 nm, and monomodal LH2 at 805 nm; the mutant ML6(B) has only the LH2; ML6(DB) has also lost the LH1; in ML6(BN9O), the LH2 is absent and concentrations of LH1 and RC are much lower than in the wild type. RCs were isolated and purified from ML6 and ML6(BN9O); LH1-RC from ML6; and LH2 from ML6, ML6(B), and ML6(DB). All protein subunits composing the complexes were found to be of typical size. Flash-induced difference spectra revealed ML6 has a fully functional photosynthetic apparatus under aerobic and microaerophilic conditions, and as is typical for AAP, there is no photosynthetic activity anaerobically. ML6(BN9O), while also functional photosynthetically aerobically, showed lower rates due to the lack of LH2 and decreased concentrations of LH1 and RC. ML6(B) and ML6(DB) showed no photoinduced electron transport. Action spectra of light-mediated reactions were also performed on ML6 and ML6(BN9O) to reveal that the majority of carotenoids are not involved in light harvesting. Finally, redox titrations were carried out on membranes of ML6 and ML6(BN9O) to confirm that midpoint redox potentials of the Q A , RC-bound cytochrome, and P + were similar in both strains. Q A midpoint potential is + 65 mV, cytochrome is + 245 mV, and P + is + 430 mV.

In the last common enzymatic step of tetrapyrrole biosynthesis, prior to the branching point leading to the biosynthesis of heme and chlorophyll, protoporphyrinogen IX (Protogen) is oxidised to protoporphyrin IX (Proto) by protoporphyrinogen IX oxidase (PPX). The absence of thylakoid-localised plastid terminal oxidase 2 (PTOX2) and cytochrome b 6 f complex in the ptox2 petB mutant, results in almost complete reduction of the plastoquinone pool (PQ pool) in light. Here we show that the lack of oxidised PQ impairs PPX function, leading to accumulation and subsequently uncontrolled oxidation of Protogen to non-metabolised Proto. Addition of 3(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) prevents the over-reduction of the PQ pool in ptox2 petB and decreases Proto accumulation. This observation strongly indicates the need of oxidised PQ as the electron acceptor for the PPX reaction in Chlamydomonas reinhardtii . The PPX-PQ pool interaction is proposed to function as a feedback loop between photosynthetic electron transport and chlorophyll biosynthesis., Competing Interests: The authors declare no competing interests.

The Arabidopsis vte1 mutant is devoid of tocopherol and plastochromanol (PC-8). When exposed to excess light energy, vte1 produced more singlet oxygen ( 1 O 2 ) and suffered from extensive oxidative damage compared with the wild type. Here, we show that overexpressing the solanesyl diphosphate synthase 1 (SPS1) gene in vte1 induced a marked accumulation of total plastoquinone (PQ-9) and rendered the vte1 SPS1oex plants tolerant to photooxidative stress, indicating that PQ-9 can replace tocopherol and PC-8 in photoprotection. High total PQ-9 levels were associated with a noticeable decrease in 1 O 2 production and higher levels of Hydroxyplastoquinone (PQ-C), a 1 O 2 -specific PQ-9 oxidation product. The extra PQ-9 molecules in the vte1 SPS1oex plants were stored in the plastoglobules and the chloroplast envelopes, rather than in the thylakoid membranes, whereas PQ-C was found almost exclusively in the thylakoid membranes. Upon exposure of wild-type plants to high light, the thylakoid PQ-9 pool decreased, whereas the extrathylakoid pool remained unchanged. In vte1 and vte1 SPS1oex plants, the PQ-9 losses in high light were strongly amplified, affecting also the extrathylakoid pool, and PQ-C was found in high amounts in the thylakoids. We conclude that the thylakoid PQ-9 pool acts as a 1 O 2 scavenger and is replenished from the extrathylakoid stock., (© 2018 John Wiley & Sons Ltd.)

Site-directed mutagenesis of chloroplast genes was developed three decades ago and has greatly advanced the field of photosynthesis research. Here, we describe a new approach for generating random chloroplast gene mutants that combines error-prone polymerase chain reaction of a gene of interest with chloroplast complementation of the knockout Chlamydomonas reinhardtii mutant. As a proof of concept, we targeted a 300-bp sequence of the petD gene that encodes subunit IV of the thylakoid membrane-bound cytochrome b 6 f complex. By sequencing chloroplast transformants, we revealed 149 mutations in the 300-bp target petD sequence that resulted in 92 amino acid substitutions in the 100-residue target subunit IV sequence. Our results show that this method is suited to the study of highly hydrophobic, multisubunit, and chloroplast-encoded proteins containing cofactors such as hemes, iron-sulfur clusters, and chlorophyll pigments. Moreover, we show that mutant screening and sequencing can be used to study photosynthetic mechanisms or to probe the mutational robustness of chloroplast-encoded proteins, and we propose that this method is a valuable tool for the directed evolution of enzymes in the chloroplast., (© 2018 American Society of Plant Biologists. All rights reserved.)

The cytochrome (cyt) b 6 f complex and Stt7 kinase regulate the antenna sizes of photosystems I and II through state transitions, which are mediated by a reversible phosphorylation of light harvesting complexes II, depending on the redox state of the plastoquinone pool. When the pool is reduced, the cyt b 6 f activates the Stt7 kinase through a mechanism that is still poorly understood. After random mutagenesis of the chloroplast petD gene, coding for subunit IV of the cyt b 6 f complex, and complementation of a Δ petD host strain by chloroplast transformation, we screened for impaired state transitions in vivo by chlorophyll fluorescence imaging. We show that residues Asn122, Tyr124, and Arg125 in the stromal loop linking helices F and G of cyt b 6 f subunit IV are crucial for state transitions. In vitro reconstitution experiments with purified cyt b 6 f and recombinant Stt7 kinase domain show that cyt b 6 f enhances Stt7 autophosphorylation and that the Arg125 residue is directly involved in this process. The peripheral stromal structure of the cyt b 6 f complex had, until now, no reported function. Evidence is now provided of a direct interaction with Stt7 on the stromal side of the membrane., Competing Interests: The authors declare no conflict of interest., (Published under the PNAS license.)

Photosynthetic reactions proceed along a linear electron transfer chain linking water oxidation at photosystem II (PSII) to CO 2 reduction in the Calvin-Benson-Bassham cycle. Alternative pathways poise the electron carriers along the chain in response to changing light, temperature and CO 2 inputs, under prolonged hydration stress and during development. We describe recent literature that reports the physiological functions of new molecular players. Such highlights include the flavodiiron proteins and their important role in the green lineage. The parsing of the proton-motive force between ΔpH and Δψ, regulated in many different ways (cyclic electron flow, ATPsynthase conductivity, ion/H + transporters), is comprehensively reported. This review focuses on an integrated description of alternative electron transfer pathways and how they contribute to photosynthetic productivity in the context of plant fitness to the environment., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Both the structure and the protein composition of thylakoid membranes have an impact on light harvesting and electron transfer in the photosynthetic chain. Thylakoid membranes form stacks and lamellae where photosystem II and photosystem I localize, respectively. Light-harvesting complexes II can be associated to either PSII or PSI depending on the redox state of the plastoquinone pool, and their distribution is governed by state transitions. Upon state transitions, the thylakoid ultrastructure and lateral distribution of proteins along the membrane are subject to significant rearrangements. In addition, quinone diffusion is limited to membrane microdomains and the cytochrome b 6 f complex localizes either to PSII-containing grana stacks or PSI-containing stroma lamellae. Here, we discuss possible similarities or differences between green algae and C3 plants on the functional consequences of such heterogeneities in the photosynthetic electron transport chain and propose a model in which quinones, accepting electrons either from PSII (linear flow) or NDH/PGR pathways (cyclic flow), represent a crucial control point. Our aim is to give an integrated description of these processes and discuss their potential roles in the balance between linear and cyclic electron flows.

Photosynthetic microorganisms typically have multiple isoforms of the electron transfer protein ferredoxin, although we know little about their exact functions. Surprisingly, a Chlamydomonas reinhardtii mutant null for the ferredoxin-5 gene (FDX5) completely ceased growth in the dark, with both photosynthetic and respiratory functions severely compromised; growth in the light was unaffected. Thylakoid membranes in dark-maintained fdx5 mutant cells became severely disorganized concomitant with a marked decrease in the ratio of monogalactosyldiacylglycerol to digalactosyldiacylglycerol, major lipids in photosynthetic membranes, and the accumulation of triacylglycerol. Furthermore, FDX5 was shown to physically interact with the fatty acid desaturases CrΔ4FAD and CrFAD6, likely donating electrons for the desaturation of fatty acids that stabilize monogalactosyldiacylglycerol. Our results suggest that in photosynthetic organisms, specific redox reactions sustain dark metabolism, with little impact on daytime growth, likely reflecting the tailoring of electron carriers to unique intracellular metabolic circuits under these two very distinct redox conditions.

The world's crop productivity is stagnating whereas population growth, rising affluence, and mandates for biofuels put increasing demands on agriculture. Meanwhile, demand for increasing cropland competes with equally crucial global sustainability and environmental protection needs. Addressing this looming agricultural crisis will be one of our greatest scientific challenges in the coming decades, and success will require substantial improvements at many levels. We assert that increasing the efficiency and productivity of photosynthesis in crop plants will be essential if this grand challenge is to be met. Here, we explore an array of prospective redesigns of plant systems at various scales, all aimed at increasing crop yields through improved photosynthetic efficiency and performance. Prospects range from straightforward alterations, already supported by preliminary evidence of feasibility, to substantial redesigns that are currently only conceptual, but that may be enabled by new developments in synthetic biology. Although some proposed redesigns are certain to face obstacles that will require alternate routes, the efforts should lead to new discoveries and technical advances with important impacts on the global problem of crop productivity and bioenergy production.

During oxygenic photosynthesis, metabolic reactions of CO2 fixation require more ATP than is supplied by the linear electron flow operating from photosystem II to photosystem I (PSI). Different mechanisms, such as cyclic electron flow (CEF) around PSI, have been proposed to participate in reequilibrating the ATP/NADPH balance. To determine the contribution of CEF to microalgal biomass productivity, here, we studied photosynthesis and growth performances of a knockout Chlamydomonas reinhardtii mutant (pgrl1) deficient in PROTON GRADIENT REGULATION LIKE1 (PGRL1)-mediated CEF. Steady state biomass productivity of the pgrl1 mutant, measured in photobioreactors operated as turbidostats, was similar to its wild-type progenitor under a wide range of illumination and CO2 concentrations. Several changes were observed in pgrl1, including higher sensitivity of photosynthesis to mitochondrial inhibitors, increased light-dependent O2 uptake, and increased amounts of flavodiiron (FLV) proteins. We conclude that a combination of mitochondrial cooperation and oxygen photoreduction downstream of PSI (Mehler reactions) supplies extra ATP for photosynthesis in the pgrl1 mutant, resulting in normal biomass productivity under steady state conditions. The lower biomass productivity observed in the pgrl1 mutant in fluctuating light is attributed to an inability of compensation mechanisms to respond to a rapid increase in ATP demand., (© 2014 American Society of Plant Biologists. All rights reserved.)

In oxygenic photosynthesis, cyclic electron flow around photosystem I denotes the recycling of electrons from stromal electron carriers (reduced nicotinamide adenine dinucleotide phosphate, NADPH, ferredoxin) towards the plastoquinone pool. Whether or not cyclic electron flow operates similarly in Chlamydomonas and plants has been a matter of debate. Here we would like to emphasize that despite the regulatory or metabolic differences that may exist between green algae and plants, the general mechanism of cyclic electron flow seems conserved across species. The most accurate way to describe cyclic electron flow remains to be a redox equilibration model, while the supramolecular reorganization of the thylakoid membrane (state transitions) has little impact on the maximal rate of cyclic electron flow. The maximum capacity of the cyclic pathways is shown to be around 60 electrons transferred per photosystem per second, which is in Chlamydomonas cells treated with 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and placed under anoxic conditions. Part I of this work (aerobic conditions) was published in a previous issue of BBA-Bioenergetics (vol. 1797, pp. 44-51) (Alric et al., 2010)., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The Chlamydomonas reinhardtii proton gradient regulation5 (Crpgr5) mutant shows phenotypic and functional traits similar to mutants in the Arabidopsis (Arabidopsis thaliana) ortholog, Atpgr5, providing strong evidence for conservation of PGR5-mediated cyclic electron flow (CEF). Comparing the Crpgr5 mutant with the wild type, we discriminate two pathways for CEF and determine their maximum electron flow rates. The PGR5/proton gradient regulation-like1 (PGRL1) ferredoxin (Fd) pathway, involved in recycling excess reductant to increase ATP synthesis, may be controlled by extreme photosystem I acceptor side limitation or ATP depletion. Here, we show that PGR5/PGRL1-Fd CEF functions in accordance with an ATP/redox control model. In the absence of Rubisco and PGR5, a sustained electron flow is maintained with molecular oxygen instead of carbon dioxide serving as the terminal electron acceptor. When photosynthetic control is decreased, compensatory alternative pathways can take the full load of linear electron flow. In the case of the ATP synthase pgr5 double mutant, a decrease in photosensitivity is observed compared with the single ATPase-less mutant that we assign to a decreased proton motive force. Altogether, our results suggest that PGR5/PGRL1-Fd CEF is most required under conditions when Fd becomes overreduced and photosystem I is subjected to photoinhibition. CEF is not a valve; it only recycles electrons, but in doing so, it generates a proton motive force that controls the rate of photosynthesis. The conditions where the PGR5 pathway is most required may vary in photosynthetic organisms like C. reinhardtii from anoxia to high light to limitations imposed at the level of carbon dioxide fixation.

The metabolism of microalgae is so flexible that it is not an easy task to give a comprehensive description of the interplay between the various metabolic pathways. There are, however, constraints that govern central carbon metabolism in Chlamydomonas reinhardtii that are revealed by the compartmentalization and regulation of the pathways and their relation to key cellular processes such as cell motility, division, carbon uptake and partitioning, external and internal rhythms, and nutrient stress. Both photosynthetic and mitochondrial electron transfer provide energy for metabolic processes and how energy transfer impacts metabolism and vice versa is a means of exploring the regulation and function of these pathways. A key example is the specific chloroplast localization of glycolysis/gluconeogenesis and how it impacts the redox poise and ATP budget of the plastid in the dark. To compare starch and lipids as carbon reserves, their value can be calculated in terms of NAD(P)H and ATP. As microalgae are now considered a potential renewable feedstock, we examine current work on the subject and also explore the possibility of rerouting metabolism toward lipid production.

The Benson-Calvin cycle enzymes are activated in vivo when disulfide bonds are opened by reduction via the ferredoxin-thioredoxin system in chloroplasts. Iodoacetamide reacts irreversibly with free -SH groups of cysteine residues and inhibits the enzymes responsible for CO2 fixation. Here, we investigate the effect of iodoacetamide on electron transport, when infiltrated into spinach leaves. Using fluorescence and absorption spectroscopy, we show that (i) iodoacetamide very efficiently blocks linear electron flow upon illumination of both photosystems (decrease in the photochemical yield of photosystem II) and (ii) iodoacetamide favors cyclic electron flow upon light excitation specific to PSI. These effects account for an NPQ formation even faster in iodoacetamide under far-red illumination than in the control under saturating light. Such an increase in NPQ is dependent upon the proton gradient across the thylakoid membrane (uncoupled by nigericin addition) and PGR5 (absent in Arabidopsis pgr5 mutant). Iodoacetamide very tightly insulates the electron current at the level of the thylakoid membrane from any electron leaks toward carbon metabolism, therefore, providing choice conditions for the study of cyclic electron flow around PSI.

Based on previous comparative genomic analyses, a set of nearly 600 polypeptides was identified that is present in green algae and flowering and nonflowering plants but is not present (or is highly diverged) in nonphotosynthetic organisms. The gene encoding one of these "GreenCut" proteins, CPLD38, is in the same operon as ndhL in most cyanobacteria; the NdhL protein is part of a complex essential for cyanobacterial respiration. A cpld38 mutant of Chlamydomonas reinhardtii does not grow on minimal medium, is high light-sensitive under photoheterotrophic conditions, has lower accumulation of photosynthetic complexes, reduced photosynthetic electron flow to P700(+), and reduced photochemical efficiency of photosystem II (ΦPSII); these phenotypes are rescued by a wild-type copy of CPLD38. Single turnover flash experiments and biochemical analyses demonstrated that cytochrome b6f function was severely compromised, and the levels of transcripts and polypeptide subunits of the cytochrome b6f complex were also significantly lower in the cpld38 mutant. Furthermore, subunits of the cytochrome b6f complex in mutant cells turned over much more rapidly than in wild-type cells. Interestingly, PTOX2 and NDA2, two major proteins involved in chlororespiration, were more than 5-fold higher in mutants relative to wild-type cells, suggesting a shift in the cpld38 mutant from photosynthesis toward chlororespiratory metabolism, which is supported by experiments that quantify the reduction state of the plastoquinone pool. Together, these findings support the hypothesis that CPLD38 impacts the stability of the cytochrome b6f complex and possibly plays a role in balancing redox inputs to the quinone pool from photosynthesis and chlororespiration.

Plants are particularly prone to photo-oxidative damage caused by excess light. Photoprotection is essential for photosynthesis to proceed in oxygenic environments either by scavenging harmful reactive intermediates or preventing their accumulation to avoid photoinhibition. Carotenoids play a key role in protecting photosynthesis from the toxic effect of over-excitation; under excess light conditions, plants accumulate a specific carotenoid, zeaxanthin, that was shown to increase photoprotection. In this work we genetically dissected different components of zeaxanthin-dependent photoprotection. By using time-resolved differential spectroscopy in vivo, we identified a zeaxanthin-dependent optical signal characterized by a red shift in the carotenoid peak of the triplet-minus-singlet spectrum of leaves and pigment-binding proteins. By fractionating thylakoids into their component pigment binding complexes, the signal was found to originate from the monomeric Lhcb4-6 antenna components of Photosystem II and the Lhca1-4 subunits of Photosystem I. By analyzing mutants based on their sensitivity to excess light, the red-shifted triplet-minus-singlet signal was tightly correlated with photoprotection in the chloroplasts, suggesting the signal implies an increased efficiency of zeaxanthin in controlling chlorophyll triplet formation. Fluorescence-detected magnetic resonance analysis showed a decrease in the amplitude of signals assigned to chlorophyll triplets belonging to the monomeric antenna complexes of Photosystem II upon zeaxanthin binding; however, the amplitude of carotenoid triplet signal does not increase correspondingly. Results show that the high light-induced binding of zeaxanthin to specific proteins plays a major role in enhancing photoprotection by modulating the yield of potentially dangerous chlorophyll-excited states in vivo and preventing the production of singlet oxygen.

Spectroscopic studies on photosynthetic electron transfer generally are based upon the monitoring of dark to light changes in the electron transfer chain. These studies, which focus on the light reactions of photosynthesis, also indirectly provide information on the redox or metabolic state of the chloroplast in the dark. Here, using the unicellular microalga Chlamydomonas reinhardtii, we study the impact of heterotrophic/mixotrophic acetate feeding on chloroplast carbon metabolism by using the spectrophotometric detection of P700(+), the photooxidized primary electron donor of photosystem I. We show that, when photosynthetic linear and cyclic electron flows are blocked (DCMU inhibiting PSII and methylviologen accepting electrons from PSI), the post-illumination reduction kinetics of P700(+) directly reflect the dark metabolic production of reductants (mainly NAD(P)H) in the stroma of chloroplasts. Such results can be correlated to other metabolic studies: in the absence of acetate, for example, the P700(+) reduction rate matches the rate of starch breakdown reported previously, confirming the chloroplast localization of the upstream steps of the glycolytic pathway in Chlamydomonas. Furthermore, the question of the interplay between photosynthetic and non-photosynthetic carbon metabolism can be addressed. We show that cyclic electron flow around photosystem I is twice as fast in a starchless mutant fed with acetate than it is in the WT, and we relate how changes in the flux of electrons from carbohydrate metabolism modulate the redox poise of the plastoquinone pool in the dark through chlororespiration.

Deletion of two of the major electron carriers, the reaction center-bound tetrahemic cytochrome and the HiPIP, involved in the light-induced cyclic electron transfer pathway of the purple photosynthetic bacterium, Rubrivivax gelatinosus, significantly impairs its anaerobic photosynthetic growth. Analysis on the light-induced absorption changes of the intact cells of the mutants shows, however, a relatively efficient photo-induced cyclic electron transfer. For the single mutant lacking the reaction center-bound cytochrome, we present evidence that the electron carrier connecting the reaction center and the cytochrome bc(1) complex is the High Potential Iron-sulfur Protein. In the double mutant lacking both the reaction center-bound cytochrome and the High Potential Iron-sulfur Protein, this connection is achieved by the high potential cytochrome c(8). Under anaerobic conditions, the halftime of re-reduction of the photo-oxidized primary donor by these electron donors is 3 to 4 times faster than the back reaction between P(+) and the reduced primary quinone acceptor. This explains the photosynthetic growth of these two mutants. The results are discussed in terms of evolution of the type II RCs and their secondary electron donors., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Photosynthetic electron transfer has been examined in whole cells, isolated membranes and in partially purified reaction centers (RCs) of Roseicyclus mahoneyensis, strain ML6 and Porphyrobacter meromictius, strain ML31, two species of obligate aerobic anoxygenic phototrophic bacteria. Photochemical activity in strain ML31 was observed aerobically, but the photosynthetic apparatus was not functional under anaerobic conditions. In strain ML6 low levels of photochemistry were measured anaerobically, possibly due to incomplete reduction of the primary electron acceptor (Q(A)) prior to light excitation, however, electron transfer occurred optimally under low oxygen conditions. Photoinduced electron transfer involves a soluble cytochrome c in both strains, and an additional reaction center (RC)-bound cytochrome c in ML6. The redox properties of the primary electron donor (P) and Q(A) of ML31 are similar to those previously determined for other aerobic phototrophs, with midpoint redox potentials of +463 mV and -25 mV, respectively. Strain ML6 showed a very narrow range of ambient redox potentials appropriate for photosynthesis, with midpoint redox potentials of +415 mV for P and +94 mV for Q(A). Cytoplasm soluble and photosynthetic complex bound cytochromes were characterized in terms of apparent molecular mass. Fluorescence excitation spectra revealed that abundant carotenoids not intimately associated with the RC are not involved in photosynthetic energy conservation.

By homology with the unique plastid terminal oxidase (PTOX) found in plants, two genes encoding oxidases have been found in the Chlamydomonas genome, PTOX1 and PTOX2. Here we report the identification of a knockout mutant of PTOX2. Its molecular and functional characterization demonstrates that it encodes the oxidase most predominantly involved in chlororespiration in this algal species. In this mutant, the plastoquinone pool is constitutively reduced under dark-aerobic conditions, resulting in the mobile light-harvesting complexes being mainly, but reversibly, associated with photosystem I. Accordingly, the ptox2 mutant shows lower fitness than wild type when grown under phototrophic conditions. Single and double mutants devoid of the cytochrome b(6)f complex and PTOX2 were used to measure the oxidation rates of plastoquinols via PTOX1 and PTOX2. Those lacking both the cytochrome b(6)f complex and PTOX2 were more sensitive to light than the single mutants lacking either the cytochrome b(6)f complex or PTOX2, which discloses the role of PTOX2 under extreme conditions where the plastoquinone pool is overreduced. A model for chlororespiration is proposed to relate the electron flow rate through these alternative pathways and the redox state of plastoquinones in the dark. This model suggests that, in green algae and plants, the redox poise results from the balanced accumulation of PTOX and NADPH dehydrogenase.

Hydrogen photoproduction by eukaryotic microalgae results from a connection between the photosynthetic electron transport chain and a plastidial hydrogenase. Algal H₂ production is a transitory phenomenon under most natural conditions, often viewed as a safety valve protecting the photosynthetic electron transport chain from overreduction. From the colony screening of an insertion mutant library of the unicellular green alga Chlamydomonas reinhardtii based on the analysis of dark-light chlorophyll fluorescence transients, we isolated a mutant impaired in cyclic electron flow around photosystem I (CEF) due to a defect in the Proton Gradient Regulation Like1 (PGRL1) protein. Under aerobiosis, nonphotochemical quenching of fluorescence (NPQ) is strongly decreased in pgrl1. Under anaerobiosis, H₂ photoproduction is strongly enhanced in the pgrl1 mutant, both during short-term and long-term measurements (in conditions of sulfur deprivation). Based on the light dependence of NPQ and hydrogen production, as well as on the enhanced hydrogen production observed in the wild-type strain in the presence of the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone, we conclude that the proton gradient generated by CEF provokes a strong inhibition of electron supply to the hydrogenase in the wild-type strain, which is released in the pgrl1 mutant. Regulation of the trans-thylakoidal proton gradient by monitoring pgrl1 expression opens new perspectives toward reprogramming the cellular metabolism of microalgae for enhanced H₂ production.

Chlamydomonas reinhardtii, a unicellular green alga, has been exploited as a reference organism for identifying proteins and activities associated with the photosynthetic apparatus and the functioning of chloroplasts. Recently, the full genome sequence of Chlamydomonas was generated and a set of gene models, representing all genes on the genome, was developed. Using these gene models, and gene models developed for the genomes of other organisms, a phylogenomic, comparative analysis was performed to identify proteins encoded on the Chlamydomonas genome which were likely involved in chloroplast functions (or specifically associated with the green algal lineage); this set of proteins has been designated the GreenCut. Further analyses of those GreenCut proteins with uncharacterized functions and the generation of mutant strains aberrant for these proteins are beginning to unmask new layers of functionality/regulation that are integrated into the workings of the photosynthetic apparatus.

Cyclic electron flow around PSI, or cyclic photophosphorylation, is the photosynthetic process which recycles the reducing equivalents produced by photosystem I in the stroma towards the plastoquinone pool. Through the activity of cytochrome b(6)f, which also transfers protons across the membrane, it promotes the synthesis of ATP. The literature dealing with cyclic electron flow in unicellular algae is far less abundant than it is for plants. However, in the chloroplast of algae such as Chlorella or Chlamydomonas, an efficient carbohydrate catabolism renders the redox poise much more reducing than in plant chloroplasts. It is therefore worthwhile highlighting the specific properties of unicellular algae because cyclic electron flow is highly dependent upon the accumulation of these stromal reducing equivalents. Such an increase of reducing power in the stroma stimulates the reduction of plastoquinones, which is the limiting step of cyclic electron flow. In anaerobic conditions in the dark, this reaction can lead to a fully reduced plastoquinone pool and induce state transitions, the migration of 80% of light harvesting complexes II and 20% of cytochrome b(6)f complex from the PSII-enriched grana to the PSI-enriched lamella. These ultrastructural changes have been proposed to further enhance cyclic electron flow by increasing PSI antenna size, and forming PSI-cyt b(6)f supercomplexes. These hypotheses are discussed in light of recently published data.

Assimilation of atmospheric CO2 by photosynthetic organisms such as plants, cyanobacteria and green algae, requires the production of ATP and NADPH in a ratio of 3:2. The oxygenic photosynthetic chain can function following two different modes: the linear electron flow which produces reducing power and ATP, and the cyclic electron flow which only produces ATP. Some regulation between the linear and cyclic flows is required for adjusting the stoichiometric production of high-energy bonds and reducing power. Here we explore, in the green alga Chlamydomonas reinhardtii, the onset of the cyclic electron flow during a continuous illumination under aerobic conditions. In mutants devoid of Rubisco or ATPase, where the reducing power cannot be used for carbon fixation, we observed a stimulation of the cyclic electron flow. The present data show that the cyclic electron flow can operate under aerobic conditions and support a simple competition model where the excess reducing power is recycled to match the demand for ATP.

Here, we describe a new imaging setup able to assess in vivo photosynthetic activity. The system specifically measures time-resolved chlorophyll fluorescence in response to light. It is composed of a fast digital camera equipped with a wide-angle lens for the analysis of samples up to 10 x 10 cm, i.e. entire plants or petri dishes. In the choice of CCD, we have opted for a 12-bits high frame rate [150 fps (frames per second)] at the expense of definition (640 x 480 pixels). Although the choice of digital camera is always a compromise between these two related features, we have designed a flexible system allowing the fast sampling of images (down to 100 micros) with a maximum spatial resolution. This image readout system, synchronized with actinic light and saturating pulses, allows a precise determination of F(0) and F(M), which is required to monitor PSII activity. This new imaging system, together with image processing techniques, is useful to investigate the heterogeneity of photosynthetic activity within leaves or to screen large numbers of unicellular algal mutant colonies to identify those with subtle changes in photosynthetic electron flow.

Three periplasmic electron carriers, HiPIP and two cytochromes c8 with low- and high-midpoint potentials, are present in the purple photosynthetic bacterium Rubrivivax gelatinosus. Comparison of the growth rates of mutants lacking one, two, or all three electron carrier proteins showed that HiPIP is the main electron donor to the photochemical reaction center and that high-potential cytochrome c8 plays a subsidiary role in the electron donation in photosynthetically growing cells. However, the triple deletion mutant was still capable of photosynthetic growth, indicating that another electron donor could be present. A new soluble cytochrome c, which can reduce the photooxidized reaction center in vitro, was purified. Based on amino acid sequence comparisons to known cytochromes, this cytochrome was identified as a diheme cytochrome c of the family of cytochromes c4. The quadruple mutant lacking this cytochrome and three other electron carriers showed about three times slower growth than the triple mutant under photosynthetic growth conditions. In conclusion, cytochrome c4 can function as a physiological electron carrier in the photosynthetic electron transport chain in R. gelatinosus.

State transitions correspond to a major regulation process for photosynthesis, whereby chlorophyll protein complexes responsible for light harvesting migrate between photosystem II and photosystem I in response to changes in the redox poise of the intersystem electron carriers. Here we disclose their physiological significance in Chlamydomonas reinhardtii using a genetic approach. Using single and double mutants defective for state transitions and/or mitochondrial respiration, we show that photosynthetic growth, and therefore biomass production, critically depends on state transitions in respiratory-defective conditions. When extra ATP cannot be provided by respiration, enhanced photosystem I turnover elicited by transition to state 2 is required for photosynthetic activity. Concomitant impairment of state transitions and respiration decreases the overall yield of photosynthesis, ultimately leading to reduced fitness. We thus provide experimental evidence that the combined energetic contributions of state transitions and respiration are required for efficient carbon assimilation in this alga.

Oxidoreductases of the cytochrome bc(1)/b(6)f family transfer electrons from a liposoluble quinol to a soluble acceptor protein and contribute to the formation of a transmembrane electrochemical potential. The crystal structure of cyt b(6)f has revealed the presence in the Q(i) site of an atypical c-type heme, heme c(i). Surprisingly, the protein does not provide any axial ligand to the iron of this heme, and its surrounding structure suggests it can be accessed by exogenous ligand. In this work we describe a mutagenesis approach aimed at characterizing the c(i) heme and its interaction with the Q(i) site environment. We engineered a mutant of Chlamydomonas reinhardtii in which Phe(40) from subunit IV was substituted by a tyrosine. This results in a dramatic slowing down of the reoxidation of the b hemes under single flash excitation, suggesting hindered accessibility of the heme to its quinone substrate. This modified accessibility likely originates from the ligation of the heme iron by the phenol(ate) side chain introduced by the mutation. Indeed, it also results in a marked downshift of the c(i) heme midpoint potential (from +100 mV to -200 mV at pH 7). Yet the overall turnover rate of the mutant cytochrome b(6)f complex under continuous illumination was found similar to the wild type one, both in vitro and in vivo. We propose that, in the mutant, a change in the ligation state of the heme upon its reduction could act as a redox switch that would control the accessibility of the substrate to the heme and trigger the catalysis.

The vertical distribution of culturable anoxygenic phototrophic bacteria was investigated at five sites at or near the Juan de Fuca Ridge in the Pacific Ocean. Twelve similar strains of obligately aerobic phototrophic bacteria were isolated in pure culture, from depths ranging from 500 to 2,379 m below the surface. These strains appear morphologically, physiologically, biochemically, and phylogenetically similar to Citromicrobium bathyomarinum strain JF-1, a bacterium previously isolated from hydrothermal vent plume waters. Only one aerobic phototrophic strain was isolated from surface waters. This strain is morphologically and physiologically distinct from the strains isolated at deeper sampling locations, and phylogenetic analysis indicates that it is most closely related to the genus Erythrobacter. Phototrophs were cultivated from three water casts taken above vents but not from two casts taken away from active vent sites. No culturable anaerobic anoxygenic phototrophs were detected. The photosynthetic apparatus was investigated in strain JF-1 and contains light-harvesting I and reaction center complexes, which are functional under aerobic conditions.

A new type of membrane-bound cytochrome c was found in a marine purple photosynthetic bacterium, Rhodovulum sulfidophilum. This cytochrome c was significantly accumulated in cells growing under anaerobic photosynthetic conditions and showed an apparent molecular mass of approximately 100 kDa when purified and analyzed by SDS-PAGE. The midpoint potential of this cytochrome c was 369 mV. Flash-induced kinetic measurements showed that this new cytochrome c can work as an electron donor to the photosynthetic reaction center. The gene coding for this cytochrome c was cloned and analyzed. The deduced molecular mass was nearly equal to 50 kDa. Its C-terminal heme-containing region showed the highest sequence identity to the water-soluble cytochrome c(2), although its predicted secondary structure resembles that of cytochrome c(y). Phylogenetic analyses suggested that this new cytochrome c has evolved from cytochrome c(2). We, thus, propose its designation as cytochrome c(2m). Mutants lacking this cytochrome or cytochrome c(2) showed the same growth rate as the wild type. However, a double mutant lacking both cytochrome c(2) and c(2m) showed no growth under photosynthetic conditions. It was concluded that either the membrane-bound cytochrome c(2m) or the water-soluble cytochrome c(2) work as a physiological electron carrier in the photosynthetic electron transfer pathway of Rvu. sulfidophilum.

Background: Lutein is the most abundant xanthophyll in the photosynthetic apparatus of higher plants. It binds to site L1 of all Lhc proteins, whose occupancy is indispensable for protein folding and quenching chlorophyll triplets. Thus, the lack of a visible phenotype in mutants lacking lutein has been surprising., Results: We have re-assessed the lut2.1 phenotypes through biochemical and spectroscopic methods. Lhc proteins from the lut2.1 mutant compensate the lack of lutein by binding violaxanthin in sites L1 and L2. This substitution reduces the capacity for regulatory mechanisms such as NPQ, reduces antenna size, induces the compensatory synthesis of Antheraxanthin + Zeaxanthin, and prevents the trimerization of LHCII complexes. In vitro reconstitution shows that the lack of lutein per se is sufficient to prevent trimerization. lut2.1 showed a reduced capacity for state I-state II transitions, a selective degradation of Lhcb1 and 2, and a higher level of photodamage in high light and/or low temperature, suggesting that violaxanthin cannot fully restore chlorophyll triplet quenching. In vitro photobleaching experiments and time-resolved spectroscopy of carotenoid triplet formation confirmed this hypothesis. The npq1lut2.1 double mutant, lacking both zeaxanthin and lutein, is highly susceptible to light stress., Conclusion: Lutein has the specific property of quenching harmful 3Chl* by binding at site L1 of the major LHCII complex and of other Lhc proteins of plants, thus preventing ROS formation. Substitution of lutein by violaxanthin decreases the efficiency of 3Chl* quenching and causes higher ROS yield. The phenotype of lut2.1 mutant in low light is weak only because rescuing mechanisms of photoprotection, namely zeaxanthin synthesis, compensate for the ROS production. We conclude that zeaxanthin is effective in photoprotection of plants lacking lutein due to the multiple effects of zeaxanthin in photoprotection, including ROS scavenging and direct quenching of Chl fluorescence by binding to the L2 allosteric site of Lhc proteins.