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Aquatic life is strongly structured by light gradients, with gradual decrease in light intensity and differential attenuation of sunlight wavelengths with depth. How phytoplankton perceive these variations is unknown. By providing the firstin vivoquantitative assessment of the action of marine diatom phytochrome photoreceptors (DPH), we show that they efficiently trigger photoreversible responses across the entire light spectrum, unlike current models of phytochrome photosensing. The distribution and activity of DPHs in the environment indicate that they are extremely sensitive detectors of spectral light variations related to depth and optical properties of the water column in temperate and polar oceans, revealing a completely novel view of how light is perceived in the marine environment.

Recurrent blooms of the toxic dinoflagellate Ostreopsis cf. ovata are frequently reported in the Northwestern Mediterranean Sea. The impact of these proliferations on other microalgal species inhabiting the same habitats is of interest from an ecological prospective. In vitro experiments were carried out to investigate the influence of O. cf. ovata on the growth of the co-occurring benthic diatoms Licmophora paradoxa, Navicula arenaria and the benthic dinoflagellates Prorocentrum lima and Coolia monotis. Overall, O. cf. ovata exhibited weak allelopathic effects towards these microalgal species, with a reduction in the cell abundance for L. paradoxa and P. lima only. Interestingly, dead cells of L. paradoxa and N. arenaria were observed embedded in the thick mucus surrounding O. cf. ovata cells, suggesting that the mucous layer could act as a toxic phycosphere, especially for non-motile cells. All competitors were further exposed for 24 h to ovatoxins, the major toxins produced by O. cf. ovata, and the maximum quantum yield efficiency of L. paradoxa, N. arenaria and P. lima was affected at a minimum concentration of 10 µg mL−1. We then hypothesized that the diffusion of solubilized ovatoxins in the culture medium affects only moderately the competitors’ growth, whereas their accumulation in the mucus would yield deleterious effects. More precisely, the competitors’ sensitivity to ovatoxins was enhanced in their stationary phase of growth and resulted from a rapid inhibition of an uncharacterized photosynthetic step downstream photosystem II. Altogether, these results emphasize the predominant role of the O. cf. ovata’s mucus in driving ecological interactions and suggest that it can affect the growth of several benthic microalgae by accumulating the potent ovatoxins.

Diatoms, the major eukaryotic phytoplankton in polar regions, are essential to sustain Arctic and Antarctic ecosystems. As such, it is fundamental to understand the physiological mechanisms and associated molecular basis of their resilience to the long polar night. Here, we report an integrative approach revealing that in prolonged darkness, diatom cells enter a state of quiescence associated with reduced metabolic and transcriptional activity during which no cell division occurs. We propose that minimal energy is provided by respiration and degradation of protein, carbohydrate, and lipid stores and that homeostasis is maintained by autophagy in prolonged darkness. We also report internal structural changes that manifest the morphological acclimation of cells to darkness. Our results further indicate that immediately following a return to light, diatom cells are able to use photoprotective mechanisms and rapidly resume photosynthesis. Cell division resumed rates similar to those before darkness. Our study demonstrates the remarkable robustness of polar diatoms to prolonged darkness at low temperatures.Graphical abstractTeaserTo survive the long winter, polar diatoms slow down metabolism and express genes to assure survival following return to light.

International audience; Like all organisms performing oxygenic photosynthesis, Chlamydomonas captures light energy in two photochemical steps to drive linear electron flow from water to NADPH and to produce ATP. However, this process alone is not sufficient to drive CO2 fixation in the Calvin–Benson cycle and to respond to environmental and metabolic constraints, for example, light availability or metabolic needs in term of ATP and NADPH. A complex network of alternative electron flows, comprises cyclic electron flow around photosystem I and various water-to-water cycles that utilize O2 as an alternative electron acceptor, provide an additional degree of freedom to face this challenge. The present chapter describes the various alternative routes of photosynthetic electron transfer in Chlamydomonas, describes how they are coordinated for optimization of photosynthesis and gives a retrospective on the early physiology work on those alternative electron flows among model photosynthetic organisms.

Organic carbon fixed through the Calvin Cycle can be diverted towards different metabolic fates within and beyond the plastids of photosynthetic eukaryotes. These include export to the cytoplasm and mitochondrial respiration; gluconeogenesis of storage compounds; and the anabolic synthesis of lipids, amino acids and cofactors via the plastidial pyruvate hub. In plants, pyruvate is principally synthesised via the lower half of glycolysis-gluconeogenesis in the cytoplasm, although a secondary plastid-targeted pathway in non-photosynthetic tissue directly links glyceraldehyde-3-phosphate to the pyruvate hub. Here, we characterize a complete plastidial lower half glycolytic-gluconeogenic pathway in the photosynthetic plastids of diatoms, obligately photosynthetic eukaryotic algae that are important contributors to marine primary production. We show that the two enzymes required to complete plastidial glycolysis-gluconeogenesis, plastidial Enolase and PGAM (bis-phospho-glycerate mutase), originated through recent duplications of mitochondria-targeted respiratory glycolytic isoforms. Through CRISPR-Cas9 mutagenesis and integrative ‘omic analyses in the diatom Phaeodactylum tricornutum, we present evidence that this pathway functions to divert excess plastidial glyceraldehyde-3-phosphate into diverse fates accessed from the pyruvate hub, and may potentially also function in the gluconeogenic direction to permit more efficient management of cellular carbon. Considering meta-genomic data, we show that this pathway is of greater importance in polar and sub-polar oceans, in which diatoms dominate primary production; and considering experimental data, we show that this principally relates to the elongated photoperiods present at high latitudes. Our data provide insights into the functions of a poorly understood yet evolutionarily recurrent plastidial metabolic pathway, and a further explanation for the success of diatoms in the contemporary ocean.

International audience; Photosynthesis in diatoms is performed using the same basic modules as cyanobacteria and plants. It can be regulated on multiple levels depending on the environmental cues, allowing diatoms to adjust their photosynthetic light reaction towards optimum whilst at the same time minimizing photodamage induced by light. In recent years, tremendous progress has been gained in understanding these acclimation processes, revealing several diatom-specific features. In this chapter, we trace several paths through the photosynthetic electron transport chain to optimize photosynthesis. We review how diatoms repair photoinactivated reaction centers and which mechanisms they have to preempt photodamage. Finally, photoprotection is set in an ecophysiological context, highlighting differences in photoprotection of diatoms from different habitats.

The mechanisms underlying interactions between diatoms and bacteria are crucial to understand diatom behaviour and proliferation, and can result in far‐reaching ecological consequences. Recently, 2‐alkyl‐4‐quinolones have been isolated from marine bacteria, both of which (the bacterium and isolated chemical) inhibited growth of microalgae, suggesting these compounds could mediate diatom–bacteria interactions. The effects of several quinolones on three diatom species have been investigated. The growth of all three was inhibited, with half‐maximal inhibitory concentrations reaching the sub‐micromolar range. By using multiple techniques, dual inhibition mechanisms were uncovered for 2‐heptyl‐4‐quinolone (HHQ) in Phaeodactylum tricornutum. Firstly, photosynthetic electron transport was obstructed, primarily through inhibition of the cytochrome b 6 f complex. Secondly, respiration was inhibited, leading to repression of ATP supply to plastids from mitochondria through organelle energy coupling. These data clearly show how HHQ could modulate diatom proliferation in marine environments., Double trouble: In the light, 2‐heptyl‐4‐quinolone (HHQ) primarily blocks photosynthetic electron flow at the cytochrome b 6 f complex in the diatom P. tricornutum. In darkness, HHQ inhibits mitochondrial respiration, resulting in a decreased ATP supply, which ultimately leads to a decreased electric field strength in the thylakoid membranes inside the chloroplast of diatoms.

International audience; Light harvesting and photochemistry is performed by photosystems coupled to specific antennae embedded in the thylakoid membrane, a common principle across diatoms, plants and green algae. Still, unique features of diatoms within this common principle have been unraveled in recent decades, likely resulting from the complex evolutionary history of diatoms. These unique features are found in (i) the lipid composition of the thylakoid membrane, ii) the spatial organization of the light harvesting complexes, and iii) their protein and pigment composition. This chapter summarizes current knowledge of these three specific features, with a focus on structural and functional properties.

Diatoms are amongst the most successful clades of oceanic phytoplankton, significantly contributing to photosynthesis on Earth. Their ecological success likely stems from their ability to acclimate to changing environmental conditions, including e.g. variable light intensity. Diatoms are outstanding at dissipating light energy exceeding the maximum photosynthetic electron transfer (PET) capacity of via Non Photochemical Quenching (NPQ). While the molecular effectors of this process, as well as the role of the Proton Motive Force (PMF) in its regulation are known, the putative regulators of the PET/PMF relationship in diatoms remain unidentified. Here, we demonstrate that the H+/K+ antiporter KEA3 is the main regulator of the coupling between PMF and PET in the model diatom Phaeodactylum tricornutum. By controlling the PMF, it modulates NPQ responses at the onset of illumination, during transients and in steady state conditions. Under intermittent light KEA3 absence results in reduced fitness. Using a parsimonious model including only two components, KEA3 and the diadinoxanthin de-epoxidase, we can describe most of the feedback loops observed between PET and NPQ. This two-components regulatory system allows for efficient responses to fast (minutes) or slow (e.g. diel) changes in light environment, thanks to the presence of a regulatory Ca2+-binding domain in KEA3 that controls its activity. This circuit is likely finely tuned by the NPQ effector proteins LHCX, providing diatoms with the required flexibility to thrive in different ocean provinces.One sentence summaryThe author(s) responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plcell/pages/General-Instructions) is Giovanni Finazzi.

International audience; Among all the chemical and biotechnological strategies implemented to extract energy from oxygenic photosynthesis, several concern the use of intact photosynthetic organisms (algae, cyanobacteria…). This means rerouting (fully or partially) the electron flow from the photosynthetic chain to an outer collecting electrode thus generating a photocurrent. While diverting photosynthetic electrons from living biological systems is an encouraging approach, this strategy is limited by the need to use an electron shuttle. Redox mediators that are able to interact with an embedded photosynthetic chain are rather scarce. In this respect, exogenous quinones are the most frequently used. Unfortunately, some of them also act as poisoning agents within relatively long timeframes. It thus raises the question of the best quinone. In this work, we use a previously reported electrochemical device to analyze the performance of different quinones. Photocurrents (maximum photocurrent, stability) were measured from suspensions of Chlamydomonas reinhardtii algae/quinones by chronoamperometry and compared to parameters like quinone redox potentials or cytotoxic concentration. From these results, several quinones were synthesized and analyzed in order to find the best compromise between bioelectricity production and toxicity.

Marine bacteria contribute substantially to nutrient cycling in the oceans and can engage in close interactions with microalgae. Many microalgae harbor characteristic satellite bacteria, many of which participate in N-acyl homoserine lactone (AHL) mediated quorum sensing. In the diffusion-controlled phycosphere, AHLs can reach high local concentrations, with some of them transforming into tetramic acids, compounds with a broad bioactivity. We tested a representative AHL, N-(3-oxododecanoyl) homoserine lactone, and its tetramic acid rearrangement product on the diatom Phaeodactylum tricornutum. While cell growth and photosynthetic efficiency of photosystem II were barely affected by the AHL, exposure to its tetramic acid rearrangement product had a negative effect on photosynthetic efficiency and led to growth inhibition and cell death in the long term, with a minimum inhibitory concentration between 20 and 50 μΜ. These results strengthen the view that AHLs may play an important role in shaping the outcome of microalgae-bacteria interactions.

International audience; Allelopathy is an efficient strategy by which some microalgae can outcompete other species. Allelochemicals from the toxic dinoflagellate Alexandrium minutum have deleterious effects on diatoms, inhibiting metabolism and photosynthesis and therefore giving a competitive advantage to the dinoflagellate. The precise mechanisms of allelochemical interactions and the molecular target of allelochemicals remain however unknown. To understand the mechanisms, the short-term effects of A. minutum allelochemicals on the physiology of the diatom Chaetoceros muelleri were investigated. The effects of a culture filtrate were measured on the diatom cytoplasmic membrane integrity (polarity and permeability) using flow-cytometry and the photosynthetic performance using fluorescence and absorption spectroscopy. Within 10 minutes, the unknown allelochemicals induced a depolarization of the cytoplasmic membranes and an impairment of photosynthesis through the inhibition of the plastoquinone-mediated electron transfer between photosystem II and cytochrome b6f. At longer time of exposure, the cytoplasmic membranes were permeable and the integrity of photosystems I, II and cytochrome b6f was compromised. Our demonstration of the essential role of membranes in this allelochemical interaction provides new insights for the elucidation of the nature of the allelochemicals. The relationship between cytoplasmic membranes and the inhibition of the photosynthetic electron transfer remains however unclear and warrants further investigation.

Diatoms possess an efficient mechanism to dissipate photons as heat in conditions of excess light, which is visualized as the Non-Photochemical Quenching of chlorophyll a fluorescence (NPQ). In most diatom species, NPQ is proportional to the concentration of the xanthophyll cycle pigment diatoxanthin formed from diadinoxanthin by the diadinoxanthin de-epoxidase enzyme. The reverse reaction is performed by the diatoxanthin epoxidase. Despite the xanthophyll cycle’s central role in photoprotection, its regulation is not yet well understood. The proportionality between diatoxanthin and NPQ allowed us to calculate the activity of both xanthophyll cycle enzymes in the model diatom Phaeodactylum tricornutum from NPQ kinetics. From there, we explored the light-dependency of the activity of both enzymes. Our results demonstrate that a tight regulation of both enzymes is key to fine-tune NPQ: (i) the rate constant of diadinoxanthin de-epoxidation is low under a light-limiting regime but increases as photosynthesis saturates, probably due to the thylakoidal proton gradient ΔpH (ii) the rate constant of diatoxanthin epoxidation exhibits an optimum under low light and decreases in the dark due to an insufficiency of the co-factor NADPH as well as in higher light through an as yet unresolved inhibition mechanism, that is unlikely to be related to the ΔpH. We observed that the suppression of NPQ by an uncoupler was due to an accelerated diatoxanthin epoxidation enzyme rather than to the usually hypothesized inhibition of the diadinoxanthin de-epoxidation enzyme.

Many cyanobacteria species can use both plastocyanin and cytochrome c

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.

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.

Microbial solar cells that mainly rely on the use of photosynthesic organisms are a promising alternative to photovoltaics for solar electricity production. In that way, we propose a new approach involving electrochemistry and fluorescence techniques. The coupled set-up Electro-Pulse-Amplitude-Modulation (“e-PAM”) enables the simultaneous recording of the produced photocurrent and fluorescence signals from the photosynthetic chain. This methodology was validated with a suspension of green alga Chlamydomonas reinhardtii in interaction with an exogenous redox mediatior (2,6-dichlorobenzoquinone; DCBQ). The balance between photosynthetic chain events (PSII photochemical yield, quenching) and the extracted electricity can be monitored overtime. More particularly, the non photochemical quenching induced by DCBQ mirrors the photocurrent. This set-up thus helps to distinguish the electron harvesting from some side effects due to quinones in real time. It therefore paves the way for future analyses devoted to the choice of the experimental conditions (redox mediator, photosynthetic organisms…) to find the best electron extraction.

International audience; Diatoms are the world's most diverse group of algae, comprising at least 100,000 species. Contributing ;20% of annual global carbon fixation, they underpin major aquatic food webs and drive global biogeochemical cycles. Over the past two decades, Thalassiosira pseudonana and Phaeodactylum tricornutum have become the most important model systems for diatom molecular research, ranging from cell biology to ecophysiology, due to their rapid growth rates, small genomes, and the cumulative wealth of associated genetic resources. To explore the evolutionary divergence of diatoms, additional model species are emerging, such as Fragilariopsis cylindrus and Pseudo-nitzschia multistriata. Here, we describe how functional genomics and reverse genetics have contributed to our understanding of this important class of microalgae in the context of evolution, cell biology, and metabolic adaptations. Our review will also highlight promising areas of investigation into the diversity of these photosynthetic organisms, including the discovery of new molecular pathways governing the life of secondary plastid-bearing organisms in aquatic environments.

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.

Many cyanobacteria species can use both plastocyanin and cytochrome c6 as lumenal electron carriers to shuttle electrons from the cytochrome b6f to either photosystem I or the respiratory cytochrome c oxidase. In Synechocystis sp. PCC6803 placed in darkness, about 60% of the active PSI centres are bound to a reduced electron donor which is responsible for the fast re-reduction of P700in vivo after a single charge separation. Here, we show that both cytochrome c6 and plastocyanin can bind to PSI in the dark and participate to the fast phase of P700 reduction, but the fraction of pre-bound PSI is smaller in the case of cytochrome c6 than with plastocyanin. Because of the inter-connection of respiration and photosynthesis in cyanobacteria, the inhibition of the cytochrome c oxidase results in the over-reduction of the photosynthetic electron transfer chain in the dark that translates into a lag in the kinetics of P700 oxidation at the onset of light. We show that this is true both with plastocyanin and cytochrome c6, indicating that the partitioning of electron transport between respiration and photosynthesis is regulated in the same way independently of which of the two lumenal electron carriers is present, although the mechanisms of such regulation are yet to be understood.

Amundsen Sea polynyas are among the most productive, yet climate-sensitive ecosystems in the Southern Ocean and host massive annual phytoplankton blooms. These blooms are believed to be controlled by iron fluxes from melting ice and icebergs and by intrusion of nutrient-rich Circumpolar Deep Water, however the interplay between iron effects and other controls, such as light availability, has not yet been quantified. Here, we examine phytoplankton photophysiology in relation to Fe stress and physical forcing in two largest polynyas, Amundsen Sea Polynya (ASP) and Pine Island Polynya (PIP), using the combination of high-resolution variable fluorescence measurements, fluorescence lifetime analysis, photosynthetic rates, and Fe-enrichment incubations. These analyses revealed strong Fe stress in the ASP, whereas the PIP showed virtually no signatures of Fe limitation. In spite of enhanced iron availability in the PIP, chlorophyll biomass remained ∼ 30–50% lower than in the Fe-stressed ASP. This apparent paradox would not have been observed if iron were the main control of phytoplankton bloom in the Amundsen Sea. Long-term satellite-based climatology records revealed that the ASP is exposed to significantly higher solar irradiance levels throughout the summer season, as compared to the PIP region, suggesting that light availability controls the magnitude of phytoplankton blooms in the Amundsen Sea. Our data suggests that higher Fe availability (e.g., due to higher melting rates of ice sheets) would not necessarily increase primary productivity in this region. Furthermore, stronger wind-driven vertical mixing in expanding ice-free areas may lead to reduction in light availability and productivity in the future.

We measured the light dependence of gross photosynthesis and oxygen uptake rates by membrane inlet mass spectrometry in two open ocean regions: the Amundsen Sea (Antarctica), dominated by Phaeocystis antarctica, and the North Atlantic, dominated by Emiliania huxleyi. In the North Atlantic, respiration was independent of irradiance and was higher than the gross photosynthetic rate at all irradiances. In contrast, in the Amundsen Sea, oxygen uptake processes were light dependent; dark respiration was one order of magnitude lower than the maximal gross photosynthetic rate, but the oxygen uptake rate increased by 10 fold at surface irradiances. Our results suggest the light dependence of oxygen uptake in Amundsen Sea has two sources: one is independent of photosynthesis, and is possibly associated with the photo-reduction of O2 mediated by dissolved organic matter; the second reflects the activity of an oxidase fueled in the light with photosynthetic electron flow. Our results highlight the importance of improving our understanding of oxygen consuming reactions in the euphotic zone.

International audience; Cyclic electron flow (CEF) is defined as a return of the reductants from the acceptor side of Photosystem I (PSI) to the pool of its donors via the cytochrome b6f. It is described to be complementary to the linear electron flow and essential for photosynthesis. However, despite many efforts aimed to characterize CEF, its pathway and its regulation modes remain equivocal, and its physiological significance is still not clear. Here we use novel spectroscopic to measure the rate of CEF at the onset of light in the green alga Chlamydomonas reinhardtii. The initial redox state of the photosynthetic chain or the oxygen concentration do not modify the initial maximal rate of CEF (60 electrons per second per PSI) but rather strongly influence its duration. Neither the maximal rate nor the duration of CEF are different in the pgrl1 mutant compared to the wild type, disqualifying PGRL1 as the ferredoxin-plastoquinone reductase involved in the CEF mechanism.

Apart from the canonical light-driven linear electron flow (LEF) from water to CO2, numerous regulatory and alternative electron transfer pathways exist in chloroplasts. One of them is the cyclic electron flow around Photosystem I (CEF), contributing to photoprotection of both Photosystem I and II (PSI, PSII) and supplying extra ATP to fix atmospheric carbon. Nonetheless, CEF remains an enigma in the field of functional photosynthesis as we lack understanding of its pathway. Here, we address the discrepancies between functional and genetic/biochemical data in the literature and formulate novel hypotheses about the pathway and regulation of CEF based on recent structural and kinetic information.

In plants, algae, and some photosynthetic bacteria, the ElectroChromic Shift (ECS) of photosynthetic pigments, which senses the electric field across photosynthetic membranes, is widely used to quantify the activity of the photosynthetic chain. In cyanobacteria, ECS signals have never been used for physiological studies, although they can provide a unique tool to study the architecture and function of the respiratory and photosynthetic electron transfer chains, entangled in the thylakoid membranes. Here, we identified bona fide ECS signals, likely corresponding to carotenoid band shifts, in the model cyanobacteria Synechococcus elongatus PCC7942 and Synechocystis sp. PCC6803. These band shifts, most likely originating from pigments located in photosystem I, have highly similar spectra in the 2 species and can be best measured as the difference between the absorption changes at 500 to 505 nm and the ones at 480 to 485 nm. These signals respond linearly to the electric field and display the basic kinetic features of ECS as characterized in other organisms. We demonstrate that these probes are an ideal tool to study photosynthetic physiology in vivo, e.g., the fraction of PSI centers that are prebound by plastocyanin/cytochrome c(6) in darkness (about 60% in both cyanobacteria, in our experiments), the conductivity of the thylakoid membrane (largely reflecting the activity of the ATP synthase), or the steady-state rates of the photosynthetic electron transport pathways.

The model green microalga Chlamydomonas reinhardtii is frequently subject to periods of dark and anoxia in its natural environment. Here, by resorting to mutants defective in the maturation of the chloroplastic oxygen-sensitive hydrogenases or in Proton-Gradient Regulation-Like1 (PGRL1)-dependent cyclic electron flow around photosystem I (PSI-CEF), we demonstrate the sequential contribution of these alternative electron flows (AEFs) in the reactivation of photosynthetic carbon fixation during a shift from dark anoxia to light. At light onset, hydrogenase activity sustains a linear electron flow from photosystem II, which is followed by a transient PSI-CEF in the wild type. By promoting ATP synthesis without net generation of photosynthetic reductants, the two AEF are critical for restoration of the capacity for carbon dioxide fixation in the light. Our data also suggest that the decrease in hydrogen evolution with time of illumination might be due to competition for reduced ferredoxins between ferredoxin-NADP(+) oxidoreductase and hydrogenases, rather than due to the sensitivity of hydrogenase activity to oxygen. Finally, the absence of the two alternative pathways in a double mutant pgrl1 hydrogenase maturation factor G-2 is detrimental for photosynthesis and growth and cannot be compensated by any other AEF or anoxic metabolic responses. This highlights the role of hydrogenase activity and PSI-CEF in the ecological success of microalgae in low-oxygen environments.

Cyclic electron flow (CEF), one of the major alternative electron transport pathways to the primary linear electron flow (LEF) in chloroplasts has been discovered in the middle of the last century. It is defined as a return of the reductants from the acceptor side of the Photosystem I (PSI) to the pool of its donors via the cytochromeb6f, and has proven essential for photosynthesis. However, despite many efforts aimed at its characterisation, the pathway and regulation of CEF remain equivocal, and its physiological significance remains to be properly defined. Here we use novel spectroscopic approaches to measure CEF in transitory conditions in the green algaChlamydomonas reinhardtii.We show that CEF operates at the same maximal rate regardless of the oxygen concentration, and that the latter influences LEF, rather than CEF in vivo, which questions the recent hypotheses about the CEF supercomplex formation. We further reveal that the pathways proposed for CEF in the literature are inconsistent with the kinetic information provided by our measurements. We finally provide cues on the regulation of CEF by light.

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

Fridman, Svetlana ... et al.-- This is contribution number 54 of Tara Oceans.-- 8 pages, 4 figures, supplementary material https://dx.doi.org/10.1038/s41564-017-0002-9, Cyanobacteria are important contributors to primary production in the open oceans. Over the past decade, various photosynthesis-related genes have been found in viruses that infect cyanobacteria (cyanophages). Although photosystem II (PSII) genes are common in both cultured cyanophages and environmental samples , viral photosystem I (vPSI) genes have so far only been detected in environmental samples . Here, we have used a targeted strategy to isolate a cyanophage from the tropical Pacific Ocean that carries a PSI gene cassette with seven distinct PSI genes (psaJF, C, A, B, K, E, D) as well as two PSII genes (psbA, D). This cyanophage, P-TIM68, belongs to the T4-like myoviruses, has a prolate capsid, a long contractile tail and infects Prochlorococcus sp. strain MIT9515. Phage photosynthesis genes from both photosystems are expressed during infection, and the resultant proteins are incorporated into membranes of the infected host. Moreover, photosynthetic capacity in the cell is maintained throughout the infection cycle with enhancement of cyclic electron flow around PSI. Analysis of metagenomic data from the Tara Oceans expedition shows that phages carrying PSI gene cassettes are abundant in the tropical Pacific Ocean, composing up to 28% of T4-like cyanomyophages. They are also present in the tropical Indian and Atlantic Oceans. P-TIM68 populations, specifically, compose on average 22% of the PSI-gene-cassette carrying phages. Our results suggest that cyanophages carrying PSI and PSII genes are likely to maintain and even manipulate photosynthesis during infection of their Prochlorococcus hosts in the tropical oceans, This work was funded by a European Commission ERC Advanced Grant (no. 321647), the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme FP7/2007-2013/ under REA Grant Agreement No. 317184, an Israel Science Foundation grant (no. 580/10) and the Louis and Lyra Richmond Memorial Chair in Life Sciences to O.B., a European Commission ERC starting grant (no. 203406) to D.L. and the Technion’s Lorry I. Lokey Interdisciplinary Center for Life Sciences and Engineering and the Russell Berrie Nanotechnology Institute.

In Chlamydomonas, unlike in flowering plants, genes coding for Nd7 (NAD7/49 kDa) and Nd9 (NAD9/30 kDa) core subunits of mitochondrial respiratory-chain complex I are nucleus-encoded. Both genes possess all the features that facilitate their expression and proper import of the polypeptides in mitochondria. By inactivating their expression by RNA interference or insertional mutagenesis, we show that both subunits are required for complex I assembly and activity. Inactivation of complex I impairs the cell growth rate, reduces the respiratory rate, leads to lower intracellular ROS production and lower expression of ROS scavenging enzymes, and is associated to a diminished capacity to concentrate CO2 without compromising photosynthetic capacity.

Diatoms, unicellular phytoplankton that account for ∼40% of marine primary productivity, often dominate coastal and open-ocean upwelling zones. Limitation of growth and productivity by iron at low light is attributed to an elevated cellular Fe requirement for the synthesis of Fe-rich photosynthetic proteins. In the dynamic coastal environment, Fe concentrations and daily surface irradiance levels can vary by two to three orders of magnitude on short spatial and temporal scales. Although genome-wide studies are beginning to provide insight into the molecular mechanisms used by diatoms to rapidly respond to such fluxes, their functional role in mediating the Fe stress response remains uncharacterized. Here, we show, using reverse genetics, that a death-specific protein (DSP; previously named for its apparent association with cell death) in the coastal diatom Thalassiosira pseudonana (TpDSP1) localizes to the plastid and enhances growth during acute Fe limitation at subsaturating light by increasing the photosynthetic efficiency of carbon fixation. Clone lines overexpressing TpDSP1 had a lower quantum requirement for growth, increased levels of photosynthetic and carbon fixation proteins, and increased cyclic electron flow around photosystem I. Cyclic electron flow is an ATP-producing pathway essential in higher plants and chlorophytes with a heretofore unappreciated role in diatoms. However, cells under replete conditions were characterized as having markedly reduced growth and photosynthetic rates at saturating light, thereby constraining the benefits afforded by overexpression. Widespread distribution of DSP-like sequences in environmental metagenomic and metatranscriptomic datasets highlights the presence and relevance of this protein in natural phytoplankton populations in diverse oceanic regimes.

Light-induced electron transfer reactions in the chlorophyll a / d -binding Photosystem I reaction centre of Acaryochloris marina were investigated in whole cells by pump-probe optical spectroscopy with a temporal resolution of ~ 5 ns at room temperature. It is shown that phyllosemiquinone, the secondary electron transfer acceptor anion, is oxidised with bi-phasic kinetics characterised by lifetimes of 88 ± 6 ns and 345 ± 10 ns. These lifetimes, particularly the former, are significantly slower than those reported for chlorophyll a -binding Photosystem I, which typically range in the 5–30 ns and 200–300 ns intervals. The possible mechanism of electron transfer reactions in the chlorophyll a/d -binding Photosystem I and the slower oxidation kinetics of the secondary acceptors are discussed.

International audience; In photosynthesis, electron transfer along the photosynthetic chain results in a vectorial transfer of protons from the stroma to the lumenal space of the thylakoids. This promotes the generation of an electrochemical proton gradient (Δμ(H)(+)), which comprises a gradient of electric potential (ΔΨ) and of proton concentration (ΔpH). The Δμ(H)(+) has a central role in the photosynthetic process, providing the energy source for ATP synthesis. It is also involved in many regulatory mechanisms. The ΔpH modulates the rate of electron transfer and triggers deexcitation of excess energy within the light harvesting complexes. The ΔΨ is required for metabolite and protein transport across the membranes. Its presence also induces a shift in the absorption spectra of some photosynthetic pigments, resulting in the so-called ElectroChromic Shift (ECS). In this review, we discuss the characteristic features of the ECS, and illustrate possible applications for the study of photosynthetic processes in vivo.

We have investigated the photosynthetic properties of Acaryochloris marina, a cyanobacterium distinguished by having a high level of chlorophyll d, which has its absorption bands shifted to the red when compared with chlorophyll a. Despite this unusual pigment content, the overall rate and thermodynamics of the photosynthetic electron flow are similar to those of chlorophyll a-containing species. The midpoint potential of both cytochrome f and the primary electron donor of photosystem I (P(740)) were found to be unchanged with respect to those prevailing in organisms having chlorophyll a, being 345 and 425 mV, respectively. Thus, contrary to previous reports (Hu, Q., Miyashita, H., Iwasaki, I. I., Kurano, N., Miyachi, S., Iwaki, M., and Itoh, S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 13319-13323), the midpoint potential of the electron donor P(740) has not been tuned to compensate for the decrease in excitonic energy in A. marina and to maintain the reducing power of photosystem I. We argue that this is a weaker constraint on the engineering of the oxygenic photosynthetic electron transfer chain than preserving the driving force for plastoquinol oxidation by P(740), via the cytochrome b(6)f complex. We further show that there is no restriction in the diffusion of the soluble electron carrier between cytochrome b(6)f and photosystem I in A. marina, at variance with plants. This difference probably reflects the simplified ultrastructure of the thylakoids of this organism, where no segregation into grana and stroma lamellae is observed. Nevertheless, chlorophyll fluorescence measurements suggest that there is energy transfer between adjacent photosystem II complexes but not from photosystem II to photosystem I, indicating spatial separation between the two photosystems.

International audience; Diatoms are one of the most ecologically successful classes of photosynthetic marine eukaryotes in the contemporary oceans. Over the past 30 million years, they have helped to moderate Earth's climate by absorbing carbon dioxide from the atmosphere, sequestering it via the biological carbon pump and ultimately burying organic carbon in the lithosphere. The proportion of planetary primary production by diatoms in the modern oceans is roughly equivalent to that of terrestrial rainforests. In photosynthesis, the efficient conversion of carbon dioxide into organic matter requires a tight control of the ATP/NADPH ratio which, in other photosynthetic organisms, relies principally on a range of plastid-localized ATP generating processes. Here we show that diatoms regulate ATP/NADPH through extensive energetic exchanges between plastids and mitochondria. This interaction comprises the re-routing of reducing power generated in the plastid towards mitochondria and the import of mitochondrial ATP into the plastid, and is mandatory for optimized carbon fixation and growth. We propose that the process may have contributed to the ecological success of diatoms in the ocean.

Summary Photosynthetic organisms have developed various photoprotective mechanisms to cope with exposure to high light intensities. In photosynthetic dinoflagellates that live in symbiosis with cnidarians, the nature and relative amplitude of these regulatory mechanisms are a matter of debate. In our study, the amplitude of photosynthetic alternative electron flows (AEF) to oxygen (chlororespiration, Mehler reaction), the mitochondrial respiration and the Photosystem I (PSI) cyclic electron flow were investigated in strains belonging to three clades (A1, B1 and F1) of Symbiodinium. Cultured Symbiodinium strains were maintained under identical environmental conditions, and measurements of oxygen evolution, fluorescence emission and absorption changes at specific wavelengths were used to evaluate PSI and PSII electron transfer rates (ETR). A light- and O2-dependent ETR was observed in all strains. This electron transfer chain involves PSII and PSI and is insensitive to inhibitors of mitochondrial activity and carbon fixation. We demonstrate that in all strains, the Mehler reaction responsible for photoreduction of oxygen by the PSI under high light, is the main AEF at the onset and at the steady state of photosynthesis. This sustained photosynthetic AEF under high light intensities acts as a photoprotective mechanism and leads to an increase of the ATP/NADPH ratio.

Diatoms are prominent phytoplanktonic organisms that contribute around 40% of carbon assimilation in the oceans. They grow and perform optimally in variable environments, being able to cope with unpredictable changes in the amount and quality of light. The molecular mechanisms regulating diatom light responses are, however, still obscure. Using knockdown Phaeodactylum tricornutum transgenic lines, we reveal the key function of a member of the light-harvesting complex stress-related (LHCSR) protein family, denoted LHCX1, in modulation of excess light energy dissipation. In contrast to green algae, this gene is already maximally expressed in nonstressful light conditions and encodes a protein required for efficient light responses and growth. LHCX1 also influences natural variability in photoresponse, as evidenced in ecotypes isolated from different latitudes that display different LHCX1 protein levels. We conclude, therefore, that this gene plays a pivotal role in managing light responses in diatoms.

Adaptation of photosynthesis in marine environment has been examined in two strains of the green, picoeukaryote Ostreococcus : OTH95, a surface/high-light strain, and RCC809, a deep-sea/low-light strain. Differences between the two strains include changes in the light-harvesting capacity, which is lower in OTH95, and in the photoprotection capacity, which is enhanced in OTH95. Furthermore, RCC809 has a reduced maximum rate of O 2 evolution, which is limited by its decreased photosystem I (PSI) level, a possible adaptation to Fe limitation in the open oceans. This decrease is, however, accompanied by a substantial rerouting of the electron flow to establish an H 2 O-to-H 2 O cycle, involving PSII and a potential plastid plastoquinol terminal oxidase. This pathway bypasses electron transfer through the cytochrome b 6 f complex and allows the pumping of “extra” protons into the thylakoid lumen. By promoting the generation of a large ΔpH, it facilitates ATP synthesis and nonphotochemical quenching when RCC809 cells are exposed to excess excitation energy. We propose that the diversion of electrons to oxygen downstream of PSII, but before PSI, reflects a common and compulsory strategy in marine phytoplankton to bypass the constraints imposed by light and/or nutrient limitation and allow successful colonization of the open-ocean marine environment.

In the original publication, Fig. 2e reports an incorrect spectrum of the Electrochromic Shift (ECS) signal in plants. The spectrum erroneously presented in this figure (obtained by Jean Alric, Institut de Biologie Physico Chimique, Paris) was measured under nonoptimum conditions to assess the ECS features. The new spectrum of the electrochromic signal in Arabidopsis thaliana leaves presented as a new panel (e) of Fig. 2 has been measured 100 ls after a flash and therefore represents a pure ECS contribution.

In changing light conditions, photosynthetic organisms develop different strategies to maintain a fine balance between light harvesting, photochemistry, and photoprotection. One of the most widespread photoprotective mechanisms consists in the dissipation of excess light energy in the form of heat in the photosystem II antenna, which participates to the Non Photochemical Quenching (NPQ) of chlorophyll fluorescence. It is tightly related to the reversible epoxidation of xanthophyll pigments, catalyzed by the two enzymes, the violaxanthin deepoxidase and the zeaxanthin epoxidase. In Phaeomonas sp. (Pinguiophyte, Stramenopiles), we show that the regulation of the heat dissipation process is different from that of the green lineage: the NPQ is strictly proportional to the amount of the xanthophyll pigment zeaxanthin and the xanthophyll cycle enzymes are differently regulated. The violaxanthin deepoxidase is already active in the dark, because of a low luminal pH, and the zeaxanthin epoxidase shows a maximal activity under moderate light conditions, being almost inactive in the dark and under high light. This light-dependency mirrors the one of NPQ: Phaeomonas sp. displays a large NPQ in the dark as well as under high light, which recovers under moderate light. Our results pinpoint zeaxanthin epoxidase activity as the prime regulator of NPQ in Phaeomonas sp. and therefore challenge the deepoxidase-regulated xanthophyll cycle dogma.