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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; 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.

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.

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.

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.

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.

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 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.

The qualitative screening method to select complex I mutants in the microalga Chlamydomonas, based on reduced growth under heterotrophic condition, is not suited for high throughput screening. In order to develop a fast screening method based on measurements of chlorophyll fluorescence, we first demonstrated that complex I mutants displayed decreased photosystem II efficiency in the genetic background of a photosynthetic mutation leading to reduced formation of the electrochemical proton gradient in the chloroplast (pgrl1 mutation). In contrast, single mutants (complex I and pgrl1 mutants) could not be distinguished from wild type by their photosystem II efficiency in the tested conditions. We next performed an insertional mutagenesis on the pgrl1 mutant. Out of ~3000 hygromycin-resistant insertional transformants, 46 had decreased photosystem II efficiency and three were complex I mutants. One of the mutants was tagged and whole genome sequencing identified the resistance cassette in NDUFAF3, a homolog of the human NDUFAF3 gene, encoding for an assembly factor involved in complex I assembly. Complemented strains showed restored complex I activity and assembly. Overall, we described here a screening method which is fast and particularly suited for identification of Chlamydomonas complex I mutants. This article is protected by copyright. All rights reserved.