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International audience; Diatoms are successful phytoplankton clades able 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 via the nonphotochemical quenching (NPQ) process. While the molecular effectors of NPQ as well as the involvement of the proton motive force (PMF) in its regulation are known, the regulators of the PET/PMF relationship remain unidentified in diatoms. We generated mutants of the H$^+$ /K$^+$ antiporter KEA3 in the model diatom $Phaeodactylum\ tricornutum$. Loss of KEA3 activity affects the PET/PMF coupling and NPQ responses at the onset of illumination, during transients and in steady-state conditions. Thus, this antiporter is a main regulator of the PET/PMF coupling. Consistent with this conclusion, a parsimonious model including only two free components, KEA3 and the diadinoxanthin de-epoxidase, describes most of the feedback loops between PET and NPQ. This simple 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 calcium ion (Ca$^{2+}$ )-binding domain in KEA3 modulating its activity. This circuit is likely tuned by the NPQ-effector proteins, LHCXs, providing diatoms with the required flexibility to thrive in different ocean provinces.

Photosynthetic algae cope with suboptimal levels of light and CO 2 . In low CO 2 and excess light, the green alga Chlamydomonas reinhardtii activates a CO 2 Concentrating Mechanism (CCM) and photoprotection; the latter is mediated by LHCSR1/3 and PSBS. How light and CO 2 signals converge to regulate photoprotective responses remains unclear. Here we show that excess light activates expression of photoprotection- and CCM-related genes and that depletion of CO 2 drives these responses, even in total darkness. High CO 2 levels, derived from respiration or impaired photosynthetic fixation, repress LHCSR3 and CCM genes while stabilizing the LHCSR1 protein. We also show that CIA5, which controls CCM genes, is a major regulator of photoprotection, elevating LHCSR3 and PSBS transcript accumulation while inhibiting LHCSR1 accumulation. Our work emphasizes the importance of CO 2 in regulating photoprotection and the CCM, demonstrating that the impact of light on photoprotection is often indirect and reflects intracellular CO 2 levels.

International audience; Algae belonging to the Microchloropsis genus are promising organisms for biotech purposes, being able to accumulate large amounts of lipid reserves. These organisms adapt to different trophic conditions, thriving in strict photoautotrophic conditions, as well as in the concomitant presence of light plus reduced external carbon as energy sources (mixotrophy). In this work, we investigated the mixotrophic responses of Microchloropsis gaditana (formerly Nannochloropsis gaditana ). Using the Biolog growth test, in which cells are loaded into multiwell plates coated with different organic compounds, we could not find a suitable substrate for Microchloropsis mixotrophy. By contrast, addition of the Lysogeny broth (LB) to the inorganic growth medium had a benefit on growth, enhancing respiratory activity at the expense of photosynthetic performances. To further dissect the role of respiration in Microchloropsis mixotrophy, we focused on the mitochondrial alternative oxidase (AOX), a protein involved in energy management in other algae prospering in mixotrophy. Knocking-out the AOX1 gene by transcription activator-like effector nuclease (TALE-N) led to the loss of capacity to implement growth upon addition of LB supporting the hypothesis that the effect of this medium was related to a provision of reduced carbon. We conclude that mixotrophic growth in Microchloropsis is dominated by respiratory rather than by photosynthetic energetic metabolism and discuss the possible reasons for this behavior in relationship with fatty acid breakdown via β-oxidation in this oleaginous alga.

Transcription of the plastid genome, one of the three genomes (nuclear, mitochondrial and plastidial) that co-exist in the plant cell, is performed by three ARN polymerases. Two NEPs (Nucleus-Encoded Plastid RNA polymerases) transcribe mainly housekeeping genes and one PEP (plastid-encoded RNA polymerase) transcribes principally photosynthesis related genes. PEP needs transcription factors of the sigma type that are nucleus-encoded. We have previously shown that all three RNA polymerases are present in dry seeds and are necessary for efficient germination. These findings raised the question of how theses RNA polymerases come into the dry seeds and what is the importance of plastid gene expression during seed formation. To answer this question my work consisted in the characterization of plastid gene expression profiles and the expression of the components of the plastid transcriptional machinery during the three phases of seed formation, i. e. embryogenesis, maturation (embryonic photosynthesis) and desiccation. The analysis of global plastid transcriptome patterns shows that mRNAs encoding proteins engaged in photosynthesis show the highest quantitative changes during seed formation. Highest mRNA levels are observed during maturation. During desiccation, photosynthesis related mRNA levels as well as the levels of the corresponding proteins strongly decrease. Concerning the expression of NEP and PEP components, we observe also a peak of protein accumulation during maturation that is followed by a strong diminution of the protein levels. On the other hand, the corresponding mRNAs increase continuously during desiccation. This means that these mRNAs accumulate without being translated. We conclude that the storage of mRNAs coding components of the plastid transcriptional machinery in dry seeds is important for efficient germination. Regarding the limited amount of biological material that is available for these types of studies, we have developed a new method for cDNA analyses on microchips that utilises quartz plates and TIRF microscopy. In this way we can visualise single molecules and the amount of necessary material is considerable diminished. Finally, we have also partially characterized the conditions under which embryonic photosynthesis is performed. These studies show that photosynthesis occurs in a special environment that is characterized by hypoxic atmosphere and green enriched light. However, the structure and functioning of the photosynthetic apparatus in seed chloroplasts seems to be very similar to that of chloroplasts in green leaves. This opens the question of how seed photosynthesis can be efficient. On the other hand we have shown that embryonic photosynthesis is indeed very important for efficient germination. Altogether, results provide new information on the functioning of plastid photosynthesis and transcription during seed formation. They underline the importance of the accumulation of NEP and PEP coding mRNAs in dry seeds. We suggest that embryonic photosynthesis influences seed germination not only by providing reserve compounds but also by producing NEP and PEP proteins. Although the majority of these proteins are degraded during desiccation, traces persist and are stored in dry seeds thus assuring immediate transcription of the plastid genome during imbibition/stratification. Our results explain how efficiency of germination is conditioned during seed formation.; L'expression du génome plastidial, un des trois génomes (nucléaire, mitochondrial et plastidial) qui coexistent dans les cellules végétales, est assurée par trois ARN polymérases. Deux NEP (Nuclear-Encoded RNA Polymerase) transcrivent la plupart des gènes de ménage tandis que la PEP (Plastid-Encoded RNA Polymerase) transcrit principalement les gènes liés à la fonction photosynthétique en s'associant à des facteurs de transcription d'origine nucléaire (facteurs sigma) importés dans le plaste. De précédents travaux dans l'équipe ont montré que, contrairement aux observations généralement admises, les trois ARN polymérases sont nécessaires pour assurer une germination efficace des graines d'Arabidopsis. L'objectif de notre travail est de comprendre comment ces enzymes ont été mises en place au cours de la formation de la graine. Pour cela, nous avons analysé l'expression de l'appareil transcriptionnel et du transcriptome plastidial durant les trois phases de formation de la graine d'Arabidopsis thaliana, c'est à dire l'embryogenèse, la maturation (phase photosynthétique) et la dessiccation. L'expression globale du transcriptome plastidial montre que les changements quantitatifs des transcrits sont les plus élevés pour les transcrits des gènes liés à la fonction photosynthétique. Ils sont très fortement exprimés pendant la phase de la maturation et diminuent ensuite, comme leurs protéines correspondantes. Nous observons également une forte accumulation des protéines codant les NEP et les sous unités de la PEP pendant la période de maturation des graines, suivie d'une forte diminution pendant la dessiccation. Cependant, les ARNm correspondants augmentent pendant la dessiccation. Le stockage de ces ARNm codant l'appareil transcriptionnel constitue une étape cruciale pour l'efficacité de la germination de la graine. La quantité de matériel biologique disponible pour ces études étant très limitée, nous avons développé une nouvelle technique de détection des ADNc sur lame de quartz, utilisant la microscopie TIRF. Cette méthode augmente la résolution (elle permet la détection de molécules uniques) et diminue considérablement la quantité de matériel nécessaire à l'hybridation. Finalement, nous avons analysé les conditions sous lesquelles se déroule la photosynthèse embryonnaire. Ces études ont montré que la photosynthèse dans l'embryon se déroule dans un environnement particulier, hypoxique, et sous un éclairement enrichi en longueurs d'onde vertes. Cependant, la structure et le fonctionnement de l'appareil photosynthétique sont semblables à ceux d'une feuille. Nous avons également montré que l'étape transitoire de la photosynthèse embryonnaire est indispensable à la vigueur germinative des graines. Les résultats obtenus lors de ce travail apportent de nouvelles informations sur le fonctionnement de la transcription plastidiale au cours de la formation de la graine. L'importance de l'accumulation d'ARNm, de certaines protéines ainsi que celle de la photosynthèse embryonnaire dans la vigueur germinative ont été soulignées. Ces données permettent de comprendre comment l'efficacité de la germination est conditionnée par la phase de formation de la graine.