Francis-André Wollman

Institute of Physical and Chemical Biology, Lutetia Parisorum, Île-de-France, France

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Publications (65)402.8 Total impact

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    ABSTRACT: We characterized two spontaneous and dominant nuclear mutations in the unicellular alga Chlamydomonas reinhardtii, ncc1 and ncc2 (for nuclear control of chloroplast gene expression), which affect two octotricopeptide repeat (OPR) proteins encoded in a cluster of paralogous genes on chromosome 15. Both mutations cause a single amino acid substitution in one OPR repeat. As a result, the mutated NCC1 and NCC2 proteins now recognize new targets that we identified in the coding sequences of the chloroplast atpA and petA genes, respectively. Interaction of the mutated proteins with these targets leads to transcript degradation; however, in contrast to the ncc1 mutation, the ncc2 mutation requires on-going translation to promote the decay of the petA mRNA. Thus, these mutants reveal a mechanism by which nuclear factors act on chloroplast mRNAs in Chlamydomonas. They illustrate how diversifying selection can allow cells to adapt the nuclear control of organelle gene expression to environmental changes. We discuss these data in the wider context of the evolution of regulation by helical repeat proteins. © 2015 American Society of Plant Biologists. All rights reserved.
    The Plant Cell 03/2015; DOI:10.1105/tpc.15.00010 · 9.58 Impact Factor
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    ABSTRACT: Photosynthetic microalgae are exposed to changing environmental conditions. In particular, microbes found in ponds or soils often face hypoxia or even anoxia and this severely impacts their physiology. Chlamydomonas reinhardtii is one amongst such photosynthetic microorganisms recognized for its unusual wealth of fermentative pathways and the extensive remodeling of its metabolism upon switch to anaerobic conditions. As regard to photosynthetic electron transfer, this remodeling encompasses a strong limitation of the electron flow downstream of Photosystem I. Here, we further characterize the origin of this limitation. We show that it stems from the strong reducing pressure that builds up upon the onset of anoxia and this pressure can be relieve either by the light-induced synthesis of ATP which promotes the consumption of reducing equivalents, or by the progressive activation of the hydrogenase pathway which provides an electron transfer pathway alternative to the CO2 fixation cycle. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 02/2015; DOI:10.1074/jbc.M114.632588 · 4.60 Impact Factor
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    ABSTRACT: Plastids have retained from their cyanobacterial ancestor a fragment of the respiratory electron chain comprising an NADPH dehydrogenase and a diiron oxidase, which sustain the so-called chlororespiration pathway. Despite its very low turnover rates compared with photosynthetic electron flow, knocking out the plastid terminal oxidase (PTOX) in plants or microalgae leads to severe phenotypes that encompass developmental and growth defects together with increased photosensitivity. On the basis of a phylogenetic and structural analysis of the enzyme, we discuss its physiological contribution to chloroplast metabolism, with an emphasis on its critical function in setting the redox poise of the chloroplast stroma in darkness. The emerging picture of PTOX is that of an enzyme at the crossroads of a variety of metabolic processes, such as, among others, the regulation of cyclic electron transfer and carotenoid biosynthesis, which have in common their dependence on the redox state of the plastoquinone pool, set largely by the activity of PTOX in darkness. Expected final online publication date for the Annual Review of Plant Biology Volume 66 is April 29, 2015. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
    Annual review of plant biology 01/2015; 66(1). DOI:10.1146/annurev-arplant-043014-114744 · 18.90 Impact Factor
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    ABSTRACT: To address possible cell-to-cell heterogeneity in growth dynamics of isogenic cell populations of Chlamydomonas reinhardtii, we developed a millifluidic drop-based device that not only allows the analysis of populations grown from single cells over periods of a week, but is also able to sort and collect drops of interest, containing viable and healthy cells, which can be used for further experimentation. In this study, we used isogenic algal cells that were first synchronized in mixotrophic growth conditions. We show that these synchronized cells, when placed in droplets and kept in mixotrophic growth conditions, exhibit mostly homogeneous growth statistics, but with two distinct subpopulations: a major population with a short doubling-time (fast-growers) and a significant subpopulation of slowly dividing cells (slow-growers). These observations suggest that algal cells from an isogenic population may be present in either of two states, a state of restricted division and a state of active division. When isogenic cells were allowed to propagate for about 1000 generations on solid agar plates, they displayed an increased heterogeneity in their growth dynamics. Although we could still identify the original populations of slow- and fast-growers, drops inoculated with a single progenitor cell now displayed a wider diversity of doubling-times. Moreover, populations dividing with the same growth-rate often reached different cell numbers in stationary phase, suggesting that the progenitor cells differed in the number of cell divisions they could undertake. We discuss possible explanations for these cell-to-cell heterogeneities in growth dynamics, such as mutations, differential aging or stochastic variations in metabolites and macromolecules yielding molecular switches, in the light of single-cell heterogeneities that have been reported among isogenic populations of other eu- and prokaryotes.
    PLoS ONE 01/2015; 10(3):e0118987. DOI:10.1371/journal.pone.0118987 · 3.53 Impact Factor
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    ABSTRACT: Starving microalgae for nitrogen sources is commonly used as a biotechnological tool to boost storage of reduced carbon into starch granules or lipid droplets, but the accompanying changes in bioenergetics have been little studied so far. Here, we report that the selective depletion of Rubisco and cytochrome b6f complex that occurs when Chlamydomonas reinhardtii is starved for nitrogen in the presence of acetate and under normoxic conditions is accompanied by a marked increase in chlororespiratory enzymes, which converts the photosynthetic thylakoid membrane into an intracellular matrix for oxidative catabolism of reductants. Cytochrome b6f subunits and most proteins specifically involved in their biogenesis are selectively degraded, mainly by the FtsH and Clp chloroplast proteases. This regulated degradation pathway does not require light, active photosynthesis, or state transitions but is prevented when respiration is impaired or under phototrophic conditions. We provide genetic and pharmacological evidence that NO production from intracellular nitrite governs this degradation pathway: Addition of a NO scavenger and of two distinct NO producers decrease and increase, respectively, the rate of cytochrome b6f degradation; NO-sensitive fluorescence probes, visualized by confocal microscopy, demonstrate that nitrogen-starved cells produce NO only when the cytochrome b6f degradation pathway is activated.
    The Plant Cell 01/2014; 26(1). DOI:10.1105/tpc.113.120121 · 9.58 Impact Factor
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    ABSTRACT: FtsH is the major thylakoid membrane protease found in organisms performing oxygenic photosynthesis. Here, we show that FtsH from Chlamydomonas reinhardtii forms heterooligomers comprising two subunits, FtsH1 and FtsH2. We characterized this protease using FtsH mutants that we identified through a genetic suppressor approach that restored phototrophic growth of mutants originally defective for cytochrome b6f accumulation. We thus extended the spectrum of FtsH substrates in the thylakoid membranes beyond photosystem II, showing the susceptibility of cytochrome b6f complexes (and proteins involved in the ci heme binding pathway to cytochrome b6) to FtsH. We then show how FtsH is involved in the response of C. reinhardtii to macronutrient stress. Upon phosphorus starvation, photosynthesis inactivation results from an FtsH-sensitive photoinhibition process. In contrast, we identified an FtsH-dependent loss of photosystem II and cytochrome b6f complexes in darkness upon sulfur deprivation. The D1 fragmentation pattern observed in the latter condition was similar to that observed in photoinhibitory conditions, which points to a similar degradation pathway in these two widely different environmental conditions. Our experiments thus provide extensive evidence that FtsH plays a major role in the quality control of thylakoid membrane proteins and in the response of C. reinhardtii to light and macronutrient stress.
    The Plant Cell 01/2014; 26(1). DOI:10.1105/tpc.113.120113 · 9.58 Impact Factor
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    Dataset: SuppMRL1
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    ABSTRACT: Photosynthesis is the biological process that feeds the biosphere with reduced carbon. The assimilation of CO2 requires the fine tuning of two co-existing functional modes: linear electron flow, which provides NADPH and ATP, and cyclic electron flow, which only sustains ATP synthesis. Although the importance of this fine tuning is appreciated, its mechanism remains equivocal. Here we show that cyclic electron flow as well as formation of supercomplexes, thought to contribute to the enhancement of cyclic electron flow, are promoted in reducing conditions with no correlation with the reorganization of the thylakoid membranes associated with the migration of antenna proteins towards Photosystems I or II, a process known as state transition. We show that cyclic electron flow is tuned by the redox power and this provides a mechanistic model applying to the entire green lineage including the vast majority of the cases in which state transition only involves a moderate fraction of the antenna.
    Nature Communications 06/2013; 4:1954. DOI:10.1038/ncomms2954 · 10.74 Impact Factor
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    ABSTRACT: Chondrus crispus is a species of red algae that grows on rocks from the middle intertidal into the subtidal zones of the North Atlantic coasts. As such, it has to cope with strongly variable abiotic conditions. Here we studied the response of the photosynthetic apparatus of this red alga to illumination. We found that, as previously described in the case of the unicellular alga Rhodella violacea (E. Delphin et al., Plant Physiol. 118 (1998) 103-113), a single multi-turnover saturating pulse of light is sufficient to induce a strong quenching of fluorescence. To elucidate the mechanisms underlying this fluorescence quenching, we combined room temperature and 77K fluorescence measurements with absorption spectroscopy to monitor the redox state of the different electron carriers in the chain. In addition, we studied the dependence of these various observables upon the excitation wavelength. This led us to identify energy spill-over from Photosystem II to Photosystem I rather than a qE-type non-photochemical quenching as the major source of fluorescence quenching that develops upon a series of 200 ms pulses of saturating light results, in line with the conclusion of Ley and Butler (Biochim. Biophys. Acta 592 (1980) 349-363) from their studies of the unicellular red alga P. cruentum. In addition, we show that the onset of this spill-over is triggered by the reduction of the plastoquinone pool.
    Biochimica et Biophysica Acta 04/2013; DOI:10.1016/j.bbabio.2013.04.004 · 4.66 Impact Factor
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    ABSTRACT: Zinc is an essential nutrient because of its role in catalysis and in protein stabilization, but excess zinc is deleterious. We distinguished four nutritional zinc states in the alga Chlamydomonas reinhardtii: toxic, replete, deficient and limited. Growth is inhibited in zinc-limited and zinc toxic cells relative to zinc-replete cells, while zinc-deficiency is visually asymptomatic but distinguished by the accumulation of transcripts encoding ZIP family transporters. To identify targets of zinc deficiency and mechanisms of zinc acclimation, we used RNA-seq to probe zinc nutrition responsive changes in gene expression. We identified genes encoding zinc-handling components, including ZIP family transporters and candidate chaperones. Additionally, we noted an impact on two other regulatory pathways, the carbon concentrating mechanism (CCM) and the nutritional copper regulon. Targets of transcription factor Ccm1 and various CAH genes are up-regulated in zinc-deficiency, likely due to reduced carbonic anhydrase activity, validated by quantitative proteomics and immunoblot analysis of Cah1, Cah3 and Cah4. Chlamydomonas is therefore not able to grow photoautotrophically in zinc-limiting conditions, but supplementation with 1% CO2 restores growth to wild-type rates, suggesting that the inability to maintain CCM is a major consequence of zinc limitation. The Crr1 regulon responds to Cu limitation and is turned on in zinc deficiency, and Crr1 is required for growth in zinc-limiting conditions. Zinc deficient cells are functionally copper deficient, even though they hyperaccumulate copper up to 50-fold over normal levels. We suggest that zinc-deficient cells sequester Cu in a bio-unavailable form, perhaps to prevent mis-metallation of critical zinc sites.
    Journal of Biological Chemistry 02/2013; DOI:10.1074/jbc.M113.455105 · 4.60 Impact Factor
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    ABSTRACT: Absorption of light in excess of the capacity for photosynthetic electron transport is damaging to photosynthetic organisms. Several mechanisms exist to avoid photodamage, which are collectively referred to as nonphotochemical quenching. This term comprises at least two major processes. State transitions (qT) represent changes in the relative antenna sizes of photosystems II and I. High energy quenching (qE) is the increased thermal dissipation of light energy triggered by lumen acidification. To investigate the respective roles of qE and qT in photoprotection, a mutant (npq4 stt7-9) was generated in Chlamydomonas reinhardtii by crossing the state transition-deficient mutant (stt7-9) with a strain having a largely reduced qE capacity (npq4). The comparative phenotypic analysis of the wild type, single mutants, and double mutants reveals that both state transitions and qE are induced by high light. Moreover, the double mutant exhibits an increased photosensitivity with respect to the single mutants and the wild type. Therefore, we suggest that besides qE, state transitions also play a photoprotective role during high light acclimation of the cells, most likely by decreasing hydrogen peroxide production. These results are discussed in terms of the relative photoprotective benefit related to thermal dissipation of excess light and/or to the physical displacement of antennas from photosystem II.
    The Plant Cell 02/2013; DOI:10.1105/tpc.112.108274 · 9.58 Impact Factor
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    ABSTRACT: Based on previous comparative genomic analyses, a set of nearly 600 polypeptides, of which ~300 have unknown physiological function, was identified that is present in green algae and flowering and nonflowering plants, but not present (or highly diverged) in non-photosynthetic organisms. The gene encoding one of these GreenCut proteins, CPLD38, is in the same operon as ndhL in most cyanobacteria; NdhL is part of a complex essential for cyanobacterial respiration. A cpld38 mutant of Chlamydomonas reinhardtii did not grow on minimal medium, was high light sensitive under photoheterotrophic conditions, had lower accumulation of photosynthetic complexes, reduced photosynthetic electron flow to P700+, and reduced photochemical efficiency of photosystem II; these phenotypes were rescued by a wild-type copy of CPLD38. Biophysical and biochemical analyses demonstrated that cytochrome b6f function was severely compromised, and levels of transcripts and polypeptide subunits associated with the cytochrome b6f complex were also significantly lower in the mutant; the subunits of the cytochrome b6f complex turned over much more rapidly in mutant than in wild-type cells. Interestingly, PTOX2 and NDA2, two major proteins involved in chlororespiration, were more than 5-fold higher in mutant relative to wild-type cells, suggesting a shift from photosynthesis toward chlororespiratory metabolism in mutant cells, which is supported by experiments that quantify the reduction state of the plastoquinone pool. These findings support the hypothesis that CPLD38 impacts the stability of the cytochrome b6f complex and may play a key role in balancing redox inputs to the quinone pool from photosynthesis and chlororespiration.
    Journal of Biological Chemistry 01/2013; DOI:10.1074/jbc.M112.427476 · 4.60 Impact Factor
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    ABSTRACT: By homology with the unique plastid terminal oxidase (PTOX) found in plants, two genes encoding oxidases have been found in the Chlamydomonas genome, PTOX1 and PTOX2. Here we report the identification of a knockout mutant of PTOX2. Its molecular and functional characterization demonstrates that it encodes the oxidase most predominantly involved in chlororespiration in this algal species. In this mutant, the plastoquinone pool is constitutively reduced under dark-aerobic conditions, resulting in the mobile light-harvesting complexes being mainly, but reversibly, associated with photosystem I. Accordingly, the ptox2 mutant shows lower fitness than wild type when grown under phototrophic conditions. Single and double mutants devoid of the cytochrome b(6)f complex and PTOX2 were used to measure the oxidation rates of plastoquinols via PTOX1 and PTOX2. Those lacking both the cytochrome b(6)f complex and PTOX2 were more sensitive to light than the single mutants lacking either the cytochrome b(6)f complex or PTOX2, which discloses the role of PTOX2 under extreme conditions where the plastoquinone pool is overreduced. A model for chlororespiration is proposed to relate the electron flow rate through these alternative pathways and the redox state of plastoquinones in the dark. This model suggests that, in green algae and plants, the redox poise results from the balanced accumulation of PTOX and NADPH dehydrogenase.
    Proceedings of the National Academy of Sciences 12/2011; 108(51):20820-5. DOI:10.1073/pnas.1110518109 · 9.81 Impact Factor
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    ABSTRACT: After endosymbiosis, organelles lost most of their initial genome. Moreover, expression of the few remaining genes became tightly controlled by the nucleus through trans-acting protein factors that are required for post-transcriptional expression (maturation/stability or translation) of a single (or a few) specific organelle target mRNA(s). Here, we characterize the nucleus-encoded TDA1 factor, which is specifically required for translation of the chloroplast atpA transcript that encodes subunit α of ATP synthase in Chlamydomonas reinhardtii. The sequence of TDA1 contains eight copies of a degenerate 38-residue motif, that we named octotrico peptide repeat (OPR), which has been previously described in a few other trans-acting factors targeted to the C. reinhardtii chloroplast. Interestingly, a proportion of the untranslated atpA transcripts are sequestered into high-density, non-polysomic, ribonucleoprotein complexes. Our results suggest that TDA1 has a dual function: (i) trapping a subset of untranslated atpA transcripts into non-polysomic complexes, and (ii) translational activation of these transcripts. We discuss these results in light of our previous observation that only a proportion of atpA transcripts are translated at any given time in the chloroplast of C. reinhardtii.
    The Plant Journal 05/2011; 67(6):1055-66. DOI:10.1111/j.1365-313X.2011.04657.x · 6.82 Impact Factor
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    ABSTRACT: Central in respiration or photosynthesis, the cytochrome bc(1) and b(6)f complexes are regarded as functionally similar quinol oxidoreductases. They both catalyse a redox loop, the Q-cycle, which couples electron and proton transfer. This loop involves a bifurcated electron transfer step considered as being mechanistically mandatory, making the Q-cycle indispensable for growth. Attempts to falsify this paradigm in the case of cytochrome bc(1) have failed. The rapid proteolytic degradation of b(6)f complexes bearing mutations aimed at hindering the Q-cycle has precluded so far the experimental assessment of this model in the photosynthetic chain. Here we combine mutations in Chlamydomonas that inactivate the redox loop but preserve high accumulation levels of b(6)f complexes. The oxidoreductase activity of these crippled complexes is sufficient to sustain photosynthetic growth, which demonstrates that the Q-cycle is dispensable for oxygenic photosynthesis.
    Nature Communications 05/2011; 2:301. DOI:10.1038/ncomms1299 · 10.74 Impact Factor
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    ABSTRACT: Organelle gene expression is characterized by nucleus-encoded trans-acting factors that control posttranscriptional steps in a gene-specific manner. As a typical example, in Chlamydomonas reinhardtii, expression of the chloroplast petA gene encoding cytochrome f, a major subunit of the cytochrome b(6)f complex, depends on MCA1 and TCA1, required for the accumulation and translation of the petA mRNA. Here, we show that these two proteins associate in high molecular mass complexes that also contain the petA mRNA. We demonstrate that MCA1 is degraded upon interaction with unassembled cytochrome f that transiently accumulates during the biogenesis of the cytochrome b(6)f complex. Strikingly, this interaction relies on the very same residues that form the repressor motif involved in the Control by Epistasy of cytochrome f Synthesis (CES), a negative feedback mechanism that downregulates cytochrome f synthesis when its assembly within the cytochrome b(6)f complex is compromised. Based on these new findings, we present a revised picture for the CES regulation of petA mRNA translation that involves proteolysis of the translation enhancer MCA1, triggered by its interaction with unassembled cytochrome f.
    The Plant Cell 02/2011; 23(1):333-49. DOI:10.1105/tpc.110.078170 · 9.58 Impact Factor
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    ABSTRACT: Chlamydomonas reinhardtii, a unicellular green alga, has been exploited as a reference organism for identifying proteins and activities associated with the photosynthetic apparatus and the functioning of chloroplasts. Recently, the full genome sequence of Chlamydomonas was generated and a set of gene models, representing all genes on the genome, was developed. Using these gene models, and gene models developed for the genomes of other organisms, a phylogenomic, comparative analysis was performed to identify proteins encoded on the Chlamydomonas genome which were likely involved in chloroplast functions (or specifically associated with the green algal lineage); this set of proteins has been designated the GreenCut. Further analyses of those GreenCut proteins with uncharacterized functions and the generation of mutant strains aberrant for these proteins are beginning to unmask new layers of functionality/regulation that are integrated into the workings of the photosynthetic apparatus.
    Photosynthesis Research 11/2010; 106(1-2):3-17. DOI:10.1007/s11120-010-9555-7 · 3.19 Impact Factor
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    Biochimica et Biophysica Acta (BBA) - Bioenergetics 07/2010; 1797:19-19. DOI:10.1016/j.bbabio.2010.04.075 · 4.83 Impact Factor

Publication Stats

2k Citations
402.80 Total Impact Points

Institutions

  • 1982–2015
    • Institute of Physical and Chemical Biology
      Lutetia Parisorum, Île-de-France, France
  • 2001–2014
    • French National Centre for Scientific Research
      • Institut de Biologie Physico-Chimique
      Lutetia Parisorum, Île-de-France, France
  • 1996–2014
    • Pierre and Marie Curie University - Paris 6
      • Institut de Biologie Physico-Chimique (IBPC) (CNRS)
      Lutetia Parisorum, Île-de-France, France
  • 2008
    • University College London
      Londinium, England, United Kingdom
  • 2006
    • Aix-Marseille Université
      Marsiglia, Provence-Alpes-Côte d'Azur, France