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Supplemental Figure S1. Experimental device used for combined measurements of gas exchange by using MIMS and chlorophyll fluorescence with a PAM. A. Picture of the experimental device. B. Schematic view of the cuvette used for simultaneous measurements of fluorescence and mass spectrometry. 2 .0 A
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Supplemental Figure S2. Representative chlorophyll fluorescence recordings obtained during a dark to light transient in anaerobically acclimated C. reinhardtii cells. (A) wild- type strain (WT). (B) flvB-208 mutant. Cells were harvested from photobioreactor cultures and placed in the MIMS cuvette. Chlorophyll fluorescence was recorded during a dark to light transient following 1h30 of dark anaerobic acclimation. Red spikes at the bottom of the figure show saturating flashes. 0 .8
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Supplemental Figure S3. Relationship between quantum yield of O2 evolution and ΔF/FM in aerobiosis at a low chlorophyll concentration. Chlorophyll fluorescence measurements were recorded using a PAM on top of a Hansatech cuvette linked to a
Membrane Inlet Mass Spectrometer (MIMS). Recorded PSII yields were correlated with O2 18 exchange measurements made with O2 using the MIMS in aerobic conditions after 10 minutes dark adaptation. Experimental relation between the ΔF/FM and the quantum yield of O2 Evolution. In red is shown the linear regression for the points with a ΔF/FM lower than 0.6. Chlorophyll concentration was set to 10µg Chl.mL-1.
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Supplemental Figure S4. Saturation curve of net photosynthesis with green light. Net O2 evolution was measured with a MIMS after 1 min illumination at different green light intensities (mean +/- SD, n=5 biological replicates). The arrow indicates the light intensity used throughout this work.
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Supplemental Figure S5. Maximum fluorescence yield (FM’) upon a shift from dark anaerobiosis to light as a control for assessing PSII absorbance changes. Maximum fluorescence level during the first ten minutes of illumination. After 1h30 of dark anaerobic acclimation in the MIMS cuvette, algal suspensions of wild type (red line) and flvB mutant (grey and dark lines) strains were illuminated (660 µmol photons m-2 s-1 green light).
Maximum chlorophyll fluorescence level was measured before illumination (FM) and each 30s upon illumination (FM’). Shown are mean values (+/-SD, n=3 for the mutants, n=6 for the WT), a star marks significant difference between the wt and the mutant strains (Pvalue <0.05) based on ANOVA analysis (Tukey adjusted P value). Fm’ value at the onset of light was normalized to 1 (r.u. relative units). A
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Supplemental Figure S8. Differences in O2 concentration present in the MIMS chamber containing flvB mutants compared to the WT during a dark to light transient in anaerobically acclimated C. reinhardtii. Differences in O2 concentration were calculated from absolute gas exchange as determined in Fig. 4. Shown are mean values of the following calculation for each of the two mutants : [O2](flvB)-[O2](WT) were [O2](flvB) and [O2](WT) are the O2 level in a mutant or a WT cell suspension respectively (+/-SD, n=3). 2 .0
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Supplemental Figure S9. Net O2 and CO2 exchange rates in the light in C. reinhardtii wild-type and two flvB mutants. After 95 min of dark anaerobic acclimation, WT (red) and two independent flvB mutant (flvB-208 and flvB-308, dark and grey respectively) cell -2 -1 suspensions were illuminated (660 µmol photons m s green light). O2 and CO2 gas exchange were measured using a MIMS. (A). Mean values of net O2 evolution (B) and net CO2 uptake rates measured between 1 min and 9 min of illumination Shown are mean values relative to the absolute WT level at the same time point (+/-SD, n=3 for the mutants, n=6 for the WT).. Letters (a, b and c) above the bars represent statistically significant differences (P value=0.05) between strains for a given gas exchange measurement based on ANOVA analysis. Values for
CO2 at 2 min are all close to 0 and were not plotted for clarity of the other data. A B
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Supplemental Figure S10. PSI and PSII activities as measured by ElectroChromic Shift (ECS) upon illumination of anaerobically-acclimated Chlamydomonas cells. ECS at 520 nm was measured after 95 min anerobic acclimation of Chlamydomonas cells (A, C) and after a subsequent 10 min illumination (200 µmol photons m-2 s-1 white light) (B, D). (A, B) flv mutants are compared to the CC-4533 parental strain. (C, D) hydA1/A2 mutants are compared to the CC-124 parental strain. Shown are means +/- SD (n=3, biological replicates). Chlorophyceae Chlamydomonas reinhardtii Core Volvox carteri f. nagariensis
Origin of hydrogenase Chlorophytes Streptophytes Chlorella variabilis NC64A Trebouxiophyceae Coccomyxa subellipsoidea C-169 Chlorella protothecoides Parachlorella kessleri
Chlorodendrophyceae Chlorophytes Tetraselmis sp. CU2551 Origin of Flvs
Bathycoccus prasinos Prasinophyceae Micromonas pusilla CCMP1545 Ostreococcus lucimarinus CCE9901 Ostreococcus tauri
Cyanidiophyceae Cyanidioschyzon merolae Galdieria sulphuraria Plantae Rhodophytes Bangiophyceae Porphyra umbilicalis Porphyridiophyceae Porphyridium purpureum
Glaucophytes Cyanophora paradoxa
Eustigmatophyceae Nannochloropsis gaditana CCMP526 Ochrophyta Phaeophyceae Ectocarpus siliculosus
Mediophyceae Thalassiosira pseudonana CCMP1335 Thalassiosira oceanica Bacillariophyta Cyclotella criptica
Harosa Bacillariophyta incertae sedis Phaeodactylum tricornutum
Cercozoa ; Chloroarachniophyceae Bigelowiella natans Chromista Cryptophyta ; Cryptophyceae Guillardia theta CCMP2712 Hacrobia
Haptophita ; Coccolithypheae Emiliania huxleyi CCMP1516 Chrysochromulina sp. CCMP291 Tisochrysis lutea
Supplemental S11. Repartition of Flavodiiron proteins (Flv) and [Fe-Fe] hydrogenase on the evolutionary tree of eukaryotic microalgae. The tree of life, representing the mains branchings between different classes of eukaryotic algae was drawn using previously published trees (Leliaert et al, 2012; Fucikova et al, 2014; Burlacot and Peltier, 2018) and sequenced organisms were placed on it. Absence of a Flavodiiron was defined as the absence of any gene meeting the requirements used in Supplemental Table 1. Presence or absence of Flvs is shown by the color of the organism name (respectively blue or red). Presence of a [Fe-Fe] hydrogenase in the genome is shown by a disc right of the organism name according to Burlacot and Peltier (2018), blue discs show the presence of hydrogenase with a known activity, purple circles show putative hydrogenases with a yet uncharacterized activity. In dashed line is shown the embranchment of streptophites with green algae (Martin and Melkonian, 2010). Proposed origin of genes are shown with arrows. Number of Organism Class Accession Reference Flvs Chlamydomonas XM_001699293.1 Chlorophyceae 2 Merchant et al, 2007 reinhardtii XM_001692864.1
Volvox carteri f. XM_002948441.1 Chlorophyceae 2 Prochnik et al, 2010 nagariensis XM_002948437.1
Chlorella variabilis XM_005849680.1 Trebouxiophyceae 2 Blanc et al, 2010 NC64A XM_005849412.1
XM_005643392.1 Coccomyxa subelisoida Trebouxiophyceae 2 Blanc et al, 2012 XM_005643394.1
GBEZ01019221.1 Tetraselmis sp. GSL018 Chlorodendrophyceae 2 D’Adamo et al, 2014 GBEZ01009242.1
XM_007514037.1 Bathycoccus prasinos Prasinophyceae 2 Moreau et al, 2012 XM_007514022.1
Micromonas pusilla XM_003057449.1 Prasinophyceae 2 Worden et al, 2009 CCMP1545 XM_003056967.1
Ostreococcus XM_001416063.1 Prasinophyceae 2 Palenik et al, 2007 lucimarinus CCE9901 XM_001416061.1
XM_003075167.1 Ostreococcus tauri Prasinophyceae 2 Derelle et al, 2006 XM_003075163.1
Supplemental Table S1. List of algae exhibiting a Flv in their genome. Genomic sequences of FlvA and FlvB from Chlamydomonas reinhardtii (https://phytozome.jgi.doe.gov/pz/portal.html Cre12.G531900 and Cre16.G691800 respectivelly) were BLAST against complete genome sequences of eukaryotic algae (https://blast.ncbi.nlm.nih.gov/Blast.cgi ). Presence of a Flavodiiron protein was defined as the presence of a gene showing a similarity score higher than 200 for one of the two reference Flvs and coding for a protein having the 3 characteristic domains of eukaryotic Flvs (Romao et al, 2016). Supplemental Material and methods:
Absorption change measurements. Carotenoid ElectroChromic Shift (ECS) was measured at 520 nm, using a JTS-10 (BioLogic, France). Cells were harvested from flasks cultures, centrifuged at 450g for 4 min and resuspended in a 3 mL glass cuvette in a buffer containing 20% Ficoll buffered with 20 mM HEPES (pH 7.2) at a final concentration of 20 µg Chl mL-1. ECS signal was measured as the rapid increase of absorbance signal at 520 nm upon a saturating laser flash (6 ns, 680 nm, 1mJ). Respective contributions of PSI and PSII to the ECS signal were obtained accordingly to Bailleul et al (2010). Prior to measurement, cells were incubated for 90 min in anaerobiosis, some were eventually illuminated for 10 minutes. Supplemental litterature cited :
Bailleul B, Cardol P, Breyton C, Finazzi G (2010) Electrochromism: a useful probe to study algal photosynthesis. Photosynth Res 106: 179-189
Blanc G, Agarkova I, Grimwood J, et al. (2012) The genome of the polar eukaryotic microalga Coccomyxa subellipsoidea reveals traits of cold adaptation. Genome Biology 13, R39.
Blanc G, Duncan G, Agarkova I, et al. (2010) The Chlorella variabilis NC64C genome reveals adaptation to photosymbiosis, coevolution with viruses, and cryptic sex. Plant Cell 22, 2943–2955.
Burlacot A, Peltier G (2018) Photosynthetic electron transfer pathways during hydrogen photoproduction in green algae: mechanisms and limitations. In M Seibert, G Torzillo, eds, Microalgal Hydrogen Production: Achievements and Perspectives. The Royal Society of Chemistry, pp 189-212
Derelle E, Ferraz C, Rombauts S et al. (2006) Genome analysis of the smallest free-living eukaryote Ostreococcus tauri unveils many unique features. Proceedings of the National Academy of Sciences, USA 103, 11647–11652.
D’Adamo S, Jinkerson RE, Boyd ES, Brown SL, Baxter BK, et al. (2014) Evolutionary and Biotechnological Implications of Robust Hydrogenase Activity in Halophilic Strains of Tetraselmis. PLoS ONE 9(1): e85812. doi:10.1371/journal.pone.0085812
Fučíková, K., Leliaert, F., Cooper, E. D., Škaloud, P., D'Hondt, S., De Clerck, O., et al. (2014). New phylogenetic hypotheses for the core Chlorophyta based on chloroplast sequence data. Front. Ecol. Evol. 2: 63. doi: 10.3389/fevo.2014.00063
Leliaert F, Smith DR, Moreau H, Herron MD, Verbruggen H, Delwiche CF, De Clerck O (2012) Phylogeny and molecular evolution of the green algae. Crit Rev Plant Sci 31: 1–46
Marin, B., and Melkonian, M. (2010). Molecular phylogeny and classification of the Mamiellophyceae class. nov. (Chlorophyta) based on sequence comparisons of the nuclear- and plastid-encoded rRNA operons. Protist 161, 304–336. doi: 10.1016/j.protis.2009.10.002
Merchant SS, Prochnik SE, Vallon O, et al. (2007) The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318, 245–251.
Moreau H, Verhelst B, Couloux A, et al. (2012) Gene functionalities and genome structure in Bathycoccus prasinos reflect cellular specialization at the base of the green lineage. Genome Biology 13, R74
Palenik B, Grimwood J, Aerts A et al. (2007) The tiny eukaryote Ostreococcus provides genomic insights into the paradox of plankton speciation. Proceedings of the National Academy of Sciences, USA 104, 7705–7710.
Prochnik, S.E., Umen, J., Nedelcu, .M. et al. (2010) Genomic analysis of organismal complexity in the multicellular green alga Volvox carteri. Science, 329, 223–226.
Worden AZ, Lee JH, Mock T et al. (2009) Green evolution and dynamic adaptations revealed by genomes of the marine picoeukaryotes Micromonas. Science 324, 268–272.
Romão C, Vicente J, Borges P, Frazão C, Teixeira M (2016) The dual function of flavodiiron proteins: oxygen and/or nitricoxide reductases. J. Biol. Inorg. Chem., DOI 10.1007/s00775-015-1329-4