Alternative photosynthesis pathways drive the algal CO2-concentrating mechanism.

Global photosynthesis consumes ten times more CO2 than net anthropogenic emissions, and microalgae account for nearly half of this consumption1. The high efficiency of algal photosynthesis relies on a mechanism concentrating CO2 (CCM) at the catalytic site of the carboxylating enzyme RuBisCO, which enhances CO2 fixation2. Although many cellular components involved in the transport and sequestration of inorganic carbon have been identified3,4, how microalgae supply energy to concentrate CO2 against a thermodynamic gradient remains unknown4-6. Here we show that in the green alga Chlamydomonas reinhardtii, the combined action of cyclic electron flow and O2 photoreduction-which depend on PGRL1 and flavodiiron proteins, respectively-generate a low luminal pH that is essential for CCM function. We suggest that luminal protons are used downstream of thylakoid bestrophin-like transporters, probably for the conversion of bicarbonate to CO2. We further establish that an electron flow from chloroplast to mitochondria contributes to energizing non-thylakoid inorganic carbon transporters, probably by supplying ATP. We propose an integrated view of the network supplying energy to the CCM, and describe how algal cells distribute energy from photosynthesis to power different CCM processes. These results suggest a route for the transfer of a functional algal CCM to plants to improve crop productivity.

Alternative electron pathways of photosynthesis power green algal CO2 capture.

Microalgae contribute to about half of global net photosynthesis, which converts sunlight into the chemical energy (ATP and NADPH) used to transform CO2 into biomass. Alternative electron pathways of photosynthesis have been proposed to generate additional ATP that is required to sustain CO2 fixation. However, the relative importance of each alternative pathway remains elusive. Here, we dissect and quantify the contribution of cyclic, pseudo-cyclic and chloroplast-to-mitochondria electron flows for their ability to sustain net photosynthesis in the microalga Chlamydomonas reinhardtii. We show that (i) each alternative pathway can provide sufficient additional energy to sustain high CO2 fixation rates, (ii) the alternative pathways exhibit cross-compensation, and (iii) the activity of at least one of the three alternative pathways is necessary to sustain photosynthesis. We further show that all pathways have very different efficiencies at energizing CO2 fixation, with the chloroplast-mitochondria interaction being the most efficient. Overall, our data lay bioenergetic foundations for biotechnological strategies to improve CO2 capture and fixation.

Hello darkness, my old friend: 3-KETOACYL-COENZYME A SYNTHASE4 is a branch point in the regulation of triacylglycerol synthesis in Arabidopsis thaliana.

Plant lipids are important as alternative sources of carbon and energy when sugars or starch are limited. Here, we applied combined heat and darkness or extended darkness to a panel of ∼300 Arabidopsis (Arabidopsis thaliana) accessions to study lipid remodeling under carbon starvation. Natural allelic variation at 3-KETOACYL-COENZYME A SYNTHASE4 (KCS4), a gene encoding an enzyme involved in very long chain fatty acid (VLCFA) synthesis, underlies the differential accumulation of polyunsaturated triacylglycerols (puTAGs) under stress. Ectopic expression of KCS4 in yeast and plants proved that KCS4 is a functional enzyme localized in the endoplasmic reticulum with specificity for C22 and C24 saturated acyl-CoA. Allelic mutants and transient overexpression in planta revealed the differential role of KCS4 alleles in VLCFA synthesis and leaf wax coverage, puTAG accumulation, and biomass. Moreover, the region harboring KCS4 is under high selective pressure and allelic variation at KCS4 correlates with environmental parameters from the locales of Arabidopsis accessions. Our results provide evidence that KCS4 plays a decisive role in the subsequent fate of fatty acids released from chloroplast membrane lipids under carbon starvation. This work sheds light on both plant response mechanisms and the evolutionary events shaping the lipidome under carbon starvation.

Absence of alka(e)nes triggers profound remodeling of glycerolipid and carotenoid composition in cyanobacteria membrane.

Alka(e)nes are produced by many living organisms and exhibit diverse physiological roles, reflecting a high functional versatility. Alka(e)nes serve as waterproof wax in plants, communicating pheromones for insects, and microbial signaling molecules in some bacteria. Although alka(e)nes have been found in cyanobacteria and algal chloroplasts, their importance for photosynthetic membranes has remained elusive. In this study, we investigated the consequences of the absence of alka(e)nes on membrane lipid composition and photosynthesis using the cyanobacterium Synechocystis PCC6803 as a model organism. By following the dynamics of membrane lipids and the photosynthetic performance in strains defected and altered in alka(e)ne biosynthesis, we show that drastic changes in the glycerolipid contents occur in the absence of alka(e)nes, including a decrease in the membrane carotenoid content, a decrease in some digalactosyldiacylglycerol (DGDG) species and a parallel increase in monogalactosyldiacylglycerol (MGDG) species. These changes are associated with a higher susceptibility of photosynthesis and growth to high light in alka(e)ne-deficient strains. All these phenotypes are reversed by expressing an algal photoenzyme producing alka(e)nes from fatty acids. Therefore, alkenes, despite their low abundance, are an essential component of the lipid composition of membranes. The profound remodeling of lipid composition that results from their absence suggests that they play an important role in one or more membrane properties in cyanobacteria. Moreover, the lipid compensatory mechanism observed is not sufficient to restore normal functioning of the photosynthetic membranes, particularly under high light intensity. We conclude that alka(e)nes play a crucial role in maintaining lipid homeostasis of thylakoid membranes, thereby contributing to the proper functioning of photosynthesis, particularly under elevated light intensities.

Acyl-CoA binding protein is required for lipid droplet degradation in the diatom Phaeodactylum tricornutum.

Diatoms (Bacillariophyceae) accumulate neutral storage lipids in lipid droplets during stress conditions, which can be rapidly degraded and recycled when optimal conditions resume. Since nutrient and light availability fluctuate in marine environments, storage lipid turnover is essential for diatom dominance of marine ecosystems. Diatoms have garnered attention for their potential to provide a sustainable source of omega-3 fatty acids. Several independent proteomic studies of lipid droplets isolated from the model oleaginous pennate diatom Phaeodactylum tricornutum have identified a previously uncharacterized protein with an acyl-CoA binding (ACB) domain, Phatrdraft_48778, here referred to as Phaeodactylum tricornutum acyl-CoA binding protein (PtACBP). We report the phenotypic effects of CRISPR-Cas9 targeted genome editing of PtACBP. ptacbp mutants were defective in lipid droplet and triacylglycerol degradation, as well as lipid and eicosapentaenoic acid synthesis, during recovery from nitrogen starvation. Transcription of genes responsible for peroxisomal ß-oxidation, triacylglycerol lipolysis, and eicosapentaenoic acid synthesis was inhibited. A lipid-binding assay using a synthetic ACB domain from PtACBP indicated preferential binding specificity toward certain polar lipids. PtACBP fused to eGFP displayed an endomembrane-like pattern, which surrounded the periphery of lipid droplets. PtACBP is likely responsible for intracellular acyl transport, affecting cell division, development, photosynthesis, and stress response. A deeper understanding of the molecular mechanisms governing storage lipid turnover will be crucial for developing diatoms and other microalgae as biotechnological cell factories.

Plant metabolomics.

Plants are a treasure trove of metabolic compounds. The chemical diversity of plant cells has developed and been maintained through evolution and metabolic regulation, and plays a crucial role in plant physiology, development, and adaption to changing environmental situations. Metabolomics, when combined with genomics and proteomics, has opened up unprecedented opportunities to address the biological importance of metabolic diversity. It has also provided an avenue for metabolic engineering to produce a particular compound of interest to meet societal and economical demands, an important effort to achieve sustainable development. This Special Issue therefore focuses on current trends in plant metabolomics research, providing examples in the development of analytical technologies, the functional study of plant metabolism, and applications to synthetic and engineering biology.

Algal photosynthesis converts nitric oxide into nitrous oxide.

Nitrous oxide (N2O), a potent greenhouse gas in the atmosphere, is produced mostly from aquatic ecosystems, to which algae substantially contribute. However, mechanisms of N2O production by photosynthetic organisms are poorly described. Here we show that the green microalga Chlamydomonas reinhardtii reduces NO into N2O using the photosynthetic electron transport. Through the study of C. reinhardtii mutants deficient in flavodiiron proteins (FLVs) or in a cytochrome p450 (CYP55), we show that FLVs contribute to NO reduction in the light, while CYP55 operates in the dark. Both pathways are active when NO is produced in vivo during the reduction of nitrites and participate in NO homeostasis. Furthermore, NO reduction by both pathways is restricted to chlorophytes, organisms particularly abundant in ocean N2O-producing hot spots. Our results provide a mechanistic understanding of N2O production in eukaryotic phototrophs and represent an important step toward a comprehensive assessment of greenhouse gas emission by aquatic ecosystems.

The phosphatidylethanolamine-binding protein DTH1 mediates degradation of lipid droplets in Chlamydomonas reinhardtii.

Lipid droplets (LDs) are intracellular organelles found in a wide range of organisms and play important roles in stress tolerance. During nitrogen (N) starvation, Chlamydomonas reinhardtii stores large amounts of triacylglycerols (TAGs) inside LDs. When N is resupplied, the LDs disappear and the TAGs are degraded, presumably providing carbon and energy for regrowth. The mechanism by which cells degrade LDs is poorly understood. Here, we isolated a mutant (dth1-1, Delayed in TAG Hydrolysis 1) in which TAG degradation during recovery from N starvation was compromised. Consequently, the dth1-1 mutant grew poorly compared to its parental line during N recovery. Two additional independent loss-of-function mutants (dth1-2 and dth1-3) also exhibited delayed TAG remobilization. DTH1 transcript levels increased sevenfold upon N resupply, and DTH1 protein was localized to LDs. DTH1 contains a putative lipid-binding domain (DTH1LBD) with alpha helices predicted to be structurally similar to those in apolipoproteins E and A-I. Recombinant DTH1LBD bound specifically to phosphatidylethanolamine (PE), a major phospholipid coating the LD surface. Overexpression of DTH1LBD in Chlamydomonas phenocopied the dth1 mutant's defective TAG degradation, suggesting that the function of DTH1 depends on its ability to bind PE. Together, our results demonstrate that the lipid-binding DTH1 plays an essential role in LD degradation and provide insight into the molecular mechanism of protein anchorage to LDs at the LD surface in photosynthetic cells.

Long-chain acyl-CoA synthetases activate fatty acids for lipid synthesis, remodeling and energy production in Chlamydomonas.

Long-chain acyl-CoA synthetases (LACSs) play many roles in mammals, yeasts and plants, but knowledge on their functions in microalgae remains fragmented. Here via genetic, biochemical and physiological analyses, we unraveled the function and roles of LACSs in the model microalga Chlamydomonas reinhardtii. In vitro assays on purified recombinant proteins revealed that CrLACS1, CrLACS2 and CrLACS3 all exhibited bona fide LACS activities toward a broad range of free fatty acids. The Chlamydomonas mutants compromised in CrLACS1, CrLACS2 or CrLACS3 did not show any obvious phenotypes in lipid content or growth under nitrogen (N)-replete condition. But under N-deprivation, CrLACS1 or CrLACS2 suppression resulted in c. 50% less oil, yet with a higher amount of chloroplast lipids. By contrast, CrLACS3 suppression impaired oil remobilization and cell growth severely during N-recovery, supporting its role in fatty acid ß-oxidation to provide energy and carbon sources for regrowth. Transcriptomics analysis suggested that the observed lipid phenotypes are likely not due to transcriptional reprogramming but rather a shift in metabolic adjustment. Taken together, this study provided solid experimental evidence for essential roles of the three Chlamydomonas LACS enzymes in lipid synthesis, remodeling and catabolism, and highlighted the importance of lipid homeostasis in cell growth under nutrient fluctuations.

Guanosine tetraphosphate (ppGpp) accumulation inhibits chloroplast gene expression and promotes super grana formation in the moss Physcomitrium (Physcomitrella) patens.

The nucleotides guanosine tetraphosphate and pentaphosphate (or (p)ppGpp) are implicated in the regulation of chloroplast function in plants. (p)ppGpp signalling is best understood in the model vascular plant Arabidopsis thaliana in which it acts to regulate plastid gene expression to influence photosynthesis, plant development and immunity. However, little information is known about the conservation or diversity of (p)ppGpp signalling in other land plants. We studied the function of ppGpp in the moss Physcomitrium (previously Physcomitrella) patens using an inducible system for triggering ppGpp accumulation. We used this approach to investigate the effects of ppGpp on chloroplast function, photosynthesis and growth. We demonstrate that ppGpp accumulation causes a dramatic drop in photosynthetic capacity by inhibiting chloroplast gene expression. This was accompanied by the unexpected reorganisation of the thylakoid system into super grana. Surprisingly, these changes did not affect gametophore growth, suggesting that bryophytes and vascular plants may have different tolerances to defects in photosynthesis. Our findings point to the existence of both highly conserved and more specific targets of (p)ppGpp signalling in the land plants that may reflect different growth strategies.

The Chlamydomonas transcription factor MYB1 mediates lipid accumulation under nitrogen depletion.

Microalgae accumulate high levels of oil under stress, but the underlying biosynthetic pathways are not fully understood. We sought to identify key regulators of lipid metabolism under stress conditions. We found that the Chlamydomonas reinhardtii gene encoding the MYB-type transcription factor MYB1 is highly induced under stress conditions. Two myb1 mutants accumulated less total fatty acids and storage lipids than their parental strain upon nitrogen (N) depletion. Transcriptome analysis revealed that genes involved in lipid metabolism are highly enriched in the wild-type but not in the myb1-1 mutant after 4 h of N depletion. Among these genes were several involved in the transport of fatty acids from the chloroplast to the endoplasmic reticulum (ER): acyl-ACP thioesterase (FAT1), Fatty Acid EXporters (FAX1, FAX2), and long-chain acyl-CoA synthetase1 (LACS1). Furthermore, overexpression of FAT1 in the chloroplast increased lipid production. These results suggest that, upon N depletion, MYB1 promotes lipid accumulation by facilitating fatty acid transport from the chloroplast to the ER. This study identifies MYB1 as an important positive regulator of lipid accumulation in C. reinhardtii upon N depletion, adding another player to the established regulators of this process, including NITROGEN RESPONSE REGULATOR 1 (NRR1) and TRIACYLGLYCEROL ACCUMULATION REGULATOR 1 (TAR1).

Fatty acid photodecarboxylase is an ancient photoenzyme that forms hydrocarbons in the thylakoids of algae.

Fatty acid photodecarboxylase (FAP) is one of the few enzymes that require light for their catalytic cycle (photoenzymes). FAP was first identified in the microalga Chlorella variabilis NC64A, and belongs to an algae-specific subgroup of the glucose-methanol-choline oxidoreductase family. While the FAP from C. variabilis and its Chlamydomonas reinhardtii homolog CrFAP have demonstrated in vitro activities, their activities and physiological functions have not been studied in vivo. Furthermore, the conservation of FAP activity beyond green microalgae remains hypothetical. Here, using a C. reinhardtii FAP knockout line (fap), we showed that CrFAP is responsible for the formation of 7-heptadecene, the only hydrocarbon of this alga. We further showed that CrFAP was predominantly membrane-associated and that >90% of 7-heptadecene was recovered in the thylakoid fraction. In the fap mutant, photosynthetic activity was not affected under standard growth conditions, but was reduced after cold acclimation when light intensity varied. A phylogenetic analysis that included sequences from Tara Ocean identified almost 200 putative FAPs and indicated that FAP was acquired early after primary endosymbiosis. Within Bikonta, FAP was retained in secondary photosynthetic endosymbiosis lineages but absent from those that lost the plastid. Characterization of recombinant FAPs from various algal genera (Nannochloropsis, Ectocarpus, Galdieria, Chondrus) provided experimental evidence that FAP photochemical activity was present in red and brown algae, and was not limited to unicellular species. These results thus indicate that FAP was conserved during the evolution of most algal lineages where photosynthesis was retained, and suggest that its function is linked to photosynthetic membranes.

The bZIP1 Transcription Factor Regulates Lipid Remodeling and Contributes to ER Stress Management in Chlamydomonas reinhardtii.

Endoplasmic reticulum (ER) stress is caused by the stress-induced accumulation of unfolded proteins in the ER. Here, we identified proteins and lipids that function downstream of the ER stress sensor INOSITOL-REQUIRING ENZYME1 (CrIRE1) that contributes to ER stress tolerance in Chlamydomonas (Chlamydomonas reinhardtii). Treatment with the ER stress inducer tunicamycin resulted in the splicing of a 32-nucleotide fragment of a basic leucine zipper 1 (bZIP1) transcription factor (CrbZIP1) mRNA by CrIRE1 that, in turn, resulted in the loss of the transmembrane domain in CrbZIP1, and the translocation of CrbZIP1 from the ER to the nucleus. Mutants deficient in CrbZIP1 failed to induce the expression of the unfolded protein response genes and grew poorly under ER stress. Levels of diacylglyceryltrimethylhomoserine (DGTS) and pinolenic acid (18:3Δ5,9,12) increased in the parental strains but decreased in the crbzip1 mutants under ER stress. A yeast one-hybrid assay revealed that CrbZIP1 activated the expression of enzymes catalyzing the biosynthesis of DGTS and pinolenic acid. Moreover, two lines harboring independent mutant alleles of Chlamydomonas desaturase (CrDES) failed to synthesize pinolenic acid and were more sensitive to ER stress than were their parental lines. Together, these results indicate that CrbZIP1 is a critical component of the ER stress response mediated by CrIRE1 in Chlamydomonas that acts via lipid remodeling.

The NanDeSyn database for Nannochloropsis systems and synthetic biology.

Nannochloropsis species, unicellular industrial oleaginous microalgae, are model organisms for microalgal systems and synthetic biology. To facilitate community-based annotation and mining of the rapidly accumulating functional genomics resources, we have initiated an international consortium and present a comprehensive multi-omics resource database named Nannochloropsis Design and Synthesis (NanDeSyn; http://nandesyn.single-cell.cn). Via the Tripal toolkit, it features user-friendly interfaces hosting genomic resources with gene annotations and transcriptomic and proteomic data for six Nannochloropsis species, including two updated genomes of Nannochloropsis oceanica IMET1 and Nannochloropsis salina CCMP1776. Toolboxes for search, Blast, synteny view, enrichment analysis, metabolic pathway analysis, a genome browser, etc. are also included. In addition, functional validation of genes is indicated based on phenotypes of mutants and relevant bibliography. Furthermore, epigenomic resources are also incorporated, especially for sequencing of small RNAs including microRNAs and circular RNAs. Such comprehensive and integrated landscapes of Nannochloropsis genomics and epigenomics will promote and accelerate community efforts in systems and synthetic biology of these industrially important microalgae.

The disassembly of lipid droplets in Chlamydomonas.

Lipid droplets (LDs) are ubiquitous and specialized organelles in eukaryotic cells. Consisting of a triacylglycerol core surrounded by a monolayer of membrane lipids, LDs are decorated with proteins and have myriad functions, from carbon/energy storage to membrane lipid remodeling and signal transduction. The biogenesis and turnover of LDs are therefore tightly coordinated with cellular metabolic needs in a fluctuating environment. Lipid droplet turnover requires remodeling of the protein coat, lipolysis, autophagy and fatty acid ß-oxidation. Several key components of these processes have been identified in Chlamydomonas (Chlamydomonas reinhardtii), including the major lipid droplet protein, a CXC-domain containing regulatory protein, the phosphatidylethanolamine-binding DTH1 (DELAYED IN TAG HYDROLYSIS1), two lipases and two enzymes involved in fatty acid ß-oxidation. Here, we review LD turnover and discuss its physiological significance in Chlamydomonas, a major model green microalga in research on algal oil.

ppGpp influences protein protection, growth and photosynthesis in Phaeodactylum tricornutum.

Chloroplasts retain elements of a bacterial stress response pathway that is mediated by the signalling nucleotides guanosine penta- and tetraphosphate ((p)ppGpp). In the model flowering plant Arabidopsis, ppGpp acts as a potent regulator of plastid gene expression and influences photosynthesis, plant growth and development. However, little is known about ppGpp metabolism or its evolution in other photosynthetic eukaryotes. Here, we studied the function of ppGpp in the diatom Phaeodactylum tricornutum using transgenic lines containing an inducible system for ppGpp accumulation. We used these lines to investigate the effects of ppGpp on growth, photosynthesis, lipid metabolism and protein expression. We demonstrate that ppGpp accumulation reduces photosynthetic capacity and promotes a quiescent-like state with reduced proliferation and ageing. Strikingly, using nontargeted proteomics, we discovered that ppGpp accumulation also leads to the coordinated upregulation of a protein protection response in multiple cellular compartments. Our findings highlight the importance of ppGpp as a fundamental regulator of chloroplast function across different domains of life, and lead to new questions about the molecular mechanisms and roles of (p)ppGpp signalling in photosynthetic eukaryotes.

CO2 supply modulates lipid remodelling, photosynthetic and respiratory activities in Chlorella species.

Microalgae represent a potential solution to reduce CO2 emission exploiting their photosynthetic activity. Here, the physiologic and metabolic responses at the base of CO2 assimilation were investigated in conditions of high or low CO2 availability in two of the most promising algae species for industrial cultivation, Chlorella sorokiniana and Chlorella vulgaris. In both species, high CO2 availability increased biomass accumulation with specific increase of triacylglycerols in C. vulgaris and polar lipids and proteins in C. sorokiniana. Moreover, high CO2 availability caused only in C. vulgaris a reduced NAD(P)H/NADP+ ratio and reduced mitochondrial respiration, suggesting a CO2 dependent increase of reducing power consumption in the chloroplast, which in turn influences the redox state of the mitochondria. Several rearrangements of the photosynthetic machinery were observed in both species, differing from those described for the model organism Chlamydomonas reinhardtii, where adaptation to carbon availability is mainly controlled by the translational repressor NAB1. NAB1 homologous protein could be identified only in C. vulgaris but lacked the regulation mechanisms previously described in C. reinhardtii. Acclimation strategies to cope with a fluctuating inorganic carbon supply are thus diverse among green microalgae, and these results suggest new biotechnological strategies to boost CO2 fixation.

Interorganelle Communication: Peroxisomal MALATE DEHYDROGENASE2 Connects Lipid Catabolism to Photosynthesis through Redox Coupling in Chlamydomonas.

Plants and algae must tightly coordinate photosynthetic electron transport and metabolic activities given that they often face fluctuating light and nutrient conditions. The exchange of metabolites and signaling molecules between organelles is thought to be central to this regulation but evidence for this is still fragmentary. Here, we show that knocking out the peroxisome-located MALATE DEHYDROGENASE2 (MDH2) of Chlamydomonas reinhardtii results in dramatic alterations not only in peroxisomal fatty acid breakdown but also in chloroplast starch metabolism and photosynthesis. mdh2 mutants accumulated 50% more storage lipid and 2-fold more starch than the wild type during nitrogen deprivation. In parallel, mdh2 showed increased photosystem II yield and photosynthetic CO2 fixation. Metabolite analyses revealed a >60% reduction in malate, together with increased levels of NADPH and H2O2 in mdh2 Similar phenotypes were found upon high light exposure. Furthermore, based on the lack of starch accumulation in a knockout mutant of the H2O2-producing peroxisomal ACYL-COA OXIDASE2 and on the effects of H2O2 supplementation, we propose that peroxisome-derived H2O2 acts as a regulator of chloroplast metabolism. We conclude that peroxisomal MDH2 helps photoautotrophs cope with nitrogen scarcity and high light by transmitting the redox state of the peroxisome to the chloroplast by means of malate shuttle- and H2O2-based redox signaling.

Deciphering Differential Life Stage Radioinduced Reproductive Decline in Caenorhabditis elegans through Lipid Analysis.

Wildlife is chronically exposed to various sources of ionizing radiations, both environmental or anthropic, due to nuclear energy use, which can induce several defects in organisms. In invertebrates, reproduction, which directly impacts population dynamics, has been found to be the most radiosensitive endpoint. Understanding the underlying molecular pathways inducing this reproduction decrease can help in predicting the effects at larger scales (i.e., population). In this study, we used a life stage dependent approach in order to better understand the molecular determinants of reproduction decrease in the roundworm C. elegans. Worms were chronically exposed to 50 mGy·h-1 external gamma ionizing radiations throughout different developmental periods (namely embryogenesis, gametogenesis, and full development). Then, in addition to reproduction parameters, we performed a wide analysis of lipids (different class and fatty acid via FAMES), which are both important signaling molecules for reproduction and molecular targets of oxidative stress. Our results showed that reproductive defects are life stage dependent, that lipids are differently misregulated according to the considered exposure (e.g., upon embryogenesis and full development) and do not fully explain radiation induced reproductive defects. Finally, our results enable us to propose a conceptual model of lipid signaling after radiation stress in which both the soma and the germline participate.

Chlorella vulgaris genome assembly and annotation reveals the molecular basis for metabolic acclimation to high light conditions.

Chlorella vulgaris is a fast-growing fresh-water microalga cultivated on the industrial scale for applications ranging from food to biofuel production. To advance our understanding of its biology and to establish genetics tools for biotechnological manipulation, we sequenced the nuclear and organelle genomes of Chlorella vulgaris 211/11P by combining next generation sequencing and optical mapping of isolated DNA molecules. This hybrid approach allowed us to assemble the nuclear genome in 14 pseudo-molecules with an N50 of 2.8 Mb and 98.9% of scaffolded genome. The integration of RNA-seq data obtained at two different irradiances of growth (high light, HL versus low light, LL) enabled us to identify 10 724 nuclear genes, coding for 11 082 transcripts. Moreover, 121 and 48 genes, respectively, were found in the chloroplast and mitochondrial genome. Functional annotation and expression analysis of nuclear, chloroplast and mitochondrial genome sequences revealed particular features of Chlorella vulgaris. Evidence of horizontal gene transfers from chloroplast to mitochondrial genome was observed. Furthermore, comparative transcriptomic analyses of LL versus HL provided insights into the molecular basis for metabolic rearrangement under HL versus LL conditions leading to enhanced de novo fatty acid biosynthesis and triacylglycerol accumulation. The occurrence of a cytosolic fatty acid biosynthetic pathway could be predicted and its upregulation upon HL exposure was observed, consistent with the increased lipid amount under HL conditions. These data provide a rich genetic resource for future genome editing studies, and potential targets for biotechnological manipulation of Chlorella vulgaris or other microalgae species to improve biomass and lipid productivity.