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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.

International audience

Nitrogen (N) deficiency is a frequently encountered situation that constrains global biomass productivity. In response to N deficiency, cell division stops and photosynthetic electron transfer are downregulated, while carbon storage is enhanced. However, the molecular mechanism downregulating photosynthesis during N deficiency and its relationship with carbon storage are not fully understood. The Proton Gradient Regulator-like 1 (PGRL1)-involved in cyclic electron flow (CEF) and Flavodiiron proteins involved in pseudo-(CEF) are major players in the acclimation of photosynthesis. To determine the role of PGRL1 or FLV in photosynthesis under N deficiency, we measured photosynthetic electron transfer, oxygen gas exchange and carbon storage in the knockout of Chlamydomonaspgrl1 and flvBmutants. Under N deficiency,pgrl1maintains higher net photosynthesis and O2photoreduction rates, whileflvBshows similar responses compared to control strains. The amount of cytochromeb6fwas maintained at a higher level inpgrl1. The photosynthetic activity ofpgrl1 flvBdouble mutants decreases in response to N deficiency similar to the control strains. Furthermore, the triacylglycerol content ofpgrl1was twice higher than the controls under N deficiency. Taken together, our results suggest that in the absence of PGRL1, FLV-mediated O2photoreduction through PCEF maintains net photosynthesis at a high level, resulting in increased triacylglycerol biosynthesis. This study reveals that PGRL1 and FLV play antagonistic roles during N deficiency. It further illustrates how nutrient status can affect the regulation of photosynthetic energy production in relation to carbon storage and provides new strategies for improving lipid productivity in algae.Significance statementNitrogen (N) deficiency, an often-encountered phenomenon in nature, triggers growth arrest and massive lipid accumulation in microalgae. The downregulation of photosynthesis is necessary to ensure cell viability. We demonstrate that a well-conserved protein in chlorophytes, the Proton Gradient Regulator-like 1 (PGRL1) is a key (down) regulator of photosynthesis. In its absence, cells exhibited sustained photosynthesis and over-accumulated lipids thanks to the Flavodiiron (FLV) protein. We propose that both PGRL1 and FLV, by having antagonistic roles in N deficiency, manage the redox landscape, carbon storage and biomass production. Our work revolves around the current paradigm of photosynthesis regulation during N deficiency and provides a new framework for improving lipid accumulation in microalgae for biotechnological purposes.

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).

Photosynthetic organisms have developed sophisticated strategies to fine-tune light energy conversion to meet the metabolic demand, thereby optimizing growth in fluctuating light environments. Although mechanisms such as energy dissipation, photosynthetic control, or the photosystem II (PSII) damage and repair have been widely studied, little is known about the regulation of protein synthesis capacity during light acclimation. By screening a Chlamydomonas reinhardtii insertional mutant library using chlorophyll fluorescence imaging, we isolated a high chlorophyll fluorescence mutant (hf0) defected in a gene encoding a putative plastid targeted DEAD-box RNA helicase called CreRH22. CreRH22 is rapidly induced upon illumination and belongs to the GreenCut, a set of proteins specific to photosynthetic organisms. While photosynthesis is slightly affected in the mutant under low light (LL), exposure to high light (HL) induces a marked decrease in both PSII and PSI, and a strong alteration of the light-induced gene expression pattern. These effects are explained by the inability of hf0 to increase plastid ribosome amounts under HL. We conclude that CreRH22, by promoting ribosomal RNA precursor maturation in a light-dependent manner, enables the assembly of extra-ribosomes required to synthesize photosystem subunits at a higher rate, a critical step in the acclimation of algae to HL.

International audience; 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.

International audience; Yonghua Li-Beisson received a B.Sc. from Henan University of Technology, China, and a Ph.D. from the University of Hull (England), where her work highlighted the importance of reducing power for lipid synthesis in oleaginous fungi. She then conducted postdoctoral research at Michigan State University focusing on understanding seed oil biosynthesis as well as dissecting the molecular pathways of lipid polyester synthesis and assembly. Since 2009, Yonghua is a staff scientist at the French Atomic and Alternative Energies Commission in France. Her current work focuses on dissecting lipid metabolism in microalgae, in particular using the model green microalga Chlamydomonas reinhardtii (Li-Beisson et al. 2019). In addition to leading her research team, she is also a director of the lipidomics platform HelioBiotec (https://www.cite-des-energies.fr/biam/plateformes-technologiques/heliobiotec/). Since 2016, Yonghua is a serving editor for Plant and Cell Physiology and handles papers in the area of plant and algal lipid metabolism and physiology

Subcellular compartmentalization confers evolutionary advantage to eukaryotic cells but entails the need for efficient interorganelle communication. Malate functions as redox carrier and metabolic intermediate. It can be shuttled across membranes through translocators. The interconversion of malate and oxaloacetate mediated by malate dehydrogenases requires oxidation/reduction of NAD(P)H/NAD(P)+; therefore, malate trafficking serves to transport reducing equivalents and this is termed the ‘malate shuttle’. Although the term 'malate shuttle' was coined more than 50 years ago, novel functions are still emerging. This review highlights recent findings on the functions of malate shuttles in photorespiration, fatty acid β-oxidation, interorganelle signaling and its putative role in CO2-concentrating mechanisms. We compare and contrast knowledge in plants and algae, thereby providing an evolutionary perspective on redox trafficking in photosynthetic eukaryotes.

Global photosynthesis consumes ten times more CO2 than net anthropogenic emissions, and microalgae account for nearly half of this consumption1. The great efficiency of algal photosynthesis relies on a mechanism concentrating CO2 (CCM) at the catalytic site of the carboxylating enzyme RuBisCO, thus enhancing CO2 fixation2. While many cellular components involved in the transport and sequestration of inorganic carbon (Ci) have been uncovered3,4, the way microalgae supply energy to concentrate CO2 against a thermodynamic gradient remains elusive4-6. Here, by monitoring dissolved CO2 consumption, unidirectional O2 exchange and the chlorophyll fluorescence parameter NPQ in the green alga Chlamydomonas, we show that the complementary effects of cyclic electron flow and O2 photoreduction, respectively mediated by PGRL1 and flavodiiron proteins, generate the proton motive force (pmf) required by Ci transport across thylakoid membranes. We demonstrate that the trans-thylakoid pmf is used by bestrophin-like Ci transporters and further establish that a chloroplast-to-mitochondria electron flow contributes to energize non-thylakoid Ci transporters, most likely by supplying ATP. We propose an integrated view of the CCM energy supply network, describing how algal cells distribute photosynthesis energy to power different Ci transporters, thus paving the way to the transfer of a functional algal CCM in plants towards improving crop productivity.One sentence summaryPhotosynthetic alternative electron flows and mitochondrial respiration drive the algal CO2 concentrating mechanism

International audience; 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 β-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.

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.

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. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

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 β-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 β-oxidation. Here, we review LD turnover and discuss its physiological significance in Chlamydomonas, a major model green microalga in research on algal oil.

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., High/low CO2 availability induces in Chlorella vulgaris and Chlorella sorokiniana specific cell responses as lipids remodelling, modulation of mitochondrial respiration and adaptations of the photosynthetic apparatus.

Light makes light work of fatty acids Photosynthetic organisms are notable for their ability to capture light energy and use it to power biosynthesis. Some algae have gone a step beyond photosynthesis and can use light to initiate enzymatic photodecarboxylation of fatty acids, producing long-chain hydrocarbons. To understand this transformation, Sorigué et al. brought to bear an array of structural, computational, and spectroscopic techniques and fully characterized the catalytic cycle of the enzyme. These experiments are consistent with a mechanism starting with electron transfer from the fatty acid to a photoexcited oxidized flavin cofactor. Decarboxylation yields an alkyl radical, which is then reduced by back electron transfer and protonation rather than hydrogen atom transfer. The wealth of experimental data explains how algae harness light energy to produce alka(e)nes and provides an appealing model system for understanding enzyme-catalyzed photochemistry more generally. Science , this issue p. eabd5687

Global photosynthesis consumes ten times more CO

Microalgae represent potential solutions to reduce the atmospheric CO2 level through photosynthesis. To boost CO2 fixation by microalgae it is essential to understand physiologic and metabolic responses at the base of CO2 assimilation and carbon flow. In this work two Trebouxiophyceae species, Chlorella sorokiniana and Chlorella vulgaris, were investigated for their metabolic responses to high and low CO2 (air level) availability. High CO2 availability resulted in an increase in biomass accumulation in both species but with a different chloroplast and mitochondrial responses. In C. sorokiniana we observed increased polar lipids and protein amount and a balanced NADPH redox state and a similar total respiration in the two conditions analysed. In contrast, in C. vulgaris high CO2 level caused an increase in TAG accumulation and a higher NADPH consumption suggesting a CO2 dependent increase of reducing power consumption in the chloroplast, which in turn influences the redox state of the mitochondria by lowering total dark respiration. Several rearrangements of the photosynthetic machinery were observed in both species, which differ from those described for the model organism Chlamydomonas reinhardtii. In the case of C. reinhardtii, adaptation of the photosynthetic apparatus to different CO2 availability relies on the translational repressor NAB1. NAB1 homologous protein could be identified only in C. vulgaris but lacked the regulation mechanisms previously described in C. reinhardtii. These findings highlight that the acclimation strategies to cope with a fluctuating inorganic carbon supply are diverse among green microalgae and point to new biotechnological strategies to boost CO2 fixation.One sentence summaryHigh/low CO2 availability induces cell responses as lipids remodelling, adaptations of the photosynthetic apparatus and modulation of mitochondrial respiration not conserved among green algae

SummaryThe nucleotides guanosine tetraphosphate and pentaphosphate (or ppGpp) are implicated in the regulation of chloroplast function in plants. ppGpp signalling is best understood in the model vascular plant Arabidopsis thaliana where it acts to regulate plastid gene expression to influence photosynthesis, plant development and immunity. However, little is known about the conservation or diversity of ppGpp signaling in other land plants. Here, 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 ppGpp signalling in the land plants that may reflect different growth strategies.

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.

International audience; 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.

SummaryChloroplasts retain elements of a bacterial stress response pathway that is mediated by the signalling nucleotides guanosine penta- and tetraphosphate, or (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 P. 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 non-targeted 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.

International audience; 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 (DTH1$^{LBD}$) with alpha helices predicted to be structurally similar to those in apolipoproteins E and A–I. Recombinant DTH1$^{LBD}$ bound specifically to phosphatidylethanolamine (PE), a major phospholipid coating the LD surface. Overexpression of DTH1$^{LBD}$ 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.

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 (DTH1(LBD)) with alpha helices predicted to be structurally similar to those in apolipoproteins E and A–I. Recombinant DTH1(LBD) bound specifically to phosphatidylethanolamine (PE), a major phospholipid coating the LD surface. Overexpression of DTH1(LBD) 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.

Due to their sessile lifestyle, plants have evolved unique mechanisms to deal with environmental challenges. Under stress, plant lipids are important as alternative sources of carbon and energy when sugars or starch are limited. Here, we applied combined heat and darkness and extended darkness to a panel of ∼ 300 Arabidopsis accessions to study lipid remodeling under carbon starvation. Natural allelic variation at3-KETOACYL-COENZYME A SYNTHASE4(KCS4), a gene encoding for an enzyme involved in very long chain fatty-acid (VLCFA) synthesis, underlies a differential accumulation of polyunsaturated triacylglycerols (puTAGs) under stress. Ectopic expression in yeast and plants proved that KCS4 is a functional enzyme localized in the ER with specificity for C22 and C24 saturated acyl-CoA. Allelic mutants and transient overexpressionin plantarevealed the differential role ofKCS4alleles in VLCFA synthesis and wax coverage, puTAG accumulation and biomass. Moreover, the region harboringKCS4is under high selective pressure and allelic variation atKCS4correlated 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.One sentence summaryNatural variation atKCS4underlies a differential accumulation of polyunsaturated triacylglycerols under carbon starvation, by acting as a regulatory branch point in the fate of fatty acids.

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

International audience; The plant kingdom contains an abundance of structurally diverse fatty acids referred to as unusual fatty acids. Unusual fatty acids on plant surfaces can form polyesters that contribute to the function of cutin as a barrier for water loss and pathogen protection. Unusual fatty acids are also found as abundant components of seed oils of selected species and often confer desirable properties for industrial and nutritional applications. Here, we review recent findings on the biosynthesis and metabolism of unusual fatty acids in cutin and seed oils and use of this information for enzyme structure-function studies and seed oil metabolic engineering. We also highlight the recent discovery of unusual fatty acids that are formed from a previously undescribed variation of fatty acid elongation.

International audience; Microalgae are continuously shaping Earth’s atmosphere through oxygenic photosynthesis, and nowadays, half of the photosynthesis is attributed to microbial photosynthesis (Field et al., 1998; Behrenfeld et al., 2005). While algal photosynthesis contributes to offsetting the CO2 footprint, it also produces nitric oxide (N2O), a potent greenhouse gas. In some ecological niches microalgae can produce hydrogen (H2), a promising energy carrier; therefore microalgae are actively explored for their potential as a platform for production of renewable energy. Measuring gas exchange between algae and the atmosphere, and understanding biological mechanisms underlying photosynthetic CO2 capture, and O2, H2, or N2O production, holds great promise—not only to better evaluate the reciprocal effects of global changes on oceanic carbon sinks, but also to explore the limits of biomass and biofuel productivity of algae. Membrane inlet mass spectrometry (MIMS) was initially developed to measure O2 exchange between algal cells and the surrounding liquid medium (Hoch and Kok, 1963), and its use has since been extended to other gases including H2 and more recently, N2O (Burlacot et al., 2020). Here we review recent breakthroughs allowed by MIMS in dissecting molecular mechanisms of gas exchange in microalgae (Fig. 1) and provide perspectives on how MIMS will be crucial to address future challenges in algal research.

International audience; Editorial Special issue "Biogenesis and Fate of Lipid Droplets" Intracellular lipid droplets (LDs) are receiving increasing interest from the scientific community, notably because of their link with metabolic diseases (obesity, diabetes) and the production of lipid-derived biofuels in microorganisms. Understanding the biogenesis and fate of LDs upon energy mobilization is essential to monitor fat storage in adipose tissue, but also to improve lipid productivity in microalgae under various conditions of growth [1]. Intracellular LDs are today considered as individual organelles since they host specific metabolic functions such as the biosynthesis of triacylglycerols (TAGs) and phospholipids [2]. In addition to the enzymes involved in these pathways, several proteins have been identified as associated with LDs and their number has been increasing with the progress of proteomics. Some of these proteins presenting hydrophobic patches and a high amphiphilicity are involved in the structure of LDs and together with polar lipids they form the membrane surrounding the neutral lipid core. Some others are involved in the budding of LDs from lipid bilayers and the interactions of LDs with membranes and other organelles. These interactions are important for the transfer of fatty acids and lipid remodelling and homeostasis. The combination of lipidomics and proteomics has led to a better description of LD composition, while microscopy has allowed the visualisation of the specific location of proteins at the surface of LDs. Nevertheless, a better knowledge of the lipid-protein interactions that lead to the biogenesis and fate of LDs is still required. In this context, cellular biologists now adopt principles and methods from biophysics and physical chemistry of lipid emulsions and colloids [3]. Emulsions made of TAGs, polar lipids and proteins can serve as models for intracellular LDs. The biodiversity of LDs in animals, plants and microorganisms and their various functions in lipid storage and transport also allows the comparison of data and knowledge from various fields that will help build a global understanding of structure-function relationships. The original articles and reviews of this special issue are a collection of topics presented at the 14th GERLI Lipidomics meeting, which was be held at St Maximin-la-Sainte Baume, France, from September 30th to October 3rd, 2018, with the aim of gathering researchers from various areas with a common interest in LDs, their physical chemistry and metabolism, their association with diseases and various applications in biotechnology, pharmacology and nutrition. The two first mini-reviews are related to lipid metabolism in microalgae. Lupette et al. cover the biosynthesis of fatty acids, acyl-glycero-lipids and sterols in diatoms and show how a metabolic intermediate common to these pathways, like acetyl-CoA, can play an essential role in directing the carbon flux from acyl-lipid to sterol biosynthesis and be determinant for the balance between TAGs and sterols. Prioretti et al. investigate how the target of rapamycin (TOR) signalling pathway could be modulated to enhance TAG production in microalgae. The use of TOR inhibitors increases TAG productivity in the marine diatom Phaeodactylum tricornutum, without stopping growth, while TAG accumulation in microalgae is usually observed under stress conditions with a low growth rate. Yuan et al. report on the characterization of three diacylglycerol acyltransferases (DGATs) from oil palm (Elaeis guineensis), the main source of vegetable oil on Earth. DGATs are key enzymes for the biosynthesis of TAGs and the formation of LDs. Using the heterologous expression of these DGATs in the yeast Saccharomyces cerevisiae, they show that two of them are true DGATs, that could restore TAG synthesis in a yeast TAG-deficient mutant, while the third one is a wax ester synthase.

Use of microbes to produce liquid transportation fuels is not yet economically viable. A key point to reduce production costs is the design a cell factory that combines the continuous production of drop-in fuel molecules with the ability to recover products from the cell culture at low cost. Medium-chain hydrocarbons seem ideal targets because they can be produced from abundant fatty acids and, due to their volatility, can be easily collected in gas phase. However, pathways used to produce hydrocarbons from fatty acids require two steps, low efficient enzymes and/or complex electron donors. Recently, a new hydrocarbon-forming route involving a single enzyme called fatty acid photodecarboxylase (FAP) was discovered in microalgae. Here, we show that in illuminated E. coli cultures coexpression of FAP and a medium-chain fatty acid thioesterase results in continuous release of volatile hydrocarbons. Maximum hydrocarbon productivity was reached under low/medium light while higher irradiance resulted in decreased amounts of FAP. It was also found that the production rate of hydrocarbons was constant for at least 5 days and that 30% of total hydrocarbons could be collected in the gas phase of the culture. This work thus demonstrates that the photochemistry of the FAP can be harnessed to design a simple cell factory that continuously produces hydrocarbons easy to recover and in pure form.

International audience; Lipid degradation processes are important in microalgae because survival and growth of microalgal cells under fluctuating environmental conditions require permanent remodeling or turnover of membrane lipids as well as rapid mobilization of storage lipids. Lipid catabolism comprises two major spatially and temporarily separated steps, namely lipolysis, which releases fatty acids and head groups and is catalyzed by lipases at membranes or lipid droplets, and degradation of fatty acids to acetyl‐CoA, which occurs in peroxisomes through the β‐oxidation pathway in green microalgae, and can sometimes occur in mitochondria in some other algal species. Here we review the current knowledge on the enzymes and regulatory proteins involved in lipolysis and peroxisomal β‐oxidation and highlight gaps in our understanding of lipid degradation pathways in microalgae. Metabolic use of acetyl‐CoA products via glyoxylate cycle and gluconeogenesis is also reviewed. We then present the implication of various cellular processes such as vesicle trafficking, cell cycle and autophagy on lipid turnover. Finally, physiological roles and the manipulation of lipid catabolism for biotechnological applications in microalgae are discussed.

Summary 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., Significance Statement Microalgae cultivation is one of the most promising strategies for novel food or biofuel production. High‐quality genome information is required for understanding algae biology, for their biotechnological optimization and for genome editing applications. Here, the genome of Chlorella vulgaris, one of the green algae species with the highest productivity potential, is presented and functionally annotated, revealing notable features among Chlorophyta.

Significance Nitrous oxide (N2O), the third most important greenhouse gas in the atmosphere, is produced in great quantities by microalgae, but molecular mechanisms remain elusive. Here we show that the green microalga Chlamydomonas reinhardtii produces N2O in the light by a reduction of NO driven by photosynthesis and catalyzed by flavodiiron proteins, the dark N2O production being catalyzed by a cytochrome p450. Both mechanisms of N2O production are present in chlorophytes, but absent from diatoms. Our study provides an unprecedented mechanistic understanding of N2O production by microalgae, allowing a better assessment of N2O-producing hot spots in aquatic environments., 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.

International audience; The use of algal biomass for biofuel production requires improvements in both biomass productivity and its energy density. Green microalgae store starch and oil as two major forms of carbon reserves. Current strategies to increase the amount of carbon reserves often compromise algal growth. To better understand the cellular mechanisms connecting cell division to carbon storage, we examined starch and oil accumulation in two Chlamydomonas mutants deficient in a gene encoding a homolog of the Arabidopsis Cell Division Cycle 5 (CDC5), a MYB DNA binding protein known to be involved in cell cycle in higher plants. The two crcdc5 mutants (crcdc5-1 and crcdc5-2) were found to accumulate significantly higher amount of starch and oil than their corresponding parental lines. Flow cytometry analysis on synchronized cultures cultivated in a diurnal light/dark cycle revealed an abnormal division of the two mutants, characterized by a prolonged S/M phase, therefore demonstrating its implication in cell cycle in Chlamydomonas. Taken together, these results suggest that the energy saved by a slowdown in cell division is used for the synthesis of reserve compounds. This work highlights the importance in understanding the interplay between cell cycle and starch/oil homeostasis, which should have a critical impact on improving lipid/starch productivity.

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International audience; Degradation of the storage compound triacylglycerol (TAG) is a crucial process in response to environmental stimuli. Failing to respond properly may be detrimental for survival. In microalgae, this process is important for re-growth when conditions become favorable after cells have experienced stresses. Mobilization of TAG is initiated by actions of lipases causing the release of glycerol and free fatty acids, which can be further broken down for energy production or recycled to synthesize membrane lipids. Although key enzymes in the process, TAG lipases remain to be characterized in the model green alga $Chlamydomonas\ reinhardtii$. Here we describe the functional analysis of a putative TAG lipase, i.e. CrLIP4, which shares 44% amino acid identity with the major TAG lipase in $Arabidopsis$ (SUGAR DEPENDENT1- SDP1). The $CrLIP4$ transcript level was down regulated during nitrogen deprivation (ND) when TAG accumulates, but was upregulated during nitrogen recovery (NR) when TAG wasdegraded. Both artificial microRNA knockdown and insertional knockout mutants showed a delay in TAG mobilization during NR. The difference in TAG degradation was more pronounced when the cultures were incubated without acetate in the dark. Furthermore, the $crlip4$ knock-out mutant over-accumulated TAG during optimal growth conditions. Taken together, the results suggest to us that CrLIP4 likely acts as a TAG lipase and plays a major role in TAG homeostasis in $Chlamydomonas$.

International audience; 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 (18:3$\Delta$5,9,12) 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.

International audience; Algal lipid metabolism fascinates both scientists and entrepreneurs due to the large diversity of fatty acyl structures that algae produce. Algae have therefore long been studied as sources of genes for novel fatty acids;and, due to their superior biomass productivity, algae are also considered a potential feed stock for biofuels. However, a major issue in a commercially viable“algal oil-to-biofuel”industry is the high production cost,because most algal species only produce large amounts of oils after being exposed to stress conditions. Recent studies have therefore focused on the identification of factors involved in TAG metabolism, on the subcellular organization of lipid pathways, and on interactions between organelles. This has been accompanied by the development of genetic/genomic and synthetic biological tools not only for the reference green alga $Chlamydomonas\ reinhardtii$ but also for $Nannochloropsis$ spp. and $Phaeodactylum\ tricornutum$. Advances in our understanding of enzymes and regulatory proteins of acyl lipid biosynthesis and turnover are described herein with a focus on carbon and energetic aspects. We also summarize how changes in environmental factors can impact lipid metabolism and describe present and potential industrial uses of algal lipids.

Nitrogen (N) starvation-induced triacylglycerol (TAG) synthesis, and its complex relationship with starch metabolism in algal cells, has been intensively studied; however, few studies have examined the interaction between amino acid metabolism and TAG biosynthesis. Here, via a forward genetic screen for TAG homeostasis, we isolated a Chlamydomonas (Chlamydomonas reinhardtii) mutant (bkdE1α) that is deficient in the E1α subunit of the branched-chain ketoacid dehydrogenase (BCKDH) complex. Metabolomics analysis revealed a defect in the catabolism of branched-chain amino acids in bkdE1α Furthermore, this mutant accumulated 30% less TAG than the parental strain during N starvation and was compromised in TAG remobilization upon N resupply. Intriguingly, the rate of mitochondrial respiration was 20% to 35% lower in bkdE1α compared with the parental strains. Three additional knockout mutants of the other components of the BCKDH complex exhibited phenotypes similar to that of bkdE1α Transcriptional responses of BCKDH to different N status were consistent with its role in TAG homeostasis. Collectively, these results indicate that branched-chain amino acid catabolism contributes to TAG metabolism by providing carbon precursors and ATP, thus highlighting the complex interplay between distinct subcellular metabolisms for oil storage in green microalgae.

We found that Chlamydomonas reinhardtii, a model microalga, produces triacylglycerols (TAGs) when the cell pellet is left overnight after centrifugation. The centrifuged cells produce three times more TAGs than those cultured in a nitrogen (N) depletion condition in the first 24 h. The chloroplast membranes of the centrifuged cells are largely disrupted. In addition, the accumulated TAGs contain only trivial amount of C18:1 (11) and C18:3 (5,9,12) fatty acids that are more abundant in TAGs accumulated under N-starved condition. Overall, TAGs that are induced by centrifugation contain more saturated fatty acids than that induced by N starvation. These suggest that the centrifugation triggers the production of TAGs through yet unknown mechanism. An importance of this finding is that not only quantity but also quality of the TAG production can be modified by alternating environmental stimuli rather than genetic means.

International audience; Microalgae have emerged as a promising platform for production of carbon-and energy-rich molecules, notably starch and oil. Establishing an economically viable algal biotechnology sector requires a holistic understanding of algal photosynthesis, physiology, cell cycle and metabolism. Starch/oil productivity is a combined effect of their cellular content and cell division activities. Cell growth, starch and fatty acid synthesis all require carbon building blocks and a source of energy in the form of ATP and NADPH, but with a different requirement in ATP/NADPH ratio. Thus, several cellular mechanisms have been developed by microalgae to balance ATP and NADPH supply which are essentially produced by photosynthesis. Major energy management mechanisms include ATP production by the chloroplast-based cyclic electron flow and NADPH removal by water-water cycles. Furthermore, energetic coupling between chloroplast and other cellular compartments, mitochondria and peroxisome, is increasingly recognized as an important process involved in the chloroplast redox poise. Emerging literature suggests that alterations of energy management pathways affect not only cell fitness and survival, but also influence biomass content and composition. These emerging discoveries are important steps towards diverting algal photosynthetic energy to useful products for biotechnological applications.