Biology subjects

Legeret, B.

Publications and source records attributed to Legeret, B..

11 recordsLinked to original sources

bioRxiv2026

DTH1-dependent remodeling of the lipid and protein environment of lipid droplets during nutrient recovery in Chlamydomonas

Lipid droplets (LDs) play a central role in cellular carbon and energy homeostasis, in particular through the roles played by numerous LD-associated proteins. A long-standing question concerns how these proteins are molecularly organized at the seemingly overcrowded LD surface. Delayed in TAG Hydrolysis 1 (DTH1) has been identified as a key protein for efficient LD degradation in Chlamydomonas reinhardtii, however the underlying molecular mechanism remains unknown. DTH1 contains several structural features including a coiled-coil domain, a phosphatidylethanolamine (PE) specific lipid-binding domain, intrinsically disordered regions, and an alpha-helical C-terminal domain, that make it a compelling candidate for acting as a molecular scaffold in protein recruitment and/or organelle tethering. In this study, we used the Chlamydomonas knock-out mutant dth1 as an entry point to begin dissecting the molecular organization of proteins at the LD surface. Comparative lipidomic and proteomic analyses of isolated LDs from both WT and dth1 mutants revealed several key features associated with LD degradation during nutrient recovery. First, PE emerged as an important lipid species involved in LD degradation. Second, active communication occurs among multiple subcellular organelles particularly between LDs, peroxisomes and mitochondria. Third, DTH1 was found to play a critical role in remodeling both the LD proteome and lipidome. Furthermore, detailed analyses of LD degradation during nitrogen recovery uncovered the formation of micro-LDs, a phenomenon not yet reported in microalgae. Collectively, these results highlight the tight coordination of metabolic activities across subcellular compartments and emphasize the importance of intracellular carbon trafficking during stress recovery.

plant biology↗

bioRxiv2025

ALGAL HOMOLOGS OF THE PLANT CER1 AND CER3 PROTEINS ARE FUNCTIONAL HYDROCARBON-FORMING ENZYMES

In plants, very-long-chain (VLC) alkanes (C25-C35) are secreted onto the epidermal surface of aerial organs, often forming the primary component of the waterproof cuticular wax layer. This secretion plays a vital role in preventing desiccation in terrestrial environments. The key to VLC alkane biosynthesis in plants lies in the complex formed by two homologous membrane-bound proteins, ECERIFERUM 3 (CER3) and ECERIFERUM 1 (CER1). This complex transforms an acyl-CoA substrate into an aldehyde intermediate, which is then converted into an alkane. The ability to synthesize and secrete alkanes is believed to have been a pivotal event in the evolution of land plants from their green algal ancestors. Interestingly, a single homolog of CER1 and CER3, known as CER1/3, has been identified in certain algae. However, the functionality of this protein remains to be investigated. In this study, we present the functional characterization in yeast of CER1/3 proteins from various algal species belonging to the green lineage. We demonstrate that CER1/3 proteins alone can efficiently mediate hydrocarbon biosynthesis. Furthermore, we demonstrate that point mutations in conserved motifs in the N- or C-terminal domains of CER1/3 lead to impaired hydrocarbon biosynthesis. Additionally, we show that coexpressing plant CER3 with algal CER1/3 results in longer alkanes being formed in yeast. Together, these findings support the hypothesis that the alkane-forming CER1/CER3 complex found in land plants evolved from a green algal CER1/3 bifunctional enzyme through a process of gene duplication followed by protein specialization.

plant biology↗

bioRxiv2024

A cytochrome P450 involved in apocarotenoid signaling enhances plant photosynthetic capacity and photooxidative stress tolerance

{beta}-cyclocitric acid and its precursor {beta}-cyclocitral are signaling apocarotenoids which trigger defense and detoxification mechanisms enhancing plant tolerance to abiotic stresses. From a transcriptomic analysis of Arabidopsis plants exposed to each apocarotenoid over several exposure times, we identified a gene (CYP81D11) encoding a cytochrome P450 that is strongly induced under all conditions and is under the control of the TGAII-SCL14 transcription regulator. Overexpressing the CYP81D11 gene in Arabidopsis led to a very tolerant phenotype to high light stress while a CYP81D11- deficient mutant was photosensitive. The transcriptomic profile of the CYP81D11 overexpressor revealed a selective upregulation of genes related to photosynthesis and the response to high light stress. Photosynthetic electron transport, CO2 fixation and biomass production were noticeably improved by high expression levels of CYP81D11. These effects occurred in high light, not in low light, and were associated with a noticeable reduction of singlet oxygen photoproduction. These findings indicate that CYP81D11 is a key component of {beta}-cyclocitral-induced stress tolerance, acting through enhancement of the photosynthetic capacity of leaves. This gene could be a new target for improving photosynthesis, particularly in sunny environments.

plant biology↗

bioRxiv2024

Joint operation of the CO2 concentrating mechanism and photorespiration in green algae during acclimation to limiting CO2

Due to low availability of CO2 in aquatic environment, microalgae have evolved a CO2 concentrating mechanism (CCM). It has long been thought that operation of CCM would suppress photorespiration by increasing the CO2 concentration at the Rubisco active site, but experimental evidence is scarce. To better explore the function of photorespiration in algae, we first characterized a Chlamydomonas reinhardtii mutant defected in low-CO2 inducible 20 (LCI20) and show that LCI20 is a chloroplast-envelope glutamate/malate transporter playing a role in photorespiration. By monitoring growth and glycolate excretion in mutants deficient in either CCM or photorespiration, we conclude that: i. CCM induction does not depend on photorespiration, ii. glycolate excretion protects algal cells from the toxicity of unmetabolized photorespiratory intermediates, iii. photorespiration is active at low CO2 when the CCM is operational. This work provides a foundation for a better understanding of the carbon cycle in the ocean where significant glycolate concentrations have been found.

plant biology↗

bioRxiv2024

Knocking out the carboxyltransferase interactor 1 (CTI1) in Chlamydomonas boosted oil content by fivefold without affecting cell growth

The first step in chloroplast de novo fatty acid synthesis is catalyzed by acetyl-CoA carboxylase (ACCase). As the rate-limiting step for this pathway, ACCase is subject to both positive and negative regulation. In this study, we identify a Chlamydomonas homolog of the plant carboxyltransferase interactor 1 (CrCTI1) and show that this protein, interacts with the Chlamydomonas -carboxyltransferase (Cr-CT) subunit of the ACCase by yeast two-hybrid protein-protein interaction assay. Three independent CRISPR-Cas9 mediated knock-out mutants for CrCTI1 each produced an "enhanced oil" phenotype, accumulating 25% more total fatty acids and storing up to five-fold more triacylglycerols (TAGs) in lipid droplets. The TAG phenotype of the crcti1 mutants was not influenced by light but was affected by trophic growth conditions. By growing cells under heterotrophic conditions, we observed a crucial function of CrCTI1 in balancing lipid accumulation and cell growth. Mutating a previously mapped in vivo phosphorylation site (CrCTI1 Ser108 to either Ala or to Asp), did not affect the interaction with Cr-CT. However, mutating all six predicted phosphorylation sites within Cr-CT to create a phosphomimetic mutant reduced significantly this pairwise interaction. Comparative proteomic analyses of the crcti1 mutants and WT suggested a role for CrCTI1 in regulating carbon flux by coordinating carbon metabolism, antioxidant and fatty acid {beta}-oxidation pathways, to enable cells adapt to carbon availability. Taken together, this study identifies CrCTI1 as a negative regulator of fatty acid synthesis in algae and provides a new molecular brick for genetic engineering of microalgae for biotechnology purposes.

plant biology↗

bioRxiv2023

The α/β hydrolase domain-containing protein 1 (ABHD1) acts as a lysolipid lipase and is involved in lipid droplet formation

Lipid droplets (LDs) are the major sites of lipid and energy homeostasis. However, few LD biogenesis proteins have been identified. Here, using Chlamydomonas as a model, we show that ABHD1, a member of the /{beta} hydrolase domain-containing protein family, is a novel type of LD-associated protein which stimulates LD formation through two distinct actions on the LD surface, one enzymatic and the other structural. ABHD1 was localized to LD surface in Chlamydomonas cells. The knockout mutants contained similar amounts of triacylglycerols (TAG) but their LDs showed an increased content in lyso- derivatives of the betaine lipid diacylglyceryl-N,N,N-trimethylhomoserine (DGTS). Over-expression of ABHD1 in Chlamydomonas induced LD formation and boosted TAG content, suggesting a key role in LD biogenesis. The purified recombinant ABHD1 protein hydrolyzed lyso-DGTS, producing a free fatty acid and a glyceryltrimethylhomoserine moiety. In vitro experiments using droplet- embedded vesicles showed that ABHD1 promoted LD emergence. Taken together, these results identify ABHD1 as a new player in LD formation by its lipase activity on lyso-DGTS and by its distinct biophysical property. This study further suggests that lipases targeted to LDs and able to act on their polar lipid coat may be interesting tools to promote LD assembly in eukaryotic cells. Significant statementLipid droplets are subcellular organelles specialized for triacylglycerol storage. Their dynamic turnover is key to managing energy homeostasis in response to cell cycle states and environmental cues. To gain insights into LD biogenesis, we characterized a putative /{beta}- hydrolase (ABHD1) in the model algae Chlamydomonas reinhardtii and show it is located at the LD surface. We found that ABHD1 overexpression promotes LD formation and acts as a lipase mainly on lyso derivatives of the betaine lipid diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), the major lipid constituent of the LD hemi-membrane. We also show that ABHD1 has a remarkable biophysical property favoring LD budding. This work thus identifies a novel type of lipase acting on betaine lipid and provides a first example of a protein with a dual function nvolved in LD formation.

biochemistry↗

bioRxiv2023

Alka(e)nes contribute to membrane lipid homeostasis and resilience of photosynthesis to high light in cyanobacteria

Alka(e)nes are produced by many living organisms and exhibit diverse physiological roles, reflecting a high functional versatility. Alka(e)nes serve as water proof 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, a possible role in photosynthesis and chloroplast function remains elusive. In this study, we investigated the consequences of the absence of alka(e)nes on membrane lipid remodeling and photosynthesis using the cyanobacteria 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 a profound remodeling of the membrane lipidome and carotenoid content 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. Under high light, this effect is accompanied in alka(e)ne deficient strains by a higher susceptibility of photosynthesis and growth, the effect being reversed by expressing an algal photoenzyme producing alka(e)nes from fatty acids. We conclude that alka(e)nes play a crucial role in maintaining lipid homeostasis of photosynthetic membranes, thereby contributing to the proper functioning of photosynthesis, particularly under elevated light intensities. Significance statementWe used cyanobacteria as a model organism to explore the role of alka(e)nes related to photosynthesis. Our findings reveal that the absence of alka(e)nes induces alterations in the composition of membrane lipids and carotenoid content, resulting in an increased susceptibility of photosynthesis. By introducing a fatty acid photodecarboxylase to produce alkanes, we could reverse these effects, highlighting the critical role of alka(e)nes in maintaining lipid balance in photosynthetic membranes and ensuring efficient photosynthesis. Uncovering the physiological role of alka(e)nes provides insights to a better understanding of the widespread presence of genes encoding alka(e)nes-synthesizing enzymes in cyanobacteria and microalgae, organisms of major ecological and evolutionary importance in the global CO2 assimilation.

microbiology↗

bioRxiv2023

An SMR cell-cycle inhibitor inducible by a carotenoid metabolite resets root development and drought tolerance in Arabidopsis

New regulatory functions in plant development and environmental stress responses have recently emerged for a number of apocarotenoids produced by enzymatic or non-enzymatic oxidation of carotenoids. {beta}-cyclocitric acid ({beta}-CCA) is one such compound derived from {beta}-carotene which triggers defense mechanisms leading to a marked enhancement of plant tolerance to drought stress. We show here that this response is associated with an inhibition of root growth affecting both root cell elongation and division. Remarkably, {beta}-CCA selectively induced cell cycle inhibitors of the SIAMESE-RELATED (SMR) family, especially SMR5, in root tip cells. Overexpression of the SMR5 gene in Arabidopsis induced molecular and physiological changes that mimicked in large part the effects of {beta}-CCA. In particular, the SMR5 overexpressors exhibited an inhibition of root development and a marked increase in drought tolerance which is not related to stomatal closure. SMR5 up-regulation induced changes in gene expression that strongly overlapped with the {beta}-CCA-induced transcriptomic changes. Both {beta}-CCA and SMR5 led to a down-regulation of many cell cycle activators (cyclins, cyclin-dependent kinases) and a concomitant up-regulation of genes related to water deprivation, cellular detoxification and biosynthesis of lipid biopolymers such as suberin and lignin. This was correlated with an accumulation of suberin lipid polyesters in the roots and a decrease in non-stomatal leaf transpiration. Taken together, our results identify the {beta}-CCA-and drought-inducible SMR5 gene as a key component of a stress signaling pathway that reorients root metabolism from growth to multiple defense mechanisms leading to drought tolerance.

plant biology↗

bioRxiv2021

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 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.

plant biology↗

bioRxiv2020

Fatty acid photodecarboxylase is an ancient photoenzyme responsible for hydrocarbon formation in the thylakoid membranes of algae

ABSTRACTFatty 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 summary FAP is present in thylakoids and conserved beyond green algae.Competing Interest StatementThe authors have declared no competing interest.View Full Text

plant biology↗

bioRxiv2018

β-cyclocitric acid: a new apocarotenoid eliciting drought tolerance in plants

{beta}-Cyclocitral ({beta}-CC) is a volatile compound deriving from 1O2 oxidation of {beta}-carotene in plant leaves. {beta}-CC elicits a retrograde signaling, modulating 1O2-responsive genes and enhancing tolerance to photooxidative stress. Here, we show that {beta}-CC is largely converted into {beta}-cyclocitric acid ({beta}-CCA) in leaves and that this metabolite is a signal involved in stress tolerance. Treatment of Arabidopsis plants with {beta}-CCA markedly enhanced plant tolerance to drought by a mechanism different from known responses such as stomatal closure, changes in osmotic potential and jasmonate signaling. Furthermore, we show that the response to {beta}-CCA does not fully overlap with the {beta}-CC-dependent signaling, indicating that {beta}-CCA induces only a branch of the {beta}-CC signaling pathway. In addition, the protective effect of {beta}-CCA is a conserved mechanism, being observed in a variety of plant species. This study provides a new bioactive agent with promising agronomic applications for protecting plants against drought.

plant biology↗