Biology subjects

Cuine, S.

Publications and source records attributed to Cuine, S..

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↗

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

Microbial photoproduction of heptane

Fatty Acid Photodecarboxylase (FAP) has emerged as a promising catalyst for the biological production of long-chain hydrocarbons. We have recently shown that purified FAP or FAP-expressing bacteria can efficiently convert octanoic acid into heptane, thus extending the potential applications of FAP to medium-chain hydrocarbons (i.e., solvent- or kerosene-type). The scarcity of natural sources of octanoic acid presents a challenge however. Here, we explore the heptane production capacity of a FAP-expressing E. coli strain engineered to biosynthesize octanoic acid via a specific thioesterase. Various FAPs and C8-specific thioesterases were tested. A blue-light-inducible promoter was used to avoid chemical inducers. We found that the expression of FAP fused with TrxA resulted in a 10-fold increase in heptane production. Coexpression of Cuphea hookeriana thioesterase and Chlorella variabilis FAP achieved the highest heptane titer (12.5 mg.L-1). Scale-up experiments in 100 mL photobioreactors allowed a constant production of heptane over two days (22 mg.L-1.day-1). Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/603920v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1dbfebforg.highwire.dtl.DTLVardef@31dbaforg.highwire.dtl.DTLVardef@14dfcd5org.highwire.dtl.DTLVardef@11234d4_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIAn E. coli strain producing heptane under blue light is described. C_LIO_LIExpression of FAP fused with TrxA increases heptane by 10-fold. C_LIO_LIA blue light-inducible promoter ensures high coexpression of FAP and thioesterase. C_LIO_LICuphea hookeriana thioesterase and Chlorella variabilis FAP give highest production C_LIO_LIHighest reported heptane productivity (22 mg.L-1.day-1) in 100 mL photobioreactors. C_LI

bioengineering↗

bioRxiv2024

DYRKP kinase regulates cell wall degradation in Chlamydomonas by inducing matrix metalloproteinase expression

The cell wall of plants and algae is an important cell structure that protects cells from changes in the external physical and chemical environment. This extracellular matrix composed of polysaccharides and glycoproteins, is needed to be remodeled continuously throughout the life cycle. However, compared to matrix polysaccharides, little is known about the mechanisms regulating the formation and degradation of matrix glycoproteins. We report here that a plant kinase belonging to the dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) family present in all eukaryotes regulates cell wall degradation in the model microalga Chlamydomonas reinhardtii by inducing the expression of matrix metalloproteinases (MMPs). In the absence of DYRKP, daughter cells fail to degrade the parental cell wall, and form multicellular structures. On the other hand, the complementation line of DYRKP was shown to degrade the parental cell wall normally. Transcriptomic and proteomic analyses indicate a marked down-regulation of MMP expression in the dyrkp mutants. Additionally, the expression of MMP was confirmed to be consistent with the expression pattern of DYRKP. Our findings show that DYRKP, by ensuring timely MMP expression, enables the successful execution of the cell cycle. Altogether, this study provides new insight into the life cycle regulation in plants and algae. IN A NUTSHELLO_ST_ABSBackgroundC_ST_ABSPlants and algae have different types of polysaccharides in their cell walls, but they have glycoproteins in common. Glycoprotein synthesis and degradation must be tightly regulated to ensure normal growth and differentiation. However, little is known about the regulatory mechanism of glycoprotein degradation in both plants and algae. The cell cycle of Chlamydomonas reinhardtii begins anew with the hatching of daughter cells, and the role of matrix metalloproteinases (MMPs) is known to be important in this process. In our previous study, we observed that a knockout mutant of the plant kinase belonging to the dual-specificity tyrosine phosphorylation-regulated kinase (DYRKP) formed a palmelloid structure and failed to hatch. QuestionsWhat is the role of DYRKP in microalgae? Specifically, why does the dyrkp mutant form a palmelloid structure? Palmelloid is usually observed in dividing cells or after exposure to stresses. We therefore hypothesized that the palmelloid phenotype observed in dyrkp mutant could either be due to a defect in cell hatching or due to an increased stress state in the mutant population. FindingsWe answered these questions by comparative studies in different culture conditions and by examining additional dyrkp knockout mutants generated by CRISPR-Cas9 in various background strains with more or less intact cell walls. Palmelloid formation in the dyrkp mutant was observed under optimal growth (mixo- or auto-trophic condition) and very low light conditions. Interestingly, unlike the parent strain, in which only cell wall fragments are observed in old cultures, the parental cell wall of the dyrkp mutant remained almost intact even after the release of daughter cells. Also, the cell division rate of the cell wall-less dyrkp mutants was similar to their background strain. These results suggest that dyrkp mutants have a problem in degrading the parental cell walls. Indeed, proteomic and transcriptomic analyses revealed reduced levels of protease families in the dyrkp mutant, and in particular with a significantly lower amount of several key members of the MMP family. Through the analysis of complementation lines, we confirmed that the DYRKP was required for strong and rapid expression of MMPs. Next stepsWe are pursuing research to understand what the phosphorylation clients of DYRKP are and how they regulate the expression of the MMPs identified in this study. One sentence summaryThe DYRKP kinase induces the expression of matrix metalloproteinases involved in the degradation of the parental cell wall, allowing prompt hatching of daughter cells after cell division.

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↗

bioRxiv2022

Boosting chloroplast ribosome biogenesis by a plastidial DEAD-box RNA helicase is critical for high light acclimation

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.

plant biology↗

bioRxiv2021

Alternative electron pathways of photosynthesis 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 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

plant biology↗

bioRxiv2021

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

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

plant biology↗

bioRxiv2020

ppGpp influences protein protection, growth and photosynthesis in Phaeodactylum tricornutum

O_LIChloroplasts 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. C_LIO_LIHere, 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. C_LIO_LIWe 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. C_LIO_LIOur 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. C_LI

plant biology↗

bioRxiv2020

ACCLIMATION OF PHOTOSYNTHESIS TO THE ENVIRONMENT 1 regulates Photosystem II Supercomplex dynamics in response to light in Chlamydomonas reinhardtii

Photosynthetic organisms require acclimation mechanisms to regulate photosynthesis in response to light conditions. Here, two mutant alleles of ACCLIMATION OF PHOTOSYNTHESIS TO THE ENVIRONMENT 1 (ape1) have been characterized in Chlamydomonas reinhardtii. The ape1 mutants are photosensitive and show PSII photoinhibition during high light acclimation or under high light stress. The ape1 mutants retain more PSII super-complexes and have changes to thylakoid stacking relative to control strains during photosynthetic growth at different light intensities. The APE1 protein is found in all oxygenic phototrophs and encodes a 25 kDa thylakoid protein that interacts with the Photosystem II core complex as monomers, dimers and supercomplexes. We propose a model where APE1 bound to PSII supercomplexes releases core complexes and promotes PSII heterogeneity influencing the stacking of Chlamydomonas thylakoids. APE1 is a regulator in light acclimation and its function is to reduce over-excitation of PSII centres and avoid PSII photoinhibition to increase the resilience of photosynthesis to high light.

plant biology↗