The DYRKP1 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, must be constantly remodeled 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 after mitosis of Chlamydomonas reinhardtii by inducing the expression of matrix metalloproteinases (MMPs). Without the plant DYRK kinase (DYRKP1), daughter cells cannot disassemble parental cell walls and remain trapped inside for more than 10 days. On the other hand, the DYRKP1 complementation line shows normal degradation of the parental cell wall. Transcriptomic and proteomic analyses indicate a marked down-regulation of MMP gene expression and accumulation, respectively, in the dyrkp1 mutants. The mutants deficient in MMPs retain palmelloid structures for a longer time than the background strain, like dyrkp1 mutants. Our findings show that DYRKP1, by ensuring timely MMP expression, enables the successful execution of the cell cycle. Altogether, this study provides insight into the life cycle regulation in plants and algae.

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

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

Catalytic Mechanism of Fatty Acid Photodecarboxylase: On the Detection and Stability of the Initial Carbonyloxy Radical Intermediate.

In fatty acid photodecarboxylase (FAP), light-induced formation of the primary radical product RCOO⋅ from fatty acid RCOO- occurs in 300 ps, upon which CO2 is released quasi-immediately. Based on the hypothesis that aliphatic RCOO⋅ (spectroscopically uncharacterized because unstable) absorbs in the red similarly to aromatic carbonyloxy radicals such as 2,6-dichlorobenzoyloxy radical (DCB⋅), much longer-lived linear RCOO⋅ has been suggested recently. We performed quantum chemical reaction pathway and spectral calculations. These calculations are in line with the experimental DCB⋅ decarboxylation dynamics and spectral properties and show that in contrast to DCB⋅, aliphatic RCOO⋅ radicals a) decarboxylate with a very low energetic barrier and on the timescale of a few ps and b) exhibit little red absorption. A time-resolved infrared spectroscopy experiment confirms very rapid, ≪300 ps RCOO⋅ decarboxylation in FAP. We argue that this property is required for the observed high quantum yield of hydrocarbons formation by FAP.

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

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

Fatty Acid Photodecarboxylase Is an Interfacial Enzyme That Binds to Lipid-Water Interfaces to Access Its Insoluble Substrate.

Fatty acid photodecarboxylase (FAP), one of the few natural photoenzymes characterized so far, is a promising biocatalyst for lipid-to-hydrocarbon conversion using light. However, the optimum supramolecular organization under which the fatty acid (FA) substrate should be presented to FAP has not been addressed. Using palmitic acid embedded in phospholipid liposomes, phospholipid-stabilized microemulsions, and mixed micelles, we show that FAP displays a preference for FAs present in liposomes and at the surface of microemulsions. The kinetics of adsorption onto phospholipid and galactolipid monomolecular films further suggests the ability of FAP to bind to and penetrate into membranes, with a higher affinity in the presence of FAs. The FAP structure reveals a potential interfacial recognition site with clusters of hydrophobic and basic residues surrounding the active site entrance. The resulting dipolar moment suggests the orientation of FAP at negatively charged interfaces. These findings provide important clues about the mode of action of FAP and the development of FAP-based bioconversion processes.

Phospholipase pPLAIIIα Increases Germination Rate and Resistance to Turnip Crinkle Virus when Overexpressed.

Patatin-related phospholipase As (pPLAs) are major hydrolases acting on acyl-lipids and play important roles in various plant developmental processes. pPLAIII group members, which lack a canonical catalytic Ser motif, have been less studied than other pPLAs. We report here the characterization of pPLAIIIα in Arabidopsis (Arabidopsis thaliana) based on the biochemical and physiological characterization of pPLAIIIα knockouts, complementants, and overexpressors, as well as heterologous expression of the protein. In vitro activity assays on the purified recombinant protein showed that despite lack of canonical phospholipase motifs, pPLAIIIα had a phospholipase A activity on a wide variety of phospholipids. Overexpression of pPLAIIIα in Arabidopsis resulted in a decrease in many lipid molecular species, but the composition in major lipid classes was not affected. Fluorescence tagging indicated that pPLAIIIα localizes to the plasma membrane. Although Arabidopsis pplaIIIα knockout mutants showed some phenotypes comparable to other pPLAIIIs, such as reduced trichome length and increased hypocotyl length, control of seed size and germination were identified as distinctive pPLAIIIα-mediated functions. Expression of some PLD genes was strongly reduced in the pplaIIIα mutants. Overexpression of pPLAIIIα caused increased resistance to turnip crinkle virus, which associated with a 2-fold higher salicylic acid/jasmonic acid ratio and an increased expression of the defense gene pathogenesis-related protein1. These results therefore show that pPLAIIIα has functions that overlap with those of other pPLAIIIs but also distinctive functions, such as the control of seed germination. This study also provides new insights into the pathways downstream of pPLAIIIα.

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

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

Branched-Chain Amino Acid Catabolism Impacts Triacylglycerol Homeostasis in Chlamydomonas reinhardtii.

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.

The Phosphate Fast-Responsive Genes PECP1 and PPsPase1 Affect Phosphocholine and Phosphoethanolamine Content.

Phosphate starvation-mediated induction of the HAD-type phosphatases PPsPase1 (AT1G73010) and PECP1 (AT1G17710) has been reported in Arabidopsis (Arabidopsis thaliana). However, little is known about their in vivo function or impact on plant responses to nutrient deficiency. The preferences of PPsPase1 and PECP1 for different substrates have been studied in vitro but require confirmation in planta. Here, we examined the in vivo function of both enzymes using a reverse genetics approach. We demonstrated that PPsPase1 and PECP1 affect plant phosphocholine and phosphoethanolamine content, but not the pyrophosphate-related phenotypes. These observations suggest that the enzymes play a similar role in planta related to the recycling of polar heads from membrane lipids that is triggered during phosphate starvation. Altering the expression of the genes encoding these enzymes had no effect on lipid composition, possibly due to compensation by other lipid recycling pathways triggered during phosphate starvation. Furthermore, our results indicated that PPsPase1 and PECP1 do not influence phosphate homeostasis, since the inactivation of these genes had no effect on phosphate content or on the induction of molecular markers related to phosphate starvation. A combination of transcriptomics and imaging analyses revealed that PPsPase1 and PECP1 display a highly dynamic expression pattern that closely mirrors the phosphate status. This temporal dynamism, combined with the wide range of induction levels, broad expression, and lack of a direct effect on Pi content and regulation, makes PPsPase1 and PECP1 useful molecular markers of the phosphate starvation response.

Chlamydomonas carries out fatty acid ß-oxidation in ancestral peroxisomes using a bona fide acyl-CoA oxidase.

Peroxisomes are thought to have played a key role in the evolution of metabolic networks of photosynthetic organisms by connecting oxidative and biosynthetic routes operating in different compartments. While the various oxidative pathways operating in the peroxisomes of higher plants are fairly well characterized, the reactions present in the primitive peroxisomes (microbodies) of algae are poorly understood. Screening of a Chlamydomonas insertional mutant library identified a strain strongly impaired in oil remobilization and defective in Cre05.g232002 (CrACX2), a gene encoding a member of the acyl-CoA oxidase/dehydrogenase superfamily. The purified recombinant CrACX2 expressed in Escherichia coli catalyzed the oxidation of fatty acyl-CoAs into trans-2-enoyl-CoA and produced H2 O2 . This result demonstrated that CrACX2 is a genuine acyl-CoA oxidase, which is responsible for the first step of the peroxisomal fatty acid (FA) ß-oxidation spiral. A fluorescent protein-tagging study pointed to a peroxisomal location of CrACX2. The importance of peroxisomal FA ß-oxidation in algal physiology was shown by the impact of the mutation on FA turnover during day/night cycles. Moreover, under nitrogen depletion the mutant accumulated 20% more oil than the wild type, illustrating the potential of ß-oxidation mutants for algal biotechnology. This study provides experimental evidence that a plant-type FA ß-oxidation involving H2 O2 -producing acyl-CoA oxidation activity has already evolved in the microbodies of the unicellular green alga Chlamydomonas reinhardtii.

Arabidopsis thaliana EPOXIDE HYDROLASE1 (AtEH1) is a cytosolic epoxide hydrolase involved in the synthesis of poly-hydroxylated cutin monomers.

Epoxide hydrolases (EHs) are present in all living organisms. They have been extensively characterized in mammals; however, their biological functions in plants have not been demonstrated. Based on in silico analysis, we identified AtEH1 (At3g05600), a putative Arabidopsis thaliana epoxide hydrolase possibly involved in cutin monomer synthesis. We expressed AtEH1 in yeast and studied its localization in vivo. We also analyzed the composition of cutin from A. thaliana lines in which this gene was knocked out. Incubation of recombinant AtEH1 with epoxy fatty acids confirmed its capacity to hydrolyze epoxides of C18 fatty acids into vicinal diols. Transfection of Nicotiana benthamiana leaves with constructs expressing AtEH1 fused to enhanced green fluorescent protein (EGFP) indicated that AtEH1 is localized in the cytosol. Analysis of cutin monomers in loss-of-function Ateh1-1 and Ateh1-2 mutants showed an accumulation of 18-hydroxy-9,10-epoxyoctadecenoic acid and a concomitant decrease in corresponding vicinal diols in leaf and seed cutin. Compared with wild-type seeds, Ateh1 seeds showed delayed germination under osmotic stress conditions and increased seed coat permeability to tetrazolium red. This work reports a physiological role for a plant EH and identifies AtEH1 as a new member of the complex machinery involved in cutin synthesis.

Saturating Light Induces Sustained Accumulation of Oil in Plastidal Lipid Droplets in Chlamydomonas reinhardtii.

Enriching algal biomass in energy density is an important goal in algal biotechnology. Nitrogen (N) starvation is considered the most potent trigger of oil accumulation in microalgae and has been thoroughly investigated. However, N starvation causes the slow down and eventually the arrest of biomass growth. In this study, we show that exposing a Chlamydomonas reinhardtii culture to saturating light (SL) under a nonlimiting CO2 concentration in turbidostatic photobioreactors induces a sustained accumulation of lipid droplets (LDs) without compromising growth, which results in much higher oil productivity than N starvation. We also show that the polar membrane lipid fraction of SL-induced LDs is rich in plastidial lipids (approximately 70%), in contrast to N starvation-induced LDs, which contain approximately 60% lipids of endoplasmic reticulum origin. Proteomic analysis of LDs isolated from SL-exposed cells identified more than 200 proteins, including known proteins of lipid metabolism, as well as 74 proteins uniquely present in SL-induced LDs. LDs induced by SL and N depletion thus differ in protein and lipid contents. Taken together, lipidomic and proteomic data thus show that a large part of the sustained oil accumulation occurring under SL is likely due to the formation of plastidial LDs. We discuss our data in relation to the different metabolic routes used by microalgae to accumulate oil reserves depending on cultivation conditions. Finally, we propose a model in which oil accumulation is governed by an imbalance between photosynthesis and growth, which can be achieved by impairing growth or by boosting photosynthetic carbon fixation, with the latter resulting in higher oil productivity.

Microalgae Synthesize Hydrocarbons from Long-Chain Fatty Acids via a Light-Dependent Pathway.

Microalgae are considered a promising platform for the production of lipid-based biofuels. While oil accumulation pathways are intensively researched, the possible existence of a microalgal pathways converting fatty acids into alka(e)nes has received little attention. Here, we provide evidence that such a pathway occurs in several microalgal species from the green and the red lineages. In Chlamydomonas reinhardtii (Chlorophyceae), a C17 alkene, n-heptadecene, was detected in the cell pellet and the headspace of liquid cultures. The Chlamydomonas alkene was identified as 7-heptadecene, an isomer likely formed by decarboxylation of cis-vaccenic acid. Accordingly, incubation of intact Chlamydomonas cells with per-deuterated D31-16:0 (palmitic) acid yielded D31-18:0 (stearic) acid, D29-18:1 (oleic and cis-vaccenic) acids, and D29-heptadecene. These findings showed that loss of the carboxyl group of a C18 monounsaturated fatty acid lead to heptadecene formation. Amount of 7-heptadecene varied with growth phase and temperature and was strictly dependent on light but was not affected by an inhibitor of photosystem II. Cell fractionation showed that approximately 80% of the alkene is localized in the chloroplast. Heptadecane, pentadecane, as well as 7- and 8-heptadecene were detected in Chlorella variabilis NC64A (Trebouxiophyceae) and several Nannochloropsis species (Eustigmatophyceae). In contrast, Ostreococcus tauri (Mamiellophyceae) and the diatom Phaeodactylum tricornutum produced C21 hexaene, without detectable C15-C19 hydrocarbons. Interestingly, no homologs of known hydrocarbon biosynthesis genes were found in the Nannochloropsis, Chlorella, or Chlamydomonas genomes. This work thus demonstrates that microalgae have the ability to convert C16 and C18 fatty acids into alka(e)nes by a new, light-dependent pathway.

Metabolism of acyl-lipids in Chlamydomonas reinhardtii.

Microalgae are emerging platforms for production of a suite of compounds targeting several markets, including food, nutraceuticals, green chemicals, and biofuels. Many of these products, such as biodiesel or polyunsaturated fatty acids (PUFAs), derive from lipid metabolism. A general picture of lipid metabolism in microalgae has been deduced from well characterized pathways of fungi and land plants, but recent advances in molecular and genetic analyses of microalgae have uncovered unique features, pointing out the necessity to study lipid metabolism in microalgae themselves. In the past 10 years, in addition to its traditional role as a model for photosynthetic and flagellar motility processes, Chlamydomonas reinhardtii has emerged as a model organism to study lipid metabolism in green microalgae. Here, after summarizing data on total fatty acid composition, distribution of acyl-lipid classes, and major acyl-lipid molecular species found in C. reinhardtii, we review the current knowledge on the known or putative steps for fatty acid synthesis, glycerolipid desaturation and assembly, membrane lipid turnover, and oil remobilization. A list of characterized or putative enzymes for the major steps of acyl-lipid metabolism in C. reinhardtii is included, and subcellular localizations and phenotypes of associated mutants are discussed. Biogenesis and composition of Chlamydomonas lipid droplets and the potential importance of lipolytic processes in increasing cellular oil content are also highlighted.

BODYGUARD is required for the biosynthesis of cutin in Arabidopsis.

The cuticle plays a critical role in plant survival during extreme drought conditions. There are, however, surprisingly, many gaps in our understanding of cuticle biosynthesis. An Arabidopsis thaliana T-DNA mutant library was screened for mutants with enhanced transpiration using a simple condensation spot method. Five mutants, named cool breath (cb), were isolated. The cb5 mutant was found to be allelic to bodyguard (bdg), which is affected in an α/ß-hydrolase fold protein important for cuticle structure. The analysis of cuticle components in cb5 (renamed as bdg-6) and another T-DNA mutant allele (bdg-7) revealed no impairment in wax synthesis, but a strong decrease in total cutin monomer load in young leaves and flowers. Root suberin content was also reduced. Overexpression of BDG increased total leaf cutin monomer content nearly four times by affecting preferentially C18 polyunsaturated ω-OH fatty acids and dicarboxylic acids. Whole-plant gas exchange analysis showed that bdg-6 had higher cuticular conductance and rate of transpiration; however, plant lines overexpressing BDG resembled the wild-type with regard to these characteristics. This study identifies BDG as an important component of the cutin biosynthesis machinery in Arabidopsis. We also show that, using BDG, cutin can be greatly modified without altering the cuticular water barrier properties and transpiration.

Microalgal lipid droplets: composition, diversity, biogenesis and functions.

Lipid droplet is the major site of neutral lipid storage in eukaryotic cells, and increasing evidence show its involvement in numerous cellular processes such as lipid homeostasis, signaling, trafficking and inter-organelle communications. Although the biogenesis, structure, and functions of lipid droplets have been well documented for seeds of vascular plants, mammalian adipose tissues, insects and yeasts, relative little is known about lipid droplets in microalgae. Over the past 5 years, the growing interest of microalgae as a platform for biofuel, green chemicals or value-added polyunsaturated fatty acid production has brought algal lipid droplets into spotlight. Studies conducted on the green microalga Chlamydomonas reinhardtii and other model microalgae such as Haematococcus and Nannochloropsis species have led to the identification of proteins associated with lipid droplets, which include putative structural proteins different from plant oleosins and animal perilipins, as well as candidate proteins for lipid biosynthesis, mobilization, trafficking and homeostasis. Biochemical and microscopy studies have also started to shed light on the role of chloroplasts in the biogenesis of lipid droplets in Chlamydomonas.

Histone H2B monoubiquitination is involved in the regulation of cutin and wax composition in Arabidopsis thaliana.

The plant cuticle is a chemically heterogeneous lipophilic layer composed of a cutin polymer matrix and waxes which covers the aerial parts of plants. This layer plays an essential role in the survival of plants by protecting them from desiccation and (a)biotic stresses. Knowledge on the gene networks and mechanisms regulating the synthesis of cuticle components during organ expansion or stress response remains limited however. Here, using five loss-of-function mutants for histone monoubiquitination, we report on the role of two RING E3 ligases, namely HISTONE MONOUBIQUITINATION 1 and 2 (HUB1 and HUB2), in the selective transcriptional activation of four cuticle biosynthesis genes in Arabidopsis thaliana. Microscopy observations showed that in hub1-6 and hub2-2 mutants irregular epidermal cells and disorganized cuticle layers were present in rosette leaves. Water loss measurements on excised rosettes demonstrated that cuticular permeability was significantly increased in the mutants. Chemical analysis of cuticle components revealed that the wax composition was changed and that cutin 16:0 dicarboxylic acid was significantly reduced in all hub mutants. Analysis of transcript levels of selected genes indicated that LACS2, ATT1 and HOTHEAD involved in cutin biosynthesis and CER1 involved in wax biosynthesis were down-regulated in the hub mutants, while the expression of LACERATA, CER3, CER6 and CER10 remained unchanged. Chromatin immunoprecipitation assays further showed that hub mutants are impaired in dynamic changes of histone H2B monoubiquitination at several loci of down-regulated genes. Taken together, these data establish that the regulation of cuticle composition involves chromatin remodeling by H2B monoubiquitination.

The green microalga Chlamydomonas reinhardtii has a single ω-3 fatty acid desaturase that localizes to the chloroplast and impacts both plastidic and extraplastidic membrane lipids.

The ω-3 polyunsaturated fatty acids account for more than 50% of total fatty acids in the green microalga Chlamydomonas reinhardtii, where they are present in both plastidic and extraplastidic membranes. In an effort to elucidate the lipid desaturation pathways in this model alga, a mutant with more than 65% reduction in total ω-3 fatty acids was isolated by screening an insertional mutant library using gas chromatography-based analysis of total fatty acids of cell pellets. Molecular genetics analyses revealed the insertion of a TOC1 transposon 113 bp upstream of the ATG start codon of a putative ω-3 desaturase (CrFAD7; locus Cre01.g038600). Nuclear genetic complementation of crfad7 using genomic DNA containing CrFAD7 restored the wild-type fatty acid profile. Under standard growth conditions, the mutant is indistinguishable from the wild type except for the fatty acid difference, but when exposed to short-term heat stress, its photosynthesis activity is more thermotolerant than the wild type. A comparative lipidomic analysis of the crfad7 mutant and the wild type revealed reductions in all ω-3 fatty acid-containing plastidic and extraplastidic glycerolipid molecular species. CrFAD7 was localized to the plastid by immunofluorescence in situ hybridization. Transformation of the crfad7 plastidial genome with a codon-optimized CrFAD7 restored the ω-3 fatty acid content of both plastidic and extraplastidic lipids. These results show that CrFAD7 is the only ω-3 fatty acid desaturase expressed in C. reinhardtii, and we discuss possible mechanisms of how a plastid-located desaturase may impact the ω-3 fatty acid content of extraplastidic lipids.

A land-plant-specific glycerol-3-phosphate acyltransferase family in Arabidopsis: substrate specificity, sn-2 preference, and evolution.

Arabidopsis (Arabidopsis thaliana) has eight glycerol-3-phosphate acyltransferase (GPAT) genes that are members of a plant-specific family with three distinct clades. Several of these GPATs are required for the synthesis of cutin or suberin. Unlike GPATs with sn-1 regiospecificity involved in membrane or storage lipid synthesis, GPAT4 and -6 are unique bifunctional enzymes with both sn-2 acyltransferase and phosphatase activity resulting in 2-monoacylglycerol products. We present enzymology, pathway organization, and evolutionary analysis of this GPAT family. Within the cutin-associated clade, GPAT8 is demonstrated as a bifunctional sn-2 acyltransferase/phosphatase. GPAT4, -6, and -8 strongly prefer C16:0 and C18:1 ω-oxidized acyl-coenzyme As (CoAs) over unmodified or longer acyl chain substrates. In contrast, suberin-associated GPAT5 can accommodate a broad chain length range of ω-oxidized and unsubstituted acyl-CoAs. These substrate specificities (1) strongly support polyester biosynthetic pathways in which acyl transfer to glycerol occurs after oxidation of the acyl group, (2) implicate GPAT specificities as one major determinant of cutin and suberin composition, and (3) argue against a role of sn-2-GPATs (Enzyme Commission 2.3.1.198) in membrane/storage lipid synthesis. Evidence is presented that GPAT7 is induced by wounding, produces suberin-like monomers when overexpressed, and likely functions in suberin biosynthesis. Within the third clade, we demonstrate that GPAT1 possesses sn-2 acyltransferase but not phosphatase activity and can utilize dicarboxylic acyl-CoA substrates. Thus, sn-2 acyltransferase activity extends to all subbranches of the Arabidopsis GPAT family. Phylogenetic analyses of this family indicate that GPAT4/6/8 arose early in land-plant evolution (bryophytes), whereas the phosphatase-minus GPAT1 to -3 and GPAT5/7 clades diverged later with the appearance of tracheophytes.

A distinct type of glycerol-3-phosphate acyltransferase with sn-2 preference and phosphatase activity producing 2-monoacylglycerol.

The first step in assembly of membrane and storage glycerolipids is acylation of glycerol-3-phosphate (G3P). All previously characterized membrane-bound, eukaryotic G3P acyltransferases (GPATs) acylate the sn-1 position to produce lysophosphatidic acid (1-acyl-LPA). Cutin is a glycerolipid with omega-oxidized fatty acids and glycerol as integral components. It occurs as an extracellular polyester on the aerial surface of all plants, provides a barrier to pathogens and resistance to stress, and maintains organ identity. We have determined that Arabidopsis acyltransferases GPAT4 and GPAT6 required for cutin biosynthesis esterify acyl groups predominantly to the sn-2 position of G3P. In addition, these acyltransferases possess a phosphatase domain that results in sn-2 monoacylglycerol (2-MAG) rather than LPA as the major product. Such bifunctional activity has not been previously described in any organism. The possible roles of 2-MAGs as intermediates in cutin synthesis are discussed. GPAT5, which is essential for the accumulation of suberin aliphatics, also exhibits a strong preference for sn-2 acylation. However, phosphatase activity is absent and 2-acyl-LPA is the major product. Clearly, plant GPATs can catalyze more reactions than the sn-1 acylation by which they are currently categorized. Close homologs of GPAT4-6 are present in all land plants, but not in animals, fungi or microorganisms (including algae). Thus, these distinctive acyltransferases may have been important for evolution of extracellular glycerolipid polymers and adaptation of plants to a terrestrial environment. These results provide insight into the biosynthetic assembly of cutin and suberin, the two most abundant glycerolipid polymers in nature.