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

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

Tackling functional redundancy of Arabidopsis fatty acid elongase complexes.

Very-long-chain fatty acids (VLCFA) are precursors for various lipids playing important physiological and structural roles in plants. Throughout plant tissues, VLCFA are present in multiple lipid classes essential for membrane homeostasis, and also stored in triacylglycerols. VLCFA and their derivatives are also highly abundant in lipid barriers, such as cuticular waxes in aerial epidermal cells and suberin monomers in roots. VLCFA are produced by the fatty acid elongase (FAE), which is an integral endoplasmic reticulum membrane multi-enzymatic complex consisting of four core enzymes. The 3-ketoacyl-CoA synthase (KCS) catalyzes the first reaction of the elongation and determines the chain-length substrate specificity of each elongation cycle, whereas the other three enzymes have broad substrate specificities and are shared by all FAE complexes. Consistent with the co-existence of multiple FAE complexes, performing sequential and/or parallel reactions to produce the broad chain-length-range of VLCFA found in plants, twenty-one KCS genes have been identified in the genome of Arabidopsis thaliana. Using CRISPR-Cas9 technology, we established an expression platform to reconstitute the different Arabidopsis FAE complexes in yeast. The VLCFA produced in these yeast strains were analyzed in detail to characterize the substrate specificity of all KCS candidates. Additionally, Arabidopsis candidate proteins were transiently expressed in Nicotiana benthamiana leaves to explore their activity and localization in planta. This work sheds light on the genetic and biochemical redundancy of fatty acid elongation in plants.

Phosphatidylinositol-4-phosphate joins the dance of plant autophagosome formation.

In plants, macroautophagy/autophagy is a key mechanism that contributes to their ability to cope with a wide range of environmental constraints such as drought, nutrient starvation or pathogen resistance. Nevertheless, the molecular mechanisms of plant autophagy, and notably that of autophagosome formation, remain poorly understood. As the starting point of our recent paper, we considered the potential functional contribution of lipids in the numerous membrane-remodeling steps involved in this process. By combining biochemistry, genetics, cell biology and high-resolution 3D imaging, we unraveled the function of the lipid phosphatidylinositol-4-phosphate (PtdIns4P) in autophagy in Arabidopsis thaliana, thus providing novel insights into the assembly of autophagosomes in plant cells.

Phosphatidylinositol-4-phosphate controls autophagosome formation in Arabidopsis thaliana.

Autophagy is an intracellular degradation mechanism critical for plant acclimation to environmental stresses. Central to autophagy is the formation of specialized vesicles, the autophagosomes, which target and deliver cargo to the lytic vacuole. How autophagosomes form in plant cells remains poorly understood. Here, we uncover the importance of the lipid phosphatidylinositol-4-phosphate in autophagy using pharmacological and genetical approaches. Combining biochemical and live-microscopy analyses, we show that PI4K activity is required for early stages of autophagosome formation. Further, our results show that the plasma membrane-localized PI4Kα1 is involved in autophagy and that a substantial portion of autophagy structures are found in proximity to the PI4P-enriched plasma membrane. Together, our study unravels critical insights into the molecular determinants of autophagy, proposing a model whereby the plasma membrane provides PI4P to support the proper assembly and expansion of the phagophore thus governing autophagosome formation in Arabidopsis.

Predictive metabolomics of multiple Atacama plant species unveils a core set of generic metabolites for extreme climate resilience.

Current crop yield of the best ideotypes is stagnating and threatened by climate change. In this scenario, understanding wild plant adaptations in extreme ecosystems offers an opportunity to learn about new mechanisms for resilience. Previous studies have shown species specificity for metabolites involved in plant adaptation to harsh environments. Here, we combined multispecies ecological metabolomics and machine learning-based generalized linear model predictions to link the metabolome to the plant environment in a set of 24 species belonging to 14 families growing along an altitudinal gradient in the Atacama Desert. Thirty-nine common compounds predicted the plant environment with 79% accuracy, thus establishing the plant metabolome as an excellent integrative predictor of environmental fluctuations. These metabolites were independent of the species and validated both statistically and biologically using an independent dataset from a different sampling year. Thereafter, using multiblock predictive regressions, metabolites were linked to climatic and edaphic stressors such as freezing temperature, water deficit and high solar irradiance. These findings indicate that plants from different evolutionary trajectories use a generic metabolic toolkit to face extreme environments. These core metabolites, also present in agronomic species, provide a unique metabolic goldmine for improving crop performances under abiotic pressure.

Biosynthesis and Functions of Very-Long-Chain Fatty Acids in the Responses of Plants to Abiotic and Biotic Stresses.

Very-long-chain fatty acids (i.e., fatty acids with more than 18 carbon atoms; VLCFA) are important molecules that play crucial physiological and structural roles in plants. VLCFA are specifically present in several membrane lipids and essential for membrane homeostasis. Their specific accumulation in the sphingolipids of the plasma membrane outer leaflet is of primordial importance for its correct functioning in intercellular communication. VLCFA are found in phospholipids, notably in phosphatidylserine and phosphatidylethanolamine, where they could play a role in membrane domain organization and interleaflet coupling. In epidermal cells, VLCFA are precursors of the cuticular waxes of the plant cuticle, which are of primary importance for many interactions of the plant with its surrounding environment. VLCFA are also major components of the root suberin barrier, which has been shown to be fundamental for nutrient homeostasis and plant adaptation to adverse conditions. Finally, some plants store VLCFA in the triacylglycerols of their seeds so that they later play a pivotal role in seed germination. In this review, taking advantage of the many studies conducted using Arabidopsis thaliana as a model, we present our current knowledge on the biosynthesis and regulation of VLCFA in plants, and on the various functions that VLCFA and their derivatives play in the interactions of plants with their abiotic and biotic environment.

Temperature Acclimation of the Picoalga Ostreococcus tauri Triggers Early Fatty-Acid Variations and Involves a Plastidial ω3-Desaturase.

Alteration of fatty-acid unsaturation is a universal response to temperature changes. Marine microalgae display the largest diversity of polyunsaturated fatty-acid (PUFA) whose content notably varies according to temperature. The physiological relevance and the molecular mechanisms underlying these changes are however, still poorly understood. The ancestral green picoalga Ostreococcus tauri displays original lipidic features that combines PUFAs from two distinctive microalgal lineages (Chlorophyceae, Chromista kingdom). In this study, optimized conditions were implemented to unveil early fatty-acid and desaturase transcriptional variations upon chilling and warming. We further functionally characterized the O. tauri ω3-desaturase which is closely related to ω3-desaturases from Chromista species. Our results show that the overall omega-3 to omega-6 ratio is swiftly and reversibly regulated by temperature variations. The proportion of the peculiar 18:5 fatty-acid and temperature are highly and inversely correlated pinpointing the importance of 18:5 temperature-dependent variations across kingdoms. Chilling rapidly and sustainably up-regulated most desaturase genes. Desaturases involved in the regulation of the C18-PUFA pool as well as the Δ5-desaturase appear to be major transcriptional targets. The only ω3-desaturase candidate, related to ω3-desaturases from Chromista species, is localized at chloroplasts in Nicotiana benthamiana and efficiently performs ω3-desaturation of C18-PUFAs in Synechocystis sp. PCC6803. Overexpression in the native host further unveils a broad impact on plastidial and non-plastidial glycerolipids illustrated by the alteration of omega-3/omega-6 ratio in C16-PUFA and VLC-PUFA pools. Global glycerolipid features of the overexpressor recall those of chilling acclimated cells.

Plastidic Δ6 Fatty-Acid Desaturases with Distinctive Substrate Specificity Regulate the Pool of C18-PUFAs in the Ancestral Picoalga Ostreococcus tauri.

Eukaryotic Δ6-desaturases are microsomal enzymes that balance the synthesis of ω-3 and ω-6 C18-polyunsaturated fatty acids (C18-PUFAs) according to their specificity. In several microalgae, including Ostreococcus tauri, plastidic C18-PUFAs are strictly regulated by environmental cues suggesting an autonomous control of Δ6-desaturation of plastidic PUFAs. Here, we identified two putative front-end Δ6/Δ8-desaturases from O tauri that, together with putative homologs, cluster apart from other characterized Δ6-desaturases. Both were plastid-located and unambiguously displayed a Δ6-desaturation activity when overexpressed in the heterologous hosts Nicotiana benthamiana and Synechocystis sp. PCC6803, as in the native host. Detailed lipid analyses of overexpressing lines unveiled distinctive ω-class specificities, and most interestingly pointed to the importance of the lipid head-group and the nonsubstrate acyl-chain for the desaturase efficiency. One desaturase displayed a broad specificity for plastidic lipids and a preference for ω-3 substrates, while the other was more selective for ω-6 substrates and for lipid classes including phosphatidylglycerol as well as the peculiar 16:4-galactolipid species occurring in the native host. Overexpression of both Δ6-desaturases in O tauri prevented the regulation of C18-PUFA under phosphate deprivation and triggered glycerolipid fatty-acid remodeling, without causing any obvious alteration in growth or photosynthesis. Tracking fatty-acid modifications in eukaryotic hosts further suggested the export of plastidic lipids to extraplastidic compartments.

AtMYB92 enhances fatty acid synthesis and suberin deposition in leaves of Nicotiana benthamiana.

Acyl lipids are important constituents of the plant cell. Depending on the cell type, requirements in acyl lipids vary greatly, implying a tight regulation of fatty acid and lipid metabolism. The discovery of the WRINKLED1 (WRI1) transcription factors, members of the AP2-EREBP (APETALA2-ethylene-responsive element binding protein) family, has emphasized the importance of transcriptional regulation for adapting the rate of acyl chain production to cell requirements. Here, we describe the identification of another activator of the fatty acid biosynthetic pathway, the Arabidopsis MYB92 transcription factor. This MYB and all the members of the subgroups S10 and S24 of MYB transcription factors can directly activate the promoter of BCCP2 that encodes a component of the fatty acid biosynthetic pathway. Two adjacent MYB cis-regulatory elements are essential for the binding and activation of the BCCP2 promoter by MYB92. Overexpression of MYB92 or WRI1 in Nicotiana benthamiana induces the expression of fatty acid biosynthetic genes but results in the accumulation of different types of acyl lipids. In the presence of WRI1, triacylglycerol biosynthetic enzymes coded by constitutively expressed genes efficiently channel the excess fatty acids toward reserve lipid accumulation. By contrast, MYB92 activates both fatty acid and suberin biosynthetic genes; hence, the remarkable increase in suberin monomers measured in leaves expressing MYB92. These results provide additional insight into the molecular mechanisms that control the biosynthesis of an important cell wall-associated acylglycerol polymer playing critical roles in plants.

A stress-response-related inter-compartmental signalling pathway regulates embryonic cuticle integrity in Arabidopsis.

The embryonic cuticle is necessary for normal seed development and seedling establishment in Arabidopsis. Although mutants with defective embryonic cuticles have been identified, neither the deposition of cuticle material, nor its regulation, has been described during embryogenesis. Here we use electron microscopy, cuticle staining and permeability assays to show that cuticle deposition initiates de novo in patches on globular embryos. By combining these techniques with genetics and gene expression analysis, we show that successful patch coalescence to form a continuous cuticle requires a signalling involving the endosperm-specific subtilisin protease ALE1 and the receptor kinases GSO1 and GSO2, which are expressed in the developing embryonic epidermis. Transcriptome analysis shows that this pathway regulates stress-related gene expression in seeds. Consistent with these findings we show genetically, and through activity analysis, that the stress-associated MPK6 protein acts downstream of GSO1 and GSO2 in the developing embryo. We propose that a stress-related signalling pathway has been hijacked in some angiosperm seeds through the recruitment of endosperm-specific components. Our work reveals the presence of an inter-compartmental dialogue between the endosperm and embryo that ensures the formation of an intact and functional cuticle around the developing embryo through an "auto-immune" type interaction.

Arabidopsis CER1-LIKE1 Functions in a Cuticular Very-Long-Chain Alkane-Forming Complex.

Plant aerial organs are coated with cuticular waxes, a hydrophobic layer that primarily serves as a waterproofing barrier. Cuticular wax is a mixture of aliphatic very-long-chain molecules, ranging from 22 to 48 carbons, produced in the endoplasmic reticulum of epidermal cells. Among all wax components, alkanes represent up to 80% of total wax in Arabidopsis (Arabidopsis thaliana) leaves. Odd-numbered alkanes and their derivatives are produced through the alkane-forming pathway. Although the chemical reactions of this pathway have been well described, the enzymatic mechanisms catalyzing these reactions remain unclear. We previously showed that a complex made of Arabidopsis ECERIFERUM1 (CER1) and CER3 catalyzes the conversion of acyl-Coenzyme A's to alkanes with strict substrate specificity for compounds containing more than 29 carbons. To learn more about alkane biosynthesis in Arabidopsis, we characterized the biochemical specificity and physiological functions of a CER1 homolog, CER1-LIKE1. In a yeast strain engineered to produce very-long-chain fatty acids, CER1-LIKE1 interacted with CER3 and cytochrome B5 to form a functional complex leading to the production of alkanes that are of different chain lengths compared to that produced by CER1-containing complexes. Gene expression analysis showed that both CER1 and CER1-LIKE1 are differentially expressed in an organ- and tissue-specific manner. Moreover, the inactivation or overexpression of CER1-LIKE1 in Arabidopsis transgenic lines specifically impacted alkane biosynthesis and wax crystallization. Collectively, our study reports on the identification of a further plant alkane synthesis enzymatic component and supports a model in which several alkane-forming complexes with distinct chain-length specificities coexist in plants.

Lipids in membrane dynamics during autophagy in plants.

Autophagy is a critical pathway for plant adaptation to stress. Macroautophagy relies on the biogenesis of a specialized membrane named the phagophore that maturates into a double membrane vesicle. Proteins and lipids act synergistically to promote membrane structure and functions, yet research on autophagy has mostly focused on autophagy-related proteins while knowledge of supporting lipids in the formation of autophagic membranes remains scarce. This review expands on studies in plants with examples from other organisms to present and discuss our current understanding of lipids in membrane dynamics associated with the autophagy pathway in plants.

Arabidopsis ketoacyl-CoA synthase 16 (KCS16) forms C36 /C38 acyl precursors for leaf trichome and pavement surface wax.

The aliphatic waxes sealing plant surfaces against environmental stress are generated by fatty acid elongase complexes, each containing a ß-ketoacyl-CoA synthase (KCS) enzyme that catalyses a crucial condensation forming a new C─C bond to extend the carbon backbone. The relatively high abundance of C35 and C37 alkanes derived from C36 and C38 acyl-CoAs in Arabidopsis leaf trichomes (relative to other epidermis cells) suggests differences in the elongation machineries of different epidermis cell types, possibly involving KCS16, a condensing enzyme expressed preferentially in trichomes. Here, KCS16 was found expressed primarily in Arabidopsis rosette leaves, flowers and siliques, and the corresponding protein was localized to the endoplasmic reticulum. The cuticular waxes on young leaves and isolated leaf trichomes of ksc16 loss-of-function mutants were depleted of C35 and C37 alkanes and alkenes, whereas expression of Arabidopsis KCS16 in yeast and ectopic overexpression in Arabidopsis resulted in accumulation of C36 and C38 fatty acid products. Taken together, our results show that KCS16 is the sole enzyme catalysing the elongation of C34 to C38 acyl-CoAs in Arabidopsis leaf trichomes and that it contributes to the formation of extra-long compounds in adjacent pavement cells.

Enrichment of hydroxylated C24- and C26-acyl-chain sphingolipids mediates PIN2 apical sorting at trans-Golgi network subdomains.

The post-Golgi compartment trans-Golgi Network (TGN) is a central hub divided into multiple subdomains hosting distinct trafficking pathways, including polar delivery to apical membrane. Lipids such as sphingolipids and sterols have been implicated in polar trafficking from the TGN but the underlying mechanisms linking lipid composition to functional polar sorting at TGN subdomains remain unknown. Here we demonstrate that sphingolipids with α-hydroxylated acyl-chains of at least 24 carbon atoms are enriched in secretory vesicle subdomains of the TGN and are critical for de novo polar secretory sorting of the auxin carrier PIN2 to apical membrane of Arabidopsis root epithelial cells. We show that sphingolipid acyl-chain length influences the morphology and interconnections of TGN-associated secretory vesicles. Our results uncover that the sphingolipids acyl-chain length links lipid composition of TGN subdomains with polar secretory trafficking of PIN2 to apical membrane of polarized epithelial cells.

Primary Fatty Alcohols Are Major Components of Suberized Root Tissues of Arabidopsis in the Form of Alkyl Hydroxycinnamates.

Suberin is a complex hydrophobic polymer that acts as a barrier controlling water and solute fluxes and restricting pathogen infections. Suberin is deposited immediately outside of the plasmalemma in the cell wall of certain tissues such as endodermis of roots, aerial and underground periderms, and seed coats. Suberin consists of a variety of fatty acid derivatives polymerized with glycerol and phenolics. In this study, we show using liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry techniques that most of the fatty alcohols not covalently linked to the suberin polymer are in the form of alkyl hydroxycinnamates (AHCs), with alkyl caffeates predominating. Such compounds are not restricted to the periderm of mature roots but also are present in the endodermis of younger roots, where they are not extracted by rapid dipping in chloroform. Analysis of several mutants affected in key enzymes involved in the biosynthesis and export of suberin monomers suggests that the formation of the suberin polymer and associated waxes involves common pathways and occurs concomitantly in Arabidopsis (Arabidopsis thaliana) roots. Although fatty alcohols represent only minor components of the suberin polymer in Arabidopsis roots, this study demonstrates that they constitute the major aliphatics of suberin-associated waxes in the form of AHCs. Therefore, our results indicate that esterified fatty alcohols, both soluble and polymerized forms, represent major constituents of Arabidopsis root suberized barriers, being as abundant as α,ω-dicarboxylic and unsubstituted fatty acids. In addition, our results show that suberized layers represent a major sink for acyl-lipid metabolism in Arabidopsis roots.

The Glycerol-3-Phosphate Acyltransferase GPAT6 from Tomato Plays a Central Role in Fruit Cutin Biosynthesis.

The thick cuticle covering and embedding the epidermal cells of tomato (Solanum lycopersicum) fruit acts not only as a protective barrier against pathogens and water loss but also influences quality traits such as brightness and postharvest shelf-life. In a recent study, we screened a mutant collection of the miniature tomato cultivar Micro-Tom and isolated several glossy fruit mutants in which the abundance of cutin, the polyester component of the cuticle, was strongly reduced. We employed a newly developed mapping-by-sequencing strategy to identify the causal mutation underlying the cutin deficiency in a mutant thereafter named gpat6-a (for glycerol-3-phosphate acyltransferase6). To this end, a backcross population (BC1F2) segregating for the glossy trait was phenotyped. Individuals displaying either a wild-type or a glossy fruit trait were then pooled into bulked populations and submitted to whole-genome sequencing prior to mutation frequency analysis. This revealed that the causal point mutation in the gpat6-a mutant introduces a charged amino acid adjacent to the active site of a GPAT6 enzyme. We further showed that this mutation completely abolished the GPAT activity of the recombinant protein. The gpat6-a mutant showed perturbed pollen formation but, unlike a gpat6 mutant of Arabidopsis (Arabidopsis thaliana), was not male sterile. The most striking phenotype was observed in the mutant fruit, where cuticle thickness, composition, and properties were altered. RNA sequencing analysis highlighted the main processes and pathways that were affected by the mutation at the transcriptional level, which included those associated with lipid, secondary metabolite, and cell wall biosynthesis.

Plant Surface Lipids and Epidermis Development.

The epidermis has a strategic position at the interface between the plant and the environment. In order to control exchanges with the environment as well as to protect the plant from external threats, the epidermis synthesises and secretes surface lipids to form a continuous, transparent and hydrophobic layer known as the cuticle. Cuticle formation is a strictly epidermal property in plants and all aerial epidermal cells produce some sort of cuticle on their surface. Conversely, all cuticularized plant surfaces are of epidermal origin. This seemingly anodyne observation has surprisingly profound implications in terms of understanding the function of the plant cuticle, since it underlies in part, the difficultly of functionally separating epidermal cell fate specification from cuticle biogenesis.

An ethoxylated surfactant enhances the penetration of the sulfated laminarin through leaf cuticle and stomata, leading to increased induced resistance against grapevine downy mildew.

Some ß-1,3-glucans and particularly sulfated laminarin (PS3) are known as resistance inducers (RIs) in grapevine against the downy mildew. However, their efficacy in vineyard is still often too low, which might be caused by a limited penetration through the leaf cuticle following spray application. We used (14) C-sucrose uptake experiments with grapevine leaves in order to select a surfactant as saccharide penetration enhancer. Our results showed that although sucrose foliar uptake was low, it was strongly enhanced by Dehscofix CO125 (DE), a highly ethoxylated surfactant. Fluorescent saccharides were then produced and laser scanning microscopy was used to analyze their foliar diffusion pattern in Arabidopsis thaliana and grapevine. Interestingly, sucrose and PS3 were seemingly able to penetrate the leaf cuticle only when formulated with DE. Diffusion could preferentially occur via stomata, anticlinal cell walls and trichomes. In grapevine, PS3 penetration rate was much higher on the stomateous abaxial surface of the leaf than on the adaxial surface. Finally, using DE allowed a higher level of downy mildew control by PS3, which corroborated diffusion observations. Our results have practical consequences for the improvement of treatments with saccharidic inducers on grape. That is, formulation of such RIs plays a critical role for their cuticular diffusion and consequently their efficacy. Also, spray application should preferentially target the abaxial surface of the leaves in order to maximize their penetration.

ECERIFERUM2-LIKE proteins have unique biochemical and physiological functions in very-long-chain fatty acid elongation.

The extension of very-long-chain fatty acids (VLCFAs) for the synthesis of specialized apoplastic lipids requires unique biochemical machinery. Condensing enzymes catalyze the first reaction in fatty acid elongation and determine the chain length of fatty acids accepted and produced by the fatty acid elongation complex. Although necessary for the elongation of all VLCFAs, known condensing enzymes cannot efficiently synthesize VLCFAs longer than 28 carbons, despite the prevalence of C28 to C34 acyl lipids in cuticular wax and the pollen coat. The eceriferum2 (cer2) mutant of Arabidopsis (Arabidopsis thaliana) was previously shown to have a specific deficiency in cuticular waxes longer than 28 carbons, and heterologous expression of CER2 in yeast (Saccharomyces cerevisiae) demonstrated that it can modify the acyl chain length produced by a condensing enzyme from 28 to 30 carbon atoms. Here, we report the physiological functions and biochemical specificities of the CER2 homologs CER2-LIKE1 and CER2-LIKE2 by mutant analysis and heterologous expression in yeast. We demonstrate that all three CER2-LIKEs function with the same small subset of condensing enzymes, and that they have different effects on the substrate specificity of the same condensing enzyme. Finally, we show that the changes in acyl chain length caused by each CER2-LIKE protein are of substantial importance for cuticle formation and pollen coat function.

The bifunctional protein TtFARAT from Tetrahymena thermophila catalyzes the formation of both precursors required to initiate ether lipid biosynthesis.

The biosynthesis of ether lipids and wax esters requires as precursors fatty alcohols, which are synthesized by fatty acyl reductases (FARs). The presence of ether glycerolipids as well as branched wax esters has been reported in several free-living ciliate protozoa. In the genome of Tetrahymena thermophila, the only ORF sharing similarities with FARs is fused to an acyltransferase-like domain, whereas, in most other organisms, FARs are monofunctional proteins of similar size and domain structure. Here, we used heterologous expression in plant and yeast to functionally characterize the activities catalyzed by this protozoan protein. Transient expression in tobacco epidermis of a truncated form fused to the green fluorescence protein followed by confocal microscopy analysis suggested peroxisomal localization. In vivo approaches conducted in yeast indicated that the N-terminal FAR-like domain produced both 16:0 and 18:0 fatty alcohols, whereas the C-terminal acyltransferase-like domain was able to rescue the lethal phenotype of the yeast double mutant gat1Δ gat2Δ. Using in vitro approaches, we further demonstrated that this domain is a dihydroxyacetone phosphate acyltransferase that uses preferentially 16:0-coenzyme A as an acyl donor. Finally, coexpression in yeast with the alkyl-dihydroxyacetone phosphate synthase from T. thermophila resulted the detection of various glycerolipids with an ether bond, indicating reconstitution of the ether lipid biosynthetic pathway. Together, these results demonstrate that this FAR-like protein is peroxisomal and bifunctional, providing both substrates required by alkyl-dihydroxyacetone phosphate synthase to initiate ether lipid biosynthesis.