Human health benefits of very-long-chain polyunsaturated fatty acids from microalgae.

Microalgae are single-cell, photosynthetic organisms whose biodiversity places them at the forefront of biological producers of high-value molecules including lipids and pigments. Some of these organisms particular are capable of synthesizing n-3 very long chain polyunsaturated fatty acids (VLC-PUFAs), known to have beneficial effects on human health. Indeed, VLC-PUFAs are the precursors of many signaling molecules in humans involved in the complexities of inflammatory processes. This mini-review provides an inventory of knowledge on the synthesis of VLC-PUFAs in microalgae and on the diversity of signaling molecules (prostanoids, leukotrienes, SPMs, EFOX, isoprostanoids) that arise in humans from VLC-PUFAs.

Functions of triacylglycerols during plant development and stress.

Plant oil in the form of triacylglycerols (TAGs) is a major storage compound used as food, feed and sustainable feedstock for biofuel production. Recent findings suggest that TAGs are more than a carbon and energy reserve in seeds and other storage tissues. In vegetative tissues, TAG metabolism is involved in cell division and expansion, stomatal opening, and membrane lipid remodeling. Moreover, in reproductive tissues, TAGs are important for both organ formation and successful pollination. Here we provide a brief overview of the physiological function and contribution of TAGs during plant development under optimal and varying environmental conditions. These roles of TAGs need to be considered during engineering attempts to further improve TAG content in different tissues.

A Plastid Phosphatidylglycerol Lipase Contributes to the Export of Acyl Groups from Plastids for Seed Oil Biosynthesis.

The lipid composition of thylakoid membranes inside chloroplasts is conserved from leaves to developing embryos. A finely tuned lipid assembly machinery is required to build these membranes during Arabidopsis thaliana development. Contrary to thylakoid lipid biosynthetic enzymes, the functions of most predicted chloroplast lipid-degrading enzymes remain to be elucidated. Here, we explore the biochemistry and physiological function of an Arabidopsis thylakoid membrane-associated lipase, PLASTID LIPASE1 (PLIP1). PLIP1 is a phospholipase A1 In vivo, PLIP1 hydrolyzes polyunsaturated acyl groups from a unique chloroplast-specific phosphatidylglycerol that contains 16:1 Δ3trans as its second acyl group. Thus far, a specific function of this 16:1 Δ3trans -containing phosphatidylglycerol in chloroplasts has remained elusive. The PLIP1 gene is highly expressed in seeds, and plip1 mutant seeds contain less oil and exhibit delayed germination compared with the wild type. Acyl groups released by PLIP1 are exported from the chloroplast, reincorporated into phosphatidylcholine, and ultimately enter seed triacylglycerol. Thus, 16:1 Δ3trans uniquely labels a small but biochemically active plastid phosphatidylglycerol pool in developing Arabidopsis embryos, which is subject to PLIP1 activity, thereby contributing a small fraction of the polyunsaturated fatty acids present in seed oil. We propose that acyl exchange involving thylakoid lipids functions in acyl export from plastids and seed oil biosynthesis.

The plant lipidome in human and environmental health.

Lipids and oils derived from plant and algal photosynthesis constitute much of human daily caloric intake and provide the basis for high-energy bioproducts, chemical feedstocks for countless applications, and even fossil fuels over geological time scales. Sustainable production of high-energy compounds from plants is essential to preserving fossil fuel sources and ensuring the well-being of future generations. As a result of progress in basic research on plant and algal lipid metabolism, in combination with advances in synthetic biology, we can now tailor plant lipids for desirable biological, physical, and chemical properties. We highlight recent advances in plant lipid translational biology and discuss untapped areas of research that might expand the application of plant lipids.

14-3-3 protein mediates plant seed oil biosynthesis through interaction with AtWRI1.

Plant 14-3-3 proteins are phosphopeptide-binding proteins, belonging to a large family of proteins involved in numerous physiological processes including primary metabolism, although knowledge about the function of 14-3-3s in plant lipid metabolism is sparse. WRINKLED1 (WRI1) is a key transcription factor that governs plant oil biosynthesis. At present, AtWRI1-interacting partners remain largely unknown. Here, we show that 14-3-3 proteins are able to interact with AtWRI1, both in yeast and plant cells. Transient co-expression of 14-3-3- and AtWRI1-encoding cDNAs led to increased oil biosynthesis in Nicotiana benthamiana leaves. Stable transgenic plants overproducing a 14-3-3 protein also displayed increased seed oil content. Co-production of a 14-3-3 protein with AtWRI1 enhanced the transcriptional activity of AtWRI1. The 14-3-3 protein was found to increase the stability of AtWRI1. A possible 14-3-3 binding motif was identified in one of the two AP2 domains of AtWRI1, which was also found to be critical for the interaction of AtWRI1 with an E3 ligase linker protein. Thus, we hypothesize a regulatory mechanism by which the binding of 14-3-3 to AtWRI1 interferes with the interaction of AtWRI1 and the E3 ligase, thereby protecting AtWRI1 from degradation. Taken together, our studies identified AtWRI1 as a client of 14-3-3 proteins and provide insights into a role of 14-3-3 in mediating plant oil biosynthesis.

Triacylglycerol Accumulation in Photosynthetic Cells in Plants and Algae.

Plant and algal oils are some of the most energy-dense renewable compounds provided by nature. Triacylglycerols (TAGs) are the major constituent of plant oils, which can be converted into fatty acid methyl esters commonly known as biodiesel. As one of the most efficient producers of TAGs, photosynthetic microalgae have attracted substantial interest for renewable fuel production. Currently, the big challenge of microalgae based TAGs for biofuels is their high cost compared to fossil fuels. A conundrum is that microalgae accumulate large amounts of TAGs only during stress conditions such as nutrient deprivation and temperature stress, which inevitably will inhibit growth. Thus, a better understanding of why and how microalgae induce TAG biosynthesis under stress conditions would allow the development of engineered microalgae with increased TAG production during conditions optimal for growth. Land plants also synthesize TAGs during stresses and we will compare new findings on environmental stress-induced TAG accumulation in plants and microalgae especially in the well-characterized model alga Chlamydomonas reinhardtii and a biotechnologically relevant genus Nannochloropsis.

Ectopic Expression of WRINKLED1 Affects Fatty Acid Homeostasis in Brachypodium distachyon Vegetative Tissues.

Triacylglycerol (TAG) is a storage lipid used for food purposes and as a renewable feedstock for biodiesel production. WRINKLED1 (WRI1) is a transcription factor that governs fatty acid (FA) synthesis and, indirectly, TAG accumulation in oil-storing plant tissues, and its ectopic expression has led to TAG accumulation in vegetative tissues of different dicotyledonous plants. The ectopic expression of BdWRI1 in the grass Brachypodium distachyon induced the transcription of predicted genes involved in glycolysis and FA biosynthesis, and TAG content was increased up to 32.5-fold in 8-week-old leaf blades. However, the ectopic expression of BdWRI1 also caused cell death in leaves, which has not been observed previously in dicotyledonous plants such as Arabidopsis (Arabidopsis thaliana). Lipid analysis indicated that the free FA content was 2-fold elevated in BdWRI1-expressing leaf blades of B. distachyon. The transcription of predicted genes involved in ß-oxidation was induced. In addition, linoleic FA treatment caused cell death in B. distachyon leaf blades, an effect that was reversed by the addition of the FA biosynthesis inhibitor cerulenin. Taken together, ectopic expression of BdWRI1 in B. distachyon enhances FA biosynthesis and TAG accumulation in leaves, as expected, but also leads to increased free FA content, which has cytotoxic effects leading to cell death. Thus, while WRI appears to ubiquitously affect FA biosynthesis and TAG accumulation in diverse plants, its ectopic expression can lead to undesired side effects depending on the context of the specific lipid metabolism of the respective plant species.

Deletion of a C-terminal intrinsically disordered region of WRINKLED1 affects its stability and enhances oil accumulation in Arabidopsis.

WRINKLED1 (WRI1) is a key transcription factor governing plant oil biosynthesis. We characterized three intrinsically disordered regions (IDRs) in Arabidopsis WRI1, and found that one C-terminal IDR of AtWRI1 (IDR3) affects the stability of AtWRI1. Analysis by bimolecular fluorescence complementation and yeast-two-hybrid assays indicated that the IDR3 domain does not determine WRI1 stability by interacting with BTB/POZ-MATH proteins connecting AtWRI1 with CULLIN3-based E3 ligases. Analysis of the WRI1 sequence revealed that a putative PEST motif (proteolytic signal) is located at the C-terminal region of AtWRI1(IDR) (3). We also show that a 91 amino acid domain at the C-terminus of AtWRI1 without the PEST motif is sufficient for transactivation. We found that removal of the PEST motif or mutations in putative phosphorylation sites increased the stability of AtWRI1, and led to increased oil biosynthesis when these constructs were transiently expressed in tobacco leaves. Oil content was also increased in the seeds of stable transgenic wri1-1 plants expressing AtWRI1 with mutations in the IDR3-PEST motif. Taken together, our data suggest that intrinsic disorder of AtWRI1(IDR3) may facilitate exposure of the PEST motif to protein kinases. Thus, phosphorylation of the PEST motif in the AtWRI1(IDR) (3) domain may affect AtWRI1-mediated plant oil biosynthesis. The results obtained here suggest a means to increase accumulation of oils in plant tissues through WRI1 engineering.

Wrinkled1, a ubiquitous regulator in oil accumulating tissues from Arabidopsis embryos to oil palm mesocarp.

Wrinkled1 (AtWRI1) is a key transcription factor in the regulation of plant oil synthesis in seed and non-seed tissues. The structural features of WRI1 important for its function are not well understood. Comparison of WRI1 orthologs across many diverse plant species revealed a conserved 9 bp exon encoding the amino acids "VYL". Site-directed mutagenesis of amino acids within the 'VYL' exon of AtWRI1 failed to restore the full oil content of wri1-1 seeds, providing direct evidence for an essential role of this small exon in AtWRI1 function. Arabidopsis WRI1 is predicted to have three alternative splice forms. To understand expression of these splice forms we performed RNASeq of Arabidopsis developing seeds and queried other EST and RNASeq databases from several tissues and plant species. In all cases, only one splice form was detected and VYL was observed in transcripts of all WRI1 orthologs investigated. We also characterized a phylogenetically distant WRI1 ortholog (EgWRI1) as an example of a non-seed isoform that is highly expressed in the mesocarp tissue of oil palm. The C-terminal region of EgWRI1 is over 90 amino acids shorter than AtWRI1 and has surprisingly low sequence conservation. Nevertheless, the EgWRI1 protein can restore multiple phenotypes of the Arabidopsis wri1-1 loss-of-function mutant, including reduced seed oil, the "wrinkled" seed coat, reduced seed germination, and impaired seedling establishment. Taken together, this study provides an example of combining phylogenetic analysis with mutagenesis, deep-sequencing technology and computational analysis to examine key elements of the structure and function of the WRI1 plant transcription factor.

Increasing the energy density of vegetative tissues by diverting carbon from starch to oil biosynthesis in transgenic Arabidopsis.

Increasing the energy density of biomass by engineering the accumulation of triacylglycerols (TAGs) in vegetative tissues is synergistic with efforts to produce biofuels by conversion of lignocellulosic biomass. Typically, TAG accumulates in developing seeds, and little is known about the regulatory mechanisms and control factors preventing oil biosynthesis in vegetative tissues in most plants. Here, we engineered Arabidopsis thaliana to ectopically overproduce the transcription factor WRINKLED1 (WRI1) involved in the regulation of seed oil biosynthesis. Furthermore, we reduced the expression of APS1 encoding a major catalytic isoform of the small subunit of ADP-glucose pyrophosphorylase involved in starch biosynthesis using an RNAi approach. The resulting AGPRNAi-WRI1 lines accumulated less starch and more hexoses. In addition, these lines produced 5.8-fold more oil in vegetative tissues than plants with WRI1 or AGPRNAi alone. Abundant oil droplets were visible in vegetative tissues. TAG molecular species contained long-chain fatty acids, similar to those found in seed oils. In AGPRNAi-WRI1 lines, the relative expression level of sucrose synthase 2 was considerably elevated and correlated with the level of sugars. The relative expression of the genes encoding plastidic proteins involved in de novo fatty acid synthesis, biotin carboxyl carrier protein isoform 2 and acyl carrier protein 1, was also elevated. The relative contribution of TAG compared to starch to the overall energy density increased 9.5-fold in one AGPRNAi-WRI1 transgenic line consistent with altered carbon partitioning from starch to oil.

Plant triacylglycerols as feedstocks for the production of biofuels.

Triacylglycerols produced by plants are one of the most energy-rich and abundant forms of reduced carbon available from nature. Given their chemical similarities, plant oils represent a logical substitute for conventional diesel, a non-renewable energy source. However, as plant oils are too viscous for use in modern diesel engines, they are converted to fatty acid esters. The resulting fuel is commonly referred to as biodiesel, and offers many advantages over conventional diesel. Chief among these is that biodiesel is derived from renewable sources. In addition, the production and subsequent consumption of biodiesel results in less greenhouse gas emission compared to conventional diesel. However, the widespread adoption of biodiesel faces a number of challenges. The biggest of these is a limited supply of biodiesel feedstocks. Thus, plant oil production needs to be greatly increased for biodiesel to replace a major proportion of the current and future fuel needs of the world. An increased understanding of how plants synthesize fatty acids and triacylglycerols will ultimately allow the development of novel energy crops. For example, knowledge of the regulation of oil synthesis has suggested ways to produce triacylglycerols in abundant non-seed tissues. Additionally, biodiesel has poor cold-temperature performance and low oxidative stability. Improving the fuel characteristics of biodiesel can be achieved by altering the fatty acid composition. In this regard, the generation of transgenic soybean lines with high oleic acid content represents one way in which plant biotechnology has already contributed to the improvement of biodiesel.

Functional analyses of cytosolic glucose-6-phosphate dehydrogenases and their contribution to seed oil accumulation in Arabidopsis.

Glucose-6-phosphate dehydrogenase (G6PDH) has been implicated in the supply of reduced nicotine amide cofactors for biochemical reactions and in modulating the redox state of cells. In plants, identification of its role is complicated due to the presence of several isoforms in the cytosol and plastids. Here we focus on G6PDHs in the cytosol of Arabidopsis (Arabidopsis thaliana) using single and double mutants disrupted in the two cytosolic G6PDHs. Only a single G6PDH isoform remained in the double mutant and was present in chloroplasts, consistent with a loss of cytosolic G6PDH activity. The activities of the cytosolic isoforms G6PD5 and G6PD6 were reciprocally increased in single mutants with no increase of their respective transcript levels. We hypothesized that G6PDH plays a role in supplying NADPH for oil accumulation in developing seeds in which photosynthesis may be light limited. G6PDH activity in seeds derived from G6PD6 and a plastid G6PDH isoform and showed a similar temporal activity pattern as oil accumulation. Seeds of the double mutant but not of the single mutants had higher oil content and increased weight compared to those of the wild type, with no alteration in the carbon to nitrogen ratio or fatty acid composition. A decrease in total G6PDH activity was observed only in the double mutant. These results suggest that loss of cytosolic G6PDH activity affects the metabolism of developing seeds by increasing carbon substrates for synthesis of storage compounds rather than by decreasing the NADPH supply specifically for fatty acid synthesis.

A heteromeric plastidic pyruvate kinase complex involved in seed oil biosynthesis in Arabidopsis.

Glycolysis is a ubiquitous pathway thought to be essential for the production of oil in developing seeds of Arabidopsis thaliana and oil crops. Compartmentation of primary metabolism in developing embryos poses a significant challenge for testing this hypothesis and for the engineering of seed biomass production. It also raises the question whether there is a preferred route of carbon from imported photosynthate to seed oil in the embryo. Plastidic pyruvate kinase catalyzes a highly regulated, ATP-producing reaction of glycolysis. The Arabidopsis genome encodes 14 putative isoforms of pyruvate kinases. Three genes encode subunits alpha, beta(1), and beta(2) of plastidic pyruvate kinase. The plastid enzyme prevalent in developing seeds likely has a subunit composition of 4alpha4beta(1), is most active at pH 8.0, and is inhibited by Glu. Disruption of the gene encoding the beta(1) subunit causes a reduction in plastidic pyruvate kinase activity and 60% reduction in seed oil content. The seed oil phenotype is fully restored by expression of the beta(1) subunit-encoding cDNA and partially by the beta(2) subunit-encoding cDNA. Therefore, the identified pyruvate kinase catalyzes a crucial step in the conversion of photosynthate into oil, suggesting a preferred plastid route from its substrate phosphoenolpyruvate to fatty acids.

Mutation of the TGD1 chloroplast envelope protein affects phosphatidate metabolism in Arabidopsis.

Phosphatidate (PA) is a central metabolite of lipid metabolism and a signaling molecule in many eukaryotes, including plants. Mutations in a permease-like protein, TRIGALACTOSYLDIACYLGLYCEROL1 (TGD1), in Arabidopsis thaliana caused the accumulation of triacylglycerols, oligogalactolipids, and PA. Chloroplast lipids were altered in their fatty acid composition consistent with an impairment of lipid trafficking from the endoplasmic reticulum (ER) to the chloroplast and a disruption of thylakoid lipid biosynthesis from ER-derived precursors. The process mediated by TGD1 appears to be essential as mutation of the protein caused a high incidence of embryo abortion. Isolated tgd1 mutant chloroplasts showed a decreased ability to incorporate PA into galactolipids. The TGD1 protein was localized to the inner chloroplast envelope and appears to be a component of a lipid transporter. As even partial disruption of TGD1 function has drastic consequences on central lipid metabolism, the tgd1 mutant provides a tool to explore regulatory mechanisms governing lipid homeostasis and lipid trafficking in plants.

EST-analysis of the thermo-acidophilic red microalga Galdieria sulphuraria reveals potential for lipid A biosynthesis and unveils the pathway of carbon export from rhodoplasts.

When we think of extremophiles, organisms adapted to extreme environments, prokaryotes come to mind first. However, the unicellular red micro-alga Galdieria sulphuraria (Cyanidiales) is a eukaryote that can represent up to 90% of the biomass in extreme habitats such as hot sulfur springs with pH values of 0-4 and temperatures of up to 56 degrees C. This red alga thrives autotrophically as well as heterotrophically on more than 50 different carbon sources, including a number of rare sugars and sugar alcohols. This biochemical versatility suggests a large repertoire of metabolic enzymes, rivaled by few organisms and a potentially rich source of thermo-stable enzymes for biotechnology. The temperatures under which this organism carries out photosynthesis are at the high end of the range for this process, making G. sulphuraria a valuable model for physical studies on the photosynthetic apparatus. In addition, the gene sequences of this living fossil reveal much about the evolution of modern eukaryotes. Finally, the alga tolerates high concentrations of toxic metal ions such as cadmium, mercury, aluminum, and nickel, suggesting potential application in bioremediation. To begin to explore the unique biology of G. sulphuraria , 5270 expressed sequence tags from two different cDNA libraries have been sequenced and annotated. Particular emphasis has been placed on the reconstruction of metabolic pathways present in this organism. For example, we provide evidence for (i) a complete pathway for lipid A biosynthesis; (ii) export of triose-phosphates from rhodoplasts; (iii) and absence of eukaryotic hexokinases. Sequence data and additional information are available at http://genomics.msu.edu/galdieria.

WRINKLED1 encodes an AP2/EREB domain protein involved in the control of storage compound biosynthesis in Arabidopsis.

The accumulation of storage compounds during seed development ensures the survival of the young seedling, and also provides nutrition to humans and animals in the form of foods and feeds. The putative AP2/EREBP transcription factor WRINKLED1 (WRI1) is involved in the regulation of seed storage metabolism in Arabidopsis. A splicing mutant allele, wri1-1, caused the reduction of seed oil accumulation. Glycolysis was compromised in this mutant, rendering developing embryos unable to efficiently convert sucrose into precursors of triacylglycerol biosynthesis. Expression of the WRINKLED1 cDNA under the control of the cauliflower mosaic virus 35S-promoter led to increased seed oil content. Moreover, the ectopic expression of the WRINKLED1 cDNA caused the accumulation of triacylglycerols in developing seedlings. This effect depended upon the presence of glucose in the growth medium or other sugars readily metabolized to glucose. Oil-accumulating seedlings showed aberrant development consistent with a prolonged embryonic state.

Loss of plastidic lysophosphatidic acid acyltransferase causes embryo-lethality in Arabidopsis.

Phosphatidic acid is a key intermediate for chloroplast membrane lipid biosynthesis. De novo phosphatidic acid biosynthesis in plants occurs in two steps: first the acylation of the sn-1 position of glycerol-3-phosphate giving rise to lysophosphatidic acid; second, the acylation of the sn-2 position of lysophosphatidic acid to form phosphatidic acid. The second step is catalyzed by a lysophosphatidic acid acyltransferase (LPAAT). Here we describe the identification of the ATS2 gene of Arabidopsis encoding the plastidic isoform of this enzyme. Introduction of the ATS2 cDNA into E. coli JC 201, which is temperature-sensitive and carries a mutation in its LPAAT gene plsC, restored this mutant to nearly wild type growth at high temperature. A green-fluorescent protein fusion with ATS2 localized to the chloroplast. Disruption of the ATS2 gene of Arabidopsis by T-DNA insertion caused embryo lethality. The development of the embryos was arrested at the globular stage concomitant with a transient increase in ATS2 gene expression. Apparently, plastidic LPAAT is essential for embryo development in Arabidopsis during the transition from the globular to the heart stage when chloroplasts begin to form.

Anionic lipids are required for chloroplast structure and function in Arabidopsis.

Photosynthetic membranes of plants primarily contain non-phosphorous glycolipids. The exception is phosphatidylglycerol (PG), which is an acidic/anionic phospholipid. A second major anionic lipid in chloroplasts is the sulfolipid sulfoquinovosyldiacylglycerol (SQDG). It is hypothesized that under severe phosphate limitation, SQDG substitutes for PG, ensuring a constant proportion of anionic lipids even under adverse conditions. A newly constructed SQDG and PG-deficient double mutant supports this hypothesis. This mutant, sqd2 pgp1-1, carries a T-DNA insertion in the structural gene for SQDG synthase (SQD2) and a point mutation in the structural gene for phosphatidylglycerolphosphate synthase (PGP1). In the sqd2 pgp1-1 double mutant, the fraction of total anionic lipids is reduced by approximately one-third, resulting in pale yellow cotyledons and leaves with reduced chlorophyll content. Photoautotrophic growth of the double mutant is severely compromised, and its photosynthetic capacity is impaired. In particular, photosynthetic electron transfer at the level of photosystem II (PSII) is affected. Besides these physiological changes, the mutant shows altered leaf structure, a reduced number of mesophyll cells, and ultrastructural changes of the chloroplasts. All observations on the sqd2 pgp1-1 mutant lead to the conclusion that the total content of anionic thylakoid lipids is limiting for chloroplast structure and function, and is critical for overall photoautotrophic growth and plant development.

Contrapuntal networks of gene expression during Arabidopsis seed filling.

We have used cDNA microarrays to examine changes in gene expression during Arabidopsis seed development and to compare wild-type and mutant wrinkled1 (wri1) seeds that have an 80% reduction in oil. Between 5 and 13 days after flowering, a period preceding and including the major accumulation of storage oils and proteins, approximately 35% of the genes represented on the array changed at least twofold, but a larger fraction (65%) showed little or no change in expression. Genes whose expression changed most tended to be expressed more in seeds than in other tissues. Genes related to the biosynthesis of storage components showed several distinct temporal expression patterns. For example, a number of genes encoding core fatty acid synthesis enzymes displayed a bell-shaped pattern of expression between 5 and 13 days after flowering. By contrast, the expression of storage proteins, oleosins, and other known abscisic acid-regulated genes increased later and remained high. Genes for photosynthetic proteins followed a pattern very similar to that of fatty acid synthesis proteins, implicating a role in CO(2) refixation and the supply of cofactors for oil synthesis. Expression profiles of key carbon transporters and glycolytic enzymes reflected shifts in flux from cytosolic to plastid metabolism. Despite major changes in metabolism between wri1 and wild-type seeds, <1% of genes differed by more than twofold, and most of these were involved in central lipid and carbohydrate metabolism. Thus, these data define in part the downstream responses to disruption of the WRI1 gene.

Arabidopsis disrupted in SQD2 encoding sulfolipid synthase is impaired in phosphate-limited growth.

The sulfolipid sulfoquinovosyldiacylglycerol is one of the three nonphosphorous glycolipids that provide the bulk of the structural lipids in photosynthetic membranes of seed plants. Unlike the galactolipids, sulfolipid is anionic at physiological pH because of its 6-deoxy-6-sulfonate-glucose (sulfoquinovose) head group. The biosynthesis of this lipid proceeds in two steps: first, the assembly of UDP-sulfoquinovose from UDP-glucose and sulfite, and second, the transfer of the sulfoquinovose moiety from UDP-sulfoquinovose to diacylglycerol. The first reaction is catalyzed by the SQD1 protein in Arabidopsis. Here we describe the identification of the SQD2 gene of Arabidopsis. We propose that this gene encodes the sulfoquinovosyltransferase catalyzing the second step of sulfolipid biosynthesis. Expression of SQD1 and SQD2 in Escherichia coli reconstituted plant sulfolipid biosynthesis in this bacterium. Insertion of a transfer DNA into this gene in Arabidopsis led to complete lack of sulfolipid in the respective sqd2 mutant. This mutant showed reduced growth under phosphate-limited growth conditions. The results support the hypothesis that sulfolipid can function as a substitute of anionic phospholipids under phosphate-limited growth conditions. Along with phosphatidylglycerol, sulfolipid contributes to maintaining a negatively charged lipid-water interface, which presumably is required for proper function of photosynthetic membranes.