BIOSYNTHESIS AND FUNCTION OF THE SULFOLIPID SULFOQUINOVOSYL DIACYLGLYCEROL

Annual Review of Plant Physiology and Plant Molecular Biology. Jun, 1998  |  Pubmed ID: 15012227

The sulfolipid sulfoquinovosyl diacylglycerol is an abundant sulfur-containing nonphosphorous glycerolipid that is specifically associated with photosynthetic membranes of higher plants, mosses, ferns, algae, and most photosynthetic bacteria. The characteristic structural feature of sulfoquinovosyl diacylglycerol is the unique head group constituent sulfoquinovose, a derivative of glucose in which the 6-hydroxyl is replaced by a sulfonate group. While there is growing evidence for the final assembly of the sulfolipid by the transfer of the sulfoquinovosyl moiety from UDP-sulfoquinovose to the sn-3 position of diacylglycerol, very little is known about the biosynthesis of the precursor UDP-sulfoquinovose. Recently, a number of mutants deficient in sulfolipid biosynthesis and the corresponding sqd genes have become available from different organisms. These provide novel tools to analyze sulfolipid biosynthesis by a combination of molecular and biochemical approaches. Furthermore, the analysis of sulfolipid-deficient mutants has provided novel insights into the function of sulfoquinovosyl diacylglycerol in photosynthetic membranes.

Digalactosyldiacylglycerol Synthesis in Chloroplasts of the Arabidopsis Dgd1 Mutant

Plant Physiology. Mar, 2002  |  Pubmed ID: 11891245

Galactolipid biosynthesis in plants is highly complex. It involves multiple pathways giving rise to different molecular species. To assess the contribution of different routes of galactolipid synthesis and the role of molecular species for growth and photosynthesis, we initiated a genetic approach of analyzing double mutants of the digalactosyldiacylglycerol (DGDG) synthase mutant dgd1 with the acyltransferase mutant, act1, and the two desaturase mutants, fad2 and fad3. The double mutants showed different degrees of growth retardation: act1,dgd1 was most severely affected and growth of fad2,dgd1 was slightly reduced, whereas fad3,dgd1 plants were very similar to dgd1. In act1,dgd1, lipid and chlorophyll content were reduced and photosynthetic capacity was affected. Molecular analysis of galactolipid content, fatty acid composition, and positional distribution suggested that the growth deficiency is not caused by changes in galactolipid composition per se. Chloroplasts of dgd1 were capable of synthesizing monogalactosyldiacylglycerol, DGDG, and tri- and tetragalactosyldiacylglycerol. Therefore, the reduced growth of act1,dgd1 and fad2,dgd1 cannot be explained by the absence of DGDG synthase activity from chloroplasts. Molecular analysis of DGDG accumulating in the mutants during phosphate deprivation suggested that similarly to the residual DGDG of dgd1, this additional lipid is synthesized in association with chloroplast membranes through a pathway independent of the mutations, act1, dgd1, fad2, and fad3. Our data imply that the severe growth defect of act1,dgd1 is caused by a reduced metabolic flux of chloroplast lipid synthesis through the eukaryotic and prokaryotic pathway as well as by the reduction of photosynthetic capacity caused by the destabilization of photosynthetic complexes.

Galactolipids Rule in Seed Plants

Trends in Plant Science. Mar, 2002  |  Pubmed ID: 11906834

Chloroplast membranes contain high levels of the galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG). The isolation of the genes involved in the biosynthesis of MGDG and DGDG, and the identification of galactolipid-deficient Arabidopsis mutants has greatly facilitated the analysis of galactolipid biosynthesis and function. Galactolipids are found in X-ray structures of photosynthetic complexes, suggesting a direct role in photosynthesis. Furthermore, galactolipids can substitute for phospholipids, as suggested by increases in the galactolipid:phospholipid ratio after phosphate deprivation. The ratio of MGDG to DGDG is also crucial for the physical phase of thylakoid membranes and might be regulated.

Arabidopsis Disrupted in SQD2 Encoding Sulfolipid Synthase is Impaired in Phosphate-limited Growth

Proceedings of the National Academy of Sciences of the United States of America. Apr, 2002  |  Pubmed ID: 11960029

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.

The Pgp1 Mutant Locus of Arabidopsis Encodes a Phosphatidylglycerolphosphate Synthase with Impaired Activity

Plant Physiology. Jun, 2002  |  Pubmed ID: 12068104

Phosphatidylglycerol is a ubiquitous phospholipid that is also present in the photosynthetic membranes of plants. Multiple independent lines of evidence suggest that this lipid plays a critical role for the proper function of photosynthetic membranes and cold acclimation. In eukaryotes, different subcellular compartments are competent for the biosynthesis of phosphatidylglycerol. Details on the plant-specific pathways in different organelles are scarce. Here, we describe a phosphatidylglycerol biosynthesis-deficient mutant of Arabidopsis, pgp1. The overall content of phosphatidylglycerol is reduced by 30%. This mutant carries a point mutation in the CDP-alcohol phosphotransferase motif of the phosphatidylglycerolphosphate synthase (EC 2.7.8.5) isoform encoded by a gene on chromosome 2. The mutant shows an 80% reduction in plastidic phosphatidylglycerolphosphate synthase activity consistent with the plastidic location of this particular isoform. Mutant plants are pale green, and their photosynthesis is impaired. This mutant provides a promising new tool to elucidate the biosynthesis and function of plastidic phosphatidylglycerol in seed plants.

Contrapuntal Networks of Gene Expression During Arabidopsis Seed Filling

The Plant Cell. Jun, 2002  |  Pubmed ID: 12084821

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.

Native Uridine 5'-diphosphate-sulfoquinovose Synthase, SQD1, from Spinach Purifies As a 250-kDa Complex

Archives of Biochemistry and Biophysics. May, 2003  |  Pubmed ID: 12706349

Sulfoquinovosyldiacylglycerol is a polar lipid present in photosynthetic membranes. It contributes to the negative surface charge of the membrane and plays a pivotal role under phosphate stress. The SQD1 protein is the key enzyme involved in the formation of the sulfolipid head group precursor, uridine 5(')-diphosphate (UDP)-sulfoquinovose, from UDP-glucose and sulfite. A cDNA encoding the spinach SQD1 protein was isolated and functionally expressed in Escherichia coli. The recombinant enzyme was compared to the native enzyme purified from isolated spinach chloroplasts. While the K(m) for UDP-glucose was indistinguishable for the two forms, the K(m) for sulfite was more than fourfold lower (< microM) for the native enzyme. Sizing by gel filtration indicated that the native form purified as a large complex of approximately 250 kDa, which is more than twice as large as the calculated size for the homodimer. It is proposed that in vivo SQD1 forms a complex with accessory proteins.

A Permease-like Protein Involved in ER to Thylakoid Lipid Transfer in Arabidopsis

The EMBO Journal. May, 2003  |  Pubmed ID: 12743031

In eukaryotes, enzymes of different subcellular compartments participate in the assembly of membrane lipids. As a consequence, interorganelle lipid transfer is extensive in growing cells. A prominent example is the transfer of membrane lipid precursors between the endoplasmic reticulum (ER) and the photosynthetic thylakoid membranes in plants. Mono- and digalactolipids are typical photosynthetic membrane lipids. In Arabidopsis, they are derived from one of two pathways, either synthesized de novo in the plastid, or precursors are imported from the ER, giving rise to distinct molecular species. Employing a high-throughput robotic screening procedure generating arrays of spot chromatograms, mutants of Arabidopsis were isolated, which accumulated unusual trigalactolipids. In one allelic mutant subclass, trigalactosyldiacylglycerol1, the primary defect caused a disruption in the biosynthesis of ER-derived thylakoid lipids. Secondarily, a processive galactosyltransferase was activated, leading to the accumulation of oligogalactolipids. Mutations in a permease-like protein of the outer chloroplastic envelope are responsible for the primary biochemical defect. It is proposed that this protein is part of a lipid transfer complex.

The Sulfolipids 2'-O-acyl-sulfoquinovosyldiacylglycerol and Sulfoquinovosyldiacylglycerol Are Absent from a Chlamydomonas Reinhardtii Mutant Deleted in SQD1

Plant Physiology. Oct, 2003  |  Pubmed ID: 14500794

The biosynthesis of thylakoid lipids in eukaryotic photosynthetic organisms often involves enzymes in the endoplasmic reticulum (ER) and the chloroplast envelopes. Two pathways of thylakoid lipid biosynthesis, the ER and the plastid pathways, are present in parallel in many species, including Arabidopsis, but in other plants, e.g. grasses, only the ER pathway is active. The unicellular alga Chlamydomonas reinhardtii diverges from plants like Arabidopsis in a different way because its membranes do not contain phosphatidylcholine, and most thylakoid lipids are derived from the plastid pathway. Here, we describe an acylated derivative of sulfolipid, 2'-O-acyl-sulfoquinovosyldiacylglycerol (ASQD), which is present in C. reinhardtii. Although the fatty acids of sulfoquinovosyldiacylglycerol (SQDG) were mostly saturated, ASQD molecular species carried predominantly unsaturated fatty acids. Moreover, directly attached to the head group of ASQD was preferentially an 18-carbon fatty acid with four double bonds. High-throughput robotic screening led to the isolation of a plasmid disruption mutant of C. reinhardtii, designated Deltasqd1, which lacks ASQD as well as SQDG. In this mutant, the SQD1 ortholog was completely deleted and replaced by plasmid sequences. It is proposed that ASQD arises from the sugar nucleotide pathway of sulfolipid biosynthesis by acylation of the 2'-hydroxyl of the sulfoquinovosyl head group. At the physiological level, the mutant showed increased sensitivity to a diuron herbicide and reduced growth under phosphate limitation, suggesting a role for SQDG and/or ASQD in photosynthesis as conducted by C. reinhardtii, particularly under phosphate-limited conditions.

Anionic Lipids Are Required for Chloroplast Structure and Function in Arabidopsis

The Plant Journal : for Cell and Molecular Biology. Dec, 2003  |  Pubmed ID: 14675442

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.

Loss of Plastidic Lysophosphatidic Acid Acyltransferase Causes Embryo-lethality in Arabidopsis

Plant & Cell Physiology. May, 2004  |  Pubmed ID: 15169931

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.

Genetic Mutant Screening by Direct Metabolite Analysis

Analytical Biochemistry. Sep, 2004  |  Pubmed ID: 15301943

Arabidopsis As a Genetic Model for Interorganelle Lipid Trafficking

Genetic Engineering. 2004  |  Pubmed ID: 15387289

WRINKLED1 Encodes an AP2/EREB Domain Protein Involved in the Control of Storage Compound Biosynthesis in Arabidopsis

The Plant Journal : for Cell and Molecular Biology. Nov, 2004  |  Pubmed ID: 15500472

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.

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

Plant Molecular Biology. May, 2004  |  Pubmed ID: 15604662

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.

Genome-wide Analysis of Glucose-6-phosphate Dehydrogenases in Arabidopsis

The Plant Journal : for Cell and Molecular Biology. Jan, 2005  |  Pubmed ID: 15634201

In green tissues of plants under illumination, photosynthesis is the primary source of reduced nicotinamide adenine dinucleotide phosphate (NADPH), which is utilized in reductive reactions such as carbon fixation and nitrogen assimilation. In non-photosynthetic tissues or under non-photosynthetic conditions, the oxidative pentose phosphate pathway contributes to basic metabolism as one of the major sources of NADPH. The first and committed reaction is catalyzed by glucose-6-phosphate dehydrogenase (G6PDH). We characterized the six members of the G6PDH gene family in Arabidopsis. Transit peptide analysis predicted two cytosolic and four plastidic isoforms. Five of the six genes encode active G6PDHs. The recombinant isoforms showed differences in substrate requirements and sensitivities to feedback inhibition. Plastidic isoforms were redox sensitive. One cytosolic isoform was insensitive to redox changes, while the other was inactivated by oxidation. The respective genes had distinct expression patterns that did not correlate with the activity of the proteins, implying a regulatory mechanism beyond the control of mRNA abundance. Two cytosolic and one plastidic isoform were detected in vivo using zymograms, and the respective genes were identified using T-DNA insertion lines. The activity of a plastidic isoform was detected in all tissues including photosynthetic tissues despite its sensitivity to reduction observed in vitro. Genomic data, gene expression, and in vivo enzyme activity data were integrated with in vitro biochemical data to propose in vivo roles for individual G6PDH isoforms in Arabidopsis.

Annotation of Genes Involved in Glycerolipid Biosynthesis in Chlamydomonas Reinhardtii: Discovery of the Betaine Lipid Synthase BTA1Cr

Eukaryotic Cell. Feb, 2005  |  Pubmed ID: 15701786

Lipid metabolism in flowering plants has been intensely studied, and knowledge regarding the identities of genes encoding components of the major fatty acid and membrane lipid biosynthetic pathways is very extensive. We now present an in silico analysis of fatty acid and glycerolipid metabolism in an algal model, enabled by the recent availability of expressed sequence tag and genomic sequences of Chlamydomonas reinhardtii. Genes encoding proteins involved in membrane biogenesis were predicted on the basis of similarity to proteins with confirmed functions and were organized so as to reconstruct the major pathways of glycerolipid synthesis in Chlamydomonas. This analysis accounts for the majority of genes predicted to encode enzymes involved in anabolic reactions of membrane lipid biosynthesis and compares and contrasts these pathways in Chlamydomonas and flowering plants. As an important result of the bioinformatics analysis, we identified and isolated the C. reinhardtii BTA1 (BTA1Cr) gene and analyzed the bifunctional protein that it encodes; we predicted this protein to be sufficient for the synthesis of the betaine lipid diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), a major membrane component in Chlamydomonas. Heterologous expression of BTA1Cr led to DGTS accumulation in Escherichia coli, which normally lacks this lipid, and allowed in vitro analysis of the enzymatic properties of BTA1Cr. In contrast, in the bacterium Rhodobacter sphaeroides, two separate proteins, BtaARs and BtaBRs, are required for the biosynthesis of DGTS. Site-directed mutagenesis of the active sites of the two domains of BTA1Cr allowed us to study their activities separately, demonstrating directly their functional homology to the bacterial orthologs BtaARs and BtaBRs.

Comparative Genomics of Two Closely Related Unicellular Thermo-acidophilic Red Algae, Galdieria Sulphuraria and Cyanidioschyzon Merolae, Reveals the Molecular Basis of the Metabolic Flexibility of Galdieria Sulphuraria and Significant Differences in Carbohydrate Metabolism of Both Algae

Plant Physiology. Feb, 2005  |  Pubmed ID: 15710685

Unicellular algae serve as models for the study and discovery of metabolic pathways, for the functional dissection of cell biological processes such as organellar division and cell motility, and for the identification of novel genes and gene functions. The recent completion of several algal genome sequences and expressed sequence tag collections and the establishment of nuclear and organellar transformation methods has opened the way for functional genomics approaches using algal model systems. The thermo-acidophilic unicellular red alga Galdieria sulphuraria represents a particularly interesting species for a genomics approach owing to its extraordinary metabolic versatility such as heterotrophic and mixotrophic growth on more than 50 different carbon sources and its adaptation to hot acidic environments. However, the ab initio prediction of genes required for unknown metabolic pathways from genome sequences is not trivial. A compelling strategy for gene identification is the comparison of similarly sized genomes of related organisms with different physiologies. Using this approach, candidate genes were identified that are critical to the metabolic versatility of Galdieria. Expressed sequence tags and high-throughput genomic sequence reads covering >70% of the G. sulphuraria genome were compared to the genome of the unicellular, obligate photoautotrophic red alga Cyanidioschyzon merolae. More than 30% of the Galdieria sequences did not relate to any of the Cyanidioschyzon genes. A closer inspection of these sequences revealed a large number of membrane transporters and enzymes of carbohydrate metabolism that are unique to Galdieria. Based on these data, it is proposed that genes involved in the uptake of reduced carbon compounds and enzymes involved in their metabolism are crucial to the metabolic flexibility of G. sulphuraria.

Ferredoxin-dependent Glutamate Synthase Moonlights in Plant Sulfolipid Biosynthesis by Forming a Complex with SQD1

Archives of Biochemistry and Biophysics. Apr, 2005  |  Pubmed ID: 15752726

UDP-sulfoquinovose synthase, SQD1, catalyzes the transfer of sulfite to UDP-glucose giving rise to UDP-sulfoquinovose, which is the head group donor for the biosynthesis of the plant sulfolipid sulfoquinovosyldiacylglyerol. The native SQD1 enzyme of spinach exists as a 250 kDa heteroprotein complex with much higher affinity for the substrate sulfite than the recombinant SQD1 protein itself. The SQD1 protein co-purified with nine proteins. Likely binding partners included rubisco activase, HSP70, and ferredoxin-dependent glutamate synthase (FdGOGAT). While the first two proteins are known to interact with many other proteins, the identification of FdGOGAT was most intriguing because this 160kDa protein contains an FMN cofactor known to bind sulfite in vitro. Using different constructs expressing recombinant forms of the multidomain protein FdGOGAT, it was demonstrated that the FMN-binding domain of FdGOGAT is essential for specific binding of the protein to SQD1. A model suggests that FdGOGAT could channel sulfite to SQD1.

Two Enzymes, BtaA and BtaB, Are Sufficient for Betaine Lipid Biosynthesis in Bacteria

Archives of Biochemistry and Biophysics. Sep, 2005  |  Pubmed ID: 16095555

Betaine lipids are non-phosphorous glycerolipid analogs of phosphatidylcholine. The biosynthesis of the betaine lipid diacylglyceryl-N,N,N-trimethylhomoserine has previously been studied in phosphate-starved cells of the purple bacterium Rhodobacter sphaeroides, and a genetic approach identified two proteins that are necessary for this process. Here, we show that all reactions of DGTS biosynthesis in R. sphaeroides are attributable to RsBtaA and RsBtaB, as co-expression of the respective genes leads to DGTS formation in Escherichia coli, which normally lacks this lipid. The recombinant RsBtaA protein was membrane-associated and showed S-adenosylmethionine/diacylglycerol 3-amino-3-carboxypropyl transferase activity. RsBtaA directed the transfer of label from 1-[(14)C]S-adenosylmethionine or [(14)C]diacylglycerol at equal rates into the betaine lipid precursor diacylglycerylhomoserine identifying both metabolites as the substrates of the reaction. Comparative analysis of RsBtaA and its bacterial orthologs revealed a motif with similarity to the AdoMet binding pocket of methyltransferases, and allowed the prediction of residues involved in substrate binding.

Mutation of the TGD1 Chloroplast Envelope Protein Affects Phosphatidate Metabolism in Arabidopsis

The Plant Cell. Nov, 2005  |  Pubmed ID: 16199613

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.

Three Enzyme Systems for Galactoglycerolipid Biosynthesis Are Coordinately Regulated in Plants

The Journal of Biological Chemistry. Jan, 2005  |  Pubmed ID: 15590685

Galactoglycerolipids, in which galactose is bound at the glycerol sn-3 position in O-glycosidic linkage to diacylglycerol, are abundant in plants and photosynthetic bacteria, where they constitute the bulk of the polar lipids of the photosynthetic membranes. Galactoglycerolipid biosynthesis in plants is highly compartmentalized involving enzymes at the endoplasmic reticulum and the two chloroplast envelopes. This peculiar organization requires extensive trafficking of lipid precursors. It is now increasingly apparent that there are three different sets of lipid galactosyltransferases capable of galactoglycerolipid biosynthesis in the model plant Arabidopsis. Two enzymes, MGD1 and DGD1, provide the bulk of galactoglycerolipids in the chloroplast and in photosynthetic tissues in general. Under phosphate-limited growth conditions and in non-photosynthetic tissues MGD2/3 and DGD2 are highly active. Moreover, galactoglycerolipids produced by this second pathway are often found in extraplastidic membranes. Although these galactosyltransferases use UDP-Gal as the galactose donor, a third pathway involves a processive enzyme, which transfers galactose from one galactolipid to another.

Non-vesicular and Vesicular Lipid Trafficking Involving Plastids

Current Opinion in Plant Biology. Jun, 2006  |  Pubmed ID: 16603410

In plants, newly synthesized fatty acids are either directly incorporated into glycerolipids in the plastid or exported and assembled into lipids at the endoplasmic reticulum (ER). ER-derived glycerolipids serve as building blocks for extraplastidic membranes. Alternatively, they can return to the plastid where their diacylglycerol backbone is incorporated into the glycerolipids of the photosynthetic membranes, the thylakoids. Thylakoid lipids are assembled at the plastid envelope membranes and are transferred to the thylakoids. Under phosphate-limited growth conditions, galactolipids are exported from the outer plastid envelope membranes to extraplastidic membranes. Proteins, such as TRIGALACTOSYLDIACYLGLYCEROL1 (TGD1) or VESICLE-INDUCING PROTEIN IN PLASTIDS1 (VIPP1), which are involved in different aspects of plastid lipid trafficking phenomena have recently been identified and mechanistic models that are based on the analysis of these components have begun to emerge.

WRI1 is Required for Seed Germination and Seedling Establishment

Plant Physiology. Jun, 2006  |  Pubmed ID: 16632590

Storage compound accumulation during seed development prepares the next generation of plants for survival. Therefore, processes involved in the regulation and synthesis of storage compound accumulation during seed development bear relevance to germination and seedling establishment. The wrinkled1 (wri1) mutant of Arabidopsis (Arabidopsis thaliana) is impaired in seed oil accumulation. The WRI1 gene encodes an APETALA2/ethylene-responsive element-binding protein transcription factor involved in the control of metabolism, particularly glycolysis, in the developing seeds. Here we investigate the role of this regulatory factor in seed germination and seedling establishment by comparing the wri1-1 mutant, transgenic lines expressing the WRI1 wild-type cDNA in the wri1-1 mutant background, and the wild type. Plants altered in the expression of the WRI1 gene showed different germination responses to the growth factor abscisic acid (ABA), sugars, and fatty acids provided in the medium. Germination of the mutant was more sensitive to ABA, sugars, and osmolites, an effect that was alleviated by increased WRI1 expression in transgenic lines. The expression of ABA-responsive genes AtEM6 and ABA-insensitive 3 (ABI3) was increased in the wri1-1 mutant. Double-mutant analysis between abi3-3 and wri1-1 suggested that WRI1 and ABI3, a transcription factor mediating ABA responses in seeds, act in parallel pathways. Addition of 2-deoxyglucose inhibited seed germination, but did so less in lines overexpressing WRI1. Seedling establishment was decreased in the wri1-1 mutant but could be alleviated by sucrose. Apart from a possible signaling role in germination, sugars in the medium were required as building blocks and energy supply during wri1-1 seedling establishment.

Phosphatidylglycerol Biosynthesis in Chloroplasts of Arabidopsis Mutants Deficient in Acyl-ACP Glycerol-3- Phosphate Acyltransferase

The Plant Journal : for Cell and Molecular Biology. Jul, 2006  |  Pubmed ID: 16774646

The biosynthesis of phosphatidylglycerol represents a central pathway in lipid metabolism in all organisms. The enzyme catalyzing the first reaction of the pathway in the plastid, glycerol-3-phosphate acyl-acyl carrier protein acyltransferase, is thought to be encoded in Arabidopsis by the ATS1 locus. A number of genetic mutants deficient in this activity have been described. However, the corresponding mutant alleles have not yet been analyzed at the molecular level and a causal relationship between the mutant phenotypes and a deficiency at the ATS1 locus has not been established. The presence in all known ats1 mutants of near wild-type amounts of phosphatidylglycerol raised the question of whether an alternative pathway of phosphatidylglycerol assembly in the plastid exists. However, detailed analysis of several independent ats1 mutant alleles revealed that all are leaky. Reduction by RNAi of ats1-1 RNA levels in the ats1-1 mutant background led to a more severe growth phenotype (small green plants and reduced seed set), but did not decrease the relative amount of phosphatidylglycerol. In contrast, when the amount of ATS2 mRNA encoding the plastidic lysophosphatidic acid acyltransferase catalyzing the second reaction of the pathway was reduced by RNAi in the ats1-1 mutant background, phosphatidylglycerol amounts decreased, leading to a growth phenotype (small pale-yellow plants) that is reminiscent of the pgp1-1 mutant deficient in a late step of plastidic phosphatidylglycerol biosynthesis. These observations indicate coordinated regulation of plastid lipid metabolism and plant development.

A Phosphatidic Acid-binding Protein of the Chloroplast Inner Envelope Membrane Involved in Lipid Trafficking

Proceedings of the National Academy of Sciences of the United States of America. Jul, 2006  |  Pubmed ID: 16818883

The biogenesis of the photosynthetic thylakoid membranes inside plant chloroplasts requires enzymes at the plastid envelope and the endoplasmic reticulum (ER). Extensive lipid trafficking is required for thylakoid lipid biosynthesis. Here the trigalactosyldiacylglycerol2 (tgd2) mutant of Arabidopsis is described. To the extent tested, tgd2 showed a complex lipid phenotype identical to the previously described tgd1 mutant. The aberrant accumulation of oligogalactolipids and triacylglycerols and the reduction of molecular species of galactolipids derived from the ER are consistent with a disruption of the import of ER-derived lipids into the plastid. The TGD1 protein is a permease-like component of an ABC transporter located in the chloroplast inner envelope membrane. The TGD2 gene encodes a phosphatidic acid-binding protein with a predicted mycobacterial cell entry domain. It is tethered to the inner chloroplast envelope membrane facing the outer envelope membrane. Presumed bacterial orthologs of TGD1 and TGD2 in Gram-negative bacteria are typically organized in transcriptional units, suggesting their involvement in a common biological process. Expression of the tgd2-1 mutant cDNA caused a dominant-negative effect replicating the tgd2 mutant phenotype. This result is interpreted as the interference of the mutant protein with its native protein complex. It is proposed that TGD2 represents the substrate-binding or regulatory component of a phosphatidic acid/lipid transport complex in the chloroplast inner envelope membrane.

Questions Remaining in Sulfolipid Biosynthesis: a Historical Perspective

Photosynthesis Research. May, 2007  |  Pubmed ID: 17334828

The plant sulfolipid sulfoquinovosyldiacylglycerol was discovered by A.A. Benson in the late 1950s. The increasing availability of radioisotope-containing biological substrates such as (35)S-sulfate provided the means to discover novel biological compounds and to sketch out their biosynthetic pathways. During this time the structure of sulfolipid with its 6-deoxy-6-sulfo-alpha-D: -glucose (sulfoquinovose) headgroup was determined. Immediately, the origin of this unusual biological sulfonic acid mystified the scientific community and several proposals for its biosynthesis were developed and tested. Strong supportive evidence for the nucleotide pathway of sulfolipid biosynthesis became available with the discovery of the bacterial and plant genes encoding the enzymes of sulfolipid biosynthesis during the 1990s. This latter work was based on the foundations laid by A.A. Benson and confirmed one initial hypothesis on sulfolipid biosynthesis. An abbreviated summary of the turning points in defining the mechanism for sulfolipid biosynthesis and remaining issues in sulfolipid biochemistry are provided.

A Heteromeric Plastidic Pyruvate Kinase Complex Involved in Seed Oil Biosynthesis in Arabidopsis

The Plant Cell. Jun, 2007  |  Pubmed ID: 17557808

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.

Digalactosyldiacylglycerol is Required for Better Photosynthetic Growth of Synechocystis Sp. PCC6803 Under Phosphate Limitation

Plant & Cell Physiology. Nov, 2007  |  Pubmed ID: 17932115

Digalactosyldiacylglycerol (DGDG) is a typical membrane lipid of oxygenic photosynthetic organisms. Although DGDG synthase genes have been isolated from plants, no homologous gene has been annotated in the genomes of cyanobacteria and the unicellular red alga Cyanidioschyzon merolae. Here we used a comparative genomics approach and identified a non-plant-type DGDG synthase gene (designated dgdA) in Synechocystis sp. PCC6803. The enzyme produced DGDG in Escherichia coli when co-expressed with a cucumber monogalactosyldiacylglycerol synthase. A DeltadgdA knock-out mutant showed no obvious phenotype other than loss of DGDG when grown in a BG11 medium, indicating that DGDG is dispensable under optimal conditions. However, the mutant showed reduced growth under phosphate-limited conditions, suggesting that DGDG may be required under phosphate-limited conditions, such as those in natural niches of cyanobacteria.

A Small ATPase Protein of Arabidopsis, TGD3, Involved in Chloroplast Lipid Import

The Journal of Biological Chemistry. Dec, 2007  |  Pubmed ID: 17938172

Polar lipid trafficking is essential in eukaryotic cells as membranes of lipid assembly are often distinct from final destination membranes. A striking example is the biogenesis of the photosynthetic membranes (thylakoids) in plastids of plants. Lipid biosynthetic enzymes at the endoplasmic reticulum and the inner and outer plastid envelope membranes are involved. This compartmentalization requires extensive lipid trafficking. Mutants of Arabidopsis are available that are disrupted in the incorporation of endoplasmic reticulum-derived lipid precursors into thylakoid lipids. Two proteins affected in two of these mutants, trigalactosyldiacylglycerol 1 (TGD1) and TGD2, encode the permease and substrate binding component, respectively, of a proposed lipid translocator at the inner chloroplast envelope membrane. Here we describe a third protein of Arabidopsis, TGD3, a small ATPase proposed to be part of this translocator. As in the tgd1 and tgd2 mutants, triacylglycerols and trigalactolipids accumulate in a tgd3 mutant carrying a T-DNA insertion just 5' of the TGD3 coding region. The TGD3 protein shows basal ATPase activity and is localized inside the chloroplast beyond the inner chloroplast envelope membrane. Proteins orthologous to TGD1, -2, and -3 are predicted to be present in Gram- bacteria, and the respective genes are organized in operons suggesting a common biochemical role for the gene products. Based on the current analysis, it is hypothesized that TGD3 is the missing ATPase component of a lipid transporter involving TGD1 and TGD2 required for the biosynthesis of ER-derived thylakoid lipids in Arabidopsis.

Arabidopsis Seedlings Deficient in a Plastidic Pyruvate Kinase Are Unable to Utilize Seed Storage Compounds for Germination and Establishment

Plant Physiology. Dec, 2007  |  Pubmed ID: 17965177

Catabolism of storage reserves and biosynthesis of metabolites necessary for growth are essential for seed germination and establishment. An Arabidopsis (Arabidopsis thaliana) mutant (pkp1) deficient in plastidic pyruvate kinase (PK(p)) and unable to accumulate storage oil to the same extent as the wild type shows delayed germination and seedling establishment dependent on an exogenous sugar supply. It appears, however, as though these phenotypes are not entirely caused specifically by lack of seed oil and may be related to reduced PK(p) activity in germinating seeds. Increasing the sucrose concentration in the medium further inhibits germination of pkp1, possibly due to the accumulation of soluble sugars in seeds. Germinating seeds of pkp1 are unable to metabolize storage oil and cannot utilize applied sucrose for hypocotyl elongation in the dark. Moreover, pkp1 contains less tocopherol and chlorophyll than the wild type. Taken together, the results are consistent with a model in which PK(p) is required for the efficient conversion of sugar into precursors for different anabolic pathways.

Functional Analyses of Cytosolic Glucose-6-phosphate Dehydrogenases and Their Contribution to Seed Oil Accumulation in Arabidopsis

Plant Physiology. Jan, 2008  |  Pubmed ID: 17993547

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.

Mutation of a Mitochondrial Outer Membrane Protein Affects Chloroplast Lipid Biosynthesis

The Plant Journal : for Cell and Molecular Biology. Apr, 2008  |  Pubmed ID: 18208519

Lipid biosynthesis in plant cells is associated with various organelles, and maintenance of cell lipid homeostasis requires nimble regulation and coordination. In plants, environmental cues such as phosphate limitation require readjustment of the lipid biosynthetic machinery to substitute phospholipids by non-phosphorous glycolipids. Biosynthesis of the galactoglycerolipids predominant in plants proceeds by a constitutive and an alternative pathway that is known to be induced in response to phosphate deprivation. Plant lipid galactosyltransferases involved in both pathways are associated with the plastid envelope membranes and are encoded by nuclear genes. To identify mechanisms governing the activity of the alternative galactoglycerolipid pathway, a genetic suppressor screen was conducted in the background of the digalactolipid-deficient dgd1 mutant of Arabidopsis. A suppressor line that partially restored digalactoglycerolipid content in the dgd1 background carries a point mutation in a mitochondrial protein, which was tentatively designated DGD1 SUPPRESSOR 1 (DGS1). Presumed orthologs of this protein are present in plants, algae and fungi, but its molecular function is not yet known. In the dgd1 dgs1 double mutant, expression of nuclear genes encoding enzymes of the alternative galactoglycerolipid pathway is increased and hydrogen peroxide levels are elevated. This increase in hydrogen peroxide is proposed to be the reason for activation of the alternative pathway in the dgd1 dgs1 double mutant. Accordingly, hydrogen peroxide and treatments producing reactive oxygen also activate the alternative pathway in the wild-type. These results likely implicate the production of reactive oxygen in the regulation of the alternative galactoglycerolipid pathway in plants.

New Connections Across Pathways and Cellular Processes: Industrialized Mutant Screening Reveals Novel Associations Between Diverse Phenotypes in Arabidopsis

Plant Physiology. Apr, 2008  |  Pubmed ID: 18263779

In traditional mutant screening approaches, genetic variants are tested for one or a small number of phenotypes. Once bona fide variants are identified, they are typically subjected to a limited number of secondary phenotypic screens. Although this approach is excellent at finding genes involved in specific biological processes, the lack of wide and systematic interrogation of phenotype limits the ability to detect broader syndromes and connections between genes and phenotypes. It could also prevent detection of the primary phenotype of a mutant. As part of a systems biology approach to understand plastid function, large numbers of Arabidopsis thaliana homozygous T-DNA lines are being screened with parallel morphological, physiological, and chemical phenotypic assays (www.plastid.msu.edu). To refine our approaches and validate the use of this high-throughput screening approach for understanding gene function and functional networks, approximately 100 wild-type plants and 13 known mutants representing a variety of phenotypes were analyzed by a broad range of assays including metabolite profiling, morphological analysis, and chlorophyll fluorescence kinetics. Data analysis using a variety of statistical approaches showed that such industrial approaches can reliably identify plant mutant phenotypes. More significantly, the study uncovered previously unreported phenotypes for these well-characterized mutants and unexpected associations between different physiological processes, demonstrating that this approach has strong advantages over traditional mutant screening approaches. Analysis of wild-type plants revealed hundreds of statistically robust phenotypic correlations, including metabolites that are not known to share direct biosynthetic origins, raising the possibility that these metabolic pathways have closer relationships than is commonly suspected.

A Role for Lipid Trafficking in Chloroplast Biogenesis

Progress in Lipid Research. Sep, 2008  |  Pubmed ID: 18440317

Chloroplasts are the defining plant organelle carrying out photosynthesis. Photosynthetic complexes are embedded into the thylakoid membrane which forms an intricate system of membrane lamellae and cisternae. The chloroplast boundary consists of two envelope membranes controlling the exchange of metabolites between the plastid and the extraplastidic compartments of the cell. The plastid internal matrix (stroma) is the primary location for fatty acid biosynthesis in plants. Fatty acids can be assembled into glycerolipids at the envelope membranes of plastids or they can be exported and assembled into lipids at the endoplasmic reticulum (ER) to provide building blocks for extraplastidic membranes. Some of these glycerolipids, assembled at the ER, return to the plastid where they are remodeled into the plastid typical glycerolipids. As a result of this cooperation of different subcellular membrane systems, a rich complement of lipid trafficking phenomena contributes to the biogenesis of chloroplasts. Considerable progress has been made in recent years towards a better mechanistic understanding of lipid transport across plastid envelopes. Lipid transporters of bacteria and plants have been discovered and their study begins to provide detailed mechanistic insights into lipid trafficking phenomena relevant to chloroplast biogenesis.

Harnessing Plant Biomass for Biofuels and Biomaterials

The Plant Journal : for Cell and Molecular Biology. May, 2008  |  Pubmed ID: 18476860

Plant Triacylglycerols As Feedstocks for the Production of Biofuels

The Plant Journal : for Cell and Molecular Biology. May, 2008  |  Pubmed ID: 18476866

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.

Lipid Trafficking Between the Endoplasmic Reticulum and the Plastid in Arabidopsis Requires the Extraplastidic TGD4 Protein

The Plant Cell. Aug, 2008  |  Pubmed ID: 18689504

The development of chloroplasts in Arabidopsis thaliana requires extensive lipid trafficking between the endoplasmic reticulum (ER) and the plastid. The biosynthetic enzymes for the final steps of chloroplast lipid assembly are associated with the plastid envelope membranes. For example, during biosynthesis of the galactoglycerolipids predominant in photosynthetic membranes, galactosyltransferases associated with these membranes transfer galactosyl residues from UDP-Gal to diacylglycerol. In Arabidopsis, diacylglycerol can be derived from the ER or the plastid. Here, we describe a mutant of Arabidopsis, trigalactosyldiacylglycerol4 (tgd4), in which ER-derived diacylglycerol is not available for galactoglycerolipid biosynthesis. This mutant accumulates diagnostic oligogalactoglycerolipids, hence its name, and triacylglycerol in its tissues. The TGD4 gene encodes a protein that appears to be associated with the ER membranes. Mutant ER microsomes show a decreased transfer of lipids to isolated plastids consistent with in vivo labeling data, indicating a disruption of ER-to-plastid lipid transfer. The complex lipid phenotype of the mutant is similar to that of the tgd1,2,3 mutants disrupted in components of a lipid transporter of the inner plastid envelope membrane. However, unlike the TGD1,2,3 complex, which is proposed to transfer phosphatidic acid through the inner envelope membrane, TGD4 appears to be part of the machinery mediating lipid transfer between the ER and the outer plastid envelope membrane. The extent of direct ER-to-plastid envelope contact sites is not altered in the tgd4 mutant. However, this does not preclude a possible function of TGD4 in those contact sites as a conduit for lipid transfer between the ER and the plastid.

A Membrane-tethered Transcription Factor Defines a Branch of the Heat Stress Response in Arabidopsis Thaliana

Proceedings of the National Academy of Sciences of the United States of America. Oct, 2008  |  Pubmed ID: 18849477

In plants, heat stress responses are controlled by heat stress transcription factors that are conserved among all eukaryotes and can be constitutively expressed or induced by heat. Heat-inducible transcription factors that are distinct from the "classical" heat stress transcription factors have also been reported to contribute to heat tolerance. Here, we show that bZIP28, a gene encoding a putative membrane-tethered transcription factor, is up-regulated in response to heat and that a bZIP28 null mutant has a striking heat-sensitive phenotype. The heat-inducible expression of genes that encode BiP2, an endoplasmic reticulum (ER) chaperone, and HSP26.5-P, a small heat shock protein, is attenuated in the bZIP28 null mutant. An estradiol-inducible bZIP28 transgene induces a variety of heat and ER stress-inducible genes. Moreover, heat stress appears to induce the proteolytic release of the predicted transcription factor domain of bZIP28 from the ER membrane, thereby causing its redistribution to the nucleus. These findings indicate that bZIP28 is an essential component of a membrane-tethered transcription factor-based signaling pathway that contributes to heat tolerance.

ENDOSPERM DEFECTIVE1 Is a Novel Microtubule-Associated Protein Essential for Seed Development in Arabidopsis

The Plant Cell. Jan, 2009  |  Pubmed ID: 19151224

Early endosperm development involves a series of rapid nuclear divisions in the absence of cytokinesis; thus, many endosperm mutants reveal genes whose functions are essential for mitosis. This work finds that the endosperm of Arabidopsis thaliana endosperm-defective1 (ede1) mutants never cellularizes, contains a reduced number of enlarged polyploid nuclei, and features an aberrant microtubule cytoskeleton, where the specialized radial microtubule systems and cytokinetic phragmoplasts are absent. Early embryo development is substantially normal, although occasional cytokinesis defects are observed. The EDE1 gene was cloned using a map-based approach and represents the pioneer member of a conserved plant-specific family of genes of previously unknown function. EDE1 is expressed in the endosperm and embryo of developing seeds, and its expression is tightly regulated during cell cycle progression. EDE1 protein accumulates in nuclear caps in premitotic cells, colocalizes along microtubules of the spindle and phragmoplast, and binds microtubules in vitro. We conclude that EDE1 is a novel plant-specific microtubule-associated protein essential for microtubule function during the mitotic and cytokinetic stages that generate the Arabidopsis endosperm and embryo.

A 25-amino Acid Sequence of the Arabidopsis TGD2 Protein is Sufficient for Specific Binding of Phosphatidic Acid

The Journal of Biological Chemistry. Jun, 2009  |  Pubmed ID: 19416982

Genetic analysis suggests that the TGD2 protein of Arabidopsis is required for the biosynthesis of endoplasmic reticulum derived thylakoid lipids. TGD2 is proposed to be the substrate-binding protein of a presumed lipid transporter consisting of the TGD1 (permease) and TGD3 (ATPase) proteins. The TGD1, -2, and -3 proteins are localized in the inner chloroplast envelope membrane. TGD2 appears to be anchored with an N-terminal membrane-spanning domain into the inner envelope membrane, whereas the C-terminal domain faces the intermembrane space. It was previously shown that the C-terminal domain of TGD2 binds phosphatidic acid (PtdOH). To investigate the PtdOH binding site of TGD2 in detail, the C-terminal domain of the TGD2 sequence lacking the transit peptide and transmembrane sequences was fused to the C terminus of the Discosoma sp. red fluorescent protein (DR). This greatly improved the solubility of the resulting DR-TGD2C fusion protein following production in Escherichia coli. The DR-TGD2C protein bound PtdOH with high specificity, as demonstrated by membrane lipid-protein overlay and liposome association assays. Internal deletion and truncation mutagenesis identified a previously undescribed minimal 25-amino acid fragment in the C-terminal domain of TGD2 that is sufficient for PtdOH binding. Binding characteristics of this 25-mer were distinctly different from those of TGD2C, suggesting that additional sequences of TGD2 providing the proper context for this 25-mer are needed for wild type-like PtdOH binding.

Mechanisms of Lipid Transport Involved in Organelle Biogenesis in Plant Cells

Annual Review of Cell and Developmental Biology. 2009  |  Pubmed ID: 19572810

Chloroplasts are the defining organelle of photoautotrophic plant cells. Photosynthetic light reactions and electron transport are the functions of an elaborate thylakoid membrane system inside chloroplasts. The lipid composition of photosynthetic membranes is characterized by a substantial fraction of nonphosphorous galactoglycerolipids reflecting the need of sessile plants to conserve phosphorus. Lipid transport and assembly of glycerolipids play an essential role in the biogenesis of the photosynthetic apparatus in developing chloroplasts. During chloroplast biogenesis, fatty acids are synthesized in the plastid and are exported to the endoplasmic reticulum, where they are incorporated into membrane lipids. Alternatively, lipids can also be assembled de novo at the inner envelope membrane of plastids in many plants. A rich repertoire of lipid exchange mechanisms involving the thylakoid membranes, the chloroplast inner and outer envelope membranes, and the endoplasmic reticulum is emerging. Studies of thylakoid biogenesis provide new insights into the general mechanisms of intermembrane lipid transfer.

FATTY ACID DESATURASE4 of Arabidopsis Encodes a Protein Distinct from Characterized Fatty Acid Desaturases

The Plant Journal : for Cell and Molecular Biology. Dec, 2009  |  Pubmed ID: 19682287

Polar membrane glycerolipids occur in a mixture of molecular species defined by a polar head group and characteristic acyl groups esterified to a glycerol backbone. A molecular species of phosphatidylglycerol specific to chloroplasts of plants carries a Delta(3-trans) hexadecenoic acid in the sn-2 position of its core glyceryl moiety. The fad4-1 mutant of Arabidopsis thaliana missing this particular phosphatidylglycerol molecular species lacks the necessary fatty acid desaturase, or a component thereof. The overwhelming majority of acyl groups associated with membrane lipids in plants contains double bonds with a cis configuration. However, FAD4 is unusual because it is involved in the formation of a trans double bond introduced close to the carboxyl group of palmitic acid, which is specifically esterified to the sn-2 glyceryl carbon of phosphatidylglycerol. As a first step towards the analysis of this unusual desaturase reaction, the FAD4 gene was identified by mapping of the FAD4 locus and coexpression analysis with known lipid genes. FAD4 encodes a predicted integral membrane protein that appears to be unrelated to classic membrane bound fatty acid desaturases based on overall sequence conservation. However, the FAD4 protein contains two histidine motifs resembling those of metalloproteins such as fatty acid desaturases. FAD4 is targeted to the plastid. Overexpression of the cDNA in transgenic Arabidopsis led to increased accumulation of the Delta(3-trans) hexadecanoyl group in phosphatidylglycerol relative to wild type. Taken together these results are consistent with the hypothesis that FAD4 is the founding member of a novel class of fatty acid desaturases.

RNA Interference Silencing of a Major Lipid Droplet Protein Affects Lipid Droplet Size in Chlamydomonas Reinhardtii

Eukaryotic Cell. Jan, 2010  |  Pubmed ID: 19915074

Eukaryotic cells store oils in the chemical form of triacylglycerols in distinct organelles, often called lipid droplets. These dynamic storage compartments have been intensely studied in the context of human health and also in plants as a source of vegetable oils for human consumption and for chemical or biofuel feedstocks. Many microalgae accumulate oils, particularly under conditions limiting to growth, and thus have gained renewed attention as a potentially sustainable feedstock for biofuel production. However, little is currently known at the cellular or molecular levels with regard to oil accumulation in microalgae, and the structural proteins and enzymes involved in the biogenesis, maintenance, and degradation of algal oil storage compartments are not well studied. Focusing on the model green alga Chlamydomonas reinhardtii, the accumulation of triacylglycerols and the formation of lipid droplets during nitrogen deprivation were investigated. Mass spectrometry identified 259 proteins in a lipid droplet-enriched fraction, among them a major protein, tentatively designated major lipid droplet protein (MLDP). This protein is specific to the green algal lineage of photosynthetic organisms. Repression of MLDP gene expression using an RNA interference approach led to increased lipid droplet size, but no change in triacylglycerol content or metabolism was observed.

The Area of Plant Biotechnology

The Plant Journal : for Cell and Molecular Biology. Jan, 2010  |  Pubmed ID: 20078843

Phosphate Regulation of Lipid Biosynthesis in Arabidopsis is Independent of the Mitochondrial Outer Membrane DGS1 Complex

Plant Physiology. Apr, 2010  |  Pubmed ID: 20181751

Galactoglycerolipids are major constituents of photosynthetic membranes in chloroplasts. At least three parallel sets of enzymes are involved in their biosynthesis that must be coordinated in response to changing growth conditions. A potential candidate for a protein affecting the activity of different galactoglycerolipid pathways is the recently described digalactosyldiacylglycerol1 (dgd1) SUPPRESSOR1 (DGS1) protein of Arabidopsis (Arabidopsis thaliana) localized in the mitochondrial outer membrane. It was discovered based on a specific gain-of-function point mutation allele, dgs1-1, that causes a partial restoration of chloroplast galactoglycerolipid deficiency in the dgd1 mutant, which is defective in the lipid galactosyltransferase, DGD1. The dgs1-1 allele causes the accumulation of hydrogen peroxide that leads to an activation of an alternative, DGD1-independent galactoglycerolipid biosynthesis pathway in chloroplasts. Analysis presented here shows that the DGS1 protein is a component of a large protein complex, which explains the previously observed dominant negative phenotype following the expression of the dgs1-1 allele. The dgs1-1 allele causes the loss of mitochondrial alternative oxidase (AOX) protein that might be related to the accumulation of hydrogen peroxide in the dgs1-1 mutant background. This effect was posttranscriptional because mRNA levels for the major form of AOX were not affected in dgs1-1 mutant seedlings. Unlike dgs1-1, a loss-of-function allele, dgs1-2, had no effect on plant growth, AOX, and lipid composition to the extent tested, leaving the quest for a possible molecular function of DGS1 open. Apparently, the DGS1 wild-type protein does not directly affect lipid metabolism in mitochondria or chloroplasts.

Arabidopsis: a Rich Harvest 10 Years After Completion of the Genome Sequence

The Plant Journal : for Cell and Molecular Biology. Mar, 2010  |  Pubmed ID: 20409265

Lipid Transport Mediated by Arabidopsis TGD Proteins is Unidirectional from the Endoplasmic Reticulum to the Plastid

Plant & Cell Physiology. Jun, 2010  |  Pubmed ID: 20410050

The transfer of lipids between the endoplasmic reticulum (ER) and the plastid in Arabidopsis involves the TRIGALACTOSYLDIACYLGLYCEROL (TGD) proteins. Lipid exchange is thought to be bidirectional based on the presence of specific lipid molecular species in Arabidopsis mutants impaired in the desaturation of fatty acids of membrane lipids in the ER and plastid. However, it was unclear whether TGD proteins were required for lipid trafficking in both directions. This question was addressed through the analysis of double mutants of tgd1-1 or tgd4-3 in genetic mutant backgrounds leading to a defect in lipid fatty acid desaturation either in the ER (fad2) or the plastid (fad6). The fad6 tgd1-1 and fad6 tgd4-3 double mutants showed drastic reductions in the relative levels of polyunsaturated fatty acids and of galactolipids. The growth of these plants and the development of photosynthetic membrane systems were severely compromised, suggesting a disruption in the import of polyunsaturated fatty acid-containing lipid species from the ER. Furthermore, a forward-genetic screen in the tgd1-2 dgd1 mutant background led to the isolation of a new fad6-2 allele with a marked reduction in the amount of digalactosyldiacylglycerol. In contrast, the introduction of fad2, affecting fatty acid desaturation of lipids in the ER, into the two tgd mutant backgrounds did not further decrease the level of fatty acid desaturation in lipids of extraplastidic membranes. These results suggest that the role of TGD proteins is limited to plastid lipid import, but does not extend to lipid export from the plastid to extraplastidic membranes.

Freezing Tolerance in Plants Requires Lipid Remodeling at the Outer Chloroplast Membrane

Science (New York, N.Y.). Oct, 2010  |  Pubmed ID: 20798281

Plants show complex adaptations to freezing that prevent cell damage caused by cellular dehydration. Lipid remodeling of cell membranes during dehydration is one critical mechanism countering loss of membrane integrity and cell death. SENSITIVE TO FREEZING 2 (SFR2), a gene essential for freezing tolerance in Arabidopsis, encodes a galactolipid remodeling enzyme of the outer chloroplast envelope membrane. SFR2 processively transfers galactosyl residues from the abundant monogalactolipid to different galactolipid acceptors, forming oligogalactolipids and diacylglycerol, which is further converted to triacylglycerol. The combined activity of SFR2 and triacylglycerol-biosynthetic enzymes leads to the removal of monogalactolipids from the envelope membrane, changing the ratio of bilayer- to non-bilayer-forming membrane lipids. This SFR2-based mechanism compensates for changes in organelle volume and stabilizes membranes during freezing.

Changes in Transcript Abundance in Chlamydomonas Reinhardtii Following Nitrogen Deprivation Predict Diversion of Metabolism

Plant Physiology. Dec, 2010  |  Pubmed ID: 20935180

Like many microalgae, Chlamydomonas reinhardtii forms lipid droplets rich in triacylglycerols when nutrient deprived. To begin studying the mechanisms underlying this process, nitrogen (N) deprivation was used to induce triacylglycerol accumulation and changes in developmental programs such as gametogenesis. Comparative global analysis of transcripts under induced and noninduced conditions was applied as a first approach to studying molecular changes that promote or accompany triacylglycerol accumulation in cells encountering a new nutrient environment. Towards this goal, high-throughput sequencing technology was employed to generate large numbers of expressed sequence tags of eight biologically independent libraries, four for each condition, N replete and N deprived, allowing a statistically sound comparison of expression levels under the two tested conditions. As expected, N deprivation activated a subset of control genes involved in gametogenesis while down-regulating protein biosynthesis. Genes for components of photosynthesis were also down-regulated, with the exception of the PSBS gene. N deprivation led to a marked redirection of metabolism: the primary carbon source, acetate, was no longer converted to cell building blocks by the glyoxylate cycle and gluconeogenesis but funneled directly into fatty acid biosynthesis. Additional fatty acids may be produced by membrane remodeling, a process that is suggested by the changes observed in transcript abundance of putative lipase genes. Inferences on metabolism based on transcriptional analysis are indirect, but biochemical experiments supported some of these deductions. The data provided here represent a rich source for the exploration of the mechanism of oil accumulation in microalgae.

Galactoglycerolipid Metabolism Under Stress: a Time for Remodeling

Trends in Plant Science. Feb, 2011  |  Pubmed ID: 21145779

Galactoglycerolipids are the predominant lipid building blocks of chloroplast membranes and are essential for plant growth. Plant chloroplasts harbor a constitutive set of UDP-Gal-dependent lipid galactosyltransferases that are responsible for the bulk of galactoglycerolipid biosynthesis. A set of paralogs is induced in response to phosphate deprivation, which leads to the remodeling of extraplastidic membranes with a partial replacement of phosphoglycerolipid by digalactosyldiacylglycerol. A third type of galactoglycerolipid biosynthetic enzyme, a UDP-Gal-independent galactoglycerolipid galactosyltransferase, was recently shown to be involved in freezing tolerance. Here, we look at how understanding of the regulation of galactoglycerolipid biosynthesis in chloroplasts by these multiple enzyme sets is rapidly evolving and discuss the increasingly recognized role of lipid remodeling in response to diverse abiotic stresses.

Arabidopsis Chloroplast Lipid Transport Protein TGD2 Disrupts Membranes and is Part of a Large Complex

The Plant Journal : for Cell and Molecular Biology. Jun, 2011  |  Pubmed ID: 21309871

In most plants the assembly of the photosynthetic thylakoid membrane requires lipid precursors synthesized at the endoplasmic reticulum (ER). Thus, the transport of lipids from the ER to the chloroplast is essential for biogenesis of the thylakoids. TGD2 is one of four proteins in Arabidopsis required for lipid import into the chloroplast, and was found to bind phosphatidic acid in vitro. However, the significance of phosphatidic acid binding for the function of TGD2 in vivo and TGD2 interaction with membranes remained unclear. Developing three functional assays probing how TGD2 affects lipid bilayers in vitro, we show that it perturbs membranes to the point of fusion, causes liposome leakage and redistributes lipids in the bilayer. By identifying and characterizing five new mutant alleles, we demonstrate that these functions are impaired in specific mutants with lipid phenotypes in vivo. At the structural level, we show that TGD2 is part of a protein complex larger than 500 kDa, the formation of which is disrupted in two mutant alleles, indicative of the biological relevance of this TGD2-containing complex. Based on the data presented, we propose that TGD2, as part of a larger complex, forms a lipid transport conduit between the inner and outer chloroplast envelope membranes, with its N terminus anchored in the inner membrane and its C terminus binding phosphatidic acid in the outer membrane.

Cardiolipin Deficiency in Rhodobacter Sphaeroides Alters the Lipid Profile of Membranes and of Crystallized Cytochrome Oxidase, but Structure and Function Are Maintained

Biochemistry. May, 2011  |  Pubmed ID: 21476578

Many recent studies highlight the importance of lipids in membrane proteins, including in the formation of well-ordered crystals. To examine the effect of changes in one lipid, cardiolipin, on the lipid profile and the production, function, and crystallization of an intrinsic membrane protein, cytochrome c oxidase, we mutated the cardiolipin synthase (cls) gene of Rhodobacter sphaeroides, causing a >90% reduction in cardiolipin content in vivo and selective changes in the abundances of other lipids. Under these conditions, a fully native cytochrome c oxidase (CcO) was produced, as indicated by its activity, spectral properties, and crystal characteristics. Analysis by MALDI tandem mass spectrometry (MS/MS) revealed that the cardiolipin level in CcO crystals, as in the membranes, was greatly decreased. Lipid species present in the crystals were directly analyzed for the first time using MS/MS, documenting their identities and fatty acid chain composition. The fatty acid content of cardiolipin in R. sphaeroides CcO (predominantly 18:1) differs from that in mammalian CcO (18:2). In contrast to the cardiolipin dependence of mammalian CcO activity, major depletion of cardiolipin in R. sphaeroides did not impact any aspect of CcO structure or behavior, suggesting a greater tolerance of interchange of cardiolipin with other lipids in this bacterial system.

Combined Genetic and Metabolic Manipulation of Lipids in Rhodobacter Sphaeroides Reveals Non-phospholipid Substitutions in Fully Active Cytochrome C Oxidase

Biochemistry. May, 2011  |  Pubmed ID: 21476580

A specific requirement for lipids, particularly cardiolipin (CL), in cytochrome c oxidase (CcO) has been reported in many previous studies using mainly in vitro lipid removal approaches in mammalian systems. Our accompanying paper shows that CcO produced in markedly CL-depleted Rhodobacter sphaeroides displays wild-type properties in all respects, likely allowed by quantitative substitution with other negatively charged lipids. To further examine the structural basis for the lipid requirements of R. sphaeroides CcO and the extent of interchangeability between lipids, we employed a metabolic approach to enhance the alteration of the lipid profiles of the CcO-expressing strains of R. sphaeroides in vivo using a phosphate-limiting growth medium in addition to the CL-deficient mutation. Strikingly, the purified CcO produced under these conditions still maintained wild-type function and characteristics, in spite of even greater depletion of cardiolipin compared to that of the CL-deficient mutant alone (undetectable by MS) and drastically altered profiles of all the phospholipids and non-phospholipids. The lipids in the membrane and in the purified CcO were identified and quantified by ESI and MALDI mass spectrometry and tandem mass spectrometry. Comparison between the molecular structures of those lipids that showed major changes provides new insight into the structural rationale for the flexible lipid requirements of CcO from R. sphaeroides and reveals a more comprehensive interchangeability network between different phospholipids and non-phospholipids.

Systems Biology Approach in Chlamydomonas Reveals Connections Between Copper Nutrition and Multiple Metabolic Steps

The Plant Cell. Apr, 2011  |  Pubmed ID: 21498682

In this work, we query the Chlamydomonas reinhardtii copper regulon at a whole-genome level. Our RNA-Seq data simulation and analysis pipeline validated a 2-fold cutoff and 10 RPKM (reads per kilobase of mappable length per million mapped reads) (~1 mRNA per cell) to reveal 63 CRR1 targets plus another 86 copper-responsive genes. Proteomic and immunoblot analyses captured 25% of the corresponding proteins, whose abundance was also dependent on copper nutrition, validating transcriptional regulation as a major control mechanism for copper signaling in Chlamydomonas. The impact of copper deficiency on the expression of several O₂-dependent enzymes included steps in lipid modification pathways. Quantitative lipid profiles indicated increased polyunsaturation of fatty acids on thylakoid membrane digalactosyldiglycerides, indicating a global impact of copper deficiency on the photosynthetic apparatus. Discovery of a putative plastid copper chaperone and a membrane protease in the thylakoid suggest a mechanism for blocking copper utilization in the chloroplast. We also found an example of copper sparing in the N assimilation pathway: the replacement of copper amine oxidase by a flavin-dependent backup enzyme. Forty percent of the targets are previously uncharacterized proteins, indicating considerable potential for new discovery in the biology of copper.

Increasing the Energy Density of Vegetative Tissues by Diverting Carbon from Starch to Oil Biosynthesis in Transgenic Arabidopsis

Plant Biotechnology Journal. Oct, 2011  |  Pubmed ID: 22003502

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.

A J-like Protein Influences Fatty Acid Composition of Chloroplast Lipids in Arabidopsis

PloS One. 2011  |  Pubmed ID: 22028775

A comprehensive understanding of the lipid and fatty acid metabolic machinery is needed for optimizing production of oils and fatty acids for fuel, industrial feedstocks and nutritional improvement in plants. T-DNA mutants in the poorly annotated Arabidopsis thaliana gene At1g08640 were identified as containing moderately high levels (50-100%) of 16∶1Δ7 and 18∶1Δ9 leaf fatty acids and subtle decreases (5-30%) of 16∶3 and 18∶3 (http://www.plastid.msu.edu/). TLC separation of fatty acids in the leaf polar lipids revealed that the chloroplastic galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) were the main lipid types affected by this mutation. Analysis of the inferred amino acid sequence of At1g08640 predicted the presence of a transit peptide, three transmembrane domains and an N-terminal J-like domain, and the gene was named CJD1 for Chloroplast J-like Domain 1. GFP reporter experiments and in vitro chloroplast import assays demonstrated CJD1 is a chloroplast membrane protein. Screening of an Arabidopsis cDNA library by yeast-2-hybrid (Y2H) using the J-like domain of CJD1 as bait identified a plastidial inner envelope protein (Accumulation and Replication of Chloroplasts 6, ARC6) as the primary interacting partner in the Y2H assay. ARC6 plays a central role in chloroplast division and binds CJD1 via its own J-like domain along with an adjacent conserved region whose function is not fully known. These results provide a starting point for future investigations of how mutations in CJD1 affect lipid composition.

The Future is Bright for The Plant Journal, Now in Its 20th Year

The Plant Journal : for Cell and Molecular Biology. Dec, 2011  |  Pubmed ID: 22150932

TGD4 Involved in Endoplasmic Reticulum-to-chloroplast Lipid Trafficking is a Phosphatidic Acid Binding Protein

The Plant Journal : for Cell and Molecular Biology. Jan, 2012  |  Pubmed ID: 22269056

The synthesis of galactoglycerolipids, which are prevalent in photosynthetic membranes, involves enzymes at the endoplasmic reticulum (ER) and the chloroplast envelope membranes. Genetic analysis of trigalactosyldiacylglycerol (TGD) proteins in Arabidopsis has demonstrated their role in polar lipid transfer from the ER to the chloroplast. The TGD1, 2, and 3 proteins resemble components of a bacterial-type ATP-binding cassette (ABC) transporter, with TGD1 representing the permease, TGD2 the substrate binding protein, and TGD3 the ATPase. However, the function of the TGD4 protein in this process is less clear and its location in plant cells remains to be firmly determined. The predicted C-terminal β-barrel structure of TGD4 is weakly similar to proteins of the outer cell membrane of Gram-negative bacteria. Here, we show that, like TGD2, the TGD4 protein when fused to DsRED specifically binds phosphatidic acid (PtdOH). As previously shown for tgd1 mutants, tgd4 mutants have elevated PtdOH content, probably in extraplastidic membranes. Using highly purified and specific antibodies to probe different cell fractions, we demonstrated that the TGD4 protein was present in the outer envelope membrane of chloroplasts, where it appeared to be deeply buried within the membrane except for the N-terminus, which was found to be exposed to the cytosol. It is proposed that TGD4 is either directly involved in the transfer of polar lipids, possibly PtdOH, from the ER to the outer chloroplast envelope membrane or in the transfer of PtdOH through the outer envelope membrane.

A Lipid Droplet Protein of Nannochloropsis with Functions Partially Analogous to Plant Oleosins

Plant Physiology. Feb, 2012  |  Pubmed ID: 22307965

As our understanding of the dynamics of lipid droplets (LDs) in animal, plant and fungal cells is rapidly evolving, still little is known about the formation and turnover of these organelles in microalgae. Yet with the growing importance of algal feedstocks for the production of biofuels and high value lipids, there is a need to understand the mechanisms of LD dynamics in microalgae. Thus, we investigated the proteins associated with LDs of the emerging heterokont model alga Nannochloropsis sp. and discovered an abundant hydrophobic Lipid Droplet Surface Protein (LDSP) with unique primary sequence, but structural similarities to other LD proteins. LDSP abundance in Nannochloropsis cells closely tracked the amount triacylglycerols during conditions of oil accumulation and degradation. Functional characterization of LDSP in an Arabidopsis thaliana OLEOSIN 1-deficient mutant allowed a separation of its physical and structural properties in its interaction with LDs from its physiological or biochemical activities. Although LDSP presence in Arabidopsis predictably affected LD size, it could not reverse the physiological impact of OLEOSIN deficiency on triacylglycerol hydrolysis during germination.

Analysis of Porphyra Membrane Transporters Demonstrates Gene Transfer Among Photosynthetic Eukaryotes and Numerous Sodium-coupled Transport Systems

Plant Physiology. Feb, 2012  |  Pubmed ID: 22337920

Membrane transporters play a central role in many cellular processes that rely on the movement of ions and organic molecules between the environment and the cell, and between cellular compartments. Transporters have been well characterized in plants and green algae, but little is known about transporters or their evolutionary histories in the red algae. Here we examined 482 expressed sequence tag (EST) contigs that encode putative membrane transporters in the economically important red seaweed Porphyra (Bangiophyceae, Rhodophyta). These contigs are part of a comprehensive transcriptome dataset from Porphyra umbilicalis and Porphyra purpurea. Using phylogenomics, we identified 30 trees that support the expected monophyly of red and green algae/plants (i.e., the Plantae hypothesis) and 19 EST contigs that show evidence of endosymbiotic/horizontal gene transfer involving stramenopiles. The majority (77%) of analyzed contigs encode transporters with unresolved phylogenies, demonstrating the difficulty in resolving the evolutionary history of genes. We observed molecular features of many sodium-coupled transport systems in marine algae, and the potential for co-regulation of Porphyra transporter genes that are associated with fatty acid biosynthesis and intracellular lipid trafficking. Although both the tissue-specific and subcellular locations of the encoded proteins require further investigation, our study provides red algal gene candidates associated with transport functions and novel insights into the biology and evolution of these transporters.