Glycerol-3-phosphate acyltransferase 4 is essential for the normal development of reproductive organs and the embryo in Brassica napus.

The enzyme sn-glycerol-3-phosphate acyltransferase 4 (GPAT4) is involved in the biosynthesis of plant lipid poly-esters. The present study further characterizes the enzymatic activities of three endoplasmic reticulum-bound GPAT4 isoforms of Brassica napus and examines their roles in the development of reproductive organs and the embryo. All three BnGPAT4 isoforms exhibited sn-2 acyltransferase and phosphatase activities with dicarboxylic acid-CoA as acyl donor. When non-substituted acyl-CoA was used as acyl donor, the rate of acylation was considerably lower and phosphatase activity was not manifested. RNA interference (RNAi)-mediated down-regulation of all GPAT4 homologues in B. napus under the control of the napin promoter caused abnormal development of several reproductive organs and reduced seed set. Microscopic examination and reciprocal crosses revealed that both pollen grains and developing embryo sacs of the B. napus gpat4 lines were affected. The gpat4 mature embryos showed decreased cutin content and altered monomer composition. The defective embryo development further affected the oil body morphology, oil content, and fatty acid composition in gpat4 seeds. These results suggest that GPAT4 has a critical role in the development of reproductive organs and the seed of B. napus.

Possible allostery and oligomerization of recombinant plastidial sn-glycerol-3-phosphate acyltransferase.

Plastidial acyl-acyl carrier protein:sn-glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) catalyzes the acyl-acyl carrier protein-dependent sn-1 acylation of sn-glycerol 3-phosphate (G3P) to produce lysophosphatic acid. Functional recombinant Erysimum asperum GPAT (EaGPAT), devoid of transit peptide, was produced in yeast. Analysis of the dependence of EaGPAT activity on increasing G3P concentration resulted in a hyperbolic response. EaGPAT exhibited a preference for 18-carbon unsaturated acyl-CoAs. Assays with concentrations of oleoyl-CoA up to 90µM revealed an exponential response to increasing concentrations of acyl donor, and the introduction of increasing concentrations of unlabeled linoleoyl-CoA into the standard reaction mixture resulted in increased incorporation of radiolabeled oleoyl moieties into lysophosphatidic acid. Collectively, the kinetic results suggest that acyl-CoA may act as both substrate and allosteric effector. EaGPAT was also shown to oligomerize to form higher molecular mass multimers, with the monomer and trimer being the predominant forms of the enzyme. Since most allosteric enzyme exhibit quaternary structure, the self-associating properties of EaGPAT are consistent with those of an allosteric enzyme. These results could have important regulatory implications when plastidial GPAT is introduced into a cytoplasmic environment where acyl-CoA is the acyl donor supporting cytoplasmic glycerolipid assembly.

Brassica napus TT16 homologs with different genomic origins and expression levels encode proteins that regulate a broad range of endothelium-associated genes at the transcriptional level.

The transcription factor TRANSPARENT TESTA 16 (TT16) plays an important role in endothelial cell specification and proanthocyanidin (PA) accumulation. However, its precise regulatory function with regard to the expression of endothelial-associated genes in developing seeds, and especially in the PA-producing inner integument, remains largely unknown. Therefore, we endeavored to characterize four TT16 homologs from the allotetraploid oil crop species Brassica napus, and systematically explore their regulatory function in endothelial development. Our results indicated that all four BnTT16 genes were predominantly expressed in the early stages of seed development, but at distinct levels, and encoded functional proteins. Bntt16 RNA interference lines exhibited abnormal endothelial development and decreased PA content, while PA polymerization was not affected. In addition to the previously reported function of TT16 in the transcriptional regulation of anthocyanidin reductase (ANR) and dihydroflavonol reductase (TT3), we also determined that BnTT16 proteins played a significant role in the transcriptional regulation of five other genes involved in the PA biosynthetic pathway (P < 0.01). Moreover, we identified two genes involved in inner integument development that were strongly regulated by the BnTT16 proteins (TT2 and δ-vacuolar processing enzyme). These results will better our understanding of the precise role of TT16 in endothelial development in Brassicaceae species, and could potentially be used for the future improvement of oilseed crops.

Transparent testa16 plays multiple roles in plant development and is involved in lipid synthesis and embryo development in canola.

Transparent Testa16 (TT16), a transcript regulator belonging to the B(sister) MADS box proteins, regulates proper endothelial differentiation and proanthocyanidin accumulation in the seed coat. Our understanding of its other physiological roles, however, is limited. In this study, the physiological and developmental roles of TT16 in an important oil crop, canola (Brassica napus), were dissected by a loss-of-function approach. RNA interference (RNAi)-mediated down-regulation of tt16 in canola caused dwarf phenotypes with a decrease in the number of inflorescences, flowers, siliques, and seeds. Fluorescence microscopy revealed that tt16 deficiency affects pollen tube guidance, resulting in reduced fertility and negatively impacting embryo and seed development. Moreover, Bntt16 RNAi plants had reduced oil content and altered fatty acid composition. Transmission electron microscopy showed that the seeds of the RNAi plants had fewer oil bodies than the nontransgenic plants. In addition, tt16 RNAi transgenic lines were more sensitive to auxin. Further analysis by microarray showed that tt16 down-regulation alters the expression of genes involved in gynoecium and embryo development, lipid metabolism, auxin transport, and signal transduction. The broad regulatory function of TT16 at the transcriptional level may explain the altered phenotypes observed in the transgenic lines. Overall, the results uncovered important biological roles of TT16 in plant development, especially in fatty acid synthesis and embryo development.

Fatty acid composition of developing sea buckthorn (Hippophae rhamnoides L.) berry and the transcriptome of the mature seed.

BACKGROUND: Sea buckthorn (Hippophae rhamnoides L.) is a hardy, fruit-producing plant known historically for its medicinal and nutraceutical properties. The most recognized product of sea buckthorn is its fruit oil, composed of seed oil that is rich in essential fatty acids, linoleic (18:2 ω-6) and α-linolenic (18:3 ω-3) acids, and pulp oil that contains high levels of monounsaturated palmitoleic acid (16:1 ω-7). Sea buckthorn is fast gaining popularity as a source of functional food and nutraceuticals, but currently has few genomic resources; therefore, we explored the fatty acid composition of Canadian-grown cultivars (ssp. mongolica) and the sea buckthorn seed transcriptome using the 454 GS FLX sequencing technology. RESULTS: GC-MS profiling of fatty acids in seeds and pulp of berries indicated that the seed oil contained linoleic and α-linolenic acids at 33-36% and 30-36%, respectively, while the pulp oil contained palmitoleic acid at 32-42%. 454 sequencing of sea buckthorn cDNA collections from mature seeds yielded 500,392 sequence reads, which identified 89,141 putative unigenes represented by 37,482 contigs and 51,659 singletons. Functional annotation by Gene Ontology and computational prediction of metabolic pathways indicated that primary metabolism (protein>nucleic acid>carbohydrate>lipid) and fatty acid and lipid biosynthesis pathways were highly represented categories. Sea buckthorn sequences related to fatty acid biosynthesis genes in Arabidopsis were identified, and a subset of these was examined for transcript expression at four developing stages of the berry. CONCLUSION: This study provides the first comprehensive genomic resources represented by expressed sequences for sea buckthorn, and demonstrates that the seed oil of Canadian-grown sea buckthorn cultivars contains high levels of linoleic acid and α-linolenic acid in a close to 1:1 ratio, which is beneficial for human health. These data provide the foundation for further studies on sea buckthorn oil, the enzymes involved in its biosynthesis, and the genes involved in the general hardiness of sea buckthorn against environmental conditions.

sn-Glycerol-3-phosphate acyltransferases in plants.

sn-Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the acylation at sn-1 position of glycerol-3-phosphate to produce lysophosphatidic acid (LPA). LPA is an important intermediate for the formation of different types of acyl-lipids, such as extracellular lipid polyesters, storage and membrane lipids. Three types of GPAT have been found in plants, localizing to the plastid, endoplasmic reticulum, and mitochondria. These GPATs are involved in several lipid biosynthetic pathways and play important biological roles in plant development. In the present review, we will focus on the recent progress in studying the physiological functions of GPATs and their metabolic roles in glycerolipid biosynthesis.

Functional and topological analysis of yeast acyl-CoA:diacylglycerol acyltransferase 2, an endoplasmic reticulum enzyme essential for triacylglycerol biosynthesis.

Acyl-CoA:diacylglycerol acyltransferase (EC 2.3.1.20) is a membrane protein present mainly in the endoplasmic reticulum. It catalyzes the final and committed step in the biosynthesis of triacylglycerol, which is the principal repository of fatty acids for energy utilization and membrane formation. Two distinct family members of acyl-CoA:diacylglycerol acyltransferase, known as DGAT1 and DGAT2, have been characterized in different organisms, including mammals, fungi, and plants. In this study, we characterized the functional role and topological orientation of signature motifs in yeast (Saccharomyces cerevisiae) DGAT2 using mutagenesis in conjunction with chemical modification. Our data provide evidence that both the N and C termini are oriented toward the cytosol and have different catalytic roles. A highly conserved motif, (129)YFP(131), and a hydrophilic segment exclusive to yeast DGAT2 reside in a long endoplasmic reticulum luminal loop following the first transmembrane domain and play an essential role in enzyme catalysis. In addition, the strongly conserved His(195) within the motif HPHG, which may play a role in the active site of DGAT2, is likely embedded in the membrane. These results indicate some similarities to the topology model of murine DGAT2 but also reveal striking differences suggesting that the topological organization of DGAT2 is not ubiquitously conserved.

Three homologous genes encoding sn-glycerol-3-phosphate acyltransferase 4 exhibit different expression patterns and functional divergence in Brassica napus.

Brassica napus is an allotetraploid (AACC) formed from the fusion of two diploid progenitors, Brassica rapa (AA) and Brassica oleracea (CC). Polyploidy and genome-wide rearrangement during the evolution process have resulted in genes that are present as multiple homologs in the B. napus genome. In this study, three B. napus homologous genes encoding endoplasmic reticulum-bound sn-glycerol-3-phosphate acyltransferase 4 (GPAT4) were identified and characterized. Although the three GPAT4 homologs share a high sequence similarity, they exhibit different expression patterns and altered epigenetic features. Heterologous expression in yeast further revealed that the three BnGPAT4 homologs encoded functional GPAT enzymes but with different levels of polypeptide accumulation. Complementation of the Arabidopsis (Arabidopsis thaliana) gpat4 gpat8 double mutant line with individual BnGPAT4 homologs suggested their physiological roles in cuticle formation. Analysis of gpat4 RNA interference lines of B. napus revealed that the BnGPAT4 deficiency resulted in reduced cutin content and altered stomatal structures in leaves. Our results revealed that the BnGPAT4 homologs have evolved into functionally divergent forms and play important roles in cutin synthesis and stomatal development.

High-level expression, purification and characterization of a recombinant medium-temperature alpha-amylase from Bacillus subtilis.

Alpha-amylases are important industrial enzymes with a wide range of applications. Although medium-temperature alpha amylase (AmyE) has some practical advantages, its low yield has limited its applications. When an amyE gene from Bacillus subtilis BF768 was cloned into vector pWB980 and over-expressed in B. subtilis WB600, high activities (723 U ml(-1)) of secreted AmyE were produced. Recombinant AmyE was purified to a specific activity of 36 U mg(-1) having optimal activity at pH 6.0 and 60 degrees C.

Antisense suppression of type 1 diacylglycerol acyltransferase adversely affects plant development in Brassica napus.

Diacylglycerol acyltransferase (DGAT) catalyzes the acyl-coenzyme A (CoA) dependent acylation of sn-1,2-diacylglycerol to form triacylglycerol in the terminal step of seed oil formation. Previous work has suggested that the level of DGAT activity may have a substantial effect on the flow of carbon into triacylglycerol, implying that the enzyme may represent a promising target for seed oil modification through biotechnological approaches. In the current study, Brassica napus DH12075 was transformed with an antisense type 1 DGAT construct, resulting in a reduction in DGAT1 gene expression, total DGAT activity and seed oil content. In addition, reduced seed yield and germination rates were observed along with severe developmental abnormalities. These data suggest that in addition to its critical role in seed oil formation, DGAT1 enzyme may also be important for normal seed development in B. napus, although the underlying mechanism(s) remain to be determined.

Acyltransferase action in the modification of seed oil biosynthesis.

Seed oils represent a major source of dietary lipid and an increasingly valuable feedstock for industrial applications. There have been several attempts to modify seed oil content and composition through biotechnological approaches, resulting in the identification of several 'bottlenecks' limiting the accumulation of unusual fatty acids in storage lipids of oilseed crops. It has been suggested that the substrate preferences of endogenous acyltransferases play an important role in the utilization of unusual fatty acids in transgenic oilseeds, and there is increasing evidence that mechanisms of 'acyl-editing' via phospholipids are also involved in substrate trafficking and utilization. In this review, we will examine acyltransferase substrate specificity and selectivity in the context of designing strategies to maximize the accumulation of unusual fatty acids using biotechnological approaches.

Metabolic control analysis is helpful for informed genetic manipulation of oilseed rape (Brassica napus) to increase seed oil content.

Top-down control analysis (TDCA) is a useful tool for quantifying constraints on metabolic pathways that might be overcome by biotechnological approaches. Previous studies on lipid accumulation in oilseed rape have suggested that diacylglycerol acyltransferase (DGAT), which catalyses the final step in seed oil biosynthesis, might be an effective target for enhancing seed oil content. Here, increased seed oil content, increased DGAT activity, and reduced substrate:product ratio are demonstrated, as well as reduced flux control by complex lipid assembly, as determined by TDCA in Brassica napus (canola) lines which overexpress the gene encoding type-1 DGAT. Lines overexpressing DGAT1 also exhibited considerably enhanced seed oil content under drought conditions. These results support the use of TDCA in guiding the rational selection of molecular targets for oilseed modification. The most effective lines had a seed oil increase of 14%. Moreover, overexpression of DGAT1 under drought conditions reduced this environmental penalty on seed oil content.