Genome-wide analysis of glycerol-3-phosphate O-acyltransferase gene family and functional characterization of two cutin group GPATs in Brassica napus.

MAIN CONCLUSION: Genome-wide identification, spatio-temporal expression analysis and functional characterization of selected Brassica napus GPATs highlight their roles in cuticular wax biosynthesis and defense against fungal pathogens. Glycerol-3-phosphate 1-O-acyltransferase (GPAT) is a key enzyme in the biosynthesis of glycerolipids, a major component of cellular membranes and extracellular protective layers, such as cuticles in plants. Brassica napus is an economically important crop and cultivated worldwide mostly for its edible oil. The B. napus GPATs (BnGPATs) are insufficiently characterized. Here, we performed genome-wide analysis to identify putative GPATs in B. napus and its diploid progenitors B. rapa and B oleracea. The 32 B. napus BnGPATs are phylogenetically divided into three major groups, cutin, suberin, and diverse ancient groups. Analysis of transcriptomes of different tissues and seeds at different developmental stages revealed the spatial and temporal expression profiles of BnGPATs. The yield and oil quality of B. napus are adversely affected by the necrotrophic fungus, Sclerotinia sclerotiorum. We showed that several BnGPATs, including cutin-related BnGPAT19 and 21, were upregulated in the S. sclerotiorum resistant line. RNAi-mediated suppression of BnGPAT19 and 21 in B. napus resulted in thinner cuticle, leading to rapid water and chlorophyll loss in toluidine blue staining and leaf bleaching assays. In addition, the RNAi plants also developed severe necrotic lesions following fungal inoculation compared to the wild-type plants, indicating that BnGPAT19 and 21 are likely involved in cuticular wax biosynthesis that is critical for initial pathogen defense. Taken together, we provided a comprehensive account of GPATs B. napus and characterized BnGPAT19 and 21 for their potential roles in cuticular wax biosynthesis and defense against fungal pathogens.

Triacylglycerol biosynthesis occurs via the glycerol-3-phosphate pathway in the insect Rhodnius prolixus.

Although triacylglycerol (TAG) stores play a critical role in organisms, mechanisms underlying TAG synthesis are poorly understood in invertebrates. In mammals, the synthesis of glycerolipids, including TAG, diacylglycerol (DAG) and phospholipids (PL), occurs predominantly by the glycerol-3-phosphate (G3P) pathway in most cell types, except for in enterocytes. In these cells, the monoacylglycerol (MAG) pathway accounts for the majority of glycerolipid production. The insect Rhodnius prolixus, a vector of Chagas' disease, exhibits a high capacity to produce glycerolipids in the midgut after a blood meal, providing substrates that are transferred to other organs, such as the fat body, which is specialized in TAG production and storage. In this report, the genes required for TAG synthesis were identified in the R. prolixus genome. The genomic data indicated that TAG is synthesized by the G3P pathway, which is the sole pathway for TAG synthesis in this organism. Furthermore, transcription of both the RpGpat and RpDgat genes were upregulated in a diverse number of organs at moments of highest lipid production. In the midgut and fat body, in vitro synthesis of glycerolipids required G3P, but not MAG, as the initial substrate. These results indicate that the G3P pathway is the only route for TAG synthesis in R. prolixus, and its regulation at the transcriptional level can be a determinant of glycerolipid synthesis and TAG formation in insect organs.

GPAT3 and GPAT4 are regulated by insulin-stimulated phosphorylation and play distinct roles in adipogenesis.

Acyl-CoA:glycerol-3-phosphate acyltransferase (GPAT) catalyzes the first step during de novo synthesis of glycerolipids. Mammals have at least four GPAT isoforms. Here we report the further characterization of the two recently identified microsomal GPAT3 and GPAT4. Both enzymes are highly expressed in adipose tissues. However, while GPAT3 is highly (approximately 60-fold) induced during adipocyte differentiation, GPAT4 induction is only modest (approximately 5-fold), leading to a lower abundance of GPAT4 mRNA in adipocytes. While overexpression of GPAT3 and GPAT4 in either insect or mammalian cells results in a comparable increase of GPAT activity, shRNA-mediated knockdown of GPAT3, but not GPAT4, in 3T3-L1 adipocytes led to a significant decrease in GPAT activity, a profound inhibition of lipid accumulation, and a lack of expression of several adipogenic markers during adipocyte differentiation. These data suggest that GPAT3 may encode the major GPAT isoform in adipocytes and play an important role in adipogenesis. Furthermore, we have shown that both GPAT3 and GPAT4 are phosphorylated by insulin at Ser and Thr residues, leading to increased GPAT activity that is sensitive to wortmannin. Our results reveal a link between the lipogenic effects of insulin and microsomal GPAT3 and GPAT4, implying their importance in glycerolipid biosynthesis.

Arabidopsis thaliana GPAT8 and GPAT9 are localized to the ER and possess distinct ER retrieval signals: functional divergence of the dilysine ER retrieval motif in plant cells.

Glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) catalyzes the committed step in the production of glycerolipids, which are major components of cellular membranes, seed storage oils, and epicuticular wax coatings. While the biochemical activities of GPATs have been characterized in detail, the cellular features of these enzymes are only beginning to emerge. Here we characterized the phylogenetic relationships and cellular properties of two GPAT enzymes from the relatively large Arabidopsis thaliana GPAT family, including GPAT8, which is involved in cutin biosynthesis, and GPAT9, which is a new putative GPAT that has extensive homology with a GPAT from mammalian cells involved in storage oil formation and, thus, may have a similar role in plants. Immunofluorescence microscopy of transiently-expressed myc-epitope-tagged GPAT8 and GPAT9 revealed that both proteins were localized to the endoplasmic reticulum (ER), and differential permeabilization experiments indicated that their N- and C-termini were oriented towards the cytosol. However, these two proteins contained distinct types of ER retrieval signals, with GPAT8 possessing a divergent type of dilysine motif (-KK-COOH rather than the prototypic -KKXX-COOH or -KXKXX-COOH motif) and GPAT9 possessing a hydrophobic pentapeptide motif (-phi-X-X-K/R/D/E-phi-; where phi are large hydrophobic amino acid residues). Notably, the divergent dilysine motif in GPAT8 only functioned effectively when additional upstream residues were included to provide the proper protein context. Extensive mutational analyses of the divergent dilysine motif, based upon sequences present in the C-termini of other GPAT8s from various plant species, further expanded the functional definition of this molecular targeting signal, thereby providing insight to the targeting signals in other GPAT family members as well as other ER-resident membrane proteins within plant cells.