Lipidome analysis and characterization of Buglossoides arvensis acyltransferases that incorporate polyunsaturated fatty acids into triacylglycerols.

Buglossoides arvensis is a burgeoning oilseed crop that contains an unique combination of ω-3 and ω-6 polyunsaturated fatty acids (PUFA), constituting ~80-85% of seed triacylglycerols (TAGs). To uncover the critical TAG biosynthetic pathways contributing for high PUFA accumulation, we performed lipidome of developing seeds and characterized acyltransferases involved in the final step of TAG biosynthesis. During seed development, distribution of lipid molecular species in individual lipid classes showed distinct patterns from an early-stage (6 days after flowering (DAF)) to the middle-stage (12 and 18 DAF) of oil biosynthesis. PUFA-containing TAG species drastically increased from 6 to 12 DAF. The expression profiles of key triacylglycerol biosynthesis genes and patterns of phosphatidylcholine, diacylglycerol and triacylglycerol molecular species during seed development were used to predict the contribution of diacylglycerol acyltransferases (DGAT1 and DGAT2) and phospholipid: diacylglycerol acyltransferases (PDAT1 and PDAT2) to PUFA-rich TAG biosynthesis. Our analysis suggests that DGATs play a crucial role in enriching TAGs with PUFA compared to PDATs. This was further confirmed by fatty acid feeding studies in yeast expressing acyltransferases. BaDGAT2 preferentially incorporated high amounts of PUFAs into TAG, compared to BaDGAT1. Our results provide insight into the molecular mechanisms of TAG accumulation in this plant and identify target genes for transgenic production of SDA in traditional oilseed crops.

Identification and functional characterization of Buglossoides arvensis microsomal fatty acid desaturation pathway genes involved in polyunsaturated fatty acid synthesis in seeds.

Buglossoides arvensis seed oil is the richest natural source of stearidonic acid (SDA), an ω-3 fatty acid with nutraceutical potential superior to α-linolenic acid (ALA). The molecular basis of polyunsaturated fatty acid synthesis in B. arvensis is unknown. Here, we describe the identification of B. arvensis fatty acid desaturase2 (BaFAD2), fatty acid desaturase3 (BaFAD3), and Delta-6-desaturase (BaD6D-1 and BaD6D-2) genes by mining the transcriptome of developing seeds and their functional characterization by heterologous expression in Saccharomyces cerevisiae. In silico analysis of their encoded protein sequences showed conserved histidine-boxes and signature motifs essential for desaturase activity. Expression profiling of these genes showed higher transcript abundance in reproductive tissues than in vegetative tissues, and their expression varied with temperature stress treatments. Yeast expressing BaFAD2 was found to desaturate both oleic acid and palmitoleic acid into linoleic acid (LA) and hexadecadienoic acid, respectively. Fatty acid supplementation studies in yeast expressing BaFAD3 and BaD6D-1 genes revealed that the encoded enzyme activities of BaFAD3 efficiently converted LA to ALA, and BaD6D-1 converted LA to γ-linolenic acid and ALA to SDA, but with an apparent preference to LA. BaD6D-2 did not show the encoded enzyme activity and is not a functional D6D. Our results provide an insight into SDA biosynthesis in B. arvensis and expand the repository of fatty acid desaturase targets available for biotechnological production of SDA in traditional oilseed crops.

Exploring triacylglycerol biosynthetic pathway in developing seeds of Chia (Salvia hispanica L.): a transcriptomic approach.

Chia (Salvia hispanica L.), a member of the mint family (Lamiaceae), is a rediscovered crop with great importance in health and nutrition and is also the highest known terrestrial plant source of heart-healthy omega-3 fatty acid, alpha linolenic acid (ALA). At present, there is no public genomic information or database available for this crop, hindering research on its genetic improvement through genomics-assisted breeding programs. The first comprehensive analysis of the global transcriptome profile of developing Salvia hispanica L. seeds, with special reference to lipid biosynthesis is presented in this study. RNA from five different stages of seed development was extracted and sequenced separately using the Illumina GAIIx platform. De novo assembly of processed reads in the pooled transcriptome using Trinity yielded 76,014 transcripts. The total transcript length was 66,944,462 bases (66.9 Mb), with an average length of approximately 880 bases. In the molecular functions category of Gene Ontology (GO) terms, ATP binding and nucleotide binding were found to be the most abundant and in the biological processes category, the metabolic process and the regulation of transcription-DNA-dependent and oxidation-reduction process were abundant. From the EuKaryotic Orthologous Groups of proteins (KOG) classification, the major category was "Metabolism" (31.97%), of which the most prominent class was 'carbohydrate metabolism and transport' (5.81% of total KOG classifications) followed by 'secondary metabolite biosynthesis transport and catabolism' (5.34%) and 'lipid metabolism' (4.57%). A majority of the candidate genes involved in lipid biosynthesis and oil accumulation were identified. Furthermore, 5596 simple sequence repeats (SSRs) were identified. The transcriptome data was further validated through confirmative PCR and qRT-PCR for select lipid genes. Our study provides insight into the complex transcriptome and will contribute to further genome-wide research and understanding of chia. The identified novel UniGenes will facilitate gene discovery and creation of genomic resource for this crop.