SummaryA full-length cDNA encoding a putative diacylglycerol acyltransferase 1 (DGAT1, EC seed-specific expression of TmDGAT1 was able to complement the low TAG/unusual fatty acid phenotype of the Arabidopsis AS11 ( DGAT1 ) mutant. Over-expression of TmDGAT1 in wild-type Arabidopsis and high-erucic-acid rapeseed (HEAR) and canola Brassica napus resulted in an increase in oil content (3.5%-10% on a dry weight basis, or a net increase of 11%-30%). Site-directed mutagenesis was conducted on six putative functional regions/ motifs of the TmDGAT1 enzyme. Mutagenesis of a serine residue in a putative SnRK1 target site resulted in a 38%-80% increase in DGAT1 activity, and over-expression of the mutated TmDGAT1 in Arabidopsis resulted in a 20%-50% increase in oil content on a per seed basis.Thus, alteration of this putative serine/threonine protein kinase site can be exploited to enhance DGAT1 activity, and expression of mutated DGAT1 can be used to enhance oil content.

BackgroundThe Arabidopsis thaliana dgat1 mutant, AS11, has an oil content which is decreased by 30%, and a strongly increased ratio of 18:3/20:1, compared to wild type. Despite lacking a functional DGAT1, AS11 still manages to make 70% of WT seed oil levels. Recently, it was demonstrated that in the absence of DGAT1, PDAT1 was essential for normal seed development, and is a dominant determinant in Arabidopsis TAG biosynthesis.MethodsBiochemical, metabolic and gene expression studies combined with genetic crossing of selected Arabidopsis mutants have been carried out to demonstrate the contribution of Arabidopsis PDAT1 and LPCAT2 in the absence of DGAT1 activity.ResultsThrough microarray and RT-PCR gene expression analyses of AS11 vs. WT mid-developing siliques, we observed consistent trends between the two methods. FAD2 and FAD3 were up-regulated and FAE1 down-regulated, consistent with the AS11 acyl phenotype. PDAT1 expression was up-regulated by ca 65% while PDAT2 expression was up-regulated only 15%, reinforcing the dominant role of PDAT1 in AS11 TAG biosynthesis. The expression of LPCAT2 was up-regulated by 50-75%, while LPCAT1 expression was not significantly affected. In vitro LPCAT activity was enhanced by 75-125% in microsomal protein preparations from mid-developing AS11 seed vs WT. Co-incident homozygous knockout lines of dgat1/lpcat2 exhibited severe penalties on TAG biosynthesis, delayed plant development and seed set, even with a functional PDAT1; the double mutant dgat1/lpcat1 showed only marginally lower oil content than AS11.ConclusionsCollectively, the data strongly support that in AS11 it is LPCAT2 up-regulation which is primarily responsible for assisting in PDAT1-catalyzed TAG biosynthesis, maintaining a supply of PC as co-substrate to transfer sn-2 moieties to the sn-3 position of the enlarged AS11 DAG pool.

Membrane lipid modulation is one of the major strategies plants have developed for cold acclimation. In this study, a combined lipidomic and transcriptomic analysis was conducted, and the changes in glycerolipids contents and species, and transcriptional regulation of lipid metabolism in maize leaves under low temperature treatment (5°C) were investigated. The lipidomic analysis showed an increase in the phospholipid phosphatidic acid (PA) and a decrease in phosphatidylcholine (PC). And an increase in digalactosyldiacylglycerol and a decrease in monogalactosyldiacylglycerol of the galactolipid class. The results implied an enhanced turnover of PC to PA to serve as precursors for galactolipid synthesis under following low temperature treatment. The analysis of changes in abundance of various lipid molecular species suggested major alterations of different pathways of plastidic lipids synthesis in maize under cold treatment. The synchronous transcriptomic analysis revealed that genes involved in phospholipid and galactolipid synthesis pathways were significantly up-regulated, and a comprehensive gene-metabolite network was generated illustrating activated membrane lipids adjustment in maize leaves following cold treatment. This study will help to understand the regulation of glycerolipids metabolism at both biochemical and molecular biological levels in 18:3 plants and to decipher the roles played by lipid remodeling in cold response in major field crop maize.

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