Abstract
Diacylglycerol acyltransferase (DGAT) catalyzes the final step in triacylglycerol (TAG) biosynthesis via the acyl-CoA-dependent acylation of diacylglycerol. This reaction is a major control point in the Kennedy pathway for biosynthesis of TAG, which is the most important form of stored metabolic energy in most oil-producing plants. In this study, Arachis hypogaea type 2 DGAT (AhDGAT2) genes were cloned from the peanut cultivar ‘Luhua 14.’ Sequence analysis of 11 different peanut cultivars revealed a gene family of 8 peanut DGAT2 genes (designated AhDGAT2a-h). Sequence alignments revealed 21 nucleotide differences between the eight ORFs, but only six differences result in changes to the predicted amino acid (AA) sequences. A representative full-length cDNA clone (AhDGAT2a) was characterized in detail. The biochemical effects of altering the AhDGAT2a sequence to include single variable AA residues were tested by mutagenesis and functional complementation assays in transgenic yeast systems. All six mutant variants retained enzyme activity and produced lipid droplets in vivo. The N6D and A26P mutants also displayed increased enzyme activity and/or total cellular fatty acid (FA) content. N6D mutant mainly increased the content of palmitoleic acid, and A26P mutant mainly increased the content of palmitic acid. The A26P mutant grew well both in the presence of oleic and C18:2, but the other mutants grew better in the presence of C18:2. AhDGAT2 is expressed in all peanut organs analyzed, with high transcript levels in leaves and flowers. These levels are comparable to that found in immature seeds, where DGAT2 expression is most abundant in other plants. Over-expression of AhDGAT2a in tobacco substantially increased the FA content of transformed tobacco seeds. Expression of AhDGAT2a also altered transcription levels of endogenous tobacco lipid metabolic genes in transgenic tobacco, apparently creating a larger carbon ‘sink’ that supports increased FA levels.
Highlights
Peanuts are used in a variety of important food products, including whole roasted peanuts, peanut butter, and as an essential ingredient of various entrees in cuisines from around the world
The expression level of AhDGAT1-1 and AhDGAT3-3 were higher in flowers than in the other tissues examined, whereas the AhDGAT1-2 transcript was more abundant in roots, seeds, and cotyledons (Chi et al, 2014)
The precise biological roles of plant DGAT3s are even less certain, but available evidence suggests it may function as part of a cytosolic pathway that balances membrane lipid and TAG biosynthesis by regulating acyl-CoA pool size and composition (Hernández et al, 2012)
Summary
Peanuts are used in a variety of important food products, including whole roasted peanuts, peanut butter, and as an essential ingredient of various entrees in cuisines from around the world. Many laboratories have focused on DGATs because of their important roles in TAG synthesis Studies of both type-1 (DGAT1) and type-2 DGAT (DGAT2) genes have provided many useful insights into the properties of these enzymes, and have shown that DGAT overexpression often greatly increases the oil content of transgenic organisms (Cao and Huang, 1986; Cases et al, 1998; Hobbs et al, 1999; Bouvier-Navé et al, 2000; Jako et al, 2001; Lardizabal et al, 2001; He et al, 2004; Kroon et al, 2006; Saha et al, 2006; Shockey et al, 2006; Wang et al, 2006; Burgal et al, 2008; Lardizabal et al, 2008; Zheng et al, 2008; Andrianov et al, 2010; Durrett et al, 2010; Cao, 2011; Cao et al, 2013; Gong et al, 2013; Guo et al, 2013; Misra et al, 2013; Niu et al, 2013; Peng et al, 2013; Xu et al, 2013; Zhang et al, 2013; Aymé et al, 2014; Chi et al, 2014; Dey et al, 2014)
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