Transient expression of avocado DGAT1 and PDAT1 in N. benthamiana leaves

Abstract

The avocado mesocarp contains up to 60-70% oil by dry weight where triacylglycerol (TAG) is the major constituent. This neutral lipid, TAG is utilized by plants for the carbon and energy source when stores in seed tissue. There is significant human nutritional demand for vegetable oil, but its use in the production of renewable biomaterials and fuels has intensified the need to increase oil production. In plants, the final and committed step in TAG biosynthesis is catalyzed by diacylglycerol acyltransferases (DGAT) and/or a phospholipid: diacylglycerol acyltransferases (PDAT). However, the regulation of TAG biosynthesis is not well-studied in nonseed tissues such as mesocarp of avocado. Based on the transcriptome data of Persea americana it is hypothesized that both DGAT and PDAT are likely to catalyze the conversion of diacylglycerol to TAG. In this study, putative DGAT1 and PDAT1 were identified and comprehensive in silico analyses were conducted to determine the respective start codons, full-length coding sequences, transmembrane domains, predicted protein structures and phylogenetic relationships with other known DGAT1s and PDAT1s. These data reveal that the putative DGAT1 and PDAT1 of a basal angiosperm species retain features that are conserved not only among angiosperms but also other eukaryotes. For transient expression, DGAT1 and PDAT1 were transformed into N. benthamiana leaves by agrobacterium-mediated transformation. Lipid droplet was visualized by Nile Red staining and lipid content and compositions were analyzed by TLC and GC-MS. It was found that avocado DGAT1 and PDAT1 increase lipid content significantly when expressed in tobacco leaves. These results suggest that avocado DGAT1 and PDAT1 are functional and synthesize TAG when expressed in planta