Ectopic overexpression of a type-II DGAT (CeDGAT2-2) derived from oil-rich tuber of Cyperus esculentus enhances accumulation of oil and oleic acid in tobacco leaves

Abstract

Background: Engineering triacylglycerol (TAG) accumulation in vegetative tissues of non-food crops has become a promising way to meet our increasing demand for plant oils, especially the renewable production of biofuels. The most important target modified in this regard is diacylglycerol acyltransferase (DGAT) enzyme responsible for the final rate-limiting step in TAG biosynthesis. Cyperus esculentus is a unique plant largely accumulating oleic acid-enriched oil in its underground tubers. We speculated that DGAT derived from such oil-rich tubers could function more efficiently than that from oleaginous seeds in enhancing oil storage in vegetative tissues of tobacco, a high-yielding biomass crops. Results: Three CeDGAT genes namely CeDGAT1, CeDGAT2-1 and CeDGAT2-2 were identified in C. esculentus by mining transcriptome of developing tubers. These CeDGATs were expressed in tissues tested, with CeDGAT1 highly in roots, CeDGAT2-1 abundantly in leaves, and CeDGAT2-2 predominantly in tubers. Notably, CeDGAT2-2 expression pattern was in accordance with oil dynamic accumulation during tuber development. Overexpression of CeDGAT2-2 functionally restored TAG biosynthesis in TAG-deficient yeast mutant H1246. Oleic acid level was significantly increased in CeDGAT2-2 transgenic yeast compared to the wild-type yeast and ScDGA1-expressed control under culture with and without feeding of exogenous fatty acids. Overexpressing CeDGAT2-2 in tobacco led to dramatic enhancements of leafy oil by 7.15- and 1.7-fold more compared to the wild-type control and plants expressing Arabidopsis seed-derived AtDGAT1. A substantial change in fatty acid composition was detected in leaves, with increase of oleic acid from 5.1% in the wild type to 31.33% in CeDGAT2-2-expressed tobacco and accompanied reduction of saturated fatty acids. Moreover, the elevated accumulation of oleic acid-enriched TAG in transgenic tobacco exhibited no significantly negative impact on other agronomic traits such as photosynthesis, growth rates and seed germination except for small decline of starch content. Conclusions: The present data indicate that CeDGAT2-2 has a high enzyme activity to catalyze formation of TAG and a strong specificity for oleic acid-containing substrates, providing new insights into understanding oil biosynthesis mechanism in plant vegetative tissues. Overexpression of CeDGAT2-2 alone can significantly increase oleic acid-enriched oil accumulation in tobacco leaves without negative impact on other agronomy traits, showing CeDGAT2-2 as the desirable target gene in metabolic engineering to enrich oil and value-added lipids in high-biomass plants for commercial production of biofuel oils.