Expression in yeast and tobacco of plant cDNAs encoding acyl CoA : diacylglycerol acyltransferase

During the course of a search for cDNAs encoding plant sterol acyltransfera ses, an expressed sequence tag clone presenting substantial identity with y east and animal acyl CoA:cholesterol acyltransferases was used to screen cD NA libraries from Arabidopsis and tobacco. This resulted in the isolation o f two full-length cDNAs encoding proteins of 520 and 532 amino acids, respe ctively. Attempts to complement the yeast double-mutant are1 are2 defective in acyl CoA:cholesterol acyltransferase were unsuccessful, showing that ne ither gene encodes acyl CoA:cholesterol acyltransferase. Their deduced amin o acid sequences were then shown to have 40 and 38% identity, respectively, with a murine acyl CoA:diacylglycerol acyltransferase and their expression in are1 are2 or wild-type yeast resulted in a strong increase in the incor poration of oleyl CoA into triacylglycerols. Incorporation was 2-3 times hi gher in microsomes from yeast transformed with these plant cDNAs than in ye ast transformed with the void vector, clearly showing that these cDNAs enco de acyl CoA:diacylglycerol acyltransferases. Moreover, during the preparati on of microsomes from the Arabidopsis DGAT-transformed yeast, a floating la yer was observed on top of the 100 000 g supernatant. This fraction was enr iched in triacylglycerols and exhibited strong acyl CoA:diacylglycerol acyl transferase activity, whereas almost no activity was detected in the corres ponding clear fraction from the control yeast. Thanks to the use of this ac tive fraction and dihexanoylglycerol as a substrate, the de novo synthesis of 1,2-dihexanoyl 3-oleyl glycerol by AtDGAT could be demonstrated. Transfo rmation of tobacco with AtDGAT was also performed. Analysis of 19 primary t ransformants allowed detection, in several individuals, of a marked increas e (up to seven times) of triacylglycerol content which correlated with the AtDGAT mRNA expression. Furthermore, light-microscopy observations of leaf epidermis cells, stained with a lipid-specific dye, showed the presence of lipid droplets in the cells of triacylglycerol-overproducer plants, thus il lustrating the potential application of acyl CoA:diacylglycerol acyltransfe rase-transformed plants.