Expression in yeast and tobacco of plant cDNAs encoding acyl CoA:diacylglycerol acyltransferase.
During the course of a search for cDNAs encoding plant sterol acyltransferases, an expressed sequence tag clone presenting substantial identity with yeast and animal acyl CoA:cholesterol acyltransferases was used to screen cDNA libraries from Arabidopsis and tobacco. This resulted in the isolation of two full-length cDNAs encoding proteins of 520 and 532 amino acids, respectively. Attempts to complement the yeast double-mutant are1 are2 defective in acyl CoA:cholesterol acyltransferase were unsuccessful, showing that neither gene encodes acyl CoA:cholesterol acyltransferase. Their deduced amino 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 incorporation of oleyl CoA into triacylglycerols. Incorporation was 2-3 times higher in microsomes from yeast transformed with these plant cDNAs than in yeast transformed with the void vector, clearly showing that these cDNAs encode acyl CoA:diacylglycerol acyltransferases. Moreover, during the preparation of microsomes from the Arabidopsis DGAT-transformed yeast, a floating layer was observed on top of the 100 000 g supernatant. This fraction was enriched in triacylglycerols and exhibited strong acyl CoA:diacylglycerol acyltransferase activity, whereas almost no activity was detected in the corresponding clear fraction from the control yeast. Thanks to the use of this active fraction and dihexanoylglycerol as a substrate, the de novo synthesis of 1,2-dihexanoyl 3-oleyl glycerol by AtDGAT could be demonstrated. Transformation of tobacco with AtDGAT was also performed. Analysis of 19 primary transformants allowed detection, in several individuals, of a marked increase (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 illustrating the potential application of acyl CoA:diacylglycerol acyltransferase-transformed plants.
Bouvier-Navé
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Institut de Biologie Moléculaire des Plantes, Strasbourg, France. Pierre.Benveniste@ibmp-ulp.u-strasbg.fr
Benveniste
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Oelkers
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Sturley
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SL
Schaller
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D000214:Acyl Coenzyme A / Q000378:metabolism*; D000217:Acyltransferases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D017124:Conserved Sequence; D051048:Diacylglycerol O-Acyltransferase; D004075:Diglycerides / Q000378:metabolism; D020224:Expressed Sequence Tags; D015870:Gene Expression; D006801:Humans; D008051:Lipid Bilayers / Q000378:metabolism; D008055:Lipids / Q000032:analysis; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D016415:Sequence Alignment; D014026:Nicotiana / Q000737:chemistry / Q000166:cytology / Q000201:enzymology* / Q000235:genetics*; D014170:Transformation, Genetic; D014280:Triglycerides / Q000096:biosynthesis / Q000378:metabolism; D015003:Yeasts / Q000166:cytology / Q000235:genetics*
D000214:Acyl Coenzyme A; D004075:Diglycerides; D008051:Lipid Bilayers; D008055:Lipids; D014280:Triglycerides; C044296:1,2-dihexanoylglycerol; D000217:Acyltransferases; D051048:Diacylglycerol O-Acyltransferase