Defective in Cuticular Ridges (DCR) of Arabidopsis thaliana, a Gene Associated with Surface Cutin Formation, Encodes a Soluble Diacylglycerol Acyltransferase

Sapa Hima Rani,T.H Anantha Krishna,Saikat Saha,Arvind Singh Negi,Ram Rajasekharan

doi:10.1074/jbc.m110.133116

Abstract

A key step in the triacylglycerol (TAG) biosynthetic pathway is the final acylation of diacylglycerol (DAG) by DAG acyltransferase. In silico analysis has revealed that the DCR (defective in cuticular ridges) (At5g23940) gene has a typical HX(4)D acyltransferase motif at the N-terminal end and a lipid binding motif VX(2)GF at the middle of the sequence. To understand the biochemical function, the gene was overexpressed in Escherichia coli, and the purified recombinant protein was found to acylate DAG specifically in an acyl-CoA-dependent manner. Overexpression of At5g23940 in a Saccharomyces cerevisiae quadruple mutant deficient in DAG acyltransferases resulted in TAG accumulation. At5g23940 rescued the growth of this quadruple mutant in the oleate-containing medium, whereas empty vector control did not. Lipid particles were localized in the cytosol of At5g23940-transformed quadruple mutant cells, as observed by oil red O staining. There was an incorporation of 16-hydroxyhexadecanoic acid into TAG in At5g23940-transformed cells of quadruple mutant. Here we report a soluble acyl-CoA-dependent DAG acyltransferase from Arabidopsis thaliana. Taken together, these data suggest that a broad specific DAG acyltransferase may be involved in the cutin as well as in the TAG biosynthesis by supplying hydroxy fatty acid.

Highlights

Triacylglycerol (TAG),3 an acyl ester glycerol, is the most efficient storage form of energy in eukaryotic cells [1, 2]
DCR Is DAG acyltransferase (DGAT)—Diacylglycerol acyltransferase catalyzes the formation of triacylglycerol from diacylglycerol using acylCoA as the acyl donor
The following findings suggest that DCR is a DGAT. (i) A typical acyltransferase motif (HX4D) and a lipid binding motif were present. (ii) Because E. coli cells do not synthesize TAG, At5g23940-transformed E. coli cells showed a significant amount of TAG. (iii) To validate further, quadruple mutant (H1246)-overexpressing At5g23940 was able to incorporate the [14C]acetate into the TAG fraction

Summary

Introduction

Triacylglycerol (TAG),3 an acyl ester glycerol, is the most efficient storage form of energy in eukaryotic cells [1, 2]. These data suggest that a broad specific DAG acyltransferase may be involved in the cutin as well as in the TAG biosynthesis by supplying hydroxy fatty acid. To evaluate the role of this gene in cuticle formation, a precursor [23] (16-hydroxyhexadecanoic acid) was fed to the quadruple mutant transformed with At5g23940, and it was observed that there was a 2-fold increase in the incorporation of hydroxy fatty acid in the total lipids compared with vector control.

Results

Conclusion