Molecular cloning and characterization of gl8, a gene involved in maize cuticular wax biosynthesis

Abstract

<p>The gl8 locus of maize was previously defined by a mutation that reduces the amount and alters the composition of seedling cuticular waxes. Sixty independently derived gl8 mutant alleles have been isolated from stocks that carried the Mutator transposon system. A DNA fragment that contains a Mu8 transposon and that co-segregates with one of these alleles, gl8-Mu3142, was identified and cloned. DNA flanking the Mu8 transposon was shown to represent the gl8 locus via allelic cross-referencing experiments. The gl8 probe reveals a 1.4-kb transcript present in wild-type seedling leaves, and in lesser amounts, in other organs and at other developmental stages. Sequence analyses reveal that the predicted GL8 protein exhibits significant sequence similarity to a class of enzymes that catalyze the reduction of ketone groups to hydroxyl groups. Polyclonal antibodies were raised against GL8 protein expressed in E. coli. Subcellular fractionation experiments indicate that the GL8 protein is associated with the endoplasmic reticulum. Furthermore, polyclonal antibodies raised against purified acyl-CoA elongase complex from leek can interact with the E. coli-expressed GL8 protein. In combination, these findings suggest that the GL8 protein is a component of the acyl-CoA elongase complex. This conclusion lends further support to the hypothesis that the GL8 protein functions as a reductase during the elongation of very long chain fatty acids required for the production of cuticular waxes.</p>