Characterization of a putative 3-deoxy-D-manno-2-octulosonic acid (Kdo) transferase gene from Arabidopsis thaliana

Abstract

The structures of the pectic polysaccharide rhamnogalacturonan II (RG-II) pectin constituent are remarkably evolutionary conserved in all plant species. At least 12 different glycosyl residues are present in RG-II. Among them is the seldom eight-carbon sugar 3-deoxy-d-manno-octulosonic acid (Kdo) whose biosynthetic pathway has been shown to be conserved between plants and Gram-negative bacteria. Kdo is formed in the cytosol by the condensation of phosphoenol pyruvate with d-arabinose-5-P and then activated by coupling to cytidine monophosphate (CMP) prior to its incorporation in the Golgi apparatus by a Kdo transferase (KDTA) into the nascent polysaccharide RG-II. To gain new insight into RG-II biosynthesis and function, we isolated and characterized null mutants for the unique putative KDTA (AtKDTA) encoded in the Arabidopsis genome. We provide evidence that, in contrast to mutants affecting the RG-II biosynthesis, the extinction of the AtKDTA gene expression does not result in any developmental phenotype in the AtkdtA plants. Furthermore, the structure of RG-II from the null mutants was not altered and contained wild-type amount of Rha-alpha(1-5)Kdo side chain. The cellular localization of AtKDTA was investigated by using laser scanning confocal imaging of the protein fused to green fluorescent protein. In agreement with its cellular prediction, the fusion protein was demonstrated to be targeted to the mitochondria. These data, together with data deduced from sequence analyses of higher plant genomes, suggest that AtKDTA encodes a putative KDTA involved in the synthesis of a mitochondrial not yet identified lipid A-like molecule rather than in the synthesis of the cell wall RG-II.