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

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.

Structural characterisation of the pectic polysaccharide rhamnogalacturonan II using an acidic fingerprinting methodology.

Rhamnogalacturonan II (RG-II) is a structurally complex cell wall pectic polysaccharide. Despite its complexity, both the structure of RG-II and its ability to dimerise via a borate diester are conserved in vascular plants suggesting that RG-II has a fundamental role in primary cell wall organisation and function. The selection and analysis of new mutants affected in RG-II formation represents a promising strategy to unravel these functions and to identify genes encoding enzymes involved in RG-II biosynthesis. In this paper, a novel fingerprinting strategy is described for the screening of RG-II mutants based on the mild acid hydrolysis of RG-II coupled to the analysis of the resulting fragments by mass spectrometry. This methodology was developed using RG-II fractions isolated from citrus pectins and then validated for RG-II isolated from the Arabidopsis mur1 mutant and irx10 irx10-like double mutant.

The synthesis of the rhamnogalacturonan II component 3-deoxy-D-manno-2-octulosonic acid (Kdo) is required for pollen tube growth and elongation.

Despite a very complex structure, the sugar composition of the rhamnogalacturonan II (RG-II) pectic fraction is extremely conserved. Among its constituting monosaccharides is the seldom-observed eight-carbon sugar 3-deoxy-D-manno-octulosonic acid (Kdo), whose phosphorylated precursor is synthesized by Kdo-8-P synthase. As an attempt to alter specifically the RG-II structure in its sugar composition and assess the consequences on the function of RG-II in cell wall and its relationship with growth, Arabidopsis null mutants were sought in the genes encoding Kdo-8-P synthase. Here, the isolation and characterization of one null mutant for the isoform 1 (AtkdsA1-S) and two distinct null mutants for the isoform 2 of Arabidopsis Kdo-8-P synthase (AtkdsA2-V and AtkdsA2-S) are described. Evidence is provided that AtkdsA2 gene expression is preferentially associated with plantlet organs displaying a meristematic activity, and that it accounts for 75% of the mRNAs to be translated into Kdo-8-P synthase. Furthermore, this predominant expression of AtKDSA2 over AtKDSA1 was confirmed by quantification of the cytosolic Kdo content in the mutants, in a variety of ecotypes. The inability to identify a double knockout mutant originated from pollen abortions, due to the inability of haploid pollen of the AtkdsA1- AtkdsA2- genotype to form an elongated pollen tube properly and perform fertilization.