Title

Characterization of the arabinose deficient mur4 mutant of Arabidopsis thaliana and cloning of a UDP-D-xylose 4-epimerase gene

Date of Completion

January 1999

Keywords

Biology, Molecular|Biology, Plant Physiology

Degree

Ph.D.

Abstract

To obtain information on the synthesis and function of arabinosylated glycans, the mur4 mutant of Arabidopsis thaliana was characterized. The mur4 mutation leads to a 50% reduction in the monosaccharide L-arabinose in most organs and to a 75% reduction in the elongation zone of inflorescence stems. The mutation affects arabinose-containing pectic cell wall polysaccharides as well as arabinogalactan-proteins. ^ Feeding of L-arabinose to mur4 plants restores the cell wall composition to wild type suggesting a partial defect in the de novo synthesis of UDP-L-arabinose, the activated sugar used by arabinosyl transferases. The defect was traced to the last step in the de novo synthesis of UDP- L-arabinose, the interconversion of UDP-D-xylose to UDP-L-arabinose, which is catalyzed by a UDP-D-xylose 4-epimerase. ^ To determine the genetic lesion underlying the mur4 phenotype, the MUR4 gene was cloned via a map-based procedure. The mur4 mutation was mapped to chromosome 1 at 1 cM above the UFO (UNUSUAL FLORAL ORGANS) gene. ^ Using the data released by the Arabidopsis sequencing project, a bacterial artificial chromosome clone end sequence with some homology to bacterial UDP-D-glucose 4-epimerases was identified in the UFO region and shown to be tightly linked to mur4. The candidate MUR4 gene was isolated from wild type plants and all four known mur4 alleles. The correct identification of the MUR4 gene was demonstrated by complementation of mutant plants by the wild type allele, and the presence of single point mutations in all four mur4 alleles. The MUR4 gene encodes a membrane-bound UDP-D-xylose 4-epimerase, an activity for which no gene has previously been described. The activity of the MUR4 gene product was demonstrated by expression in S. cerevisiae . It is a novel type of enzyme which provides L-arabinose moieties for the arabinosylation of cell wall polysaccharides and wall-resident glycoproteins and proteoglycans. ^ Although the mur4 mutation does not lead to any visible phenotype, it interacts synergistically with the mur1 mutation (defect in fucose synthesis in the shoot), yielding extremely dwarfed plants with defects in cell elongation. ^