Genetic and biochemical analysis of the mur2 mutant of Arabidopsis thaliana

Date of Completion

January 2001

Keywords

Biology, Molecular

Degree

Ph.D.

Abstract

To identify mutants that are affected in the fucosylation of specific cell wall polymers, we investigated the mur2 mutant of A. thaliana, which has a 50% reduction in total cell wall L-fucose content. mur2 plants have no visible phenotype, with the exception of altered trichome papillae, which appear collapsed when compared to wild type plants. Of the three possible fucosylated polymers in plants (glycoproteins, pectins, and xyloglucan), fractionation studies indicate that the mutation is specific to the polysaccharide xyloglucan. Extensive characterization of mur2 xyloglucan indicates that it contains less than 2% as much L-fucose as wild type xyloglucan. Even with the virtual absence of fucose-containing xyloglucan, mur2 plants have tensile properties indistinguishable from wild type. ^ To determine the molecular basis of the mur2 mutation the MUR2 gene was positionally cloned and shown to be identical to AtFT1, a fucosyltransferase acting on xyloglucan. The mur2 mutation results in an aspartic acid close to the carboxy terminus being replaced by an asparagine, which creates a potential N-glycosylation site. Assays of recombinant protein indicated that wild type MUR2 was able to convert non-fucosylated tamarind xyloglucan to its fucosylated form, while the mutant mur2 protein was enzymatically inactive. ^ A site directed mutagenesis project was undertaken to identify domains of the MUR2 protein important to enzymatic activity. In general, mutations closest to the C-terminal portion of MUR2 had the greatest affect on catalysis. We hypothesize that this portion of MUR2 may be involved in the binding or proper coordination of xyloglucan, since the mur2 mutation (D550N) did not affect GDP binding. ^ The decreased xyloglucan fucosylation in all organs of mur2 plants, the ability of MUR2 proteins to fucosylate xyloglucan in vitro , and the normal fucosylation of glycoproteins and pectins confirms that the MUR2 protein is both necessary and sufficient for xyloglucan fucosylation, and does not appear to be involved in the fucosylation of cell wall components other than xyloglucan. The normal growth habit and wall strength of mur2 plants necessitates a re-evaluation of models regarding the role of fucosylated xyloglucan in serving as a source of signal molecules, and in establishing a strong cellulose-xyloglucan network. ^

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