Title

Genetic and biochemical analysis of L-fucose biosynthesis in Arabidopsis thaliana

Date of Completion

January 2000

Keywords

Biology, Molecular|Biology, Botany|Biology, Cell|Chemistry, Biochemistry

Degree

Ph.D.

Abstract

An understanding of L-fucose biosynthesis and the role of this monosaccharide in higher plants began with the characterization of the L-fucose deficient mutant, mur1 of Arabidopsis thaliana. Biochemical assays revealed that mur1 is unable to synthesize GDP-L-fucose from GDP- D-mannose. A reduction in mur1 fertility, as determined by the analysis of progeny from test crosses, suggests that L-fucose is important for reproduction in Arabidopsis. ^ The MUR1 gene was cloned and shown to encode an isoform of GDP-D-mannose 4,6-dehydratase, catalyzing the first step in the de novo synthesis of GDP-L-fucose. Furthermore, nucleotide sequences encoding another isoform of this enzyme, designated GMD1, were isolated from cDNA and genomic libraries. Root-specific gene expression of GMD1 correlates with 4,6-dehydratase activity in mur1 roots, accounting for the L-fucose found in this tissue. Plants expressing the GUS gene under the control of the GMD1 promoter showed that the reporter gene was expressed in the root tip and in pollen grains just prior to anthesis. Analysis of MUR1::GUS plants revealed that MUR1 is expressed everywhere except a portion of the root tip. ^ To isolate and clone genes encoding the 3,5-epimerase and 4-reductase activities in GDP-L-fucose biosynthesis, data on bacterial genes involved in the synthesis of lipopolysaccharides and capsules were used to identify similar genes in Arabidopsis via database searches. An expressed sequence tag (EST) was identified and the corresponding gene, now designated GER1, was cloned from a genomic library. The GER1 gene is expressed throughout the plant. Assays of the recombinant protein expressed in E. coli clearly established that the GER1 gene product exhibits GDP-4-keto-6-deoxy- D-mannose 3,5-epimerase-4-reductase activity, completing the de novo synthesis of GDP-L-fucose. Moreover, GER1 antisense plants have decreased 3,5-epimerase-4-reductase activity. ^ Isolation and characterization of all of the genes involved in the de novo synthesis of GDP-L-fucose should provide a framework by which other nucleotide sugar interconversion enzymes may be studied and allow for the isolation of mutants completely deficient in L-fucose. ^