The biosynthesis of L-arabinose in plants: molecular cloning and characterization of a Golgi-localized UDP-D-xylose 4-epimerase encoded by the MUR4 gene of Arabidopsis

Abstract

The mur4 mutant of Arabidopsis shows a 50% reduction in the monosaccharide L-Ara in leaf-derived cell wall material because of a partial defect in the 4-epimerization of UDP-D-Xyl to UDP-L-Ara. To determine the genetic lesion underlying the mur4 phenotype, the MUR4 gene was cloned by a map-based procedure and found to encode a type-II membrane protein with sequence similarity to UDP-D-Glc 4-epimerases. Enzyme assays of MUR4 protein expressed in the methylotropic yeast Pichia pastoris indicate that it catalyzes the 4-epimerization of UDP-D-Xyl to UDP-L-Ara, the nucleotide sugar used by glycosyltransferases in the arabinosylation of cell wall polysaccharides and wall-resident proteoglycans. Expression of MUR4-green fluorescent protein constructs in Arabidopsis revealed localization patterns consistent with targeting to the Golgi, suggesting that the MUR4 protein colocalizes with glycosyltransferases in the biosynthesis of arabinosylated cell wall components. The Arabidopsis genome encodes three putative proteins with >76% sequence identity to MUR4, which may explain why mur4 plants are not entirely deficient in the de novo synthesis of UDP-L-Ara.

Figure 1.

De Novo Pathway for the Synthesis of UDP-l-Ara and Other UDP Sugars from UDP-d-Glc via NAD(P)⁺-Dependent Dehydrogenation, Decarboxylation, and 4-Epimerization Reactions.

Figure 2.

Molecular Cloning and Structure of the MUR4 Gene. (A) Exon-intron structure of the MUR4 coding region based on a comparison of the genomic and cDNA sequences. The location of the translational initiation codon was inferred from the presence of a 419–amino acid open reading frame on the cDNA preceded by an in-frame stop codon. The positions of missense and splice site mutations within the four known mur4 alleles are indicated. (B) Relative Ara content of wild-type (WT) plants (white bars), the mur4 mutant (black bars), and mur4 plants transformed with the wild-type allele (gray bars). The latter line represents a T2 population segregating for the MUR4 transgene. Leaf material from individual plants was analyzed for the Ara content of its cell walls by gas-liquid chromatography of alditol acetates. The height of the bars indicates the number of plants with a specific percentage of Ara content.

Figure 3.

Assay of the Microsomal Fractions from mur4 Plants and a mur4 Line Transformed with the Wild-Type MUR4 Allele for the Conversion of UDP-d-Glucuronate to UDP-d-Xyl and UDP-l-Ara. Radiolabeled UDP sugars produced at different time points were hydrolyzed, and the monosaccharides were separated and quantified by thin layer chromatography. Data points represent means of three samples ± sd.

Figure 4.

Alignment of the Amino Acid Sequences of the MUR4 and UGE1 Proteins from Arabidopsis and the galE Gene Product from B. subtilis. The putative transmembrane domain within MUR4 is boxed, and the highly conserved GxxGxxG motif involved in the binding of the NAD(P)⁺ cofactor is indicated above the sequence alignment. The amino acid substitutions in mur4-1 and mur4-3 also are shown.

Figure 5.

RNA Gel Blot Analysis of Total RNA Extracted from Roots, Flowers, Leaves, Stems, and Siliques and Probed with a ³²P-Labeled MUR4 Fragment.

Figure 6.

Autoradiograph of a Thin Layer Chromatogram of the ¹⁴C-Labeled Reaction Products from Assays for 4-Epimerase Activities of the MUR4 Protein Expressed in P. pastoris. The types of protein extracts used are indicated at top, and the nucleotide sugar substrates are indicated at bottom. Nucleotide sugars were hydrolyzed to monosaccharides before thin layer chromatography analysis. The only interconversion reaction observed was the reversible 4-epimerization of UDP-d-Xyl to UDP-l-Ara in the presence of native MUR4 protein.

Figure 7.

MUR4-GFP Localization in Arabidopsis Root Protoplasts. (A) Confocal image of a single protoplast showing MUR4-GFP fluorescence consistent with a Golgi-localized protein. (B) Confocal image of a single protoplast showing numerous punctate bodies indicating vesiculation and collapse of the Golgi stacks after brefeldin A treatment (2 h at 100 μg/mL). Bar in (A) = 3 μm for (A) and (B).