Site-directed mutagenesis of IRX9, IRX9L and IRX14 proteins involved in xylan biosynthesis: glycosyltransferase activity is not required for IRX9 function in Arabidopsis.

Samuel P. Hazen,Yanfang Ren,Jane Lau,Berit Ebert,Henrik Vibe Scheller,Sara Fasmer Hansen

doi:10.1371/journal.pone.0105014

Abstract

Xylans constitute the main non-cellulosic polysaccharide in the secondary cell walls of plants. Several genes predicted to encode glycosyltransferases are required for the synthesis of the xylan backbone even though it is a homopolymer consisting entirely of β-1,4-linked xylose residues. The putative glycosyltransferases IRX9, IRX14, and IRX10 (or the paralogs IRX9L, IRX14L, and IRX10L) are required for xylan backbone synthesis in Arabidopsis. To investigate the function of IRX9, IRX9L, and IRX14, we identified amino acid residues known to be essential for catalytic function in homologous mammalian proteins and generated modified cDNA clones encoding proteins where these residues would be mutated. The mutated gene constructs were used to transform wild-type Arabidopsis plants and the irx9 and irx14 mutants, which are deficient in xylan synthesis. The ability of the mutated proteins to complement the mutants was investigated by measuring growth, determining cell wall composition, and microscopic analysis of stem cross-sections of the transgenic plants. The six different mutated versions of IRX9 and IRX9-L were all able to complement the irx9 mutant phenotype, indicating that residues known to be essential for glycosyltransferases function in homologous proteins are not essential for the biological function of IRX9/IRX9L. Two out of three mutated IRX14 complemented the irx14 mutant, including a mutant in the predicted catalytic amino acid. A IRX14 protein mutated in the substrate-binding DxD motif did not complement the irx14 mutant. Thus, substrate binding is important for IRX14 function but catalytic activity may not be essential for the function of the protein. The data indicate that IRX9/IRX9L have an essential structural function, most likely by interacting with the IRX10/IRX10L proteins, but do not have an essential catalytic function. Most likely IRX14 also has primarily a structural role, but it cannot be excluded that the protein has an important enzymatic activity.

Highlights

Xylan is the most abundant non-cellulosic polysaccharide in plants, comprising around 30% of the biomass
The phylogenetic tree shows that IRX14 and IRX14L in Arabidopsis result from a recent duplication, whereas IRX9 and IRX9L are products of a more ancient duplication in plants (Fig. 1) as reported by Wu et al [12]
This study shows that amino acid residues that have been shown to be essential in mammalian GT43 proteins are not important for the biological function of the Arabidopsis IRX9 and IRX9L proteins

Summary

Introduction

Xylan is the most abundant non-cellulosic polysaccharide in plants, comprising around 30% of the biomass. The structure of xylan is a b-1,4-linked backbone of D-xylose residues, decorated with various substituents. Glucuronic acid residues and arabinose substitutions are the most common monosaccharide substituents. The glucuronic acid residues are often 4-O-methylated and acetylation of the xylose residues at C-2 or C-3 positions is a common modification [1]. Because of the importance of xylan, many studies have addressed genes and proteins involved in the biosynthesis, but it has turned out to be unexpectedly challenging and progress has been very slow. Identification of enzymes required for adding the substitutions has been more successful, and several glucuronosyltransferases, arabinosyltransferases, xylosyltransferases and actetyltransferases have been reported [1,2,3,4,5,6,7]

Methods

Results

Conclusion