Comparison of five xylan synthesis mutants reveals new insight into the mechanisms of xylan synthesis

Abstract

SummaryPrevious studies using co-expression analysis have identified a large number of genes likely to be involved in secondary cell-wall formation. However, the function of very few of these genes is known. We have studied the cell-wall phenotype of irx7, irx8 and irx9, three previously described irregular xylem (irx) mutants, and irx14 and parvus-3, which we now show also to be secondary cell-wall mutants. All five mutants, which have mutations in genes encoding putative glycosyltransferases, exhibited large decreases in xylan. In addition, all five mutants were found to have the same specific defect in xylan structure, retaining MeGlcUA but lacking GlcUA side branches. Polysaccharide analysis by carbohydrate gel electrophoresis (PACE) was used to determine the xylan structure in Arabidopsis, and revealed that side branches are added to approximately one in every eight xylose residues. Interestingly, this ratio is constant in all the lines analysed despite the wide variation in xylan content and the absence of GlcUA branches. Xylanase digestion of xylan from wild-type plants released a short oligosaccharide sequence at the reducing end of the xylan chain. MALDI-TOF MS analysis indicated that this sequence of sugars was absent in xylan from irx7, irx8 and parvus-3 mutants, but was present in irx9 and irx14. This is consistent with previous NMR analysis of xylan from irx7, irx8 and irx9, and suggests that PARVUS may be involved in the synthesis of a xylan primer whereas IRX14 may be required to synthesize the xylan backbone. This hypothesis is supported by assays showing that irx9 and irx14 are both defective in incorporation of radiolabel from UDP 14 C-xylose. This study has important implications for both our understanding of xylan biosynthesis and the functional analysis of cell-wall biosynthesis genes.

RNA Interference Suppression of Genes in Glycosyl Transferase Families 43 and 47 in Wheat Starchy Endosperm Causes Large Decreases in Arabinoxylan Content

“…Since Arabidopsis mutants lacking functional IRX9 or IRX10 have decreased glucuronoxylan chain length (Brown et al, 2007(Brown et al, , 2009Pena et al, 2007), we determined the distribution of WE-AX chain size by TaGT43_2 RNAi lines (lines 1, 3, and 6) and three TaGT47_2 RNAi lines (lines 1, 4, and 6). A to E, Average of azygous controls (A) and homozygous transgenics (H) for three lines; error bars represent SE, and asterisks denote significant differences at P , 0.05 from Student's t test.…”

“…The effects can be compared with those in Arabidopsis xylan due to mutations in the respective homologs of TaGT43_2 and TaGT47_2, IRX9 and IRX10. Arabidopsis plants carrying these mutations also continue to produce xylan (due to the presence of functionally redundant genes), but in severely decreased amounts and with shorter average chain length (Brown et al, 2007(Brown et al, , 2009Pena et al, 2007;Wu et al, 2010;Lee et al, 2012). However, important differences were identified here: (1) Ara substitution was increased in the transgenic wheat lines, whereas GlcA substitution remains constant in the Arabidopsis mutants; (2) a clear difference in phenotype was identified in the loss of long AX chains in TaGT43_2 but not TaGT47_2 RNAi endosperm, whereas no clear difference between the xylan from irx9 and irx10 Arabidopsis mutants has been reported.…”

“…] in a microsomal complex from wheat seedlings with xylan synthase activity (Zeng et al, 2010). From studies in Arabidopsis, it appears that there is a requirement for all three components to maintain xylan synthase activity (Brown et al, 2007(Brown et al, , 2009Pena et al, 2007;Wu et al, 2010;Lee et al, 2012). Since homologous poplar genes are able to complement for the lack of functional IRX9 or IRX14 (Lee et al, 2011), their function seems to have been conserved in evolution.…”

“…Also, GT61 proteins have been shown to be xylan arabinosyl transferases in grasses, including wheat (Anders et al, 2012). While only a IRX14 homolog was detected in this complex, of great interest is whether a IRX9 homolog is also required in grasses for xylan backbone extension, as appears to be the case in woody and herbaceous dicots (Brown et al, 2007;Pena et al, 2007;Lee et al, 2011Lee et al, , 2012.…”

“…Plants carrying mutations in the GT43 family genes IRREGULAR XYLEM9 (IRX9) and IRX14 and in the GT47 family gene IRX10 all have decreased xylan content, with the remaining xylan having a shorter chain length compared with the wild type but the reducing end oligosaccharide still being present (Brown et al, 2007(Brown et al, , 2009Pena et al, 2007;Wu et al, 2009). Closely related genes exist for all these (IRX9-L, IRX10-L, and IRX14-L) but are less expressed and are functionally redundant with the more highly expressed counterpart (Brown et al, 2009;Wu et al, 2010).…”