Loss of TaIRX9b gene function in wheat decreases chain length and amount of arabinoxylan in grain but increases cross‐linking

Abstract

Wheat contains abundant xylan in cell walls of all tissues, but in endosperm, there is an unusual form of xylan substituted only by arabinose (arabinoxylan; AX) that has long chains and low levels of feruloylation, a fraction of which is extractable in water (WE-AX). WE-AX acts as soluble dietary fibre but also gives rise to viscous extracts from grain, a detrimental trait for some nonfood uses of wheat. Here, we show that a glycosyl transferase family 43 wheat gene abundantly expressed in endosperm complements the Arabidopsis irx9 mutant and so name the three homoeologous genes TaIRX9b. We generated wheat lines with a constitutive knockout of TaIRX9b by stacking loss-of-function alleles for these homeologues from a mutagenized hexaploid wheat population resulting in decreases in grain extract viscosity of 50%-80%. The amount and chain length of WE-AX molecules from grain of these triple-stack lines was decreased accounting for the changes in extract viscosity. Imaging of immature wheat grain sections of triple-stacks showed abolition of immunolabelling in endosperm with LM11 antibody that recognizes epitopes in AX, but also showed apparently normal cell size and shape in all cell types, including endosperm. We identified differentially expressed genes from endosperm of triple-stacks suggesting that compensatory changes occur to maintain this endosperm cell wall integrity. Consistent with this, we observed increased ferulate dimerization and increased crosslinking of WE-AX molecules in triple-stacks. These novel wheat lines lacking functional TaIRX9b therefore provide insight into control of wheat endosperm cell walls.

RNAi suppression of xylan synthase genes in wheat starchy endosperm

“…The proportions of short AXOS released by GH11 digestion were decreased more in the single GT43_1 and triple lines (to 33%, 35%) lines than total AX, as was the proportion of WE-X (decreased to 21%, 20%) ( Table 2 ). This was also the case in our previous studies of RNAi suppression of GT43_2 and GT47_2 and of a triple mutant of GT43_2, where suppression of these genes induced a change in AX composition such that the solubility and digestibility with the GH11 endoxylanase were decreased [ 10 , 15 , 28 ]. This could be because suppression of these genes knock-outs xylan synthase complexes which synthesis a specific type of AX, or because the loss of cell AX in the endosperm cell walls results in compensatory increases in AX cross-linking: there was evidence of such compensation in our previous studies [ 15 , 29 ] but that does not rule out the first hypothesis.…”

“…RNAi down-regulation of the single xylan synthase subunit encoded by GT43_1 and simultaneous down-regulation of all three subunits (encoded by GT43_1, GT43_2, GT47_2) resulted in similar decreases in total AX, to 46% and 47% of the total X determined by monosaccharide analysis ( Table 2 ). This compares with previously reported RNAi down-regulation of single xylan synthase subunit genes (GT43_2 or GT47_2) which resulted in decreases in total AX [ 10 ] to 50–60% of those in the control lines and the development of a triple knock-out mutant of GT43_2 (stacking mutations in A,B,D homeologues of GT43_2A,B,D/TaIRX9b) in which total AX was reduced to about 65% of that in the control line [ 15 ]. The proportions of short AXOS released by GH11 digestion were decreased more in the single GT43_1 and triple lines (to 33%, 35%) lines than total AX, as was the proportion of WE-X (decreased to 21%, 20%) ( Table 2 ).…”

“…In wheat endosperm, the genes that are most likely to encode the components of xylan synthase are GT43_1 (IRX14 homologue), GT43_2 (known to be a functional IRX9; [ 15 ]) and GT47_2 (IRX10 homologue), as these are the most highly expressed IRX14, IRX9 and IRX10 genes in endosperm [ 8 ]. Consistent with this, the suppression of either GT43_2 or GT47_2 by RNAi in wheat endosperm led to up to 50% decrease in total AX content [ 10 ] while a triple mutant knockout of the three homeologues of GT43_2 (or TaIRX9b) had a 35% decrease in total AX [ 15 ].…”

“…In wheat endosperm, the genes that are most likely to encode the components of xylan synthase are GT43_1 (IRX14 homologue), GT43_2 (known to be a functional IRX9; [ 15 ]) and GT47_2 (IRX10 homologue), as these are the most highly expressed IRX14, IRX9 and IRX10 genes in endosperm [ 8 ]. Consistent with this, the suppression of either GT43_2 or GT47_2 by RNAi in wheat endosperm led to up to 50% decrease in total AX content [ 10 ] while a triple mutant knockout of the three homeologues of GT43_2 (or TaIRX9b) had a 35% decrease in total AX [ 15 ]. Although the role of GT43_1 in wheat endosperm has not so far been confirmed the GT43_1 protein has been identified as being a key component of a xylan synthase complex from wheat seedlings ([ 13 ]; called “TaGT43-4” in their nomenclature) as, unlike GT43_2 and GT47_2, it is highly expressed in vegetative tissues.…”

Probing the role of cell wall feruloylation during maize development by differential expression of an apoplast targeted fungal ferulic acid esterase

“…The xax1 rice mutants which had reduced xylan and a lower degree of diferulate cross-links, also exhibited ~10% increase in terminal arabinose [ 54 ]. More recently Pellnny et al (2020) [ 55 ] have shown that knocking down expression of three Ta IRX9B (orthologs of Arabidopsis irx9) genes in wheat endosperm resulted in reduced levels of AX and increased ferulate dimerization. These observations, which are in line with the data presented here, points to a compensatory mechanism in plants that indicates a level of interdependence between different groups of cell wall components.…”