Arabidopsis Family GT43 Members are Xylan Xylosyltransferases Required for the Elongation of the Xylan Backbone

Abstract

Xylan is the second most abundant polysaccharide in plant biomass targeted for biofuel production. Therefore, it is imperative to understand the biochemical mechanism underlying xylan biosynthesis. Although previous genetic studies have identified several genes implicated in xylan biosynthesis, biochemical proof of any of their encoded proteins as a xylan xylosyltransferase (XylT) responsible for xylan backbone biosynthesis is still lacking. In this study, we investigated the enzymatic activities of two Arabidopsis thaliana GT43 members, IRX9 (Irregular Xylem9) and IRX14, which have been genetically shown to be non-redundantly involved in the elongation of the xylan backbone. IRX9 and IRX14, alone or simultaneously, were heterologously expressed in tobacco BY2 cells, and microsomes isolated from the transgenic BY2 cells were tested for XylT activity using xylotetraose (Xyl(4)) as an acceptor and UDP-[(14)C]xylose as a donor. It was found that although microsomes with expression of IRX9 or IRX14 alone exhibited little incorporation of radiolabeled xylose, a high level of incorporation of radiolabeled xylose onto Xyl(4) was conferred by microsomes with co-expression of IRX9 and IRX14. Further analysis using fluorescent anthranilic acid-labeled xylotetraose (Xyl(4)-AA) as an acceptor revealed that up to five β-(1,4)-linked xylosyl residues were able to be transferred onto Xyl(4)-AA by microsomes with co-expression of IRX9 and IRX14. Furthermore, it was shown that xylooligomers ranging from Xyl(3)-AA to Xyl(6)-AA could all be used as acceptors for the xylosyl transfer by microsomes with co-expression of IRX9 and IRX14. Together, these findings provide the first biochemical evidence that IRX9 and IRX14 are xylosyltransferases that operate cooperatively in the elongation of the xylan backbone.

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

“…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.…”

“…It is unclear how the products from the very different GT43 and GT47 gene families, which appear to be equally essential in Arabidopsis, cooperate to synthesize the xylan backbone. By overexpressing IRX9 and IRX14 together in tobacco (Nicotiana tabacum) BY2 cells, xylan xylosyl transferase activity was stimulated, suggesting that they encode the key proteins mediating the activity, although an endogenous IRX10 ortholog would presumably also be present in these cells (Lee et al, 2012). On the other hand, abundant xylan is synthesized in psyllium (Plantago afra) seed mucilage, which has abundant transcripts of an IRX10 ortholog but only very low levels of transcripts of IRX9 and IRX14 orthologs (Jensen et al, 2011).…”

“…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.…”

Genetic Determinants for Enzymatic Digestion of Lignocellulosic Biomass Are Independent of Those for Lignin Abundance in a Maize Recombinant Inbred Population

“…Saccharification-related QTL include genes that encode cellulose-and xylan-related enzymes such as UDPglucuronosyltransferases, CELLULOSE SYNTHASE2, the nucleotide interconversion pathway gene UDP-XYLOSE EPIMERASE1 (MUR4), and IRREGULAR XYLEM14 (Supplemental Table S5). IRREGULAR XYLEM14 coexpressed with IRREGULAR XYLEM9 represent synthases of secondary wall xylan backbones (Lee et al, 2012). In addition, signaling-related genes such as wall-associated kinases and homeobox transcription factor regulators and several different types of transcription factors are also overexpressed in the dominant parent, notably, a hecate transcription factor (HEC1), a homeobox-leucine zipper gene (HAT14), and a cell wall-associated kinase (WAK1; Supplemental Table S5).…”