β-1,4-Xylan backbone synthesis in higher plants: How complex can it be?

Abstract

Xylan is a hemicellulose present in the cell walls of all land plants. Glycosyltransferases of the GT43 (IRX9/IRX9L and IRX14/IRX14L) and GT47 (IRX10/IRX10L) families are involved in the biosynthesis of its β-1,4-linked xylose backbone, which can be further modified by acetylation and sugar side chains. However, it remains unclear how the different enzymes work together to synthesize the xylan backbone. A xylan synthesis complex (XSC) has been described in the monocots wheat and asparagus, and co-expression of asparagus AoIRX9, AoIRX10 and AoIRX14A is required to form a catalytically active complex for secondary cell wall xylan biosynthesis. Here, we argue that an equivalent XSC exists for the synthesis of the primary cell wall of the eudicot Arabidopsis thaliana, consisting of IRX9L, IRX10L and IRX14. This would suggest the existence of distinct XSCs for primary and secondary cell wall xylan synthesis, reminiscent of the distinct cellulose synthesis complexes (CSCs) of the primary and secondary cell wall. In contrast to the CSC, in which each CESA protein has catalytic activity, the XSC seems to contain proteins with non-catalytic function with each component bearing potentially unique but crucial roles. Moreover, the core XSC formed by a combination of IRX9/IRX9L, IRX10/IRX10L and IRX14/IRX14L might not be stable in its composition during transit from the endoplasmic reticulum to the Golgi apparatus. Instead, potential dynamic changes of the XSC might be a means of regulating xylan biosynthesis to facilitate coordinated deposition of tailored polysaccharides in the plant cell wall.

Keywords: IRX10/IRX10L; IRX14/IRX14L; IRX9/IRX9L; Xylan biosynthesis; enzyme complex; primary cell wall.

Figure 1

The XSC in primary cell wall xylan synthesis in Arabidopsis. (A) Anti-GFP immunoprecipitation of the XSC in Arabidopsis root callus. Mascot protein scores for IRX9L, IRX10L, IRX14 and STL1 (control) are shown for three biological replicates using IRX14-GFP (left) or STL1-GFP (control, right) as bait, respectively. (B) AlphaFold-Multimer model of a heterotrimer of the IRX9L (blue), IRX10L (violet), and IRX14 (yellow) globular domain. A putative nucleotide-binding residue, Asp296 (Wilson et al., 2022), is shown in IRX10L. Note the predicted α-helix of IRX14 at the interaction surfaces of both IRX9L and IRX10L (asterisk). (C) Phenotype of the adherent mucilage of XSC mutants. Mucilage is stained with ruthenium red. Wt: Columbia Col-0 wildtype control. Scale bar = 100 µm.

Figure 2

The CTS of GT43s, their potential interactions and impact of interactions on Golgi localization. (A). Sequence logos showing conserved motifs in the predicted transmembrane helices (TMH) of orthologs of IRX9/IRX9L (top) and IRX14/IRX14L (bottom). (B) Close-ups of AlphaFold-Multimer models of the transmembrane dimers for an IRX9 homodimer (top), an IRX9–IRX14 heterodimer (middle) and an IRX14 homodimer (bottom). Residues of the GAS_right motif and conserved cysteines are highlighted in magenta or dark blue, respectively. (C) Subcellular localization of Arabidopsis IRX9-GFP, transiently expressed in tobacco leaves. Top panel (left to right), IRX9-GFP (green), ManI-mCherry from soy (Golgi marker, red), merge of the two former, and merge including differential interference contrast (DIC) image. Bottom panel shows the same localization analysis in presence of co-expressed, untagged IRX10 and IRX14. Note the change of localization of IRX9-GFP in presence of IRX10 and IRX14.