Composition, Assembly, and Trafficking of a Wheat Xylan Synthase Complex.

Nan Jiang,Richard E Wiemels,Michael Held,Aaron Soya,Rebekah Whitley,Ahmed Faik

doi:10.1104/pp.15.01777

Abstract

Xylans play an important role in plant cell wall integrity and have many industrial applications. Characterization of xylan synthase (XS) complexes responsible for the synthesis of these polymers is currently lacking. We recently purified XS activity from etiolated wheat (Triticum aestivum) seedlings. To further characterize this purified activity, we analyzed its protein composition and assembly. Proteomic analysis identified six main proteins: two glycosyltransferases (GTs) TaGT43-4 and TaGT47-13; two putative mutases (TaGT75-3 and TaGT75-4) and two non-GTs; a germin-like protein (TaGLP); and a vernalization related protein (TaVER2). Coexpression of TaGT43-4, TaGT47-13, TaGT75-3, and TaGT75-4 in Pichia pastoris confirmed that these proteins form a complex. Confocal microscopy showed that all these proteins interact in the endoplasmic reticulum (ER) but the complexes accumulate in Golgi, and TaGT43-4 acts as a scaffold protein that holds the other proteins. Furthermore, ER export of the complexes is dependent of the interaction between TaGT43-4 and TaGT47-13. Immunogold electron microscopy data support the conclusion that complex assembly occurs at specific areas of the ER before export to the Golgi. A di-Arg motif and a long sequence motif within the transmembrane domains were found conserved at the NH2-terminal ends of TaGT43-4 and homologous proteins from diverse taxa. These conserved motifs may control the forward trafficking of the complexes and their accumulation in the Golgi. Our findings indicate that xylan synthesis in grasses may involve a new regulatory mechanism linking complex assembly with forward trafficking and provide new insights that advance our understanding of xylan biosynthesis and regulation in plants.

Highlights

Xylans play an important role in plant cell wall integrity and have many industrial applications
Phylogenetic analysis showed that the identified proteins clustered with TaGT43-4 and TaGT47-13, but no proteins clustered with Arabidopsis IRX7 (Fig. 1B) or with IRX9/IRX9-L (Fig. 1A)
This result is in agreement with western-blotting analysis in which purified anti-TaGT43-4 and anti-TaGT47-13 antibodies detected the presence of TaGT43-4 and TaGT47-13 in the purified wheat xylan synthase (XS) activity (Zeng et al, 2010)

Summary

Introduction

Xylans play an important role in plant cell wall integrity and have many industrial applications. It is becoming increasingly evident that understanding the mechanisms controlling protein-protein interaction, sorting, and trafficking of polysaccharide synthases (including XSCs) will help elucidate how plants regulate cell wall synthesis and deposition during their development. To this end, we believe that the purified wheat XS activity (Zeng et al, 2010) is an excellent model for this type of study. Characterization of the wheat XSC and its trafficking furthers our understanding of xylan biosynthesis in grasses and helps elucidate how polysaccharide synthase complexes are assembled, sorted, and maintained in different compartments of the secretory pathway

Methods

Results

Conclusion