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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. [ABSTRACT FROM AUTHOR]

Glucuronoarabinoxylans (GAXs) are the major hemicelluloses in grass cell walls, but the proteins that synthesize them have previously been uncharacterized. The biosynthesis of GAXs would require at least three glycosyltransferases (GTs): xylosyltransferase (XylT), arabinosyltransferase (AraT), and glucuronosyltransferase (GlcAT). A combination of proteomics and transcriptomics analyses revealed three wheat (Triticum aestivum) glycosyltransferase (TaGT) proteins from the GT43, GT47, and GT75 families as promising candidates involved in GAX synthesis in wheat, namely TaGT43-4, TaGT47-13, and TaGT75-4. Coimmunoprecipitation experiments using specific antibodies produced against TaGT43-4 allowed the immunopurification of a complex containing these three GT proteins. The affinity-purified complex also showed GAX-XylT, GAX-AraT, and GAX-GlcAT activities that work in a cooperative manner. UDP Xyl strongly enhanced both AraT and GlcAT activities. However, while UDP arabinopyranose stimulated the XylT activity, it had only limited effect on GlcAT activity. Similarly, UDP GIcUA stimulated the XylT activity but had only limited effect on AraT activity. The [14C]GAX polymer synthesized by the affinity-purified complex contained Xyl, Ara, and GlcUA in a ratio of 45:12:1, respectively. When this product was digested with purified endoxylanase III and analyzed by high-pH anion-exchange chromatography, only two oligosaccharides were obtained, suggesting a regular structure. One of the two oligosaccharides has six Xyls and two Aras, and the second oligosaccharide contains Xyl, Ara, and GIcUA in a ratio of 40:8:1, respectively. Our results provide a direct link of the involvement of TaGT43-4, TaGT47-13, and TaGT75-4 proteins (as a core complex) in the synthesis of GAX polymer in wheat. [ABSTRACT FROM AUTHOR]

The article focuses on xylan biosynthesis in grasses. It mentions that xylan is one plant cell wall polysaccharide that plays a role in the well-being of a person. Unique features of xylans from grasses are given which include the presence of Xyl-arabinofuranosyl side chains and feruloyl groups on the C-5 position of Araf residues. It concludes that a cooperative mechanism is likely used by plant cells to synthesize several hemicellulosic polymers in primary cell walls.

Two homologous plant-specific Arabidopsis thaliana genes, RGXT1 and RGXT2, belong to a new family of glycosyltransferases (CAZy GT-family-77) and encode cell wall (1,3)-α-D-xylosyltransferases. The deduced amino acid sequences contain single transmembrane domains near the N terminus, indicative of a type II membrane protein structure. Soluble secreted forms of the corresponding proteins expressed in insect cells showed xylosyltrensferase activity, transferring D-xylose from UDP-α-xylose to L-fucose. The disaccharide product was hydrolyzed by α-xylosidase, whereas no reaction was catalyzed by β-xylosidase. Furthermore, the regio- and stereochemistry of the methyl xylosyl-fucoside was determined by nuclear magnetic resonance to be an α-(1,3) linkage, demonstrating the isolated glycosyltransferases to be (1,3)-α-D-xylosyltransferases. This particular linkage is only known in rhamnogalacturonan-II, a complex polysaccharide essential to vascular plants, and is conserved across higher plant families. Rhamnogalacturonan-II isolated from both RGXT1 and RGXT2 T-DNA insertional mutants functioned as specific acceptor molecules in the xylosyltransferase assay. Expression of RGXT1- and RGXT2-enhanced green fluorescent protein constructs in Arabidopsis revealed that both fusion proteins were targeted to a Brefeldin A-sensitive compartment and also colocalized with the Golgi marker dye BODIPY TR ceramide, consistent with targeting to the Golgi apparatus. Taken together, these results suggest that RGXT1 and RGXT2 encode Golgi-localized (1,3)-α-D-xylosyltransferases involved in the biosynthesis of pectic rhamnogalacturonan-II. [ABSTRACT FROM AUTHOR]

Plant cell walls have two constituent parts with different components and developmental stages. Much of the mystery concerning the mechanisms of synthesis, decomposition, modification, and so forth, has been resolved using omics and microscopic techniques. However, it still remains to be determined how cell wall development progresses over time after leaf emergence. Our focus in the present study was to expand our knowledge of the molecular mechanisms associated with cell wall synthesis in rice leaf blade during three distinct stages (sink, sink-to-source transition, and source). The RNA-seq, quantitative reverse transcription PCR (qRT-PCR) and carbohydrate concentrations were evaluated using developing fifth leaf blades harvested at different time points. The results revealed that some of the essential genes for the primary cell wall (PCW) were highly upregulated in the sink-to-source transition compared to the sink stage, whereas those essential to the secondary cell wall (SCW) displayed relatively higher levels (p < 0.05) during the source stage. The concentrations of soluble carbohydrates differed via type rather than stage; we observed higher monosaccharides during the sink stage and higher di- and oligo-saccharides during the sink-to-source transition and source stages. In conclusion, our findings suggest that the transcriptional regulation of plant cell wall biosynthesis genes are both synchronistic with and independent of, and directly and indirectly governed by, the abundance of soluble carbohydrates in the developing leaf blade, and, finally, raffinose is likely to play a transport role comparable to sucrose. [ABSTRACT FROM AUTHOR]