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• Biochemistry of plant cell wall biosynthesis lacks high-throughput methods. • Overview of new high-throughput technologies to benefit plant cell wall biosynthesis. • Use of protein array technology in enzyme assays and protein–protein interaction. • Adapting recent advances in analytical chemistry for rapid product identification. Progress in the functional biochemical analysis of plant glycosyltransferases (GTs) has been slow because plant GTs are generally membrane proteins, operate as part of larger, multimeric complexes, and utilize a vast complexity of substrate acceptors. Therefore, the field would benefit from development of adequate high throughput expression as well as product detection and characterization techniques. Here we review current approaches to tackle such obstacles and suggest a new path forward: nucleic acid programmable protein arrays (NAPPA) with liquid sample desorption ionization (LS-DESI-MS) mass spectrometry. NAPPA utilizes in vitro transcription and translation to produce epitope-tagged fusion proteins from cloned GT cDNAs. LS-DESI is a soft ionization technique that allows rapid and sensitive MS-based product characterization in situ. Coupling both approaches provides the opportunity to examine individual GT functions as well as protein–protein interactions. Furthermore, advances in automated oligosaccharide synthesis and lipid nanodisc technology should allow testing of plant GT activity in presence of numerous substrate acceptors and lipid environments in a high throughput fashion. Thus, NAPPA-DESI-MS has great potential to make headway in biochemical characterization of the large number of plant GTs. [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.

Examines the role of an Arabidopsis gene encoding an α-xylosyltransferase on xyloglucan biosynthesis. Importance of microsomal membranes on the formation of xyloglucan; Presence of cello-oligosaccharide-dependent xylosyltransferase activity; Characterization of the radiolabeled products.

Microsomal membranes from etiolated wheat (Triticum aestivum) seedlings cooperatively incorporated xylose (Xyl), arabinose, and glucuronic acid residues from their corresponding uridine 5′-diphosphosugars into an ethanol-insoluble glucurono(arabino)xylan (GAX)-like product. A glucuronyltransferase activity that is enhanced by the presence of UDP-Xyl was also identified in these microsomes. Wheat glucuronyltransferase activity was optimal at pH 7 and required manganese ions, and several lines of evidence suggest its involvement in GAX-like biosynthesis. The GAX characteristics of the 14C-product were confirmed by digestion with a purified endo-xylanase from Aspergillus awamori (endo-xylanase III) and by total acid hydrolysis, resulting in a Xyl:arabinose:glucuronic acid molar ratio of approximately 105:34:1. Endoxylanase III released only three types of oligosaccharides in addition to free Xyl. No radiolabel was released as xylobiose, xylotriose, or xylotetraose, indicating the absence of long stretches of unbranched Xyl residues in the nascent GAX-like product. High-pH anion exchange chromatography analysis of the resulting oligosaccharides along with known arabinoxylan oligosaccharide standards suggests that a portion of the nascent GAX-like product has a relatively regular structure. The other portion of the [14C]GAX-like polymer was resistant to proteinase K, endo-polygalacturonase, and endo-xylanase III (GH11 family) but was degraded by Driselase, supporting the hypothesis that the xylan backbone in this portion of the product is most likely highly substituted. Size exclusion chromatography indicated that the nascent GAX-like polymer had an apparent molecular mass of approximately 10 to 15 kD; however, mature GAXs from wheat cell walls had larger apparent molecular masses (>66 kD). [ABSTRACT FROM AUTHOR]

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]

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]