Arabinoxylan Biosynthesis in Wheat. Characterization of Arabinosyltransferase Activity in Golgi Membranes

• Published in: Plant physiology (Bethesda) 2002-09, Vol.130 (1), p.432-441
• Main Authors: Porchia, Andrea Celia, Sørensen, Susanne Oxenbøll, Scheller, Henrik Vibe
• Format: Article
• Language: English
• Subjects: Agronomy. Soil science and plant productions; Biochemical Processes and Macromolecular Structures; Biological and medical sciences; Biosynthesis; Carbon Radioisotopes; Cell biochemistry; Cell physiology; Cell walls; Chromatography, Gel; Economic plant physiology; Enzymes; Fundamental and applied biological sciences. Psychology; Golgi Apparatus - enzymology; Golgi Apparatus - metabolism; Intracellular Membranes - drug effects; Intracellular Membranes - metabolism; Net assimilation, photosynthesis, carbon metabolism. Photorespiration, respiration, fermentation (anoxia, hypoxia); Nutrition. Photosynthesis. Respiration. Metabolism; Pentosyltransferases - metabolism; Plant cells; Plant physiology and development; Plants; Polysaccharides; Radioactive decay; Seedlings; Triticum - drug effects; Triticum - enzymology; Triticum - metabolism; Uridine Diphosphate Sugars - pharmacology; Wheat; Xylans; Xylans - biosynthesis; Xylose - metabolism
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.003400

Arabinoxylan arabinosyltransferase (AX-AraT) activity was investigated using microsomes and Golgi vesicles isolated from wheat (Triticum aestivum) seedlings. Incubation of microsomes with UDP-[14C]-β-L-arabinopyranose resulted in incorporation of radioactivity into two different products, although most of the radioactivity was present in xylose (Xyl), indicating a high degree of UDP-arabinose (Ara) epimerization. In isolated Golgi vesicles, the epimerization was negligible, and incubation with UDP-[14C]Ara resulted in formation of a product that could be solubilized with proteinase K. In contrast, when Golgi vesicles were incubated with UDP-[14C]Ara in the presence of unlabeled UDP-Xyl, the product obtained could be solubilized with xylanase, whereas proteinase K had no effect. Thus, the AX-AraT is dependent on the synthesis of unsubstituted xylan acting as acceptor. Further analysis of the radiolabeled product formed in the presence of unlabeled UDP-Xyl revealed that it had an apparent molecular mass of approximately 500 kD. Furthermore, the total incorporation of [14C]Ara was dependent on the time of incubation and the amount of Golgi protein used. AX-AraT activity had a pH optimum at 6, and required the presence of divalent cations, Mn2+ being the most efficient. In the absence of UDP-Xyl, a single arabinosylated protein with an apparent molecular mass of 40 kD was radiolabeled. The [14C]Ara labeling became reversible by adding unlabeled UDP-Xyl to the reaction medium. The possible role of this protein in arabinoxylan biosynthesis is discussed.