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Arabinoxylan biosynthesis in wheat. Characterization of arabinosyltransferase activity in Golgi membranes

Author:
Porchia, A.C., Sorensen, S.O., Scheller, H.V.
Source:
Plant physiology 2002 v.130 no.1 pp. 432-441
ISSN:
0032-0889
Subject:
Triticum aestivum, wheat, food crops, seedlings, Golgi apparatus, microsomes, plant proteins, transferases, enzyme activity, xylan, biosynthesis, pH, manganese, cations, solubilization, carbon, isotopes, isotope labeling, chemical constituents of plants, arabinoxylan
Abstract:
Arabinoxylan arabinosyltransferase (AX-AraT) activity was investigated using microsomes and Golgi vesicles isolated from wheat (Triticum aestivum) seedlings. Incubation of microsomes with UDP-[14C]-beta-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.
Agid:
671317