Intracellular feruloylation of arabinoxylan in wheat: evidence for feruloyl-glucose as precursor.

Incorporation of [(3)H]arabinose and [(14)C]ferulic acid into soluble and polymeric fractions from suspension-cultured wheat (Triticum aestivum L.) cells and the corresponding extracellular medium was studied. The major part of these products was identified as arabinoxylan and two proteins of 40 and 100 kDa. The time course suggests an intracellular synthesis of feruloylated arabinoxylan with feruloyl-glucose as substrate. In contrast, synthesis of feruloylated proteins appears to occur with feruloyl-CoA as precursor. Intracellular formation of ferulic acid dimers is limited to 8,5'-diferulic acid, while other dimers appear to be formed extracellularly. [(3)H]Arabinose was incorporated into polymeric material in both the cellular and in the medium fraction while [(14)C]ferulic was only found in polymers from the cellular fraction, indicating synthesis of both feruloylated and non-feruloylated arabinoxylan by the cells.

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

Arabinoxylan arabinosyltransferase (AX-AraT) activity was investigated using microsomes and Golgi vesicles isolated from wheat (Triticum aestivum) seedlings. Incubation of microsomes with UDP-[(14)C]-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-[(14)C]Ara resulted in formation of a product that could be solubilized with proteinase K. In contrast, when Golgi vesicles were incubated with UDP-[(14)C]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 [(14)C]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, Mn(2+) 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 [(14)C]Ara labeling became reversible by adding unlabeled UDP-Xyl to the reaction medium. The possible role of this protein in arabinoxylan biosynthesis is discussed.

Dynamic changes in cell wall polysaccharides during wheat seedling development.

Changes in arabinoxylan content and composition during development of wheat seedlings were investigated. The cell walls isolated from the seedlings showed an increasing content of arabinoxylan during development, which could be correlated to increased activity of xylan synthase and arabinoxylan arabinosyltransferase. Arabinoxylan changed from initially having a high degree of arabinose substitution to a much lower degree of substitution. beta-Glucan was present in the walls at the early stages of development, but was actively degraded after day 4. Increased deposition of arabinoxylan did not take place until beta-glucan had been fully degraded. Ferulic and p-coumaric acid esters were present at all points but increased significantly from day 3 to 6, where lignification began. Ferulic acid dimers did not appear in the cell wall until day three and the different ferulic acid dimers varied in the course of accumulation. The ratio of ferulic acid dimers to free ferulic acid was maximal at the time when the wall had been depleted for beta-glucan, which had not yet been fully replaced by arabinoxylan. This pattern suggests a role for ferulic acid dimers in stabilizing the wall during the transition from a flexible to a more rigid structure. To investigate if the same changes could be observed within a single seedling, 7 day old seedlings were divided into four sections and the walls were analyzed. Some of the changes observed during the seedling development could also be observed within a single seedling, when analyzing the segments from the elongation zone at the base to the top of the leaf. However, the expanding region of older seedlings was much richer in hydroxycinnamates than the expanding region of younger seedlings. Diferulic acids are stabilizing the wall in the transition phase from an expanding to a mature wall. This transition can take place in different manners depending on the cell and tissue type.