Extracellular cross‐linking of maize arabinoxylans by oxidation of feruloyl esters to form oligoferuloyl esters and ether‐like bonds

Abstract

Primary cell walls of grasses and cereals contain arabinoxylans with esterified ferulate side chains, which are proposed to cross-link the polysaccharides during maturation by undergoing oxidative coupling. However, the mechanisms and control of arabinoxylan cross-linking in vivo are unclear. Non-lignifying maize (Zea mays L.) cell cultures were incubated with l-[1-(3)H]arabinose or (E)-[U-(14)C]cinnamate (radiolabelling the pentosyl and feruloyl groups of endogenous arabinoxylans, respectively), or with exogenous feruloyl-[(3)H]arabinoxylans. The cross-linking rate of soluble extracellular arabinoxylans, monitored on Sepharose CL-2B, peaked suddenly and transiently, typically at approximately 9 days after subculture. This peak was not associated with appreciable changes in peroxidase activity, and was probably governed by fluctuations in H(2)O(2) and/or inhibitors. De-esterified arabinoxylans failed to cross-link, supporting a role for the feruloyl ester groups. The cross-links were stable in vivo. Some of them also withstood mild alkaline conditions, indicating that they were not (only) based on ester bonds; however, most were cleaved by 6 m NaOH, which is a property of p-hydroxybenzyl-sugar ether bonds. Cross-linking of [(14)C]feruloyl-arabinoxylans also occurred in vitro, in the presence of endogenous peroxidases plus exogenous H(2)O(2). During cross-linking, the feruloyl groups were oxidized, as shown by ultraviolet spectra and thin-layer chromatography. Esterified diferulates were minor oxidation products; major products were: (i) esterified oligoferulates, released by treatment with mild alkali; and (ii) phenolic components attached to polysaccharides via relatively alkali-stable (ether-like) bonds. Thus, feruloyl esters participate in polysaccharide cross-linking, but mainly by oligomerization rather than by dimerization. We propose that, after the oxidative coupling, strong p-hydroxybenzyl-polysaccharide ether bonds are formed via quinone-methide intermediates.