The habituation of cultured cells to cellulose biosynthesis inhibitors such as dichlobenil (dichlorobenzonitrile, DCB) has proven a valuable tool to elucidate the mechanisms involved in plant cell wall structural plasticity. Our group has demonstrated that maize cells cope with DCB through a modified cell wall in which cellulose is replaced by a more extensive network of highly cross-linked feruloylated arabinoxylans. In order to gain further insight into the contribution of phenolics to the early remodelling of cellulose-deficient cell walls, a comparative HPLC-PAD analysis was carried out of hydroxycinnamates esterified into nascent and cell wall polysaccharides obtained from non-habituated (NH) and habituated to low DCB concentrations (1.5 μM; H) maize suspensioncultured cells. Incipient DCB-habituated cell walls showed significantly higher levels of esterified ferulic acid and p-coumaric acid throughout the culture cycle. In terms of cell wall fortification, ferulic acid is associated to arabinoxylan crosslinking whereas the increase of p-coumaric suggests an early lignification response. As expected, the level of hydroxycinnamates esterified into nascent polysaccharides was also higher in DCB-habituated cells indicating an overexpression of phenylpropanoid pathway. Due to their key role in cell wall strengthening, special attention was paid into the dimerization pattern of ferulic acid. A quantitative comparison of diferulate dehydrodimers (DFAs) between cell lines and cell compartments revealed that an extra dimerization took place in H cells when both nascent and mature cell wall polysaccharides were analysed. In addition, qualitative differences in the ferulic acid coupling pattern were detected in H cells, allowing us to suggest that 8-O-4′-DFA and 8-5′-DFA featured the ferulic acid dimerization when it occurred in the protoplasmic and cell wall fractions respectively. Both qualitative and quantitative differences in the phenolic profile between NH and H cells point to a regioselectivity in the ferulate dehydrodimerization.

Introduction

Primary cell walls of commelinoid monocots (also known as type II cell walls) such as maize are composed of a framework of cellulose microfibrils embedded in a hemicellulosic matrix of (glucurono)arabinoxylans and a smaller amount of pectins and glycoproteins (Carpita and Gibeaut, 1993). These cell walls are also characterised by the presence of cell wall phenolics, mainly the hydroxycinnamates, ferulic (FA) and p-coumaric (CA) acids, which are ester-linked to α–L-arabinosyl residues of (glucurono)arabinoxylans (Smith and Hartley, 1983; Wende and Fry, 1997). Although phenolics are minor components of the primary cell wall (~1% of cell wall dry weight), their contribution to cell wall assembly seems to be crucial. In vitro experiments have demonstrated that hydroxycinnamates are susceptible to oxidative coupling in the presence of peroxidases and hydrogen peroxide, cross-linking adjacent arabinoxylan (AX) molecules and thus contributing to cell wall assembly (Geissman and Neukom, 1971; Fry et al., 2000; Fry, 2004; Encina and Fry, 2005; Parker et al., 2005; Burr and Fry, 2009). Due to its polysaccharide cross-linking role, phenolic coupling is involved in a number of cell wall properties, causing cell wall stiffening and growth cessation, promoting tissue cohesion, strengthening cell wall structure in response to biotic and abiotic stresses and hindering cell wall degradability (de O Buanafina, 2009).