Plant Cell, Tissue and Organ Culture 37: 121-127, 1994. (~) 1994 Klawer Academic Publishers. Printed in the Netherlands. Differential expression of a cell wall-localized peroxidase isoe~e capable of oxidizing 4-hydroxystilbenes during the cell culture of grapevine (Vitis vinifera cv. Airen and Monastrell) A. A. Calder6n, J. M. Zapata & A. Ros Barcel6* Department of Plant Biology (Plant Physiology), University of Murcia, Campus of Espinardo, E-30100 Murcia, Spain (*requests for offprints) Received 31 March 1993; accepted in revised form 1 December 1993 Key words: cell wall localization, differential expression, H202 production, 4-hydroxystilbene oxidizing activity, peroxidase isoenzyme A1, viniferin synthesis Abstract Differential expression and localization of peroxidase isoenzymes capable of oxidizing 4-hydroxystilbenes was studied during establishment of callus cultures from Vitis vinifera 'Airen' (anthocyanin-non accumulating) and 'Monastrell' (anthocyanin-accumulating) berries. Callus formation from mesocarp tissues was accompanied by differential expression of several peroxidase isoenzyemes located in cell walls, among which only peroxidase isoenzyme A1 was capable of oxidizing 4-hydroxystilbene to any great extent. Likewise, grape cell cultures were capable of accumulating the grape stilbene phytoalexin, resveratrol. However, e-viniferin, the most powerful phytoalexin in grapevines and previously considered as the product of peroxidase-mediated oxidative coupling of two resveratrol moieties, was only detectable in trace amounts. Since grapevine suspension cell cultures were unable to produce H202 as revealed by the luminol test, H202 production by the cultured cells appears to be one of the main factors which limits resveratrol oxidation in the cell walls of grapevine cells cultured in suspension. Introduction Studies on the expression of resistance mechanisms have involved whole plants, tissue slices, callus cul- tures and protoplasts (Bell 1981; Gotthardt & Gram- bow 1992). Resistance in callus cultures depends upon critical concentrations of plant growth regulators, organic supplements and on temperature (Holliday & Klarman 1979). Even under optimal conditions, the level of disease resistance in callus cultures is less than that in the whole plants (Holliday & Klarman 1979). Until now cell cultures have offered little advantage over whole plants for integrated studies on resistance, probably due to the fact that they lack structural barri- ers (Bell 1981; Daub 1986). However, they are capa- ble of expressing all of the individual molecular bio- chemical mechanisms, which participate in defence, especially those reactions of the secondary metabolism involved in disease resistance (Rolfs et al. 1987). Thus, it has been shown that suspension cultured grapevine cells are capable to inducing stilbene synthase, the key enzyme in resveratrol synthesis (SchrOder & SchrOder 1990), in response to fungal elicitors (Liswidowati et al. 1991; Melchior & Kindl 1991). e-Viniferin is the most potent phytoalexin in Vitaceae (Langcake & Pryce 1977), and is synthe- sized from resveratrol by peroxidase (Langcake & Pryce 1977). We have employed grapevine cell cul- tures as a standardized system to investigate the activ- ity to peroxidases in catalyzing the oxidative cou- pling of two 4-hydroxystilbene backbones to viniferin- type compounds (Calder6n et al. 1990a) using 4- hydroxystilbene, a stilbene compound that shows the minimal structural characteristics common to resver- atrol and pterostilbene. The aim of the present work was to study the expression of peroxidase isoenzymes capable of oxidizing 4-hydroxystilbenes in grapevine cell cultures in relation to their expression in the plant