Plant Science 130 (1997) 97–105 The subcellular localization of peroxidase and the implication of oxidative stress in hyperhydrated leaves of regenerated carnation plants Enrique Olmos, Abel Piqueras, Juan Ramo ´n Martı ´nez-Solano, Eladio Hellı ´n * Departamento de Nutricio ´n y Fisiologı ´a Vegetal, Centro de Edafologı ´a y Biologı ´a Aplicada del Segura, CSIC, Aenida de la Fama 1, P.O. Box 4195, 30080 Murcia, Spain Received 20 February 1997; received in revised form 23 September 1997; accepted 29 September 1997 Abstract The immunolocalization and distribution of peroxidases from hyperhydrated leaves of carnation were studied. A lower lignification and higher water content were observed in hyperhydrated leaves. To analyze the possible involvement of oxidative stress in the process of hyperhydration, peroxidase activity and lipid peroxidation products (MDA) were measured. Hyperhydrated leaves differed from normal ones in total peroxidase activity, in the isozyme pattern and distribution of peroxidase activity by immunogold labelling. These results suggest the existence of an oxidative stress during hyperhydricity in carnation leaves. © 1997 Elsevier Science Ireland Ltd. Keywords: Carnation; Hyperhydricity; Immunogold; Lignification; Peroxidase pattern 1. Introduction Hyperhydricity is a physiological disorder fre- quently related to the in vitro environment during micropropagation. Although many factors able to induce hyperhydricity have been identified, it is not completely predictable. When it occurs, tissues undergo changes in their metabolic processes, lead- ing to an abnormal morphology and anatomy [1]. The hyperhydricity-related alterations affect primarily the surface of the tissues whose cells have low lignin content with an abnormal epider- mis and alterations in epicuticular wax deposition [2,3]. It has been suggested that lignification is deficient in these cells because of a reduced PAL and peroxidase activity in the different cell com- partments mediated by ethylene [4]. Hypolignifi- cation causes malformations in the vascular tissues, frequently tracheids are missing in stems, buds and leaves. In these tissues, the elongation * Corresponding author. Tel.: +34 68 215717; fax: +34 68 266613; e-mail: ehellin@natura.cebas.csic.es 0168-9452/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S0168-9452(97)00214-8