Changes in the water binding characteristics of the cell walls from transgenic Nicotiana tabacum leaves with enhanced levels of peroxidase activity Jose´ A. Mercado a , Antonio J. Matas b , Antonio Heredia b , Victoriano Valpuesta b and Miguel A. Quesada a, * a Departamento de Biologı´a Vegetal, Universidad de Ma ´laga, 29071 Ma ´laga, Spain b Departamento de Biologı´a Molecular y Bioquı´mica, Universidad de Ma ´laga, 29071 Ma ´laga, Spain *Corresponding author, e-mail: quefe@uma.es Received 28 July 2004 Pore size in the cell wall matrix may affect cell wall–water relations, particularly under osmotic stress. Cross linkage of plant cell wall matrix polymers is an important step in the formation of this structure and peroxidases have been pro- posed to catalyse the cross-linking of phenolic constituents. Transgenic tobacco (Nicotiana tabacum) plants expressing a basic tomato peroxidase gene (TPX2) showed increased apo- plastic ferulic acid peroxidase activity in mature leaves. This enhanced activity was not associated with a decreased leaf growth. Differential scanning calorimetry (DSC) of control dried cell walls showed a putative glass transition, after Ca 21 removal, that was absent in the transgenic line. This would indicate that transgenic walls were more rigid. DSC analysis of water-hydrated cell wall preparations distinguished two pools of water, freezable and non-freezable water. The amount of non-freezable water, which corresponds to strongly bound water, was higher in the transgenic line (64 versus 55%). DSC thermograms of the transgenic cell wall were displaced to lower temperatures, and this may be interpreted as the result of a stronger interaction between this freezable water and this wall. Water sorption and desorption isotherms, obtained at relative humidity ranging from 5 to 93%, demon- strated the presence of very strongly bound water in the transgenic cell walls that was absent in controls. Water sorp- tion–desorption hysteresis of the isotherms was evident in the control wall but not in the transgenic line. These changes in cell wall–water interaction seem to be relevant at the organ level because leaf discs of transgenic plants maintained higher relative water content than control discs, at water potentials between 1.05 and2.31 MPa. Introduction Plant peroxidases are enzymes that catalyse the oxida- tion of a variety of substrates using hydrogen peroxide as oxidant. These enzymes have been involved in several physiological processes, from cell growth and differentia- tion to biotic and abiotic stress responses (Gaspar et al. 1991, Mohan et al. 1993). Transgenic plants with mod- ified peroxidase expression have been also generated in an attempt to elucidate the role of specific peroxidase isoenzymes (Lagrimini et al. 1990, 1992, El Mansouri et al. 1999). The present work focuses the effect that cell wall peroxidases may exert on the water-holding capacity of the cell walls. It is noteworthy that the role of peroxidase activity in cell walls has been intensively studied, as well as its correlation with growth, but the above-mentioned subject has generally been overlooked. In general, studies of the chemical composition of the plant cell wall distinguish two main phases: a micro- fibrillar phase, mainly cellulosic in nature, and a matrix phase which is extremely complex. This matrix phase includes pectins, hemicelluloses, proteins and phenolics. An important phenolic constituent of the cell wall is ferulic acid, which is esterified to arabinose and galactose in pectins and hemicelluloses and may have an important role in the cross-linking of these polymers (Brett and Waldron 1996). In addition, walls of some differentiated cells also contain the phenolic polymer lignin PHYSIOLOGIA PLANTARUM 122: 504–512. 2004 doi: 10.1111/j.1399-3054.2004.00429.x Printed in Denmark – all rights reserved Copyright #Physiologia Plantarum 2004 Abbreviations – DSC, differential scanning calorimetry; MS, Murashige and Skoog basal medium; RWC, relative water content. 504 Physiol. Plant. 122, 2004