Hydrogen Peroxide Is Required for Poly(phenolic) Domain Formation during Wound-Induced Suberization FAWZI A. RAZEM AND MARK A. BERNARDS* Department of Plant Sciences, The University of Western Ontario, London, ON, N6A 5B7 Canada The requirement for hydrogen peroxide (H 2 O 2 ) during suberization was demonstrated in wound- induced potato tubers by monitoring the extent of phenolic polymerization after the inhibition of H 2 O 2 production using diphenyleneiodonium (DPI). In DPI-treated tissues the extent of phenolic polymer- ization in suberized tissues, measured using DFRC (Derivatization Followed by Reductive Cleavage) and thioglycolic acid analyses, was greatly reduced relative to untreated controls. Concomitantly, a large quantity of new soluble phenolics accumulated in the DPI-treated tissue some of which were not present in the controls. We suggest that the inhibition of H 2 O 2 production prevented these phenolics from being oxidized by cell wall peroxidases. As a result, these phenolics were left unpolymerized and accumulated in the tissue. Several of the soluble phenolics were identified as hydroxycinnamic acid derivatives. From the data presented, it was concluded that H 2 O 2 is required for the polymerization step in the formation of the poly(phenolic) domain of suberized potato tubers. KEYWORDS: Solanum tuberosum; Derivatization Followed by Reductive Cleavage (DFRC); diphenyl- eneiodonium; hydrogen peroxide; hydroxycinnamic acids; NADPH-dependent oxidase; poly(phenolic) domain; soluble phenolics; thioglycolic acid; wall-bound phenolics; wound-induced suberization INTRODUCTION Over the course of evolution, plants have developed effective defense mechanisms to overcome environmental challenges, including stresses caused by injuries, pathogens, and a desic- cating environment. Plant defenses tend to be site specific such that the affected cells or tissues are healed or isolated from healthy, unaffected ones. In the case of injury, this is usually accomplished by producing high quantities of toxic chemicals (e.g., H 2 O 2 , secondary metabolites) to kill pathogens, followed by the formation of a polymeric barrier next to the infected site (e.g., a suberized layer). For example, in potato tubers challenged with pathogens, large amounts of free radicals are produced (1) and rapid suberization is necessary to provide an effective resistance against dehydration and pathogen penetration (2). Apart from the direct toxic effect of H 2 O 2 on pathogens, other roles for this reactive oxygen species (ROS) have also been proposed, including the oxidative cross-linking of cell wall components, (reviewed in 3). This latter role is potentially important during the wound-healing process, since it would allow the formation of a physical barrier to prevent water loss and further pathogen penetration, (reviewed in 4, 5). Further- more, in wound healing potato tubers, suberizing cells not only accumulate H 2 O 2 , but also actively produce it via the dispro- portionation of O 2 - (1, 6). This production and accumulation of H 2 O 2 is an indication of the potential involvement of H 2 O 2 in suberization. Suberization is the name given to the deposition of a specific cell wall modification in periderm, wound periderm, and endo- and exodermal cells, that is characterized by both a poly- (phenolic) domain and wax-embedded poly(aliphatic) domain. The term suberin, however, is often associated with the aliphatic component of suberized tissues, while the poly(phenolic) domain has been labeled a lignin. And while there is an element of truth to this description, two recent major breakthroughs have reshaped our understanding of suberization: (i) direct evidence that the poly(phenolic) domain contains a significant amount of covalently cross-linked hydroxycinnamic acids (7, 8), and (ii) that glycerol is a significant component of the poly(aliphatic) domain (9, 10). Based on these seminal results, and many supporting papers, a new conceptual picture of suberized cells has emerged (10, 11). In this model, the poly(phenolic) domain (herein referred to as the SPPD) contains a significant amount of nonlignin precursors (principally hydroxycinnamic acids and their derivatives) that are covalently linked to one another and embedded in the primary cell wall. Suberin, which is covalently linked to the SPPD at the cell wall surface, is depicted as a linear, 3-dimensional, glycerol-bridged polyester network. The polymerization of phenolics into the SPPD of potato has been hypothesized to occur via a peroxidase/H 2 O 2 mediated free radical coupling process (5) and a specific anionic peroxidase has been implicated (12, 13). In order for this hypothesis to be correct, there has to be a supply of H 2 O 2 at the site of polymerization. It should be noted, however, that H 2 O 2 involve- ment in suberization has not yet been demonstrated and no H 2 O 2 generating system has been linked to it. Furthermore, the * To whom correspondence should be addressed. E-mail: bernards@ uwo.ca, Phone: 519-661-2111, ext 86477, Fax: 519-661-3935. J. Agric. Food Chem. 2002, 50, 1009-1015 1009 10.1021/jf0110248 CCC: $22.00 © 2002 American Chemical Society Published on Web 02/01/2002