Zinc-induced oxidative stress in Verbascum thapsus is caused by an accumulation of reactive oxygen species and quinhydrone in the cell wall

Abstract

Oxidative stress is one aspect of metal toxicity. Zinc, although unable to perform univalent oxido-reduction reactions, can induce the oxidative damage of cellular components and alter antioxidative systems. Verbascum thapsus L. plants that were grown hydroponically were exposed to 1 and 5 mM Zn²+. Reactive oxygen species (ROS) accumulation was demonstrated by the fluorescent probe H₂ DCFDA and EPR measurements. The extent of zinc-induced oxidative damage was assessed by measuring the level of protein carbonylation. Activities and isoform profiles of some antioxidant enzymes and the changes in ascorbate and total phenolic contents of leaves and roots were determined. Stunted growth because of zinc accumulation, preferentially in the roots, was accompanied by H₂O₂ production in the leaf and root apoplasts. Increased EPR signals of the endogenous oxidant quinhydrone, •CH₃ and •OH, were found in the cell walls of zinc-treated plants. The activities of the antioxidative enzymes ascorbate peroxidase (APX) (EC 1.11.1.11), soluble superoxide dismutase (SOD) (EC 1.15.1.1), peroxidase (POD), (EC 1.11.1.7) and monodehydroascorbate reductase (EC 1.6.5.4) were increased; those of glutathione reductase (EC 1.6.4.2), dehydroascorbate reductase (EC 1.8.5.1) and ascorbate oxidase (AAO) (EC 1.10.3.3) were decreased with zinc treatment. Zinc induced a cell-wall-bound SOD isoform in both organs. Leaves accumulated more ascorbate and phenolics in comparison to roots. We propose a mechanism for zinc-promoted oxidative stress in V. thapsus L. through the generation of charge transfer complexes and quinhydrone because of phenoxyl radical stabilisation by Zn²+ in the cell wall. Our results suggest that the SOD and APX responses are mediated by ROS accumulation in the apoplast. The importance of the POD/Phe/AA (ascorbic acid) scavenging system in the apoplast is also discussed.