Oxidative defence reactions in sunflower roots induced by methyl-jasmonate and methyl-salicylate and their relation with calcium signalling

Abstract

Ca²⁺ plays a critical role as second messenger in the signal–response coupling of plant defence responses, and methyl-jasmonate and methyl-salicylate are important components of signal transduction cascades activating plant defences. When intact axenic non-induced seedling roots of sunflower were treated with different Ca²⁺ concentrations up to 1 mM, there was no significant increase in O ^.−₂ generation or DMAB–MBTH peroxidase (extracellular, ECPOX) activities in the apoplast, probably because these roots had enough Ca²⁺ in their exo- and endocellular reservoirs. Both activities were strongly inhibited by the RBOH–NADPH oxidase inhibitor DPI and by the Ca²⁺ surrogate antagonist La³⁺, but the voltage-dependent Ca²⁺ channel blocker verapamil was only inhibitory at concentrations higher than those active on animal L-type Ca²⁺ channels. Concentrations >5 mM EGTA (chelating Ca²⁺ in the apoplast) and Li⁺ (inhibiting PI cycle dependent endogenous Ca²⁺ fluxes) also inhibited both activities. W7, inhibitor of binding of Ca–CaM to its target protein, enhanced both activities, but the inactive analogue W5 showed a similar effect. Our data suggest that Ca²⁺ from exocellular and, to a lesser extent, from endocellular stores is involved in oxidative activities, and that RBOH–NADPH oxidase is the main system supporting them. Ca²⁺ activation of the PM cytosolic side of RBOH–NADPH oxidase is probably the key to Ca²⁺ involvement in these processes. Roots induced by MeJA or MeSA showed significant enhancement of both oxidative activities, as corresponding to the oxidative burst evoked by the two phytohormones in the root apoplast. But while ECPOX activity showed a response to the effectors similar to that described above for non-induced roots, O ^.−₂ generation activity in the apoplast of induced roots was insensitive to EGTA, verapamil and Li⁺, the inhibitors of exogenous and endogenous Ca²⁺ fluxes; only DPI and La³⁺ were inhibitory. As exogenously added 0.1 mM Ca²⁺ also increased O ^.−₂ generation, we propose that, in these roots, activation of RBOH–NADPH oxidase by Ca²⁺ could be regulated by Ca²⁺ sensors in the apoplast.