ABA-dependent amine oxidases-derived H2O2 affects stomata conductance

Abstract

Recently we showed that ABA is at least partly responsible for the induction of the polyamine exodus pathway in Vitis vinifera plants. Both sensitive and tolerant plants employ this pathway to orchestrate stress responses, differing between stress adaptation and programmed cell death. Herein we show that ABA is an upstream signal for the induction of the polyamine catabolic pathway in Vitis vinifera. Thus, amine oxidases are producing H2O2 which signals stomata closure. Moreover, the previously proposed model for the polyamine catabolic pathway is updated and discussed.

Figure 1

Polyamine oxidase-induced H₂O₂ accumulation by Spermidine oxidation in Vitis vinifera. (A) In situ H₂O₂ epifluorescne in control grapevine cells (a), in cells treated with 10 mM Spd for 1 h (b), in cells treated with 10 mM Spd, 0.5 mM aminoguanidine and 0.5 mM guazatine for 1 h (c) and in cells treated with 10 mM Spd and 1 mM ascorbate for 1 h (d). (B) In situ DAB detection of H₂O₂ in fully developed leaves (15^th) of grapevine. Control leaf (a); leaf infiltrated with 10 mM Spd for 10 min prior to DAB infiltration (b); leaf infiltrated with 10 mM Spd for 1 h prior to DAB infiltration (c); close-up pictures of (b) showing H₂O₂ accumulation in secondary veins (d and e) and in closed stomata (f). (C) Hydrogen peroxide detection by transmission Electron microscopy (TEM) in control leaf (a), leaf treated with 10 mM Spd for 1 h (b) and with 10 mM Spd, 0.5 mM aminoguanidine and 0.5 mM guazatine for 1 h (c). Hydrogen peroxide was observed as cerium perhydroxide deposits (CPD) in vascular parenchyma cells. In (b), staining was heavy in cell corner middle lamella and significantly increased in cell walls and in plasma membrane (arrow heads). Staining was sparse in (a) and even lower in (c). CML, cell corner middle lamella; CW, cell wall; IS, intercellular space. (D) Scanning electron microscopy (SEM) of control leaves (a), leaves treated with 10 mM Spd for 1 h (b) and leaves treated with Spd supplemented with 0.5 mM aminoguanidine and 0.5 mM guazatine (c and d). (E) In situ Spd-induced H₂O₂ accumulation in guard cells and stomatal closure. DCFH-DA fluorescence in control strips (a), in leaf strips treated with 10 mM Spd (c) and in strips treated with 0.5 mM aminoguanidine and 0.5 mM guazatine (e). (b, d and f) bright field images of (a, c and e), respectively. Methods were described previously in Moschou et al.

Figure 2

Proposed model for PAO-derived H₂O₂ participation in stress and development. That PAO correlates with development was previously highlighted by Paschalidis and Roubelakis-Angelakis, and Paschalidis et al. During development, intrinsic clues induce PA catabolism and generation of H₂O₂. This molecule seems to act as signal to activate Ca²⁺ channels, and stomata closure (herein). Higher Spd titers or increased PAO activity results to high levels of H₂O₂ that activate Ca²⁺ channels, but the Ca²⁺ influx is either not restricted or the influx is exceedingly high leading to deleterious effects. Under stress, PA exodus into the apoplast is signaled where it is oxidized by PAO. The generated H₂O₂ induces either expression of tolerance-effector genes or the PCD syndrome. The decision depends both, on the size of the H₂O₂ and on the restoration of intracellular PA homeostasis, which is brought about by the simultaneous induction of the PA biosynthetic genes. All the previous reinforce the view that only optimal oxidation of Spd by PAO can lead to normal molecular responses. Moreover, in the case of the control of cation channels the back-conversion pathway seems to possess significant role, but yet a role for this pathway in other responses, like effector-genes induction remains to be established.