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NBT staining illustrated that SA elicited O2�C production (Fig.?2b). SHAM significantly suppressed SA-induced O2�C production (P? (Fig.?2b). These results indicate that O2�C production is attributed to activation of SHAM-sensitive peroxidases. To clarify that ROS acts find more as second messenger in SA signalling, we examined SA-induced ROS accumulation in guard cells using 2��,7��-dichlorodihydrofluorescein diacetate (H2DCF-DA). SA increased ROS levels in guard cells compared with control (P? investigate involvement of NO in SA signalling, we examined the effect of a NO specific scavenger [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO)] on SA-induced stomatal closure. SA-induced stomatal closures were significantly suppressed by pre-treatment with cPTIO (P?AZD6738 chemical structure (P?=?0.99) (Fig.?4b). In addition, in SHAM (P?MASP1 proportion of Ca2+ is localized in extracellular spaces. External application of Ca2+ has been shown to promote stomatal closure and induces [Ca2+]cyt oscillation in guard cells (McAinsh et?al. 1995; Allen et?al. 2001). In order to clarify involvement of extracellular Ca2+ in SA-induced stomatal closure, we investigated the effect of Ca2+ chelator (EGTA) and calcium ion channel blocker (LaCl3), in SA-induced stomatal closure. Both EGTA and LaCl3 significantly suppressed SA-induced stomatal closure (P?