Role and interrelationship of MEK1-MPK6 cascade, hydrogen peroxide and nitric oxide in darkness-induced stomatal closure

- The role and interrelationship of MEK1-MPK6 cascade, H2O2 and NO in dark-induced stomatal closure were studied.
- Darkness activates MPK6 and MPK3 in Arabidopsis leaves via MEK1- and H2O2-dependent and NO-independent manners.
- MEK1-MPK6 cascade, AtRBOHD/F-sourced H2O2 and NIA1-origined NO all participate in the dark-induced stomatal closure.
- H2O2 mediates dark-induced NO generation in guard cells and subsequent stomatal closure.
- MEK1-MPK6 cascade mediates H2O2-induced NO production in darkness guard cell signaling.

Pharmacological data have suggested the involvement of mitogen-activated protein kinase (MPK) cascades in dark-induced stomatal closure, but which specific MPK cascade participates in the darkness guard cell signaling and its relationship with hydrogen peroxide (H2O2) and nitric oxide (NO) remain unclear. In this paper, we observed that darkness induced activation of MPK6 in leaves of wild-type Arabidopsis (Arabidopsis thaliana) and mutants for nitrate reductase 1 (NIA1), but this effect was inhibited in mutants for MPK Kinase 1 (MEK1) and ATRBOHD/F. Mutants for MEK1, MPK6 and NIA1 showed defect of dark-induced NO production in guard cells and stomatal closure, but were normal in the dark-induced H2O2 generation, while stomata of mutant AtrbohD/F showed defect of dark-induced H2O2 and NO production and subsequent closure. Moreover, exogenous NO rescued the defect of dark-induced stomatal closure in mutants of AtrbohD/F, mek1 and mpk6, while exogenous H2O2 could not rescue the defect of dark-induced stomatal closure in mutants of mek1, mpk6 and nia1. These genetic and biochemical evidences not only show that MEK1-MPK6 cascade, AtRBOHD/F-dependent H2O2 and NIA1-dependent NO are all involved in dark-induced stomatal closure in Arabidopsis, also indicate that MEK1-MPK6 cascade functions via working downstream of H2O2 and upstream of NO.