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Summary: Respiration in leaves and the continued elevation in the atmospheric CO2 concentration cause CO2‐mediated reduction in stomatal pore apertures. Several mutants have been isolated for which stomatal responses to both abscisic acid (ABA) and CO2 are simultaneously defective. However, there are only few mutations that impair the stomatal response to elevated CO2, but not to ABA. Such mutants are invaluable in unraveling the molecular mechanisms of early CO2 signal transduction in guard cells. Recently, mutations in the mitogen‐activated protein (MAP) kinase, MPK12, have been shown to partially impair CO2‐induced stomatal closure. Here, we show that mpk12 plants, in which MPK4 is stably silenced specifically in guard cells (mpk12 mpk4GC homozygous double‐mutants), completely lack CO2‐induced stomatal responses and have impaired activation of guard cell S‐type anion channels in response to elevated CO2/bicarbonate. However, ABA‐induced stomatal closure, S‐type anion channel activation and ABA‐induced marker gene expression remain intact in the mpk12 mpk4GC double‐mutants. These findings suggest that MPK12 and MPK4 act very early in CO2 signaling, upstream of, or parallel to the convergence of CO2 and ABA signal transduction. The activities of MPK4 and MPK12 protein kinases were not directly modulated by CO2/bicarbonate in vitro, suggesting that they are not direct CO2/bicarbonate sensors. Further data indicate that MPK4 and MPK12 have distinguishable roles in Arabidopsis and that the previously suggested role of RHC1 in stomatal CO2 signaling is minor, whereas MPK4 and MPK12 act as key components of early stomatal CO2 signal transduction. Significance statement: We show that MPK4 and MPK12 are required for CO2‐triggered stomatal responses but not for ABA‐induced stomatal closure. These results suggest that these two MAP kinases function upstream of the convergence of CO2 and ABA signaling. However, activities of MPK4 and MPK12 were not modulated by CO2/bicarbonate and, thus, direct HCO3−/CO2 sensing mechanisms remain to be identified. [ABSTRACT FROM AUTHOR]

Stomatal pores are formed between a pair of guard cells and allow plant uptake of CO2 and water evaporation. Their aperture depends on changes in osmolyte concentration of guard cell vacuoles, specifically of K+ and Mal2−. Efflux of Mal2− from the vacuole is required for stomatal closure; however, it is not clear how the anion is released. Here, we report the identification of ALMT4 (ALUMINUM ACTIVATED MALATE TRANSPORTER4) as an Arabidopsis thaliana ion channel that can mediate Mal2− release from the vacuole and is required for stomatal closure in response to abscisic acid (ABA). Knockout mutants showed impaired stomatal closure in response to the drought stress hormone ABA and increased whole-plant wilting in response to drought and ABA. Electrophysiological data show that ALMT4 can mediate Mal2− efflux and that the channel activity is dependent on a phosphorylatable C-terminal serine. Dephosphomimetic mutants of ALMT4 S382 showed increased channel activity and Mal2− efflux. Reconstituting the active channel in almt4 mutants impaired growth and stomatal opening. Phosphomimetic mutants were electrically inactive and phenocopied the almt4 mutants. Surprisingly, S382 can be phosphorylated by mitogen-activated protein kinases in vitro. In brief, ALMT4 likely mediates Mal2− efflux during ABA-induced stomatal closure and its activity depends on phosphorylation. [ABSTRACT FROM AUTHOR]