Extracellular malate induces stomatal closure via direct activation of guard-cell anion channel SLAC1 and stimulation of Ca²⁺ signalling.

Plants secrete malate from guard cells to apoplast under stress conditions and exogenous malate induces stomatal closure. Malate is considered an extracellular chemical signal of stomatal closure. However, the molecular mechanism of malate-induced stomatal closure is not fully elucidated. We investigated responses of stomatal aperture, ion channels, and cytosolic Ca²⁺ to malate. A treatment with malate induced stomatal closure in Arabidopsis thaliana wild-type plants, but not in the mutants deficient in the slow (S-type) anion channel gene SLOW ANION CHANNEL-ASSOCIATED 1 (SLAC1). The treatment with malate increased S-type anion currents in guard-cell protoplasts of wild-type plants but not in the slac1 mutant. In addition, extracellular rather than intracellular application of malate increased the S-type currents of constitutively active mutants of SLAC1, which have kinase-independent activities, in a heterologous expression system using Xenopus oocytes. The treatment with malate transiently increased cytosolic Ca²⁺ concentration in the wild-type Arabidopsis guard cells and the malate-induced stomatal closure was inhibited by the Ca²⁺ channel blocker and the Ca²⁺ chelator. These results indicate that extracellular malate directly activates SLAC1 and simultaneously stimulates Ca²⁺ signalling in guard cells, resulting in steady and solid activation of SLAC1 for stomatal closure.