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

Abstract

Summary 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.

A drought stress-responsive metabolite malate modulates stomatal responses through G-protein-dependent pathway

“…To explore the modulation of VvSLAC1 activity by TCA cycle metabolites, we used a two-electrode voltage-clamp technique on Xenopus oocytes expressing VvSLAC1. The negative currents in oocytes expressing VvSLAC1 were minimal (Supplementary Figure 3), resembling the characteristics of the inactive form of AtSLAC1 (Mimata et al, 2022a). Constitutively active AtSLAC1 mutants, such as AtSLAC1T513D and AtSLAC1F450A, exhibit substantial basal activity and their activity were further enhanced by malate in oocytes (Chen et al, 2010; Maierhofer et al, 2014; Mimata et al, 2022a).…”

“…The negative currents in oocytes expressing VvSLAC1 were minimal (Supplementary Figure 3), resembling the characteristics of the inactive form of AtSLAC1 (Mimata et al, 2022a). Constitutively active AtSLAC1 mutants, such as AtSLAC1T513D and AtSLAC1F450A, exhibit substantial basal activity and their activity were further enhanced by malate in oocytes (Chen et al, 2010; Maierhofer et al, 2014; Mimata et al, 2022a). Therefore, we expressed VvSLAC1T503D, corresponding to AtSLAC1T513D, and VvSLAC1F440A, corresponding to AtSLAC1F450A, in oocytes.…”

“…To further investigate the malate signaling pathway, we conducted a series of pharmacological experiments (Supplemental Table 1). Malate-induced stomatal closure in both V. vinifera and A. thaliana was abolished by anion channel blockers, 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) and 9-anthracenecarboxylic acid (9-AC), an extracellular Ca 2+ chelator 1,2-bis(2-aminophenoxy)ethane- N , N , N’ , N’ -tetraacetic acid (BAPTA), and Ca 2+ channel blockers, nifedipine and La 3+ (Figure 4C–4F; Mimata et al, 2022a). These results suggest anion channels and Ca 2+ signaling are essential for malate-induced stomatal closure in V. vinifera and A. thaliana , consistent with the results for [Ca 2+ ] cyt and SLAC1 activity (Figure 2A, 2D and 3).…”

“…Amino acids, lipids, terpenoids, phenolic acids, alkaloids, and flavonoids were the primary categories exhibiting changes (Figure 1D and Supplemental Figure 1B). Among the upregulated metabolites, we specifically examined three TCA cycle metabolites—malate, isocitrate, and citrate—since malate functions as a signaling molecule inducing stomatal closure (Mimata et al, 2022a). Malate, isocitrate, and citrate increased after 12 or 24 h of dehydration stress (Figure 1E).…”

“…Previous studies have shown that malate increases [Ca 2+ ] cyt and ROS production in guard cells and directly activates SLAC1, leading to stomatal closure in A. thaliana (Mimata et al, 2022a; 2022b). In this study, we demonstrate that several TCA cycle metabolites accumulate in grapevine leaves during dehydration stress, among which malate most effectively regulates stomatal response via a G-protein signaling cascade.…”