Vacuolar CAX1 and CAX3 influence auxin transport in guard cells via regulation of apoplastic pH.

CATION EXCHANGERs CAX1 and CAX3 are vacuolar ion transporters involved in ion homeostasis in plants. Widely expressed in the plant, they mediate calcium transport from the cytosol to the vacuole lumen using the proton gradient across the tonoplast. Here, we report an unexpected role of CAX1 and CAX3 in regulating apoplastic pH and describe how they contribute to auxin transport using the guard cell's response as readout of hormone signaling and cross talk. We show that indole-3-acetic acid (IAA) inhibition of abscisic acid (ABA)-induced stomatal closure is impaired in cax1, cax3, and cax1/cax3. These mutants exhibited constitutive hypopolarization of the plasma membrane, and time-course analyses of membrane potential revealed that IAA-induced hyperpolarization of the plasma membrane is also altered in these mutants. Both ethylene and 1-naphthalene acetic acid inhibited ABA-triggered stomatal closure in cax1, cax3, and cax1/cax3, suggesting that auxin signaling cascades were functional and that a defect in IAA transport caused the phenotype of the cax mutants. Consistent with this finding, chemical inhibition of AUX1 in wild-type plants phenocopied the cax mutants. We also found that cax1/cax3 mutants have a higher apoplastic pH than the wild type, further supporting the hypothesis that there is a defect in IAA import in the cax mutants. Accordingly, we were able to fully restore IAA inhibition of ABA-induced stomatal closure in cax1, cax3, and cax1/cax3 when stomatal movement assays were carried out at a lower extracellular pH. Our results suggest a network linking the vacuolar cation exchangers to apoplastic pH maintenance that plays a crucial role in cellular processes.

The Arabidopsis vacuolar anion transporter, AtCLCc, is involved in the regulation of stomatal movements and contributes to salt tolerance.

In plant cells, anion channels and transporters are essential for key functions such as nutrition, resistance to biotic or abiotic stresses, and ion homeostasis. In Arabidopsis, members of the chloride channel (CLC) family located in intracellular organelles have been shown to be required for nitrate homeostasis or pH adjustment, and previous results indicated that AtCLCc is involved in nitrate accumulation. We investigated new physiological functions of this CLC member in Arabidopsis. Here we report that AtCLCc is strongly expressed in guard cells and pollen and more weakly in roots. Use of an AtCLCc:GFP fusion revealed localization to the tonoplast. Disruption of the AtCLCc gene by a T-DNA insertion in four independent lines affected physiological responses that are directly related to the movement of chloride across the tonoplast membrane. Opening of clcc stomata was reduced in response to light, and ABA treatment failed to induce their closure, whereas application of KNO3 but not KCl restored stomatal opening. clcc mutant plants were hypersensitive to NaCl treatment when grown on soil, and to NaCl and KCl in vitro, confirming the chloride dependence of the phenotype. These phenotypes were associated with modifications of chloride content in both guard cells and roots. These data demonstrate that AtCLCc is essential for stomatal movement and salt tolerance by regulating chloride homeostasis.

Phosphorylation of the vacuolar anion exchanger AtCLCa is required for the stomatal response to abscisic acid.

Eukaryotic anion/proton exchangers of the CLC (chloride channel) family mediate anion fluxes across intracellular membranes. The Arabidopsis thaliana anion/proton exchanger AtCLCa is involved in vacuolar accumulation of nitrate. We investigated the role of AtCLCa in leaf guard cells, a specialized plant epidermal cell that controls gas exchange and water loss through pores called stomata. We showed that AtCLCa not only fulfilled the expected role of accumulating anions in the vacuole during stomatal opening but also mediated anion release during stomatal closure in response to the stress hormone abscisic acid (ABA). We found that this dual role resulted from a phosphorylation-dependent change in the activity of AtCLCa. The protein kinase OST1 (also known as SnRK2.6) is a key signaling player and central regulator in guard cells in response to ABA. Phosphorylation of Thr(38) in the amino-terminal cytoplasmic domain of AtCLCa by OST1 increased the outward anion fluxes across the vacuolar membrane, which are essential for stomatal closure. We provide evidence that bidirectional activities of an intracellular CLC exchanger are physiologically relevant and that phosphorylation regulates the transport mode of this exchanger.