Vacuolar channel CLCa is involved in guard cell pH homeostasis (PNAS)

In plants, stomata are involved in a host of responses, controlled by ion homeostasis in guard cells. In an attempt to understand the function of a plant chloride channel (CLC), CLCa, which transports chloride (Cl^–) and nitrate (NO₃^–) in guard cells, Demes and colleagues sought to establish a relationship between ion transport and pH regulation by the channel. To this end, they used a fluorescent sensor named ClopHensor, developed for use in mammalian cells. Briefly, it uses a chloride- and pH-sensitive GFP that is fused to DsRed. Excitation with different wavelengths of light and ratiometric analysis allow for the simultaneous measurement of Cl^– (or other anions), and pH. In plant cells, the authors found that ClopHensor is a good indicator for the concentration of NO₃^–, but not sensitive to the physiological levels of Cl^– . They then used this probe to explore how guard cells behave when the vacuolar chloride channel is knocked out. In wild-type plants, the authors found that NO₃^– uptake directly correlates with acidification of guard cell cytosol. Contrarily in clca mutants, it was found that there is no significant acidification upon addition of NO₃^–, hinting at a role for NO₃^‑/H⁺ exchange activity of CLCa. Going deeper, the authors applied fungal toxin fusicoccin, which activates the plasma-membrane H⁺ pump, to wild type and mutant guard cells; while the pH in both increased after fusicoccin treatment, that of mutant plants failed to recover, showing that CLCa can induce changes in pH (through H⁺ transport) independent of anion transport. (Summary by Pavithran Narayanan @pavi_narayanan) Proc. Natl. Acad. Sci. USA 10.1073/pnas.2007580117