Dynamic measurement of cytosolic pH and [NO(3)(−)] uncovers the role of the vacuolar transporter AtCLCa in cytosolic pH homeostasis

Ion transporters are key players of cellular processes. The mechanistic properties of ion transporters have been well elucidated by biophysical methods. Meanwhile, the understanding of their exact functions in cellular homeostasis is limited by the difficulty of monitoring their activity in vivo. The development of biosensors to track subtle changes in intracellular parameters provides invaluable tools to tackle this challenging issue. AtCLCa (Arabidopsis thaliana Chloride Channel a) is a vacuolar NO(3)(−)/H(+) exchanger regulating stomata aperture in A. thaliana. Here, we used a genetically encoded biosensor, ClopHensor, reporting the dynamics of cytosolic anion concentration and pH to monitor the activity of AtCLCa in vivo in Arabidopsis guard cells. We first found that ClopHensor is not only a Cl(−) but also, an NO(3)(−) sensor. We were then able to quantify the variations of NO(3)(−) and pH in the cytosol. Our data showed that AtCLCa activity modifies cytosolic pH and NO(3)(−). In an AtCLCa loss of function mutant, the cytosolic acidification triggered by extracellular NO(3)(−) and the recovery of pH upon treatment with fusicoccin (a fungal toxin that activates the plasma membrane proton pump) are impaired, demonstrating that the transport activity of this vacuolar exchanger has a profound impact on cytosolic homeostasis. This opens a perspective on the function of intracellular transporters of the Chloride Channel (CLC) family in eukaryotes: not only controlling the intraorganelle lumen but also, actively modifying cytosolic conditions.