Dynamic measurement of cytosolic pH and [NO3-] uncovers the role of the vacuolar transporter AtCLCa in the control of cytosolic pH

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 the whole cell homeostasis is limited by the difficulty to monitor their activity in vivo . The development of biosensors to track subtle changes in intracellular parameters provides an invaluable key to tackle this challenging issue. Here, we adapted the use of a dual biosensor using guard cells as experimental model to visualize the impact on the cytosol of anion transport from intracellular compartments. To image the activity of AtCLCa, a vacuolar NO 3 - /H + exchanger regulating stomata aperture in Arabidopsis thaliana , we expressed a genetically encoded biosensor, ClopHensor allowing monitoring the dynamics of cytosolic anion concentration and pH. We first show that ClopHensor is not only a Cl - but also a NO 3 - sensor. We were then able to unravel and quantify the variations of NO 3 - and pH in the cytosol. Our data show that AtCLCa activity modifies cytosolic pH and NO 3 - , demonstrating that the transport activity of a vacuolar exchanger has a profound impact on cytosolic homeostasis. We propose that a major function of this endomembrane transporter is to adjust cytosolic conditions to cellular needs. This opens a novel perspective on the function of intracellular transporters of the CLC family in eukaryotes: not only controlling the intra organelle lumen but also actively modifying cytosolic conditions. Significance Intracellular transporters are key actors in cell biological processes. Their disruption causes major physiological defects. The role of intracellular ion transporters is usually seen through an “intra organelle” lens, meanwhile their potential action on cytosolic ion homeostasis is still a black box. The case of a plant CLC is used as a model to uncover the missing link between the regulation of conditions inside the vacuole and inside the cytosol. The development of an original live imaging workflow to simultaneously measure pH and anion dynamics in the cytosol reveals the role of an Arabidopsis thaliana CLC, AtCLCa, in the modification of cytosolic pH. Our data highlight an unsuspected function of endomembrane transporters in the regulation of cytosolic pH.