Ion flux coordination and signaling in plant cells (Influx) team

 

Intracellular transporters are key actors in cell biological processes. Their disruption causes major physiological defects. Intracellular ion transporters are usually thought to control luminal conditions in organelles; meanwhile, their potential action on cytosolic ion homeostasis is still a black box. The case of a plant Chloride Channel (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 importance of an Arabidopsis thaliana CLC, AtCLCa, in cytosolic pH homeostasis. Our data highlight an unsuspected function of endomembrane transporters in the regulation of cytosolic pH.

Variation of cytosolic nitrate as a function of external NO3–

Abstract
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 NO3–/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 NO3– sensor. We were then able to quantify the variations of NO3– and pH in the cytosol. Our data showed that AtCLCa activity modifies cytosolic pH and NO3–. In an AtCLCa loss of function mutant, the cytosolic acidification triggered by extracellular NO3– 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.

Article
Demes E, Besse L, Cubero-Font P, Satiat-Jeunemaître B, Thomine S, De Angeli A✉ (2020) Dynamic measurement of cytosolic pH and [NO3–] uncovers the role of the vacuolar transporter AtCLCa in cytosolic pH homeostasis. P. Natl. Acad. Sci. USA, (in press)