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

Abstract

SignificanceIntracellular 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. AbstractIon 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 Clbut also a NO 3sensor. We were then able to unravel and quantify the variations of NO 3and 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. 3 \body

K+ and pH homeostasis in plant cells is controlled by a synchronized K+/H+ antiport at the plasma and vacuolar membrane

“…As next, we tested whether chloride or nitrate transport alters guard cells' pH homeostasis differentially since cellular pH modifications were reported to follow NO 3 À transport in guard cells (Demes et al, 2020), while our results indicated a minor role of anion transport in the KCl-induced pH changes (Fig. 2b,c).…”

“…These findings suggested a larger NO 3 À /H + symport into the vacuole that attenuated or even balanced the dominance of the K + /H + -antiport of the K-homeostats at both membranes. The toxicity of [NO 3 À ] cyt may force the cell to provide a sequestration mechanism into the vacuole that is more efficient for nitrate than for Cl À , which could also manifest itself in the differences in the anion/H + antiport properties of CLCs (De Angeli et al, 2006;Jossier et al, 2010;Demes et al, 2020).…”

Transcriptome and Small RNA Sequencing Reveal the Mechanisms Regulating Harvest Index in Brassica napus

“…ATH13 affects the lipid composition of chloroplast membranes and regulates iron distribution within chloroplasts ( Manara et al, 2014 , 2015 ). CLC-A regulates the accumulation of nitrate within vacuoles and plays an important role in modifying cytosolic conditions ( De Angeli et al, 2006 ; Manara et al, 2015 ; Demes et al, 2020 ). Additional regulators of nitrate balance and transport include members of the NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER (NPF) family, such as AtNPF3.1 (At1g68570) ( Yang et al, 2017 ) and AtNPF6.2 (At2g26690) ( Tong et al, 2016 ); AtNRT1.7/NPF2.13 (At1g69870) also plays important roles in source-to-sink remobilization of nitrate in Arabidopsis ( Fan et al, 2009 ; Liu et al, 2017 ).…”

MtNPF6.5 mediates chloride uptake and nitrate preference in Medicago roots

“…The exposure of these phenotypes required nitrate free conditions, reflecting the dominant preference for plants to accumulate NO 3 − over Cl − ; when NO 3 − is replete, Cl − uptake is essentially abolished, making Cl − uptake phenotypes difficult to detect. Several other key Cl − transporters have been identified, including AtNPF2.4 and AtNPF2.5, which passively efflux Cl − , and CLC transporters involved in Cl − and NO 3 − compartmentalization in the vacuole (Geelen et al , 2000; De Angeli et al , 2006; Jossier et al , 2010; Li et al , 2016, 2017; Hu et al , 2017; Demes et al , 2020). It was not until recently that ZmNPF6.4 and ZmNPF6.6 from maize were shown to have both NO 3 − and Cl − uptake activities in Xenopus oocytes, but their role in Cl − transport in plants was not explored (Wen et al , 2017).…”