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Therefore, vacuolar ion transporters are key actors of stomata responses. Turgor changes in GCs depend on the accumulation/release of ions into/from the vacuole. Increase and decrease of the turgor pressure in GCs open and close the stomata, respectively. The regulation of the stomata pore aperture is based on the capacity of GCs to change their turgor pressure and consequently, their shape. Two GCs delimit the stomata pore and regulate its aperture according to environmental conditions. Stomata control gas exchanges between the photosynthetic tissues and the atmosphere, including water loss by transpiration. The VM delimits the largest intracellular compartment of GCs, the vacuole ( 17, 20). Their biological function relies on the regulation of ion transport systems residing in the plasma membrane (PM) and vacuolar membrane (VM) ( 17– 19). In plants, GCs are specialized cells gating the stomata pores at the leaf surface. Plant guard cells (GCs) constitute an appropriate experimental model to unravel CLC functions at the subcellular level.
#CLC SEQUENCE VIEWER 7.5 WORKFLOW FULL#
However, we still lack a molecular interpretation of the role of the CLC exchangers within cells, preventing a full understanding of the defects observed in organisms carrying mutations in CLC genes ( 2). In the last few decades, many studies addressed the biophysical properties of intracellular CLCs and provided a solid ground to understand the transport mechanisms of these exchangers ( 12– 16). In plants, CLCs regulate nutrient storage and photosynthesis and participate in drought and salt stress tolerance ( 5– 11). In mammals, mutations in intracellular CLCs lead to severe genetic diseases affecting bones, kidneys, and the brain ( 2). In eukaryotes, all of the CLCs localized in intracellular membranes behave as anion/H + exchangers. The members of the CLC family function as anion channels or anion/H + exchangers sharing a similar structural fold ( 3, 4). Among the different families of ion transporters identified, the CLC (Chloride Channel) family, which has been widely investigated in the last decades, constitutes a group of membrane proteins present in all organisms ( 2). The localization of transport systems in intracellular membranes prevents the use of in vivo electrophysiological approaches, considerably limiting our understanding of their cellular functions. Defects in the transport systems residing in intracellular membranes result in major physiological failures at the cellular and the whole-organism levels ( 1). The fluxes of ions between cell compartments are driven by membrane proteins forming ion channels, exchangers, symporters, and pumps. 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. 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. Our data showed that AtCLCa activity modifies cytosolic pH and NO 3 −. We were then able to quantify the variations of NO 3 − and pH in the cytosol. We first found that ClopHensor is not only a Cl − but also, an NO 3 − sensor. 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. AtCLCa ( Arabidopsis thaliana Chloride Channel a) is a vacuolar NO 3 −/H + exchanger regulating stomata aperture in A. The development of biosensors to track subtle changes in intracellular parameters provides invaluable tools to tackle this challenging issue. Meanwhile, the understanding of their exact functions in cellular homeostasis is limited by the difficulty of monitoring their activity in vivo. The mechanistic properties of ion transporters have been well elucidated by biophysical methods. Ion transporters are key players of cellular processes.