Chloride (Cl^–) is a micronutrient that accumulates to macronutrient levels since it is normally available in nature and actively taken up by higher plants. Besides a role as an unspecific cell osmoticum, no clear biological roles have been explicitly associated with Cl^– when accumulated to macronutrient concentrations. To address this question, the glycophyte tobacco (Nicotiana tabacum L. var. Habana) has been treated with a basal nutrient solution supplemented with one of three salt combinations containing the same cationic balance: Cl^–-based (CL), nitrate-based (N), and sulphate+phosphate-based (SP) treatments. Under non-saline conditions (up to 5mM Cl^–) and no water limitation, Cl^– specifically stimulated higher leaf cell size and led to a moderate increase of plant fresh and dry biomass mainly due to higher shoot expansion. When applied in the 1–5mM range, Cl^– played specific roles in regulating leaf osmotic potential and turgor, allowing plants to improve leaf water balance parameters. In addition, Cl^– also altered water relations at the whole-plant level through reduction of plant transpiration. This was a consequence of a lower stomatal conductance, which resulted in lower water loss and greater photosynthetic and integrated water-use efficiency. In contrast to Cl^–, these effects were not observed for essential anionic macronutrients such as nitrate, sulphate, and phosphate. We propose that the abundant uptake and accumulation of Cl^– responds to adaptive functions improving water homeostasis in higher plants.

Chloride (Cl⁻) has traditionally been considered a micronutrient largely excluded by plants due to its ubiquity and abundance in nature, its antagonism with nitrate (NO₃⁻), and its toxicity when accumulated at high concentrations. In recent years, there has been a paradigm shift in this regard since Cl⁻ has gone from being considered a harmful ion, accidentally absorbed through NO₃⁻ transporters, to being considered a beneficial macronutrient whose transport is finely regulated by plants. As a beneficial macronutrient, Cl⁻ determines increased fresh and dry biomass, greater leaf expansion, increased elongation of leaf and root cells, improved water relations, higher mesophyll diffusion to CO₂, and better water- and nitrogen-use efficiency. While optimal growth of plants requires the synchronic supply of both Cl⁻ and NO₃⁻ molecules, the NO₃⁻/Cl⁻ plant selectivity varies between species and varieties, and in the same plant it can be modified by environmental cues such as water deficit or salinity. Recently, new genes encoding transporters mediating Cl⁻ influx (ZmNPF6.4 and ZmNPF6.6), Cl⁻ efflux (AtSLAH3 and AtSLAH1), and Cl⁻ compartmentalization (AtDTX33, AtDTX35, AtALMT4, and GsCLC2) have been identified and characterized. These transporters have proven to be highly relevant for nutrition, long-distance transport and compartmentalization of Cl⁻, as well as for cell turgor regulation and stress tolerance in plants.

Significance: 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.

Land plants regulate their photosynthesis and water transpiration by exchanging gases (CO₂ and H₂O_vapour) with the atmosphere. These exchanges take place through microscopic valves, called stomata, on the leaf surface. The opening of the stomata is regulated by two guard cells that actively and reversibly modify their turgor pressure to modulate the opening of the stomatal pores. Stomatal function depends on the regulation of the ion transport capacities of cell membranes as well as on the modification of the subcellular organisation of guard cells. Here we report how the vacuolar and cytosolic compartments of guard cells quantitatively participate in stomatal opening. We used a genetically encoded biosensor to visualise changes in ionic concentration during stomatal opening. The 3D reconstruction of living guard cells shows that the vacuole is the responsible for the change in guard cell volume required for stomatal opening.