Chloride as a macronutrient increases water-use efficiency by anatomically driven reduced stomatal conductance and increased mesophyll diffusion to CO 2 .

Chloride (Cl-) has been recently described as a beneficial macronutrient, playing specific roles in promoting plant growth and water-use efficiency (WUE). However, it is still unclear how Cl- could be beneficial, especially in comparison with nitrate (NO3-), an essential source of nitrogen that shares with Cl- similar physical and osmotic properties, as well as common transport mechanisms. In tobacco plants, macronutrient levels of Cl- specifically reduce stomatal conductance (g(s)) without a concomitant reduction in the net photosynthesis rate (A(N)). As stomata-mediated water loss through transpiration is inherent in the need of C-3 plants to capture CO2, simultaneous increase in photosynthesis and WUE is of great relevance to achieve a sustainable increase in C-3 crop productivity. Our results showed that Cl--mediated stimulation of larger leaf cells leads to a reduction in stomatal density, which in turn reduces g(s) and water consumption. Conversely, Cl- improves mesophyll diffusion conductance to CO2 (g(m)) and photosynthetic performance due to a higher surface area of chloroplasts exposed to the intercellular airspace of mesophyll cells, possibly as a consequence of the stimulation of chloroplast biogenesis. A key finding of this study is the simultaneous improvement of A(N) and WUE due to macronutrient Cl- nutrition. This work identifies relevant and specific functions in which Cl- participates as a beneficial macronutrient for higher plants, uncovering a sustainable approach to improve crop yield.