Summary 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 (gs) without a concomitant reduction in the net photosynthesis rate (AN). As stomata‐mediated water loss through transpiration is inherent in the need of C3 plants to capture CO2, simultaneous increase in photosynthesis and WUE is of great relevance to achieve a sustainable increase in C3 crop productivity. Our results showed that Cl−‐mediated stimulation of larger leaf cells leads to a reduction in stomatal density, which in turn reduces gs and water consumption. Conversely, Cl− improves mesophyll diffusion conductance to CO2 (gm) 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 AN 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.

Chloride (Cl − ) has traditionally been considered harmful to agriculture because of its toxic effects in saline soils and its antagonistic interaction with nitrate (NO 3 − ), which impairs NO 3 − nutrition. It has been largely believed that Cl − antagonizes NO 3 − uptake and accumulation in higher plants, reducing crop yield. However, we have recently uncovered that Cl − has new beneficial macronutrient functions that improve plant growth, tissue water balance, plant water relations, photosynthetic performance, and water-use efficiency. The increased plant biomass indicates in turn that Cl − may also improve nitrogen use efficiency (NUE). Considering that N availability is a bottleneck for the growth of land plants excessive NO 3 − fertilization frequently used in agriculture becomes a major environmental concern worldwide, causing excessive leaf NO 3 − accumulation in crops such as vegetables, which poses a potential risk to human health. New farming practices aimed to enhance plant NUE by reducing NO 3 − fertilization should promote a healthier and more sustainable agriculture. Given the strong interaction between Cl − and NO 3 − homeostasis in plants, we have verified if indeed Cl − affects NO 3 − accumulation and NUE in plants. For the first time to our knowledge, we provide a direct demonstration which shows that Cl − , contrary to impairing NO 3 − nutrition, facilitates NO 3 − utilization and improves NUE in plants. This is largely due to Cl − improvement of the N-NO 3 − utilization efficiency (NU T E), having little or moderate effect on N-NO 3 − uptake efficiency (NU P E) when NO 3 − is used as the sole N source. Clear positive correlations between leaf Cl − content vs. NUE/NU T E or plant growth have been established at both intra-and interspecies levels. Optimal NO 3 − vs. Cl − ratios become a useful tool to increase crop yield and quality, agricultural sustainability and to reduce the negative ecological impact of NO 3 − on the environment and on human health.

Phosphoenolpyruvate carboxylase (PEPC) activity was detected in aleurone-endosperm extracts of barley (Hordeum vulgare) seeds during germination, and specific anti-sorghum (Sorghum bicolor) C 4 PEPC polyclonal antibodies immunodecorated constitutive 103-kD and inducible 108-kD PEPC polypeptides in western analysis. The 103-and 108-kD polypeptides were radiolabeled in situ after imbibition for up to 1.5 d in 32 P-labeled inorganic phosphate. In vitro phosphorylation by a Ca 2؉ -independent PEPC protein kinase (PK) in crude extracts enhanced the enzyme's velocity and decreased its sensitivity to L-malate at suboptimal pH and [PEP]. Isolated aleurone cell protoplasts contained both phosphorylated PEPC and a Ca 2؉ -independent PEPC-PK that was partially purified by affinity chromatography on blue dextran-agarose. This PK activity was present in dry seeds, and PEPC phosphorylation in situ during imbibition was not affected by the cytosolic protein-synthesis inhibitor cycloheximide, by weak acids, or by various pharmacological reagents that had proven to be effective blockers of the light signal transduction chain and PEPC phosphorylation in C 4 mesophyll protoplasts. These collective data support the hypothesis that this Ca 2؉ -independent PEPC-PK was formed during maturation of barley seeds and that its presumed underlying signaling elements were no longer operative during germination.

C 4 phosphoenolpyruvate carboxylase (PEP-Case: EC 4.1.1.31) is subjected to in vivo regulatory phosphorylation by a light up-regulated, calciumindependent protein kinase. Salt stress greatly enhanced phosphoenolpyruvate carboxylase-kinase (PEPCase-k) activity in leaves of Sorghum. The increase in PEPCase-k anticipated the time course of proline accumulation thereby suggesting that water stress was not involved in the kinase response to salt. Moreover, osmotic stress seemed not to be the main factor implicated, as demonstrated by the lack of effect when water availability was restricted by mannitol. In contrast, LiCl (at a concentration of 10 mM in short-term treatment of both excised leaves and whole plants) mimicked the effects of 172 mM NaCl salt-acclimation, indicating that the rise in PEPCase-k activity resulted primarily from the ionic stress. Both NaCl and LiCl treatments increased the activity of a Ca 2+ -independent, 35 kDa kinase, as demonstrated by an in-gel phosphorylation experiment. Short-term treatment of excised leaves with NaCl or LiCl partially reproduces the effects of whole plant treatments. Finally, salinization also increased PEP-Case-k activity and the phosphorylation state of PEP-Case in darkened Sorghum leaves. This fact, together with increased malate production during the dark period, suggests a shift towards mixed C 4 and crassulacean acid metabolism types of photosynthesis in response to salt stress.

In C4 plants, the photosynthetic enzyme phosphoenolpyruvate carboxylase (PEPCase; EC 4.1.1.31) is subjected to a phosphorylation process via the light-dependent up-regulation of a Ca2+-independent PEPCase-kinase. The present work aimed to study the effect of salt stress on PEPCase phosphorylation in Sorghum vulgare Pers. leaves. The growth of salt-treated plants was reduced compared with that of the control plants. PEPCase activity modestly increased (around 20-40%) whereas PEPCase phosphorylation was markedly enhanced, on a protein basis, in extracts from illuminated leaves. The enhanced protein kinase activity was found to display a low molecular mass in the range 32-35 kDa, to be independent of Ca2+ and to be up-regulated by light. Furthermore, up-regulation was blocked in vivo by the cytosolic protein synthesis inhibitor cycloheximide. Collectively, these data demonstrated that salinity stress altered the Ca2+-independent PEPCase-kinase, presumably by increasing the mesophyll content of the enzyme. Potassium chloride, but not abscisic acid, mimicked the effect of NaCl on PEPCase-kinase activity.