Chloride as a macronutrient increases water-use efficiency by anatomically driven reduced stomatal conductance and increased mesophyll diffusion to CO2

Chloride as a macronutrient increases water-use efficiency by anatomically driven reduced stomatal conductance and increased mesophyll diffusion to CO 2 Juan D. Franco-Navarro 1,† , Miguel A. Rosales 1,† , Paloma Cubero-Font 1,2 , Purificaci on Calvo 3 , Rosario  Alvarez 4 , Antonio Diaz-Espejo 1 and Jos e M. Colmenero-Flores 1 1 Instituto de Recursos Naturales y Agrobiolog ıa,CSIC, Avda Reina Mercedes 10, 41012, Sevilla, Spain, 2 Biochimie et Physiologie Mol eculaire des Plantes (BPMP), Univ. Montpellier, CNRS, INRA, SupAgro, 2 Place P. Viala, Montpellier 34060, France, 3 Departamento de Microbiolog ıa, Facultad de Biolog ıa, Universidad de Sevilla, Avda Reina Mercedes 6,41012, Sevilla, Spain, and 4 Departamento de Biolog ıa Vegetal y Ecolog ıa, Facultad de Biolog ıa, Universidad de Sevilla, Avda. Reina Mercedes 6, 41012, Sevilla, Spain Received 21 September 2018; revised 17 May 2019; accepted 22 May 2019; published online 31 May 2019. *For correspondence (e-mail [email protected]). † Both authors contributed equally to this work. 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 (NO 3 ), 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 CO 2 , 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 CO 2 (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. Keywords: chloride nutrition, beneficial macronutrient, nitrate, water-use efficiency, photosynthesis, stomatal conductance, stomatal density, mesophyll diffusion conductance to CO 2 , chloroplast, Nicotiana tabacum. Linked article: This paper is the subject of a Research Highlight article. To view this Research Highlight article visit https://doi.org/10.1111/tpj.14498. INTRODUCTION Especially in the agronomic context, chloride (Cl  ) has been traditionally considered a toxic anion rather than a plant nutrient. Due to its abundance and ubiquity in nature, it has been largely believed that Cl  deficiency is a very rare phenomenon in plants growing in their natural habitats or in agriculture (White and Broadley, 2001). Given the particular precautions that were required by earlier investigators to induce Cl  deficiency symptoms in different plant species, the relevance of Cl  in plant growth has been largely neglected (Xu et al., 2000). However, in addition to being an essential micronutrient (Johnson et al., 1957; Xu et al., 2000; White and Broadley, 2001; Marschner, 2011), we have recently shown that, Cl  is a beneficial macronutrient for plants (Franco-Navarro et al., 2016), a definition further supported by others (Raven, 2017; Wege et al., 2017; Geilfus, 2018). ©2019 The Authors The Plant Journal ©2019 John Wiley & Sons Ltd 815 The Plant Journal (2019) 99, 815–831 doi: 10.1111/tpj.14423 When supplied to tobacco plants (Nicotiana tabacum L.) at concentrations in excess of those needed to satisfy the micronutrient requirements, but insufficient to cause toxicity (1–5m M Cl  ), Cl  treatments resulted in leaf accumulation levels that are typical of the content of a macronutrient (Franco-Navarro et al., 2016). Under these conditions, Cl  has played specific roles in regulating leaf osmotic potential and turgor. Besides improving leaf water balance parameters, Cl  also alters water relations at the whole-plant level through reduction in plant transpiration. This was a consequence of the reduction in stomatal conductance (g s ), which resulted in lower water loss and greater WUE, measured as intrinsic WUE (WUEi; the ratio of net photosynthesis rate to stomatal conductance) and integrated WUE (long-term ratio of plant biomass production to total water consumed). Plant WUE has become a key measure of the efficiency of the use of water resources and a target for crop selection and breeding (Linderson et al., 2012; Gago et al., 2014; Tomas et al., 2014). As stomata-mediated water loss through transpiration is inherent in the need for C 3 plants to capture CO 2 , there is an urgent need for simultaneously increasing photosynthesis/yield and WUE in C 3 crops (Morison et al., 2008; Flexas et al., 2016). Improved photosynthetic efficiency has played only a minor role in the remarkable increases in agricultural productivity achieved in the last half century. Therefore, it is expected that further increases in crop yield potential will rely in a large part on improved photosynthesis (Long et al., 2006; Zhu et al., 2010; Ort et al., 2015). Modern agriculture requires that the increase in productivity be made in a sustainable way, minimizing the raise of cultivated area and maximizing the efficiency in the use of resources like fertilizers and, especially, water (Gilbert, 2012; Ort et al., 2015; Flexas et al., 2016). Given the effect of Cl  nutrition on WUE regulation (Franco-Navarro et al., 2016), Cl  fertilization arises as a potential strategy to manipulate whole WUE in crops, as previously proposed for other agronomic (Gregory, 2004) and biotechnological approaches (Boyer, 1996; Parry et al., 2005; Flexas, 2016; Flexas et al., 2016). Despite reducing water consumption, the presence of Cl  has led to a moderate increase in plant fresh and dry biomass, a phenomenon that occurred in parallel with an increase in leaf cell size (including epidermal, mesophyll and stomatal cells) and shoot expansion (Franco-Navarro et al., 2016). Reduction in g s is expected to reduce atmospheric CO 2 capture, resulting in concomitant reduction in the net photosynthesis rate (A N ). Unexpectedly, the Cl  treatment did not result in a reduction in A N when compared with control tobacco plants treated with equivalent amounts of phosphate +sulphate salts (Franco-Navarro et al., 2016). This apparent inconsistency could be explained if the Cl  treatment also improved the overall carboxylation rate of ribulose-1,5-bisphosphate (RuBP) as a result of: (i) higher ribulose-1,5-bisphosphate carboxylase/ oxygenase (RuBisCo) activity; and (ii) higher diffusion rate of atmospheric CO 2 into leaf mesophyll cells and chloroplasts. In the past, it was believed that virtually all the resistance to CO 2 diffusion was caused by stomata. However, it is now clear that the mesophyll diffusion conductance to CO 2 from substomatal cavities to the sites of carboxylation (g m ) accounts actually for more than 40% of the decrease in CO 2 (Flexas et al., 2008; Warren, 2008; Galm es et al., 2013). Therefore, g m is of great relevance for A N in many species (Flexas et al., 2012; Muir et al., 2014), also affecting WUE (Flexas et al., 2016). As CO 2 diffuses into the leaf, it must pass through several anatomical and cell barriers: the boundary layer, the stomatal pore, the airfilled pore space within the leaf, the cell wall, the plasma membrane and cytoplasm as well as the chloroplast membrane. Alterations in anatomical and cell parameters such as leaf thickness, cell packing, shape and wall thickness and chloroplasts arrangement to intercellular airspaces can influence the conductance to CO 2 at each step of this diffusion pathway (Terashima et al., 2011; Tosens et al., 2012; Tomas et al., 2013). Leaf anatomical variations induced by macronutrient levels of Cl  in tobacco leaves, including higher leaf thickness and bigger mesophyll cells could potentially affect g m (Franco-Navarro et al., 2016). This possibility adds further complexity to attempts to understand the mechanism of genotypic and phenotypic variability of g m (Raven, 2017). Positive effects on cell size, biomass increase, osmotic regulation and WUE were specific for Cl  relative to other mineral macronutrient anions (Franco-Navarro et al., 2016). But it is still unclear how Cl  could be beneficial, especially in comparison with nitrate (NO 3 ), which in addition to being an essential source of nitrogen, shares with Cl  similar physical and osmotic properties, as well as common transport mechanisms (Flowers, 1988; Cubero- Font et al., 2016; Li et al., 2017; Wege et al., 2017). Therefore, in this study, we aimed: (i) to confirm whether macronutrient levels of Cl  in plants reduces g s without a concomitant reduction in A N ; (ii) to identify physiological/ anatomical mechanisms responsible for the observed Cl  - dependent g s reduction; (iii) to verify whether Cl  nutrition modifies g m and which physiological/anatomical alterations rely behind this phenomenon. Understanding these phenomena can be of interest in two respects. The ability to manipulate relevant physiological processes in individuals with the same genetic background is a valuable tool to better understand the studied phenomena. Conversely, a simple and inexpensive agronomic practice like Cl  fertilization could potentially increase WUE without reducing the photosynthetic efficiency. It is therefore important to understand how Cl  improves plant growth through the coordinated regulation of WUE and photosynthetic performance. ©2019 The Authors The Plant Journal ©2019 John Wiley & Sons Ltd, The Plant Journal, (2019), 99, 815–831 816 Juan D. Franco-Navarro et al. RESULTS Effect of Cl  on plant growth, photosynthesis, leaf water balance and WUE First, we verified that the 5 m M Cl  treatment (CL) reproduced the previously reported Cl  -specific responses on plant growth, photosynthesis, water balance and WUE (Franco-Navarro et al., 2016). With this aim the effects of the CL treatment on the parameters of interest were compared with those of plants subjected to low Cl  treatments (SP and N). Leaf Cl  concentration in CL-treated plants was around 45–50 mg g 1 dry weight (DW) (Figure S1a and Table 1), which is typical of the content of a macronutrient. In SP and N plants, Cl  concentration was about 100 times smaller, although above the critical level of micronutrient deficiency (Franco-Navarro et al., 2016). No deficiency symptoms like wilting, chlorosis or bronzing were observed in SP- and N-treated plants. In fact, NO 3 -supple- mented plants exhibited the best performance in terms of dry biomass and leaf area (Figure S1b–d), ruling out the possibility that plants containing low Cl  (N and SP plants), were experiencing Cl  deficiency. The fact that N-treated plants showed the highest growth and biomass was expected given the essential role of nitrogen on plant development (Hawkesford et al., 2012). The positive effect of the Cl  treatment on growth and biomass was confirmed in the plants used in this work since CL plants showed higher DW and leaf growth than SP plants (Figure S1b–d). The effect of the Cl  treatment on leaf water parameters was confirmed in the plants used in this work since CL plants showed higher water content (Figure S2a) and higher relative water content (RWC) (Figure S2b). In consequence, leaves of CL plants were more succulent (Figure S2c) and thicker (Figure S2d). As previously reported (Franco-Navarro et al., 2016) CL plants exhibited more negative osmotic potential than SP and N plants (Figure S2e) and higher turgor potential (Figure S2f). Compared with N and SP treatments, CL plants showed around 33% reduction in g s (Figure 1a), producing a concomitant reduction on leaf transpiration as indicated by the Fresh Weight loss assay (Figure 1b). Accordingly, CL plants consumed less water throughout the experiment (Figure 1c). Interestingly, despite the lower g s ,A N of CL plants was not impaired in comparison with SP plants (Figure 1d), determining a 27% higher WUEi (Figure 1e). Consistent with the lower water consumption, the integrated WUE of CL plants was 25% higher than that of SP and N plants (Figure 1f). No significant differences among treatments were found in other photosynthetic parameters like the maximum rate of RuBP carboxylation (V cmax ) and the maximum rate of electron transport (J max ). Altogether, the results confirmed the beneficial effect of macronutrient Cl  nutrition on tobacco plant growth, water balance and whole-plantwater relationships. Effect of Cl  on stomatal aperture, stomatal density and stomatal index To identify which physiological mechanism is responsible for the observed Cl  -dependent g s reduction, different functional and anatomical parameters influencing stomatal-de- pendent gas-exchange capacity were studied. The opening ratio, pore size, stomatal density and index values were quantified in plants subjected to CL, SP and N treatments. After 3 h illumination, CL and SP plants presented the same stomatal aperture ratio (width/length), around 0.24, whereas N plants had 22.5% higher aperture ratio (Figure 2b), around 0.31. These values are within the expected range of fully open stomata (0.18–0.35), previously reported by Willmer and Fricker (1996). During the following 4 h, light-incubated peelings exhibited a progressive stomatal closure, in line with the natural circadian closure occurring in our plants between 15:00 and 19:00 h (Figure 2a). Addition of ABA to the peelings incubation medium determined that plants did not respond to the light treatment (Figure 2c), remaining the stomata as closed as those from non-illuminated control peelings (Figure 2b), with aperture ratio values around 0.045, within the expected range of fully closed stomata (below 0.05) reported by Willmer and Fricker (1996). Interestingly, N plants remained partially open under the ABA treatment, Table 1 Ion concentration and total nitrogen content in leaves of plants subjected to different treatments Treatment Ion concentration (mg g 1 DW) TNCK + Ca 2+ Mg 2+ Cl  NO 3 PO 43 SO 42 SP 49.61 3.27 28.10 5.87 8.89 2.08 0.54 0.07 b 8.78 1.29 b 14.28 1.10 a 34.21 4.06 a 63.07 6.66 b N 50.43 3.27 24.42 3.20 8.11 1.31 0.60 0.08 b 51.03 7.57 a 11.01 1.23 b 19.21 4.15 ab 212.69 19.79 a CL 49.54 1.88 25.26 4.82 8.15 1.47 52.98 3.40 a 2.59 0.49 b 10.52 0.55 b 14.64 2.07 b 48.82 3.11 b P-value ns ns ns *** *** * * * Treatment consisted of the basal nutrient solution supplemented with 5 m M chloride (CL), 5 m M nitrate (N) or the sulphate +phosphate (SP) salt mixture containing the same cationic balance as in the CL and N treatments. Total nitrogen content (TNC) was calculated as the sum of NO 3 plus organic nitrogen content (mg g 1 DW). Mean values SE, n=6. ‘Homogeneous group’ statistics was calculated through ANOVA test, where mean values with different letters are significantly different according to Tukey’s test at P≤0.05. Levels of significance: P≤0.05 (*); P≤0.001 (***); and P>0.05 (‘ns’, no significant differences). ©2019 The Authors The Plant Journal ©2019 John Wiley & Sons Ltd, The Plant Journal, (2019), 99, 815–831 Cl  simultaneously reduces g s and increases g m 817 with aperture ratios around 0.075, 40% higher than those of ABA-treated SP and CL plants (Figure 2c). In addition, when the aperture ratios were compared with those of untreated plants (grown in basal nutrient solution, BS), we observed that all nutritional treatments (SP, N and CL) were able to stimulate stomatal aperture (Figure S3). We also quantified the absolute stomatal aperture through the measurement of the stomatal pore area and we observed that N and CL plants showed similar pore area values, around 45 lm 2 , whereas SP plants showed around 39% less aperture area, corresponding to 27.5 lm 2 (Figure 2d). In summary, results showed that: (i) the N treatment induced the highest aperture ratio; (ii) SP and CL treatments induced lower and similar aperture ratios; and (iii) N and CL treatments exhibited similar stomatal pore areas. The smaller aperture ratio to compensate for larger Figure 1. Effect of Cl  nutrition on gas-exchange parameters and water-use efficiency. Plants were alternatively treated with the following salt supplements: 5 m M chloride salts (CL); 5 m M nitrate salts (N); and a mixture of sulphate + phosphate salts (SP) containing the same cationic balance as in the CL and N treatments. (a) Effect on stomatal conductance (g s ). (b) Effect on leaf transpiration measured as the fresh weight loss (FW loss) of detached leaves over time. (c) Effect on total water consumed throughout the experiment relative to the final fresh biomass (FW). (d) Effect on net photosynthesis rate (A N ). (e) Effect on intrinsic water-use efficiency (WUEi). (f) Effect on integrated water-use efficiency (WUE). (g) Effect on maximum rate of carboxylation (V cmax ). (h) Effect on maximum rate of electron transport (J max ). Mean values SE, n= 6. ‘Homogeneous group’ statistics were calculated through ANOVA (a, c–f) AND MANOVA (b), where mean values with different letters are significantly different according to Tukey’s test at P≤0.05. Levels of significance: **P≤0.01 and ***P≤0.001. ©2019 The Authors The Plant Journal ©2019 John Wiley & Sons Ltd, The Plant Journal, (2019), 99, 815–831 818 Juan D. Franco-Navarro et al. pore surface in CL plants is consistent with the higher stomata size in Cl  -treated plants. Therefore, we ruled out the possibility that the lower g s of CL plants (compared with SP and N plants) were the consequence of a reduced stomatal opening capacity. These results are consistent with the observation that Cl  specifically increases the size of different leaf cell types including epidermal, guard, mesophyll and trichome cells (Franco-Navarro et al., 2016). This may alter the distribution of the stomata on the leaf surface. Therefore, Cl  -me- diated reduction in g s can be a consequence of a lower stomatal density (number of stomata per unit area) or a lower stomatal index (percentage of stomata out of the total number of epidermal cells plus stomata). To determine possible alterations in stomatal density and/or stomatal index in CL plants, these parameters were quantified in these cell types (Figure 3). We first confirmed the occurrence of larger epidermal (Figure 3a) and guard (Figure 3b) cells in CL plants. As a direct consequence of the higher cell size, we could verify that epidermal and stomatal density of CL plants were significantly lower than those of SP and N plants (Figure 3c,d, respectively), whereas the stomatal index remained the same in all three treatments (Figure 3e), indicating that the CL treatment regulates stomatal cell size, but not stomatal differentiation. To ascertain if g s reduction in CL plants (Figure 1a) can be explained by the lower stomatal density, despite the greater pore area (Figure 2d), the theoretical g w,max , which computes the interaction of the stomatal size with the Figure 2. Effect of Cl  nutrition on stomatal aperture. Plants were alternatively treated with the following salt supplements: 5 m M chloride salts (CL); 5m M nitrate salts (N); and a mixture of sulphate + phosphate salts (SP) containing the same cationic balance as in the CL and N treatments. (a) Real-time measurement of leaf turgor on a representative plant of the assay using the non-invasive magnetic leaf patch clamp pressure probes (Zimmermann et al., 2008). Patch-pressure (P p ) is inversely correlated with leaf turgor pressure and positively correlated with leaf water potential and plant transpiration (Zimmermann et al., 2008, 2010). P p values were recorded for 24 h, and the time interval in which the stomatal opening assays were performed, between 11:00 and 19:00 h, is indicated (dotted line box), including the maximal stomata aperture period (lowest leaf turgor between 11:00 and 15:30 h) and the progressive stomatal closure (increasing leaf turgor between 15:30 and 19:00 h). (b, c) Stomatal opening given as the width/length ratio (w/l) quantified from optical microscope images of epidermal peelings obtained at 940. After synchronizing stomatal closure through a 2 h dark period between, the opening ratios were quantified: in peelings illuminated for 3 h (15:00), 5 h (17:00) and 7 h (19:00) or in control peelings kept in the dark during the same time intervals (b); in peelings treated with 50 l M ABA and illuminated for 3 h (15:00), 5 h (17:00) and 7 h (19:00) (c). (d) Average aperture area (lm 2 ) of stomata from peelings subjected to 3 h illumination (quantified from the same stomata pictures used for the opening ratio measurements shown in panel (b)). Mean values SE are shown. For each time point or treatment and light condition, 30–40 pores were measured. Each experiment was repeated on three separate days using fresh plants (n=90–120). ‘Homogeneous group’ statistics were calculated through ANOVA (d) and MANOVA (b, c), where mean values with different letters are significantly different according to Tukey’s test at P≤0.05. Levels of significance: *P≤0.05 and ***P≤0.001. ©2019 The Authors The Plant Journal ©2019 John Wiley & Sons Ltd, The Plant Journal, (2019), 99, 815–831 Cl  simultaneously reduces g s and increases g m 819 maximum pore area and the stomatal density, was calculated according to the procedure reported by Franks et al. (2009). Using the experimentally obtained values of the pore area (Figure 2d), the stomatal cell size (Figure 3b) and the stomatal density (Figure 3d), g w,max was calculated, showing 36.8% lower values for CL plants in comparison with N and SP plants, indicating that a reduction in g s measured in CL plants was consistent with the reduction in stomatal density. Effect of Cl  on mesophyll diffusion conductance to CO 2 , mesophyll anatomy and chloroplasts distribution To uncover the reasons why the specific reduction in of g s observed in CL plants (Figure 1a) did not result in the expected reduction in A N (Figure 1d), g m was estimated. When compared with SP plants, CL plants exhibited significantly higher values of g m (Figure 4a). N plants presented intermediate values, not significantly different to those of SP and CL treatments. Simultaneous improvement of WUE and A N in CL plants indicated that Cl  regulates g s and g m independently and, therefore, WUEi should correlate on the ratio g m /g s rather than on g m itself (Flexas et al., 2013). Therefore, Figure 4(b) shows the expected correlation, indicating that Cl  -mediated g m enhancement was a key factor determining the higher WUE found in this treatment. To determine which factors were in the origin of the gm enhancement, different anatomical and cellular parameters of the mesophyll were measured. No differences were found in the surface area of mesophyll cells exposed to the intercellular airspaces (Smes; Figure 5a). However, Cl-trea- ted plants showed significantly higher values of Sc and Sc/ Smes than SP and N plants (Figure 5b,c), indicating that the CL treatment determined a higher proportion of chloroplasts covering the mesophyll cell surface as a consequence of a higher surface area of chloroplasts exposed to the intercellular airspace of mesophyll cells Figure 3. Effect of Cl  nutrition on epidermal leaf cells size and density. Plants were alternatively treated with the following salt supplements: 5 m M chloride salts (CL); 5 m M nitrate salts (N); and a mixture of sulphate + phosphate salts (SP) containing the same cationic balance as in the CL and N treatments. (a) Effect on epidermal cell size. (b) Effect on stomatal cell size determined by measuring 60 random stomata per treatment. (c) Effect on epidermal cell density. (d) Effect on stomatal cell density. (e) Effect on stomatal index. (f) Modelling of the maximum stomatal conductance according to Franks et al. (2009). Mean values SE, n= 6. ‘Homogeneous group’ statistics were calculated through ANOVA (a–f), where mean values with different letters are significantly different according to Tukey’s test at P≤0.05. NS, not significant. ©2019 The Authors The Plant Journal ©2019 John Wiley & Sons Ltd, The Plant Journal, (2019), 99, 815–831 820 Juan D. Franco-Navarro et al. Quantification of the size and number of chloroplasts showed that mesophyll cells of CL plants contain a higher number of smaller chloroplasts (Figure 6a,b). Photomicrographs of leaf sections also revealed the occurrence in CL plants of chloroplasts containing bigger starch granules in lacunar and palisade parenchyma cells (Figure S4). Furthermore, measurement of starch content in tobacco leaves confirmed higher content in CL plants in comparison to SP and N plants (Figure 7). To determine for each treatment which components among the diffusional and biochemical parameters played a major role in modifying A N , individual contributions Figure 4. Effect of Cl  nutrition on mesophyll diffusion conductance to CO 2 . Plants were alternatively treated with the following nutritional supplements: 5 m M chloride salts (CL); 5 m M nitrate salts (N); and a mixture of sulphate + phosphate salts (SP) containing the same cationic balance as in the CL and N treatments. Fully expanded photosynthetically active leaves from tobacco plants were used. (a) Effect of nutritional treatments on mesophyll diffusion conductance to CO 2 (g m ). g m was estimated from simultaneous measurements of leaf gas exchange and fluorescence following the method by Harley et al. (1992). (b) Relationship between intrinsic water-use efficiency (A N /g s ; WUEi) and g m /g s . Mean values SE, n= 5. ‘Homogeneous group’ statistics were calculated through ANOVA (a), where mean values with different letters are significantly different according to Tukey’s test at P≤0.05. Figure 5. Effect of Cl  nutrition on mesophyll anatomical parameters. Plants were alternatively treated with the following nutritional supplements: 5 m M chloride salts (CL); 5 m M nitrate salts (N); and a mixture of sulphate + phosphate salts (SP) containing the same cationic balance as in the CL and N treatments. (a) Surface area of mesophyll cells exposed to the intercellular airspaces per unit leaf area (S mes ). (b) Surface area of chloroplasts exposed to the intercellular airspace per unit leaf area (S c ). (c) Proportion of chloroplasts covering the mesophyll cell surface (S c /S mes ). Mean values SE, n=10–12. ‘Homogeneous group’ statistics were calculated through ANOVA (a–c), where mean values with different letters are significantly different according to Tukey’s test at P≤0.05. Figure 6. Effect of Cl  nutrition on the number and size of chloroplasts. Plants were alternatively treated with the following salt supplements: 5 m M chloride salts (CL); 5 m M nitrate salts (N); and a mixture of sulphate + phosphate salts (SP) containing the same cationic balance as in the CL and N treatments. (a) Chloroplast size. (b) Chloroplast density. Mean values SE, n= 500 chloroplasts analysed in five plants per treatment. ‘Homogeneous group’ statistics were calculated through ANOVA (a, b), where mean values with different letters are significantly different according to Tukey’s test at P≤0.05. ©2019 The Authors The Plant Journal ©2019 John Wiley & Sons Ltd, The Plant Journal, (2019), 99, 815–831 Cl  simultaneously reduces g s and increases g m 821 directly related to CO 2 uptake and fixation were calculated according to Buckley and Diaz-Espejo (2015). With this aim, the g s ,g m , and V c,max /J max contributions were quantified in CL and SP plants using as reference the values of N plants (the treatment with the highest photosynthetic rate; Figure 8). Regarding the diffusional components, the higher limitation imposed by g s reduction in CL plants (9.0% compared to SP plants) was compensated by a lower g m limitation (+13.2% in CL plants compared with SP plants), resulting in total diffusional limitations (q diff )of6.4% for CL plants and 9.2% for SP plants. The biochemical components (q Vcmax/Jmax ) determined a greater limitation for CL plants (17.2%) than for SP plants (13.5%), probably as a consequence of the lower leaf nitrogen content of CL plants (Table 1). Therefore, total photosynthetic limitation (q total ) of CL plants (23.4%) was very similar to that of SP plants (22.7%), in agreement to the similar A N values experimentally obtained for both treatments (Figure 1d). Altogether, these results showed that Cl  improves CO 2 conductance in the mesophyll, which compensates for the limitation produced by the reduction in stomatal conductance. DISCUSSION We demonstrated that Cl  supplementation in the range 1– 5m M increased DW, WUE, cell size and leaf growth in tobacco plants (Franco-Navarro et al., 2016). This work attempts to reveal what is the specific beneficial role of Cl  on plant growth and development when it is provided to macronutrient levels, and how it can be reconciled with the g s reduction observed in these plants. We first verified that the previously reported effects of Cl  on the growth and water relations of tobacco plants (Franco-Navarro et al., 2016) were clearly reproduced in the plants used in this study. The 5 m M Cl  treatment resulted in: (i) higher dry biomass and greater leaf area (Figure S1); (ii) improved leaf water balance parameters, including Water Content (WC), RWC, succulence, leaf thickness, tissue osmolarity and turgor (Figure S2); and (iii) greater WUE as a consequence of a lower g s that reduced leaf transpiration and increased both photosynthetic and intrinsic WUE (Figure 1). This work elucidates: the physiological mechanism responsible for the observed Cl  -dependent g s reduction; how this phenomenon can be, in turn, compatible with the stimulation of higher growth in tobacco plants; and the physiological and cellular basis behind the beneficial effect of Cl  on plant growth when this element accumulates to levels that are typical of the content of a macronutrient. Effect of nutritional treatments on stomatal opening Stomatal opening is a crucial mechanism in the regulation of gas exchange. It has been proposed that rather than anatomical alterations, changes of stomatal opening is the most important factor regulating g s and WUEi (Paoleti and Gellini, 1993). In fact, the role of anion transporters on the regulation of stomatal closure is a well known phenomenon at the cellular and molecular level. Particularly, anion channels such as SLAC1 and SLAH3, which mediate Cl  and NO 3 release from guard cells, are the components that connect the ABA-mediated signalling of environmental cues with the physical events that trigger stomatal closure (Negi et al., 2008; Vahisalu et al., 2008; Meyer et al., 2010; Geiger et al., 2011). High content of Cl  in leaf tissues of CL plants, for example, concentrations ranging 100–150 m M Cl  (Franco-Navarro et al., 2016), might alter Figure 7. Effect of Cl  nutrition on leaf starch content.Plants were alternatively treated with the following nutritional supplements: 5 m M chloride salts (CL); 5 m M nitrate salts (N); and a mixture of sulphate + phosphate salts (SP) containing the same cationic balance as in the CL and N treatments. Mean values SE, n= 5. ‘Homogeneous group’ statistic was calculated through ANOVA, where mean values with different letters are significantly different according to Tukey’s test at P≤0.05. Figure 8. Relative limitations on A N . Contributions (q) of individual variables (g s , stomatal conductance; g m , mesophyll diffusion conductance to CO 2 ; and V c,max , maximum velocity of carboxylation or J max , maximum rate of electron transport) to the reduction in net photosynthesis rate (A N ) shown by CL and SP plants using the values of N plants as reference. q Vcmax/Jmax does not represent the contribution of the ratio V cmax /J max but the contribution of each value. ©2019 The Authors The Plant Journal ©2019 John Wiley & Sons Ltd, The Plant Journal, (2019), 99, 815–831 822 Juan D. Franco-Navarro et al. the flow of anions from epidermal cells, for example due to high extracellular accumulation of the anion, therefore modifying regulatory properties of stomatal activity. However, we proved that the Cl  -dependent reduction in g s did not result from a lower stomatal opening (Figure 2). First, plants supplemented with any of the nutritional treatments SP, N and CL, increased their stomatal opening capacity in comparison with plants grown with the BS (Figure S3). Second, the CL and SP treatments resulted in the same stomatal opening (width to length ratio; Figure 2b,c). Third, the absolute stomatal pore area of CL plants was 39% greater than that of SP plants (Figure 2d). Therefore, we could conclude that Cl  does not induce a greater stomatal closure. The nutritional treatments (SP, N and CL) clearly pointed to a role of inorganic ions in the induction in stomatal aperture according to their availability (Figure S4). Mineral nutrition is one of the factors affecting plant growth and water relations (Cramer et al., 2009). The three nutritional supplements have in common the same concentration of cations, including potassium (K + , 2.5 m M ), calcium (Ca 2+ , 0.6 m M ), and magnesium (Mg 2+ , 0.6 m M ). The importance of potassium (K + ) in plant performance and photosynthetic efficiency is a well known phenomenon. The nutritional treatments (SP, N and CL) increased K + concentration from 2.1 m M (in the BS solution) to 4.6 m M (Table S1). In Laurus nobilis,K + application resulted in a 45% increase in the transpiration rate 24 h after fertilization (Oddo et al., 2011). It is not clear, however, which cellular, biochemical or physiological factors are directly involved in this response. Losch et al. (1992) concluded a beneficial effect of K + supply on WUE as a consequence of altered stomatal sizes and densities. However, in this work K + was applied as KCl and the specific effects of Cl  on the parameters measured were not controlled. Interestingly, the NO 3 treatment (N) induced an even greater stomatal opening ratio compared to the other supplements applied (SP and CL; Figure 2b), even in the presence of ABA (Figure 2c). This phenomenon is possibly related to the higher A N observed in N plants (Figure 1d). Plants treated with an additional supply of 5 m M NO 3 (N plants), presented higher leaf NO 3 and total nitrogen content (TNC; Table 1). The bulk of leaf nitrogen is associated with photosynthetic enzymes, particularly ribulose-1,5-bis- phosphate carboxylase/oxygenase (RuBisCo), resulting in a strong positive correlation between A N and leaf TNC (Schulze et al., 1994; Wright et al., 2003), as observed in Figure 1(d), where N plants presented 17% higher A N than SP and CL plants. It has been proposed that higher leaf TNC increases the availability of photosynthetic protein, which improves the assimilation rate of CO 2 (Cramer et al., 2009). Although N plants showed higher V cmax and J max values (Figure 1g,h), no statistically significant differences with CL and SP plants were observed. It has been in turn been proposed as a positive regulation of g s by leaf nitrogen content, which has been quantified using both ecological (Schulze et al., 1994) and physiological (Wilkinson et al., 2007) approaches. It is not clear, however, if the proposed nitrogen-dependent g s regulation relies on the control of the stomatal aperture, the stomatal density, or both. Our results indicate that the increase in leaf TNC observed in N plants leads to a higher stomatal aperture (Figure 2b, c), not affecting the stomatal density (Figure 3d). Effect of Cl  nutrition on epidermal cell size and stomatal density While macronutrient Cl  nutrition did not induce stomatal closure, it decreased stomatal density (number of stomata per unit area; Figure 3d). Stomatal density is an important eco-physiological parameter that affects gas exchange and photosynthesis (Pompelli et al., 2010). During leaf ontogenesis, stomatal density is firstly determined by stomatal initiation and later by epidermal cell expansion. Therefore, stomatal density is a function of cell size (Salisbury, 1928), which explains that the increase in the size of epidermal cells in CL plants (Figure 3a,b) is in the origin of the reduced stomatal density (Figure 3d). Conversely, the stomata index (percentage of stomata out of the total number of epidermal cells plus stomata), which might imply a biological impact on stomatal development (Ticha, 1982; Lake et al., 2001), is independent of cell size (Salisbury, 1928), and was not affected by the nutritional treatments (Figure 3e). Various environmental factors such as irradiance, water deficit and salinity modify the absolute values of stomatal density and stomatal sizes, without affecting the ontogenic pattern of the changes (Ticha, 1982). Our results showed that rather than the biogenesis of stomata, Cl  affects the size and the density of different leaf cell types, including that of guard cells (Figure 3). The larger size of stomata in CL plants is also in agreement with its greater stomatal pore area (39% compared with the SP plants; Figure 2d). Although this may seem contradictory to the decreased g s , the calculation of the maximum pore area according to Franks et al. (2009), showed that the effect of a lower stomatal density in CL plants is more determinant than that of a larger pore size to explain the eventual effect on decreasing g s (Figure 3f). This g w,max reduction value (36.8%) was similar to the 33.3% reduction in g s calculated for CL plants (Figure 1a). Therefore we could conclude that the lower stomatal density accounts for the lower g s quantified in Cl  -treated plants. 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