Nitrogen (N) is the main limiting nutrient for land plants and, therefore, has been classified as an essential macronutrient. Nitrate (NO₃^–) represents the major N source and a signal molecule involved in the control of many physiological and developmental processes, strongly improving crop yield (Frink et al., 1999; Wang et al., 2012; Krapp et al., 2014; Guan, 2017). The decisive role of N in crop yield has led to excessive use of NO₃^– in agriculture over decades generating serious environmental problems like water pollution, which is harmful to people and nature (Nitrates Directive, 1991; Kant et al., 2011). In addition, when the application rate of NO₃^– exceeds the plant growth needs, overaccumulation of NO₃^– in leaves reduces the nutritional quality of crops (Prasad and Chetty, 2008; Xing et al., 2019). Many large-leaved plants such as beets, cabbage, celery, lettuce, or spinach tend to store huge amounts of NO₃^– (MAFF, 1998), posing a serious risk to human health. When ingested, NO₃^– is rapidly converted to nitrite and N-nitrous compounds as nitrosamines or nitric oxide causing methemoglobinemia or “blue baby syndrome” in infants and gastric cancer among other pathological disorders (Comly, 1945; Santamaria et al., 1999; Mensinga et al., 2003).

Considering that the growing world population is predicted to reach 9.8 billion in 2050, global efforts are being made to increase food resources improving crop or agronomic practices (Tilman et al., 2002; Godfray et al., 2010). Since only 30–40% of the N applied to soil is used by plants, a greater N use efficiency (NUE) could improve the yield and quality of crops, reducing economic costs as well as decreasing environmental degradation (Baligar et al., 2001). NUE can be defined as the vegetative or reproductive biomass yield per unit of N available in the soil (Moll et al., 1982; Woodend and Glass, 1993; Ríos et al., 2010). This concept has many variants that can be split into two main elements: (i) N uptake efficiency (NU_PE), defined as the capacity of plant roots to take N from soil, and (ii) N utilization efficiency (NU_TE), defined as the fraction of plant-acquired N to be converted to total biomass or grain yield (Xu et al., 2012). Both are considered important traits in agriculture to reduce the abusive use of N fertilizers or when N availability constrains plant growth, with substantial benefits for farmers and to the environment (Baligar et al., 2001; Han et al., 2016). Crops with higher NUE promote greater yields under limited N in soil, or require lower N to produce the same yield as those with lower NUE capacity (Ruiz et al., 2006; Kant et al., 2011; Rubio-Wilhelmi et al., 2012). Therefore, when NUE is increased, both crop-production costs and the harmful input of NO₃^– into ecosystems are reduced.

Traditionally, chloride (Cl^–) has been considered an essential micronutrient for plants (White and Broadley, 2001; Broadley et al., 2012). But recently, Cl^– has been uncovered as beneficial when accumulated to macronutrient levels in plant tissues (Franco-Navarro et al., 2016; Raven, 2017; Wege et al., 2017; Colmenero-Flores et al., 2019), with new biological functions that improve tissue water balance, whole-plant water relations, photosynthesis performance, and water-use efficiency (Franco-Navarro et al., 2016, 2019; Nieves-Cordones et al., 2019). Chloride represents the dominant inorganic anion in the vacuole, with leaf contents that can be similar to those of the macronutrient K⁺, promoting cell osmoregulation, turgor-driven processes, leaf cell elongation, and a reduction in stomatal conductance (g_s; Franco-Navarro et al., 2016). In addition, Cl^– specifically increases mesophyll diffusion conductance to CO₂ (g_m) as a consequence of the greater surface area of chloroplasts exposed to the intercellular airspace of mesophyll cells, which in turn points towards Cl^– playing a role in chloroplast performance (Franco-Navarro et al., 2019). Thus, Cl^– specifically reduces g_s and water loss through transpiration without affecting the photosynthetic capacity due to g_m stimulation, resulting in overall higher water-use efficiency (Franco-Navarro et al., 2016, 2019; Maron, 2019). Nitrate and Cl^– are the most abundant inorganic anions, having similar physical and osmoregulatory properties and sharing transport mechanisms (Colmenero-Flores et al., 2019). This is probably the reason why NO₃^– and Cl^– show strong dynamic interactions in plants (Wege et al., 2017), a phenomenon that has been described as a competitive interaction between these two monovalent anions. Different studies have reported a negative effect of Cl^– on root NO₃^– uptake and accumulation (Siddiqi et al., 1990; Cerezo et al., 1997; Xu et al., 2000). For this reason and because of the toxicity generated by excessive Cl^– accumulation in sensitive crops under salt–stress conditions (Li et al., 2017; Geilfus, 2018), Cl^– has been considered detrimental to agriculture. Overall, Cl^– is believed to reduce NUE by limiting NO₃^– uptake and accumulation in plant tissues, reducing in turn its availability for plant metabolism (Xu et al., 2000; Anjana and Iqbal, 2007; Wege et al., 2017). However, Cl^– is a non-metabolized anion whose vacuolar accumulation requires a lower energy cost than the accumulation of NO₃^– (Wege et al., 2017). Thus, considering the close interactions between these two anions, it has been hypothesized that preferential Cl^– compartmentalization may reduce vacuolar NO₃^– storage in leaves (Flowers, 1988), allowing higher NO₃^– availability for plant metabolism and, consequently, promoting more efficient use of this N source, meaning higher NUE (Colmenero-Flores et al., 2019). Therefore, the goal of this study was to verify whether Cl^– reduces leaf NO₃^– accumulation while promoting more efficient use of N–NO₃^–. In order to prove this, different plant species with contrasting Cl^–-accumulating abilities have been used in this work: three leafy herbaceous species with strong Cl^–-including capacity (chard, spinach, and lettuce), two herbaceous Cl^–-including Solanaceae species (tobacco and tomato), and two Cl^–-excluding woody species (olive and the salt-tolerant citrus rootstock Cleopatra mandarin). To directly ascertain the effect of Cl^– on NO₃^– nutrition, plant growth and different NUE parameters have been quantified, using NO₃^– as the sole N source.

NO₃^–, an essential source of N, and Cl^–, an important osmoregulatory molecule and beneficial macronutrient, are the most abundant inorganic anions in plants, and both must be coordinately incorporated during the active growth of plants (Cubero-Font et al., 2016; Colmenero-Flores et al., 2019). Both anions play important roles in charge balance and turgor regulation, showing strong dynamic interactions in land plants (Wege et al., 2017; Geilfus, 2018; Colmenero-Flores et al., 2019). Since NO₃^– and Cl^– also present similar physical properties in solution, they share ion transport mechanisms with uncertain selectivities for both anions. NO₃^–, as a source of the essential macronutrient N, is assimilated during anabolic metabolism, while Cl^–, which is not metabolized, becomes accumulated in plant tissues. Interaction between NO₃^– and Cl^– has been traditionally understood as antagonistic. For instance, a high tissue content of Cl^– is believed to reduce the content of NO₃^– and vice versa (Xu et al., 2000; Umar and Iqbal, 2007). The presence of external NO₃^– has been shown to inhibit root Cl^– uptake (Glass and Siddiqi, 1985; Iglesias et al., 2004), and on the contrary, high Cl^– content reduces NO₃^– accumulation in plants, suggesting that common transport mechanisms could facilitate the influx of both anions (Xu et al., 2000; Teakle and Tyerman, 2010). This antagonism between NO₃^– and Cl^– has been widely reported for many crops, pointing to a clear detrimental effect of Cl^– on NO₃^– nutrition (transport, accumulation, and/or assimilation; Buwalda and Smith, 1991; Cerezo et al., 1997; Xu et al., 2000). As a result, Cl^– is considered harmful to crop productivity, to the extent that its presence in some types of plant fertilizers is considered as a negative indicator of their quality (EU Regulation 2019/1009). However, Cl^– has been recently defined as a beneficial macronutrient that improves plant development, water relations, CO₂ assimilation, and water-use efficiency when supplemented at concentrations higher than those necessary to satisfy micronutrient requirements but insufficient to cause toxicity (e.g., in the beneficial range of 1–5 mM Cl^–; Colmenero-Flores et al., 2019; Franco-Navarro et al., 2019). The fact that Cl^– specifically promotes plant biomass due to these beneficial effects (Franco-Navarro et al., 2016) is difficult to reconcile with a detrimental effect on NO₃^– nutrition.

Consistent with our previous findings (Franco-Navarro et al., 2016, 2019), tobacco plants accumulated Cl^– at levels that are typical of a macronutrient, stimulating plant growth when applied at concentrations above 1 mM Cl^– (Figures 1A, 2A). Furthermore, although leaf Cl^– content was ∼120 times lower in SP and N plants in comparison to CL, it remained over the critical threshold of Cl^– deficiency reported for non-halophytic plants (<0.2 mg g^–1 shoot DW; Flowers, 1988; Xu et al., 2000; White and Broadley, 2001; Franco-Navarro et al., 2016), which ruled out the occurrence of Cl^– deficiency in SP and N treatments. Moreover, the higher growth of N plants (Figure 1A) confirmed this point and strengthens the well-known fact that NO₃^– has a strong impact on plant growth and development (Glass, 2003; Hawkesford et al., 2012; Wang et al., 2012; Krapp et al., 2014; Guan, 2017).

NUE is an important crop trait described as a useful tool to improve agricultural systems (Fageria et al., 2008). This work clearly states that, contrary to what was previously believed, Cl^– improves NUE in plants, at least when NO₃^– is used as the sole N source. The decline in leaf biomass has been directly correlated to N deficiency particularly in tobacco plants (Balachandran et al., 1997), since this crop requires high quantities of NO₃^– for maximum vegetative yield (Ruiz et al., 2006). Considering that N is not only an essential nutrient for optimal crop yield but also an environmental concern, adequate management of N fertilization regimes to enhance NUE remains critical for crop breeding. Our results confirm that Cl^– significantly increases NUE, not only in tobacco plants (Figures 1, 2) but also in different crop species (Figure 4), when accumulated at macronutrient levels. NUE improvement in tobacco plants was a consequence of more efficient use of the NO₃^– taken up by the plant (NU_TE; Figures 2D, 3C), meaning that Cl^– improves NO₃^– assimilation, as observed in other crop species (Figure 4D). A significant positive correlation (r² = 0.995) between leaf Cl^– content and NU_TE was established in tobacco plants (Figure 2F). Interestingly, this positive correlation was also observed in different plant species with contrasting abilities to accumulate Cl^– (Figure 4D). Thus, NU_TE gain by Cl^– application was minimal in Cl^– excluding species (0–22% NU_TE increment in olive and the citrus rootstock Cleopatra mandarin plants, respectively) and maximal in Cl^– including ones (60–80% NU_TE increment in tomato and tobacco plants, respectively), indicating a positive ecophysiological correlation between leaf Cl^– accumulation and NU_TE. However, this correlation was lost in strong Cl^– including vegetables (30–55% NU_TE increment in the large-leaved spinach, chard, and lettuce plants), suggesting the occurrence of a saturable response, possibly as a consequence of excessive Cl^– accumulation. This positive correlation between Cl^– content and NU_TE can be a selection criterion to identify new cultivars or genotypes obtained from breeding programs, with potentially improved NUE capacity. Thus, genotypes that, in the presence of 5 mM Cl^–, show leaf Cl^– contents between 20 and 50 mg g^–1 DW (Table 1), within the positive linear response range shown in Figure 4D, could be good candidates not only for improved NUE but also for higher efficiency in the use of water and CO₂ (Colmenero-Flores et al., 2019).

These results were obtained comparing SP and CL treatments, both containing the same NO₃^– concentration (5 mM NO₃^–). However, NU_TE stimulation by Cl^– was much higher when the CL treatment was compared with the N treatment (10.25 mM NO₃^–). The increase in NU_TE in CL vs. N tobacco plants was ∼250% (Figure 1F), suggesting that NO₃^– fertilization in the field can be efficiently regulated if optimal supplies of NO₃^–/Cl^– ratios are used. Thus, increasing the Cl^–/NO₃^– ratio showed two positive effects on plants: reduction in leaf NO₃^– content (Figure 3A) while at the same time increasing plant biomass (Figure 3B). Different studies have proposed a negative effect of Cl^– on NO₃^– uptake and accumulation (Siddiqi et al., 1990; Cerezo et al., 1997; Li et al., 2017), which is supposed to reduce NUE. Nevertheless, our results ruled out the possibility that Cl^– impairs N use because the CL treatment increased plant biomass (Figure 3B), while the effective reduction in NO₃^– in the nutrient solution produced a strong reduction in plant biomass (Figure 3E). This clearly indicates that the loss of leaf NO₃^– content through Cl^– application is not a consequence of lower root NO₃^– uptake (e.g., lower NO₃^– availability as a consequence of Cl^– antagonism; Figure 2C) but of a greater NO₃^– assimilation capacity. The NO₃^– vs. Cl^– antagonism must be understood in terms of the selectivity of anion transporters. Given the great relevance of N for plant nutrition, plants prioritize NO₃^– uptake over Cl^– uptake when NO₃^– is available in the soil. This means that active transport mechanisms are normally more selective for NO₃^– than for Cl^– (Glass and Siddiqi, 1985; Wege et al., 2017; Wen et al., 2017). Consequently, increasing the NO₃^– concentration in the nutrient solution reduces Cl^– content in plants (Glass and Siddiqi, 1985; Iglesias et al., 2004). However, the opposite situation is not necessarily true. Although widely reported (Xu et al., 2000; and references therein), Cl^– application in the low millimolar range should not impair NO₃^– uptake given the high selectivity for NO₃^– over Cl^–. The total N content of plants does not decrease in response to Cl^– application (Figure 1D; Ourry et al., 1992; Liu and Shelp, 1996; Inal et al., 1998). However, in Figure 4C, a moderate reduction in NU_PE can be observed in different plant species in response to Cl^– application. Rather than an effective reduction in NO₃^– uptake transport through transmembrane transporters at the soil–root interface, NU_PE reduction can be a consequence of the calculation procedure. The NU_PE formula computes the NO₃^– content in plant tissues, which is lower in plants treated with Cl^– because NO₃^– is more efficiently assimilated, as also proposed by Liu and Shelp (1996). It is very likely, however, that under salinity stress conditions, Cl^– antagonizes NO₃^– influx in plant cells, significantly reducing root NO₃^– uptake (Cerezo et al., 1997; Li et al., 2017).

Therefore, our results strongly support the previously suggested role of Cl^– as preferred plant osmoregulatory molecule in plants (Flowers, 1988; Franco-Navarro et al., 2016; Colmenero-Flores et al., 2019). Thus, we propose that, on the one hand, Cl^– is preferably compartmentalized in the vacuole. On the other hand, NO₃^–, an essential N source for land plants, is preferentially assimilated, which is not possible when this molecule is sequestered in the vacuole to carrry out an osmotic function. Only when Cl^– is not sufficiently available in the soil, or as a result of excessive NO₃^– availability, NO₃^– could be preferentially compartmentalized (Siddiqi et al., 1991; Radcliffe et al., 2005). Therefore, macronutrient accumulation of Cl^– reduces NO₃^– compartmentalization in the vacuole, facilitating its assimilation, which increases NUE and plant biomass. Under the same premise, Cl^– should also play an adaptive role to improve plant growth under conditions of low N availability, which is also explained in terms of differential transport selectivity. When little NO₃^– is available, root Cl^– uptake through active anion transporters is less inhibited (Wen et al., 2017), increasing cell Cl^– content and replacing NO₃^– in the vacuole, which facilitates NO₃^– assimilation and NUE. A clear demonstration that the relationship between Cl^– and NO₃^– homeostasis in higher plants is not limited to an antagonistic interaction has been recently shown by Cubero-Font et al. (2016). This work describes a molecular mechanism that determines the rate of NO₃^–/Cl^– accumulation in aerial organs of Arabidopsis thaliana based on the Cl^– conductance of the AtSLAH3 channel, which is, in turn, regulated by environmental cues.

Agronomic and scientific communities have traditionally believed that little amounts of Cl^– are required to achieve suitable crop yields (Geilfus, 2018). Nevertheless, some studies have shown that the application of Cl^–-enriched fertilizers to the soil increases the vegetative yield in different crops (Christensen et al., 1981; Timm et al., 1986; Inal et al., 1998; Xu et al., 2000). However, it was not clear to what extent plant yield improvement was due to the accompanying cations or whether other anions could replace Cl^– in the reported growth-promoting effects. In accordance with the recently revealed functions of Cl^– as a beneficial macronutrient (Franco-Navarro et al., 2016; Colmenero-Flores et al., 2019), it has been proven that a number of physiological perturbations impairing the growth and yield of durum wheat under field conditions are specifically due to soil Cl^– deficiency (Schwenke et al., 2015). Hence, we investigated how crops could benefit from certain levels of Cl^– fertilization. In the herbaceous species studied (i.e., tomato, lettuce, spinach, and chard), the 5 mM Cl^– treatment determined plant biomass gains following the leaf Cl^– content within the beneficial macronutrient range (40–110 mg g^–1 DW; Colmenero-Flores et al., 2019; Figure 3B). These Cl^– content values are up to an order of magnitude above what was classically considered toxic concentrations in plants (Xu et al., 2000), largely dismantling this view of Cl^– as detrimental to agriculture (Colmenero-Flores et al., 2019).

Given the high NO₃^– content in fertilizers and its often abusive use in agriculture, NO₃^– can be excessively accumulated in leaves of most horticultural crops, resulting in food safety problems (e.g., methemoglobinemia and cancer) because of its transformation into nitrites and nitrosamines (Colla et al., 2018). This is particularly harmful in leafy vegetables, for which the European Commission has developed severe regulations (1881/2006 and 1258/2011) to reduce the excessive dietary intake of NO₃^–, especially that of vulnerable people such as infants, the elderly, and vegetarians. As previously stressed, increasing the Cl^–/NO₃^– ratios reduced the leaf NO₃^– content (Figure 3A) without impairing, or even increasing, plant biomass (Figure 3B). In our study, the NO₃^– content in leafy species (lettuce, spinach, and chard) treated with SP ranged between 577 and 1,035 mg NO₃^– kg^–1 FW (Supplementary Table S4), proving to be much lower than the maximum permitted levels, which are set at 3,500 and 2,500 mg NO₃^– kg^–1 FW in spinach and iceberg lettuce, respectively. It should be noted, however, that the SP treatment contains 5 mM NO₃^–, probably well below the levels applied in the field by farmers. Chloride reduced about 25–70% the NO₃^– content in the plant species assayed (compared to SP plants; Figure 4B). These results are in accordance with those reported by Urrestarazu et al. (1998) in lettuce, Inal et al. (1998) in carrot, and Borgognone et al. (2016) in cardoon. Therefore, Cl^– nutrition is expected to considerably improve the nutritional quality of vegetables and brings to light the important benefits of using Cl^–-enriched fertilizers in human health. Interestingly, Cl^–-treated tobacco plants showed the strongest decrease in NO₃^– content (∼70% compared to SP plants; Figure 4B). Considering that NO₃^– is the main inducer of nitrogen oxides and nitrosamines in flue-cured tobacco during smoking (Hoffmann and Hecht, 1985), Cl^– nutrition could also help to reduce the nitrosamine levels in cigarettes, improving the quality of this crop.

We provide for the first time a direct demonstration which shows that Cl^–, contrary to impairing NO₃^– nutrition, facilitates NO₃^– utilization and improves NUE in plants. This is largely due to Cl^– improvement of NU_TE, having a little or moderate effect on NU_PE when NO₃^– is used as the sole N source in the nutrient solution. Clear positive correlations between leaf Cl^– content vs. NU_TE or vs. plant growth have been established at both intra- and interspecies levels: in tobacco plants treated with growing Cl^– concentrations and comparing different species with contrasting abilities to accumulate Cl^–. Our results strongly suggest that macronutrient Cl^– nutrition reduces NO₃^– sequestration in plant leaf tissues (e.g., vacuolar compartmentalization), making this valuable N source available for assimilation and biosynthesis of organic N. Our results give light to a brand-new interpretation of Cl^– properties as a beneficial macronutrient for higher plants that promote more efficient use of water, carbon, and nitrogen, becoming a potential resource to improve agricultural production and quality, reducing NO₃^– inputs in the field and unhealthy leaf NO₃^– content in vegetables.

The datasets generated for this study are available on request to the corresponding author.

JF-N performed the experiments, analyzed the data, and participated in the writing of the manuscript. PP-T and PD-R participated in the experiments. RÁ participated in the conception of research plans. MR and JC-F conceived research plans, supervised the experiments, and wrote the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.