Macronutrient chloride nutrition improves drought resistance by enhancing water deficit avoidance and tolerance mechanisms

Journal of Experimental Botany, Vol. 72, No. 14 pp. 5246–5261, 2021 doi:10.1093/jxb/erab143 Advance Access Publication 30 March 2021 Abbreviations:ABA, abscisic acid; AN, net photosynthetic rate; Cl−, chloride; CTR, control; DAS, days after sowing; gm, mesophyll diffusion conductance to CO2; gs, stomatal conductance; NO3−, nitrate; NUE, nitrogen-use efficiency;PO43−, phosphate; Pp, patch output pressure; Qy, PSII quantum yield; RWC, relative water content; SLA, specific leaf area; SO42−, sulfate; WD, water deficit; WUE, water-use efficiency;WUEi, intrinsic water-use efficiency; Ψ π, osmotic potential; Ψ p, turgor potential; Ψ W, water potential. © The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. RESEARCH PAPER Chloride nutrition improves drought resistance by enhancing water deficit avoidance and tolerance mechanisms JuanD.Franco-Navarro1,, PabloDíaz-Rueda1,, CarlosM.Rivero-Núñez1, JavierBrumós2, AlfredoE.Rubio-Casal3,, AlfonsodeCires3, JoséM.Colmenero-Flores1,4,*, and MiguelA.Rosales1,4,*, 1 Group of Plant Ion and Water Regulation, Instituto de Recursos Naturales y Agrobiología, Consejo Superior de Investigaciones Científicas (CSIC), 41012 Seville, Spain 2 Instituto Valenciano de Investigaciones Agrarias, Centro de Genómica, Moncada, 46113 Valencia, Spain 3 Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla, 41012 Seville, Spain 4 Laboratory of Plant Molecular Ecophysiology, Instituto de Recursos Naturales y Agrobiología, Consejo Superior de Investigaciones Científicas (CSIC), 41012 Seville, Spain * Correspondence: mr[email protected] or [email protected] Received 30 September 2020; Editorial decision 24 March 2021; Accepted 25 March 2021 Editor: Ian Dodd, Lancaster University,UK Abstract Chloride (Cl−), traditionally considered harmful for agriculture, has recently been defined as a beneficial macronutrient with specific roles that result in more efficient use of water (WUE), nitrogen (NUE), and CO2 in well-watered plants. When supplied in a beneficial range of 1–5mM, Cl− increases leaf cell size, improves leaf osmoregulation, and reduces water consumption without impairing photosynthetic efficiency, resulting in overall higher WUE. Thus, adequate management of Cl− nutrition arises as a potential strategy to increase the ability of plants to withstand water deficit. To study the relationship between Cl− nutrition and drought resistance, tobacco plants treated with 0.5–5mM Cl− salts were subjected to sustained water deficit (WD; 60% field capacity) and water deprivation/rehydration treatments, in comparison with plants treated with equivalent concentrations of nitrate, sulfate, and phosphate salts. The results showed that Cl− application reduced stress symptoms and improved plant growth during water deficit. Drought resistance promoted by Cl− nutrition resulted from the simultaneous occurrence of water deficit avoidance and tolerance mechanisms, which improved leaf turgor, water balance, photosynthesis performance, and WUE. Thus, it is proposed that beneficial Cl− levels increase the ability of crops to withstand drought, promoting a more sustainable and resilient agriculture. Keywords: Beneficial macronutrient, chloride, drought resistance, photosynthesis, turgor, water deficit, water relations, WUE. applyparastyle "fig//caption/p[1]" parastyle "FigCapt" This paper is available online free of all access charges (see https://academic.oup.com/jxb/pages/openaccess for further details) Downloaded from https://academic.oup.com/jxb/article/72/14/5246/6204166 by UNIVERSIDAD DE SEVILLA user on 06 September 2021 Chloride nutrition improves drought tolerance | 5247 Introduction In the context of the climate change and strong water demand from intensive agriculture, drought is certainly the abiotic stress that most severely affects crop productivity (Comas etal., 2013; FAO, 2016). Understanding how plants respond to water availability and how water is used for optimal biomass production and yield has gained enormous importance in agriculture (Davies and Bennett, 2015; Maurel and Nacry, 2020). In general, lower availability of soil water during drought leads to a decrease of leaf relative water content (RWC) and leaf water potential (Ψ w) that causes abscisic acid (ABA) biosynthesis (McAdam and Brodribb, 2016, 2018; Sack etal., 2018), triggering complex plant acclimatization responses at molecular, cellular, and physiological levels. These responses include water deficit (WD) avoidance and tolerance mechanisms, according to the nomenclature of Levitt (1972). Avoidance responses include mechanisms that maintain plant water content and Ψw close to unstressed levels, mainly by increasing water uptake or limiting water loss. Induction of stomatal closure reduces water loss through transpiration (Rosales etal., 2012; Koevoets etal., 2016; Buckley, 2019), but leads to a reduction of CO2 availability and photosynthesis and, consequently, to the decrease of vegetative growth and yield (Galmés etal., 2007; Chaves etal., 2009; Ferguson etal., 2018). In addition, mechanisms that improve soil water uptake are also stimulated (Rosales etal., 2019; Scharwies and Dinneny, 2019). When WD avoidance mechanisms are overcome and plant tissues experience cellular dehydration, tolerance mechanisms must ensure cell survival and the plant ability to resume growth, including the induction of cell osmotic adjustment and the biosynthesis of protective solutes and proteins (Verslues etal., 2006). Considering that 80% of the available freshwater resources are currently consumed by agriculture, the improvement of water-use efficiency (WUE), defined as the amount of carbon fixed in photosynthesis per unit of water transpired, remains essential for establishing a balance between agriculture and water resources (Condon etal., 2004; Flexas etal., 2016). Because of the urgent need to improve the world’s crop production, WUE is considered an essential trait to minimize the loss of water in plants. As a consequence, considerable efforts have been made to elucidate physiological and genetic factors associated with this trait (Condon etal., 2004; Blum, 2009; Hessini etal., 2009; Medrano etal., 2015). Several strategies have focused on obtaining new crop varieties with higher WUE and on better management of water resources, such as: (i) improving the irrigation processes and reducing the water loss through soil evaporation or leakage; (ii) increasing the efficiency of fixing carbon in relation to water transpired; and (iii) partitioning more of the achieved biomass into the harvested product (reviewed in Condon etal., 2004). However, due to the complexity of these traits, simpler and more specific aspects of WUE are required to identify single targets of manipulation (Flexas etal., 2016). Chloride (Cl−) has been well characterized as a micronutrient, playing an essential role as a cofactor for PSII and regulating the activity of some enzymes (Broadley etal., 2012). In addition, Cl− is a major osmotically active solute in the vacuole (Flowers, 1988). As a counter anion, Cl− plays relevant roles in regulating the electrical potential of different membranes, the organellar pH gradients, and the electrical excitability of plant cells (White and Broadley, 2001). However, Cl− has been traditionally considered harmful for agriculture, for two main reasons: (i) the toxicity resulting from excessive Cl− accumulation in sensitive crops under salt stress conditions (Li etal., 2017; Geilfus, 2018); and (ii) the generalized belief that Cl− antagonizes nitrate (NO3−) homeostasis, impairing the ability of crops to transport and accumulate NO3− (Kafkafi etal., 1982; Siddiqi etal., 1990; Xu etal., 2000; Wege etal., 2017). However, Cl− nutrition to typical macronutrient levels has been recently uncovered as beneficial for plant growth under well-watered conditions, with new biological functions that improve cell water balance, whole-plant water relations, photosynthesis performance, WUE, and nitrogen-use efficiency (NUE; i.e. the vegetative or reproductive biomass yield per unit of nitrogen available in the soil) in plants (Franco-Navarro et al., 2016, 2019; Rosales etal., 2020). Thus, Cl− has been proposed as a beneficial macronutrient (Franco-Navarro etal., 2016), a definition further supported by others (Raven, 2017; Wege etal., 2017; Geilfus, 2018; Orieux etal., 2018; Bazihizina etal., 2019; Raven, 2020). Firstly, when supplied above the micronutrient requirement and below the toxicity threshold (e.g. 1–5mM Cl−), Cl− plays specific roles in the regulation of cell osmolarity and turgor, stimulating leaf cell size and leaf water balance. The resulting enlargement of leaf cell size reduces the stomatal density, which in turn lowers stomatal conductance (gs) and water consumption. Secondly, Cl− also increases mesophyll diffusion conductance to CO2 (gm), which makes it possible to maintain the plant photosynthetic capacity despite the reduction of gs, resulting in overall higher WUE in well-watered plants (Franco-Navarro etal., 2019). Therefore, adequate management of Cl− nutrition to improve crop yield while also reducing water consumption is particularly challenging in C3 plants (Maron etal., 2019). Cl− fluxes are also relevant for adequate regulation of stomatal closure (Nieves-Cordones etal., 2019) and specifically required for cell osmotic adjustment in response to osmotic stress (Shabala and Lew, 2002). Therefore, through its role in the regulation of cell osmolarity, water balance, and WUE under well-watered conditions, Cl− homeostasis arises as a potential adaptive mechanism that might increase the ability of plants to withstand drought stress. So far, all previously reported functions of Cl− nutrition as a beneficial macronutrient have been experimentally performed under wellwatered conditions. No direct relationship between Cl− and drought resistance in glycophyte plants has been established to date. Therefore, the aim of this work is to elucidate this Downloaded from https://academic.oup.com/jxb/article/72/14/5246/6204166 by UNIVERSIDAD DE SEVILLA user on 06 September 2021 5248 | Franco-Navarro etal. question by: (i) quantifying the degree of WD resistance of Cl−-treated plants compared with plants treated with equivalent concentrations of anionic macronutrients such us NO3−, phosphate, and sulfate; and (ii) identifying relevant physiological mechanisms regulated by Cl− nutrition that improve WD resistance in plants. Materials andmethods Plant cultivation and experimentaldesign Tobacco (Nicotiana tabacum L. var. Havana) plants were grown under greenhouse experimental conditions (temperature of 25/17±2 °C day/night, relative humidity of 60±10%, and a 16 h/8h photoperiod with a photosynthetic photon flux density of 300–350μmol m−2 s−1). Plants were grown in 7.5 liter pots (20×17×25cm) containing a mix of perlite:vermiculite (4:6), and watered with a basal nutrient solution supplemented with three nutritional treatments: 5mM Cl− salts (CL), 5mM NO3− salts (N), and a mix of sulfate+phosphate (SO42−+PO43−) salts (SP), as previously reported in Franco-Navarro etal. (2016). The CL treatment was performed with the application of 5mM Cl−: 2.5mM KCl, 0.625mM MgCl2, and 0.625mM CaCl2. To evaluate the specificity of Cl− in the studied phenomena, two additional treatments were used: N treatment containing 2.5mM KNO3, 0.625mM Mg(NO3)2, and 0.625mM Ca(NO3)2; and SP treatment containing 1.25mM KH2PO4, 0.625mM K2SO4, 0.625mM MgSO4, and 0.625mM CaSO4. All treatments (CL, N, and SP) contained the same cationic balance as shown in Franco-Navarro etal. (2016). Nutrients present in the basal nutrient solution were as follows: 1.25mM KNO3, 0.625mM KH2PO4, 0.053mM K2HPO4, 2 mM Ca(NO3)2, 1 mM MgSO4, 0.1 mM FeNa-EDTA, 0.1 mM H3BO3, 0.1 mM MnSO4, 29 μM ZnSO4, 0.11 μM CoCl2, 53μM KCl, 0.1μM CuSO4, 1μM Na2MoO4, and 5μM KI. All experimental solutions were adjusted to pH 5.7 withKOH. After 30 d (45days after sowing; DAS), in addition to the three nutritional treatments, plants were subjected to two irrigation treatments: optimal irrigation (control; CTR), in which pots containing tobacco plants were irrigated up to 100% field capacity (3.5 ml g−1 substrate) throughout the experiment, and WD, with pots irrigated every 2–3 d up to 60% of field capacity (2.1ml g−1 substrate) for 20 d (65 DAS). During the WD treatment, the resulting average soil water content ranged between 60% and 10% of field capacity (Supplementary Fig. S1A). Another set of experiments with increasing concentrations of Cl− and SO42−+PO43− salts, in combination with CTR and WD regimes (100% and 60% of field capacity, respectively) as explained above, was performed for 26 d under similar experimental conditions to those previously described (Supplementary Fig. S1B). For CL treatments, 0.5, 2, and 5mM Cl− salts were applied to the basal solution, whereas the equivalent SP treatments were also added to ensure the same cationic balance as in different CL treatments (as described in Franco-Navarro etal., 2016). Plant sampling and determination of biomass and leaf parameters Samplings were performed from each combination of nutritional and irrigation treatments after 20 d or 26 d of water restriction, in which all plants were non-senescent and at the early reproductive stage. Different plant tissues were harvested separately and leaf area was measured as explained below. Subsequently, FW values from different plant tissues were obtained, and samples were dried in a forced-air oven at 75°C for 48h to obtain the DW values, both parameters recorded as grams perplant. After obtaining FW values, detached leaves of each tobacco plant were photographed and their leaf area was measured through pixel quantification with ImageJ2 Software with a high precision of 99.95– 100% (Rasband, 1997; Rueden etal., 2017). Data were obtained in cm2. Specific leaf area (SLA) was calculated as follows (Marcelis etal., 1998): SLA=(total leaf area)(total leaf DW)−1. Nutrient content determination Oven-dried leaf tissue was ground to powder using a homogenizer (Taurus, 25790, Barcelona, Spain) and the concentration of Cl−, NO3−, SO42−, and PO43− was determined as previously reported (FrancoNavarro etal., 2016). Water parameters Leaf water content, RWC, succulence, leaf osmotic potential (Ψ π), leaf Ψ w, and leaf turgor (or pressure) potential (Ψ p) were determined as previously described in Franco-Navarro etal. (2016). Water consumption was quantified gravimetrically by recording the weight loss of each pot, equivalent to the volume of solution consumed and lost by evapotranspiration by each plant. In WD-treated plants, water consumption was quantified as the volume of water needed to maintain field capacity up to 60%. Integrated WUE (WUEi) was calculated as the increase of plant DW over time related to the accumulated water consumption (g DW ml−1 H2O), as well as the DW obtained throughout the experiment and after harvesting related to total water consumption (g DW ml−1 H2O) (Abbate etal., 2004). Water deprivation and rehydration assay: quantum yield and pressureprobes Six tobacco plants of each nutritional treatment (SP, CL, and N) were maintained under CTR conditions up to 73 DAS, when water deprivation was applied for 4 d, and, at 77 DAS, plants were rehydrated at 100% of field capacity and monitored until 80 DAS. Three plants from each nutritional treatment were monitored every day by gravimetric methods to verify the water content in the soil, and PSII quantum yield (Qy) measurements were performed. For Qy determination, chlorophyll fluorescence in light-adapted plants was measured using a portable fluorometer (FluorPen FP-100; Photon System Instruments, Brno, Czech Republic), as described in Franco-Navarro etal. (2016). For each treatment, 3–5 photosynthetically active and fully expanded intermediate leaves from six plants were used. Qy measurements were conducted every day between 10 h and 12h from the beginning of the water restriction treatment (46–64DAS). For the other three plants, each plant was monitored with 2–3 LPCP probes (so-called ZIM probes; ZIM Plant Technology GmbH, Hennigsdorf, Germany), a non-invasive technique that records leaf turgor pressure in real-time (described in detail in Zimmermann etal., 2008, 2010). The leaf patch output pressure (Pp) is recorded in a leaf that is patched between a metallic sensing chip and a magnetic pad. Pp is inversely correlated with the leaf turgor pressure (Ehrenberger etal., 2012). Signals are sent wirelessly by transmitters to a controller that transfers the data to a GPRS modem linked to an Internet server. Probes and the Internet-based data transfer system were purchased from ZIM Plant Technology GmbH. Probes were clamped on 2–3 photosynthetically active and fully expanded intermediate leaves (fifth–sixth leaves from the top of the plant, at ~0.80 m above the ground), between the central vascular bundle and the edge of the leaves (~3cm away from the edge), and in the middle part of those leaves, in order to establish a uniform contact with the leaf tissue avoiding nerves (Fernández etal., 2011). The clamping was performed pre-dawn at maximum turgidity as recommended by Zimmermann etal. (2008, 2010, 2013). Pressure signals were appropriately adjusted between 10 kPa and 25 kPa, changing the distance between the two Downloaded from https://academic.oup.com/jxb/article/72/14/5246/6204166 by UNIVERSIDAD DE SEVILLA user on 06 September 2021 Chloride nutrition improves drought tolerance | 5249 magnets. The pressure sensor magnet was placed on the abaxial side of the leaves. When pressure probe recordings became stable at 68 DAS, variation in the diurnal amplitude of Pp was found because of possible differences in the initial clamp pressure, leaf thickness, or compressibility variations as reported in Zimmermann etal. (2008). Leaf gas exchange parameters Net photosynthetic rate (AN) and stomatal conductance (gs) were measured between 12.00h and 14.00h using an open gas exchange system (LI-6400, LI-COR, Lincoln, NE, USA) equipped with a 2×3cm LED chamber (LI-6400-02B) as described in Franco-Navarro et al. (2016). The WUEi was calculated as the ratio between the rate of photosynthesis and stomatal conductance (AN/gs). Statistical analyses Statistical analyses were performed using STATGRAPHICS Centurion XVI software (StatPoint Technologies, Warrenton, VA, USA). Shapiro– Wilk (W) test was used to verify the normality of the data sets. One-way ANOVA and multivariate analysis of variance (MANOVA) were performed to determine significant differences between groups of samples, and levels of significance were described by asterisks: *P≤0.05; **P≤0.01; ***P≤0.001. Non-significant (ns) differences were indicated when P was >0.05. Multiple comparisons of means were determined by the Tukey’s HSD (honestly significant difference) and MRT (multiple range test) statistical tests included in the mentioned software. Analysis of covariance (ANCOVA) was performed with R software (https://www.rproject.org/) to compare the slopes of the relationship of total biomass with water consumption between CL and SP treatments. Values represent the mean of at least six tobacco plants in each treatment, which were reproduced in at least three independent experiments (Supplementary Table S1). Results The effect of Cl− on plant growth during waterdeficit To study whether Cl− nutrition participates in plant adaptive responses to drought stress, greenhouse experiments were performed under two irrigation regimes: optimal irrigation (CTR) and sustained water deficit (WD). For the WD treatment, plants were watered every 2–3 d with the three nutritional treatments (CL, N, and SP) until the substrate reached 2.1 ml g−1 (60% of field capacity). The WD treatment was maintained for 20 d, whereas watering up to 100% of field capacity was established for the CTR treatment (3.5ml g−1). The time course of the substrate water loss throughout a representative experiment is presented in Supplementary Fig. S1A. First, we verified whether the effects of the 5mM Cl− treatment (CL) on plant growth were consistent with those previously obtained in Franco-Navarro etal. (2016, 2019). With this aim, we conducted new sets of experiments (Supplementary Table S1) and compared different nutritional and physiological effects of the CL treatment with those of plants subjected to low Cl− (SP and N treatments). Consistently, leaf anion contents (Cl−, NO3−, SO42−, and PO43−) were differentially accumulated in plants according to the respective nutritional treatments (CL, N, and SP) under both irrigation regimes (Table 1; Supplementary Table S2). Nutritional and irrigation treatments and their interaction significantly affected Cl− and NO3− contents in tobacco leaves (Table 1; Supplementary Table S2). Under control conditions, the Cl− concentration in CL-treated leaves was 106.5mM (i.e. 55.7mg g−1 DW), reaching typical macronutrient levels. In SP and N plants, Cl− content was 100 times lower, although far exceeding the critical levels of deficiency required to fulfil essential micronutrient functions (Broadley etal., 2012; Colmenero-Flores etal., 2019). Interestingly, the Cl‒ content significantly increased in drought-stressed CL and SP plants (1.12 and 2.5 times, respectively), whereas no changes were observed in N plants. In addition, the NO3− concentration was strongly decreased by WD in SP and N plants (2.1 and 3.3 times, respectively), whereas no relevant changes in SO42− and PO43− contents were observed (Table 1). Table 1. Anion concentration in leaves subjected to different nutritional and irrigation treatments Cl− (mM) NO3− (mM) PO43− (mM) SO42− (mM) CTR WD PCTR WD PCTR WD PCTR WD P SP 1.02±0.08 b 2.56±0.61 b * 6.71±1.18 b 3.20±0.78 b * 15.7±1.51 a 13.8±1.62 a ns 33.5±1.80 a 30.8±4.06 a ns CL 106.5±3.85 a 118.9±2.50 a * 2.48±0.28 b 2.00±0.42 b ns 6.97±0.58 b 8.76±0.29 b ** 12.0±2.55 b 11.1±0.38 b ns N1.01±0.16 b 1.05±0.10 b ns 46.9±7.22 a 14.2±1.19 a ** 9.02±0.28 b 9.01±0.24 b ns 15.5±3.56 b 17.2±1.20 b ns P*** *** *** *** *** *** *** *** I** *** ns ns NT *** *** *** *** I×NT ** *** ns ns Nutritional treatment (NT) consisted of a basal nutrient solution supplemented with 5mM chloride (CL), 5mM nitrate (N), or the sulfate+phosphate (SP) salt mixture containing the same cationic balance as in the CL and N treatments. Irrigation treatment (I) consisted of a control treatment of well-watered plants (CTR; 100% field capacity) and sustained water deficit (WD; 60% field capacity) treatments. Mean values ±SE, n=6. Levels of significance: ***P≤0.001,**P≤0.01, *P≤0.05, and P >0.05 (‘ns’, non-significant). ‘Homogeneous group’ statistics were calculated through ANOVA and MANOVA tests, where mean values with different letters are significantly different according to Tukey’s test. Downloaded from https://academic.oup.com/jxb/article/72/14/5246/6204166 by UNIVERSIDAD DE SEVILLA user on 06 September 2021 5250 | Franco-Navarro etal. As demonstrated in Franco-Navarro etal. (2016), the application of 5mM Cl− under control conditions promoted plant growth when compared with SP plants (Supplementary Figs S2, S3A–C), mainly due to higher leaf expansion and shoot growth (Supplementary Fig. S3D), which was in turn a consequence of the stimulatory effect of Cl− on cell expansion (Franco-Navarro etal., 2016). On the other hand, the N treatment strongly stimulated plant growth and leaf expansion as a result of a higher rate of both cell division and metabolic activity given the important role of nitrogen in plant metabolism, growth, and development (Hawkesford et al., 2012; FrancoNavarro et al., 2016, 2019; Supplementary Figs S2, S3A–C). When evaluating growth responses to drought, we found that plants subjected to WD showed reduced total, leaf, and root biomass under all nutritional treatments, with significant interactions between irrigation and nutritional treatments (Fig. 1A; Supplementary Figs S2, S3A–C). However, the Cl− application caused lower reduction of plant growth (35–45% reductions of total and organs biomass) than SP and N treatments (45–55% and 50–60% reductions, respectively) during WD (Fig. 1A). To further explore the role of Cl− in plant acclimatization to WD, different morphological parameters widely used as key leaf traits were measured: leaf area, number of leaves, and SLA (i.e. the leaf area per unit of biomass invested). Under control conditions, N plants showed the significantly highest leaf area due to the occurrence of larger and more numerous leaves, while CL plants presented higher leaf area than SP plants (Supplementary Fig. S3D, E). However, non-significant differences in SLA between the three nutritional treatments were observed (Supplementary Fig. S3F). The WD treatment caused a strong reduction in both the area and number of leaves in SP and N plants, which was more significant in N-treated plants (Fig. 1B; Supplementary Fig. S3D, E). Interestingly, WD caused no changes in the number of leaves in CL plants, exhibiting a smaller reduction of leaf area in comparison with the SP and N treatments (Fig. 1B). Furthermore, whereas SP and N plants showed a similar SLA reduction under WD, Cl− application significantly increased it (Fig. 1B; Supplementary Fig. S3F). Taken together, our results validate the beneficial effect of Cl− nutrition on plant growth under both well-watered and WD conditions in tobacco plants, whereas N-treated plants exhibited the highest sensitivity toWD. The effect of Cl− nutrition on whole-plant water-use efficiency and water balance during waterdeficit Considering that Cl− nutrition improves whole-plant WUE and water balance in well-watered plants (Franco-Navarro et al., 2016, 2019), and alleviates detrimental effects of WD on plant growth (Fig. 1), we wondered whether Cl− nutrition induces plant physiological responses linked to water relations during WD. Measurement of the total plant weight relative to accumulated water consumed showed higher integrated WUE values in Cl‒-treated plants during both CTR (Fig. 2A) and WD (Fig. 2B) treatments. Interestingly, when compared with well-watered plants, we observed that WUE values exhibited a >2-fold increase during WD, remaining higher always in CL plants (Fig. 2B). To better compare differences between Fig. 1. Effect of Cl− nutrition and sustained water deficit on plant growth. Plants were alternatively treated with (i) three nutritional treatments: 5mM chloride salts (CL), 5mM nitrate salts (N), and a mixture of sulfate+phosphate salts (SP) containing the same cationic balance as in the CL and N treatments; and (ii) two irrigation treatments: 100% field capacity (CTR, control) and 60% field capacity (WD, water deficit). (A) Effect on total, leaf, and root DW (%) in WD plants normalized to CTR plants. (B) Effect on leaf area and number and specific leaf area (SLA) in WD plants normalized to CTR plants. Absolute values of CTR treatments were as follows: total DW (g), SP=23.2±1.19, CL=27.9±0.58, N=43.8±1.66; leaf DW (g), SP=7.60±0.34, CL=9.43±0.40, N=16.4±0.63; root DW (g), SP=2.47±0.16, CL=2.96±0.13, N=4.82±0.25; leaf area (cm2), SP=2156±44.5, CL=2575±49.7, N=4198±75.1; number of leaves, SP=17.8±0.47, CL=14.3±0.43, N=26.5±0.73; SLA (cm2 g-1 DW), SP=273.5±6.72, CL=269.4±1.53, N=263.1±4.45. Mean values ±SE. n=6. ‘Homogeneous group’ statistics were calculated through ANOVA, where mean values with different letters are significantly different according to Tukey’s test at P≤0.05. Levels of significance: ***P≤0.001. Downloaded from https://academic.oup.com/jxb/article/72/14/5246/6204166 by UNIVERSIDAD DE SEVILLA user on 06 September 2021 Chloride nutrition improves drought tolerance | 5251 high and low Cl− treatments, biomass versus water use relationships were plotted in Fig. 2C (CTR) and Fig. 2D (WD). Given that the biomass of N plants differs greatly from that of the other treatments (Supplementary Fig. S3), only the CL versus SP ionic treatments were compared. Using ANCOVA (as reported in Puértolas etal., 2017), significant differences between CL and SP treatments were observed in both CTR and WD treatments, showing that Cl−-treated plants have a greater capacity to produce biomass in relation to the amount of water consumed (Fig. 2C, D). It is noteworthy that under control conditions, CL plants maintained higher growth with less water consumed than SP plants (Fig. 2C). However, under WD conditions, CL plants maintained higher WUE (Fig. 2B) despite consuming more water, due to higher biomass production (Fig. 2D). When control and WD values were plotted together (Supplementary Fig. S4), the ANCOVA showed that the slopes of the relationship varied between CL and SP treatments, further supporting WUE differences between ionic treatments. When we delved into leaf-level responses to WD, our results showed that WD reduced leaf water content, RWC, and succulence in plants subjected to all nutritional treatments (Fig. 3). However, these water parameters exhibited a significant Cl−-dependent stimulation in comparison with SP and N treatments under both control and WD conditions, showing that Cl− alleviates negative effects of WD on plant water balance. To further investigate the role of Cl− in regulating wholeplant water relations and, particularly, turgor maintenance during water deprivation/rehydration, we monitored the turgor pressure changes of tobacco leaves by using magnetic leaf patch-clamp pressure probes (ZIM-probe; Zimmermann etal., 2008). This non-invasive technique allows the real-time monitoring of the turgor pressure of intact leaves with high precision (Fig. 4A). The measured leaf patch output pressure Pp is inversely proportional to the leaf turgor. Before water deprivation, Pp values recorded in the three treatments (SP, CL, and N) gradually increased during the day, indicating turgor loss after sunrise, and abruptly decreased during sunset, indicating leaf Fig. 2. Effect of Cl− nutrition and sustained water deficit on integrated water-use efficiency. Plants were alternatively treated with (i) three nutritional treatments (NT): 5mM chloride salts (CL); 5mM nitrate salts (N); and a mixture of sulfate+phosphate salts (SP) containing the same cationic balance as in the CL and N treatments; and (ii) two irrigation treatments were also applied: 100% field capacity (CTR, control) and 60% field capacity (WD, water deficit). Effect on integrated water-use efficiency (WUE) in plants subjected to CTR (A) and WD (B) treatments. Relationship between total biomass and accumulated water consumption in plants during CTR (C) and WD (D) treatments. Mean values ±SE, n=6. ‘Homogeneous group’ statistics were calculated through ANOVA and MANOVA, where mean values with different letters are significantly different according to Tukey’s test at P≤0.05. The regression line for each SP and CL pool is shown in both panels (C and D), where P-values and ANCOVA to compare regression slopes are shown. Levels of significance: ***P≤0.001. Downloaded from https://academic.oup.com/jxb/article/72/14/5246/6204166 by UNIVERSIDAD DE SEVILLA user on 06 September 2021 5252 | Franco-Navarro etal. turgor recovery during the night. Although some differences in amplitude were found between treatments, the kinetics of the Pp curves from different plants showed the same circadian trends. Irrigation with the three nutritional treatments (SP, CL, and N) was withheld for 4 d until the water content of drought-stressed pots reached between 10% and 20% of the water content measured in well-watered pots. Subsequently, irrigation was restored to control water levels. After WD imposition, strong loss of turgor (increase in Pp values) was observed in plants subjected to SP and N treatments. However, turgor values were not significantly altered by WD in CL plants, which maintained a Pp pattern similar to that of wellwatered plants (Fig. 4A). To quantify cell damage produced by the resulting leaf tissue dehydration, the photosynthetic efficiency of PSII was measured with a chlorophyll fluorometer in a dark-adapted state. The CL treatment determined much greater protection of the photosynthetic machinery under severe WD, with significantly higher Qy values than those of SP and N treatments (Fig. 4A). After rehydration, CL plants, but not SP and N plants, fully recovered Pp and Qy values to those of control conditions. To determine whether improved water balance parameters of CL plants were associated with the Cl− osmoregulatory properties and the resulting stimulation of leaf turgor observed in well-watered plants (Franco-Navarro etal., 2016; Colmenero-Flores etal., 2019), Ψ π, Ψ w, and Ψ p were measured in leaves of tobacco plants. Cl–-treated plants showed more negative values of Ψ π under both control and WD conditions (Fig. 4B), indicating greater osmoregulatory capacity due to higher accumulation of osmotically active solutes in their leaf tissues. This in turn led to significantly higher Ψ p values in CL plants (Fig. 4B) and, consequently, to a better tolerance to WD. Higher turgor of CL plants was also a consequence of less negative Ψ w values under both control and WD conditions (Fig. 4B), caused by the higher leaf water content of Cl−-treated plants (Fig. 3A). Interestingly, the more positive leaf Ψ w of CL plants, in comparison with SP and N plants, indicates that other events affecting plant water relations might be regulated by Cl−, as described below. The effect of Cl− nutrition on gas exchange and photosynthetic water-use efficiency during waterdeficit To better understand the role of Cl− on the regulation of plant water relations, gs was quantified under CTR and WD conditions (Fig. 5A). As previously shown (Franco-Navarro etal., Fig. 3. Effect of Cl− nutrition and sustained water deficit on water parameters. Plants were alternatively treated with (i) three nutritional treatments (NT): 5mM chloride salts (CL), 5mM nitrate salts (N), and a mixture of sulfate+phosphate salts (SP) containing the same cationic balance as in the CL and N treatments; and (ii) two irrigation treatments (I): 100% field capacity (CTR, control) and 60% field capacity (WD, water deficit). Effect on leaf water content (A), leaf relative water content (RWC) (B), and leaf succulence (C) in CTR and WD treatments. Mean values ±SE, n=6. ‘Homogeneous group’ statistics were calculated through ANOVA, where mean values with different letters are significantly different according to Tukey’s test at P≤0.05. Levels of significance: ***P≤0.001, **P≤0.01, and ‘ns’ P>0.05. Downloaded from https://academic.oup.com/jxb/article/72/14/5246/6204166 by UNIVERSIDAD DE SEVILLA user on 06 September 2021 Chloride nutrition improves drought tolerance | 5253 2016), the CL treatment gave rise to lower gs in well-watered plants due to the lower stomatal density. The lower gs did not impair the net photosynthetic rate when compared with SP plants (AN; Fig. 5B) as a consequence of the positive effect of Cl− on the gm (Franco-Navarro etal., 2019), leading to higher photosynthetic or intrinsic WUEi (AN/gs; Fig. 5C). As a result, Fig. 4. Effect of Cl− nutrition on water status during water deprivation and rehydration treatments. Plants were alternatively treated with three nutritional treatments (NT): 5mM chloride salts (CL); 5mM nitrate salts (N); and a mixture of sulfate+phosphate salts (SP) containing the same cationic balance as in the CL and N treatments. Tobacco plants of each NT (SP, CL, and N) were maintained under optimal irrigation (CTR) up to 73 DAS, then water deprivation was applied for 4 d and, at 77 DAS, plants were rehydrated up to 100% of field capacity and further monitored until 80 DAS. (A) Effect on field capacity, efficiency of PSII, and real-time measurement of leaf turgor using the non-invasive magnetic leaf patch-clamp pressure probes (Zimmermann etal., 2008). Patch pressure (Pp) is inversely correlated with leaf turgor pressure and positively correlated with leaf water potential and plant transpiration (Zimmermann etal., 2008, 2010). (B) Effect of Cl− nutrition and sustained water deficit on leaf osmotic potential (Ψ π), leaf water potential (Ψ w), and leaf turgor (or pressure) potential (Ψ p) in CL, N, and SP plants, which were treated for 20 d with two irrigation regimes (I): 100% field capacity (CTR, control) and 60% field capacity (WD, water deficit). Mean values ±SE, n=6. ‘Homogeneous group’ statistics were calculated through ANOVA and MANOVA, where mean values with different letters are significantly different according to Tukey’s test at P≤0.05. Levels of significance: ***P≤0.001; **P≤0.01; and *P≤0.05. Downloaded from https://academic.oup.com/jxb/article/72/14/5246/6204166 by UNIVERSIDAD DE SEVILLA user on 06 September 2021 5254 | Franco-Navarro etal. the better water balance (Fig. 4) and WUE of Cl−-treated plants increased their tolerance to WD, as evidenced by the lower cell damage suffered in photosynthetic tissues (Fig. 5D). Therefore, SP and N plants, with more dehydrated and less turgid leaves (Fig. 4B), became more stressed by the WD treatment (Fig. 5D), leading to stronger gs reduction (Fig. 5A) and greater loss Fig. 5. Effect of Cl− nutrition and sustained water deficit on gas exchange parameters, water-use efficiency, and stability of PSII. Plants were alternatively treated with (i) three nutritional treatments (NT): 5mM chloride salts (CL); 5mM nitrate salts (N); and a mixture of sulfate+phosphate salts (SP) containing the same cationic balance as in the CL and N treatments; and (ii) two irrigation treatments (I) were also applied: 100% field capacity (CTR, control) and 60% field capacity (WD, water deficit). Effect on (A) stomatal conductance (gs), (B) net photosynthetic rate (AN), and (C) photosynthetic or instantaneous water-use efficiency (WUEi) measured in fully expanded photosynthetically active leaves from plants between 51 and 65days after sowing (DAS). (D) Effect on the highly sensitive physiological stress marker quantum yield (Qy; stability of PSII) measured in fully expanded photosynthetically active leaves from plants between 46 and 65 DAS. Mean values ±SE. n=6. ‘Homogeneous group’ statistics were calculated through ANOVA and MANOVA, where mean values with different letters are significantly different according to Tukey’s test at P≤0.05. Levels of significance: ***P≤0.001; **P≤0.01; *P≤0.05; and ‘ns’ P>0.05. Downloaded from https://academic.oup.com/jxb/article/72/14/5246/6204166 by UNIVERSIDAD DE SEVILLA user on 06 September 2021 Chloride nutrition improves drought tolerance | 5261 WegeS, GillihamM, HendersonSW. 2017. Chloride: not simply a ‘cheap osmoticum’, but a beneficial plant macronutrient. Journal of Experimental Botany 68, 3057–3069. WhitePJ, BroadleyMR. 2001. Chloride in soils and its uptake and movement within the plant: a review. Annals of Botany 88, 967–988. XuGH, MagenH, TarchitzkyJ, KafkafiU. 2000. Advances in chloride nutrition of plants. In: Sparks DL, ed. Advances in agronomy, Vol. 68. Amsterdam: Elsevier, 97–150. Zimmermann U, Bitter R, Ribeiro-Marchiori PE, Rüger S, EhrenbergerW, SukhorukovVL, SchüttlerA, Vasconcelos-RibeiroR. 2013. A non-invasive plant-based probe for continuous monitoring of water stress in real time: a new tool for irrigation scheduling and deeper insight into drought and salinity stress physiology. Theorical and Experimental Plant Physiology 25, 2–11. Zimmermann D, Reuss R, Westhoff M, Gessner P, Bauer W, BambergE, BentrupFW, ZimmermannU. 2008. A novel, non-invasive, online-monitoring, versatile and easy plant-based probe for measuring leaf water status. Journal of Experimental Botany 59, 3157–3167. ZimmermannU, RügerS, ShapiraO, et al. 2010. Effects of environmental parameters and irrigation on the turgor pressure of banana plants measured using the non-invasive, online monitoring leaf patch clamp pressure probe. Plant Biology 12, 424–436. Downloaded from https://academic.oup.com/jxb/article/72/14/5246/6204166 by UNIVERSIDAD DE SEVILLA user on 06 September 2021