Using Tomato Recombinant Lines to Improve Plant Tolerance to Stress Combination Through a More Efficient Nitrogen Metabolism

Using Tomato Recombinant Lines to Improve Plant Tolerance to Stress Combination Through a More Efficient Nitrogen Metabolism María Lopez-Delacalle 1 , Daymi M. Camejo 1 , María García-Martí 1 , Pedro A. Nortes 2 , Manuel Nieves-Cordones 1 , Vicente Martínez 1 , Francisco Rubio 1 , Ron Mittler 3,4 and Rosa M. Rivero 1 * 1 Department of Plant Nutrition, CEBAS-CSIC, Murcia, Spain, 2 Department of Irrigation, CEBAS-CSIC, Murcia, Spain, 3 The Division of Plant Sciences, College of Agriculture, Food and Natural Resources, Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO, United States, 4 University of Missouri School of Medicine, Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO, United States The development of plant varieties with a better nitrogen use efficiency (NUE) is a means for modern agriculture to decrease environmental pollution due to an excess of nitrate and to maintain a sufficient net income. However, the optimum environmental conditions for agriculture will tend to be more adverse in the coming years, with increases in temperatures, water scarcity, and salinity being the most important productivity constrains for plants. NUE is inherently a complex trait, as each step, including N uptake, translocation, assimilation, and remobilization, is governed by multiple interacting genetic and environmental factors. In this study, two recombinant inbred lines (RIL-66 and RIL-76) from a cross between Solanum lycopersicum and Solanum pimpinellifoilum with different degree of tolerance to the combination of salinity and heat were subjected to a physiological, ionomic, amino acid profile, and gene expression study to better understand how nitrogen metabolism is affected in tolerant plants as compared to sensitive ones. The ionomics results showed a different profile between the two RILs, with K + and Mg 2+ being significantly lower in RIL-66 (low tolerant) as compared to RIL-76 (high tolerant) under salinity and heat combination. No differences were shown between the two RILs in N total content; however, N-NO 3 − was significantly higher in RIL-66, whereas N-N org was lower as compared to the other genotype, which could be correlated with its tolerance to the combination of salinity and heat. Total proteins and total amino acid concentration were significantly higher in RIL-76 as compared to the sensitive recombinant line under these conditions. Glutamate, but more importantly glutamine, was also highly synthesized and accumulated in RIL-76 under the combination of salinity and heat, which was in agreement with the upregulation of the nitrogen metabolism related transcripts studied (SlNR,SlNiR,SlGDH,SlGLT1,SlNRT1.2,SlAMT1,and SlAMT2). This study emphasized the importance of studying abiotic stress in combination and how recombinant material with different degrees of tolerance can be Frontiers in Plant Science | www.frontiersin.org January 2020 | Volume 10 | Article 17021 Edited by: Sonia Negrao, University College Dublin, Ireland Reviewed by: Amalia Barone, University of Naples Federico II, Italy Mingku Zhu, Jiangsu Normal University, China *Correspondence: Rosa M. Rivero [email protected] Specialty section: This article was submitted to Plant Abiotic Stress, a section of the journal Frontiers in Plant Science Received: 16 September 2019 Accepted: 03 December 2019 Published: 17 January 2020 Citation: Lopez-Delacalle M, Camejo DM, García-Martí M, Nortes PA, Nieves-Cordones M, Martínez V, Rubio F, Mittler R and Rivero RM (2020) Using Tomato Recombinant Lines to Improve Plant Tolerance to Stress Combination Through a More Efficient Nitrogen Metabolism. Front. Plant Sci. 10:1702. doi: 10.3389/fpls.2019.01702 ORIGINAL RESEARCH published: 17 January 2020 doi: 10.3389/fpls.2019.01702 highly important for the improvement of nitrogen use efficiency in horticultural plants through the targeting of N-related markers. Keywords: nitrogen metabolism, abiotic stress combination, tomato recombinant lines, photosynthesis, chlorophyll fluorescence, ionomics, amino acids, gene expression INTRODUCTION Environmental conditions will tend to be more adverse in the coming years due to global warming, with increases in temperatures, water scarcity, and salinity. The climate predictions are devastating for agriculture, which point to plant growth inhibition and a reduction in productivity to never-before-seen maximum levels. Moreover, it has been shown that when these devastating conditions act in combination, i.e., salinity plus high temperature, the economic losses in agriculture triple (Rizhsky et al., 2004;Mittler, 2006; Suzuki et al., 2014). This situation makes necessary to act urgently in order to ensure that there will be enough food for the growing world population. Thus, the study of the physiological, biochemical, and molecular mechanisms implicated in the plant's response to abiotic stress are crucial. In the last few years, the study of the plant's response to abiotic stresses acting in combination has become important in the elucidation of the mechanisms implicated in the plant's tolerance and their interconnections within plant metabolism (Rivero et al., 2014;Martinez et al., 2016;Zandalinas et al., 2016; Martinez et al., 2018). Martinez et al. (2016) demonstrated that when tomato plants were subjected to the combination of salinity and heat, the metabolic, biochemical, and molecular profile obtained was different from that observed when these stresses were applied individually. Similarly, Rivero et al. (2014) concluded that there was a specificaccumulationof osmoprotectants, more specifically, trehalose and glycine betaine, when tomato plants were grown under the combination of salinity and heat, that was not observed when applying the single stresses. Aside from the different responses observed under stress combination as compared to single stresses, it was found that when abiotic stresses acted jointly, antagonistic responses could also be observed (Cheong et al., 2002;Rizhsky et al., 2004; Mittler, 2006). For example, when plants were subjected to heat stress, their stomata were opened to reduce leaf temperature. But, if at the same time these plants were subject to drought, their stomata won't be able to be opened, and an increase in the leaf temperature and cell death were observed. Nitrogen (N), together with phosphorus and potassium, is one of the primary macronutrients needed by plants for their survival. During the last 50 years, the application of N fertilizers has increased 20 times and its application is expected to increase to 180 million tons by 2030 (Spiertz, 2009;Rütting et al., 2018). Also, the prices of N fertilizers have risen more than 2.5 times in the last decade (Spiertz, 2009). This has caused problems to the farmers' costs and also to the environment. Thus, the development of plant varieties with a better nitrogen use efficiency (NUE) may be a mean for modern agriculture to decrease environmental pollution due to the excess use of nitrates and to maintain a sufficient N net income. NUE is inherently a complex trait, as each step, including N uptake, translocation, assimilation, and remobilization, is governed by multiple interacting genetic and environmental factors. N enters to the root through an active symport mechanism that is dependent on nitrate or ammonium transporters (NRTs or AMTs, respectively) with H + and ATP consumption by the activity of an ATPase H + -P. This NO 3 − can be stored in the vacuole or reduced to NO 2 − by the enzyme nitrate reductase (NR) and subsequently to NH 4+ by the enzyme nitrite reductase (NiR). This NH 4+ is then added to C skeletons within the N assimilation pathway, resulting in the synthesis of certain important amino acids for plant metabolism. The remaining NO 3 − is transported by the xylem to mesophyll cells, where it is temporarilystoredinthevacuole(Masclaux-Daubresseetal.,2010). The NH 4+ ions resulting from NO 3 − reduction (or those directly incorporated into the cells from the soil environment) serve as a substrate for the enzymes glutamine synthetase (GS) andglutamate synthase (GOGAT) to produce glutamic acid (Glu) and glutamine (Gln),both ofwhichareprecursorsforthesynthesis oftherestofthe amino acids (Suzuki and Knaff, 2005). It is known that N metabolism and its remobilization processes within the plant are regulated by internal factors, such as NR and GS activities (Tian et al., 2016)andby external factors, such as the abiotic stresses of heat, drought, and salinity (Zandalinas et al., 2018). Hossain et al. (2012) demonstrated that tomato plants subjected to salinity conditions, in addition to limiting NO 3 − acquisition by the roots, also restricted their ability to reduce and assimilate N by inhibiting the synthesis and activities of the assimilation enzymes, such as NR, NiR, GS, and GOGAT. NR activity under saline stress conditions decreases significantly in tomato (Debouba et al., 2007), sugar beet (Ghoulam et al., 2002) maize (Baki et al., 2000), and beans (Gouia et al., 1994). However, it has been shown that salinity can also have a stimulating effect on NR activity in the roots of tomato (Debouba et al., 2006) or soybean (Bourgeais-Chaillou et al., 1992). Similarly, heat stress decreases the levels of absorption of nutrients and assimilation proteins in tomato plants (Giri et al., 2017), limiting the development, growth, and reproduction of plants (Boyer, 1982;Zinn et al., 2010;Williams et al., 2013). Heat stress can also alter the enzymes involved in N metabolism, altering the assimilation of NO 3 − and NH 4+ (Klimenko et al., 2006;Hungria and Kaschuk, 2014;Giri et al., 2017). In this sense, Argentel-Martínez et al. (2019) concluded that heat stress in wheat alters the activity of the enzymes NR and GS involved in the assimilation of N. When abiotic stresses act in combination, these results could change, Lopez-Delacalle et al. Nitrogen Metabolism and Climate Change Frontiers in Plant Science | www.frontiersin.org January 2020 | Volume 10 | Article 17022 and could be very different that the observed under the single stress described previously. For example, Jin et al. (2016) found that in Portulaca oleracea L. plants there was a specific accumulation of certain amino acids, such as Gln, ornithine, tyrosine (Tyr), valine, and tryptophan (Trp), when subjected to drought and heat stress acting in combination, which suggests that the plant is making a cellular osmotic adjustment aimed at maintaining leaf turgor during this particular stress condition. It has also been shown that under drought or UV-B stress, cucumber plants increased the activity of the NR enzyme, while decreasing their total cellular NO 3 − content (Rybus-Zając and Kubiś, 2018). At present, there are very little information about the effect of abiotic stress combination on the N uptake and assimilation pathway (Cui et al., 2019). Thus, in order to identify agronomic strategies that can be used to fight against the consequences of climate change in crops, detailed studies related to nitrogen metabolism should be developed at the physiological, biochemical, and molecular levels, which is also the primary metabolism related to growth and production in plants. The results from these studies will provide data on the agronomic responses to the environmental scenario predicted for the coming years. On the other hand, the use of varieties with different sensitivity to the abiotic stresses acting jointly could be a great strategy to identify these tolerance-related mechanisms. Traditionally, the selection of the different agronomical plant varieties has been done accordingly to their vigor and yield. However, the new climate scenario is forcing us to select plant varieties based on their tolerance to new environmental conditions. Thus, in this study, two recombinant lines (RIL-66 and RIL-76) from a cross between Solanum lycopersicum and Solanum pimpinellifoilum with different degrees of tolerance to the combination of salinity and heat were subjected to a physiological, ionomic, amino acids profile and gene expression study related to the N acquisition and assimilation pathway to better understand how nitrogen metabolism may be affected by stress combination. The identification of some metabolic and/or molecular markers may help to advance knowledge on how to improve NUE in plants for an improved and more sustainable agriculture. MATERIALS AND METHODS Plant Material and Growth Conditions for the Preliminary Recombinant Inbred Lines Selection Experiments A scheme summarizing the experimental design followed for the selection of sensitive and tolerant tomato RILs to the combination of salinity and heat can be found in Supplementary Material (Supplemental Scheme 1). In brief, our experiments began with a population of 84 recombinant inbred lines (RILs) resulting from a cross between S. lycopersicum (CLN2498E) x Solanum pimpinellifolium (LA1579). This recombinant inbred collection was kindly ceded by Prof. Perez-Alfocea (CEBAS-CSIC, Spain). All seeds were germinated in vermiculite and irrigated with distilled water until 95% of the seeds had germinated. Seven days after germination all seedlings were transferred to two different greenhouse modules (module A and B). Nine plants from each recombinant line were transplanted into rock wool sacs (1 m long; three plants per sac). Plants were irrigated with a modified Hoagland's solution with the following composition: KNO 3 (3 mM), Ca(NO 3 ) 2 (2 mM), MgSO 4 (0.5 mM), KH 2 PO 4 (0.5 mM), Fe-EDTA (10 µM), H 3 BO 3 (10 µM), MnSO 4 ·H 2 O(1µM), ZnSO 4 ·7H 2 O(2µM),CuSO 4 ·5H 2 O(0.5µM),and(NH 4 ) 6 Mo 7 O 24 ·4H 2 O. The pH and electric conductivity (EC) of the nutrient solution were measured and maintained within the range of 5.2–5.6 and 1.4–1.7 mS/cm, respectively. The environmental conditions of each greenhouse module were the same for 10 days (25°C/20°C day/night, 65–75% RH). The greenhouse climate control system operated as described in Rodriguez-Ortega et al. (2017). After 10 days under these conditions, the treatments were started. For these, the air temperature of module B was increased to 35°C/20°C (day/ night) and half of the plants growing in this received 75 mM NaCl in their irrigation solution. Therefore, the plants growing in module A were considered as the control plants, and plants growing in module B were considered as the stress combination (salinity+heat) treated plants. The treatments were maintained for 2 weeks, after which the plants were sampled. The fresh weight (FW) and the dry weight (DW) of the shoots were determined. For the DW, the plant material was oven-dried for 72 h at 70°C and weighed. With the values obtained for FW % tolerance was determined as follows: % tolerance = 100 −FWcontrol FWstressðÞ=FWcontrol From the results obtained (Figures 1B, C), three levels of tolerance were established: low, intermediate, and high tolerance to the combination of salinity+heat. Then, 12 RILs (4 lines from each level of tolerance) were selected and their percentage of tolerance to the stress combination was validated under growth chamber conditions. Plants were germinated as described previously. Seven days after germination all seedlings were transferred to growth chamber (chamber A) with 16/8 h photoperiod (day/night) at 25°C/20°C (day/night) and 65% RH. Plants were grown in 20 L containers filled with modified Hoagland nutrient solution. The composition, chemical concentration, pH, and CE used for the nutrient solution for plant irrigation were as described previously. After 8 days of the transplanting and 15 days after germination, salinity and temperature treatments were imposed. For this, half of the plants from each RILs were transferred to another growth chamber (chamber B) where the temperature was set at 35°C/ 20°C (day/night) and the irrigation nutrient solution was supplemented with 75 mM NaCl. After 15 days of growing under these conditions, plants were sampled and % tolerance to salinity+heat was calculated as described previously. After validating the results obtained for % tolerance, two RILs were selected for the experiments: the ones having the lowest (RIL-66) and the highest (RIL-76) % tolerance to the combination of salinity+heat. Lopez-Delacalle et al. Nitrogen Metabolism and Climate Change Frontiers in Plant Science | www.frontiersin.org January 2020 | Volume 10 | Article 17023 Experimental Design and Plant Sampling Two recombinant lines (RIL-66 and RIL-76) differing in their tolerance to the combination of salinity and heat were selected for this experiment as described previously. Germination and growth chamber conditions were the same as described previously for chamber A for 8 days. After 8 days of transplanting and 15 days after germination, salinity and temperature treatments were imposed. Half of the plants were transferred to another growth chamber (chamber B) with the same environmental conditions as those described previously but FIGURE 1 | Tomato recombinant inbred lines (RILs) selection for their tolerance to the combination of salinity and heat. Eighty four RILs lines were grown under control (A) and under the combination of salinity and heat (B) and fresh weight (FW) was recorded. Twelve RILs were selected for each grade of tolerance (low, intermediate, and high tolerance) to the combination of salinity and heat as compared to control plants (C). The selected lines (C, red circle) were grown again under growth chamber conditions to validate the different grade of tolerance obtained previously under greenhouse conditions. RIL-66 (low tolerant) and RIL-76 (high tolerant) were selected for further experiments (D). Values represented in each figure are means ± SE (n = 6). Lopez-Delacalle et al. Nitrogen Metabolism and Climate Change Frontiers in Plant Science | www.frontiersin.org January 2020 | Volume 10 | Article 17024 with an air temperature set at 35°C/20°C (day/night). In parallel, half of the plants from each growth chamber received 75 mM NaCl in their nutrient solution, so this time individual stresses were also applied in order to better dissect the specificity of the stress combination. Thus, the treatments that were applied were: a) control (25°C 0 mM NaCl), b) salinity (25°C and 75 mM NaCl), c) heat (35°C 0 mM NaCl), and d) heat+salinity (35°C and 75 mM NaCl). Plants were grown under these conditions for 10 days, and at the end of the experiments, leaves, and roots were sampled. For each determination described below six biological replicates for each treatment were used. Gas Exchange and Chlorophyll Fluorescence Parameters The different photosynthetic and chlorophyll fluorescence parameters were measured at the end of the experiment in the youngest fully-expanded leaf of each plant (six plants per treatment), using a LI-6400XT photosynthesis system (Li-Cor, Inc., Lincoln, NE, USA). The conditions established in the LICOR were as described in Martinez et al. (2018). Mineral Nutrients and Anions Analysis 0.1 g of dried plant material from six biological replicates were digested with HNO 3 :HClO 4 (2:1, v:v), and cations concentrations were determined by atomic absorption spectrometry with an ICP-OES (Iris Intrepid II, Thermo Electron Corporation, Franklin, USA). Cations concentration were expressed as log 2 of the normalized values against control plants. Absolute values obtained for the concentration of the main cations can be found in Supplementary Table S1. Hydrogen-Nuclear Magnetic Resource Analysis for Amino Acid Concentration Six biological replicates of tomato leaves for each treatment were used for metabolite profiling by 1 H-NMR. The extraction and quantification of the amino acid were performed as described in Blanco-Ulate et al. (2015). Amino acids concentration was expressed as log 2 of the normalized values against control plants. Absolute values obtained for the concentration of the amino acids assayed can be found in Supplementary Table S2. Enzymatic Activities Crude Extract For the determination of the activities of NR, NiR, glutamate dehydrogenase (GDH), and GOGAT, leaf tissues from six biological replicates were homogenized with 50 mM KH 2 PO 4 buffer (pH 7.5), containing 2 mM ethylenediaminetetraacetic acid (EDTA), 2 mM DL-dithiothreitol (DTT), and 1% polyvinylpolypyrrolidone (PVPP). For the determination of the activity of GS, leaf tissues were homogenized with 50 mM TrisHCl (pH 7.5) containing 5 mM EDTA, 1 mM DTT, and 1% of PVPP. The homogenate was centrifuged at 15,000 rpm for 20 min and the supernatant collected was used for measuring the enzyme activity. Total soluble proteins were measured with the Bradford method (Bradford, 1976). Nitrate Reductase Assay NR (EC 1.7.1.1) activity was measured by the decrease in absorbance (A 340 )duetoNADHoxidation(Campbell and Smarrelli, 1978). Nitrite Reductase Assay NiR activity was determined by the absorbance change at 540 nm as described by Lillo (1984). Glutamate Synthase/Glutamine Synthase Assays GOGAT (EC 2.6.1.53) activity was assayed by determining the rate of glutamine-dependent NADH oxidation as described by Groat and Vance (1981) with some modifications. The reaction assay was carried out in a mixture reaction containing 25 mM KH 2 PO 4 buffer (pH 7.5), 2.5 mM 2-oxoglutarate, 1 mM hydroxylamine, 0.1 mM NADH,and50–100 µgtotalprotein,withthereactionstartedbythe addition of 10 mM of L-glutamine. GOGAT activity was assayedby determining the rate of glutamine-dependent NADH oxidation at 340 nm for 180 s. GS (EC 6.3.1.2) activity was assayed according to Slawyk and Rodier (1986) with some modification. Enzymatic extract (150 µg of total protein) was incubated in a mixture reaction containing 15 mM magnesium chloride, 0.5 mM EDTA, 20 mM potassium-glutamate, 4 mM ammonic acetate, and 1 mM adenosine triphosphate (ATP) at 30°C for 15 min. The reaction was stopped by the addition of 1 N sulfuric acid and then the product was centrifuged at 11,000 rpm for 2 min. GS activity was determined by measuring the ATP-dependent phosphate concentration. The phosphate concentration was measured with a Phosphate Assay kit (Sigma-Aldrich) at 620 nm. A phosphate standard curve was used to determine the amount of free phosphate in the sample. Glutamate Dehydrogenase Assay GDH (EC 1.4.1.2) activity was assayed by measuring the rate of 2-oxoglutarate-dependent NADH oxidation at 340 nm for 180 s (Robinson et al., 1991) Gene Expression Total RNA was isolated from whole tomato leaves from six biological replicates, as described by Martinez et al. (2018). The primer sequences used for the different genes tested as well as the primer sequences of the internal controls are available in Supplementary Table S3. As reference gene, SlEF1awas used as described previously by Lacerda et al. (2015) and Cheng et al. (2017). The normalization of transcript expression against housekeeping genes, reaction components, and PCR settings were carried out as described by Martinez et al. (2018). Expression and log 2 values of the different genes can be found in Supplementary Table S4. Statistical Analysis The software package SPSS ® (ver.10.0, SAS Institute) was used for statistical analyses of the above-mentioned parameters. A two-way ANOVA using Tukey's test was used to compare between treatments and recombinant lines for significant Lopez-Delacalle et al. Nitrogen Metabolism and Climate Change Frontiers in Plant Science | www.frontiersin.org January 2020 | Volume 10 | Article 17025 differences at p < 0.05 for all cases and standard error (SE) values for the different treatments and genotypes was calculated and added as shown in figures. RESULTS Recombinant Inbred Lines Selection Under the Combination of Salinity and Heat Eighty-four tomato recombinant inbred lines (from here on RILs) were grown in two independent greenhouses at different environmental conditions: A) control conditions (25°C, 16/8 h day/night photoperiod, 65% relative humidity) where the plants were irrigated with a modified Hoagland's nutrient solution and 0 mM NaCl; and B) salinity+heat conditions (35°C, 16/8 day/ night photoperiod, 65% relative humidity) where the plants were irrigated with a modified Hoagland's solution supplemented with 75 mM NaCl. Plants were grown under these conditions for 1 month and fresh weight and dry weight of the plants were recorded (Figure 1). In order to evaluate the degree of tolerance of each line to the combination of salinity and heat, the percentage of tolerance was calculated as described in the Materials and Methods section (Figure 1C). After analyzing the results obtained and shown in Figure 1C, three different groups were identified: lines with low tolerance (from 25 to 50% tolerance) intermediate tolerance (from 50 to 75% tolerance) and high tolerance (from 75 to 100% tolerance). From each group, 4 plants were selected (a total of 12 plants) for the next experiment (marked with a red circle, Figure 1C) based on the fresh weight obtained under control conditions, which had to be 300 g per plant or higher (fresh weight average obtained under control conditions). These 12 lines were grown again in hydroponics, but this time in two growth chambers, with same characteristics as those mentioned above for the greenhouses (see full description in Materials and Methods section) in order to have full control of the environmental and nutrient solution conditions. As Figure 1D showed, the results for the % tolerance obtained in the growth chambers for these lines were very similar to those obtained previously in the greenhouse experiment. Based on these results, a RIL line with a low tolerance (RIL-66) and a RIL line with high tolerance (RIL76) to the combination of salinity and heat, were selected in order to elucidate the mechanisms implicated in this tolerance. In a third experiment, RIL-66 (sensitive to salinity+heat combination) and RIL-76 (high tolerant to salinity+heat combination) were both grown in two different growth chambers set with the same environmental conditions, except for the temperature, set at 25°C in chamber A and 35°C in chamber B. Also, half of the plants in each chamber received 75 mM NaCl in their nutrient solution. Therefore, in this FIGURE 2 | Phenotypes and tolerance rate in tomato recombinant inbred line (RIL)-66 and RIL-76. (A) Phenotypes obtained in tomato RIL-66 and RIL-76 under control, salinity, heat stress, and the combination of salinity + heat stress. (B) Fresh weight (FW) of the tomato RIL-66 and RIL-76 at the end of the experiment (15 days of treatments). (C) Percentage of tolerance to salinity, heat, and the combination of salinity and heat obtained in RIL-66 and RIL-76 respect to their controls (red line = 100% of tolerance). Bars are mean values ± SE (n = 6), and bars with different letters are significantly different at p < 0.05 according to Tukey's test. (n = 6). Lopez-Delacalle et al. Nitrogen Metabolism and Climate Change Frontiers in Plant Science | www.frontiersin.org January 2020 | Volume 10 | Article 17026 experiment, four treatments were applied: control (25°C + 0 mM NaCl), salinity (25°C + 75 mM NaCl), heat (35°C + 0 mM NaCl), and salinity+heat (35°C + 75 mM NaCl), in order to characterize the specificity of the tomato plants response to the combination to salinity and heat as compared to the single stress responses (Figure 2). As shown in Figure 2A, under control conditions, RIL-66 showed a similar phenotype as RIL-76, which was confirmed with the fresh weight data obtained for these plants. Thus, under control conditions, RIL-66 and RIL-76 did not show significant differences in their fresh weight (Figure 2B). However, under salinity, heat, and the combination of salt+heat, RIL-76 had a significant biomass increase with respect to RIL-66 (Figure 2A), being three times higher (in case of salinity and salt+heat) and two times higher (in case of heat) as compared to the biomass obtained in RIL-66 under these conditions (Figure 2B). These data translated in an increase in the percentage of stress tolerance of RIL-76 as compared to RIL-66 (Figure 2C). Interestingly, RIL76 had an increased biomass under salinity and under heat with respect to the same plants grown under control conditions (Figure 2A), and similar observations were made for RIL 66 under heat, which translated into a higher percentage of tolerance to these stresses with respect to their controls threshold (100%) (Figure 1C). Lastly, it should be highlighted that RIL-76 showed 100% tolerance under the combination of salinity and heat, which meant that this line was able to maintain a good biomass production under stress combination conditions. Photosynthetic Performance There are several physiological stress markers that can be measured in plants and that can provide insights on how photosynthesis and photosynthetic components behave under stress conditions, such as gas exchange parameters (CO 2 assimilation rate, stomatal conductance, transpiration rate), and chlorophyll fluorescence parameters (Fv/Fm, FPSII, and ETR) (Figure 3). In relation to gas exchange parameters (Figures 3A–C), the CO 2 assimilation rate was higher in RIL-76 as compared to RIL-66 for salinity and the combination of salinity and heat treatments, obtaining values for RIL-76 that were similar to those obtained in control plants for this parameter under these two treatments (Figure 3A). The heat treatment induced an increase in the CO 2 assimilation rate of about 25% with respect to control plants in both recombinant lines studied. On the contrary, salinity or the combination of salinity and heat reduced net photosynthesis by 42 and 35%, respectively, in RIL-66 as compared to control plants (Figure 3A). Similar results to those observed for net photosynthesis were also obtained for stomatal conductance (Figure 3B). Curiously, the transpiration rate was higher and similar in both recombinant lines under heat and the combination of salinity and heat treatments (Figure 3C) with respect to control plants. Under control conditions, RIL-76 had a significant lower transpiration rate as compared to RIL-66. However, the lowest transpiration values were obtained when salinity was applied as a sole stress in the sensitive line RIL-66. Fluorescence of the chlorophylls has been commonly used as a good marker for quantifying damage due to stress in plants. In this sense, quantum yield efficiency (Fv´/Fm´), PSII efficiency (ɸPSII), and electron transport rate (ETR) were measured in our sensitive and tolerant recombinant lines under simple or combined stress conditions (Figures 3D, E). Quantum yield efficiency (Fv´/Fm´) showed a similar response as that observed for the net photosynthesis for both lines, sensitive and tolerant (Figure 3D), with the most significant differences observed when salinity and salinity+heat were applied as compared to control or heat-stressed plants. PSII efficiency was lower in the sensitive line RIL-66 than in the tolerant RIL-76 line under control condition, which may be indicative of an important physiological difference between these two tomato lines (Figure 3E). Salinity was again the stress that resulted in the lowest values of the chlorophyll fluorescence parameters in the sensitive line as compared to the tolerant. Lastly, the ETR showed similar values to those observed for PSII efficiency in both recombinant lines under the different stresses applied (Figure 3F). Cations and Nitrogen Forms Content Salinity, heat, or their combination have also been known to induce ionic and other nutrient imbalances in plants, with NO − 3 being one of the nutrients that is most affected by Na + intracellular concentration. With the aim of observing if there were any differences in the leaf's ion concentration between a sensitive or tolerant tomato plant under the different stresses applied, but especially under the combination of salinity+heat, the most important physiologically-active cations (as Na + ,K + , Ca 2+ , and Mg 2+ ) and different nitrogen forms (total N, NO − 3 , and organic N) were measured (Figure 4). As was expected, Na + concentration was higher in the treatments where NaCl was applied exogenously (salinity and salinity+heat) in both recombinant lines as compared to their controls. As shown in Figure 4A, the ionomic profile obtained for each of the two RILs was different depending on the type of stress applied, with K + and Mg 2+ concentrations being significantly lower in the sensitive RIL-66 line as compared to the tolerant RIL-76 line under salinity or salinity+heat combination (Figure 4A; Supplementary Table S1). K + concentration was significantly lower in RIL-76 under salinity as compared to control plants, but higher than the concentrations found in RIL-66. On the other hand, Ca 2+ only showed a significant lower concentration in the tolerant RIL-76 line under the combination of salinity and heat (Figure 4A;Supplementary Table S1). With respect to the different nitrogen forms found in these recombinant lines, total N content and organic nitrogen (Figures 4A, B, D) showed significant differences between the tolerant and the sensitive recombinant lines only when salinity and heat were applied in combination, with concentrations for these two nitrogen forms significantly lower in the sensitive RIL-66 line. Also, organic N was lower in RIL-66 under salinity as compared to control plants. On the contrary, N-NO 3 − was significantly lower in RIL-76 under any of the stresses studied with respect to control plants, whereas in the sensitive RIL-66 line, the N-NO 3 − concentration was significantly lower than control plants when Lopez-Delacalle et al. Nitrogen Metabolism and Climate Change Frontiers in Plant Science | www.frontiersin.org January 2020 | Volume 10 | Article 17027 salinity and heat were applied in combination, but moreover when heat was applied as a single stress (Figures 4A, C). Proteins and amino acids are the most important and the major sources of organic nitrogen in plants, and for this reason these were an object of our study (Figures 4E, F). Total soluble proteins were significantly higher in the tolerant RIL-76 under heat stress as compared to control plants and to the sensitive RIL-66 plants (Figure 4E). Salinity induced a reduction in the total soluble proteins in both RIL-66 and RIL-76. However, when salinity and heat were applied together, RIL-66 showed an important reduction of more than 50% with respect to control plants, whereas the total soluble protein concentration found in RIL-76 was similar to that obtained in control plants (Figure 4E). In the case of total amino acids concentration, RIL-76 had a higher concentration under any of the stresses applied as compared to the concentration obtained under control conditions, with the maximum (two-fold) found when salinity and heat were applied jointly. RIL-66 only had an increase in amino acids concentration under the combination of FIGURE 3 | Gas exchange parameters (A–C) and chlorophyll fluorescence parameters (D–F) in tomato recombinant inbred line (RIL)-66 and RIL-76 under control, salinity, heat stress, and the combination of salinity and heat. Each figure is representative of three different measurements during the experiment. Bars are mean values ± SE (n = 6), and bars with different letters are significantly different at p < 0.05 according to Tukey's test. (n = 6). Lopez-Delacalle et al. Nitrogen Metabolism and Climate Change Frontiers in Plant Science | www.frontiersin.org January 2020 | Volume 10 | Article 17028 salinity and heat (Figure 4F). Due to these results obtained, a complete accumulation profile of the most physiologicallyimportant amino acids in the tolerant and sensitive tomato lines under the different stresses studied was performed (Figure 5). The results are shown in Figure 5A and these were expressed as log 2 value of the normalized concentration obtained under the different stresses applied against the control (Supplementary Table S2). Amino Acids Profile As observed in Figure 4F, the different stresses resulted in a different amino acid accumulation profiles. Heat applied as a unique stress was the most similar to control plants and only gamma-aminobutyric acid (GABA), alanine (Ala), and glutamine (Glu) showed an inhibition of their accumulation in the sensitive line RIL-66, whereas proline (Pro) and asparagine (Asn) increased significantly in both tolerant and sensitive recombinant lines as compared to their controls (Figure 5A; Supplementary Table S2). On the other hand, salinity and the combination of salinity and heat induced changes in most of the amino acids studied. In general terms, salinity seems to significantly reduce the accumulation of most of the amino acids in both recombinant lines, except for Pro (in RIL-66 and RIL-76), and Tyr and Glu (in RIL-76) (Figure 5A). The combination of salinity and heat induced a specific response in FIGURE 4 | Ionomics in leaves of tomato recombinant inbred line (RIL)-66 and RIL-76 under salinity, heat, or the combination of salinity and heat. (A) Heat map of the concentration (expressed as log2) of the main cations and nitrogen (N) forms in tomato leaves of RIL-76 (salinity+heat tolerant) and RIL-66 (salinity+heat sensitive) under salinity, heat stress, and the combination of salinity+heat as compared to their controls. (B–F) Absolute concentration of total nitrogen (B), nitrates (C), organic nitrogen (D), soluble proteins (E), and total amino acids (F) obtained in RIL-66 and RIL-76. Bars are mean values ± SE (n = 6), and bars with different letters are significantly different at p < 0.05 according to Tukey's test. (n = 6). Stars are representative of significant differences p < 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001) respect to control plants. Lopez-Delacalle et al. Nitrogen Metabolism and Climate Change Frontiers in Plant Science | www.frontiersin.org January 2020 | Volume 10 | Article 17029 There are other works that showed how high salinity inhibited the activity of many enzymes involved in N assimilation in maize, mung bean, and tomato (Khan and Srivastava, 1998;Chakrabarti and Mukherji, 2003;Debouba et al., 2006;Debouba et al., 2007). Also, increased levels of salinity were shown to reduce the activities of various nitrogen assimilation-related enzymes such as NR, NiR, GS, GOGAT, and GDH in Brassica juncea (Nathawat et al., 2005;Siddiqui et al., 2009). Likewise, the activities of NR and GS in barley under drought stress were found to be reduced (Robredo et al., 2011). As for wheat, the translocation of N during the grain filling period was shown to be limited under drought stress, resulting in lower grain yields (Kirda et al., 2001). Also, in creeping bentgrass, wheat, and rice high temperature was shown to inhibit the uptake and assimilation of NO 3 − (Ercoli et al., 2008;Ito et al., 2009). In another study which utilized NR-gene overexpressing transgenic tobacco under drought stress, the plants retained 50% of NR activity as compared to untransformed plants, with NR activity not detected in untransformed plants (Ferrario-Méry et al., 1998). Lastly, in a study where chloroplast GS2 was overexpressed, enhanced salt tolerance in transgenic rice was found as a result (Hoshida et al., 2000), while the ectopic overexpression of pine cytoplasmic GS1 in transgenic poplar conferred these plants with an enhanced tolerance to drought stress (el-Khatib et al., 2004). On the contrary in rice, the overexpression of the OsGS1;2 gene resulted in a higher sensitivity to salt, drought and cold stress (Cai et al., 2009). Also in rice, the overexpression of the OsGS gene enhanced tolerance to cadmium stress due to oxidative stress response modulation (Lee et al., 2013). Overexpression of Escherichia coli gdhA conferred tolerance under water deficit conditions in transgenic tobacco and maize (Mungur et al., 2005). Most of these studies also correlated an improvement of growth and plant production when N assimilation increased. Our results showed that GOGAT, GS, and GDH showed different behaviors depending if the tomato line was sensitive or tolerant to the combination of salinity and heat. Thus, GOGAT was inhibited in RIL-66 and overexpressed in RIL76; GS (cytoplasmic, SlGTS1) was strongly inhibited in RIL-76 under stress combination, whereas GDH was upregulated in both recombinant lines under these conditions. Our results of the gene expression, translated into enzymatic activity of the transcribed protein, revealed that GOGAT and GS were regulated at the transcriptional level, whereas GDH might have suffered a post-translational modification, as the activity found was much lower that the expression of their respective transcripts. Also, it is possible that under the combination of salinity and heat, the low accessibility of glutamate by GS may be due to glutamate being diverted to the synthesis of osmolytes under such conditions, such as Pro (Figure 7)(Ashraf et al., 2018). The clear reduction of the NO 3 − absorption and the osmotic changes induced by salinity but also under salt+heat combination in RIL-66 may also be responsible for the inhibition of the activities of most of the N-assimilation related enzymes under these conditions, as Ashraf et al. (2018) reported previously. CONCLUSION Traditionally, the selection of the different agronomical plant varieties has been done accordingly to their vigor and yield. However, the new climate scenario is forcing us to select plant varieties based on their tolerance to new environmental conditions. In our experiments, the improvement in NR, NiR, GS, and ASN enzymatic activities found in the tolerant line RIL76 clearly correlated with the high levels of Gln, Glu, and Asn, as well as with a better performance of the photosynthetic apparatus and with a higher growth under the combination of salinity and heat [see principal component analysis (PCA) and dendrograms in Supplementary Figures S1–S3]. Our results demonstrate that these can be used as molecular markers for the selection of varieties with improved NUE and thus, increasing plant productivity under abiotic stress combination. These results can help scientific community to alleviate the negative effects of climate change on plant yield and can also serve for reducing the abuse of N in plant fertilizers by improving N use efficiency under these unfavorable conditions. DATA AVAILABILITY STATEMENT All datasets generated for this study are included in the article/ Supplementary Material. AUTHOR CONTRIBUTIONS ML-D and RMR conceived the idea and wrote the manuscript. ML-D also participated and supervised the physiological, ionomics and biochemistry experiments. DC and MG-M carried out the biochemistry experiments. PN, MN-C, VM, and RMR carried out the physiological experiments. FR and RMR carried out the ionomics experiments, while ML-D, RM, and RMR made the gene expression analyses. FUNDING This work was supported by the Ministry of Economy and Competitiveness from Spain (Grant No. AGL2015-66033-R and Grant No. PGC2018-095731-B-I00, MCIU/AEI/FEDER, UE). ACKNOWLEDGMENTS We sincerely acknowledge Mario G. Fon for his corrections and suggestions for the English grammar in this manuscript. We also thank Prof. Perez-Alfocea for the supply of the RILs population for this research. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2019. 01702/full#supplementary-material Lopez-Delacalle et al. Nitrogen Metabolism and Climate Change Frontiers in Plant Science | www.frontiersin.org January 2020 | Volume 10 | Article 170216 REFERENCES Argentel-Martínez, L., Arredondo, T., Yépez, E. A., and Garatuza-Payán, J. (2019). Effects of experimental warming on peroxidase, nitrate reductase and glutamine synthetase activities in wheat. Ashraf, M., Shahzad, S. M., Imtiaz, M., and Rizwan, M. S. (2018). Salinity effects on nitrogen metabolism in plants–focusing on the activities of nitrogen metabolizing enzymes: A review. J. Plant Nutr. 41 (8), 1065–, 1081. doi: 10.1080/01904167.2018.1431670 Baki, G. K. A. E., Siefritz, F., Man, H. M., Weiner, H., Kaldenhoff, R., and Kaiser, W. M. (2000). Nitrate reductase in Zea mays L. under salinity. Plant Cell Environ. 23 (5), 515–521. doi: 10.1046/j.1365-3040.2000.00568.x Blanco-Ulate, B., Amrine, K. C., Collins, T. S., Rivero, R. M., Vicente, A. R., Morales-Cruz, A., et al. (2015). Developmental and metabolic plasticity of white-skinned grape berries in response to botrytis cinerea during noble rot. Plant Physiol. 169 (4), 2422–2443. doi: 10.1104/pp.15.00852 Bohnert, H. J., and Jensen, R. G. (1996). Strategies for engineering water-stress tolerance in plants. Trends Biotechnol. 14 (3), 89–97. doi: doi.org/10.1016/ 0167-7799(96)80929-2 Botella, M. A., Martínez, V., Nieves, M., and Cerdá, A. (1997). Effect of salinity on the growth and nitrogen uptake by wheat seedlings. J. Plant Nutr. 20 (6), 793– 804. doi: 10.1080/01904169709365295 Bourgeais-Chaillou, P., Perez-Alfocea, F., and Guerrier, G. (1992). Comparative effects of N–sources on growth and physiological responses of soyabean exposed to NaCI–stress. J. Exp. Bot. 43 (9), 1225–1233. doi: 10.1093/jxb/ 43.9.1225 Boyer, J. S. (1982). Plant productivity and environment. Sci. (New York NY) 218 (4571), 443–448. doi: 218/4571/443 Bradford, M. M. (1976). A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72 (1-2), 248–254. doi: 10.1006/abio.19769999 Cai, H., Zhou, Y., Xiao, J., Li, X., Zhang, Q., and Lian, X. (2009). Overexpressed glutamine synthetase gene modifies nitrogen metabolism and abiotic stress responses in rice. Plant Cell Rep. 28 (3), 527–537. doi: 10.1007/s00299-008-0665-z Campbell, W. H., and Smarrelli, J. (1978). Purification and kinetics of higher plant NADH : nitrate reductase. Plant Physiol. 61 (4), 611–616. doi: 10.1104/pp.61.4.611 Chakrabarti, N., and Mukherji, S. (2003). Effect of phytohormone pretreatment on nitrogen metabolism in vigna radiata under salt stress. Biol. Plantarum 46 (1), 63–66. doi: 10.1023/a:1022358016487 Cheng, Y., Bian, W., Pang, X., Yu, J., Ahammed, G. J., Zhou, G., et al. (2017). Genome-wide identification and evaluation of reference genes for quantitative RT-PCR analysis during tomato fruit development. Front. Plant Sci. 8, 1440. doi: 10.3389/fpls.2017.01440 Cheong, Y. H., Chang, H.-S., Gupta, R., Wang, X., Zhu, T., and Luan, S. (2002). Transcriptional profiling reveals novel interactions between wounding, pathogen, abiotic stress, and hormonal responses in Arabidopsis. Plant Physiol. 129 (2), 661–677. doi: 10.1104/pp.002857 Cui, J., Abadie, C., Carroll, A., Lamade, E., and Tcherkez, G. (2019). Responses to Kdeficiency and waterlogging interact via respiratory and nitrogen metabolism. Plant Cell Environ. 42 (2), 647–658. doi: 10.1111/pce.13450 Debouba, M., Gouia, H., Suzuki, A., and Ghorbel, M. H. (2006). NaCl stress effects on enzymes involved in nitrogen assimilation pathway in tomato “Lycopersicon esculentum”seedlings. J. Plant Physiol. 163 (12), 1247–1258. doi: 10.1016/j.jplph.2005.09.012 Debouba, M., Maâroufi-Dghimi, H., Suzuki, A., Ghorbel, M. H., and Gouia, H. (2007). Changes in growth and activity of enzymes involved in nitrate reduction and ammonium assimilation in tomato seedlings in response to NaCl stress. Ann. Bot. 99 (6), 1143–, 1151. doi: 10.1093/aob/mcm050 Di Gioia, F., De Bellis, P., Mininni, C., Santamaria, P., and Serio, F. (2017). Physicochemical, agronomical and microbiological evaluation of alternative growing media for the production of rapini (Brassica rapa L.) microgreens. J. Sci. Food Agric. 97 (4), 1212–1219. doi: 10.1002/jsfa7852 Du, S-t, Zhang, Y-s, and Lin, X-y (2007). Accumulation of nitrate in vegetables and its possible implications to human health. Agric. Sci. China 6 (10), 1246–1255. doi.org/10.1016/S1671-2927(07)60169-2 Dutt, M. C., Lim, H. Y., and Chew, R. K. H. (1987). Nitrate consumption and the incidence of gastric cancer in Singapore. Food Chem. Toxicol. 25 (7), 515–520. doi.org/10.1016/0278-6915(87)90202-X Ehlting, B., Dluzniewska, P., Dietrich, H., Selle, A., Teuber, M., Hansch, R., et al. (2007). Interaction of nitrogen nutrition and salinity in Grey poplar (Populus tremula x alba). Plant Cell Environ. 30 (7), 796–811. doi: 10.1111/j.13653040.2007.01668.x el-Khatib, R. T., EP, Hamerlynck, Gallardo, F., and Kirby, E. G. (2004). Transgenic poplar characterized by ectopic expression of a pine cytosolic glutamine synthetase gene exhibits enhanced tolerance to water stress. Tree Physiol. 24 (7), 729–736. doi: 10.1093/treephys/24.7.729 Ercoli, L., Lulli, L., Mariotti, M., Masoni, A., and Arduini, I. (2008). Post-anthesis dry matter and nitrogen dynamics in durum wheat as affected by nitrogen supply and soil water availability. Eur. J. Agron. 28 (2), 138–147. doi: 10.1016/ j.eja.2007.06.002 Ferrario-Méry, S., Valadier, M.-H., and Foyer, C. H. (1998). Overexpression of nitrate reductase in tobacco delays drought-induced decreases in nitrate reductase activity and mRNA. Plant Physiol. 117 (1), 293–302. doi: 10.1104/pp.117.1.293 Flores, P., Botella, M. A., Cerdá, A., and Martínez, V. (2011). Influence of nitrate level on nitrate assimilation in tomato (Lycopersicon esculentum) plants under saline stress. Can. J. Bot. 82, 207–213. doi: 10.1139/b03-152 Ghoulam, C., Foursy, A., and Fares, K. (2002). Effects of salt stress on growth, inorganic ions and proline accumulation in relation to osmotic adjustment in five sugar beet cultivars. Environ. Exp. Bot. 47 (1), 39–50. doi: 10.1016/S00988472(01)00109-5 Giri, A., Heckathorn, S., Mishra, S., and Krause, C. (2017). Heat stress decreases levels of nutrient-uptake and-assimilation proteins in tomato roots. Plants 6 (1), 6. doi: 10.3390/plants6010006 Goel, P., and Singh, A. K. (2015). Abiotic stresses downregulate key genes involved in nitrogen uptake and assimilation in brassica juncea L. PloS One 10 (11), e0143645. doi: 10.1371/journal.pone.0143645 Gouia, H., Ghorbal, M. H., and Touraine, B. (1994). Effects of NaCl on flows of N and mineral ions and on NO3-reduction rate within whole plants of saltsensitive bean and salt-tolerant cotton. Plant Physiol. 105 (4), 1409–1418. doi: 10.1104/pp.105.4.1409 Gouiaa, S., and Khoudi, H. (2015). Co-expression of vacuolar Na+/H+ antiporter and H+-pyrophosphatase with an IRES-mediated dicistronic vector improves salinity tolerance and enhances potassium biofortification of tomato. Phytochemistry 117, 537–546. doi.org/10.1016/j.phytochem.2015.05.016 Groat, R. G., and Vance, C. P. (1981). Root nodule enzymes of ammonia assimilation in alfalfa (Medicago sativa L.): Developmental patterns and responce to applied nitrogen. Plant Physiol. 67 (6), 1198–1203. doi: 10.1104/ pp.67.61198 Hayat, S., Hayat, Q., Alyemeni, M. N., Wani, A. S., Pichtel, J., and Ahmad, A. (2012). Role of proline under changing environments: a review. Plant Signal Behav. 7 (11), 1456–1466. doi: 10.4161/psb.21949 Hemon, P., Robbins, M. P., and Cullimore, J. V. (1990). Targeting of glutamine synthetase to the mitochondria of transgenic tabacco. Plant Mol. Biol. 15 (6), 895–904. doi: 10.1007/bf00039428 Hoshida, H., Tanaka, Y., Hibino, T., Hayashi, Y., Tanaka, A., Takabe, T., et al. (2000). Enhanced tolerance to salt stress in transgenic rice that overexpresses chloroplast glutamine synthetase. Plant Mol. Biol. 43 (1), 103–111. doi: 10.1023/A:1006408712416 Hossain, M. A., Uddin, M. K., Ismail, M. R., and Ashrafuzzaman, M. (2012). Responses of glutamine synthetase-glutamate synthase cycle enzymes in tomato leaves under salinity stress. Int. J. Agric. Biol. 14, 4. Huber, S. C., Huber, J. L., and Kaiser, W. M. (1994). Differential response of nitrate reductase and sucrose-phosphate synthase-activation to inorganic and organic salts, in vitro and in situ.Physiol. Plantarum 92 (2), 302–310. doi: 10.1111/ j.1399-3054.1994.tb05341.x Hungria, M., and Kaschuk, G. (2014). Regulation of N2 fixation and NO3–/NH4 assimilation in nodulated and N-fertilized Phaseolus vulgaris L. exposed to high temperature stress. Environ. Exp. Bot. 98, 32–39. doi: 10.1016/ j.envexpbot.2013.10.010 Ito, S., Hara, T., Kawanami, Y., Watanabe, T., Thiraporn, K., Ohtake, N., et al. (2009). Carbon and Nitrogen Transport during Grain Filling in Rice Under High-temperature Conditions. J. Agron. Crop Sci. 195 (5), 368–376. doi: 10.1111/j.1439-037X.2009.00376.x Jin, R., Wang, Y., Liu, R., Gou, J., and Chan, Z. (2016). Physiological and metabolic changes of purslane (Portulaca oleracea L.) in response to drought, heat, and combined stresses. Front. Plant Sci. 6, 1123. doi: 10.3389/fpls.2015.01123 Lopez-Delacalle et al. Nitrogen Metabolism and Climate Change Frontiers in Plant Science | www.frontiersin.org January 2020 | Volume 10 | Article 170217 Khan, M. G., and Srivastava, H. S. (1998). Changes in growth and nitrogen assimilation in maize plants Induced by NaCl and growth regulators. Biol. Plantarum 41 (1), 93–99. doi: 10.1023/a:1001768601359 Kirda, C., Derici, M. R., and Schepers, J. S. (2001). Yield response and N-fertiliser recovery of rainfed wheat growing in the Mediterranean region. Field Crops Res. 71 (2), 113–122. doi.org/10.1016/S0378-4290(01)00153-8 Klimenko, S. B., Peshkova, A. A., and Dorofeev, N. V. (2006). Nitrate reductase activity during heat shock in winter wheat. J. Stress Physiol. Biochem. 2(1),50–55. Krapp, A. (2015). Plant nitrogen assimilation and its regulation: a complex puzzle with missing pieces. Curr. Opin. Plant Biol. 25, 115–122. doi.org/10.1016/ j.pbi.2015.05.010 Lacerda, A. L. M., Fonseca, L. N., Blawid, R., Boiteux, L. S., Ribeiro, S. G., and Brasileiro, A. C. M. (2015). Reference gene selection for qPCR analysis in tomato-bipartite begomovirus interaction and validation in additional tomatovirus pathosystems. PloS One 10 (8), e0136820–e0136820. doi: 10.1371/ journal.pone.0136820 Lee, H. J., Abdula, S. E., Jang, D. W., Park, S. H., Yoon, U. H., Jung, Y. J., et al. (2013). Overexpression of the glutamine synthetase gene modulates oxidative stress response in rice after exposure to cadmium stress. Plant Cell Rep. 32 (10), 1521–1529. doi: 10.1007/s00299-013-1464-8 Lillo, C. (1984). Diurnal variations of nitrite reductase, glutamine synthetase, glutamate synthase, alanine aminotransferase and aspartate aminotransferase in barley leaves. Physiol. Plantarum 61 (2), 214–218. doi: 10.1111/j.13993054.1984.tb05899.x Martinez, V., Mestre, T. C., Rubio, F., Girones-Vilaplana, A., Moreno, D. A., Mittler, R., et al. (2016). Accumulation of flavonols over hydroxycinnamic acids favors oxidative damage protection under abiotic stress. Front. Plant Sci. 7, 838. doi: 10.3389/fpls.2016.00838 Martinez, V., Nieves-Cordones, M., Lopez-Delacalle, M., Rodenas, R., Mestre, T., Garcia-Sanchez, F., et al. (2018). Tolerance to stress combination in tomato plants: New insights in the protective role of melatonin. Molecules 23 (3), 535. doi: 10.3390/molecules23030535 Masclaux-Daubresse, C., Daniel-Vedele, F., Dechorgnat, J., Chardon, F., Gaufichon, L., and Suzuki, A. (2010). Nitrogen uptake, assimilation and remobilization in plants: challenges for sustainable and productive agriculture. Ann. Bot. 105 (7), 1141–1157. doi: 10.1093/aob/mcq028 Misra, N., and Dwivedi, U. N. (1990). Nitrogen assimilation in germinating phaseolus aureus seeds under saline stress. J. Plant Physiol. 135 (6), 719–724. doi.org/10.1016/S0176-1617(11)80886-2 Mittler, R. (2006). Abiotic stress, the field environment and stress combination. Trends Plant Sci. 11 (1), 15–19. doi: 10.1016/j.tplants.2005.11.002 Mungur, R., Glass, A. D. M., Goodenow, D. B., and Lightfoot, D. A. (2005). Metabolite fingerprinting in transgenic nicotiana tabacum altered by the escherichia coli glutamate dehydrogenase gene. J. Biomed. Biotechnol. 2005, 2. doi: 10.1155/jbb.2005.198 Naliwajski, M. R., and Skłodowska, M. (2018). The relationship between carbon and nitrogen metabolism in cucumber leaves acclimated to salt stress. PeerJ 6, e6043–e6043. doi: 10.7717/peerj6043 Nathawat, N. S., Kuhad, M. S., Goswami, C. L., Patel, A. L., and Kumar, R. (2005). Nitrogen-metabolizing enzymes: effect of nitrogen sources and saline irrigation. J. Plant Nutr. 28 (6), 1089–1101. doi: 10.1081/PLN-200058911 Plaza, B. M., Paniagua, F., Ruiz, M. R., Jiménez-Becker, S., and Lao, M. T. (2017). Nutritional responses of Cordyline fruticosa var. ‘Red Edge' to fertigation with leachates vs. conventional fertigation: Sodium, potassium, calcium and magnesium. Scientia Hortic. 215, 157–163. doi.org/10.1016/j.scienta.2016.12.009 Rivero, R. M., Ruiz, J. M., and Romero, L. M. (2004). Importance of N source on heat stress tolerance due to the accumulation of proline and quaternary ammonium compounds in tomato plants. Plant Biol. (Stuttg) 6 (6), 702–707. doi: 10.1055/s-2004-821293 Rivero, R. M., Gimeno, J., Van Deynze, A., Walia, H., and Blumwald, E. (2010). Enhanced cytokinin synthesis in tobacco plants expressing P-SARK:IPT prevents the degradation of photosynthetic protein complexes during drought. Plant Cell Physiol. 51 (11), 1929–1941. doi: 10.1093/pcp/pcq143 Rivero, R. M., Mestre, T. C., Mittler, R., Rubio, F., Garcia-Sanchez, F., and Martinez, V. (2014). The combined effect of salinity and heat reveals a specific physiological, biochemical and molecular response in tomato plants. Plant Cell Environ. 37 (5), 1059–1073. doi: 10.1111/pce.12199 Rizhsky, L., Liang, H., Shuman, J., Shulaev, V., Davletova, S., and Mittler, R. (2004). When defense pathways collide. The response of Arabidopsis to a combination of drought and heat stress. Plant Physiol. 134 (4), 1683–1696. doi: 10.1104/pp.103.033431 Robinson, S. A., Slade, A. P., Fox, G. G., Phillips, R., Ratcliffe, R. G., and Stewart, G. R. (1991). The role of glutamate dehydrogenase in plant nitrogen metabolism. Plant Physiol. 95 (2), 509–516. doi: 10.1104/pp.95.2.509 Robredo, A., Pérez-López, U., Miranda-Apodaca, J., Lacuesta, M., Mena-Petite, A., and Muñoz-Rueda, A. (2011). Elevated CO2 reduces the drought effect on nitrogen metabolism in barley plants during drought and subsequent recovery. Environ. Exp. Bot. 71 (3), 399–408. doi.org/10.1016/j.envexpbot. 2011.02.011 Rodriguez-Ortega, W. M., Martinez, V., Rivero, R. M., Camara-Zapata, J. M., Mestre, T., and Garcia-Sanchez, F. (2017). Use of a smart irrigation system to study the effects of irrigation management on the agronomic and physiological responses of tomato plants grown under different temperatures regimes. Agric. Water Manage 183, 158–168. doi: 10.1016/j.agwat.2016.07.014 Ruiz, J. M., Blasco Leon, M. B., Rivero, R., and Romero, L. (2005). Nicotine-free and salt-tolerant tobacco plants obtained by grafting to salinity-resistant rootstocks of tomato. Physiol. Plantarum 124, 465–475. doi: 10.1111/j.13993054.2005.00532.x Rütting, T., Aronsson, H., and Delin, S. (2018). Efficient use of nitrogen in agriculture. Rybus-Zając, M., and Kubiś, J. (2018). Nitrogen metabolism in cucumber cotyleons and leaves exposed to the drought stress and excessive uv-B radiation. Acta Sci. Polonorum-Hortorum Cultus 17 (5), 23–36. doi: 10.24326/asphc.2018.5.3 Siddiqui, M. H., Mohammad, F., and Khan, M. N. (2009). Morphological and physio-biochemical characterization of Brassica juncea L. Czern. & Coss. genotypes under salt stress. J. Plant Interact. 4(1),67–80. doi: 10.1080/ 17429140802227992 Slawyk, G., and Rodier, M. (1986). Glutamine synthetase ase actyvity in in chaetoceros affinis (bacillariophyceae): comparison with other estimates of nitrogen utilization during nutrient perturbation1. J. Phycol. 22 (3), 270–275. doi: 10.1111/j.1529-8817.1986.tb00023.x Spiertz, J. H. J. (2009). “Nitrogen, sustainable agriculture and food security: a review.”In Lichtfouse, E., Navarrete, M., Debaeke, P., Véronique, S., and Alberola, C. (eds) Sustainable Agriculture (Dordecht: Springer), 635–651. doi: 10.1007/978-90-481-2666-8_39 Suzuki, A., and Knaff, D. B. (2005). Glutamate synthase: structural, mechanistic and regulatory properties, and role in the amino acid metabolism. Photosynth. Res. 83 (2), 191–217. doi: 10.1007/s11120-004-3478-0 Suzuki, N., Rivero, R. M., Shulaev, V., Blumwald, E., and Mittler, R. (2014). Abiotic and biotic stress combinations. New Phytol. 203 (1), 32–43. doi: 10.1111/ nph.12797 Tian, Z., Li, Y., Liang, Z., Guo, H., Cai, J., Jiang, D., et al. (2016). Genetic improvement of nitrogen uptake and utilization of winter wheat in the Yangtze River Basin of China. Field Crops Res. 196, 251–260. doi: 10.1016/ j.fcr.2016.07.007 Wang, M., Zheng, Q. S., Shen, Q. R., and Guo, S. W. (2013). The critical role of potassium in plant stress response. Int. J. Mol. Sci. 14 (4), 7370–7390. doi: 10.3390/ijms14047370 Wang, X., Cai, X., Xu, C., Wang, S., Dai, S., and Wang, Q. (2018). Nitrate accumulation and expression patterns of genes involved in nitrate transport and assimilation in spinach. Molecules (Basel Switzerland) 23, 9. doi: 10.3390/ molecules23092231 Williams, A. P., Allen, C. D., Macalady, A. K., Griffin, D., Woodhouse, C. A., Meko, D. M., et al. (2013). Temperature as a potent driver of regional forest drought stress and tree mortality. Nat. Clim. Change 3 (3), 292. doi: 10.1038/ nclimate1693 Yancey, P. H. (2005). Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high osmolarity and other stresses. J. Exp. Biol. 208 (Pt 15), 2819–2830. doi: 10.1242/jeb.01730 Yildirim, E., Karlidag, H., and Turan, M. (2009). Mitigation of salt stress in strawberry by foliar K, Ca and Mg nutrient supply. Plant Soil Environ. 55, 213– 221. doi: 10.1007/s10343-017-0393-9 Yu, C., Lv, D. G., Qin, S. J., Yang, L., Ma, H. Y., and Liu, G. C. (2010). Changes in photosynthesis, fluorescence, and nitrogen metabolism of hawthorn Lopez-Delacalle et al. Nitrogen Metabolism and Climate Change Frontiers in Plant Science | www.frontiersin.org January 2020 | Volume 10 | Article 170218 (Crataegus pinnatifida) in response to exogenous glutamic acid. Photosynthetica 48 (3), 339–347. doi: 10.1007/s11099-010-0044-1 Zandalinas, S. I., Rivero, R. M., Martínez, V., Gómez-Cadenas, A., and Arbona, V. (2016). Tolerance of citrus plants to the combination of high temperatures and drought is associated to the increase in transpiration modulated by a reduction in abscisic acid levels. BMC Plant Biol. 16 (1), 105. doi: 10.1186/s12870-0160791-7 Zandalinas,S.I.,Mittler,R.,Balfagón,D.,Arbona,V.,andGómez-Cadenas, A. (2018). Plant adaptations to the combination of drought and high temperatures. Physiologia Plantarum 162 (1), 2–12. doi: 10.1111/ppl. 12540 Zhou, Y-h, Zhang, Y-l, Wang, X-m, Cui, J-x, Xia, X-j, Shi, K., et al. (2011). Effects of nitrogen form on growth, CO₂assimilation, chlorophyll fluorescence, and photosynthetic electron allocation in cucumber and rice plants. J. Zhejiang Univ. Sci. B. 12 (2), 126–134. doi: 10.1631/jzus.B1000059 Zinn, K. E., Tunc-Ozdemir, M., and Harper, J. F. (2010). Temperature stress and plant sexual reproduction: uncovering the weakest links. J. Exp. Bot. 61 (7), 1959–1968. doi: 10.1093/jxb/erq053 Conflict of Interest: 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. Copyright © 2020 Lopez-Delacalle, Camejo, García-Martí, Nortes, Nieves-Cordones, Martínez, Rubio, Mittler and Rivero. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Lopez-Delacalle et al. Nitrogen Metabolism and Climate Change Frontiers in Plant Science | www.frontiersin.org January 2020 | Volume 10 | Article 170219