Changes in the salinity tolerance of sweet pepper plants as affected by nitrogen form and high CO2 concentration

Journal of Plant Physiology 200 (2016) 18–27

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Changes in the salinity tolerance of sweet pepper plants as affected by nitrogen form and high CO2 concentration ˜ María C. Pinero, Margarita Pérez-Jiménez, Josefa López-Marín, Francisco M. del Amor ∗ Departamento de Hortofruticultura. Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario (IMIDA), C/Mayor s/n, 30150 Murcia, Spain

a r t i c l e

i n f o

Article history: Received 27 April 2016 Received in revised form 17 May 2016 Accepted 25 May 2016 Available online 8 June 2016 Keywords: Climate change Ammonium Nitrate Carbon dioxide Abiotic stress Gas exchange

a b s t r a c t The assimilation and availability of nitrogen in its different forms can significantly affect the response of primary productivity under the current atmospheric alteration and soil degradation. An elevated CO2 concentration (e[CO2 ]) triggers changes in the efficiency and efficacy of photosynthetic processes, water use and product yield, the plant response to stress being altered with respect to ambient CO2 conditions (a[CO2 ]). Additionally, NH4 + has been related to improved plant responses to stress, considering both energy efficiency in N-assimilation and the overcoming of the inhibition of photorespiration at e[CO2 ]. Therefore, the aim of this work was to determine the response of sweet pepper plants (Capsicum annuum L.) receiving an additional supply of NH4 + (90/10 NO3 − /NH4 + ) to salinity stress (60 mM NaCl) under a[CO2 ] (400 ␮mol mol−1 ) or e[CO2 ] (800 ␮mol mol−1 ). Salt-stressed plants grown at e[CO2 ] showed DW accumulation similar to that of the non-stressed plants at a[CO2 ]. The supply of NH4 + reduced growth at e[CO2 ] when salinity was imposed. Moreover, NH4 + differentially affected the stomatal conductance and water use efficiency and the leaf Cl− , K+ , and Na+ concentrations, but the extent of the effects was influenced by the [CO2 ]. An antioxidant-related response was prompted by salinity, the total phenolics and proline concentrations being reduced by NH4 + at e[CO2 ]. Our results show that the effect of NH4 + on plant salinity tolerance should be globally re-evaluated as e[CO2 ] can significantly alter the response, when compared with previous studies at a[CO2 ]. © 2016 Elsevier GmbH. All rights reserved.

1. Introduction The functioning and composition of ecosystems are changing due to the effect of humankind, which is producing modifications in the climate and global biochemistry (IPCC, 2014). Thus, the atmospheric [CO2 ] has been increased by the burning of fossil fuel in the last two centuries, stimulating an acceleration of global climate change (IPCC, 2014). In principle, C3 plants increase their rates of photosynthesis and growth as much as 35% under elevated [CO2 ] (e[CO2 ]) (Russell et al., 2014), as these plants are generally not photosynthetically saturated at current [CO2 ]. However, despite the improvement in yields, the efficiency of use of CO2 is reduced at e[CO2 ], due in part to the reduction of photosynthetic stimulation after a certain time of exposure, associated with various aspects of a phenomenon known as “acclimatization” or “down-regulation” (Reich et al., 2006). Several studies indicate that the acclimatization under e[CO2 ] is a consequence of an insufficient sink capacity caused by a limiting N supply (Rogers and Ainsworth, 2006), where

∗ Corresponding author. E-mail address: [email protected] (F.M. del Amor). http://dx.doi.org/10.1016/j.jplph.2016.05.020 0176-1617/© 2016 Elsevier GmbH. All rights reserved.

inhibition of photorespiration may play a crucial role (Bloom et al., 2010). Effects of the N input (as NO3 − or NH4 + ) on photosynthesis were observed, especially in relation to stomatal conductance and the intercellular partial pressure (Ci) of CO2 : plants supplied with NH4 + showed higher rates of assimilation and stomatal conductance than those receiving N exclusively as NO3 − (Bloom et al., 2002). Thus, the growth of many plant species is affected by the form of nitrogenous nutrition (Fernández-Crespo et al., 2012), the photosynthetic response to e[CO2 ] also depending on the balance between the supply and the new demand for N, the distribution of biomass, and the source-sink balance (Sanz-Sáez et al., 2010). Salinity is one of the main stressors limiting plant development and crop productivity worldwide, and could be exacerbated by global climate change. Salinity affects plants at different levels – morphological, physiological, and molecular (Dajic, 2006) – and decreases their growth and development (Zhu, 2002). Salinity can decrease the amount of N in plants whilst increasing the leaf Cl− concentration (Munns and Tester, 2008). However, the negative effects of salinity could, in part, be compensated by the greater plant growth potential at e[CO2 ] and increased water use efficiency caused by reduced transpiration (Geissler et al., 2009). Additionally, some studies pointed out that the contribu-

M.C. Pi˜ nero et al. / Journal of Plant Physiology 200 (2016) 18–27

tion of NO3 − as the sole N source may not be beneficial under salt stress, since salinity reduces its uptake rate (Kant et al., 2007) and decreases the N content. Consequently, the growth response of plants to N fertilization under saline stress varies depending on whether N is supplied as NO3 − or NH4 + as well as on the species considered (Misra and Dwivedi, 1990). Moreover, Cruz et al. (1997) pointed out that the response of carob to high [CO2 ] was strongly dependent on the N source. Thus, the complexity of plant N metabolism under high-CO2 conditions is still far from being understood in detail (Kant et al., 2011), as the uptake of different N-forms, translocation, reduction of NO3 − , and amino acid synthesis are all regulation points which are potentially affected by changed growth and demand at elevated [CO2 ] (Tausz et al., 2013). Sweet pepper (Capsicum annuum L.) is an important crop species for greenhouse cultivation in Europe, and is generally considered to be salt sensitive (del Amor and Cuadra-Crespo, 2012). However, there is little information about the interactive effects of different N forms and high atmospheric [CO2 ] under saline conditions. Consequently, it is necessary to gain an insight into the plant growth responses under such new conditions. Therefore, the main objective of this work is to evaluate the extent to which the form in which N is supplied can modulate salinity tolerance under e[CO2 ]. Specifically, our aims were (i) to identify the extent to which plant growth and leaf ion concentrations are affected by different N forms under salinity-stress conditions and whether the response is altered under high [CO2 ], (ii) to determine the water relations and gas exchange response under such conditions, and (iii) to ascertain the extent to which each specific response implicates changes in reactive oxygen species activity and leaf amino-acid metabolism. Thus, in addition to a better understanding of the key physiological processes and mechanisms altered under high CO2 and salinity stress, the information from this study will help to improve Nfertilizer management strategies for this important crop, under a climate change scenario.

19

both ambient (a[CO2 ]) (400 ␮mol mol−1 CO2 ) and elevated [CO2 ] (800 ␮mol mol−1 CO2 ). 2.2. Plant growth Eight plants per treatment were used. Forty-four days after transplanting, pepper plants were harvested and divided into leaves and stems. Part of the plant material was weighed for fresh weight (FW) determination, oven dried for 72 h at 70 ◦ C, and weighed to determine the dry weight (DW). The specific leaf area (SLA) was calculated as the ratio between the area and the dry weight of leaf discs of 6.91 cm2 . In order to analyze plant growth, we used the methodology proposed by Poorter and Pérez-Soba (2001). Thus, the BER (biomass enhancement ratio) was determined as an indicator of the stimulatory effect of elevated CO2 on plant biomass, while GRS (growth reduction due to stress) is an indicator of the stress experienced by the plants due to a non-optimal level of the environmental factor under study (salinity). The intensity of the stress was calculated as the reduction in biomass at ambient CO2 of plants grown at the non-optimal level, compared to the biomass of the plants grown at the optimal level. As these ratios are lnnormally distributed by nature, we first ln-transformed the BER values obtained under optimal and non-optimal conditions and then scaled the difference between these two values by the growth reduction caused by the interacting stress factor applied (salinity), SLB being an acronym for ‘slope of the line connecting the two BER values’ (Poorter and Pérez-Soba, 2001). 2.3. Osmolarity and leaf water potential The osmolarity of the sap (mmol kg−1 ) was measured with a Wescor Vapro5520 osmometer (Wescor Inc., South Logan, Utah, USA). Leaf water potential (w) was measured using a Scholander pressure chamber (Model 3000; Soil Moisture Equipment Corp., Santa Barbara, CA, USA).

2. Material and methods

2.4. Gas exchange measurements

2.1. Plant material, growth conditions, and treatments

At the end of each experimental period and for each [CO2 ], the net CO2 assimilation (ACO2 ), stomatal conductance to H2 O (gs), and evapotranspiration (E) were measured in the youngest fully expanded leaf of each plant, using a CIRAS-2 (PP system, Amesbury, MA, USA) with a PLC6 (U) Automatic Universal Leaf Cuvette, measuring both sides of the leaves. The cuvette provided light (LED) with a photon flux of 1300 ␮mol m−2 s−1 , 400 or 800 ␮mol mol−1 CO2 , and a leaf temperature of 25 ◦ C. The instantaneous water-use efficiency (WUEi) was calculated as the ratio ACO2 /E. Soil (root) respiration was measured after the photosynthesis readings, with the CIRAS-2 and the SRC-1 Soil Respiration Chamber. The chamber (volume: 1171 cm3 ; area: 78.5 cm2 ) was placed on the substrate at the top of each container, after holding the chamber in air to flush it out with the a[CO2 ] (15 s). Calibration was performed before each measurement, and readings were taken after CO2 stabilization (maximum 60 s).

Pepper (Capsicum annuum L.), cv. Melchor (Zeta Seeds S.A.) Lamuyo type, seeds were germinated on a mixture of peat and perlite (3:1), with a nutrient solution of 0.5 mM CaSO4 . Pepper seedlings were selected for uniformity after 26 days, and transplanted to 5-L black containers filled with coconut coir fiber (Pelemix, Alhama de Murcia, Spain). Each container was rinsed with 2 L of water after transplanting. Irrigation was supplied by selfcompensating drippers (2 L h−1 ) and fresh nutrient solution was applied with a minimum of 35% drainage. The experiment was carried out in a climate chamber designed by our department specifically for plant research purposes (del Amor et al., 2010), with fully-controlled environmental conditions: 50% relative humidity, 16-h photoperiod, day/night temperature of 26/18 ◦ C, and a photosynthetically-active radiation (PAR) of 250 ␮mol m−2 s−1 provided by a combination of fluorescent lamps (TL-D Master reflex 830 and 840, Koninklijke Philips Electronics N.V., the Netherlands) and high-pressure sodium lamps (SonT Agro, Philips). Pepper plants were irrigated with a modified Hoagland solution containing NO3 − as the sole N source (mM); NO3 − : 12.0; H2 PO4 − : 1.0; SO4 2− : 3.5; K+ : 7.0; Ca2+ : 4.5; Mg2+ : 2.0, or a nutrient solution where NO3 − was partially substituted by NH4 + as (NH4 )2 SO4 (NO3 − /NH4 + concentration percentages of 100/0 or 90/10). The pH of both irrigation solutions was maintained at 5.8. Salinity stress was imposed by adding salt (60 mM NaCl) to each nutrient solution, and the experiment was run at

2.5. Photosynthetic pigments and fluorescence Chlorophylls a and b were extracted from one leaf disc (6.91 cm2 ) per sample with 4 mL of N,N-dimethylformamide, for 72 h at 4 ◦ C in darkness. Subsequently, the absorbance was measured with a BioMate spectrophotometer (Thermo Spectronic) at 750, 664, and 647 nm (Porra et al., 1989). On the leaf used for gas exchange measurements, the dark-adapted maximum fluorescence (Fm) and minimum fluorescence (Fo) and the lightadapted, steady-state chlorophyll fluorescence (F) and maximum

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fluorescence (Fm0) were measured with a portable modulated fluorometer, model OS-30P (Opti-Science, USA). The ratio between the variable fluorescence from a dark-adapted leaf (Fv) and the maximal fluorescence from a dark-adapted, youngest, fully expanded leaf (Fm) – called the maximum potential quantum efficiency of photosystem II (Fv/Fm) – was calculated. A special leaf clip holder was allocated to each leaf to maintain dark conditions for at least 30 min before reading. 2.6. Leaf mineral concentrations Pepper leaves (of similar size and age to those selected for photosynthesis determination) were dried at 65 ◦ C in a heater for 72 h. The NO3 − and Cl− were extracted from ground material (0.4 g) with 20 mL of deionized water. The NO3 − and Cl− were analyzed in an ion chromatograph (METROHM 861 Advanced Compact IC; METROHM 838 Advanced Sampler, Metrohm Ltd., Herisau, Switzerland) and the column used was a METROHM Metrosep A Supp7 250/4.0 mm. The leaf Na+ and K+ concentrations were determined by inductively coupled plasma-optical emission spectrometry (ICP-OES) (Varian Vista-MPX, Varian Australia, Mulgrave, Vic., Australia), after acid digestion in a microwave oven (Milestone Ethos 1 Advanced Microwave Digestion System, Italy). 2.7. Leaf phenolic concentration, lipid peroxidation, and ascorbate peroxidase activity The total phenolic compounds were extracted from 0.4 g of frozen leaves (−80 ◦ C) with 4 mL of methanol and 0.1 mol L−1 HCl. The homogenate was centrifuged at 15,000g for 20 min, at 4 ◦ C. For the determination, Folin–Ciocalteu reagent was used, diluted with distilled water (1:10). The diluted reagent (2 mL) was mixed with 400 ␮L of supernatant and then 1600 ␮L of sodium carbonate (7.5%) were added. The mixture was kept for 30 min in the dark and then centrifuged at 5000g for 5 min. The supernatant was separated and its absorbance at 765 nm was measured according to Kähkönen et al. (1999). The total phenolic content was expressed as gallic acid equivalents, in mg/100 g fresh weight. Lipid peroxidation was determined using the method described by our group in 2009 (del Amor et al., 2009). Briefly, fresh leaves were homogenized and centrifuged. The supernatant was separated and a mixture of trichloroacetic acid (TCA), thiobarbituric acid (TBA), and butylated hydroxytoluene (BHT) was added. The mixture was heated and then quickly cooled on ice. The contents were centrifuged and the absorbance was measured at 532 nm. The value for nonspecific absorption at 600 nm was subtracted. The concentration of TBARS was calculated using an extinction coefficient of 155 mmol L−1 cm−1 . Ascorbate peroxidase (APOX) activity was determined according to del Amor et al. (2009). Briefly, extracts for the determination of APOX activity was homogenized and centrifuged and the supernatant was used for the assays. The H2 O2 dependent oxidation of ascorbate was followed as a decrease in absorbance at 290 nm (e, 2.8 mmol L−1 cm−1 ). 2.8. Leaf amino acids concentration Free amino acids were extracted from leaves frozen at −80 ◦ C: the sap was extracted after centrifuged at 5000g (10 min, 4 ◦ C) and analyzed by the AccQ·Tag-ultra ultra-performance liquid chromatography (UPLC) method (Waters, UPLC Amino Acid Analysis Solution. Waters Corporation, Milford, MA (2006)). For derivatization, 70 ␮L of borate buffer were added to the hydrolyzed sample or to 10 ␮L of the leaf sap. Next, 20 ␮L of reagent solution were added. The reaction mixture was mixed immediately and heated at 55 ◦ C for 10 min. After cooling, an aliquot of the reaction mixture was used for UPLC injection. The UPLC was performed on an Acquity

system (Waters, Milford, MA, USA) equipped with a fluorescence detection (FLR) system. A BEH C18 100 mm × 2.1 mm, 1.7 ␮m column (Waters) was used. The flow rate was 0.7 mL min−1 and the column temperature was kept at 55 ◦ C. The injection volume was 1 ␮L. The excitation (␭ex) and emission (␭em) wavelengths were set at 266 and 473 nm, respectively. The solvent system consisted of two eluents: (A) AccQ·Tag-ultra eluent A concentrate (5%, v/v) and water (95%, v/v); (B) AccQ·Tag ultra eluent B. The following elution gradient was used: 0–0.54 min, 99.9% A–0.1% B; 5.74 min, 90.9% A–9.1% B; 7.74 min, 78.8% A–21.2% B; 8.04 min, 40.4% A–59.6% B; 8.05–8.64 min, 10% A–90% B; 8.73–10 min, 99.9% A–0.1% B. Empower 2 (Waters) software was used for system control and data acquisition. External standards (Thermo Scientific) were used for quantification of (Ala) alanine; (Asp) aspartic acid; (Arg) arginine; (Cys) cysteine; (Gly) glycine; (Ser) serine; (Glu) glutamic acid; (Ile) isoleucine; (Leu) leucine; (Lys) lysine; (Phe) phenylalanine; (Pro) proline; (Met) methionine; (Thr) threonine; (Tyr) tyrosine; and (Val) valine. 2.9. Statistical analysis The experiment had a completely randomized design, with a 2 × 2 × 2 factorial arrangement composed of two nitrogen source ratios (NO3 − /NH4 + 100/0, 90/10), two salinity conditions (absence and presence of NaCl at 60 mM), and two CO2 concentrations (800 or 400 ␮mol mol−1 ). The analyses were performed using eight repetitions per treatment (an individual plant per pot was considered one repetition). The data were tested first for homogeneity of variance and normality of distribution. Significance was determined by analysis of variance (ANOVA), and the significance (P < 0.05) of differences between mean values was tested by Duncan’s New Multiple Range Test, using Statgraphics Centurion® XVI (StatPoint Technologies, Inc. Warrenton, VA, USA). 3. Results 3.1. Plant growth After 44 days of treatment, in comparison with their nonsalinized counterparts, the salinized plants (60 mM NaCl) had a significantly decreased total shoot DW at a[CO2 ]: it declined from 26.67 g to 9.10 g with the 100/0 NO3 − /NH4 + ratio, and from 23.03 g to 11.66 g with the 90/10 NO3 − /NH4 + ratio. However, this dramatic effect was not observed for the plants that grew at e[CO2 ]; under these conditions, the plants were able to maintain shoot growth despite the salinity (Fig. 1A). The NH4 + supply did not significantly increase plant DW accumulation (at both CO2 concentrations) under salinity: the response was, in general, a reduction in DW at e[CO2 ] and no effect at a[CO2 ]. In the absence of stress, this response was more accentuated at a[CO2 ]. However, this effect depended on the organ considered: thus, leaves were more affected by salinity than stems (Fig. 1CD). Although e[CO2 ] increased plant height with the 100/0 ratio, under saline and non-saline conditions, it was maintained with the 90/10 ratio (Fig. 1B). The growth response was analyzed more specifically through the BER and GRS (Fig. 2). The 100/0 + NaCl treatment showed a GRS of 0.58 whilst 90/10 + NaCl gave a value of 0.54. The 100/0 + NaCl treatment had a BER of 2.77, and 90/10 + NaCl gave a value of 2.36, representing increases of 164.61% and 125.90%, respectively, compared with the non-salinized control (100/0). Thus, the differential response of the pepper plants to both [CO2 ] and the composition of the nutrient solution was also seen when the slope relating the CO2 × different irrigations (SLB) was calculated. Hence, all the different nutrient solutions showed a positive SLB, and the salinizing treatments (100/0 + NaCl, and 90/10 + NaCl), with SLB values of 1.68

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21

Fig 1. Effect of salinity and two nitrogen forms under an elevated CO2 concentration on sweet pepper plants: (A) total plant dry weight; (B) plant height; (C) leaf dry weight, and (D) stem dry weight. Data are means ± SE of eight plants.

Fig. 2. Effect of salinity and two nitrogen forms under elevated an CO2 concentration on sweet pepper plants: BER (growth stimulation by elevated CO2 ), GRS (growth reduction due to stress), and SLB (slope of the line connecting the two BER values), the latter values are between parentheses. Data are means ± SE of eight plants.

and 1.50, respectively, showed the greatest responses. The nonsalinized 90/10 treatment showed a slightly lower SLB (1.47). 3.2. Leaf water relations Under non-salinized conditions, the supply of NH4 + increased the osmolarity of the sap at both a[CO2 ] and e[CO2 ], but under

saline stress no significant differences were found with respect to the supply of N as NO3 − alone (Table 1). The leaf water potential was marginally affected by [CO2 ] but not by the N-source; however, leaf morphology, studied as SLA, was more affected. In general, e[CO2 ] and salinity reduced SLA, but the addition of NH4 + only affected SLA in the absence of salinity stress. The e[CO2 ] increased WUEi and this effect was greater with the saline solution. However, the

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Table 1 Effect of salinity and two regimes of N-nutrition under an elevated CO2 concentration on sweet pepper plants: osmolarity, water potential, SLA (specific leaf area), and WUEi (instantaneous water-use efficiency). Data are means ± SE of eight plants. Nitrogen form

Osmolarity (mmol kg−1 )

Water potential (-MPa)

SLA (m2 g−1 )

WUEi

400

100/0 90/10 100/0 + NaCl 90/10 + NaCl

a

322.57 ± 6.9 365.50 ± 7.9b 404.17 ± 6.2cd 408.40 ± 5.4d

ab

0.77 ± 0.4 0.69 ± 0.2a 0.79 ± 0.5ab 0.85 ± 0.5bc

8.9 ± 0.45c 12.6 ± 0.40b 10.4 ± 0.39b 8.4 ± 0.40b

8.01 ± 0.4ab 7.09 ± 0.3a 9.30 ± 0.4bc 6.39 ± 0.3a

800

100/0 90/10 100/0 + NaCl 90/10 + NaCl

350.29 ± 3.7b 389.43 ± 6.5c 441.33 ± 4.2e 444.56 ± 5.3e

0.94 ± 0.4c 0.85 ± 0.4bc 0.77 ± 0.4ab 0.83 ± 0.3bc

15.6 ± 0.43c 5.6 ± 0.30a 5.5 ± 0.32a 5.4 ± 0.32a

10.92 ± 0.4c 13.78 ± 0.7d 15.14 ± 0.9d 17.58 ± 0.5e

[CO2 ]

ANOVAb CO2 NFc NaCl CO2 × NF CO2 × NaCl NF × NaCl CO2 × NF × NaCl a

***

*

***

***

***

ns ns ns

***

ns

***

***

**

***

*

ns

**

ns ns

***

ns ns ***

*

***

ns

ns

*

Different letters within a column indicate significant (P ≤ 0.05) differences between treatments. b Analysis of variance: ns. not significant. c Nitrogen form. * P ≤ 0.05. ** P ≤ 0.001. *** P ≤ 0.0001.

supply of NH4 + produced a significant increase in WUEi at e[CO2 ], under both control and saline conditions, but this effect was not observed at a[CO2 ] (Table 1). 3.3. Gas exchange, chlorophyll content, and maximum potential quantum efficiency of PSII Under non-stressing conditions, NH4 + increased the leaf net CO2 assimilation rate at a[CO2 ] (Fig. 3A). However, with the addition of NaCl, the differential N-supply did not alter the response of this parameter. The e[CO2 ] produced a higher rate of photosynthesis after 44 days of treatment, with respect to a[CO2 ] (38.2% and 25.7% higher, respectively, in the presence and absence of NH4 + ), this effect being much more dramatic under saline conditions (66.7% and 84.8% higher, respectively). It was also relevant that salinity-stressed plants grown at e[CO2 ] achieved a higher rate of photosynthesis than non-stressed plants at a[CO2 ]. Although the addition of NH4 + had a marginal effect on this parameter, both gs and E were significantly increased when NH4 + was added at ambient but not at e[CO2 ], under both saline and non-saline conditions (Fig. 3BC). Additionally, root respiration was higher at e[CO2 ] than at a[CO2 ], with a positive effect of NH4 + for non-stressed roots (Fig. 3D). Under salinity stress, the inverse pattern was found: the addition of NH4 + increased the root respiration rate at a[CO2 ], and decreased it at e[CO2 ]. In general, the treatments had a higher impact on chlorophyll b than on chlorophyll a (Table 2). The level of chlorophyll b increased when saline stress was imposed at e[CO2 ], and at a[CO2 ]. Provision of NH4 + increased the chlorophyll b concentration by 26.5% but no effect was found under saline conditions. NH4 + addition decreased the ratio a/b by 19.6% and 6.5% at a[CO2 ] and e[CO2 ], respectively. However, the CO2 had the contrary effect with respect to the supply of N used; it was increased with NO3 − alone (6.1%), and decreased with NH4 + (9.2%). Also, NH4 + significantly increased the Fv/Fm value for both stressed and non-stressed plants at e[CO2 ], compared with the plants that only received NO3 − . 3.4. Leaf ion concentrations Under salinity stress, the leaf Cl− concentration was lower at e[CO2 ] than at a[CO2 ]. However, the addition of NH4 + did not reduce the concentration of this anion in the leaves (Fig. 4A). Additionally,

e[CO2 ] significantly reduced the leaf [NO3 − ] under both saline and non-saline conditions; this was not offset by the addition of NH4 + to the nutrient solution (Fig. 4B). However, the leaf [K+ ] was increased only by salinity and e[CO2 ] (Fig. 4D). The [CO2 ] had a more dramatic effect on the leaf [Na+ ] (Fig. 4D), which was 58.2% and 56.4% lower (with or without NH4 + addition, respectively) at e[CO2 ] than at a[CO2 ]. 3.5. Total phenolic compounds, ascorbate peroxidase activity, and lipid peroxidation At a[CO2 ], addition of NH4 + under saline conditions produced a lower leaf total phenolics concentration than NO3 − alone, and this trend was similar under e[CO2 ] (Table 3). However, the opposite effect occurred under non-saline conditions: NH4 + increased the concentration of total phenolics. Ascorbate peroxidase activity was increased by salinity at both [CO2 ], whilst it was decreased at e[CO2 ] under salinity. Lipid peroxidation was increased under salinity at a[CO2 ]. Under salinity stress, e[CO2 ] and the addition of NH4 + reduced lipid peroxidation, compared with a[CO2 ]. Lipid peroxidation was less affected under the studied conditions. 3.6. Leaf free amino acid concentration The total amino acid concentration in the leaves of salinitystressed plants and plants supplied with NH4 + was consistently lower than in the leaves of plants that received only NO3 − , with a marginal effect of [CO2 ] (Fig. 5). However, in a more detailed analysis under salinity, and considering each amino acid individually, the proline concentration was significantly reduced by e[CO2 ] and the supply of NH4 + also triggered a reduced concentration of this amino acid. Moreover, alanine, glutamic acid, and serine had similar patterns but were less affected by [CO2 ]. 4. Discussion The effect of the N form on the growth of plants has been studied by many authors (Ghanem et al., 2011; Houdusse et al., 2005), with different results. This variability could be attributed to the direct effects of the environmental conditions (such as light intensity, root medium temperature, N concentration, medium pH, and K+ supply), the proportions of the distinct N forms (NO3 − versus NH4 + )

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Fig. 3. Effect of salinity and two regimes of N-nutrition under an elevated CO2 concentration on sweet pepper plants: (A) net photosynthesis rate; (B) stomatal conductance; (C) transpiration rate, and (D) root respiration. Data are means ± SE of eight plants.

Table 2 Effect of salinity and two regimes of N-nutrition under an elevated CO2 concentration on sweet pepper plants: chlorophyll a, chlorophyll b, total chlorophyll content, chlorophyll a/b ratio, and chlorophyll fluorescence. Data are means ± SE of eight plants. Nitrogen form

Chl a (mg L−1 g−1 FW)

Chl b (mg L−1 g−1 FW)

Chl a + b (mg L−1 g−1 FW)

Ratio a/b

Fv/Fm

400

100/0 90/10 100/0 + NaCl 90/10 + NaCl

121 ± 4.5 104 ± 4.2b 109 ± 2.6b 108 ± 4.6b

69.1 ± 3.8 87.4 ± 4.3cd 92.6 ± 3.7d 77.3 ± 4.2abc

170 ± 8.2a 191 ± 7.9ab 195 ± 7.3b 186 ± 8.3ab

1.48 ± 0.02e 1.19 ± 0.03b 1.18 ± 0.03b 1.40 ± 0.04de

0.79 ± 0.0026bcd 0.79 ± 0.0003bc 0.80 ± 0.0004de 0.80 ± 0.0003cde

800

100/0 90/10 100/0 + NaCl 90/10 + NaCl

99.9 ± 3.6b 98.9 ± 1.8b 98.4 ± 3.4b 87.8 ± 2.0a

72.2 ± 2.2a 75.9 ± 1.2ab 84.1 ± 3.3bcd 88.4 ± 3.6d

172 ± 5.4a 175 ± 2.7ab 182 ± 6.7ab 174 ± 4.1a

1.39 ± 0.04d 1.30 ± 0.02c 1.17 ± 0.01b 1.04 ± 0.03a

0.78 ± 0.0037ab 0.80 ± 0.0044de 0.77 ± 0.0047a 0.80 ± 0.0020e

**

ns ns

*

***

*

**

***

***

*

ns ns

ns ns ns ns

** *

[CO2 ]

ANOVAb CO2 NFc NaCl CO2 × NF CO2 × NaCl NF × NaCl CO2 × NF × NaCl a

b

ns ns ns *

ns ns

a

**

*

***

***

*

*

***

ns

***

ns ns

Different letters within a column indicate significant (P ≤ 0.05) differences between treatments. b Analysis of variance: ns. not significant. c Nitrogen form. * P ≤ 0.05. ** P ≤ 0.001. *** P ≤ 0.0001.

used, and the plant species tested (Helali et al., 2010). The NH4 + ion is an intermediate in many metabolic reactions and fundamental processes of plants, such as NO3 − reduction, photorespiration, degradation of amides, and catabolism of proteins (Joy, 1988). Houdusse et al. (2008) underlined that pepper plants are sensitive to NH4 + nutrition; however, the low energy cost associated with the mechanisms of adaptation to salinity in which NH4 + is involved may result beneficial with regard to improving tolerance.

On the other hand, Frechilla et al. (2001) found that the salinityinduced growth reduction of pea plants was more pronounced in NH4 + -fed plants than in those fed with NO3 − . Our previous studies, where the specific effect of e[CO2 ] was analyzed (del Amor et al., ˜ 2010; Pinero et al., 2014; Pérez-Jiménez et al., 2015), indicated that the beneficial effect of CO2 fertilization counterbalanced (partially or totally) specific abiotic stresses. In sweet pepper, under control (non-saline) and a[CO2 ] conditions, the different ratios of NH4 + to

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Fig. 4. Effect of salinity and two regimes of N-nutrition under an elevated CO2 concentration on sweet pepper plants: (A) leaf NO3 − concentration; (B) leaf Cl− concentration; (C) leaf K+ concentration, and (D) leaf Na+ concentration. Data are means ± SE of eight plants. Table 3 Effect of salinity and two regimes of N-nutrition under an elevated CO2 concentration on sweet pepper plants: total phenolic compounds, ascorbate peroxidase, and lipid peroxidation. Data are means ± SE of eight plants. [CO2 ]

Nitrogen form

[Gallic acid] mg/100 g FW

APOX (␮mol g−1 FW)

TBARS (nmol g−1 FW)

400

100/0 90/10 100/0 + NaCl 90/10 + NaCl

190.67 ± 10.7cd 208.61 ± 9.4de 203.08 ± 12.8de 127.87 ± 8.1a

0.97 ± 0.1a 0.91 ± 0.1a 7.03 ± 0.4d 6.35 ± 0.3cd

3.3 ± 0.2a 2.8 ± 0.2a 4.2 ± 0.2bc 4.6 ± 0.4c

800

100/0 90/10 100/0 + NaCl 90/10 + NaCl

167.29 ± 9.1bc 232.90 ± 12.9e 179.35 ± 10.7cd 137.66 ± 11.1ab

1.44 ± 0.1a 1.50 ± 0.2a 6.06 ± 0.3bc 5.55 ± 0.3b

3.5 ± 0.3ab 3.8 ± 0.3ab 3.6 ± 0.2ab 3.5 ± 0.3ab

ns ns

ns

ns ns

***

***

**

*

ns

ns

ns

***

**

***

*

ns

ns

ns ns

ANOVAb CO2 NFc NaCl CO2 × NF CO2 × NaCl NF × NaCl CO2 × NF × NaCl a

*

Different letters within a column indicate significant (P ≤ 0.05) differences between treatments. b Analysis of variance: ns. not significant. c Nitrogen form. * P ≤ 0.05. ** P ≤ 0.001. *** P ≤ 0.0001.

NO3 − had significant effects on dry weight. Plants irrigated with NH4 + had decreased biomass production, as found previously by Li et al. (2007) for tomato. The ability of plants to respond to environmental factors such as e[CO2 ] and salinity depends on their capacity to take up and assimilate nutrients, N being a major factor limiting plant production. In our investigation, leaf tissue levels of ions like Cl− and Na+ were increased by salinity and were decreased by CO2 . However, NH4 + nutrition produced different trends in these ions, increasing

Cl− while decreasing Na+ under salinity and a[CO2 ]. In this work, the better acclimation to salinity of the plants grown with e[CO2 ] was attributable directly to low Na+ accumulation rather than to the increased K+ concentration. In other important crops, such as Sorghum (Miranda et al., 2013) and citrus (Fernández-Crespo et al., 2012), the effect of NH4 + represented an important strategy to withstand salinity, since it greatly limited the Na+ accumulation and improved the K+ /Na+ homeostasis in short-term salinity exposures. Kant et al. (2007) also pointed out that competition between NH4 +

M.C. Pi˜ nero et al. / Journal of Plant Physiology 200 (2016) 18–27

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Fig. 5. Effect of salinity and two regimes of N-nutrition under an elevated CO2 concentration on sweet pepper plant amino acid profiles. Data are means ± SE of eight plants.

and Na+ for root uptake sites could reduce Na+ uptake and transport from roots to shoots. Our study partially agrees, as this effect was not observed at e[CO2 ]. On the other hand, e[CO2 ] reduced the leaf [K+ ] under non-saline conditions but, as indicated by Helali et al. (2010), the K+ concentration in leaf tissues was strongly diminished only in the medium containing NH4 + (in our experiment, salinity and e[CO2 ] produced the inverse response). Dluzniewska et al. (2007) found that the N balance of NH4 + -fed plants was more severely affected by salt stress than that of plants supplied with NO3 − . Carlisle et al. (2012) indicated that, in general, NH4 + -supplied wheat plants had higher [K+ ], which suggests that nutrient concentrations are differentially affected by the inorganic N form supplied to the plants and CO2 enrichment. Bar-Tal et al. (2001) observed that decreasing the NO3 − /NH4 + ratio sharply decreased the uptake of cations by pepper. That response was also reported by Marti and Mills (1991), each increment in NH4 + decreasing the uptake of K+ . However, we observed that this effect cannot be directly attributed to a dilution effect derived from the greater biomass at e[CO2 ] under salinity stress. Furthermore, the vital nature of K+ transport systems and cytosolic K+ homeostasis under saline conditions is supported by the importance of maintaining a low cytosolic Na+ /K+ ratio for salt-tolerance mechanisms, which was observed at e[CO2 ]. The leaf NO3 − concentration was reduced by the provision of NH4 + under control conditions at e[CO2 ]. Matt et al. (2001) indicated that, under e[CO2 ], NH4 + supply increased NH4 + uptake and inhibited nitrate reductase activity. On the other hand, the leaf NO3 − concentration observed can be attributed to an important decrease in Rubisco without limitation of photosynthesis (Makino et al., 2000). PérezLópez et al. (2013) suggested reasons for the decrease in leaf NO3 − concentration under salinity: (1) an antagonism between Cl− and NO3 − for NO3 − transporters and/or inactivation of NO3 − transporters by the toxic effects of salinity; and (2) an absence of ATP,

which is required for active NO3 − transport. Furthermore, the fact that ACO2 was increased in plants grown at e[CO2 ], in spite of their lower foliar NO3 − concentrations, indicates that less N is required for photosynthesis. As regards gas exchange, the stimulation of photosynthesis under a[CO2 ] and control conditions (non-salinized) occurred to a greater extent with NH4 + ; however, the greater growth stimulation observed indicates a higher efficiency of the photosynthetic process under NO3 − nutrition. Nevertheless, under e[CO2 ] and both types of N nutrition, the rate of photosynthesis was similar, even under salinity stress. Surprisingly, this maintenance of the photosynthetic rate under high CO2 and NH4 + nutrition occurred despite the stomatal closure observed. It has been suggested that stomatal closure in response to e[CO2 ] increases the WUEi. Vega-Mas et al. (2015) also found, in tomato plants, that WUEi was higher under e[CO2 ], especially for plants supplied with NH4 + . In addition, the higher leaf proline and K+ levels detected under salinity and e[CO2 ] could have played an adaptive role, decreasing the negative impact of the salinity on the photosynthetic machinery, which was observed also in drought stress by Gimeno et al. (2014). It has been described that plants suffering from salt stress have a larger internal requirement for K+ , likely because K+ is required for maintenance of photosynthetic CO2 fixation (Gimeno et al., 2014). It has been suggested that the increased rates of carbon assimilation detected at e[CO2 ], due to increased photosynthesis, could raise the availability of substrates and thus increase the osmolarity, allowing for greater osmotic adjustment and thereby conserving a high water potential (Pérez-López et al., 2009). Conversely, gs was strongly increased by NH4 + nutrition under a[CO2 ], which may be due to a lower concentration of K+ , because this is an essential element in the regulation of stomatal movement (Zhang et al., 2010).

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In addition, our data also show that the supply of NH4 + increased root respiration under non-saline conditions and e[CO2 ] and at a[CO2 ] under salinity. Niu et al. (2013) attributed a differential root response to root morphology, since NO3 − -fed plants had more lateral roots but a shorter taproot under e[CO2 ], compared with a[CO2 ], whereas NH4 + -fed plants had longer roots with fewer lateral roots under e[CO2 ]. Such differences in root architecture could also explain the observed differences in nutrient uptake. Additionally, Bar-Tal et al. (2001) found that a decreasing N NO3 − :N NH4 + ratio reduced transpiration, probably as a result of the compacted canopy; our data also show a reduced leaf DW under e[CO2 ]. During salinity-induced oxidative stress, the availability of atmospheric CO2 is reduced, because of increased stomatal closure, and consumption of NADPH by the Calvin Cycle is decreased (Turkan and Demiral, 2009). Consequently, increased CO2 concentrations could ameliorate the redox homeostasis, photosynthesis, and damaged cell structure of plants exposed to salinity (PérezLópez et al., 2013). However, the magnitude and direction of change of the concentrations of phenolic compounds in response to [CO2 ] ˜ elevation may be species-specific (Penuelas et al., 1996). In our case, NH4 + -fed sweet pepper presented a higher free radicals scavenging capacity and more efficient protection against salt stress under e[CO2 ], as revealed by the lower level of lipid peroxidation and improved plant water status as well as the activities of some antioxidants (APOX). In this study, NH4 + nutrition did not have much effect on antioxidant enzymes. Under a[CO2 ], the APOX activity was not affected by the contribution of NH4 + . However, the phenolic content was decreased by the addition of low doses of NH4 + (90/10) under salinity stress. This increase in the content of phenolic compounds was reflected in a reduction of the lipid peroxidation. As expected, salinity caused oxidative damage, which can be observed as the increase in lipid peroxidation, but interestingly, this oxidative damage disappeared at high [CO2 ]. The total amino acids data demonstrate that the salinity stimulated their accumulation in leaves. The most abundant amino acids were Ser, Arg, Asp, Glu, and Pro. Dluzniewska et al. (2007) found that the large accumulation of amino compounds in the leaves of salt-exposed plants is a notable response of N metabolism to the saline environment. Frechilla et al. (2001) found that the N source was a major factor affecting pea responses to saline stress and that NH4 + -fed plants contained more free amino acids than NO3 − -fed plants. By contrast, in our case, the NH4 + -fed plants had a lower concentration of total amino acids. High CO2 produced opposite effects in NH4 + -fed plants under control and saline conditions. Under e[CO2 ] and induced salinity stress, the NH4 + addition reduced the total leaf amino acid content, while under control conditions it increased the content. Miranda et al. (2016) reported that, in plants exposed to salinity, the burst of amino acids will help to maintain the water balance, osmoprotection, ROS scavenging, and amelioration of salinity-induced K+ efflux.

5. Conclusion The results obtained in this work point out the complexity and importance of a multifactorial approach in relation to the response of sweet pepper plants to NH4 + nutrition and salinity stress at high [CO2 ]. Thus, the reduction in the leaf ion concentration caused by reduced transpiration at high [CO2 ] was altered by NH4 + , but the supposed higher nutrient N efficiency (in terms of energy) was not reflected in substantial changes in ascorbate peroxidase activity or lipid peroxidation under salinity. Moreover, the provision of NH4 + did not alter leaf osmotic or water potential, compared with the supply of NO3 − alone. However, in addition to the related effect of high [CO2 ] on WUEi, NH4 + nutrition was able to significantly improve this efficiency under the studied stress. The amino acid

profile was also modified by salinity, and NH4 + had an effect, reducing the leaf Pro concentration at both [CO2 ]. Our study shows the complexity of the response of sweet pepper to NH4 + supply under salinity and differing [CO2 ], and can be contrasted with previous studies in which only the current a[CO2 ] was considered when determining the ability of different plant strategies to withstand salinity in the future climate change scenario. Acknowledgements ˜ M.C Pinero is the recipient of a pre-doctoral fellowship from the INIA-CCAA. The authors thank G. Otálora and M. Marín for their technical assistance. This work has been supported by the Instituto Nacional de Investigaciones Agrarias (INIA), through project RTA2011-00026-C02-01. Part of this work was also funded by the European Social Fund. References Bar-Tal, A., Aloni, B., Karni, L., Rosenberg, R., 2001. Nitrogen nutrition of greenhouse pepper. II. Effects of nitrogen concentration and NO3 − : NH4 + ratio on growth, transpiration, and nutrient uptake. Hortscience 36 (7), 1252–1259. Bloom, A.J., Smart, D.R., Nguyen, D.T., Searles, P.S., 2002. Nitrogen assimilation and growth of wheat under elevated carbon dioxide. Proc. Natl. Acad. Sci. U. S. A. 99 (3), 1730–1735. Bloom, A.J., Burger, M., Rubio-Asensio, J.S., Cousins, A.B., 2010. Carbon dioxide enrichment inhibits nitrate assimilation in wheat and arabidopsis. Science 328 (5980), 899–903. Carlisle, E., Myers, S., Raboy, V., Bloom, A., 2012. The effects of inorganic nitrogen form and CO2 concentration on wheat yield and nutrient accumulation and distribution. Front. Plant Sci., 3. Cruz, C., Lips, S.H., Martins-Loucao, M.A., 1997. Changes in the morphology of roots and leaves of carob seedlings induced by nitrogen source and atmospheric carbon dioxide. Ann. Bot. 80 (6), 817–823. Dajic, Z., 2006. Salt stress. In: Madhava Rao, K.V., Raghavendra, A.S., Janardhan Reddy, K. (Eds.), Physiology and Molecular Biology of Stress Tolerance in Plants. Springer, Netherlands, pp. 41–99. del Amor, F.M., Cuadra-Crespo, P., 2012. Plant growth-promoting bacteria as a tool to improve salinity tolerance in sweet pepper. Funct. Plant Biol. 39 (1), 82–90. del Amor, F.M., Cuadra-Crespo, P., Varo, P., Gómez, M.C., 2009. Influence of foliar urea on the antioxidant response and fruit color of sweet pepper under limited N supply. J. Sci. Food Agric. 89 (3), 504–510. del Amor, F.M., Cuadra-Crespo, P., Walker, D.J., Cámara, J.M., Madrid, R., 2010. Effect of foliar application of antitranspirant on photosynthesis and water relations of pepper plants under different levels of CO2 and water stress. J. Plant Physiol. 167 (15), 1232–1238. Dluzniewska, P., Gessler, A., Dietrich, H., Schnitzler, J.P., Teuber, M., Rennenberg, H., 2007. Nitrogen uptake and metabolism in Populus x canescens as affected by salinity. New Phytol. 173 (2), 279–293. Fernández-Crespo, E., Camanes, G., García-Agustín, P., 2012. Ammonium enhances resistance to salinity stress in citrus plants. J. Plant Physiol. 169 (12), 1183–1191. Frechilla, S., Lasa, B., Ibarretxe, L., Lamsfus, C., Aparicio-Tejo, P., 2001. Pea responses to saline stress is affected by the source of nitrogen nutrition (ammonium or nitrate). Plant Growth Regul. 35 (2), 171–179. Geissler, N., Hussin, S., Koyro, H.-W., 2009. Elevated atmospheric CO2 concentration ameliorates effects of NaCl salinity on photosynthesis and leaf structure of Aster tripolium L. J. Exp. Bot. 60 (1), 137–151. Ghanem, M.E., Hichri, I., Smigocki, A.C., Albacete, A., Fauconnier, M.L., Diatloff, E., Martínez-Andujar, C., Lutts, S., Dodd, I.C., Pérez-Alfocea, F., 2011. Root-targeted biotechnology to mediate hormonal signalling and improve crop stress tolerance. Plant Cell Rep. 30 (5), 807–823. Gimeno, V., Díaz-López, L., Simón-Grao, S., Martínez, V., Martínez-Nicolás, J.J., García-Sánchez, F., 2014. Foliar potassium nitrate application improves the tolerance of Citrus macrophylla L. seedlings to drought conditions. Plant Physiol. Biochem. 83, 308–315. Helali, S.M.R., Nebli, H., Kaddour, R., Mahmoudi, H., Lachaal, M., Ouerghi, Z., 2010. Influence of nitrate-ammonium ratio on growth and nutrition of Arabidopsis thaliana. Plant Soil 336 (1–2), 65–74. Houdusse, F., Zamarreno, A.M., Garnica, M., García-Mina, J., 2005. The importance of nitrate in ameliorating the effects of ammonium and urea nutrition on plant development: the relationships with free polyamines and plant proline contents. Funct. Plant Biol. 32 (11), 1057–1067. Houdusse, F., Garnica, M., Zamarreno, A.M., Yvin, J.C., García-Mina, J., 2008. Possible mechanism of the nitrate action regulating free-putrescine accumulation in ammonium fed plants. Plant Sci. 175 (5), 731–739. Intergovernmental Panel on Climate Change, 2014. Climate Change 2014: Synthesis Report. Contribution of Working Groups I, II and III to the Fifth Assessment Report of the Intergovernmental Panel on climate change [Core

M.C. Pi˜ nero et al. / Journal of Plant Physiology 200 (2016) 18–27 Writing Team, Pachauri, R.K., Meyer, L.A., (eds.)]. IPCC, Geneva, Switzerland, p. 151. Joy, K.W., 1988. Ammonia, glutamine and asparagine—a carbon nitrogen interface. Can. J. Bot. 66 (10), 2103–2109. Kähkönen, M.P., Hopia, A.I., Vuorela, H.J., Rauha, J.P., Pihlaja, K., Kujala, T.S., Heinonen, M., 1999. Antioxidant activity of plant extracts containing phenolic compounds. J. Agric. Food Chem. 47 (10), 3954–3962. Kant, S., Kant, P., Lips, H., Barak, S., 2007. Partial substitution of NO3 − by NH4 + fertilization increases ammonium assimilating enzyme activities and reduces the deleterious effects of salinity on the growth of barley. J. Plant Physiol. 164 (3), 303–311. Kant, S., Bi, Y.M., Rothstein, S.J., 2011. Understanding plant response to nitrogen limitation for the improvement of crop nitrogen use efficiency. J. Exp. Bot. 62 (4), 1499–1509. Li, J., Zhou, J.M., Duan, Z.Q., 2007. Effects of elevated CO2 concentration on growth and water usage of tomato seedlings under different ammonium/nitrate ratios. J. Environ. Sci. 19 (9), 1100–1107. Makino, A., Nakano, H., Mae, T., Shimada, T., Yamamoto, N., 2000. Photosynthesis, plant growth and N allocation in transgenic rice plants with decreased Rubisco under CO2 enrichment. J. Exp. Bot. 51, 383–389. Marti, H.R., Mills, H.A., 1991. Calcium-uptake and concentration in bell pepper plants as influenced by nitrogen form and stages of development. J. Plant Nutr. 14 (11), 1177–1185. Matt, P., Geiger, M., Walch-Liu, P., Engels, C., Krapp, A., Stitt, M., 2001. The immediate cause of the diurnal changes of nitrogen metabolism in leaves of nitrate-replete tobacco: a major imbalance between the rate of nitrate reduction and the rates of nitrate uptake and ammonium metabolism during the first part of the light period. Plant Cell Environ. 24 (2), 177–190. Miranda, R.D.S., Alvarez-Pizarro, J.C., Silva Araujo, C.M., Prisco, J.T., Gomes-Filho, E., 2013. Influence of inorganic nitrogen sources on K+ /Na+ homeostasis and salt tolerance in sorghum plants. Acta Physiol. Plant. 35 (3), 841–852. Miranda, R.D.S., Gomes-Filho, E., Prisco, J.T., Alvarez-Pizarro, J.C., 2016. Ammonium improves tolerance to salinity stress in Sorghum bicolor plants. Plant Growth Regul. 78 (1), 121–131. Misra, N., Dwivedi, U.N., 1990. Nitrogen assimilation in germination phaseolus-aureus seeds under saline stress. J. Plant Physiol. 135 (6), 719–724. Munns, R., Tester, M., 2008. Mechanisms of salinity tolerance, annual review of plant biology. Ann. Rev., 651–681, Palo Alto. Niu, Y.F., Chai, R.S., Jin, G.L., Wang, H., Tang, C.X., Zhang, Y.S., 2013. Responses of root architecture development to low phosphorus availability: a review. Ann. Bot. 112 (2), 391–408. ˜ Pérez-Jiménez, M., López-Pérez, A.J., Otálora-Alcón, G., Marín-Nicolas, D., Pinero, M.C., del Amor, F.M., 2015. A regime of high CO2 concentration improves the acclimatization process and increases plant quality and survival. Plant Cell Tissue Organ Cult. 121 (3), 547–557.

27

˜ Pérez-López, U., Robredo, A., Lacuesta, M., Mena-Petite, A., Munoz-Rueda, A., 2009. The impact of salt stress on the water status of barley plants is partially mitigated by elevated CO2 . Environ. Exp. Bot. 66 (3), 463–470. ˜ Pérez-López, U., Robredo, A., Miranda-Apodaca, J., Lacuesta, M., Munoz-Rueda, A., Mena-Petite, A., 2013. Carbon dioxide enrichment moderates salinity-induced effects on nitrogen acquisition and assimilation and their impact on growth in barley plants. Environ. Exp. Bot. 87, 148–158. ˜ Penuelas, J., Ribas-Carbo, M., Giles, L., 1996. Effects of allelochemicals on plant respiration and oxygen isotope fractionation by the alternative oxidase. J. Chem. Ecol. 22 (4), 801–805. ˜ Pinero, M.C., Houdusse, F., García-Mina, J.M., Garnica, M., del Amor, F.M., 2014. Regulation of hormonal responses of sweet pepper as affected by salinity and elevated CO2 concentration. Physiol. Plant. 151 (4), 375–389. Poorter, H., Pérez-Soba, M., 2001. The growth response of plants to elevated CO2 under non-optimal environmental conditions. Oecologia 129 (1), 1–20. Porra, R.J., Thompson, W.A., Kriedemann, P.E., 1989. Determination of accurate extinction coefficients and simultaneous-equations for assaying chlorophyll-b extracted with 4 different solvents –verification of the concentration of chlorophyll standards by atomic-absorption spectroscopy. Biochim. Biophys. Acta 975 (3), 384–394. Reich, P.B., Hungate, B.A., Luo, Y., 2006. Carbon-nitrogen interactions in terrestrial ecosystems in response to rising atmospheric carbon dioxide. Ann. Rev. Ecol. Evol. Syst. 37, 611–636. Rogers, A., Ainsworth, E.A., 2006. The response of foliar carbohydrates to elevated CO(2). Managed Ecosyst. CO2 187, 293–308. Russell, K., Lee, C., McCulley, R.L., Van Sanford, D., 2014. Impact of Climate Change on Wheat Production in Kentucky. Sanz-Sáez, A., Erice, G., Aranjuelo, I., Nogues, S., Irigoyen, J.J., Sánchez-Díaz, M., 2010. Photosynthetic down-regulation under elevated CO2 exposure can be prevented by nitrogen supply in nodulated alfalfa. J. Plant Physiol. 167 (18), 1558–1565. Tausz, M., Tausz-Posch, S., Norton, R.M., Fitzgerald, G.J., Nicolas, M.E., Seneweera, S., 2013. Understanding crop physiology to select breeding targets and improve crop management under increasing atmospheric CO2 concentrations. Environ. Exp. Bot. 88, 71–80. Turkan, I., Demiral, T., 2009. Recent developments in understanding salinity tolerance. Environ. Exp. Bot. 67 (1), 2–9. Vega-Mas, I., Marino, D., Sánchez-Zabala, J., González-Murua, C., María Estavillo, J., Begona González-Moro, M., 2015. CO2 enrichment modulates ammonium nutrition in tomato adjusting carbon and nitrogen metabolism to stomatal conductance. Plant Sci. 241, 32–44. Zhang, F., Niu, J., Zhang, W., Chen, X., Li, C., Yuan, L., Xie, J., 2010. Potassium nutrition of crops under varied regimes of nitrogen supply. Plant Soil 335 (1–2), 21–34. Zhu, J.K., 2002. Salt and drought stress signal transduction in plants. Annu. Rev. Plant Biol. 53, 247–273.