Combined effects of salinity and nitrogen levels on some physiological and biochemical aspects at the halophytic forage legume Sulla carnosa

ABSTRACT Salinity and low nitrogen availability are two major abiotic stresses that often coexist and disturb plant productivity. This study aimed at investigating the combined effects of salinity (0 and 100 mM NaCl) and nitrogen (0.5 and 5.0 mM NH4NO3) levels on the morphological parameters, the mineral status, nitrogen use efficiency, photosynthesis, and nitrate reductase (NR, E.C. 1.6.6.1) and glutamine synthetase (GS, EC 6.3.1.2) activities at Sulla carnosa.‎ Salinity enhanced growth rate (66%), the relative growth rate (RGR), root volume (RV), Na+ and Cl− accumulations and nitrogen use efficiency (NUE). It had no detrimental effect on photosynthetic assimilation rate (A) and leaf NR and GS activities under high nitrogen level condition. High nitrogen availability under saline condition enhanced growth rate, RGR, shoot/root ratio, photosynthesis activity, NR activity in roots, and the accumulation of NO3 − in different plant organs and all cations and anions in roots. In brief, salinity and high nitrogen availability represented an adequate condition for optimal growth and high Na+ and NO3 − accumulations at S. carnosa. This study suggested that S. carnosa could be a suitable forage legume that can be widely cultivated in the discarded, salinized and nitrogen-contaminated agricultural soils.


Introduction
Salinity is the major significant environmental stress that limits plant establishment, development and productivity worldwide (Song et al. 2009;Silva et al. 2018). It induces several physiological and biochemical changes in plants (Hasegawa et al. 2000) including osmotic stress caused by the high concentration of Na + and Cl − in the soil solution decreasing water availability to plants (Maia et al. 2010), nutritional imbalance due to the impaired nutrient uptake and allocation inducing nutrient/ ions deficiency (Ashraf et al. 2017), and specific ion toxicity resulted to the high level of accumulated sodium (Flowers et al. 2015). According to their responses to salinity, plants can be divided into two major groups; glycophytes, which are sensitive to salinity and cannot survive in saline soils and halophytes that are plants that can survive and complete their life cycle in saline environments.
The salinized agricultural soils are increasing annually worldwide (Gupta and Huang 2014) especially in arid and semi-arid regions where precipitation is low and temperatures and evaporation rates are high. Estimates reported that 0.25-0.5 million ha of agricultural land is lost annually (Qadir et al. 2014). A substantial part of the agricultural lands accumulates salt over time mainly due to the intensive agricultural practices and the inadequate irrigation-water quality. Consequently, the productivity declined progressively and thus the fertilization becomes the most prevailing solution. Increasing the use of fertilizers for enhancing agricultural production has been under consideration for more than 50 years. Nitrogen form is the major used fertilizer and contributed substantially to the yield enhancement in agriculture (Zahoor et al. 2014).
Nitrogen (N) is one of the major nutrients that influences plant growth since it is an integral component of proteins that construct cell materials and plant tissues. It is required by plants as NO 3 − and/or NH 4 + which are the main available forms of N in the soils. The better vegetative growth of the main plant species was obtained under a mixture of NO 3 − and NH 4 + (Ashraf et al. 2018). These two N-forms are absorbed and assimilated in plant tissues, nitrate reductase and glutamine synthetase are the key enzymes in NO 3 − and NH 4 + assimilation, respectively. The activity of these two enzymes was influenced by several environmental conditions; including salinity and nitrogen deficiency (Barhoumi et al. 2016;Ashraf et al. 2018). The doubling of crop yield over the last half century is related to sevenfold rise in nitrogen fertilizer usage (Han et al. 2016). The used nitrogen fertilizers increased from 10.8 million metric tons in 1960 to 82 million metric tons in 2000 and anticipated to rise further to 249 million metric tons by 2050 (Ahmed et al. 2017).
Unfortunately, 40 to 50% of the applied nitrogen fertilizers to agricultural systems is not absorbed by plants and then lost to the environment as ammonia, nitrate, nitrous oxide or molecular nitrogen (Coskun et al. 2017). Thus, nitrogen fertilizer in agriculture practices has not only underpinned food production but also lead to a number of adverse impacts on environment and human health (Wang and Lu 2020). The management of these salinized and nitrogen contaminated agricultural lands is difficult and frequently results in their abandonment (Cuevas et al. 2019). Currently and due to the exponential increase of saline lands through the world, halophytes are receiving more attention and become a promising group to be domesticated for their multiple interests and uses (Li et al. 2019;Daba et al. 2019). In this context, the identification of halophytic species that can desalinize soils and use efficiently the high available nitrogen is a great issue.
Sulla carnosa is a spontaneous halophytic legume, which received a wide focus in the last years. Several studies evaluated its response to various abiotic stresses (Rouached et al. 2013;Rabhi et al. 2017;Elkhouni et al. 2018). Also, some encouraging tests to improve its productivity with microbial inoculation were conducted (Hmaeid et al. 2019). According to these investigations, S. carnosa could be a promising species that can be domesticated for its forage interest. However, up to day its response to nitrogen availability in salinized soils is not yet inspected. Therefore, we attempt in the current work to investigate the combined effects of nitrogen and salinity levels on S. carnosa with the ultimate goal to improve the productivity of the halophytic forage legumes and explore the discarded salinized and nitrogen-contaminated agricultural soils.

Plant culture and treatments
Seeds of Sulla carnosa were sterilized with 3% (w/v) calcium hypochlorite solution for 10 min, washed thoroughly with distilled water and germinated in Petri dishes at 25°C. Eight uniform seedlings were transferred into 5 L pots filled with twenty-fold diluted solution (Hewitt 1966). After two weeks of pretreatment, only five homogeneous plantlets were maintained per pot. A total of four pots per treatment were irrigated with the same complete nutrient solution containing 1.5 mM MgSO 4 , 1.6 mM KH 2 PO 4 , 0.6 mM K 2 HPO 4 , 3.5 mM CaCl 2 , 3 mM KCl and 3.0 µM Fe-K-EDTA. Trace elements were supplied as follows (µM): 0.04 Cu, 0.05 Zn, 0.02 Mo, 0.5 Mn and 0.05 B. NaCl salt treatments were imposed by adding (S) or not (C) 100 mM to the nutrient solution. NH 4 NO 3 nitrogen form was supplied to the nutrient solution at 0.5 mM (Low N; LN) or 5.0 mM (High N; HN). The nutritive solution was continuously aerated and changed every week, the pH was adjusted to 6.5 ± 0.3.
Plants were harvested 0 and 45 days after treatment and divided into leaves, stems and roots. Fresh weights of different organs were immediately determined. Dry matter was determined after drying for 72 h in an oven at 65°C. The relative growth rate (RGR) was determined according to Khan et al. (2000).

Mineral analysis
Main cations were extracted from homogenized powder of leaves, stems and roots with HNO 3 (0.5%). Ca 2+ and Mg 2+ concentrations were determined using atomic absorption spectrophotometer (Varian 06). K + and Na + contents in different plant organ tissues were determined using flame spectrophotometry (Corning). Major anions were extracted with boiling M-Q water (Drihem and Pilbeam 2002) and the concentrations were determined with ion chromatography on a Metrohm Model 761 ion Chromatograph equipped with Metrosep anion dual 2 (6.1006.100) using 2.0 mM NaHCO 3 /1.3 mM Na 2 CO 3 as the eluent. The reduced nitrogen was determined using Kjeldahl method (Bremner 1965). Total nitrogen estimated as the sum of nitrate and reduced nitrogen. Nitrogen use efficiency was calculated according to the following formula: NUE = SDW/SN (SDW = Shoot dry weight, SN = total shoot nitrogen).

Photosynthetic parameter measurements
Photosynthetic parameters (A, net photosynthetic rate, E; transpiration rate, g s ; stomatal conductance, Ci; internal CO 2 concentration) were measured from 10 to 12 a.m. with a portable system LCpro +. Measurements were conducted under the following conditions; PAR, incident on leaf surface, 850 µmol m −2 s −1 ; leaf chamber temperature, 28°C; CO 2 reference, 430 ppmv and boundary resistance to H 2 O, 0.3 m 2 s mol −1 . Intrinsic water use efficiency (iWUE) calculated as A/g s ratio. Chlorophyll a (Chl a), chlorophyll b (Chl b) and carotenoids (Car) were extracted by 80% acetone and determined spectrophotometrically according to Torrecillas et al. (1984).

Nitrate reductase and glutamine synthetase extract and assays
Frozen leaves and roots samples were homogenized at 4°C in an extraction buffer containing: 0.1 M potassium phosphate buffer (pH 7.4), 2.5% (w/v) casein, 1 mM EDTA, and 7.5 mM cysteine were used to measure nitrate reductase (NR) activity as follows: After filtration, the homogenate was centrifuged at 30,000×g for 15 min at 4°C. The activity of NR was determined according to Wray and Filner (1970) method. Sample extract was incubated in 0.1 M potassium buffer phosphate (pH 7.4), 1 M KOH, 332 mM EDTA, 0.15 mM NADH at 30°C for 30 min. After ending the reaction with 1 M zinc acetate, absorbance of the supernatant was measured at 540 nm after diazotation of nitrite ions with 5.8 mM sulfanilamide and 0.8 mM N-(1-naphthyl)-ethylenediaminedihydrochloride.
To measure glutamine synthetase (GS) activity, frozen leaf and root samples were homogenized at 4°C in grinding medium containing 50 mM Tris-HCl buffer (pH 7.6), 1 mM MgCl 2 , 1 mM EDTA, 1% (w/v) polyvinypolypyrrolidone (PVPP). The homogenate was centrifuged at 15,000 × g for 30 min at 4°C. GS activity was determined using hydroxylamine as substrate, and the formation of α-glutamylhydroxamate (α-GHM) was determined with acidified ferric chloride according to Wallsgrove et al. (1979). Quantification of α-GHM was done by using commercial glutamine as a standard after reading the absorbance of the incubation medium at 540 nm.

Statistical analysis
Data were subjected to two-way ANOVA analysis using salinity (S) and nitrogen (N) as factors for each parameter. Significance between means was evaluated by LSD post hoc test at p˂0.05 with the statistical package SPSS (version 16.00) for Windows (SPSS Inc., Chicago, IL, USA). Principal component analyses (PCA) were performed using the same software.

Growth and morphological parameters
Salt treatment (100 mM NaCl) had no significant effect on leaf, stem and root dry weights of LNtreated plants (Figure 1 A). Under high nitrogen (HN) availability condition, the supply of 100 mM NaCl had no significant effect on dry weight of leaves and roots, while it increased significantly that of stems by 274% compared to the control (Figure 1 A). Under non-saline condition, HN availability increased significantly leaves dry weight by 274% compared to the control, while it had no effect on stems and roots (Figure 1 A). Under saline condition, HN availability increased leaf, stem and root dry weights by 77%, 392% and 28%, respectively, compared to the controls (Figure 1 A).
Application of 100 mM NaCl had no significant effect on whole dry weight of plants receiving LN concentration however, it increased that of HN-treated ones by 66% (Figure 1 B). HN availability enhanced the whole plant dry weight under non-saline and saline conditions by 209% and 155%, respectively, compared to the controls (Figure 1 B).
Salinity reduced shoot and root dry weights ratio at LN-treated plants by 36%, however, it increased that of the HN-treated ones by 86% (Figure 1 C). Increasing N availability had no effect on shoot and root dry weights ratio at non-saline-treated plants, while increased it at saline-treated ones by 175% (Figure 1 C).  Concerning the relative growth rate (RGR), the two-way ANOVA result showed that salinity and nitrogen factors and their interaction (S × N) had significant effects on such parameter (F = 12.285, 0.257 and 102.574, P˂0.01, respectively; Table 1). Salt treatment increased RGR at LN-treated plants by 100% and at the HN-treated ones by 25% (Table 1). Similarly, HN availability enhanced RGR of control and saline-treated plants by 162.5% and 64.5%, respectively, compared to the controls ( Table 1).
The two-way ANOVA results showed that S, N and S x N had no significant effects on stem and root lengths (Table 1). The leaf number (LfN) was significantly influenced by S (F = 17.853, P˂0.001), N (F = 4.800, P˂0.05) and S × N (F = 7.078, P˂0.05). LfN increased by salt treatment under HN availability condition and by HN level under saline condition (Table 1).
Root volume (RV) was significantly influenced by S (F = 10.981, P˂0.01) and N (F = 5.013, P˂0.05) however, the interaction 'S x N' had no effect (Table 1). Application of 100 mM NaCl increased RV of LN-and HN-treated plants by 129% and 62%, respectively, compared to the controls (Table 1). Increasing N availability enhanced RV of saline-treated plants by 31% compared to the control (Table 1).

Mineral status and nitrogen use efficiency
Major cations content results are summarized in Table 2. Sodium concentration in leaves, stems and roots were significantly influenced by salinity (F = 552.705, 0.012 and 709.908, P˂0.001 respectively), while the effects of N and S × N were significant only on roots (F = 9.972 and 18.910, P˂0.01 respectively; Table 2). Salt application increased Na + content in leaves, stems and roots of both LNand HN-treated plants (Table 2). Increasing N availability had no significant effect on Na + accumulation in different organs of control plants. For saline-treated plants, HN availability had no significant effect on Na + accumulation in leaves and stems, while increased it in roots (Table 2).
Potassium accumulation in leaves, stems and roots were significantly influenced by S (F = 26.050, 90.179 and 18.495, P˂0.001, respectively; Table 2). Significant effects of N and S × N were noted on K + accumulation in leaves (F = 18.544 and 21.594, P˂0.001, respectively, Table 2). At LN-treated plants, the supply of 100 mM NaCl had no significant effect on K + accumulation in leaves, while reduced it in stems and roots by 45% and 31%, respectively (Table 2). Under HN availability condition, the supply of 100 mM NaCl reduced K + accumulation in leaves and stems by 38% and 47%, respectively, and it had no effect on roots (Table 2). HN availability increased K + accumulation in leaves of non-saline- Table 1. The combined effects of salinity (C = 0 mM NaCl, S = 100 mM NaCl) and nitrogen (LN = 0.5 mM NH 4 NO 3 , HN = 5.0 mM NH 4 NO 3 ) on the relative growth rate (RGR) and the morphological parameters (SL, stem length; RL, root length; LfN, leaf number; RV, root volume) of Sulla carnosa after 45 days of treatment.

Treatment
Relative growth rate and morphological parameters Data are means ± SE of seven measurements, ns: non significance; * significance at 0.05 probability level;** significance at 0.01 probability level; *** significance at 0.001 probability level Data are means ± SE of seven measurements, ns: non significance; * significance at 0.05 probability level;** significance at 0.01 probability level; *** significance at 0.001 probability level treated plants by 53%, while it had no effect on stems and roots (Table 2). At salt-treated plants, increasing N availability enhanced K + accumulation in roots by 27% compared to the control (Table 2). Calcium accumulation in leaves was not influenced by S, N and their interaction however, a significant effect was noted on roots (F = 8.469, 251.41 and 62.205, P˂0.01, respectively; Table 2). Salinity increased Ca 2+ content in stems (104%) and roots (239%) of LN-treated plants, while decreased them (by 34.5% and 17.5%, respectively) at HN-treated ones ( Table 2). The highest values of Ca 2+ content in stems and roots were noted at C/HN-treated plants. Under control condition, HN availability had no significant effect on Ca 2+ accumulation in leaves and increased it in stems and roots by more than two-and five-folds, respectively (Table 2). Under saline condition, increasing N availability reduced Ca 2+ accumulation in leaves and increased it in roots by 53% compared to the control (Table 2). Increasing N availability had no significant effect on leaf Ca 2+ content at control plants, while decreased it at salt-treated ones ( Table 2). HN availability increased Ca 2+ content in roots under control and saline conditions ( Table 2).
Salinity had a significant effect on Mg 2+ accumulation in leaves and stems (F = 18.423 and 11.509, P˂0.01, respectively), while it had no effect on roots (Table 2). N had significant effect on Mg 2+ accumulation in leaves, stems and roots (F = 5.078, 11.065 and 11.209, P˂0.05, respectively; Table 2) however, S × N had no effect (Table 2). Salt treatment reduced Mg 2+ accumulation in leaves of LNtreated plants, while it had no effect on HN-treated ones. The supply of 100 mM NaCl reduced Mg 2+ content in stems of both LN-and HN-treated plants and had no effect on roots (Table 2). Under control condition, HN availability reduced Mg 2+ accumulation in leaves and stems, while increased it in roots by 24% compared to the control (Table 2). For saline-treated plants, increasing N availability had no effect on Mg 2+ accumulation in leaves, decreased it in stems, and enhanced it in roots ( Table 2).
The main anions contents in different plant organs and the related two-way ANOVA results were summarized in Table 3. Salinity had significant effect on NO 3 − accumulation in leaves (F = 76.638, P˂0.001) and stems (F = 134.84, P˂0.001), while it had no effect on roots (Table 3). N and S × N had significant effects on NO 3 − accumulation in different plant organs (Table 3). S had no effect on NO 3 − accumulation in leaves and stems of LN-treated plants, while decreased it in roots. Under HN condition, salinity decreased NO 3 − accumulation in leaves and stems, while it had no effect on roots (Table 3). Under control condition, HN availability increased NO 3 − accumulation in leaves by 45folds and in stems by more than 8-folds however, it had no effect on roots (Table 3). Under saline condition, increasing N availability enhanced NO 3 − accumulation in different plant organs and the highest enhancement (54-folds) was noted in roots (Table 3).
Phosphate accumulation in leaves and stems were significantly influenced by S (F = 152.533 and 88.310 P˂0.001, respectively), while no effect was noted for roots (Table 3). N and S × N had significant effect on PO 4 3accumulation in different plant organs (Table 3). The supply of 100 mM NaCl decreased PO 4 3accumulation in leaves of LN-treated plant, increased it in stems and it had no effect on roots (Table 3). Under HN availability condition, S had no effect on PO 4 3accumulation in leaves and stems, while increased it in roots (Table 3). Under control condition, HN availability decreased PO 4 3content in leaves, increased it in stems, and it had no effect on roots (Table 3). Under saline condition, HN availability decreased PO 4 3accumulation in leaves, increased it in roots and it had no effect on stems (Table 3). S and N factors had significant effects on SO 4 2accumulation in different plant organs (Table 3), the interaction S x N had a significant effect only on leaves and stems (F = 45.631 and 40.800, P˂0.001 respectively; Table 3). Salinity effect on SO 4 2accumulation was dependent on plant organ; application of 100 mM NaCl had no effect on SO 4 2accumulation in leaves of LN-treated plants, increased it in stems and decreased it in roots. Under HN availability condition, salinity reduced the accumulations of SO 4 2in leaves and roots, while it had no effect on stems (Table 3). Under control condition, HN availability increased SO 4 2accumulation in different plant organs (Table 3). Under 100 mM NaCl  Data are means ± SE of seven measurements, ns: non significance; * significance at 0.05 probability level;** significance at 0.01 probability level; *** significance at 0.001 probability level treatment, increasing N availability enhanced SO 4 2accumulation in leaves and roots, while it had no effect on stems (Table 3).
Concerning chloride content, S had a significant effect on the accumulation of Clin leaves, stems and roots (F = 33.609, 117.863 and 29.930, P˂0.001 respectively; Table 3). N had a highly significant effect on Claccumulation in leaves and stems (F = 17.212 and 47.862, P˂0.001, respectively), while it had no effect on roots (Table 3). S × N had a highly significant effect on Claccumulation in roots (F = 86.606, P˂0.001; Table 3). Application of 100 mM NaCl increased Claccumulation in leaves and stems of LN-treated plants, while decreased it in roots (Table 3). Under HN availability condition, S increased Clcontent in leaves, stems and roots by 102%, 97% and 144% respectively compared to the controls (Table 3). Under control condition, HN availability reduced Claccumulation in leaves, stems and roots by 41%, 39% and 44%, respectively (Table 3). Under salt treatment condition, increasing N availability reduced Claccumulation in leaves and stem, while increased it in roots (Table 3).
Total nitrogen content (TNC) in different plant organs, nitrogen use efficiency (NUE) and the twoway ANOVA results in response to S and N treatments were summarized in Table 4. S had highly significant effect on TNC in stems and roots (F = 17.624 and 42.575, P˂0.001, respectively). Equally, N had significant effect on TNC in leaves and roots (F = 7.708 and 15.906, P˂0.05 respectively; Table  4). S × N had a significant effect on TNC only on stems (F = 5.623, P˂0.05; Table 4). The supply of 100 mM NaCl had no significant effect on TNC in leaves and stems of LN-treated plants, while decreased it in roots (Table 4). Under HN availability condition, S had no significant effect on TNC in leaves, and decreased it in stems and roots (Table 4). Increasing N availability enhanced TNC in leaves, stems and roots of control plants by 15%, 22% and 23%, respectively, however, it had no significant effect on different organs of salt-treated plants (Table 4). NUE was significantly influenced by S (F = 6.102, P˂0.05), while maintained invariant by N and S × N ( Table 4). The supply of 100 mM NaCl increased this parameter at LN and HN availability treated plants by 127% and 61%, respectively, compared to the controls, however, increasing N availability had no significant effect on this parameter either at control or saline-treated plants (Table 4).

Photosynthetic parameters and pigment contents
Net photosynthetic rate (A) was influenced by N (F = 16.124, P˂0.001), but not by S and S × N (Table  5). HN availability increased A by 57.4% and 44.2% at control and saline-treated plants, respectively (Table 5). Transpiration rate (E) and stomatal conductance (g s ) were significantly influenced by S (F = 5.82 and 12.36, P˂0.05, respectively), but not by N and S × N ( Table 5). The supply of 100 mM NaCl reduced E and g s at HN-treated plants by 33.3% and 29.7%, respectively, while it had Table 4. The combined effects of salinity (C = 0 mM NaCl, S = 100 mM NaCl) and nitrogen (LN = 0.5 mM NH 4 NO 3 , HN = 5.0 mM NH 4 NO 3 ) on total nitrogen contents (TNC) in leaves (L), stem (S) and root (R) and nitrogen use efficiency (NUE) in Sulla carnosa after 45 days of treatment. Data are means ± SE of seven measurements, ns: non significance; * significance at 0.05 probability level;** significance at 0.01 probability level; *** significance at 0.001 probability level no effect on LN-treated plants ( Table 5). The internal CO 2 concentration (C i ) was influenced by S and N (F = 3.99 and 5.04, P˂0.05, respectively), but not by S × N ( Table 5). Application of 100 mM NaCl reduced Ci in LN-treated plants, but no significant effect was noted at HN-treated ones (Table 5). HN availability decreased C i in control plants, while it had no effect on salt-treaded ones. The intrinsic water use efficiency (iWUE) was significantly influenced by S and N (F = 3.26 and 4.48, P˂0.05, respectively), but not by S × N ( Table 5). The supply of 100 mM NaCl increased iWUE in HN-treated plants by 67.8%, however no significant effect was noted on LN-treated ones (Table 5). Concerning N effect, increasing the availability of such nutrient enhanced iWUE at saline-treated plants by 79.2%, while it had no effect on control plants (Table 5).
According to the two-way ANOVA results, S and N had significant effects on Chl a (F = 7.265 and 33.34, respectively), Chl b (F = 2.044 and 11.601, respectively) and Car (F = 2.285 and 70.446, respectively, P˂0.05) contents (Table 5). S × N had no effect on Chl a content, but it has significant effect on Chl b and Car (F = 41.536 and 24.358, P˂0.001, respectively, Table 5). The supply of 100 mM NaCl had no significant effect on Chl a content at LN-treated plants, while it reduced Chl b and Car (Table 5). At HN-treated plants, S had no effect on Chl a content, while it increased Chl b and Car (Table 5). Under control condition, increasing N availability enhanced Chl a and Car contents, while it reduced Chl b. Under saline condition, N availability increased the three photosynthetic pigment contents (Table 5).

Nitrate reductase and glutamine synthetase activities
Nitrate reductase (NR; EC 1.7.1.1) activity in leaves was insensitive to S, N and S × N however, in roots, it was significantly influenced by the both factors and their interaction (Table 6). S had no significant effect on NR activity in roots of LN-treated plants, while increased it in HN-treated ones by 100% compared to the control (Table 6). HN availability enhanced NR activity in roots of control and salinetreated plants by 55% and 223%, respectively (Table 6).
Glutamine synthetase (GS; EC 6.3.1.2) activity in leaves was significantly influenced by S and N (F = 5.765 and 57.978, P˂0.05, respectively; Table 6). In roots, GS activity was significantly influenced by S and S × N (F = 10.515 and 51.460, P˂0.01, respectively; Table 6). Application of 100 mM NaCl had no significant effect on GS activity in leaves of both LN and HN-treated plants. At the root level, S increased GS activity at LN-treated plants by 226% and decreased it at HN-treated ones by 31.5% (Table 6). Increasing N availability reduced GS activity in leaves of control and salinetreated plants. N availability increased GS activity in roots of the control plants and declined it at the salt-treated ones (Table 6). Table 5. The combined effects of salinity (C = 0 mM NaCl, S = 100 mM NaCl) and nitrogen (LN = 0.5 mM NH 4 NO 3 , HN = 5.0 mM NH 4 NO 3 ) on the photosynthetic parameters (A, net photosynthetic assimilation; E, transpiration; g s , stomatal conductance; Ci, internal CO 2 concentration; iWUE, intrinsic water use efficiency) and pigments (Chl a, Chl b and Car) in Sulla carnosa after 45 days of treatment.

Discussion
Understanding salinity and nitrogen availability effects on plant is of great interest and exploring the physiological and biochemical responses may help in ameliorating plant productivity in saline soils. Legume plant growth and productivity are dependent on several factors mainly water availability, nutrients uptake and assimilation, photosynthesis activity and environmental conditions. Salinity has been shown to reduce productivity of several legume plants; Lotus corniculatus and Lotus tenuis (Teakle et al. 2006), Leucaena leucocephala and Acacia saligna (Tadros 2011) and Melilotus siculus (Kotula et al. 2019). Moreover, the major commercially legume forages have been reported to be sensitive to salt stress (Nichols et al. 2008). In the current study, salinity of 100 mM NaCl had no significant effect on growth of Sulla carnosa when nitrogen was limiting, while enhanced it by 66% when N is highly available. Thus, the effect of salinity on S. carnosa growth was dependent on N level available. We expect that after application of 100 mM NaCl and increasing Na + and Cl − contents in plant tissues (Tables 2 and 3), S. carnosa must sequestrate these toxic ions in vacuoles to prevent the deleterious effects on the intracellular biochemistry and consequently it synthetized compatible solutes with high levels in the cytoplasm to maintain the osmotic adjustment. The compatible solutes are mainly nitrogen-containing compounds including amino acids, amides, polyamines, proteins and quaternary ammonium compounds (Parida and Das 2005). Thus, under HN availability condition (i.e. N is a non-limiting factor), salinity of 100 mM NaCl induced cells extension growth with an adequate water uptake (Figure 1 D) leading to an increase of S. carnosa biomass production as assumed by the dry weight accumulation. Nitrogen occupies a prominent position among crucial nutrients that absorbed by plants and it is vital for growth and development. It is largely used to enhance productivity of glycophytes (Varadi Hirişcău et al. 2019) and halophytes (Guan et al. 2019). In the present study, the beneficial effect of HN availability on growth of S. carnosa was noted under control and saline conditions. Under control condition, growth enhancement which was 209% might be due to the increase of (i) N nutrition that could be confirmed by the high TNC in different plant organs, (ii) the net photosynthetic rate, which enhanced by 104%, and (iii) the NR and GS activities especially in roots. This result was in disagreement with that reported at the cultivated legumes, Trifolium alexandrinum receiving the same N concentration (Barhoumi et al. 2016). Herein, we presume that N concentration (5 mM NH 4 NO 3 ) is greater than the requirement of T. alexandrinum (2.5 mM NH 4 NO 3 ), which induced a decline in biomass production. This explanation is in accordance with the statement that an amount of N greater than the required may slow or inhibit growth (Iqbal et al. 2015). However, our result was in agreement with those reported at several other halophytes (Song et al. 2006;Barhoumi et al. 2010). Under saline condition, HN availability also enhanced growth of S. carnosa but with a lesser degree (155%) than under non saline condition. Among the four treatments, salinity and high nitrogen availability (S/HN) appeared the most convenient for the growth of S. carnosa. Table 6. The combined effects of salinity (C = 0 mM NaCl, S = 100 mM NaCl) and nitrogen (LN = 0.5 mM NH 4 NO 3 , HN = 5.0 mM NH 4 NO 3 ) on nitrate reductase (NR) and glutamine synthetase (GS) in leaves (L) and roots (R) of Sulla carnosa after 45 days of treatment. Mineral nutrients are fundamental for plant growth since they are implied in different vital processes. Their availability and absorption by plants are sensitive to salinity, which has been reported to affect nutrient uptake, induce nutrient deficiency or disorders in plants (Ashraf et al. 2017) and reduce photosynthetic activity and biomass production (Gong et al. 2018). The current investigation showed that 100 mM NaCl increased the accumulation of Na + and Cl − in all plant organs, however the effects on other ions were dependent on plant organ and N level. At the photosynthetic organ, salinity had either no effect or reduced the accumulation of other major ions. The decline concerned Mg 2+ and PO 4 3accumulations when N is limiting and K + , NO 3 − and SO 4 2− when N is highly available. The reduction of some cation and anion absorptions was mainly attributed to their antagonisms with Na + and Cl -, respectively (Netondo et al. 2004;Hu and Schmidhalter 2005). According to the two-way ANOVA results, N availability had variable effects on mineral nutrition of S. carnosa depending on the mineral element, the plant organ and the salt treatment with a general feature that the effect was more pronounced on anions than on cations. In this context, the evident results are that N availability had no significant effect on Na + accumulation and decreased Cl − content in leaves and stems under both control and saline conditions. Equally, increasing N availability enhanced NO 3 − accumulation in different plant organs under control and saline conditions. The noteworthy finding in the current mineral nutrition study was that HN availability increased the accumulation of all cations and anions in roots under saline condition. Herein, we hypothesized that S. carnosa might use this strategy (decreasing root osmotic potential) to overcome water retention in the saline rhizosphere and thus increasing its uptake by plants.
Nitrogen (N) is an essential nutrient for plants and is a constituent of amino acids, proteins, nucleic acids, chlorophyll, and various primary and secondary metabolites, thus it is vital for plant growth and development. The major inorganic N forms in the soil solution are NO 3 − and NH 4 + which are usable by plants. They are assimilated into amino acids and proteins that play, with carbon metabolism, a pivotal role in plant growth and development. N uptake was well reported to be sensitive to salinity (Iqbal et al. 2015). In this study, the application of 100 mM NaCl had no effect on TNC in leaves of S. carnosa however, a deleterious effect was noted on T. alexandrinum (Barhoumi et al. 2016). This is a sign that could substantiate the ability of S. carnosa to maintain an adequate N provision to the photosynthetic organ under saline condition, this aptitude was not shown at the commercial legume T. alexandrinum (Barhoumi et al. 2016). The deleterious effect of salinity on N uptake and assimilation was attributed to the antagonisms between Na + and NH 4 + in one hand and between Cl − and NO 3 − on the other hand (Shawer 2014) and to the down regulation of genes that are responsible for N assimilation (Wang et al. 2012). N availability effect on TNC was dependent on whether plants are treated or not with salinity. HN availability increased N accumulation in different organs of control plants, while it had no effect on saline-treated ones.
According to NUE result, S. carnosa used N more efficient under saline condition than under control condition independently to N level. NUE increases by salinity were 127% and 61% under LN and HN conditions, respectively. This finding was inconsistent with those reported at other halophytes (Barhoumi et al. 2010;Huez-Lopez et al. 2011), while it was in disagreement with report at the commercial legume forage, T. alexandrinum at which NUE decreased with salinity when N is highly available (Barhoumi et al. 2016). Increasing N availability had no effect on NUE in S. carnosa under control and saline conditions however, a significant decline was previously reported at T. alexandrinum (Barhoumi et al. 2016). This result denoted an interesting feature which was the ability of S. carnosa to use N efficiently even it existed with a high concentration. Evidently, this trait has crucial economic and environmental benefits since it allows avoiding N fertilizer excess losses and higher N leaching fractions especially N-nitrate form to the groundwater.
Photosynthesis is a major physiological process that having an important role in plant growth, development and productivity. Photosynthetic activity is often considered as a good marker of plant response to the environmental stresses (Liu et al. 2008). Several studies reported a decline of this parameter with salinity (Barhoumi 2019;Ullah et al. 2020). In the current work, the supply of 100 mM NaCl had no effect on A under LN and HN level treatments. This result was in disagreement with that reported in our previous study at the commercial legume forage, T. alexandrinum at which A was drastically reduced by a same salt concentration (Barhoumi et al. 2016). Our results showed also that salinity effects on E, g s , Ci and iWUE were N level dependent. Concerning the effect of N availability on photosynthetic parameters in legume was not well studied and further investigations are needed. The limited literature reported that N availability increase had no effect on A and g s at Glycine max (Moreira et al. 2015), increased A and decreased g s at Cicer arietinum (Tak et al. 2010), and increased A and maintained invariant g s at Phaseolus vulgaris (Jifon and Wolfe 2002). Similarly, contrasting findings were reported for cereal grains and dicot non-legumes (Lopes et al. 2004;Lopes and Araus 2006). In our study, HN availability enhanced A and maintained invariant g s under control and saline conditions however, the effect on E, Ci and iWUE was dependent on salt concentration. In contrast, at the cultivated legume forage T. alexandrinum, N availability was shown to have no significant effect on A (Barhoumi et al. 2016). In the current study, A and iWUE showed high correlations (r = 0.957 and 0.932, respectively) with the growth response to salinity, nitrogen and their interaction ( Figure S1 -Supplementary materials).
Concerning photosynthetic pigments, salinity reduced Chl b and Car contents in LN-treated plants and increased them under HN availability condition. Increasing N availability enhanced Chl a and Car contents in control and saline-treated plants. In contrast, the same salt and N levels were shown to have no significant effects on photosynthetic pigments at T. alexandrinum (Barhoumi et al. 2016).
NR and GS are the key enzymes that implied in NO 3 and NH 4 + assimilations, their activities were shown to be influenced by salinity (Ullah et al. 2020) and N concentration (Saghaiesh et al. 2019). In our study, application of 100 mM NaCl had no effect on NR and GS activities in leaves of S. carnosa independently to the N level. This result was in disagreement with that reported at T. alexandrinum at which leaf NR and GS activities were influenced by the same salt concentration (Barhoumi et al. 2016). In contrast, NR and GS activities in roots were enhanced by salinity under HN and LN treatments, respectively. Generally, the available literature reported depressive effect of salinity on NR and GS activities in plants (Queiroz et al. 2012;Ahanger and Agarwal 2017) and when plants were subjected to different N concentrations, different responses could be obtained (Barhoumi et al. 2016). Increasing N availability had no effect on NR activity in leaves under both control and salinity conditions, whilst increased it in roots. HN availability decreased GS activity in leaves of control and saline-treated plants, however its effect on root GS was salt-treatment dependent. According to the correlation analysis, a high positive correlation was noted between S. carnosa growth and root NR activity (r = 0.953), however a high negative one was recorded with leaf GS activity (r = -0.970) in S. carnosa ( Figure S2 -Supplementary materials).
The principal component analysis (PCA) could offer an overview of all parameters assessed in the current study. The first two principal component vectors (PC1 and PC2) explained 70.58% of the total variation ( Figure S3 -Supplementary materials). The first and second axis of PCA accounted for 39.13 and 31.45% of the total variance. The first factorial axis (PC1) was high positively associated with some parameters (oval shape with continuous line) mainly leaf and stem nitrate content (0.999 and 0.977, respectively), root and stem total nitrogen contents (0.972 and 0.953, respectively) and leaf sulphate and potassium contents (0.967 and 0.951, respectively), while the second factorial axis (PC2) was related with some others parameters (oval shape with discontinuous line) mainly root dry weight (0.936), root nitrate reductase (0.912) and whole plant dry weight (0.897). According to the principal component analysis, the most important variables, which explained more than 70% of variance were mainly related to the mineral status of plant especially nitrogen nutrient and to the morphological character principally the dry weight and root volume ( Figure S3 -Supplementary materials).
In summary, this study showed that Sulla carnosa preferred salinity and nitrogen availability to produce an optimal biomass. Increasing N availability under saline condition had a beneficial effect on biomass production and vice-versa. Salinity enhanced the relative growth rate, the intrinsic water use efficiency and nitrogen use efficiency at this species when N was highly available. Moreover, leaf NR and GS and the photosynthetic activities were highly preserved under saline treatment especially when N was highly available. Therefore, we suggest that S. carnosa could be a potential spontaneous halophytic legume species that can be domesticated for the forage interest and could be a pioneer candidate to be cultivated in the abandoned salinized and nitrogen-contaminated agricultural lands.