Effects of salinity on photosynthetic traits, ion homeostasis and nitrogen metabolism in wild and cultivated soybean

Plant materials and salt treatments

The experiment was conducted outdoors and the pots were sheltered from rain, at Northeast Normal University Changchun, Jilin, Northeast China (43°05′∼45°15′N, 124°18′∼127°05′E).The mean ± standard error (SE) temperature was 18.5 ± 1.5 °C at night and 26 ± 2 °C in the daytime, and the humidity was 60% ±5%. The seeds of the wild soybean genotype (W; Huinan 06116) and the cultivated soybean accession (C; Jinong 24) were provided by Jilin Province Crop Breeding Center of New Varieties. The seeds were sown in plastic pots (15 cm in diameter) with a hole in the bottom ( two cm in diameter) and filled with 2.5 kg of thoroughly washed sand. Initially, the seedlings were watered with full-strength Hoagland solution every morning. At the third compound leaf stage, thirty-five pots of uniform seedlings were selected and split into seven sets (per accession) for stress treatment: three sets of five pots each for saline stress (NaCl and Na₂SO₄, at a 1:1 molar ratio), three sets of five pots each for alkaline stress three (Na₂CO₃ and NaHCO₃, at a 1:1 molar ratio) and one set of five pots for the positive control (untreated). Both saline and alkaline stresses consisted of three concentrations (10, 20 and 30 mmol L⁻¹, n = 5). All solutions were prepared in 1 ×Hoagland solution. The stress was gradually implemented with 10 mmol L⁻¹ of saline or alkaline stress solution for the first two days to allow the seedlings to adapt to salt stress, followed by the respective treatments, i.e., 10, 20 or 30 mmol L⁻¹ for two weeks. The leaves of the two soybean accessions were then harvested from three randomly selected pots per treatment and immediately frozen in liquid nitrogen and stored at −80 °C for further analysis (see below).

Growth parameters

After the soybean seedlings were harvested, shoot height, root length, shoot and root fresh weight, and shoot and root dry weight were measured (Shao et al., 2016).

Gas exchange parameters

After two weeks of stress treatment, gas exchange parameters of fully expanded leaves were measured at photosynthetically active radiation (PAR) of 1,200 ± 50 µmolm⁻² s⁻¹, using a portable open flow gas exchange system LI-6400 (LI-COR, USA) at 11:00 am. The concentration of CO₂ in the atmosphere was 380 ± 5 cm³ m⁻¹. Air humidity and temperature were about 50% and 24 ° C, respectively. The results for net CO₂ assimilation rate (P_n), transpiration rate (E), stomatal conductance (G_s) and ratio of sub-stomatal to atmospheric CO₂ concentration (C_i/C_a) were expressed in units of µmol CO₂m⁻² s⁻¹, µmol H₂O m⁻² s⁻¹, mol m⁻² s⁻¹, and cm³m⁻³, respectively. The water-use efficiency parameter (WUE) was calculated as the ratio of P_n/E. Measurements were carried out for each treatment from each of three replicate pots and the mean values were recorded.

Measurement of photosynthetic pigments

Dry leaf samples (0.1 g) were immersed in 80% acetone/anhydrous ethanol mixture (1:1) to extract the photosynthetic pigments completely. Photosynthetic pigments were determined using a spectrophotometer (Type UV-754; Shanghai Accurate Scientific Instrument Co., China) to measure absorbance at 440, 645 and 663 nm.

Photosynthetic pigment concentrations (mg g⁻¹) were calculated using the following equations: ${\displaystyle \text{Chlorophyll a (Chl a)}=9.784A_{663}-0.990A_{645}}$ ${\displaystyle \text{Chlorophyll b (Chl b)}=21.426A_{645}-4.650A_{663}}$ ${\displaystyle \text{Total chlorophyll (Chl t)}=\mathrm{Chla}+\mathrm{Chlb}=5.134A_{663}-20.436A_{645}}$ ${\displaystyle \text{Carotenoids (Car)}=4.695A_{440}-0.268Chlt}$ ${\displaystyle \text{Content}\left({\mathrm{mgg}}^{-1}\right)=\mathrm{concentration}\times \mathrm{volume}\left(10\mathrm{mL}\right)∕\mathrm{sample dry weight}\left(50\times 10^{-3}\mathrm{g}\right)}$ (Holm, 1954).

Mineral ion concentration determination

Each dry root samples (0.05 g) was treated with deionized water (four mL) at 100 °C for 40 min and then centrifuged at 3000 × g for 15 min.The supernatant was collected and the extraction was repeated twice, with the extracts combined and adjusted to 15 mL. This combined supernatant was used for the determination of NO₃⁻, Cl⁻, SO₄²⁻, H₂PO⁴⁻ and oxalate (C₂O₄²⁻) anions, using ion chromatography (DX-300 ion chromatographic system, AS4A-SC chromatographic column, CDM-II electrical conductivity detector, mobile phase: Na₂CO₃/NaHCO₃ = 1.7/1.8 mM; Dionex, Sunnyvale, CA, USA). An atomic absorption spectrophotometer (Super 990F, Beijing Purkinje General Instrument Co. Ltd. Beijing, China) was used to determine the concentrations of Na⁺, K⁺, Ca²⁺, Mg²⁺, Fe²⁺, B³⁺, Cu²⁺, Mn²⁺, Zn²⁺, and P³⁺ cations (Jiao et al., 2018)

Enzyme extract preparation

Fresh leaf samples were ground in a chilled mortar and homogenized with 50 mM Tris-HCl buffer (pH 7.5), containing 500 mM EDTA, 1 mM MgCl₂, 10 mM β-mercaptoethanol, and 0.5% polyvinylpyrrolidone (PVP). The homogenate was centrifuged at 12,000 × g for 10 min at 4 °C and the supernatant was retained to assay in vitro activities of nitrate reductase (NR), glutamine synthetase (GS), glutamine synthase (GOGAT), glutamate dehydrogenase (GDH), alanine aminotransferase (AlaAT) and aspartate aminotransferase (AspAT), as well as protein concentration.

Nitrate reductase  (NR) activity

NR activity was determined by the sulfamate colorimetric method (Cruz & Martins Loução, 2002). The reaction mixture, containing 200 mM KNO₃, 5 mM EDTA, and 0.15 mM NADH in 100 mM phosphate buffer (pH 7.5) was incubated for 1 h at 30 °C. Then two mL of sulfanilamide and two mL of α-naphthylamine reagents were added and the absorbance was read at 540 nm. The enzyme activity was expressed in units of µmol g⁻¹ protein.

Glutamine synthetase (GS) activity

GS activity was assayed according to the method of Oaks et al. (1980). The reaction mixture, which included 100 mM Tris–HCl buffer (pH 7.4), 80 mM MgSO₄, 20 mM sodium glutamate, 20 mM cysteine, 2 mM EGTA, ATP solution (0.7 mL) and enzyme extract, was incubated for 30 min at 37 °C . Then, one mL of ferric chloride reagent (0.37 M FeCl₃ and 0.2 M trichloroacetic acid in 0.5 M HCl) was added and centrifuged at 5,000 × g for 10 min at 4 °C. The absorbance was read at 540 nm and enzyme activity was expressed in units of µmol g⁻¹ protein.

Glutamine synthase (GOGAT) activity

GOGAT activity was assayed according to the method of Rachim & Nicholas (1985). The reaction mixture contained 20 mM L-glutamine, 100 mM α-ketoglutaric acid, 10 mMKCl, and 3 mM NADH in 25 mM Tris–HCl (pH 7.6). The reaction was started by adding the enzyme extract. The NADH oxidation was monitored by continuously recording the absorbance at 340 nm. The oxidation of 1 µmol NADH per min was considered to be an enzyme unit (µmol g⁻¹ protein).

Glutamate dehydrogenase (GDH) activities

Aminating glutamate dehydrogenase (NADH-GDH) activity was evaluated according to the method of Groat & Vance (1981). For deaminating glutamate dehydrogenase (NAD-GDH) activity, the reaction mixture consisted of 23.1 mM α-ketoglutaric acid, 231 mM NH4Cl, 30 mM CaCl2, and 6 mM NADH in 100 mM Tris–HCl buffer (pH 8.0), and the reaction was started by adding the enzyme extract. The reaction mixture for NAD-GDH consisted of 100 mM L-glutamic acid, and 1 mM NAD in 100 mM Tris–HCl buffer (pH 8.8), plus the enzyme extract. The NADH oxidation (NADH-GDH) or NAD reduction (NAD-GDH) activity was measured spectrophotometrically at 340 nm. The enzyme activity was expressed in units of µmol g-1 protein.

Aminotransferase activities

The alanine aminotransferase (AlaAT) and aspartate aminotransferase activities were measured spectrophotometrically at 340 nm in the alanine-pyruvate/aspartate-oxaloacetate directions coupled with oxidation of NADH by lactate dehydrogenase and malate dehydrogenase, respectively (De Sousa & Sodek, 2003). For AlaAT assay, the enzyme extract was mixed with a buffer solution of 500 M L-alanine, 15 mM α-oxoglutarate, 0.15 mM NADH and 5 units of lactate dehydrogenase in 100 mM Tris–HCl buffer (pH 7.5) (De Sousa & Sodek, 2003). The reaction mixture for AspAT consisted of 200 M L-aspartate, 5 mM EDTA, 12 mM 2-oxoglutarate, 0.15 mM NADH, and five units of malate dehydrogenase in 100 mM Tris–HCl buffer (pH 7.2), to which was added the enzyme extract (De Sousa & Sodek, 2003). The aminotransferases were measured using the absorption coefficient for NADH (ε = 6.22 mM⁻¹ cm⁻¹). They were expressed in units expressed in µmol g⁻¹protein.

Protein determination

Protein concentration was measured following the method of Bradford (1976), using bovine serum albumin as the standard.

Data analysis

All measurements were repeated three times, and the data organized in Microsoft Excel 2007. The data values are presented as mean ± standard error (SE). The data were analyzed statistically by two-way analysis of variance (ANOVA) in SPSS (v.13.0;IBM, Armonk, NY, USA) and significant differences among treatment means were detected at P < 0.05 by pairwise multiple comparisons, using Duncan’s multiple range test. Sigma Plot 10.0. was used to draw the figure graphics, all of which show data points and error bars as the mean ± SE. To aid interpretation, principal component analysis (PCA) of the ionome variation analysis, photosynthetic parameters and nitrogen assimilation enzyme activities were performed for both species.

Changes in plant growth performance

The growth performances of the seedlings of the wild and cultivated soybean accessions exhibited obvious differences in response to stress treatment (Tables 1 and 2). In response to increasing concentrations of either salt treatment, the shoot and root fresh and dry weights of both soybean accessions all decreased significantly. The two stresses significantly reduced shoot and root length in both the wild and cultivated accessions (Tables 1 and 2; P < 0.05). Growth reduction was greater in the cultivated soybean accession than in the wild soybean accession, and the negative impact of alkaline stress was more obvious than that of saline stress, i.e 12.67% decrease (in 30 mmol L⁻¹ saline stress) and 18.17% decrease (in 30 mmol L⁻¹ alkaline stress) in the wild accession, compared with 34.96% (in 30 mmol L⁻¹ saline stress) and 34.96% (in 30 mmol L⁻¹ alkaline stress) in the cultivated soybean accession. Root length in the wild soybean accession increased significantly by 1.99% under 10 and 20 mmol L⁻¹ saline stress, relative to the unstressed control (Tables 1 and 2).

Analysis of the ionome

PCA showed different responses to saline and alkaline salt stress with respect to the accumulation of ions by roots of the two soybean accessions (Figs. 1A–1D). The Na⁺ concentration in both soybean accessions showed trends of significant increases in response to increases in the concentrations of either stress. Wild soybean roots accumulated significantly (P < 0.05) more Na⁺ than did cultivated soybean roots, i.e., 238.77% relative to CK versus 196.90%, respectively, for 30 mmol L⁻¹ alkaline stress and 144.00% versus 111.72%, respectively, for 30 mmol L⁻¹ saline stress (Table 3).

For both stresses, Ca²⁺, K⁺, Mg²⁺ and P³⁺ concentrations in the roots of both species showed trends of significant decreases in response to increasing stress, in comparison with the control (Table 3, P < 0.05). A more pronounced decrease in Ca²⁺, K⁺, Mg²⁺ and P³⁺ concentrations was observed in plants subjected to alkali stress (38.20, 29.96, 26.20 and 46.48%, respectively, in wild soybean and 55.53, 51.17, 42.54 and 51.50%, respectively, in cultivated soybean under 30 mmol L⁻¹ alkaline stress), than in plants subjected to saline stress (20.29, 15.45, 15.66 and 21.03%, respectively, in wild and 38.74, 35.95, 30.20 and 41.77%, respectively, in cultivated soybean under 30 mmol L⁻¹ saline stress). Both salt stresses significantly increased B³⁺ concentration in the roots of the two accessions (Table 4, P < 0.05). The 10 and 20 mmol L⁻¹ concentrations of both stresses exhibited similar increases in B⁺ concentration in both soybean accessions. A more pronounced increase was observed in plants subjected to 30 mmol L⁻¹ alkaline stress (81.55% in wild soybean and 63.01% in cultivated soybean) than in plants subjected to 30 mmol L⁻¹ saline stress (58.68% in wild soybean and 52.24% in cultivated soybean).

Low saline and alkaline stress concentrations (10 mmol L⁻¹) significantly increased Fe³⁺ concentrations in roots of both accessions, whereas medium (20 mmol L⁻¹) and high (30 mmol L⁻¹) saline or alkaline concentrations significantly increased Fe³⁺ concentration in wild soybean roots but significantly reduced them in cultivated soybean roots (Table 4, P < 0.05). The Zn²⁺ and Mn²⁺ concentrations in the roots of both soybean accessions showed significant decreasing trends in response to increasing concentrations of either salt treatment (Table 4). The root Zn²⁺ and Mn²⁺ concentrations of both soybean accessions showed significant decreasing trends under both salt treatments (Table 4, P < 0.05). The concentrations of Zn²⁺ and Mn²⁺ decreased more in plants subjected to 30 mmol L⁻¹ alkaline stress (28.74% and 25.86% in wild soybean, respectively, and 40.86% and 50.45% in cultivated soybean, respectively) than in plants subjected to 30 mmol L⁻¹ saline stress (29.51% and 20.98% in wild soybean, respectively, and 36.38% and 32.71% in cultivated soybean, respectively). With increasing concentrations of both stresses, the root Cu²⁺ concentration increased in both soybean accessions, significantly so in the case of wild soybean, where the increase for alkali stress was significantly greater than that for saline stress (Table 4, P < 0.05).

With respect to changes in anion concentration in response to salt stress, the Cl⁻ concentration increased significantly in salt-treated roots of both soybean accessions, with the increase in wild soybean roots being clearly greater (P < 0.05) than in cultivated soybean roots under the same level of saline stress (Table 4, P < 0.05). Similarly, the NO₃⁻ concentration in roots of both soybean accessions and the SO₄²⁻ concentration in cultivated soybean roots showed significant decreasing trends with increasing concentrations of the two stresses, with a more pronounced impact being observed under alkaline than under saline stress (Table 4, P < 0.05). Moreover, H₂PO₄⁻ concentration decreased in response to increasing stress in roots of cultivated soybean grown under both stresses and in wild soybean under alkaline stress. The SO₄²⁻ and H₂PO₄⁻ concentrations of wild soybean roots showed significant increasing trends under saline stress. Moreover, in wild soybean, the increase in SO₄²⁻ concentration (5.95, 10.62 and 30.31%) was greater than that of H₂PO₄⁻ (0.78, 6.81 and 13.03%, relative to that of the control) under 10, 20 and 30 mmol L⁻¹ saline stress, respectively. When subjected to alkaline stress, SO₄²⁻ concentration in wild soybean increased at low alkaline stress (10 mmol L⁻¹) by 1.47% whereas it decreased by 0.47% and 10.25% at medium and high alkaline stress (10 and 20 mmol L⁻¹), respectively (Table 4).

Additionally, the C₂O₄²⁻ concentration accumulated significantly in roots of both soybean accessions exposed to salt stress, with a greater increase in plants subjected to alkaline stress than to saline stress. The wild soybean accession accumulated significantly more C₂O₄²⁻ than did the cultivated soybean accession under all concentrations of both stresses,with a more pronounced increase in plants subjected to 30 mmol L⁻¹ alkaline stress (80.85% in wild soybean and 36.23% in cultivated soybean) than in plants subjected to 30 mmol L⁻¹ saline stress (9.66% in wild soybean and 5.26% in cultivated soybean) (Table 5) (Dataset S1 and Dataset S2).

Changes in photosynthesis parameters

PCA showed an obvious distinction between the photosynthetic capacities of the two soybean species in response to increasing salt and alkali concentrations (Figs. 2A–2D). Concentrations of photosynthetic pigments (Chla, Chlb, and Chl t) of both accessions showed significantly decreasing trends in response to increasing salt stress (Table 6, P < 0.05). The decrease was significantly higher in cultivated soybean than wild soybean, particularly in plants subjected to alkaline stress. Moreover, significant decreasing trends were also observed in the Car concentrations of cultivated and wild soybean under both salt and alkaline stress, respectively (Table 4, P < 0.05).The Car concentration in wild soybean did not increase significantly under saline stress. Similarly, photosynthetic gaseous exchange parameters of the two soybean accessions responded differently to the two salt stresses. The P_n, G_s, C_i/ C_a and E in cultivated soybean showed significant decreasing trends in response to increasing salt stress (Table 7, P < 0.05), while water-use efficiency (WUE) did not decrease significantly under both stresses, regardless of the level of stress (Table 7). The wild soybean accession showed significantly higher P_n and G_s at 10, 20 and 30 mmol L⁻¹saline stress, higher WUE at 10, 20 and 30 mmol L ⁻¹saline stress and higher E at 10 mmol L ⁻¹saline stress, relative to that of the control (Table 7, P < 0.05) (Dataset S1 and Dataset S2).

Changes in nitrogen assimilation enzyme activities

PCA and Loading plot results (Figs. 3A–3D) showed different responses of nitrogen assimilation enzyme activities exhibited by roots of the two soybean species exposed to saline/alkaline stresses. In comparison with the control, nitrate reductase (NR) activity in leaves of the cultivated soybean accession decreased significantly in response to stress by 14.02, 32.14 and 44.02% in plants subjected to 10, 20 and 30 mmol L⁻¹ alkaline stress, respectively) and by 8.62, 19.61 and 32.15% in plants subjected to 10, 20 and 30 mmol L⁻¹ saline stress, respectively (Fig. 4, P < 0.05). In the wild accession NR activity increased by 6.02% at 10 mmol L-¹saline stress but decreased at 20 and 30 mmol L⁻¹ saline stress by 0.62% and 8.40%. The NR activity in the wild soybean also decreased in response to 10, 20 and 30 mmol L⁻¹ alkaline stress, by 3.74%, 12.51% and 21.06%, respectively, compared with the unstressed control (Fig. 4).

In response to increasing concentrations of either salt treatment, glutamine synthetase/glutamine synthase (GS/GOGAT ) activities decreased significantly in leaves of both accessions (Figs. 5A, 5B; P < 0.05). The decrease was clearly greater in the cultivated soybean than in the wild soybean. The decrease in GS and GOGAT activities was more pronounced in plants subjected to 30 mmol L⁻¹ alkali stress (33.96% and 41.18%, respectively, in the wild soybean and 48.84% and 60.87%, respectively, in the cultivated soybean) than in the corresponding plants subjected to saline stress (16.22% and 29.41%, respectively, in the wild accession and 40.70% and 43.48%, respectively, in the cultivated accession) (Figs. 5A, 5B). In response to increases in salt concentrations of both stresses, the activities of glutamate dehydrogenase (GDH), alanine aminotransferase (AlaAT) and aspartate aminotransferase (AspAT) all increased significantly in leaves of both soybean accessions, with the increase in wild soybean leaves being higher than that in cultivated soybean (Figs. 6A, 6B, Figs. 7A, 7B; P < 0.05).The increases in glutamate dehydrogenase (NAD-GDH, NADH-GDH), AlaAT and AspAT activities were more pronounced in plants subjected to 30 mmol L⁻¹ saline stress (51.70, 48.15, 40.12 and 65.36%, respectively), in wild soybean relative to the control plants, than in plants subjected to alkaline stress (23.56, 29.63, 23.30 and 41.83%, respectively). Similarly, the increases in NAD-GDH, NADH-GDH, AlaAT and AspAT activities in cultivated soybean were more pronounced in plants subjected to 30 mmol L⁻¹ saline stress (21.14, 20.00, 16.98 and 29.55%, respectively), relative to the control plants) than in alkaline stress (11.66, 20.00, 17.11 and 18.75%, respectively) (Figs. 6A, 6B, 7A, 7B; P < 0.05) (Dataset S1 and Dataset S2).