Nitric oxide alleviates salt stress in seed germination and early seedling growth of pakchoi (Brassica chinensis L.) by enhancing physiological and biochemical parameters

Ecotoxicology and Environmental Safety 187 (2020) 109785

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Nitric oxide alleviates salt stress in seed germination and early seedling growth of pakchoi (Brassica chinensis L.) by enhancing physiological and biochemical parameters

T

Yanfang Rena,b, Wei Wanga, Junyu Hea,b,∗, Luyun Zhanga, Yuanjuan Weib, Min Yangb a b

School of Environmental and Safety Engineering, Changzhou University, Changzhou, Jiangsu, 213164, China College of Agriculture, Guizhou University, Guiyang, 550025, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Nitric oxide Pakchoi Salt stress Antioxidant defense Osmolyte

The germination and seedling vigor of crops is negatively affected by soil salinity. Nitric oxide (NO) has emerged as a key molecule involved in many physiological events in plants. The objective of present study was to evaluate the impact of exogenous sodium nitroprusside (SNP, a NO donor) at different concentrations on the seed germination and early seedling growth characteristics of pakchoi (Brassica chinensis L.) under NaCl stress. 100 mM NaCl stress markedly inhibited the seed germination potential, germination index, vitality index and growth of radicles and plumules. SNP pretreatment attenuated the salt stress effects in a dose-dependent manner, as indicated by enhancing the characteristics of seed germination and early seedling growth parameters, and the mitigating effect was most pronounced at 10 μM SNP. Efficient antioxidant systems were activated by SNP pretreatment, and which effectively increased the activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX), and reduced contents of malondialdehyde (MDA) and hydrogen peroxide (H2O2) and the production rate of superoxide anion radical (O2·-) in radicles and plumules, thereby preventing oxidative damage from NaCl stress. SNP pre-treatment also increased the contents of proline and soluble sugar in radicles and plumules under NaCl stress. In addition, SNP pre-treatment significantly increased the K+ contents and decreased Na+ contents in radicles and plumules, resulting in the increased level of K+/Na+ ratio. Our results demonstrated that SNP application on pakchoi seeds may be a good option to improve seed germination and seedling growth under NaCl stress by modulating the physiological responses resulting in better seed germination and seedling growth.

1. Introduction Soil salinity is one of the most severe environmental stresses effecting croplands worldwide (Farhangi-Abriz and Ghassemi-Golezani, 2018; Hasanuzzaman et al., 2018). It has been estimated that approximately 20% of agricultural lands and nearly 50% of irrigated lands are affected by high salinity in the world (Fan et al., 2013; Siddiqui et al., 2017). In addition to primary salinization caused by natural processes, the areas subjected to secondary salinization of agricultural lands are rapidly increasing in recent decades due to anthropogenic actions, such as long-term and heavy fertilization, irrigation with saline water and improper irrigation methods (Fan et al., 2017; da Silva et al., 2017). Other major reasons for secondary salinization of agricultural soil are the lack of rainfall leaching and strong surface evaporation in protected cultivation (Siddiqui et al., 2017). Many previous studies have reported on the adverse effects of ∗

salinity on the physical and chemical properties of soil and on plant growth and yield (Deinlein et al., 2014; Meena et al., 2019). Salinity caused soil quality deterioration, such as high soil impedance, low hydraulic quality and oxygen availability, high salt content, organic matter scarcity, etc (Iwai et al., 2012). Such problems affect water and air movement, plant-available water holding capacity, root penetration, runoff, erosion and tillage and sowing operations; subsequently, severe water limitation and imbalances in plant-available nutrients have a negative influence on plant growth, development and yield (Deinlein et al., 2014; Farhangi-Abriz and Ghassemi-Golezani, 2018). In addition, the accumulation of salt ions in plants could cause osmotic stress, ionic toxicity, nutritional deficiency, inactivation of photosynthetic electron transport, suppression of enzymatic activity, and oxidative damage (Matthees et al., 2018; Paul et al., 2017), and therefore leads to a series of metabolic disorders and abnormal cellular functions and reduced productivity (Ahanger and Agarwal, 2017; Fan et al., 2017; Negrão

Corresponding author. School of Environmental and Safety Engineering, Changzhou University, Changzhou, Jiangsu, 213164, China. E-mail address: [email protected] (J. He).

https://doi.org/10.1016/j.ecoenv.2019.109785 Received 6 June 2019; Received in revised form 16 September 2019; Accepted 8 October 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.

Ecotoxicology and Environmental Safety 187 (2020) 109785

Y. Ren, et al.

NO in alleviating salt stress in pakchoi via modifying the physiological and biochemical responses. The results would provide more evidence to improve our understanding about the underlying mechanisms by which NO improved salt tolerance during seed germination. On the other hand, the expected results would also make that SNP, as a NO donor, might be used as seed coating to induce pakchoi seedling tolerance to salt stress.

et al., 2017; Hasanuzzaman et al., 2018; Gadelha et al., 2017). To ensure their own survival, plants have developed multiple strategies including accumulation of osmolytes like soluble sugars, organic acids and free amino acids, excluding and exporting out Na+ and accumulating K+ (Negrão et al., 2017; Paul et al., 2017; Farhangi-Abriz and Torabian, 2017). Seed germination is usually the most critical stage in seedling establishment, determining successful crop production (Ali et al., 2017; He et al., 2014; Kataria et al., 2017) and is a complicated process and the most critically vulnerable stage to adverse environmental conditions (Fan et al., 2013; Kataria et al., 2017). Salinity exerts adverse effects on seed germination and seedling growth through physiological and biochemical changes such as osmotic stress, ionic toxicity and oxidative damage (Ali et al., 2017; Alsaeedi et al., 2017; Fang et al., 2017; Lin et al., 2012; Yu et al., 2013). Thus, it is importance to develop suitable measures to alleviate the negative effects of salinity on seed germination and early seedling development. Seed pre-treatment with certain protective agents could stimulate germination metabolic processes and improve seed performance under diverse environmental conditions (Kataria et al., 2017; Fan et al., 2013; Paul et al., 2017). Recently, it has been found that application of plant growth regulators is an effective way for this purpose (Ali et al., 2017; Manai et al., 2014). Nitric oxide (NO), as a reactive nitrogen species, is now recognized for its well-known role in signaling in plant, and has been reported to participate in the process of plant growth and development (Siddiqui et al., 2011; Ren et al., 2017), as well as the response to various environmental stresses, including salinity (Ali et al., 2017; Fan et al., 2013), UV-B (Yan et al., 2016), water deficit (Silveira et al., 2016), heat (Song et al., 2013), and heavy metals (He et al., 2014), etc. Earlier studies have suggested that exogenous NO application improved salt tolerance and mitigated the adverse effects of salt stress in diverse plant species by altering their cellular metabolism and invoking various defense mechanisms (Ali et al., 2017; Fan et al., 2013). For example, pretreatment with sodium nitroprusside (SNP, a NO donor) ameliorated toxic effects of salt on cotton, tomato and wheat by improving the antioxidant system (Liu et al., 2014; Manai et al., 2014; Ali et al., 2017). NO enhanced salt tolerance in cucumber seedlings by regulating free polyamine content (Fan et al., 2013). Application of NO facilitated the maintenance of ion homeostasis and promoted the accumulation of osmolytes for plants under salt stress (Tian et al., 2015). These evidences provide an implication that developing NO-associated technology may be feasible in improving crop production that suffer from salinity. However, the previous studies suggested that the impact of NO on plant growth and development depends on the concentration of salt, the sensitivity of crops and the genetic makeup of plants for resisting and overcoming the adverse effects of salt stress (Siddiqui et al., 2017). Pakchoi (Brassica chinensis L.) is one of the most popular and important leafy vegetables because of its high nutritional value, rapid growth, and low production cost across china (Liang et al., 2016), but its cultivation faces risk from salinity (Xu et al., 2017). When pakchoi seeds were sown directly in the field, salinity can often not only delay seed germination and seedling emergence, but also cause nonuniform pakchoi seedling emergence. The problem is aggregated because secondary salinization of agricultural lands becomes greater (Fan et al., 2017; da Silva et al., 2017). Therefore, early and uniform germination, seedling emergence and establishment are critical stage for the commercial cultivation of pakchoi. Based on reported protective role of NO against salt-induced damages in plants, it was hypothesized that NO application might increase the tolerance of pakchoi against salinity by modulating multiple stress-responsive pathways. However, little information is available regarding the alleviation of adverse effects of salt stress in pakchoi through seed pre-treatment with exogenous NO. Therefore, the objectives of the present study were to explore (1) whether SNP pretreatment ameliorates salt toxicity in pakchoi seed germination and early seedling growth, (2) the optimal concentration of SNP to alleviate salt toxicity, and (3) the regulatory mechanisms of

2. Materials and methods 2.1. Plant materials and culture condition Seeds of pakchoi (Brassica chinensis L. cv Tiancuiqing) were purchased from Guizhou Guifeng Seed Industry Co. Ltd, China. All chemical reagents used in the work were of analytical grade. Sodium nitroprusside (SNP) was purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd, China. Pakchoi seeds were selected for uniformity by choosing those homogeneous and identical in size and colour, and were surface-sterilized for 10 min in 1% (v/v) sodium hypochlorite solution, and then rinsed thoroughly with deionized water. Subsequently, the seeds were soaked in varying levels (5, 10, 25, 50, 100, and 200 μM) of SNP solution or distilled water for 2 h in dark at 25 ± 1 °C in plastic cups. After pre-soaking, all seeds were rinsed with distilled water and evenly dispersed in petri dishes containing two sheets of Whatman No. 1 filter paper moistened with 7 mL 100 mM NaCl solution (the stress level was chosen on the basis of about 50% of the inhibitory of radicle length by NaCl in the preliminary experiment), respectively. Controls (CK) were obtained by moistening the filter papers with 7 mL deionized water. Each dish contained 50 seeds. There were 8 treatments in the experiment: Control (CK), 100 mM NaCl treatment (NaCl), 100 mM NaCl + 5 μM SNP pre-treatment (SNP5), 100 mM NaCl + 10 μM SNP pre-treatment (SNP10), 100 mM NaCl + 25 μM SNP pre-treatment (SNP25), 100 mM NaCl + 50 μM SNP pre-treatment (SNP50), 100 mM NaCl + 100 μM SNP pre-treatment (SNP100) and 100 mM NaCl + 200 μM SNP pre-treatment (SNP200). Each treatment included at least three replicates. The seeds were incubated in dark at 25 ± 1 °C with 70–75% relative humidity for 7 d in a growth chamber. The treatment solutions were renewed each day to maintain unaltered concentrations. During 7 days of the germination, germinated seeds were counted every day. After 7 days of incubation, the germination characteristics (germination percentage, germination potential, germination index, and vitality index), the length and fresh weight of radicle and plumule were determined. In addition, another batch of seeds were sowed for each treatment and each treatment had three replicates with 8 petri dishes per replicate. Samples of radicles and plumules were collected after 7 days of incubation and immediately frozen in liquid nitrogen and stored at −80 °C for biochemical analysis. 2.2. Determination of germination parameters Seeds were considered to have germinated when radicle had emerged and elongated by at least 2 mm. The number of seeds germinated was recorded daily. Germination characteristics were determined:

Germination percentage (%) = number of germinated seeds in 7th day/total seed number for testing×100 Germination potential (%) = number of germinated seeds in 3rd day/total seed number for testing×100

Germination index  = 2

∑ (Gt/Dt)

Ecotoxicology and Environmental Safety 187 (2020) 109785

Y. Ren, et al.

Vitality index =

∑ (Gt/Dt) ×S

amount of enzyme required to cause a 50% inhibition in the reduction of NBT monitored at 560 nm. SOD activity is expressed as U/g FW. Catalase (CAT) activity was determined according to He et al. (2014). The reaction mixture contained 25 mM sodium phosphate buffer (pH 7.0), 10 mM H2O2 and 100 μL of enzyme extract. The decrease in absorbance was measured at 240 nm. Enzyme activity is expressed as U/g FW. Peroxidase (POD) activity was determined according to He et al. (2014). The reaction solution contained 3 mL of 25 mM (pH 7.0) sodium phosphate buffer, 0.1 mM EDTA, 0.05% guaiacol, 1.0 mM H2O2, and 100 μL enzyme extract. The increase in absorbance due to oxidation of guaiacol was measured at 470 nm. Enzyme activity is expressed as U/ g FW. Ascorbate peroxidase (APX) activity was determined according to He et al. (2014). 3 mL reaction mixture consisted of 25 mM (pH 7.0) sodium phosphate buffer, 0.1 mM EDTA, 0.25 mM ascorbate, 1.0 mM H2O2 and 100 μL enzymes extract. H2O2-dependent oxidation of ascorbate was followed by a decrease in the absorbance at 290 nm. Enzyme activity is expressed as U/g FW.

where Gt is the germination number at day t, Dt is day t, and S is the average radicle length of the seedlings. 2.3. Growth parameters After 7 days of incubation, lengths of radicles and plumules were measured from 20 plants of each petri dish. The radicle and plumule samples were blotted the surface water with a paper towel and then determined fresh weight (FW). The average length and fresh weight of radicles and plumules for 20 plants was a replicate. 2.4. Determination of lipid peroxidation Membrane lipid peroxidation was determined in terms of malondialdehyde (MDA) content using the thiobarbituric acid (TBA) method (He et al., 2014). 0.5 g sample was homogenized with a precooled mortar and pestle in 2.5 mL 5% trichloroacetic acid (TCA) and centrifuged at 15,000×g for 20 min at 4 °C. Then 2.0 mL of the supernatant was added to 4.0 mL of thiobarbituric acid (TBA, 0.67%) with 20% TCA, heated at 100 °C for 30 min, and then quickly cooled to room temperature. Absorbance of the supernatant was measured spectrophotometrically (Shimadzu Corp., Kyoto, Japan) at 450 nm, 532 nm, and 600 nm after centrifugation at 10,000×g for 10 min. MDA contents are expressed as μmol/g FW.

2.7. Determination of osmolytes Proline content was determined according to the method described by Zhang et al. (2018). Approximately 0.2 g sample was homogenized with the addition of 5 mL 3% sulphosalicylic acid solution. The homogenate was centrifuged at 3000×g for 20 min. The supernatant (2 mL) was combined with 2 mL of glacial acetic acid, 2 mL of acid ninhydrin, and 2 mL of water, boiled at 100 °C for 1 h, and then allowed to cool at room temperature. The mixture was then extracted with 4 mL of toluene and thoroughly mixed. The free toluene was quantified spectrophotometrically at 520 nm using L-proline as a standard. Proline content was expressed as μg/g FW. Soluble sugar content was measured using anthrone-sulfuric acid method and its absorbance was measured at 630 nm (Zhang et al., 2018). Samples were extracted with 4 mL 80% methanol at 80 °C for 40 min and were then centrifuged at 2000×g for 15 min. The methanol supernatants of three successive centrifugations were used for the sugar analyses. About 4 mL of anthrone reagent was then added. The mixture was heated in a boiling water bath for 10 min, and then cooled. Absorbance was measured spectrophotometrically at 620 nm. Standard curve was prepared using D-glucose and amount was expressed as mg/g FW.

2.5. Estimation of reactive oxygen species (ROS) generation Superoxide anion radical (O2·-) production rate was determined following Zhang et al. (2018) with some modifications. 0.2 g sample was homogenized in an ice bath with 2 mL 50 mM phosphate buffer (pH 7.8), and centrifuged at 15,000×g for 10 min at 4 °C. An aliquot of the supernatant was added to incubation buffer consisting of 50 mM potassium phosphate buffer (pH 7.8) and 1 mM hydroxylamine hydrochloride, incubating the mixture at 25 °C for 20 min. Then it was added 17 mM aminobenzene sulphonic acid, and 7 mM α-naphthylamine, and incubated again at 25 °C for 20 min. Absorbance was measured spectrophotometrically at 530 nm, and NaNO2 was used to prepare a standard curve. Hydrogen peroxide (H2O2) level was assayed according to the method described by Zhang et al. (2018). H2O2 was extracted by homogenizing 0.2 g sample with 3 mL of phosphate buffer (50 mM, pH 6.5) at 4 °C. The homogenate was centrifuged at 6000×g for 25 min. A supernatant of 3 mL was mixed with 1 mL of 0.1% TiCl4 in 20% H2SO4 (v/v), and the mixture was then centrifuged at 6000×g for 15 min at room temperature. The peroxide-titanium complex was dissolved in 3 mL 1 M H2SO4. The absorbance was measured spectrophotometrically at 410 nm. The H2O2 content was calculated from a standard curve prepared in a similar way.

2.8. Ion analysis After harvesting, seedlings were divided into radicles and plumules. The samples of radicle and plumule were washed thoroughly with 0.1 M HCl to ensure that any adhered materials are totally removed, rinsed in deionized water, and then dried at 70 °C for 48 h. The dry samples were ground by a stainless steel grinder and passed through a 60-mesh sieve. For determination of Na+ and K+, 0.1 g of the samples was digested with solution containing (3:1) H2SO4–H2O2 (He et al., 2014). The concentration of Na+ and K+ were determined by flame photometer (Shanghai Precision and Scientific Instrument Co., Ltd., 6400 A type) with standard sample (Tianjin Yongda Reagent Development Center, Tianjin, China).

2.6. Assay of antioxidant enzymes For enzymatic assay, 0.3 g sample was homogenized with a precooled mortar and pestle in 3 mL 50 mM potassium phosphate buffer (pH 7.8) containing 1% (w/v) polyvinylpolypyrrolidone (PVP), 0.1 mM ethylenediaminetetraacetic acid (EDTA) and 0.3% (w/v) Triton X100. The homogenate samples were centrifuged at 15,000×g for 20 min at 4 °C and the supernatant was collected and used for the enzyme assays. Superoxide dismutase (SOD) activity was assayed by monitoring the inhibition of the photochemical reduction of nitro-blue tetrazolium (NBT) according to the method of He et al. (2014). Each 3 mL reaction mixture contained 50 mM potassium phosphate buffer (pH 7.8), 13 mM methionine, 75 μM NBT, 20 μM riboflavin, 0.1 mM EDTA, and 100 μL enzyme extract. The reaction mixtures were illuminated for 20 min at a light intensity of 4000 lx. One unit (U) of SOD activity is defined as the

2.9. Statistical analysis All experiments were carried out at least three independent replicates. All of the data were expressed as mean ± standard error (n = 3). All data were performed by one-way analysis of variance (ANOVA) and the differences among the means were compared by Duncan's multiple range test with a significance of P < 0.05 using SPSS 16.0 statistical program (SPSS Inc., Chicago, IL). To further evaluate the responses of seed germination and seedling 3

Ecotoxicology and Environmental Safety 187 (2020) 109785

Y. Ren, et al.

Table 1 Effects of different concentrations of SNP on seed germination parameters and seedling growth of pakchoi under 100 mM NaCl stress. Treatment

Germination percentage (%)

Germination potential (%)

Germination index

Vitality index

Radicle length (cm)

Plumule length (cm)

Radicle fresh weight (mg)

Plumule fresh weight (mg)

CK NaCl SNP5 SNP10 SNP25 SNP50 SNP100 SNP200

91.33 86.67 90.67 92.67 91.33 90.00 89.33 88.00

88.00 80.67 85.33 87.33 86.00 85.33 84.00 82.67

39.40 34.08 37.22 38.82 37.91 36.17 35.26 34.57

178.00 ± 12.55a 81.40 ± 9.66e 113.89 ± 10.71c 147.90 ± 12.28b 124.72 ± 10.98bc 101.64 ± 10.97cd 92.03 ± 8.76d 79.51 ± 7.88e

4.52 2.39 3.06 3.81 3.29 2.81 2.61 2.30

2.94 2.22 2.51 2.76 2.64 2.45 2.38 2.25

20.30 10.50 14.30 16.50 15.00 13.13 12.10 10.30

58.3 ± 3.32a 42.40 ± 3.06d 49.90 ± 2.91c 54.30 ± 3.12 ab 51.67 ± 2.38b 48.30 ± 2.83c 46.50 ± 3.17c 44.25 ± 2.61cd

± ± ± ± ± ± ± ±

3.06a 3.06a 3.05a 5.03a 4.16a 3.46a 3.05a 3.46a

± ± ± ± ± ± ± ±

2.00a 2.66c 3.06b 3.06 ab 2.00 ab 2.31b 2.00b 3.06bc

± ± ± ± ± ± ± ±

2.10a 1.68c 1.93b 2.09a 1.90 ab 1.21b 1.38bc 1.32c

± ± ± ± ± ± ± ±

0.43a 0.31e 0.31bc 0.33b 0.27bc 0.23c 0.21d 0.22e

± ± ± ± ± ± ± ±

0.17a 0.15d 0.15b 0.18 ab 0.16b 0.17c 0.14cd 0.14d

± ± ± ± ± ± ± ±

1.82a 1.27e 1.70bc 1.66b 1.32b 1.43c 1.51d 0.63e

Values are the mean ± SD of three replicates, and different letters following them in the same column indicate significant difference among treatments at P < 0.05 according to Duncan's multiple range test.

growth to the NaCl stress and SNP pre-soaking, principal component analysis (PCA) was performed using SPSS Statistics 16.0. The PCA allowed the ordination of the parameters to discover potential groupings within the parameters. Plots were generated using principal components (PC) 1 and 2 as axes. 3. Results 3.1. Seed germination parameters As shown in Table 1, NaCl stress did not significantly affect pakchoi seed germination percentage (P > 0.05). The germination potential, germination index, and vitality index were significantly decreased by 100 mM NaCl stress and it caused 8.33%, 13.50%, and 54.27% reduction in germination potential, germination index, and vitality index of pakchoi seeds as compared to the control, respectively. However, presoaking seed with 5–100 μM SNP showed higher germination potential, germination index, and vitality index under NaCl stress, and 10 μM SNP was the most effective in this experiment among all the treatments (P < 0.05)(Table 1). Germination potential, germination index, and vitality index of pakchoi under 100 mM NaCl stress were increased by 7.67%, 14.20% and 74.51% after 10 μM SNP pre-treatment as compared to NaCl treatment alone, respectively (Table 1). However, 200 μM SNP had no obvious effect on the germination potential, germination index, and vitality index of pakchoi under NaCl stress.

Fig. 1. Effect of exogenous SNP at different concentrations on MDA content in radicels and plumules of pakchoi under 100 mM NaCl stress.

by 36.45% and 22.49% compared to the NaCl treatment alone, respectively. This means that exogenous SNP at appropriate concentration significantly declined the lipid peroxidation of pakchoi seedling under NaCl stress. However, pre-treatment with 200 μM SNP resulted in no obvious changes in MDA content, compared to the NaCl treatment alone.

3.2. Seedling growth parameters

3.4. ROS generation

As shown in Table 1, NaCl stress strongly inhibited the growth of radicles and plumules of pakchoi (P < 0.05), and the length and fresh weight were decreased by 47.12% and 48.28% in radicles, 24.49% and 27.27% in plumules, as compared to the control, respectively. Seed pretreatment with 5–100 μM SNP significantly mitigated the inhibition caused by NaCl (P < 0.05), especially at 10 μM SNP, which increased radicle and plumule length by 59.41% and 24.32% (Table 1), radicle and plumule fresh weight by 57.14% and 28.07% (Table 1), respectively, compared to the NaCl treatment alone. However, pre-treatment with 200 μM SNP did not have any significant effects on the length and weight of radicles and plumules compared with the NaCl treatment alone.

The effects of SNP pre-treatment on O2.- production rate and H2O2 content in radicles and plumules of pakchoi are indicated in Fig. 2. NaCl treatment resulted in a significant increase of O2.- production rate and H2O2 content by 152.02% and 125.45% in radicels and 70.82% and 46.09% in plumules, respectively, when compared to control (Fig. 2A and B). Pre-treatment with different concentration (5–50 μM) of SNP significantly reduced O2.- production rate and H2O2 content in radicles and plumules of pakchoi under NaCl stress (P < 0.05). In all treatments, 10 μM SNP treatment showed the most maximum decrease in O2.- production rate and H2O2 content which were only 48.89% and 42.74%, 33.73% and 23.74% of the salt stress treatment in radicles and plumules, respectively (Fig. 2A and B). However, there was no any effect of application of 200 μM SNP on the inhibition of O2.- production rate and H2O2 content in radicles and plumules of pakchoi seedling.

3.3. MDA content Malondialdehyde (MDA), a cytotoxic product of membrane lipid peroxidation, has been considered as an indicator of oxidative damage. The NaCl treatment dramatically increased the MDA content in radicles and plumules of pakchoi by 93.39% and 47.58%, respectively, compared with the control (Fig. 1). Pre-treatment with 5, 10, 25, and 50 μM SNP effectively reversed these effects (P < 0.05) (Fig. 1), especially at 10 μM SNP, which decreased the MDA content of radicles and plumules

3.5. Antioxidant enzymes activities The changes in SOD, POD, CAT and APX activities in radicles and plumules of pakchoi under NaCl stress are shown in Fig. 3. Compared to the control, SOD and POD activities were dramatically increased by 4

Ecotoxicology and Environmental Safety 187 (2020) 109785

Y. Ren, et al.

Fig. 2. Effects of exogenous SNP at different concentrations on O2.- production rate and H2O2 content in radicles and plumules of pakchoi under 100 mM NaCl stress.

plumules of pakchoi compared to the NaCl treatment alone (P < 0.05) (Fig. 3 C and D). The maximum increase in activities of CAT and APX under NaCl conditions was observed in 10 μM SNP pre-treatment. 200 μM SNP pre-treatment showed no significant effects on these enzymes activities in pakchoi radicles and plumules under NaCl stress.

37.16% and 70.68% in radicles (Fig. 3A), 28.56% and 60.23% in plumules (Fig. 3B), in the presence of NaCl alone, respectively, while exogenous application of different concentration of SNP as pre-soaking seed treatment further improved SOD activities of radicles and plumules under NaCl stress in a dose proportional manner. The maximal increase in SOD activity was found in plants raised from seed pretreated with 10 μM level of SNP. There was no significant effect of application of 200 μM SNP on SOD activity under NaCl conditions (Fig. 3A). Exogenous SNP treatment could lower POD activity under NaCl stress and the effect was more pronounced in seeds treated with 10 μM SNP (P < 0.01) (Fig. 3B). NaCl treatment caused a significant decrease in CAT and APX activities in radicles and plumules of pakchoi (Fig. 3 C and D). The activities of CAT and APX were decreased by 45.35% and 29.29% in radicles, 31.68% and 25.75% in plumules under NaCl treatment alone, respectively, compared with the control. However, 5–100 μM SNP pretreatment significantly enhanced CAT and APX activities in radicels and

3.6. Osmolytes content Proline contents in radicles and plumules of pakchoi under NaCl stress significantly increased compared with the control (P < 0.05) (Fig. 4A). Proline contents further increased in various degree in radicles and plumules under SNP pre-treatment at lower concentrations, and the most significant effects were also observed in 10 μM SNP pretreatment, in which proline contents were increased by 27.89% in radicles and 24.36% in plumules compared to the NaCl treatment alone. However, 200 μM SNP pre-treatment had no significant effect on proline contents in radicles and plumules when compared to the NaCl

Fig. 3. Effects of exogenous SNP at different concentrations on SOD, POD, CAT and APX activities in radicles and plumules of pakchoi under 100 mM NaCl stress. 5

Ecotoxicology and Environmental Safety 187 (2020) 109785

Y. Ren, et al.

Fig. 4. Effects of exogenous SNP at different concentrations on the proline and soluble sugar contents of pakchoi radicles and plumules treated with 100 mM NaCl stress.

increase in Na+ content (Fig. 5A) and a 14.58% and 15.19% decrease in K+ content (Fig. 5B) in radicles and plumules of pakchoi compared with those of the control, respectively. The application of 5–100 μM SNP under NaCl stress decreased Na+ content by 3.42%–21.83% in radicles and 4.78%–24.65% in plumules (Fig. 5A), but increased K+ content by 2.04%–12.06% in radicles and 3.53%–13.26% in plumules (Fig. 5B) compared to NaCl stress alone. Moreover, the changes of Na+ and K+ contents disturbed ion homeostasis in pakchoi by altering the K+/Na+ ratio. As compared with the control, K+/Na+ ratio in radicles and plumules decreased by 92.88% and 87.52% (Fig. 5C), respectively, under NaCl stress alone. However, it significantly increased in both radicles and plumules of pakchoi pre-treated with 5–100 μM SNP under NaCl stress. Among the concentrations tested, 10 μM SNP pre-treatment

treatment alone. Soluble sugar contents in radicles and plumules of pakchoi increased under NaCl stress (Fig. 4B). SNP pre-treatment improved soluble sugar contents in radicles and plumules of pakchoi under NaCl stress and the response of soluble sugar to SNP differed depending on the concentration of SNP. The maximum increase in soluble sugar contents in radicles and plumules of pakchoi was observed in 10 μM SNP, viz. 1.23 times and 1.29 times respectively, compared with NaCl treatment alone (Fig. 4B). 3.7. Na+ and K+ contents and the ratio of K+/Na+ As shown in Fig. 5, NaCl stress resulted in a 12.03-fold and 6.75-fold

Fig. 5. Effects of exogenous SNP at different concentrations on the Na+, K+ content and K+/Na+ ratio of pakchoi radicles and plumules treated with 100 mM NaCl stress. 6

Ecotoxicology and Environmental Safety 187 (2020) 109785

Y. Ren, et al.

indicate that NO plays important roles in plant tolerance to environmental stress including salt stress (Lin et al., 2012; Fan et al., 2013; Gadelha et al., 2017), drought stress (Silveira et al., 2016), heavy metal toxicity (He et al., 2014), etc. Our study showed that SNP (as an exogenous NO donor) pre-treatment at a lower concentration increased NaCl tolerance during pakchoi seed germination and seedling establishing (Table 1). Among which, 10 μM SNP showed the strongest effect to alleviate NaCl stress, which significantly promoted seed germination and the growth of radicle and plumule under NaCl stress (Table 1). Likewise, SNP was shown to improve the seed germination and early seedling growth in various plants affected by salt stress (Yadu et al., 2017; Gadelha et al., 2017; Fan et al., 2013; Lin et al., 2012). Although lower concentrations of NO can exert a protective effect by increasing plant tolerance to stress conditions, high concentrations of NO may be toxic to plants due to its high reactivity (Reda et al., 2018). In this study, higher concentration of SNP-pretreated seeds (200 μM) showed no significant difference in seed germination and early seedling growth parameters compared with the NaCl treatment alone (Table 1). Numerous studies have shown that salt stress may cause imbalance of the inner cellular ions and thus resulting in ion toxicity and osmotic stress in plant cells (Deinlein et al., 2014; Negrão et al., 2017). Maintaining Na+ and K+ balance is greatly important for detoxifying excessive Na+ ionic damages in saline-affected plants (Deinlein et al., 2014; Ma et al., 2016). In this study, NaCl stress caused a drastic increase in Na+ content and simultaneously decrease in K+ content in pakchoi radicles and plumules (Fig. 5). Similar to our findings, Ashraf et al. (2018) have reported that more accumulation of Na+ and lower accumulation of K+ in different parts of maize under salinity, which consequently reduced growth of root and leaves. Under the saline conditions, high level of external Na+ not only interferes with K+ uptake by the radicles, but also may disrupt the integrity of radicle membranes and may alter their selective permeability (Silva et al., 2015; Farhangi-Abriz and Torabian, 2017). In addition, previous reports showed that ROS could activate potassium permeable cation channels at the plasma membrane resulting in K+ leakage (Demidchik et al., 2014; Ahanger and Agarwal, 2017). Assaha et al. (2017) indicated that the decrease in Na+ accumulation and the maintenance of a high K+/Na+ ratio are key salt tolerance traits, because they decrease the ionic toxicity and allow re-establishment of several metabolic processes. Our results showed SNP pre-treatment significantly decreased Na+ contents and simultaneously increased K+ contents with improved K+/Na+ ratio in radicles and plumules of pakchoi under NaCl stress (Fig. 5). Similar results have already been proved in wheat (Tian et al., 2015) and Jatropha curcas (Gadelha et al., 2017) under salinity stress. The decrease in Na+ content and the increase in K+/Na+ ratio in NOtreated plants under salinity might be related to inhibition in vacuolar Na compartmentalization, or due to Na influx through the plasma membrane of radicle (Zhang et al., 2006). The increase in K+ content and K+/Na+ ratio might be related to the increase in competitive absorption sites and the decline in K+ efflux induced by Na+, further compromising the homeostasis of this ion (Assaha et al., 2017). Another factor that may be involved in increasing the K+ concent in NO-treated plants under salinity is the decrease in the H2O2 concentration; when ROS stimulates the activity of K+ permeable channels, K+ efflux is observed (Campos et al., 2019). Moreover, NO as a signal molecular, could induce the expression of H+-ATPase and H+-PPase which forces the Na+/H+ ion exchange to detoxify the cell, and the expression of AKT1-type K+ channel, thus partaking to increase salinity tolerance (Chen et al., 2013; Hasanuzzaman et al., 2018). Osmolyte adjustment was found in many plant species in response to a broad range of stress conditions (Fang et al., 2017; Negrão et al., 2017). Proline and soluble sugar are important osmotic adjustment substances in plant cells, which play a role in balancing cell metabolism and maintaining the relative stability of the intracellular environment under various stress conditions (Kanu et al., 2019). In addition, the accumulation of proline and soluble sugar in cytoplasm can also

Fig. 6. Principal component analysis (PCA) plots of seed germination, seedling growth, physiological and biochemical parameters for different treatments.

resulted in the greatest increase in K+ content and K+/Na+ ratio and decrease in Na+ content under NaCl stress. However, compared with NaCl stress alone, 200 μM SNP pre-treatment had no significant effects on contents of Na+ and K+ as well as K+/Na+ ratio in radicles and plumules under NaCl stress. 3.8. Principal components analysis Principal component analysis (PCA) was performed to further evaluate the effects of SNP pre-treatment on seed germination, seedling growth, and physiological and biochemical parameters under NaCl stress. Principal component 1 (PC1) and principal component 2 (PC2) explained 72.46% and 21.71% of the total variability, respectively (Fig. 6). PC1 tended to separate the effects of salt stress and different concentrations of SNP, and PC2 further segregated the differences of SNP. According to these results, the lower concentration of SNP had a greater alleviative effect on the NaCl stress than higher concentration of SNP. 4. Discussion Seed germination and seedlings stage of plants are most sensitive stage to adverse environmental conditions (He et al., 2014). They contribute significantly for better crop stand establishment and final crop yield. Salt stress greatly affects seed germination, leading to a reduction in germination rate and a delay in the initiation of germination and seedling establishment (Ali et al., 2017; Fan et al., 2013; Kataria et al., 2017). The germination percentage, germination potential, germination index and vitality index of seeds and radicle length are commonly used as indicators, which reveal seed germination and vigor (Li et al., 2019). The results of the present study showed that germination potential, germination index and vitality index of pakchoi seeds as well as radicle length significantly decreased under NaCl stress (Table 1), which further confirmed the negative effect of salinity on the seed germination and seedling establishment. Inhibition in seed germination and seedling growth of pakchoi under NaCl stress could be attributed to inhibition of water absorption by seeds (Alsaeedi et al., 2017), the increased oxidative stress, excessive accumulation of Na+ with a simultaneous decrease of K+ content in plants (Kataria et al., 2017; Li et al., 2019). Exogenous use of signal molecules has been shown to improve salt tolerance by activating biochemical and physiological defence systems in several plant species (Savvides et al., 2016; Gadelha et al., 2017; Farhangi-Abriz and Ghassemi-Golezani, 2018). Increasing evidences 7

Ecotoxicology and Environmental Safety 187 (2020) 109785

Y. Ren, et al.

NaCl treatment alone. However, the detailed mechanism of SNP reduced ROS generation still needs to be further explored. To protect themselves against oxidative stress, plants have evolved ROS scavenging systems (Zhou et al., 2017). It is well known that the coordinated action of antioxidant enzymes such as SOD, CAT, APX and POD, plays a significant role in scavenging ROS to protect cell membranes, which is thought to be a major mechanism of resistance to salt stress in plants (Farhangi-Abriz and Torabian, 2017; Fan et al., 2013; Ali et al., 2017). As shown in Fig. 3, NaCl treatment dramatically increased the activities of SOD and POD, while it significantly decreased the activities of CAT and APX in pakchoi radicle and plumule (Fig. 3). However, the activities of POD and SOD in Capsicum annum L. seedlings were decreased by salinity stress (Shams et al., 2019). In addition, salt stress significantly inhibited the activities of SOD, CAT, POD and APX in cucumber hypocotyls, but increased APX activity in radicles (Lin et al., 2012). These diverse results suggested that not all antioxidant enzymes followed the same response mode under salt stress, which could depend on treatment time, plant tissues, plant species (Zhu et al., 2004; Hasanuzzaman et al., 2014; Ahmadi et al., 2018). Previous studies found that the function of NO on alleviating oxidative stress was attributed to induction of various ROS-scavenging enzyme activity (Ali et al., 2017; Fan et al., 2013; Li et al., 2008; Kopyra and Gwóźdź, 2003; Siddiqui et al., 2017; Manai et al., 2014; Zhou et al., 2017). In accordance with previous researches, our results further confirmed that SNP pre-treatment with a suitable concentration significantly improved the activities of SOD, CAT and APX and decreased the activities of POD in pakchoi radicle and plumule under NaCl stress (Fig. 3). The decreased POD activities and increased SOD, APX and CAT activities matched the lower H2O2 level and O2·- production rate and reduced oxidative damage (Fig. 2). These results give strong evidence that the higher antioxidant defense capacity is one of the important mechanisms for exogenous SNP to alleviate oxidative stress and improve seed germination and growth parameters in pakchoi under salt stress. Some researches implied that exogenous NO may act as a signal molecule for activating the expression of antioxidant enzymes-related biosynthetic genes, thereby providing salt tolerance (Ahmad et al., 2016; Gadelha et al., 2017). For example, NO enhanced expression of the SOD, CAT and APX genes in salt-affected chickpea plants, contributing to decrease H2O2 and MDA levels and improve growth parameters during salt-induced stress (Ahmad et al., 2016). NO treatment also increased the APX gene expression in the leaves of maize under NaCl stress (Boldizsar et al., 2013). In addition, the increase of antioxidant enzymes could be due to role of NO in increasing K+ content and K+/Na+ ratio (Fig. 5), and this is in line with the results of Ahanger and Agarwal (2017) who reported the positive role of K+ on increasing the enzyme activity in wheat under salinity.

prevent protein denaturation and reduce ROS-induced structural damage of cells (Lalelou and Fateh, 2014). Salinity often caused the accumulation of proline and soluble sugar as evident in Glycyrrhiza uralensis (Zhang et al., 2018), Brassica napus (Ahmadi et al., 2018), cucumber (Yu et al., 2013) and bean (Farhangi-Abriz and Torabian, 2017). In this study, a significant increase in the contents of proline and soluble sugar in pakchoi radicles and plumules was also observed under NaCl stress (Fig. 4), while SNP pre-treatment further increased proline and soluble sugar levels under NaCl stress (Fig. 4). These results were in accordance with the reports in SNP-treated P. sativum (Yadu et al., 2017) and Lactuca sativa (Campos et al., 2019) under saline stress. The NO-induced improvement in proline content could be attributed to enhanced activity of proline synthesizing enzymes, together with a reduction in proline catabolism under stress conditions (Khan et al., 2015; Farhangi-Abriz and Ghassemi-Golezani, 2018; Yu et al., 2013). In addition, previous studies also showed that the enhanced proline content under salt stress is strongly related with enhanced activities of antioxidant enzymes (Hasanuzzaman et al., 2014; Ali et al., 2017). This viewpoint was further supported in the present study, since pakchoi radicles and plumules with SNP pre-treatment had higher antioxidant enzymes (SOD, CAT and APX) activities under NaCl stress than those in NaCl treatment alone (Fig. 3). Enhancing soluble sugar under salt stress may be attributed to rising invertase activity (Farhangi-Abriz and Ghassemi-Golezani, 2018; Yu et al., 2013). All these results indicated that the increased accumulation of proline and soluble sugar in pakchoi radicles and plumules incude by SNP pre-treatment with proper concentration exhibited a highly protective mechanism against salt stress, and could harmonize the balance between intracellular and extracellular osmotic pressure, which was favorable to seed germination and plant growth under salinity. Salt toxicity can cause formation and accumulation of ROS, such as H2O2 and O2·-, which directly or indirectly damages the cellular components, enzymes and biological membranes and imposes damage to the plants and even cell death (Ali et al., 2017; Farhangi-Abriz and Torabian, 2017; Yu et al., 2013; Fan et al., 2013). MDA is formed by the reaction of ROS with lipid molecules of tissues and is toxic for biomacromolecules. It is well known that MDA, H2O2, and O2·- are often used as indicators for oxidative damage in plants under various environmental conditions (Siddiqui et al., 2017; Ahmadi et al., 2018; He et al., 2014). Our results showed that NaCl stress induced oxidative stress of pakchoi as indicated by the increase in MDA, H2O2, and O2·- levels in the radicles and plumules (Figs. 1–2). These findings are in line with salinity-induced oxidative stress described in previous studies in Brassica napus (Ahmadi et al., 2018), wheat (Ali et al., 2017), cucumber (Fan et al., 2013; Yu et al., 2013), bean (Farhangi-Abriz and Torabian, 2017), tomato (Siddiqui et al., 2017; Manai et al., 2014; Zhou et al., 2017). In the present study, we observed that SNP pre-treatment with a suitable concentration significantly decreased the contents of MDA and H2O2, as well as the O2·- production rate in pakchoi radicles and plumules under salt stress (Fig. 1–2). These results suggested that SNP with a suitable concentration can partially prevent the oxidative damage of pakchoi radicles and plumules caused by NaCl stress. A similar NOinduced down-regulation of MDA and H2O2 contents and the O2·- production rate was also observed in wheat (Ali et al., 2017), tomato (Manai et al., 2014), and cucumber (Fan et al., 2013) under saline stress. Many mechanisms which may explain NO protective action against oxidative damage have been widely reported. Firstly, NO can detoxify ROS by reacting with O2·- and generating peroxynitrite ion (ONOO-), which is less toxic and thus limit cellular damage (Manai et al., 2014). Secondly, NO could function as a signaling molecule, which activates cellular antioxidant system (Ali et al., 2017; Fan et al., 2013). Thirdly, the decrease in Na+ accumulation and the maintenance of ion balance both contributed to a lower ROS generation (Campos et al., 2019). Our results also support the latter two viewpoints, since radicles and plumules of pakchoi in SNP pre-treatment had lower Na+ content (Fig. 5) and higher antioxidant activities (Fig. 3) than those in

5. Conclusion In summary, the present study clearly demonstrate that low levels of SNP pre-soaking treatment performed an advantageous effect on attenuation of inhibition of seed germination and growth of pakchoi radicles and plumules under NaCl stress. The mechanism might be attributed to the maintenance of ion homeostasis, improvement of osmolytes accumulation, and the induced better antioxidant system for the effective removal of ROS in plants. However, the alleviating effect exhibited a significant concentration-dependent pattern and among the SNP concentration tested, 10 μM SNP was found to be the most effective concentration inducing the highest level of tolerance to salinity. These results suggested that pre-soaking seed with SNP could be a potential method to withstand the detrimental effects of salt stress. Acknowledgments This work was funded by the National Natural Science Foundation of China (31460100, 31660477, 31360413 and 41261095) and 8

Ecotoxicology and Environmental Safety 187 (2020) 109785

Y. Ren, et al.

Scientific Research Foundation for Talents of Changzhou University of China (201709 and 201710). We want to appreciate Dr. Guoqiang Ren from the University of Washington for the correction of language mistakes in the manuscript.

under cadmium toxicity. Environ. Sci. Pollut. Res. https://doi.org/10.1007/s11356019-05779-7. Kataria, S., Baghel, L., Guruprasad, K.N., 2017. Pre-treatment of seeds with static magnetic field improves germination and early growth characteristics under salt stress in maize and soybean. Biocatalysis Agric. Biotechnol. 10, 83–90. Khan, M.I.R., Nazir, F., Asgher, M., Per, T.S., Khan, N.A., 2015. Selenium and sulfur influence ethylene formation and alleviate cadmium-induced oxidative stress by improving proline and glutathione production in wheat. J. Plant Physiol. 173, 9–18. Kopyra, M., Gwóźdź, E.A., 2003. Nitric oxide stimulates seed germination and counteracts the inhibitory effect of heavy metals and salinity on root growth of Lupinus luteus. Plant Physiol. Biochem. 41, 1011–1017. Lalelou, F.S., Fateh, M., 2014. Effects of different concentrations of zinc on chlorophyll, starch, soluble sugars and proline content in Cucurbita pepo. Int. J. Biosci. 4, 6–12. Li, Q.Y., Niu, H.B., Yin, J., Wang, M.B., Shao, H.B., Deng, D.Z., Chen, X.X., Ren, J.P., Li, Y.C., 2008. Protective role of exogenous nitric oxide against oxidative-stress induced by salt stress in barley (Hordeum vulgare). Colloids Surfaces B Biointerfaces 65, 220–225. Li, Z., Pei, X., Yin, S., Lang, X., Zhao, X., Qu, G.Z., 2019. Plant hormone treatments to alleviate the effects of salt stress on germination of Betula platyphylla seeds. J. For. Res. 30 (3), 779–787. Lin, Y., Yan, L., Liu, Z., Shi, Q., Wang, X., Wei, M., Yang, F., 2012. Exogenous nitric oxide (NO) increased antioxidant capacity of cucumber hypocotyl and radicle under salt stress. Sci. Hortic. 142, 118–127. Liang, T., Ding, H., Wang, G., Kang, J., Pang, H., Lv, J., 2016. Sulfur decreases cadmium translocation and enhances cadmium tolerance by promoting sulfur assimilation and glutathione metabolism in Brassica chinensis L. Ecotoxicol. Environ. Saf. 124, 129–137. Liu, S., Dong, Y., Xu, L., Kong, J., 2014. Effects of foliar applications of nitric oxide and salicylic acid on salt-induced changes in photosynthesis and antioxidative metabolism of cotton seedlings. Plant Growth Regul. 73, 67–78. Ma, X., Zhang, J., Huang, B., 2016. Cytokinin-mitigation of salt-induced leaf senescence in perennial ryegrass involving the activation of antioxidant systems and ionic balance. Environ. Exp. Bot. 125, 1–11. Manai, J., Gouia, H., Corpas, F.J., 2014. Redox and nitric oxide homeostasis are affected in tomato (Solanum lycopersicum) roots under salinity-induced oxidative stress. J. Plant Physiol. 171, 1028–1035. Matthees, H.L., Thom, M.D., Gesch, R.W., Forcella, F., 2018. Salinity tolerance of germinating alternative oilseeds. Ind. Crops Prod. 113, 358–367. Meena, M.D., Yadav, R.K., Narjary, B., Yadav, G., Jat, H.S., Sheoran, P., Meena, M.K., Antil, R.S., Meena, B.L., Singh, H.V., Meena, V.S., Rai, P.K., Ghosh, A., Moharana, P.C., 2019. Municipal solid waste (MSW): strategies to improve salt affected soil sustainability: a review. Waste Manag. 84, 38–53. Negrão, S., Schmöckel, S.M., Tester, M., 2017. Evaluating physiological responses of plants to salinity stress. Ann. Bot. 119, 1–11. Paul, S., Roychoudhury, A., Banerjee, A., Chaudhuri, N., Ghosh, P., 2017. Seed pretreatment with spermidine alleviates oxidative damages to different extent in the salt (NaCl)-stressed seedlings of three indica rice cultivars with contrasting level of salt tolerance. Plant Gene 11, 112–123. Reda, M., Golicka, A., Kabała, K., Janicka, M., 2018. Involvement of NR and PM-NR in NO biosynthesis in cucumber plant subjected to salt stress. Plant Sci. 267, 55–64. Ren, Y., He, J., Liu, H., Liu, G., Ren, X., 2017. Nitric oxide alleviates deterioration and preserves antioxidant properties in ‘Tainong’ mango fruit during ripening. Hortic. Environ. Biotechnol. 58 (1), 27–37. Savvides, A., Ali, S., Tester, M., Fotopoulos, V., 2016. Chemical priming of plants against multiple abiotic stresses: mission possible? Trends Plant Sci. 21, 329–340. Shams, M., Ekinci, M., Ors, S., Turan, M., Agar, G., Kul, R., Yildirim, E., 2019. Nitric oxide mitigates salt stress effects of pepper seedlings by altering nutrient uptake, enzyme activity and osmolyte accumulation. Physiol. Mol. Biol. Plants. https://doi.org/10. 1007/s12298-019-00692-2. Siddiqui, M.H., Alamri, S.A., Al-Khaishany, M.Y., Al-Qutami, M.A., Ali, H.M., AL-Rabiah, H., Kalaji, H.M., 2017. Exogenous application of nitric oxide and spermidine reduces the negative effects of salt stress on tomato. Hortic. Environ. Biotechnol. 58 (6), 537–547. Siddiqui, M.H., Al-Whaibi, M.H., Basalah, M.O., 2011. Role of nitric oxide intolerance of plants to abiotic stress. Protoplasma 248, 447–455. Silva, E.N., Silveira, J.A.G., Rodrigues, C.R.F., Viégas, R.A., 2015. Physiological adjustment to salt stress in Jatropha curcas is associated with accumulation of salt ions, transport and selectivity of K+, osmotic adjustment and K+/Na+ homeostasis. Plant Biol. 17, 1023–1029. Silveira, N.M., Frungillo, L., Marcos, F.C.C., Pelegrino, M.T., Miranda, M.T., Seabra, A.B., Salgado, I., Machado, E.C., Ribeiro, R.V., 2016. Exogenous nitric oxide improves sugarcane growth and photosynthesis under water deficit. Planta 244, 181–190. Song, L.L., Yue, L.L., Zhao, H.Q., Hou, M.F., 2013. Protection effect of nitric oxide on photosynthesis in rice under heat stress. Acta Physiol. Plant. 35, 3323–3333. Tian, X.Y., He, M.R., Wang, Z.L., Zhang, J.W., Song, Y.L., He, Z.L., Dong, Y.J., 2015. Application of nitric oxide and calcium nitrate enhances tolerance of wheat seedlings to salt stress. Plant Growth Regul. 77, 343–356. Xu, F., Ye, L., Xia, X., 2017. Physiology response of roots of pakchoi varieties to salt stress. Agric. Res. Arid Areas 35 (3), 178–181 189 (in chinese). Yadu, S., Dewangan, T.L., Chandrakar, V., Keshavkant, S., 2017. Imperative roles of salicylic acid and nitric oxide in improving salinity tolerance in Pisum sativum L. Physiol. Mol. Biol. Plants 23 (1), 43–58. Yan, F., Liu, Y., Sheng, H., Wang, Y., Kang, H., Zeng, J., 2016. Salicylic acid and nitric oxide increase photosynthesis and antioxidant defense in wheat under UV-B stress. Biol. Plant. 60 (4), 686–694. Yu, L., Zhang, C., Shang, H., Wang, X., Wei, M., Yang, F., Shi, Q., 2013. Exogenous

References Ahanger, M.A., Agarwal, R.M., 2017. Salinity stress induced alterations in antioxidant metabolism and nitrogen assimilation in wheat (Triticum aestivum L) as influenced by potassium supplementation. Plant Physiol. Biochem. (Paris) 115, 449–460. Ahmadi, F.I., Karimi, K., Struik, P.C., 2018. Effect of exogenous application of methyl jasmonate on physiological and biochemical characteristics of Brassica napus L. cv. Talaye under salinity stress. South Afr. J. Bot. 115, 5–11. Ahmad, P., Latef, A.A.A., Hashem, A., Abd Allah, E.F., Gucel, S., Tran, L.S., 2016. Nitric oxide mitigates salt stress by regulating levels of osmolytes and antioxidant enzymes in chickpea. Front. Plant Sci. 7, 347. Ali, Q., Daud, M.K., Haider, M.Z., Ali, S., Rizwan, M., Aslam, N., Noman, A., Iqbal, N., Shahzad, F., Deeba, F., Ali, I., Zhu, S.J., 2017. Seed priming by sodium nitroprusside improves salt tolerance in wheat (Triticum aestivum L.) by enhancing physiological and biochemical parameters. Plant Physiol. Biochem. (Paris) 119, 50–58. Alsaeedi, A.H., El-Ramady, H., Alshaal, T., El-Garawani, M., Elhawat, N., Almohsen, M., 2017. Engineered silica nanoparticles alleviate the detrimental effects of Na+ stress on germination and growth of common bean (Phaseolus vulgaris). Environ. Sci. Pollut. Res. 24, 21917–21928. Ashraf, M.A., Akbar, A., Parveen, A., Rasheed, R., Hussain, I., Iqbal, M., 2018. Phenological application of selenium differentially improves growth, oxidative defense and ion homeostasis in maize under salinity stress. Plant Physiol. Biochem. (Paris) 123, 268–280. Assaha, D.V.M., Ueda, A., Saneoka, H., Al-Yahyai, R., Yaish, M.W., 2017. The role of Na+ and K+ transporters in salt stress adaptation in glycophytes. Front. Physiol. 8, 509. Boldizsar, A., Simon-Sarkadi, L., Szirtes, K., Soltesz, A., Szalai, G., Keyster, M., Ludidi, N., Galiba, G., Kocsy, G., 2013. Nitric oxide affects salt-induced changes in free amino acid levels in maize. J. Plant Physiol. 170, 1020–1027. Campos, F.V., Oliveira, J.A., Pereira, M.G., Farnese, F.S., 2019. Nitric oxide and phytohormone interactions in the response of Lactuca sativa to salinity stress. 2019. Planta. https://doi.org/10.1007/s00425-019-03236-w. Chen, J., Xiong, D.Y., Wang, W.H., Hu, W.J., Simon, M., Xiao, Q., Chen, J., Liu, T.W., Liu, X., Zheng, H.L., 2013. Nitric Oxide mediatesroot K+/Na+ balance in a mangrove plant, Kandelia obovata, by enhancing the expression of AKT1-type K+ channel and Na+/H+ antiporter under high salinity. PLoS One 8, 71543. da Silva, C.J., Batista Fontes, E.P., Modolo, L.V., 2017. Salinity-induced accumulation of endogenous H2S and NO is associated with modulation of the antioxidant and redox defense systems in Nicotiana tabacum L. cv. Havana. Plant Sci. 256, 148–159. Deinlein, U., Stephan, A.B., Horie, T., Luo, W., Xu, G., Schroeder, J.I., 2014. Plant salttolerance mechanisms. Trends Plant Sci. 19 (6), 371–379. Demidchik, V., Straltsova, D., Medvedev, S.S., Pozhvanov, G.A., Sokolik, A., Yurin, V., 2014. Stress-induced electrolyte leakage: the role of K+-permeable channels and involvement in programmed cell death and metabolic adjustment. J. Exp. Bot. 65, 1259–1270. Fan, H., Ding, L., Xu, Y., Du, C., 2017. Seed germination, seedling growth and antioxidant system responses in cucumber exposed to Ca(NO3)2. Hortic. Environ. Biotechnol. 58 (6), 548–559. Fan, H.F., Du, C.X., Ding, L., Xu, Y.L., 2013. Effects of nitric oxide on the germination of cucumber seeds and antioxidant enzymes under salinity stress. Acta Physiol. Plant. 35, 2707–2719. Fang, Y., Li, J., Jiang, J., Geng, Y., Wang, J., Wang, Y., 2017. Physiological and epigenetic analyses of Brassica napus seed germination in response to salt stress. Acta Physiol. Plant. 39, 128. Farhangi-Abriz, S., Torabian, S., 2017. Antioxidant enzyme and osmotic adjustment changes in bean seedlings as affected by biochar under salt stress. Ecotoxicol. Environ. Saf. 137, 64–70. Farhangi-Abriz, S., Ghassemi-Golezani, K., 2018. How can salicylic acid and jasmonic acid mitigate salt toxicity in soybean plants? Ecotoxicol. Environ. Saf. 147, 1010–1016. Gadelha, C.G., Miranda, R.S., Alencar, N.L.M., Costa, J.H., Prisco, J.T., Gomes-Filho, E., 2017. Exogenous nitric oxide improves salt tolerance during establishment of Jatropha curcas seedlings by ameliorating oxidative damage and toxic ion accumulation. J. Plant Physiol. 212, 69–79. Hasanuzzaman, M., Oku, H., Nahar, K., Bhuyan, M.H.M.B., Mahmud, L.A., Baluska, F., Fujita, M., 2018. Nitric oxide-induced salt stress tolerance in plants: ROS metabolism, signaling, and molecular interactions. Plant Biotechnol. Rep. 12 (2), 77–92. Hasanuzzaman, M., Alam, M.M., Rahman, A., Hasanuzzaman, M., Nahar, K., Fujita, M., 2014. Exogenous proline and glycine betaine mediated upregulation of antioxidant defense and glyoxalase systems provides better protection against salt-induced oxidative stress in two rice (Oryza sativa L.) varieties. 2014. BioMed Res. Int. 757219. https://doi.org/10.1155/2014/757219. He, J., Ren, Y., Chen, X., Chen, H., 2014. Protective roles of nitric oxide on seed germination and seedling growth of rice (Oryza sativa L.) under cadmium stress. Ecotoxicol. Environ. Saf. 108, 114–119. Iwai, C.B., Oo, A.N., Topark-ngarm, B., 2012. Soil property and microbial activity in natural salt affected soils in an alternating wet–dry tropical climate. Geoderma 189–190, 144–152. Kanu, A.S., Ashraf, U., Mo, Z., Baggie, I., Charley, C.S., Tang, X., 2019. Calcium amendment improved the performance of fragrant rice and reduced metal uptake

9

Ecotoxicology and Environmental Safety 187 (2020) 109785

Y. Ren, et al.

Zhou, Y., Wen, Z., Zhang, J., Chen, X., Cui, J., Xu, W., Liu, H., 2017. Exogenous glutathione alleviates salt-induced oxidative stress in tomato seedlings by regulating glutathione metabolism, redox status, and the antioxidant system. Sci. Hortic. 220, 90–101. Zhu, Z., Wei, G., Li, J., Qian, Q., Yu, J., 2004. Silicon alleviates salt stress and increases antioxidant enzymes activity in leaves of salt-stressed cucumber (Cucumis sativus L.). Plant Sci. 167, 527–533.

hydrogen sulfide enhanced antioxidant capacity, amylase activities and salt tolerance of cucumber hypocotyls and radicles. J. Integr Agric. 12 (3), 445–456. Zhang, W., Yu, X., Li, M., Lang, D., Zhang, X., Xie, Z., 2018. Silicon promotes growth and root yield of Glycyrrhiza uralensis under salt and drought stresses through enhancing osmotic adjustment and regulating antioxidant metabolism. Crop Protect. 107, 1–11. Zhang, Y., Wang, L., Liu, Y., Zhang, Q., Wei, Q., Zhang, W., 2006. Nitric oxide enhances salt tolerance in maize seedlings through increasing activities of proton-pump and Na+/H+ antiport in the tonoplast. Planta 224, 545–555.

10