Seeds of Z. xanthoxylum were collected from wild plants within the Alxa League (39°05′N, 105°34′E at an elevation of 1,360 m) in the Inner-Mongolia Autonomous Region, China. Healthy seeds were washed several times under running water using magnetic stirrers to remove any substances that might inhibit their germination, soaked in distilled water for 1 day at 4°C, and germinated for 2 days in the dark at 25°C. Vigorous germinating seeds with uniform radicle length were selected and transplanted into plastic trays (15 cm × 24 cm × 11 cm; 12 seedlings per container) filled with 2-mm diameter white quartz grits. The quartz grits were washed with standard procedure to remove Na⁺ as much as possible: firstly, soaked in 2N sulfuric acid and 1N hydrochloric acid for 2 days, then washed with running water four times, and lastly with distilled water six times. It should be noted that there was still trace amounts of Na⁺ left in washed quartz grits (0.8 ± 0.1 micromoles⋅g^-1 dry wt.). And this size of grit is too coarse to maintain or absorb water. The plants were irrigated with modified half-strength Hoagland nutrient solution containing 2 mM KNO₃, 0.5 mM NH₄H₂PO₄, 0.25 mM MgSO₄⋅H₂O, 0.1 mM Ca (NO₃)₂⋅4H₂O, 50 μM Fe-citrate, 92 μM H₃BO₃, 18 μM MnCl₂⋅4H₂O, 1.6 μM ZnSO₄⋅7H₂O, 0.6 μM CuSO₄⋅5H₂O, and 0.7 μM (NH₄)₆Mo₇O₂₄⋅4H₂O. Prior to planting, the weight of each grit-filled container was recorded using an electronic scale to control and record the proper amount of solution added to each tray daily. In order to prevent drowning damage to roots, the amount of culture solution was limited to depths of between 1 and 2 cm, approximately one-seventh the height of each cultivation tray. Seedlings were grown in a culture room at 32°C/26°C (day/night) under a light/dark cycle of 16 h/8 h and at a light intensity of 400 μm mol m^-2 s^-1. Relative humidity was maintained at approximately 65% in growth room.

The culture boxes containing 2-week-old Z. xanthoxylum seedlings were randomly separated into two groups and treated for 1 week with modified half-strength Hoagland nutrient solution supplemented with, or without, 50 mM NaCl. The NaCl concentration used in this study was based on previous work where 50 mM NaCl was shown to be optimum among a series of concentration gradients to promote growth and alleviate the deleterious impacts of drought stress (Ma et al., 2012; Yue et al., 2012). Distilled water was supplemented every day to maintain a relatively constant culture solution concentration.

In order to determine whether or not NaCl treatment had a positive effect on drought tolerance in Z. xanthoxylum, plant watering was curtailed after each treatment. The culture solutions quickly became dried out within 2 days through evapotranspiration, plant deaths occurred following unrecoverable wilt, and the survival rate was calculated as (live plants/total plants) × 100%. In addition, the amount of evapotranspiration was recorded in each case on the 7th day of treatment, calculated as the total weight of each tray containing plants following water supplementation at the end of the sixth treatment day minus the total weight of the tray containing plants at the end of the seventh treatment day.

In order to further verify the effects of NaCl, a mimic water deficit experiment was performed using -0.5 MPa mannitol (Michel et al., 1983) prepared with half-strength Hoagland nutrient solution. This combination was applied over a 24-h period to plants that had been treated with NaCl for 7 days, and a number of biochemical indicators were assayed using leaves from the third and fourth nodes counted from the bottom of plants (the same below). Thus, the superoxide radical was determined using hydroxylamine oxidation (Elstner and Heupel, 1976), and lipid peroxidation was assessed by monitoring malondialdehyde (MDA) production in leaves, quantified as thiobarbituric acid reactive substances (Dhindsa et al., 1981). Leaf electrolyte leakage was determined by means of relative membrane permeability as measured using a conductivity meter (Murray et al., 1989), while superoxide dismutase (SOD) was assayed using the nitroblue tetrazolium method (Dhindsa et al., 1981), and catalase (CAT) and peroxidase (POD) activities were measured following the method outlined by Cakmak et al. (1993). The ascorbate peroxidase (APX) activity was determined by Nakano and Asada (1981). Glutathione reductase (GR) activity was determined as described by Rao et al. (1996). The Reduced glutathione (GSH) and oxidized glutathione (GSSG) were determined according to Nagalakshmi and Prasad (2001). Briefly, 0.2 g fresh leaves were homogenized by grinder (BioSpec Mini-Beadbeater-96, United States) in liquid N₂. For MDA, 5 ml 0.1% (w/v) trichloroacetic acid was used to extract the sample and then centrifuged; the resulted supernatant was added 5 ml of 0.5% thiobarbituric acid solution and heated for 10 min at 100°C. After cooling, the precipitate was removed by centrifugation. The absorbance of the sample was measured at 450, 535, and 600 nm. For SOD, CAT, POD, and APX, 8 ml 50 mM sodium phosphate buffer (pH 7.8) was used to extract the sample, centrifuged to get the crude enzyme solution, and then measured accordingly. For GR, the sample extracted by 1.5 ml 100 mM potassium phosphate buffer (pH 7.8), 0.1% (v/v) Triton X-100 and 2 mM MgCl₂ was centrifuged 10 min at 12,000 g. The supernatant was crude extract. For GSH and GSSG, 5 ml of 5% (w/v) sulphosalicylic acid was used to extract and then centrifuged.

Contents of Na⁺, K⁺, and Ca²⁺ were measured following the method outlined by Wang et al. (2007). Briefly, these ions were extracted from dried plant samples soaked in 100 mM acetic acid at 99°C for 4 h; ion analysis was then performed using a polarization Zeeman atomic absorption spectrophotometer (Hitachi Z-2000, Hitachi High-Technologies Co., Japan). As for chloride (Cl^-), 0.2 g fresh leaves oven-dried were grinded (BioSpec Mini-Beadbeater-96, United States) into powder, extracted by 1.2 ml ddH₂O twice within boiled water bath for 2 h, and determined by Mohr titration (Korkmaz, 2001).

Total soluble sugar content was measured using the anthrone method (Dubois et al., 1956; Chow and Landhäusser, 2004). Briefly, 0.2 g samples of fresh leaves were dried, ground in liquid nitrogen, and double-extracted using 4 ml of 80% ethanol by boiling in capped polypropylene tubes at 95°C for 30 min. Extraction supernatants were then centrifuged at 12,000 rpm for 10 min and combined for sugar analysis; each 0.5 ml extraction was diluted in 1.5 ml of distilled water, and 0.5 ml of 2% (w/v) anthrone ethyl acetate reagent was rapidly added, followed by 2.5 ml of concentrated sulfuric acid (H₂SO₄). Absorbance was then measured at 630 nm (Hitachi ultraviolet spectrophotometer U-3900, Hitachi High-Technologies Co., Japan) following 10 min of color development in the dark.

The content of free proline was determined using the sulphosalicylic acid method (Bates et al., 1973). Briefly, 0.2 g samples of fresh leaves were dried and homogenized (Mini-Beadbeater-96, Bio Spec Products Inc. United States) in 5 ml of 3% (w/v) sulphosalicylic acid and centrifuged; ninhydrin and glacial acetic acid were then added to the supernatants, and the mixture was heated at 95°C for 60 min in a water bath. Reactions were then stopped using an ice bath, the mixture was extracted using toluene, and the absorbance was read at 520 nm.

The variable Ψ_s was measured using a freezing-point osmometer (FM-8P, Shanghai Yida Instrument Co., Japan). Briefly, fresh leaves were frozen and thawed several times in liquid N and then squeezed using a syringe. Leaf sap was then collected, centrifuged, and then used for Ψ_s determination at 25°C. These readings (mmol kg^-1) were used to calculate Ψ_s in Mpa, as follows:

In this expression, R = 0.008314 and K = 298.8. Similarly, Ψ_s values for each inorganic and organic solute were calculated using the Van’t Hoff equation, as follows:

In this expression, n denotes the number of solute molecules, while R = 0.008314, and T = 298.8. The contributions of each solute to leaf Ψ_s were then estimated, as follows:

At the end of treatments, whole plants were washed with distilled water and tissue samples were rapidly taken and immediately weighed to determine fresh weight (FW). Additional tissues were oven-dried at 80°C for 72 h and dry weight (DW) was determined. Thus, tissue and absolute water contents were calculated as follows:

The measurement method used to calculate leaf volume was as follows:

This equation was taken from the manual of a patented volumenometer (ZL 2015207858757) with a measurement precision of 0.1 mm³; in this expression, V_leaf refers to leaf volume, while M_leaf is leaf FW, M₁ is the weight of the volumenometer fully filled with pure water, M₂ denotes the weight of this instrument fully filled with pure water plus the leaf, and ρ_water is the density of pure water at ambient temperature.

In order to assess the water loss rate, leaves from the same position as those sampled above were carefully excised from the plant with a scalpel and the cut petiole section was sealed with white vaseline to prevent water loss. These cut leaves were then placed on filter paper in plastic dishes in a dark chamber at constant temperature and humidity and their weights were recorded every 4 h. Water loss rates were calculated as follows:

In this expression, FW_Tn is the weight at each time point, while ΔT denotes the time interval between consecutive measurements.

The extraction and purification of ABA from leaves was carried out following previously described procedures (Dobrev and Vankova, 2012). Briefly, an approximately 1 g sample of fresh leaves was homogenized and extracted using a methanol/double-distilled water/formic acid (15:4:1, v:v:v) mixture. This extract was then passed through a SPE C18 column (T1616-2008, Tianjin BoJin Technology Co., Japan) and the eluate was collected and evaporated using a N₂-pressure blowing concentrator at room temperature in the dark to about one-tenth of its original volume. The residue was then dissolved in 2 mL of 80% methanol and filtered through a 0.45-μm filter, prior to the determination of ABA content using liquid chromatography mass spectrometry (Applied Biosystems PE Sciex API 2000, ABSciex, United States).

Leaves from Control and Na treatments were fixed using 2.5% (v/v) glutaraldehyde in phosphate-buffered saline (PBS, pH 7.4) for 12 h at 4°C. Samples were then washed with PBS, dehydrated using a graded ethanol series, and embedded in LR White resin (London Resin Co., United Kingdom). The resin was then polymerized at 55°C for 3 days, and 0.5 and 0.1 μm sections were cut with a diamond knife using a Leica EM UC7 ultramicrotome (Leica Mikrosysteme GmbH, Austria).

Semi-thin 0.5 μm sections were placed onto glass slides for light microscope observations, stained with 1% (w/v) Toluidine Blue O (with 1% [wt/vol] sodium borate) for 5 min, and observed using an OLYMPUS BX51 system (Olympus Corporation, Japan).

Leaves were fixed with 2.5% (v/v) glutaraldehyde in pH 7.4 PBS for 12 h at 4°C before being re-washed with PBS, dehydrated using a graded ethanol series, immersed in isoamyl acetate, and fully dried with a critical point dryer for SEM observations. Treated samples were then mounted on copper stubs and sputter-coated with gold-palladium; specimens were observed and photographed using a field emission (FE) SEM (S-4800, HITACHI, Japan) at 10 kV.

Each experiment was repeated at least twice and comprised three replicates. All values are reported in this study as means ± standard deviation (SD); analysis of variance (ANOVA) was performed for experiments using the software Excel 2016 applying the Student’s t-test for simple one-way comparisons of two samples.

Two-week-old Z. xanthoxylum seedlings were randomly treated either with NaCl, or without NaCl (control), for 1 week. The results revealed that water loss by evapotranspiration from trays of NaCl-treated plants was significantly less than from control plants on the 7th day after treatment (Figure 1A). Watering was stopped following treatment and time-course analyses of survival rate and durability were performed. These experiments showed that because of rapid evapotranspiration and the fact that large quartz grit particles cannot retain moisture, the solution quickly ran out in the culture trays within 2 days or less. On the 3rd day after watering was stopped, the control plants started to visibly wilt; as shown in Figure 1B, the survival rate of this group also sharply decreased between the 4th and 6th day after watering was stopped because of unrecoverable wilt, while this rate did not change in the NaCl- treated group. It is also noteworthy that the plants in the control group were totally dried out by between 6 and 8 days after watering was stopped, while this took between 35 and 45 days in the treatment group (Figures 1C,D). The results corroborated the fact that NaCl treatment enhanced the survival rate and durability of Z. xanthoxylum under absolute drought conditions.

In order to test whether, or not, NaCl treatment enhances osmotic stress resistance, plants that had been subject to 7 days of treatment were exposed for 24 h to a further culture solution containing an additional -0.5 MPa of mannitol. Observations revealed that less of this solution remained in the control trays than in those treated with NaCl, while visible wilt wrinkles appeared on the leaves of control plants but not on treated individuals. Consistent with these observations, biochemical assays demonstrated that the content of the superoxide radical (O₂^-⋅), a reactive oxygen species (ROS) produced in plants under stress, was significantly less in NaCl-treated plants than in the control group (Figure 2A). In addition, the content of MDA, an end product of lipid peroxidation that is stimulated by ROS, was also significantly lower in NaCl-treated plants (Figure 2B), while the relative conductivity or electrolyte leakage, proxy for the degree of cell membrane damage due to stress, was also markedly reduced in NaCl-treated plants (Figure 2C). The activity of enzymes involved in removing ROS, such as SOD, POD, CAT, APX, and GR were also significantly reduced in NaCl-treated plants (Figures 2D–H). The Ratio of the non-enzymatic antioxidant reduced/oxidized glutathione (GSH/GSSG) was lower in NaCl-treated plants, too, indicating the treated leaves with more efficiency to use reduced glutathione (Figure 2I). All of these results demonstrated that treatment with NaCl reduced the damage caused by osmotic stress.

In order to investigate the function of Na⁺ in drought tolerance, leaf solute potential (Ψ_s) and the contents of Na⁺, K⁺, Ca²⁺, Cl^-, soluble sugar, and proline in leaves were measured. The concentration of each solute, as well as its relative contribution to Ψ_s, were also calculated. Results showed (Figure 3A) that Ψ_s was significantly lowered in NaCl-treated plants compared to the control group and the same was true for K⁺, Ca²⁺, soluble sugar, and proline, while opposite pattern was seen in Na⁺ and Cl^- content (Figure 3B). Na⁺ was greatly accumulated to more than 50 mM in control group plants (Figure 3B), even though these ions were not added to the culture solution and a great deal of effort was expended to clean the quartz grit used in these experiments. At the same time, the contribution of inorganic solutes, such as Na⁺, K⁺, Ca²⁺, and Cl^-, to Ψ_s was as high as 87.36% in the control plants, while the contribution of Na⁺ was just 11.2% ± 1.1%. In treated plants, the contribution of inorganic solutes was 76.37%, and the Na⁺ contribution was definitely higher, 63.9% ± 10.4% (Figure 3C). These results suggested that inorganic solutes of Na⁺, K⁺, Ca²⁺, and Cl^- are the preferred osmolytes of Z. xanthoxylum and that the first of them is favored overall. Thus, when available, Na⁺ is rapidly accumulated in leaves, significantly decreasing their solute potential.

In order to further determine the effects of accumulated Na⁺ on leaf growth, mature leaf characteristics pruned from the third and fourth nodes of plants were examined. The results showed that leaf FW and DW, absolute water content, leaflet length, thickness, and volume all markedly increased in treated plants (Figures 4A–F). Results also suggested that NaCl treatment had a great impact on promoting leaf succulence.

Further, to examine the effect of NaCl on leaf succulence at the cellular level, semi-thin cross-sections of mature leaves from the same positions on plants were investigated. Compared with the control, both palisade and spongy cells were enlarged dramatically in NaCl-treated plants (Figure 5); because of cell enlargement, morphological differences between palisade and spongy tissues became less obvious in treated plants (Figures 5A,B). Thus, to quantify the effect of Na⁺ on cell enlargement, areas of the three largest palisade and spongy cells in each different section were measured. These results demonstrated that the areas of these cells were significantly increased by 91.3% ± 18.8% and 89.2% ± 19.5% in NaCl-treated plants compared with the control, respectively (Figures 4G,H, 5C–F). Because the number of leaf cell layers did not differ between the control and treatment (Figure 4I), these results suggested that succulence promoted by Na⁺ was mainly the result of cell enlargement rather than cell division.

In order to gain a deeper understanding of the significance of Na⁺ in Z. xanthoxylum, the distributions of both water and Na⁺ at the organ level in plants were also investigated. These results showed that Na⁺ sequestered in plants was overwhelmingly allocated to leaves, irrespective of treatment or control (Figure 6A), and that the same distribution pattern was seen for water in different organs (Figure 6B). And the Na⁺ concentration was the highest in leaves (Figure 6C). Correlation analysis showed that the Na⁺ distribution varies across organs at the whole plant level, but was positively correlated with the corresponding water distribution (Figure 6D). Thus, absolute plant water content increased by 188.0% ± 33. 5% in NaCl-treated plants compared to the control and that this increment comprised as much as a 99.4% ± 19.0% increase in leaves. These results also showed strongly that Na⁺ absorbed by Z. xanthoxylum was mainly allocated to leaves so that cells could be enlarged for water storage.

Because the results of this study showed that water loss via evapotranspiration at the plant tray level was reduced by NaCl treatment (Figure 1A), it was further hypothesized that this might also act to reduce the plant water loss rate. Thus, to test this, the water loss rates of detached third and fourth node leaves from 2-week-old seedlings treated for 7 days with NaCl and without NaCl (control) were investigated (Figure 7). The results of this comparison showed that when leaves were detached from their parent plant their water content generally declined; however, the rate of water content loss was clearly less in NaCl-treated leaves than in those from control plants (Figure 7A). Results showed that the water loss rate of detached leaves from NaCl-treated plants was only about one eighth of that from their control counterparts after a period of up to 4 h (Figure 7B) and that the rate in leaves from the latter remained significantly higher throughout this period even though overall water content was lower (Figure 7A,B). This suggested that the water loss rate of detached leaves was independent of cell sap concentration; this rate also fell to almost zero in NaCl-treated detached leaves within 12 h, while that in control leaves continued to decline (Figure 7B). This suggested that NaCl treatment promoted water retention and greatly reduced leaf water losses.

To further understand how this treatment influences water loss, leaf stomata from the third and fourth nodes of 2-week-old plants subjected to treatment with, and without, NaCl for 7 days were examined via SEM. These observations showed that although the stomata of control group leaves were mostly open, those on both the adaxial and abaxial surfaces of treatment group plants were closed (Figure 8). Further SEM analysis revealed that 69.5 and 94.4% of adaxial and abaxial epidermal stomata were open in control plants, respectively, while just 5.7 and 6.1% were open in their NaCl-treated counterparts (Supplementary Table S1). Statistical analysis also showed that stomatal density on both the adaxial and abaxial surfaces of NaCl-treated plants was significantly reduced compared to the control (Figure 7C); however, as these leaves were completely developed on the third and fourth nodes of 2-week old-seedlings, this decrease in stomatal density might also be due to the enlargement of epidermal cells in treated plants (Figures 5A,B, 8A–D). Taken together, these results demonstrated that NaCl treatment influenced water loss reduction mainly via stomatal closure and decreases in the density of these structures.

It is likely that levels of the stress signaling molecule ABA, which promotes stomatal closure, increase when plants are subject to water stress. Thus, to detect whether, or not, reductions in stomatal aperture size can be linked to changes in the proportions of this molecule, ABA content was measured. The data showed that ABA content did not vary significantly between the control and NaCl-treated plants (Figure 7D); this suggested that the leaves of NaCl-treated plants may not suffer stress and that stomatal closure was probably independent of ABA.