Tobacco (Nicotiana tabacum cv. NC89) was used for salt stress response assays, and the transgenic lines (resulting from self-fertilization), ‘T₁-68’ and ‘T₁-71,’ were produced during previous research (Li et al., 2015). Based on the germination results, the seeds of WT tobacco plants were sown 2 days earlier than those of transgenic plants. All of the seeds were sterilized in 2.5% NaClO and were sown into plastic plots (10 cm × 10 cm). Then, 2 weeks later, robust seedlings were transferred to plastic plugs (50 holes) filled with nursery substrate (peat:vermiculite:perlite = 2:1:1). The above experiment was carried out in a controlled environment in an illuminated incubator with an air temperature of 28°C during the day and 23°C during the night, a light intensity of 140 mmol m^-2 s^-1, and a relative humidity of 60%. Four weeks later, batches of five seedlings of WT and CsPLDα overexpression plants (‘T₁-68’ and ‘T₁-71’) were transferred to a 5-L (33 cm × 25 cm × 11 cm) plastic tank filled with an aerated complete nutrient solution (pH 6.0–6.5) containing 4.0 mM Ca(NO₃)₂⋅4H₂O, 6 mM KNO₃, 1 mM NH₄H₂PO₄, 2 mM MgSO₄⋅7H₂O, 46.3 μM H₃BO₃, 10 μM MnSO₄⋅H₂O, 0.77 μM ZnSO₄⋅7H₂O, 0.02 μM (NH₄)₆Mo₇O₂⋅4H₂O, 0.32 μM CuSO₄⋅5H₂O, and 54 μM EDTA-Fe Na in a glass greenhouse at Shandong Agricultural University. The experiment was carried out under natural conditions with an air temperature of 25–30°C during the day and 18–25°C at night. After 2 weeks, young tobacco seedlings were used as experimental materials for the treatments as described below.

According to the method described by Gaxiola et al. (2001), the nutrient solution was supplemented with NaCl, incrementally increasing with each successive watering from 50, through 100, 150 and 200, to a final concentration of 250 mM NaCl. Tanks were arranged in a completely randomized block design with three replicates, providing a total of three tanks with a total of 15 plants per treatment. The samples were collected after 0 and 5 h treatments. The PLD activity and PA content in leaves, the expression of endogenous NtPLDα1 and mitogen activated protein kinase (NtMAPK), the Na⁺ and K⁺ contents and the expression of several genes related to ion exchange (NtNHX1, NtNKT1, NtHAK1, NtNHA1, and NtVAG1) in leaves and roots were measured.

Based on the aforementioned method, when NaCl reached the treatment concentration, replacing the nutrient solution to form a final NaCl concentration of 250 mM, the nutrient solutions in all the plastic tanks were completely renewed every 3 days. Tanks were arranged in a completely randomized block design with five replicates, providing a total of five tanks with a total of 25 plants per treatment. The samples were collected before treatment (CK) and after 8 and 16 days of the 250 mM NaCl treatment. Electrolyte leakage (EL), malondialdehyde (MDA) content, antioxidant enzyme [superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), and catalase (CAT)] activities, ROS (O₂^•− and H₂O₂) generation, Na⁺ and K⁺ contents, and the contents of proline, soluble protein, and soluble sugar in leaves were measured. In addition, fresh weights (FWs) and dry weights (DWs) of roots under conditions of control and stress (250 mM NaCl) were measured 16 days after treatment.

Cytoplasmic extracts from treated plant leaves were measured for PLD activity using a PLD ELISA kit as per the manufacturer’s instructions (Bangyi, China). The PA content was analyzed using a PA ELISA kit per the manufacturer’s instructions (Fangcheng, China).

The Na⁺ and K⁺ contents of leaves and roots were determined using a flame photometer (F-300, Shanghai Metash Instruments, Co. Ltd, Shanghai, China) (Essah et al., 2003).

Total RNA was extracted from young leaves and roots of tobacco using the TRIzol method according to the manufacturer’s instructions (CWBIO). Total RNA (1 μg) from each sample was used as a template for the reverse transcription reaction to synthesize cDNA using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen). The NtActin primers, 5′-TTAAAGAGAAACTGGCATATGTTG-3′ (forward) and 5′-GCCCATCTGGTAACTCATAGC-3′ (reverse), were used to amplify the internal control. The PCR primers were designed to avoid the conserved region and to amplify products of 150- to 300-bp products. Primer sequences are shown in detail in Supplementary Table S1. Quantitative RT-PCR was performed using the TransStart^® Tip Green qPCR SuperMix, and 25 μL of the reaction system was mixed for the determination of salt stress-associated gene expression using a ABI 7500 RT PCR instrument (Thermo Fisher Scientific, USA) as described in the literature (Li et al., 2012). The procedure of Real-time PCR was: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 1 min. Each expression profile was independently verified in three replicate experiments. Data were analyzed using SDS 2.0 software (ABI), and the relative gene expression levels were calculated using the method of 2^-ΔΔCt (Livak and Schmittgen, 2001).

Under conditions of control and stress (250 mM NaCl) after 16 days of treatment, three plants each of WT, ‘T₁-68’ and ‘T₁-71’ were collected. The FWs of tobacco roots were measured immediately. Then, the root samples were dried at 80°C for 48 h and then measured to obtain the DWs.

The histochemical staining of O₂^•− and H₂O₂ was performed as previously described (Xia et al., 2009). The O₂^•− productivity rate and H₂O₂ content were quantified according to Patterson (Rauckman et al., 1979; Patterson et al., 1984). The EL and MDA content were determined according to Wang et al. (2010).

Superoxide dismutase activity was assayed by measuring its ability to inhibit the photochemical reduction of nitrobluetetrazolium following the method of Stewart and Bewley (1980). CAT activity was measured as the decline in absorbance at 240 nm due to the decrease in the extinction of H₂O₂, according to the method of Patra et al. (1978). POD activity was measured by the increase in absorbance at 470 nm due to guaiacol oxidation (Nickel and Cunningham, 1969). APX activity was measured by the decrease in absorbance at 290 nm as ascorbic acid was oxidized according to previous studies (Nakano and Asada, 1981).

Fresh leaf samples of 0.5 g were extracted with 5 mL of 3% sulfo-salicylic acid at 100°C for 10 min with shaking. The other processes were performed as previously described (Xu et al., 2011). Soluble sugar was measured by the sulfuric acid–anthrone colorimetry method (Dubois et al., 1951). The protein content was measured using bovine serum albumin as the standard, according to Bradford (1976).

Values presented are means ±1 standard deviation (SD) of three replicates. Statistical analyses were carried out by analysis of variance (ANOVA) using SAS (SAS Institute, Cary, NC, USA) software. Differences between treatments were separated by the least significant difference (LSD) test at P < 0.05 and P < 0.01.

As shown in Figure 1A, PLD activity in transgenic plant leaves increased as the 250 mM NaCl treatment time increased, while there was a decrease in the WT plants. When the NaCl concentration reached 250 mM (0 h) gradually, PLD activities in transgenic plants of ‘T₁-68’ and ‘T₁-71’ leaves were both 17.9% significantly higher than that of WT plants. After 5 h of the 250 mM NaCl treatment, the PLD activities in ‘T₁-68’ and ‘T₁-71’ leaves were 36.3 and 44.9% higher, respectively, than of the activity in WT plants. Moreover, to determine the involvement of endogenous NtPLDα1 in salt responses, the expression of NtPLDα1 in leaves and roots of WT and transgenic plants was also measured (Figures 1C,D). The results indicated that there was no difference between WT and transgenic plants under short-term NaCl stress. Thus, the overexpression of CsPLDα could enhance the sensitivity of plants to high salinity stress in a short time.

The change in the PA content was similar to that of the PLD activity (Figure 1B). The 250 mM NaCl treatment for 5 h significantly induced PA formation in CsPLDα-overexpression plants compared with WT plants. The PA content in ‘T₁-68’ and ‘T₁-71’ transgenic plants increased by 37.7 and 74.3%, respectively. When the NaCl concentration reached 250 mM (0 h) gradually, the PA content in leaves of ‘T₁-68’ and ‘T₁-71’ transgenic plants was also 15.8 and 43.4% higher, respectively, than its content in WT plants. As a result, accumulated PA could generate a range of reactions, especially triggering the salt-tolerance pathway in tobacco plants.

The MAPK cascades are major pathways by which extracellular signals, such as growth factors, hormones and stress stimuli, are transduced into intracellular responses in plants. To identify the relationship between the CsPLDα and MAPK, the expression of NtMAPK in leaves and roots of WT and transgenic plants were detected. Figure 2 shows that the mRNA abundances of the NtMAPK gene in both leaves and roots of overexpression plants were remarkable induced after the 5 h treatment. Compared with those of WT plants, the levels of expression in the leaves and roots of ‘T₁-68’ and ‘T₁-71’ were higher by 2.5 or 3.0 times (Figure 2A) and 1.7 or 4.8 times (Figure 2B), respectively. When the NaCl concentration reached 250 mM (0 h) instantly, the transcript levels of MAPK gene in leaves and roots of transgenic plants did not undergo relatively large increases compared with the 5 h treatment, however, there was significant difference between transgenic and WT plants. Thus, in general, NtMAPK may be an important indicator in tobacco salt tolerance.

To determine the involvement of CsPLDα in salt responses to modulate Na⁺–K⁺ homeostasis, RT-PCR was conducted to analyze the expression levels of ion exchange-related genes, including NtNHX1, NtNKT1, NtHAK1, NtNHA1 and NtVAG1, in leaves and roots of transgenic and WT plants. NtNHX1, a major gene controlling sodium ion regionalization, could prompt the transportation of Na⁺ into vacuoles. NtNKT1 is a gene encoding a K⁺ channel protein, while NtHAK1 encodes a K⁺ transporter protein. Both NtNHA1 and NtVAG1 belong to proton pump genes, with one being located on the cytoplasmic membrane and the other on the vacuolar membrane. As shown in Figures 3A–J, after 5 h of stimulation with stress, the mRNA abundances of these regulatory genes in both roots and leaves of transgenic plants were dramatically induced compared with that of WT plants. When the NaCl concentration reached 250 mM (0 h) gradually, the transcript levels of genes related to ion exchange in leaves and roots of transgenic plants did not undergo a relatively large rise compared with after the 5 h of treatment.

The significant changes in the expression of ion exchange-related genes resulted in alterations in the Na⁺ and K⁺ contents. Treatment with 250 mM NaCl for 5 h caused an obvious decrease in K⁺ content in roots compared with treatment for 0 h (Figure 4B). This decrease in ‘T₁-68’ (13.6%) and ‘T₁-71’ (14.5%) transgenic plants was more significant than that in WT plants (11.0%). Meanwhile, the K⁺ content in the roots of ‘T₁-68’ and ‘T₁-71’ transgenic plants was lower by 4.3 and 12.4%, respectively, than that of WT. Additionally, the K⁺ content in leaves was also decreased after treatment (Figure 4A). The decrease in the WT plants (20.0%) was more significant than that in the ‘T₁-68’ (7.1%) and ‘T₁-71’ (3.7%) transgenic plants. However, the K⁺ content in the leaves of ‘T₁-68’ and ‘T₁-71’ transgenic plants was 21.4 and 38.6% higher, respectively, than that of the WT.

Distinct from the altered patterns in the K⁺ content, the Na⁺ content was increased in both leaves and roots after treatment (Figures 4C,D). The transgenic tobacco cells absorbed more Na⁺ than WT cells after 0 and 5 h of the 250 mM NaCl treatment. Additionally, the Na⁺ contents in leaves and roots of ‘T₁-68’ and ‘T₁-71’ transgenic plants were significantly higher by 25.0 or 68.0% and 28.0 or 45.5%, respectively, after 5 h of the 250 mM NaCl treatment.

Based on the above findings, the ratio of Na⁺/K⁺ was calculated, and the results revealed that the ratio increased in both leaves and roots as the treatment time increased, and the ratio of the Na⁺/K⁺ concentration in transgenic plants was higher than in WT plants, especially in roots after 5 h of the 250 mM NaCl treatment (Figures 4E,F). The ratios of Na⁺/K⁺ in the roots of ‘T₁-68’ and ‘T₁-71’ transgenic plants were significantly higher by 33.7 and 66.1%, respectively, after 5 h of the 250 mM NaCl treatment. These findings suggested that CsPLDα-overexpression tobacco plants absorbed more Na⁺ than K⁺, which resulted in an increased ratio of Na⁺/K⁺ and consequently maintained Na⁺–K⁺ homeostasis. Combined with the results obtained from the expression analyses of the ion exchange genes, we concluded that CsPLDα negatively regulated salinity stress through the regulation of key genes involved in the maintenance of ion homeostasis at the transcriptional level and the resistance of salinity-induced osmotic stress over a short time.

After 16 days of treatment, the photographs showed that overexpression of CsPLDα in tobacco significantly promoted the growth of seedlings under 250 mM NaCl stress (Figures 5A,B). The root growth of both WT and transgenic seedlings were severely suppressed during the 16 days exposure to 250 mM NaCl. In particular, the growth of WT seedlings was more significantly inhibited. The FWs (Figure 5C) and DWs (Figure 5D) of WT, ‘T₁-68’ and ‘T₁-71’ roots were significantly lower by 301.3, 134.0 and 141.9%, and 239.2, 77.9, and 106.5%, respectively, compared with the control. The root FWs and DWs of ‘T₁-68’ and ‘T₁-71’ transgenic plants increased by 75.5 or 81.3% and 92.9 or 77.6%, respectively, compared with WT plants after undergoing the 16-days treatment. Thus, the transgenic tobacco plants exhibited stronger capabilities of resisting salt stress, while CsPLDα may play an important role in alleviating the stress damage caused by high salt.

Figure 6 shows that the PLD activity levels were not significantly different between WT and transgenic plants before treatment (CK). However, the PLD activities in transgenic plants were induced to a greater degree than that in the WT under high NaCl conditions. The PLD activities in leaves of ‘T₁-68’ and ‘T₁-71’ transgenic plants were significantly higher by 15.3 and 29.3%, respectively, than that in WT after 8 days of the 250 mM NaCl treatment. In addition, the high salt treatment slightly enhanced the PLD activities in WT and transgenic plants as the treatment time increased. Thus, PLD may be positively induced by salt stress and CsPLDα-overexpression may have sensitized plant responses to the long-term salt stress.

Under a long-time exposure to salts, Na⁺ concentrations in both the leaves and roots of WT and transgenic plants increased significantly (Figures 7C,D), while the K⁺ content decreased as shown in Figures 7A,B. Compared with WT plants, Na⁺ content after the 16-days treatment increased by more than 29.3% in leaves, but only 17.8% in roots, of ‘T₁-71’ transgenic plants. Meanwhile, the K⁺ content increased by 57.7% in leaves and 40.6% in roots. Consequently, the Na⁺/K⁺ ratio increased in both the leaves and roots after 16 days of salt stress (Figures 7E,F). Unlike WT plants, both transgenic plants still maintained the highest levels of Na⁺ or K⁺ in their organisms, but the lowest Na⁺/K⁺ ratio after the 16-days salt stress, and the difference was more distinct in the ‘T₁-71’ plants. Thus, the patterns of ion influx and efflux in all of the tobacco lines were consistent when the responses to salt stress. However, the overexpression of CsPLDα could protect against stress-induced changes in ion concentrations to maintain Na⁺–K⁺ homeostasis during a long-term treatment.

Figure 8 shows that the 250 mM NaCl treatment generated more EL and MDA than the CK. Compared with WT lines, the EL of ‘T₁-68’ in leaves decreased by 16.8% and that of ‘T₁-71’ significantly decreased by 11.7% (P < 0.05) after 8 days of the 250 mM NaCl treatment (Figure 8A). The ELs in ‘T₁-68’ and ‘T₁-71’ transgenic plant leaves decreased by 8.4 and 8.9%, respectively, after the 16-days treatment; however, there were no significant differences between the transgenic and WT lines (P < 0.05).

After undergoing the same procedures, the MDA content in ‘T₁-68’ and ‘T₁-71’ transgenic plant leaves decreased by 19.3 and 30.0%, respectively, after 16 days of the 250 mM NaCl treatment (Figure 8B). This was a trend distinct from that of the EL. After 16 days of 250 mM NaCl, the MDA content in transgenic tobacco plants was lower than that 8 days after treatment. However, the MDA content in WT plants was maintained at a stable level. Thus, the expression of CsPLDα in tobacco plants enhanced the tolerability of plants to the salt stress associated with membrane lipid peroxidation.

With time, after NaCl stimulation, abundant levels of H₂O₂ and O₂^•− accumulated in the plants and peaked at day 16 (Figures 9A–D). Overall, the degrees of H₂O₂ and O₂^•− accumulation in the leaves of ‘T₁-68’ and ‘T₁-71’ plants were lower than in WT plants. The H₂O₂ contents in leaves of ‘T₁-68’ and ‘T₁-71’ lines decreased by 14.2 and 18.1%, respectively, when compared with WT plants. As for O₂^•−, the production rated in leaves of ‘T₁-68’ and ‘T₁-71’ plants decreased by 28.2 and 36.9%, respectively, compared with in leaves of WT plants.

To evaluate the antioxidant defense system in plants exposed to salt stress, the activities of four well-recognized kinds of antioxidant enzymes, SOD, CAT, POD and APX, were measured in leaves of both WT and transgenic plants. There were opposing trends for H₂O₂ and O₂^•−. As shown in Figures 10A–D, antioxidant enzyme activities between WT and transgenic plants without a salt treatment showed no significant differences. Nevertheless, a NaCl treatment increased the enzyme activities in all of the plants, with the enzyme activities being higher in ‘T₁-71’ and ‘T₁-68’ than in WT plants. During the experimental period, SOD, POD, CAT, and APX activities peaked at 8 days, were maintained at high levels, and finally decreased at day 16, but their levels remained high compared with the CK. When compared with WT plants after 8 days of treatment, the SOD activities in leaves of ‘T₁-68’ and ‘T₁-71’ plants increased by 7.1 and 12.3%, respectively (P < 0.01; Figure 10A), and the POD activities increased by 41.9 and 45.9%, respectively (P < 0.05; Figure 10B). The CAT activities increased by 26.2 and 27.1%, respectively (P < 0.05; Figure 10C), and the APX activities increased by 32.7 and 40.0% (P < 0.01; Figure 10D), respectively. Thus, CsPLDα-overexpression in tobacco plants facilitated the growth and salt-stress tolerance of plants.

The contents of soluble sugar, proline, and soluble protein in the leaves of the lines were determined (Figure 11), and their contents under salt stress were higher than in the CK. However, there were no significant differences between WT and transgenic plants at CK. The contents of proline and soluble sugar increased in a time-dependent manner and finally peaked at day 16, while the soluble protein content peaked at 8 days, was maintained at a high level, and finally decreased at day 16. After the 16-days treatment, the contents of proline and soluble sugar in leaves of ‘T₁-68’ and ‘T₁-71’ transgenic lines increased by 26.5 and 25.3% (Figure 11A), and 16.3 and 16.4% (Figure 11B), respectively, compared with WT. After the 8-days treatment, the content of soluble protein in leaves of ‘T₁-68’ and ‘T₁-71’ transgenic lines increased by 19.4% (P < 0.05) and 29.7% (Figure 11C), respectively, compared with WT. These results indicated that the substances capable of causing major osmotic adjustments were significantly upregulated by the overexpression of CsPLDα.