The seeds of P. tenuiflora were grown on the hydroponic culture sieves covered with bibulous paper and wetted with distilled water in rectangular trays in dark for 7 days. After germination, seedlings were irrigated with Hoagland nutrient solution (5 mM L^-1 KNO₃, 0.5 mM L^-1 NH₄H₂PO₄, 0.25 mM L^-1 MgSO₄.7H₂O, 1.5 mM L^-1 Ca(NO₃)₂.4H₂O, 0.5 mM L^-1 Fe-citrate, 92 μM L^-1 H₃BO₃, 18 μM L^-1 MnCl₂.4H₂O, 1.6 μM L^-1 ZnSO₄.7H₂O, 0.6 μM L^-1 CuSO₄.5H₂O, 0.7 μM L^-1 (NH₄)₆Mo₇O₂₄.4H₂O) that was renewed every 3 days. When seedling reached 5 cm in height, they were transferred into black containers with the Hoagland nutrient solution and cultivated in a greenhouse with the temperature of 28°C/23°C (day/night), the daily photoperiod of 16/8 h (light/dark, the flux density of about 600 μmol m^-2 s^-1) and the relative humidity of 60–80%. Solution was renewed every 3 days. Given that the growth of P. tenuiflora was inhibited slightly when external NaCl concentration increased to 25 mM, and became severely under 150 mM NaCl treatments (Wang et al., 2009, 2015; Guo et al., 2012), 4-week-old seedlings were treated with 25 (mild salt stress) or 150 mM NaCl (severe salt stress) for 0, 1, 6, 24, and 48 h before harvest.

Total RNA was extracted from roots and shoots of above harvested seedlings using the RNAprep pure plant Kit (Tiangen Biotech Co., Ltd, Beijing, China). The first-strand cDNA was synthesized with PrimeScript^TM RT Master Mix (Perfect Real Time; Takara Biotech Co., Ltd, Dalian, China).

The primers of PtACTIN, PtSOS1, PtHKT1;5, and PtNHX1 were designed by using the Primer 5.0 program (Premier Biosoft International, Palo Alto, CA, USA; Table 1). The expression levels of SOS1, HKT1;5, and NHX1 genes in different tissues of P. tenuiflora under different salt treatments (25 or 150 mM NaCl) were detected by ABI PRISM 7500 sequence detection system. PtACTIN was used for RNA normalization. SYBR Green PCR master mix (Takara Biotech Co., Ltd, Dalian, China) was used for 20 μL PCR with the program as follows: 95°C for 30 s, 40 cycles of 95°C for 5 s, and 60°C for 34 s. All reactions were performed with three replicates. The relative expression levels of all genes were calculated using the 2^-ΔΔCt method (Zhang et al., 2016).

All the data were presented as means with standard deviation, and data analysis was performed by one-way analysis of variance (ANOVA) using SPSS Statistics 19 software (SPSS Inc., Chicago, IL, USA), Duncan’s multiple range test was used to detect the differences among means at a significance level of P < 0.05.

In order to investigate the tissue-specific expressions of PtHKT1;5 and PtNHX1 in P. tenuiflora, we performed qRT-PCR to test the relative expressions of the two genes in shoots and roots. The results showed that PtHKT1;5 expressed mainly in roots and was barely detected in shoots (Figure 1A); however, the expression level of PtNHX1 in shoots was significantly higher than that in roots (Figure 1B).

The expressions of PtSOS1 and PtHKT1;5 in roots were investigated under different concentrations of NaCl (Figure 2). Under 25 mM NaCl, the expression of PtSOS1 showed a sharp increase and reached the peak value at 6 h which was 5.8-folds higher than that under control condition (0 h) and then a decrease trend, still 1.3-folds higher than that of control at 48 h (Figure 2A). The 150 mM NaCl significantly induced the expression of PtSOS1 only by 1.4-folds at 6 h compared to control, then it decreased to the same expression level as control at 24–48 h (Figure 2A). Notably, the expression level of PtSOS1 was always significantly higher under 25 mM NaCl than that under 150 mM NaCl from 1 to 48 h of treatments (Figure 2A).

PtHKT1;5 was induced by both 25 and 150 mM NaCl with a similar pattern: the transcription levels of PtHKT1;5 showed an increase before 6 h then a decrease trend, dropping to the control level at 48 h (Figure 2B). It is worth noting that the expression of PtHKT1;5 was induced by 2.2-folds under 150 mM NaCl while by 1.2-folds under 25 mM NaCl at 6 h compared to control (Figure 2B).

Here the relative expression level of PtNHX1 was analyzed only in shoots since above result showed that it expressed dominantly in shoots (Figure 1B). PtNHX1 in shoots were up-regulated by 25 and 150 mM NaCl with apparent different patterns (Figure 3). Under 25 mM NaCl, the transcript of PtNHX1 was significantly increased, peaking at 1 h and 1.9-folds higher than control, then dropped to control level at 24 h after treatment (Figure 3). Under 150 mM NaCl, the transcript of PtNHX1 was rapidly up-regulated and reached a maximum level at 6 h, 7.1-folds higher than control, and maintained significantly 5.4- and 5-folds higher levels at 24 and 48 h after treatment, respectively (Figure 3). In general, the transcript of PtNHX1 under 150 mM NaCl was much higher than that under 25 mM NaCl from 1 to 48 h of treatments, especially, 2.6-, 5.8-, and 5.1-folds higher at 6, 24, and 48 h after treatments, respectively (Figure 3).

Plasma membrane Na⁺/H⁺ transporter SOS1 play important roles in Na⁺ transport. It has been found that AtSOS1 was expressed mainly in roots and significantly induced by NaCl (Shi et al., 2000, 2002b). Similar expression pattern was observed for O. sativa OsSOS1 (Martínez-Atienza et al., 2007), Triticum aestivum TaSOS1 (Xu et al., 2008), Phragmites australis PhaNHA1 (Takahashi et al., 2009), Thellungiella salsuginea ThSOS1 (Oh et al., 2009), etc. Our previous studies showed that PtSOS1 exhibited a higher expression level in roots (Guo et al., 2012), and the present study also showed that its expression in roots was strongly up-regulated by NaCl (Figure 2A). Shi et al. (2002b) found that AtSOS1 preferentially expressed in parenchyma cells at the xylem/symplast boundary of roots, the encoding protein functions in loading Na⁺ into xylem for controlling Na⁺ delivery to the shoots and storage in leaf mesophyll cells in A. thaliana, and the atsos1 mutant accumulated less Na⁺ in shoots than the Wild Type (WT) under moderate salinity. Similar result was obtained from the xerophyte Z. xanthoxylum: ZxSOS1-silenced plants accumulated more Na⁺ in roots but less in shoots than WT under 50 mM NaCl, indicating that ZxSOS1 was involved in long-distance transport and spatial distribution of Na⁺ among plant tissues (Ma et al., 2014). In the present study, the expression of PtSOS1 displayed a rapid and persistent increase trend under mild salinity, but an instantaneously (only at 6 h) increase under severe salt treatment (Figure 2A), suggesting that PtSOS1 functions in delivery Na⁺ to shoots for osmotic adjustment by loading Na⁺ into the xylem mainly under 25 mM NaCl (Shi et al., 2002b; Guo et al., 2012; Yuan et al., 2015). While under 150 mM NaCl, excessive Na⁺ was accumulated in shoots, which in turn restricted long-distance Na⁺ transport from roots to shoots by reducing Na⁺ loading into the xylem (Guo et al., 2012).

HKT-like proteins are known to play significant roles in regulating Na⁺ and K⁺ transport and maintaining their homeostasis in higher plants (Schachtman and Schroeder, 1994; Munns and Tester, 2008; Horie et al., 2009). HKT transporters encoded by HKT1;5-like gene were identified as Na⁺ transporters and mediate Na⁺ retrieval from xylem in rice (Ren et al., 2005) and wheat (Byrt et al., 2007). It is noteworthy that OsHKT1;5 is preferentially expressed in the parenchyma cells surrounding the xylem vessels of roots and the voltage-clamp analysis showed that OsHKT1;5 mediates Na⁺ exclusion from leaves by removing Na⁺ from the xylem sap in roots to prevent Na⁺ over-accumulation in shoots (Ren et al., 2005). Moreover, Sunarpi et al. (2005) reported that AtHKT1, renamed as AtHKT1;1, selectively unloaded Na⁺ directly from xylem vessels to XPCs and thus reduced Na⁺ content in xylem vessels in roots and leaves, thereby playing a crucial role in protecting leaves from Na⁺ toxicity. Similar results were observed for Nax2 (TmHKT1;5-A) and Kna1 (TaHKT1;5-D) involved in limiting massive Na⁺ transport from xylem to leaves (Munns et al., 2012). TmHKT1;5-A and TaHKT1;5-D were both expressed in the roots but not in the leaves of T. monococcum and T. aestivum, respectively, and the expression of TmHKT1;5-A was up-regulated by salinity (Byrt et al., 2007; Munns et al., 2012). Our present results also showed that PtHKT1;5 mainly expressed in roots (Figure 1A), and it was up-regulated by NaCl, especially at higher concentration (150 mM) (Figure 2B), implying PtHKT1;5 plays an important role in unloading Na⁺ from xylem to parenchyma cells in roots mainly under high concentration of NaCl. Guo et al. (2012) found that when P. tenuiflora was exposed to severe salt conditions, Na⁺ in vacuoles of its leaves reached the maximum concentration, which in turn regulated HKT to unload Na⁺ from xylem. Our results further confirmed this opinion: shoot Na⁺ accumulation in P. tenuiflora was significantly increased in P. tenuiflora under 150 mM NaCl (Wang et al., 2009), which in turn strongly induced the expression of PtHKT1;5, facilitated excessive Na⁺ unloading into XPCs of roots (Figure 2B), and consequently alleviated Na⁺ toxicity in plants.

Sequestering Na⁺ into vacuoles is one of crucial strategies for plants to reduce Na⁺ toxicity in cytoplasm under salt stress (Nass et al., 1997; Martinoia et al., 2000; Gong et al., 2005). The tonoplast Na⁺/H⁺ antiporter NHX1 is a ubiquitous transmembrane protein playing a key role in compartmentalizing Na⁺ into vacuoles to maintain Na⁺ homeostasis and thus to enhance plant salt tolerance (Shi and Zhu, 2002). Many studies revealed that the transcript level of NHX1 in shoots was observably increased during salt treatments. The transcript level of Mesembryanthemum crystallinum McNHX1 increased and reached a high and stable level in leaves but not in roots 10 days after onset of salt stress (Cosentino et al., 2010). A similar trend was observed in cotton and chrysanthemum (Wu et al., 2004; Zhang et al., 2012). Wu et al. (2011) found that ZxNHX also preferentially expressed in the leaf tissue and was significantly induced by salt treatments. In Z. xanthoxylum and D. morifolium, a positive correlation existed between the expression of NHX1 and Na⁺ accumulation in leaves when plants were exposed to salinity (Wu et al., 2011; Zhang et al., 2012). In this study, although the expression of PtNHX1 in shoots was significantly up-regulated by both 25 and 150 mM NaCl (peaked at 1 and 6 h after treatments, 1.9- and 7.1-folds higher than that of control), its expression under 150 mM NaCl was 2.6-, 5.8-, and 5.1-folds higher than that under 25 mM NaCl at 6, 24, and 48 h, respectively (Figure 3), indicating that PtNHX1 sequestrated Na⁺ in shoots mainly under higher NaCl concentration. It was proposed that under mild salinity, Na⁺ accumulation in the leaves of plants was probably below vacuole capacity for sequestering Na⁺ (Blumwald et al., 2000), therefore, in our study PtNHX1 was induced transiently (Figure 3). Under severe salinity, excessive Na⁺ was accumulated in shoots (Wang et al., 2009), here, PtNHX1 expressed higher and more persistently (Figure 3) so that a great quantity of Na⁺ could be sequestered into the vacuoles as soon as possible.

The survival of plants under salinity was attributed to many genes and a complex genetic regulatory network, including extruding excessive Na⁺ or sequestering Na⁺ into vacuoles and controlling Na⁺ long-distance transport (Shi and Zhu, 2002; Shi et al., 2002a,b; Ren et al., 2005; Apse and Blumwald, 2007; Ghars et al., 2008). These processes were controlled by corresponding Na⁺ transporters. It was confirmed that SOS1 and HKT1;5 have opposite roles in regulating Na⁺ transport from roots to shoots by mediating Na⁺ efflux and influx, respectively, across the plasma membranes of XPCs, hence, the cooperation of them contributes to Na⁺ homeostasis in the whole plant (Hauser and Horie, 2010; Guo et al., 2012; Yuan et al., 2015). Moreover, it was found that Z. xanthoxylum ZxNHX determined Na⁺ accumulation in vacuoles of mesophyll cells and controlled the opposite Na⁺ fluxes across the plasma membranes of XPCs mediated by ZxSOS1 and ZxHKT1;1 through a feedback regulation (Yuan et al., 2015). Our previous study showed that under 25 mM NaCl, P. tenuiflora maintained a significantly lower net Na⁺ uptake rate and there was no significant increase in Na⁺ accumulation (Wang et al., 2009). Under the same condition, here, PtNHX1 was induced transiently (Figure 3), indicating that Na⁺ was compartmentalized into vacuoles slowly. While under severe salt condition (150 mM NaCl), the net Na⁺ uptake rate and Na⁺ concentration increased dramatically in tissues of P. tenuiflora (Wang et al., 2009). Here, PtNHX1 expressed higher and more persistently (Figure 3), indicating that a great quantity of Na⁺ could be sequestered into vacuoles rapidly. The expressions of PtSOS1 and PtHKT1;5 in roots were significantly induced and peaked at 6 h after both 25 and 150 mM NaCl treatments, but the expression of PtSOS1 was 5.8-folds whereas that of PtHKT1;5 1.2-folds higher under 25 mM NaCl than under control condition (Figure 2). It was proposed that under mild salinity, Na⁺ accumulation in leaves is below vacuole capacity for sequestering Na⁺ by NHX1 (Blumwald et al., 2000; Wang et al., 2009), thus, Na⁺ transport ability of SOS1 outweigh that of HKT1;5 and Na⁺ could be loaded into the xylem (Guo et al., 2012; Yuan et al., 2015). On the contrary, the transcript of PtSOS1 increased by only 1.4-folds whereas that of PtHKT1;5 by 2.2-folds in roots 6 h after plants were treated with 150 mM NaCl (Figure 2), suggesting that under severe salinity, Na⁺ accumulation reached or even exceeded vacuole capacity for sequestering Na⁺ in shoots and the transport ability of HKT1;5 overwhelm SOS1 so that Na⁺ could be unloaded from xylem into XPCs (Guo et al., 2012; Yuan et al., 2015).