Soil salinity is a major constraint of crop productivity because it reduces yield and limits expansion of agriculture onto uncultivated land (Flowers and Yeo 1995). Trifolium repens L. (white clover) is one of the most commonly cultivated legume forages due to its high protein content which makes its productivity essential for sheep meat, beef cattle and dairy production industries (Wang et al. 2010). In addition, the high nitrogen fixing ability of white clover makes it adaptable to a wide range of soil and environmental conditions and combines well with many perennial grasses (Tang et al. 2010). However, T. repens is a salt-sensitive species (Rogers et al. 1993) and suffers a yield decline at soil salinity levels of around 2 dS m−1 (Mehanni and Repsys 1986). Therefore, the objective of cultivating a salt tolerant T. repens has long been pursued. To overcome the toxicity of salinity in plant species, many curative and management practices have been adopted. One of these methods is to apply exogenous silicon (Si) in salinity soil.

Si is the second most abundant element on the surface of the earth and can particularly alleviate both biotic and abiotic stresses in higher plant, although it has not been listed among the generally essential elements of plants (Epstein 1994; Liang et al. 1996). Si is known to be involved in plant protection against salt stress. Yeo et al. (1999) reported that under salt stress, the deposition and polymerization of Si in the endodermis and rhizodermis blocked Na+ influx through the apoplastic pathway in the roots of rice, thereby reducing the entrance of Na+ without significantly affecting the overall transpiration flux and plant growth. Romero-Aranda et al. (2006) found that silicate crystals deposited in the epidermal cells form a barrier that reduces water loss through cuticles and improves water relation in tomato plant tissue which contributes to salt dilution within plants, mitigating salt toxicity effects. In addition, the increased uptake and transport of K+ and decreased uptake and transport of Na+ from roots to shoots in plants may be attributable to Si-induced stimulation of the root plasma membrane (PM) H+-ATPase under salt stress (Liang et al. 2005, 2006). This indicated that restriction of Na+ influx either into root cells or into the xylem stream is one way of regulating the optimum cytosolic K+/Na+ ratio (Chinnusamy et al. 2005). Taken together, the responses vary depending not only on Na and Si concentrations but also on plant species. However, Si involved in alleviating Na toxicity in T. repens has not been explored.

The objective of this study was to reveal the role of Si in the resistance to salt stress. In view of this, effects of Si on the growth parameters and Na+, K+ accumulations in T. repens plants exposed to 0 or 120 mM NaCl with or without 1.5 mM Si was investigated.

Materials and Methods

Seeds of white clover were sterilized with sodium hypochlorite solution (5 %) for 5 min and rinsed thoroughly with distilled water, then were germinated on moistened filter paper for 7 days at 25°C in the dark. When the plumule emerged, seedlings were selected for uniformity and were subsequently transferred into plastic containers (7 cm × 7 cm × 7 cm) filled with 0.6 L modified Hoagland solution (2 mM KNO3, 1 mM Ca(NO3)2, 0.5 mM MgSO4, 0.25 mM NH4H2PO4, 11.9 μM Fe-citrate, 11.5 μM H3BO3, 1.25 μM MnSO4, 0.2 μM ZnSO4, 0.075 μM CuSO4, and 0.025 μM NH4Mo7O24) for 4 weeks. The nutrient solution was adjusted to pH 6.8 with 0.1 M HCl/NaOH, and was renewed every 2 days. Plants were grown in a growth chamber at a day/night cycle 16/8 h, at 22/18°C, respectively, a relative humidity between 50 % and 60 %, and a light intensity of 120 μmol m−2 s−1 PAR.

Si was added to the Hoagland solution as K2SiO3. KNO3 was reduced, in the preparation of the Hoagland solution, proportionally to the K supply provided by K2SiO3, and additional K introduced by K2SiO3 was subtracted from KNO3 and the resultant nitrate loss was supplemented with dilute nitric acid. Notably, in a preliminary experiment, adding different concentrations of Si (0, 0.5, 1.0, 1.5 and 2.0 mM) partially alleviated the negative impacts of salt stress on growth in white clover, but 1.5 mM Si was found to be the most effective. Therefore, 1.5 mM was used in all subsequent experiments. A randomized block design consisting of a control and three treatments (1.5 mM Si, 120 mM NaCl and 1.5 mM Si plus120 mM NaCl) was used. NaCl concentrations were incrementally increased with 30 mM NaCl day−1 increments until final concentrations (120 mM NaCl) were achieved. Four-week-old white clover seedlings were harvested after 7 days of receiving the silicon and salinity treatments. Three plants for white clover were pooled in each replicate. The experiment was repeated five times.

The relative growth rate (RGR) of whole plants was calculated using the formula RGR = (lnWj − lnWi)/Δt, where Wj and Wi are final (after 7 days of treatments) and initial (before treatments) dry weights (DW), respectively, and Δt is the time elapsed (days) between the two measurements; initial dry weight was determined before treatments (Martínez et al. 2005).

At the end of each treatment, plant roots were washed twice for 8 min in ice-cold 20 mM CaCl2 to exchange cell wall-bound Na+ and shoots rinsed in deionized water to remove surface salts (Wang et al. 2007). Harvested plants were washed thoroughly with running distilled water, separated into shoots and roots; fresh weights were determined immediately and then oven dried at 80°C for 3 days to obtain dry weights. Na+ and K+ were extracted from dried plant tissue in 100 mM acetic acid at 90°C for 2 h and ion analysis was performed using an atomic absorption spectrophotometer (AA-6300C, Shimadza, Kyoto, Japan).

According to Wang et al. (2009), the net Na+, K+ uptake rate was calculated for each time interval, as net Na+, K+ uptake rate (nmol g RFW−1 min−1) = (C2 − C1)/R2/(t2 − t1), where C is Na+, K+ content in whole plant, R2 is the root fresh weight (RFW), and t is the time at two harvests, respectively. Sub-indexes 1 and 2 are indicated before treatments and after 7 days of treatments, respectively.

Selective transport capacity for K+ over Na+ (ST) value indicates the net capacity of selection for transport of K+ over Na+ from roots to shoots (Guo et al. 2012). ST values were estimated according to the following equation as described by Guo et al. (2012) where ST = (K+/Na+ in shoots)/(K+/Na+ in roots).

All the data are presented as means with standard errors (SE). Statistical analyses, one-way ANOVA, and Duncan’s multiple range tests were performed by statistical software (Ver.13.0, SPSS Inc., Chicago, IL, USA).

Results and Discussion

As shown in Table 1, application of Si alone did not influence the DW and RGR in plants compared to the control. Added NaCl alone significantly reduced shoots and roots DW by 54.47 % and 60.34 %, respectively, compared to the control. However, when plants were treated with NaCl plus Si, the reductions of shoots and roots DW were only 26.98 % and 24.59 % relative to the additional Si alone, respectively. Based on these results, in the presence of NaCl, the addition of Si significantly increased RGR by 69.71 % in T. repens plants. Furthermore, in the presence of NaCl, added Si decreased roots Na+ concentrations by 48.19 % and increased shoots K+ concentration by 48.40 % in T. repens plants (Fig. 1). Meanwhile, NaCl plus Si caused a 114.11 nmol g RFW−1 min−1 reduction in net Na+ uptake rate and 61.71 nmol g RFW−1 min−1 increase net K+ uptake rate compared to NaCl treatments alone, leading to enhanced whole plant K+/Na+ ratios in T. repens (Table 2). Similar results were also reported in other plants exposed to salt stress (Liang 1999; Ashraf et al. 2010). The ameliorative effect of added Si in alleviating deleterious effects of NaCl could be related to Si being irreversibly precipitated as amorphous silica (SiO2·nH2O) in cell walls and lumens, leading to Si inducing a reduction of Na+ in transpiration rate (Matoh et al. 1986) and to the partial blockage of the transpirational bypass flow (Yeo et al. 1999).

Table 1 Effects of Si on DW of shoots and roots and RGR in white clover under salt stress
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Fig. 1
figure 1

Effects of Si on Na+ and K+ concentration in white clover under salt stress. Four-week-old white clover seedlings were exposed to 0 or 120 mM NaCl with or without 1.5 mM Si for 7 days. Values are mean ± SE. Each value is a mean of five replicates (n = 5). Bars indicate ±SE. Mean values (±SE) with different letters are significantly different at p < 0.05 (Duncan’s test)

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Table 2 Effects of Si on whole plant K+/Na+ ratio, net Na+, K+ uptake and ST value in white clover under salt stress
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On the other hand, Si reduced Na+ uptake by roots due to Si-induced stimulation of the root PM H+-ATPase under salt stress (Liang et al. 2005, 2006), which contributed to increased K+ uptake and -transport in plants (Liang 1999). It is known that the proton motive force created by PM H+-ATPases drives Na+ effluxes from plant cells through Na+/H+ antiporters in the PM (Blumwald et al. 2000). The most likely candidate is a putative PM Na+/H+ antiporter (SOS1), and SOS1 also is the major component of selective transport capacity for K+ over Na+ (ST) in plants (Guo et al. 2012). Interestingly, we observed the ST values in white clover plants treated with NaCl plus Si were 1.60 times higher than that of NaCl treatment alone (Table 2). As for higher plants, cation/H+ antiporters are the main transport systems involved in Na+ and K+ homeostasis through PM associated transport processes (Olías et al. 2009), thereby contributing to maintaining higher K+/Na+ selectivity in plants (Hasegawa et al. 2000). Therefore, Si might stimulate activity of PM H+-ATPase which would provide additional energy needed for PM Na+/H+ antiporter and exclude more Na+ from the plant cell. This would further enhance the selective transport capacity for K+ over Na+, hence regulating K+/Na+ homeostasis in plants under salt stress.

In conclusion, Si could alleviate Na toxicity in white clover plants subjected to salt stress due to enhance the selective transport capacity for K+ over Na+, hence regulating K+/Na+ homeostasis and improving the plant growth.