Effects of NaCl and silicon on ion distribution in the roots, shoots and leaves of two alfalfa cultivars with different salt tolerance

Abstract

The effects of exogenous NaCl and silicon on ion distribution were investigated in two alfalfa (Medicago sativa. L.) cultivars: the high salt tolerant Zhongmu No. 1 and the low salt tolerant Defor. The cultivars were grown in a hydroponic system with a control (that had neither NaCl nor Si added), a Si treatment (1 mmol L⁻¹ Si), a NaCl treatment (120 mmol L⁻¹ NaCl), and a Si and NaCl treatment (120 mmol L⁻¹ NaCl + 1 mmol L⁻¹ Si). After 15 days of the NaCl and Si treatments, four plants of the cultivars were removed and divided into root, shoot and leaf parts for Na⁺, K⁺, Ca²⁺, Mg²⁺, Fe³⁺, Mn²⁺, Cu²⁺ and Zn²⁺ content measurements. Compared with the NaCl treatment, the added Si significantly decreased Na⁺ content in the roots, but notably increased K⁺ contents in the shoots and leaves of the high salt tolerant Zhongmu No.1 cultivar. Applying Si to both cultivars under NaCl stress did not significantly affect the Fe³⁺, Mg²⁺ and Zn²⁺ contents in the roots, shoots and leaves of Defor and the roots and shoots of Zhongmu No.1, but increased the Ca²⁺ content in the roots of Zhongmu No.1 and the Mn²⁺ contents in the shoots and leaves of both cultivars, while it decreased the Ca²⁺ and Cu²⁺ contents of the shoots and leaves of both cultivars under salt stress. Salt stress decreased the K⁺, Ca²⁺, Mg²⁺ and Cu²⁺ contents in plants, but significantly increased Zn²⁺ content in the roots, shoots and leaves and Mn²⁺ content in the shoots of both cultivars when Si was not applied. Thus, salt affects not only the macronutrient distribution but also the micronutrient distribution in alfalfa plants, while silicon could alter the distributions of Na⁺ and some trophic ions in the roots, shoots and leaves of plants to improve the salt tolerance.

Key words:

• alfalfa
• distribution
• ion
• NaCl
• silicon
• stress

INTRODUCTION

Soil salinization is a worldwide problem that markedly reduces food production. Approximately 20% of irrigated agricultural land is adversely affected by salinity (Viswanathan et al. 2005), which is one of the major abiotic stresses that limit plant growth. Reduction of growth response to salinity is usually attributed to either ion cytotoxicity (mainly because of Na⁺, Cl⁻ and SO²⁻ ₄) and/or low external osmotic potential (Munns and Termeat 1986). Ion cytotoxicity is caused by the replacement of K⁺ by Na⁺ in biochemical reactions and conformational changes, and by the loss of function of proteins as Na⁺ and Cl⁻ penetrate the hydration shells and interfere with the noncovalent interaction between their amino acids (Zhu 2002). Salinity also affects nutrient balance in plant tissues (Pessarakli et al. 1991). Increased salt treatment causes Na⁺ and Cl⁻ contents to rise and K⁺, Ca²⁺ and Mg²⁺ contents to diminish in a number of plants (John et al. 2003b; Khan et al. 1999, 2000; Khan 2001). However, it is still unclear how salinity affects the micronutrient content and composition of plants. Hu and Schmidhalter (2001) declared that micronutrients are generally less affected by salt stress than macronutrients. However, salinity increased the Zn²⁺ content in the roots and leaves of pepper (Cornillon and Palloix 1997) and the Zn²⁺, Cu²⁺ and Mn²⁺ contents in wheat and rice (Alpaslan et al. 1998). Concentration of Fe³⁺, Mn²⁺ and Zn²⁺ in the leaves of zucchini increased with rising salinity, but Cu²⁺ decreased (Villora et al. 2000). In addition to this, Sanchez-Raya and Delgado (1996) reported that salinity reduced the Fe³⁺ and Mn²⁺ contents in sunflower seedlings.

Silicon (Si), which is abundant in the soil, is a major constituent of many plants, but its roles in plant biology have been poorly understood (Liang 1999). Although Si has not been listed among the generally essential elements of higher plants, there have been reports of interactions between Si supply and the responses of members of the Poaceae to biotic and abiotic stresses (Epstein 1994; Gong et al. 2006; Kaya et al. 2006; Liang et al. 1996; Rodrigues et al. 2003). Matoh et al. (1986) reported that silicate at 0.89 mmol L⁻¹ reduces the translocation of Na⁺ to the shoots and increases dry matter production of salt-stressed rice plants compared to the control. Liang (1999), Yeo et al. (1999) and Gong et al. (2006) claimed that the addition of Si was found to reduce Na⁺ in shoots and roots of salt-stressed plants. However, fewer reports have examined the effects of Si on the distribution of trophic ions in roots, shoots and leaves of leguminous plants under salt stress.

Alfalfa, a leguminous plant, often suffers from a significant reduction in biomass under severe salt stress and different cultivars respond differently to salt stress (Al-Khatib and Collins 1994). We hypothesize that Si alters the distributions of Na⁺ and some trophic ions in alfalfa plants to improve the salt tolerance in salt stress environments. Thus, the present study was conducted to determine the effect of Si and NaCl on the growth of two alfalfa cultivars, the content change and distribution of ions such as Na⁺, K⁺, Ca²⁺, Mg²⁺, Fe³⁺, Mn²⁺, Cu²⁺ and Zn²⁺ in roots, shoots and leaves for two cultivars under salt or salt and Si treatment, and to determine the possible mechanisms of Si-enhancement of salt tolerance in alfalfa.

MATERIALS AND METHODS

Two alfalfa (Medicago sativa L.) cultivars, Zhongmu No. 1 (high salt tolerance) and Defor (low salt tolerance) (Wu 1999), were used in this study. The seeds were sterilized with 6% sodium hypochlorite solution for 5 min. Following germination in a sand medium at 25/20°C for 8 h/16 h in a dark room, four seedlings fixed into the holes of a foam quadrat were transplanted into plastic vessels (13 cm high, 28 cm wide, 35 cm long). The vessels were wrapped with aluminum foil to minimize irradiation-induced heating and to suppress algal growth. Each vessel contained 4.4 L of a nutrient solution composed of: 2.5 mmol L⁻¹ Ca(NO₃)₂, 2.5 mmol L⁻¹ KNO₃, 1 mmol L⁻¹ MgSO₄, 0.5 mmol L⁻¹ (NH₄)H₂PO₄, 2 × 10⁻⁴ mmol L⁻¹ CuSO₄, 1 × 10⁻³ mmol L⁻¹ ZnSO₄, 0.1 mmol L⁻¹ ethylenediaminetetraacetic acid (EDTA) Fe Na, 2 × 10⁻² mmol L⁻¹ H₃BO₃, 5 × 10⁻⁶ mmol L⁻¹ (NH)₄Mo₂₇O₄, and 1 × 10⁻³ mmol L⁻¹ MnSO₄. The plants were grown in a growth chamber with 45% relative humidity, alternative temperatures of day and night of 30°C and 25°C, 13 h light with 450 µmol m⁻² s⁻¹ from bio-sodium lamps. Plants were cultivated hydroponically in the standard nutrient solution for 15 days. Then, four treatments of the cultivars with applications of 1.0 mmol L⁻¹ Si, 120 mmol L⁻¹ NaCl and 120 mmol L⁻¹ NaCl + 1.0 mmol L⁻¹ Si, in addition to a control, each with four replications, were established. The NaCl and Si concentrations were those used according to Liang et al. (1996) in barley. Plants were exposed to salinity by adding NaCl to the growth medium in 60 mmol L⁻¹ increments every 12 h, until the final concentrations of 120 mmol L⁻¹ were reached. Silicon treatments began by adding potassium silicate (K₂SiO₃), and additional K⁺ introduced by K₂SiO₃ was subtracted from KNO₃, and the resultant nitrate loss was supplemented with dilute nitric acid (Zhu et al. 2004). The pH of the nutrient solution was adjusted to 6.0 daily using 0.01 mol L⁻¹ KOH and/or H₂SO₄ (Zhu et al. 2004). The nutrient solutions were renewed every 7 days and deionized water was added daily to replace the water lost by transpiration. The nutrient solutions were continuously aerated using an air pump.

Fifteen days after the NaCl and Si treatments began, four plants were removed from each treatment and separated into roots, shoots and leaves for parameter measurements. Dry biomass was measured at 60°C for 48 h. Finely ground oven-dried tissue (0.1 g) was digested overnight with 25 mL of 0.1 mol L⁻¹ HNO₃ at room temperature (John et al. 2003a). Contents of ion in the acid extract were determined using an inductive coupled plasma emission spectrometer (ICP, OPTIMA 3,300DV, Perkin Elmer, Kleve, Germany).

Root, shoot and leaf dry weights and the ion content in plant roots, shoots and leaves were determined by anovas using SPSS software (10.0). Differences between treatment means were separated using the least significant difference (LSD) at a 0.05 probability level.

RESULTS

As shown in Table 1, the dry weights of the shoots of the two cultivars and the leaves and roots of Defor were drastically reduced by NaCl stress; however, this inhibition was alleviated to various extents by Si supplement. Under salt stress, the added Si apparently increased the dry weight of shoots of both cultivars. While under non-salt stress, the added Si did not significantly increase the dry weight of the roots, shoots and leaves of both cultivars.

The Na⁺ content in roots, shoots and leaves was markedly higher for both cultivars in the NaCl treatment

Table 1 Dry weight (mg plant−1) of roots, shoots and leaves of Zhongmu No. 1 and Defor grown under the four treatments after 15 days

Treatments	Zhongmu No.1				Defor
	Control	Si	NaCl	NaCl + Si	Control	Si	NaCl	NaCl + Si
Root	67.6a ± 3.5	69.7a ± 10.8	56.0ab ± 9.9	64.9a ± 7.4	64.3a ± 5.9	58.9a ± 7.3	33.1c ± 7.9	41.2bc ± 6.3
Shoot	83.2a ± 19.0	91.4a ± 11.2	58.3b ± 1.7	77.6a ± 12.9	73.9a ± 8.0	75.7a ± 14.6	32.2d ± 3.8	41.4c ± 3.0
Leaf	128.7ab ± 6.9	140.1a ± 11.6	100.0b ± 23.5	129.8ab ± 22.3	111.3b ± 14.8	114.4b ± 14.5	60.5c ± 12.6	60.7c ± 7.9
Data are expressed as mean ± standard deviation of four replicates (n = 4). Means marked with a different letter in the same line are significantly different using a least significant difference test (P < 0.05).

Table 2 Sodium (mmol g−1) contents in roots, shoots and leaves of Zhongmu No. 1 and Defor grown under the four treatments after 15 days

Treatments	Zhongmu No.1				Defor
	Control	Si	NaCl	NaCl + Si	Control	Si	NaCl	NaCl + Si
Root	0.04c ± 0.00	0.07c ± 0.01	1.09a ± 0.10	0.82b ± 0.08	0.05c ± 0.01	0.11c ± 0.01	1.18a ± 0.07	0.79b ± 0.09
Shoot	0.02d ± 0.00	0.04d ± 0.00	0.76bc ± 0.09	0.74bc ± 0.09	0.02d ± 0.00	0.16d ± 0.03	1.06a ± 0.08	1.00ab ± 0.09
Leaf	0.02c ± 0.00	0.03c ± 0.00	0.40b ± 0.06	0.37b ± 0.04	0.02c ± 0.00	0.14c ± 0.02	0.63a ± 0.05	0.60a ± 0.10
Data are expressed as mean ± standard error of four replicates (n = 4). Means marked with a different letter in the same line are significantly different using a least significant difference test (P < 0.05).

than in the control without Si treatment. Adding Si significantly decreased Na⁺ contents in roots, but had no significant effect on Na⁺ contents in shoots and leaves in either cultivar (Table 2). It is also noted that under salt stress without Si treatment, less Na⁺ accumulation was found in the roots, shoots and leaves of the high salt tolerant cultivar compared with the low salt tolerant cultivar. Under salt stress without Si treatment, Na⁺ content in roots was higher than that in shoots and leaves in both cultivars; and a lower ratio of root Na⁺ to shoot Na⁺, and of shoot Na⁺ to leaf Na⁺, was found in the low salt tolerant cultivar compared with the high salt tolerant cultivar.

The salt treatment reduced K⁺ contents in roots, shoots and leaves of both cultivars without Si in the nutrient solution (Table 3). Yet, the K⁺ content in the high salt tolerant cultivar shoots and leaves under salt treatment with Si was much higher than the K⁺ content of shoots and leaves of the low salt tolerant cultivar under salt treatment with Si.

The Ca²⁺ contents of the roots, shoots and leaves of salt-treated plants were still lower than the Ca²⁺ content of plants not treated with salt and Si, although no significant difference was observed in the roots of Zhongmu No. 1 and the shoots of Defor (Table 3). Adding Si increased the root Ca²⁺ content in Zhongmu No. 1, but decreased the shoot and leaf Ca²⁺ contents of both cultivars under salt stress (Table 3).

As shown in Table 3, the Mg²⁺ contents of roots, shoots and leaves in both cultivars were reduced by NaCl stress. However, this inhibition was not significantly alleviated by Si supplement. It is interesting to note that higher root K⁺, Ca²⁺ and Mg²⁺ contents were found in the low salt tolerant cultivar compared with the high salt tolerant cultivar under NaCl stress without additional Si; however, lower shoot and leaf K⁺, Ca²⁺ and Mg²⁺ contents were found in the low salt tolerant cultivar compared with the high salt tolerant cultivar under the same condition.

For Zhongmu No. 1, salt stress significantly decreased Fe³⁺ contents of roots, shoots and leaves irrespective of Si treatment. For Defor, salt stress also decreased the Fe³⁺ contents of roots, but slightly increased shoot and leaf Fe³⁺ contents irrespective of Si treatment. However, the addition of Si did not significantly affect Fe³⁺ and Mg²⁺ contents in roots, shoots and leaves for both cultivars under salt stress (Tables 3,4). Shoot and leaf Fe³⁺ contents were much higher for Defor than for Zhongmu No. 1 under salt stress (Table 4).

Irrespective of Si treatment, salt stress decreased the Mn²⁺ contents in roots and leaves of both cultivars, but significantly increased their shoot Mn²⁺ content, with slight differences between them. However, the addition of Si significantly increased Mn²⁺ contents in shoots and leaves of both cultivars under salt stress (Table 4).

Salt led to a decline in the Cu²⁺ content in roots and shoots of both cultivars, with no significant differences in root, shoot and leaf Cu²⁺ contents between the two cultivars (Table 4). The addition of Si reduced Cu²⁺ contents in shoots and leaves in both cultivars and in roots in Defor under salt stress.

Contrary to other nutrient ion changes, Zn²⁺ contents in the roots, shoots and leaves significantly increased in both alfalfa cultivars under salt stress irrespective of Si treatment. However, the degressive effects of Si on the Zn²⁺ contents in roots, shoots and leaves in both cultivars were observed under salt stress. Under salt stress alone, the Zn²⁺ content in leaves of Zhongmu No. 1 was much higher than that in Defor (Table 4).

DISCUSSION

Salinity is a major environmental factor that limits plant growth and crop productivity (Asish and Anath 2005). Improvement of salt tolerance by the addition of Si has been reported in several plants (Ahmad et al. 1992; Al-Aghabary et al. 2004; Bradbury and Ahmad 1990; Liang et al. 1996; Matoh et al. 1986). In the present study, the growth of salt-stressed alfalfa plants is improved by the addition of Si. It has been reported that the alleviation of salt toxicity by Si addition results from a reduction in the Na⁺ content in the shoots of rice (Gong et al. 2006; Matoh et al. 1986; Yeo et al. 1999), Prosopis juliflora (Bradbury and Ahmad 1990) and barley (Liang 1999).

In the present study, Defor (low salt tolerant) had higher Na⁺ contents in roots, shoots and leaves compared with Zhongmu No. 1 (high salt tolerant), and the latter cultivar showed higher exclusion and/or efflux capacity in the NaCl without Si treatment. Both cultivars maintained the highest Na⁺ contents within their roots; and the low salt tolerant cultivar had a lower ratio of root Na⁺ to shoot Na⁺ and shoot Na⁺ to leaf Na⁺ than the high salt tolerant cultivar under salt stress without Si, which indicated that transportation of Na⁺ from root to shoot and from shoot to leaf were two important physiological contributions to salt tolerance. The experiments of testing rice (Matoh et al. 1986), wheat (Ahmad et al. 1992) and barley (Liang 1999) showed that adding Si to the nutrient solution diminished the Na⁺ content in the shoots. However, our study showed that the Na⁺ contents in roots of both cultivars only decreased significantly with added Si under salt stress. This result suggested that the additional Si in the nutrient solution might inhibit alfalfa roots from absorbing Na⁺ under salt stress. Thus, our present study supports the idea that Si deposition and polymerization in alfalfa endodermis

Table 3 Potassium (mmol g−1), Ca (mmol g−1) and Mg (mmol g−1) contents in roots, shoots and leaves of Zhongmu No. 1 and Defor grown under the four treatments after 15 days

Treatments	Zhongmu No.1				Defor
	Control	Si	NaCl	NaCl + Si	Control	Si	NaCl	NaCl + Si
K contents
Root	1.87a ± 0.07	1.66b ± 0.15	0.91c ± 0.07	0.77cd ± 0.10	1.49b ± 0.14	1.55b ± 0.08	0.57d ± 0.03	0.92c ± 0.02
Shoot	1.72b ± 0.06	1.89a ± 0.04	1.49c ± 0.03	1.70b ± 0.08	1.72b ± 0.04	1.76ab ± 0.05	1.25d ± 0.02	1.36cd ± 0.07
Leaf	1.64ab ± 0.03	1.53c ± 0.04	1.52c ± 0.01	1.67a ± 0.02	1.47c ± 0.04	1.55bc ± 0.04	1.15d ± 0.02	1.24d ± 0.05
Ca contents
Root	0.12ab ± 0.01	0.12ab ± 0.01	0.09d ± 0.00	0.11bc ± 0.00	0.13a ± 0.01	0.11abc ± 0.01	0.10cd ± 0.01	0.11bcd ± 0.01
Shoot	0.41a ± 0.01	0.45a ± 0.02	0.36b ± 0.02	0.36bc ± 0.02	0.36bc ± 0.02	0.41a ± 0.01	0.32cd ± 0.00	0.29d ± 0.01
Leaf	0.54a ± 0.02	0.55a ± 0.03	0.49ab ± 0.01	0.45bc ± 0.02	0.55a ± 0.03	0.53a ± 0.02	0.41c ± 0.02	0.33d ± 0.02
Mg contents
Root	78.10a ± 6.74	76.57a ± 4.06	60.79c ± 5.04	67.50abc ± 5.89	77.83a ± 6.22	81.15a ± 4.31	67.96ab ± 2.63	69.94ab ± 1.51
Shoot	84.99a ± 3.72	86.01a ± 1.74	67.71cd ± 4.35	72.20bc ± 5.62	81.02ab ± 1.78	86.43a ± 4.63	59.18d ± 2.18	67.83cd ± 1.13
Leaf	90.53ab ± 3.08	97.09a ± 2.83	73.82c ± 3.97	74.86c ± 5.21	99.35a ± 3.84	101.99a ± 2.67	71.02c ± 5.03	79.71bc ± 5.86
Data are expressed as mean ± standard error of four replicates (n = 4). Means marked with a different letter in the same line are significantly different using a least significant difference test (P < 0.05).

Table 4 Iron (mmol g−1), Mn (µmol g−1), Cu (µmol g−1) and Zn (µmol g−1) contents in roots, shoots and leaves of Zhongmu No. 1 and Defor grown under the four treatments after 15 days

Treatments	Zhongmu No.1				Defor
	Control	Si	NaCl	NaCl + Si	Control	Si	NaCl	NaCl + Si
Fe contents
Root	37.71a ± 3.57	30.77ab ± 3.62	19.88cd ± 2.64	26.46bc ± 1.95	37.24a ± 3.40	28.49b ± 0.52	18.85cd ± 0.42	17.93d ± 0.43
Shoot	1.44abc ± 0.21	1.01cd ± 0.09	0.92d ± 0.13	0.95d ± 0.08	1.17bcd ± 0.20	1.19bcd ± 0.23	1.54ab ± 0.14	1.78a ± 0.25
Leaf	2.71a ± 0.27	2.35ab ± 0.07	1.99b ± 0.13	2.08b ± 0.16	2.50ab ± 0.18	2.39ab ± 0.25	2.74a ± 0.23	2.35ab ± 0.20
Mn contents
Root	3.19a ± 0.13	2.63ab ± 0.21	2.08b ± 0.23	2.58ab ± 0.33	3.03a ± 0.49	2.92ab ± 0.37	2.72ab ± 0.37	2.76ab ± 0.21
Shoot	0.49de ± 0.01	0.46e ± 0.01	0.56cd ± 0.01	0.66ab ± 0.02	0.46e ± 0.02	0.55cd ± 0.02	0.62bc ± 0.03	0.69a ± 0.05
Leaf	1.74a ± 0.13	1.67a ± 0.07	1.33c ± 0.09	1.64ab ± 0.04	1.38bc ± 0.10	1.75a ± 0.14	1.33c ± 0.09	1.68a ± 0.04
Cu contents
Root	0.23a ± 0.02	0.19b ± 0.01	0.15cd ± 0.00	0.15cd ± 0.02	0.21ab ± 0.01	0.21ab ± 0.00	0.18bc ± 0.00	0.14d ± 0.01
Shoot	0.22a ± 0.01	0.21ab ± 0.01	0.19b ± 0.01	0.15c ± 0.00	0.19b ± 0.01	0.21ab ± 0.00	0.18b ± 0.01	0.15c ± 0.01
Leaf	0.17b ± 0.01	0.17b ± 0.01	0.17b ± 0.01	0.14c ± 0.01	0.17b ± 0.01	0.20a ± 0.01	0.18ab ± 0.01	0.17b ± 0.00
Zn contents
Root	1.14ab ± 0.02	1.17ab ± 0.03	1.32a ± 0.06	1.19ab ± 0.12	1.23ab ± 0.08	1.09b ± 0.05	1.29a ± 0.08	0.87c ± 0.02
Shoot	0.88b ± 0.04	0.97ab ± 0.01	1.13a ± 0.08	1.02ab ± 0.06	0.67c ± 0.05	1.02ab ± 0.10	0.99ab ± 0.03	0.95ab ± 0.07
Leaf	0.74b ± 0.04	0.75b ± 0.04	0.93a ± 0.04	0.75b ± 0.02	0.62c ± 0.01	0.74b ± 0.02	0.82b ± 0.03	0.76b ± 0.02
Data are expressed as mean ± standard error of four replicates (n = 4). Means marked with a different letter in the same line are significantly different using a least significant difference test (P < 0.05).

and rhizodermis block Na⁺ influx through the apoplastic pathway in the root (Yeo et al. 1999).

High salinity can affect essential cation uptake and nutrient balance. The present study showed that lower root, shoot and leaf K⁺, Ca²⁺ and Mg²⁺ contents were found in the low salt tolerant cultivar compared with the high salt tolerant cultivar under NaCl stress without additional Si treatment. These significant differences in cation content between cultivars were, in part, a result of the shoots’ different capacity to exclude Na⁺. The leaf Na⁺/Mg⁺ ratio is considered to be an index of Na⁺ toxicity (Jeschke 1987) and the higher values found in Defor (low salt tolerant) could impair the key cell functions. The higher leaf Na⁺/Ca²⁺ ratio in Defor could also indicate the displacement of Ca²⁺ by Na⁺ and consequent K⁺ leakage from the cytosol (Cramer 2002). A study of several species showed that low Na⁺/K⁺ ratios were favorable to the plant (Jeschke 1987). The present study indicated that Zhongmu No.1 has a better capacity to maintain a physiological cytosolic K⁺/Na⁺ ratio than Defor.

Shoot and leaf K⁺ contents for Zhongmu No.1 were notably increased in response to additional Si under salt stress. The possible mechanism causing Si to stimulate the plant to absorb K⁺ under salt stress was the activation of H⁺-ATPase in the membranes (Liang 1999). The interaction between Si and Ca²⁺ varies with the plant species and the stressed environments to which the plants are exposed (Liang 1999). In this study, added Si decreased the shoot and leaf Ca²⁺ concentration in Defor under salt stress. This corresponds with the results from a study of rice (Ma and Takahashi 1993), which suggested that this was the result of a low transpiration rate caused by Si. Inanaga and Okasaka (1996) reported that Ca and Si could combine and be compounds in cell walls of rice shoots. We assumed that the increasing root Ca²⁺ content of both cultivars by added Si under salt stress may result from combining the compounds of Ca and Si in the cell walls. However, further study is required to determine whether or not Ca and Si might combine into compounds in alfalfa roots.

Little knowledge is available about micronutrient uptake and translocation in plants under salt stress. Therefore, we have also determined the contents of micronutrients such as Fe³⁺, Mn²⁺, Cu²⁺ and Zn²⁺ in roots, shoots and leaves under salt stress. The Fe³⁺ contents of shoots and leaves in Zhongmu No. 1 markedly decreased under salt stress without Si treatment, but for Defor, salt stress slightly enriched shoot and leaf Fe³⁺ contents irrespective of Si treatment. In addition, the Fe³⁺ contents in shoots and leaves were much higher in Defor than in Zhongmu no.1 under salt stress. These differences in Fe³⁺ contents in shoots and leaves between the two cultivars signify that different transport mechanisms of Fe³⁺ may exist in the two cultivars. The addition of Si did not markedly affect Fe³⁺ and Mg²⁺ contents of roots, shoots and leaves for both cultivars under salt stress, which indicates that these important cations in both cultivars are not altered by additional Si under salt stress.

Salinity induced an increase in Mn²⁺ contents in the shoots of the plants of both alfalfa cultivars without Si treatment. This result was consistent with previous findings for wheat, rice (Alpaslan et al. 1998) and zucchini (Villora et al. 2000). However, Cramer et al. (1991) determined that salinity reduced the Mn²⁺ contents in shoots of barley. We also found that salinity reduced the contents of Mn²⁺ in roots and leaves of both cultivars without Si treatment. The addition of Si significantly increased the Mn²⁺ contents of shoots and leaves of both cultivars under salt stress. Salt led to a decline in Cu²⁺ contents in roots and shoots (Table 4), but an increase in Zn²⁺ in all organs of both cultivars under salt stress without Si. Cramer et al. (1985) reported that NaCl treatment could increase Zn²⁺ translocation into leaves, which is emphatically pronounced due to a higher plasma membrane permeability caused by Na⁺. In experiments using rice, wheat (Alpaslan et al. 1998), zucchini (Villora et al. 2000) and strawberry (Ece and Atilla 2005), salinity led to an increase in Zn²⁺ content in the root parts of plants. In most cases, salinity increased the Cu²⁺ content in plant organs, for example, in rice and wheat (Alpaslan et al. 1998), pepper (Cornillon and Palloix 1997) and zucchini (Villora et al. 2000). However, Esechie and Rodriguez (1999) reported that salinity decreased Cu²⁺ content in alfalfa leaf. In the present study, additional Si decreased Zn²⁺ and Cu²⁺, but increased the Ca²⁺ and Mn²⁺ content in plants under salt stress, which might be attributed to metal cations, including Ca²⁺ and Mn²⁺, inhibiting plant uptake of Cu²⁺ and Zn²⁺ by competition on the carrier site (Safwan et al. 2003).

In conclusion, salinity reduced plant growth in both cultivars. However, the added Si apparently increased the dry weight of shoots of both cultivars under salt stress. Summing up the results of the study, it is possible to conclude that physiologically salt tolerance of alfalfa is associated with a low accumulation of Na⁺, but high K⁺, Ca²⁺, Mg²⁺ and Zn²⁺ contents in shoots and leaves. Salt stress decreased the K⁺, Ca²⁺, Mg²⁺ and Cu²⁺ contents in plants, but significantly increased Na⁺ and Zn²⁺ contents in the roots, shoots and leaves and Mn²⁺ in the shoots of both cultivars when Si was not applied. However, compared with the NaCl treatment, the added Si significantly decreased the Na⁺ content in the roots, but notably increased the K⁺ contents in the shoots and leaves of the high salt tolerant Zhongmu No.1 cultivar. Applying Si to both cultivars under NaCl stress increased the Ca²⁺ content of roots of Zhongmu No.1 and the Mn²⁺ contents in the shoots and leaves of both cultivars, while it decreased the Ca²⁺ and Cu²⁺ contents of the shoots and leaves of both cultivars. Thus, we conclude that Si may act to alleviate salt stress in alfalfa by inhibiting Na⁺ uptake by roots and affecting the uptake, transportation and/or distribution of some nutritional ions in plants under salt conditions.

ACKNOWLEDGMENTS

We thank Dr Zhang Yingjun for constructive comments on the manuscript and Cheng Wen for checking the language. This study was supported financially by the educational committee of Beijing and construction project of key lab and subject of Beijing (project numbers XK100190552 and JD100190537).