Effects of EDTA and Zn Stress on Physiological Characteristics of Medicago sativa L.

Nan HOU, Hui JING, Shuhe WEI, Huiping DAI*, Xiaona HOU*

1. College of Biological Science & Engineering, Shaanxi Province Key Laboratory of Bio-resources, Shaanxi University of Technology, Hanzhong 723001, China; 2. Key Laboratory of Pollution Ecology and Environment Engineering, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China

Abstract [Objectives] The paper was better understand the mechanisms of Zn plant uptake in the presence of EDTA and to evaluate the contributions of Zn-EDTA complexes to Zn uptake. [Methods] Three alfalfa cultivars were cultivated for 60 d before exposure to 0, 250 μg Zn and 250 μg Zn +10 μg EDTA per kg soil for 50 d. Zn concentrations in tissues were analyzed by flame atomic absorbance spectrometry. Subsequently, Zn amount per plant, translocation factor (TF) and bio-concentration factor (BCF) were calculated. Nonenzymatic compounds in tissues were analyzed spectrophotometrically. [Results] Application of Zn+ EDTA expressively increased biomass of different tissues of three alfalfa cultivars. Among the three alfalfa cultivars, Medicago sativa ssp. displayed the highest Zn concentration in tissues, the largest Zn amount in aerial parts, and the highest BCF in aerial parts under Zn+EDTA exposure. Under Zn+ EDTA stress, increases in free proline in roots, stem, and leaves of M. sativa ssp. were found. Inhibited O2·- production in stem and leaves, increases in soluble sugar, but decreases in soluble protein were observed in M. sativa ssp. [Conclusions] M. sativa ssp. is superior to other two cultivars for Zn phyto-remediation, and its well-coordinated physiological changes under Zn+EDTA exposure confer the great Zn tolerance of this cultivar.

Key words Zn-EDTA; Zn uptake; Medicago sativa L.

1 Introduction

Metal pollution of soil is widespread across the globe. Acute poisoning may occur in humans due to a high intake of toxic metals[1], while their presence in the ecological cycles may cause chronic illness due to metal bio-accumulation. The heavy metal zinc (Zn) rapidly accumulates in soil and water because of anthropogenic activities (e.g.mining, smelting and fertilization with sewage sludge) that lead to Zn contamination[2-3]. Zn is an essential element for plants that can be taken up by roots. Conventional remediation methods usually involve excavation and removal of soil layer, physical stabilization and/or washing of soils with strong acids, which are not cost effective and ecofriendly, and will change the soil properties[4]. In contrary to this, plant-based clean up technology (phytoremediation) offers a number of advantages on traditional clean up methods[5]. Thus, selection of appropriate plants is a crucial requirement for efficient phytoremediation and a plant with ability to accumulate several metals in a significant manner, high biomass production, having a short life span, and easy to harvest is suitable for phytoremediation. Several plants have been identified for Zn hyperaccumulation, includingArabidopsishalleri,Noccaea(formerlyThlaspi)caerulescens, andSedumalfredii. However, in these plants, the amount of accumulated Zn is limited by slow growth and low biomass. Thus, fast-growing plants, such as alfalfa species, which have a large aboveground biomass, a deep root system and the ability to accumulate intermediate metal concentrations, have been proposed for the phytoremediation of Zn-contaminated soil[2].

It is well documented that phytoextration of heavy metal is dependent on metal availability and several chelating agents are reported to enhance metal availability in soil matrix[6]. Among chelators, EDTA is used most widely because it has high efficiency in removing heavy metals[7]. Even though, the effect of EDTA regarding Zn accumulation and uptake through plants has not been clearly described in the past. EDTA enhanced metal uptake and root to stem translocation of Zn was observed in many plants[8]. In our earlier studies, we have identified high Zn accumulation of different alfalfa cultivars for contrasting soils sufficient in Zn[2]. To elucidate Zn distribution and physiological regulation mechanisms in the roots, stem and leaves of alfalfa cultivars were exposed to 0, 250 μg Zn and 250 μg Zn+10 μg EDTA per kg soil. The objectives of this study were as follows: (i) to elucidate the physiological mechanisms by which alfalfa responds to Zn exposure and; (ii) to examine whether EDTA addition alleviates Zn accumulation in alfalfa. For these purposes, Zn accumulation, physiological regulation mechanisms of alfalfa plant involved in Zn uptake and translocation, and antioxidants were analyzed in roots, stem and leaves of different alfalfa cultivars, in order to understand their physiological responses to Zn and EDTA exposure.

2 Materials and methods

2.1 Plant materials and Zn exposureThe experiments were performed in the orchard of Shaanxi University of Technology, Hanzhong (33°34′ N, 107°28′ E), China. Alfalfa (Medicagosativassp., ‘Vitoria’ alfalfa,M.sativacv.) seeds were purchased from Northwest A&F University. Seeds were surface-sterilized with 0.1% (W/V) HgCl2for 10 min. Alfalfa (M.sativassp., ‘Vitoria’ alfalfa,M.sativacv.) seeds were sown in plastic pots filled with mixture of peat/vermiculite (W/W1:1) and grown in greenhouse with 12/12 h light/dark, day/night temperature of 25/15 ℃ and 500 μmol/(m2·s) photosynthetic active radiation. The nutrient solution contains 5 mM Ca(NO3)2·4H2O, 5 mM KNO3, 2 mM MgSO4·4H2O, 1 mM KH2PO4, 0.1 mM EDTA-Fe, 461 mM H3BO3, 9.11 mM MnCl2·4H2O, 0.321 mM CuSO4·5H2O, 0.761 mM ZnSO4·7H2O and 0.51 mM H2MoO4·H2O. After 2-month cultivation, the plants were treated by the nutrient solution with 0, 250 μg Zn and 250 μg Zn+10 μg EDTA, respectively. Six replicates were designed for each treatment.

2.2 Sampling and measurementsThe seedlings were harvested after 50 d of exposure. The roots were immersed in the 5 mM CaCl2for 15 min and then the whole plants were rinsed with deionized water. The leaves and stems were divided and dried in an air oven at 80 ℃ until a constant dry weight was gained. The effects of Zn treatment on the dry weight of roots, stem and leaves were assessed by two-way ANOVA.

2.3 Analysis of Zn concentrationSamples of leaves, stem and roots were separately dried at 80 ℃ in oven and ground to powder in a laboratory mill. 0.3 g of plant materials were mineralized in HNO3/HClO4mixture (5∶1,V/V)[9]and clarified with ultrapure water for measurements of total Zn by an atomic absorption spectrophotometer (Atomic Absorption Spectrometer 373, Perkin-Elmer, Norwalk, CT, USA).

2.4 Bioconcentration factor and translocation factorBCF (bioconcentration factor) was defined as the ratio of metal concentration in plant roots or aerial tissues to that in the soil or solution[10]. The translocation factor (TF) indicated the ability of plants to translocate Zn from the root to the shoot[10-11]. TF was calculated as the Zn concentration in aerial tissues of a plant divided by the Zn concentration in roots[10].

2.5 Determination of malondialdehyde (MDA), soluble protein, soluble sugar, O2·-and proline contentThe MDA, soluble protein, soluble sugar and O2·-concentration was measured according to the method proposed by Daietal.[2]. The absorbance rate at 530 nm was measured and the O2·-level was calculated according to a standard curve. Proline content was determined spectrophotometrically according to the method proposed by Daietal.[2].

2.6 Statistical analysisA completely randomized design was used for each time point with 6 replicates. Data were subjected to analysis of variance (ANOVA) to examine the effects of time, treatment and organs. Statistical analysis was conducted by using STATISTICA 5.1 software (Statsoft Inc., United States of America). Separation of means was carried out by using Fisher’sLSDtest atP<0.05 andP<0.01 significance levels.

3 Results

3.1 BiomassThe reaction of different tissue biomass is shown in Table 1. Application of Zn stress increased the biomass of roots, stem and leaves ofM.sativassp., however, Zn stress decreased different tissues biomass of ‘Vitoria’ alfalfa andM.sativacv. Compared with Zn stress alone, the addition of EDTA expressively increased different tissues biomass of three alfalfa cultivars.

Table 1 Biomass of three alfalfa cultivars grown with 0 (CK), Zn, and Zn+EDTA for 50 d

3.2 Zn concentration, BCF, and TFTo evaluate the ability of plant to accumulate Zn, bioconcentration factor (BCF), translocation factor (TF) and concentrations of Zn in roots, stem and leaves of different alfalfa cultivars were analyzed (Table 2). Zn accumulation in roots showed more accumulation than stem and leaves at 250 μg/kg Zn concentration and exposure 50 d (Table 2). Compared with Zn stress alone, the addition of EDTA (10 μg) remarkably increased the Zn accumulation in leaves. Among the three alfalfa cultivars,M.sativassp. displayed the highest Zn concentrations in the analyzed tissues of Zn-exposed plants. Zn concentration in leaves ofM.sativassp. was 41.2 mg/kg DW, which is above the threshold value defined for Zn hyperaccumulation. To further evaluate the ability of different alfalfa cultivars to accumulate Zn, their BCF and was determined (Table 2). Compared with Zn stress alone, the BCFs successively wereM.sativassp.>M.sativacv.> ‘Vitoria’ alfalfa after the addition of EDTA. BCF was significantly higher in the leave ofM.sativassp. than those of the other two cultivars. BCF in the roots varied from 1.91 to 2.83 in the three cultivars and relatively larger BCFs were found in ‘Vitoria’ alfalfa andM.sativassp. than inM.sativacv. The translocation factor (TF) is often used to evaluate the ability of plants to translocate heavy metals from roots to the aerial tissues. Compared with Zn stress alone, TF varied from 0.34 to 1.08 among the three alfalfa cultivars after the addition of EDTA(Table 2). TF were significantly higher inM.sativacv. than those in the other two cultivars. TF were significantly higher inM.sativacv. (up to 1.08) andM.sativassp. (up to 0.89) than that in the leaves of ‘Vitoria’ alfalfa.

Table 2 Effect of Zn and Zn +EDTA on the Zn concentrations, bio-concentration factor and translocation factor of three alfalfa cultivars of Medicago sativa after 50 d of exposure

3.3 MDA and O2·-MDA, an indicator for membrane lipid oxidation, is often used to evaluate the ability of plants to tolerate abiotic stresses. Interestingly, no effects of Zn exposure on MDA concentrations were found in these alfalfa plants (Fig.1). However, compared to those under Zn exposure alone, cultivar-specific differences in MDA concentrations were found after the addition of EDTA. MDA concentrations in roots ofM.sativacv. were higher than those inM.sativassp., and ‘Vitoria’ alfalfa. Similarly, MDA concentrations in stem and leaves differed among cultivars. The highest MDA concentrations were found in stem and leaves of ‘Vitoria’ alfalfa and the lowest in stem ofM.sativassp. and leaves ofM.sativacv. ROS such as O2·-is often accumulated in plants exposed to heavy metals. Compared to those under Zn exposure alone, O2·-concentrations were significantly elevated by 18.3% in roots of ‘Vitoria’ alfalfa after the addition of EDTA, but remained unchanged in roots ofM.sativassp.(Fig.2). Compared to those under Zn exposure alone, O2·-concentrations were significantly inhibited in stem and leaves of ‘Vitoria’ alfalfa,M.sativacv. andM.sativassp. after the addition of EDTA. Among the analyzed alfalfa cultivars, O2·-production was the highest in roots, stem, and leaves of ‘Vitoria’ alfalfa.

Fig.1 MDA concentration of three alfalfa cultivars grown with 0 (CK), Zn, and Zn+EDTA for 50 days

Fig.2 O2·- concentration of three alfalfa cultivars grown with 0 (CK), Zn, and Zn+EDTA for 50 d

4 Discussion

Although Zn is a nutrient element for alfalfa, excess Zn in the soil can be toxic for these plants[2,12]. In this study, we have integrated plant biomass and Zn accumulation capacity to assess Zn tolerance in the three alfalfa cultivars. Among the three alfalfa cultivars, compared to those under Zn exposure alone, the Zn concentrations in roots and leaves ofM.sativassp. showed that the alfalfa cultivar had the ability to accumulate relatively higher Zn in roots and leaves than the other two cultivars after the addition of EDTA (Table 1). Furthermore, compared with Zn stress alone, the addition of EDTA and the largest Zn amount in aerial parts ofM.sativassp. suggested that the alfalfa cultivar is superior to the other two alfalfa cultivars for Zn phyto-remediation because aboveground Zn accumulation is ideal for easy harvests[12]. Additionally, the addition of EDTA, the highest BCF in aerial parts and a relatively high TF ofM.sativacv. (Table. 1) also demonstrated that this alfalfa cultivar has a stronger capacity to accumulate and translocate Zn to aboveground parts (stem and leaves) of plants than the other analyzed alfalfa cultivars.

To avoid ROS-induced injury, O2·-need to be scavenged by antioxidants (e.g., free proline, soluble sugar, soluble protein and antioxidative enzymes). Free proline is an important nonenzymatic antioxidative compound in bean seedlings under Zn stress[13]. It was also noticed that application of EDTA and Zn stress increased the accumulation of free proline in both roots and leaves of three alfalfa cultivars. Application of EDTA remarkably improved both proline and lipid peroxidation levels with increasing Zn concentration, which is determinative of a correlation between ROS generation (hydroxyl radicals mostly) and ROS scavenging by proline. Our results are in similarity with the findings of Najeebetal.[14], who observed similar increase in content of proline under EDTA applications along with metal stress.

5 Conclusion

It can be deduced that EDTA plays a vital role to enhance growth and development of alfalfa exposed to heavy metal stress. In the study, we have identified that toxicity of Zn can cause reduction in the growth, biomass, Zn accumulation, bioconcentration factor, translocation factor, and oxidant detoxification capacity. However, EDTA addition significantly improves the Cd uptake and translocation, osmotic regulation, membrane lipid biosynthesis and oxidant detoxification capacity. Interestingly, in view of these results, it can be concluded that EDTA has favorable role on different alfalfa cultivars grown under Zn toxicity. Our results also demonstrated that alfalfa might uptake a substantial amount of toxicant like Zn and it is also considered as hyper accumulator plant. Additionally, our experiment was carried out in soil conditions. In order to evaluate the improving role of EDTA more effectively in phytoextraction of Zn from polluted soils, more soil-based environment study is necessary.