Introduction

From a global view after climate, soil crust is considered as one of the important parts of the human environment. Soil is not only considered as the living environment for terrestrial organism, especially human, but also a unique environment for the other living organisms such as plants. Soil contamination by inorganic pollutants such as heavy metals is one of environmental hazards that threaten human life today. Soil pollution causes loss of vegetation, reducing the plants growth and development, rangeland degradation, soil erosion, and finally desertification. Excessive accumulation of heavy metals in soil reduces biological activity and fertility resulting in reduced yield and quality loss of products that can be dangerous to human health or animal consumption (Stingu et al. 2012).

Cadmium (Cd) is the most prevalent pollution heavy metal in soil which is toxic to human and animal. Cadmium is used in some kinds of batteries, plastic byproducts, fertilizers, and pesticides (Amini et al. 2005). The total amount of Cd in agricultural soils should not exceed 0.4–0.5 mg kg−1 and higher values reflect the excessive use of phosphatic fertilizers as well as sewage sludge (Bjelkova et al. 2011; Stingu et al. 2012). During the past 30 to 40 years, using of fertilizers containing phosphoric acid (superphosphate and ammonium phosphate) to agricultural soil in the Iran has increased. Most of agricultural lands in Iran received 100–300 kg superphosphate per hectare annually for more than four decades (Jalali and Khanlari 2008). Amini et al. (2005) assessed 255 top soil samples from central Iran; they reported the total Cd concentration in most of tested samples were more than recommended thresholds of 0.8 mg kg−1. Therefore, ways to remove or degrade this pollutant has been a great concern. Phytoremediation is a green, simple, eco-friendly, sustainable, and cost-effective remediation technology that uses plants to remove and degrade contaminants from the soil. In this regard, Medicago species has been widely adopted for the phytoremediation of polluted soils with heavy metals (Lopez et al. 2005; Fan et al. 2008; Wei and Pan 2010). Medicago sativa L. has shown the ability to hyperaccumulate heavy metals in its tissues (Angle and Linacre 2005) and to grow well in highly contaminated soils (Amer et al. 2013).

Arid and semi-arid regions comprise more than 60% of Iran (Fakhari and Sadeghi 2016). The average annual rainfall in Iran is about 251 mm and recently, due to increased drought risks, need for crops with higher water stress tolerance has increased strongly (Sadeghi and Robati 2015). Medicago scutellata (L.) Mill. is a Fabaceae widely cultivated for it capacity to produce high-quality forage (Fakhari and Sadeghi 2016). Annual Medicago is found in almost all regions of Iran. More than 556,000 ha of Medicago species are grown as continuous cropping in Iran. This suggests that these plants are appropriate for Iranian pastures. Medicago has several annual species, such as Medicago rigidula L., Medicago polymorpha L., and Medicago scutellata L., which can produce high numbers of seeds. It establishes relatively easily but its early growth in autumn is rapid and erect, making it susceptible to overgrazing (Sadeghi and Fakhari 2016). The barrel medic is adapted to different soil texture types, from sandy to clay, and particularly to well-drained neutral-to-alkaline soils with a pH of 6 to 8; it is suited to warm temperate conditions, especially Mediterranean-type climates, has 250–600 mm annual rainfall with dry hot summers and mild moist winters, and is intolerant of winter frosts. It has poor regrowth for a second harvest, but has a good ability to self-regenerate from seeds present in the soil seed bank (Fakhari and Sadeghi 2016). Legumes are a good choice for phytoremediation since they have the capability of N2 fixation (Amer et al. 2013). A review of current literature indicated that no research, to best of our knowledge, has yet tested the co-exposure of Cd and water stress on the phytoremediation ability of M. scutellata. Therefore, the present study was designed with the intention to evaluate the biochemical response of M. scutellata to Cd–water stress co-exposures and its utilization potential for phytoremediation.

Materials and methods

Study site and plant material

This research was conducted as a factorial experiment based on completely randomized design in a greenhouse at the College of Agriculture, Shiraz University (29° 43′ N and 52° 35′ W), Shiraz, Iran, during 2014. The temperatures of day and night in the greenhouse were set at 28 and 22 °C. The seeds of Medicago scutellata were prepared from Pakan Bazr Co. and stored at 5 °C.

Experimental design

Silty clay loam soil collected from the surface layer (0–30 cm) of soils (fine mixed, mesic Typic Calcixerpets soil) in the College of Agriculture was selected for the pot experiments. Some of soil characteristics are presented in Table 1 and the heavy metals of original soil were as follows: absorbable Cd, 0.2 mg kg−1; absorbable Pb, 0.4 mg kg−1; absorbable Cu, 1.31 mg kg−1; and absorbable Ni, 0.15 mg kg−1.

Table 1 Chemical and physical properties of the pot soil
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Silty clay loam soil gathered from the top layer (0–30 cm) of soils in the College of Agriculture was selected for the pot experiments. Some of soil characteristics are presented in Table 1. Soil samples were air dried and grounded to pass 2-mm nylon sieve prior to use. The soils were spiked with sulfuric acid cadmium salt at the rate of 0, 5, 25, 50, 100, and 125 mg kg−1 and incubated for 2 weeks. After incubation, each plastic pot was filled with 4 kg of the treated soil. Ten seeds were planted in each pot. After emergence, six plants per pot were kept. Pots were watered at field capacity. Water stress was imposed on the plants 20 days after planting by applying different irrigation regimes (100% as control, 75% and 50% field capacity (FC)) for 3 months. At the end of the trial, different plant organs (roots, shoot, leaves) were separated and sampled for analysis.

Analysis and measurement

The harvested plant parts were washed completely with distilled water, dried at 70 °C, and weighed. For the measurement of mineral elements, plant samples were dried at 550 °C for 4 h to obtain ashes, then extracted with 2 M HCl, filtered through Whatman No. 42 filter paper, and analyzed for Cd, Fe, and Zn by an atomic absorption spectrophotometer (model Perkin Elmer 4110) (Azizian et al. 2011). Potassium concentrations were determined by flame photometry (INESA, FP6410, Shanghai, China). Available metals were determined using diethylene-triamine-pentaacetic acid (DTPA) buffered at pH 7.3. Cadmium was determined by atomic absorption spectrophotometry (AAS).

For the proline measurements, plant’s part samples were grounded in liquid nitrogen. The concentration of proline was estimated based on the acid-ninhydrin method (Bates et al. 1973) by spectrophotometer with minor modifications (Fakhari and Sadeghi 2016).

Crude protein was estimated from nitrogen by multiplying total N by 6.25, according to the Kjeldahl method described in the Association of Official Analytical Chemists (2002). The translocation factor (TF) from root to shoot was calculated as follows (Su et al. 2014):

$$ \mathrm{TF}=\frac{\left[\mathrm{Cd}\right]\mathrm{shoot}}{\left[\mathrm{Cd}\right]\mathrm{root}} $$

Statistical analysis

This experiment was conducted as a factorial experiment based on a completely randomized design with three replications. Analysis was performed with SAS Program Version 9.1.3 (2004). Different cadmium levels were the first factor and included 0, 5, 25, 50, 100, and 125 mg kg−1. Second factor was the water stress, which had three levels (100, 75, and 50% FC).

Results

Some physiochemical properties of the selected soil for the pot experiment are presented in Table 1. The tested soil has a medium texture (silty clay loam) with no acidity and salinity problem and its cadmium (Cd) level was very low.

ANOVA (Table 2) showed that the interaction effect of the cadmium application and water stress ([CD] × water stress) was significant on all the measured factors of Medicago scutellata, with the exception of potassium concentration in root and proline content, at 1% probability level. However, the main effect of cadmium application and water stress was significant at 1% probability level about potassium concentration in root and proline content (Table 2).

Table 2 Analysis variance of the effect of cadmium [Cd] and water stress on measured factors of M. scutellata
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According to Table 3, with increasing the level of cadmium of up to 100 mg kg−1, Cd concentration in roots, stems, and leaves increased but then decreased at the level of 125 mg kg−1 and the Cd concentration of different organs (roots, stems, and leaves) reduced with increasing water stress level from 100 to 50% FC (Table 3). The highest Cd average concentration of root was observed at 100 mg kg−1 and 100% FC which increased 54-fold compared with the control (0 mg kg−1 cadmium and 100% FC). Cadmium concentration of roots from 100 to 125 mg kg−1 decreased 7% (Table 3). The highest Cd average of shoot and leaves belonged to 100 mg kg−1 and 100% FC which increased 35-fold and 58-fold compared with the control, respectively (Table 3). The average concentrations of Cd in leaves, stems, and roots were 63.16, 30.12, and 20.45 mg kg−1, respectively. In other words, the average concentrations of Cd in leaves and shoots were 3.08 and 1.47 times higher than roots, respectively. The highest amount of Cd in soil was observed at 125 mg kg−1 Cd under 50% FC which was 298 times higher than control (0 mg kg−1 cadmium and 100% FC) (Table 3).

Table 3 Cadmium concentration (mg kg−1) concentration in different organs (root, shoot, leaves) of M. scutellata at various water stress and cadmium levels
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The highest translocation factor (TF) was observed at the concentration of 50 mg kg−1 under 75% FC while the lowest one, with 50% reduction, was observed at 125 mg kg−1 under 100% FC (Table 4). In all treatments, the TF was more than 1 which reflected the high ability of M. scutellata in translocation Cd from root to shoot (Table 4).

Table 4 The effect of cadmium [Cd] and water stress on Translocation factor (TF) of M. scutellata
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According to Table 5, the average of iron (Fe) concentration in different organs (root, shoot, and leaves) decreased with the increasing Cd level from 0 to 125 mg kg−1. Under water stress, the Fe concentration of different organs significantly increased. In all tested organs, the highest Fe concentration belonged to the 0 mg kg−1 Cd and 50% FC while the lowest one was observed at 100 mg kg−1 and 100% FC (Table 5).

Table 5 Iron [Fe] concentration in different organs (root, shoot, leaves) of M. scutellata at various water stress and cadmium levels
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Table 6 showed that Zinc (Zn) concentration in different organs increased significantly with the increasing water stress level from 100 to 50% FC. Similar to Fe concentration, in all organs (roots, shoots, and leaves), the highest Zn concentration belonged to the 0 mg kg−1 Cd under 50% FC while the lowest one was observed at 100 mg kg−1 and 100% FC. Also, the average of Zn concentration in different organs decreased with increasing Cd level from 0 to 125 mg kg−1 (Table 6).

Table 6 Zinc [Zn] concentration in different organs (root, shoot, leaves) of M. scutellata at various water stress and cadmium levels
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Potassium (K) concentration of different organs significantly decreased with the increasing Cd level up to 100 mg kg−1 but increased at 125 mg kg−1 compared with 100 (Table 7). K concentration of roots decreased while in leaves and shoots increased by increasing water stress level. The highest amount of K concentration in all organs belonged to the 5 mg kg−1 Cd under 50% FC while the lowest one was observed at 100 mg kg−1 Cd and 100% FC in the case of shoots and leaves and 100 mg kg−1 and 50% FC in the case of roots (Table 7).

Table 7 Potassium [K] concentration in different organs (root, shoot, leaves) of M. scutellata at various water stress and cadmium levels
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According to Table 8, proline content increased significantly with the increasing Cd level and water stress. The highest amount of proline content was observed at 125 mg kg−1 Cd under 50% FC while the lowest one belonged to the control treatment. Unlike proline, the percentage of crude protein decreased significantly with the increasing Cd level and water stress. The highest percentage of crude protein was observed in control treatment while the lowest belonged to 125 mg kg−1 Cd and 50% FC. By consuming Cd, protein content decreased significantly (Table 8).

Table 8 Proline and crude protein content of M. scutellata at various water stress and cadmium levels
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The result of correlation analysis (Table 9) showed that the highest correlation coefficient was observed between Zn concentrations in roots with Zn concentration in shoot (0.991**). Protein had negative and significant correlation with proline and Cd concentration in different organs; however, it showed a positive and significant correlation with other measured elements (Fe, Zn, and K). K showed a negative and significant correlation with Cd and positive and significant correlation with Zn and Fe concentration in different organs. Fe and Cd showed a negative and significant correlation within different organs (Table 9).

Table 9 Correlation coefficient between the measured variables
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Discussion

In this study, the effect of different Cd levels of soil on the biochemical response of Medicago scutellata was investigated under water stress. According to the results, Cd uptake in different organs increased with increasing Cd levels up to 100 mg kg−1 while the water stress had a negative effect on Cd uptake by M. scutellata. These results are supported by Shen et al. (2002) who founded that Cd uptake of corn (Zea mays L.) increased with the increasing Cd level of soil. Azizian et al. (2011) also found the same results about Cd concentration of corn (Zea mays L.) and oat (Avena sativa L.) under different irrigation treatments. Gardea-Torresdey et al. (2000) reported that nickel uptake of (Medicago sativa L.) increased with increasing soil nickel and shoots showed to have more nickel concentration than the roots. In contrast, Bjelkova et al. (2011) observed that the most Cd was accumulated by roots in linseed (Linum usitatissimum L.) cultivars. These phenomena may be due to a difference in chemical functional groups in the plants species roots and shoots.

Suitable plants for phytoremediation should be able to prevent the transportation of heavy metals to the aerial parts and keep them at the root level thus preventing them from entering to the food chain (Stingu et al. 2012), or they are grown for industrial, non-food purpose such as using in textile or furniture industry. In our study, the average concentrations of Cd in leaves and shoots were 3.08 and 1.47 times higher than roots, respectively. The high value of translocation factor (TF) confirms the high ability of M. scutellata in translocation Cd from root to shoot. Since the main application of M. scutellata is for forage production for feeding animals, this factor should be taken into consideration. Studies have shown that cattle which graze on metal contaminated plants will accumulate the toxic metals in their bodies which could then be passed to humans (Chamberlain and Miller 1982). The standard limit of Cd is 0.1 mg kg−1 of the weight of the plant for human consumption and 10 mg kg−1 for animal consumption, while in our study, it was 63.16 mg kg−1 just in leaves.

In our study, Cd concentration of different organs (roots, stems, and leaves) reduced with increasing water stress level; similar findings were reported by Angle et al. (2003) who observed higher metal uptake as well as biomass production at higher soil moisture.

Fe, Zn, and K concentration of different organs significantly decreased with the increasing Cd level. Wong et al. (1986) observed a significant reduction in Zn and Fe by increasing Cd level and stated it can be due to the antagonistic effects of accumulation of Cd in plant tissues on the uptake of these essential metals. Yildiz (2005) reported that Zn concentration in tomato (Solanum lycopersicum L.) and corn (Zea mays L.) plant decreased by increasing Cd level. Also, similar findings were reported by Veselov et al. (2003) and Sandalio et al. (2001) in wheat (Triticum aestivum L.) and pea (Pisum sativum L.) plants. Cd toxicity in plants can be due to the interaction with essential nutrients for the plant which cab affect the balance of nutrients and reduced plants fertility. Cadmium and zinc are chemically similar; hence, they are competing with each other for absorbing by plants (Nazar et al. 2012).

Fe and Zn concentration increased by increasing water stress level in all organs and K concentration of roots decreased while in leaves and shoots increased by increasing water stress level. These results are in line with the findings of Alizadeh (2010) in corn (Zea mays L.). Osmotic advantage of K in improving cell water saturation under water stress is well documented which can explain the reason of its translocation from roots to the aerial parts under water stress (Cakmak 2005; Zhao 2000).

According to the result, proline content increased significantly with increasing Cd level and water stress. Proline acts as an osmolyte as well as an osmoprotectant under stressed condition which helps in protecting of enzymes, biological membranes, and photosynthetic apparatus from oxidative damages (Batish et al. 2006). Since proline supplies energy for growth and survival, its accumulation during stress helps the plant to tolerate the stressed condition better and easier (Gill and Toteja 2010). The observations made in this study are parallel with that in earlier studies (Shah et al. 2001; Mehta and Gaur 1999; Parida et al. 2008). According to the results, protein content decreased significantly by increasing Cd level. Cd alters the conformation of proteins such as enzymes and transporter proteins due to its strong affinity as ligands to sulfhydryl and carboxylic group (Nazar et al. 2012). Studies have shown the activity of reactive oxygen species during stress can damage biological molecules such as proteins and lipids (Molassiotis et al. 2006).

Conclusion

The forage crop M. scutellata can be considered as a Cd accumulator plant species. It also showed a good performance under the co-exposure of water stress and Cd indicated by accumulating proline and K in leaves. Its translocation factor was more than 1 which reflected the high ability of M. scutellata in translocation Cd from root to shoot. The amount of Cd accumulation in leaves is about 63 mg kg−1 which is 6 times more than standard limit for animal consumption. Since the main application of M. scutellata is for forage production for feeding animals and it does not have any remarkable non-food uses, it cannot be a good choice, due to the further transfer into the food chain. However, the ability enhancement of Cd translocation to M. scutellata shoot in higher concentrations of soil Cd indicates a great performance of the plant for Cd phytoextraction and could be introduced as Cd hyperaccumulator plant.