1. Introduction
Soil contamination with trace metals (TM), caused by industrial sewage and agricultural production, has become a worldwide problem [
1]. The contamination of TM negatively affects the environment as well as decreases agricultural productivity and eventually causing a serious health risk to the consumers [
2,
3]. Through many natural processes, the contamination of soil is determined, especially by anthropogenic, lithogenic and pedogenic factors [
4]. Plants absorb and accumulate TM, such as Cu, Mn, Fe and Zn from the soil, which is required for the normal growth and development at trace level since they are the catalytic and structural components of some enzymes and proteins [
5,
6]. In contrast, TM (Al, Cr, Pb and Cd) and even those essential for growth and metabolism, may affect different metabolic and physiological processes at high concentrations, such as hindering of functional groups of essential molecules, e.g., enzymes, polynucleotides, transport systems for essential ions and nutrients, the substitution of important ions from cellular sites, inactivation and denaturation of enzymes and distraction of cell and organelle membrane integrity [
7]. Among toxic TM, Cd is one of the most mobile non-recyclable pollutants, which is taken up by plants easily and distributed to the above-ground parts where it can accumulate to high levels. Thereby, it can easily enter the food chain [
8] and become very detrimental to humans as well as animal’s health [
9].
The accumulation of Cd greatly varies amongst plant species and cultivars [
10]. Root accumulates a higher amount of Cd than shoot [
11]. Several studies revealed that Cd might hamper with different chemical processes such as impregnation through ammonia, compounds of nitrogen and different microbial contact and distresses yield of the plants [
12]. According to Ahmad et al. [
13], the accumulation of Cd brings complex changes in plants at physiological, biochemical and genetic levels. In plants, Cd is not an essential nutrient element and caused toxic effects at high concentrations. Excessive Cd produces free radicals and reactive oxygen species (ROS), which can oxidize proteins, lipids, DNA and carbohydrates, hence disturbing some physical and biological processes in plants [
14]. Toxic effects of Cd are shown in inhibition of photosynthesis and oxidative stress leading to membrane damage as well as altered cellular metabolism [
15]. Likewise, the presence of extreme amounts of Cd in the environment causes a variety of plant responses, such as stunted growth, leaf chlorosis, reduced plant fresh and dry biomass and even death [
16,
17]. Moreover, an excessive amount of Cd may cause inhibition of various enzymes activities and decreased the uptake of nutrient elements (Fe, Cu, Zn and Mn), which can weaken the passage of these elements from roots to the above-ground organs, thus generated to reduce the photosynthetic pigments [
18,
19]. The primary site of Cd toxicity is unknown; however, the mitochondrial electron transfer chain of plant cells is expected to be one of the major targets of Cd toxicity and is the site of the most rapid Cd-induced ROS production [
20].
Underlying the toxic effects of Cd, plants adopt different tolerance mechanisms which include active exclusion, vacuolar restoration, retaining in the roots and immobilization of cell walls and complexation by binding metal to low molecular-weight proteins [
21,
22]. It has been shown that the decreasing of Cd accumulation by exclusion in roots of
Thlaspi arvense consults greater tolerance in the Cd-tolerance ecotype [
23]. One persistent mechanism for TM metals detoxification within plants is chelation by a ligand. In wheat, Cd binds to the Cd–phytochelatins complexes (Cd–PCs), reducing free Cd
2+ in the cytosol, and the Cd–PCs complexes are in turn transported into the vacuole or out of the cell by ATP binding cassette transporters [
24]. The retention of Cd in the cell wall of the roots is another mechanism of tolerance which might be performed by the carboxyl groups of cell wall proteins or the thiols groups of soluble proteins and non-protein thiols in root cells [
25]. Studies have shown that vacuolar compartmentalization prevents the free moments of Cd ions in the cytoplasm and restricts them to limited areas [
26]. Besides, transcriptomic in combination with genetic approaches as different to physiological and biochemical studies, may help in understanding the mechanism of metal tolerance. Such as tolerant species in comparison with non-tolerant ones revealed high expression of genes, e.g.,
HMA3 might play a role in vacuolar sequestration and metals detoxification [
27]. Furthermore, studies have shown that vitamin E (alpha-tocopherol), an important antioxidant, is crucial to oxidative stress tolerance induced by Cd in
Arabidopsis thaliana, while a chromatin remodeling factor, named
OXS3, is recognized for Cd tolerance of
Brassica juncea cDNA library in
Schizosaccharomyces pombe [
20].
Chickpea (
Cicer arietinum L., family: Fabaceae) is an important pulse crop amongst the legume crops and ranked third after beans and pea plants, which surpasses the production of 8.40 million tons/year in the world [
28]. Chickpea is a rich and cheap source of protein, fats and carbohydrates in developing countries and used as a green manure and fodder for animals worldwide [
13]. Earlier studies revealed that legume crops are less resistant to Cd toxicity than cereals and grasses and encounter severe suppression of biomass production even at very low levels of Cd [
29]. Though, compared with other species, little information is available concerning the ability of tolerance and accumulation in chickpea cultivars under Cd stress. Therefore, the present work has been conducted to screen the chickpea cultivars under Cd stress. The main objectives of the present experiment include: (1) to understand the effect of Cd on growth, and Cd accumulation of chickpea cultivars, (2) to understand differential responses of Cd stress among different chickpea cultivars and (3) to screen Cd-tolerant and non-tolerant chickpea cultivars for further studies.
2. Materials and Methods
2.1. Chickpea Seeds and Growth Conditions
The seeds of chickpea cultivars (NC234 (NC2), ICCV89310 (IC8) and ICCV89323-B (IC8-B)), were provided for this study by the Crop Genetic Resources Institute, Xinjiang Academy of Agricultural Science, Urumchi, China.
A pot experiment was conducted in the Key Laboratory of Plant Ecology, Department of Botany, Northeast Forestry University, Harbin, China. The selection of the three cultivars was entirely based on the germination percentage and vigorous growth among ten chickpea cultivars (data not shown).
2.2. Plants Materials and Cadmium Treatments
Healthy seeds of chickpea were surface sterilized with 75% ethanol solution for 20 s and transferred to 6% NaOCl for 10 min to make them free from infection followed by washing several times with sterile distilled water. The sterilized seeds were then kept in Petri dishes having some distilled water for 24 h under fluorescent white light in a
germinator. For further growth, one day old ten surface-sterilized seeds were sown in trays containing vermiculite and watered with distilled water to maintain the moisture content efficiently. After 12 days of growth, uniform seedlings were transferred hydroponically to a 1 L plastic container (6 plants per pot) for more 20 days using 20% of a modified Hoagland’s nutrient solution [
30]. The compositions of the modified nutrient solution were (Ca(NO
3)
2·4H
2O, 1180 mg L
−1; KNO
3, 505 mg L
−1; MgSO
4·7H
2O, 493 mg L
−1; NH
4NO
3, 80 mg L
−1; KH
2PO
4, 68 mg L
−1, FeEDTA, 22.5 mg L
−1; H
3BO
3, 2.86 mg L
−1; MnCl
2·4H
2O, 1.81 mg L
−1; ZnSO
4·7H
2O, 0.22 mg L
−1; H
2MoO
4·H
2O, 0.09 mg L
−1; Na
2MoO
4·2H
2O, 0.12 mg L
−1 and CuSO
4·5H
2O, 0.051 mg L
−1).
The growth room was climate-controlled with a temperature ranged 22−25 °C, 70% relative humidity and 14-h photoperiod. The nutrient solution was renewed every four days. On the 20th day of seedling transplanting, Cd was supplemented to the nutrient medium as CdCl
2 in two concentrations, 25 and 50 µM (Cd1 and Cd2, respectively). Simultaneously, control plants were grown in the same nutrient solution without Cd supplementation. In total, the seedlings remained in the nutrient solution for 25 days, including the Cd stress. Cd concentrations were selected based on previously predicted toxicity in chickpea [
17,
31].
2.3. Measurements of Plant Growth and Cd Accumulation
On the 25th day, the seedlings were collected and soaked with 20 mM Na2-EDTA solution for 15 min, washed with sanitized water 3–4 times to take away Cd on the root surfaces and separated into roots and shoots. The length of roots and shoots along with total plant height was done by the measuring tape. To calculate the fresh (FW) and dry (DW) weights of the biomasses, an automatic weighing scale was used. For DW determination, samples were oven-dried at 120 °C for 20 min and continued for three days in the oven at 60 °C. From individual groups of treatments all the biomasses were assessed.
Subsequently, for biochemical analysis, the oven-dried samples were ground with mortar and pestle into powder. The grinded samples were weighed (0.3 g) and digested with a mixture of acids (HNO3 + HClO4 in the ratio of 5:1 (v/v)). The concentrations of Cd were then quantified by ICP-OES (Optima-8300 DV; PerkinElmer, Inc., Waltham, MA, USA). At least three times every experiment was repeated.
The accumulation of Cd in plant tissues, total Cd accumulation and distribution proportion of Cd in roots were calculated as follows [
22]:
Cd accumulation = biomass (DW) × Cd concentration in plant tissues,
Total Cd accumulation = Cd accumulation in root + Cd accumulation in shoot,
Cadmium distribution proportion in root = Cd accumulation in root/total Cd accumulation, while the translocation factor (TF) of Cd from root to shoot, bioconcentration factor (BCF) and bioaccumulation coefficient (BAC) were determined according to the method of Amin et al. [
32]. Where Cd
s and Cd
r represents the Cd concentration in shoot and root respectively, while Cd
ns reveals the Cd concentration in nutrient solution.
TF = Cds/Cdr,
BCF = Cdr/Cdns,
BAC = Cds/Cdns.
2.4. Growth Tolerance Indices
Tolerance index (TI) was calculated separately based on dry plant biomass, containing root biomass, shoot biomass and total plant dry biomass according to Wiszniewska et al. [
33] with slight modification. Where TI
r, TI
s and TI
tp indicates the tolerance indices of root, shoot and total plant, respectively; Cd
r, Cd
s and Cd
tp represents the dry weight of root, shoot and total plant under Cd stress, respectively; while MV
rc, MV
sc and MV
tpc reveals mean values of root, shoot and total plant dry weight in control, respectively.
2.5. Shoot Water Content Measurement
Water content (WC) was determined after the Cd stress period. For calculating the WC, the FW of the examined plant shoots were recorded. Subsequently, the samples were oven-dried at 60 °C for 72 h and their DW were determined. WC in the shoot was calculated as follows [
34]:
2.6. Statistical Analysis
The experiment was accomplished following Completely Randomized Design (CRD) with nine treatments and it was repeated three times under the same condition. The data were subjected to analysis of variance (ANOVA) using COSTAT computer package ver. 6.4 (CoHort software in Monterey, CA, USA). The significance of variances between means was taken using the Duncan’s Multiple Range Test (DMRT) at p < 0.05. The values were displayed as the mean ± standard error (SE), (n = 3).