Genotypic variation for cadmium tolerance in common bean (Phaseolus vulgaris L.)

Ecotoxicology and Environmental Safety 190 (2020) 110178

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Genotypic variation for cadmium tolerance in common bean (Phaseolus vulgaris L.)

T

Ramin Bahmania,∗, Mahsa Modareszadehb, Mohammad reza Bihamtac a

School of Biological Sciences, Seoul National University, Seoul, 08826, South Korea Department of Molecular Biology, Sejong University, Seoul, 143-747, South Korea c Department of Agronomy, University of Tehran, Karaj, Iran b

A R T I C LE I N FO

A B S T R A C T

Keywords: Bean Cadmium Genotype Oxidative stress Plant hormones

Given the limitation of crop production in Cd-polluted areas, the identification and selection of plant genotypes tolerant to Cd stress are of great significance. In the present work, we show the existence of genotypic variation for Cd tolerance in common bean. The laboratory screening of 25 bean genotypes indicated a significant positive correlation of the mean productivity (MP) and the geometric mean productivity (GMP) with plant fresh weight both in control and Cd-treated plants. A principal component analysis further confirmed this variation and, together with other analyses, led to the selection of genotypes G-11867, Taylor, Emerson, and D-81083 as tolerant genotypes. A total of six bean genotypes with different degrees of Cd tolerance were selected, and their long-term physiological responses to Cd (0, 45, and 90 mg/kg soil) were evaluated. Increasing Cd concentrations led to higher Cd accumulation both in roots and shoots, and to significant rises in the levels of the oxidative stress biomarkers malondialdehyde (MDA), dityrosine (D-T), and 8-hydroxy-2′-deoxyguanosine (8-OH-2′-dG). Remarkable reductions in plant hormone levels and chlorophyll contents, as well as in dry and fresh weight, were observed in Cd-treated plants. Among the examined genotypes, Emerson, Taylor, and G-11867 were found to be more tolerant to Cd owing to lower Cd accumulation and lower oxidative stress levels, as well as higher chlorophyll and hormone contents. Our results contribute to the understanding of the physiological and biochemical basis of Cd tolerance in bean plants and may therefore, be useful for breeding programs directed towards obtaining bean varieties showing low Cd accumulation.

1. Introduction Heavy metals pollution is a growing concern due to the possibility of metal accumulation in the food chain (An et al., 2012; Schreck et al., 2012). Cadmium is a non-essential element recognized as a major hazardous environmental pollutant since toxicity effects take place even at low concentrations (Sanita di Toppi and Gabbrielli, 1999; Clemens et al., 2013). In plants, cadmium is readily uptaken by roots and transported to aerial organs, causing inhibition of plant growth and organ development (Besson-Bard et al., 2009; Mohamed et al., 2012; Xue et al., 2013). Cd accumulation in the soil is favored by diverse anthropogenic activities such as unconventional application of phosphate fertilizers and irrigation with wastewaters, as well as mining activities (Panda et al., 2011; Wuana and Okieimen, 2011). General Cdtoxicity symptoms have been widely reported; they include reduction of growth and photosynthesis (Per et al., 2016; Rizwan et al., 2016; Figlioli et al., 2019), lipid peroxidation (Gallego et al., 1999;

∗

Muradoglu et al., 2015; Bahmani et al., 2017), over-accumulation of reactive oxygen species (ROS) (He et al., 2015; Bahmani et al., 2019a), and altered antioxidant enzymes activities (Schutzendubel et al., 2001; Bahmani et al., 2019b). Plants' tolerance to environmental stresses is highly dependent on their ROS detoxification/scavenging capacity (Foyer et al., 1994). Hence, an equilibrium between ROS production and ROS detoxification achieved through efficient antioxidant systems is required to maintain the physiological processes and organisms’ health (Valavanidis et al., 2006). It has been indicated that the activation and inhibition mechanisms of antioxidant enzymes depend on the plant species and tissue type (Dai et al., 2006; Mobin and Khan, 2007; Zhang et al., 2009). Phytohormones participate in various physiological and developmental processes in plants and also regulate Cd toxicity, either positively or negatively, through the regulation of their biosynthetic pathways (Peleg and Blumwald, 2011; Zhu et al., 2013). A large body of evidence indicates that abscisic acid (ABA) and auxin concentrations

Corresponding author. E-mail address: [email protected] (R. Bahmani).

https://doi.org/10.1016/j.ecoenv.2020.110178 Received 8 October 2019; Received in revised form 3 January 2020; Accepted 5 January 2020 0147-6513/ © 2020 Elsevier Inc. All rights reserved.

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Emerson, Taylor, and G-11867 (as tolerant), Akhtar and G-01437 (as semi-sensitive), and G-14088 (as sensitive). Sterilized seeds were sown in polyethylene pots containing 7 kg of soil (sandy loam texture; electric conductivity: 5.71; pH: 6.9; total nitrogen and organic matter: 0.54% and 0.061%, respectively). Seven seeds were sown in each pot; after emergence, seedlings were thinned to 4 per pot. Cadmium (as CdCl2) was prepared at a final concentration of 45 or 90 mg/kg of soil, mixing thoroughly with the soil. According to a preliminary experiment in which different Cd doses were tested, these concentrations were expected to cause, respectively, moderate and high toxicity in bean plants. Control pots without cadmium treatment were set in parallel. To allow cadmium stabilization in the soil, sowing was carried out 5 days after treatment. Plants were kept in an experimental greenhouse (day/night temperatures: 26/16 ± 2 °C; relative humidity: 50 ± 4%) and were irrigated when necessary to keep soil moisture constant. The experiment was set up as a factorial in a completely randomized design (CRD) with three replicates per treatment. Measurements were performed after 10 weeks.

increase in response to cadmium addition in various plant species (Hu et al., 2013; Stroinski et al., 2013; Zhu et al., 2013; Bahmani et al., 2016); however, the experimental conditions strongly affect plant responses. Likewise, it has been reported that rice seedlings subjected to Cd treatment increased their ABA level, and this response was closely linked with Cd tolerance (Hsu and Kao, 2005). Besides, auxins have been shown to promote Cd tolerance by immobilizing Cd in the roots and inducing antioxidant enzymes activity in Arabidopsis and wheat plants (Agami and Mohamed, 2013; Zhu et al., 2013). Plant species are distinct in terms of cadmium tolerance and uptake. In contrast to cereals and grasses, legumes are more sensitive to cadmium toxicity and exhibit a severe biomass reduction, even at low Cd concentrations (Inouhe et al., 1994). Although several experiments have been carried out with legumes to evaluate their genetic variation for heavy metals tolerance and accumulation (Horst, 1983; Belimov et al., 2003; Metwally et al., 2005; Namdjoyan et al., 2011), the physiological responses and Cd accumulation patterns of the common bean are poorly understood. The common bean (Phaseolus vulgaris L.) is one of the most important food crops in the world, with a total production worldwide of over 4.6 million tons in 2017 (FAO, 2017). Due to its remarkable nutritional value (rich in proteins, minerals, and fiber), bean contributes to a great extent to the diet of many individuals, particularly in developing countries (Castro-Guerrero et al., 2016). In several central areas of Iran (Markazi and Isfahan provinces) showing bean cultivation, the increasing industrialization resulted in the contamination of the soil and groundwater with various heavy metals such as cadmium and arsenic. Therefore, it is of great interest to study the response of different bean genotypes to Cd toxicity. We previously reported that growth and physiological traits of bean genotypes were strongly affected by a shortterm cadmium treatment (Bahmani et al., 2014). In the present study, the genotypic variation of common bean in terms of physiological responses to Cd treatment as well as Cd accumulation were investigated. A preliminary screening of 25 bean genotypes was performed under laboratory conditions, and six genotypes with different degrees of tolerance were selected to be included in further studies conducted in a greenhouse.

2.3. Plant fresh and dry mass Plants were harvested, rinsed with distilled water, and blotted using kimwipes. Then, plants were weighed to determine the fresh mass, subsequently oven-dried for 2 days at 70 °C and finally weighed again to determine the dry mass. The calculation of the tolerance index (%) was performed based on the following formula:

Weight (fresh or dry) in stress condition TI(%) = ⎡ × 100⎤ − 100 ⎢ ⎥ ⎣ Weight (fresh or dry) in normal condition ⎦ In this formula, first the ratio between fresh or dry weight was calculated with respect to its control and the obtained value was decreased from 100 to show the degree of reduction caused by Cd treatment respect to the control seedlings as a percentage. For other measured traits, including chlorophyll content, oxidative stress level, and plant hormone contents, the same calculation was performed in which the value for each trait in control condition was considered as 100% and the reduction value compared to control plants was shown for Cdtreated plants.

2. Materials and methods 2.1. Laboratory experiment

2.4. Chlorophyll content A screening experiment was carried out using 25 bean genotypes to evaluate growth and physiological responses to Cd stress, and to select the most tolerant and the most sensitive bean genotypes under laboratory conditions, as previously described (Bahmani et al., 2014). Seeds of bean were obtained from the Seed and Plant Improvement Institute, Khomeyn Branch, Iran (Supplementary Table S1). Seeds were surface sterilized by immersing them in 2.5% (v/v) sodium hypochlorite for 15 min and rinsed (at least 3 times) with distilled water. The sterilized seeds were germinated between two layers of Whatman filter paper, then treated with a CdCl2 solution (4 mg/L) or distilled water (control), and further incubated at 24 °C. Ten days after germination, the seedlings were harvested, and growth and physiological traits were evaluated following published methods (Bahmani et al., 2014). Several stress tolerance indices, including mean productivity (MP), geometric mean productivity (GMP), tolerance (TOL), and stress susceptibility index (SSI) were calculated as previously reported (Fischer and Maurer, 1978; Rosielle and Hamblin, 1981; Fernandez, 1992).

Chlorophyll a (Chl a), chlorophyll b (Chl b), and chlorophyll a + b contents were determined as described previously (Ni et al., 2009). In brief, fresh leaves (0.3 g) were ground in liquid nitrogen; the homogenates obtained were transferred to Falcon tubes containing 5 mL of 80% acetone and mixed in the dark for 15–30 min. After centrifugation (3000 rpm) at 4 °C for 15 min, the supernatants were transferred to clean centrifuge tubes and kept in the dark; this centrifugation step was repeated twice. Finally, the absorbance (A) in the supernatants was measured at the wavelength of 645 (Chl b) and 663 (Chl a) nm in a spectrophotometer (UV-1280, Shimadzu, Japan). The chlorophyll content was then calculated by the following equation: Chl a (mg/gFW) = [12.7 × A663 + 2.69 × A645] × V/1000 × W Chl b (mg/gFW) = [22.9 × A645 + 4.86 × A663] × V/1000 × W Chl a + b (mg/gFW) = [8.02 × A663 + 20.20 × A645] × V/ 1000 × W (Chlorophyll a + b), where V = volume of the extract (mL) and W = Weight of fresh leaves (g).

2.2. Greenhouse experiment 2.5. Plant hormones 2.2.1. Plant material, growth conditions, and treatments Based on the results obtained in the screening assay under laboratory conditions, six bean genotypes were selected to be further evaluated in the greenhouse experiments. The genotypes selected were

Fresh leaf samples were immediately ground to a fine powder in liquid nitrogen using a pre-chilled mortar and pestle. The resulting powder was kept at −80 °C until use. Auxins, gibberellins, and 2

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Table 1 Assessment of the cadmium tolerance in common bean genotypes using stress tolerance indices. Genotypes

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

YP

YS

MP

TOL

SSI

GMP

Value

Rank

Value

Rank

Value

Rank

Value

Rank

Value

Rank

Value

Rank

28.68 17.80 21.97 15.71 24.26 23.03 20.85 25.37 18.62 27.42 16.88 22.12 20.26 21.33 19.79 17.70 19.49 14.77 21.11 20.09 17.58 25.12 11.55 14.18 10.66

1 17 8 21 5 6 11 3 16 2 20 7 12 9 14 18 15 22 10 13 19 4 24 23 25

15.05 9.69 9.74 8.41 13.17 13.19 11.53 17.02 8.79 14.92 8.68 13.84 11.13 14.27 10.64 9.81 11.48 8.17 12.21 9.48 9.34 13.66 11.54 9.33 9.94

2 18 17 24 8 7 11 1 22 3 23 5 13 4 14 16 12 25 9 19 20 6 10 21 15

21.86 13.74 15.85 12.06 18.71 18.11 16.19 21.19 13.70 21.17 12.78 17.98 15.69 17.80 15.21 13.75 15.48 11.47 16.66 14.78 13.46 19.39 11.54 11.75 10.30

1 17 11 21 5 6 10 2 18 3 20 7 12 8 14 16 13 24 9 15 19 4 23 22 25

13.63 8.11 12.23 7.30 11.09 9.84 9.32 8.35 9.38 12.49 8.21 8.28 9.12 7.06 9.14 7.89 8.01 6.60 8.89 10.61 8.23 11.46 0.01 4.84 0.72

25 9 23 6 21 19 17 13 18 24 10 12 15 5 16 7 8 4 14 20 11 22 2 3 1

1.118 1.072 1.310 1.093 1.076 1.005 1.052 0.774 1.242 1.073 1.143 0.881 1.060 0.779 1.088 1.049 0.967 1.051 0.992 1.243 1.103 1.074 0.002 0.805 0.159

21 14 25 19 17 9 12 3 23 15 22 6 13 4 18 10 7 11 8 24 20 16 1 5 2

20.77 13.13 14.63 11.49 17.87 17.43 15.51 20.78 12.79 20.23 12.10 17.50 15.02 17.45 14.51 13.18 14.96 10.99 16.05 13.80 12.81 18.52 11.54 11.50 10.29

2 17 13 23 5 8 10 1 19 3 20 6 11 7 14 16 12 24 9 15 18 4 21 22 25

YP; yield in normal condition, YS; yield in stress condition, MP; Mean Productivity, TOL; Tolerance Index, SSI; Stress Susceptibility Index, and GMP; Geometric Mean Productivity.

2.7. Cadmium accumulation in plant tissues

cytokinins were extracted and purified, following the method described by Shindy and Smith (1975). Quantification was carried out using twodimensional HPLC, as previously described (Dobrev et al., 2005).

Roots and shoots were separated, washed with ice-cold 5 mM of CaCl2, and oven-dried at 65 °C for 72 h. The resulting samples were digested in an acid mixture consisting of HClO4 and HNO3 [3:5 (v/v)]. Cadmium concentration was measured by inductively coupled plasma mass spectrometry (ICP-MS) (Agilent 7500c, USA). Triplicate samples from three independent experiments were analyzed; mean values were obtained.

2.6. Oxidative damage biomarkers Leaf samples were prepared as described in hormone determination section. Malondialdehyde (MDA) content was measured using highperformance liquid chromatography (HPLC) (Bird et al., 1983). Trichloracetic acid (TCA, 20% w/v) was used to precipitate proteins from tissue homogenates. The estimation of MDA was based on the condensation of thiobarbituric acid (TBA) with MDA, which leads to the formation of a TBA + MDA complex. Tetraethoxypropane (TES) was used to provide the MDA + TBA standards; absorbance was measured at 540 nm. MDA levels were expressed as nM/mg of protein after normalization based on the protein contents of the samples. As an indicator of protein oxidation, dityrosine was measured. Leaf samples (1.5–2.0 g) were homogenized with 5 mL of ice-cold extraction buffer (50 mM HEPES-KOH pH 7.2, 10 mM EDTA, 2 mM PMSF, 0.1 mM p-chloromercuribenzoic acid, 0.1 mM DL-norleucine, and 0.1 g polyclar AT), centrifuged at 6000 g for 1 h, and o,o'-dityrosine was purified from the clear supernatant using preparative HPLC (HP Agilent 1100 series system, USA), as described earlier (Orhan et al., 2004). To prepare the dityrosine standard, the method of Amadò et al. (1984) was followed. The production of dityrosine derived from the reaction between tyrosine and H2O2 catalyzed by horseradish peroxidase (HRP) was quantified based on the absorptivity coefficient Ɛ315 = 4.5 mM−1 cm−1. As a biomarker of oxidative DNA damage, 8-hydroxy-2′-deoxyguanosine (8-OH-2′-dG) was measured in leaf tissues as reported previously (Bogdanov et al., 1999). An automated column-switching LCEC method was applied, based on the ability of the carbon column to detect selectively 8-OH-2′-dG and allow the exclusion of interfering peaks, eventually extracting 8-OH-2-DG with an analytical C18 column. Protein contents in the samples were determined as previously described (Bradford, 1976).

2.8. Statistical analysis A two-way ANOVA was employed for data analysis and means comparison was conducted according to Tukey's test (HSD) at P ≤ 0.05 using SAS software (Institute Inc., Version 9.1). Pearson correlation coefficients between stress tolerance indices, and principal component analysis (PCA) was carried out using the Minitab (version 16) software. 3. Results 3.1. Selection of tolerant genotypes based on stress tolerance indices For this purpose, we first determined the fresh weight of the seedlings (which may be considered as yield under laboratory-testing conditions) and evaluated the correlation between several indices of stress tolerance/sensitivity. As shown in Supplementary Table S2, fresh weight (yield) in normal condition (YP) had a significant positive correlation with all the evaluated indices. Bean yield under Cd stress condition (YS) showed a significant positive correlation with MP and GMP, a significant negative correlation with SSI, and no significant correlation with TOL. Thus, in the selection of tolerant genotypes, higher values for MP and TOL, and lower values for SSI were considered desirable. Next, we evaluated the bean genotypes for cadmium tolerance based on those indices (Table 1). According to YP, MP, and GMP indices, genotypes 1, 8, 10, and 22 were considered tolerant genotypes. 3

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The YS index indicated as tolerant genotypes 1, 8, 10, and 14, while TOL and SSI pointed out as tolerant genotypes 18, 23, 24, and 25, and genotypes 3, 9, 11, and 20, respectively.

3.5. Cd effects on chlorophyll content Cd exposure reduced chlorophyll concentration in a dose-dependent manner. A significant decrease in chlorophyll a (17.9%), chlorophyll b (20.1%), and chlorophyll a+b (18.4%) was recorded for plants grown in the presence of Cd at the dose of 45 mg/kg, and this adverse effect was more significant when Cd dose was 90 mg/kg: decreases of 39.49%, 29.7%, and 36.4%, respectively, were observed (Fig. 2A, C, and E). As shown in Fig. 2B, across both Cd doses tested, chlorophyll a concentration was less diminished in genotypes Emerson (13% and 28% for Cd at 45 mg/kg and 90 mg/kg, respectively), Taylor (10% and 25%), and G-11867 (14.8% and 30.4%) than in genotype G-14088 (27.7% and 62.3%). A similar pattern was observed for chlorophyll b and chlorophyll a+b contents, with Emerson, Taylor, and G-11867 genotypes exhibiting ~2-fold less reduction as compared to that observed in genotype G-14088 (Fig. 2D, F).

3.2. Principal components analysis (PCA) Results of the PCA showed that only two components had eigenvalues > 1, and they together explained 99.4% of the total variance (Supplementary Table S3). The first component had a significant correlation with YP, YS, MP, TOL, SSI, and GMP indices, accounting for 75.7% of the variance. Thus, this component was considered to be related to cadmium stress tolerance, and therefore, a higher amount of this component would be favorable. The second component, which explained roughly 24% of the variance, had a positive correlation with YP, TOL, SSI, and GMP, while it had a negative correlation with YS and MP; consequently, it was considered as the sensitive component respect to cadmium stress. A lower amount of this component would be preferable. These components are related to stress tolerance indices as follows: First component = 0.45 YP+0.366 YS+0.451 MP+ 0.38 TOL +0.378 SSI+0.514 GMP Second component = 0.025 YP−0.716 YS−0.159 MP+0.659 TOL +0.012 SSI+0.164 GMP Accordingly, genotypes 1 (G-11867), 8 (Taylor), 10 (Emerson), and 22 (D-81083) were selected as tolerant genotypes based on the comparison with all other genotypes (Supplementary Figure S1A). MP and GMP were found to be dominant indices (Supplementary Figure S1B). Taken together, the results of the laboratory test indicated that cadmium stress negatively affected the growth and development of bean seedlings, but their responses were highly dependent on genotypes. G-11867, Taylor, Emerson, and D-81083 genotypes were more tolerant than other genotypes (Supplementary Figure S2). Based on the laboratory findings, six bean genotypes with different degrees of tolerance were selected and included in the subsequent study carried out in a greenhouse.

3.6. Cd effects on oxidative stress biomarkers It is widely known that Cd stress induces ROS over-accumulation, which results in oxidative stress and damage to DNA, RNA, proteins, and lipids (Sandalio et al., 2012; Chmielowska-Bak et al., 2018). In order to determine the oxidative stress level in bean plants under Cd stress, MDA (a lipid peroxidation index), dityrosine (a biomarker of oxidative protein damage), and 8-OHdG (a biomarker of oxidative DNA damage) contents were examined. The content of all oxidative-damage biomarkers increased in Cd-exposed plants in a dose-dependent manner (Fig. 3A, C, and E). These biomarkers showed increases over the control of about 27% and 50% at Cd doses of 45 mg/kg and 90 mg/kg, respectively. Significant differences among the six bean genotypes selected were found in terms of the biomarkers studied. As shown in Fig. 3B, the MDA level remarkably raised in genotype G-14088 at both Cd doses, 45 mg/ kg and 90 mg/kg (41.3% and 88.3%, respectively). However, MDA increases were less pronounced in genotypes Emerson, Taylor, and G11867: 17%–20% (45 mg/kg) and 33%–37% (90 mg/kg). Similarly, the higher dityrosine and 8-OHdG contents were observed in genotype G14088 (37.2% and 44.7% increase at the lower Cd dose, respectively; 76.6% and 70.8% increase at the higher Cd dose, respectively). The genotypes Emerson, Taylor, and G-11867 showed less pronounced increases in the range of 15%–23% for dityrosine and 35%–39% for 8OHdG (Fig. 3 D, F).

3.3. Greenhouse experiment Considerable differences among bean genotypes and between control and cadmium treated plants for all traits studied were found (Supplementary Table S4). Besides, a significant interaction genotype × cadmium was detected, indicating a distinct response of bean genotypes to Cd stress and the occurrence of enough genotypic variation for the identification of tolerant genotypes.

3.7. Cd effects on plant hormones Cadmium treatment significantly affected auxin (indoleacetic acid, IAA), gibberellin, and cytokinin contents, causing decreases of 17%–20% and 30%–33% when applied at 45 mg/kg or 90 mg/kg, respectively (Fig. 4A, C, and E). Both Cd doses significantly declined the auxin content in the genotype G-14088 (26.3% and 55.6%, respectively); in genotypes Emerson, Taylor, and G-11867, reductions were considerably lower: 11%–15% and 22%–27% at Cd doses of 45 mg/kg and 90 mg/kg, respectively (Fig. 4B). Likewise, cytokinin content was highly diminished in the genotype G-14088 (30% and 51.7% at Cd concentrations of 45 mg/kg and 90 mg/kg, respectively), while in the genotypes Emerson, Taylor, and G-11867, reductions were less marked (12%–16% and 19%–24% at 45 mg/kg and 90 mg/kg, respectively) (Fig. 4D). There was not any significant difference among genotypes in gibberellin content at the lower Cd dose; however, the differences were evident when Cd concentration was 90 mg/kg. At this Cd dose, genotype G-14088 showed a 45.6% reduction in gibberellin concentration, in contrast to genotypes Emerson, Taylor, and G-11867, which exhibited decreases of about 23% (Fig. 4F).

3.4. Plant growth and cadmium tolerance index Growth reduction is a common symptom of Cd toxicity; for this reason, the growth of the selected bean genotypes cultivated under different Cd concentrations was studied. Cd treatment significantly declined the fresh and dry weight in all bean genotypes (Fig. 1A, C). These parameters were decreased respect to non-treated plants by 20% and 19% at the Cd dose of 45 mg/kg, and by 36.5% and 33.3% at the dose of 90 mg/kg, respectively. As shown in Fig. 1B, across both Cd treatments, Emerson, Taylor, and G-11867 were found as the more tolerant genotypes (with TIs ranging from 9.9% to 14% at 45 mg/kg and from 19% to 26% at 90 mg/kg of Cd treatments), while G-14088 was the less tolerant genotype (TIs: 36.8% at 45 mg/kg and 66.5% at 90 mg/kg of Cd treatments). A similar pattern was observed for dry weight: genotypes Emerson, Taylor, and G-11867 exhibited TIs in the range of 11%–16% (Cd dose: 45 mg/kg) and 18%–21% (Cd dose: 90 mg/kg), contrasting with genotype G-14088, which showed TIs of 31.5% (45 mg/kg) and 60.2% (90 mg/kg) (Fig. 1D). 4

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Fig. 1. Cd-tolerance indices. Fresh and dry weights of cadmium-treated seedlings (A and C), stress tolerance indices based on fresh and dry weights (B and D). To calculate the tolerance index of each bean genotype, the growth in the presence of Cd (45 and 90 mg/kg) respect to that in the absence of the metal was considered. Data are means ± SE (n = 3). Different letters over the bars indicate significant differences, according to Tukey's test (P < 0.05).

3.8. Cadmium concentration in plant tissues and translocation factor (TF)

4. Discussion

Cd accumulation was remarkably increased in both roots and shoots of bean plants as Cd concentration in the soil augmented. In control plants, Cd accumulation in plant tissues was negligible (Fig. 5A and B). Cd accumulation in the roots was in the range of 304 ppm (45 mg/kg) to 447.5 ppm (90 mg/kg) (Fig. 5A), while in the shoots, values ranged from 188.6 ppm (45 mg/kg) to 269.3 ppm (90 mg/kg) (Fig. 5B). Plants grown in the presence of Cd at 90 mg/kg showed about a 1.5-fold increase for Cd accumulation both in roots and shoots compared to plants grown at the dose of 45 mg/kg. In addition, roots accumulated 1.7-fold more Cd than shoots, suggesting that the majority of Cd was retained in the root tissue and not transferred to the aerial parts, which can be considered a tolerance mechanism to Cd stress (Zornoza et al., 2002). At both Cd doses, genotype G-14088 demonstrated a significantly higher Cd accumulation in roots and shoots than the other genotypes (Fig. 5A and B). These results were further confirmed by the evaluation of the Cd translocation factor (TF). As shown in Fig. 5C, the TF for all the examined genotypes was < 1, indicating that bean plants are Cd excluders rather than Cd hyperaccumulators. Interestingly, TFs showed no changes under increasing Cd concentrations in the soil, showing values ranging from 0.45 to 0.82 at the lowest Cd dose and from 0.49 to 0.71 at the highest Cd dose. Although all genotypes presented TFs < 1, genotype G-14088 had the highest TF across both Cd doses (Fig. 5C).

During the last decades, Cd environmental pollution has received great attention mostly due to its potential hazardous effects on all living organisms, as well as contamination of the food chains (An et al., 2012; Schreck et al., 2012). Several factors account for the fate, transformation, and mobility of Cd in the soil environment, including biodegradation, photolysis, chemical degradation, organic matter, soil structure, and pH (Pachana et al., 2010; Clemens and Ma, 2016). Despite the above processes, most of the Cd present in soils can be absorbed by the plants. One efficient strategy to decrease Cd entrance into the food chain is to use plant varieties that tend to accumulate reduced amounts of this metal. Therefore, understanding the genotypic variation in physiological traits and Cd accumulation patterns is of great significance and can pave the way when breeding programs directed to generate low Cdaccumulating cultivars are designed. Although the genotypic variation in response to Cd stress has been studied for several plant species including pea (Metwally et al., 2005; Rahman et al., 2016), bermudagrass (Cynodon dactylon (L.) Pers.) (Xie et al., 2014), and wheat (Naeem et al., 2016), this topic has been scarcely addressed for common bean, an important edible plant. In the present work, the response to Cd exposure of 25 common bean genotypes was first investigated at the germination stage, and remarkable differences among the examined genotypes were detected. Further evaluation of this genotypic variation based on the stress 5

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Fig. 2. Chlorophyll contents. Chlorophyll a (A), chlorophyll b (C), and chlorophyll a+b (E) contents in the control and cadmium-treated bean plants. Chlorophyll a (B), chlorophyll b (D), and chlorophyll a+b (F) contents in cadmium-treated plants expressed as percentage respect to those in non-treated plants. Data are means ± SE (n = 3). Different letters over bars indicate significant differences, according to Tukey's test (P < 0.05).

stress condition (Rosielle and Hamblin, 1981). For instance, the comparison of YP and YS for genotypes 23 and 25 (selected as tolerant based on TOL) suggests that rather the lower YP value than the higher YS value led to the selection of them (Table 1). It has been stated that a suitable index to compare the stress tolerance of different genotypes should have a significant positive correlation with YP and YS (Blum, 1988). Considering what is shown in Table 1, MP and GMP indices fulfilled this requirement and were considered the most suitable. Therefore, based on these indices, genotypes 1, 8, 10, and 22 were selected as tolerant genotypes. Overall, our findings show that genotypes 1 (G-11867), 8 (Taylor), 10 (Emerson),

tolerance indices mean productivity (MP), geometric mean productivity (GMP), yield in normal conditions (YP), and yield in stress conditions (YS), for which higher values indicate higher tolerance (Rosielle and Hamblin, 1981; Fernandez, 1992), showed that genotypes 1, 8, 10, and 22 were more tolerant compared to the others (Table 1). Moreover, according to the TOL and SSI indices, for which lower values imply higher tolerance to the stress (Fischer and Maurer, 1978; Rosielle and Hamblin, 1981), genotypes 18, 23, 24, and 25, and genotypes 3, 9, 11, and 20, respectively, would have been selected (Table 1). However, it should be noted that the single finding of a lower TOL or SSI index for a given genotype does not mean the suitability of that genotype to the 6

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Fig. 3. Oxidative stress biomarkers. MDA (A), dityrosine (C), and 8-OH-dG (E) contents in the control and cadmium-treated plants. MDA (B), dityrosine (D), and 8OH-dG (F) contents in cadmium-treated plants expressed as percentage respect to those in non-treated plants. Data are means ± SE (n = 3). Different letters over the bars indicate significant differences, according to Tukey's test (P < 0.05).

2016; Gong et al., 2017; Bahmani et al., 2019b), Cd treatment reduced the fresh and dry weight of bean plants (Fig. 1). Among the studied genotypes, Emerson, Taylor, and G-11867 were found to be more tolerant to Cd as compared to other genotypes, with TIs > −30%) (Fig. 1). Plants’ tolerance to Cd stress generally depends on both metal accumulation rates and detoxification mechanisms to cope with the harmful effects of this metal. Plant growth inhibition caused by Cd exposure can be due to cell division or cell enlargement retardation (Yuan and Huang, 2016). Besides, photosynthesis is sensitive to Cd, and

and 22 (D-81083) were more tolerant to Cd stress than other genotypes (Table 1, Supplementary Figure S1; Supplementary Figure S2). Accordingly, six genotypes with variable degrees of Cd tolerance were selected, and their tolerance and physiological responses to the metal were studied in more detail under greenhouse conditions. The results of the greenhouse experiment correlated with those of the screening test under laboratory conditions and further confirmed that the selected genotypes were significantly distinct in terms of Cd tolerance. Similarly to that observed for other plant species and communicated in previous reports (Perez-Chaca et al., 2014; Rahman et al., 7

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Fig. 4. Plant hormone levels. Indoleacetic acid (IAA) (A), cytokinin (C), and gibberellin (E) in the control and cadmium-treated plants. IAA (B), cytokinin (D), and gibberellin (F) contents in cadmium-treated plants expressed as percentage respect to those in non-treated plants. Data are means ± SE (n = 3). Different letters over the bars indicate significant differences, according to Tukey's test (P < 0.05).

which act as cofactors in various enzymatic reactions and structural components of the photosynthetic machinery (Khan et al., 2013), may occur. In this study, the genotypes Emerson, Taylor, and G-11867 showed less reduction in chlorophyll contents (< 30%) compared to the rest of the genotypes (Fig. 2). This pattern matched exactly with the results in terms of tolerance index (Fig. 1), suggesting that photosynthesis is closely associated with bean plants’ growth and can be considered a determinant factor for Cd tolerance. The decrease in chlorophyll content in this study was in accordance with the results of previous studies (Verma et al., 2013; Li et al., 2016). A growing body of evidence indicates that Cd causes over-

disruption in the photosynthesis process may also result in growth retardation (Yu et al., 2013). In the present work, Cd addition resulted in decreased chlorophyll contents in a dose dependent manner. A significant (P ≤ 0.05) decline in chlorophyll contents (Chl a, Chl b, and Chl a+b) was observed at both Cd doses tested (45 and 90 mg/kg) compared to non-treated control plants (Fig. 2). It has been reported that Cd can inhibit the photosynthetic process in several ways: chloroplasts may be damaged through oxidative stress (Ali and Hadi, 2015), thylakoid membranes may be injured resulting in accelerated leaf senescence (Jin et al., 2008), and interactions with divalent nutrients such as Fe, Zn, and Mn, 8

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accumulation of reactive oxygen species (ROS), thereby inducing lipid peroxidation in plants (Garnier et al., 2006; Perez-Chaca et al., 2014; Lv et al., 2017; Song et al., 2017). In the present work, the mean values of the examined oxidative stress indicators (MDA, dityrosine, and 8OHdG) showed that Cd caused oxidative stress in bean plants in a dosedependent manner, with significant increases in comparison with nontreated control plants (Fig. 3). Interestingly, oxidative stress was less induced in the tolerant genotypes (Emerson, Taylor, and G-11867) than in the sensitive genotype (G-14088), where a higher level of oxidative stress was observed (Fig. 3B, D, and F). Cd-induced ROS accumulation in plant cells has been linked to indirect mechanisms such as inhibition of antioxidant enzymes (Romero-Puertas et al., 2019), and the variability in the oxidative stress levels among bean genotypes detected in this study may be attributed to a differential alteration in the activity of the antioxidant enzymes. To some extent, the results obtained in this work are consistent with those of our previous study, in which higher activities for superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) were found in some bean genotypes after Cd exposure (Foroozesh et al., 2012). It has been indicated that plant hormones have dual functions in Cd signaling (Kong et al., 2017), and auxin concentration was found to be enhanced (Bahmani et al., 2016) or reduced (Hu et al., 2013) by Cd addition in different plant species. In this experiment, Cd treatment at both concentrations tested declined auxin, gibberellin, and cytokinin levels (Fig. 4A, C, and E). However, in the tolerant genotypes (Emerson, Taylor, and G-11867), these reductions were less pronounced compared to that exhibited by the sensitive genotype (G-14088), suggesting that phytohormones may play a positive role in Cd tolerance in bean plants (Fig. 4B, D, and F). In support of this hypothesis, auxin has been shown to diminish Cd toxicity by decreasing Cd translocation from the root to the aerial part and inducing the activity of antioxidant enzymes, which are essential for Cd-produced ROS scavenging (Agami and Mohamed, 2013; Zhu et al., 2013). Furthermore, lower concentration of auxin and higher accumulation of the ROS hydrogen peroxide and superoxide were detected in the rice mutant OsAUX1 subjected to Cd stress compared to control plants (Yu et al., 2015). In Arabidopsis, the exogenous treatment with gibberellic acid (GA) mitigated Cd toxicity by repressing the expression of the Cd importer gene IRT1 through the inhibition of NO accumulation (Zhu et al., 2012). GA can also ameliorate the deleterious effects of Cd by regulating the oxidative stress and the activity of some enzymes, including proteases, catalases, and peroxidases (El-Monem et al., 2009; Meng et al., 2009). Kinetin application promoted the growth and photosynthesis of pea plants in the presence of Cd (Al-Hakimi, 2007). In addition, the application of auxins, cytokinins, and gibberellins declined Cd biosorption and enhanced the activity of antioxidant enzymes in the green alga Chlorella vulgaris (Piotrowska-Niczyporuk et al., 2012). Cd accumulation and Cd translocation from root to shoot are the main factors involved in plant tolerance to Cd stress; these traits were extensively studied in various plant species (Belimov et al., 2003; Ci et al., 2010; Henson et al., 2013). In this study, Cd accumulation was remarkably enhanced in both root and shoot of Cd-treated plants, and to a higher degree as the metal concentration in the soil increased (Fig. 5). All genotypes accumulated more Cd in roots than in shoots; however, the genotypes Emerson, Taylor, and G-11867 showed lower Cd accumulation in both plant organs than genotype G-14088 (Fig. 5A and B). A very similar pattern was also observed for the translocation factor (Fig. 5C), indicating that in the tolerant genotypes, not only less amount of Cd is accumulated in the root, but also most of this Cd is retained in the root. In support of this concept, the genotypic variation for Cd tolerance observed between two different rice subspecies (O. sativa ssp. japonica and O. sativa ssp. indica) was attributed to the difference in their root-to-shoot Cd translocation rates (Uraguchi et al., 2009; Ishikawa et al., 2011). A lower Cd accumulation may be the result of either less metal uptake or higher metal export to external media, as indicated for the model plants Arabidopsis and rice (Connolly

Fig. 5. Cadmium accumulation and translocation factors. Cadmium concentrations in root (A) and shoot (B) after treatment with 0, 45, and 90 mg/kg of cadmium chloride (CdCl2). Root to shoot translocation factor in control and cadmium-treated plants (C). Data are means ± SE (n = 3). Different letters over the bars indicate significant differences, according to Tukey's test (P < 0.05).

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Fig. 6. Heat-map graph Cd tolerance.

et al., 2002; Nakanishi et al., 2006; Kim et al., 2007; Takahashi et al., 2011; Sasaki et al., 2012). To elucidate the molecular mechanisms underlying the lower Cd accumulation of these tolerant bean genotypes, we are studying putative metal transporters. 5. Conclusions Our results show the occurrence of genotypic variation for cadmium tolerance in common bean. Genotypes Emerson, Taylor, and G-11867 behaved as tolerant genotypes, compared to the other genotypes tested (Fig. 6). The higher tolerance demonstrated by these genotypes was attributed to lower Cd accumulation and oxidative stress levels, as well as less adverse effects on plant hormones and chlorophyll contents, resulting in less impairment of growth. The results of this study are relevant for future breeding programs, given the need to reduce the potential risk of food contamination with Cd, thus contributing to human health safety. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. CRediT authorship contribution statement Ramin Bahmani: Investigation, Writing - original draft, Formal analysis. Mahsa Modareszadeh: Investigation, Writing - original draft, Visualization. Mohammad reza Bihamta: Supervision, Conceptualization. Declaration of competing interest None. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoenv.2020.110178. Bean genotypes were classified based on their Cd tolerance using all the studied traits. Scores were assigned based on the percentage of change calculated for each genotype grown in the presence of Cd at 90 mg/kg relative to the control condition and are shown in green to 10

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