Subcellular Distribution and Chemical Forms of Cadmium in Two Hot

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Subcellular Distribution and Chemical Forms of Cadmium in Two Hot Pepper Cultivars Differing in Cadmium Accumulation Junliang Xin and Baifei Huang* Department of Safety and Environmental Engineering, Hunan Institute of Technology, Hengyang 421002, China ABSTRACT: A greenhouse experiment was conducted to compare the subcellular distribution and chemical forms of cadmium (Cd) in roots, stems, leaves, and fruits between a low-Cd cultivar (Yeshengchaotianjiao, YCT) and a high-Cd cultivar (Jinfuzaohuangjiao, JFZ) of hot pepper (Capsicum annuum L.). The Cd concentrations in the root’s subcellular fractions, and in all chemical forms in roots, were 1.85−4.88- and 1.84−4.90-fold higher, respectively, in YCT than in JFZ. Compared with JFZ, YCT had significantly lower Cd concentrations in the subcellular fractions (1.10−2.42-fold) of stems and leaves and in almost all chemical forms (1.17−2.97-fold) in the stems and leaves. Also, in fruits, the concentrations of Cd in the cell wall and soluble fractions were 1.18−2.24-fold significantly lower in YCT than in JFZ, and there were lower Cd concentrations (1.36−2.08-fold) in the chemical forms in YCT than in JFZ. KEYWORDS: hot pepper (Capsicum annuum L.), cadmium (Cd), subcellular distribution, chemical form, low-Cd cultivar

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INTRODUCTION Cadmium (Cd) accumulation in agricultural soils as a result of industrial and agricultural activities has become a worldwide environmental problem.1 Cadmium is readily taken up by the roots of crop plants and transported to edible plant parts because of its high mobility and bioavailability. Consequently, Cd-contaminated foodstuffs have become the major source of Cd intake by humans.2 There are a variety of serious health risks associated with Cd intake, including lung cancer, osteoporosis, cardiovascular disease, and renal dysfunction.3 The accumulation of Cd in crops varies greatly not only among crop species but also among cultivars or genotypes within the same species.4−6 Therefore, many researchers have suggested that the production of low-Cd cultivars could be useful tools in reducing the amount of Cd entering the human food chain.2,7−9 However, the reasons for the variations in Cd uptake and accumulation among cultivars are still poorly understood. There is some evidence that the subcellular distribution and chemical forms of Cd greatly affect the concentrations of free Cd ions in plant cells. Therefore, these factors can explain some of the differences in uptake and translocation of Cd among cultivars or genotypes.10,11 Qiu et al. reported that the proportion of Cd bound to the cell walls was higher in a low-Cd cultivar than in a high-Cd cultivar of Chinese flowering cabbage (Brassica parachinensis L.).12 However, the proportions of Cd bound to cell walls in the stems and young leaves were similar in low-Cd and high-Cd cultivars of water spinach (Ipomoea aquatica Forsk.).13 Zhou et al. compared the chemical forms of Cd between low- and high-Cd genotypes of amaranth (Amaranthus spp.) and found that only the low-Cd genotype’s stems had a lower proportion of Cd integrated into pectates and proteins.14 In contrast, the shoots and roots of a low-Cd cultivar of Chinese flowering cabbage had higher proportions of Cd in the pectate/protein-integrated form, and lower proportions of Cd in the inorganic and other water-soluble forms, compared with a high-Cd cultivar.12 Such studies have © 2013 American Chemical Society

not yet provided consistent results regarding the differences in Cd uptake and storage among cultivars/species. Furthermore, these previous studies mainly focused on the roots, stems, and leaves of plants; there is little information available on other plant organs, including fruits, grains, or flowers. Hot pepper (Capsicum annuum L.) is one of the most important vegetable crops worldwide. In China, hot pepper is cultivated on approximately 1.3 million hm2 annually, producing 27 million tons of pepper.15 It was reported that pepper has a medium tolerance to Cd, and accumulates Cd more readily in its fruits, when compared with the other two crops from the Solanaceae family, tomato (Lycopersicon esculentum) and aubergine (Solanum melongena).16 Furthermore, the shoot portion of chili peppers maintained higher Cd uptake levels than the roots,17 which is beneficial to the movement of Cd into fruits. Therefore, to minimize Cd accumulation in hot pepper fruits, it is extremely important to screen for low-Cd cultivars and elucidate the mechanisms for lower Cd accumulation. In our previous study, we identified some typical low- and high-Cd cultivars of hot pepper and found that the difference in fruit Cd concentrations between the low- and high-Cd cultivars was due to differences in Cd translocation rather than differences in Cd uptake by the roots.18 However, the mechanisms of Cd translocation via the xylem or phloem to fruits, which are responsible for the differences in fruit Cd concentrations between low- and highCd cultivars, remained unclear. To study these mechanisms, we conducted a greenhouse pot experiment to investigate the subcellular distribution and chemical forms of Cd in the roots, stems, leaves, and fruits of two cultivars of hot pepper with different Cd accumulation characteristics. Received: Revised: Accepted: Published: 508

October 4, 2013 December 25, 2013 December 30, 2013 December 30, 2013 dx.doi.org/10.1021/jf4044524 | J. Agric. Food Chem. 2014, 62, 508−515

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mortar and pestle.21 The homogenate was sieved through nylon cloth (80 μm), and the liquid was squeezed from the residue. The residue on the cloth was washed twice with homogenization buffer and was designated fraction I (FI); it contained mainly cell walls and cell wall debris. The filtrate was centrifuged (Beckman JA-25.50 rotor, Fullerton, CA, USA) at 20000g for 45 min. The supernatant solution was referred to as the soluble fraction (including the vacuoles) and was designated fraction II (FII). The deposit was treated as the organelles (excluding the vacuoles) and was designated fraction III (FIII). All steps were performed at 4 °C. The subcellular fractions were dried at 70 °C to a constant weight and then digested at 145 °C for 24 h with an oxidative acid mixture of HNO3/HClO4 (2:1, v/v). Extraction of Chemical forms of Cd. The chemical forms of Cd were determined using the method described by Wu et al.10 Cd in different chemical forms was extracted by the specific solutions, in the following order: (1) 80% ethanol, which extracts inorganic Cd, giving priority to nitrate/nitrite, chloride, and aminophenol forms of Cd; (2) d-H2O (Milli-Q, Millipore, Bedford, MA, USA), which extracts watersoluble Cd−organic acid complexes and Cd(H2PO4)2; (3) 1 M NaCl, which extracts pectate- and protein-integrated Cd; (4) 2% acetic acid (HAc), which extracts undissolved Cd phosphates including CdHPO4, Cd3(PO4)2, and other Cd−phosphate complexes; and (5) 0.6 M HCl, which extracts Cd oxalate. Cd was not detected in residues because of its low concentration in the samples. Frozen tissues were homogenized in extraction solution with a mortar and pestle, diluted at the ratio of 1:10 (w/v), and shaken for 22 h at 25 °C. Then, the homogenate was centrifuged at 5000g for 10 min, and the first supernatant was removed and transferred to a conical beaker. The sediment was resuspended twice in the same extraction solution, shaken for 2 h at 25 °C, and centrifuged at 5000g for 10 min, and the supernatant was collected. The three supernatants were pooled. The extraction procedure was the same for each of the five extraction solutions. Each of the pooled supernatant solutions was then evaporated on an electric plate at 70 °C to a constant weight and digested at 145 °C with an oxidative acid mixture of HNO3/HClO4 (2:1, v/v). Chemical Analysis. The Cd concentrations in the digests were determined with an atomic absorption spectrophotometer (Shimadzu AA-6300C, Kyoto, Japan). A Certified Reference Material (CRM) of plant GBW07605 (provided by the National Research Center for CRM, China) was used for quality assurance and quality control (QA/ QC) of the Cd analytical procedure. Statistical Analysis. All Cd concentrations were calculated on the basis of the fresh weights of samples before separation or extraction. Data were statistically analyzed with the independent samples t test and least significant difference (LSD) test based on one-way ANOVA using Excel 2003 and SPSS 13.0. The data were checked for heteroscedasticity using Levene’s test before the ANOVA was performed and showed no heteroscedasticity.

The objectives of this study were to analyze the subcellular distribution, chemical forms, and amounts of Cd in various organs of a low-Cd cultivar and a high-Cd cultivar of hot pepper. We hypothesized that the subcellular distribution and chemical forms of Cd differ significantly among different tissues as well as between the low-Cd and high-Cd cultivars.

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MATERIALS AND METHODS

Plant Materials. Two cultivars, Yeshengchaotianjiao (YCT) and Jinfuzaohuangjiao (JFZ), were selected from 30 hot pepper cultivars used in our previous study. The fruit Cd concentration in JFZ (0.19− 3.22 mg kg−1 dry weight, DW) was 2.1−2.7-fold higher than that in YCT (0.07−1.54 mg kg−1, DW) when grown in Cd-contaminated soils (0.28−2.69 mg kg−1, DW).18 Therefore, YCT and JFZ were identified as low-Cd and high-Cd cultivars, respectively. Chemicals and Reagents. In the present study, 2-(Nmorpholino)ethanesulfonic acid (MES), tris(hydroxymethyl)aminomethane (Tris), and 1,4-dithioerythritol (C4H10O2S2, DTE) were purchased from Sigma (St. Louis, MO, USA). All other chemicals and reagents, except deionized water (d-H2O), were of analytical grade and purchased from Guangzhou Chemical Regent Factory (Guangzhou, China). Experimental Site and Soil. We conducted the pot experiment at the experimental garden of Hunan Institute of Technology (112°41′ E, 26°52′ N), Hunan Province, China. The experimental soil was collected from nearby farmland and was air-dried and ground to pass through a sieve with a 5 mm mesh size. The soil was a sandy loam, and its main physical and chemical properties were determined using the analytical methods described by Lu.19 The soil pH, organic matter content, cation exchange capacity, total nitrogen, available P, available K, and total Cd were 6.42 g kg−1, 19.3 g kg−1, 91.0 mmol kg−1, 1.6 g kg−1, 103 mg kg−1, 129 mg kg−1, and 1.22 mg kg−1, respectively. According to the Farmland Environmental Quality Evaluation Standards for Edible Agricultural Products (HJ 332-2006), the maximum level (ML) for Cd in soil is 0.3 mg kg−1; therefore, the tested soil is considered as Cd-contaminated and served as a treatment (T1) without additional Cd2+ in this experiment. Two other Cd treatments (T2 and T3) consisted of soil with target Cd concentrations of 2.0 and 3.0 mg kg−1, respectively, which were created by mixing T1 soil with appropriate amounts of Cd in the form of Cd(NO3)2·4H2O. Each of the T2 and T3 soils was placed in a large basin, watered, and left to equilibrate outdoors under a waterproof tarpaulin for approximately 6 months. This long period allowed for the equilibration of the various sorption mechanisms in the soils. For T2 and T3 treatments, the final soil total Cd concentrations were 2.02 and 3.26 mg kg−1, respectively. Experimental Design. The pot experiment was conducted in a greenhouse with an air temperature of 28−35 °C. Plastic pots (top diameter, 18 cm; bottom diameter, 13 cm; height, 15 cm) were filled with 3.0 kg (DW) of prepared soil. For each treatment, three pots (n = 3) were planted for each cultivar. The seeds were sown in the soil in the pots on March 10, 2013, and watered daily with tap water. The experiment was arranged in a completely randomized design. Within 15 days after germination, the seedlings were thinned to one per pot. A solid compound fertilizer (N/P/K = 15:15:15) was applied to the soil at the rate of 3.0 g pot−1 every 2 weeks. After the 120 day growth period, the plants were harvested. Fruits, leaves, stems, and roots were separately rinsed with tap water, and roots were desorbed for 15 min in ice-cold 5 mM CaCl2 solution (5 mM MES-Tris, pH 6.0). All samples were thoroughly washed with deionized water, and the fresh weights were recorded. The fresh plant samples were divided into two portions: one for the analysis of the subcellular distribution of Cd and the other for the analysis of the chemical forms of Cd. Each portion was weighed (approximately 5 g), then immediately frozen in liquid N2, and kept frozen until analyzed. Separation of Tissue Fractions. Frozen plant samples were pretreated according to the method of Lozano-Rordriguez et al.20 Each sample was homogenized in precooled extraction buffer (50 mM TrisHCl, 250 mM sucrose, and 1.0 mM DTE, pH 7.5) with a chilled

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RESULTS Effect of Soil Cd Concentrations on Plant Biomass. For both cultivars, there were no significant differences (p > 0.05) among the treatments in terms of the biomasses of roots, stems, and leaves (Table 1). However, the fruit biomass of JFZ in the T2 and T3 treatments was higher than that in the T1 treatment, and the fruit biomass of JFZ in the T2 treatment was significantly higher (p < 0.05) than that in the T1 treatment (Table 1). There was no significant difference (p > 0.05) in the fruit biomass of YCT among the three treatments (Table 1). Subcellular Distribution of Cd. The subcellular distribution of Cd in fruits, leaves, stems, and roots of YCT (low-Cd cultivar) and JFZ (high-Cd cultivar) was analyzed by determining the Cd concentration in each of the different tissues. The Cd concentrations in the subcellular fractions of various tissues increased with increasing concentrations of Cd in the soil (Table 2). The concentrations of FI-Cd and FII-Cd in fruits were always significantly higher (p < 0.01 or p < 0.05) 509

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Table 1. Biomass of Two Hot Pepper (Capsicum annuum L.) Cultivars, YCT and JFZ, under Different Cadmium (Cd) Treatments (T1−T3)

those in JFZ fruit (22−26 and 15−18%, respectively). In leaves, most of the Cd was in FI (57−63% for YCT and 56−64% for JFZ), followed by FIII (20−28% for YCT and 23−24% for JFZ), and the smallest proportion of Cd was in FII (14−18% for YCT and 13−20% for JFZ) (Figure 1B). In the stems, there were higher proportions of FI-Cd in YCT (71−75%) than in JFZ (63−70%) and lower proportions of FIII-Cd (13−15%) in YCT than in JFZ (18−21%) (Figure 1C). Furthermore, with increasing soil Cd concentrations, the proportions of FI-Cd decreased in both YCT (from 75 to 71%) and JFZ (from 70 to 63%), but the proportions of FII-Cd increased in both YCT (from 10 to 15%) and JFZ (from 10 to 19%). The proportions of FIII-Cd remained fairly constant (Figure 1C). In roots, the largest proportion of Cd (77−82% for YCT and 82−87% for JFZ) was stored in FII, and the proportions of FIICd slightly increased as the soil Cd concentration increased in both cultivars (Figure 1D). In both cultivars, only small proportions of Cd (12−17% for YCT and 8−10% for JFZ) were bound to the cell wall, and even smaller proportions of Cd were partitioned into the organelles (5−6% for YCT and 4−8% for JFZ) (Figure 1D). Chemical Forms of Cd. Overall, the concentrations of Cd in different chemical forms in various organs of hot pepper increased with increasing soil Cd concentrations (Table 3). In fruits, the concentrations of Cd in all chemical forms were consistently and significantly higher in JFZ (p < 0.01 or p < 0.05) than in YCT (Table 3), except there was no significant difference in the level of Cd extracted by 2% HAc between the two cultivars in the T2 treatment (Table 3). In addition, the Cd form extracted by 0.6 M HCl was not detected in fruits of either cultivar. In leaves, the concentration of Cd extracted by 80% ethanol was higher in JFZ (p < 0.05) than in YCT in the T1 and T3

biomassa (g FW) organ

cultivar

T1

T2

T3

fruit

YCT JFZ

10.9 ± 2.0 23.7 ± 6.7b

10.6 ± 1.2 38.4 ± 7.6a

9.2 ± 0.7 32.4 ± 7.0ab

leaf

YCT JFZ

41.2 ± 1.2 33.7 ± 1.4

41.2 ± 1.6 31.0 ± 4.0

39.6 ± 0.7 31.3 ± 3.0

stem

YCT JFZ

32.8 ± 2.7 19.1 ± 0.5

35.1 ± 0.4 19.8 ± 1.6

33.1 ± 0.6 19.2 ± 2.5

root

YCT JFZ

35.6 ± 4.3 36.2 ± 4.0

39.6 ± 3.6 31.6 ± 1.7

37.4 ± 2.8 31.6 ± 1.7

Values are the mean ± SD (n = 3). Different letters in a row indicate significant difference at p < 0.05. a

in JFZ than in YCT (Table 2). However, there were no significant differences (p > 0.05) in the concentrations of FIIICd in fruits between the two cultivars in all treatments (Table 2). In each treatment, the concentrations of FI-Cd, FII-Cd, and FIII-Cd in the leaves and stems of JFZ were significantly higher (p < 0.01 or p < 0.05) than those of their counterparts in the leaves and stems of YCT (Table 2). In contrast, the concentrations of FI-Cd, FII-Cd, and FIII-Cd in roots were always lower (p < 0.01) in JFZ than in YCT (Table 2). In fruits, the majority of Cd was associated with FI, followed by FII and then FIII (Figure 1A). The proportions of FI-Cd in YCT fruit (46−51%) were lower than those in JFZ fruit (57− 63%), but the proportions of FII-Cd and FIII-Cd in YCT fruit (27−30 and 22−24%, respectively) were slightly higher than

Table 2. Subcellular Distribution of Cadmium (Cd) in Different Organs of Low-Cd (YCT) and High-Cd (JFZ) Cultivars of Hot Pepper (Capsicum annuum L.) Cd concentrationa (mg kg−1, FW) subcellular fraction cell wall (FI)

treatment

cultivar

T1

YCT JFZ YCT JFZ YCT JFZ

0.02 0.05 0.04 0.08 0.06 0.11

± ± ± ± ± ±

0.00* 0.00 0.00* 0.00 0.01** 0.01

0.34 0.55 0.55 1.33 1.21 2.46

± ± ± ± ± ±

0.04** 0.04 0.08** 0.15 0.07** 0.25

0.52 0.75 0.79 1.03 1.46 1.61

± ± ± ± ± ±

0.02* 0.08 0.09* 0.08 0.02* 0.08

0.39 0.08 0.60 0.14 0.97 0.34

± ± ± ± ± ±

0.03** 0.01 0.08** 0.02 0.16** 0.00

YCT JFZ YCT JFZ YCT JFZ

0.01 0.02 0.02 0.03 0.04 0.05

± ± ± ± ± ±

0.00** 0.00 0.00** 0.00 0.00** 0.00

0.10 0.19 0.15 0.31 0.28 0.49

± ± ± ± ± ±

0.00** 0.01 0.02** ** 0.03 0.03* * 0.08

0.07 0.11 0.16 0.19 0.31 0.48

± ± ± ± ± ±

0.00** 0.01 0.00* 0.01 0.02** 0.04

1.76 0.67 2.76 1.48 6.64 3.58

± ± ± ± ± ±

0.15** 0.11 0.06** 0.20 0.37** 0.22

YCT JFZ YCT JFZ YCT JFZ

0.01 0.01 0.02 0.02 0.03 0.03

± ± ± ± ± ±

0.00ns 0.00 0.00ns 0.00 0.00ns 0.00

0.11 0.23 0.27 0.53 0.43 0.87

± ± ± ± ± ±

0.02** 0.02 0.0**2 0.01 0.04** 0.06

0.10 0.21 0.14 0.33 0.27 0.46

± ± ± ± ± ±

0.01** 0.03 0.01** 0.01 0.02 0.07

0.14 0.07 0.22 0.10 0.44 0.18

± ± ± ± ± ±

0.01** 0.01 0.02** 0.01 0.07** 0.02

T2 T3

soluble fraction (FII)

T1 T2 T3

organelle (FIII)

T1 T2 T3

fruit

leaf

stem

root

Values shown are the mean ± SD, n = 3. ns, *, and ** indicate that the difference between the two cultivars in the same treatment is not significant, significant at p < 0.05, and significant at p < 0.01, respectively. a

510

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Figure 1. Percentages of cadmium (Cd) in subcellular fractions of fruits (A), leaves (B), stems (C), and roots (D) of hot pepper (Capsicum annuum L.) grown in soil with three different Cd concentrations. Cd percentage (%) = Cd concentration in fraction/(sum of Cd concentrations in all fractions) × 100.

Cd extracted by d-H2O, 2% HAc, or 0.6 M HCl in the stems, leaves, and roots were similar between YCT and JFZ.

treatments, but there was no significant difference (p > 0.05) between the two cultivars in the T2 treatment (Table 3). Also, the concentrations of Cd in other forms were always significantly higher in JFZ (p < 0.05 or p < 0.01) than in YCT (Table 3). In stems, the concentrations of Cd in all chemical forms were significantly higher in JFZ (p < 0.05 or p < 0.01) than in YCT, except for the chemical form extracted by dH2O in the T1 treatment (Table 3). In roots, the Cd concentration of each chemical form was significantly lower in JFZ (p < 0.05 or p < 0.01) than in YCT (Table 3). In terms of the distribution of the different chemical forms of Cd, the Cd form extracted by 1 M NaCl accounted for the largest proportion of Cd in fruits, leaves, and stems, but the form extracted by 80% ethanol was the predominant form in roots (Figure 2). In addition, with increasing soil Cd concentrations, the proportion of the Cd form extracted by 1 M NaCl decreased in the leaves, stems, and roots, whereas those of the forms extracted by 0.6 M HCl, 2% HAc, and 80% ethanol markedly increased in the leaves, stems, and roots, respectively (Figure 2B−D). The proportions of each Cd form in the fruits were similar between YCT and JFZ (Figure 2A). However, in leaves, the proportions of the Cd form extracted by 80% ethanol were slightly higher in YCT (11−14%) than in JFZ (7−10%) in all treatments, but the proportions of the Cd form extracted by 1 M NaCl were lower in YCT than in JFZ in the T2 and T3 treatments (Figure 2B). In the stems and roots, the proportion of the Cd form extracted by 1 M NaCl was higher in YCT than in JFZ, whereas the proportion of the Cd form extracted by 80% ethanol was lower in YCT than in JFZ (Figure 2C). In most cases, the proportions of other forms of

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DISCUSSION The subcellular distribution of Cd has extremely important effects on the accumulation, migration, and detoxification of Cd in different species or cultivars.12,13 In this study, most of the Cd (77−87%) in hot pepper roots was stored in the soluble fraction. The vacuole is a dynamic organelle that occupies as much as 90% of the total cell volume in some cell types.22 Our results imply that the vacuole is the predominant sink for Cd in hot pepper. Similarly, Vázquez et al. reported that Cd was primarily associated with the vacuoles of bean roots, and no Cd was detected in the cell walls.23 Wu et al.10 reported that 51% of total Cd was present in the soluble fraction in barley roots, and a similar proportion of Cd was associated with the soluble fraction in rice roots, as reported by Yu et al.11 Sequestration of Cd into the vacuole would greatly decrease its concentration in the cytosol, thereby preventing damage not only to organelles but also to many physiological and biochemical processes in the cells. Plant cell walls, which are the first barrier protecting the protoplast against Cd toxicity, are mainly composed of polyose (including cellulose, hemicellulose, and pectin) and proteins, which provide functional groups, such as carboxyl, hydroxyl, amino, and aldehyde groups, on their surfaces to bind Cd ions and restrict their transport across the cell membrane.1 Wang et al. reported that a large proportion of Cd (48.2−57.6%) in Bechmeria nivea (L.) Gaud. roots was bound to the cell walls.24 However, in the present study, only a small proportion (8− 511

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Table 3. Concentrations of Different Chemical Forms of Cadmium (Cd) in Two Hot Pepper (Capsicum annuum L.) Cultivars Cd concentrationa (mg kg−1, FW) extractant

treatment

cultivar

80% ethanol

T1

YCT JFZ YCT JFZ YCT JFZ

0.01 0.01 0.01 0.02 0.02 0.03

± ± ± ± ± ±

0.00** 0.00 0.00** 0.00 0.00** 0.00

0.07 0.09 0.12 0.14 0.21 0.25

± ± ± ± ± ±

0.01* 0.01 0.03ns 0.01 0.01* 0.01

0.05 0.13 0.09 0.20 0.18 0.35

± ± ± ± ± ±

0.01** 0.02 0.01** 0.01 0.02** 0.03

1.16 0.57 2.61 1.20 5.36 2.84

± ± ± ± ± ±

0.15** 0.08 0.23** 0.13 0.24** 0.09

YCT JFZ YCT JFZ YCT JFZ

0.01 0.02 0.02 0.04 0.04 0.06

± ± ± ± ± ±

0.00* 0.00 0.00** 0.00 0.00** 0.00

0.03 0.08 0.06 0.13 0.09 0.27

± ± ± ± ± ±

0.00** 0.01 0.00** 0.01 0.01** 0.04

0.06 0.06 0.09 0.11 0.17 0.26

± ± ± ± ± ±

0.01ns 0.01 0.01* 0.01 0.00* 0.04

0.28 0.08 0.39 0.19 0.90 0.49

± ± ± ± ± ±

0.02** 0.01 0.05** 0.01 0.02** 0.07

YCT JFZ YCT JFZ YCT JFZ

0.02 0.03 0.04 0.05 0.05 0.08

± ± ± ± ± ±

0.00** 0.00 0.01* 0.01 0.00* 0.01

0.30 0.54 0.49 1.00 0.76 1.73

± ± ± ± ± ±

0.02** 0.07 0.05** 0.02 0.05** 0.20

0.55 0.76 0.76 1.01 1.29 1.56

± ± ± ± ± ±

0.04** 0.02 0.09* 0.09 0.08* 0.08

0.63 0.21 0.77 0.30 1.83 0.67

± ± ± ± ± ±

0.07** 0.03 0.12** 0.04 0.09** 0.04

YCT JFZ YCT JFZ YCT JFZ

0.01 0.01 0.01 0.01 0.01 0.02

± ± ± ± ± ±

0.00* 0.00 0.00ns 0.00 0.00** 0.00

0.03 0.08 0.06 0.11 0.15 0.25

± ± ± ± ± ±

0.00** 0.01 0.00* 0.02 0.01* 0.04

0.01 0.03 0.03 0.06 0.14 0.22

± ± ± ± ± ±

0.00** 0.00 0.00** 0.00 0.01* 0.04

0.02 0.00 0.02 0.01 0.05 0.01

± ± ± ± ± ±

0.00** 0.00 0.01* 0.00 0.00** 0.00

YCT JFZ YCT JFZ YCT JFZ

ND ND ND ND ND ND

0.07 0.14 0.32 0.61 0.65 1.29

± ± ± ± ± ±

0.01* 0.03 0.05** 0.06 0.05** 0.16

0.03 0.04 0.03 0.04 0.11 0.17

± ± ± ± ± ±

0.00** 0.00 0.00* 0.00 0.02* 0.02

0.01 0.00 0.01 0.00 0.10 0.03

± ± ± ± ± ±

0.00* 0.00 0.00* 0.00 0.01** 0.00

T2 T3

d-H2O

T1 T2 T3

1 M NaCl

T1 T2 T3

2% HAc

T1 T2 T3

0.6 M HCl

T1 T2 T3

fruit

leaf

stem

root

Values shown are the mean ± SD (n = 3). ND, not detected. ns, *, and ** indicate that the difference between the two cultivars in the same treatment is not significant, significant at p < 0.05, and significant at p < 0.01, respectively. a

study, a large proportion of Cd (63−75%) in stems was bound to the cell wall fraction. This could effectively reduce Cd accumulation in the cytoplasm and, hence, decrease the amount of Cd transported to the leaves. In addition, 56−64% of the total Cd in leaves was in the cell wall fraction. Therefore, large amounts of Cd were immobilized by the cell walls in stem and leaf tissues, thus decreasing the amount of Cd translocated to fruits. In the subcellular distribution of Cd between the two cultivars, YCT consistently had lower Cd concentrations in all three fractions of the stems and leaves and had a higher or similar proportion of Cd in FI, which contributed to the decrease of Cd mobility into the fruits from the stems and leaves. These attributes met the characteristics of a low-Cd cultivar. A high Cd concentration in the soluble fraction of rice leaves facilitates Cd translocation from leaves to grains.11 Therefore, the reason for the high concentrations of Cd in fruits of JFZ might be that it has high concentrations of Cd in the FII of stem and leaf tissues. A decrease in the concentration of free Cd in the cytosol of plants is one of the defense strategies against Cd toxicity and is achieved via various mechanisms.10 The plant cell decreases the amount of Cd in the cytosol by sequestering Cd in subcellular

17%) of Cd was bound to the cell wall fraction in the roots of hot pepper. These conflicting results presumably reflect differences in experimental conditions and differences among species that have specific mechanisms and characteristics of Cd tolerance, uptake, translocation, and compartmentalization. There was a higher Cd concentration in YCT’s roots than in JFZ’s roots, indicating that the low-Cd cultivar could retain more Cd in the root tissues. This may be associated with protection mechanisms by which excessive accumulation of a toxic substance will alleviate further uptake of that substance, such as NH4-N in N-fixing symbiotic associations.25 Similarly, a low-Cd water spinach cultivar accumulated more Cd in the roots than did a high-Cd cultivar.13 However, Yu et al. reported that the root Cd concentration in a low-Cd rice cultivar was significantly lower than that in a high-Cd cultivar.11 Therefore, the low Cd translocation from roots to aboveground parts in the low-Cd hot pepper cultivar, YCT, might be an additional mechanism to decrease uptake of Cd from the soil. Further research is needed to clarify this point. When Cd is translocated from roots to aerial parts, the stem acts as a cation exchange column and, thus, it effectively reduces the amount of Cd that accumulates in leaves.26 In the present 512

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Figure 2. Percentages of different chemical forms of cadmium (Cd) in fruits (A), leaves (B), stems (C), and roots (D) of hot pepper (Capsicum annuum L.) grown in soil with three different Cd concentrations. Cd percentage (%) = Cd concentration in fraction/(sum of Cd concentrations in all fractions) × 100.

H2O) migrate more readily than other forms and have strong negative effects on plant cells. Other forms of Cd, such as pectate/protein-integrated Cd (extracted by 1 M NaCl), insoluble cadmium phosphate (extracted by 2% HAc), and cadmium oxalate (extracted by 0.6 M HCl) are the least mobile and the least toxic to plant cells.24 The largest proportion of Cd in roots of hot pepper was in the inorganic form, which is consistent with the highest proportion of Cd in FII. In root cells, Cd ions can induce phytochelatins (PCs) synthesis in the cytosol.29,30 The PC−Cd complexes are sequestered into vacuoles by ATP-binding cassette transporters and can also be transported into vacuoles by heavy metal ATPases (e.g., HMA3) and proton/cation exchange transporters.28 Thus, the amount of Cd2+ in the cytosol that can be loaded into the xylem by HMA2 and HMA4 and then translocated from root to shoot31,32 is greatly decreased. In this sense, compartmentalization into the vacuoles may be crucial for Cd detoxification. Therefore, we can assume that more of the water-soluble Cd, extracted by 80% ethanol and d-H2O from the roots, could be sequestered into vacuoles in YCT compared with that in JFZ. Also, the Cd concentration in the cytosol of root cells might not be higher in YCT than in JFZ. Further research is required to explore this point in detail. More importantly, HMA2 and HMA4 have been identified as key transporters mediating rootto-shoot Cd translocation via the xylem in rice.31 Therefore, the Cd concentrations in aerial parts in the two hot pepper cultivars

compartments. Our results showed that compared with YCT, JFZ accumulated a greater proportion of Cd in FI of fruits, thus reducing the proportion of Cd stored in FII. As a consequence, JFZ and YCT had similar concentrations of Cd in FIII of fruits. Also, the Cd concentrations in FIII of the stems and leaves were significantly higher in JFZ than in YCT, but the Cd concentrations in FIII of roots were significantly lower in JFZ than in YCT. However, the biomasses of stems, leaves, and roots did not decrease with increasing soil Cd concentrations in either cultivar. These results suggest that both cultivars are tolerant to Cd at the levels used in this experiment and that the Cd toxicity may be alleviated by sequestering Cd in cell walls. However, it is necessary to state that not all hot pepper cultivars have the same tolerance to Cd. León et al. reported that there were significant differences in tolerances to Cd2+ ions among pepper cultivars, and the tolerance to Cd is mainly dependent on the availability of nicotinamide adenine dinucleotide phosphate (NADPH) in pepper plants.27 It was noteworthy that the highest proportion of Cd associated with organelles was generally in leaves, followed by fruits and then stems, and the lowest was in roots for both cultivars in all treatments. This may be because of the preferential accumulation of Cd in chloroplasts.28 The biological activity of Cd in plants is also dependent on its chemical forms, which can be extracted with various extractants.10 For example, water-soluble Cd in inorganic forms (extracted by 80% ethanol) and organic forms (extracted by d513

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might be strongly associated with that of the xylem sap, rather than that of the root’s water-soluble form. In the stems of YCT and JFZ, most of the Cd was integrated into pectates/proteins, and the Cd proportion in this form was higher in YCT than in JFZ; this might be partly related to the lower concentration of water-soluble Cd in the stem tissues of YCT. As a result, less Cd was translocated to leaves from the stem via the xylem in YCT than in JFZ. In leaves, most of the Cd was integrated with pectates/proteins, but the proportion of cadmium oxalate dramatically increased with higher soil Cd concentrations. The higher Cd concentrations in leaves might induce an increase in the production of oxalate to immobilize Cd and, thus, reduce Cd translocation to fruits via the phloem. Phloem-mediated transport might play a key role in delivering Cd to hot pepper fruits, and xylem-mediated Cd transport due to the transpiration of the fruits might also play an important role. Physiological experiments revealed that Cd is transported into seeds of grains mainly via the phloem in wheat and rice plants.33,34 Analyses of different plant species have shown that the main Cd-ligand molecules in phloem sap are glutathione (GSH) and PCs.35,36 Thus, it is assumed that in JFZ, which had higher Cd concentrations in fruits, a greater level of Cd translocation from the roots to aboveground parts via the xylem leads to higher levels of Cd in the stems and leaves (source organs). Subsequently, more Cd is available for transport from the source organs to sink organs (fruits) via the phloem, leading to higher Cd concentrations in fruits of JFZ than in fruits of YCT. Certainly, it is also possible that the greater Cd concentration in the xylem is transported directly into fruits after the xylem−phloem transfer, which may also cause more Cd accumulation in fruits. The concentrations of all forms of Cd in fruits were lower in YCT than in JFZ, but the proportions of Cd in each form were similar in the two cultivars. Wu et al. reported that a Cdsensitive barley genotype had higher Cd concentrations in inorganic and water-soluble forms, but lower concentrations of Cd in pectate/protein-integrated forms as compared with the Cd-resistant genotypes.10 Because there were no differences in the proportions of Cd in each chemical form between YCT and JFZ and because the fruit biomass of the two cultivars did not decrease with increasing soil Cd concentrations, we assumed that the fruits of both hot pepper cultivars had a high tolerance to Cd at the levels used in these experiments. In conclusion, our data support the hypothesis that there is a significant difference in the subcellular distribution and chemical forms of Cd among different tissues between the low-Cd and high-Cd hot pepper cultivars. Although most of the Cd is stored in the soluble fraction in roots of the two cultivars, the difference in the ability to translocate Cd from the roots to the stems is the main reason for the genotypic difference in Cd accumulation. In the stems, leaves, and fruits of both cultivars, the cell wall is the most crucial subcellular fraction for immobilization of Cd. Compared with the high-Cd cultivar, the low-Cd cultivar had lower concentrations of inorganic Cd (extracted by 80% ethanol), water-soluble Cd (extracted by dH2O), and pectate/protein-integrated Cd (extracted by 1 M NaCl) in both the stems and leaves; this resulted in less Cd being translocated from the stems and leaves to fruits via the phloem.

Article

AUTHOR INFORMATION

Corresponding Author

*(B.H.) Phone: +84-734-3452095. Fax: +84-734-3452008. Email: [email protected]. Funding

This work was supported by the National Natural Science Foundation of China (Grants 41101303 and 41201320) and the Hunan Provincial Natural Science Foundation of China (Grants 11JJ6013 and 14JJ7082). Notes

The authors declare no competing financial interest.

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