JXB Advance Access originally published online on July 26, 2006
Journal of Experimental Botany 2006 57(12):2955-2965; doi:10.1093/jxb/erl056
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RESEARCH PAPER |
Characteristics of cadmium accumulation and tolerance in novel Cd-accumulating crops, Avena strigosa and Crotalaria juncea
1Graduate School of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan
2Department of Public Health, School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
*To whom correspondence should be addressed. E-mail: ushim{at}cc.tuat.ac.jp
Received 18 March 2006; Accepted 16 May 2006
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Abstract |
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Characteristics of accumulation and tolerance of cadmium (Cd) in green manure crops were investigated to identify Cd-accumulating crops and to clarify the mechanisms involved in Cd accumulation and tolerance. Seedlings of eight crop species were treated with Cd (1 mg l–1 or 5 mg l–1) in the growing medium for 4 d. Cd concentration in leaves of Avena strigosa Schreb. cv. New-oat, Crotalaria juncea L. and Tagetes erecta L. cv. African-tall was greater than values used to define Cd-hyperaccumulation (>100 mg Cd kg–1 DW). However, in leaves of T. erecta, lipid peroxidation level increased significantly, and the activities of superoxide dismutase, ascorbate peroxidase, glutathione reductase, and catalase were depressed by both Cd treatments. By contrast, A. strigosa and C. juncea exhibited high Cd tolerance. Avena strigosa leaves showed higher activities of antioxidative enzymes such as superoxide dismutase and ascorbate peroxidase than those of other species tested. Crotalaria juncea showed higher amounts of total soluble phenolics which, in leaves, were doubled by 5 mg l–1 Cd treatment. When two Cd-tolerant accumulators (A. strigosa and C. juncea) and the non-accumulator (C. spectabilis) were treated with lower Cd concentrations for 4 weeks, A. strigosa and C. juncea exhibited superior Cd accumulation in the shoots with greater biomass production compared with C. spectabilis. These results indicate that A. strigosa and C. juncea possess the greater potential for Cd accumulation and tolerance than common crops.
Key words: Avena strigosa, cadmium, Crotalaria juncea, hyperaccumulation
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Introduction |
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Cadmium (Cd) is a major pollutant metal that is extremely toxic to organisms. Vast areas of agricultural soils are contaminated with Cd through the use of phosphate fertilizers and sludge, and inputs from mining and smelting industries (Sanità di Toppi and Gabbrielli, 1999; McGrath et al., 2001). Agricultural crops grown in Cd-polluted environments contain Cd to varying degrees (Wolnik et al., 1983; Watanabe et al., 1996). Daily consumption of Cd-contaminated foods poses a risk to human health. In Japan, Cd-contaminated rice caused Itai-itai disease near the Jinzu River basin in the middle of the 20th century; even in recent years, rice is the major source of Cd intake of people in Japan (Watanabe et al., 2000). Techniques are required to remediate agricultural soils that have moderate and widespread metal-contamination to make food produced on these soils safe for human consumption.
Phytoextraction has been viewed as a promising technique to remediate metal-contaminated soils because it offers advantages of being in situ, cost effective, and non-destructive (McGrath et al., 2001). Application of phytoextraction can reduce phyto-available metals in the soil and thereby diminish toxic metal contents in agricultural products. The principal approach of phytoextraction is the use of metal-hyperaccumulator plants such as Thlaspi caerulescens, which is known to be a Cd/Zn hyperaccumulator (Baker et al., 2000). However, Cd-hyperaccumulation in higher plants is a rare phenomenon. In addition to T. caerulescens, Arabidopsis halleri (Küpper et al., 2000), Sedum alfredii (Yang et al., 2004), and Athyrium yokoscense, a fern which is common in metal-contaminated areas in Asia (Morishita and Boratynski, 1992; S Uraguchi, unpublished results) have been reported as evident Cd-hyperaccumulator plants. The exceeding bioconcentration factor of Cd and Zn in T. caerulescens shoots enables a remarkable yield of both metals from contaminated soil (Robinson et al., 1998; McGrath et al., 2001; McGrath and Zhao, 2003). These wild metal-accumulators, for example A. yokoscense, tend to grow slowly and heterogeneously and would be difficult to cultivate in fields.
Chemically assisted phytoextraction using crop species has been studied to overcome these problems (Lombi et al., 2001), but the addition of chelated substances to the soil poses a risk of leaching toxic metals into the groundwater (Lombi et al., 2001; McGrath et al., 2001; Römkens et al., 2002). Moreover, crop species generally show low Cd accumulation and less tolerance against Cd toxicity. Further exploration of Cd-accumulating crop plants will provide alternatives for phytoremediation of Cd-polluted soils. Considering the application of phytoextraction in Japan, it is also preferable that plants are able to adapt to slightly acidic soils and the humid climate of Japan. Both T. caerulescens and A. halleri are endemic to metalliferous alkaline soils in Europe (Baker et al., 2000; Dahmani-Muller et al., 2000; Lombi et al., 2000).
The Cd in plant cells tends to be stored in the apoplast and in vacuoles, which might contribute to Cd tolerance in hyperaccumulator plants and common crops (Lozano-Rodriguez et al., 1997; Küpper et al., 2000; Boominathan and Doran, 2003b; Cosio et al., 2005; Ma et al., 2005). However, free Cd ions in the cytosol can engender toxicity to plant cells. Unlike Cu and Fe, Cd is a redox-inactive metal that is incapable of producing reactive oxygen species (ROS) directly; nevertheless, Cd can indirectly promote ROS generation by disrupting physiological processes (Sanità di Toppi and Gabbrielli, 1999). Several studies have demonstrated that Cd treatment increases lipid peroxidation levels in common crops, indicating that oxidative stress is induced by Cd (Somashekaraiah et al., 1992; Gallego et al., 1996; Choui et al., 1997; Sandalio et al., 2001). These authors have also reported altered antioxidative system activity. In plants, the antioxidative system consists of enzymes including superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), and catalase (CAT), along with low molecular-weight antioxidants such as glutathione (GSH), carotenoids, and phenolics (Rice-Evans et al., 1996). In addition to the metal-compartmentation mechanism, antioxidative ability might play an important role in the tolerance of Cd-hyperaccumulator plants. Boominathan and Doran (2003a) reported that in the hairy root of T. caerulescens, SOD activity and GSH concentration were higher than in Nicotiana tabacum, and Cd treatment (178 µM) did not inhibit growth. Notwithstanding, little is known about the involvement and the response of antioxidative systems in leaves of Cd-accumulating plants.
This study investigated characteristics of Cd uptake and tolerance of eight green manure crops to seek new Cd-accumulating plant candidates and to elucidate the mechanisms involved in Cd accumulation. Three possible Cd-accumulators were identified and their tolerance against Cd examined by comparing the responses and the capabilities of their antioxidative systems. Furthermore, the ability to accumulate Cd in these Cd-accumulating candidates was evaluated by long-term Cd treatments with low Cd concentrations in a hydroponic system.
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Materials and methods |
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Plant materials, culture conditions, and Cd treatment
Kaneko Seeds Co. Ltd. (Maebashi, Gunma, Japan) supplied seeds of the green manure crops. Eight common green manure crops were tested: three Gramineae species (Avena strigosa Schreb. cv. New-oat, Sorghum bicolor Moench cv. Milo-sorgo, Echinochloa utilis Ohwi et Yabuno cv. Greenmillet-bansei), three Leguminosae species (Crotalaria juncea L., Crotalaria spectabilis Roth cv. Nemaclean, Sesbania rostrata Bremek. & Oberm.), and two Compositae species (Helianthus annuus L. cv. Hybrid-sunflower and Tagetes erecta L. cv. African-tall). The experiments were carried out in a growth chamber (14 h day with light intensity of 400 µmol photons m–2 s–1, supplied by a fluorescent lamp, and day/night temperatures of 25/20 °C).
Seeds were germinated on filter paper moistened with deionized water. Following germination, seedlings were transferred to a plastic pot and a full nutrient solution of the following composition was provided and renewed every other day: 5 mM KNO3, 3 mM Ca(NO3)2, 2 mM NH4H2PO4, 2 mM MgSO4, 25 µM H3BO3, 20 µM FeSO4, 2 µM MnCl2, 2 µM ZnSO4, 0.5 µM H2MoO4, and 0.3 µM CuSO4. The solution pH was adjusted to 5.6 with 0.1 M KOH. Plants were grown for 4 weeks and then transferred to vessels containing the nutrient medium described above with 0.75% w/v agar (gelling temperature 30–31 °C) supplemented with 1 and 5 mg l–1 Cd2+ as CdCl2 (9 and 45 µM, respectively). Media without Cd2+ were prepared for controls. Each treatment was replicated in five vessels. After treatment for 4 d, plants were harvested as explained below. Roots were washed carefully using tap water. Then whole plants were rinsed three times using distilled water. Plants were separated into leaves, stems and roots; the fresh weights of respective organs were measured. Analyses of pigments and soluble phenolics were conducted immediately using fresh samples; other fresh leaf samples were frozen in liquid nitrogen and stored at –85 °C until biochemical analyses were performed. For elemental analyses, the leaves, the stem, and the roots of one seedling from each vessel were dried at 60 °C for 3 d.
Avena strigosa, C. juncea (Cd accumulators with tolerance), and C. spectabilis (non-accumulator of Cd) were exposed to long-term Cd exposure. Seeds were germinated and the seedlings were grown for 2 weeks on vermiculite. Plants were then transferred to vessels containing the nutrient solution supplemented with Cd2+. The Cd concentrations in the solution were 1.1 mg l–1 (10 µM) for A. strigosa and 0.56 mg l–1 (5 µM) for others. The solution without Cd2+ was prepared as a control. Plants were grown for 4 weeks and then harvested as described above. Harvested samples were dried at 60 °C for 3 d and the dry weights were measured before elemental analyses.
Pigments and soluble phenolics analyses
Chlorophylls and carotenoid were extracted using N,N-dimethylformamide from fresh leaf samples; they were then determined spectrophotometrically (Porra et al., 1989; Doong et al., 1993). The concentration of total soluble phenolics in leaves, stem, and roots was determined using Folin–Ciocalteu reagent (Singleton and Rossi, 1965) after extraction using 80% methanol. Chlorogenic acid was used as a standard to calculate the concentration of soluble phenolics in samples.
Lipid peroxidation assay
Lipid peroxidation levels in the leaves were estimated as the amount of 2-thiobarbituric acid-reactive substances (TBA-rs), as described previously (Heath and Packer, 1968; Dixit et al., 2001). The absorbance of the reaction mixture was measured at 532 nm. Correction of the non-specific turbidity was made by subtracting the absorbance at 600 nm. The concentration of TBA-rs was calculated using an extinction coefficient of 155 mM–1 cm–1.
Enzymatic analyses
Leaves stored at –85 °C (0.3–0.5 g FW) were ground to a fine powder in liquid nitrogen with a chilled mortar and pestle under ice-cold conditions. They were subsequently homogenized in 50 mM K-phosphate buffer (pH 7.0) containing 0.4 mM EDTA-4H, 5 mM ascorbic acid, and 2% polyvinyl polypyrrolidone. Homogenates were centrifuged at 15 000 g for 20 min at 4 °C, and the supernatant was used for protein and enzyme determination. Protein concentration in the supernatant was measured according to the method of Bradford (1976), with bovine serum albumin as a standard. All enzymic activities were measured spectrophotometrically (UV-1200; Shimadzu Corp., Kyoto, Japan) at 25 °C.
SOD (EC 1.15.1.1 [EC] ) activity was assayed using the method described previously (McCord and Fridovich, 1968). The reduction rate of cytochrome c in the reaction mixture was measured using the rate of increase of the absorbance at 550 nm. APX (EC 1.11.1.11 [EC] ) activity was determined as described previously (Nakano and Asada, 1987). The oxidation of ascorbic acid in the reaction mixture was measured using the rate of decrease in absorbance at 290 nm; enzyme activity was calculated using an extinction coefficient of 2.8 mM–1 cm–1. GR (EC 1.6.4.2 [EC] ) activity was determined as described previously (Halliwell and Foyer, 1979). The oxidation of NADPH in the reaction mixture was measured using the rate of decrease in absorbance at 340 nm; enzyme activity was calculated using an extinction coefficient of 6.1 mM–1 cm–1. CAT (EC 1.11.1.6 [EC] ) activity was determined using the method of Aebi (1979). The disappearance of H2O2 in the reaction mixture was measured using the rate of decrease in absorbance at 240 nm. The enzyme activity was calculated using an extinction coefficient of 0.036 mM–1 cm–1. Guaiacol peroxidase (GPX; EC 1.11.1.7 [EC] ) activity was determined using the method of Choui et al. (1997). The polymerization of guaiacol in the reaction mixture was measured using the rate of decrease in absorbance at 470 nm; enzyme activity was calculated using an extinction coefficient of 26.6 mM–1 cm–1.
All enzyme activities were expressed as units per milligram of protein. One SOD unit was defined as the amount of enzyme to inhibit cytochrome c reduction by 50% at 25 °C. For APX, GR, and CAT activity, one unit was defined as the amount of enzyme to decompose 1 µmol min–1 of each substrate at 2 5 °C. One GPX unit was defined as the amount of enzyme to produce 1 µmol min–1 of tetraguaiacol at 25 °C.
Elemental analyses
Dried samples (up to 0.1 g DW) of each organ were digested by 2 ml of nitric acid in a microwave system. After dilution and filtration, element concentrations of Mg, Mn, Cu, Zn, and Cd were analysed using an inductively coupled plasma mass spectrometer (ICP-MS; Agilent 7500; Agilent Technologies Japan Ltd., Tokyo, Japan). Rhodium was used as the internal standard. Concentrations of K, Ca, and Fe were analysed using an atomic absorption spectrophotometer (AAS; Hitachi Z-5310; Hitachi Ltd., Tokyo, Japan). The accuracy and the precision of the analyses were assessed using a standard reference material NIES No.1 (National Institute for Environmental Studies, Japan). Recovery was 101% for Cd and 89–105% for the other elements analysed.
Statistical analyses
Data are presented as mean values ±standard error. To verify the statistical significance of differences among treatments and species, data obtained from short-term treatments were analysed using SPSS computer software (SPSS Inc., Tokyo, Japan) by one-way ANOVA, followed by Tukey's multiple range test (P <0.05). The data obtained from long-term treatments were analysed by Student's t-test (P <0.05).
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Results |
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Effect of short-term Cd treatment on Cd accumulation
After treatment with 5 mg l–1 Cd for only 4 d, the respective Cd concentrations of leaves of A. strigosa, C. juncea, and T. erecta—171, 118, and 157 mg kg–1 DW—were higher than 100 mg kg–1 DW (Fig. 1a), which is the value used to define Cd hyperaccumulation in leaves of plants grown in soils (Baker et al., 2000). These three species showed higher Cd contents in their leaves than did other species, even at 1 mg l–1 Cd treatment, but the level was below the threshold of hyperaccumulation (Fig. 1a). Foliar Cd contents in other species were much lower than 100 mg kg–1 DW (Fig. 1a). The highest Cd concentration in the stem was observed in C. juncea (270 mg kg–1 DW), but differences in stem Cd contents between Cd-accumulating plants and non-accumulating plants were unclear (Fig. 1b). The Cd content in roots was also generally much higher in all species tested than the contents of the shoots (Fig. 1a–c), probably because of Cd binding to the root cell walls.
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The leaf:stem concentration ratios of Cd and nutrient elements are shown in Table 1. Differences between Cd-accumulators and non-accumulators were not clear in the stem:root Cd ratio (data not shown). However, the respective leaf:stem Cd ratios of Cd-accumulators were higher than those of other species. In A. strigosa, the leaf:stem ratios of Cd, as well as those of Ca and Mg, were greater than the respective values of other species. Among the plants tested, C. juncea showed the highest leaf:stem Ca ratio. However, its leaf:stem Cd ratio was only slightly higher than that of non-accumulating plants. It is also noteworthy that T. erecta exhibited higher leaf:stem ratios of Cd, Mn, and Zn.
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Effect of short-term Cd treatment on the growth, pigments, and soluble phenolic contents, and lipid peroxidation
Fresh weights of A. strigosa were not significantly affected by Cd treatment (Fig. 2). Nevertheless, among the three Cd-accumulating plants, in C. juncea and T. erecta, a dose-dependent reduction of fresh weight was observed, especially in the leaves (Fig. 2). The amount of Chl, carotenoid, and total soluble phenolics in T. erecta leaves showed dose-dependent decreases with Cd treatment (Fig. 3a–c). Remarkable increases in TBA-rs levels occurred in the leaves of T. erecta with both Cd treatments, but the TBA-rs levels did not increase in the leaves of the other Cd-accumulators, C. juncea and A. strigosa (Fig. 3d). No significant reduction in Chl and carotenoid levels was observed in A. strigosa leaves (Fig. 3a, b). In C. juncea leaves, fresh weights decreased with Cd treatment (Fig. 2a), but Chl and carotenoid contents tended to increase with Cd exposure (Fig. 3a, b). Amounts of soluble phenolics in the leaves of C. juncea were doubled by treatment with 5 mg l–1 Cd (Fig. 3c). Similar effects of Cd on the amount of phenolics were apparent in the leaves and stems of C. spectabilis (data not shown). The amount of phenolics in the leaves of two Crotalaria plants was 1.3–4.3 times higher than those of other plants of control (data not shown).
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Antioxidative enzyme activity
Antioxidative enzyme activities in the leaves of plants exposed to Cd are shown in Table 2. SOD activity was most affected by Cd treatments. In particular, with Cd treatment SOD activity of A. strigosa increased to twice that of the control. Significant increases in SOD activity with Cd treatment were also observed in C. spectabilis (5 mg l–1 Cd), Sesbania rostrata (5 mg l–1 Cd), and E. utilis (1 and 5 mg l–1 Cd). On the other hand, SOD activity of T. erecta and H. annuus decreased significantly, even in the presence of 1 mg l–1 Cd. In T. erecta, Cd treatment depressed CAT activity significantly, and APX and GR activity tended to decrease by 5 mg l–1 Cd treatment. In C. juncea, SOD activity did not alter with Cd treatment, however, CAT and GPX activity increased by 4-fold and 3-fold, respectively, in comparison with the control values as a result of exposure to 5 mg l–1 Cd. In addition to the increased SOD activity induced by Cd treatment, A. strigosa showed higher activities of SOD, GPX, APX, and GR than those of other species (Table 2). By contrast, T. erecta showed lower activities of those enzymes. Furthermore, C. juncea exhibited lower SOD, CAT, and GPX activity in the control condition, but Cd treatment resulted in induction of CAT and GPX activity.
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Nutrient accumulation
Fe content increased 4-fold in C. juncea leaves as a result of Cd treatments (Fig. 4a), and 3-fold in the roots in the presence of 5 mg l–1 Cd compared with the control value of each organ, but they did not increase in the stems (data not shown). Fe accumulation in leaves induced by Cd treatments was also observed in C. spectabilis (data not shown). By contrast to the response in Crotalaria plants, Fe depletion, a well-known feature of Cd toxicity in plants, was observed in Helianthus annuus leaves (data not shown). No significant change in Fe concentration occurred in any of the other plants, including A. strigosa and T. erecta (Fig. 4a). The Mn and Zn concentrations in the leaves of C. juncea and T. erecta were reduced by Cd treatment, but not those in leaves of A. strigosa (Fig. 4b, c). Ca concentration was not significantly changed by exposure to Cd in any of the plants tested (Fig. 4d).
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Levels of nutrient elements in the plant bodies analysed in this study varied depending on species, although the nutrient concentration in the growth medium was the same. Iron concentrations in the leaves of C. juncea grown without Cd (control) were much lower than in those of other plants tested (Fig. 5a). The Mg levels in leaves of the three Cd-accumulators were higher than in the leaves of others, except H. annuus (Fig. 5b). The Ca content in the leaves differed among the three Gramineae species (Fig. 5c): A. strigosa accumulated Ca in leaves at the level of 13 mg g–1 DW, close to the level of Leguminosae and Compositae species (about 15–27 mg g–1 DW); by contrast, Ca levels in leaves of the other Gramineae species were lower (about 5–7 mg g–1 DW). However, in the stem, this difference in Ca content was not as clear as that in the leaves (data not shown).
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Effect of long-term Cd treatment on Cd accumulation and growth
As A. strigosa and C. juncea represented superior Cd accumulation with Cd tolerance, these two species were given a 4 week Cd treatment with lower Cd concentrations (Table 3). Crotalaria juncea, which was treated with 0.56 mg l–1 (5 µM) Cd, accumulated 103 mg Cd kg–1 DW in leaves and 267 mg Cd kg–1 DW in stems. Avena strigosa accumulated 74 mg Cd kg–1 DW in leaves and 140 mg Cd kg–1 DW in stems when treated with 1.1 mg l–1 (10 µM) Cd. The Cd concentration in leaves of C. spectabilis, which represented lower Cd accumulation in short-term Cd treatment (Fig. 1a), was much lower than in those of C. juncea and A. strigosa. Moreover, dry weights of C. spectabilis were remarkably depressed by Cd treatment (85%, 72%, and 65% decrease in leaves, stems, and roots, respectively), while no significant reduction with Cd treatment in biomass was observed in A. strigosa (P >0.05, Student's t-test). As a result of Cd treatment, dry weights of C. juncea were reduced (by 58%, 37%, and 57% in leaves, stems, and roots, respectively); however, biomass of these tissues obtained with Cd treatment was larger in C. juncea than in C. spectabilis.
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The amounts of Cd accumulated in A. strigosa shoots were higher than in other plants tested (29.3 µg and 47µg in leaves and stems, respectively). In C. juncea, which showed higher Cd concentrations in shoots than A. strigosa, Cd amounts in the shoots were lower than those of A. strigosa (20.6 µg and 24.1 µg in leaves and stems, respectively). Reflecting the reduced biomass, Cd amounts in C. spectabilis were much lower than in the two Cd-accumulating species.
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Discussion |
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This study first undertook a short-term Cd treatment to investigate novel Cd-accumulating crops and to clarify interspecific differences in Cd accumulation and tolerance of eight common manure crops. Cd concentrations used in short-term experiments (1 and 5 mg l–1) were designed with the total and exchangeable Cd concentrations found in agricultural soils in Cd-contaminated areas in Japan (Xian et al., 1988; Asami et al., 1995; S Uraguchi, unpublished results). Three species (A. strigosa, C. juncea, and T. erecta) were found to represent superior Cd accumulation abilities, especially to their leaves (Fig. 1a; Table 1). Cd concentrations in their leaves surpassed the threshold of Cd hyperaccumulation (Baker et al., 2000) despite treatment for only 4 d with 5 mg l–1 Cd, while accumulated Cd levels of other species tested were much lower in the leaves than in the roots (Fig. 1a–c), indicating that these plants showed a typical Cd-accumulation ability of non-accumulating plants to maintain Cd in the roots, probably by binding Cd to the cell walls (Baker, 1981; Boominathan and Doran, 2003b). The results obtained from short-term Cd treatments suggest A. strigosa, C. juncea, and T. erecta as candidates for Cd-accumulator crops, although further experiments using Cd-polluted soils are needed to evaluate the Cd-accumulation abilities of these plants.
Cd accumulation and tolerance in T. erecta
Thlaspi caerulescens and Arabidopsis halleri are tolerant of Cd at hyperaccumulation concentrations in shoots (Küpper et al., 2000; Lombi et al., 2000; Roosens et al., 2003). This is another reason why these species are suggested for phytoextraction applications. However, tolerance and hyperaccumulation of Cd have been revealed as genetically independent characteristics in A. halleri (Bert et al., 2003). In fact, T. erecta, one of the Cd-accumulating plants obtained in this study, reflected Cd toxicity in biomass (Fig. 2), pigments and phenolics contents (Fig. 3a–c), and the TBA-rs level (Fig. 3d). Activities of antioxidative enzymes—SOD, CAT, APX, and GR—were generally depressed by exposure to Cd (Table 2). These results for T. erecta suggest that photosynthesis, secondary metabolism, and antioxidative systems are disrupted by Cd itself, while ROS is indirectly induced by Cd, engendering oxidative damage to the cells. Previous reports showed similar Cd toxicity in common crop species (Somashekaraiah et al., 1992; Gallego et al., 1996; Larsson et al., 1998; Dixit et al., 2001). The leaf:stem ratios in T. erecta were relatively high in Mg, Mn, and Zn as well as in Cd (Table 1), indicating that, in T. erecta, Cd was partly transported opportunistically by carriers for these nutrient metals (Welch and Norvell, 1999), and that may have resulted in the Cd toxicity observed in this study. In addition to the lack of mechanisms for Cd homeostasis, the lower potential in antioxidative systems (Table 2) might be conducive to inefficient ROS scavenging and might promote Cd toxicity. It is concluded that T. erecta exhibits superior Cd accumulation ability, but its tolerance against Cd appears to resemble that of common crop species.
Cd accumulation and tolerance in A. strigosa
Avena strigosa leaves showed no toxic symptoms in the 5 mg l–1 Cd medium despite amounts of 171 mg Cd kg–1 DW. Cd toxicity was not observed in the resulting TBA-rs level, fresh weight, pigment contents, and soluble phenolics content in the leaves. These results indicate that A. strigosa is tolerant of Cd in its leaves at the level of hyperaccumulation. Enhanced SOD activity and higher activities of SOD, GPX, APX, and GR in A. strigosa leaves (Table 2) appear to be involved in effective scavenging of ROS generated by Cd toxicity. Actually, the Cd/Zn hyperaccumulator T. caerulescens showed higher activities of SOD and CAT than a non-accumulating plant (Boominathan and Doran, 2003a). In T. caerulescens, Cd has been found in the apoplast and in the vacuoles (Boominathan and Doran, 2003b; Cosio et al., 2005; Ma et al., 2005); such compartmentation might serve to protect cells from Cd toxicity. Therefore, aside from antioxidative systems, Cd compartmentation mechanisms might exist in A. strigosa representing Cd tolerance. But the mechanisms mediating Cd transport to the vacuoles are little understood, especially in hyperaccumulator plants. A transporter for Cd–phytochelatin complexes like HMT1 from yeast (Ortiz et al., 1995) and Ca2+/H+ antiporter on tonoplast AtCAX2 (Hirschi et al., 2000) may sequester Cd to the vacuoles. However, in T. caerulescens, the principal involvement of phytochelatins in Cd tolerance was not shown (Ebbs et al., 2002), but Cd in T. caerulescens leaves was shown to be associated with organic acids (Küpper et al., 2004). No investigations have addressed the function of CAX in Cd-accumulating plants.
Pathways for Ca or Mg transport appear to be responsible for Cd uptake by non-accumulating plants and T. caerulescens Prayon ecotype (Clemens et al., 1998; Zhao et al., 2002; Roosens et al., 2003; Cosio et al., 2004). Avena strigosa showed higher leaf:stem ratios of Ca and Mg as well as Cd in leaves (Table 1). Contents of Ca in A. strigosa leaves were also higher than in those of other Gramineae species known to be rich in silicate (Fig. 5c). These results indicate that the superior ability of Ca or Mg accumulation in A. strigosa might be involved in Cd accumulation. The LCT1 transporter cloned from wheat can mediate both Cd and Ca uptake (Clemens et al., 1998), and Ca channels might contribute to Cd uptake in the T. caerulescens Prayon ecotype (Zhao et al., 2002). In addition, AtCAX2 on the vacuolar membrane can transport Cd, Ca, and Mn from the cytosol to the vacuoles (Hirschi et al., 2000). Recently, HMA2 and HMA4, members of the P-type ATPase family cloned from Arabidopsis thaliana (Eren and Argüello, 2004; Verret et al., 2004) and T. caerulescens (Bernard et al., 2004; Papoyan and Kochian, 2004) have been shown to mediate Cd and Zn transport. The involvement of these transporters in Cd accumulation and tolerance of A. strigosa should be investigated further.
Cd accumulation and tolerance in C. juncea
Another Cd-accumulating plant, C. juncea showed characteristic responses to Cd. In C. juncea leaves, by strong contrast to those of T. erecta, Cd did not cause serious oxidative damage to C. juncea leaves but, as with T. erecta, the fresh weight of C. juncea leaves decreased dose-dependently according to Cd exposure (Fig. 2a). The remarkable increase of soluble phenolics and enhanced activities of GPX and CAT in C. juncea leaves as a result of Cd treatment might explain these unmatched results (Table 2; Fig. 3d). Increased lignification can decrease cell wall plasticity and cell elongation, following growth inhibition at the plant tissue level (Maksymiec, 1997; Diaz et al., 2001). In pepper (Capsicum annuum) seedlings, Cu treatment decreased the biomass, and increased total soluble phenolics and lignin contents mediated by enhanced activities of GPX, shikimate dehydrogenase (both involved in phenolic metabolism), and CAT (Diaz et al., 2001). The present study did not analyse lignin contents and shikimate dehydrogenase activity; however, the results of phenolics content and related enzyme activities suggest Cd in C. juncea leaves may increase lignification as a mechanism for Cd tolerance, probably by maintaining Cd in the cell wall fraction and, in consequence, as observed in Cu-treated pepper plants (Diaz et al., 2001), decrease its biomass. Another possible role of abundant and increased phenolics in Cd accumulation of C. juncea may be the formation of a Cd–phenolics complex in vacuoles, as indicated in Cd-tolerant species (Küpper et al., 2004; Lavid et al., 2001a, b).
The ZIP-family transporter IRT1 responsible for Fe uptake in the root can also transport Cd (Korshunova et al., 1999). Therefore, Cd treatment generally inhibits Fe uptake by plants (Yoshihara et al., 2006). In fact, the Fe content was decreased in H. annuus (data not shown) and T. caerulescens Ganges ecotypes by Cd exposure (Roosens et al., 2003). The results for C. juncea showed that Cd treatment remarkably enhanced Fe accumulation in leaves (Fig. 4a). The initial Fe content in C. juncea leaves was much lower than in any of the plants analysed (Fig. 5a) and lower than the critical deficient content of Fe in the leaves (0.05–0.15 g kg–1 DW; Marschner, 1995) without showing chlorosis. Iron deficiency increased the IRT1 transcript level in the roots and enhanced Cd accumulation in T. caerulescens (Lombi et al., 2002). It can be speculated that C. juncea is under a pseudo-Fe-deficient condition and Cd treatment might stimulate the expression of IRT1-like transporter in C. juncea root, thereby enhancing Fe uptake and probably Cd uptake. It should also be noted that C. juncea showed higher leaf:stem Ca ratios and Mg contents in leaves.
Cd accumulation and tolerance in A. strigosa and C. juncea under long-term Cd exposure
From the results of short-term Cd exposure, A. strigosa and C. juncea were shown to be superior in Cd accumulation and Cd tolerance in the leaves. Therefore, to evaluate the possibility of these two species being used in phytoextraction, long-term Cd treatments were conducted using possible accumulators (A. strigosa and C. juncea) and the non-accumulator (C. spectabilis). Foliar Cd concentration was 103 mg kg–1 DW in C. juncea, which was equivalent to the threshold of Cd hyperaccumulation (Baker et al., 2000); however, in A. strigosa, Cd concentration in the leaves was slightly below that value (Table 3). These Cd concentrations in the leaves were much lower than those of T. caerulescens treated with the same Cd concentration for 8 weeks (Cosio et al., 2005), but higher than that of C. spectabilis. The amount of Cd in shoots (leaf+stem), which will be important in phytoextraction, was 44.7 µg in C. juncea treated with 5 µM Cd and that was comparable to the value for T. caerulescens Prayon ecotype (41.4 µg) calculated from Cd concentration and the dry weight of the shoot reported previously (Cosio et al., 2005). The amount of Cd in the shoot of A. strigosa treated with 10 µM Cd was 76.3 µg which was also equivalent to the calculated value of T. caerulescens Prayon ecotype (75.8 µg) treated with 10 µM Cd for 8 weeks. However, Cd amounts in A. strigosa and C. juncea observed in this study were approximately half as much as the values of T. caerulescens Ganges ecotype treated with the same Cd concentrations applied for each species in this study (Cosio et al., 2005). With regard to tolerance, A. strigosa and C. juncea exhibited much more tolerance to Cd than C. spectabilis considering the dry weights of organs.
Crop species typically present advantages of seed availability, controlled growth in agricultural fields, and growth rates compared with wild Cd-hyperaccumulating plants. This study revealed two crop species (A. strigosa and C. juncea) which present superior Cd accumulation and tolerance capabilities compared with non-accumulating crops. Further investigations using these two species may lead to further understanding of Cd accumulation in plants. For instance, it would appear interesting to examine the effects of root exudates from A. strigosa and C. juncea on metal mobilization in the rhizosphere soils, because both species have characteristic root exudates (Uraguchi et al., 2004).
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We gratefully acknowledge Takuji Yasuda and Shigeo Kobayashi (Kaneko Seeds Co. Ltd.) for providing seeds and valuable information, and Kazutaka Murayama, Takeshi Izuta, and Yoshihiro Katayama (Tokyo University of Agriculture and Technology) for helpful advice regarding enzyme assays.
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