Characteristics of Cd uptake, translocation and accumulation in a novel Cd-accumulating tree, star fruit (Averrhoa carambola L., Oxalidaceae)

Environmental and Experimental Botany 71 (2011) 352–358

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Characteristics of Cd uptake, translocation and accumulation in a novel Cd-accumulating tree, star fruit (Averrhoa carambola L., Oxalidaceae) J.T. Li, B. Liao, R. Zhu, Z.Y. Dai, C.Y. Lan, W.S. Shu ∗ School of Life Sciences and State Key Laboratory of Biocontrol, Sun Yat-Sen University, No. 135, Xing-Gang-Xi Road, Guangzhou 510275, PR China

a r t i c l e

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Article history: Received 20 December 2009 Received in revised form 23 December 2010 Accepted 2 February 2011 Keywords: Averrhoa carambola Cd hyperaccumulation Translocation Uptake Zn pathway

a b s t r a c t Cadmium (Cd) accumulation by terrestrial higher plants is an intriguing phenomenon that may be exploited for phytoextraction of Cd-contaminated soils. Characterizing the physiological processes responsible for elevated concentrations of Cd in shoots is a first step towards a comprehensive understanding of the mechanisms underlying Cd accumulation in plants and may eventually improve the efficiency of phytoextraction. Woody species that can accumulate Cd have been recently recommended as good candidates for phytoextraction of Cd-contaminated soils. However, little is known about the mechanisms of Cd accumulation by woody species. In an attempt to understand the physiological processes contributing to Cd accumulation in woody species, Cd uptake and translocation by a novel tropical Cd-accumulating tree, star fruit (Averrhoa carambola) were characterized and compared with those of a non-Cd-accumulating tree (Clausena lansium). Our results showed that A. carambola had higher Cd uptake and root-to-shoot translocation efficiencies than C. lansium, which might account for its greater Cd-accumulating capacity. Furthermore, Cd accumulation by A. carambola was not significantly affected by zinc (Zn), whereas Zn accumulation was greatly lowered by Cd. This phenomenon could not be fully explained by a simple competition between Cd2+ and Zn2+ , implying the existence of a transport system with a preference for Cd over Zn. Collectively, our results indicate that A. carambola has noteworthy physiological traits associated with accumulation of Cd to high levels. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Cadmium (Cd) is regarded as one of the most toxic environmental pollutants and is readily absorbed by certain crops from soils (Wagner, 1993). Elevated concentrations of Cd in agricultural soils have posed a significant threat to safe food production and have therefore become a worldwide concern (Wagner, 1993; McLaughlin et al., 1999; Järup and Åkesson, 2009). The last two decades have witnessed an increasing need for the remediation of Cd-contaminated soils (Maejima et al., 2007; Leˇstan et al., 2008). Phytoextraction, i.e. the use of plants to remove pollutants (including Cd) from contaminated soils, has been proposed as a promising technology that is both low cost and environmentally friendly (Baker et al., 2000). A general approach for phytoextraction of Cd-contaminated soils is to make use of Cd-hyperaccumulating plants (Baker et al., 2000; McGrath and Zhao, 2003). However, to date, only a few herbaceous species have been identified as Cd-hyperaccumulators, including Thlaspi caerulescens, Arabidopsis halleri, Viola baoshanensis, Sedum alfredii, Sedum plumbizincicola and

∗ Corresponding author. Tel.: +86 20 39332933; fax: +86 20 39332944. E-mail addresses: [email protected], [email protected] (W.S. Shu). 0098-8472/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2011.02.001

Thlaspi praecox (Baker et al., 1994; Bert et al., 2002; Liu et al., 2004; Yang et al., 2004; Jiang et al., 2010; Vogel-Mikuˇs et al., 2010). Furthermore, in most cases, hyperaccumulators tend to grow slowly and produce a relatively low biomass (Krämer, 2010), which greatly limits the speed of Cd removal (McGrath et al., 2006; Wu et al., 2007; Zhuang et al., 2007). In recent years, interest has grown in the potential use of woody species for metal phytoextraction (Pulford and Watson, 2003; Dickinson and Pulford, 2005; Mertens et al., 2006; Komárek et al., 2008; Jensen et al., 2009). Woody species generally have high biomass and extensive root systems, which make them potentially suitable for phytoextraction (Pulford and Watson, 2003). In addition, a dense tree canopy can also prevent the spread of contaminated soil by wind erosion. There are a very few naturally occurring Cd-accumulating trees identified so far that may be efficient at removing Cd from contaminated soils, such as Salix caprea, Salix fragilis and Salix striandra (Mertens et al., 2006; Wieshammer et al., 2007). However, for most of the other woody species tested, their capacities to extract Cd from contaminated soils are relatively low, which makes their large-scale applications currently non-feasible (van Nevel et al., 2007; Jensen et al., 2009; Wang and Jia, 2010). One possible strategy to improve the efficiency of Cd phytoextraction would be the transfer of appropriate genes involved in Cd

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accumulation to fast-growing and high-biomass species (Cherian and Oliveira, 2005; Doty, 2008). This avenue, however, requires a greater understanding of mechanisms underlying Cd accumulation in plants (Verbruggen et al., 2009). Root uptake of Cd and root-to-shoot Cd translocation are the two key physiological processes related to Cd accumulation in plants (Clemens, 2006). A better understanding of these processes is therefore essential for a comprehensive understanding of the mechanisms underlying Cd accumulation in plants (Clemens, 2006). There is increasing evidence that enhanced root uptake and/or root-to-shoot translocation of Cd are responsible for the high accumulation of Cd in T. caerulescens, A. halleri and S. alfredii (Lombi et al., 2001; Zhao et al., 2002, 2006; Lu et al., 2008). For example, Zhao et al. (2002) found that symplastic root uptake of Cd is 4.5 times higher in the high Cd-accumulating T. caerulescens ecotype (Ganges) than in the low Cd-accumulating ecotype (Prayon), while Lu et al. (2008) reported that the rate of root-to-shoot translocation of Cd in a hyperaccumulating S. alfredii ecotype is >10-fold higher than in a non-hyperaccumulating ecotype. On the other hand, no highaffinity Cd transporter has been identified, although evidence has shown that radial symplastic passage and rapid xylem loading are required for efficient Cd uptake and translocation in the plants (Verbruggen et al., 2009). Alternatively, Cd uptake and translocation in Cd-accumulating plants may at least partly be achieved through the Zn pathway (Zhao et al., 2002, 2006; Wong and Cobbett, 2008; Verbruggen et al., 2009). The significant progress mentioned above was made in Cd-accumulating herbaceous species, whereas little is known about the physiology of Cd accumulation in woody species. Averrhoa carambola L., Oxalidaceae (star fruit) is a novel tropical Cd-accumulating tree, which has potential for Cd phytoextraction (Li et al., 2009). This tree can accumulate considerable amounts of Cd in its shoots (approximately 500 mg kg−1 , on a dry weight basis, DW) without significant biomass reduction, when growing in solutions containing 5 mg Cd L−1 for 3 months (Li et al., 2010). In the present study, the characteristics of root uptake and root-to-shoot translocation of Cd in A. carambola were investigated and compared with those of a non-Cd-accumulating tree [Clausena lansium (Lour.) Skeels, Rutaceae], to unravel the physiological processes contributing to the Cd-accumulating capacity of this woody species. C. lansium was selected to be compared with A. carambola; because this tree is a non-Cd-accumulating species (Li et al., 2006), while most close relatives of A. carambola are herbaceous (Huang et al., 1998). Interactions between transport of Cd and Zn in A. carambola were characterized to determine whether or not the root uptake and root-to-shoot translocation of Cd in this species were mediated via transporters for Zn. As root uptake of Cd is the first step of Cd accumulation in plants (Clemens, 2006), the characteristics of root uptake have frequently been investigated to explain the differences in Cd-accumulating ˜ capacity between species, ecotypes and cultivars (Pineros et al., 1998; Lombi et al., 2001; Zhao et al., 2002; Lu et al., 2008; Redjala et al., 2009). The previous studies have shown that root uptake of Cd can be partitioned into an apoplastic and a symplastic component, which represent Cd binding to cell walls and trans-plasma membrane transport of Cd, respectively. It is therefore important to distinguish the symplastic Cd fraction from the apoplastic one in the characterization of Cd influx into roots. One widely used approach for estimating Cd binding in root apoplast is to determine the apparent uptake of Cd at 2 ◦ C, assuming that symplastic root uptake of Cd is negligible at this low temperature (Lombi et al., 2001; Zhao et al., 2002; Lu et al., 2008; Redjala et al., 2009). In the present study, this approach was therefore applied to characterize apoplastic Cd binding in roots of the two species.

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2. Materials and methods 2.1. Plant material and culture conditions Uniform 3-month-old seedlings of A. carambola (cv. Malaysia) and C. lansium (cv. Ji-Xin) were provided by the Guangzhou Institute of Fruit Sciences (Guangzhou, China). For hydroponic cultures, seedlings were transferred to 5.0 L vessels containing basal nutrient solution and placed in a glasshouse (25 ± 1 ◦ C). The composition of the basal nutrient solution was as follows: 2.5 mM KNO3 , 2.5 mM Ca(NO3 )2 , 1 mM MgSO4 , 0.5 mM KH2 PO4 , 100 ␮M H3 BO3 , 40 ␮M MnCl2 , 2 ␮M CuSO4 , 1 ␮M ZnCl2 , 0.4 ␮M MoO4 Na2 , and 100 ␮M NaFe-EDTA. The pH of the solution was maintained at 6.0 ± 0.1 using HCl or NaOH, as required. The nutrient solution was renewed weekly. After 2 weeks of preculture, healthy seedlings with new roots were used for further experiments. 2.2. Cd-uptake experiments Cadmium uptake by roots of A. carambola and C. lansium was investigated using a depletion technique similar to that previously described by Zhao et al. (2002). In detail, seedlings were pretreated with a solution containing 2 mM MES (2morpholinoethanesulphonic acid, pH 6.0) and 0.5 mM CaCl2 for 24 h before the uptake experiment. The uptake experiment was initiated at 2 h after the beginning of the light period and performed at 25 ± 1 ◦ C. The seedlings were transferred to 125 mL vessels (one seedling per vessel) filled with 100 mL of uptake solution containing 2 mM MES (pH 6.0), 0.5 mM CaCl2 and 10 ␮M CdCl2 . For each species, 4 replications were set up and each replication comprised 1 seedling. In order to monitor the depletion of Cd in uptake solutions, at 0, 0.5, 1, 2, 3, 4 h after the initiation of uptake experiment, 1.0 mL solution was taken from each vessel for Cd determination. The removed solutions were compensated by addition of deionized water. At the end of the uptake experiment, roots of the seedlings were collected and weighed. Concentrations of Cd in the roots were calculated from the depletion of Cd in the uptake solutions and expressed on a fresh weight basis (FW). For example, concentration of Cd in the root exposed to Cd for 1 h (C1 h-root ) could be calculated according to this equation: C1 h-root = [(C0 h-solution − C1 h-solution ) × the total volume of uptake solution]/root weight, where C0 h-solution stands for the initial Cd concentration in uptake solution and C1 h-solution is the Cd concentration in uptake solution sampled at 1 h after the initiation of uptake experiment. The root uptake of Cd at 25 ◦ C represents the total amount of Cd taken by the roots, which comprises apoplastic and symplastic components (Zhao et al., 2002). To estimate the apoplastic Cd binding in roots of the two woody species, the uptake experiment described above was also carried out at 2 ◦ C (Zhao et al., 2002), in which the seedlings were transferred to an ice-cold (2 ◦ C) pretreatment solution for 30 min and then kept in vessels that contained 100 mL uptake solution. During the 4-h uptake experiment, the vessels were placed in an ice bath and shaded from light. The other experimental procedures were the same as those used previously at 25 ± 1 ◦ C. 2.3. Cd-translocation experiment To investigate Cd translocation from roots to shoots, timecourse experiments involving Cd accumulation in the shoots were conducted according to Xing et al. (2008). Seedlings of the two species were transferred to 2.0 L vessels (four seedlings per vessel) and pretreated with the basal nutrient solutions spiked with 10 ␮M CdCl2 for 24 h. Four replications were set up for each species and each replication comprised 4 seedlings. After pretreatment with Cd for 24 h, the roots of the seedlings were immersed into the basal

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nutrient solutions for 20 min to remove Cd from the root surfaces. Following this procedure, 4 seedlings (one seedling from each replication) of each species were harvested and the others were grown in the basal nutrient solutions without Cd supplement. For each species, 4 seedlings were harvested after culture in the Cd-free nutrient solutions for 1, 5 and 9 days, respectively. After harvest, all plants were rinsed with deionized water; twigs (twigs + stems) and leaves were oven-dried at 70 ◦ C to a constant weight and then their weights were recorded. 2.4. Interactions between transport of Cd and Zn To determine whether A. carambola accumulates Cd through the Zn pathway, the interactions between Cd and Zn transport were characterized using the method described by Zhao et al. (2006). To test the effects of Cd on Zn transport in A. carambola, seedlings were exposed to 2.0 L basal nutrient solutions containing 0 (control), 1, 5, 15 or 50 ␮M CdCl2 and a constant 5 ␮M Zn(NO3 )2 . Likewise, seedlings of A. carambola were grown in 2.0 L basal nutrient solutions containing 1 (control), 5, 50 or 500 ␮M Zn(NO3 )2 and a constant 5 ␮M CdCl2 , to investigate the effects of Zn on Cd accumulation. Each treatment was replicated 4 times and each replication comprised 4 seedlings in a vessel. All nutrient solutions were renewed weekly. The vessels were placed randomly in a glasshouse (25 ± 1 ◦ C) and repositioned once a week. After 8 weeks of growth, all plants were harvested and divided into roots, twigs (twigs + stems) and leaves. The 4 seedlings per vessel were combined into one sample. To remove the Cd adsorbed onto root surfaces, roots of each seedling were immersed into 20 mM Na2 EDTA (disodium ethylenediaminetetraacetate) solutions for 0.5 h. The plant materials were then treated as described above. In addition, the dry biomasses of different tissues were weighed. 2.5. Chemical analyses For heavy metal analyses, dried plant material was ground and digested with a mixture of HNO3 /HClO4 (5:1, v/v). Concentrations of the metals in digests or uptake solutions were measured using a graphite furnace atomic absorption spectrophotometer (Hitachi Z5300; Tokyo, Japan). To investigate, preliminarily, the interactions between transport of Cd, calcium (Ca) and iron (Fe) in A. carambola, Ca and Fe concentrations in different plant tissues from experiments regarding the interactions between transport of Cd and Zn were also determined. 2.6. Statistical analysis Statistical analysis was performed using SPSS version 16.0 for windows (SPSS Inc., USA). Student’s t-test was used to assess significant differences in Cd uptake and translocation between A. carambola and C. lansium. In the experiments concerning interactions between transport of Cd and Zn in A. carambola, we measured substantial variations in biomass production and tissue heavy metal concentrations among Cd or Zn treatments by a one-way ANOVA followed by Fisher’s least significant difference (LSD) test. 3. Results 3.1. Time-dependent Cd uptake by roots The symplastic root uptake of Cd in A. carambola and C. lansium was calculated by subtracting apoplastic Cd binding in root (root uptake of Cd at 2 ◦ C) from the total Cd uptake in roots (root uptake of Cd at 25 ◦ C). During the 4-h uptake experiment, the symplastic root uptake of Cd in the two species increased gradually with time (Fig. 1). However, the symplastic root uptake of Cd in A. carambola

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SU-Cd in A. carambola SU-Cd in C. lansium AB-Cd in A. carambola AB-Cd in C. lansium

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Time (h) Fig. 1. Time-dependent symplastic root uptake of Cd (SU-Cd) and apoplastic Cd binding in root (AB-Cd) of A. carambola and C. lansium treated with 10 ␮M CdCl2 for 4 h. Data are presented as means ± S.D. (n = 4). Asterisks indicate that the root SU-Cd in A. carambola is significantly (P < 0.05, t-test) higher than that of C. lansium at the same time.

was consistently higher than that of C. lansium; significant (P < 0.05) differences between the two species were found at all times after 1 h of uptake (Fig. 1). At the end of the uptake experiment, the symplastic root uptake of Cd was 2.1 times higher in A. carambola than in C. lansium. In contrast, there were no significant (P > 0.05) differences between the two species in apoplastic Cd binding in roots (Fig. 1). 3.2. Time-dependent Cd accumulation in shoots After pretreatment with Cd solutions for 24 h (day 0 in Fig. 2), the Cd concentrations in twigs and leaves of A. carambola were 2.96 and 3.16 mg kg−1 (DW), being 3.5 and 3.3 times higher than those in C. lansium, respectively (Fig. 2). Afterwards, the Cd concentrations in twigs and leaves of A. carambola increased rapidly with time over the following 9 days. However, those in C. lansium increased very slowly (Fig. 2). Consequently, on day 9 after culture in Cd-free solutions, the Cd concentrations in twigs and leaves of A. carambola were 4.5 and 9.8 times higher than those in C. lansium, respectively (Fig. 2). 3.3. The effects of Cd on Zn accumulation in A. carambola Increasing Cd concentrations in solutions with a constant concentration of Zn (5 ␮M) did not significantly decrease biomasses of A. carambola; instead, a significant increase in root biomass at 15 ␮M Cd treatment was recorded (Fig. 3a). The Cd concentrations in different tissues of A. carambola increased significantly (P < 0.05) with increasing solution Cd, while the highest tissue Cd concentration (469 mg kg−1 , DW) was recorded in the leaves at 50 ␮M Cd (Fig. 4a). By contrast, the Zn concentrations in different tissues of A. carambola were significantly decreased (P < 0.05) by increasing Cd concentrations in solutions (Fig. 4b). The Zn concentrations in roots, twigs and leaves were 1.9, 3.1 and 3.7 times lower at 50 ␮M Cd than at 0 ␮M Cd, respectively.

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Fig. 3. The effects of Cd (a) and Zn (b) treatments on tissue biomass of A. carambola grown in Cd or Zn solutions for 8 weeks. Data are presented as means ± S.D. (n = 4). Bars marked by the same letters do not differ significantly (LSD, P < 0.05).

3.4. The effects of Zn on Cd accumulation in A. carambola Tissue biomass of A. carambola was not significantly (P > 0.05) reduced by increasing Zn in solutions with a constant concentration of Cd (5 ␮M), with the only exception being the root biomass obtained in the 500 ␮M Zn treatment (Fig. 3b). Increasing solution Zn resulted in significant (P < 0.05) increases in Zn concentrations in tissues of A. carambola, while the maximum tissue Zn concentration (2121 mg kg−1 , DW) was obtained in leaves at 500 ␮M Zn treatment (Fig. 5a). On the other hand, 5 and 50 ␮M Zn slightly (P > 0.05) increased the Cd concentrations in different tissues of A. carambola, whereas the 500 ␮M Zn treatment significantly (P < 0.05) decreased the tissue Cd concentrations (Fig. 5b). 3.5. The effects of Cd on Ca and Fe accumulation in A. carambola Concentrations of Ca and Fe in different tissues of A. carambola were in the normal ranges for plants (Fig. 6). On the other hand, they were not significantly (P > 0.05) influenced by the increasing Cd levels in nutrient solutions (Fig. 6, details on results of LSD tests not shown). 4. Discussion In the present study, based on the results of the uptake experiment at 2 ◦ C, there were no significant differences between the Cd-accumulator A. carambola and the non-accumulator C. lansium in apoplastic Cd binding in roots (Fig. 1). Similarly, no significant difference was found between the two contrasting T. caerulescens ecotypes Ganges and Prayon in Cd binding in root apoplast (Zhao et al., 2002). However, an inconsistent result was recently reported by Lu et al. (2008) who found that the apoplastic Cd binding in root

is greater in the non-Cd-hyperaccumulating S. alfredii ecotype than in the Cd-hyperaccumulating one. These results suggest that there is no consistent link between apoplastic Cd binding in roots and the Cd-accumulating capacity of plants. In contrast, it has always been found that Cd-hyperaccumulating plants take up more Cd symplastically into roots than non-Cd-hyperaccumulating ones (Lombi et al., 2001; Zhao et al., 2002; Lu et al., 2008). In line with the previous results, the present study showed that the time-dependent symplastic root uptake of Cd was significantly greater in the Cdaccumulator A. carambola than in the non-accumulator C. lansium (Fig. 1). This result implies that the greater Cd-accumulating capacity of A. carambola compared to C. lansium might, at least partly, be attributed to the greater symplastic root uptake of Cd in A. carambola. Cadmium accumulation in shoots is a direct result of root-toshoot translocation of Cd, which is an important physiological process subsequent to Cd uptake into the root symplasm (Clemens, 2006). It is well known that Cd-hyperaccumulators are characterized by high Cd translocation factors (the shoot-to-root Cd quotients > 1). However, there are contrasting opinions about the extent to which Cd hyperaccumulation in plants can be explained by their more efficient root-to-shoot translocation of Cd. Lombi et al. (2001) found that the concentration of Cd in xylem sap collected from T. caerulescens Ganges ecotype is 5 times higher than that of Prayon. These authors, however, believed that this difference was largely ascribed to the 5 times difference in root uptake of Cd between the two ecotypes. Therefore, they concluded that the divergence between the two T. caerulescens ecotypes in Cdaccumulating capacity is largely explained by their difference in root uptake of Cd, rather than in translocation of Cd from root to shoot (Lombi et al., 2001). In contrast, Lu et al. (2008) found

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Solution Cd (μM) Fig. 4. The effects of solution Cd on Cd (a) and Zn (b) concentrations in different tissues of A. carambola grown in solutions with 0, 1, 5, 15 or 50 ␮M Cd, and a constant 5 ␮M Zn for 8 weeks. Data are presented as means ± S.D. (n = 4).

that there is 46 times higher concentration of Cd in shoots of a Cd-hyperaccumulating S. alfredii ecotype than in the non-Cdhyperaccumulating one, which cannot be explained by the twice higher symplastic root uptake of Cd in the Cd-hyperaccumulating S. alfredii ecotype. On this basis, it was concluded that Cd hyperaccumulation in the Cd-hyperaccumulating S. alfredii ecotype was largely attributed to greater root-to-shoot translocation of Cd, rather than enhanced symplastic root uptake of Cd (Lu et al., 2008). Interestingly, our results suggest that the greater Cd-accumulating capacity of A. carambola compared with C. lansium can be ascribed not only to the enhanced symplastic root uptake of Cd, but also to the greater root-to-shoot translocation of Cd. In the Cdtranslocation experiment of our study, the Cd concentrations in twigs and leaves of A. carambola on day 0 were 3.5 and 3.3 times higher than those of C. lansium, respectively (Fig. 2). It is unlikely that these about 3 times differences between the two species in shoot Cd concentrations would change greatly in the following 9 days without further supply of Cd in solutions, if there was no difference between the two species in root-to-shoot translocation of Cd. However, at the end of the Cd-translocation experiment (day 9, Fig. 2), there were 4.5 and 9.8 times higher concentrations of Cd in twigs and leaves of A. carambola than in those of C. lansium which could not be fully explained by the twice higher symplastic root uptake of Cd in A. carambola (Fig. 1). These results show that the root-to-shoot translocation of Cd was more pronounced in A. carambola than in C. lansium, which may be due to more xylem loading of Cd (Clemens, 2006). No high-affinity Cd transporter has been identified, although a growing body of physiological evidence suggests that more efficient root uptake and/or root-to-shoot translocation of Cd have evolved in Cd-accumulating plants (Verbruggen et al., 2009). Alternatively,

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Solution Zn (μM) Fig. 5. The effects of solution Zn on Zn (a) and Cd (b) concentrations in different tissues of A. carambola grown in solutions with 1, 5, 50 or 500 ␮M Zn, and a constant 5 ␮M Cd for 8 weeks. Data are presented as means ± S.D. (n = 4).

as a non-essential but highly toxic element for plant growth, Cd has always been demonstrated to be transported in plants by transporters for essential elements, especially for Zn (Clemens, 2006). Zhao et al. (2006) found that the Cd hyperaccumulation in A. halleri is at least partly mediated by the Zn pathway. However, it was recently reported that Cd uptake by a Cd-hyperaccumulating S. alfredii ecotype is likely regulated by Ca transporters/channels rather than Zn transporters (Lu et al., 2008). In the present study, the possibility that Cd accumulation in A. carambola was also related to the Zn pathway was tested, in order to shed light on the role of Zn transporters in Cd accumulation in woody species. Our results showed that increasing Cd concentrations in solutions significantly decreased the Zn concentrations in different tissues, while they greatly increased Cd concentrations in tissues of A. carambola (Fig. 4). At first glance, this phenomenon was not surprising, because, the decreased tissue Zn concentrations might largely be explained by the well-documented competitive interactions between Cd and Zn ions for entry into the active sites of Zn transporters (Clemens, 2006). The increased Cd concentrations in the tissues could mainly be related to the increases in the concentrations of free Cd ions in corresponding solutions (Zhao et al., 2006). However, upon examination of Zn effects on Cd transport in A. carambola, it was interesting to find that the interactions between transport of Cd and Zn in this species could not be fully interpreted by simple competition between the two ions. In contrast to the expectation, Cd concentrations in tissues of A. carambola were slightly increased by increasing concentrations of Zn in solutions (Fig. 5), with the exception of decreases recorded at the highest Zn treatment (500 ␮M Zn) which might be attributed to toxicity of Zn in this species, considering that the root biomass of A. carambola at

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5. Conclusions

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This study showed that the root uptake and root-to-shoot translocation of Cd in A. carambola were more efficient than those in C. lansium, which possibly contributed to the Cd-accumulating capacity of A. carambola. Furthermore, it was found that Cd transport in A. carambola was not significantly affected by Zn, whereas Zn transport was greatly inhibited by Cd. This finding implies that Cd accumulation in this species is mediated at least partially by Zn transporters which, however, seem to have higher affinity for Cd than for Zn. Collectively, our results primarily indicate that A. carambola has developed noteworthy physiological mechanisms to accumulate Cd at a high level.

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We thank Professor Alan J.M. Baker of the University of Melbourne, Professor Hans Lambers of the University of Western Australia and the two reviewers for their help in improving the final version of this manuscript. This work was partially supported by the International Foundation for Science, Stockholm, Sweden, through a grant to Jin-tian Li (No. C/4785-1). We are also grateful for financial support from the Science and Technology Planning Project of Guangdong Province (2009B030802005) and the China Postdoctoral Science Foundation (No. 20080440793).

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References

Solution Cd (μM) Fig. 6. The effects of solution Cd on Ca (a) and Fe (b) concentrations in different tissues of A. carambola grown in solutions with 0, 1, 5, 15 or 50 ␮M Cd, and a constant 5 ␮M Zn for 8 weeks. Data are presented as means ± S.D. (n = 4).

the highest Zn treatment was significantly lowered (Fig. 3b). This result was distinct from those of previous studies showing that Cd concentrations in tissues of the Cd-hyperaccumulator A. halleri and non-accumulating crops (such as Triticum aestivum and Triticum turgidum) are significantly lowered by increasing solution Zn (Hart et al., 2002; Zhao et al., 2006). There are two possible explanations for this interesting finding of our study, although the elaborate underlying mechanisms remain to be demonstrated. The first is that upon exposure to increasing Zn concentrations in solutions, the expression and/or activity of Zn transporters in A. carambola is enhanced which is sufficient for transport of both Zn and Cd ions from the solution to tissues. However, most available evidence argues against such a possibility. As reviewed by Verbruggen et al. (2009), most identified transporters involved in Zn/Cd uptake and translocation (such as ZNT1 and AtHMA4) are generally expressed independently of the Zn supply in hyperaccumulators, and induced by Zn deficiency in non-accumulators. An alternative explanation is the existence of a transport system with a preference for Cd over Zn. As seen in Fig. 5b, Cd concentrations in tissues were not significantly lowered, even when the molar ratio of solution Zn to Cd reached 10, suggesting that Cd was bound better than or at least as good as Zn to a transporter. Similarly, a putative Cd-preferred transport system was assumed to function in Cd hyperaccumulation in the Ganges population of T. caerulescens (Lombi et al., 2001; Zhao et al., 2002). In addition, it was found that the increasing solution Cd did not significantly (P > 0.05) alter the Ca and Fe concentrations in tissues of A. carambola (Fig. 6), indicating that the transport systems for Ca and Fe were not involved in Cd accumulation in this species (Clemens, 2006; Dong et al., 2006).

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