Characteristics of cadmium uptake in two contrasting ecotypes of the hyperaccumulator Thlaspi caerulescens

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1 Journal of Experimental Botany, Vol. 53, No. 368, pp , March 2002 Characteristics of cadmium uptake in two contrasting ecotypes of the hyperaccumulator Thlaspi caerulescens Fang-Jie Zhao 1,3, Rebecca E. Hamon 2, Enzo Lombi 1, Mike J. McLaughlin 2 and Steve P. McGrath 1 1 IACR-Rothamsted, Agriculture and the Environment Division, Harpenden, Hertfordshire AL5 2JQ, UK 2 CSIRO Land and Water, PMB 2, Glen Osmond, SA 5064, Australia Received 17 May 2001; Accepted 5 November 2001 Abstract Uptake of Cd and Zn by intact seedlings of two contrasting ecotypes of the hyperaccumulator Thlaspi caerulescens was characterized using radioactive tracers. Uptake of Cd and Zn at 2 8C was assumed to represent mainly apoplastic binding in the roots, whereas the difference in uptake between 22 8C and 2 8C represented metabolically dependent influx. There was no significant difference between the two ecotypes in the apoplastic binding of Cd or Zn. Metabolically dependent uptake of Cd was 4.5-fold higher in the high Cd-accumulating ecotype, Ganges, than in the low Cd-accumulating ecotype, Prayon. By contrast, there was only a 1.5-fold difference in the Zn uptake between the two ecotypes. For the Ganges ecotype, Cd uptake could be described by Michaelis Menten kinetics with a V max of 143 nmol g 1 root FW h 1 and a K m of 0.45 mm. Uptake of Cd by the Ganges ecotype was not inhibited by La, Zn, Cu, Co, Mn, Ni or Fe(II), and neither by increasing the Ca concentration. By contrast, addition of La, Zn or Mn, or increasing the Ca concentration in the uptake solution decreased Cd uptake by Prayon. Uptake of Ca was larger in Prayon than in Ganges. The results suggest that Cd uptake by the low Cd-accumulating ecotype (Prayon) may be mediated partly via Ca channels or transporters for Zn and Mn. By contrast, there may exist a highly selective Cd transport system in the root cell membranes of the high Cd-accumulating ecotype (Ganges) of T. caerulescens. Key words: Cd, hyperaccumulation, Thlaspi caerulescens, uptake, Zn. Introduction Hyperaccumulation of cadmium is a rare phenomenon in higher plants. So far, only Thlaspi caerulescens J. & C. Presl (Brassicaceae) has been identified as a Cd hyperaccumulator, which is defined as being able to accumulate more than 100 mg Cd kg 1 in the shoot dry weight (Baker et al., 2000). Another possible Cd hyperaccumulator is Arabidopsis halleri (L) O Kane & Al-Shehbaz ( previously known as Cardaminopsis halleri) and it has been shown that A. halleri was capable of hyperaccumulating Cd under hydroponic culture conditions (Küpper et al., 2000). T. caerulescens is also a well-known Zn hyperaccumulator, with the ability to accumulate and tolerate up to mg Zn kg 1 dry weight in the shoots without suffering phytotoxicity (Brown et al., 1995; Shen et al., 1997). It has been suggested that there exist common mechanisms of absorption and transport of several metals, including Zn and Cd, in this species (Baker et al., 1994). However, Lombi et al. recently identified two contrasting populations of T. caerulescens, which differed greatly in Cd accumulation but not in Zn accumulation (Lombi et al., 2000). The population from southern France, named the Ganges ecotype, can accumulate up to mg Cd kg 1 in the shoot dry matter without suffering phytotoxicity under hydroponic conditions. By contrast, the population from Belgium, named the Prayon ecotype, accumulated much less Cd and was also less tolerant to this metal. Further studies showed that the two ecotypes differed markedly in the kinetics of Cd uptake, but not in Zn uptake (Lombi et al., 2001). The level of Cd hyperaccumulation and tolerance shown by the Ganges ecotype is probably unprecedented in living organisms, considering the non-essential and toxic nature of Cd. Due to the large uptake, this ecotype also offers 3 To whom correspondence should be addressed. Fax: q44 (0) Fangjie.Zhao@bbsrc.ac.uk ß Society for Experimental Biology 2002

2 536 Zhao et al. a useful model to study the mechanism of Cd transport. The extraordinary ability to hyperaccumulate both Cd and Zn may have a great potential for future use in phytoremediation of contaminated soils. It is generally believed that Cd uptake by nonaccumulator plants represents opportunistic transport by a carrier for another divalent cation such as Zn 2q, Cu 2q or Fe 2q, or via cation channels for Ca 2q and Mg 2q (Welch and Norvell, 1999). Though it should be noted that most studies to date have examined Cd transport at uncharacteristically high concentrations of Cd in the experimental medium. There is evidence that in certain types of animal cells, Cd enters the cell via a voltagedependent, L-type, Ca channel (Hinkle et al., 1987, 1992). The wheat cdna LCT1 has been shown to mediate uptake of both Ca and Cd, when expressed in Saccharomyces cerevisiae (Clemens et al., 1998). Members of the ZIP gene family are capable of transporting transition metals including Fe(II), Zn, Mn, and Cd (Guerinot, 2000). The Fe(II) transporters such as IRT1 (a member of ZIP) and Nramp have been shown to be capable of transporting several metals including Cd in Arabidopsis thaliana (Korshunova et al., 1999; Thomine et al., 2000). Furthermore, the Zn transporter, ZNT1, recently cloned from the Prayon ecotype of T. caerulescens, has been shown to mediate low-affinity uptake of Cd (Pence et al., 2000). There are also numerous studies showing inhibitory effects of Ca, Zn, Cu or Mn on Cd uptake by higher plants or algae (Smeyers-Verbeke et al., 1978; Cataldo et al., 1983; Karez et al., 1990; Costa and Morel, 1993; Tripathi et al., 1995). It is not known if these mechanisms are also responsible for Cd uptake in the hyperaccumulator T. caerulescens, particularly the Ganges ecotype. The present study extends the investigation of the mechanisms of Cd hyperaccumulation in T. caerulescens (Lombi et al., 2000, 2001). The main objective was to characterize the uptake of Cd further in the two contrasting ecotypes of T. caerulescens. In particular, the kinetics of Cd uptake was investigated using a depletion technique similar to that used for measuring uptake of K (Claassen and Barber, 1974) and of Zn (Mullins and Sommers, 1986) by maize (Zea mays). Furthermore, the effects of Ca, Ca channel blockers, and several other divalent cations on Cd uptake by the two contrasting ecotypes of T. caerulescens were investigated. Materials and methods Plant culture Seeds of the Prayon and Ganges ecotypes of T. caerulescens were sown in trays containing a mixture of vermiculite and perlite moistened with deionized water. After germination in the dark (about 1 week), seedlings were provided with a full nutrient solution of the following composition: 3.55 mm Ca(NO 3 ) 2, 1.2 mm KNO 3, mm K 2 HPO 4, 1.45 mm MgSO 4,75mM Fe-HBED (di-(hydroxybenzoyl)-ethylenediamine-diacetic acid), 5 mm ZnSO 4, 10 mm MnCl 2, 0.2 mm CuSO 4, 10 mm HBO 3, 0.2 mm Na 2 MoO 4, 0.5 mm NiCl 2, and 10 mm NaCl. Solution ph was buffered at 6.0"0.2 with 2 mm MES (2-morpholinoethanesulphonic acid). Three weeks after germination, vermiculite and perlite were washed from the roots, and three seedlings were transferred to each 50 ml pot, which was wrapped with aluminium foil and contained full nutrient solution. The nutrient solution was topped up every day, completely renewed twice every week, and aerated continuously. Plants were grown in the pots for 20 d in a controlled environment growth cabinet (dayunight period 14u10 h, dayunight temperatures 22u16 8C, and a light intensity of 300 mmol m 2 s 1 ). Comparison of 109 Cd and 65 Zn uptake Twelve hours before the uptake experiment, the nutrient solution was replaced with a pretreatment solution containing 0.5 mm CaCl 2, buffered at ph 6.0 with 2 mm MES (Lasat et al., 1996). The uptake experiment started at 2 h after the light period began. The pretreatment solution was replaced with uptake solutions containing 0.5 mm CaCl 2, 2 mm MES (ph 6.0) and either 5 mm CdCl 2 or 5 mm ZnCl 2. Cadmium and zinc were labelled with the isotopes 109 Cd (17.5 kbq pot 1 ) and 65 Zn (75 kbq pot 1 ), respectively. Spiking with 65 Zn increased the total Zn concentration in the uptake solution to 5.59 mm, whereas the increase in the total Cd concentration after spiking with 109 Cd was negligible. The uptake solution was aerated continuously. To follow the depletion of 109 Cd or 65 Zn, at 0, 5, 10, 20, and 30 min, and thereafter at 15 min intervals until 210 min, 100 ml uptake solution was taken from each pot for the determination of radioactivities. Total amounts of 109 Cd and 65 Zn removed by sampling of the uptake solution were less than 3% of the initial amounts of 109 Cd and 65 Zn in each pot. After each sampling, 100 ml of deionized water was added to each pot. Transpiration loss of water was measured by weighing at 30 min intervals and compensated by additions of deionized water. At 210 min, plants were rinsed with deionized water, separated into roots and shoots, blotted dry with tissue paper, and weighed. Roots and shoots were digested with 10 ml HNO 3 (5.25 M). The radioactivity of 109 Cd and 65 Zn in the uptake solutions and plant samples were determined using a c counter (EG&G Wallac, Turku, Finland). Concentrations of Cd and Zn in the uptake solution at the end of the uptake experiment were also determined by inductively coupled plasma atomic emission spectrometry (ICP-AES; Spectro Instruments). There was an excellent agreement between the concentrations determined by ICP-AES and concentrations calculated from the radioactivities (n ¼ 60, R 2 ¼ 0.98, and 0.99 for Zn and Cd, respectively). The uptake experiment was conducted at both 22 8C and 2 8C (ice-cold). For the experiment at 2 8C, plants were transferred to ice-cold pretreatment solution 30 min prior to the uptake. Pots were placed in an ice bath and shaded from light. Each ecotype of T. caerulescens was replicated 4-fold. Effects of Ca and Ca channel blockers on uptake of 109 Cd The experimental procedure was the same as described above. There were four treatments for each ecotype: control (0.5 mm CaCl 2 ), high Ca (5 mm CaCl 2 ), qla (0.5 mm CaCl 2 q50 mm LaCl 3 ), and qverapamil (0.5 mm CaCl 2 q100 mm verapamil). Uptake solutions contained 5 mm Cd labelled with 109 Cd, and

3 2 mm MES to buffer the ph at 6.0. The experiment was performed at both 22 8C and 2 8C. Depletion of 109 Cd was followed at 0, 7.5, 15, 30, 60, 90, 120, 150, and 180 min. Each treatment was replicated 3-fold. At the end of the uptake experiment (180 min), 109 Cd in the roots and shoots was determined as described above. The concentration of La in the qla treatment at the end of the uptake experiment was determined by ICP-AES. To quantify the effect of the Ca channel blockers La and verapamil on Ca uptake by the two ecotypes of T. caerulescens, an uptake experiment was conducted using 45 Ca. There were three treatments: control (0.5 mm CaCl 2 ), qla (0.5 mm CaCl 2 q50 mm LaCl 3 ) and qverapamil (0.5 mm CaCl 2 q 100 mm verapamil), each with four replicates. The radioactivity of 45 Ca was 74 kbq pot 1. Solution ph was buffered at 6.0 with 2 mm MES. The uptake experiment was performed at both 22 8C and 2 8C. Uptake of 45 Ca was terminated at 60 min after the exposure of roots to the uptake solutions. Roots were rinsed briefly with deionized water, and then transferred to pots each containing 50 ml desorption solution that consisted of 10 mm unlabelled CaCl 2 and 2 mm MES (ph 6.0), and which was maintained at ice-cold conditions. The duration for desorption of apoplastic 45 Ca was 15 min. Roots and shoots were then rinsed with deionized water, separated, blotted dry, and weighed. Plant tissues were digested with concentrated HNO 3 at 70 8C. The radioactivity of 45 Ca was determined using liquid scintillation. Effects of Zn, Ni, Co, Cu, Mn, and Fe on 109 Cd uptake The experimental procedure was similar to that described previously (Lombi et al., 2001). Three seedlings each (40 45-d-old) of the two ecotypes of T. caerulescens were placed in separate 50 ml pots containing the pretreatment solution (0.5 mm CaCl 2, 2 mm MES, ph 6.0) 12 h before the uptake experiment. The uptake solution contained 0.5 mm CaCl 2, 2 mm MES (ph 6.0) and 5 mm CdCl 2 labelled with 3.7 kbq 109 Cd pot 1. Treatments included control, qzn (5 mm ZnCl 2 ), qni (5 mm NiCl 2 ), qco (5 mm CoCl 2 ), qcu (5 mm CuCl 2 ), and qmn (5 mm MnCl 2 ). Each treatment was replicated 4-fold. After uptake for 20 min, roots were rinsed briefly with deionized water, and the apoplastic 109 Cd was desorbed for 15 min under ice-cold conditions with 100 mm unlabelled CdCl 2 and 5 mm CaCl 2, buffered at ph 6.0 with 2 mm MES. Roots were blotted dry and weighed, and the radioactivity of 109 Cd was determined. In a separate experiment, the effect of Fe(II) on Cd uptake by the Ganges and Prayon ecotypes was investigated. There were two treatments, i.e. control (5 mm CdCl 2 ) and qfe (5 mm CdCl 2 q5 mm FeCl 2 ), each being replicated 5-fold. The procedure for this experiment was the same as described above. Statistical analysis Analysis of variance (ANOVA) was performed on all data sets, and least significant difference (LSD) was used to compare treatments. Results Cadmium uptake in Thlaspi 537 Differences between the two ecotypes of T. caerulescens in Cd and Zn uptake The accumulation of Cd and Zn by plants (Fig. 1) was calculated, on a root fresh weight (FW) basis, from the depletion of 109 Cd and 65 Zn in the uptake solutions. At 2 8C, the uptake of both Cd and Zn rapidly plateaued, Fig. 1. Cumulative uptake of Cd and Zn at 2 8C and 22 8C, as determined from the depletion of 109 Cd and 65 Zn in the uptake solution. (a) Uptake of Cd by Prayon; (b) uptake of Cd by Ganges; (c) uptake of Zn by Prayon; and (d) uptake of Zn by Ganges. Error bars represent "SEs (n ¼ 4).

4 538 Zhao et al. suggesting that the depletion from solution was likely to be due predominantly to saturable apoplastic binding of the metals in the root cell walls, henceforth described as apparent uptake. It is clear that the Prayon and Ganges ecotypes differed little in the apparent uptake of either Cd or Zn at 2 8C. The apparent uptake of Zn at 2 8C (70 80 nmol g 1 root FW) was smaller than that of Cd ( nmol g 1 root FW), and the former also reached a plateau earlier than the latter. At 22 8C, the Ganges ecotype accumulated Cd at a much faster rate than the Prayon ecotype (Fig. 1). By 210 min, almost all of the Cd in the external solution had been taken up by Ganges, but only about half of the Cd had been taken up by Prayon. The difference between the uptakes at 22 8C and 2 8C can be assumed to represent true uptake into the symplast. The net accumulation of Cd, i.e. 22 8C 2 8C, increased linearly (R 2 )0.97, P-0.001) between 45 and 120 min. The slopes were 18 and 82 nmol Cd g 1 root FW h 1 for the Prayon and Ganges ecotypes, respectively, indicating that the rate of the true uptake of Cd was 4.5-fold higher in the latter ecotype. For the Ganges ecotype that depleted Cd in the uptake solution completely (to less than the detection limit of 2 nm) within 210 min, it was possible to construct a graph of Cd influx versus Cd concentration (Fig. 2). In Fig. 2, only depletion data from the 45 min sampling onwards were used, because uptake prior to 45 min was likely to be dominated by apoplastic binding. The relationship between Cd influx and Cd concentration in the uptake solution could be fitted satisfactorily (R 2 ¼ 0.91, P-0.001) with the Michaelis Menten kinetic equation. This produced a V max of 143.2"6.6 nmol g 1 root FW h 1 and K m of 0.45"0.07 mm. The range of the Cd concentration (0 2.5 mm) shown in Fig. 2 was likely to be more realistic for plants growing on soils than that used previously (Lombi et al., 2001). At 22 8C, the Ganges ecotype also accumulated Zn faster than the Prayon ecotype (Fig. 1), reaching a plateau (due to the depletion of 65 Zn in the uptake solution) earlier than the latter. However, the difference between the two ecotypes in Zn uptake was much less marked than that in Cd uptake. During the linear phase of true uptake (between 30 and 105 min), the slopes were 150 and 228 nmol Zn g 1 root FW h 1 for Prayon and Ganges, respectively. This represents a 1.5-fold difference only. At the end of the experiment (210 min), little (-0.4%) of the 109 Cd taken up by the two ecotypes had been transported to the shoots at either uptake temperature. By contrast, a significant proportion of the 65 Zn taken up was distributed to the shoots at 22 8C, but not at 2 8C. At 22 8C, the proportions of 65 Zn in the shoots were similar in both Prayon (7.3"0.9%) and Ganges (6.8"1.5%). Effects of Ca and Ca channel blockers on Cd uptake Increasing the concentration of CaCl 2 from 0.5 mm to 5 mm or adding 50 mm LaCl 3 decreased the apparent uptake of Cd by both ecotypes at 2 8C (Table 1), indicating that both CaCl 2 and LaCl 3 reduce the apoplastic binding of Cd. Addition of verapamil (100 mm) had no effect on the apparent uptake of Cd at 2 8C. Figure 3a, b shows the cumulative net uptake of Cd in the presence of the different potential Ca channel blockers, which were calculated from the differences between 22 8C and 2 8C using the data for 109 Cd depletion from the uptake solution. In addition, Fig. 3c, d shows the net uptake of Cd at the end of the experiment (180 min), which was calculated from the differences between 22 8C and 2 8C using the data of 109 Cd in the roots and shoots. Regardless of the treatments, Cd uptake by Ganges was much greater than that by Prayon (Fig. 3; note the different scales of the y-axis used). In the control treatment, the total net uptake of Cd by Ganges was 4.8-fold higher than that by Prayon at the end of the experiment. The two ecotypes also showed marked differences in their responses to LaCl 3 and increasing CaCl 2 in the uptake solution. In the Ganges ecotype, addition of Table 1. Apparent uptake of Cd (nmol g 1 root FW) by the two ecotypes of Thlaspi caerulescens at 2 8C, measured from the depletion of 109 Cd from the uptake solution after 180 min Treatment Prayon Ganges Fig. 2. Relationship between Cd influx in the Ganges ecotype and the concentration of Cd in the uptake solution. Control (0.5 mm CaCl 2 ) 159"11 192"2 qlacl 3 125"6 143"4 qverapamil 154"6 187"11 5 mm CaCl 2 72"6 115"20

5 Cadmium uptake in Thlaspi 539 Fig. 3. Effects of La, verapamil and high Ca concentration on the cumulative net uptake of Cd in the Prayon ecotype (a) and the Ganges ecotype (b), as determined from the depletion of 109 Cd in the uptake solution, and presented as the difference between 22 8C and 2 8C. Net uptake of Cd (22 8C 2 8C) in the roots and shoots of Prayon (c) and Ganges (d) at the end of the uptake experiment. Error bars represent "SEs (n ¼ 3). LaCl 3 or increasing CaCl 2 from 0.5 mm to 5 mm had no significant effect on the net uptake of Cd (Fig. 3b, d). By contrast, the addition of LaCl 3 suppressed the net uptake of Cd markedly in the Prayon ecotype (Fig. 3a, c). At the end of the uptake experiment, net uptake of Cd in the roots and shoots was decreased by 67% in the qlacl 3 treatment compared to the control (P-0.05 according to analysis of variance after log transformation to stabilize the variance). Increasing CaCl 2 concentration also decreased the net uptake of Cd by Prayon, although the effect did not reach a significant level. In both ecotypes, the addition of 100 mm verapamil had little effect on the net uptake of Cd. In the qlacl 3 treatment, apparent uptake of La was determined from the depletion of La in the uptake solution at the end of experiment. On average, the concentration of La in the uptake solution was decreased from the initial 50 mm to 12.4 mm. There were no significant differences in uptake of La between the two ecotypes, or between 2 8C and 22 8C (Table 2). In the experiment with 45 Ca, the two ecotypes differed significantly (P-0.01) in net uptake of Ca, with Prayon accumulating on average nearly double the amount of Ca as Ganges (Fig. 4) in the control treatments. The addition of 50 mm LaCl 3 decreased the net uptake of Ca in both Table 2. Apparent uptake of La (mmol g 1 root FW) by the two ecotypes of Thlaspi caerulescens at 2 8C and 22 8C, measured from the depletion of La from the uptake solution after 180 min Uptake temperature Prayon Ganges 2 8C 3.16" " C 3.59" " Ca Fig. 4. Effects of La and verapamil on the net uptake of (22 8C 2 8C) by the two ecotypes of T. caerulescens. Error bars represent SEs (n ¼ 4).

6 540 Zhao et al. ecotypes to a similar extent (;50%; P-0.001), whereas addition of 100 mm verapamil had no significant effect. Effects of Zn, Co, Cu, Mn, Ni, and Fe on Cd uptake In the two experiments that investigated the effects of other divalent cations on Cd uptake, net 109 Cd uptake was determined after desorption for 15 min with a 20-fold higher concentration of unlabelled Cd solution and a higher concentration of Ca. On average, Cd uptake by the Ganges ecotype was 73% (Fig. 5a) or 92% (Fig. 5b) greater than that by the Prayon ecotype. Addition of Zn, Co, Cu, Mn or Ni at equal molar concentrations to Cd (5 mm) had no significant effect on Cd uptake by Ganges (Fig. 5a). By contrast, addition of Mn decreased Cd uptake by Prayon by 36% compared to the control (P-0.05). Addition of Zn also decreased Cd uptake by Prayon by 24% compared to the control (P ¼ 0.07; the differences between the qzn and qco or qni treatments were significant at P-0.05). Addition of Co, Cu and Ni had no significant effect on the Cd uptake by Prayon. Also, Fe(II) did not affect Cd uptake by the two ecotypes significantly (Fig. 5b). Fig. 5. Effects of Zn, Co, Cu, Mn, Mn or Ni (a) and Fe(II) (b) on Cd uptake by the two ecotypes of T. caerulescens. The concentrations of Cd and other divalent cations were 5 mm. Uptake of 109 Cd was for 20 min at 20 8C, followed by a desorption step with unlabelled Cd for 15 min at 2 8C. Error bars represent SEs (n ¼ 4 5). Discussion Apoplastic binding versus symplastic uptake of Cd A common difficulty encountered in the studies of root uptake of divalent cations is how to differentiate apoplastic binding from uptake into symplast. Cataldo et al. found that the Cd bound in the root apoplast of soybean (Glycine max) included both exchangeable and non-exchangeable fractions (Cataldo et al., 1983). The size of the two fractions appeared to be dependent on the concentration of Cd in the uptake solution. The exchangeable and non-exchangeable fractions accounted for up to 32% and 45% of the total uptake of Cd. Costa and Morel showed that, even after 2 h desorption with unlabelled Cd at a concentration 40-fold higher than that of the labelled Cd used in the uptake phase, about 25% of the 109 Cd uptake by the roots of Lupinus albus was due to an apoplastically bound fraction (Costa and Morel, 1993). A prolonged desorption step may also result in significant losses of Cd from the symplast through efflux or leakage. Therefore, complete removal of apoplastically bound Cd by desorption, yet without risking efflux of symplastic Cd, is probably unachievable. From these considerations and from results of previous studies (Hart et al., 1998; Lombi et al., 2001), it is probable that the desorption step used in the divalent cation competition experiments (Fig. 5) did not fully remove apoplastic Cd. Apoplastically bound Cd may be indirectly estimated from the apparent uptake at 2 8C on the assumption that metabolically dependent uptake would be negligible at low temperatures. Thus, the difference in uptake between 22 8C and 2 8C represents the metabolically dependent uptake of Cd, most likely into the symplast. Similar approaches were used by other researchers in the studies of Fe uptake by Chlorella vulgaris (Allnutt and Bonner, 1987), of Zn uptake by cultured cells of carrot (Daucus carota L.) (Hamon, 1995), and of Cd and Ca uptake by yeast (Clemens et al., 1998). Errors may arise if symplastic uptake is still significant at 2 8C, anduor if apoplastic binding of Cd is significantly affected by temperature. The rate of apoplastic binding is likely to increase with increasing temperature, with a Q 10 of about (Marschner, 1995). However, the capacity for apoplastic binding appears to have been less affected by temperature. The evidence for this is that the apparent uptake of La, which mostly likely involves apoplastic binding only (Läuchli, 1976), differed little between 2 8C and 22 8C for both ecotypes of T. caerulescens (Table 2). Results from the depletion experiments (Fig. 1) suggest that apoplastic binding of Cd and Zn reached a plateau at 45 and 30 min, respectively, at 2 8C. At 22 8C, the rate of appoplastic binding may be increased by 20 40% (Q 10 ¼ ), thus reaching the plateau earlier. It is clear that the two ecotypes of T. caerulescens had a similar capacity for

7 apoplastic binding of Cd, and of Zn. At the end of the uptake experiment (210 min, Fig. 1), apoplastically bound Cd accounted for 75% and 43% of the total uptake measured at 22 8C in the Prayon and Ganges ecotypes, respectively. In contrast, apoplastically bound Zn represented 12% and 14% of the total uptake at 22 8C in Prayon and Ganges, respectively. The higher amounts of apoplastically bound Cd ( 109 Cd-labelled) than Zn ( 65 Zn-labelled) can probably be explained by the presence of Zn (5 mm) and the absence of Cd in the nutrient solution used to grow the seedlings before the uptake experiment. Contrast between the Ganges and Prayon ecotype of T. caerulescens in Cd uptake Metabolically dependent uptake of Cd was 4.5-fold faster in the Ganges ecotype than in the Prayon ecotype (Figs 1, 3). A smaller difference between the two ecotypes was obtained in the experiments involving divalent cation competition (Fig. 5). This was probably because the desorption step used did not remove the apoplastic Cd completely (see above). The kinetic parameters obtained for the Ganges ecotype (Fig. 2, V max ¼ 143 nmol g 1 root FW h 1 and K m ¼ 0.45 mm) were similar to the values reported earlier using a different experimental procedure (Lombi et al., 2001), i.e. short-term concentrationdependent uptake followed by a desorption step. For the Prayon ecotype, it was not possible to obtain the kinetic parameters from this work, because Cd in the uptake solution was far from depletion at the end of the experiment. Because experimental procedures and conditions have a considerable influence on the kinetics of ion uptake, V max and K m reported by different authors are not strictly comparable. Nevertheless, it is interesting to observe that the V max for the Ganges was about 5 10-fold greater than the values reported for nonaccumulating plants, including soybean (Cataldo et al., 1983), lupin (Costa and Morel, 1993) and wheat (Hart et al., 1998). By contrast, the K m for the Ganges ecotype obtained in this study was 5 10-fold higher than those reported for the non-accumulating plants. It is likely that a high density on the root cell membranes of a transporter that can mediate Cd uptake contributes to the ability of Cd hyperaccumulation in this special ecotype of T. caerulescens (Lombi et al., 2000). This is consistent with the concept that for metal hyperaccumulating plants growing on highly metalliferous soils, a high V max is probably more important than a low K m for hyperaccumulation. A Cd specific transporter? The second important difference in the Cd uptake between the two ecotypes of T. caerulescens can be seen in the competitive effects of other divalentutrivalent Cadmium uptake in Thlaspi 541 cations. La 3q, a potent Ca channel inhibitor (Piñeros and Tester, 1997; also Fig. 4), suppressed the metabolically dependent Cd uptake substantially in the Prayon ecotype, but not in the Ganges ecotype. Increasing the concentration of Ca also appeared to suppress Cd uptake in Prayon but not in Ganges (Fig. 3). Addition of LaCl 3 or increasing CaCl 2 concentration in the uptake solution increased the concentration of Cl. This would decrease the activity of free Cd 2q as a result of complexation of Cd 2q with Cl. For example, free Cd 2q activity was decreased by 50%, from 3.73 mm in the control to 1.86 mm in the 5 mm CaCl 2 treatment (computed using GEOCHEM-PC; Parker et al., 1995). However, as this effect was consistent for both ecotypes, and yet neither increased CaCl 2 nor LaCl 3 led to a decrease in Cd uptake by Ganges, it is reasonable to assume that the decrease in Cd uptake by Prayon engendered by these agents was due to a direct effect of the cations rather than the increased complexation with Cl. The results suggest that Ca channels may contribute to the Cd uptake by the low accumulating Prayon ecotype, but play no significant role in Cd hyperaccumulation by the Ganges ecotype. Contrary to the difference in Cd uptake, Ca uptake was higher in the Prayon than in the Ganges ecotype (Fig. 4). In this study, verapamil at 100 mm was found to have no effect on the uptake of Ca or Cd by either ecotype of T. caerulescens. It has been shown that verapamil at mm concentrations blocked voltage-dependent Ca and Cd influx in animal cells (Hinkle et al., 1987; Piñeros and Tester, 1997). The reported effects of verapamil are inconsistent in plant studies. It was found that verapamil at 100 mm had no effect on Ca influx into the plasma membrane vesicles isolated from wheat roots (Huang et al., 1994) or oat seedlings (Babourina et al., 2000). By contrast, verapamil was found to decrease Ca uptake, but to increase Cd uptake in the aquatic plants Cricosphaera elongata (Karez et al., 1990) and Ceratophyllum demersum (Tripathi et al., 1995). T. caerulescens is well known for its ability to hyperaccumulate Zn. It has been suggested that a common mechanism may explain hyperaccumulation of multiple heavy metals by T. caerulescens (Baker et al., 1994). Recently, Pence et al. cloned a Zn transporter cdna, ZNT1, from the Prayon ecotype of T. caerulescens (Pence et al., 2000). ZNT1 was found to mediate highaffinity Zn 2q uptake (K m ¼ 7.5 mm) as well as low-affinity Cd 2q uptake. In a yeast mutant expressing ZNT1, Cd influx increased linearly with Cd concentration in the medium and did not conform to Michaelis Menten kinetics, although the level of influx was comparable to that for Zn 2q. ZNT1 may indeed be partially responsible for Cd uptake in the low accumulating Prayon ecotype. This is consistent with the inhibitory effect of Zn on Cd uptake observed in Prayon (Fig. 5; Lombi et al., 2001). In

8 542 Zhao et al. the high Cd-accumulating Ganges ecotype, however, Cd uptake probably does not share the same system as that for Zn uptake for two reasons: (1) Zn, as well as several other divalent cations, did not inhibit Cd uptake (Fig. 5); and (2) the difference between the two ecotypes in Cd uptake was much greater than that for Zn uptake (Figs 1, 3; Lombi et al., 2001). The physiological evidence obtained from this and the previous study (Lombi et al., 2001) suggests the existence of a highly selective Cd transport system, which is highly expressed in the root cells of the Ganges ecotype of T. caerulescens. This hypothesis is being tested using molecular biological tools. It is speculated that some analogues of the metal transporters in the ZIP family (Guerinot, 2000) may be involved. Recently, Rogers et al. showed that a single amino acid substitution on the Fe(II) transporter protein, IRT1, of Arabidopsis thaliana altered the selectivity of the protein for Fe, Zn or Mn (Rogers et al., 2000). Whether a mutated IRT1, or other metal transporters with some modifications of the amino acid sequence, is responsible for the much enhanced Cd accumulation in the Ganges ecotype remains to be investigated. Acknowledgements We thank the Australian GRDC for a fellowship to FJZ. We gratefully acknowledge financial support from DG XII of the European Commission for the PHYTOREM Project. IACR-Rothamsted receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom. References Allnutt FCT, Bonner Jr WD Characterization of iron uptake from Ferrioxamine B by Chlorella vulgaris. Plant Physiology 85, Babourina O, Shabala S, Newman I Verapamil-induced kinetics of ion flux in oat seedlings. 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