Author for correspondence: Christopher S. Cobbett Tel: +61 3 8344 6246 Fax: +61 3 8344 5139 Email:firstname.lastname@example.org
• The Zn/Cd-transporting ATPases, HMA2 and HMA4, essential for root-to-shoot Zn translocation, are also able to transport Cd. Phytochelatins (PCs) are a major mechanism of Cd detoxification through the sequestration of PC-Cd complexes in vacuoles. The roles of HMA2 and HMA4 in root-to-shoot Cd translocation and Cd tolerance were investigated in the PC-deficient, cad1-3 mutant and CAD1 backgrounds.
• Six lines, with all possible combinations of hma2, hma4 and cad1 mutations, were constructed. The lines were tested for Cd-sensitivity on agar medium, and radioactive 109Cd was used to measure Cd uptake and translocation from root to shoot over periods of up to 6 d.
• In hma4 and hma2,hma4, but not hma2, root-to-shoot Cd translocation was decreased to about 60 and 2%, respectively, of that in the wild-type. Cd sensitivity increased approximately twofold in the hma2,hma4 mutant in both CAD1 and cad1 backgrounds. PC deficiency resulted in an increase in shoot Cd concentrations.
• The near-complete abolition of root-to-shoot Cd translocation resulting from the loss of function of HMA2 and HMA4 demonstrates they are the major mechanism for Cd translocation in Arabidopsis thaliana.
A further well-characterized mechanism of Cd detoxification in plants is the phytochelatin (PC) peptides. PCs are synthesized enzymatically by PC synthase from glutathione and are induced in planta by exposure to a wide range of heavy metals. PCs have the structure (γ-GluCys)n-Gly and form thiolate bonds with heavy metal ions, such as Cd, in complexes that accumulate in vacuoles. In Arabidopsis a PC synthase-deficient, cad1, mutant is hypersensitive to a number of heavy metals (Howden & Cobbett, 1992; Howden et al., 1995). PCs have been implicated in the root-to-shoot translocation of Cd. When PC synthase was expressed in the roots of the cad1-3 mutant, PCs were detected in shoot tissue demonstrating root-to-shoot translocation (Gong et al., 2003). Experiments where a wild-type shoot was grafted on to cad1-3 root stock have also demonstrated PC translocation in the opposite direction from shoot to root (Chen et al., 2006). In Brassica napus plants exposed to Cd, high concentrations of Cd and PCs are found in phloem sap (Mendoza-Cozatl et al., 2008).
In this study, we investigate the effect of combinations of hma2, hma4 and cad1 mutations on Cd sensitivity and root-to-shoot Cd translocation in A. thaliana. While loss of function of HMA2 and HMA4 together had only a small effect on Cd sensitivity in both the CAD1 and cad1 backgrounds, the near-complete loss of root-to-shoot translocation in the hma2,hma4 double mutant demonstrates that the HMAs are the major mechanism mediating Cd translocation.
Materials and Methods
Plant material and growth conditions
The Arabidopsis thaliana (L.) Heynh strains used in this study were wild-type (Col0), hma2-4 and hma4-2 (Hussain et al., 2004) and cad1-3 (Howden et al., 1995) mutants (all in the Columbia ecotype). The hma2-4,hma4-2 double mutant was described previously (Hussain et al., 2004). The cad1,hma mutant combinations were generated by crossing of the hma single and double mutants with cad1-3. F1 plants were allowed to self-fertilize and F2 progeny were screened as follows. F2 populations segregating for hma2 and cad1 or hma4 and cad1 were tested for sensitivity to 0.5 µm Cd on which the cad1 but not the hma mutant parents showed sensitivity. Cd-sensitive individuals were presumed to be cad1 mutants and were screened for their hma genotype by PCR, as previously described (Hussain et al., 2004). The cad1-3 mutation is a G to C single base-pair substitution that abolishes a NlaIV restriction site. This polymorphism was used to confirm the cad1 genotype The population segregating for hma2, hma4 and cad1 was first allowed to grow in soil to identify Zn-deficient hma2, hma4 double mutant individuals, as previously described (Hussain et al., 2004). These were then rescued by the application of excess Zn to the soil and tested for their cad1 genotype using the NlaIV polymorphism. Seed stocks were generated by growing the hma2,hma4 genotypes in the presence of excess Zn. Seeds were surface-sterilized and germinated on agar medium under conditions as previously described (Howden et al., 1995). MM solution comprised 5 mm KNO3, 2.5 mm KH2PO4, 2 mm MgSO4, 2 mm Ca(NO3)2, 50 µm Fe(III)-EDTA, 70 µm H3BO3, 14 µm MnSO4, 10 µm NaCl, 1 µm ZnSO4, 0.5 µm CuSO4, 0.2 µm (NH4)6Mo7O24, and 0.01 µm CoCl2. MM agar medium comprised MM solution, 2% (w/v) sucrose and 0.7 or 0.8% Bacto agar.
Tests for Cd sensitivity
Seeds were germinated and grown on 0.8% agar MM containing different concentrations of added CdSO4. Shoot FW was measured 12 or 14 d post-imbibition. For root growth analysis, seeds were germinated and grown for 7 d on 0.7% agar medium positioned vertically to promote downwards growth of roots along the surface of the medium before transfer to medium containing added CdSO4. Root growth was measured at 24 h intervals for 4 d.
109Cd translocation analysis
For the short-term assay, seedlings were germinated and grown on 0.7% agar medium for 10 d before being transferred on to fresh medium for an additional 6 d. Seedlings were gently removed from the agar and placed in 2 ml of radioactive 109Cd uptake solution (quarter-strength MM without Fe(III)-EDTA + 0.5 µm CdSO4) in a 24-well plate and placed in a plant growth cabinet (20°C, 150–250 µmol m−2 s−1). At each time-point, six plants were transferred into 2 ml of washing solution (quarter-strength MM without Fe(III)-EDTA + 20 mm Ca(NO3)2) and the roots were washed for 2 min. The shoots and roots were separated, blotted dry, weighed and counted for radioactivity using a LKB-Wallac1282 Compugamma instrument. Each experiment was carried out on two separate occasions.
For the longer-term assay, seedlings were grown for 12 d and transferred on to 0.7% agar plates containing 0.2 or 0.5 µm 109Cd. Sterile toothpicks were used to prevent direct contact between leaves and the agar medium. At each time-point, roots and shoot tissues of six plants were separated. Shoot tissues were weighed and directly assayed for radioactivity. Adhering agar on root tissues was removed by a jet of water and the roots were then washed in 2 ml of washing solution for 1 min, blotted dry, weighed and assayed for radioactivity.
Statistical analysis was carried out using Microsoft Excel 2004 for Mac. Quantitative values are presented as mean ± standard error of the mean (SE). Significance values were adjusted for multiple comparisons using the Bonferroni correction. Statistical significance was at the 0.05 level.
hma mutations increase Cd-sensitivity to a small extent
Earlier studies of single hma mutants or transgenic, overexpression lines have indicated a role for HMA2 and HMA4 in Cd tolerance and translocation. To test this further, we compared wild-type (Col0) with single and double hma2 and hma4 mutants previously isolated in the Col background (Hussain et al., 2004). In the previous study we had also generated hma, cad1 mutant combinations. These, however, were derived from crosses between hma mutants isolated in the Ws ecotype and the cad1 mutant in the Col background. It was possible that apparent Cd-sensitive phenotypes may have been due, in part, to background effects, particularly the HMA3 locus, which is nonfunctional in Col0 but is believed to be active in Ws. To overcome these uncertainties, the various hma,cad1 mutant combinations were reconstructed from crosses between the parental mutant lines, hma2-4, hma4-2 and cad1-3, previously identified in the Col0 background.
To measure the relative sensitivity of the different lines to Cd, seeds were germinated directly on agar mineral salts medium with added CdSO4 and allowed to grow for 14 d, after which shoot FW was measured. The growth of the wild-type compared with the different CAD1,hma mutant combinations is shown in Fig. 1. In repeated experiments when grown on control medium in the absence of Cd, the hma2,hma4 line gave significantly greater (c. 20%) FW than the wild-type or single hma mutants (data not shown). Because of this, even though on some concentrations of Cd the hma2,hma4 plants showed a distinct Cd-sensitive phenotype compared with the wild-type (Fig. 1b), there was no significant difference in shoot FW from the wild-type. To take this into account, Fig. 1a shows growth relative to that on Cd-free medium rather than absolute FW values. In the presence of 1.0 and 1.2 µm Cd, both the CAD1,hma4 and CAD1, hma2,hma4 lines were relatively more sensitive to Cd than the CAD1,HMA wild-type control. No significant difference was observed at higher Cd concentrations or between the control and the CAD1,hma2 line at any Cd concentration tested. We also tested the cad1,hma mutants compared with the cad1,HMA control (Fig. 2a,b). Because cad1 mutants are Cd-hypersensitive, a lower range of Cd concentrations was used (0.6, 0.12 and 0.18 µm). In this case, no significant differences were observed between the lines when grown on control medium without Cd, so the actual FW data are shown in Fig. 2. No significant differences between the cad1,HMA control and cad1,hma2 were observed, while the cad1,hma4 line was more sensitive than the control at the higher concentrations of Cd tested. The cad1,hma2,hma4 mutant was more sensitive than the cad1,HMA control at all concentrations tested.
To measure the sensitivity of root growth to Cd, seedlings were germinated and grown on agar plates in the absence of Cd, then transferred to medium containing Cd, and root growth was measured over the ensuing 4 d. To observe an effect of Cd on root growth under these conditions, considerably higher concentrations of Cd than in the shoot growth assay were required. For the CAD1 lines, growth relative to that in the absence of Cd is shown in Fig. 1c. A small but significant difference in root growth was observed between the CAD1,HMA and CAD1,hma4 lines in the presence of 60 and 80 µm Cd. No difference between the HMA and hma2,hma4 lines was observed. For the cad1 lines (Fig. 2c), the cad1,hma4 and cad1,hma2,hma4 lines showed greater inhibition of root growth than the cad1,HMA control at the higher Cd concentrations tested (15–25 µm).
While mutation of hma4 or both hma2 and hma4 conferred an increase in Cd sensitivity in most cases, that increase was relatively small in comparison to the effect of the cad1 mutation. For the cad1 mutant, the same degree of inhibition of shoot growth can occur with c. 10- to 20-fold less Cd compared with the wild-type (Ha et al., 1999); this study). In contrast, the concentration of Cd required for an approximately 50% decrease in shoot FW of the hma2,hma4 line was only two- to threefold less than that required to inhibit the corresponding HMA wild-type. This shows that in both the CAD1 and cad1 backgrounds, loss of HMA2 and HMA4 has only a limited influence on Cd sensitivity. The influence of the hma mutations on Cd sensitivity was more consistently observed in the cad1 background compared with CAD1.
Loss of both HMA2 and HMA4 has a major effect on root-shoot Cd translocation
To determine the relationship between Cd sensitivity and Cd uptake and translocation, we measured the uptake of the radioisotope 109Cd into roots and its translocation into shoots over a period of hours. The amount of Cd g−1 FW in roots increased approximately linearly over the course of 7 h and no difference between wild-type and hma2,hma4 plants was detected (Fig. 3a). In shoots, Cd g−1 FW accumulated approximately exponentially over the same period and concentrations in hma2,hma4 shoots were approximately half of those in the wild-type (Fig. 3b). The percentage translocation into the shoots of the total Cd taken up into the hma mutant plants was therefore also approximately half that in the wild-type (Fig. 3c). A similar effect was observed when comparing the cad1 and cad1,hma2,hma4 lines (Fig. 3d).
Previous observations have shown that Zn accumulates in the roots of the hma4 and hma2,hma4 mutants (Hussain et al., 2004). Cd accumulates in the roots of the hma4 mutant, but the hma2,hma4 mutant has not been previously tested. In this study, no significant difference in Cd accumulation in roots between wild-type and hma mutant plants was observed in the short-term uptake experiment. To test this further, Cd accumulation was assayed over a number of days. In this case, plants were grown in agar medium in the absence of Cd and transferred to the same medium containing 109Cd and assayed after periods of 2, 4 and 6 d. CAD1 lines were first assayed in the presence of 0.5 µm Cd (Fig. 4). Because cad1 lines were significantly inhibited by this concentration of Cd, subsequently all lines were assayed in the presence of 0.2 µm Cd. These data are shown in Fig. 5 and show only the results obtained after 4 d. (The full data sets are shown in Supporting Information, Figs S1 and S2.) In all experiments shown, the FW of both roots and shoots of all lines increased two- to threefold from 2 to 6 d (data not shown), indicating that growth was not significantly affected by the Cd concentration during this period. Furthermore, the total Cd g−1 FW per plant did not differ significantly between the different genotypes (data not shown), indicating that the genotype did not influence the uptake of Cd from the medium. A summary of the main observations is as follows: only small and a few statistically significant differences between HMA and hma2 lines were observed; hma2,hma4 lines showed increased Cd accumulation in roots and < 3% Cd translocation to shoots; the hma4 lines showed an intermediate effect in both root accumulation and translocation to the shoot. In general, the same effects of the hma mutations were observed in the CAD1 and the cad1 backgrounds at the concentration(s) tested.
PC deficiency has a minor effect on shoot Cd accumulation
In the short-term experiment, no significant difference between the CAD1 and cad1 lines was observed for Cd uptake or translocation (Fig. 3c,d and data not shown). In the longer-term experiment in the presence of 0.2 µm Cd (Fig. 5, Fig. S2), the concentration of root Cd in the cad1 mutants was significantly less than in the corresponding CAD1 line for all HMA genotypes at all three time-points, except for the hma2,hma4 double mutant, where the cad1 and CAD1 lines showed no significant difference. Differences in the concentrations of shoot Cd between CAD1 and cad1 lines were significant for some HMA genotypes at some time-points, but no consistent trend was observed, while the percentage translocation was less in the CAD1 lines compared with the corresponding cad1 lines at all time-points for all HMA genotypes, including the hma2,hma4 double mutant, except at 6 d. These data indicate that the presence of PCs allows a greater proportion of the Cd to be retained in the roots rather than being translocated to shoots.
In the short-term uptake and translocation assay in which all lines were exposed to the same concentration of Cd, the concentration of Cd in roots generally was not significantly affected by the hma genotype, while in shoots, the hma2,hma4 mutant accumulated 40–70% of the level in the corresponding HMA wild-type. In contrast, in the longer-term assay, the hma genotype had a significant effect on both root and shoot Cd concentrations. In these experiments, Cd accumulation in roots increased in hma4 lines and further increased in hma2,hma4 lines. Correspondingly, Cd accumulation in shoots decreased in the hma4 lines and further decreased in the hma2,hma4 lines to < 3% of the HMA wild-type.
These data demonstrate that HMA2 and HMA4 together provide the major mechanism by which Cd is translocated from root to shoot. The almost complete loss of Cd translocation in the hma double mutant suggests that, at these concentrations of Cd, no other metal transporters can allow significant adventitious transport of Cd in the same cells and tissues in which the HMAs are expressed. Similarly, these HMAs are the primary mechanism for Zn translocation, as indicated by the previously described Zn-deficient phenotype of the hma2,hma4 double mutant (Hussain et al., 2004). The Zn-deficient phenotype can be rescued by the application of additional Zn to the soils, suggesting that at higher Zn concentrations there are additional mechanisms for Zn transport. The same may be true for Cd but cannot be readily tested, owing to the toxicity of Cd at higher concentrations.
In contrast to the significant role of the HMAs, PCs appeared to have a relatively small effect on root-to-shoot Cd translocation. In general, the loss of PCs in the cad1 mutants resulted in lower Cd accumulation in roots if either HMA was functional and an increased percentage translocation to shoots regardless of HMA genotype. This may reflect the decreased ability of PC-deficient root cells to sequester Cd, resulting in an increase in free Cd and allowing a greater amount of root-to-shoot translocation by the HMAs. An alternative is that PCs are important in shoot-to-root translocation as suggested by a recent analysis of phloem sap in Brassica napus (Mendoza-Cozatl et al., 2008). However, the difference in root Cd concentrations or the percentage translocation to shoots between the CAD1 and cad1 genotypes was not greater than twofold, indicating that PCs have only a small effect on Cd translocation.
Previous studies comparing Cd uptake and translocation in cad1 and wild-type plants have shown different effects. Larsson et al. (2002), using 0.5 µm Cd over a 2 h period, found similar amounts of Cd accumulated in the mutant and wild-type. Although the concentrations of Cd accumulated in shoots and roots were not significantly different when calculated as percentage translocation, the cad1 mutant tended to translocate less Cd to the shoot than the wild-type (Larsson et al., 2002). Gong et al. (2003), using 20 µm Cd, found less Cd in shoots and more in roots of the cad1 mutant compared with the wild-type in the later stages of a 72 h period. In the former study, the cad1 mutant was reported to be more sensitive than the wild-type to the concentration of Cd used when growth was measured over an extended period of 10 d. A similar effect was observed in the latter study during the assay period of 72 h. Because of the differences in the conditions used in these and the present study, it is difficult to make direct comparisons. While root-specific expression of PC synthase in a cad1 mutant background (Gong et al., 2003), grafting experiments between wild-type and cad1 mutant plants (Chen et al., 2006) and analysis of phloem sap in Brassica napus (Mendoza-Cozatl et al., 2008) have shown that PCs can be translocated, it is not clear to what extent translocation of PCs themselves contributes to Cd detoxification. While PCs may play a role in Cd translocation, it appears that any such role is minor compared with the role of the HMAs.
Previous observations of increased Cd-sensitivity of the hma4 mutant have been confirmed here and extended by the analysis of the hma double mutant. The effect of the hma4 and hma2,hma4 mutations on the sensitivity of root growth to Cd was more consistently apparent in the cad1 background compared with the CAD1 lines. Similarly, the effect of Cd on shoot growth was less apparent in the CAD1 lines where a difference between the wild-type and hma4 or hma2,hma4 was only observed at the lowest concentration tested. Since the effect of hma2 and hma4 is to cause increased Cd accumulation in roots but decreased Cd in shoots, the inhibition of shoot growth is presumably primarily the result of inhibition of root growth or metabolism. One possibility is that Cd accumulation in roots specifically inhibits translocation of Zn, or possibly some other metal ions, to shoots. Although the absence of PCs in the cad1 lines coupled with the loss of HMA function resulted in a more consistently observable effect on growth inhibition, the effect of the hma mutations was to increase Cd-sensitivity by c. two- to threefold at most. In contrast, PC deficiency results in a 10- to 20-fold increase in Cd sensitivity.
Thus, while the HMAs have a major role in Cd translocation, their loss had relatively little effect on Cd sensitivity. In contrast, previous observations have shown that over-expression of HMA4 from a heterologous promoter can confer increased Cd tolerance, while, in general, over-expression of CAD1 has little effect. In A. halleri, a species which hyperaccumulates and tolerates high concentrations of Zn and Cd, a major QTL locus conferring Cd tolerance corresponded to a highly expressed HMA4 gene (Courbot et al., 2007) and a recent study using RNAi has shown that HMA4 is required for Cd tolerance (Hanikenne et al., 2008). This demonstrates that an increased capacity to translocate Cd from roots to shoots via the HMAs is required to confer a significant capacity for increased Cd accumulation and tolerance. In contrast, as shown here, the loss of the HMAs by mutation has only a small effect on tolerance even though there is a large effect on root-to-shoot translocation.