In this study, we investigated the accumulation of phytochelatins (PCs) and other low molecular weight (LMW) thiols in response to Cd exposure in two contrasting ecotypes differing in Cd accumulation. Using a root elongation test, we found that the highly accumulating ecotype Ganges was more tolerant to Cd than the low Cd-accumulation ecotype Prayon. l-buthionine-(S,R)-sulphoximine (BSO), a potent inhibitor of the γ-glutamylcysteine synthetase (γ-ECS) (an enzyme involved in the PC biosynthetic pathway), increased the Cd sensitivity of Prayon, but had no effect on Ganges. Although PC accumulation increased in response to Cd exposure, no significant differences were observed between the two ecotypes. Cd exposure induced a dose-dependent accumulation of both Cys and a still unidentified LMW thiol in roots of both ecotypes. Root accumulation of Cys and this thiol was higher in Ganges than in Prayon; the ecotypic differences were more pronounced when the plants were treated with BSO. These findings suggest that PCs do not contribute to the Cd hypertolerance displayed by the Ganges ecotype of Thlaspi caerulescens, whereas Cys and other LMW thiols might be involved.
In recent years, a great deal of research has been carried out on the physiological and molecular mechanisms responsible for metal hyperaccumulation and tolerance (see reviews by Lasat 2002; McGrath, Zhao & Lombi 2002; McGrath & Zhao 2003; Krämer 2005; Pilon-Smits 2005). However, the full picture is far from complete, and much more knowledge is still needed before hyperaccumulating traits can be transferred to high-biomass plants, in an attempt to develop an economically feasible technology to phytoremediate metal-polluted soils.
Regarding metal hyperaccumulation, the Zn and Cd hyperaccumulator Thlaspi caerulescens has been suggested as a model species for research on metal uptake, accumulation and tolerance (Assunção, Schat & Aarts 2003). While Zn hyperaccumulation and tolerance have received considerable attention, the mechanisms for Cd hyperaccumulation and tolerance have only recently started to be elucidated (Ebbs et al. 2002; Papoyan & Kochian 2004; Ma et al. 2005; Ueno et al. 2005). In part, this is due to the identification of a T. caerulescens ecotype (named Ganges) from southern France that shows a much higher Cd accumulation capacity than other T. caerulescens populations tested so far (Lombi et al. 2000; Roosens et al. 2003). Most interestingly, the high and low Cd ecotypes appear to have a similar capacity to hyperaccumulate Zn (Lombi et al. 2000, 2001; Zhao et al. 2002), providing an excellent opportunity to unravel the mechanisms behind Cd hyperaccumulation and tolerance in T. caerulescens.
Phytochelatins (PCs) are small metal-binding peptides with the general structure (γ-glu-cys)n-gly in which n varies from 2 to 11 (Grill, Winnacker & Zenk 1985). PC accumulation is induced in higher plants in response to Cd exposure (Schat et al. 2002). PC-based Cd sequestration is generally considered essential for constitutive Cd tolerance in organisms with functional PC synthase genes (Cobbett & Goldsbrough 2002; Schat et al. 2002). Likewise, overexpression of some enzymes and transporters involved in PC-based metal sequestration has been studied in an attempt to understand the implication of PCs and other related thiols on Cd detoxification (Ortíz et al. 1995; Clemens et al. 1999; Zhu et al. 1999a,b). A role for PCs in Cd detoxification was supported by the isolation of Arabidopsis thaliana mutants that are deficient in PC and glutathione (GSH) synthesis, but which are more sensitive to Cd exposure (Howden et al. 1995a,b; Cobbet et al. 1998). Vögeli-Lange & Wagner (1989) proposed a model in which PCs act as transporters or shuttles of Cd to the vacuole. Vacuolar accumulation of PC–Cd complexes was demonstrated in tobacco plants (Vögeli-Lange & Wagner 1989). Similarly, Cd sequestration in vacuoles as Cd–S crystallites in high molecular weight PC–Cd–S complexes has been observed in tomato (Reese, White & Winge 1992), Silene vulgaris (De Knecht et al. 1994) and Brassica juncea (Speiser & Abrahamson 1992). In B. juncea, Salt et al. (1995) observed that the majority of Cd appeared bound to S ligands, with a probable Cd–S4 coordination and a bond-length coincident with that of the purified PC–Cd complex.
The main objective of this work was to investigate the accumulation of PCs and other low molecular weight (LMW) thiols in response to Cd exposure in two contrasting ecotypes of T. caerulescens differing in Cd accumulation.
MATERIALS AND METHODS
Plant material and growth conditions
Seeds of the high (Ganges) and low (Prayon) Cd ecotypes of Thlaspi caerulescens J. & C. Presl were germinated on a mixture of perlite and vermiculite moistened with deionized water for 1 week. Subsequently, the seedlings were provided with a nutrient solution as previously described (Shen et al. 1997) (in µM): Ca(NO3)2, 1000.0; MgSO4, 500.0; K2HPO4, 50.0; KCl, 100.0; H3BO3, 10.0; MnSO4, 1.8; Na2MoO4, 0.2; CuSO4, 0.31; NiSO4, 0.5; Fe(III)–ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDHA), 50.0; and ZnSO4, 5.0. Solution pH was kept at around 6.0 with 2000 µm 2-morpholinoethanesulphonic acid (MES, 50% mol mol−1, potassium salt). After 20 d, perlite and vermiculite were carefully washed off from the roots, and the seedlings were transferred to 350 mL black pots (1 seedling per pot) filled with the same nutrient solution. The plants were grown for 15 d before Cd treatments in a controlled environment room under the following conditions: 16 h day length, light intensity of 350 µmol photons m−2 s−1 supplied by sodium vapour lamps, 20:16 °C day:night temperature, and 60–70% relative humidity. The hydroponic solution was continuously aerated and renewed every 3 d. All experiments were conducted under the same controlled conditions.
Plants of approximately 2 g fresh weight (FW) were exposed to the following Cd concentrations (in µM) for 5 d: 0, 5, 25, 50, 100, 250, 500 and 1000. Each concentration was replicated in 12 pots (1 plant per pot). Cd was added to the nutrient solutions as CdCl2. Phosphate concentration in the nutrient solution was lowered to 10 µm to avoid excessive Cd precipitation.
To examine the effect of an inhibitor of PC synthesis on Cd tolerance and accumulation, half of the pots were supplied with 300 mm BSO according to Schat et al. (2002). BSO (300 µm) was added to the BSO-treated pots three days before Cd treatments and during the Cd exposure.
Root growth and biomass measurements
Five days after Cd treatments, root elongation was determined using the charcoal staining method (Schat & Ten Bookum 1992). Initially, the roots were stained black by dipping them into a stirred suspension of finely powdered active charcoal (Sigma, St Louis, MO, USA), followed by rinsing in deionized water. New growth of roots, appearing in white, were measured after 5 d of Cd exposure. At harvest, the roots and shoots were separated, rinsed once with tap water and then twice with deionized water, and blotted dry with tissue paper. FWs of roots and shoots were recorded. Dry weights (DWs) were determined after the plant materials were dried in an oven at 70 °C for 48 h.
Analysis of LMW thiols
After 5 d of Cd exposure, the roots were desorbed in an ice-cold solution of 5 mm Pb(NO3)2 for 30 min to remove apoplastically bound Cd. Then, the roots and shoots were separated, washed once with tap water and twice with deionized water, covered in muslin cloth and immediately frozen in liquid nitrogen. Frozen tissues (roots and shoots) were freeze dried for 72 h and stored at −80 °C.
Freeze-dried samples (approximately 50 mg for roots and 200 mg for shoots) were homogenized using a mortar and pestle by adding 1–2 mL of a cold extraction solution containing 0.36% HCl and 5 mm diethylenetriamine-pentaacetic acid (DTPA) to the mortar. The slurry was then centrifuged at 13 000 g for 10 min at 4 °C, and the supernatant (tissue extract) was then collected. Four hundred microlitres of tissue extract was mixed with 565 mL of CHES-DTPA buffer (i.e. 200 mm CHES plus 5 mm DTPA, pH 7.8) [2-(N-cyclohexylamino)-ethanesulphonic acid], before derivatization of thiol groups with a sulfhydril-specific fluorescent probe [i.e. monobromobimane (MBB)]. After the addition of 10 µL of 9 mm MBB (in acetonitrile), the samples were kept in the dark for 30 min at 45 °C before the reaction was stopped with the addition of 50 µL of 36% HCl. Subsequently, derived samples were placed over 0.45 µm organic filters (13 mm Ø; Millipore, Billerica, MA, USA) using a syringe. Thiol-containing metabolites were separated by reverse-phase high-performance liquid chromatography (RP-HPLC; Waters, Milford, MA, USA) on a C-18 Nova-Pak column (3.9 × 150 mm, Waters) with a Resolve C18 Guard-Pak pre-column (Waters). The mobile phase was composed of solvent A, methanol (100%) and solvent B, water:methanol: acetic acid (89.75:10:0.25). With a flow rate of 1.5 ml min−1, the following elution profile, controlled by Millenium software (Milford, MA, USA), was used: 0–15 min, 0–4.5% A; 15–22 min, 4.5–40% A; 22–26 min, 40–100% A; 26–32 min, 100–0% A; 32–36 min, isocratic 100% B. Cys, γ-glutamylcysteine (γ-EC), GSH and PCs were determined using a post-column fluorescence detector (SFD 474, Waters) and a photodiode array detector (PDA-996, Waters). Initially identified peaks were further confirmed by spiking with the appropriate standards. Total PC content was estimated as the total area of assigned PC oligopeptide peaks and was expressed as nmol GSH equivalents g−1 DW, based on peak areas of reduced GSH (Artetxe et al. 2002).
Freeze-dried samples (approximately 50 mg for roots and 200 mg for shoots) were finely ground and digested in 5 mL of a mixture of concentrated HClO4 and HNO3 (13:87 v/v) (Zhao, McGrath & Crosland 1994). The concentrations of Cd and other elements (S, Zn, Fe, Mn, etc.) in the digest were determined using inductively coupled plasma-atomic emission spectrometry (ICP-AES; Fisons-ARL Accuris, Ecublens, Switzerland). The reliability of the digestion and analytical procedure was tested including blanks and standards of spinach leaves (SRM 1570a; National Institute of Standards & Technology 1998) with every batch of sample digest.
All data sets were statistically analysed using three-way (ecotype × Cd × BSO) analysis of variance (anova). Statistical analyses were performed using Genstat 5 for Windows (VSN International, Hemel Hempstead, UK).
The data relating to root growth in response to Cd concentration were fitted to a logistic curve, with the following equation:
where y is the root length, x is log(Cd concentration), and a, b and x0 are the parameters to be fitted.x0 represents the log[Cd concentration that inhibits root-length growth by 50% of the control (EC50)] value for Cd. Curve fitting was performed using SigmaPlot (SPSS Inc, Chicago, IL, USA).
Root growth was inhibited at high concentrations of Cd (Fig. 1). In the absence of BSO, the Cd EC50 value for Ganges was 75% higher than that for Prayon (Table 1). The BSO treatment sensitized Prayon towards Cd exposure, decreasing the EC50 by almost fivefold. In contrast, the BSO treatment had no significant effect on the EC50 value in Ganges.
Table 1. Cd concentration that inhibits root-length growth by 50% (EC50) in l-buthionine-(S,R)-sulphoximine (BSO)-treated (+BSO) and BSO-untreated (–BSO) Ganges and Prayon ecotypes of Thlaspi caerulescens
Cd EC50 (µM)
SEs in parentheses.
Exposure to Cd for 5 d did not significantly affect shoot biomass, whereas root biomass was decreased slightly by Cd at high concentrations (data not shown).
Under our experimental conditions, both T. caerulescens ecotypes accumulated similar high levels of Cd in their tissues (Fig. 2). Higher Cd concentrations were detected in roots than in shoots. In roots, the BSO treatment had no significant effect on Cd accumulation. In contrast, Cd accumulation in shoots was decreased significantly (P < 0.05 for the Cd–BSO interaction) by the BSO treatment.
Accumulation of LMW thiols
Table 2 shows the effect of Cd, in the presence or absence of BSO, on the concentrations of identified LMW thiols in roots and shoots of the two ecotypes. Figure 3 shows high-performance liquid chromatography (HPLC) chromatograms obtained from the analysis of PCs and other PC-related LMW thiols in roots of Ganges, in the presence or absence of BSO, at 0 or 250 µm Cd. Root PC accumulation increased markedly with Cd exposure (Table 2), although in all cases the PC:Cd molar ratio was lower than 0.4. There were no significant differences between Ganges and Prayon in the accumulation of PCs in roots in response to Cd exposure. As expected, root PC accumulation was inhibited by the addition of BSO, and the extent of this inhibition was similar in both ecotypes. Regarding other LMW thiols (Table 2), high levels of Cys accumulation were found in the roots of both ecotypes in response to Cd exposure. Cys accumulation was significantly (P < 0.001) higher in Ganges than in Prayon. The BSO treatment increased the Cys concentration in Ganges roots in the Cd treatments of 5–500 µm Cd, but had no significant effect in Prayon. Root γ-EC and GSH concentrations also increased in Ganges and Prayon roots in response to Cd exposure (Table 2). The γ-EC concentrations were small compared with other thiols. The concentration of GSH followed a biphasic pattern: at lower Cd concentrations, root GSH concentrations increased in both ecotypes, whereas at higher Cd levels (above 50 µm Cd), root GSH concentrations decreased. The BSO treatment decreased root γ-EC and GSH concentrations in both ecotypes.
Table 2. Accumulation of low molecular weight (LMW) thiols in l-buthionine-(S,R)-sulphoximine (BSO)-treated (+BSO) and BSO-untreated (–BSO) roots of Ganges and Prayon ecotypes of Thlaspi caerulescens subjected to different Cd concentrations
The concentrations of the identified LMW thiols were low in shoots (data not shown) than in roots, and similar in both ecotypes, with the exception of GSH and Cys, which were present at significantly higher concentrations (P < 0.001) in Ganges than in Prayon. In addition, although BSO treatment significantly increased (P < 0.05) the Cd-induced Cys accumulation in Ganges shoots, the Cys concentrations in shoots were fairly low (below 250 nmol g−1 DW).
In the HPLC chromatograms, an unidentified peak (at 7.3 min retention time) was detected in roots and shoots of both ecotypes (Fig. 4). In the absence of Cd, roots of both BSO-treated and BSO-untreated plants contained a substantial amount (about 1 µmol g−1 DW) of this SH-containing metabolite (named X7.3) (Fig. 4). In the presence of Cd, root X7.3 concentration was significantly (P < 0.01) higher in Ganges than in Prayon. Increasing Cd concentration in solution up to 500 µm also increased the concentration of X7.3 in Ganges roots in the absence of BSO. In the presence of BSO, root X7.3 concentration increased initially with increasing Cd, followed by a decrease at > 250 µm Cd. At 250 µm Cd, X7.3 represented more than 60 and 70% of the total LMW thiol content in BSO-treated and BSO-untreated Ganges roots, respectively. Root X7.3 concentration in Prayon was less responsive to Cd exposure. Shoot X7.3 concentrations were lower than those in roots. In the presence of Cd, X7.3 concentration was significantly (P < 0.01) higher in BSO-treated and BSO-untreated Ganges shoots than in Prayon.
Finally, Ganges roots appear to display a higher active basal S metabolism than Prayon roots, as shown by a 30% higher concentration of LMW thiols (the sum of Cys, X7.3, γ-EC, GSH and PCs) in the control plants (in the absence of Cd and BSO). Furthermore, total root LMW thiol accumulation in response to Cd exposure was always higher in Ganges than in Prayon, probably reflecting the long-term exposure of plants to high S levels at the Prayon site. For example, at 250 µm Cd, the total root LMW thiol concentration was 1.5-fold higher in Ganges than in Prayon. At concentrations above the EC50, such as 500 and 1000 µm Cd, the total root LMW thiol concentration was 2.1- and 1.9-fold higher in Ganges than in Prayon, respectively. These differences between ecotypes were even higher when plants were treated with BSO (e.g. at 250 and 1000 µm Cd, total root LMW thiol content was 2.8- and 4.7-fold higher in Ganges than in Prayon, respectively). These higher total root LMW thiol contents found in Ganges are mainly due to the exceptionally high concentrations of Cys and especially, X7.3 detected in both BSO-treated and BSO-untreated Ganges plants in response to Cd treatments. In fact, at 1000 µm Cd, the sum of Cys and X7.3 accounted for 88 and 79% of the total root LMW thiol content for BSO-treated and BSO-untreated Ganges plants, respectively.
Different ecotypes of T. caerulescens differ considerably in their capacity to accumulate and tolerate Cd, with the Ganges population from southern France being far superior than the other populations, including Prayon (Lombi et al. 2000; Roosens et al. 2003). This higher capacity for Cd accumulation in the Ganges ecotype has been partially attributed to a high affinity transporter in its roots (Lombi et al. 2001; Zhao et al. 2002). The ecotypic difference was more pronounced at lower than at higher Cd concentrations in the growth medium (Lombi et al. 2001; Roosen et al. 2003). This may explain why the two ecotypes showed similar levels of Cd accumulation in our experiment, in which Cd was supplied at very high concentrations in order to impose Cd toxicity within a short-term exposure. Consistent with previous reports from longer term (several weeks) exposure (Lombi et al. 2001; Roosen et al. 2003), our experiment showed that Ganges was more tolerant to Cd than Prayon. This hypertolerance must be achieved through internal detoxification. In shoots, there is evidence that Cd is sequestered in the vacuoles (Vázquez et al. 1992; Ma et al. 2005) with the formation of the Cd-malate complex (Ueno et al. 2005). Mechanisms responsible for Cd detoxification in T. caerulescens roots are less clear.
PCs have been shown to be essential for constitutive tolerance to Cd in A. thaliana (Cobbett et al. 1998; Ha et al. 1999). However, the role of PCs in the adaptive Cd hypertolerance of T. caerulescens has been questioned (Ebbs et al. 2002; Schat et al. 2002; Wójcik et al. 2005). Our results show that PC synthesis was induced by Cd exposure in both Ganges and Prayon ecotypes of T. caerulescens. However, root PC:Cd molar ratios were lower (below 1.0:2.5) than those expected for a PC-mediated Cd sequestration (ratios of 1:1–3:1) (Rauser 1990; De Knecht et al. 1994; Salt et al. 1995). In addition, despite their difference in Cd tolerance, both ecotypes showed similar PC concentrations in their root tissues. Furthermore, Cd tolerance in Ganges was unaffected by the BSO treatment, suggesting that PC synthesis is not a key determinant of the Cd hypertolerance observed in this ecotype. In contrast, BSO addition significantly increased Cd toxicity to Prayon, suggesting that PC-mediated Cd detoxification may play a certain role in this less tolerant ecotype. Previous studies have reported a Cd sensitization effect because of BSO addition in other populations of T. caerulescens, such as the population from Monte Prinzera (Italy) which is relatively sensitive to Cd (Schat et al. 2002), and a population from Plombières (Belgium, near Prayon) (Wójcik et al. 2005), which is probably similar to the Prayon ecotype used in this study.
Recently, Freeman et al. (2005) have proposed a model for the involvement of salicylic acid in the Ni tolerance of Thlaspi hyperaccumulators. Indeed, salicylic acid activates Ser-acetyltransferase (Freeman et al. 2004) post-translationally causing accumulation of GSH and activation of GSH reductase (Knörzer, Durner & Boger 1996) to maintain an enhanced pool of reduced GSH. In addition, salicylic acid potentially blocks PC synthase activity (Pál et al. 2002), inhibiting PC biosynthesis in response to Ni and conserving GSH to act as an antioxidant (Freeman et al. 2005), thereby preventing Ni-induced oxidative stress in Thlaspi hyperaccumulators. Tolerance of Thlaspi hyperaccumulators to other metals, such as Cd, could be affected by a similar mechanism. In any case, the role of salicylic acid and GSH to prevent oxidative stress in Thlaspi ecotypes showing different levels of Cd tolerance still needs to be elucidated.
The more Cd-tolerant Ganges ecotype appeared to synthesize more Cys in roots than Prayon in response to Cd exposure, suggesting its possible involvement in Cd tolerance. The differences between ecotypes were even more pronounced in the presence of BSO (BSO inhibits PC synthesis at the level of γ−ECS). Several reports have previously suggested the possible role of Cys in cellular Cd detoxification. Domínguez-Solís et al. 2001) investigated the expression of the A. thaliana gene (Atcys-3 A) encoding the cytosolic O-acetylserine(thiol)lyase, which catalyses the last step of Cys biosynthesis through the incorporation of sulphide into the O-acetyl-L-Ser molecule, under Cd stress conditions. They found a sevenfold induction of this gene after 18 h exposure to 50 mm Cd. In addition, Arabidopsis-transformed plants overexpressing Atcys-3 A showed an increased tolerance to Cd when grown in a medium containing 250 µm CdCl2, suggesting that increased Cys availability might be responsible for Cd tolerance. Howarth et al. (2003) reached a similar conclusion through expression studies of other A. thaliana genes implicated in Cys biosynthesis.
In our studies, Cys was not the main LMW thiol accumulated in T. caerulescens roots when exposed to Cd. A still unidentified LMW thiol, named X7.3, accumulated in Cd-treated roots and shoots of both ecotypes, especially in Ganges. In this ecotype, the response of X7.3 to Cd exposure was increased by the BSO treatment, suggesting that this compound is most likely a thiol-containing metabolite derived from Cys, but not from γ-EC or GSH. BSO induced a considerable increase of X7.3 in Prayon only under relatively low Cd exposure (i.e. 5 and 50 µm Cd in the nutrient solution).
Hu, Lau & Wu (2001) suggested a possible link between a higher Cys synthesis and a high molecular weight PC–Cd–S2– complex formation through CdS incorporation. These authors suggested that a dramatic increase in Cys levels could either reflect an increased supply of sulphide inside the cell or indicate an increment in a labile sulphide donor. Although the identity of the putative S donor remains unknown, the utilization of labile sulphide to chelate Cd in high molecular weight PC–Cd–S2– complexes has been widely reported in higher plants (Cobbett & Goldsbrough 2002). Speiser et al. (1992) reported a Schizosaccharomyces pombe mutant defective in PC–Cd–S2– complex production and proposed a model in which Cys sulphinate (a S-containing non-protein amino acid, analogue to aspartate) was linked to the purine biosynthetic pathway as well as to the production of acid labile sulphide for high molecular weight complex formation. The incorporation of labile sulphide within a PC–Cd complex would (1) increase the stability of that PC–Cd complex (Cobbett & Goldsbrough 2002); (2) make the dissociation of Cd less likely (Hu et al. 2001); and (3) allow a greater number of Cd atoms to be detoxified per PC molecule (Ebbs et al. 2002). We speculate that the unidentified X7.3 LMW thiol could be a cytoplasmic S donor for Cd chelation derived from Cys. This remains to be investigated further. In any case, regarding the identification of X7.3, some compounds, such as Na2S2O3, homocysteine, Cys–Gly and N-acetyl-Cys were unsuccessfully tested in this respect. In turn, other compounds such as l-cysteine-S-sulphate, cysteinesulphinic, S-methylcysteine and l-methionine were not detected by our HPLC method.
To what extent the accumulation of LMW thiols (such as Cys and X7.3) are involved in Cd tolerance or are just a secondary response to Cd exposure is a question that still remains unanswered. The higher activity of the S reduction pathway observed in the more tolerant Ganges ecotype might hold the key to this puzzle. The results presented here are in the line with a combined detoxification system consisting of enhanced root-to-shoot transport together with an active detoxification system through Cd (CdS?) sequestration or inactivation in the roots, and Cys may play an important role in this system.
J. Hernández-Allica and O. Barrutia received a fellowship from the Ministry of Science and Technology of Spain. This research was supported in part by research grants: Universidad País Vasco (UPV) 0018.310-135331/2001 and ETORTEK Programme of the Basque Goverment. Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the UK.