Cadmium hyperaccumulation leads to an increase of glutathione rather than phytochelatins in the cadmium hyperaccumulator Sedum alfredii

ARTICLE IN PRESS Journal of Plant Physiology 164 (2007) 1489—1498

www.elsevier.de/jplph

Cadmium hyperaccumulation leads to an increase of glutathione rather than phytochelatins in the cadmium hyperaccumulator Sedum alfredii Qin Suna,b,d, Zhi Hong Yec,d, Xiao Rong Wangb, Ming Hung Wongd, a

Ministry of Education Key Laboratory of Integrated Regulation and Resource Development on Shallow Lakes, College of Environmental Science and Engineering, Hohai University, Nanjing 210098, PR China b State Key Laboratory of Pollution Control and Resources Reuse, School of the Environment, Nanjing University, Nanjing 210093, PR China c School of Life Sciences, Zhongshan (Sun Yat-sen) University, Guangzhou 510275, PR China d Croucher Institute for Environmental Sciences, Department of Biology, Hong Kong Baptist University, Hong Kong SAR, PR China Received 13 June 2006; accepted 16 October 2006

KEYWORDS Cadmium hyperaccumulator; Glutathione; Heavy metal tolerance; Phytochelatins; Sedum alfredii

Summary Sedum alfredii has been reported to be a cadmium (Cd) hyperaccumulator. Phytochelatins (PCs) and other thiol (SH)-containing compounds have been proposed to play an important role in the detoxification and tolerance of some heavy metals, but it is not clear whether PCs are responsible for Cd hyperaccumulation and tolerance in S. alfredii. In this study, two geographically isolated populations of S. alfredii were studied: one population grew on an old Pb/Zn mine site, while the other on a non-mine site. The mine population of this species exhibited a stronger heavy metal tolerance than in the other population. Root-to-shoot transport of Cd was higher in population located at the mine site than at the non-mine site. Considerable amounts of Cd were accumulated in leaves and stems of mine plants, while most Cd was distributed in roots of non-mine plants. Non-protein SH in plant tissues of two populations were further investigated by a HPLC pre-column derivatization system. Upon exposure to Cd, no PCs were detected in all tissues of mine population, while an appreciable amount of glutathione (GSH) was observed in the descending order of stem4root4leaf. The concentrations of GSH consistently increased with the increase of exogenous Cd concentrations and time. On the

Abbreviations: ACN, acetonitrile; Cd, cadmium; Cys, L-cysteine; d. wt, dry weight; DTPA, diethylenetriamine-pentaccetic acid; gEC, g-Glu-Cys; f. wt, fresh weight; GSH, glutathione; HEPPS, N-[2-hydroxyethyl]piperazine-N0 -[3-propane sulfonic acid]; HPLC, highperformance liquid chromatography; LSD, least significance deviation; mBrB, monobromobimane; MSA, methanesulfonic acid; PCs, phytochelatins; PC2, (g-GluCys)2-Gly; PC3, (g-GluCys)3-Gly; PC4, (g-GluCys)4-Gly; SH, thiol; TFA, trifluoroacetic acid Corresponding author. Tel.: +852 34117746; fax: +852 34117743. E-mail address: [email protected] (M.H. Wong). 0176-1617/$ - see front matter & 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2006.10.001

ARTICLE IN PRESS 1490

Q. Sun et al. contrary, Cd exposure strongly induced the production of PCs (mainly PC2 and PC3) and GSH in plant tissues of non-mine population, and the concentrations of GSH showed an initial drop over the duration of 7-d exposure. The present results provided strong evidence that PCs are not involved in Cd transport, hyperaccumulation and tolerance in mine population of S. alfredii. & 2007 Elsevier GmbH. All rights reserved.

Introduction Recently, considerable interest has been attracted to hyperaccumulators because of their potential use in phytoremediation, as they are able to extract metals from the soil and to concentrate them in their aboveground parts. A cadmium (Cd) hyperaccumulator Sedum alfredii at an old Pb/Zn mine site in Zhejiang Province of China has been identified (Ni and Wei, 2003; Yang et al., 2004). Compared with a well-known Zn/Cd hyperaccumulator Thlaspi caerulescens, S. alfredii has characteristics of fast growth, large biomass, asexual reproduction and perennial, and can grow up to 40 cm in height, propagate 3–4 times in a year if environmental conditions are favorable (Yang et al., 2001). Furthermore, the maximum Cd concentrations in stem and leaf of this plant, grown at 400 mM Cd nutritive solution without phytotoxicity, were 4512 and 3317 mg kg1 (dry weight (d. wt)), respectively (Ni and Wei, 2003), which largely surpassed the threshold concentration of 100 mg kg1 (d. wt) in Cd hyperaccumulators reported (Baker and Brooks, 1989). This phenomenon of Cd hyperaccumulation by S. alfredii and its growth predominance has attracted considerable scientific attention in the field of physiological and ecological studies in China. Investigation on Cd hyperaccumulation and tolerance in mine population of S. alfredii and its survival mechanism in a severe environment can offer important information on the application of this plant for effective soil remediation. To date, the mechanisms of Cd hyperaccumulation and tolerance in this plant have not been fully understood. Metal detoxification and tolerance in plants can be achieved by numerous mechanisms such as chelation by metal-binding compounds, metal deposition in vacuoles, alterations of membrane structures, and synthesis of stress metabolites. Phytochelatins (PCs), a class of small thiol (SH)-rich peptides, comprise one of the mechanisms involved in the chelation of heavy metals. Due to their ability to bind metals by thiolate coordination, PCs are generally considered to be important cellular chelating agents. Many heavy metals can induce PC production by plants and Cd has been found to be

the most effective inducer of PCs (Grill et al., 1985). Several biochemical (Clemens et al., 1999; Ha et al., 1999) and mutant (Howden et al., 1995a, b) studies showed that PC production is the main responsive mechanism to Cd stress in higher plants and plays a critical role in Cd detoxification by sequestering Cd into a less toxic form. It has also been suggested that increased Cd tolerance is associated with higher concentrations of PCs and accumulation of high molecular weight PCs (Rauser, 1999). There is by now considerable debate on the role of PCs in metal detoxification and tolerance of higher plants. Earlier studies have shown that tolerant plants or cell lines have higher levels of PCs than sensitive ones exposed to Cd (Steffens et al., 1986). It was also reported that the overexpression of bacterial g-glutamylcysteine synthase or glutathione synthase in Brassica juncea enhances PC synthesis and Cd tolerance (Zhu et al., 1999a, b). Moreover, PCs might be needed for Hg tolerance (Gupta et al., 1998). Recently, it has been suggested that PC synthesis is essential for As hypertolerance in naturally selected As-hypertolerant Holcus lanatus (Hartley-Whitaker et al., 2001, 2002). However, not all studies supported the role of PCs in metal tolerance. In fact, there is so far a lack of convincing evidence for naturally selected enhanced PC synthesis in tolerant/hypertolerant plants from metal-contaminated environments. Silene vulgaris has been widely used as a model plant for studies on PCs as a general detoxifying agent. Most studies showed that Cd/Cu/Zn tolerance in this plant does not rely on differential PC production (De Knecht et al., 1992, 1994, 1995; De Vos et al., 1992; Schat and Kalff, 1992; Harmens et al., 1993). More recently, it was found that the amounts of PC synthesis in most hyperaccumulators are not correlated with metal accumulation rates (Ebbs et al., 2002; Schat et al., 2002; Zhao et al., 2003). In view of the ambiguous role of PCs in metal detoxification and tolerance, as well as various detoxification mechanisms in different plant species, the main objective of this study is to investigate if PCs can be induced in the Cd hyperaccumulator S. alfredii, and whether they

ARTICLE IN PRESS Cadmium hyperaccumulation in Sedum alfredii play a role in Cd detoxification and tolerance. To assess the contributions of PCs in metal tolerance, we compared the concentrations of PCs and other low molecular weight SH in different plant tissues of mine and non-mine populations of S. alfredii under controlled laboratory conditions.

Materials and methods Plant materials and chemicals Samples of S. alfredii Hance (sedum) were collected from an old Pb/Zn mine site in Quzhou, Zhejiang Province (mine plants/population), and a non-mine site in Guangzhou, Guangdong Province (non-mine plants/population), China. The following chemicals were obtained: N-[2-hydroxyethyl]piperazine-N0 -[3-propane sulfonic acid] (HEPPS, 499.5%), glutathione (GSH,498%), g-Glu-Cys (gEC, 499% ), L-cysteine (Cys, 498%) and trifluoroacetic acid (TFA, 499%) from Sigma; monobromobimane (mBrB, 495%), methanesulfonic acid (MSA, 499%) and diethylenetriamine-pentaccetic acid (DTPA, 499%) from Fluka; acetonitrile (ACN, 499.9%, HPLC grade) from Lab-Scan; and PCn (n ¼ 2, 3, 4, 495%) standards from Biopeptide Co., LLC. USA. The other chemicals used were of reagent grade or of the commercially highest grade available. Milli-Q water (18.3 MO) was used to prepare aqueous solutions throughout. A fluorescent probe mBrB was prepared daily. Other solutions were prepared weekly and stored in the dark at 4 1C. Conditions of plant culture Healthy and uniform-sized shoots of mine and nonmine plants were chosen, cleared, and transplanted in 2.5 L containers (6 plants pot1) containing a modified Hoagland solution (Hewitt, 1966) with half-strength macronutrients and full-strength micronutrients (Fe was supplied as Fe-EDTA). Solution pH value was adjusted to 5.8 (according to pH value of soil in the Pb/Zn mine area) with 0.1 M HCl or NaOH once every 3 d. The nutrient solution was aerated continuously and renewed once every 6 d. Plants were precultured for 30 d for the initiation of new roots before experiments. All experiments were conducted in a greenhouse (natural photoperiod, temperature was controlled at 20–25 1C) under a random block design. Experimental design Experiment 1 After 30-d preculture, the young plants of both populations were exposed to a range of Cd (supplied as CdCl2) treatment solutions: 0, 10, 20, 40, 80, 160, and 320 mM. The composition of nutrients was the same as the preculture. There were three replicates for each treatment. The treated solution was renewed once every 2 d.

1491 After 7 d, the roots of all plants were immersed in icecold 10 mM CaCl2 for 10 min to remove adhering Cd from root surfaces. After washing with tap water and deionized water, the plants were air-dried and separated into roots, stems, and leaves. Each tissue part was subdivided into two portions, one was immediately weighed, frozen in liquid nitrogen (196 1C), and stored at 80 1C for analysis of PCs and other low molecular weight SH, while the other portion was left for analysis of Cd concentrations. Experiment 2 After 30-d preculture, the mine plants were exposed to 0, 25 and 100 mM Cd, and sampled at 0.5, 1, 2, 3, 4, 5, 6 and 7 d. These two concentrations were chosen because they were not toxic to this plant as reported previously (Ni and Wei, 2003; Yang et al., 2004). The non-mine plants were exposed to 0, 25 and 75 mM (instead of 100 mM to avoid the extreme toxicity) and sampled at the same time intervals. The composition of nutrients was the same as the preculture. The treatment solution was renewed once every 2 d. On each sampling, plants from three replicate pots were harvested and treated as described above. Analytical methods Determination of PCs and other low molecular weight thiols Step 1 (extraction): Extraction and analysis of PCs and other low molecular weight SH were performed according to the method described by Sneller et al. (2000) with a slight modification. Frozen plant tissues were homogenized with mortar and pestle with quartz sand in 2 mL 6.3 mM DTPA with 0.1% TFA at 4 1C. The homogenate (14,000g) was centrifuged at 4 1C for 12 min. The clear supernatants were collected and immediately used for the assay of PCs and other low molecular weight SH by high-performance liquid chromatography (HPLC), using pre-column derivatization with a fluorescent probe mBrB. Step 2 (derivatization): 250 mL of supernatant was mixed with 450 mL of 200 mM HEPPS buffer, at pH 8.2, with 6.3 mM DTPA, and 10 mL of 25 mM mBrB. Derivatization was carried out in the dark at 45 1C for 30 min. The reaction was terminated by adding 300 mL of 1 M MSA. The samples were stored in the dark at 4 1C until HPLC analysis. Reagent blanks were used to identify the reagent peaks. Step 3 (detection with HPLC): The bimane derivatives were separated using a binary gradient of mobile phase A (0.1% TFA) and B (100% ACN) at room temperature (2272 1C). Fluorescence was detected at 380 nm excitation and 470 nm emission wavelengths. The flow rate was 0.5 mL min–1. The derivatized sample (50 mL) was run in a linear gradient from 12% to 25% B for 15 min, then 25–35% B for 14 min, and subsequently 35–50% B for 21 min. Before injecting a new sample, the column was cleaned (5 min, 100% B) and equilibrated (10 min, 12% B). The post time was 5 min resulting in a total analysis time of

ARTICLE IN PRESS 1492

Determination of total Cd Fresh tissues were weighed accurately to 3 g for leaves and stems, and 1 g for roots and then oven-dried (70 1C) to a constant weight. The dried tissues were digested by mixed acid [HNO3/HClO4 (85/15, v/v)] and tested for Cd using Inductively Coupled Plasma-Optical Emission Spectrometer (ICP-OES, Perkin Elmer, Model 3000DV). A standard reference plant material (GBW07602) from the Department of Earth and Mine, Guangdong Province, China, was used to verify the accuracy of metal determination. The recovery rates for all metals were within 90710%. Statistical analyses Statistical analyses were performed using the SPSS statistical package (version 10.0 for Windows). Data were tested at significant levels of Po0:05 or Po0:01 by oneway ANOVA. The subsequent multiple comparisons among means were examined based on the least significance deviation (LSD) (Little and Hills, 1978).

leaf

8

stem

root

(a)

7 6 5 4 3

Fresh biomass (g plant –1 f. wt)

70 min. All solvents were degassed before use. The analytical data were integrated by using HP Chemstation. The retention times of PCs and other low molecular weight SH (GSH, Cys) in biological samples were checked using (g-GluCys)2-Gly (PC2), (g-GluCys)3-Gly (PC3), (g-GluCys)3-Gly (PC4), GSH, gEC, and Cys standards. Individual PC subtype was quantified by using the relationship peak vs. concentrations of GSH standard solutions. Corrections for differential derivatization efficiencies were made according to the methods stated by Sneller et al. (2000).

Q. Sun et al.

2 1 0 6

(b)

5 4 3 2 1 0

0

10

20

40

80

160

320

Cd concentrations, µM Figure 1. Fresh weights of different tissues of mine (a) and non-mine (b) populations of Sedum alfredii exposed to a range of Cd for 7 d (mean7SD, n ¼ 3).

Cadmium accumulation

Results Growth of plants During the period of Cd treatments, the mine plants grew well without any Cd toxicity symptoms. However, the non-mine plants showed significant toxicity symptoms such as wilting or drooping leaves and root necrosis. The toxicity symptoms became more serious with the increase of Cd concentrations. Fresh weights (f. wt) of different tissues of mine plants were nearly unaffected by increasing Cd concentrations in nutrient solution (Fig. 1a). Unlike mine plants, f. wt of leaves, stems, and roots of non-mine plants were significantly (Po0:05) decreased by 19%, 18%, and 31% from 0 to 10 mM, respectively, and higher decreases were observed with the increase of Cd concentrations (Fig. 1b). It was indicated that the mine population of S. alfredii was highly tolerant to Cd exposure than plant population from the non-mine site.

Concentrations of Cd in plants after Cd treatments were calculated based on tissue f. wt. Concentrations of Cd in leaves, stems, and roots of mine and non-mine plants showed an almost linear increase in response to the increase of Cd concentrations in nutrient solution (Fig. 2a, b). However, there were significant differences in Cd distribution between the two populations. Substantial amounts of Cd (465%) were transported to leaves and stems of mine plants (Fig. 2a), while most Cd was accumulated in roots of nonmine plants (Fig. 2b), which was 2–8 times higher than those of mine population. In mine plant, much Cd was transported to leaves, reaching levels even 23 times higher than those of non-mine plant. Cd accumulation by two populations with time is presented in Fig. 3. Cd in nutrient solution was rapidly translocated to stems and leaves of both mine and non-mine plants within 12 h after Cd treatments. Concentrations of Cd in leaves, stems, and roots of both plants increased with time over a

ARTICLE IN PRESS Cadmium hyperaccumulation in Sedum alfredii root

(a)

120

stem

1493

Relationship between Cd accumulation and production of glutathione

leaf

100

GSH concentrations increased to varying degrees in leaves, stems, and roots of both mine and nonmine plants in response to a range of Cd, whereby the non-mine plants produced more GSH than the mine plants (Fig. 5). Positive linear relationships were observed between GSH concentrations and Cd accumulation in leaves (r 2 ¼ 0:866,  Po0:05), stems (r 2 ¼ 0:925,  Po0:01), and roots (r 2 ¼ 0:942,  Po0:01) of mine plants, but not in non-mine plants. During the course of 7-d period, the concentrations of GSH in leaves, stems, and roots of mine plants steadily and consistently increased with increasing Cd accumulation, while GSH concentrations in non-mine plants showed an initial decrease during the first few days followed by an increase afterwards (Fig. 6).

80

Cd con centrations (mg kg–1 f. wt)

60 40 20 0 800

(b)

700 600 500 400 300 200

Discussion

100 0

0

10

20 40 80 160 Cd concentrations, µM

320

Figure 2. Concentrations of Cd in different tissues of mine (a) and non-mine (b) populations of Sedum alfredii exposed to a range of Cd for 7 d (mean7SD, n ¼ 3).

7-d period. The leaves of mine plants were found to have considerably higher Cd concentrations than those of non-mine plants at all times, indicating that large amounts of Cd in shoots were further transported to the leaves of mine plants.

Relationship between Cd accumulation and production of phytochelatins HPLC fluorescence analysis clearly showed that the mine plants were not capable of synthesizing PCs in response to varying Cd concentrations (data not shown). Kinetic experiment also confirmed that no PCs were induced in leaves, stems, and roots of mine plants throughout the course of 7-d exposure (data not shown). On the contrary, substantial amounts of PCs were induced in non-mine plants, particularly in stems and roots, and significantly (Po0:01) increased with increasing Cd concentrations and time (Table 1 and Fig. 4). At higher concentrations of Cd in nutrient solution, the PC accumulation in roots of non-mine plants was drastically decreased, mainly due to severe Cd toxicity.

It was observed in this study that there were marked differences in Cd uptake and accumulation in two populations of S. alfredii. Considerable amounts of Cd were accumulated in leaves and stems of mine plants, while most Cd was distributed in roots of non-mine plants. Root-to-shoot transport of Cd was higher in mine population than in the non-mine population. Moreover, a significant difference in Cd hyperaccumulation existed between mine populations (Deng et al., 2006). It was indicated that S. alfredii exhibited a populationspecific variation in Cd hyperaccumulation. A similar intraspecific variation was observed in populations of T. caerulescens (Lombi et al., 2002) and those of Arabidopsis halleri (Bert et al., 2002). However, it has been shown that one non-metallicous population of T. caerulescens has the ability to hyperaccumulate Cd (Roosens et al., 2003). Bert et al. (2002) also found that 17% of the plant individuals of metallicous populations of A. halleri and 10% of the plant individuals of non-metallicous populations of A. halleri could accumulate 100 mg Cd kg1 in their shoots. By contrast, the present study showed that only mine population of S. alfredii possessed the Cd hyperaccumulation trait. The distinct intraspecific variation of S. alfredii may provide potential material for studying the mechanisms of Cd hyperaccumuation trait. The present results clearly showed that S. alfredii from the mine site did not synthesize PCs, although a large amount of Cd was taken up and stored in leaves and stems. On the contrary, a

ARTICLE IN PRESS Q. Sun et al.

Cd concentrations (mg kg–1 f. wt)

1494 70 60 50 40 30 20 10 0 80 70 60 50 40 30 20 10 0 80 70 60 50 40 30 20 10 0

ck

25

12

10 0

10

Leaf

ck

25

75

Leaf

8 6 4 2 0 100

Stem

Stem

80 60 40 20 0

700 600 500 400 300 200 100 0

Root

0.5 1

2

3

4

5

6

7

Root

0. 5 1

2

3

4

5

6

7

Time of exposure (d)

Figure 3. Time-course of Cd accumulation in different tissues of mine (left) and non-mine (right) populations of Sedum alfredii exposed to Cd (mean7SD, n ¼ 3). Table 1. Phytochelatin concentrations in non-mine population of Sedum alfredii exposed to a range of Cd for 7 d (nmol SH g1 f. wt) Cd concentrations (mM)

Leaves

10 20 40 80 160 320

28 30 35 71 37 36

(2.9) (2.3) (3.4) (2.7) (2.3) (6.6)

Stems d cd bc a b bc

582 736 779 864 878 1106

(109) (112) (148) (141) (132) (137)

Roots c bc bc b ab a

442 829 1059 1184 322 283

(57) c (24) b (169) a (124) a (50) cd (31) c

1 Phytochelatin data are reported as a molar concentration (normalized to f. wt) of the sum of PC2, PC3, and unidentified isophytochelatins being eluted after PC4. 2 Data in the same column with the same letters are not significantly different from each other at Po0.05. 3 Data in parentheses are SD of three replicates.

substantial amount of PCs was detected in nonmine plants, but they could not prevent Cd toxicity. Furthermore, over the duration of Cd exposure, no PCs were induced in any tissue of mine plants, while PCs were rapidly synthesized in leaves, stems, and roots of non-mine plants. Therefore, Cd hyperaccumulation and tolerance in mine population of S. alfredii were not related to PC synthesis. Consistent with our results, it was found that PC accumulation was higher in non-metallicolocus plants than in hypertolerant plants of T. caerulescens, suggesting that PC-based sequestration is not

essential for constitutive tolerance or hypertolerance to these metals, and furthermore, adaptive Cd hypertolerance is not dependent on PC-based sequestration (Schat et al., 2002). Similar results were also reported by Ebbs et al. (2002). Moreover, it was reported that PCs were not involved in metal detoxification in Ni hyperaccumulating tree Sebertia acuminate (Sagner et al., 1998) and Co hyperaccumulator Crotalaria cobalticola (Oven et al., 2002). Recently, Zhao et al. (2003) observed that the molar ratios of PC-SH to As were 0.09 and 0.03 for shoots and roots, respectively, suggesting that PCs play a limited role in the hypertolerance of

ARTICLE IN PRESS Cadmium hyperaccumulation in Sedum alfredii 80

1495 leaf

25

70

300

75

60

(a)

200

40

GSH concentrations (nmol g –1 f. wt)

30 20

Phytochelatin concentrations (nmol –SH g –1 f. wt)

root

250

Leaf

50

stem

10 0 2000

Stem

1500

1000

150 100 50 900 800

(b)

700 600 500 400

500

300 200

0

100

2000

0

Root

0

10

20

40

80

160

320

Cd concentrations, µM 1500

Figure 5. Concentrations of GSH in different tissues of mine (a) and non-mine (b) populations of Sedum alfredii exposed to a range of Cd for 7 d (mean7SD, n ¼ 3).

1000

500

0 0.5

1

2 3 4 5 Time of exposure (d)

6

7

Figure 4. Time-course of phytochelatin synthesis in different tissues of non-mine population of Sedum alfredii exposed to 25 and 75 mM Cd. Phytochelatin data are reported as a molar concentration (normalized to fresh weight) of the sum of PC2, PC3, and unidentified isophytochelatins being eluted after PC4 (mean7SD, n ¼ 3).

As in an As hyperaccumulator, Pteris vittata, in which only a small proportion (1–3%) of As is chelated with PCs. Similar to the results commonly obtained for hyperaccumulators, De Knecht et al. (1992) reported that sensitive plants of S. vulgaris produce more PCs than tolerant plants of the same species when exposed to the same external Cd concentration. In addition, the in vivo acid-labile sulphide contents of the Cd–PC complexes and PC chain length distributions were identical (De Knecht et al., 1994). Furthermore, De Knecht et al. (1995) obtained equal capacities and activation constants for Cd-induced PC synthesis in crude protein

extracts prepared from roots of Cd-tolerant and non-metallicolous S. vulgaris. Cu/Zn-sensitive plants of S. vulgaris also produced more PCs than tolerant ones (De Vos et al., 1992; Harmens et al., 1993). Recently, it has been observed that Cd cannot induce PC synthesis in Cd-tolerant Salix viminalis (Landberg and Greger, 2004). All these findings from a number of hyperaccumulators and metal-tolerant plants provide convincing evidence that metal hyperaccumulation and tolerance in plants may be controlled by PC-independent mechanisms. Steffens (1990) suggested that an over-production of PCs in plants chronically exposed to heavy metals would expend a large amount of energy required for sulfate reduction, that a mechanism of this kind of increased tolerance is unlikely to be evolved. Since Cd did not induce PC synthesis in mine population of S. alfredii, this population must have developed a new detoxification mechanism, instead of PCs, in order to prevent the toxic effect of Cd ions on normal cellular metabolism. An increase of GSH concentrations was observed in two populations of S. alfredii exposed to a range of Cd concentrations. In contrast to mine population, the non-mine population could not tolerate high Cd concentrations (Fig. 5). The kinetic results also

ARTICLE IN PRESS 1496

Q. Sun et al. ck

GSH concentrations (nmol g–1 f. wt)

140 120 100 80 60 40 20 0

220 200 180 160 140 120 100 80 60 40 20 0 20 0 18 0 16 0 14 0 12 0 10 0 80 60 40 20 0

25

100

ck

120

25

75

100 80 60 40

Leaf

Leaf

20 0

500

Stem

Stem

400 300 200 100 0 50 0

Root

Root

40 0 30 0 20 0 10 0

0.5 1

2

3

4

5

0

6 7 0.5 1 Time of exposure (d)

2

3

4

5

6

7

Figure 6. Time-course of GSH synthesis in different tissues of mine (left) and non-mine (right) populations of Sedum alfredii exposed to Cd (mean7SD, n ¼ 3).

showed that GSH concentrations consistently increased with the increase of Cd exposure time in mine population, compared with non-mine population (Fig. 6). An increase of GSH in metal-tolerant plants was also found in Arabidopsis trichome cells (Gutie´rrez et al., 2000). GSH is the most abundant cellular SH in most living organisms and is involved in metal stress in a number of ways. Firstly, GSH is a well-known antioxidant playing a prominent role in the defense system against radicals caused by metal stress in plants. Freeman et al. (2004) reported that elevated GSH concentrations are involved in conferring tolerance to Ni-induced oxidative stress in Thlaspi Ni hyperaccumulators. Secondly, GSH has a relatively high affinity (KdCd41010) for binding Cd2+ (Perrin and Watt, 1971), making it a potential cytosolic chelator of this metal, and further reduces metal toxicity (Vo ¨geli-Lange and Wagner, 1996). Mehra and Mulchandani (1995) presented the role of GSH as an effective donor of heavy metal ion, and it is probably the in vivo donor of heavy metals to PCs (potentially toxic heavy metal ions are firstly chelated by GSH and then transferred to PCs for eventual sequestration). Thirdly, it was commonly suggested that GSH serves as a precursor in PC biosynthesis. PC synthesis induced

by metals is accompanied by a rapid depletion of total GSH in plant cell suspension and intact plant (Jackson et al., 1992; Gupta et al., 1998). Based on this background and the fact that no PCs were induced in the mine population of S. alfredii, it is speculated that GSH might serve as an antioxidant or a metal chelator involved in Cd detoxification and tolerance in mine population of S. alfredii. The role of GSH in plant metal tolerance, however, is controversial. Working with mutants of Arabidopsis, Xiang et al. (2001) demonstrated that GSH is essential in protecting plants from heavy metal toxicity. On the contrary, normal plants (tobacco and transgenic poplar) have higher GSH levels, but are much more sensitive to oxidative stress (Noctor et al., 1996; Creissen et al., 1999). Treatment of roots with the glutathione synthesis inhibitor, buthionine sulfoximine (BSO), exacerbated H2O2 accumulation in Cd-treated Nicotiana tabacum, but had a relatively minor effect on H2O2 levels and did not reduce Cd tolerance in T. caerulescens, suggesting that GSH-dependent tolerance is of minor importance in Cd hyperaccumulator (Boominathan and Doran, 2003). Therefore, future studies are needed to clarify the role of GSH in Cd detoxification and tolerance in mine population of S. alfredii.

ARTICLE IN PRESS Cadmium hyperaccumulation in Sedum alfredii It has been proposed that PCs act as a potential biomarker for evaluating metal toxicity. For particular metals, e.g. Cd and Cu, linear relationships between metal toxicity and expression of PCs have been found in S. vulgaris, Zea mays, and Triticum aestivum L. (Schat and Kalff, 1992; De Knecht et al. 1995; Keltjens and Van Beusichem, 1998; Sneller et al., 1999), and in certain alga (Pawlik-Skowron ´ska, 2002). The present results further confirmed this. At the same Cd treatment solution, significant toxic effects were observed in non-mine plants, accompanied by the over-production of PCs. Kinetic studies showed PCs could be detected at 12 h after Cd exposure and dramatically increased with increasing exposure time. It was suggested that PC concentrations can be used as a quantitative tolerance-independent measurement of the degree of Cd toxicity experienced by plants. In conclusion, there were significant differences in Cd uptake and accumulation in two populations of S. alfredii, suggesting that Cd hyperaccumulation trait may be a property of a population, and not of an entire species. Therefore, Cd hyperaccumulation and tolerance in mine population of S. alfredii was not related to PC synthesis.

Acknowledgments This work was supported by the Research Council of University Grants Committee, Hong Kong (RGC: HKBU-2072/01M and HKBU-2181/03M), the National Natural Science Foundation of China (No. 20237010), the Natural Science Foundation of Jiangsu Province, China (No. 2004091), and the fund of the Key Laboratory of Integrated Regulation and Resource Development on Shallow Lakes, Hohai University (No. 2006KJ007).

References Baker AJM, Brooks RR. Terrestrial higher plants which hyperaccumulate metallic elements – a review of their distribution, ecology and phytochemistry. Biorecovery 1989;1:81–126. Bert V, Bonnin I, Saumitou-Laprade P, de Laguerie P, Petit D. Do Arabidopsis halleri from non-metallicolous populations accumulate zinc and cadmium more effectively than those from metallicolous populations? New Phytol 2002;155:47–57. Boominathan R, Doran PM. Cadmium tolerance and antioxidative defenses in hairy roots of the cadmium hyperaccumulator, Thlaspi caerulescens. Biotechnol Bioeng 2003;83:158–67. Clemens S, Kim EJ, Neumann D, Schroeder JI. Tolerance to toxic metals by a gene family of phytochelatin

1497 synthases from plants and yeast. EMBO J 1999;18: 3325–33. Creissen G, Firmin J, Fryer M, Kula B, Leyland N, Reynolds H, et al. Elevated glutathione biosynthetic capacity in the chloroplasts of transgenic tobacco plants paradoxically causes increased oxidative stress. Plant Cell 1999;11:1277–91. De Knecht JA, Koevoets PLM, Verkleij JAC, Ernst WHO. Evidence against a role for phytochelatins in naturally selected increased cadmium tolerance in Silene vulgaris (Moench) Garcke. New Phytol 1992;122:681–8. De Knecht JA, Van Dillen M, Koevoets PLM, Schat H, Verkleij JAC, Ernst WHO. Phytochelatins in cadmiumsensitive and cadmium-tolerant Silene vulgaris: chain length distribution and sulphide incorporation. Plant Physiol 1994;104:255–61. De Knecht JA, Van Baren N, Ten Bookum WM, Wong Fong Sang HW, Koevoets PLM, Schat H, et al. Synthesis and degradation of phytochelatins in cadmium-sensitive and cadmium-tolerant Silene vulgaris. Plant Sci 1995;106:9–18. De Vos CHR, Vonk MJ, Vooijs R, Schat H. Glutathione depletion due to copper-induced phytochelatin synthesis causes oxidative stress in Silene cucubalus. Plant Physiol 1992;98:853–8. Deng DM, Shu WS, Zhang J, Zou HL, Ye ZH, Wong MH. Zinc and cadmium accumulation and tolerance in populations of Sedum alfredii. Environ Pollut 2007;147: 381–6. Ebbs S, Lau I, Ahner B, Kochian L. Phytochelatin synthesis is not responsible for Cd tolerance in the Zn/Cd hyperaccumulator Thlaspi caerulescens (J&C Presl). Planta 2002;214:635–40. Freeman JL, Persans MW, Nieman K, Albrecht C, Peer W, Pickering IJ, et al. Increased glutathione biosynthesis plays a role in nickel tolerance in Thlaspi nickel hyperaccumulators. Plant Cell 2004;16:2176–91. Grill E, Winnacker EL, Zenk MH. Phytochelatins: the principal heavy metal complexing peptides of higher plants. Science 1985;230:674–6. Gupta M, Tripathi RD, Rai UN, Chandra P. Role of glutathione and phytochelatin in Hydrilla verticillata Royle and Valliseria spiralis L. under mercury stress. Chemosphere 1998;37:785–800. Gutie´rrez AG, Gotor C, Meyer AJ, Fricker M, Vega JM, Romero LC. Glutathione biosynthesis in Arabidopsis trichome cells. Proc Natl Acad Sci USA 2000;97: 11108–13. Ha SB, Smith AP, Howden R, Dietrich WM, Bugg S, ` connell MJ, et al. Phytochelatin synthase genes from O Arabidopsis and the yeast Schizosaccharomyces pombe. Plant Cell 1999;11:1153–63. Harmens H, Hartog PRD, Ten Bookum WM, Verkleij JAC. Increased zinc tolerance in Silene vulgaris (Moench) Garcke is not due to increased production of phytochelatins. Plant Physiol 1993;103:1305–9. Hartley-Whitaker J, Ainsworth G, Vooijs R, Ten Bookum W, Schat H, Meharg AA. Phytochelatins are involved in differential arsenate tolerance in Holcus lanatus. Plant Physiol 2001;126:299–306.

ARTICLE IN PRESS 1498 Hartley-Whitaker J, Woods C, Meharg AA. Is differential phytochelatin production related to decreased arsenate influx in arsenate tolerant Holcus lanatus. New Phytol 2002;155:219–25. Hewitt EJ. Sand and water culture methods used in the study of plant nutrition. Slough: Commonwealth Agricultural Bureaux; 1966. Howden R, Goldsbrough PB, Anderson CR, Cobbett CS. Cadmium-sensitive, cad1 mutants of Arabidopsis thaliana are phytochelatin-deficient. Plant Physiol 1995a;107:1059–66. Howden R, Goldsbrough PB, Anderson CR, Cobbett CS. A cadmium-sensitive glutathione-deficient mutant of Arabidopsis thaliana. Plant Physiol 1995b;107: 1067–73. Jackson PJ, Delhaize E, Kuske CR. Biosynthesis and metabolic roles of cadystins (g-EC)n-G and their precursors in Datura innoxia. Plant Soil 1992;146:281–9. Keltjens WG, Van Beusichem ML. Phytochelatins as biomarker for heavy metal stress in maize (Zea mays L.) and wheat (Triticum aestivum L.): combined effects of copper and cadmium. Plant Soil 1998;203: 119–26. Landberg T, Greger M. No phytochelatin (PC2 and PC3) detected in Salix viminalis. Physiol Plant 2004;121: 481–7. Little TM, Hills JJ. Agricultural experimention: design and analysis. Chichester: Wiley; 1978. Lombi E, Tearall KL, Homwarth JR, Zhao FJ, Hawkesford MJ, McGrath SP. Influence of iron status on cadmium and zinc uptake by different ecotypes of the hyperaccumulator Thlaspi caerulescens. Plant Physiol 2002;128:1359–67. Mehra RK, Mulchandani P. Glutathione-mediated transfer of Cu (I) into phytochelatins. Biochem J 1995;307: 697–705. Ni TH, Wei YZ. Subcellular distribution of cadmium in mining ecotype Sedum alfredii. Acta Bot Sin 2003;45: 925–8. Noctor G, Strohm M, Jouanin L, Kunert KJ, Foyer CH, Rennenberg H. Synthesis of glutathione in leaves of transgenic poplar overexpressing g-glutamylcysteine synthease. Plant Physiol 1996;112:1071–8. Oven M, Grill E, Golan-Goldhirsh A, Kutchan TM, Zenk MH. Increase of free cysteine and citric acid in plant cells exposed to cobalt ions. Phytochemistry 2002;60: 467–74. Pawlik-Skowron ´ska B. Correlations between toxic Pb effects and production of Pb-induced thiol peptides in the microalga Stichococcus bacillaris. Environ Pollut 2002;119:119–27. Perrin DD, Watt AE. Complex formation of zinc and cadmium with glutathione. Biochem Biophys Acta 1971;230:96–104. Rauser WE. Structure and function of metal chelators produced by plants: the case for organic acids, amino acids, phytin and metallothioneins. Cell Biochem Biophys 1999;32:19–48. Roosens N, Verbruggen N, Meerts P, Ximenez-enbu ` n P, Smith JAC. Natural variation in cadmium tolerance

Q. Sun et al. and its relationship to metal hyperaccumulation for seven populations of Thlaspi caerulescens from western Europe. Plant Cell Environ 2003;26:1657–72. Sagner S, Kneer R, Wanner G, Cosson JP, Deus-Neumann B, Zenk MH. Hyperaccumulation, complexation and distribution of nickel in Sebertia acuminata. Phytochemistry 1998;47:339–47. Schat H, Kalff MMA. Are phytochelatins involved in differential metal tolerance or do they merely reflect metal-imposed strain? Plant Physiol 1992;99:1475–80. Schat H, Llugany M, Vooijs R, Hartley-Whitaker J, Bleeker PM. The role of phytochelatins in constitutive and adaptive heavy metal tolerances in hyperaccumulator and non-hyperaccumulator metallophytes. J Exp Bot 2002;379:2381–92. Sneller FEC, Noordover ECM, Bookum WMT, Schat H, Bedaux JJM, Verkleij JAC. Quantitative relationship between phytochelatin accumulation and growth inhibition during prolonged exposure to cadmium in Silene vulgaris. Ecotoxicology 1999;8:167–75. Sneller FEC, Van Heerwaarden LM, Koevoets PLM, Vooijs R, Schat H, Verkleij JAC. Derivatization of phytochelatins from Silene vulgaris, induced upon exposure to arsenate and cadmium: comparison of derivatization with Ellman’s reagent and monobromobimane. J Agric Food Chem 2000;48:4014–9. Steffens JC. The heavy metal-binding peptides of plants. Annu Rev Plant Physiol Plant Mol Biol 1990;41: 553–75. Steffens JC, Hunt DF, Williams BG. Accumulation of nonprotein metal-binding polypeptides (g-glutamyl-cysteinyl)n-glycines in selected cadmium resistant tomato cells. J Biol Chem 1986;261:13879–82. Vo ¨geli-Lange R, Wagner GJ. Relationship between cadmium, glutathione and cadmium-binding peptides (phytochelatins) in leaves of intact tobacco seedlings. Plant Sci 1996;114:11–8. Xiang CB, Werner BL, Christensen EM, Oliver DJ. The biological functions of glutathione revisited in Arabidopsis transgenic plants with altered glutathione levels. Plant Physiol 2001;126:564–74. Yang XE, Long XX, Ni WZ, Ni SF. Zinc tolerance and hyperaccumulation in a new ecotype of Sedum alfredii Hance. Acta Phytoecol Sin 2001;25:670–7. Yang XE, Long XX, Ye HB, He ZL, Calvert DV, Stoffella PJ. Cadmium tolerance and hyperaccumulation in a new Zn-hyperaccumulating plant species (Sedum alfredii Hance). Plant Soil 2004;259:181–9. Zhao FJ, Wang JR, Barker JHA, Schat H, Bleeker PM, McGrath SP. The role of phytochelatins in arsenic tolerance in the hyperaccumulator Pteris vittata. New Phytol 2003;159:403–10. Zhu YL, Pilon-Smits EAH, Jouanin L, Terry N. Cadmium tolerance and accumulation in Indian mustard is enhanced by overexpressing g-glutamylcysteine synthase. Plant Physiol 1999a;121:1169–77. Zhu YL, Pilon-Smits EAH, Jouanin L, Terry N. Overexpression of glutathione synthase in Indian mustard enhanced cadmium accumulation and tolerance. Plant Physiol 1999b;119.