Effects of copper and mineral nutrition on growth, copper accumulation and mineral element uptake in two Rumex japonicus populations from a copper mine and an uncontaminated field sites

Environmental and Experimental Botany 59 (2007) 59–67

Effects of copper and mineral nutrition on growth, copper accumulation and mineral element uptake in two Rumex japonicus populations from a copper mine and an uncontaminated field sites Wenshan Ke a,b , Zhi-Ting Xiong a,∗ , Shijian Chen c , Jianjun Chen b a

c

Department of Environment Sciences, Wuhan University, Wuhan 430079, PR China b College of Life Science, Hubei University, Wuhan 430062, PR China Institute of Geodesy and Geophysics, Chinese Academy of Sciences, Wuhan 430077, PR China Received 21 October 2004; received in revised form 18 May 2005; accepted 20 October 2005

Abstract Two Rumex japonicus populations, one from a copper mine and the other from an uncontaminated site, were studied in hydroponic experiments for the plant growth, copper accumulation and mineral nutrient content under excess copper and nutrient deficiency conditions. The tolerance indices of the contaminated population were significantly higher than that of the uncontaminated population, indicating the evolution of Cu resistance in the former. At control and low Cu treatment, there was no difference in Cu accumulation in roots between the two populations. At high Cu (100 ␮M) treatment, however, the contaminated population accumulated less Cu in roots than the uncontaminated one, suggesting the root exclusion mechanism existing in the former. The contaminated population was also more tolerant to general nutrient deficiency than the uncontaminated one. The results indicated that the contaminated population had evolved not only Cu-tolerance but also tolerance to low nutrient supply. Under Cu stress, the contaminated population had less change in nutrient composition than the uncontaminated one. The similar result was observed in general nutrient deficiency experiment. The results indicated that the mineral composition homeostasis under the stresses was important in metal tolerance and colonizing the Cu-enriched soils for the Cu-tolerant population. At high Cu (100 ␮M) treatment and general nutrient deficiency treatment, the contaminated population accumulated significantly lower copper and higher phosphorus in both roots and shoots than the uncontaminated one. This was not the case for other mineral nutrients Ca, Mg and Fe except for root Mg and root Fe at Cu treatment. The result suggested that the high Ca-metabolism in R. japonicus was uncorrelated with high Cu-tolerance and that P might play an important role in governing Cu bioaccumulation. © 2005 Elsevier B.V. All rights reserved. Keywords: Copper tolerance; Nutrient deficiency; Mineral element accumulation; Rumex japonicus

1. Introduction Copper is an essential micronutrient for normal plant growth and metabolism. In plants, copper plays a vital role in both photosynthetic and respiratory electron transport, and functions as a cofactor for a variety of enzymes such as superoxide dismutase, cytochrome c, oxidase and plastocyanin (Clemens, 2001). However, if in excess it is harmful to most plants (Fernandes and Henriques, 1991). Copper can cause chlorosis, inhibition of root growth and damage to plasma ∗

Corresponding author. Tel.: +86 27 67866643. E-mail address: [email protected] (Z.-T. Xiong).

0098-8472/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2005.10.007

membrane permeability, leading to ion leakage (De Vos et al., 1991; Ouzounidou et al., 1992). However, some plants can grow in Cu-contaminated soils and are unaffected by the excessive Cu. These plants have evolved heavy metal tolerance (Ernst et al., 2000). The evolution of metal-tolerant populations in higher plants in nature is a classic example of local adaptation and microevolution (Antonovics et al., 1971; Shaw, 1990). Specific mechanisms may have developed in the resistant populations via natural selection in response to heavy metal contamination of the soils (Liu et al., 2004). Baker (1981) proposed that some plant species growing in environments with elevated metal concentrations had evolved two basic strategies of metal tolerance: (1) exclusion, and (2)

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accumulation and sequestration. The former avoids excessive metal uptake and transport in plant tissues by enhancing border cell formation and higher mucilage production by the border cells (Miyasaka and Hawes, 2001; Llugany et al., 2003), immobilizing heavy metal in the cell wall (Nishizono et al., 1987), or limiting metal ions transport via the endodermal casparian strip (MacFarlane and Burchett, 2000). In contrast, accumulators or hyperaccumulators can accumulate large amounts of heavy metals in plant tissues even in aerial parts, in which plants detoxify free metal ions by compartmentation of metals in vacuoles, complexation of metal ions with organic acids, amino acids and metal-binding peptides (Clemens, 2001; Hall, 2002; Lou et al., 2004; Tong et al., 2004). Reports have shown that copper and other heavy metals can induce deficiency of essential elements in plants (Biddappa et al., 1987; Godbold and Kettner, 1991; Monni et al., 2000). A deficiency of these elements in plants is often a manifestation of toxic effects (Antosiewicz, 1995). However, compared to the sensitive plants, the metal-tolerant plants are more tolerant to mineral deficiency (Antonovics et al., 1971; Baker, 1987; Antosiewicz, 1995). Many previous studies focused on the relationship between deficiency of some nutrient elements (e.g. Ca, P and Fe) and heavy metals uptake, accumulation and tolerance (Antosiewicz, 1995; Chen et al., 2004; Wallace and Cha, 1989). Very little is known about the relationship between the Cu-tolerance and general nutrient deficiency tolerance in populations from copper waste heap. In Elsholtzia splendens, Cu-tolerance perhaps is correlated with its ability to maintain high mineral nutrient level in plants under Cu stress (Yang et al., 2002). However, it is unclear whether the Cu-tolerant population is also tolerant to general nutrient deficiency. Rumex japonicus is a herb with fast growth and higher biomass, and grows easily in the wetland habitat. This species has been used for phytoremediation of eutrophic water (Huang et al., 1998). Interestingly, this species is also found in the old Cu mine heap at Tonglushan Hill, a copper mine that has been in development for about 3000 years in Hubei Province of China (Hua and Lu, 1996). It is unclear about the strategy of Cu uptake, accumulation and tolerance of this species growing in the Cu mine heap. Knowing the strategy, we can determine that the species may be used as metal excluder in revegetating bare soil areas (e.g. in phytostabilisation technology) where the lack of vegetation results from excessive Cu or as metal-(hyper)accumulator in phytoremediation of high Cu-contaminated soils (e.g. in phytoextraction technology) (Dahmani-Muller et al., 2000). On the other hand, it is important to consider that contam-

inated soils, such as mine soils, may have several problems to plant growth except for heavy metal contamination. Additional growth-limiting factors, which may be encountered in such soils, include a lack of soil structure, poor drainage, or nutrient deficiency (Kidd et al., 2004). Reports show that nutrient can affect the uptake and accumulation of Cu and other metals in plants (Xiong et al., 2002; Wu et al., 2004). Cu concentration of the soil in the Cu mine heap is considerably elevated and nutrient of the soil is also deficient (Chen et al., 2000; Ke et al., 2001; Huang and Luo, 2003; Liu et al., 2004). We hypothesize that the population of this species growing on Cu mine spoil has evolved Cu-tolerance as well as tolerance to nutrient deficiency. In order to test the hypothesis and to determine the pattern of Cu uptake and accumulation in R. japonicus, we focused on the study on the responses of two R. japonicus populations, respectively from the contaminated and non-contaminated sites, to Cu stress and general nutrient deficiency in two hydroponic experiments: Cu stress experiment and general nutrient deficiency experiment. The objectives of this study were: (1) to determine possible differences in Cu uptake, accumulation and tolerance between the two populations; (2) to determine difference in nutrient deficiency tolerance between the two populations; and (3) to reveal the relationship between copper tolerance and nutrient status.

2. Materials and methods 2.1. Plant material and preculture Two natural populations of R. japonicus Houtt. were selected for study, one population at Cu-contaminated site, the other at non-contaminated site. Seeds of the contaminated site population (CS population) were collected from the outcrop of Cu mine at Tonglushan Hill in Hubei Province, China, and those of the uncontaminated site population (UCS population) were collected from a field on the campus of Hubei University. In each population, a large number of seeds were randomly collected from 30 plants and were pooled. The seeds for the pot experiments were randomly taken from the pooled ones. Background values of copper concentration in the soils of the two sites (three replicates per site) are listed in Table 1. Seeds from the two populations were sterilized in 0.3% of hypochlorite for 30 min and washed three times with redistilled water. Then the seeds were transferred into a pyrex dish and displayed on a filter paper sheet. Germination took place in a growth chamber with the following condition: photoperiod

Table 1 Copper concentration (mg/kg soil), organic matter, phosphorus content and pH in the soils supporting the Rumex japonicus (0–10 cm depth) Locality

Cutotal

CuH2 O

Corg (%)

pHH2 O

PEDTA (mg/kg soil)

Ptotal (%)

CS UCS

1044.5 ± 273.4 62.5 ± 5.3

468.6 ± 86.3 2.7 ± 1.1

1.68 ± 0.1 3.65 ± 1.5

6.8 ± 0.4 7.2 ± 0.2

7.0 ± 3.2 33.9 ± 11.3

0.06 ± 0.01 0.13 ± 0.02

CS, contaminated site; UCS, uncontaminated site; results are mean ± S.D. (n = 3).

W. Ke et al. / Environmental and Experimental Botany 59 (2007) 59–67

16 h light/8 h darkness, day/night temperature 25 ◦ C/20 ◦ C. After germination, seedlings were evenly planted in 15-cmdiameter and 6-cm-depth plastic containers filled with acid washed quartz sands and grown in a greenhouse under natural light at a temperature between 25 and 30 ◦ C. Seedlings were irrigated every day with 1/2 strength Hoagland’s solution (HS) containing (in mM/L) 5KNO3 , 5Ca(NO3 )·H2 O, 2MgSO4 ·7H2 O, 1KH2 PO4 , mixture of 0.02FeSO4 ·7H2 O and 0.02Na2 -EDTA, 0.045H3 BO3 , 0.01MnCl2 ·4 H2 O, and (in ␮M/L) 0.8ZnSO4 , 0.3CuSO4 ·5H2 O, and 0.1NaMoO4 ·2 H2 O. Solution pH was adjusted to 5.5 ± 0.3 by 0.1 M NaOH or 0.1 M HCl. 2.2. Copper and nutrition deficiency treatments After 4 weeks culture, healthy and uniform seedlings were selected and transplanted to vessels filled with 500 ml of HS. Each vessel was covered with a foam plastic plate, four holes in it, one plant per hole. After 5 days culture, two experiments were carried out. Experiment 1 was performed with three levels of Cu treatments: 0.3 (control), 25 and 100 ␮M Cu for 14 days of growth. Solution Cu2+ was supplemented as CuSO4 ·5H2 O. Experiment 2 was performed with two levels of nutrient treatments: full HS (control), general nutrition deficiency (1 part HS + 49 parts H2 O) in medium for 21 days of growth. Solution pH was adjusted to 5.5 ± 0.3 as the above description. Each treatment was conducted in triplicate, four seedlings per replicate. The culture medium was changed every 3 days. 2.3. Tolerance index determination The longest root of each seedling was measured after 14 days of Cu treatments in the Experiment 1. The tolerance index (TI) was calculated according to Wilkins (1978). The following equation was used to calculate TI: TI% = 100 × (mean root length of longest root developed in Cu treatment)/(mean root length of longest root developed in the control).

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weighed from the pooled sample per replicate was digested in a mixture of HNO3 /HClO4 (3/1, v/v), at 150 ◦ C for 2 h and 210 ◦ C for 1 h, and then dissolved in HCl (0.5 N). P was measured colorimetrically; Cu, Ca, Mg and Fe were analyzed by ICP-OES. 2.5. Statistical analysis The results were analyzed statistically by the SPSS (10.0). Two-way ANOVA was performed to test the significant differences for population and Cu treatments and for population and nutrient treatment, respectively. If the F-value showed significant differences (P < 0.05), means of plant biomass, Cu and other mineral elements content in plant were compared with the Duncan test. All the values presented in this paper were the means of three replicates.

3. Results 3.1. Copper tolerance The tolerance indexes showed significant differences in Cu-tolerance between the two R. japonicus populations (Fig. 1). The TI values of the population from the contaminated (copper mine) site were significantly higher than those from the uncontaminated site. 3.2. Effect of copper on biomass Significant difference in biomass was observed between the two populations. In the control, the root and shoot biomass of UCS population were higher than those of CS population (Table 2). However, with copper added at two concentrations (25 ␮M and 100 ␮M Cu), the root (percent of control) and shoot biomasses of UCS population were significantly lower than that of CS population. At the lower Cu treatment

In Experiment 2, all four seedlings per replicate were pooled and the dry weight (DW) of biomass was weighed. The tolerance of the plants to general nutrition deficiency was performed on the basis of dry weights of experimental plants relative to controls (Antosiewicz, 1995). 2.4. Elements analysis Plants were harvested after treatment and roots were washed with 5 mM cool Pb(NO3 )2 for 30 min to desorb the Cu adsorbed on the root surface. Each seedling was washed with deionized water three times, divided into shoot and root, and then oven-dried at 70 ◦ C for 48 h to obtain the dry weight of each seedling. Dried plant tissues from the same container were pooled as one sample and were ground to fine powder. Approximately 0.1 g the powdered material

Fig. 1. Tolerance index during 14 days exposure to different Cu in two Rumex japonicus populations from contaminated site (CS) and uncontaminated site (UCS). Values are mean ± SD (n = 3); (*) significantly different between two ecotypes within each group treatment (P < 0.05).

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W. Ke et al. / Environmental and Experimental Botany 59 (2007) 59–67

Table 2 Biomass of two populations of Rumex japonicus (mg DW/plant) after 14 days of hydroponic culture with various copper concentrations Populations

Treatment (␮M)

Biomass Root

Percent of control

Shoot

Percent of control

Root/shoot

CS

Control 25 100

45.3 ± 3.6 b 53.7 ± 3.7 a 43.7 ± 2.1 b

100.0 ± 7.8 b 118.5 ± 8.3 a 96.5 ± 4.6 b

166.3 ± 4.7 c 190.2 ± 11.1 b 141.9 ± 3.8 d

100.0 ± 2.3 b 114.4 ± 6.7 a 85.3 ± 2.3 c

0.27 ± 0.02 cd 0.28 ± 0.05 cd 0.31 ± 0.01 c

UCS

Control 25 100

54.5 ± 2.0 a 53.6 ± 2.4 a 45.7 ± 0.5 b

100.0 ± 3.7 b 98.3 ± 4.4 b 83.9 ± 0.9 c

227.4 ± 12.3 a 168.4 ± 8.4 c 76.6 ± 6.4 e

100.0 ± 5.4 b 74.1 ± 3.7 d 33.7 ± 2.8 e

0.24 ± 0.01 d 0.32 ± 0.01 b 0.60 ± 0.05 a

Results are mean ± S.D. (n = 3). Means with different letters in the same column are significant (P < 0.05) according to Duncan multiple range test.

(25 ␮M), the shoot and root biomass of CS population were significantly higher, and the shoot biomass of UCS population was distinctly lower as compared with their controls, respectively. At the higher Cu treatment (100 ␮M), the shoot biomass of the two populations and the root biomass of the UCS population reduced markedly. Significant differences of root/shoot biomass were also found between the two populations. The root/shoot ratios of the CS population at Cu treatments were significantly lower than that of the UCS population (Table 2), indicating clearly that the biomass allocation between root and shoot in CS population was different from UCS population under Cu stress conditions. 3.3. Effect of copper on mineral elements uptake The copper concentrations in plant tissues of the two populations increased with increasing copper concentration in the solution (Table 3). In 25 ␮M Cu-treated plants, the difference of Cu concentration between the two populations was only found in shoots. The Cu concentration in shoots of CS population was lower as compared with the UCS population. When the plants were treated with 100 ␮M Cu, Cu concentrations in roots and shoots in the CS population were significantly lower than those in the UCS population. In roots, the copper concentrations of both populations were several-fold higher than in their shoots. The root/shoot ratios of Cu concentration increased significantly in the both populations, but no clear difference between them was found. Phosphorus concentrations in tissues of both populations declined with increasing Cu concentrations in the solution (Table 4). Clear differences between populations were found

in 100 ␮M Cu treatment. The P levels in both root and shoot of the CS population were significantly higher than those of the UCS population. The magnesium concentrations in different Cu-treated plants did not change significantly in comparison with control except for Mg content of root in UCS population (Table 4). Difference in Mg content between populations was only found in root, the CS population kept higher Mg level in root than the UCS population. The calcium concentrations of both populations increased significantly in shoot, but not in roots (Table 4). At 25 ␮M Cu treatment, the CS population had a significantly higher Ca level in root and shoot than the UCS population. At 100 ␮M treatment, however, the CS population had a significantly lower Ca level in root and shoot than the UCS population. Fe concentrations in tissues of both populations declined significantly with increasing external Cu concentrations. At 25 ␮M Cu treatment, Fe concentration of CS population was significantly higher than that of UCS population. At 100 ␮M Cu treatment, however, no difference was found between the two populations (Table 4). 3.4. Effects of general nutrient deficiency (1/50 HS) on biomass Table 5 shows the influence of nutrient deficiency on biomass of two R. japonicus populations. Under general nutrient deficiency, the shoot biomass was obviously decreased in the two populations. However, the root biomass of both populations increased sharply in comparison with the controls, being 145.18% of the control for UCS population

Table 3 Copper concentration (␮g/g DW) in plant tissues of two Rumex japonicus populations after 14 days of hydroponic culture with various copper concentrations Populations

Cu in solution (␮M)

Concentration Root

Shoot

Root/shoot

CS

Control 25 100

20.72 ± 0.32 d 137.23 ± 3.27 c 210.55 ± 14.25 b

12.58 ± 1.85 d 34.63 ± 1.67 c 38.66 ± 1.55 bc

1.67 ± 0.27 c 3.97 ± 0.27 b 5.46 ± 0.57 a

UCS

Control 25 100

17.74 ± 1.83 d 147.52 ± 39.14 c 279.14 ± 19.12 a

11.22 ± 1.24 d 40.06 ± 1.92 b 47.69 ± 4.47 a

1.59 ± 0.21 c 3.71 ± 1.1 b 5.86 ± 0.23 a

Results are mean ± S.D. (n = 3). Values followed by the same letter in the same column are not significantly different (P < 0.05) according to the Duncan test.

W. Ke et al. / Environmental and Experimental Botany 59 (2007) 59–67

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Table 4 Mineral concentrations (␮g/g DW) in plant tissues of two Rumex japonicus populations after 14 days of hydroponic culture with various copper concentrations Tissues

Elements (␮g/g)

Cu in solution (␮M) Control

25

100

Root

P-CS P-UCS Ca-CS Ca-UCS Mg-CS Mg-UCS Fe-CS Fe-UCS

7007.34 6946.32 3465.51 3095.62 6736.12 7779.48 330.08 318.84

± ± ± ± ± ± ± ±

708.06 a 84.99 a 441.70 a 112.34 ab 455.09 a 122.66 a 37.42 a 15.47 a

6917.84 6493.82 3540.25 1838.39 6622.54 5546.60 240.05 144.76

± ± ± ± ± ± ± ±

222.51 a 326.92 a 652.00 a 187.00 c 605.26 a 195.53 b 23.56 b 4.36 d

5427.52 4619.10 2630.28 3800.95 7179.86 4102.13 232.12 183.41

± ± ± ± ± ± ± ±

129.52 b 397.84 c 134.16 b 371.63 a 135.46 a 565.40 c 42.08 c 28.10 c

Shoot

P-CS P-UCS Ca-CS Ca-UCS Mg-CS Mg-UCS Fe-CS Fe-UCS

7416.94 7472.54 8328.14 9026.59 6707.95 6106.96 241.04 206.79

± ± ± ± ± ± ± ±

79.39 a 398.87 a 694.52 d 99.23 cd 586.91 a 395.85 a 8.20 a 5.33 b

6646.11 6732.68 11390.9 8410.78 5868.76 5839.19 143.78 120.63

± ± ± ± ± ± ± ±

260.47 b 216.71 b 351.18 bc 388.26 d 628.40 a 114.19 a 8.90 c 8.60 d

6172.23 5516.70 10186.8 12593.6 6316.90 6970.82 124.05 105.77

± ± ± ± ± ± ± ±

451.99 b 191.45 c 680.67 ab 1475.0 a 256.99 a 644.93 a 16.80 d 4.84 d

Root/shoot

P-CS P-UCS Ca-CS Ca-UCS Mg-CS Mg-UCS Fe-CS Fe-UCS

0.94 0.93 0.42 0.34 1.01 1.28 1.36 1.55

± ± ± ± ± ± ± ±

0.09 a 0.05 a 0.02 a 0.01 ab 0.13 b 0.09 a 0.12 bc 0.04 b

1.04 0.96 0.35 0.22 1.13 0.95 1.67 1.20

± ± ± ± ± ± ± ±

0.07 a 0.04 a 0.07 ab 0.03 d 0.11 ab 0.05 b 0.16 b 0.12 c

0.83 0.81 0.23 0.31 1.14 0.60 1.51 2.20

± ± ± ± ± ± ± ±

0.02 b 0.03 b 0.02 cd 0.06 bc 0.03 ab 0.14 c 0.42 b 0.46 a

Results are mean ± S.D. (n = 3). Different letters in same row and between populations of the same element indicate a significant difference (P < 0.05) with respect to treatment levels and populations according to Duncan test.

and 165.15% for CS population. Significant differences between populations were observed under general nutrient deficiency according to the dry mass of the plants in comparison with control (Table 5). The root and shoot biomass in the CS population was markedly higher than in the UCS population. 3.5. Effects of general nutrient deficiency on mineral nutrient accumulation Under general nutrient deficiency, the differences of Cu concentrations in roots and shoots between the two populations were significant (Fig. 2). Cu concentration in both roots and shoots of the UCS population was higher than that of the

CS population. For shoot Cu concentrations, UCS population was about twice that of the CS population. P concentrations in roots and shoots of both populations were three to five times lower than those of the controls. However, differences of P concentrations between populations were also observed (Fig. 2). The CS population had significantly higher P content in both roots and shoots than the UCS population. There were significant differences in Ca, Mg and Fe concentrations between two populations. In UCS population, root-Ca and root-Mg concentrations were significantly higher than those of CS population. In shoots, Ca and Mg concentrations in the UCS population were significantly higher in comparison to the controls; nevertheless, the CS population

Table 5 Biomass of two populations of Rumex japonicus (mg DW/plant) after 21 days growth under general nutrient deficiency (1/50 HS) condition Population

Treatment

Biomass Root

Percent of control

Shoot

Percent of control

Root/shoot

CS

Control 1/50 HS

66.9 ± 1.3 d 66.9 ± 1.3 d

100.0 ± 3.7 c 185.2 ± 15.6 a

383.0 ± 11.6 b 119.1 ± 3.0 c

100.0 ± 4.1 a 31.1 ± 1.0 b

0.18 ± 0.01 c 1.04 ± 0.06 b

UCS

Control 1/50 HS

73.0 ± 1.1 c 105.9 ± 2.8 b

100.0 ± 6.1 c 145.1 ± 4.7 b

406.0 ± 11.3 a 88.0 ± 1.8 d

100.0 ± 3.4 a 21.7 ± 0.5 c

0.18 ± 0.01 c 1.20 ± 0.03 a

Results are mean ± S.D. (n = 3). Means with different letters in the same column are significant (P < 0.05) according to Duncan multiple range test.

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Fig. 2. Cu, P, Ca, Mg, Fe concentrations (␮g/g DW) of two populations of Rumex japonicus after 21 days growth under general nutrient deficiency (1/50 HS) condition. Values are mean ± S.D. (n = 3). Different letters of root (or shoot) indicate a significant difference (P < 0.05) with respect to populations and treatments according to Duncan test.

maintained the level comparable to the control treatment (Fig. 2).

4. Discussion Heavy metal contamination of soils or sediments can constitute a powerful selective force in plant evolution (Antonovics et al., 1971; Ye et al., 2003). Populations of plants growing there are often genetically distinct from populations of the same species in soils of low heavy metal content. In this study, root TI values (Fig. 1) and biomasses (Table 2) of R. japonicus population from the copper mine site are significantly higher than that from an uncontaminated site. The results indicate that the CS population has developed into a copper-tolerant population under long-term copper stress. This could be the reason why R. japonicus can colonize the Cu-enriched soils of Cu mine at Tonglushan Hill. It is generally regarded that underground stresses usually lead to increasing root/shoot ratios of biomass (Szaniawski,

1987), allowing the plant to have a greater root surface area for absorption of water and nutrients (Xiong et al., 2002). Our study show that the CS population has less change in the root/shoot ratios of biomasses than the UCS population does under copper stress (Table 2) and under general nutrient deficiency (Table 5). The results may be correlated to a higher Cu-tolerance of CS population than UCS population. High Cu-tolerance of CS population may be due to the low accumulation of Cu in shoots of CS population (Table 3). A similar result was observed in Rumex dentatus (Liu et al., 2004). In contrast, the shoot of UCS population accumulates high concentration of Cu (Table 3), which results in significantly low biomass and high root/shoot ratio (Table 2) due to Cu phytotoxicity. Shoot/root ratios of tissue metal have been used as a measure of restriction of metal transport for comparing the behavior of metal-tolerant and less tolerant plant species and races (Reilly and Reilly, 1973). In most cases, the plants from metal-contaminated soils have significantly lower ratios than the plants from uncontaminated soils, which suggests that metal tolerance in these species (Wu et al., 1975; Baker, 1981). However, in R. japonicus no difference of shoot/root ratio of tissue Cu (Table 3) was detected between the coppertolerant and non-tolerant plants. This result suggests that copper tolerance in R. japonicus is not dependent on different copper transport from root to shoot. Qureshi et al. (1985) and Baker and Walker (1990) reported that tolerant races generally showed great root uptake of the metal and less metal accumulation in the shoots as compared with non-tolerant ones. Metal accumulation and immobilization in the roots has been shown to be a mechanism of metal tolerance in plants (Peterson, 1983; Wu and Antonovics, 1976; Kruckeberg and Wu, 1992). In Lotus purshianus, no difference is detected in either the pattern of Cu uptake or concentration in plant tissue between tolerant and non-tolerant populations (Wu and Lin, 1990). In the present study, copper concentration in roots of the tolerant population of R. japonicus at high Cu treatment was significantly lower than that of the non-tolerant one (Table 3). This result suggests that the root exclusion mechanism may exist in the tolerant population of R. japonicus. Efficient exclusion of Cu from whole roots also has been found in Cu-tolerant ecotypes of Silene vulgaris (De Vos et al., 1991; Lolkema and Vooijs, 1986) and Silene armeria (Llugany et al., 2003). Obviously, metal tolerance mechanisms are complicated and different mechanisms may exist in different species (or different ecotypes). In S. vulgaris, the root exclusion in copper tolerant ecotype from Cu mine seems to be due to increased Cu efflux from root cells because of the overexpression of an efflux transporter controlled by a major tolerance gene and of the overexpression of a metallotionein gene, SvMT2b, which behaves as a hypostatic enhancer of the degree of tolerance (Van Hoof et al., 2001a,b). In S. aemeria, Llugany et al. (2003) suggests that in copper tolerance population from Cu mine, enhanced border cells may also play an important role in Cu resistance in addition to the Cu efflux mechanism. In

W. Ke et al. / Environmental and Experimental Botany 59 (2007) 59–67

the serpentine population of S. aemeria, Cu exclusion from roots may be a mere consequence of root impermeabilization of enhanced border cells in response to metal toxicity (Llugany et al., 2003; Poschenrieder and Barcel´o, 1999), rather than the result of an effective Cu efflux mechanism. Other reports show that mucilages of plant root exudates may play a role of filter in heavy metal absorption (Deianaa et al., 2003). The mucilages are deposited outside the root hairs forming a gelatinous envelope known as “mucigel” (Floyd and Ohlrogge, 1970; Paull et al., 1975; Wright and Northcote, 1976). Mucilages constitute an important fraction of the apoplast and rhizosphere, and possess a high ability to complex and immobilize metals in the reticulating sites of the root free space. Whether Cu efflux or root impermeabilization of enhanced border cells or filter of “mucigel” or all these mechanisms operates in R. japonicus from Cu mine heap needs further studies. Heavy metals may interfere with essential nutrient uptake and transport and thereby disturb mineral nutrition composition of plants. Consequently, heavy metals may result in phytotoxic effects by influencing mineral nutrition metabolisms of the plants (Monni et al., 2000). In our study, Cu reduces the uptake and transport of elements P and Fe and increases shoot-Ca concentration in R. japonicus plant. However, in the CS population, mineral nutrients such as P and Fe are less affected than that in the UCS population. Root-Mg concentration was significantly reduced in UCS population and was unchanged in CS population (Table 4). Fluctuating of Ca concentration caused by excess Cu in UCS population was also greater than that in CS one. These results indicate that the capacity of CS population in maintaining mineral nutrient stabilization is stronger than that of UCS population. Some investigations also show that some mineral elements are less affected in tolerant population (Macnair, 1993) or not affected in tolerant reed species (Ali et al., 2002) by Cu and other heavy metals. It is generally regarded that damage to the plasmalemma of root cells constituted the first effect of Cu toxicity (Woolhouse, 1983), causing a loss of ions and lipid peroxidation (De Vos et al., 1993). Thus, the degree of Cu-tolerance may depend on the capacity of the plant to prevent this effect (Ali et al., 2002). The tolerant population of R. japonicus may have a strong capacity to prevent a loss of ions and therefore, can maintain the relative stabilization of mineral composition. In general nutrient deficiency experiment, mineral element concentrations of shoots, such as P, Ca and Mg are less change in CS population than in UCS one, indicating further the strong capacity of Cutolerant population in maintaining the stabilization of mineral composition. Antosiewicz (1995) showed that high Pb-tolerant species, Silene inflata, was high resistant to general nutrient deficiency. In the present study, root and shoot biomass of the Cu-tolerant population in R. japonicus was significantly higher than that of the non-tolerant one under nutrient deficiency (Table 5), indicating a higher tolerance to general nutrient deficiency in the Cu-tolerance population than that

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in the sensitive one. The copper tolerance and the nutrient deficiency tolerance of CS population may be evolved independently, but are selected together by the Cu mine soils which are high in Cu and low in nutrients. Whether they are genetically and physiologically linked need further studies. In general nutrient deficiency experiment, Cu accumulation in shoots of both R. japonicus populations significantly increases (Fig. 2), and the Cu increase in the shoots of UCS population (315.1% of the control) is more pronounced than that in the CS one (151.6% of the control). The nutrient deficiency promotes obviously Cu transport from root to shoot and the promotion is more significant in UCS population than in CS one. Excessive Cu accumulation in shoot leads to Cu phytotoxicity and inhibition of plant growth. Less Cu accumulation in shoot may be the reason of tolerance to general nutrient deficiency in CS population. Changes in calcium concentrations have been found to be a general physiological response of plants against metal toxicity (Nieminen and Helmisaari, 1996). High Ca-metabolism in Silene is in correlation with high Pb-tolerance (Antosiewicz, 1995). For the R. japonicus, at 25 ␮M Cu treatment, Ca concentration in the CS population is significantly higher than in UCS population. However, at 100 ␮M Cu treatment, root Ca concentration is significantly lower in the CS population than in UCS population. Shoot Ca concentration in CS population is slightly lower and no difference is detected between the two populations. A similar result, i.e. Cu and Ca concentration in shoots is higher in UCS population than in CS population, was also observed in general nutrient deficiency experiments. High Ca-metabolism in R. japonicus may be uncorrelated with high Cu-tolerance. Study on L. purshianus L. indicates that there is no significant difference in phosphorus concentration of the plant between the tolerant and non-tolerant lines (Lin and Wu, 1994). In R. japonicus, we find that the phosphorus element maintains higher levels in the CS population than in the UCS population in high Cu (100 ␮M) stress and nutrient deficiency experiments. A similar result is also found in our phosphorus deficiency experiment (data not shown). Positive correlation between the Cu-tolerance and the phosphorus accumulation suggests that phosphorus may play an important role in controlling Cu accumulation and transport in R. japonicus. Some studies show that phosphorus plays an important role in governing Cu accumulation in Bush bean (Wallace and Cha, 1989) and Brassica pekinensis (Xiong et al., 2002). When plants are cultured in growth medium deficient in P, their roots can extrude protons to acidify the rhizosphere, thus improving P availability (Grinsted et al., 1982; Logan et al., 2000). Consequently, rhizosphere acidification may improve cation absorption. Plants with the exclusion strategy are currently used to revegetate bare soil areas with high metal concentration; plants with the accumulation strategy are used for phytoextraction of high metal soils (Dahmani-Muller et al., 2000). R. japonicus plant from Cu mine heap belongs to the former

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and has potential to be used in recovery of vegetation in Cu-contaminated soil areas.

5. Conclusion As compared with those in uncontaminated site, the plants of R. japonicus growing at a copper contaminated mine site has great growth rate, copper tolerance, and mineral nutrient deficiency tolerance. Their mineral composition is less affected by the Cu stress. It suggests that stability and homeostasis of mineral composition under nutrient deficiency stress plays an important role in Cu-tolerance of plants.

Acknowledgements This work was funded by the National Natural Science Foundation of China (Project 20477032) and Natural Science Foundation of Zhejiang Province (Project Y504256).

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