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Arsenate reduces copper phytotoxicity in gametophytes of Pteris vittata
ARTICLE IN PRESS Journal of Plant Physiology 165 (2008) 1906—1916
www.elsevier.de/jplph
Arsenate reduces copper phytotoxicity in gametophytes of Pteris vittata Yongqiang Zhenga,b, Xiaojing Daia,b, Lei Wanga,b, Wenzhong Xua, Zhenyan Hea, Mi Maa, a
Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, PR China b The Graduate School of Chinese Academy of Sciences, Beijing 100039, PR China Received 9 January 2008; received in revised form 17 April 2008; accepted 17 April 2008
KEYWORDS Arsenic; Copper; Hyperaccumulator; Phytotoxicity; Pteris vittata
Summary The fern Pteris vittata is an arsenic (As) hyperaccumulator and can take up very high concentrations of arsenic from the soil. However, little is known about its response to co-contamination with arsenic and copper (Cu). In this study, we used an in vitro model system of P. vittata gametophytes to investigate the impact of changes in As and Cu status on growth, chlorophyll (chl) concentration, metal accumulation, and subcellular localization. A remarkable inhibition of growth occurred when gametophytes were exposed to concentrations X1.0 mM Na3AsO4 or X0.5 mM CuSO4. chl concentration decreased significantly when gametophytes were exposed to 40.25 mM of CuSO4, but increased steadily with concentration to p2 mM Na3AsO4. Interestingly, the inhibitory effect caused by Cu was reduced in the presence of 0.25 mM Na3AsO4. However, the inhibition caused by exposure to 1.0 mM Na3AsO4 was not alleviated by 0.25 mM CuSO4. Further studies showed that 0.25 mM Na3AsO4 increased cell viability (CV) and chl concentration, while decreasing cell membrane permeability (CMP) of gametophytes with 1.0 mM CuSO4 stress. In contrast, 0.25 mM CuSO4 decreased CV and chl concentration, while increasing CMP when gametophytes were treated with 1.0 mM Na3AsO4. In addition, the subcellular distribution of As and Cu in P. vittata gametophytes differed. As was found primarily in the cytoplasm, while Cu was mainly localized in the cell wall. These results suggest that As can reduce Cu phytotoxicity in the As hyperaccumulator P. vittata, and that this may serve as a biological mechanism for the fern to adapt to soils cocontaminated with As and Cu. Crown Copyright & 2008 Published by Elsevier GmbH. All rights reserved.
Abbreviations: As, arsenic; BA, benzylaminopurine; chl, chlorophyll; CMP, cell membrane permeability; Cu, copper; CV, cell viability; DW, dry weight; FW, fresh weight; HM, heavy metal; GA, gibberellin; P. vittata, Pteris vittata. Corresponding author. Tel.: +86 10 8259 9422; fax: +86 10 6259 0833. E-mail address: [email protected] (M. Ma). 0176-1617/$ - see front matter Crown Copyright & 2008 Published by Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2008.04.008
ARTICLE IN PRESS Arsenate-reduced copper toxicity in fern
Introduction Heavy metal (HM) and arsenic (As) pollution has become a major problem resulting from the increase in mining and smelting of metal ores, industrial waste, mineral fertilizers, pesticides, vehicle exhausts, and municipal sewage sludge. Investigators have begun to pay more attention to plants that can hyperaccumulate HMs and As due to their unique physiology and potential use in phytoremediation of polluted soils (Bondada et al., 2004; Kramer, 2005). The Chinese brake fern Pteris vittata (L.), the first plant species to be reported as an As hyperaccumulator (Ma et al., 2001), can take up extremely high concentrations of As (up to 2.3% dry plant weight) from soil and allocate most of it to the aboveground fronds for final storage (Tu et al., 2004). Thus, it has great potential for use in phytoremediation of As-contaminated sites. Previous work has focused on the tolerance and hyperaccumulation of As in P. vittata. A few studies have described the zinc (Zn) tolerance of P. vittata, as well as the relationship between As and other elements with respect to their effect on growth of the organism. For example, An et al. (2006) found that P. vittata has a very high tolerance to Zn and can grow normally at sites with high Zn concentrations. Li et al. (2006) investigated the impact of As on chloroplast ultrastructure and calcium (Ca) distribution in P. vittata using histochemical methods. They found that the Ca concentration in fronds was not significantly affected by the addition of As, but that the Ca concentration in mature pinnae increased significantly after addition of As, consistent with As toxicity (Li et al., 2006). These findings indicate that there is a close relationship between Ca concentration and As toxicity in P. vittata. Fayiga and Ma (2006) determined that rock phosphate was effective in increasing the uptake of As and decreasing the uptake of other metals (e.g., cadmium [Cd], Zn) by P. vittata. Recently, attention has focused on the physiological and biochemical effects of HM interaction (antagonism and synergism). Several reports have shown that Zn can compete for Cd, both in animals (in vitro) (Kingsley and Frazier, 1979) and in plants (in vivo) (Smilde et al., 1992). Wang et al. (2001) reported that copper (Cu) can reduce the phytotoxicity of As in soybean (Glycine max) based on changes in respiration rate, proteinase activity, and lipid peroxidase (POD) activity. As-stress significantly decreased respiration rate and proteinase activity, while lipid POD activity increased significantly upon Cu addition. Sun et al. (2008) found that lead (Pb) and nickel (Ni) had a synergistic effect in inducing oxidative stress in the moss
1907 Hypnum plumaeforme, especially when exposed to both metals simultaneously at relatively high concentrations (0.1 mM Ni and 1.0 mM Pb). Exposure to one or both metals decreased catalase (CAT) activity and increased POD activity, indicating that moss POD plays an important role in resisting the oxidative stress induced by Pb and Ni. The effect of a combination of As and Cu on growth and HM accumulation of P. vittata has not been examined. This may be because the sporophyte of P. vittata is a slow-growing perennial plant with a large genome. As such, it is less than ideal for investigating the basic mechanisms underlying As hyperaccumulation in plants. However, Gumaelius et al. (2004) demonstrated that the gametophyte of P. vittata is an easily manipulated model system useful for study of the molecular mechanisms involved in As hyperaccumulation. Recently, we developed a plant regeneration system that can easily propagate plenty of freeliving, autotrophic haploid gametophytes consisting of a small (o1 mm) single-layered sheet of cells (Zheng et al., 2008). In the current study, we examined the growth of P. vittata gametophytes in MS culture medium (Murashige and Skoog, 1962) exposed to different concentration of As, Cu, or a combination of the two. The aim of this study was to identify subcellular Cu and As co-tolerance, accumulation, and distribution in P. vittata gametophytes and the interactive effects of the combination of the two metals.
Materials and methods Plant material and culture P. vittata Linn. sporophytes were obtained from As mine spoil in Hubei Province, China, and maintained in a glass greenhouse for 3 years before experiments commenced. Gametophytes of P. vittata were obtained according to Zheng et al. (2008). Mature spores of P. vittata were collected from the greenhouse and surface-sterilized in a 1.5-mL tube by immersion in a solution containing 0.3% NaClO for 10 min. The sterile spores were added to half-strength MS containing 20 g L1 sucrose. After 25 d, spores began to germinate and form gametophytes. Gametophytes were subcultured for 25 d in half-strength MS containing 20 g L–1 sucrose. Experimental design and implementation The sterile gametophytes of P. vittata were transplanted into MS media containing 0 (control), 0.25, 0.5, 1.0, and 2.0 mM of Na3AsO4 or CuSO4. All gametophytes used in these experiments were of similar size, with an approximate fresh weight (FW) of about 0.46–0.50 g in
ARTICLE IN PRESS 1908 each culture bottle. Gametophytes were picked from these treatments every 10 d to measure FW and observe the growth with a stereomicroscope (SZX9; Olympus, Tokyo, Japan). Gametophytes were harvested on the 40th day to measure Chl concentration, CV, CMP, and the concentration of Cu and As in subcellular fractionation of gametophytes. Gametophytes were picked on the 50th day to dry at 60 1C for 3 d, and then the accumulation of Cu and As was measured. The As and Cu interaction experimental design was as follows: the gametophytes were subcultured on solid MS medium containing Na3AsO4 1.0 mM, Na3AsO41.0 mM+CuSO4 0.25 mM, CuSO4 1.0 mM, and CuSO4 1.0 mM+Na3AsO4 0.25 mM. Gametophytes were picked from these treatments every 10 d to measure FW. Gametophytes were picked on 40th day to measure the concentrations of Cu and As. The equation used to calculate the relative increase in FW was FW change over 10 d ðgÞ ¼ FWnext FWinitial Relative increase in FW ¼ ðFWnext FWinitial Þ=FWinitial
Chlorophyll concentration Gametophytes were mechanically disintegrated using glass beads in a centrifuge tube and extracted in 90% acetone for chlorophyll (chl) determination. Acetonesoluble pigments were separated from colorless debris by centrifugation for 10 min at 1000g. Absorbance was determined at 647, 664, and 470 nm using a U 2010 spectrophotometer (Hitachi, Japan). The equations (Moran, 1982) used to calculate the concentration of chl were Ca ¼ 12:21 OD664 2:81 OD647 Cb ¼ 20:13 OD647 5:03 OD664 Cc ¼ ð1000 OD470 3:27Ca 104Cb Þ=229 C ðconcentration of chlÞ ¼ ðCa þ Cb þ Cc Þ V extraction dilution factor=FWðmg g1 FWÞ. where Ca, Cb, and Cc are chl a, chl b, and carotenoids, respectively; OD is the optical density; and Vextraction is the extraction volume. Subcellular fractionation of gametophytes Subcellular fractionation of gametophytes (Chen et al., 2005) was carried out according to Hans (1980), Kramer et al. (2000), and Pathore et al. (1972). One gram of cultured gametophytes was homogenized with a mortar and pestle and then with a glass tissue grinder. The homogenization solution contained 0.25 M sucrose, 50 mM Tris-maleate buffer (pH 7.8), 1 mM MgCl2, and 10 mM cysteine. The final pH of the solution was adjusted to 7.8. All procedures were performed at 4 1C. The homogenate was brought to a final volume of 15–20 mL, transferred to a 50-mL tube and centrifuged at 300g for 30 s. The resulting pellet containing primarily cell walls
Y. Zheng et al. (including cell wall interspaces) was designated the cell wall fraction (F1). The supernatant was then centrifuged at 20 000g for 45 min to sediment organelles. This pellet was designated as the organellar fraction (F2). The resultant supernatant was designated the cytoplasmic supernatant fraction (F3) and included macromolecular organic matter and inorganic ions from the cytoplasm and vacuoles. Each of these fractions was used for analysis of As and Cu concentration. Analysis of metal concentration by graphite furnace atomic absorption spectrometry Samples of the above subcellular fractions were carefully diluted up to 5 mL distilled water before analysis, and the oven-dried gametophytes were ground to a fine powder in a Wiley mill to pass through a 1-mm sieve. Then 10 mL concentrated HNO3 was added to each sample and digested using EPA Method 3051 (USEPA, 1994) in a Hot Block Digestion System (Environmental Express, Mt. Pleasant, SC). After complete mineralization, samples were diluted to 10 mL with MilliQ water (Millipore, Billerica, MA, USA) and centrifuged at 3000g for 15 min at 25 1C. The supernatants were then diluted to 50 mL with 0.2% HNO3. Chemical analyses were carried out using a graphite furnace atomic absorption spectrometer (GFAAS; SIMA 6000; Perkin-Elmer, Waltham, MA, USA) using the USEPA Method 7060A (Chen and Ma, 1998). The standard reference material was carried out through the supernatants of those oven-dried gametophytes and analyzed as part of the quality assurance–quality control protocol. Reagent blanks and internal standards were used where appropriate to ensure accuracy and precision in the analysis of As and Cu. Determination of cell viability (CV)—Evans blue staining Determination of CV was carried out as described by Baker and Mock (1994). After treatment with Na3AsO4 and/or CuSO4, 1.0 g of gametophytes was washed three times with ultrapure water and collected by filtration. The gametophytes of each sample were stained in 0.25% (w/v) Evans blue in water for 5 min at room temperature and subsequently washed in water as treatment. Evans blue only enters cells with a freely permeable plasma membrane, i.e., dead cells. Whilst, gametophytes of each sample were stained in 0.25% (w/v) Evans blue in water for 10 min at 60 1C as a control. Care was taken to prevent gametophytes from adhering and drying onto the filter membranes. Filtered gametophytes were carefully transferred to 2.0-mL Eppendorf tubes containing quartz sand (o0.1 g). To stop cell uptake of Evans blue and reduce duplication errors, 0.5 mL of 1% SDS was added. Gametophytes were pulverized with a plastic stirring rod in 0.5 mL distilled water. The reaction tubes were mixed and then centrifuged at 8800g for 3 min. Absorbance was determined at 600 nm using at least 0.8 mL of supernatant. And CV can be estimated by the absorbance changes of each sample between control and treatment.
ARTICLE IN PRESS Arsenate-reduced copper toxicity in fern
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Determination of cell membrane permeability (CMP) Non-electrolytes (such as amino acids and polysaccharides) of most plant material have an absorption peak at 264 nm (Bewley and Black, 1982). Determination of CMP was performed according to Xie and Xu (1986), transferring 2.0 g of thoroughly washed gametophytes to 10 mL distilled water and shaking every 20 min. After 3 h, the absorbance of the supernatant at 264 nm was measured using a UV spectrophotometer [unit: OD264 nm/(g h)]. Data analysis All experiments were repeated at least three times, and the data presented here are expressed as the mean7S.D. of three independent experiments. All statistical analyses were performed with SPSS 13.0 (SPSS Inc., Chicago, IL, USA). One-way analysis of variance was used to compare means. After analysis using Levene’s test, means with equal variance were tested by leastsquares determination, whereas means with unequal variance were analyzed using Dunnett’s T3 test. Differences at Po0.05 were considered significant.
Results Phytotoxicity of Cu2+ The time course of the relative increase in FW of gametophytes exposed to different concentrations of CuSO4 in solid MS medium is shown in Figure 1A. The relative increase in FW for all treatments decreased significantly with an increase in culture time. However, the relative increase in FW for the 0.25 mM CuSO4 treatment was much higher than that observed at other concentrations (Po0.05), and no growth retardation or other visible symptoms of phytotoxicity were seen for a period of at least 40 d. Growth retardation and visible symptoms of phytotoxicity appeared after 40–50 d of growth in 0.5 mM CuSO4, and after 30–40 d with 1.0 or 2.0 mM CuSO4 (Figure 1B). The average total FWs of the gametophytes at the end of the culture period after treatment with 0, 0.25, 0.5, 1.0, and 2.0 mM CuSO4 were 5.86, 9.78, 5.12, 3.40, and 0.94 g, respectively. When gametophytes received more than 1.0 mM CuSO4, their growth was significantly inhibited and FW decreased by about 42% compared to that of control cultures (0 mM CuSO4). In P. vittata gametophytes grown on solid MS medium plus control, 0.25, 0.5, 1.0, or 2.0 mM CuSO4, the concentration of chl decreased slightly (P40.05) with increase in Cu2+ concentration up to 0.25 mM; however, the concentration of carotenoids increased significantly (Po0.05). When the Cu2+ concentration exceeded 0.5 mM, the chl and
Figure 1. Effects of CuSO4 on growth and chlorophyll concentration of P. vittata gametophytes. (A) Changes in FW of gametophytes under CuSO4 stress. (B) Time course of changes in FW of gametophytes exposed to different concentrations of CuSO4. (C) Changes in chlorophyll and carotenoids concentration of gametophytes under CuSO4 stress. Each value is the mean of three individual replicates (7SD). Different lowercase letters indicate significant differences (po0.05).
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carotenoids concentration in gametophytes decreased significantly (Figure 1C).
Phytotoxicity of As (V) The relative increase in FW of gametophytes exposed to different concentrations of Na3AsO4 is shown in Figure 2A. The relative increase in FW for all treatments decreased significantly with an increase in culture time. The relative increase in FW of cultures exposed to either 1.0 or 2.0 mM Na3AsO4 was much lower than that for other concentrations over a 40-d culture period (Po0.05). The relative increase in FW was much lower in cultures exposed to 2 mM Na3AsO4 than that observed for cultures exposed to 1 mM or control (Po0.05). The increase in FW was significantly higher for cultures exposed to either 0.25 or 0.5 mM Na3AsO4 than that observed for any of the other concentrations tested (Figure 2B). No growth retardation or visible symptoms of phytotoxicity were seen within 40 d in gametophytes exposed to less than 1.0 mM Na3AsO4, but appearing during the next 10 d of exposure. In contrast, growth retardation and visible symptoms of phytotoxicity appeared between 20 and 30 d in cultures exposed to 2.0 mM Na3AsO4. The average total FW of gametophytes at the end of the culture period and exposed to control, 0.25 mM, 0.5 mM, 1.0 mM, or 2.0 mM Na3AsO4 was 5.86, 10.9, 8.33, 5.68, and 1.24 g, respectively. The growth of gametophytes was significantly inhibited only when more than 2.0 mM Na3AsO4 was added to the MS medium. Growth was inhibited by more than 20% when gametophytes were grown with 2.0 mM Na3AsO4 compared to the growth observed in the absence of Na3AsO4. The amount of chl present in P. vittata gametophytes grown on solid MS medium plus control, 0.25, 0.5, 1.0, or 2.0 mM Na3AsO4 is shown in Figure 2C. The chl concentration increased slightly in gametophytes exposed to 0.25 mM Na3AsO4 (Po0.05); however, cultures exposed to 0.5, 1.0, and 2.0 mM Na3AsO4 had chl concentrations that were significantly higher than those observed upon exposure to control (Po0.05). And the carotenoids concentration increased significantly with the As level increasing from 0 to 2.0 mM (Figure 2C).
Effect of As(V) status on Cu2+ tolerance of P. vittata gametophytes When gametophytes were exposed to different concentrations of As in the presence of Cu, the relative increase in FW was much higher at a
Figure 2. Effects of Na3AsO4 on the growth and chlorophyll concentration of P. vittata gametophytes. (A) Changes in FW of gametophytes under Na3AsO4 stress. (B) Time course changes in FW of gametophytes exposed to different concentrations of Na3AsO4. (C) Changes in chlorophyll and carotenoids concentration of gametophytes under Na3AsO4 stress. Each value is the mean of three individual replicates (7SD). Different lowercase letters indicate significant differences (po0.05).
ARTICLE IN PRESS Arsenate-reduced copper toxicity in fern concentration of 0.25 mM Na3AsO4 or CuSO4 than control; there were no significant differences in the concentration of chl. In contrast, cultures exposed to 1.0 mM Na3AsO4 or CuSO4 demonstrated a significantly inhibited relative increase in FW compared to control. The time course of the relative increase in gametophyte FW and concentration of chl upon exposure to 0.25 and 1.0 mM of Na3AsO4, and CuSO4 in various combinations is shown in Figure 3A and B. The relative increase in gametophyte FW was significantly greater with exposure to 0.25 mM Na3AsO4 and 0.25 mM CuSO4 than with other treatments. When gametophytes were cultured in the presence of 1.0 mM CuSO4+0.25 mM Na3AsO4, the relative increase in FW was much greater than that observed in cultures exposed to 1.0 mM CuSO4 alone (Po0.05). Conversely, cultures exposed to 1.0 mM Na3AsO4+0.25 mM CuSO4 had a much lower relative increase in FW than cultures grown in the presence of 1.0 mM Na3AsO4 alone (Figure 3A). Although gametophytes grown in 1.0 mM CuSO4 and 1.0 mM CuSO4+0.25 mM Na3AsO4 showed no indication of growth retardation after 40 d, visible symptoms of phytotoxicity were observed at 40 and 20 d, respectively, when exposed to 1.0 mM CuSO4+0.25 mM Na3AsO4 and 1.0 mM CuSO4. In contrast, P. vittata gametophytes grown in the presence of 1.0 mM Na3AsO4 and 1.0 mM Na3AsO4+0.25 mM CuSO4 exhibited both growth retardation and visible symptoms of phytotoxicity after 40 and 20 d, respectively (Figure 3B). The average total FW of gametophytes at the end of the culture period in the presence of 1.0 mM Na3AsO4, 1.0 mM Na3AsO4+0.25 mM CuSO4, 1.0 mM CuSO4, and 1.0 mM CuSO4+0.25 mM Na3AsO4 was 4.77, 3.70, 2.91, and 7.94 g, respectively. In P. vittata gametophytes grown on solid MS medium containing 1.0 mM CuSO4+0.25 mM Na3AsO4, the chl and carotenoids concentration increased significantly (Po0.05) compared to those grown with 1.0 mM CuSO4 alone (Figure 3C). However, the chl and carotenoids concentration in cultures grown in the presence of 1.0 mM Na3AsO4+0.25 mM CuSO4 decreased significantly compared to those grown with 1.0 mM Na3AsO4 or 0.25 mM CuSO4 alone.
Impact of As and Cu on cell viability and cell membrane permeability Stress from reactive oxygen species (ROS) can lead to cell membrane damage and cell death (Chattopadhyay et al., 2002; Lynn et al., 2000). A widely used method for detection of membrane
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Figure 3. Effects of Na3AsO4+CuSO4 on the growth and chlorophyll concentration of P. vittata gametophytes exposed to different combinations of Na3AsO4+CuSO4 concentrations. (A) Changes in FW of gametophytes under combined Na3AsO4+CuSO4 stress. (B) Time course of changes in FW of gametophytes exposed to different combinations of Na3AsO4+CuSO4 concentrations. (C) Changes in chlorophyll and carotenoids concentration of gametophytes under combined Na3AsO4+CuSO4 stress. Each value is the mean of three individual replicates (7SD). Different lowercase letters indicate significant differences (po0.05).
ARTICLE IN PRESS 1912 integrity (electrolyte leakage) and cell death (Evans blue staining) (Mur et al., 2005) was used to assess changes in the CMP and CV of P. vittata gametophytes upon exposure to As and Cu, and effects on CMP and CV could be a subsection of phytotoxic effects. The effect of As and Cu on CV and CMP of As hyperaccumulator P. vittata gametophytes is shown in Figure 4A and B, respectively. Upon exposure to 0.25 mM Na3AsO4 in 1.0 mM CuSO4, gametophytes were visibly chlorotic and/or dead, CV was significantly increased, and CMP significantly decreased compared to cultures exposed to 1.0 mM CuSO4 alone.
Figure 4. Effects of different combinations of Na3AsO4+CuSO4 on cell viability (CV) and cell membrane permeability (CMP) of P. vittata gametophytes. CV was determined by monitoring the uptake of Evans blue by P. vittata gametophytes exposed to different combinations of As+Cu. The relative increase in cell death was estimated by spectrophotometric monitoring of Evans blue retention. (A) Effects of different combinations of As+Cu on CV. (B) Effects of different combinations of As+Cu on CMP. Each value is the mean of three individual replicates (7SD). Different lowercase letters indicate significant differences (po0.05).
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Influence of concomitant exposure to CuSO4 and Na3AsO4 on Cu and As concentrations in P. vittata gametophytes The effect of concomitant exposure of P. vittata gametophytes to CuSO4 and Na3AsO4 on Cu concentration is shown in Figure 5A. When cultures were provided with 1.0 mM CuSO4 in the medium, the Cu concentration in the gametophytes was 2730 mg kg–1 dry weight (DW). Upon exposure to 1.0 mM CuSO4+0.25 mM Na3AsO4, however, the Cu concentration in the gametophytes decreased significantly to 2350 mg kg–1 DW. Gametophytes cultured in 1.0 mM Na3AsO4 had As concentrations of 526.9 mg kg–1 DW. Those cultured in 1.0 mM Na3AsO4+0.25 mM CuSO4 showed significantly decreased As concentrations of 323.8 mg kg–1 DW (Figure 5B).
Figure 5. Effects of combined Na3AsO4+CuSO4 on accumulation of As and Cu in P. vittata gametophytes. (A) Cu concentration in gametophytes exposed to different Cu+As concentrations in the culture medium. (B) As concentration in gametophytes exposed to different Cu+As concentrations in the culture medium. Each value is the mean of three individual replicates (7SD). Different lowercase letters indicate significant differences (po0.05).
ARTICLE IN PRESS Arsenate-reduced copper toxicity in fern
As status and impact on Cu distribution in gametophyte subcellular fractions Copper distribution in the subcellular fraction of P. vittata gametophytes grown under different Cu2+ and As (V) treatments is shown in Table 1. The Cu concentration in the cell wall fraction (F1) and soluble fraction (F3) decreased significantly (Po0.05) with exposure to 1.0 mM Cu2++0.25 mM Na3AsO4. The Cu concentration in the organelle fraction (F2) was not significantly decreased. In addition, the percentage of Cu distributed in each subcellular fraction did not change significantly. The Cu distribution percentages in F1, F2, and F3 were 72.16%, 11.29%, and 16.55%, respectively, when the culture medium contained 1.0 mM CuSO4. Thus, Cu in P. vittata gametophytes was primarily sequestered in the cell wall layer.
Cu status and impact on As distribution in gametophyte subcellular fractions Arsenic distribution in the subcellular fraction of P. vittata gametophytes grown in the presence of varying As concentrations is shown in Table 2. The amount of As in F1 decreased significantly with exposure to 1.0 mM As(V)+0.25 mM CuSO4. The concentration of As in F2 and F3 did not change significantly. The percentage distribution of As in Table 1. and As
1913 all subcellular fractions was unchanged. The percentage distribution of As in F1, F2, and F3 was 23.08%, 13.27%, and 63.65%, respectively, when gametophytes were cultured in the presence of 1.0 mM As. Therefore, As in P. vittata gametophytes was located mainly in the cytoplasmic supernatant fraction (including vacuoles), followed by the cell wall layer, and least of all in the organelles.
Discussion Plants from metalliferous soils often exhibit tolerance to different HMs. This tolerance may be due either to a combination of different metalspecific tolerance mechanisms (multiple tolerance) or less-specific mechanisms that confer pleiotropic tolerance to different metals (co-tolerance) (Gregory and Bradshaw, 1965; Schat and Ten Bookum, 1992). In this study we found that cultured gametophytes of the As hyperaccumulator fern P. vittata can tolerate higher concentrations of Na3AsO4 (1.0 mM) than CuSO4 (0.5 mM; Figures 1 and 2) as assessed by changes in FW and chl concentration. The FW of gametophytes declined significantly and symptoms of chlorosis began to appear when the culture medium contained over 1.0 mM Na3AsO4 or
Cu distribution in subcellular fractions of P. vittata gametophytes exposed to different combinations of Cu
Treatment (mM)
As 1.0 Cu 1.0 Cu 1.0+As 0.25 As 1.0+Cu 0.25
Cu concentration (mg/kg FW)
Percentage distribution
F1
F2
F3
F1
F2
F3
5.20d 656.66a 386.4b 113.79c
5.05d 102.74a 90.77ab 69.24c
3.82d 150.61a 106.17b 90.31bc
31.63c 72.16a 66.24ab 41.63c
30.76a 11.29c 15.56c 25.33ab
23.23b 16.55c 18.20c 33.04a
Note: Lowercase letters indicate values that differ significantly among cell fractions (po0.05). F1, cell wall fraction; F2, organelle fraction; F3, soluble fraction.
Table 2. and As
As distribution in subcellular fractions of P. vittata gametophytes exposed to different combinations of Cu
Treatment (mM)
As 1.0 Cu 1.0+As 0.25 As 1.0+Cu 0.25
As concentration (mg/kg FW)
Percentage distribution
F1
F2
F3
F1
F2
F3
40.54a 27.89b 31.09b
23.31a 9.94b 18.83a
111.79a 61.17c 92.76ab
23.08a 28.17b 22.01a
13.27a 10.04a 13.33a
63.65a 61.79a 65.66a
Note: Lowercase letters indicate values that differ significantly among cell fractions (po0.05). F1, cell wall fraction; F2, organelle fraction; F3, soluble fraction.
ARTICLE IN PRESS 1914 0.5 mM CuSO4. The concentration of chl in gametophytes cultured with 2.0 mM Na3AsO4 was higher than in control plants. This is due to the fact that although the concentration of chla (Ca) and chlb (Cb) decreased slightly (P40.05), the concentration of co-extracted carotenoids (Cc) increased significantly upon exposure to 2.0 mM Na3AsO4 compared to control (0 mM Na3AsO4) (Figure 2C). However, the concentration of chl in gametophytes cultured with 0.5 mM CuSO4 decreased significantly compared to control, suggesting that Cu is more phytotoxic than As to P. vittata gametophytes. This may be due to the fact that Cu can be transported to the cytoplasm and then to the chloroplasts where it inhibits photosynthesis by uncoupling electron transport to NADP (Femandes and Henriques, 1991; Sandmann and Boger, 1980). Cu is bound to various cellular proteins including some of those found in the chloroplast membranes resulting in an increase in ion concentration that leads to degradation of chl and other pigments, preventing photosynthesis and eventually causing cell death (Barn et al., 1995). Previous studies have shown that As is toxic to most plants by interfering with the physiological functions normally performed by phosphorus (P) (Meharg and Macnair, 1990, 1991). However, the results presented here indicate that low concentrations of As (0.5 mM Na3AsO4) can actually stimulate P. vittata gametophyte growth (FW and DW). This observation is in agreement with the results of Tu and Ma (2005) with regard to P. vittata sporophyte growth and preferential As accumulation in young fronds of up to 50 mg As kg–1 DW, suggesting that As may act as a nutrient similar to phosphorus in P. vittata. Interactive effects (synergism or antagonism) among different HMs are well documented. Landberg and Greger (1994) found that selenium does not reduce the toxicity of HMs to plants, but instead enhances metal uptake and toxicity. Here we found that As can reduce the phytotoxicity of Cu in P. vittata gametophytes. Moreover, Cu does not reduce the toxicity of As to P. vittata gametophytes, in contrast to results reported for soybean (Wang et al., 2001). Analogously to the P. vittata sporophyte, it is possible that As reduces the toxicity of Cu in P. vittata gametophytes because at low levels of As exposure, enzymatic antioxidants (superoxide dismutase, CAT, ascorbate POD, guaiacol POD) are important for As detoxification and accumulation, while non-enzymatic antioxidants (glutathione, acidsoluble thiol) are more important at high As levels. More specifically, the concentration of non-enzymatic antioxidants increases with soil As concentrations, resulting in greater accumulation in the fronds than in the roots especially when exposed to high As
Y. Zheng et al. concentrations (450 mg kg1). Activity of enzymatic antioxidants in P. vittata sporophyte increased until soil As concentrations reached mg kg1 levels, and then decreased (Cao et al., 2004). The work described here demonstrates that appropriate As concentrations (o1.0 mM of Na3AsO4) reduce CMP, thereby inhibiting Cu2+ entry and altering the distribution of Cu in subcellular compartments by increasing resistance to ROS. Additionally, exposure of cultured gametophytes to 1.0 mM CuSO4+0.25 mM Na3AsO4 together not only significantly decreased chlorosis and death compared to those cultured in the presence of 1.0 mM CuSO4 alone, it also significantly increased CV and reduced CMP. Interestingly, while Cu can also inhibit As entry, it does not reduce the phytotoxicity of As. When cultures were exposed to 1.0 mM Na3AsO4+0.25 mM CuSO4, chlorosis and death were not significantly reduced, CV decreased significantly, and CMP increased significantly. These results suggest a unique mechanism of HM tolerance in cultured P. vittata gametophytes. Sporophytes and gametophytes of P. vittata may have evolved a set of analogous physiological and molecular mechanisms specifically for the escape of As toxicity. Subcellular As was primarily distributed in the soluble fraction (cytoplasm including vacuoles), while Cu was mainly located in cell walls, demonstrating that this organism avoids Cu absorption as much as possible. However, gametophytes of P. vittata transported As from the cell wall to the cytoplasm or vacuole. Although little is known about whether phosphate transporters can be charged for the absorption of As, P. vittata did transport excess As (over 100 mg kg1 FW) to the cytoplasm or vacuole of gametophyte, and adopting a dynamic equilibrium may be important for the prevention of HM toxicity. In summary, this study provides novel findings that As can stimulate growth and reduce Cu phytotoxicity in gametophytes of P. vittata, a plant species that shows high tolerance for As, Cu, and Zn and is an As hyperaccumulator. This process may have evolved as a biological mechanism allowing the fern to adapt to co-contamination with As and Cu. Additionally, we have shown that P. vittata gametophytes are ideal candidates for studying the physiological and biochemical mechanisms of As hyperaccumulation.
Acknowledgments This work was supported by the State High-tech Project (2007AA021404, 2007AA091704) and the
ARTICLE IN PRESS Arsenate-reduced copper toxicity in fern National Natural Science Foundation of China (30670171, 30500307).
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ARTICLE IN PRESS 1916 USEPA. SW 846 Method 3051, Microwave assisted acid digestion of sediments, Sludges, Soils, and Oils. US Environmental Protection Agency; 1994 Available online at /http://www.epa.gov/epaoswer/hazwaste/ test/pdfs/3051.pdfS. Wang Y, Liu D, Zhang L, Guo H. Effect of Cu and As and their combination pollution on Glycine max. Chin J Appl Ecol 2001;12:117–20.
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www.elsevier.de/jplph
Arsenate reduces copper phytotoxicity in gametophytes of Pteris vittata Yongqiang Zhenga,b, Xiaojing Daia,b, Lei Wanga,b, Wenzhong Xua, Zhenyan Hea, Mi Maa, a
Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, PR China b The Graduate School of Chinese Academy of Sciences, Beijing 100039, PR China Received 9 January 2008; received in revised form 17 April 2008; accepted 17 April 2008
KEYWORDS Arsenic; Copper; Hyperaccumulator; Phytotoxicity; Pteris vittata
Summary The fern Pteris vittata is an arsenic (As) hyperaccumulator and can take up very high concentrations of arsenic from the soil. However, little is known about its response to co-contamination with arsenic and copper (Cu). In this study, we used an in vitro model system of P. vittata gametophytes to investigate the impact of changes in As and Cu status on growth, chlorophyll (chl) concentration, metal accumulation, and subcellular localization. A remarkable inhibition of growth occurred when gametophytes were exposed to concentrations X1.0 mM Na3AsO4 or X0.5 mM CuSO4. chl concentration decreased significantly when gametophytes were exposed to 40.25 mM of CuSO4, but increased steadily with concentration to p2 mM Na3AsO4. Interestingly, the inhibitory effect caused by Cu was reduced in the presence of 0.25 mM Na3AsO4. However, the inhibition caused by exposure to 1.0 mM Na3AsO4 was not alleviated by 0.25 mM CuSO4. Further studies showed that 0.25 mM Na3AsO4 increased cell viability (CV) and chl concentration, while decreasing cell membrane permeability (CMP) of gametophytes with 1.0 mM CuSO4 stress. In contrast, 0.25 mM CuSO4 decreased CV and chl concentration, while increasing CMP when gametophytes were treated with 1.0 mM Na3AsO4. In addition, the subcellular distribution of As and Cu in P. vittata gametophytes differed. As was found primarily in the cytoplasm, while Cu was mainly localized in the cell wall. These results suggest that As can reduce Cu phytotoxicity in the As hyperaccumulator P. vittata, and that this may serve as a biological mechanism for the fern to adapt to soils cocontaminated with As and Cu. Crown Copyright & 2008 Published by Elsevier GmbH. All rights reserved.
Abbreviations: As, arsenic; BA, benzylaminopurine; chl, chlorophyll; CMP, cell membrane permeability; Cu, copper; CV, cell viability; DW, dry weight; FW, fresh weight; HM, heavy metal; GA, gibberellin; P. vittata, Pteris vittata. Corresponding author. Tel.: +86 10 8259 9422; fax: +86 10 6259 0833. E-mail address: [email protected] (M. Ma). 0176-1617/$ - see front matter Crown Copyright & 2008 Published by Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2008.04.008
ARTICLE IN PRESS Arsenate-reduced copper toxicity in fern
Introduction Heavy metal (HM) and arsenic (As) pollution has become a major problem resulting from the increase in mining and smelting of metal ores, industrial waste, mineral fertilizers, pesticides, vehicle exhausts, and municipal sewage sludge. Investigators have begun to pay more attention to plants that can hyperaccumulate HMs and As due to their unique physiology and potential use in phytoremediation of polluted soils (Bondada et al., 2004; Kramer, 2005). The Chinese brake fern Pteris vittata (L.), the first plant species to be reported as an As hyperaccumulator (Ma et al., 2001), can take up extremely high concentrations of As (up to 2.3% dry plant weight) from soil and allocate most of it to the aboveground fronds for final storage (Tu et al., 2004). Thus, it has great potential for use in phytoremediation of As-contaminated sites. Previous work has focused on the tolerance and hyperaccumulation of As in P. vittata. A few studies have described the zinc (Zn) tolerance of P. vittata, as well as the relationship between As and other elements with respect to their effect on growth of the organism. For example, An et al. (2006) found that P. vittata has a very high tolerance to Zn and can grow normally at sites with high Zn concentrations. Li et al. (2006) investigated the impact of As on chloroplast ultrastructure and calcium (Ca) distribution in P. vittata using histochemical methods. They found that the Ca concentration in fronds was not significantly affected by the addition of As, but that the Ca concentration in mature pinnae increased significantly after addition of As, consistent with As toxicity (Li et al., 2006). These findings indicate that there is a close relationship between Ca concentration and As toxicity in P. vittata. Fayiga and Ma (2006) determined that rock phosphate was effective in increasing the uptake of As and decreasing the uptake of other metals (e.g., cadmium [Cd], Zn) by P. vittata. Recently, attention has focused on the physiological and biochemical effects of HM interaction (antagonism and synergism). Several reports have shown that Zn can compete for Cd, both in animals (in vitro) (Kingsley and Frazier, 1979) and in plants (in vivo) (Smilde et al., 1992). Wang et al. (2001) reported that copper (Cu) can reduce the phytotoxicity of As in soybean (Glycine max) based on changes in respiration rate, proteinase activity, and lipid peroxidase (POD) activity. As-stress significantly decreased respiration rate and proteinase activity, while lipid POD activity increased significantly upon Cu addition. Sun et al. (2008) found that lead (Pb) and nickel (Ni) had a synergistic effect in inducing oxidative stress in the moss
1907 Hypnum plumaeforme, especially when exposed to both metals simultaneously at relatively high concentrations (0.1 mM Ni and 1.0 mM Pb). Exposure to one or both metals decreased catalase (CAT) activity and increased POD activity, indicating that moss POD plays an important role in resisting the oxidative stress induced by Pb and Ni. The effect of a combination of As and Cu on growth and HM accumulation of P. vittata has not been examined. This may be because the sporophyte of P. vittata is a slow-growing perennial plant with a large genome. As such, it is less than ideal for investigating the basic mechanisms underlying As hyperaccumulation in plants. However, Gumaelius et al. (2004) demonstrated that the gametophyte of P. vittata is an easily manipulated model system useful for study of the molecular mechanisms involved in As hyperaccumulation. Recently, we developed a plant regeneration system that can easily propagate plenty of freeliving, autotrophic haploid gametophytes consisting of a small (o1 mm) single-layered sheet of cells (Zheng et al., 2008). In the current study, we examined the growth of P. vittata gametophytes in MS culture medium (Murashige and Skoog, 1962) exposed to different concentration of As, Cu, or a combination of the two. The aim of this study was to identify subcellular Cu and As co-tolerance, accumulation, and distribution in P. vittata gametophytes and the interactive effects of the combination of the two metals.
Materials and methods Plant material and culture P. vittata Linn. sporophytes were obtained from As mine spoil in Hubei Province, China, and maintained in a glass greenhouse for 3 years before experiments commenced. Gametophytes of P. vittata were obtained according to Zheng et al. (2008). Mature spores of P. vittata were collected from the greenhouse and surface-sterilized in a 1.5-mL tube by immersion in a solution containing 0.3% NaClO for 10 min. The sterile spores were added to half-strength MS containing 20 g L1 sucrose. After 25 d, spores began to germinate and form gametophytes. Gametophytes were subcultured for 25 d in half-strength MS containing 20 g L–1 sucrose. Experimental design and implementation The sterile gametophytes of P. vittata were transplanted into MS media containing 0 (control), 0.25, 0.5, 1.0, and 2.0 mM of Na3AsO4 or CuSO4. All gametophytes used in these experiments were of similar size, with an approximate fresh weight (FW) of about 0.46–0.50 g in
ARTICLE IN PRESS 1908 each culture bottle. Gametophytes were picked from these treatments every 10 d to measure FW and observe the growth with a stereomicroscope (SZX9; Olympus, Tokyo, Japan). Gametophytes were harvested on the 40th day to measure Chl concentration, CV, CMP, and the concentration of Cu and As in subcellular fractionation of gametophytes. Gametophytes were picked on the 50th day to dry at 60 1C for 3 d, and then the accumulation of Cu and As was measured. The As and Cu interaction experimental design was as follows: the gametophytes were subcultured on solid MS medium containing Na3AsO4 1.0 mM, Na3AsO41.0 mM+CuSO4 0.25 mM, CuSO4 1.0 mM, and CuSO4 1.0 mM+Na3AsO4 0.25 mM. Gametophytes were picked from these treatments every 10 d to measure FW. Gametophytes were picked on 40th day to measure the concentrations of Cu and As. The equation used to calculate the relative increase in FW was FW change over 10 d ðgÞ ¼ FWnext FWinitial Relative increase in FW ¼ ðFWnext FWinitial Þ=FWinitial
Chlorophyll concentration Gametophytes were mechanically disintegrated using glass beads in a centrifuge tube and extracted in 90% acetone for chlorophyll (chl) determination. Acetonesoluble pigments were separated from colorless debris by centrifugation for 10 min at 1000g. Absorbance was determined at 647, 664, and 470 nm using a U 2010 spectrophotometer (Hitachi, Japan). The equations (Moran, 1982) used to calculate the concentration of chl were Ca ¼ 12:21 OD664 2:81 OD647 Cb ¼ 20:13 OD647 5:03 OD664 Cc ¼ ð1000 OD470 3:27Ca 104Cb Þ=229 C ðconcentration of chlÞ ¼ ðCa þ Cb þ Cc Þ V extraction dilution factor=FWðmg g1 FWÞ. where Ca, Cb, and Cc are chl a, chl b, and carotenoids, respectively; OD is the optical density; and Vextraction is the extraction volume. Subcellular fractionation of gametophytes Subcellular fractionation of gametophytes (Chen et al., 2005) was carried out according to Hans (1980), Kramer et al. (2000), and Pathore et al. (1972). One gram of cultured gametophytes was homogenized with a mortar and pestle and then with a glass tissue grinder. The homogenization solution contained 0.25 M sucrose, 50 mM Tris-maleate buffer (pH 7.8), 1 mM MgCl2, and 10 mM cysteine. The final pH of the solution was adjusted to 7.8. All procedures were performed at 4 1C. The homogenate was brought to a final volume of 15–20 mL, transferred to a 50-mL tube and centrifuged at 300g for 30 s. The resulting pellet containing primarily cell walls
Y. Zheng et al. (including cell wall interspaces) was designated the cell wall fraction (F1). The supernatant was then centrifuged at 20 000g for 45 min to sediment organelles. This pellet was designated as the organellar fraction (F2). The resultant supernatant was designated the cytoplasmic supernatant fraction (F3) and included macromolecular organic matter and inorganic ions from the cytoplasm and vacuoles. Each of these fractions was used for analysis of As and Cu concentration. Analysis of metal concentration by graphite furnace atomic absorption spectrometry Samples of the above subcellular fractions were carefully diluted up to 5 mL distilled water before analysis, and the oven-dried gametophytes were ground to a fine powder in a Wiley mill to pass through a 1-mm sieve. Then 10 mL concentrated HNO3 was added to each sample and digested using EPA Method 3051 (USEPA, 1994) in a Hot Block Digestion System (Environmental Express, Mt. Pleasant, SC). After complete mineralization, samples were diluted to 10 mL with MilliQ water (Millipore, Billerica, MA, USA) and centrifuged at 3000g for 15 min at 25 1C. The supernatants were then diluted to 50 mL with 0.2% HNO3. Chemical analyses were carried out using a graphite furnace atomic absorption spectrometer (GFAAS; SIMA 6000; Perkin-Elmer, Waltham, MA, USA) using the USEPA Method 7060A (Chen and Ma, 1998). The standard reference material was carried out through the supernatants of those oven-dried gametophytes and analyzed as part of the quality assurance–quality control protocol. Reagent blanks and internal standards were used where appropriate to ensure accuracy and precision in the analysis of As and Cu. Determination of cell viability (CV)—Evans blue staining Determination of CV was carried out as described by Baker and Mock (1994). After treatment with Na3AsO4 and/or CuSO4, 1.0 g of gametophytes was washed three times with ultrapure water and collected by filtration. The gametophytes of each sample were stained in 0.25% (w/v) Evans blue in water for 5 min at room temperature and subsequently washed in water as treatment. Evans blue only enters cells with a freely permeable plasma membrane, i.e., dead cells. Whilst, gametophytes of each sample were stained in 0.25% (w/v) Evans blue in water for 10 min at 60 1C as a control. Care was taken to prevent gametophytes from adhering and drying onto the filter membranes. Filtered gametophytes were carefully transferred to 2.0-mL Eppendorf tubes containing quartz sand (o0.1 g). To stop cell uptake of Evans blue and reduce duplication errors, 0.5 mL of 1% SDS was added. Gametophytes were pulverized with a plastic stirring rod in 0.5 mL distilled water. The reaction tubes were mixed and then centrifuged at 8800g for 3 min. Absorbance was determined at 600 nm using at least 0.8 mL of supernatant. And CV can be estimated by the absorbance changes of each sample between control and treatment.
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1909
Determination of cell membrane permeability (CMP) Non-electrolytes (such as amino acids and polysaccharides) of most plant material have an absorption peak at 264 nm (Bewley and Black, 1982). Determination of CMP was performed according to Xie and Xu (1986), transferring 2.0 g of thoroughly washed gametophytes to 10 mL distilled water and shaking every 20 min. After 3 h, the absorbance of the supernatant at 264 nm was measured using a UV spectrophotometer [unit: OD264 nm/(g h)]. Data analysis All experiments were repeated at least three times, and the data presented here are expressed as the mean7S.D. of three independent experiments. All statistical analyses were performed with SPSS 13.0 (SPSS Inc., Chicago, IL, USA). One-way analysis of variance was used to compare means. After analysis using Levene’s test, means with equal variance were tested by leastsquares determination, whereas means with unequal variance were analyzed using Dunnett’s T3 test. Differences at Po0.05 were considered significant.
Results Phytotoxicity of Cu2+ The time course of the relative increase in FW of gametophytes exposed to different concentrations of CuSO4 in solid MS medium is shown in Figure 1A. The relative increase in FW for all treatments decreased significantly with an increase in culture time. However, the relative increase in FW for the 0.25 mM CuSO4 treatment was much higher than that observed at other concentrations (Po0.05), and no growth retardation or other visible symptoms of phytotoxicity were seen for a period of at least 40 d. Growth retardation and visible symptoms of phytotoxicity appeared after 40–50 d of growth in 0.5 mM CuSO4, and after 30–40 d with 1.0 or 2.0 mM CuSO4 (Figure 1B). The average total FWs of the gametophytes at the end of the culture period after treatment with 0, 0.25, 0.5, 1.0, and 2.0 mM CuSO4 were 5.86, 9.78, 5.12, 3.40, and 0.94 g, respectively. When gametophytes received more than 1.0 mM CuSO4, their growth was significantly inhibited and FW decreased by about 42% compared to that of control cultures (0 mM CuSO4). In P. vittata gametophytes grown on solid MS medium plus control, 0.25, 0.5, 1.0, or 2.0 mM CuSO4, the concentration of chl decreased slightly (P40.05) with increase in Cu2+ concentration up to 0.25 mM; however, the concentration of carotenoids increased significantly (Po0.05). When the Cu2+ concentration exceeded 0.5 mM, the chl and
Figure 1. Effects of CuSO4 on growth and chlorophyll concentration of P. vittata gametophytes. (A) Changes in FW of gametophytes under CuSO4 stress. (B) Time course of changes in FW of gametophytes exposed to different concentrations of CuSO4. (C) Changes in chlorophyll and carotenoids concentration of gametophytes under CuSO4 stress. Each value is the mean of three individual replicates (7SD). Different lowercase letters indicate significant differences (po0.05).
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carotenoids concentration in gametophytes decreased significantly (Figure 1C).
Phytotoxicity of As (V) The relative increase in FW of gametophytes exposed to different concentrations of Na3AsO4 is shown in Figure 2A. The relative increase in FW for all treatments decreased significantly with an increase in culture time. The relative increase in FW of cultures exposed to either 1.0 or 2.0 mM Na3AsO4 was much lower than that for other concentrations over a 40-d culture period (Po0.05). The relative increase in FW was much lower in cultures exposed to 2 mM Na3AsO4 than that observed for cultures exposed to 1 mM or control (Po0.05). The increase in FW was significantly higher for cultures exposed to either 0.25 or 0.5 mM Na3AsO4 than that observed for any of the other concentrations tested (Figure 2B). No growth retardation or visible symptoms of phytotoxicity were seen within 40 d in gametophytes exposed to less than 1.0 mM Na3AsO4, but appearing during the next 10 d of exposure. In contrast, growth retardation and visible symptoms of phytotoxicity appeared between 20 and 30 d in cultures exposed to 2.0 mM Na3AsO4. The average total FW of gametophytes at the end of the culture period and exposed to control, 0.25 mM, 0.5 mM, 1.0 mM, or 2.0 mM Na3AsO4 was 5.86, 10.9, 8.33, 5.68, and 1.24 g, respectively. The growth of gametophytes was significantly inhibited only when more than 2.0 mM Na3AsO4 was added to the MS medium. Growth was inhibited by more than 20% when gametophytes were grown with 2.0 mM Na3AsO4 compared to the growth observed in the absence of Na3AsO4. The amount of chl present in P. vittata gametophytes grown on solid MS medium plus control, 0.25, 0.5, 1.0, or 2.0 mM Na3AsO4 is shown in Figure 2C. The chl concentration increased slightly in gametophytes exposed to 0.25 mM Na3AsO4 (Po0.05); however, cultures exposed to 0.5, 1.0, and 2.0 mM Na3AsO4 had chl concentrations that were significantly higher than those observed upon exposure to control (Po0.05). And the carotenoids concentration increased significantly with the As level increasing from 0 to 2.0 mM (Figure 2C).
Effect of As(V) status on Cu2+ tolerance of P. vittata gametophytes When gametophytes were exposed to different concentrations of As in the presence of Cu, the relative increase in FW was much higher at a
Figure 2. Effects of Na3AsO4 on the growth and chlorophyll concentration of P. vittata gametophytes. (A) Changes in FW of gametophytes under Na3AsO4 stress. (B) Time course changes in FW of gametophytes exposed to different concentrations of Na3AsO4. (C) Changes in chlorophyll and carotenoids concentration of gametophytes under Na3AsO4 stress. Each value is the mean of three individual replicates (7SD). Different lowercase letters indicate significant differences (po0.05).
ARTICLE IN PRESS Arsenate-reduced copper toxicity in fern concentration of 0.25 mM Na3AsO4 or CuSO4 than control; there were no significant differences in the concentration of chl. In contrast, cultures exposed to 1.0 mM Na3AsO4 or CuSO4 demonstrated a significantly inhibited relative increase in FW compared to control. The time course of the relative increase in gametophyte FW and concentration of chl upon exposure to 0.25 and 1.0 mM of Na3AsO4, and CuSO4 in various combinations is shown in Figure 3A and B. The relative increase in gametophyte FW was significantly greater with exposure to 0.25 mM Na3AsO4 and 0.25 mM CuSO4 than with other treatments. When gametophytes were cultured in the presence of 1.0 mM CuSO4+0.25 mM Na3AsO4, the relative increase in FW was much greater than that observed in cultures exposed to 1.0 mM CuSO4 alone (Po0.05). Conversely, cultures exposed to 1.0 mM Na3AsO4+0.25 mM CuSO4 had a much lower relative increase in FW than cultures grown in the presence of 1.0 mM Na3AsO4 alone (Figure 3A). Although gametophytes grown in 1.0 mM CuSO4 and 1.0 mM CuSO4+0.25 mM Na3AsO4 showed no indication of growth retardation after 40 d, visible symptoms of phytotoxicity were observed at 40 and 20 d, respectively, when exposed to 1.0 mM CuSO4+0.25 mM Na3AsO4 and 1.0 mM CuSO4. In contrast, P. vittata gametophytes grown in the presence of 1.0 mM Na3AsO4 and 1.0 mM Na3AsO4+0.25 mM CuSO4 exhibited both growth retardation and visible symptoms of phytotoxicity after 40 and 20 d, respectively (Figure 3B). The average total FW of gametophytes at the end of the culture period in the presence of 1.0 mM Na3AsO4, 1.0 mM Na3AsO4+0.25 mM CuSO4, 1.0 mM CuSO4, and 1.0 mM CuSO4+0.25 mM Na3AsO4 was 4.77, 3.70, 2.91, and 7.94 g, respectively. In P. vittata gametophytes grown on solid MS medium containing 1.0 mM CuSO4+0.25 mM Na3AsO4, the chl and carotenoids concentration increased significantly (Po0.05) compared to those grown with 1.0 mM CuSO4 alone (Figure 3C). However, the chl and carotenoids concentration in cultures grown in the presence of 1.0 mM Na3AsO4+0.25 mM CuSO4 decreased significantly compared to those grown with 1.0 mM Na3AsO4 or 0.25 mM CuSO4 alone.
Impact of As and Cu on cell viability and cell membrane permeability Stress from reactive oxygen species (ROS) can lead to cell membrane damage and cell death (Chattopadhyay et al., 2002; Lynn et al., 2000). A widely used method for detection of membrane
1911
Figure 3. Effects of Na3AsO4+CuSO4 on the growth and chlorophyll concentration of P. vittata gametophytes exposed to different combinations of Na3AsO4+CuSO4 concentrations. (A) Changes in FW of gametophytes under combined Na3AsO4+CuSO4 stress. (B) Time course of changes in FW of gametophytes exposed to different combinations of Na3AsO4+CuSO4 concentrations. (C) Changes in chlorophyll and carotenoids concentration of gametophytes under combined Na3AsO4+CuSO4 stress. Each value is the mean of three individual replicates (7SD). Different lowercase letters indicate significant differences (po0.05).
ARTICLE IN PRESS 1912 integrity (electrolyte leakage) and cell death (Evans blue staining) (Mur et al., 2005) was used to assess changes in the CMP and CV of P. vittata gametophytes upon exposure to As and Cu, and effects on CMP and CV could be a subsection of phytotoxic effects. The effect of As and Cu on CV and CMP of As hyperaccumulator P. vittata gametophytes is shown in Figure 4A and B, respectively. Upon exposure to 0.25 mM Na3AsO4 in 1.0 mM CuSO4, gametophytes were visibly chlorotic and/or dead, CV was significantly increased, and CMP significantly decreased compared to cultures exposed to 1.0 mM CuSO4 alone.
Figure 4. Effects of different combinations of Na3AsO4+CuSO4 on cell viability (CV) and cell membrane permeability (CMP) of P. vittata gametophytes. CV was determined by monitoring the uptake of Evans blue by P. vittata gametophytes exposed to different combinations of As+Cu. The relative increase in cell death was estimated by spectrophotometric monitoring of Evans blue retention. (A) Effects of different combinations of As+Cu on CV. (B) Effects of different combinations of As+Cu on CMP. Each value is the mean of three individual replicates (7SD). Different lowercase letters indicate significant differences (po0.05).
Y. Zheng et al.
Influence of concomitant exposure to CuSO4 and Na3AsO4 on Cu and As concentrations in P. vittata gametophytes The effect of concomitant exposure of P. vittata gametophytes to CuSO4 and Na3AsO4 on Cu concentration is shown in Figure 5A. When cultures were provided with 1.0 mM CuSO4 in the medium, the Cu concentration in the gametophytes was 2730 mg kg–1 dry weight (DW). Upon exposure to 1.0 mM CuSO4+0.25 mM Na3AsO4, however, the Cu concentration in the gametophytes decreased significantly to 2350 mg kg–1 DW. Gametophytes cultured in 1.0 mM Na3AsO4 had As concentrations of 526.9 mg kg–1 DW. Those cultured in 1.0 mM Na3AsO4+0.25 mM CuSO4 showed significantly decreased As concentrations of 323.8 mg kg–1 DW (Figure 5B).
Figure 5. Effects of combined Na3AsO4+CuSO4 on accumulation of As and Cu in P. vittata gametophytes. (A) Cu concentration in gametophytes exposed to different Cu+As concentrations in the culture medium. (B) As concentration in gametophytes exposed to different Cu+As concentrations in the culture medium. Each value is the mean of three individual replicates (7SD). Different lowercase letters indicate significant differences (po0.05).
ARTICLE IN PRESS Arsenate-reduced copper toxicity in fern
As status and impact on Cu distribution in gametophyte subcellular fractions Copper distribution in the subcellular fraction of P. vittata gametophytes grown under different Cu2+ and As (V) treatments is shown in Table 1. The Cu concentration in the cell wall fraction (F1) and soluble fraction (F3) decreased significantly (Po0.05) with exposure to 1.0 mM Cu2++0.25 mM Na3AsO4. The Cu concentration in the organelle fraction (F2) was not significantly decreased. In addition, the percentage of Cu distributed in each subcellular fraction did not change significantly. The Cu distribution percentages in F1, F2, and F3 were 72.16%, 11.29%, and 16.55%, respectively, when the culture medium contained 1.0 mM CuSO4. Thus, Cu in P. vittata gametophytes was primarily sequestered in the cell wall layer.
Cu status and impact on As distribution in gametophyte subcellular fractions Arsenic distribution in the subcellular fraction of P. vittata gametophytes grown in the presence of varying As concentrations is shown in Table 2. The amount of As in F1 decreased significantly with exposure to 1.0 mM As(V)+0.25 mM CuSO4. The concentration of As in F2 and F3 did not change significantly. The percentage distribution of As in Table 1. and As
1913 all subcellular fractions was unchanged. The percentage distribution of As in F1, F2, and F3 was 23.08%, 13.27%, and 63.65%, respectively, when gametophytes were cultured in the presence of 1.0 mM As. Therefore, As in P. vittata gametophytes was located mainly in the cytoplasmic supernatant fraction (including vacuoles), followed by the cell wall layer, and least of all in the organelles.
Discussion Plants from metalliferous soils often exhibit tolerance to different HMs. This tolerance may be due either to a combination of different metalspecific tolerance mechanisms (multiple tolerance) or less-specific mechanisms that confer pleiotropic tolerance to different metals (co-tolerance) (Gregory and Bradshaw, 1965; Schat and Ten Bookum, 1992). In this study we found that cultured gametophytes of the As hyperaccumulator fern P. vittata can tolerate higher concentrations of Na3AsO4 (1.0 mM) than CuSO4 (0.5 mM; Figures 1 and 2) as assessed by changes in FW and chl concentration. The FW of gametophytes declined significantly and symptoms of chlorosis began to appear when the culture medium contained over 1.0 mM Na3AsO4 or
Cu distribution in subcellular fractions of P. vittata gametophytes exposed to different combinations of Cu
Treatment (mM)
As 1.0 Cu 1.0 Cu 1.0+As 0.25 As 1.0+Cu 0.25
Cu concentration (mg/kg FW)
Percentage distribution
F1
F2
F3
F1
F2
F3
5.20d 656.66a 386.4b 113.79c
5.05d 102.74a 90.77ab 69.24c
3.82d 150.61a 106.17b 90.31bc
31.63c 72.16a 66.24ab 41.63c
30.76a 11.29c 15.56c 25.33ab
23.23b 16.55c 18.20c 33.04a
Note: Lowercase letters indicate values that differ significantly among cell fractions (po0.05). F1, cell wall fraction; F2, organelle fraction; F3, soluble fraction.
Table 2. and As
As distribution in subcellular fractions of P. vittata gametophytes exposed to different combinations of Cu
Treatment (mM)
As 1.0 Cu 1.0+As 0.25 As 1.0+Cu 0.25
As concentration (mg/kg FW)
Percentage distribution
F1
F2
F3
F1
F2
F3
40.54a 27.89b 31.09b
23.31a 9.94b 18.83a
111.79a 61.17c 92.76ab
23.08a 28.17b 22.01a
13.27a 10.04a 13.33a
63.65a 61.79a 65.66a
Note: Lowercase letters indicate values that differ significantly among cell fractions (po0.05). F1, cell wall fraction; F2, organelle fraction; F3, soluble fraction.
ARTICLE IN PRESS 1914 0.5 mM CuSO4. The concentration of chl in gametophytes cultured with 2.0 mM Na3AsO4 was higher than in control plants. This is due to the fact that although the concentration of chla (Ca) and chlb (Cb) decreased slightly (P40.05), the concentration of co-extracted carotenoids (Cc) increased significantly upon exposure to 2.0 mM Na3AsO4 compared to control (0 mM Na3AsO4) (Figure 2C). However, the concentration of chl in gametophytes cultured with 0.5 mM CuSO4 decreased significantly compared to control, suggesting that Cu is more phytotoxic than As to P. vittata gametophytes. This may be due to the fact that Cu can be transported to the cytoplasm and then to the chloroplasts where it inhibits photosynthesis by uncoupling electron transport to NADP (Femandes and Henriques, 1991; Sandmann and Boger, 1980). Cu is bound to various cellular proteins including some of those found in the chloroplast membranes resulting in an increase in ion concentration that leads to degradation of chl and other pigments, preventing photosynthesis and eventually causing cell death (Barn et al., 1995). Previous studies have shown that As is toxic to most plants by interfering with the physiological functions normally performed by phosphorus (P) (Meharg and Macnair, 1990, 1991). However, the results presented here indicate that low concentrations of As (0.5 mM Na3AsO4) can actually stimulate P. vittata gametophyte growth (FW and DW). This observation is in agreement with the results of Tu and Ma (2005) with regard to P. vittata sporophyte growth and preferential As accumulation in young fronds of up to 50 mg As kg–1 DW, suggesting that As may act as a nutrient similar to phosphorus in P. vittata. Interactive effects (synergism or antagonism) among different HMs are well documented. Landberg and Greger (1994) found that selenium does not reduce the toxicity of HMs to plants, but instead enhances metal uptake and toxicity. Here we found that As can reduce the phytotoxicity of Cu in P. vittata gametophytes. Moreover, Cu does not reduce the toxicity of As to P. vittata gametophytes, in contrast to results reported for soybean (Wang et al., 2001). Analogously to the P. vittata sporophyte, it is possible that As reduces the toxicity of Cu in P. vittata gametophytes because at low levels of As exposure, enzymatic antioxidants (superoxide dismutase, CAT, ascorbate POD, guaiacol POD) are important for As detoxification and accumulation, while non-enzymatic antioxidants (glutathione, acidsoluble thiol) are more important at high As levels. More specifically, the concentration of non-enzymatic antioxidants increases with soil As concentrations, resulting in greater accumulation in the fronds than in the roots especially when exposed to high As
Y. Zheng et al. concentrations (450 mg kg1). Activity of enzymatic antioxidants in P. vittata sporophyte increased until soil As concentrations reached mg kg1 levels, and then decreased (Cao et al., 2004). The work described here demonstrates that appropriate As concentrations (o1.0 mM of Na3AsO4) reduce CMP, thereby inhibiting Cu2+ entry and altering the distribution of Cu in subcellular compartments by increasing resistance to ROS. Additionally, exposure of cultured gametophytes to 1.0 mM CuSO4+0.25 mM Na3AsO4 together not only significantly decreased chlorosis and death compared to those cultured in the presence of 1.0 mM CuSO4 alone, it also significantly increased CV and reduced CMP. Interestingly, while Cu can also inhibit As entry, it does not reduce the phytotoxicity of As. When cultures were exposed to 1.0 mM Na3AsO4+0.25 mM CuSO4, chlorosis and death were not significantly reduced, CV decreased significantly, and CMP increased significantly. These results suggest a unique mechanism of HM tolerance in cultured P. vittata gametophytes. Sporophytes and gametophytes of P. vittata may have evolved a set of analogous physiological and molecular mechanisms specifically for the escape of As toxicity. Subcellular As was primarily distributed in the soluble fraction (cytoplasm including vacuoles), while Cu was mainly located in cell walls, demonstrating that this organism avoids Cu absorption as much as possible. However, gametophytes of P. vittata transported As from the cell wall to the cytoplasm or vacuole. Although little is known about whether phosphate transporters can be charged for the absorption of As, P. vittata did transport excess As (over 100 mg kg1 FW) to the cytoplasm or vacuole of gametophyte, and adopting a dynamic equilibrium may be important for the prevention of HM toxicity. In summary, this study provides novel findings that As can stimulate growth and reduce Cu phytotoxicity in gametophytes of P. vittata, a plant species that shows high tolerance for As, Cu, and Zn and is an As hyperaccumulator. This process may have evolved as a biological mechanism allowing the fern to adapt to co-contamination with As and Cu. Additionally, we have shown that P. vittata gametophytes are ideal candidates for studying the physiological and biochemical mechanisms of As hyperaccumulation.
Acknowledgments This work was supported by the State High-tech Project (2007AA021404, 2007AA091704) and the
ARTICLE IN PRESS Arsenate-reduced copper toxicity in fern National Natural Science Foundation of China (30670171, 30500307).
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