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The effects of Pb chelater (EDTA-Pb) and ionic Pb (Pb(NO(3))(2)) on root cell death, Pb accumulation, changes of ROS, activities of antioxidant enzymes and uptake of mineral elements in response to Pb toxicity in Sedum alfredii H. were compared. Loss of plasma membrane integrity became serious by increasing Pb concentration in the medium, 200 microM Pb + 200 microM EDTA has alleviated the root cell death. The biomass was significantly affected by high concentration of Pb, and root growth was also affected by EDTA-Pb compared with ionic Pb. Lead accumulation was higher in the samples treated with ionic lead than that of the control. The concentration of reactive oxygen species (ROS) was determined by fluorescence microscopy, which indicates that the Pb stress increased the content of ROS significantly, whereas the EDTA-Pb decreased the burst of H(2)O(2). High Pb concentrations increase the activity of SOD and LOX. The Cu concentration in root increased significantly under Pb and EDTA-Pb treatment, and 200 microM Pb markedly increased the Fe content in roots. Under ionic Pb condition, the contents of Mg, Ca and K in shoots decreased, whereas they were significantly increased in case of EDTA-Pb. These results suggested that accumulating ecotype of S. alfredii roots were inefficient in uptake of higher concentration of EDTA-chelated Pb for long treatment duration, and that lead toxicity could be alleviated by EDTA.

Plant growth, ultrastructural and antioxidant adaptations and glutathione biosynthesis in Cd-hyperaccumulating ecotype Sedum alfredii Hance (HE) countering high Cd environment were investigated and compared with its non Cd-hyperaccumulating ecotype (NHE). Cadmium exposure resulted in significant ultrastructural changes in root meristem and leaf mesophyll cells of S. alfredii, but damage was more pronounced in NHE even when Cd concentrations were one-tenth of those applied to HE. Cadmium stress damaged chloroplasts causing imbalanced lamellae formation coupled with early leaf senescence. Histochemical results revealed that glutathione (GSH) biosynthesis inhibition led to overproduction of hydrogen peroxide (H(2)O(2)) and superoxide radical (O(2)(*-)) in HE but not in NHE. Differences were noted in both HE and NHE for catalase (CAT), guaiacol peroxidase (GPX), ascorbate peroxidase (APX) and glutathione reductase (GR) activities under various Cd stress levels. No relationship was found between antioxidative defense capacity including activities of superoxide dismutase (SOD), CAT, GPX, APX and GR as well as ascorbic acid (AsA) contents and Cd tolerance in the two ecotypes of S. alfredii. The GSH biosynthesis induction in root and shoot exposed to elevated Cd conditions may be involved in Cd tolerance and hyperaccumulation in HE of S. alfredii H.

Hydroponics studies were conducted to investigate the antioxidant adaptations, ascorbate and glutathione metabolism in hyperaccumulating ecotype of Sedum alfredii (HE) exposed to high Cd environment, when compared with its nonhyperaccumulating ecotype (NHE). Exposure to Cd induced a burst of oxidative stress in both ecotypes which was evident by the sharp increase in hydrogen peroxide (H(2)O(2)) contents and lipid peroxidation. Buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, did not affect H(2)O(2) concentrations as well as growth of both ecotypes in the absence of Cd. However, compared with Cd application alone, BSO combined with Cd treatment caused a substantial augmentation of H(2)O(2) accumulation accompanied by a reduction in Cd concentrations in roots and leaves of HE at the end of treatment, which may rule out the possibility that GSH biosynthesis may play an important role as a signal of the stress regulation. No efficient and superior enzymatic antioxidant defense mechanisms against Cd-imposed oxidative stress existed in both NHE and HE, but the essential nonenzymatic components like ascorbic acid (AsA) and GSH played a prominent role in tolerance against Cd. Cadmium stimulated a notable rise in AsA concentration in both ecotypes soon after the application of treatment. A preferential Cd-stress response in HE was suggested to changes in the GSH pool, where acclimation was marked by increased GSH concentrations.

Lab scale and pot experiments were conducted to compare the effects of synthetic chelators and low molecular weight organic acids (LMWOA) on the phytoextraction of multi-contaminated soils by two ecotypes of Sedum alfredii Hance. Through lab scale experiments, the treatment dosage of 5 and 10 mM for synthetic chelators and LMWOA, respectively, and the treatment time of 10 days were selected for pot experiment. In pot experiment, the hyperaccumulating ecotype (HE) was found more tolerant to the metal toxicity compared with the non-hyperaccumulating ecotype (NHE). EDTA for Pb, EDDS for Cu, and DTPA for Cu and Cd were found more effective to enhance heavy metal accumulation in the shoots of S. alfredii Hance. Compared with synthetic chelators, the phytoextraction ability of LMWOA was lesser. Considering the strong post-harvest effects of synthetic chelators, it is suggested that higher dosage of LMWOA could be practiced during phytoextraction, and some additional measures could also be taken to lower the potential environmental risks of synthetic chelators in the future studies.

A hydroponic study was conducted to investigate the biomass, root morphology, and zinc (Zn), calcium (Ca) and sulfur (S) contents of two Sedum alfredii ecotypes under effects of different concentration calcium (Ca2+) addition. The results showed that with increasing exogenous Ca2+ concentration, the dry mass of the two S. alfredii ecotypes increased, and the shoot dry mass of hyperaccumulation ecotype increased significantly (P <0.05). The root length and root surface area of hyperaccumulation ecotype increased with increasing exogenous Ca2+ concentration, while those of non-hyperaccumulation ecotype were in adverse. The Zn accumulation in the root, stem and leaf of hyperaccumulation ecotype was increased with increasing exogenous Ca2+ concentration though the differences among Ca2+ treatments were not significant (P >0.05), while the Zn accumulation in the shoot of non-hyperaccumulation ecotype was significantly low (P <0.05). The Ca accumulation in the root, stem and leaf of non-hyperaccumulation ecotype had a significant positive correlation with the concentration of exogenous Ca2+ (P <0.05), so as the S accumulation in the root of hyperaccumulation ecotype (P <0.01). Exogenous Ca2+ promoted the Zn absorption and accumulation of hyperaccumulation ecotype, while inhibited the Zn accumulation of non-hyperaccumulation ecotype. Appropriate concentration of exogenous Ca2+ could promote the growth of hyperaccumulation ecotype S. alfredii, and improve its ability of accumulating more zinc.

Sedum alfredii Hance has been identified as zinc (Zn) and cadmium (Cd) co-hyperaccumulator. In this paper the relationships of Zn or Cd hyperaccumulation to the generation and the role of H2O2 in Sedum alfredii H. were examined. The results show that Zn and Cd contents in the shoots of Sedum alfredii H. treated with 1000 micromol/L Zn2+ and/or 200 micromol/L Cd2+ increased linearly within 15 d. Contents of total S, glutathione (GSH) and H2O2 in shoots also increased within 15 d, and then decreased. Total S and GSH contents in shoots were higher under Cd2+ treatment than under Zn2+ treatment. However, reverse trends of H2O2 content in shoots were obtained, in which much higher H2O2 content was observed in Zn2+-treated shoots than in Cd2+-treated shoots. Similarly, the microscopic imaging of H2O2 accumulation in leaves using H2O2 probe technique showed that much higher H2O2 accumulation was observed in the Zn2+-treated leaf than in the Cd2+-treated one. These results suggest that there are different responses in the generation of H2O2 upon exposure to Zn2+ and Cd2+ for the hyperaccumulator Sedum alfredii H. And this is the first report that the generation of H2O2 may play an important role in Zn hyperaccumulation in the leaves. Our results also imply that GSH may play an important role in the detoxification of dissociated Zn/Cd and the generation of H2O2.

The phytotoxicity and antioxidative adaptations of lead (Pb) accumulating ecotype (AE) and non-accumulating ecotype (NAE) of Sedum alfredii Hance were investigated under different Pb treatments involving 0, 0.02 mmol/L Pb, 0.1 mmol/L Pb and 0.1 mmol/L Pb/0.1 mmol/L ethylenediaminetetraacetic acid (EDTA) for 6 days. With the increasing Pb level, the Pb concentration in the shoots of AE plants enhanced accordingly, and EDTA supply helped 51% of Pb translocation to shoots of AE compared with those treated with 0.1 mmol/L Pb alone. Moreover, the presence of EDTA alleviated Pb phytotoxicity through changes in plant biomass, root morphology and chlorophyll contents. Lead toxicity induced hydrogen peroxide (H2O2) accumulation and lipid peroxidation in both ecotypes of S. alfredii. The activities of superoxide dismutase (SOD), guaiacol peroxidase (G-POD), ascorbate peroxidase, and dehydroascorbate reductase elevated in both leaves and roots of AE as well as in leaves of NAE with the increasing Pb levels, but SOD and G-POD declined in roots of NAE. Enhancement in glutathione reductase activity was only detected in roots of NAE while a depression in catalase activity was recorded in the leaves of NAE. A significant enhancement in glutathione and ascorbic acid (AsA)levels occurred in both ecotypes exposed to Pb and Pb/EDTA treatment compared with the control, however, the differences between these two treatments were insignificant. The dehydroascorbate (DHA) contents in roots of both ecotypes were 1.41 to 11.22-fold higher than those in leaves, whereas the ratios of AsA to DHA (1.38 to 6.84) in leaves altering more to the reduced AsA form were much higher than those in roots. These results suggested that antioxidative enzymes and antioxidants play an important role in counteracting Pb stress in S. alfredii.

Phytochelatins (PCs) are known to play an essential role in the heavy metal detoxification of some higher plants and fungi by chelating heavy metals. However, three recent papers reported that no PCs could be detected in the hyperaccumulator Sedum alfredii Hance upon cadmium, lead or zinc treatment, respectively. In this paper, PC synthesis was assayed again in the mine population of S. alfredii with the help of reversed phase high-performance liquid chromatography (HPLC), HPLC-mass spectrometry, and HPLC-tandem mass spectrometry. Our data showed that PC formation could be induced in the leaf, stem and root tissues of S. alfredii upon exposure to 400 microM cadmium, and only in the stem and root when exposed to 700 microM lead. However, no PCs were found in any part of S. alfredii when it was subjected to exposure to 1600 microM zinc.

This study was the first attempt to examine the possible role of the naturally occurring rhizospheric bacteria in heavy metal removal by Sedum alfredii Hance, a terrestrial Zn/Cd hyperaccumuluator, from Zn, Cd, Cu and Pb contaminated water using antibiotic ampicillin. Moreover, the toxicity symptom in plants under heavy metal stress expressed as total chlorophyll, chlorophyll a and b content, growth inhibition, root length, and N, P contents were studied, and the possible relationship among them were also discussed. These results indicate that rhizospheric bacteria may play an important role in the uptake of N and P by S. alfredii, and consequently result in the increase of Chlorophyll content in the leaves and plant biomass due to improved photosynthesis. At the same time, root length significantly decreased under the treatment with ampicillin, which suggested that rhizospheric bacteria appeared to protect the roots against heavy metal toxicity. The Pb, Zn, Cu and Cd concentrations in the roots, stems and leaves of S. alfredii were much higher than those exposed to ampicillin. Accordingly, metal concentrations in the contaminated water without ampicillin treatment were lower than those treated with ampicillin. These results suggest that the rhizospheric bacteria may be useful in plant tolerance to heavy metal toxicity, and also accelerate the metal removal from contaminated water.

Sedum alfredii has been reported to be a cadmium (Cd) hyperaccumulator. Phytochelatins (PCs) and other thiol (SH)-containing compounds have been proposed to play an important role in the detoxification and tolerance of some heavy metals, but it is not clear whether PCs are responsible for Cd hyperaccumulation and tolerance in S. alfredii. In this study, two geographically isolated populations of S. alfredii were studied: one population grew on an old Pb/Zn mine site, while the other on a non-mine site. The mine population of this species exhibited a stronger heavy metal tolerance than in the other population. Root-to-shoot transport of Cd was higher in population located at the mine site than at the non-mine site. Considerable amounts of Cd were accumulated in leaves and stems of mine plants, while most Cd was distributed in roots of non-mine plants. Non-protein SH in plant tissues of two populations were further investigated by a HPLC pre-column derivatization system. Upon exposure to Cd, no PCs were detected in all tissues of mine population, while an appreciable amount of glutathione (GSH) was observed in the descending order of stem>root>leaf. The concentrations of GSH consistently increased with the increase of exogenous Cd concentrations and time. On the contrary, Cd exposure strongly induced the production of PCs (mainly PC(2) and PC(3)) and GSH in plant tissues of non-mine population, and the concentrations of GSH showed an initial drop over the duration of 7-d exposure. The present results provided strong evidence that PCs are not involved in Cd transport, hyperaccumulation and tolerance in mine population of S. alfredii.

Hydroponics and pot experiments were conducted to study the effects of ethylenediaminetetraacetic acid (EDTA) on Pb transportation and accumulation by two contrasting ecotypes of Sedum alfredii Hance. In hydroponics experiments, the accumulating ecotype (AE) showed more ability to tolerate Pb toxicity compared with the non-accumulating ecotype (NAE). When treated with equimolar mixtures of EDTA and Pb, maximum Pb accumulation occurred without any phytotoxicity symptoms. Pot experiments with Pb contents of 400 mg kg-1 showed that 5 mM EDTA is the optimum dose for the phytoextraction of soils contaminated with relatively low Pb levels; in contrast, increasing EDTA addition resulted in increased Pb accumulation in the shoots of AE in soils with high Pb content (1200 mg kg(-1)). The post-harvest effects of EDTA on available Pb were strong compared with those without addition of EDTA (CK). Within the initial 7 days almost no differences of water-soluble Pb were noted in soils contaminated with both levels of Pb but after 2 weeks, water-soluble Pb started to decrease significantly compared with before. Considering the toxicity and biodegradability of synthetic chelators, it can be concluded that the chelate-assisted technique is more suitable for soils contaminated with low Pb levels and to avoid environment risks; a suitable dose of chelators must be considered before application.

To investigate the variation of Zn and Cd accumulation and tolerance of Sedum alfredii (a newly reported Zn/Cd hyperaccumulator), field surveys and hydroponic experiments were conducted among three populations of this species: two originating from old Pb/Zn mines in Zhejiang (ZJ) and Hunan (HN) Provinces and one from a "clean" site in Guangdong (GD) Province, China. Under field conditions, up to 12,524 and 12,253 mg kg(-1) Zn, and 1400 and 97 mg kg(-1) Cd in shoots of ZJ and HN plants were recorded respectively. Under hydroponic conditions, ZJ and HN plants accumulated significantly higher Zn and Cd in their leaves and stems, and possessed significantly higher Zn and Cd tolerance than GD plants. Among the two contaminated populations, ZJ plants showed higher Cd tolerance and accumulation (in leaves) than HN plants. The present results indicate that significant differences in Zn and Cd accumulation and tolerance exist in populations of S. alfredii.

Sedum alfredii Hance, a newly discovered hyperaccumulator, could serve as a good material for phytoremediation of Cd polluted sites. Malondialdehyde (MDA), reactive oxygen species (ROS) and antioxidases (catalase (CAT); superoxide dismutase (SOD); peroxidase (POD)) in the leaf were determined when S. alfredii was treated for 15 d with various CdCl2 concentrations ranging from 0 to 800 micromol/L. The results showed that the production rate of 2',7'-dichlorofluorescein (DCF), which is an indicator of ROS level, reached up to the maximum at 400 micromol/L CdCl2 and then declined with the increase of CdCl2 concentration, while MDA accumulation tended to increase. CAT activity was significantly inhibited at all tested CdCl2 concentrations and SOD activity was sharply suppressed at 800 micromol/L CdCl2. However, the enhancement of POD activity was observed when CdCl2 concentration was higher than 400 micromol/L. In addition, its activity increased when treated with 600 micromol/L CdCl2 for more than 5 d. When sodium benzoate, a free radical scavenger, was added, S. alfredii was a little more sensitive to Cd toxicity than that exposed to Cd alone, and the Cd accumulation tended to decline with the increase of sodium benzoate concentration. It came to the conclusions that POD played an important role during Cd hyperaccumulation, and the accumulation of ROS induced by Cd treatment might be involved in Cd hyperaccumulation.

Sedum alfredii Hance can accumulate Zn in shoots over 2%. Leaf and stem Zn concentrations of the hyperaccumulating ecotype (HE) were 24- and 28-fold higher, respectively, than those of the nonhyperaccumulating ecotype (NHE), whereas 1.4-fold more Zn was accumulated in the roots of the NHE. Approximately 2.7-fold more Zn was stored in the root vacuoles of the NHE, and thus became unavailable for loading into the xylem and subsequent translocation to shoot. Long-term efflux of absorbed 65Zn indicated that 65Zn activity was 6.8-fold higher in shoots but 3.7-fold lower in roots of the HE. At lower Zn levels (10 and 100 microM), there were no significant differences in 65Zn uptake by leaf sections and intact leaf protoplasts between the two ecotypes except that 1.5-fold more 65Zn was accumulated in leaf sections of the HE than in those of the NHE after exposure to 100 microM for 48 h. At 1,000 microM Zn, however, approximately 2.1-fold more Zn was taken up by the HE leaf sections and 1.5-fold more 65Zn taken up by the HE protoplasts as compared to the NHE at exposure times >16 h and >10 min, respectively. Treatments with carbonyl cyanide m-chlorophenylhydrazone (CCCP) or ruptured protoplasts strongly inhibited 65Zn uptake into leaf protoplasts for both ecotypes. Citric acid and Val concentrations in leaves and stems significantly increased for the HE, but decreased or had minimal changes for the NHE in response to raised Zn levels. These results indicate that altered Zn transport across tonoplast in the root and stimulated Zn uptake in the leaf cells are the major mechanisms involved in the strong Zn hyperaccumulation observed in S. alfredii H.

Chelant-enhanced phytoextraction is one of the most promising technologies to remove heavy metals from soil. The key of the technology is to choose suitable additives in combination with a suitable plant. In the present study, laboratory batch experiment of metal solubilization, cress seeds germination were undertaken to investigate the metal-mobilizing capability and the phytotoxicity of organic additives, including ethylene diamine triacetic acid (EDTA), citric acid, acetic acid, oxalic acid, glutamine and monosodium glutamate waste liquid (MGWL) from food industry. Experiments in pots were carried out to study the effects of the additives on Zn and Cd phytoextraction. Furthermore, a leaching experiment with lysimeter was performed to evaluate the environmental risks of additive-induced leaching to underground water. The results showed that EDTA had a strong mobilizing ability for Zn and Cd, followed by mixed reagent (MR) and MGWL. MGWL and acetic acid at 5 mmol equivalent per liter resulted in seed germination index less than 2%. Experiments in pots verified the phytotoxicity of acetic acid and MGWL. Addition of the mixed reagent at 6-10 mmol/kg significantly increased Zn phytoextraction by Thlaspi caerulescens. The same for EDTA and the mixed reagent at 10 mmol/kg by Sedum alfredii. But only mixed reagents could significantly increase Cd phytoextraction by the studied hyperaccumulators. This suggested that the strong chelant was not always the good agent to enhance phytoextraction. S. alfredii combined with 2-10 mmol/kg soil MR was preferred for phytoremediation of Cd/Zn contaminated soils in southern China, this could result in high phytoextraction of Cd/Zn and reduce the leaching risk to underground water than EDTA assisted phytoextration.

A hypothetical adaptive response of succulent plants to drought-stress is the redistribution of water from old to young leaves. We examined the effects of possible movement of water from old to young leaves in three succulent species, Carpobrotus edulis (weak CAM-inducible), Kalanchoe tubiflora (CAM) and Sedum spectabile (possibly a CAM-cycler or CAM-inducible). Old leaves were removed from plants, and photosynthesis, transpiration, f. wt : d. wt ratios, diurnal acid fluctuations, stomatal conductance and internal CO2 concentrations of the remaining young leaves were measured during drought-stress. Comparison was made with plants retaining old leaves. There was no evidence that water moved from old to young leaves during drought-stress as previously hypothesized. Only in drought-stressed plants of K. tubiflora, were photosynthetic and transpiration rates of young leaves greater on shoots with old leaves removed compared with attached. There was a trend in all species for greater fluctuations in acidity in young leaves on shoots that lacked older leaves. For two of the three species studied, the f. wt : d. wt ratios of young leaves were greater under drought-stress, on shoots with old leaves removed than with them attached. Absence of old leaves may reduce competition for water with young leaves, which consequently have higher water content and greater photosynthetic rates.