Stem and leaf sequestration of zinc at the cellular level in the hyperaccumulator Sedum alfredii.

* Sedum alfredii is a fast-growing, high-biomass zinc (Zn) hyperaccumulator native to China. Here, the characteristics of in vivo Zn distribution in stems and leaves of the hyperaccumulating (HE) and nonhyperaccumulating ecotypes (NHE) of S. alfredii were investigated by synchrotron radiation X-ray fluorescence (SRXRF) analysis, together with a Zn probe. * Preferential Zn accumulation in leaf and stem epidermis was observed in both ecotypes, but to a much greater extent for HE. Epidermal Zn increased largely in leaves and stems of HE as exposure time was prolonged, while Zn saturation occurred relatively early in HE leaf mesophyll cells and stem vascular bundles. A second peak of Zn enrichment in stem and leaf vascular systems was shown in both ecotypes. However, the proportion of Zn accumulated in stem vascular bundles relative to other tissues was much greater for HE than for NHE. * Leaf and stem distribution patterns of phosphorus (P) and sulphur (S) in the HE were very like that for Zn, while the calcium (Ca) distribution pattern was the reverse of that for Zn. No such relationship was observed in NHE. * Our study mainly suggested that epidermal layers serve as important storage sites for accumulated Zn in the S. alfredii HE.

Enhanced root-to-shoot translocation of cadmium in the hyperaccumulating ecotype of Sedum alfredii.

Sedum alfredii (Crasulaceae) is the only known Cd-hyperaccumulating species that are not in the Brassica family; the mechanism of Cd hyperaccumulation in this plant is, however, little understood. Here, a combination of radioactive techniques, metabolic inhibitors, and fluorescence imaging was used to contrast Cd uptake and translocation between a hyperaccumulating ecotype (HE) and a non-hyperaccumulating ecotype (NHE) of S. alfredii. The K(m) of (109)Cd influx into roots was similar in both ecotypes, while the V(max) was 2-fold higher in the HE. Significant inhibition of Cd uptake by low temperature or metabolic inhibitors was observed in the HE, whereas the effect was less pronounced in the NHE. (109)Cd influx into roots was also significantly decreased by high Ca in both ecotypes. The rate of root-to-shoot translocation of (109)Cd in the HE was >10 times higher when compared with the NHE, and shoots of the HE accumulated dramatically higher (109)Cd concentrations those of the NHE. The addition of the metabolic inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP) resulted in a significant reduction in Cd contents in the shoots of the HE, and in the roots of the NHE. Cd was distributed preferentially to the root cylinder of the HE but not the NHE, and there was a 3-5 times higher Cd concentration in xylem sap of the HE in contrast to the NHE. These results illustrate that a greatly enhanced rate of root-to-shoot translocation, possibly as a result of enhanced xylem loading, rather than differences in the rate of root uptake, was the pivotal process expressed in the Cd hyperaccumulator HE S. alfredii.

Differential generation of hydrogen peroxide upon exposure to zinc and cadmium in the hyperaccumulating plant species (Sedum alfredii Hance).

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.

Changes of root morphology and Pb uptake by two species of Elsholtzia under Pb toxicity.

Elsholtzia argyi and Elsholtzia splendens, which are Chinese endemic Pb/Zn mined and Cu mined ecotype respectively, were investigated in the aspect of their response to Pb toxicity in the presence or absence of EDTA addition. After 8 d's Pb treatment, root length, root surface area and root volume of E. splendens decreased much more than those of E. argyi, and reduced considerably with increase of Pb, while no marked change was noted for root average diameter. Compared to E. argyi, length of root with diameter (D)<0.2 mm was significantly reduced for E. splendens as Pb increasing. Root with cross-sectional area of D<0.1 mm for E. splendens was at Pb> or =10 mg/L, while for E. argyi, it was at Pb> or =25 mg/L. DW of E. splendens decreased much more than that of E. argyi with increase of Pb. E. argyi exhibited much more tolerance to Pb toxicity than E. splendens. Treatment with 100 mg/L Pb plus 50 mmol/L EDTA significantly decreased the length and surface area of D< or =0.2 mm root, increased the length and surface area of 0.2< or =D< or =0.8 mm root for the case of E. argyi, while for E. splendens, length and surface area of D<0.6 mm root reduced, as compared to 100 mg/L Pb treatment, alone. At 100 mg/L Pb, shoot Pb accumulation in E. splendens and E. argyi were 27.9 and 89.0 microg/plant DW respectively, and much more Pb was uptaken by the root and translocated to the stem of E. argyi as compared to E. splendens. Treatment of the plant with 100 mg/L Pb plus 50 mmol/L EDTA increase leaf Pb accumulation from 16.8 to 84.9 g/plant for E.splendens and from 18.8 to 52.5 g/plant for E. argyi, while both root and stem Pb pronouncedly reduced for both Elsholtzia species. The increased translocation of Pb to the leaf of E. splendens than that of E. argyi at the treatment of 100 mg/L Pb plus 50 mmol/L EDTA should be further investigated.

Accumulation and ultrastructural distribution of copper in Elsholtzia splendens.

Copper accumulation and intracellular distribution in Elsholtzia splendens, a native Chinese Cu-tolerant and accumulating plant species, was investigated by transmission electron microscope (TEM) and gradient centrifugation techniques. Copper concentrations in roots, stems and leaves of E. splendens increased with increasing Cu levels in solution. After exposure to 500 micromol/L Cu for 8 d, about 1000 mg/kg Cu were accumulated in the stem and 250 mg/kg Cu in the leaf of E. splendens. At 50 micromol/L Cu, no significant toxicity was observed in the chloroplast and mitochondrion within its leaf cells, but separation appeared at the cytoplasm and the cell wall within the root cells. At >250 micromol/L Cu, both root and leaf cell organelles in E. splendens were damaged heavily by excessive Cu in vivo. Copper subcellular localization in the plant leaf after 8 days' exposure to 500 micromol/L Cu using gradient centrifugation techniques was found to be decreased in the order: chloroplast>cell wall>soluble fraction>other organelles. The plant root cell wall was found to be the site of highest Cu localization. Increase of Cu exposure time from 8 d to 16 d, increased slightly Cu concentration in cell wall fraction in roots and leaves, while that in the chloroplast fraction decreased in leaves of the plants grown in both 0.25 micromol/L and 500 micromol/L Cu. TEM confirmed that much more Cu localized in cell walls of E. splendens roots and leaves, but also more Cu localized in E. splendens' chloroplast when the plant is exposed to Cu levels>250 micromol/L, as compared to those in the plant grown in 0.25 micromol/L Cu. Copper treatment at levels>250 micromol/L caused pronounced damage in the leaf chloroplast and root organelles. Copper localization in cell walls and chloroplasts could mainly account for the high detoxification of Cu in E. splendens.

Gama-aminobutyric acid accumulation in Elsholtzia splendens in response to copper toxicity.

A solution with different Cu supply levels was cultured to investigate gama-aminobutyric acid (GABA) accumulation in Elsholtzia splendens, a native Chinese Cu-tolerant and accumulating plant species. Increasing Cu from 0.25 to 500 micromol/L significantly enhanced levels of GABA and histidine (His), but considerably decreased levels of aspartate (Asp) and glutamate (Glu) in the leaves. The leaf Asp level negatively correlated with leaf Cu level, while leaf GABA level positively correlated with leaf Cu level. The leaf Glu level negatively correlated with leaf GABA level in Elsholtzia splendens. The depletion of leaf Glu may be related to the enhanced synthesis of leaf GABA under Cu stress.

Improved cadmium uptake and accumulation in the hyperaccumulator Sedum alfredii: the impact of citric acid and tartaric acid.

The elucidation of a natural strategy for metal hyperaccumulation enables the rational design of technologies for the clean-up of metal-contaminated soils. Organic acid has been suggested to be involved in toxic metallic element tolerance, translocation, and accumulation in plants. The impact of exogenous organic acids on cadmium (Cd) uptake and translocation in the zinc (Zn)/Cd co-hyperaccumulator Sedum alfredii was investigated in the present study. By the addition of organic acids, short-term (2 h) root uptake of (109)Cd increased significantly, and higher (109)Cd contents in roots and shoots were noted 24 h after uptake, when compared to controls. About 85% of the (109)Cd taken up was distributed to the shoots in plants with citric acid (CA) treatments, as compared with 75% within controls. No such effect was observed for tartaric acid (TA). Reduced growth under Cd stress was significantly alleviated by low CA. Long-term application of the two organic acids both resulted in elevated Cd in plants, but the effects varied with exposure time and levels. The results imply that CA may be involved in the processes of Cd uptake, translocation and tolerance in S. alfredii, whereas the impact of TA is mainly on the root uptake of Cd.

The impact of EDTA on lead distribution and speciation in the accumulator Sedum alfredii by synchrotron X-ray investigation.

The in vivo localization and speciation of lead (Pb) in tissues of the accumulator Sedum alfredii grown in EDTA-Pb and Pb(NO(3))(2) was studied by synchrotron X-ray investigation. The presence of EDTA-Pb in solution resulted in a significant reduction of Pb accumulation in S. alfredii. Lead was preferentially localized in the vascular bundles regardless of treatments but the intensities of Pb were lower in the plants treated with EDTA. Lead was predominantly presented as a Pb-cell wall complex in the plants regardless of its supply form. However, a relatively high proportion of Pb was observed as Pb-EDTA complex when the plant was treated with EDTA-Pb, but as a mixture of Pb(3)(PO(4))(2), Pb-malic, and Pb-GSH when cultured with ionic Pb. These results suggest that EDTA does not increase the internal mobility of Pb, although the soluble Pb-EDTA complex could be transported and accumulated within the plants of S. alfredii.

Cadmium uptake and xylem loading are active processes in the hyperaccumulator Sedum alfredii.

Sedum alfredii is a well known cadmium (Cd) hyperaccumulator native to China; however, the mechanism behind its hyperaccumulation of Cd is not fully understood. Through several hydroponic experiments, characteristics of Cd uptake and translocation were investigated in the hyperaccumulating ecotype (HE) of S. alfredii in comparison with its non-hyperaccumulating ecotype (NHE). The results showed that at Cd level of 10 microM measured Cd uptake in HE was 3-4 times higher than the implied Cd uptake calculated from transpiration rate. Furthermore, inhibition of transpiration rate in the HE has no essential effect on Cd accumulation in shoots of the plants. Low temperature treatment (4 degrees C) significantly inhibited Cd uptake and reduced upward translocation of Cd to shoots for 9 times in HE plants, whereas no such effect was observed in NHE. Cadmium concentration was 3-4-fold higher in xylem sap of HE, as compared with that in external uptake solution, whereas opposite results were obtained for NHE. Cadmium concentration in xylem sap of HE was significantly reduced by the addition of metabolic inhibitors, carbonyl cyanide m-chlorophenylhydrazone (CCCP) and 2,4-dinitrophenol (DNP), in the uptake solutions, whereas no such effect was noted in NHE. These results suggest that Cd uptake and translocation is an active process in plants of HE S. alfredii, symplastic pathway rather than apoplastic bypass contributes greatly to root uptake, xylem loading and translocation of Cd to the shoots of HE, in comparison with the NHE plants.

[Effects of exogenous Ca2+ on the growth and Zn accumulation of two Sedum alfredii Hance ecotypes].

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.