Guiping Wang

Cadmium-induced accumulation of hydrogen peroxide in the leaf apoplast of Phaseolus aureus and Vicia sativa and the roles of different antioxidant enzymes

The effects of cadmium (Cd) on the accumulation of hydrogen peroxide (H(2)O(2)) and superoxide anion (O(2)(-)) in leaves of Phaseolus aureus and Vicia sativa were investigated. Cadmium at 100 microM significantly increased the production of O(2)(-) and H(2)O(2), as well as the activities of plasma membrane-bound nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and the symplastic and apoplastic activities of superoxide dismutase and ascorbate peroxidase in the leaves of both species. Apoplastic guaiacol peroxidase activity was significantly induced in the leaves of both species, particularly in P. aureus exposed to 100 microM Cd. Experiments with diphenylene iodonium as an inhibitor of NADPH oxidase and NaN(3) as an inhibitor of peroxidase showed that the majority of Cd-induced reactive oxygen species production in the leaves of both species may involve plasma membrane-bound NADPH oxidase and apoplastic peroxidase. Compared to V. sativa, increases in Cd-induced production of O(2)(-) and H(2)O(2) and activities of NADPH oxidase and apoplastic peroxidase were more pronounced in P. aureus. In contrast, V. sativa had higher leaf symplastic superoxide dismutase and ascorbate peroxidase activities than P. aureus. The results indicated that V. sativa was more tolerant to Cd than P. aureus.

Excess copper induces production of hydrogen peroxide in the leaf of Elsholtzia haichowensis through apoplastic and symplastic CuZn-superoxide dismutase

The effects of excess copper (Cu) on the production of reactive oxygen species (ROS) and antioxidant enzyme activities in leaves of the Cu-accumulator Elsholtzia haichowensis Sun were investigated. The addition of 100 microM of copper significantly increased the accumulation of hydrogen peroxide (H(2)O(2)) and the activities of superoxide dismutase (SOD) and other antioxidant enzymes. The increase in SOD activity was attributable to an increase in apoplastic and symplastic copper-zinc superoxide dismutase (CuZn-SOD) activity. Induction of CuZn-SOD proteins was demonstrated by immunoblot analysis. This study also provides the first cytochemical evidence of an accumulation of superoxide anion in the chloroplasts of mesophyll cells, and H(2)O(2) in the mesophyll cell walls and extracellular space, as a consequence of Cu treatment. Experiments with diphenyleneiodonium as an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and N-N-diethyldithiocarbamate as an inhibitor of SOD showed that the source of H(2)O(2) in the cell wall could be, in part, NADPH oxidase. Apoplastic guaiacol peroxidase (POD) and symplastic ascorbate peroxidase (APX) activities were induced in leaves of E. haichowensis with 100 microM Cu, suggesting that apoplastic POD and symplastic APX may be important in avoiding the buildup of toxic H(2)O(2) concentrations.

Overexpression of Elsholtzia haichowensis metallothionein 1 (EhMT1) in tobacco plants enhances copper tolerance and accumulation in root cytoplasm and decreases hydrogen peroxide production

To evaluate the functional roles of metallothionein (MT) in copper tolerance, we generated transgenic tobacco plants overexpressing EhMT1 from the Cu-accumulator Elsholtzia haichowensis Sun. Overexpression of EhMT1 in tobacco plants imparted increased copper (Cu) tolerance based on seedling dry biomass when compared to wild-type plants. Plants expressing EhMT1 accumulated more Cu in roots, which was mainly attributable to an increase of the soluble fraction. Levels of lipid peroxidation and production of hydrogen peroxide were lower in roots of transgenic tobacco than in wild-type plants. EhMT1 was suggested to bind Cu in the cytoplasm, thereby decreasing activity of free Cu(2+) ions and blocking Cu(2+) from interacting with cytoplasmic components, which in turn decreases the production of reactive oxygen species. In addition, our results also indicate that EhMT1-overexpressing tobacco has a more efficient antioxidant system, with improved peroxidase activity to better cope with oxidative stress.

Influence of the application of chelant EDDS on soil enzymatic activity and microbial community structure.

The present study evaluated the effects of a biodegradable chelant, S,S-ethylenediaminedisuccinic acid (EDDS), on enzyme activities and microbial community composition in copper (Cu)-contaminated soils, planted with either corn or beans. Results showed that the application of EDDS did not affect urease and acid phosphatase activities in the soil, but greatly reduced catalase and saccharase activities, and increased β-glucosidase activity on the seventh day after EDDS application. On the 28th day, no significant difference was observed in the enzyme activities (except for β-glucosidase) of EDDS-treated soils compared to the controls. Analysis of phospholipid fatty acids (PLFAs) showed that the application of 3 mmol kg(-1) EDDS did not cause significant stress to soil microbial communities. However, PCR-denaturing gradient gel electrophoresis (PCR-DGGE) fingerprint revealed that EDDS influenced the bacterial communities in the soils, and the effects on bean soils were more significant than that with corn. In general, the enzyme activities and bacterial communities were influenced by the application of EDDS, but the impact became weaker or even disappeared with the biodegradation of EDDS.

Proteomic analysis of copper-binding proteins in excess copper-stressed rice roots by immobilized metal affinity chromatography and two-dimensional electrophoresis

Copper (Cu) is an essential micronutrient required for plant growth and development. However, excess Cu can inactivate and disturb protein structure as a result of unavoidable binding to proteins. To understand better the mechanisms involved in Cu toxicity and tolerance in plants, we developed a new immobilized metal affinity chromatography (IMAC) method for the separation and isolation of Cu-binding proteins extracted from roots of rice seedling exposed to excess Cu. In our method, IDA-Sepharose or EDDS-Sepharose column (referred as pre-chromatography) and Cu-IDA-Sepharose column (referred as Cu-IMAC) were connected in tandem. Namely, protein samples were pre-chromatographed with IDA-Sepharose column to removal metal ions, then protein solution was flowed into Cu-IMAC column for enriching Cu-binding proteins in vitro. Compared with the control (Cu-IMAC without any pre-chromatography), IDA-Sepharose pre-chromatography method markedly increased yield of the Cu-IMAC-binding proteins, and number of protein spots and the abundance of 40 protein spots on two-dimensional electrophoresis (2-DE) gels. Thirteen protein spots randomly selected from 2-DE gel and 11 proteins were identified using MALDI-TOF-TOF MS. These putative Cu-binding proteins included those involved in antioxidant defense, carbohydrate metabolism, nucleic acid metabolism, protein folding and stabilization, protein transport and cell wall synthesis. Ten proteins contained one or more of nine putative metal-binding motifs reported by Smith et al. (J Proteome Res 3:834–840, 2004) and seven proteins contained one or two of top six motifs reported by Kung et al. (Proteomics 6:2746–2758, 2006). Results demonstrated that more proteins specifically bound with Cu-IMAC could be enriched through removal of metal ions from samples by IDA-Sepharose pre-chromatography. Further studies are needed on metal-binding characteristics of these proteins in vivo and the relationship between Cu ions and protein biological activities to fully understand the mechanisms of Cu tolerance and toxicity in plants.

Heating treatment schemes for enhancing chelant‐assisted phytoextraction of heavy metals from contaminated soils

Recent research has shown that chelant-assisted phytoextraction approaches often require a high dosage of chelant applied to soil. The present study focused on optimization of phytoremediation processes to increase the phytoextraction efficiency of metals at reduced chelant applications. Pot experiments were carried out to investigate the effects of increased soil temperature on shoot uptake of heavy metals by corn (Zea mays L.) and mung bean (Vigna radiat L. Wilczek) from heavy metal-contaminated soils. After the application of S,S-ethylenediaminedisuccinic acid or ethylenediaminetetra-acetic acid, soils were exposed to high temperatures (50 or 80 degrees C) for 3 h, which significantly increased the concentration of heavy metals in shoots. The heating treatment 2 d after the chelant addition resulted in higher concentrations of metals compared with those treatments 2 d before or simultaneously with the chelant application. Irrigation with 100 degrees C water 2 d after the chelant addition, or irrigation with 100 degrees C chelant solutions directly, also resulted in significantly higher phytoextraction of metals in the two crops compared with 25 degrees C chelant solutions. In addition, a novel application method to increase soil temperature using underground polyvinyl chloride tubes would increase the chelant-assisted extraction efficiency of Cu approximately 10- to 14-fold in corn and fivefold in mung bean compared with those nonheating treatments. In a field experiment, increasing soil temperature 2 d after chelant addition also increased the shoot Cu uptake approximately fivefold compared with those nonheating treatments. This new technique may represent a potential, engineering-oriented approach for phytoremediation of metal-polluted soils.

Copper-Resistant Bacteria Enhance Plant Growth and Copper Phytoextraction

In this study, we investigated the role of rhizospheric bacteria in solubilizing soil copper (Cu) and promoting plant growth. The Cu-resistant bacterium DGS6 was isolated from a natural Cu-contaminated soil and was identified as Pseudomonas sp. DGS6. This isolate solubilized Cu in Cu-contaminated soil and stimulated root elongation of maize and sunflower. Maize was more sensitive to inoculation with DGS6 than was sunflower and exhibited greater root elongation. In pot experiment, inoculation with DGS6 increased the shoot dry weight of maize by 49% and sunflower by 34%, and increased the root dry weight of maize by 85% and sunflower by 45%. Although the concentrations of Cu in inoculated and non-inoculated seedlings did not differ significantly, the total accumulation of Cu in the plants increased after inoculation. DGS6 showed a high ability to solubilize P and produce iron-chelating siderophores, as well as significantly improved the accumulation of P and Fe in both maize and sunflower shoots. In addition, DGS6 produced indole–3–acetic acid (IAA) and ACC deaminase, which suggests that it may modulate ethylene levels in plants. The bacterial strain DGS6 could be a good candidate for re-vegetation of Cu-contaminated sites. Supplemental materials are available for this article. Go to the publishers online edition of International Journal of Phytoremediation to view the supplemental file.

CADMIUM AND MANGANESE ACCUMULATION IN PHYTOLACCA AMERICANA L. AND THE ROLES OF NON-PROTEIN THIOLS AND ORGANIC ACIDS

Phytolacca americana L. can accumulate large amounts of heavy metals in its aerial tissues, especially cadmium (Cd) and manganese (Mn). It has great potential for use in phytoextraction of metals from multi-metal-contaminated soils. This study was conducted to further investigate the Cd- and Mn-tolerance strategies of this plant. Concentrations of non-protein thiols (NPTs) and phytochelatins (PCs) in leaves and roots increased significantly as the concentration of Cd in solution increased. The molar ratios of PCs:soluble Cd ranged from 1.8 to 3.6 in roots and 8.1 to 31.6 in leaves, suggesting that the cellular response involving PC synthesis was sufficient to complex Cd ions in the cytosol, especially that of leaves. In contrast, excess Mn treatments did not result in a significant increase in NPT or PC concentrations in leaves or roots. Oxalic acid concentrations in leaves of plants exposed to 2 or 20 mM Mn reached 69.4 to 89.3 mg (0.771 to 0.992 mmol) g–1 dry weight, respectively, which was approximately 3.7- to 8.6-fold higher than the Mn level in the 0.6 M HCl extract. Thus, oxalic acid may play an important role in the detoxification of Mn.

Cloning and characterization of a type 1 metallothionein gene from the copper-tolerant plant Elsholtzia haichowensis

EhMT1, a type 1 metallothionein (MT) gene, was cloned from Elsholtzia haichowensis, a Cu-tolerant plant. Typical of plant type 1 MTs, EhMT1 encodes a putative peptide of 74 amino acid residues containing cysteine-rich domains. Sequence comparisons with various databases revealed strong similarities at the nucleotide and amino acid levels between EhMT1 and the type 1 MT of Mimulus guttatus. EhMT1 transcription was greater in the roots than in the leaves, and was markedly increased by treatments with Cu, H2O2, and heat shock. EhMT1-GUS was localized to the cytoplasm of onion epidermal cells. Escherichia coli cells expressing pET-30a-EhMT1 were more tolerant to Cu and accumulated more Cu than control cells. Our results show that EhMT1 is involved in Cu tolerance and accumulation in E. haichowensis.

Differences in Copper Absorption and Accumulation between Copper-Exclusion and Copper-Enrichment Plants: A Comparison of Structure and Physiological Responses.

Differences in copper (Cu) absorption and transport, physiological responses and structural characteristics between two types of Cu-resistant plants, Oenothera glazioviana (Cu-exclusion type) and Elsholtzia haichowensis (Cu-enrichment type), were investigated in the present study. The results indicated the following: (1) After 50 μM Cu treatment, the Cu ratio in the xylem vessels of E. haichowensis increased by 60%. A Cu adsorption experiment indicated that O. glazioviana exhibited greater resistance to Cu, and Cu absorption and the shoot/root ratio of Cu were significantly lower in O. glazioviana than in E. haichowensis. (2) An analysis of the endogenous abscisic acid (ABA) variance and exogenous ABA treatment demonstrated that the ABA levels of both plants did not differ; exogenous ABA treatment clearly reduced Cu accumulation in both plants. (3) The leaf stomatal density of O. glazioviana was significantly less than that of E. haichowensis. Guard cells in E. haichowensis plants were covered with a thick cuticle layer, the epidermal hair was more numerous and longer, and the number of xylem conduits in the root was small. (4) The transpiration rate and the stomatal conductance of O. glazioviana were both significantly lower than those of E. haichowensis, regardless of whether the plants were treated with Cu. Taken together, these results indicate that the differences in the structural characteristics between these two plant species, particularly in the characteristics related to plant transpiration, are important factors that govern whether plants acquire or exclude Cu.