Yahua Chen

Metal leaching along soil profiles after the EDDS application – A field study

One concern about the chelant-enhanced phytoextraction is the potential metal leaching associated with chelant application. A field study was carried out and the metal leaching along the 60-cm depth soil profiles were evaluated within 36 days after the biodegradable chelant EDDS was applied. Results showed EDDS significantly increased soluble Cu in the top 5 cm soil layer 1 day after the application, and the increase of soluble metals was generally limited in the top 20 cm soil. Metal speciation analysis indicated all Cu and Zn were in forms of Cu-EDDS and Zn-EDDS complexes in soil solution, and Ca was the major competitor with trace metals to EDDS. The soluble metals decreased quickly with time, and no significant difference was observed in the extractable Cu between EDDS treatments and the controls 22 days after the EDDS addition. The potential leaching associated with biodegradable EDDS addition may be controlled under field conditions.

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.

Residual effects of EDDS leachates on plants during EDDS-assisted phytoremediation of copper contaminated soil.

In this study, a novel experimental setup (one pot placed above another) was used to investigate the residual effects of EDDS application on plant growth and metal uptake. Two plant species, garland chrysanthemum and ryegrass, were grown in the upper pots (mimicking the upper soil layers) and were harvested 7 days after EDDS application. During this period the upper pots were watered twice. The lower pots (mimicking the subsoil under the upper soil layers) served as leachate collectors. Thereafter, the two pots were separated, and the same plants were grown in the upper and lower pots in two continuous croppings. Results showed that EDDS application restrained the growth of the first crop and resulted in a dramatic enhancement of Cu accumulation in plants grown in the upper pots. However, no negative growth effects were identified for the second and third crops, which were harvested 81 and 204 days after the EDDS application, respectively. In the lower pots, the leachate from the upper pots after EDDS application exhibited the increased total and CaCl(2)-extractable Cu concentrations in the soil. However, the growth of garland chrysanthemum and ryegrass, and their shoot Cu concentrations were unaffected. These data suggest that the residual risk associated with EDDS application was limited, and that subsoil to which EDDS leachate was applied may exhibit reduced Cu bioavailability for plants due to the biodegradation of EDDS.

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.

Using Contaminated Plants Involved in Phytoremediation for Anaerobic Digestion

This study investigated the anaerobic digestion capability of five plants and the effects of copper (Cu) and S,S’-ethylenediaminedisuccinic acid (EDDS, a chelator widely used in chelant-assisted phytoremediation) on biogas production to determine a feasible disposal method for plants used in remediation. The results showed that in addition to Phytolacca americana L., plants such as Zea mays L., Brassica napus L., Elsholtzia splendens Nakai ex F. Maekawa, and Oenothera biennis L. performed well in biogas production. Among these, O. biennis required the shortest period to finish anaerobic digestion. Compared to normal plants with low Cu content, the plants used in remediation with increased Cu levels (100 mg kg−1) not only promoted anaerobic digestion and required a shorter anaerobic digestion time, but also increased the methane content in biogas. When the Cu content in plants increased to 500, 1000, and 5000 mg kg−1, the cumulative biogas production decreased by 12.3%, 14.6%, and 41.2%, respectively. Studies also found that EDDS conspicuously restrained biogas production from anaerobic digestion. The results suggest that anaerobic digestion has great potential for the disposal of contaminated plants and may provide a solution for the resource utilization of plants used in remediation.

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.

Growth and photosynthetic responses of ectomycorrhizal pine seedlings exposed to elevated Cu in soils

It is still controversial whether ectomycorrhizal (ECM) mycelia filter out toxic metals in nutrient absorption of host trees. In this study, pine (Pinus densiflora) seedlings colonized by Cu-sensitive and Cu-tolerant ECM species were exposed to a wide spectrum of soil Cu concentrations to investigate functions of ECM fungi under Cu stress. The photosynthetic rates of intact needles were monitored in situ periodically. The biomass and elements of plants were also measured after harvest. The ameliorating effect of ECM infection on host plants exposed to toxic stress was metal concentration specific. Under lower-level Cu stress, ECM fungi increased seedling performance, while ECM seedlings accumulated more Cu than nonmycorrhizal (NM) seedlings. Under higher-level Cu stress, photosynthesis decreased well before visible symptoms of Cu toxicity appeared. The reduced photosynthesis and biomass in ECM seedlings compared to NM seedlings under higher Cu conditions were also accompanied by lower phosphorus in needles. There was no marked difference between the two fungal species. Our results indicate that the two ECM fungi studied in our system may not have an ability to selectively eliminate Cu in nutrient absorption and may not act as effective barriers that decrease toxic metal uptake into host plants.

Cloning and characterization of the Oryza sativa wall-associated kinase gene OsWAK11 and its transcriptional response to abiotic stresses

AimsTo examine heavy metal-induced regulatory mechanisms at the transcriptional level, a cell wall-associated receptor kinase (WAK) gene, OsWAK11 and its upstream promoter region (−946/+28) were isolated from Oryza sativa. OsWAK11 expression in response to abiotic stress was examined using a β-glucuronidase (GUS) gene fusion.MethodsSemi-quantitative RT-PCR was used to analyze expression of the OsWAK11 gene. Histochemical detection of GUS was conducted by X-gluc staining methods, and fluorometric measurements of GUS activity were made with 4-methyl umbelliferyl glucuronide (MUG) substrate.ResultsThe WAK promoter (−946/+28) responded to aluminum chloride, sodium chloride, and copper (II) sulfate with 3.0-, 2.2-, or 6.4-fold induction of GUS activity, respectively. Sodium nitroprusside and wounding treatment stimulated GUS activity. A histochemical analysis revealed strong GUS staining in the hypocotyls, cotyledons, first leaf, and petiole of cotyledons in transgenic tobacco seedlings. Strong GUS staining was also observed in the stigma and ovary of mature flowers, but not in the stamens.ConclusionOsWAK11 expression is regulated by aluminum, sodium, and copper. The GUS expression observed in transgenic tobacco carrying WAK11 promoter demonstrated significant tissue-specificity. The OsWAK11 promoter was strongly upregulated in response to metals and wounding.