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In this study, the effects of excess NH4+-N on the subcellular accumulation of O2 − and H2O2 in submerged plant Hydrilla verticillata (L.f.) Royle were investigated using both histochemical and cytochemical methods. Treatments with ≥ 2.00 and ≥ 5.00 mg L−1 NH4+-N for 5 d significantly increased production of O2 − and H2O2, respectively. The activities of plasma membrane-bound NADPH (nicotinamide adenine dinucleotide phosphate) oxidases and antioxidant enzymes (superoxide dismutase, peroxidase, ascorbate peroxidase, catalase, dehydroascorbate reductase and glutathione reductase) were also increased correspondingly. This study also provides the first cytochemical evidence of subcellular accumulation of O2 − and H2O2 in the submerged plants. In the leaves of H. verticillata treated with 20.0 mg L−1 NH4+-N, O2 − dependent DAB precipitates were found primarily on the inner side of the plasma membrane, extracellular space and chloroplasts. H2O2-CeCl3 precipitates were mainly localized on the inner side of the plasma membrane and extracellular space of the mesophyll cells. Treatments with the inhibitors of NADPH oxidase (diphenylene iodonium and imidazole) indicate that NH4+-N-induced production of O2 − and H2O2 in H. verticillata leaves may involve plasma membrane-bound NADPH oxidase. Moreover, low-light treatment decreased NH4+-induced O2 − production, suggesting that alterations in the photosynthetic electron transfer chain due to NH4+ toxicity could lead to O2 − production.

Along with the widespread use of copper (Cu)-based fungicides and fertilizers, Cu-contaminated soil has been a major issue resulting in the loss of crop yield. To improve Cu tolerance in Oryza sativa, we previously carried out a screen of Cu sensitivity and obtained the Cu tolerant (B1139) and the Cu sensitive (B1195) rice varieties. Here, by analyzing an RNA-Seq transcriptome profile of root tips, 20 glutathione S-transferase (GST) genes were differently expressed between the Cu tolerant B1139 and the Cu sensitive B1195 when exposed to excess Cu2+. A more rapid decrease of root GST activity occurred in Cu-treated B1195 than in Cu-treated B1139, suggesting that different GST activity might affect Cu tolerance in rice. This is further supported by the observation of Cu hypersensitivity in rice seedlings deficient of the most abundant GST protein, OsGSTF2, demonstrating that GST proteins modulate Cu tolerance in rice. This, at least partially, leads to different Cu tolerance between B1139 and B1195. Furthermore, homologous modeling showed that a putative Cu2+ binding site, OsGSTF2H41 locates at the active center of GST enzyme. In vitro analysis demonstrated that Cu2+ interferes with the GST activity of OsGSTF2 protein, indicating that GST activity reduction by Cu2+ might be the binding of Cu2+ to the OsGSTF2H41 site. Consistently, amino acid replacement of histidine 41 with glycine (OsGSTF2H41G) resulted in decreased GST activity of OsGSTF2. Additionally, ectopic expression of OsGSTF2 enhances Cu tolerance in Escherichia coli, suggesting that the GST proteins might alleviate Cu toxicity by the binding of free Cu2+ in cells.

Pisolithus sp1 is an ectomycorrhizal (ECM) fungi that was chosen during a screening test of six strains of ECM fungi due to its ability to tolerate and remove hexavalent chromium (Cr(VI)). The physiological responses of Pisolithus sp1 to Cr(VI) exposure, the relationship between Pisolithus sp1 and exogenously added organic acids (EAOAs) or Na3VO4 (H+-ATPase inhibitor) and the ability of Pisolithus sp1 to reduce Cr(VI) in liquid culture were also investigated. Hydrogen ions (H+), which were produced directly by Pisolithus sp1, reduced the pH of the medium and played an important role in Cr(VI) reduction; however, Na3VO4 significantly inhibited this process and resulted in a decrease in the Cr(VI) reduction rates. Organic acids were secreted after the reduction in Cr(VI) by Pisolithus sp1, and EAOAs did not significantly affect Cr(VI) reduction; those results revealed the secondary role of organic acids in Cr(VI) reduction. The Cr(VI) removal rate of Pisolithus sp1 approached 99% after Cr(VI) treatment for 12 days. Overall, 75% of the Cr(VI) removal was due to extracellular reduction and 24% was due to adsorption. The results of this study provide a strong basis for using Cr(VI)-tolerant and Cr(VI)-reducing fungi, as well as ectomycorrhiza, in the remediation of Cr(VI)-contaminated sites.

Methionine sulfoxide reductases (MSRs), a family of enzymes catalyzing the conversion of methionine from its oxidized form into its reduced form, has a pivotal role in responding to oxidative stress. In the present study, we found that OsMSRB5 plays a crucial role in rice adaption to Cu stress. OsMSRB5 was mainly expressed in leaves, with low transcriptional levels of OsMSRB5 observed in seeds, stems, and roots; this gene was also induced by the excess copper (Cu) treatment. The functions of OsMSRB5 were analyzed through ectopic expression in Escherichia coli and functional disruption in rice. An in vitro enzymatic activity assay showed that OsMSRB5 had the ability to reduce free methionine- R -sulfoxide (Met- R -SO) and protein-bound-like Met-SO (dabsyl-Met-SO) to Met and dabsyl-Met, respectively. Ectopic expression of OsMSRB5 conferred E. coli cells higher tolerance to excess Cu and methyl viologen (MV). Furthermore, OsMSRB5 mutation decreased Cu and MV tolerance in rice. Our results demonstrate that rice OsMSRB5 is a functional methionine sulfoxide reductase and is involved in defense against Cu toxicity. In addition, rice seedlings responding to MV-induced oxidative stress showed a similar phenotype with excess Cu.

Several pharmacological experiments were performed to investigate the possible roles of heme oxygenase 1 (HO1) and its catalytic products on hemin-induced attenuation of excess zinc toxicity. The results showed that hemin markedly reduced concentrations of zinc in rice seedlings (including roots and shoots); this effect may be achieved by down-regulating the relative expression of OsZIP1, OsZIP3, OsZIP7, and OsZIP8. However, zinc protoporphyrin IX (Znpp, an HO1 inhibitor) application could reverse hemin-induced decreases in zinc accumulation and upregulate the relative expression of the above four genes. These results showed that HO1 is indeed involved in the mitigation of zinc stress. The following experiments were conducted to illustrate the contributive effect of different hemin by-products in the hemin-mediated attenuating effect. The results showed that bilirubin (BR) does not have any alleviative effect, whereas carbon monoxide (CO) has a similar effect as hemin; the alleviative effect of ferrous ion (Fe2+) is even better than that of hemin. Altogether, these findings suggested that hemin, through the HO1/Fe2+, CO system, may reduce zinc accumulation in rice seedlings and ultimately strengthen Zn tolerance of rice seedlings.

Cadmium (Cd) is one of the most toxic heavy metals. Cd tolerance ability differs among varieties in plants, but the underlying molecular mechanisms remain largely unknown. In this study, we identified genes that are involved in Cd stress responses and different Cd tolerances of two V. sativa varieties (Cd-tolerant variety (L3) and Cd-sensitive variety (ZM)). Transcriptomic analysis using Illumina pair-end sequencing was carried out on root tissues of L3 and ZM grown with 5 μM and 50 μM of Cd treatments. A de novo assembled V. sativa transcriptome was generated. Differentially expressed genes (DEGs) were assigned to Gene Ontology (GO) functions and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and enrichment analysis was performed. The expression of selected DEGs were confirmed by quantitative reverse transcription–polymerase chain reaction (qRT-PCR). A total of 49,062 sequences were identified as unigenes. In the 5 μM Cd treatment, 69 and 28 differentially expressed unigenes were found as compared with the control in the L3 and ZM respectively, while in the 50 μM Cd treatment, 1036 and 335 differentially expressed unigenes were found in comparison with the control in L3 and ZM. Pathway enrichment analysis suggested that genes related to the cell wall, stress response, the glutathione pathway, metal transporters, and transcription factors are commonly up-regulated in response to Cd stress in both varieties. However, the expression of metal transporter genes and transcription factor genes showed significant differential responses to Cd stress. In addition to the regulation of transcription by transcription factors, metal transporters play a vital role in controlling the different Cd tolerances of V. sativa varieties L3 and ZM.

The soybean sprout is a nutritious and delicious vegetable that is rich in ascorbic acid (AsA). Hydrogen gas (H2) may have potential applications in the vegetable processing industry. To investigate whether H2 is involved in the regulation of soybean sprouts AsA biosynthesis under UV irradiation, we set 4 different treatments: white light(W), W+HRW, UV-A and UV-A+HRW. The results showed that H2 significantly blocked the UV-A-induced accumulation of ROS, decreased TBARS content and enhanced SOD and APX activity in soybean sprouts. We also observed that the UV-A induced accumulation of AsA was enhanced more intensely when co-treated with HRW. Molecular analyses showed that UV-A+HRW significantly up-regulated AsA biosynthesis and recycling genes compared to UV-A in soybean sprouts. These data demonstrate that the H2 positively regulates soybean sprouts AsA accumulation under UV-A and that this effect is mediated via the up-regulation of AsA biosynthesis and recycling genes.

Hydrogen gas (H2) has recently been recognized as an important plant bio-regulator, inducing tolerance to several abiotic stresses. However, any role for H2 in amelioration of high-temperature-induced stress remains largely unknown. We investigated the mechanism of hydrogen-rich water (HRW)-mediated enhancement of heat-tolerance in cucumber seedlings exposed to high-temperature stress. The 3 weeks seedlings were pretreated with 50 or 100% HRW for 7 days, the effects on photosynthesis, chlorophyll content, chlorophyll fluorescence parameters, electrolyte leakage, lipid peroxidation, and antioxidant activity were examined when cucumber seedlings subjected for 3 days to heat stress treatment. With respect to samples treat with high temperature stress alone, HRW pretreatment remarkably alleviated stress-induced effects on the above parameters. Furthermore, after 3 days of treatment, HRW pretreatment also significantly increased the activities of antioxidative enzymes, promoted high-level accumulation of osmoprotectants, and upregulated HSP70 expression in cucumber leaves. All of these data suggest that pretreatment with exogenous HRW partially alleviated the detrimental effects of high-temperature stress on the growth of cucumber seedlings by improving the photosynthetic capacity, increasing the antioxidant response, and promoting the accumulation of HSP70 and osmolytes. Therefore, HRW pretreatment may improve cucumber seedlings heat-tolerance.

This study aimed to investigate the effect of Se (Selenium) treatment on nutritional quality in radish sprouts. The results showed that 15 µM sodium selenite significantly increased phenolics compounds, flavonoids compounds, anthocyanins, and some essential amino acid content, while improving the total antioxidant capacity of radish sprouts. Besides, the Se-enriched radish sprouts significantly alleviated the liver damage caused by carbon tetrachloride (CCl4 ) in mice and improved the antioxidant capacity of the liver in mice, whereas the Se-enriched radish sprouts alleviated the inflammatory reaction and apoptosis caused by CCl4 . These results imply that Se-enriched radish sprouts have a positive impact on mice with CCl4 -induced liver injury, and that in future Se-enriched radish sprouts could be developed into an effective food and health care product for the liver injury prevention. PRACTICAL APPLICATION: Because selenium is an essential trace element in the human body, selenium-enriched sprouts can help eliminate free radicals in the body, relieve aging, and selenium-deficient diseases. They are easy to grow and have low costs. Hence, selenium-enriched sprouts have a great potential of being widely consumed.

An important factor in periodontitis pathogenesis relates to a network of interactions of various cytokines. Thrombospondin-1 (TSP-1) is upregulated in several inflammatory diseases. We previously found that Porphyromonas gingivalis lipopolysaccharide (P. gingivalis LPS)-induced TSP-1 production, and that TSP-1 simultaneously and effectively elevated inflammatory cytokines in THP-1 macrophages. This suggests that TSP-1 plays an important role in the pathology of periodontitis. However, the function of TSP-1 on oral cells is largely unknown. This study aimed to elucidate the underlying molecular mechanisms of TSP-1 in human periodontal fibroblasts (hPDLFs). We demonstrated that TSP-1 is highly expressed in the gingival crevicular fluid of patients with chronic periodontitis and in the inflammatory gingival tissues of rats. TSP-1 overexpression or treatment with recombinant human TSP-1(rTSP-1) promoted the expression of MMP-2, MMP-9 and RANKL/OPG in hPDLFs, while anti-TSP-1 inhibited cytokines production from P. gingivalis LPS-treated hPDLFs. Additional experiments showed that SB203580 (a special p38MAPK inhibitor) inhibited MMP-2, MMP-9 and RANKL/OPG expression induced by rTSP-1. Thus, TSP-1 effectively promoted P. gingivalis LPS-induced periodontal tissue (extracellular matrix (ECM) and alveolar bone) destruction by the p38MAPK signalling pathway, indicating that it may be a potential therapeutic target against periodontitis.

Arbuscular mycorrhizal fungi (AMF) are widespread soil fungi that can form endosymbiotic structures with the root systems of most plants and can improve the tolerance of host plants to heavy metals. In the present study, we investigated the effects of AMF (Glomus coronatum) inoculation on the tolerance of Tagetes patula L. to Cu. Almost all of the non-mycorrhizal plants exposed to 100 μM Cu died after 3 d, whereas phytotoxicity was only observed in mycorrhizal plants that were exposed to Cu concentrations greater than 100 μM. Analysing the dynamic accumulation of Cu indicated that, after 7 d of Cu exposure, less Cu was absorbed or accumulated by mycorrhizal plants than by control plants, and significantly less Cu was translocated to the shoots. Meanwhile, analysing the root morphology, the integrity of the root plasma membranes, the photosynthesis rate, and the content of essential elements of plants growing in cultures with 50 μM Cu revealed that AMF inoculation markedly alleviated the toxic effects of Cu stress on root system activity, photosynthesis rate, and mineral nutrient accumulation. In addition, to understand the Cu allocation, an energy spectrum analysis of Cu content at the transverse section of root tips was conducted and subsequently provided direct evidence that intraradical hyphae at the root endodermis could selectively immobilise large amounts of Cu. Indeed, the sorption and barrier mechanisms of AMF hyphae reduce Cu toxicity in the roots of T. patula and eventually enhance the plants' Cu tolerance.

A copper-tolerant phenanthrene (PHE)-degrading bacterium, strain Sphingobium sp. PHE-1, was newly isolated from the activated sludge in a wastewater treatment plant. Two key genes, ahdA1b-1 encoding polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase (PAH-RHDɑ) and xyLE encoding catechol-2,3-dioxygenase (C23O), involved in the PHE metabolism by strain PHE-1 were identified. The PAH-RHD gene cluster showed 96% identity with the same cluster of Sphingomonas sp. P2. Our results indicated the induced transcription of xylE and ahdA1b-1 genes by PHE, simultaneously promoted by Cu(II). For the first time, high concentration of Cu(II) is found to encourage the expression of PAH-RHDɑ and C23O genes during PHE degradation. Applying Sphingomonas PHE-1 in PHE-contaminated soils for bioaugmentation, the abundance of xylE gene was increased by the planting of ryegrass and the presence of Cu(II), which, in turn, benefited ryegrass growth. The best performance of PHE degradation and the highest abundance of xylE genes occurred in PHE-copper co-contaminated soils planted with ryegrass.

In this study, we investigated the possible role of hemin in alleviating zinc (Zn), lead (Pb) and chromium (Cr) toxicity in rice seedlings grown hydroponically by analyzing the morphological and physiological parameters. Our results showed that exposure of rice seedlings to excess Zn, Pb or Cr could cause severe leaf chlorosis, inhibit photosynthetic activity and consequently suppress plant growth. The concentration of O2 •− and H2O2 significantly increased and the activities of antioxidative enzymes decreased in roots of rice seedlings under metal exposure. The combined treatments (hemin + ZnSO4, hemin + Pb(NO3)2 and hemin + K2Cr2O7), on the other hand, significantly enhanced the photosynthesis- and plant growth-related parameters compared with their corresponding heavy-metal-stress alone. Combined treatments dramatically stimulated the activities of superoxide dismutase (SOD), ascorbic peroxidase (APX) and glutathione reductase (GR) as well as the concentrations of ascorbic acid (AsA) and glutathione (GSH) as compared with the metal- stress alone. The concentrations of reactive oxygen species (ROS, e.g. O2 •− and H2O2) were significantly reduced in the metal plus hemin treatments. Hemin addition also reduced metal accumulation in rice seedlings especially in root tissues. These findings suggest that hemin-elevated levels of antioxidants, activities of antioxidative enzymes and hemin-reduced accumulation of heavy-metal could confer resistance against Zn, Pb, and Cr stress in rice seedlings, resulting in improved pigments accumulation, photosynthetic attributes and plant growth.

Phytotoxicity and accumulation of Cu in mature and young leaves of submerged macrophyte Hydrilla verticillata (L.f.) Royle were investigated by analyzing the chlorophyll contents, chloroplast ultrastructure and leaf surface structure under different Cu treatments (0, 0.01, 0.05 and 0.1 f mg L−1). The results showed that 0.05 and 0.1 mg L−1 Cu treatment decreased the contents of Chl a and Chl b, and caused damage on leaf surface structure and chloroplast ultrastructure compared with control (0 mg L−1 Cu treatment). Higher concentration of Cu induced Chlorophyll decreases and the damages on the leaf surface structure and chloroplast ultrastructure were more pronounced in mature than in young leaves. It was observed that leaf Cu concentration increased almost linearly with exposure time and majority of the Cu accumulated in the cell walls. Among different cell wall fractions, the majority of Cu accumulated in cell walls was bound to the hemicellulose 1 and cellulose, followed by the pectin, hemicellulose 2. Mature leaves had significantly higher the concentrations of total Cu and bound-Cu in cell walls due to higher uronic acid content in their cell wall fractions (pectin, hemicellulose 1 and cellulose) than young leaves. Distinct cell wall composition might partially contribute to the different Cu toxicity and accumulation between mature and young leaves of submerged macrophyte H. verticillata. Our results show that mature leaves are more efficient in the uptake and accumulation of Cu than young leaves, which might explain why mature leaves sustain more severe damage.

Lignin accumulation is a general response to heavy metal toxicity; however, the underlying mechanism is unclear. We isolated two rice l ignin- d efective m utants (ldm1 and ldm2); both showed reduced mechanical strength and lignin accumulation. Also, the levels of the syringyl and guaiacyl units of lignin were decreased in both mutants. Both mutants were more hypersensitive to excess Cu than wild-type plants, possibly resulting from more Cu in leaf sheaths and roots of the mutants than wild-type plants. Moreover, Cu retention in the cell wall was altered in the ldm1 and ldm2 mutants. Genotypic analysis confirmed that ldm1 has a T-DNA insert in the last exon of OsCCoAMT1 and ldm2 in the second exon of OsCCoAMT20. Consistently, both mutants displayed significantly decreased CCoAOMT enzymatic activity in all tissues examined, which catalyzed the transfer of methyl groups from S-adeosyl-Lmethionin (SAM) to benzene carbon 3 position of caffeoyl-CoA or 5-hydroxyferuloyl-CoA. Furthermore, the expression of OsCCoAOMT1 and CCoAOMT20 and CCoAOMT activity were upregulated in leaf sheaths and roots under excess Cu, suggesting that lignin synthesis mediated by OsCCoAOMT1 and OsCCoAOMT20 plays an important role in the adaptation to excess Cu stress. Based on the role of lignin in the sequestration of heavy metals in the plant cell wall, we speculate that enhancement of lignin synthesis may be an adaptive strategy to alleviate the toxicity of Cu ions.

Copper (Cu) toxicity has a deleterious effect on plant growth, and different nitrogen (N) forms have significantly different impacts on the uptake and accumulation of heavy metals by plants. However, it remains unclear how excess Cu inhibits the growth of rice seedlings under different N forms. Here, we examined the mechanism of Cu toxicity inhibiting the growth of rice supplied with different N forms. Rice seedlings were grown in a nutrient solution with 0.81 mmol L−1 N, as ammonium (NH4+), nitrate (NO3−) and NH4+ + NO3−, or without N (0 N) in the presence of 0.2 μmol L−1 CuSO4 or 10 μmol L−1 CuSO4. The inhibition of shoot growth under excess Cu was more pronounced in plants that were supplied with NO3− than NH4+; such inhibition was not induced by higher Cu concentration in shoots. Applied with NO3− alone increased solution pH value up to 6.2, but supplied with NH4+ alone and NH4+ + NO3− decreased solution pH value to 4.0 and 4.2, respectively. The increment of solution pH reduced Cu concentration in shoots of rice supplied with NO3− alone. Copper toxicity decreased NO3− concentrations in rice seedlings that were supplied with NO3− alone but increased the NH4+ concentrations in plants that were supplied with NH4+ or NH4+ + NO3−. High Cu levels reduced the uptake of NO3− in roots by the analysis of net NO3− flux and NO3− assimilation enzymes activity. Under excess Cu, the transcript levels of OsNPF6.5, OsNPF2.2 and OsNPF2.4 genes were suppressed, while OsNRT2.1, OsNRT2.2 and OsNAR2.1 were raised in roots. In conclusion, Cu toxicity inhibits NO3− uptake and upward translocation by modulating the expression level of NO3− transporter genes. The reduction in the concentrations of NO3− and total N decreased shoot growth of rice seedlings when N was supplied as NO3−. Hence, rice seedlings supplied with NO3− had lower shoot biomass than those with NH4+ under Cu stress.

Background Hydrogen gas (H2) is the most abundant element in the universe, and has been reported to act as a novel beneficial gaseous molecule in plant adaptive responses. Radish sprouts are popular because they contain substantial amounts of antioxidants and health-promoting compounds, such as anthocyanin and glucosinolates. Although radish sprouts accumulated more anthocyanin under UV-A after treatment with hydrogen-rich water (HRW), the molecular mechanism responsible is still elusive. To explore these mechanisms, RNA-seq analysis was used. Results Four cDNA libraries from radish sprout hypocotyls were constructed, and a total of 14,564 differentially expressed genes (DEGs) were identified through pairwise comparisons. By Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, these unigenes were found to be implicated in light signal perception and transduction, starch and sucrose metabolism, photosynthesis, nitrogen metabolism and biosynthesis of secondary metabolites. The MYB-bHLH-WD40 complex accounted for the majority of the transcription factors found to be involved in anthocyanin biosynthesis, and levels of transcripts for this complex were in accordance with the anthocyanin concentrations observed. In addition, other transcription factors (such as NAC, bZIP and TCP) might participate in HRW-promoted anthocyanin biosynthesis. Furthermore, the signaling processes of plant hormones, MAPKs and Ca2+ might be involved in HRW-promoted anthocyanin biosynthesis under UV-A. The expression patterns of 16 selected genes were confirmed using qRT-PCR analysis. Conclusions Taken together, the results of this study may expand our understanding of HRW-promoted anthocyanin accumulation under UV-A in radish sprouts. Electronic supplementary material The online version of this article (10.1186/s12870-018-1449-4) contains supplementary material, which is available to authorized users.

We examined the effects of Cd on growth, lipid peroxidation, reactive oxygen species (ROS) accumulation, antioxidant enzymatic activity, and lignin content in the roots of two varieties of Vicia sativa. Treatment with Cd decreased plant growth and increased ROS and lipid peroxidation levels to a greater extent in the Cd-sensitive variety ZM than in the Cd-tolerant variety L3. Most hydrogen peroxide (H2O2) and superoxide anion (O2(•-)) were accumulated in the cell walls and extracellular spaces in response to Cd treatments. Chemical assays and experiments using inhibitors showed that larger increases in H2O2 and O2(•-) production in ZM than in L3 were probably attributed to elevated Cd-induced nicotinamide adenine dinucleotide-peroxidase (NADH-POD) activity. Cd treatment increased the accumulation of lignin and the guaiacol peroxidase (GPOD) activities in the apoplast more significantly in ZM root than in L3. Howerver, root laccase activity was higher in L3 than in ZM. Thus Cd toxicity induced significant lignification in the roots of V. sativa, and increases in H2O2 accumulation and apoplastic GPOD activity were likely responsible for this effect.

Metallothioneins (MTs) are low-molecular-weight, cysteine-rich metal-binding proteins found in numerous genera and species, but their functions in abiotic stress tolerance remain unclear. Here, a MT gene from Oryza sativa, OsMT2c, was isolated and characterized, encoding a type 2 MT, and observed expression in the roots, leaf sheathes, and leaves, but only weak expression in seeds. OsMT2c was upregulated by copper (Cu) and hydrogen peroxide (H2O2) treatments. Excessive Cu elicited a rapid and sustained production and release of H2O2 in rice, and exogenous H2O2 scavengers N,N'-dimethylthiourea (DMTU) and ascorbic acid (Asc) decreased H2O2 production and OsMT2c expression. Furthermore, the expression of OsMT2c increased in the osapx2 mutant in which the H2O2 levels were higher than in wild-type (WT) plants. These results showed that Cu increased MT2c expression through the production and accumulation of Cu-induced H2O2 in O. sativa. In addition, the transgenic OsMT2c-overexpressing Arabidopsis displayed improved tolerance to Cu stress and exhibited increased reactive oxygen species (ROS) scavenging ability compared to WT and empty-vector (Ev) seedlings.

In this study, the effectiveness of six soil amendments (ferrihydrite, manganese dioxide, gibbsite, calcium carbonate, biochar, and organic fertilizer) was investigated to assess the feasibility of minimizing possible environmental contaminant leaching during S,S-ethylenediaminedisuccinic acid (EDDS)-enhanced phytoextraction process based on 0.01-M CaCl2 extraction. Results showed that the application of EDDS could significantly increase Cu concentrations in the leaching solution. Compared with control, incorporation of six amendments (excluding organic fertilizer) significantly decreased CaCl2-extractable Cu concentrations in both soils. When EDDS-containing solutions leached from the soil columns (mimicking the upper soil layers) were added to soils with different amendments (mimicking the subsoil), CaCl2-extractable Cu in the soils amended with ferrihydrite, manganese dioxide, gibbsite, and calcium carbonate was significantly lower than that in the control soil (no amendments) and remained relatively constant during the first 14 days. Incorporation of biochar or organic fertilizer had no positive effect on the immobilization of Cu in EDDS leachates in soils. After 14 days, CaCl2-extractable Cu concentration decreased rapidly in soils incorporated with various amendments. Integrating soil washing with biodegradable chelating agents or chelant-enhanced phytoextraction and immobilization of heavy metals in subsoil could be used to rapidly reduce the concentration of bioavailable metal fractions in the upper soil layers and minimize environmental risks of secondary pollution.

The present study investigated the effects of the biodegradable chelant S , S -ethylenediaminedisuccinic acid (EDDS) and the plant-growth-promoting bacterium DGS6 on pollutant uptake by corn from e-waste–contaminated soils. The highest concentration and total uptake of Cu and Zn in corn shoots were observed in the presence of EDDS and DGS6 + EDDS, respectively. The ΣPCB concentrations in shoots ranged from 0.53 to 0.72 ng g −1 , and the highest PCB concentration was observed in the presence of EDDS. This could be ascribed to the enhanced dissolved organic carbon, increased dissolution and efficient translocation of PCBs from roots to shoots, as well as potential root damage due to increased soluble metal levels in soil solution. In contrast, the highest total uptake of PCBs in shoots was observed in the presence of DGS6, likely due to enhanced shoot biomass and high levels of air deposition.

It was reported that fulvic acid (FA) has a positive effect on enhancing plant tolerance to various environmental stresses, including salinity stress and drought stress and so on. However, there is little study regarding the effects of FA on plants in response to heavy metal stress. Hence, the objective of this study was to investigate the potential effects of fulvic acid (FA) on cadmium (Cd) toxicity alleviation in lettuce seedlings. Our results showed that application of 0.5 g/L FA significantly mitigate Cd-induced toxic symptoms in lettuce seedlings. Cd stress triggered plant growth inhibition, photosynthetic pigment reduction, destruction of the photosynthesis apparatus, reactive oxygen species (ROS) accumulation, and nutrient elemental imbalance. We observed that FA promoted the growth in lettuce under Cd stress, mainly reflected in those alterations that the increase of biomass, chlorophyll content and photosynthesis capacity and reduction of the Cd content and lipid peroxidation in plant tissue. Foliar spraying of FA significantly alleviated these detrimental symptoms and facilitated nutrient element translocation from root to shoot, particularly the absorption of elements involved in photosynthesis, including iron (Fe), zinc (Zn), and manganese (Mn). In summary, foliar application of FA conferred Cd toxicity tolerance to lettuce by increasing ROS-scavenging capacity, inhibiting Cd uptake and the transport of elemental nutrients to shoots, which in turn protected the photosynthetic apparatus and promoted plant growth.

The present manuscript reviews the research progresses on the phytoextraction of metals using biodegradable chelants in our research group. It mainly focuses on the theoretical considerations including metal solubilization, root uptake and translocation upward to the shoots, the effects of chelant application on metal leaching and soil microbes, and the optimization of the chelant-assisted phytoextraction. In addition, the potential of phytoextraction of heavy metals and persistent organic pollutants (POPs) with biodegradable chelant in the heavy metal-POPs co-contaminated is reviewed as well.

The in situ phytoremediation, using plants to restore the deteriorated soils, is a promising technology in cleanup of polluted sites. The potential use of high-biomass plants was investigated for the EDTA or EDDS-enhanced phytoextraction of metals from contaminated soils. The approaches for enhancing the efficiency of chelant-assisted phytoextraction and the environmental risk (metals leaching) of applying EDTA and EDDS were also investigated. The results showed that, compared to the monocotyledon species (e.g., barley, wheat, sorghum, corn, vetiver grass, etc.), the dicotyledon species (i.e., mung bean, buckwheat, pea, sunflower oil, mustard, etc.) had a higher sensitivity to the EDTA or EDDS treatments, which could be reflected by the severer toxicity of seedlings and the higher concentrations of heavy metals in the shoots. Considering the three factors (the metal level of the shoot, the biomass of plants, and the growing seasons of plants), mustard and pea were more suitably used in the cool seasons, while corn, sunflower, mung bean, and buckwheat could be selected in the warm seasons. Vetiver grass might be suitably used in the phytostabilization of heavy metals in soils. There was a new approach that the increase of soil temperature could improve the efficiency of heavy metal accumulation of plants from soils by 10–40 times in comparison with the normal chelator application. This was the highest efficiency of chelator application reported in the literature up to now. Soil column leaching tests and a field study were carried out to investigate the leaching behavior of heavy metals in chelant-enhanced phytoextraction. Results showed that the soluble heavy metals in the contaminated soils after EDTA application could be persistent in the soils for a long time (at least 32 weeks), which could be easily leached down to the deep soils under the large rainfall percolation. On the contrary, EDDS application had a little effect on the movement of heavy metals in the soils. The results showed that the potential leaching associated with EDDS addition may be controlled under field conditions. The use of biodegradable chelators for enhancing phytoextraction of heavy metal-contaminated soils is generally regarded as an environmentally acceptable approach.

Proteomic studies were performed to identify proteins involved in the response of Oenothera glazioviana seedlings under Cu stress. Exposure of 28-d-old seedlings to 50 μM CuSO4 for 3 d led to inhibition of shoot and root growth as well as a considerable increase in the level of lipid peroxidation in the roots. Cu absorbed by O. glazioviana accumulated more easily in the root than in the shoot. Label-free proteomic analysis indicated 58 differentially abundant proteins (DAPs) of the total 3,149 proteins in the roots of O. glazioviana seedlings, of which 36 were upregulated and 22 were downregulated under Cu stress conditions. Gene Ontology analysis showed that most of the identified proteins could be annotated to signal transduction, detoxification, stress defence, carbohydrate, energy, and protein metabolism, development, and oxidoreduction. We also retrieved 13 proteins from the enriched Kyoto Encyclopaedia of Genes and Genomes and the protein-protein interaction databases related to various pathways, including the citric acid (CA) cycle. Application of exogenous CA to O. glazioviana seedlings exposed to Cu alleviated the stress symptoms. Overall, this study provided new insights into the molecular mechanisms of plant response to Cu at the protein level in relation to soil properties.

Cadmium (Cd) is one of the most toxic heavy metal elements in nature, and it causes serious damage to plant cells. Here, we report that a transcription factor OsMYB45 is involved in Cd stress response in rice. OsMYB45 is highly expressed in rice leaves, husks, stamens, pistils, and lateral roots, and its expression is induced by Cd stress. OsMYB45 fused to green fluorescent protein localized to the cell nucleus in onion epidermal cells. Mutation of OsMYB45 resulted in hypersensitivity to Cd treatment, and the concentration of H2O2 in the leaves of mutant nearly doubled, while catalase (CAT) activity was halved compared with the wild-type. Moreover, gene expression analysis indicated that OsCATA and OsCATC expression is significantly lower in the mutant than in the wild-type. In addition, overexpression of OsMYB45 in the mutant complemented the mutant phenotype. Taken together, OsMYB45 plays an important role in tolerance to Cd stress in rice.

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.

Induction of lignin biosynthesis is an adaptive response of plants subjected to many abiotic stresses. In this study, we examined the response of lignin biosynthesis to copper (Cu) stress, with a particular focus on the regulatory mechanism. We performed a transcriptomic analysis of rice (Oryza sativa L.) roots, and the microarray data on lignin biosynthesis pathway genes were corroborated by quantitative reverse transcription–polymerase chain reaction (qRT-PCR) analysis. Physiological analyses of rice seedlings treated with Cu(II) sulfate (CuSO4) were used to confirm the relationship between excess Cu and lignin biosynthesis. In addition, we examined the role of hydrogen peroxide (H2O2) in Cu-induced lignin biosynthesis through pretreatments with an NADPH oxidase inhibitor (diphenyleneiodonium, DPI) and a H2O2 scavenger (dimethylthiourea, DMTU). Lignin biosynthesis pathway genes were upregulated under Cu stress. The lignin content of rice roots increased significantly with increasing concentrations and durations of Cu treatment; elevations in root lignin content were correlated with marked inhibitions in root growth. Pretreatments with DPI and DMTU inhibited the activities of Cu-induced lignin polymerization enzymes (peroxidase, POD and laccase, LAC) and lignin accumulation in rice roots. Conversely, exogenous H2O2 increased the root lignin content. Rice roots under Cu stress accumulate lignin through enhanced polymerization of lignin monolignol, a mechanism that requires Cu stress induced H2O2.

Traditional metal chelators, such as ethylenediaminetetraacetic acid (EDTA), have been gradually replaced due to their poor biodegradability in soil and high risk of heavy metal leaching into groundwater, which pose high environmental risks to the health of humans and animals. In this study, a liquid amino acid fertilizer (LAAF, waste proteins from hydrolysates of animal carcasses) and polyaspartate (PASP) were used as additives to enhance the phytoextraction of cadmium (Cd) and lead (Pb) from contaminated soil. We conducted pot experiments to investigate the phytoextraction capacity of Solanum nigrum, a Cd accumulator, grown on soil highly contaminated with Cd and Pb in the absence (as controls) or presence of PASP and LAAF. Both PASP and LAAF significantly improved plant growth, Cd accumulation, and total Cd and Pb content in S. nigrum shoots and roots. PASP and LAAF application promoted Cd translocation from roots to shoots in S. nigrum and Cd bio-accessibility in rhizosphere soils, but this was not the case for Pb. Both PASP and LAAF increased Cd and Pb phytoextraction by S. nigrum plants, and Cd phytoextraction was more effective in LAAF-assisted S. nigrum than in PASP-assisted S. nigrum. These findings demonstrate that the low cost and ecofriendly features of recycled waste proteins make them good candidates for chelant-enhanced phytoextraction from heavy metal-contaminated soils.

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.

The effects of environmental factors on the paeonol accumulation in cortex moutan of herbal Paeonia suffruticosa cv. Fengdan were investigated. Cortex moutan of ‘Fengdan’ and the corresponding soil samples were collected from Tongling (Anhui Province, China). A pot experiment was also conducted to investigate the responses of plant growth and paeonol concentrations to copper (Cu) supply. The paeonol and Cu concentrations in cortex moutan samples, the basic soil properties and soil’s bacterial diversity were analyzed. The results showed that moderate levels of Cu in the soil enhanced the paeonol concentration in the cortex moutan. Moreover, the paeonol concentration was related significantly to soil bacterial diversity. The results of the pot experiment showed that increasing Cu supply from 63 to 263 mg·kg−1 did not produce any significant effect on the dry weight of ‘Fengdan’ cortex moutan, duramen, and total plant. Increasing Cu supply significantly increased the concentrations of paeonol in cortex moutan and duramen; but at 263 mg·kg−1 Cu there was a slight decline in their concentrations than those obtained at 156 mg·kg−1 Cu. In all treatments, the concentrations of paeonol were much higher in the cortex moutan than those in the duramen. Due to its ecological benefits, ‘Fengdan’ can be planted in soil contaminated with moderate Cu.

Laccases are encoded by a multigene family and widely distributed in plant genomes where they play roles oxidizing monolignols to produce higher-order lignin involved in plant development and stress responses. We identified 30 laccase genes (OsLACs) from rice, which can be divided into five subfamilies, mostly expressed during early development of the endosperm, growing roots, and stems. OsLACs can be induced by hormones, salt, drought, and heavy metals stresses. The expression level of OsLAC10 increased 1200-fold after treatment with 20 μM Cu for 12 h. The laccase activities of OsLAC10 were confirmed in an Escherichia coli expression system. Lignin accumulation increased in the roots of Arabidopsis over-expressing OsLAC10 (OsLAC10-OX) compared to wild-type controls. After growth on 1/2 Murashige and Skoog (MS) medium containing toxic levels of Cu for seven days, roots of the OsLAC10-OX lines were significantly longer than those of the wild type. Compared to control plants, the Cu concentration decreased significantly in roots of the OsLAC10-OX line under hydroponic conditions. These results provided insights into the evolutionary expansion and functional divergence of OsLAC family. In addition, OsLAC10 is likely involved in lignin biosynthesis, and reduces the uptake of Cu into roots required for Arabidopsis to develop tolerance to Cu.

The ameliorative effect of H2 on aluminum (Al)-induced stress remains poorly understood. We treated maize seedlings with Al and hydrogen-rich water (HRW) to determine the roles of H2 in the alleviation of Al toxicity. Our results demonstrated that Al stress triggered damage to the photosynthetic apparatus, plant growth inhibition, and reactive oxygen species (ROS) production, and boosted lipid peroxidation. However, the addition of HRW at 75% saturation markedly alleviated Al toxicity symptoms through the promotion of root elongation. These responses were related to the significantly increased activities of typical antioxidant enzymes (CAT, APX, SOD, and POD). In vivo imaging of plasma membrane integrity, lipid peroxidation, and the level of ROS provided further evidence that HRW could improve Al tolerance. Our results also indicate that 100% HRW mitigated Al toxicity less than 75% HRW. Moreover, different concentrations of HRW significantly improved photosynthesis and increased nutrient uptake. We conclude that exogenous H2 supplementation could enhance Al tolerance by reestablishing redox homeostasis and maintaining nutrient homeostasis.

Copper (Cu) is an essential micronutrient for plants, but excess Cu can inactivate and disturb the protein function due to unavoidable binding to proteins at the cellular level. As a redox-active metal, Cu toxicity is mediated by the formation of reactive oxygen species (ROS). Cu-binding structural motifs may alleviate Cu-induced damage by decreasing free Cu(2+) activity in cytoplasm or scavenging ROS. The identification of Cu-binding proteins involved in the response of plants to Cu or ROS toxicity may increase our understanding the mechanisms of metal toxicity and tolerance in plants. This study investigated change of Cu-binding proteins in radicles of germinating rice seeds under excess Cu and oxidative stress using immobilized Cu(2+) affinity chromatography, two-dimensional electrophoresis, and mass spectra analysis. Quantitative image analysis revealed that 26 protein spots showed more than a 1.5-fold difference in abundances under Cu or H2O2 treatment compared to the control. The identified Cu-binding proteins were involved in anti-oxidative defense, stress response and detoxification, protein synthesis, protein modification, and metabolism regulation. The present results revealed that 17 out of 24 identified Cu-binding proteins have a similar response to low concentration Cu (20 μM Cu) and H2O2 stress, and 5 out of 24 were increased under low and high concentration Cu (100 μM Cu) but unaffected under H2O2 stress, which hint Cu ions can regulate Cu-binding proteins accumulation by H2O2 or no H2O2 pathway to cope with excess Cu in cell. The change pattern of these Cu-binding proteins and their function analysis warrant to further study the roles of Cu ions in these Cu-binding proteins of plant cells.

To study possible cadmium (Cd) resistance mechanisms in cabbage (Brassica oleracea L.), several parameters of metal uptake, distribution, and complexation were compared between two varieties Chunfeng [CF (Cd-tolerant)] and Lvfeng [LF (Cd-sensitive)]. Results showed that CF contained significantly lower Cd concentrations in leaves and higher Cd concentrations in roots than LF. Approximately 70 to 74 % and 66 to 68 % of Cd taken up by LF and CF, respectively, was transported to shoots. More Cd was bound to the cell walls of leaves, stems, and roots in CF than in LF. The higher capacity of CF to limit Cd uptake into shoots could be explained by immobilization of Cd in root cell walls. Compared with control groups, Cd treatment also significantly increased concentrations of nonprotein thiols, phytochelatins (PCs), and citric acid in the leaves and roots of the two varieties; the increases were more pronounced in CF than in LF. Taken together, the results suggest that the greater Cd resistance in CF than in LF may be attributable to the greater capacity of CF to limit Cd uptake into shoots and complex Cd in cell walls and metal binding ligands, such as PCs and citric acid. However, the contributions of PCs and citric acid to Cd detoxification might be smaller than those in cell walls.

Copper (Cu) is an essential micronutrient required for growth and development of plants. However, excess Cu is toxic to plants. To understand the mechanisms involved in copper stress response, a proteomic approach was used to investigate the differences in Cu stress-induced protein expression between a Cu-tolerant variety (B1139) and a Cu-sensitive one (B1195) of rice. Rice seedlings were exposed to 8 μM Cu for 3 days, with plants grown in the normal nutrient solution containing 0.32 μM Cu serving as the control. Proteins were extracted from the roots and separated by two-dimensional PAGE. Thirty four proteins were identified using MALDI-TOF mass spectrometry. Thirty-four protein spots were found to be differently expressed in the Cu-stressed roots in at least one variety of rice, including those involved in antioxidative defense, redox regulation, stress response, sulfur and glutathione (GSH) metabolism, carbohydrate metabolism, signal transduction, and some other proteins with various functions. Nine proteins, including putative cysteine synthase, probable serine acetyltransferase 3, L-ascorbate peroxidase 1, putative glutathione S-transferase 2, and thioredoxin-like 3-3, exhibited a greater increase in response to Cu stress in the Cu-tolerant variety B1139 compared with the Cu–sensitive variety B1195. The majority of the proteins showing differential expression in response to Cu exposure are involved in the redox regulation, and sulfur and GSH metabolism, suggesting that these proteins, together with antioxidant enzymes, play an important role in the detoxification of excess Cu and maintaining cellular homeostasis.

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.

Protein extraction is a critical step in a two-dimensional electrophoresis (2-DE)-based proteomic analysis. Dimethyl sulfoxide (DMSO), a small organic molecule, is widely used as a solvent in biological sciences. In this study, we modified the cleanup step of the commonly used trichloroacetic acid (TCA)/acetone method of protein extraction by using 20 % DMSO/acetone as a solvent to wash TCA–acetone-precipitated pellets. The improved protocol (TCA/acetone–DMSO) was compared with the TCA/acetone and phenol extraction method based on the protein yield, the number of spots, and resolution on 2-DE maps. TCA/acetone–DMSO washing increased protein yield, and improved the resolution in 2-DE with less background, streaking, and smearing. This method also produced the highest number of protein spots. To our knowledge, the present study is the first to use DMSO as a cleanup solvent for TCA/acetone-precipitated proteins to enhance their quality prior to 2-DE.

Aims Many studies have proved that EDTA (ethylenediaminetetraacetic acid), EDDS ([S, S’]-ethylenediamine disuccinic acid), and other chelating agents significantly enhance phyto-extraction of copper (Cu) from soil. However, some key factors, such as changes in membrane permeability of root cells and subcellular distribution of Cu and Cu-EDDS complex in leaves and roots, remain unresolved.

We examined ameliorative effects of salicylic acid (SA) on two cadmium (Cd)-stressed legume crops with different Cd tolerances, viz. Phaseolus aureus (Cd sensitive) and Vicia sativa (Cd tolerant). Cd at 50 μM significantly increased the production of hydrogen peroxide (H2O2) and superoxide anion (O 2 ·− ) in root apoplasts of P. aureus and V. sativa. When comparing the two species, we determined that Cd-induced production of H2O2 and O 2 ·− was more pronounced in P. aureus root apoplasts than in V. sativa root apoplasts. V. sativa had higher activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) than P. aureus in root symplasts and apoplasts. Seed-soaking pretreatment with 100 μM SA decreased Cd-induced production of H2O2 and O 2 ·− in apoplasts of both species, and increased activities of symplastic and apoplastic SOD, symplastic APX, and apoplastic CAT under Cd stress. Hence, SA-induced Cd tolerances in P. aureus and V. sativa are likely associated with increases in symplastic and apoplastic antioxidant enzyme activities.

To study the mechanism of chelant-metal complexes to be absorbed into plant roots in the presence of different concentration chelating agents, the sites, pathways, and mechanism of absorption of Cu-EDDS complex ([S, S’]-ethylene diamine disuccinic acid) in maize (Zea mays L.) primary roots were systematically studied. The results showed that, at low concentrations of the Cu-EDDS complex (

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.

The concentrations of heavy metals in Phytolacca americana L. and corresponding soil samples from three contaminated sites and an uncontaminated site were studied. Hydroponic experiments were also conducted to investigate the Cd uptake ability and mechanism of P. americana. The field results showed that the average Cd concentration was 42 mg kg(-1) in P. americana leaves, with the highest concentration of 402 mg kg(-1) found at Datianwan. A significant relationship was observed between the concentrations of Cd in leaves and those of corresponding soils on a logarithmic scale. Under laboratory hydroponic conditions, the maximum Cd concentration in aerial tissues of P. americana was 637 mg kg(-1), under treatment with 100 microM Cd. The population from the uncontaminated site (Zijinshan) also had a remarkable ability to accumulate Cd in shoots to concentrations well in excess of 100 microM in the hydroponic experiment, similar to the population from contaminated site, suggesting that Cd accumulation is a constitutive trait of P. americana. In the presence of 100 microM Cd, the addition of polyethylene glycol decreased leaf transpiration, the shoot Cd concentration, and the shoot/root Cd concentration ratio. There was a significantly positive relationship between the shoot Cd concentration and the leaf transpiration of P. americana. A similar significant positive correlation was also obtained between the shoot/root Cd concentration and leaf transpiration. Moreover, pretreatment with 5 microM abscisic acid or 5 microM HgCl(2) significantly decreased the Cd concentration in P. americana shoots. These results suggest that transpiration has an important role in Cd accumulation in shoots of P. americana.

Using both histochemical and cytochemical methods, we investigated the effects of copper (Cu) on the production of hydrogen peroxide (H2O2) and superoxide anion (O2(.-)) in the leaves of the moss Plagiomnium cuspidatum. Cu treatment significantly increased the contents of total thiobarbituric acid-reactive substances and H2O2, as well as the activity of guaiacol peroxidase and superoxide dismutase (SOD). Native PAGE detected all three forms of SOD (Mn-SOD, Fe-SOD and CuZn-SOD) in P. cuspidatum, and the increase in the total SOD activity appeared to be mainly caused by an increase in CuZn-SOD activity. According to cytochemical results, H2O2-dependent CeCl3 precipitates were primarily localized in the plasma membranes and cell walls, and O2(.-) was chiefly localized on the inner side of the plasma membrane and in the cytoplasm surrounding the chloroplasts. Experiments using imidazole as an inhibitor of NADPH oxidase, N-N-diethyldithiocarbamate as an inhibitor of CuZn-SOD, and 1,2-dihydroxybenzene-3,5-disulphonic acid as an O2(.-) scavenger indicated that a partial source of H2O2 in the cell walls may be NADPH oxidase. The results also showed that peroxidase (POD) is involved in the detoxification of H2O2. Increased POD activity induced by Cu may remove excess H2O2 caused by Cu.

To study tolerance of rice (Oryza sativa L.) to excessive copper (Cu), 167 varieties were screened for Cu tolerance or sensitivity. Based on the elongation of the root, No. 1139 and No. 1195 were chosen as Cu-tolerant and Cu-sensitive variety. Compared with the control (0.32 μ mol L− 1 Cu), treatments with 5 and 8 μ mol L− 1 Cu for 10 days had no significant effect on the root and shoot dry masses of Cu-tolerant variety No. 1139, but significantly decreased root and shoot dry masses of variety No. 1195. Copper at 5 and 8 μ mol L− 1 also significantly decreased contents of chlorophyll and carotenoid and the ratio of variable to maximal chlorophyll fluorescence (Fv/Fm) in the leaves of the Cu-sensitive variety No. 1195. Roots and shoots of variety No. 1139 contained significantly higher the concentration of Cu than variety No. 1195 exposed to 5 and 8 μ mol L− 1 Cu. However, variety No. 1139 had higher Cu proportions on the cell walls in shoots and roots than variety No. 1195.

The effect of Al stress on H2O2 production of rice (Oryza sativa L.) seedlings and difference in responses of antioxidant enzymes between Al-tolerant variety (Azucena) and Al-sensitive rice one (IR 64) were investigated. Aluminum-induced H2O2 production and malondialdehyde (MDA) content were more pronounced for IR 64 than for Azucena. In the presence of 2 mM Al, addition of 10 mM imidazole (inhibitor of NADPH oxidase) and 1 mM azide (inhibitor of peroxidase) significantly decreased H2O2 production by 16% and 43% for Azucena, and 21% and 68% for IR 64, respectively. Under Al treatment, the Al-tolerant variety Azucena had significantly higher activities of catalase, ascorbate peroxidase, dehydroascorbate reducase, glutathione peroxidase and glutathione reductase, and higher concentrations of reduced glutathione than the Al-sensitive one IR 64. Treatment with buthionine sulfoximine, a specific inhibitor of GSH synthesis, significantly increased H2O2 production in both varieties in the presence and absence of Al. In contrast, the treatment with GSH significantly decreased the production of H2O2 induced by Al stress. Results suggest that GSH may play an important role in scavenging H2O2 caused by Al stress.

Aquatic macrophytes can absorb heavy metals either from sediments via the root system, from the water phase by leaves, or from both sources. In this study, cadmium accumulation and distribution in the aquatic plant Hydrilla verticillata were investigated, with a focus on the role of roots. Results showed that leaves of H. verticillata had a higher Cd concentration than roots when intact plants were grown in sediments and solutions containing Cd. Cadmium can significantly decrease the leaf chlorophyll content, and the leaves of intact plants with roots had lower chlorophyll contents than the leaves of detached ones without roots due to the transfer effect of roots. The majority of the Cd accumulated in leaves of H. verticillata was bound to the cell walls. When roots were submerged in a solution containing Cd, with shoots in a control solution without Cd, the Cd concentrations in leaves were considerably lower than in roots. In contrast, Cd was almost undetectable in roots when the shoots were submerged in a solution containing Cd, with roots in the control solution. Compared to the leaves and stems of detached shoots without roots, the concentrations of Cd were much higher in the leaves and stems of intact plants with roots. It is suggested that the roots of intact plants absorb Cd and transfer it to leaves and that more Cd is removed from the solution by intact plants.

Soil that is co-contaminated with heavy metals (HMs) and polycyclic aromatic hydrocarbons (PAHs) is difficult to bioremediate due to the ability of toxic metals to inhibit PAH degradation by bacteria. We demonstrated the resistance mechanisms to Cu(II) and Cd(II) of two newly isolated strains of Sphingobium sp. PHE-SPH and Ochrobactrum sp. PHE-OCH and further tested their potential application in the bioremediation of HM-phenanthrene (PhA) co-contaminated sites. The PHE-SPH and PHE-OCH strains tolerated 4.63 and 4.34 mM Cu(II) and also showed tolerance to 0.48 and 1.52 mM Cd(II), respectively. Diverse resistance patterns were detected between the two strains. In PHE-OCH cells, the maximum accumulation of Cu(II) occurred in the cell wall, while the maximum accumulation was in the cytoplasm of PHE-SPH cells. This resulted in a sudden suppression of growth in PHE-OCH and a gradual inhibition in PHE-SPH as the concentration of Cu(II) increased. Organic acid production was markedly higher in PHE-OCH than in PHE-SPH, which may also have a role in the resistance mechanisms, and contributes to the higher Cd(II) tolerance of PHE-OCH. The factors involved in the absorption of Cu(II) or Cd(II) in PHE-SPH and PHE-OCH were identified as proteins and carbohydrates by Fourier transform infrared (FT-IR) spectroscopy. Furthermore, both strains showed the ability to efficiently degrade PhA and maintained this high degradation efficiency under HM stress. The high tolerance to HMs and the PhA degradation capacity make Sphingobium sp. PHE-SPH and Ochrobactrum sp. PHE-OCH excellent candidate organisms for the bioremediation of HM-PhA co-contaminated sites.

The non-selective apoplastic passage of Cu and Cu-citrate complexes into the root stele of monocotyledonous corn and dicotyledonous soybean was investigated using an inorganic-salt-precipitation technique. Either Cu ions or Cu-citrate complexes were drawn into root through the apoplast from the root growth medium, and K4[Fe(CN)6] was subsequently perfused through xylem vessels or the entire root cross section. Based on microscopic identification of the reddish-brown precipitates of copper ferrocyanide in the cell walls of the xylem of corn and soybean roots, Cu(2+) passed through the endodermal barrier into the xylem of both species. When the solution containing 200 μM CuSO4 and 400 μM sodium citrate (containing 199.98 μM Cu-citrate, 0.02 μM Cu(2+)) was drawn via differential pressure gradients into the root xylem while being perfused with K4[Fe(CN)6] through the entire root cross-section, reddish-brown precipitates were observed in the walls of the stele of soybean, but not corn root. However, when a CuSO4 solution containing 0.02 or 0.2 μM free Cu(2+) was used, no reddish-brown precipitates were detected in the stele of either of the two plants. Results indicated that endodermis was permeable to Cu-citrate complexes in primary roots of soybean, but not corn. The permeability of the endodermal barrier to the Cu-citrate complex may vary between dicotyledonous and monocotyledonous plants, which has considerable implications for chelant-enhanced phytoextraction.

Cadmium (Cd) toxictity and possible role of salicylic acid (SA) in alleviating Cd-induced toxicity were investigated on ornamental hydrophyte Iris hexagona. Compared to the control, treatments with 100 and 500 µM Cd for 7 days significantly decreased dry weight, the contents of chlorophyll, photosynthetic parameters, and increased the content of thiobarbituric acid reactive substance. Pretreatment of the roots of I. hexagona seedlings with 1 µM SA before Cd exposure may increase dry weight, photosynthetic rate, activities of antioxidant enzymes, improve the cell ultrastructure and protect plants from Cd-induced oxidative stress damage. However, SA pretreatment had no significant effect on Cd concentrations in the leaves and roots. It is suggested that SA-induced Cd tolerances in I. hexagona are likely associated with increases in antioxidant enzyme activities and vacuolar compartmentation, rather than Cd uptake.