Bacterial heavy metal transporter MerC increases mercury accumulation in Arabidopsis thaliana

This study evaluated the feasibility of transgenic Arabidopsis engineered to express the bacterial heavy metal transporter MerC for the phytoremediation of mercury pollution. MerC, MerC–SYP121, or MerC–AtVAM3 proteins were found to be expressed in leaf segments of transgenic plants using an anti-MerC antibody immunostaining method. By sucrose density gradient centrifugation and immunoblotting analyses, MerC, MerC–SYP121, and MerC–AtVAM3 were found to localized in the Golgi apparatus, plasma membrane, and vacuole membrane, respectively. Transgenic Arabidopsis plants that expressed merC–SYP121 were more resistant to mercury and accumulated significantly more of this metal than wild-type Arabidopsis. These results demonstrated that expression of the bacterial heavy metal transporter MerC promoted the transport and accumulation of mercury in transgenic Arabidopsis, which may be a useful method for improving plants for the phytoremediation of mercury pollution.     

AtATM3 is involved in heavy metal resistance in Arabidopsis

AtATM3, an ATP-binding cassette transporter of Arabidopsis (Arabidopsis thaliana), is a mitochondrial protein involved in the biogenesis of iron-sulfur clusters and iron homeostasis in plants. Our gene expression analysis showed that AtATM3 is up-regulated in roots of plants treated with cadmium [Cd(II)] or lead (II); hence, we investigated whether this gene is involved in heavy metal tolerance. We found that AtATM3-overexpressing plants were enhanced in resistance to Cd, whereas atatm3 mutant plants were more sensitive to Cd than their wild-type controls. Moreover, atatm3 mutant plants expressing 35S promoter-driven AtATM3 were more resistant to Cd than wild-type plants. Since previous reports often showed that the cytosolic glutathione level is positively correlated with heavy metal resistance, we measured nonprotein thiols (NPSH) in these mutant plants. Surprisingly, we found that atatm3 contained more NPSH than the wild type under normal conditions. AtATM3-overexpressing plants did not differ under normal conditions, but contained less NPSH than wild-type plants when exposed to Cd(II). These results suggest a role for AtATM3 in regulating cellular NPSH level, a hypothesis that was further supported by our gene expression study. Genetic or pharmacological inhibition of glutathione biosynthesis led to the elevated expression of AtATM3, whereas expression of the glutathione synthase gene GSH1 was increased under Cd(II) stress and in the atatm3 mutant. Because the closest homolog of AtATM3 in fission yeast (Schizosaccharomyces pombe), HMT1, is a vacuolar membrane-localized phytochelatin-Cd transporter, it is tempting to speculate that glutathione-Cd(II) complexes formed in the mitochondria are exported by AtATM3. In conclusion, our data show that AtATM3 contributes to Cd resistance and suggest that it may mediate transport of glutamine synthetase-conjugated Cd(II) across the mitochondrial membrane.     

Mechanisms of metal resistance in plants: aluminum and heavy metals

Plants have evolved sophisticated mechanisms to deal with toxic levels of metals in the soil. In this paper, an overview of recent progress with regards to understanding fundamental molecular and physiological mechanisms underlying plant resistance to both aluminum (Al) and heavy metals is presented. The discussion of plant Al resistance will focus on recent advances in our understanding of a mechanism based on Al exclusion from the root apex, which is facilitated by Al-activated exudation of organic acid anions. The consideration of heavy metal resistance will focus on research into a metal hyperaccumulating plant species, the Zn/Cd hyperaccumulator, Thlaspi caerulescens, as an example for plant heavy metal research. Based on the specific cases considered in this paper, it appears that quite different strategies are used for Al and heavy metal resistance. For Al, our current understanding of a resistance mechanism based on excluding soil-borne Al from the root apex is presented. For heavy metals, a totally different strategy based on extreme tolerance and metal hyperaccumulation is described for a hyperaccumulator plant species that has evolved on naturally metalliferous soils. The reason these two strategies are the focus of this paper is that, currently, they are the best understood mechanisms of metal resistance in terrestrial plants. However, it is likely that other mechanisms of Al and/or heavy metal resistance are also operating in certain plant species, and there may be common features shared for dealing with Al and heavy resistance. Future research may uncover a number of novel metal resistance mechanisms in plants. Certainly the complex genetics of Al resistance in some crop plant species, such as rice and maize, suggests that a number of presently unidentified mechanisms are part of an overall strategy of metal resistance in crop plants.     

Resistance to and uptake of heavy metals in mushrooms

Resistance levels to heavy metals in 21 mushrooms obtained mainly from Japan, but also from Thailand, were tested. The mushrooms were 7 Pleurotus species, 2 Pycnoporus and Pholiota species and one each of Agrocybe, Cryptoporus, Coriolus, Inonotus, Lampteromyces, Grifola, Flammulina, Lyophyllum, Agaricus, and Polyporus species. The Pleurotus species strains showed higher resistance to the heavy metals, copper, cadmium, zinc, nickel, cobalt, mercury than the other species. Pleurotus ostreatus exhibited the highest resistance to all these heavy metals. Pholiota species, Flammulina veltipes, Lyophyllum ulmarium, Agaricus bisporus and Polyporus arcularius were rather sensitive to all the metals tested. The accumulation of copper, zinc and cadmium in Pleurotus ostreatus was studied. The uptake of heavy metals into the mycelia of P. ostreatus increased proportionally to an increasing concentration of these metals in the medium.     

Wounding enhances expression of AtSUC3, a sucrose transporter from Arabidopsis sieve elements and sink tissues

The Arabidopsis AtSUC3 gene encodes a sucrose (Suc) transporter that differs in size and intron number from all other Arabidopsis Suc transport proteins. Each plant species analyzed so far possesses one transporter of this special type, and several functions have been discussed for these proteins, including the catalysis of transmembrane Suc transport, and also Suc sensing and regulation of other Suc transporters. Here, we show that the AtSUC3 protein is localized in the sieve elements of the Arabidopsis phloem and is not colocalized with the companion cell-specific AtSUC2 phloem loader. Even stronger AtSUC3 expression is observed in numerous sink cells and tissues, such as guard cells, trichomes, germinating pollen, root tips, the developing seed coat, or stipules. Moreover, AtSUC3 expression is strongly induced upon wounding of Arabidopsis tissue. The physiological role of AtSUC3 in these different cells and tissues is discussed.     

Functional characterization and expression analysis of a glutathione transporter, BjGT1, from Brassica juncea: evidence for regulation by heavy metal exposure

Glutathione and its derivatives play an important role in the tolerance of plants against heavy metals. A glutathione transporter, BjGT1 (AJ561120), was cloned and functionally characterized from Brassica juncea, a plant which may be used for phytoremediation. The full‐length BjGT1 cDNA showed homology with the high affinity glutathione transporter HGT1 from Saccharomyces cerevisiae and shares 92% identity with a putative glutathione transporter from A. thaliana (At4g16370). When expressed in the S. cerevisiae hgt1Δ strain, BjGT1 complemented the mutant on medium with glutathione as the only sulphur source and mediated the uptake of [³H]GSH. Immunoblot analysis with a peptide‐specific antiserum directed against a C‐terminal sequence revealed high BjGT1 expression in leaf tissue and relatively low expression in stem tissue, whereas BjGT1 protein was not detectable in root tissue. The amounts of BjGT1 mRNA and protein were analysed during a 6 d exposure of B. juncea to 25 µm Cd(NO₃)₂. BjGT1 mRNA was strongly induced by cadmium in stems and leaves. Unexpectedly, the amount of BjGT1 protein in leaves showed a pronounced decrease with a minimum after 96 h of Cd exposure, followed by partial recovery. The strong regulation of BjGT1 by cadmium suggests a role of this glutathione transporter during heavy metal exposure.     

种子内生菌增强宿主植物重金属抗性的功能机制研究进展

种子是植物的繁殖器官,其内定殖有一定数量的内生菌,种子内生菌通过垂直传播成为新生植物组织内最早定殖的微生物,对连续几代植物内生菌群落的形成起着决定性作用,并在植物抗逆方面发挥着重要作用.本文对种子内生菌与宿主植物重金属抗性之间的关系及其功能机制进行综述,并对下一步研究方向予以展望.     

Retromobilization of heavy metal resistance genes in unpolluted and heavy metal polluted soil

Abstract: Retromobilization of the nonconjugative (Tra⁻Mob⁺) IncQ vector, pMOL155, and the non-mobilizable (Tra⁻Mob⁻) vector, pMOL149, by means of the IncP plasmids RP4 and pULB113 (RP4::Mu3A), was studied in plate matings and in soil microcosms, and compared with direct and triparental mobilization. Both vectors harbour the czc genes, originating from Alcaligenes eutrophus , which code for resistance to Co, Zn, and Cd. The donor of the czc genes was Escherichia coli which did not express these genes. The recipient, Alcaligenes eutrophus , expressed the czc genes very well. Retromobilization, direct and triparental mobilization of pMOL155 was observed in sterile soil. Both the addition of nutrients and heavy metals significantly enhanced the number of (retro)transconjugants. Retromobilization was also detected in nutrient amended nonsterile soil, but the presence of the autochthonous soil biota strongly reduced the number of retrotransconjugants and also prevented their increase upon application of heavy metals to the soil. Retromobilization of the czc genes, cloned in pMOL149, by using pULB113 was also observed, yet only in sterile, nutrient amended, heavy metal polluted soil.     

Remobilization of toxic heavy metals adsorbed to bacterial wall-clay composites

Significant quantities of Ag(I), Cu(II), and Cr(III) were bound to isolated Bacillus subtilis 168 walls, Escherichia coli K-12 envelopes, kaolinite and smectite clays, and the corresponding organic material-clay aggregates (1:1, wt/wt). These sorbed metals were leached with HNO3, Ca(NO3)2, EDTA, fulvic acid, and lysozyme at several concentrations over 48 h at room temperature. The remobilization of the sorbed metals depended on the physical properties of the organic and clay surfaces and on the character and concentration of the leaching agents. In general, the order of remobilization of metals was Cr much less than Ag less than Cu. Cr was very stable in the wall, clay, and composite systems; pH 3.0, 500 microM EDTA, 120-ppm [mg liter-1] fulvic acid, and 160-ppm Ca remobilized less than 32% (wt/wt) of sorbed Cr. Ag (45 to 87%) and Cu (up to 100%) were readily removed by these agents. Although each leaching agent was effective at mobilizing certain metals, elevated Ca or acidic pH produced the greatest overall mobility. The organic chelators were less effective. Lysozyme digestion of Bacillus walls remobilized Cu from walls and Cu-wall-kaolinite composites, but Ag, Cr, and smectite partially inhibited enzyme activity, and the metals remained insoluble. The extent of metal remobilization was not always dependent on increasing concentrations of leaching agents; for example, Ag mobility decreased with some clays and some composites treated with high fulvic acid, EDTA, and lysozyme concentrations. Sometimes the organic material-clay composites reacted in a manner distinctly different from that of their individual counterparts; e.g., 25% less Cu was remobilized from wall- and envelope-smectite composites than from walls, envelopes, or smectite individually in 500 microM EDTA. Alternatively, treatment with 160-ppm Ca removed 1.5 to 10 times more Ag from envelope-kaolinite composites than from the individual components. The particle size of the deposited metal may account for some of the stability changes; those metals that formed large, compact aggregates (Cr and Ag) as seen by transmission electron microscopy were less likely to be remobilized. In summary, it is apparent that remobilization of toxic heavy metals in sediments, soils, and the vadose zone is a complicated issue. Predictions based on single inorganic or organic component systems are too simplistic.     

Bacterial resistance and detoxification of heavy metals

Microbial cells have resistances to essentially all of the toxic heavy metals of the Periodic Table. In bacterial cells, the genetic determinants of these resistances are frequently found on small extrachromosomal plasmids and transposons. Sometimes the resistances are associated with detoxifying enzymes. This is true for the Hg²⁺ → Hg⁰ reductase, the As³⁺ → As⁵⁺ oxidase and the Cr⁶⁺ → Cr³⁺ reductase. In other cases, such as As⁵⁺, Ag⁺ and Cd²⁺, no change in redox state occurs but, rather, uptake and transport differences accompany resistance determinants. This article summarizes what is known of bacterial metal resistances for which enzymatic detoxification is known to be the mechanism of resistance. The characteristics and functions of the enzymes are described, as well as a summary of the newer DNA sequence analysis (basic science) and bench-scale efforts (applied science) for the mercuric resistance system.     

Development of a broad-spectrum fluorescent heavy metal bacterial biosensor

Bacterial biosensors can measure pollution in terms of their actual toxicity to living organisms. A recombinant bacterial biosensor has been constructed that is known to respond to toxic levels of Zn²⁺, Cd²⁺ and Hg²⁺. The zinc regulatory gene zntR and zntA promoter from znt operon of E. coli have been used to trigger the expression of GFP reporter protein at toxic levels of these ions. The sensor was induced with 3–800?ppm of Zn²⁺, 0.005–4?ppm of Cd²⁺ and 0.001–0.12?ppm of Hg²⁺ ions. Induction studies were also performed in liquid media to quantify GFP fluorescence using fluorimeter. To determine the optimum culture conditions three different incubation periods (16, 20 and 24?h) were followed. Results showed an increased and consistent fluorescence in cells incubated for 16?h. Maximum induction for Zn²⁺, Cd²⁺ and Hg²⁺ was observed at 20, 0.005 and 0.002?ppm, respectively. The pPROBE-zntR-zntA biosensor reported here can be employed as a primary screening technique for aquatic heavy metal pollution.