Zinc Tolerance Research Articles

Metal accumulation by Arabidopsis halleri subsp. gemmifera at a limestone mining site

Arabidopsis halleri subsp. gemmifera, a close wild relative of A. thaliana in eastern Asia, is an important model species of Brassicaceae used to study cadmium (Cd) and zinc (Zn) tolerance and hyperaccumulation in plants. To investigate the effects of soil factors on metal accumulation in this plant, we collected plants and rhizosphere soil samples from a limestone mining site on Mount Ibuki, and compared them with those collected from non-calcareous soil in Japan. Irrespective of the sampling site, all the plants efficiently accumulated Cd in shoot tissues. The plants growing on non-calcareous soil also accumulated Zn in shoot tissues, but shoot Zn concentration in plants growing on calcareous soils was below the level required for hyperaccumulators. The pH of calcareous soil samples was between 7.68 and 8.21. Total Zn contents were similar in calcareous and non-calcareous soils, but the amounts of Zn extractable by 0.1 M HCl were lower in calcareous than in non-calcareous soil. These results indicate that the properties of calcareous soil affect metal accumulation in plants.

A moonlighting function of Mycobacterium smegmatis Ku in zinc homeostasis?

Ku protein participates in DNA double-strand break repair via the nonhomologous end-joining pathway. The three-dimensional structure of eukaryotic Ku reveals a central core consisting of a β-barrel domain and pillar and bridge regions that combine to form a ring-like structure that encircles DNA. Homologs of Ku are encoded by a subset of bacterial species, and they are predicted to conserve this core domain. In addition, the bridge region of Ku from some bacteria is predicted from homology modeling and sequence analyses to contain a conventional HxxC and CxxC (where x is any residue) zinc-binding motif. These potential zinc-binding sites have either deteriorated or been entirely lost in Ku from other organisms. Using an in vitro metal binding assay, we show that Mycobacterium smegmatis Ku binds two zinc ions. Zinc binding modestly stabilizes the Ku protein (by ∼3°C) and prevents cysteine oxidation, but it has little effect on DNA binding. In vivo, zinc induces significant upregulation of the gene encoding Ku (∼sixfold) as well as a divergently oriented gene encoding a predicted zinc-dependent MarR family transcription factor. Notably, overexpression of Ku confers zinc tolerance on Escherichia coli. We speculate that zinc-binding sites in Ku proteins from M. smegmatis and other mycobacterial species have been evolutionarily retained to provide protection against zinc toxicity without compromising the function of Ku in DNA double-strand break repair.

Effects of nitric oxide on zinc tolerance of the submerged macrophyte Hydrilla verticillata

Heavy metal pollution has become an increasingly recognized threat to the aquatic environment. In response to heavy metal stresses, various terrestrial plants produce the hormone nitric oxide (NO), but minimal information is available about the role of NO in aquatic macro- phytes under high zinc (Zn) stress. In this study, we measured physiochemical parameters in the leaves of Hydrilla verticillata exposed to Zn 2+ (10 mg l �1 ) alone or combined with NO (supplied as sodium nitroprusside, SNP) for 1 d and 4 d. Excess Zn disturbed nutrient uptake and altered the redox status of the aquatic plants. NO application partially reversed negative impacts of Zn stress on the contents of photosynthetic pigments, malondialdehyde, nutrient concentration, ascorbic acid, and the activity of antioxidant enzymes. However, the metabolites of SNP did not have similar effects. After treatments for 4 d, plants were further cultured in a solution containing Zn 2+ (10 mg l �1 ) for 10 d. NO application partially reversed Zn-induced negative effects in parameters such as nutrient uptake, antioxidant enzymatic activities, and biomass, as compared to treatments with Zn alone. These results indicate that NO supply could mitigate Zn stress in H. verticillata, as a defense mechanism of the plant against Zn toxicity.

Neotyphodium Endophyte Changes Phytoextraction of Zinc in Festuca arundinacea and Lolium perenne

The effect of Neotyphodium endophytes on growth parameters and zinc (Zn) tolerance and uptake was studied in two grass species of Festuca arundinacea and Lolium perenne. Plants were grown under different Zn concentrations (control, 200, 400, 800, and 1800 mg kg−1) in potted soil for 5 months. The results showed that the number of plant tillers was 85 and 51% greater in endophyte infected Festuca (FaEI) and Lolium (LpEI), respectively, compared to their endophyte free (EF) plants. Roots and shoots dry weights in infected Festuca were 87 and 9% greater than non-infected counterparts but in opposite, EF Lolium had 47 and 8% greater root and shoot dry weights than LpEI. Endophyte infected Festuca and Lolium improved chlorophyll fluorescence as Fv/Fm at high concentrations of Zn, showing their better chlorophyll functions and significant reduction of Zn stress in endophyte infected plants. Shoots of endophyte infectedFestuca had 82% greater concentration of Zn than EF Festuca when grown in soil containing 1800 mg kg−1 Zn. Festuca and Lolium may tolerate high Zn concentration in soil without reduction in shoot and root growth. Endophyte infection in Festuca may help the grass accumulate and transport more Zn in aboveground parts under Zn-stress, thereby aiding phytoremediation of contaminated soils.

The nuclear magnetic resonance solution structure of the synthetic AhPDF1.1b plant defensin evidences the structural feature within the γ-motif.

Plant defensins (PDF) are cysteine-rich peptides that are major actors in the innate immunity in plants. Besides their antifungal activity, some PDF such as Arabidopsis halleri PDF1.1b confer zinc tolerance in plants. Here we present (i) an efficient protocol for the production of AhPDF1.1b by solid-phase peptide synthesis followed by controlled oxidative folding to obtain the highly pure native form of the defensin and (ii) the three-dimensional (3D) nuclear magnetic resonance structure of AhPDF1.1b, the first 3D structure of plant defensin obtained with a synthetic peptide. Its fold is organized around the typical cysteine-stabilized α-helix β-sheet motif and contains the γ-core motif involved in the antifungal activity of all plant defensins. On the basis of our structural analysis of AhPDF1 defensins combined with previous biological data for antifungal and zinc tolerance activities, we established the essential role of cis-Pro41 within the γ-core. In fact, the four consecutive residues (Val39-Phe40-Pro41-Ala42) are strictly conserved for plant defensins able to tolerate zinc. We hypothesized that structural and/or dynamic features of this sequence are related to the ability of the defensin to chelate zinc.

Zinc Accumulation and Tolerance in Solanum nigrum are Plant Growth Dependent

Zinc tolerance, accumulation, and organic acid production by Solanum nigrum, a known Zn accumulator, was studied during pre- and post-flowering stages of development. The plants, when challenged with Zn concentrations lethal to plantlets, showed an increase in tolerance from pre-flowering to post-flowering, which was accompanied by a reduction of Zn translocation to the aerial plant parts. Treatment with Zn induced a differential response in organic acids according to the plant organ and developmental stage. In the roots, where Zn concentrations were similar in pre- and post-flowering plants, a general decrease in organic acid in pre-flowering roots contrasted with the increase observed in post-flowering plants. In the stems, Zn induced a generalized increase in organic acids at both growth stages while in the leaves, a slight increase in malic and shikimic was observed in pre-flowering plants and only shikimic acid levels were significantly increased in post-flowering plants. This work shows that Zn accumulation and tolerance in S. nigrum vary during plant development – an observation that may be important to improve the efficiency of phytoremediation approaches. Furthermore, the data suggest the involvement of specific organic acids in this response.

Genomic analyses of metal resistance genes in three plant growth promoting bacteria of legume plants in Northwest mine tailings, China

To better understand the diversity of metal resistance genetic determinant from microbes that survived at metal tailings in northwest of China, a highly elevated level of heavy metal containing region, genomic analyses was conducted using genome sequence of three native metal-resistant plant growth promoting bacteria (PGPB). It shows that: Mesorhizobium amorphae CCNWGS0123 contains metal transporters from P-type ATPase, CDF (Cation Diffusion Facilitator), HupE/UreJ and CHR (chromate ion transporter) family involved in copper, zinc, nickel as well as chromate resistance and homeostasis. Meanwhile, the putative CopA/CueO system is expected to mediate copper resistance in Sinorhizobium meliloti CCNWSX0020 while ZntA transporter, assisted with putative CzcD, determines zinc tolerance in Agrobacterium tumefaciens CCNWGS0286. The greenhouse experiment provides the consistent evidence of the plant growth promoting effects of these microbes on their hosts by nitrogen fixation and/or indoleacetic acid (IAA) secretion, indicating a potential in-site phytoremediation usage in the mining tailing regions of China.

Zinc tolerance and zinc removal ability of living and dried biomass of Desmodesmus communis.

Effects of zinc on growth, cell morphology, oxidative stress, and zinc removal ability of the common phytoplankton species Desmodesmus communis were investigated at a concentration range of 0.25-160 mg L(-1) zinc. Cell densities and chlorophyll content decreased in treated cultures, changes in coenobia morphology and elevated lipid peroxidation levels appeared above 2.5 mg L(-1) zinc. The most effective zinc removal was observed at 5 mg L(-1) zinc concentration, while maximal amount of removed zinc appeared in 15 mg L(-1) zinc treated culture. Removed zinc is mainly bound on the cell surface. Dead biomass adsorbed more zinc than living biomass relative to unit of dry mass, but living biomass was more effective, relative to initial zinc content. This study comprehensively examines the zinc tolerance and removal ability of D. communis and demonstrates, in comparison with published literature, that these characteristics of different isolates of the same species can vary within a wide range.

Bioavailability and toxicity of zinc from contaminated freshwater sediments: Linking exposure-dose–response relationships of the freshwater bivalve Hyridella australis to zinc-spiked sediments

To evaluate the use of the freshwater bivalve Hyridella australis as a potential biomonitor for zinc contamination in freshwater sediments, the bioavailability and toxicity of zinc contaminated sediments (low 44±5, medium 526±41, high 961±38μg/g dry mass) were investigated in laboratory microcosms for 28 days by examining H. australis exposure-dose–response relationships. Zinc concentrations in sediments and surface waters were measured as zinc exposure. Zinc in whole organism soft body tissues and five individual tissues were measured as organism zinc dose. Sub-cellular localisation of zinc in hepatopancreas tissues was investigated to further understand the zinc handling strategies and tolerance of H. australis. Total antioxidant capacity, lipid peroxidation and lysosomal membrane stability were measured in hepatopancreas tissues as zinc induced biomarker responses. Accumulated zinc concentrations in whole body tissues of H. australis reflected the zinc exposure and exhibited exposure dependent zinc accumulation at day 28. Gills accumulated significantly higher zinc concentrations than other tissues, however, no significant differences in zinc accumulation between treatments were detected for any of the individual tissues analysed. Analysis of individual tissue zinc concentrations, therefore, may not offer any advantages for monitoring bioavailable zinc in freshwater environments with this organism. Relationships between tissue zinc and calcium concentration suggest accumulation of zinc by H. australis may have occurred as an analogue of calcium which is a major constituent in shell and granules of unionid bivalves. A high percentage of accumulated zinc in the hepatopancreas tissues was detoxified and stored in metallothionein like proteins and metal rich granules. Of the zinc accumulated in the biologically active metal pool, 59–70% was stored in the lysosome+microsome fraction. At the concentrations tested, increasing zinc exposure resulted in decreasing total antioxidant capacity and measurable increases in the sublethal effects, lipid peroxidation and lysosomal membrane destabilisation, were observed. Based on exposure-dose analysis, H. australis partially regulates zinc uptake and weakly exhibits bioavailability of zinc in freshwater environments, however, exposure-response analysis shows zinc induced toxicological effects, suggesting the potential of this organism as a biomonitor for zinc in heavily contaminated freshwater environments.

Variation in copper and zinc tolerance and accumulation in 12 willow clones: implications for phytoextraction.

Willows (Salix spp.) have shown high potential for the phytoextraction of heavy metals. This study compares variations in copper (Cu) and zinc (Zn) tolerance and accumulation potential among 12 willow clones grown in a nutrient solution treated with 50 μmol/L of Cu or Zn, respectively. The results showed differences in the tolerance and accumulation of Cu and Zn with respect to different species/clones. The biomass variation among clones in response to Cu or Zn exposure ranged from the stimulation of growth to inhibition, and all of the clones tested showed higher tolerance to Cu than to Zn. The clones exhibited less variation in Cu accumulation but larger variation in Zn accumulation. Based on translocation factors, it was found that most of the Cu was retained in the roots and that Zn was more mobile than Cu for all clones. It is concluded that most willow clones are good accumulators of Zn and Cu.

Ionising Radiation in Modulating Zinc Tolerance Potential of Aspergillus niger

The present study describes the potential of gamma rays in enhancing zinc tolerance of Aspergillus niger. Gamma exposed group of A. niger showed 1.82 times more zinc tolerance than the unirradiated ones. Gamma exposed A. niger grown in different zinc enriched media, showed increase in growth (expressed in terms of colony forming unit), higher efficiency in zinc uptake and removal, when compared to that of their unirradiated counterparts. The present investigation throws light towards utilizing gamma radiation for formulating more metal-tolerant fungi for bioremediation.

Root-secreted nicotianamine from Arabidopsis halleri facilitates zinc hypertolerance by regulating zinc bioavailability.

Hyperaccumulators tolerate and accumulate extraordinarily high concentrations of heavy metals. Content of the metal chelator nicotianamine (NA) in the root of zinc hyperaccumulator Arabidopsis halleri is elevated compared with nonhyperaccumulators, a trait that is considered to be one of the markers of a hyperaccumulator. Using metabolite-profiling analysis of root secretions, we found that excess zinc treatment induced secretion of NA in A. halleri roots compared with the nonhyperaccumulator Arabidopsis thaliana. Metal speciation analysis further revealed that the secreted NA forms a stable complex with Zn(II). Supplying NA to a nonhyperaccumulator species markedly increased plant zinc tolerance by decreasing zinc uptake. Therefore, NA secretion from A. halleri roots facilitates zinc hypertolerance through forming a Zn(II)-NA complex outside the roots to achieve a coordinated zinc uptake rate into roots. Secretion of NA was also found to be responsible for the maintenance of iron homeostasis under excess zinc. Together our results reveal root-secretion mechanisms associated with hypertolerance and hyperaccumulation.

The sensitivity of two Monoraphidium species to zinc: their possible future role in bioremediation

Effects of zinc on growth, cell morphology, oxidative stress responses and zinc removal activity of two common phytoplankton species, Monoraphidium pusillum (Printz) Komárková-Legnerová and Monoraphidium griffithii (Berkeley) Komárková-Legnerová were investigated at a concentration range of 0.2–160 mg l−1 zinc. Cell densities and chlorophyll content decreased compared with controls in cultures of both species, effective concentrations causing 50 % growth inhibition within 72 h on the basis of cell numbers were 33.69 and 25.63 mg l−1 zinc for M. pusillum and M. griffithii, respectively. Changes in cell morphology and elevated lipid peroxidation levels appeared in zinc-treated cultures of both species, but only at higher (>10 mg l−1) zinc concentrations. The most effective zinc removal appeared at 20 and 10 mg l−1 zinc concentration for M. pusillum and M. griffithii, respectively. Removed zinc is mainly bound on the cell surface in the case of both species. This study provides new data for the zinc tolerance and zinc removal ability of the green algae M. pusillum and M. griffithii and shows that green algal species common in surface waters could have zinc tolerance and zinc-binding abilities, which makes them feasible in treatment of waters contaminated with 10–20 mg l−1 zinc.

Improved zinc tolerance of tobacco by transgenic expression of an allene oxide synthase gene from hexaploid wheat

Allene oxide synthase (AOS, EC 4.2.1.92) is the first specific jasmonate biosynthetic pathway gene. In this study, a full-length cDNA of AOS gene was cloned from common wheat nannong 9918. The gene contains an open reading frame (1,446 bp) encoding 418 amino acids. Comparative and bioinformatic analysis revealed that the deduced protein of TaAOS was highly homologous to AOSs from other plant species. The transcript of TaAOS was found to be abundantly expressed in the flag leaves, and was up-regulated by the inoculation of B. Graminis (DC.) E.O. Speer f. sp. Tritici (Bgt) in wheat leaves. In addition, it was also induced by high concentration of NaCl and ZnCl2. When TaAOS was overexpressed in tobacco leaves via Agrobacterium tumefaciens infection, the transgenic tobacco plants displayed stronger tolerance to ZnCl2 stress compared to transgenic GFP plants. Taken together, the above facts demonstrated that TaAOS may play a role in response to diverse stresses in plants.

Intron retention in the 5'UTR of the novel ZIF2 transporter enhances translation to promote zinc tolerance in arabidopsis.

Root vacuolar sequestration is one of the best-conserved plant strategies to cope with heavy metal toxicity. Here we report that zinc (Zn) tolerance in Arabidopsis requires the action of a novel Major Facilitator Superfamily (MFS) transporter. We show that ZIF2 (Zinc-Induced Facilitator 2) localises primarily at the tonoplast of root cortical cells and is a functional transporter able to mediate Zn efflux when heterologously expressed in yeast. By affecting plant tissue partitioning of the metal ion, loss of ZIF2 function exacerbates plant sensitivity to excess Zn, while its overexpression enhances Zn tolerance. The ZIF2 gene is Zn-induced and an intron retention event in its 5′UTR generates two splice variants (ZIF2.1 and ZIF2.2) encoding the same protein. Importantly, high Zn favours production of the longer ZIF2.2 transcript, which compared to ZIF2.1 confers greater Zn tolerance to transgenic plants by promoting higher root Zn immobilization. We show that the retained intron in the ZIF2 5′UTR enhances translation in a Zn-responsive manner, markedly promoting ZIF2 protein expression under excess Zn. Moreover, Zn regulation of translation driven by the ZIF2.2 5′UTR depends largely on a predicted stable stem loop immediately upstream of the start codon that is lost in the ZIF2.1 5′UTR. Collectively, our findings indicate that alternative splicing controls the levels of a Zn-responsive mRNA variant of the ZIF2 transporter to enhance plant tolerance to the metal ion.

Nitrogen metabolism and gas exchange parameters associated with zinc stress in tobacco expressing an ipt gene for cytokinin synthesis

Increased endogenous plant cytokinin (CK) content through transformation with an isopentyl transferase (ipt) gene has been associated with improved plant stress tolerance. The impact of zinc (tested levels Zn1=250, Zn2=500, Zn3=750mgkg(-1)soil) on gas exchange parameters (net photosynthetic rate, transpiration rate, stomatal conductance, intercellular CO2 concentration) and nitrogen utilization by plants resulted in changes of free amino acid concentrations (glutamic acid, glutamine, asparagine, aspartate, glycine, serine, cystein) and differed for transformed and non-transformed tobacco plants. For pot experiments, tobacco plants (Nicotiana tabacum L., cv. Wisconsin 38) transformed with a construct consisting of SAG12 promoter fused with the ipt gene for cytokinin synthesis (SAG plants) and its wild type (WT plants as a control) were used. Physiological analyses confirmed that SAG plants had improved zinc tolerance compared with the WT plants. The enhanced Zn tolerance of SAG plants was associated with the maintenance of accumulation of amino acids and with lower declines of photosynthetic and transpiration rates. In comparison to WT plants, SAG plants exposed to the highest Zn concentration accumulated lower concentrations of asparagine, which is a major metabolic product during senescence.

Evolutionary tinkering of the expression of PDF1s suggests their joint effect on zinc tolerance and the response to pathogen attack

Multigenic families of Plant Defensin type 1 (PDF1) have been described in several species, including the model plant Arabidopsis thaliana as well as zinc tolerant and hyperaccumulator A. halleri. In A. thaliana, PDF1 transcripts (AtPDF1) accumulate in response to pathogen attack following synergic activation of ethylene/jasmonate pathways. However, in A. halleri, PDF1 transcripts (AhPDF1) are constitutively highly accumulated. Through an evolutionary approach, we investigated the possibility of A. halleri or A. thaliana species specialization in different PDF1s in conveying zinc tolerance and/or the response to pathogen attack via activation of the jasmonate (JA) signaling pathway. The accumulation of each PDF1 from both A. halleri and A. thaliana was thus compared in response to zinc excess and MeJA application. In both species, PDF1 paralogues were barely or not at all responsive to zinc. However, regarding the PDF1 response to JA signaling activation, A. thaliana had a higher number of PDF1s responding to JA signaling activation. Remarkably, in A. thaliana, a slight but significant increase in zinc tolerance was correlated with activation of the JA signaling pathway. In addition, A. halleri was found to be more tolerant to the necrotrophic pathogen Botrytis cinerea than A. thaliana. Since PDF1s are known to be promiscuous antifungal proteins able to convey zinc tolerance, we propose, on the basis of the findings of this study, that high constitutive PDF1 transcript accumulation in A. halleri is a potential way to skip the JA signaling activation step required to increase the PDF1 transcript level in the A. thaliana model species. This could ultimately represent an adaptive evolutionary process that would promote a PDF1 joint effect on both zinc tolerance and the response to pathogens in the A. halleri extremophile species.

CadA of Mesorhizobium metallidurans isolated from a zinc-rich mining soil is a PIB-2-type ATPase involved in cadmium and zinc resistance

Mesorhizobium metallidurans STM 2683T is a nitrogen-fixing bacterium that nodulates Anthyllis vulneraria in mine tailings highly contaminated in zinc, lead and cadmium. To study the mechanisms whereby this bacterium copes with metals, we functionally screened a cosmid genomic library of M. metallidurans for zinc or cadmium tolerance. A cosmid clone harbored a gene encoding PIB-type ATPase homologous to CadA that leads to cadmium and zinc resistance in Escherichia coli. The CadA protein structure presents one duplication of the two N-terminal metal binding domains (i.e. a heavy metal-associated domain followed by a histidine-rich domain) which allows specific binding to zinc and cadmium cations. A cadA-deleted strain of M. metallidurans failed to grow at high zinc concentrations (2mM) and its growth was delayed at lower zinc concentrations. Expression studies using a transcriptional fusion of cadA promoter to gfp showed that cadA is specifically induced in a dose-dependent manner by zinc and cadmium in M. metallidurans in vitro conditions and into A. vulneraria nodules after Zn stress. Metal induction sensitivity was increased in the strain where cadA gene was deleted.This study identified cadA as a first mesorhizobial resistance determinant involved in detoxification of cadmium and zinc and which confers upon M. metallidurans greater capacity for coping with high zinc concentrations. This improves the knowledge of this bacterium for potential use as a symbiotic inoculant of Anthyllis in phytostabilization strategies of metal-rich sites.

Nickel and zinc accumulation capacities and tolerance to these metals in the excluder Thlaspi arvense and the hyperaccumulator Noccaea caerulescens

Representatives of Brassicaceae species—the hyperaccumulator Noccaea caerulescens F.K. Mey and the metal excluder Thlaspi arvense L.—were compared in terms of their ability to accumulate nickel (Ni) and zinc (Zn) and their tolerance to these metals. Four ecotypes of N. caerulescens were used: the ecotypes La Calamine (LC, Belgium) and Saint Felix de Pallieres (SF, France) grow naturally on calamine soils rich in Zn, Cd, and Pb; the ecotype Monte Prinzera (MP, Italy) originates from serpentine soils rich in Ni, Co, and Cr; and the ecotype Lellingen (LE, Luxembourg) inhabits non-metalliferous soils. The plants of N. caerulescens were grown for 8 weeks in a half-strength Hoagland solution supplemented with 25, 100, 200, 300, and 400 μM Ni(NO3)2 (ecotypes LC, SF, MP, LE) or 100, 200, 400, 800, and 1000 μM Zn(NO3)2 (ecotypes LC, SF, LE); the plants of T. arvense were grown in the presence of 10, 20, 25, and 30 μM Ni(NO3)2 or 40, 50, 60, 70, 80 μM Zn(NO3)2. The toxic effect of Ni and Zn was assessed from changes in dry matter of roots and shoots of treated plants compared to untreated. The content of metals in roots and shoots was determined by means of atomic absorption spectrophotometry. The Ni-accumulating capacity of N. caerulescens ecotypes increased in the order: LC < SF < LE < MP, and the Zn-accumulating capacity increased in the row: LC < SF < LE. In the hyperaccumulating plant N. caerulescens, the increments of biomass started to decrease at a lower metal content in roots than in shoots, whereas the opposite pattern was observed in the metal excluder T. arvense. Since T. arvense plants accumulated Ni and Zn in roots, whereas N. caerulescens accumulated these metals in shoots, one may assume that the greater sensitivity of root growth compared with shoots in N. caerulescens was determined by more effective mechanisms of metal detoxification in shoots. Conversely, the higher sensitivity of shoot growth compared to root growth in T. arvense was determined by more effective mechanisms of metal detoxification in roots. Being more tolerant to Ni and Zn than T. arvense plants, the N. caerulescens ecotypes differed substantially in terms of metal-accumulating capacity and their tolerance to heavy metals. The ecotype originating from non-metalliferous soils (LE) accumulated larger amounts of Zn, but was less tolerant compared with ecotypes growing naturally on calamine soils (SF and LC), whereas the ecotype occurring on serpentine soils (MP) exhibited a markedly greater tolerance to Ni, compared with other ecotypes examined, as well as the largest accumulation of this metal. The results indicate the existence of different mechanisms responsible for plant tolerance to Ni and Zn; the study of these mechanisms is a promising direction for future research.

Expression of metallothionein of freshwater crab (Sinopotamon henanense) in Escherichia coli enhances tolerance and accumulation of zinc, copper and cadmium

Metallothioneins (MTs) are ubiquitous metal-binding, cysteine-rich, small proteins and play a major role in metal homeostasis and/or detoxification in all organisms. In a previous study, a novel full length MT gene was isolated from the freshwater crab (Sinopotamon henanense), a species widely distributed in Shanxi and Henan Provinces, China. In this report, the gene for the crab MT was inserted into a PET-28a-6His-SUMO vector and recombinant soluble MT was over-expressed as fusions with SUMO in Escherichia coli. The recombinant fusion protein was purified by affinity chromatography and its biochemical properties were analyzed. In addition, on the basis of constructing SUMO-MT, two mutants, namely SUMO-MTt1 and SUMO-MTt2, were constructed to change the primary structure of SUMO-MT using site-directed mutagenesis techniques with the amino acid substitutions D3C and S37C in order to increase metal-binding capacity of MT. E. coli cells expressing SUMO-MT and these single-mutant proteins exhibited enhanced metal tolerance and higher accumulation of metal ions than control cells. The results showed that the bioaccumulation and tolerance of Zn(2+), Cu(2+) and Cd(2+) in these strains followed the decreasing order of SUMO-MTt1 > SUMO-MTt2 > SUMO-MT. E. coli cells have low tolerance and high accumulation towards cadmium compared to zinc and copper. These results show that the MT of S. henanense could enhance tolerance and accumulation of metal ions. Moreover, we were able to create a novel protein based on the crab MT to bind metal ions at high density and with high affinity. Therefore, SUMO-MT and its mutants can provide potential candidates for heavy metal bioremediation. This study could help further elucidate the mechanism of how the crab detoxifies heavy metals and provide a scientific basis for environment bioremediation of heavy metal pollution using the over-expression of the crab MT and mutant proteins.