Increased metallothionein gene expression in 5-aza-2'-deoxycytidine-induced resistance to cadmium cytotoxicity

The pyrimidine analog, 5-azacytidine (AZA-CR), has been shown to increase the expression of the metallothionein (MT) gene and to induce tolerance to cadmium toxicity. Since incorporation into DNA of AZA-CR appears to be required for this effect, the deoxynucleoside of AZA-CR should also be effective. Therefore, this study was undertaken to assess the effect of 5-aza-2'-deoxycytidine (AZA-CdR) pretreatment on cadmium-induced cytotoxicity and MT expression in cultured cells. TRL 1215 cells in log phase of growth were exposed to AZA-CdR (0.4, 0.8, 4.0, 8.0 microM) followed 48 h later by the addition of cadmium (10 microM). MT concentrations were measured 24 h after the addition of cadmium. AZA-CdR alone caused modest, dose-related increases in MT levels (2.3-fold maximum), while cadmium alone resulted in a 9.5-fold increase. Pretreatment with AZA-CdR in combination with cadmium caused a 19--24-fold increase in cellular MT at all doses of AZA-CdR. Addition of the DNA synthesis inhibitor, hydroxyurea (HU), to the incubation medium during AZA-CdR exposure prevented the enhancing effect of the analog on cadmium induction of MT accumulation. Time course studies revealed that AZA-CdR pretreatment reduced the time required for cadmium to induce MT levels from 4--8 h to 0--2 h. AZA-CdR pretreated cells placed in suspension with cadmium (125 microM) showed a marked reduction in cadmium-induced cytotoxicity as reflected by reduced glutamic-oxaloacetic transaminase (GOT) loss. Uptake studies showed that AZA-CdR pretreatment had no effect on cadmium transport during the initial phases of exposure, indicating that an alteration in the toxicokinetics of the metal did not account for the reduction in toxicity. AZA-CdR did, however, cause hypomethylation of the MT-I gene. These results suggest that AZA-CdR pretreatment induces tolerance to cadmium toxicity by increasing the genetic expression of MT possibly through hypomethylation of the MT gene.     

The cytosolic O-acetylserine(thiol)lyase gene is regulated by heavy metals and can function in cadmium tolerance

Regulation of the expression of the cytosolic O-acetylserine(thiol)lyase gene (Atcys-3A) from Arabidopsis thaliana under heavy metal stress conditions has been investigated. Northern blot analysis of Atcys-3A expression shows a 7-fold induction after 18 h of cadmium treatment. Addition of 50 microm CdCl(2) to the irrigation medium of mature Arabidopsis plants induces a rapid accumulation of the mRNA throughout the leaf lamina, the root and stem cortex, and stem vascular tissues when compared with untreated plants, as observed by in situ hybridization. High pressure liquid chromatography analysis of GSH content shows a transient increase after 18 h of metal treatment. Our results are compatible with a high cysteine biosynthesis rate under heavy metal stress required for the synthesis of GSH and phytochelatins, which are involved in the plant detoxification mechanism. Arabidopsis-transformed plants overexpressing the Atcys-3A gene by up to 9-fold show increased tolerance to cadmium when grown in medium containing 250 microm CdCl(2), suggesting that increased cysteine availability is responsible for cadmium tolerance. In agreement with these results, exogenous addition of cystine can, to some extent, also favor the growth of wild-type plants in cadmium-containing medium. Cadmium accumulates to higher levels in leaves of tolerant transformed lines than in wild-type plants.     

Lead, zinc and cadmium accumulation from two metalliferous soils with contrasting calcium contents in heavy metal-hyperaccumulating and non-hyperaccumulating metallophytes: a comparative study

Aims and background

We previously compared metallicolous (M) and non-metallicolous (NM) populations of Noccaea (=Thlaspi) caerulescens, Silene vulgaris, and Matthiola flavida for their abilities to tolerate and (hyper)-accumulate lead (Pb) in hydroponics. In the present study we aimed 1) to check the hyperaccumulation and tolerance abilities of these populations in controlled experiments using metalliferous soils, 2) to test the M. flavida M population for Zn and Cd hypertolerance in hydroponics.

Methods

Plants were grown in hydroponics and fertilized metalliferous substrates, collected from a Zn/Pb smelter sinter deposit near Plombières, Belgium (low pH, low Ca), and a tailing of the Irankouh Zn/Pb mine, Iran (high pH, high Ca). Metal tolerance was assessed from root growth inhibition in hydroponics, or mortality, stunting or chlorosis in the experiments with soil.

Results

Metallicolous M. flavida did not show hypertolerance or hyperaccumulation of Cd or Zn in hydroponics. Only one of the N. caerulescens M populations and the native S. vulgaris M population were able to grow in Plombières soil, whereas the others stopped growing or died within 40?days. All the populations survived and maintained growth for 40?days in Irankouh soil. When grown in Irankouh soil, the M population of M. flavida hyperaccumulated Pb. N. caerulescens hyperaccumulated Zn from Plombières soil, but not from Irankouh soil.

Conclusions

The M. flavida M population is non-Pb-hypertolerant. It hyperaccumulates Pb from Irankouh soil, but not from Pb-amended nutrient solution. N. caerulescens does not hyperaccumulate Zn from the calcareous Irankouh soil.     

Local Adaptation of Metallicolous and Non‐Metallicolous Anthyllis vulneraria Populations: Their Utilization in Soil Restoration

Restoration of metalliferous mine soils requires using plant species tolerant to high metal concentrations and adapted to nutrient‐poor soil. Legumes can increase plant productivity through N₂‐fixation, but they are often scarce in metalliferous sites. We examined survival, growth, and tolerance of four populations of a legume, Anthyllis vulneraria, from two metalliferous (MET) Zn‐Pb mine sites, Avinières (AV) ([Zn‐EDTA] = 26,000 mg/kg) and Eylie (EY) ([Zn‐EDTA] = 4,632 mg/kg), and two non‐metalliferous (NMET) sites located in the south of France with the aim to select the most appropriate populations for restoration of mined soils. In a common garden experiment, plants from each population were reciprocally grown in soil from the provenance of each population. The two NMET populations exhibited high mortality and low growth rates in soil from the mined sites. The AV MET exhibited a high growth rate in metalliferous soils, but showed high mortality in non‐metalliferous soils. The growth of the EY MET was very low in the AV‐contaminated soil, but was the highest of all populations in moderately and non‐metalliferous soils. Plants from the AV MET population showed a high growth and survival in metalliferous soil and would be appropriate in the restoration of metal‐contaminated sites (>30,000 mg Zn kg^?1). The EY MET population would be adapted to the restoration of moderate metal‐contaminated soils (<30,000 mg Zn kg^?1). Taking into account the broad distribution of A. vulneraria, these two populations could be suitable for the restoration of derelict mine sites in mediterranean and temperate regions of Europe and North America.     

Cd/Zn exposure interactions on metallothionein response in Eisenia fetida (Annelida, Oligochaeta)

We studied metallothionein (MT) response in the manure worm Eisenia fetida after exposures to cadmium (Cd), zinc (Zn) or cadmium and zinc spiked media. MT was studied both at the protein level by Dot Immunobinding Assay, (DIA) and at the expression level by Northern blotting. Cd was highly accumulated by worms whereas Zn body concentration was regulated. In addition, Zn would limit Cd accumulation in worms exposed to low Cd concentrations (1 and 8 mg Cd kg(-1) of dry soil). Exposure to a mixture of Cd and Zn at high concentrations increased cytosolic MT levels. This increase would allow worms to regulate body Zn concentrations and also to limit Cd toxicity. Cd exposures increased gene expression of Cd-binding MT isoform (MT 2A) whereas Zn did not. However, when both metals were at high concentrations in the exposure medium, this expression was further increased. Several hypotheses are proposed to explain the results and the best approach to estimate metal exposure of this earthworm species is given. Further experiments have now to be performed to evaluate the usefulness of these MT responses for field contaminated soils toxicity assessment using this earthworm species.     

The influence of cadmium,copper, lead,and zinc on the distribution and evolution of metallophytes in the British Isles

Mine spoils and other soils contaminated with cadmium, copper, lead and zinc show natural colonization by species which have strategies of avoidance or tolerance of metal toxicities. The distribution of plants on such substrata in the British Isles is examined in the light of present knowledge of such strategies. Evolutionary processes mediating the selection of tolerant individuals and ecotypic differentiation of adapted populations on metalliferous soils are considered. Other factors determining which species can and which cannot evolve tolerance include constitutional differences in species sensitivity to toxic metals, and phenotypic (environmentally-induced) tolerances. The importance of constitutional properties and phenotypic responses in providing explanations for plant distribution on metalliferous soils is assessed.     

Response of sugarcane to increasing concentrations of copper and cadmium and expression of metallothionein genes

Sugarcane (Saccharum spp.) offers the potential to be a phytoremediator species due to its outstanding biomass production, but its prospective metal accumulation and tolerance have not been fully characterized. Sugarcane plantlets were able to tolerate up to 100microM of copper in nutrient solution for 33 days, with no significant reduction in fresh weight, while accumulating 45mgCukg(-1) shoot dry weight. Higher levels of copper in solution (250 and 500microM) were lethal. Sugarcane displayed tolerance to 500microM Cd without symptoms of toxicity, accumulating 451mgCdkg(-1) shoot dry weight after 33 days, indicating its potential as Cd phytoremediator. DNA gel blot analyses detected 8 fragments using a metallothionein (MT) Type I probe, while 10 were revealed for the MT Type II and 8 for MT Type III. The number of genes for each type of MT in sugarcane might be similar to the ones identified in rice considering the interspecific origin of sugarcane cultivars. MT Type I gene appeared to present the highest level of constitutive expression, mainly in roots, followed by MT Type II, corroborating the expression pattern described based on large-scale expressed sequence tags sequencing. MT Type II and III genes were more expressed in shoots, where MT I was also importantly expressed. Increasing Cu concentration had little or no effect in modulating MT genes expression, while an apparent minor modulation of some of the MT genes could be detected in Cd treatments. However, the level of response was too small to explain the tolerance and/or accumulation of Cd in sugarcane tissues. Thus, cadmium tolerance and accumulation in sugarcane might derive from other mechanisms, although MT may be involved in oxidative responses to high levels of Cd. Sugarcane can be considered a potential candidate to be tested in Cd phytoremediation.     

Gene expression of the ericoid mycorrhizal fungus Oidiodendron maius in the presence of high zinc concentrations

A heavy metal tolerant strain of the ericoid mycorrhizal species Oidiodendron maius, isolated from roots of Vaccinium myrtillus growing in soil heavily contaminated with zinc, was previously shown to tolerate high concentrations of zinc and cadmium ions in the growth medium. We have investigated the genetic basis of this fungal strain tolerance to high zinc concentrations by using an untargeted approach. From a cDNA library constructed by using mRNA from Zn-treated O. maius mycelia, 444 clones were randomly selected and 318 were sequenced. Sequence analysis identified 219 unique clones: 117 showed homology to previously identified genes, 26 matched unknown protein coding regions found in other organisms, and 76 were novel. Variation in the gene expression level after a 20-day treatment with high concentrations of Zn was monitored on 130 unigenes by reverse northern blot hybridisation. Sixteen unigenes were shown to be either up- (9) or down- (7) regulated. The putative function of these genes and their involvement in stress tolerance is discussed.     

Organic Acids Accumulation and Antioxidant Enzyme Activities in Thlaspi caerulescens under Zn and Cd Stress

Growth, organic acid and phytochelatin accumulation, as well as the activity of several antioxidative enzymes, i.e. superoxide dismutase (SOD), ascorbate peroxidase (APX) guaiacol peroxidase (POX) and catalase (CAT) were investigated under Zn and Cd stress in hydroponically growing plants of Thlaspi caerulescens population from Plombières, Belgium. Tissue Zn and Cd concentration increased (the highest concentration of both was in roots) as the concentration of these metals increased in the nutrient solution. Increasing Zn concentration enhanced plant growth, while with Cd it declined compared to the control. Both metals stimulated malate accumulation in shoots, Zn also caused citrate to increase. Zn did not induce phytochelatin (PC) accumulation. In plants exposed to Cd, PC concentration increased with increasing Cd concentration, but decreased with time of exposure. Under Zn stress SOD activity increased, but APX activity was higher at 500 and 1000 μM Zn and CAT activity only at 500 μM Zn in comparison with the control. CAT activity decreased in Cd- and Zn-stressed plants. The results suggest that relative to other populations, a T. caerulescens population from Plombières, when grown in hydroponics, was characterized by low Zn and Cd uptake and their translocation to shoots and tolerance to both metals. The accumulation of malate and citrate, but not PC accumulation was responsible for Zn tolerance. Cd tolerance seems to be due to neither PC production nor accumulation of organic acids.     

大蒜植物络合素合酶基因转化对酵母重金属抗性的提高

重金属污染是全球面临的亟待解决的生态问题。利用植物对重金属的富集作用来清除环境重金属污染即植物修复已成为重要的环境生物技术之一。这一技术的长远发展有赖于在重金属富集或耐受中起关键作用的基因的克隆和应用。植物络合素是植物体内一类重要的对重金属起螯合作用的多肽, 其合成受植物络合素合酶的催化。该文取得了如下研究结果:1)通过原子吸收测定表明,在大蒜(Allium sativum)的根部可以积累3 000 mg·kg^-1的重金属镉;2)将克隆的大蒜植物络合素合酶基因(AsPCS)置于酵母表达启动子之下,构建酵母表达载体,并将其分别转入了因CUP1和acr3基因缺失而对重金属镉和砷敏感的酵母突变体菌株后,发现来自大蒜的AsPCS基因的表达使酵母CUP1缺失菌株对镉的耐受性提高了4倍, acr3缺失菌株对砷的耐受性提高了两倍;3)表达AsPCS基因酵母的生长模式证实了AsPCS基因的表达是酵母对重金属耐受性提高的原因。这些结果暗示, 大蒜植物络合素合酶基因在大蒜对重金属的抗性及大蒜根部对镉的积累中起关键作用,可作为重要的基因元件应用到修复污染的植物基因工程中。     

Enhanced accumulation of cadmium in Linum usitatissimum L. plants due to overproduction of metallothionein α-domain as a fusion to β-glucuronidase protein

As most cultivars of flax and linseed (Linum usitatissimum L.) are capable of accumulating cadmium (Cd), they are suitable candidates for phytoextraction of the metal from contaminated soils. In an attempt to enhance the phytoextraction capacity of L. usitatissimum through overproduction of an efficient heterologous Cd-binding peptide, we engineered linseed breeding line AGT 917 to constitutively express genetic fusion of α-domain of mammalian metallothionein 1a (αMT1a) and β-glucuronidase gus gene under the control of CaMV 35S promoter. An improved transformation protocol was developed, which involved co-cultivation of AGT 917 hypocotyl segments with partially removed epidermis with Agrobacterium suspension for 10 min in the presence of 200 mg l^?1 cellulase. The enzyme treatment increased the transformation efficiency (TE) 1.6-fold as compared to agroinfection without cellulase. Less pronounced impact on TE exerted 100 mg l^?1 acetosyringone, increasing TE 1.3-fold. When tested in soils amended with Cd at 20 and 360 mg kg^?1, the mature αMT1a::gus plants accumulated more Cd than parental AGT 917: the stem Cd concentrations in the best performing αMT1/2 line were 3.3- and 1.9-fold higher, respectively. Moreover, hypocotyl explants of αMT1/2 line showed 1.7-fold higher biomass than those of AGT 917 on media containing 15 mg Cd l^?1, indicating that αMT1a::gus did confer higher Cd tolerance to engineered plant. Overproduction of metal-binding peptides thus appears to be a viable strategy for the production of L. usitatissimum with improved phytoremediation capacity.     

Cadmium tolerance in Thlaspi caerulescens: I. Growth parameters,metal accumulation and phytochelatin synthesis in response to cadmium

A population of the metallophyte, Thlaspi caerulescens, originating from a Cd–Pb–Zn old mining and smelter site at Plombières (Belgium) was studied. T. caerulescens was cultivated hydroponically to investigate Cd uptake and tolerance. Cd was added to Hoagland’s medium at concentration range from 5 to 500 μM. The plants could tolerate 500 μM Cd in the solution showing only minor visible symptoms of toxicity but with a 32% decrease in fresh weight. After 14 days at 500 μM, Cd content in roots and shoots was 707 and 602 mg kg⁻¹ of dry weight (d.w.), respectively. Application of Cd to hydroponically cultivated T. caerulescens induced the accumulation of PCs in plant roots and shoots. Buthionine sulfoximine (BSO) application almost completely reduced (by 98–100%) the accumulation of PCs without simultaneous increase in plants sensitivity to Cd. These results suggest a minor if any role of PCs in tolerance to Cd of the studied population of T. caerulescens in hydroponics. On the other hand, no PC accumulation was detected either in T. caerulescens plants growing in their natural environment at Plombierès or in plants growing in their native soil in a greenhouse. These results suggest that naturally selected tolerance in T. caerulescens population from Plombières is not associated with enhanced PCs synthesis.     

Additive genetic variation of transcriptional regulation: metallothionein expression in the soil insect Orchesella cincta

Field-selected metal tolerance in Orchesella cincta is correlated with overexpression of the single copy cadmium (Cd) inducible metallothionein (mt). Previously, we have demonstrated large phenotypic variation in mt gene expression, and a higher frequency of high-expression phenotypes in a tolerant population. Here, we describe midparent-offspring regression analysis of mt gene expression in a laboratory culture originating from a noncontaminated natural population. Families were either not exposed (n=47) or exposed to 0.5 micromol Cd per gram dry food (n=46). Mean mt gene expressions normalized to 28S rRNA and beta-actin RNA were generated using real-time RT-PCR applied to parents and offspring RNA and subjected to regression analysis. A significant heritability (h2) for mt gene expression was estimated between 0.36 (beta-actin normalized) and 0.46 (28S normalized) in Cd exposed families. Nontreated families did not yield a significant h2 value. Restriction Fragment Length Polymorphism analysis of the metallothionein promoter sequence revealed eight promoter alleles that show structural variation. Three alleles show increased frequencies in families with high mt expression. Another gene, croquemort (isolated from a differential screening for 1 micromole Cd treatment) showed no h2 of gene expression in response to 0.5 micromol Cd. This gene codes for a receptor-protein involved in recognition of apoptotic cells and may participate in the general stress response. The present data suggest that evolution of metal tolerance in O. cincta can occur in the field by selection for high mt expression due to structural changes in mt cis-regulation.