Tobacco transformants expressing antisense sequence of proline dehydrogenase gene possess tolerance to heavy metals

Analysis of resistance of genetically modified tobacco plants bearing antisense suppressor of proline dehydrogenase gene and characterized with higher content of proline to elevated concentrations of heavy metals was performed. It was demonstrated that progeny of transgenic plants have high resistance to lead, nickel and cadmium ions.     

Elevated proline content in maize plants expressing a fragment of the proline dehydrogenase gene in antisense orientation

A fragment of the first exon of the proline dehydrogenase gene of arabidopsis (Arabidopsis thaliana L.) in antisense orientation was transferred to maize (Zea mays L.) genome by agrobacterial transformation in planta to elevate the content of free proline in maize cells. The presence of genetic structure carrying an antisense sequence of the fragment of the proline dehydrogenase gene (ASPG) from the genome of diploid maize seedlings was corroborated by PCR method. T-DNA insertions comprising ASPG were found in the genomes of 30 plants (1.2% of 2409 examined seedlings) belonging to the generation T0. A reliable 4.6-fold increase in proline content was registered in the leaves of transformed maize plants.     

Evaluation of salt tolerance in Nicotiana tabacum plants bearing an antisense suppressor of the proline dehydrogenase gene

We studied the stress resistance of genetically modified (GM) tobacco plants bearing an antisense suppressor of the gene for proline dehydrogenase. Such plants are characterized by elevated proline content. The progeny of the transgenic plants were shown to have elevated salt tolerance.     

Evaluation of salt tolerance in Nicotiana tabacum plants bearing an antisense suppressor of the proline dehydrogenase gene

We studied the stress resistance of genetically modified (GM) tobacco plants bearing an antisense suppressor of the gene for proline dehydrogenase. Such plants are characterized by elevated proline content. The progeny of the transgenic plants were shown to have elevated salt tolerance.     

Partial suppression of gene encoding proline dehydrogenase enhances plant tolerance to various abiotic stresses

The role of gene of proline dehydrogenase (PDH) in the maintenance of stress tolerance was investigated using the model transgenic plants of tobacco (Nicotiana tabacum L.) carrying an antisense suppressor of PDH gene (a fragment of Arabidopsis PDH gene under the control of cauliflower mosaic virus 35S promoter in antisense orientation) and notable for a low activity of PDH and elevated content of proline. The progeny of transgenic plants belonging to the 5th generation (T₅) with partially suppressed PDH activity was more resistant to various types of stress as compared with the control plants of tobacco, cv. Petit Havana SR-1 (SR1). The seedlings of transgenic lines cultured in Petri dishes on agar media supplemented with stress agents were resistant to high NaCl concentrations (200–300 mM) and water deficit simulated by an increased agar content in the medium (14 g/l) as compared to the control seedlings of cv. SR1. Juvenile plants of transgenic lines grown in pots filled with a mixture of vermiculite and perlite also manifested the higher resistance to water deficit and low temperatures (2°C and −2°C) than the control plants. Thus, the partial PDH suppression correlated with an increase in nonspecific resistance to different types of abiotic stress: salinity, water deficit, and low temperatures. Such transgenic lines of tobacco are promising genetic models for thorough investigation of molecular mechanisms of stress resistance in plants.     

Physiological and biochemical analysis of the transformants of aerobic methylobacteria expressing the dcm A gene of dichloromethane dehydrogenase

The transformants of Methylobacterium dichloromethanicum DM4 (DM4-2cr-/pME8220 and DM4-2cr-/pME8221) and of Methylobacterium extorquens AM1 (AM1/pME8220 and AM1/pME8221) that express the dcm A gene of dichloromethane dehalogenase undergo lysis when incubated in the presence of dichloromethane and are sensitive to acidic shock. The lysis of the transformants was found to be related neither to the accumulation of Cl- ions, CH2O, and HCOOH, nor to the impairment of glutathione synthesis or to the maintenance of intracellular pH. The (exo-) Klenow fragment-mediated incorporation of [alpha-32P]dATP into the DNA of the transformants DM4-2cr-/pME8220 and AM1/pME8220 was considerably greater when the transformed cells were incubated with CH2Cl2 than when they were incubated with CH3OH, indicating the occurrence of a significant increase in the total length of gaps. At the same time, the strain AM1 (which lacks dichloromethane dehalogenase) and the dichloromethane-degrading strain DM4 incubated with CH2Cl2 showed an insignificant increase in the total length of the gaps. The transformed cells are likely to lyse due to the relatively inefficient repair of DNA lesions that are induced in response to the alkylating action of S-chloromethylglutathione, an intermediate product of CH2Cl2 degradation. The data obtained suggest that the bacterial mineralization of dichloromethane requires an efficient DNA repair system.     

Patterns of tolerance to heavy metals among methane-utilizing bacteria

The susceptibility of obligate methane-utilizing bacteria, including 14 reference strains and 175 environmental isolates, to five readily available heavy metal pollutants was determined. The chloride salts of Hg(II), Cd(II), Cu(II), Cr(III) and Zn(II) were tested in a system free from organic matter. Methane-utilizers appeared to have relatively discrete metal tolerance patterns with resistances to all metals varying with isolation site. Methanotrophs proved to be quite sensitive to mercury and cadmium but relatively resistant to copper, chromium and zinc     

Expression of the mouse metallothionein-I gene in Escherichia coli: increased tolerance to heavy metals

The cDNA of mouse metallothionein, a small metal-binding protein rich in cysteine, has been cloned downstream from a bacterial inducible promoter and expressed in Escherichia coli. Upon induction, E. coli harboring this cDNA clone contained a protein species readily labelled by [35S]cysteine in vivo and incorporated 10-times as much 109Cd from the medium than would otherwise be the case. We show that expression of metallothionein endows resistance in E. coli to heavy metal ions such as mercury, silver, copper, cadmium and zinc by sequestering rather than exclusion or conversion, common mechanisms of metal resistance in bacteria.     

Increase in the level of proline and osmotic pressure of cytoplasm in transformed tobacco bearing an antisense suppressor of the proline dehydrogenase gene

The antisense suppressor was constructed for proline dehydrogenase gene (PDH; a fragment of PDH from Arabidopsis in antisense orientation and under the control of 35S promoter of cauliflower mosaic virus, CMV). In Nicotiana tabacum SR1 tobacco transformants bearing antisense suppressor for PDH, the proline content and the cytoplasm osmotic pressure were increased. The proline content in these transformants varied, whereas cytoplasm osmotic pressure was stable, which seems to reflect complicated relationships between these characteristics of the plant cell.     

Bioaccumulation of heavy metals in Escherichia coli expressing an inducible synthetic human metallothionein gene

A synthetic DNA coding for human hepatic metallothionein (HMT), fraction MT-2, has been cloned in the plasmid vector pING 1 and expressed in E. coli. Upon induction with arabinose, an araB′-HMT fusion protein is synthesized. The fusion protein is produced to a level of approximately 8% of the total protein in E. coli, with an apparent half-life of 50 min. Without arabinose, no fusion protein is expressed, demonstrating that the expression system is tightly regulated. In the presence of ¹⁰⁹Cd, the fusion protein binds the metal ion in vitro. Concurrently, a direct correlation is found between the expression in E. coli of the araB′-HMT fusion protein which binds Cd²⁺ in vitro and the bioaccumulation of Cd²⁺ in vivo. The bioaccumulation of Cu⁺ in E. coli, when the fusion protein is produced, has also been established.     

Biological cost of tolerance to heavy metals in the mosquito Anopheles gambiae

The global rate of heavy metal pollution is rapidly increasing in various habitats. Anopheles malaria vector species (Diptera: Culicidae) appear to tolerate many aquatic habitats with metal pollutants, despite their normal proclivity for ‘clean’ water (i.e. low levels of organic matter). Investigations were conducted to establish whether there are biological costs for tolerance to heavy metals in Anopheles gambiae Giles sensu stricto and to assess the potential impact of heavy metal pollution on mosquito ecology. Anopheles gambiae s.s. were selected for cadmium, copper or lead tolerance through chronic exposure of immature stages to solutions of the metals for three successive generations. Biological costs were assessed in the fourth generation by horizontal life table analysis. Tolerance in larvae to cadmium (as cadmium chloride, CdCl₂), copper [as copper II nitrate hydrate, Cu(NO₃)₂ 2.5 H₂O] and lead [as lead II nitrate, Pb(NO₃)₂], monitored by changes in LC₅₀ concentrations of the metals, changed from 6.07 µg/L, 12.42 µg/L and 493.32 µg/L to 4.45 µg/L, 25.02 µg/L and 516.69 µg/L, respectively, after three generations of exposure. The metal‐selected strains had a significantly lower magnitude of egg viability, larval and pupal survivorship, adult emergence, fecundity and net reproductive rate than the control strain. The population doubling times were significantly longer and the instantaneous birth rates lower in most metal‐selected strains relative to the control strain. Our results suggest that although An. gambiae s.s. displays the potential to develop tolerance to heavy metals, particularly copper, this may occur at a significant biological cost, which can adversely affect its ecological fitness.     

Molecular mechanisms of proline-mediated tolerance to toxic heavy metals in transgenic microalgae

Pro has been shown to play an important role in ameliorating environmental stress in plants and microorganisms, including heavy metal stress. Here, we describe the effects of the expression of a mothbean delta(1)-pyrroline-5-carboxylate synthetase (P5CS) gene in the green microalga Chlamydomonas reinhardtii. We show that transgenic algae expressing the mothbean P5CS gene have 80% higher free-Pro levels than wild-type cells, grow more rapidly in toxic Cd concentrations (100 microM), and bind fourfold more Cd than wild-type cells. In addition, Cd-K edge extended x-ray absorption fine structure studies indicated that Cd does not bind to free Pro in transgenic algae with increased Pro levels but is coordinated tetrahedrally by sulfur of phytochelatin. In contrast to P5CS-expressing cells, Cd is coordinated tetrahedrally by two oxygen and two sulfur atoms in wild-type cells. Measurements of reduced/oxidized GSH ratios and analyses of levels of malondialdehyde, a product of the free radical damage of lipids, indicate that free Pro levels are correlated with the GSH redox state and malondialdehyde levels in heavy metal-treated algae. These results suggest that the free Pro likely acts as an antioxidant in Cd-stressed cells. The resulting increased GSH levels facilitate increased phytochelatin synthesis and sequestration of Cd, because GSH-heavy metal adducts are the substrates for phytochelatin synthase.     

Tobacco chloroplast transformants expressing genes encoding dehydroascorbate reductase, glutathione reductase, and glutathione-S-transferase, exhibit altered anti-oxidant metabolism and improved abiotic stress tolerance

One approach to understanding the Reactive Oxygen Species (ROS)-scavenging systems in plant stress tolerance is to manipulate the levels of antioxidant enzyme activities. In this study, we expressed in the chloroplast three such enzymes: dehydroascorbate reductase (DHAR), glutathione-S-transferase (GST) and glutathione reductase (GR). Homoplasmic chloroplast transformants containing either DHAR or GST, or a combination of DHAR:GR and GST:GR were generated and confirmed by molecular analysis. They exhibited the predicted changes in enzyme activities, and levels or redox state of ascorbate and glutathione. Progeny of these plants were then subjected to environmental stresses including methyl viologen (MV)-induced oxidative stress, salt, cold and heavy metal stresses. Overexpression of these different enzymes enhanced salt and cold tolerance. The simultaneous expression of DHAR:GR and GST:GR conferred MV tolerance while expression of either transgene on its own didn't. This study provides evidence that increasing part of the antioxidant pathway within the chloroplast enhances the plant's ability to tolerate abiotic stress.     

Enhancement of heavy metal tolerance and accumulation efficiency by expressing Arabidopsis ATP sulfurylase gene in alfalfa

Abstract

Transgenic alfalfa (Medicago sativa L.) plants overexpressing the Arabidopsis ATP sulfurylase gene were generated using Agrobacterium-mediated genetic transformation to enhance their heavy metal accumulation efficiency. The ATP sulfurylase gene was cloned from Arabidopsis, following exposure to vanadium (V) and lead (Pb), and transferred into an Agrobacterium tumefaciens binary vector. This was co-cultivated with leaf explants of the alfalfa genotype Regen SY. Co-cultivated leaf explants were cultured on callus and somatic embryo induction medium, followed by regeneration medium for regenerating complete transgenic plants. The transgenic nature of the plants was confirmed using PCR and southern hybridization. The expression of Arabidopsis ATP sulfurylase gene in the transgenic plants was evaluated through RT-PCR. The selected transgenic lines showed increased tolerance to a mixture of five heavy metals and also demonstrated enhanced metal uptake ability under controlled conditions. The transgenic lines were fertile and did not exhibit any apparent morphological abnormality. The results of this study indicated an effective approach to improve the heavy metal accumulation ability of alfalfa plants which can then be used for the remediation of contaminated soil in arid regions.     

Observations on tolerance to heavy metals of four green algae in relation to pH

Abstract

The inhibition of growth by different concentration of eight heavy metals: Cd, Cr, Co, Cu, Hg, Ni, Se and Tl, in inorganic medium at pH 3 and 6.5, was studied in four green algae: Chlorella protothecoides Krüger, Chlorella saccharophila (Krüger) Migula, Coenochloris sp. and Stichococcus bacillaris Naegeli.

The results suggest that pH has an important effect on heavy metal toxicity in algae although it is difficult to establish a relationship between pH and heavy metals.     

Nucleotide sequence of a mutation in the proB gene of Escherichia coli that confers proline overproduction and enhanced tolerance to osmotic stress

We determined the nucleotide (nt) sequence of a mutation that confers proline overproduction and enhanced tolerance of osmotic stress on bacteria. The mutation, designated as proB74, is an allele of the Escherichia coli proB gene which results in a loss of allosteric regulation of the protein product, gamma-glutamyl kinase. Our sequencing indicated that the proB74 mutation is a substitution of an A for a G at nt position 319 of the coding strand of the gene, resulting in a change of an aspartate to an asparagine at amino acid (aa) residue 107 of the predicted protein product. Rushlow et al. [Gene 39 (1984) 109-112] determined that another proB mutation (designated as DHPR), that resulted in a loss of allosteric inhibition by proline of the E. coli gamma-glutamyl kinase, was due to a substitution of an alanine for a glutamate at aa residue 143. Therefore, even though both the DHPR and the proB74 mutations caused a loss of allosteric inhibition of gamma-glutamyl kinase, they are due to different amino acid substitutions.     

Physiological responses of plants to heavy metals and the quantification of tolerance and toxicity

Abstract

Techniques available for assessing the tolerance of plants to heavy metal toxins are reviewed. All are based on physiological responses and range from long-term growth trials in metal-contaminated substrates, to rapid cytological tests. Problems associated with the ecophysiological interpretation of in vitro measurements of tolerance are considered. The implications of multiple tolerance, co-tolerance, constitutional tolerance, inducible tolerance and possible stimulatory effects of metals on plant responses are discussed.