Phytoremediation potential of Arabidopsis thaliana, expressing ectopically a vacuolar proton pump, for the industrial waste phosphogypsum

Phosphogypsum (PG) is a by-product of the phosphorus–fertiliser industry and represents an environmental concern since it contains pollutants such as cadmium (Cd). We have recently shown that the overexpression of a proton pump gene (TaVP1) in transgenic tobacco (Nicotiana tabacum) led to an enhanced Cd tolerance and accumulation. The aim of this study was to evaluate the potential of transgenic Arabidopsis thaliana plants harbouring the TaVP1 gene to phytoremediate phosphogypsum. A pot experiment was carried out under greenhouse conditions. Transgenic A. thaliana plants harbouring the TaVP1 gene were grown on various substrates containing phosphogypsum (0, 25, 50 and 100 %) for 40 days. At the end of the growth period, we examined the growth (germination, root length, fresh weight) and physiological parameters (chlorophyll and protein contents, catalase activity and proteolysis) as well as the cadmium, Mg, Ca, and P contents of the A. thaliana plants. In order to evaluate Cd tolerance of the A. thaliana lines harbouring the TaVP1 gene, an in vitro experiment was also carried out. One week-old seedlings were transferred to Murashige and Skoog agar plates containing various concentrations of cadmium; the germination, total leaf area and root length were determined. The growth and physiological parameters of all A. thaliana plants were significantly altered by PG. The germination capacity, root growth and biomass production of wild-type (WT) plants were more severely inhibited by PG compared with the TaVP1 transgenic A. thaliana lines. In addition, TaVP1 transgenic A. thaliana plants maintained a higher antioxidant capacity than the WT. Interestingly, elemental analysis of leaf material derived from plants grown on PG revealed that the transgenic A. thaliana line accumulated up to ten times more Cd than WT. Despite its higher Cd content, the transgenic A. thaliana line performed better than the WT counterpart. In vitro evaluation of Cd tolerance showed that TaVP1 transgenic A. thaliana lines were more Cd-tolerant than the WT plants. These results suggested that ectopic expression of a vacuolar proton pump in A. thaliana plants can lead to various biotechnological applications including the phytoremediation of industrial wastes.     

Phytoremediation of 2,4-dichlorophenol using wild type and transgenic tobacco plants

Introduction

Transgenic plant strategies based on peroxidase expression or overexpression would be useful for phenolic compound removal since these enzymes play an important role in phenolic polymerizing reactions.

Material and methods

Thus, double transgenic (DT) plants for basic peroxidases were obtained and characterized in order to compare the tolerance and efficiency for 2,4-dichlorophenol (2,4-DCP) removal with WT and simple transgenic plants expressing TPX1 or TPX2 gene. Several DT plants showed the expression of both transgenes and proteins, as well as increased peroxidase activity.

Results

DT lines showed higher tolerance to 2,4-DCP at early stage of development since their germination index was higher than that of WT seedlings exposed to 25?mg/L of the pollutant. High 2,4-DCP removal efficiencies were found for WT tobacco plants. TPX1 transgenic plants and DT (line d) reached slightly higher removal efficiencies for 10?mg/L of 2,4-DCP than WT plants, while DT plants (line A) showed the highest removal efficiencies (98%). These plants showed an increase of 21% and 14% in 2,4-DCP removal efficiency for solutions containing 10 and 25?mg/L 2,4-DCP, respectively, compared with WT plants. In addition, an almost complete toxicity reduction of postremoval solutions using WT and DT plants was obtained through AMPHITOX test, which indicates that the 2,4-DCP degradation products would be similar for both plants.

Conclusion

These results are relevant in the field of phytoremediation application and, moreover, they highlight the safety of using DT tobacco plants because nontoxic products were formed after an efficient 2,4-DCP removal.     

Transgenic poplar trees expressing yeast cadmium factor 1 exhibit the characteristics necessary for the phytoremediation of mine tailing soil

Genetic engineering of plants for phytoremediation is thought to be possible based on results using model plants expressing genes involved in heavy metal resistance, which improve the plant’s tolerance of heavy metals and accumulation capacity. The next step of progress in this technology requires the genetic engineering of plants that produce large amounts of biomass and the testing of these transgenic plants in contaminated soils. Thus, we transformed a sterile line of poplar Populus alba X P. tremula var. glandulosa with a heavy metal resistance gene, ScYCF1 (yeast cadmium factor 1), which encodes a transporter that sequesters toxic metal(loid)s into the vacuoles of budding yeast, and tested these transgenic plants in soil taken from a closed mine site contaminated with multiple toxic metal(loid)s under greenhouse and field conditions. The YCF1-expressing transgenic poplar plants exhibited enhanced growth, reduced toxicity symptoms, and increased Cd content in the aerial tissue compared to the non-transgenic plants. Furthermore, the plants accumulated increased amounts of Cd, Zn, and Pb in the root, because they could establish an extensive root system in mine tailing soil. These results suggest that the generation of YCF1-expressing transgenic poplar represents the first step towards producing plants for phytoremediation. The YCF1-expressing poplar may be useful for phytostabilization and phytoattenuation, especially in highly contaminated regions, where wild-type plants cannot survive.     

Tolerance of transgenic canola plants (Brassica napus) amended with plant growth-promoting bacteria to flooding stress at a metal-contaminated field site

The growth of transgenic canola (Brassica napus) expressing a gene for the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase was compared to non-transformed canola exposed to flooding and elevated soil Ni concentration, in situ. In addition, the ability of the plant growth-promoting bacterium Pseudomonas putida UW4, which also expresses ACC deaminase, to facilitate the growth of non-transformed and transgenic canola under the above mentioned conditions was examined. Transgenic canola and/or canola treated with P. putida UW4 had greater shoot biomass compared to non-transformed canola under low flood-stress conditions. Under high flood-stress conditions, shoot biomass was reduced and Ni accumulation was increased in all instances relative to low flood-stress conditions. This is the first field study to document the increase in plant tolerance utilizing transgenic plants and plant growth-promoting bacteria exposed to multiple stressors.     

Biotransformation of metamitron by human p450 expressed in transgenic tobacco cell cultures

In the present investigation, the oxidative metabolism of 14C-labeled metamitron was examined in plant cell cultures of tobacco overexpressing human P450 enzymes CYP1A1 or CYP1A2; special interest was in the aromatic hydroxylation of the herbicide. The oxidative metabolites deaminometamitron (DAM) and 4-hydroxydeaminometamitron (4-HDAM) were found in the untransformed control culture as well as in the transgenic culture. The transgenic cultures, however, exhibited higher turnover rates after 48 h of incubation with 20 microg 14C-metamitron per assay (untransformed: 40%, CYP1A1: 80%, CYP1A2: 100%). Primary metabolite 4-HDAM was partially found in glucosylated form in the transgenic cultures. As minor oxidative metabolites, 6-hydroxyphenyl-3-methoxymethyl-1,2,4-triazine-5(4H)-one and 3-hydroxymethyl-6-phenyl-1,2,4-triazine-5(4H)-one were identified in the transgenic cultures by GC-MS, LC-MS. Additionally, it could be demonstrated that both foreign enzymes (CYP1A1, CYP1A2) also catalyzed the deamination of metamitron. In a large-scale study (up to 400 microg per assay) with the transgenic culture expressing CYP1A2, the high efficiency of this P450 system toward metamitron was demonstrated: turnover of the xenobiotic was almost complete with 400 microg. Since large portions of unglucosylated 4-H-DAM were found, the activity of foreign CYP1A2 apparently exceeded that of endogenous O-glucosyltransferases of the tobacco cell culture. We concluded that in comparison to the nontransformed cell culture, the extent of metabolism was considerably higher in the transgenic cultures. The transgenic cell cultures expressing human CYP1A1 or CYP1A2 are thus suitable tools for the production of large quantities of primary oxidized metabolites of metamitron.     

Biotransformation of Metamitron by Human P450 Expressed in Transgenic Tobacco Cell Cultures

In the present investigation, the oxidative metabolism of ¹⁴C-labeled metamitron was examined in plant cell cultures of tobacco overexpressing human P450 enzymes CYP1A1 or CYP1A2; special interest was in the aromatic hydroxylation of the herbicide. The oxidative metabolites deaminometamitron (DAM) and 4-hydroxydeaminometamitron (4-HDAM) were found in the untransformed control culture as well as in the transgenic culture. The transgenic cultures, however, exhibited higher turnover rates after 48 h of incubation with 20 μg ¹⁴C-metamitron per assay (untransformed: 40%, CYP1A1: 80%, CYP1A2: 100%). Primary metabolite 4-HDAM was partially found in glucosylated form in the transgenic cultures. As minor oxidative metabolites, 6-hydroxyphenyl-3-methoxymethyl-1,2,4-triazine-5(4H)-one and 3-hydroxymethyl-6-phenyl-1,2,4-triazine-5(4H)-one were identified in the transgenic cultures by GC-MS, LC-MS. Additionally, it could be demonstrated that both foreign enzymes (CYP1A1, CYP1A2) also catalyzed the deamination of metamitron. In a large-scale study (up to 400 μ g per assay) with the transgenic culture expressing CYP1A2, the high efficiency of this P450 system toward metamitron was demonstrated: turnover of the xenobiotic was almost complete with 400 μ g. Since large portions of unglucosylated 4-H-DAM were found, the activity of foreign CYP1A2 apparently exceeded that of endogenous O-glucosyltransferases of the tobacco cell culture. We concluded that in comparison to the nontransformed cell culture, the extent of metabolism was considerably higher in the transgenic cultures. The transgenic cell cultures expressing human CYP1A1 or CYP1A2 are thus suitable tools for the production of large quantities of primary oxidized metabolites of metamitron.     

Sucrose amendment enhances phytoaccumulation of the herbicide atrazine in Arabidopsis thaliana

Growth in the presence of sucrose was shown to confer to Arabidopsis thaliana (thale cress or mustard weed) seedlings, under conditions of in vitro culture, a high level of tolerance to the herbicide atrazine and to other photosynthesis inhibitors. This tolerance was associated with root-to-shoot transfer and accumulation of atrazine in shoots, which resulted in significant decrease of herbicide levels in the growth medium. In soil microcosms, application of exogenous sucrose was found to confer tolerance and capacity to accumulate atrazine in Arabidopsis thaliana plants grown on atrazine-contaminated soil, and resulted in enhanced decontamination of the soil. Application of sucrose to plants grown on herbicide-polluted soil, which increases plant tolerance and xenobiotic absorption, thus appears to be potentially useful for phytoremediation.     

Hydrocarbon degradation, plant colonization and gene expression of alkane degradation genes by endophytic Enterobacter ludwigii strains

The genus Enterobacter comprises a range of beneficial plant-associated bacteria showing plant growth promotion. Enterobacter ludwigii belongs to the Enterobacter cloacae complex and has been reported to include human pathogens but also plant-associated strains with plant beneficial capacities. To assess the role of Enterobacter endophytes in hydrocarbon degradation, plant colonization, abundance and expression of CYP153 genes in different plant compartments, three plant species (Italian ryegrass, birdsfoot trefoil and alfalfa) were grown in sterile soil spiked with 1% diesel and inoculated with three endophytic E. ludwigii strains. Results showed that all strains were capable of hydrocarbon degradation and efficiently colonized the rhizosphere and plant interior. Two strains, ISI10-3 and BRI10-9, showed highest degradation rates of diesel fuel up to 68% and performed best in combination with Italian ryegrass and alfalfa. All strains expressed the CYP153 gene in all plant compartments, indicating an active role in degradation of diesel in association with plants.     

污泥耐受性草本植物的筛选及其对重金属Cu、Zn的富集

为探索植物修复污泥中重金属污染的可行性,配制不同污泥含量的介质土开展盆栽实验,对从河南、云南和上海等地收集的抗逆性强的19种草本植物进行污泥适应性筛选;研究了植物对污泥的生理响应及其对重金属Cu、Zn的吸收和分布.结果表明,上海市浦东新区白龙港污水处理厂污泥农用是可行的,农用时污泥比例应控制在60％以下;筛选出三叶鬼针草、稗子、荆芥和金叶马兰4种污泥耐受性植物;三叶鬼针草具有修复重金属Cu、Zn污染土壤的潜力;稗子具有修复重金属Zn污染土壤的能力;金叶马兰具有修复重金属Cu、Zn污染土壤的能力.     

Overexpressing GSH1 and AsPCS1 simultaneously increases the tolerance and accumulation of cadmium and arsenic in Arabidopsis thaliana

The goal of this study was to develop transgenic plants with increased tolerance for and accumulation of heavy metals and metalloids from soil by simultaneous overexpression of AsPCS1 and GSH1 (derived from garlic and baker's yeast) in Arabidopsis thaliana. Phytochelatins (PCs) and glutathione (GSH) are the main binding peptides involved in chelating heavy metal ions in plants and other living organisms. Single-gene transgenic lines had higher tolerance to and accumulated more Cd and As than wild-type. Compared to single-gene transgenic lines, dual-gene transformants exhibited significantly higher tolerance to and accumulated more Cd and As. One of the dual-gene transgenic lines, PG1, accumulated twice the amount of Cd as single-gene transgenic lines. Simultaneous overexpression of AsPCS1 and GSH1 led to elevated total PC production in transgenic Arabidopsis. These results indicate that such a stacking of modified genes is capable of increasing Cd and As tolerance and accumulation in transgenic lines, and represents a highly promising new tool for use in phytoremediation efforts.     

Effects of copper sulfate on growth and physiological responses of Limoniastrum monopetalum

A glasshouse study of the coastal shrub Limoniastrum monopetalum was carried out to evaluate its tolerance and capacity to accumulate copper. We investigate the effects of Cu from 0 to 60 mmol l^?1 on the growth, photosynthetic apparatus, and nutrient uptake of L. monopetalum, by measuring gas exchange, chlorophyll fluorescence parameters, photosynthetic pigments, and total copper, nitrogen, phosphorus, sulfur, calcium, and magnesium content in the plant tissues. Although L. monopetalum did not survive at 60 mmol l^?1 Cu, the species demonstrated a high tolerance to Cu-induced stress, since all plants survived external Cu concentrations of up to 35 mmol l^?1 and displayed similar growth in the Cu-enriched medium as in the control treatment of up to the external level of 15 mmol Cu l^?1 (1,000 mg Cu l^?1). The reduced growth registered in plants exposed to 35 mmol Cu l^?1 can be attributed to reduced photosynthetic carbon assimilation associated with the adverse effect of the metal on the photochemical apparatus and a reduction in the absorption of essential nutrients. Copper tolerance was associated with the capacity of the plant to accumulate the metal in its roots and effectively prevent its translocation to photosynthetic tissues. L. monopetalum has the characteristics of a Cu-excluder plant and could be used in the revegetation of Cu-contaminated soils.     

Recombinant human AhR-mediated GUS reporter gene assays for PCB congeners in transgenic tobacco plants in comparison with recombinant mouse and guinea pig AhRs

Four expression plasmids for recombinant human aryl hydrocarbon receptor (hAhR) consisting of a ligand binding domain of hAhR, a DNA-binding domain of LexA and a transactivation domain of VP16 as well as β-glucuronidase (GUS) reporter genes were constructed. All the expression plasmids were transformed into tobacco plants. The selected transgenic tobacco plants were used to assay. PCB congeners showed GUS activity in a TEF-dependent manner. The selected transgenic tobacco plant XhD4V17 was compared with the transgenic tobacco plants XmD4V26 and XgD2V23 containing recombinant mouse (m) AhR-mediated GUS reporter gene expression cassette and recombinant guinea pig (g) AhR-mediated GUS reporter gene expression cassette for PCB congener-inducible GUS activity. The data revealed that the tobacco plant XgD2V23 was the most active in PCB congener-inducible GUS activity. In a 1:1 mixture of PCB126 and PCB80 a reduced PCB126-induced GUS activity was observed in plant XgD2V23, which could possibly be due to interaction between PCB126 and PCB80.     

Ca(2+) -dependent plant response to Pb(2+) is regulated by LCT1

Tobacco plants transformed with TaLCT1 were cultured on Knop's medium with modified calcium concentrations (0.01-3 mM) in the presence of Pb(2+), and in soil contaminated by lead. A 4-5 microM Pb(2+) administered in the presence of 1 mM Ca(2+) inhibited the root growth of transgenic plants to much lesser degree than of control plants, whereas in the presence of 3mM Ca(2+) no differences were found between the studied lines. The reduction of Pb(2+) toxicity in the presence of 1 mM Ca(2+) was not accompanied by a change in the lead tissue concentration. However, when Ca(2+) level in the medium was lowered to 0.01 mM, several fold higher root/shoot Pb ratio in transgenic plants was observed, twofold increase in the total amount of metal accumulated, and lower concentration of Pb in the xylem sap. Results suggest the involvement of TaLCT1 in the regulation of Ca-dependent Pb-detoxification, and under conditions of low calcium in lead uptake and distribution.     

Recombinant human AhR-mediated GUS reporter gene assays for PCB congeners in transgenic tobacco plants in comparison with recombinant mouse and guinea pig AhRs

Four expression plasmids for recombinant human aryl hydrocarbon receptor (hAhR) consisting of a ligand binding domain of hAhR, a DNA-binding domain of LexA and a transactivation domain of VP16 as well as β-glucuronidase (GUS) reporter genes were constructed. All the expression plasmids were transformed into tobacco plants. The selected transgenic tobacco plants were used to assay. PCB congeners showed GUS activity in a TEF-dependent manner. The selected transgenic tobacco plant XhD4V17 was compared with the transgenic tobacco plants XmD4V26 and XgD2V23 containing recombinant mouse (m) AhR-mediated GUS reporter gene expression cassette and recombinant guinea pig (g) AhR-mediated GUS reporter gene expression cassette for PCB congener-inducible GUS activity. The data revealed that the tobacco plant XgD2V23 was the most active in PCB congener-inducible GUS activity. In a 1:1 mixture of PCB126 and PCB80 a reduced PCB126-induced GUS activity was observed in plant XgD2V23, which could possibly be due to interaction between PCB126 and PCB80.     

Effects of biosurfactants on assays of PCB congeners in transgenic arabidopsis plants carrying a recombinant guinea pig AhR-mediated GUS reporter gene expression system

The transgenic Arabidopsis plants carrying a recombinant guinea pig (g) aryl hydrocarbon receptor (AhR)-mediated β-glucuronidase (GUS) reporter gene expression system were generated for assays of polychlorinated biphenyl (PCB) congeners. The selected transgenic Arabidopsis plant XgD2V11-6 exhibited a correlation between uptake of PCB126 and PCB126-induced GUS activity. Also, the plants showed induced GUS activity towards the supplemental indole 3-acetic acid (IAA). Thus, the GUS assay may reflect induction by both endogenous and exogenous AhR ligands. When biosurfactants, MEL-B, produced in the culture of yeast isolated from plants were used for assays of PCB congeners in the transgenic Arabidopsis plants, they showed marked PCB126 dose-dependent and toxic equivalency factor (TEF) dependent GUS activities. The effects of biosurfactants were clearer when the plants were cultivated on soils containing PCB congeners for 7 days as compared with on soils for 3 days as well as in the medium for 3 days. Threfore, it was estimated that biosurfactants form micellae with PCB congeners, which are easily uptaken by the plants in a mode of passive diffusion, transport into the aerial parts and then induce GUS activity.     

Isolation and characterization of a heavy metal-resistant Burkholderia sp. from heavy metal-contaminated paddy field soil and its potential in promoting plant growth and heavy metal accumulation in metal-polluted soil

A heavy metal-resistant bacterial strain was isolated from heavy metal-contaminated soils and identified as Burkholderia sp. J62 based on the 16S rDNA gene sequence analysis. The heavy metal- and antibiotic resistance, heavy metal solubilization of the isolate were investigated. The isolate was also evaluated for promoting plant growth and Pb and Cd uptakes of the plants from heavy metal-contaminated soils in pot experiments. The isolate was found to exhibit different multiple heavy metal and antibiotic resistance characteristics. Atomic absorption spectrometer analysis showed increased bacterial solubilization of lead and cadmium in solution culture and in soils. The isolate produced indole acetic acid, siderophore and 1-aminocyclopropane-1-carboxylate deaminase. The isolate also solubilized inorganic phosphate. Inoculation with the isolate was found to significantly (p < 0.05) increase the biomass of maize and tomato plants. Increase in tissue Pb and Cd contents varied from 38% to 192% and from 5% to 191% in inoculated plants growing in heavy metal-contaminated soils compared to the uninoculated control, respectively. These results show that heavy metal-solubilizing and plant growth promoting bacteria are important for plant growth and heavy metal uptake which may provide a new microbial enhanced-phytoremediation of metal-polluted soils.     

Effects of biosurfactants on assays of PCB congeners in transgenic arabidopsis plants carrying a recombinant guinea pig AhR-mediated GUS reporter gene expression system

The transgenic Arabidopsis plants carrying a recombinant guinea pig (g) aryl hydrocarbon receptor (AhR)-mediated β-glucuronidase (GUS) reporter gene expression system were generated for assays of polychlorinated biphenyl (PCB) congeners. The selected transgenic Arabidopsis plant XgD2V11-6 exhibited a correlation between uptake of PCB126 and PCB126-induced GUS activity. Also, the plants showed induced GUS activity towards the supplemental indole 3-acetic acid (IAA). Thus, the GUS assay may reflect induction by both endogenous and exogenous AhR ligands. When biosurfactants, MEL-B, produced in the culture of yeast isolated from plants were used for assays of PCB congeners in the transgenic Arabidopsis plants, they showed marked PCB126 dose-dependent and toxic equivalency factor (TEF) dependent GUS activities. The effects of biosurfactants were clearer when the plants were cultivated on soils containing PCB congeners for 7 days as compared with on soils for 3 days as well as in the medium for 3 days. Therefore, it was estimated that biosurfactants form micellae with PCB congeners, which are easily uptaken by the plants in a mode of passive diffusion, transport into the aerial parts and then induce GUS activity.     

Xenobiotics: Substrates and inhibitors of the plant cytochrome P450

The ability of a plant cytochrome P450 to bind and metabolise plant endogenous molecules and xenobiotics was investigated. The work was performed on the yeast-expressed CYP73A1, a cinnamate 4-hydroxylase isolated fromHelianthus tuberosus. CYP73 controls the general phenylpropanoid pathway and is likely to be one of the most abundant sources of P450 in the biosphere. The enzyme shows a high selectivity toward plant secondary metabolites. Nevertheless, it oxygenates several small and planar xenobiotics with low efficiency, including an herbicide (chlorotoluron). One xenobiotic molecule, 2naphthoic acid, is hydroxylated with an efficiency comparable to that of the physiological substrate. This reaction was used to devise a fluorimetric test for the rapid measurement of enzyme activity. A series of herbicidal molecules (hydroxybenzonitriles) are shown to bind the active site without being metabolised. These molecules behave as strong competitive inhibitors of CYP73 with a K_i in the same micromolar range as the K_m for the physiological substrate. It is proposed that their inhibition of the phenylpropanoid pathway reinforces their other phytotoxic effects at the level of the chloroplasts. All our results indicate a strong reciprocal interaction between plant P450s and xenobiotics.