Engineering the metabolism of the phenylurea herbicide chlortoluron in genetically modified Arabidopsis thaliana plants expressing the mammalian cytochrome P450 enzyme CYP1A2

Transgenic Arabidopsis thaliana plants were generated by introduction of the human P450 CYP1A2 gene, which metabolizes a number of herbicides, insecticides and industrial chemicals. Transgenic A. thaliana plants expressing CYP1A2 gene showed remarkable resistance to the phenylurea herbicide chlortoluron (CTU) supplemented either in plant growth medium or sprayed on foliar parts of the plants. HPLC analyses showed a strong reduction in CTU accumulation in planta supporting the tolerance of transgenic lines to high concentrations of CTU. Besides increased herbicide tolerance, expression of CYP1A2 resulted in no other visible phenotype in transgenic plants. Our data indicate that CYP1A2 can be used as a selectable marker for plant transformation, allowing efficient selection of transgenic lines in growth medium and/or in soil-grown plants. Moreover, these transgenic plants appear to be useful for herbicide resistance as well as phytoremediation of environmental contaminants.     

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.     

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.     

Effect of BPA on the germination,root development,seedling growth and leaf differentiation under different light conditions in Arabidopsis thaliana

Bisphenol A (BPA) is a well-known environmental toxic substance, which exerts unfavorable effects through endocrine disruptor (ER)-dependent and ER-independent mechanisms to threaten ecological systems seriously. BPA may also interact with other environmental factors, such as light and heavy metals, to have a synergetic effect in plants. However, there is little data concerning the toxic effect of BPA on the primary producers-plants and its possible interaction with light-dependent response. Here, the effects of BPA on germination, fresh weight, tap root length, and leaf differentiation were studied in Arabidopsis thaliana under different parts of light spectrum (dark, red, yellow, green, blue, and white light). Our results showed that low-dose BPA (1.0, 5.0 μM) caused an increase in the fresh weight, the tap root length and the lateral root formation of A. thaliana seedlings, while high-dose BPA (10.0, 25.0 μM) show an inhibition effect in a dose-dependent manner. Unlike karrikins, the effects of BPA on germination fresh weight and tap roots length under various light conditions are similar, which imply that BPA has no notable role in priming light response in germination and early seedling growth in A. thaliana. Meanwhile, BPA exposure influences the differentiation of A. thaliana leaf blade significantly in a light-dependent manner with little to no effect in dark and clear effect under red illumination.     

Characterization and cadmium-resistant gene expression of biofilm-forming marine bacterium Pseudomonas aeruginosa JP-11

Biofilm-forming marine bacterium Pseudomonas aeruginosa JP-11 was isolated from coastal marine sediment of Paradeep Port, Odisha, East Coast, India, which resisted up to 1,000 ppm of cadmium (Cd) as cadmium chloride in aerobic conditions with a minimal inhibitory concentration of 1,250 ppm. Biomass and extracellular polymeric substances (EPS) secreted by the cells effectively removed 58.760?±?10.62 and 29.544?±?8.02 % of Cd, respectively. The integrated density of the biofilm-EPS observed under fluorescence microscope changed significantly (P?≤?0.05) in the presence of 50, 250, 450, 650 and 850 ppm Cd. ATR-FTIR spectroscopy showed a peak at 2,365.09/cm in the presence of 50, 250, 450 and 650 ppm Cd which depicts the presence of sulphydryl group (–SH) within the EPS, whereas, a peak shift to 2,314.837/cm in the presence of 850 ppm Cd suggested the major role of this functional group in the binding with cadmium. On exposure to Cd at 100, 500 and 1,000 ppm, the expression profiles of cadmium resistance gene (czcABC) in the isolate showed an up-regulation of 3.52-, 17- and 24-fold, respectively. On the other hand, down-regulation was observed with variation in the optimum pH (6) and salinity (20 g l^?1) level. Thus, the cadmium resistance gene expression increases on Cd stress up to the tolerance level, but an optimum pH and salinity are the crucial factors for proper functioning of cadmium resistance gene.     

Ecotoxicological effects of cadmium on three ornamental plants

Ecotoxicological effects of cadmium (Cd) on three ornamental plants African marigold (Tagetes erecta), scarlet sage (Salvia splendens) and sweet hibiscus (Abelmoschus manihot) were investigated. Seeds of these plants were exposed to five different concentrations of Cd (0-50 mgl(-1)). Ecotoxicological indexes based on inhibition rate (IC) of seed germination, root and shoot elongation, biomass (fresh weight and dry weight), as well as IC50 (Cd concentration when 50% plants show inhibition) and tolerance indexes (the ratio of maximum root length in an experimental group to that in a control group) were determined. The results indicated that Cd had little effects (p>0.05) on seed germination of the three plants and shoot elongation of scarlet sage (S. splendens). Cadmium had significant (p<0.05) inhibitory effects on root elongation of the three plants and shoot elongation of African marigold (T. erecta). The fresh weight biomass of scarlet sage (S. splendens) was most sensitive to Cd, while that of sweet hibiscus (A. manihot) was least sensitive. On a dry weight basis, African marigold (T. erecta) was the least sensitive, and scarlet sage (S. splendens) was the most sensitive to Cd. Based on IC50 of seed germination, sweet hibiscus (A. manihot) was the most insensitive plant with an IC50 value as high as 428.0 gl(-1). According to Cd-tolerance indexes under the same Cd concentration, sweet hibiscus (A. manihot) was the most tolerant plant whereas scarlet sage (S. splendens) was the most sensitive one.     

Characterization of endophytic Rahnella sp. JN6 from Polygonum pubescens and its potential in promoting growth and Cd,Pb, Zn uptake by Brassica napus

Microbe-assisted phytoremediation has been considered as a promising measure for the remediation of heavy metal-polluted soils. In this study, a metal-tolerance and plant growth-promoting endophytic bacterium JN6 was firstly isolated from roots of Mn-hyperaccumulator Polygonum pubescens grown in metal-contaminated soil and identified as Rahnella sp. based on 16S rDNA gene sequence analysis. Strain JN6 showed very high Cd, Pb and Zn tolerance and effectively solubilized CdCO₃, PbCO₃ and Zn₃(PO₄)₂ in culture solution. The isolate produced plant growth-promoting substances such as indole-3-acetic acid, siderophore, 1-aminocyclopropane-1-carboxylic deaminase, and also solubilized inorganic phosphate. Based upon its ability in metal tolerance and solubilization, the isolate JN6 was further studied for its effects on the growth and accumulation of Cd, Pb and Zn in Brassica napus (rape) by pot experiments. Rape plants inoculated with the isolate JN6 had significantly higher dry weights, concentrations and uptake of Cd, Pb and Zn in both above-ground and root tissues than those without inoculation grown in soils amended with Cd (25 mg kg^?1), Pb (200 mg kg^?1) or Zn (200 mg kg^?1). The isolate also showed a high level of colonization in tissue interior of rapes. The present results demonstrated that Rahnella sp. JN6 is a valuable microorganism, which can cost-effectively improve the efficiency of phytoremediation in soils contaminated by Cd, Pb and Zn.     

Oxidative stress and arsenic toxicity: Role of NADPH oxidases

The effect of arsenic (25 and 50 μM As for 1 and 5 d) was analysed in wild type (WT) and Arabidopsis thaliana (L.) Heynh plants deficient in NADPH oxidase C (AtrbohC). The content of H₂O₂ and malondialdehyde (MDA) increased with the As concentration, while the opposite effect was found for NO in WT and AtrbohC plants. The As treatment reduced catalase and increased glutathione reductase activities to the same extent in WT and AtrbohC plants, although the induction of all SOD isoforms (mainly CuZn–SODs) was observed in WT plants, the opposite effects being found in AtrbohC plants. Glycolate oxidase (H₂O₂ producers) considerably increased with the concentration and time of treatment with As in WT and AtrbohC mutants. Arsenic induced the uptake and translocation of P, S, Cu, Zn, and Fe in WT plants, while in AtrbohC plants the opposite trend was noted and the uptake of As became considerably lower than in WT plants. These results suggest that As causes oxidative stress by inducing glycolate oxidase, while NADPH oxidase does not appear to participate in ROS overproduction but could be critical in regulating antioxidant defences as well as the transport and translocation of As and macro/micronutrients.     

Assessment of cadmium accumulation,toxicity, and tolerance in Brassicaceae and Fabaceae plants—implications for phytoremediation

This study, based on a greenhouse pot culture experiment conducted with 15-day-old rapeseed (Brassica campestris L. cv. Pusa Gold; family Brassicaceae) and moong bean (Vigna radiata L. Wilczek cv. Pusa Ratna; family Fabaceae) plants treated with cadmium (Cd) concentrations (0, 50, and 100 mg kg^?1 soil), investigates their potential for Cd accumulation and tolerance, and dissects the underlying basic physiological/biochemical mechanisms. In both species, plant dry mass decreased, while Cd concentration of both root and shoot increased with increase in soil Cd. Roots harbored a higher amount of Cd (vs. shoot) in B. campestris, while the reverse applied to V. radiata. By comparison, root Cd concentration was higher in B. campestris than in V. radiata. The high Cd concentrations in B. campestris roots and V. radiata shoots led to significant elevation in oxidative indices, as measured in terms of electrolyte leakage, H₂O₂ content, and lipid peroxidation. Both plants displayed differential adaptation strategies to counteract the Cd burden-caused anomalies in their roots and shoots. In B. campestris, increasing Cd burden led to a significantly decreased reduced glutathione (GSH) content but a significant increase in activities of GSH reductase (GR), GSH peroxidase (GPX), and GSH sulfotransferase (GST). However, in V. radiata, increasing Cd burden caused significant increase in GSH content and GR activity, but a significant decline in activities of GPX and GST. Cross talks on Cd burden of tissues and the adapted Cd tolerance strategies against Cd burden-accrued toxicity indicated that B. campestris and V. radiata are good Cd stabilizer and Cd extractor, respectively, wherein a fine tuning among the major components (GR, GPX, GST, GSH) of the GSH redox system helped the plants to counteract differentially the Cd load-induced anomalies in tissues. On the whole, the physiological/biochemical characterization of the B. campestris and V. radiata responses to varying Cd concentrations can be of great help in elaborating the innovative plant-based remediation technologies for metal/metalloid-contaminated sites.     

Cd-tolerant Suillus luteus: A fungal insurance for pines exposed to Cd

Soil metal pollution can trigger evolutionary adaptation in soil-borne organisms. An in vitro screening test showed cadmium adaptation in populations of Suillus luteus (L.: Fr.) Roussel, an ectomycorrhizal fungus of pine trees. Cadmium stress was subsequently investigated in Scots pine (Pinus sylvestris L.) seedlings inoculated with a Cd-tolerant S. luteus, isolated from a heavy metal contaminated site, and compared to plants inoculated with a Cd-sensitive isolate from a non-polluted area. A dose-response experiment with mycorrhizal pines showed better plant protection by a Cd-adapted fungus: more fungal biomass and a higher nutrient uptake at high Cd exposure. In addition, less Cd was transferred to aboveground plant parts. Because of the key role of the ectomycorrhizal symbiosis for tree fitness, the evolution of Cd tolerance in an ectomycorrhizal partner such as S. luteus can be of major importance for the establishment of pine forests on Cd-contaminated soils.     

Screening of Cd tolerant genotypes and isolation of metallothionein genes in alfalfa (Medicago sativa L.)

In order to evaluate Cd tolerance in wide-ranging sources of alfalfa (Medicago sativa) and to identify Cd tolerant genotypes which may potentially be useful for restoring Cd-contaminated environments, thirty-six accessions of alfalfa were screened under hydroponic culture. Our results showed that the relative root growth rate varied from 0.48 to 1.0, which indicated that different alfalfa accessions had various responses to Cd stress. The candidate fragments derived from differentially expressed metallothionein (MT) genes were cloned from leaves of two Cd tolerant genotypes, YE and LZ. DNA sequence and the deduced protein sequence showed that MsMT2a and MsMT2b had high similarity to those in leguminous plants. DDRT-PCR analysis showed that MsMT2a expressed in both YE and LZ plants under control and Cd stress treatment, but MsMT2b only expressed under Cd stress treatment. This suggested that MsMT2a was universally expressed in leaves of alfalfa but expression of MsMT2b was Cadmium (Cd) inducible.     

Effect of octylphenol on physiologic features during growth in Arabidopsis thaliana

Alkylphenol ethoxylates are widely used as detergents, emulsifiers, solubilizers, wetting agents and dispersants. Octylphenol (OP) ethoxylates, one of alkylphenol ethoxylates, represent 15–20% of the market, and their metabolic residues may be discharged to surface waters, sediments and soils as a persistent and ubiquitous pollutant. We tested the response of Arabidopsis thaliana to different concentrations of OP. OP affected the germination percentage and mean germination period. 10 d treatment with OP, especially high concentration (10 and 50 mg L⁻¹), decreased shoot and root biomass and root length of 30 d-old A. thaliana. Content of chlorophyll was decreased but that of proline was increased in leaves with OP treatment. OP caused oxidative stress in leaves; malondialdehyde content was increased, and the activities of ascorbate peroxidase, catalase and superoxide dismutase were induced. OP affects the physiologic and morphologic features of A. thaliana during growth. Because plants might be exposed to OP for a long time in the surroundings, more attention needs to be paid to the effect of OP on plants.     

The negative effects of cadmium on Bermuda grass growth might be offset by submergence

Revegetation in the water-level-fluctuation zone (WLFZ) could stabilize riverbanks, maintain local biodiversity, and improve reservoir water quality in the Three Gorges Reservoir Region (TGRR). However, submergence and cadmium (Cd) may seriously affect the survival of transplantations. Bermuda grass (Cynodon dactylon) is a stoloniferous and rhizomatous prostrate weed displaying high growth rate. A previous study has demonstrated that Bermuda grass can tolerate deep submergence and Cd stress, respectively. In the present study, we further analyzed physiological responses of Bermuda grass induced by Cd-and-submergence stress. The ultimate goal was to explore the possibility of using Bermuda grass for revegetation in the WLFZ of China’s TGRR and other riparian areas. The Cd-and-submergence-treated plants had higher malondialdehyde contents and peroxidase than control, and both increased with the Cd concentration increase. All treated plants catalase activity increased with the experimental duration increases, and their superoxide dismutase also gradually increased with the Cd concentration from 1 day to 15 days. Total biomass of the same Cd-and-submergence plants increased along the experimental duration as well. Plants exposed to Cd-and-submergence stress showed shoot elongation. The heights of all treated plants were taller than those of the control. Leaf chlorophyll contents, maximum leaf length, and soluble sugars contents of all the Cd-and-submergence-treated plants were more than those of the untreated control. Although Cd inhibits plants growth, decreases chlorophyll and biomass content, and with the submergence induced the leaf and shoot elongation, more part of the Cd-and-submergence stress plants appeared in the air, exhibited fast growth with maintenance of leaf color, which guaranteed the plants’ photosynthesis, and ensured the total biomass and carbohydrate sustainability, further promoting Cd-and-submergence tolerance. The results imply that the negative effects of cadmium on Bermuda grass growth might be offset by submergence.     

Saline irrigation and Zn amendment effect on Cd phytoavailability to Swiss chard (Beta vulgaris L.) grown on a long-term amended agricultural soil: a human risk assessment

Crops, particularly in the Northeast region of Mexico, have to cope with increasing soil salinization due to irrigation. Chloride (Cl^?) concentration has been strongly related to enhance cadmium (Cd) uptake by plants due to increased solubility in the soil solution. The effect of irrigation with slightly saline water from a local well was evaluated in this work on the accumulation and translocation of Cd in Swiss chard (Beta vulgaris L.) grown in soil historically amended with stabilized sewage sludge under a regime of phosphorus and zinc fertilization. A factorial pot experiment was conducted with two phosphate fertilizer levels (PF, 0 and 80 kg ha^?1 dry soil, respectively), two Zn levels (0 and 7 kg ha^?1 dry soil), and two sources of water for irrigation deionized water (DW) and slightly saline well water (WW) from an agricultural site. Additionally, a human risk assessment for Cd ingestion from plants was assessed. Results showed that Cl^? salinity in the WW effectively mobilized soil Cd and increased its phytoavailability. A higher level of Cd was found in roots (46.41 mg kg^?1) compared to shoots (10.75 mg kg^?1). Although the total content of Cd in the edible parts of the Swiss chard irrigated with WW exceeded permissible recommended consumption limit, bioavailable cadmium in the aboveground parts of the plant in relation to the total cadmium content was in the range from 8 to 32 %. Therefore, human health risks might be overestimated when the total concentration is taken into account.     

Phosphorus and cadmium interactions in Kandelia obovata (S. L.) in relation to cadmium tolerance

This study focused on the cadmium (Cd) tolerance of mangroves with application of phosphate (P) in order to explore whether exogenous P can alleviate Cd stress on these intertidal species. Kandelia obovata (S. L.) seedlings were cultivated in rhizoboxes under different levels of Cd and P concentrations. The speciation distributions of Cd in the rhizosphere and non-rhizosphere sediments were examined by sequential extraction procedures; organic acid in plant tissues and soil solution was measured by high-performance liquid chromatography; Cd and P accumulation in the plants was also determined. Results showed that considerable differences existed in Cd speciation distributions between rhizosphere and non-rhizosphere sediments. Root activity influenced the dynamics of Cd, P application increased the organic acid content in root tissues, P also increased Cd accumulation in roots whilst lowering Cd translocation from root to the above-ground tissues, and a significant positive correlation was found between Cd and P in roots (r?=?0.905). It is postulated that Cd detoxification of K. obovata (S. L.) is associated with higher Cd immobilization in the presence of higher P and organic acid contents in root tissue.     

Drought-tolerant <Emphasis Type="Italic">Streptomyces pactum</Emphasis> Act12 assist phytoremediation of cadmium-contaminated soil by <Emphasis Type="Italic">Amaranthus hypochondriacus</Emphasis>: great potential application in arid/semi-arid areas

Microbe-assisted phytoremediation provides an effective approach to clean up heavy metal-contaminated soils. However, severe drought may affect the function of microbes in arid/semi-arid areas. Streptomyces pactum Act12 is a drought-tolerant soil actinomycete strain isolated from an extreme environment on the Qinghai-Tibet Plateau, China. In this study, pot experiments were conducted to assess the effect of Act12 on Cd tolerance, uptake, and accumulation in amaranth (Amaranthus hypochondriacus) under water deficit. Inoculated plants had higher Cd concentrations (root 8.7–33.9 %; shoot 53.2–102.1 %) and uptake (root 19.9–95.3 %; shoot 110.6–170.1 %) than non-inoculated controls in Cd-treated soil. The translocation factor of Cd from roots to shoots was increased by 14.2–75 % in inoculated plants, while the bioconcentration factor of Cd in roots and shoots was increased by 10.2–64.4 and 53.9–114.8 %, respectively. Moreover, inoculation with Act12 increased plant height, root length, and shoot biomass of amaranth in Cd-treated soil compared to non-inoculated controls. Physiochemical analysis revealed that Act12 enhanced Cd tolerance in the plants by increasing glutathione, elevating superoxide dismutase and catalase activities, as well as reducing malondialdehyde content in the leaves. The drought-tolerant actinomycete strain Act12 can enhance the phytoremediation efficiency of amaranth for Cd-contaminated soils under water deficit, exhibiting potential for application in arid and semi-arid areas.     

Cadmium phytoremediation potential of turnip compared with three common high Cd-accumulating plants

Phytoextraction is a phytoremediation technique used for remediating polluted soils and it greatly relies on the plants’ capacities to accumulate contaminants. Turnip is a high cadmium (Cd)-accumulating plant. We compared the Cd tolerance, growth, and Cd accumulation characteristics of two turnip landraces with three additional commonly known high Cd-accumulating species to systematically estimate its Cd phytoremediation potential. Results showed that the turnips could tolerate relatively lower Cd concentrations than other plants. Growth characteristics analyses indicated that the turnips initially grew rapidly and then gradually slowed down, and their photosynthetic parameters indicated that biomass accumulation was easily affected by light. However, the Cd uptake and translocation capacities of the two turnip landraces were higher than those of Phytolacca americana Linn. and Bidens pilosa Linn. but close to that of Brassica napus Linn.. Ultimately, large amounts of Cd accumulated in turnips during early growth and slightly increased as the fleshy roots increased in size. Based on these findings, the present turnip landraces have potential for soil remediation, but additional research is needed before these landraces can be practically used. Moreover, turnips are good candidates for studying the molecular mechanism of high Cd accumulation in plants.