Are Phragmites australis enzymes involved in the degradation of the textile azo dye acid orange 7?

The role of antioxidant and detoxification enzymes of Phragmites australis, in the degradation of an azo dye, acid orange 7 (AO7), was studied. Activities of several enzymes involved in plant protection against stress were assayed through the activity characterization of superoxide dismutase (SOD), peroxidases (POD), catalase (CAT), ascorbate peroxidase (APOX), dehydroascorbate reductase (DHAR) and glutathione S-transferase (GST), obtained from P. australis crude extracts of leaves, stems and roots. A sub-surface vertical flow constructed wetland, planted with P. australis was used to test the plants response to the AO7 exposure at two different concentrations (130 and 700 mg l(-1)). An activity increase was detected for an AO7 concentration of 130 mg l(-1) for most enzymes studied (SOD, CAT and APOX), especially in leaves, suggesting a response of the reactive oxygen species scavenging enzymes to the chemical stress imposed. GST activity increase in this situation can also be interpreted as an activation of the detoxification pathway and subsequent AO7 conjugation. A totally different behaviour was observed for AO7 at 700 mg l(-1). An evident decrease in activity was observed for SOD, CAT, APOX and GST, probably due to enzymatic inhibition by AO7. Contrarily, DHAR activity augmented drastically in this situation. POD activity was not greatly affected during trial. Altogether these results suggest that P. australis effectively uses the ascorbate-glutathione pathway for the detoxification of AO7.     

Responses induced by high concentration of cadmium in Phragmites australis roots

Cadmium is an important environmental pollutant with high toxicity to plants. We report the effects of high-dose Cd (100 µ M for 21 days) on the root apparatus of Phragmites australis plants, which are characterized by elevated water detoxification capacity and widely used in phytoremediation programmes. The examination of root sections by light and electron microscopy failed to reveal any significant cadmium-induced structural or ultra-structural modifications. However, histochemical localization of Cd disclosed accumulation of the metal in the parenchyma cells below the exodermis. Phytochelatins (PC) are thiol-rich peptides whose synthesis is induced by a range of metals. Our results indicate that total PC production increases after exposure to Cd, which suggests a pivotal role for phytochelatins in the sequestration of metal. Cd treatment also induced lignin deposition and marked stimulation of root antioxidant systems, suggesting that, because of its ability to adopt different strategies against the harmful effects of cadmium, Phragmites australis is a plant with high detoxification potential.     

Enhanced tolerance of transgenic poplar plants overexpressing gamma-glutamylcysteine synthetase towards chloroacetanilide herbicides.

A wild-type poplar hybrid and two transgenic clones overexpressing a bacterial gamma-glutamylcysteine synthetase in the cytosol or in the chloroplasts were exposed to the chloroacetanilide herbicides acetochlor and metolachlor dispersed in the soil. The transformed poplars contained higher gamma-glutamylcysteine and glutathione (GSH) levels than wild-type plants and therefore it was supposed that they would have an elevated tolerance towards these herbicides, which are detoxified in GSH-dependent reactions. Phenotypically, the transgenic and wild-type plants did not differ. The growth and the biomass of all poplar lines were markedly reduced by the two chloroacetanilide herbicides. However, the decrease of shoot and root fresh weights caused by the herbicides was significantly smaller in the transgenic than in wild-type plants. In addition, the growth rate of poplars transformed in the cytosol was reduced to a significantly lesser extent than that of wild-type plants following herbicide treatments. The effects of the two herbicides were similar. Herbicide exposures markedly increased the levels of gamma-glutamylcysteine and GSH in leaves of each poplar line. The increase in the foliar amounts of these thiols was stronger in the transgenic lines than in the wild type, particularly in the upper leaves. Considerable GST activities were detected in leaves of all poplar plants. Exposure of poplars to chloroacetanilide herbicides resulted in a marked induction of GST activity in upper leaf positions but not in middle and lower leaves. The extent of enzyme induction did not differ significantly between transgenic and wild-type poplars. Although the results show that the transgenic poplar lines are good candidates for phytoremediation purposes, the further improvement of their detoxification capacity, preferably by transformation using genes encoding herbicide-specific GST isoenzymes, seems to be the most promising way to obtain plants suitable for practical application.     

Biochemical Characterization and Distribution of Glutathione S-Transferases in Leaping Mullet (Liza saliens)

In this study, feral leaping mullet (Liza saliens) liver cytosolic glutathione S-transferases (GSTs) were investigated and characterized using 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (EA) as substrates. The average GST activities towards CDNB and EA were found to be 1365 +/- 41 and 140 +/- 20 nmol/min per mg protein, respectively. The effects of cytosolic protein amount and temperature ranging from 4 to 70 degrees C on enzyme activities were examined. While both activities towards CDNB and EA showed similar dependence on protein amount, temperature optima were found as 37 and 42 degrees C, respectively. In addition, the effects of pH on GST-CDNB and -EA activities were studied and different pH activity profiles were observed. For both substrates, GST activities were found to obey Michaelis-Menten kinetics with apparent V(max) and K(m) values of 1661 nmol/min per mg protein and 0.24 mM and 157 nmol/min per mg protein and 0.056 mM for CDNB and EA, respectively. Distribution of GST in Liza saliens tissues was investigated and compared with other fish species. Very high GST activities were measured in tissues from Liza saliens such as liver, kidney, testis, proximal intestine, and gills. Moreover, our results suggested that GST activities from Liza saliens would be a valuable biomarker for aquatic pollution.     

Development of transgenic tobacco plants overexpressing maize glutathione S-transferase I for chloroacetanilide herbicides phytoremediation

Glutathione S-transferases (GSTs, EC 2.5.1.18) are a multigene family of detoxification enzymes that biotransform a wide variety of endogenous and exogenous electrophilic substrates, including herbicides. The isozyme GST I from maize exhibits significant catalytic activity for the chloroacetanilide herbicide alachlor and appears to be involved in its detoxifying process. To establish the in planta ability of GST I to detoxify from alachlor, transgenesis studies were carried out. The gene gstI-6His, which encodes for 6His-tagged GST I, was used for the construction of a binary vector suitable for genetic engineering of tobacco plants (Nicotiana tabacum). Through biolistic method transgenic tobacco plants were obtained. Integration of gstI-6His gene in transgenic tobacco plants genome was confirmed by polymerase chain reaction and Southern blot hybridization. The expression of active GST I was established by Western blot analysis, using anti-6His antibody, and by direct purification of 6-His tagged GST I on Ni-NTA agarose. Primary transformed plants harboring the gstI-6His gene were transferred to MS medium supplemented with alachlor and their phenotype was evaluated. The transgenic plants showed substantially higher tolerance to alachlor compared to non-transgenic plants in terms of root, leaves and vigorous development. These transgenic plants are potentially useful biotechnological tools for the development of phytoremediation system for the degradation of herbicide pollutants in agricultural fields.     

Phytoremediation of organic xenobiotics - Glutathione dependent detoxification in Phragmites plants from European treatment sites

Studies on the uptake of several organic xenobiotics and on their subsequent conjugation to biomolecules have been performed to elucidate the use of reed plants in phytoremediation of polluted water. Phragmites australis plants were able to accumulate organic xenobiotics in their rhizomes. The uptake was correlated to the logKOW and pKa of the xenobiotics and highest with compounds exhibiting logKOWs between 1 and 3. Detoxification of xenobiotics was demonstrated when the activity of glutathione S-transferase was determined in plants from various treatment sites. Enzyme activities were strongly dependent on the provenience of the plant and the history of the stand. Detoxification enzymes were also inducible. Naphthylic acetic acid (NAA), 2,4-dichlorophenol and BION were tested as potential inducers. BION was able to induce the GST activity 5-fold, albeit only for a short period of hours. The mechanism of induction and the flexibility of the detoxification system of certain ecotypes of reed toward stress or the pollution level will require further investigation.     

Glutathione S-transferase activities in phytophagous insects: Induction and inhibition by plant phototoxins and phenols

The effects of two plant phototoxins (xanthotoxin and harmine) and three plant phenols (quercetin, ellagic acid, and juglone) on detoxification enzymes were studied in the polyphagous cabbage looper, Trichoplusia ni, and the oligophagous black swallowtail, Papilio polyxenes. In P. polyxenes, glutathione S-transferase (GST) activities toward 1-chloro-2,4-dinitrobenzene (CDNB) were 1840 and 1750 nmol CDNB conjugate/mg protein/min in the cytosolic fraction of midgut and fat body, respectively. Dietary xanthotoxin (0.1% fw) increased the activity 2.5 and 2.9-fold in the midgut and fat body, respectively. Xanthotoxin-conjugating GST activity was absent in both tissues. In T. ni, GST activity, 513 nmol CDNB conjugate/mg protein/min in the cytosolic fraction of midgut, was increased almost twofold by dietary xanthotoxin and harmine. Plant phenols effectively inhibited in vitro GST and Se-independent glutathione peroxidase (GPOX) activities in a dose-dependent manner in the two species. Both GST and GPOX of P. polyxenes were 2-fold less sensitive to phenol inhibitors than T. ni. GST inhibition differed according to the nature of the inhibitor in P. polyxenes. Quercetin is competitive with CDNB and is non-competitive with respect to GSH. In contrast, inhibition by ellagic acid is non-competitive with CDNB and competitive with GSH. Juglone showed competitive inhibition with both GSH and CDNB.     

芦苇抗镉污染机理研究

研究了芦苇幼苗体内 Cd的积累、亚细胞微区分布、存在形态和其诱导蛋白以及植物络合素合成抑制剂 (BSO)对芦苇光合作用和生长的影响。在 Cd污染条件下 ,芦苇幼苗植株和根皮层细胞中可积累大量的Cd,但 Cd在芦苇各器官和根皮层细胞亚细胞结构中的分布显著不均 ;Cd在芦苇幼苗体内的分配为 :根 >叶片 >茎 >地下茎 ,在根皮层细胞中的分布为 :细胞间隙 >细胞壁 >液泡 >细胞质。受 Cd污染的芦苇幼苗体内的 Cd以不同化学形态存在 ,其中 Na Cl提取态的 Cd在根和叶片中占的比例均为最大 ,其次为根内的醋酸提取态 ;在叶片中以水提取态为主 ,其它形态的含量相对较低。层析结果表明 ,根和叶片中各存在一种Cd结合蛋白 ,其中根内的 Cd结合蛋白可能是一种植物络合素聚合体。受 Cd诱导 ,芦苇幼苗根中还新合成了一种小分子蛋白或多肽 ,但另有一种蛋白因 Cd影响而消失。此外 ,BSO实验证明了植物络合素对 Cd的解毒作用。可见 ,芦苇的抗 Cd机理与以下几个方面有关 :根部截留 ,细胞间隙积累 ,细胞壁沉淀 ,液泡区域化 ,形成活性较低的难溶化合物 ,形成 Cd结合蛋白     

Phytoremediation of selenium using subsurface-flow constructed wetland

The potential of two plant species, Phragmites australis (common reed) and Typha latifolia (cattail), in the phytoremediation process of selenium (Se) was studied in subsurface-flow constructed wetland (SSF). Se was supplemented continuously at a concentration of 100 microg Se L(-1) in the inlet of the cultivation beds of the SSF. Water samples collected from the outlet of the Phragmites bed after 1, 3, 6, 9, and 12 wk of treatments showed that Se content was under detectable limits. Water samples collected from the Typha bed at the same five periods showed that Se concentrations in the outlet were 55, 47, 65, 76, and 25 microg/L, respectively. The results of bioaccumulation in the biomass of both species after 12 wk of treatment indicated that Typha plants accumulated Se mainly in fine roots. Phragmites accumulated Se mainly in leaves and rhizomes, and moderate levels were found in stems and fine organic materials. The results indicate that common reed is a very good species for Se phytoextraction and phytostabilization (immobilization) and that cattail is only a phytostabilization species. The use of common reed and cattail for Se phytoremediation in a SSF system and in constructed wetland models are discussed.     

A Mu-class glutathione S-transferase from gills of the marine shrimp Litopenaeus vannamei: purification and characterization

Glutathione S-transferases (GSTs) are a family of detoxifying enzymes that catalyze the conjugation of glutathione (GSH) to electrophiles, thereby increasing the solubility of GSH and aiding its excretion from the cell. In this study, a glutatione S-transferase from the gills of the marine shrimp Litopenaeus vannamei was purified by affinity chromatography using a glutathione-agarose affinity column. GST was purified to homogeneity as judged by reducing SDS-PAGE and zymograms. This enzyme is a homodimer composed of approximately 25-kDa subunits and identified as a Mu-class GST based on its activity against 1-chloro-2,4-dinitrobenzene (CDNB) and internal peptide sequence. The specific activity of purified GST was 440.12 micromol/(min mg), and the K(m) values for CDNB and GSH are very similar (390 and 335 microM, respectively). The intersecting pattern of the initial velocities of this enzyme in the Lineweaver-Burke plot is consistent with a sequential steady-state kinetic mechanism. The high specific activity of shrimp GST may be related to a highly effective detoxification mechanism necessary in gills since they are exposed to the external and frequently contaminated environment.     

Plants for waste water treatment--effects of heavy metals on the detoxification system of Typha latifolia

Upon treatment with Cd and As cattail (Typha latifolia) showed induced catalase, monodehydroascorbate reductase and ascorbate peroxidase activities in leaves but strong inhibition in rhizomes. Peroxidase activity in leaves of the same plants was inhibited whereas linear increase was detected after Cd treatment in rhizomes.Glutathione S-transferase measurements resulted in identical effects of the trace elements on the substrates CDNB, DCNB, NBC, NBoC, fluorodifen. When GST was assayed with the model substrate DCNB, a different pattern of activity was observed, with strongly increasing activities at increasing HM concentrations. Consequently, to improve the success rates, future phytoremediation plans need to preselect plant species with high antioxidative enzyme activities and an alert GST pattern capable of detoxifying an array of organic xenobiotics.     

Insulin-like growth factor binding protein-3 in patients with liver cirrhosis

Aim of the study was to evaluate serum levels of insulin-like growth factor binding protein-3 in patients with liver cirrhosis and to compare serum IGFBP-3 levels with other liver function tests. Fifty-one patients with liver cirrhosis were selected for our study. We measured IGFBP-3 (1.67+/-1.06 mg/l, mean+/-SD), albumin (32+/-8 g/l), prealbumin (0.22+/-0.14 g/l), AST (2.29+/-2.38 microkat/l), ALT (2.11+/-4.83 microkat/l) and cholinesterase (mean 78.6+/-45.2 microkat/l) in the serum. There was a significant positive correlation of serum IGFBP-3 with serum albumin and serum cholinesterase. The correlation coefficient was much lower between serum IGFBP-3 and serum prealbumin. There was no significant correlation between serum AST, ALT and IGFBP-3. Serum IGFBP-3 proves to be a better marker for the hepatic synthetic capacity than serum albumin or cholinesterase.     

Glutathione transferase catalyzed conjugation of benzo[a]pyrene 7,8-diol 9,10-epoxide with glutathione in human skin

Glutathione transferase (GST) activity towards racemic as well as the resolved enantiomers of 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene (anti-BPDE) and 1-chloro-2,4-dinitrobenzene (CDNB) was measured in post-microsomal supernatants (PMS) obtained from eight human skin samples. All preparations showed significant activity towards anti-BPDE and an almost exclusive preference for the more tumourigenic (+)-enantiomer. The specific activity towards (+)-anti-BPDE varied about five-fold between different PMS (range 147-781 pmol/min per mg protein) whereas the variation in specific activities towards CDNB was about two-fold (range 30-71 nmol/min per mg protein). The activities obtained with PMS at saturating concentrations of racemic anti-BPDE were about half of the activity towards the (+)-enantiomer indicating that (-)-anti-BPDE competitively inhibits conjugation of the (+)-form. No correlation was evident between the activities towards (+)-anti-BPDE and CDNB implying that different classes of GST isoenzymes participated in the two different reactions. Immunoblot analysis revealed the presence of Class Alpha and Pi isoenzymes whereas Class Mu isoenzymes seemed to be absent in the human skin samples analyzed. Quantitatively, the Class Pi isoenzyme(s) predominated in all skin samples and the amount of enzyme was about 1-3 micrograms GST Pi/mg PMS protein. The almost exclusive conjugation of (+)-anti-BPDE by PMS and previous results with GST Pi enzymes from human placenta suggested that this type of enzymes catalysed the conjugation reaction. The five-fold variation in specific activity towards (+)-anti-BPDE observed among the different PMS may be explained by individual differences in GST Pi content or by the presence of endogenous modifiers of GST activity towards the diol-epoxide.     

Effects of extract of Ginkgo biloba leaves and its constituents on carcinogen-metabolizing enzyme activities and glutathione levels in mouse liver

The effects of a standardized extract of Ginkgo biloba L. leaves (EGb) and its terpene constituents, bilobalide and ginkgolides, on the activities of detoxification enzymes, i.e., glutathione S-transferases (GSTs) and DT-diaphorase, and glutathione contents, were investigated in the mouse liver. Oral treatment with EGb (100-1,000 mg/kg) and bilobalide (10-30 mg/kg) once a day for 4 days caused a dose-dependent elevation in GST activity. Ginkgolide A (30 mg/kg, for 4 days) also significantly elevated GST activity, whereas ginkgolide B and ginkgolide C at the same dose had no effects. EGb significantly increased the protein level of GST pi, and bilobalide significantly increased those of GST alpha and GST mu Moreover, EGb-treatment and bilobalide-treatment caused significant elevations in DT-diaphorase activity and in hepatic glutathione contents.     

Efficacy of Imazapyr and Glyphosate in the Control of Non-Native Phragmites australis

The cosmopolitan common reed ( Phragmites australis ) has been expanding into previously unoccupied wetland habitats throughout North America. This invasion by a non-native haplotype of Phragmites has become a major concern due to a reduction in plant diversity, reduction of faunal biodiversity, and changes in ecosystem structure. A randomized complete block design was used to compare the efficacy of two herbicides, glyphosate (Rodeo, Dow AgroSciences, IN, U.S.A.) and imazapyr (Habitat, BASF Corporation, NC, U.S.A.), on 1-ha Phragmites monoculture in a shallow borrow pit. Six foliar experimental treatments were applied consisting of (1) 2% glyphosate formulation, June application; (2) 2% glyphosate formulation, September application; (3) 2% imazapyr formulation, June application; (4) 2% imazapyr formulation, September application; (5) 5% imazapyr formulation, June application; and (6) 5% imazapyr, September application. Experimental plots were monitored yearly for two years after treatment. Relative importance values (RIV) were determined to assess the efficacy of herbicide treatments. We report that imazapyr foliar application is statistically superior to glyphosate in reducing Phragmites RIV, with no significant differences between the 2 and 5% formulations. Both herbicides are more effective in reducing Phragmites RIV if applied early in the growing season (June). No significant differences in non- Phragmites plant recolonization were observed between herbicide treatments over the two-year time course. These results suggest that imazapyr is superior in reducing Phragmites RIV, and that earlier applications of herbicides may be more effective on Phragmites . However, managers must note that adjacent nontarget plant species may be negatively affected by earlier treatments.     

Isoprene emission and primary metabolism in Phragmites australis grown under different phosphorus levels

Aquatic plants are generally used for wastewater purification and phytoremediation, but some of them also emit large amounts of isoprene, the most abundant biogenic volatile organic compound. Since isoprenoid biosynthesis requires high amounts of phosphorylated intermediates, the emission may also be controlled by inorganic phosphorus concentration (Pi) in leaves. We carried out experiments to determine the emission of isoprene from Phragmites australis plants used in reconstructed wetlands to phytoremediate elevated levels of phosphorus contributed by urban wastes. Four groups of plants were grown hydroponically in water containing different levels of KH(2)PO(4). High levels of phosphorus in the water resulted in high Pi in the leaves. High Pi stimulated photosynthesis at intercellular CO(2) concentrations lower and higher than ambient, implying higher ribulose 1,5-bisphosphate carboxylase (Rubisco) activity and higher ribulose 1,5-bisphosphate regeneration rates, respectively. However, isoprene emission was substantially lower at high Pi than at low Pi, and was not associated to photosynthesis rates at high Pi. This surprising result suggests that isoprene is limited by processes other than photosynthetic intermediate availability or by energetic (ATP) requirements under high Pi levels. Irrespective of the mechanism responsible for the observed reduction of isoprene emission, our results show that Phragmites plants may effectively remove phosphorus from water without concurrently increase isoprene emission, at least on a leaf area basis. Thus, Phragmites used in reconstructed wetlands for phytoremediation of urban wastes rich of phosphates will not contribute high loads of hydrocarbons which may influence air quality over urban and peri-urban areas.     

Comparison of glutathione S-transferase and general esterase in two bugs, Eurygaster integriceps Puton (Heteroptera: Scutelleridae) and Brachynema germari Kolenati (Heteroptera: Pentatomidae)

Glutathione S -transferases (GSTs) and general esterases play important roles in the detoxification of many substances including allelochemicals from plants and chemical pesticides. In this study, to determine the feeding status and effects of agronomical practices on GST and general esterase, two bug species, Eurygaster integriceps and Brachynema germari , were selected with differences in these cases. GST and general esterase responses varied when switching from 1-chloro-2,4-dinitrobenzene (CDNB) and α -naphtyl to 1,2-dichloro-4-nitro-benzene (DCNB) and β -naphtyl in E. integriceps and B. germari , respectively. The activities of GST and esterase by using CDNB and α -naphtyl were higher than that of DCNB and β -naphtyl for both insects. It was shown that the optimal pH for GST and general esterase activity varied between pH 5.5 and 6. While the Michaelis constant (K_m) value related to E. integriceps toward CDNB and α -naphtyl was lower than K_m for B. germari , K_m values calculated for E. integriceps toward DCNB and β -naphtyl corresponding to those related to A. hejeri were similar to CDNB and α -naphtyl values. The maximal reaction velocity (V_max) values related to E. integriceps for both substrates (CDNB and α -naphtyl) were always higher than those from B. germari and showed significant differences. At the basement of the native-PAGE electrophoresis, five bands were observed in for GST and three were visualized for B. germari , with a large, darker band for E. integriceps in the case of esterases. Studies on the detoxification enzymes of herbivores should be undertaken to determine accurately the effect of the host plants on the organisms eating them, particularly in terms of biochemical and ecological advantages.     

Modulation of substrate-specific glutathione S-transferase activity in Daphnia magna with concomitant effects on toxicity tolerance

Glutathione S-transferase (GST) activity was measured in Daphnia magna and Ceriodaphnia reticulata using 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (EA) as conjugation substrates. Levels of GST activity were comparable between species with CDNB; however, D. magna had nearly twice the GST activity with EA as compared to C. reticulata. GST activity with CDNB was elevated from exposure of daphnids to either CDNB or sodium pentachlorophenate (PCP), but not from exposure to EA. GST activity with EA could not be modulated from exposure to CDNB or EA. GST activity towards CDNB and EA was biochemically separated into different protein fractions suggesting the existence of two distinct isozymes. Preexposure of daphnids to CDNB or PCP increased the organisms' tolerance to the toxic effects of PCP, but not CDNB.     

Study on the biochemical characterization of herbicide detoxification enzyme, glutathione S-transferase

To gain further insight into herbicide detoxification, we studied the herbicide activity and specificity toward glutathione S-transferases from human and rice. In this study, the genes of the plant specific phi and tau class GST enzymes from Oryza sativa (OsGST) and human pi class GST enzyme (hGSTP1-1) were cloned and expressed in Escherichia coli with the pET and pKK vector systems, respectively. The gene products were purified to homogeneity by GSH Sepharose affinity column chromatography. The herbicide specificity of the enzymes was investigated by enzyme-catalyzed conjugation of GSH with chloroacetanilide, diphenylether and chloro-s-triazine herbicides. The hGSTP1-1 showed very high specific activity toward atrazine. On the other hand, the phi class OsGST enzymes showed high specific activity toward chloroacetanilide herbicides, acetochlor, alachlor and metolachlor. The tau class GST enzymes displayed remarkable activity toward the diphenylether herbicide, fluorodifen. From these results, we conclude that the phi and the tau class GST enzymes show herbicide specificities and also they play an important role in the detoxification reaction of plant toward herbicides.