TY - JOUR
AU - Rennenberg, Heinz
AB - Abstract A wild‐type poplar hybrid and two transgenic clones overexpressing a bacterial γ‐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 γ‐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 γ‐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. Chloroacetanilide herbicides, γ‐glutamylcysteine synthetase, glutathione, herbicide detoxification, transgenic poplar. CDNB, 1‐chloro‐2,4‐dinitrobenzene, γ‐EC, γ‐l‐glutamyl‐l‐cysteine, γ‐ECS, γ, ‐glutamylcysteine synthetase, GSH, glutathione, GSSG, oxidized glutathione, GST, glutathione S‐transferase Introduction Phytoremediation is an emerging new technology that uses plants to remove or degrade various pollutants from soils. A number of plant species are able to accumulate high amounts of heavy metals in their above‐ground tissues or to degrade various organic soil pollutants (Salt et al., 1998; Kömives and Gullner, 2000). Poplar trees are good candidates for phytoremediation purposes due to their extensive root system, high water uptake, rapid growth, and large biomass production. Poplar plants have already been used to remove atrazine (Burken and Schnoor, 1997), trichloroethylene (Newman et al., 1997) and selenium (Pilon‐Smits et al., 1998) from polluted soils. The remedial capacity of plants can be significantly increased by genetic manipulation. Transgenic yellow poplar plants overexpressing the bacterial gene encoding mercuric reductase were developed for the phytoremediation of mercury pollution (Rugh et al., 1998). Recently poplar plants were transformed to overexpress the bacterial gene encoding γ‐glutamylcysteine synthetase (γ‐ECS), which is the rate‐limiting regulatory enzyme in the biosynthesis of the ubiquitous tripeptide thiol compound glutathione (GSH, γ‐l‐glutamyl‐l‐cysteinyl‐glycine) (Rennenberg, 1997; Noctor and Foyer, 1998). The transformed poplars contained higher levels of GSH and its precursor γ‐l‐glutamyl‐l‐cysteine (γ‐EC) than the wild type (Noctor et al., 1996, 1998a) and showed elevated phloem and xylem transport of these thiols as compared to wild‐type plants (Herschbach et al., 1998). Tobacco plants overexpressing γ‐ECS showed chlorotic and necrotic symptoms (Creissen et al., 1999). In contrast to these tobacco plants, γ‐ECS overexpressing poplars did not show a particular phenotype or physiological symptoms of oxidative stress (Noctor et al., 1998a). GSH is a principal component of the antioxidative and detoxification defence reactions in plant tissues (Rennenberg, 1997; Noctor et al., 1998b). Elevated GSH levels were found in plants exposed to a wide range of environmental stresses, chemicals and microbial infections (Mauch and Dudler, 1993; Fodor et al., 1997; Foyer and Rennenberg, 2000). The increased production of GSH contributes to the antioxidative protection of plant cells against oxidative stress caused by various environmental factors (Noctor and Foyer, 1998). GSH, as the metabolic precursor of the heavy metal chelating phytochelatins, also plays an important role in the heavy metal detoxification in plants (Zenk, 1996; Cobbett, 2000). The overexpression of γ‐ECS activity in Indian mustard plants resulted in improved phytochelatin biosynthesis and, hence, reduced sensitivity towards cadmium (Zhu et al., 1999). Transgenic poplars overexpressing γ‐ECS exposed to cadmium also contained higher amounts of phytochelatins and accumulated higher amounts of cadmium in their roots than wild‐type plants. However, the enhanced phytochelatin synthesis did not prevent injury from acute cadmium exposure (Rennenberg and Will, 2000). Another recent report confirmed that although the overexpression of γ‐ECS in poplar allows greater tissue cadmium accumulation, it has only a marginal effect on cadmium tolerance (Arisi et al., 2000). GSH and the glutathione S‐transferase isoenzyme family (GST, EC 2.5.1.18) play crucial roles in the degradation of several herbicides. GSTs are able to catalyse conjugation reactions between a number of xenobiotics (including herbicides) and GSH. The herbicide–GSH conjugates are generally much less toxic and more water‐soluble than the original herbicide molecules (Edwards et al., 2000; Pascal et al., 2000). Chloroacetanilide herbicides are widely used for the control of annual grasses and broad‐leaf weeds in a variety of major crops such as maize and soybeans. Conjugation with GSH or its homologue homoglutathione has long been established as the major metabolic reaction by which these commercially important herbicides are detoxified in higher plants (O'Connell et al., 1988; Scarponi et al., 1991; Jablonkai and Hatzios, 1991). In the present study the suitability of a wild‐type poplar hybrid and two transgenic poplar lines overexpressing γ‐ECS was investigated for phytoremediation of soils artificially contaminated with the chloroacetanilide herbicides acetochlor or metolachlor. It was supposed that transgenic poplar plants containing elevated GSH levels could reasonably be used for the removal of chloroacetanilide herbicides from soils. The aims of the investigations were to elucidate if transgenic poplar lines are more tolerant towards acetochlor and metolachlor than the wild type and whether the anticipated difference in herbicide tolerance is related to improved detoxification capacity. Materials and methods Plant material An untransformed poplar hybrid (Populus tremula×Populus alba), INRA‐clone No. 717–1‐B4 (in the following designated as the wild type) and two genetically transformed poplar lines overexpressing γ‐ECS in the cytosol (11ggs) or in the chloroplasts (6LgI) were used for the experiments. Vector construction, transformation, identification, and characterization of transformants were published earlier (Noctor et al., 1996, 1998a). Following their micropropagation in vitro, poplar plants were transferred to a greenhouse and were grown in plastic pots with supplementary light (150 μmol m−2 s−1) for 16 h at 26 °C and 60% relative humidity as described earlier (Strohm et al., 1999). Approximately 75–80‐d‐old‐plants that had attained a height of 55–64 cm were treated by the herbicides acetochlor or metolachlor. Herbicide treatments Aqueous solutions (1.5 mM) of acetochlor [2‐chloro‐N‐(ethoxymethyl)‐N‐(2‐ethyl‐6‐methylphenyl)‐acetamide] and metolachlor [2‐chloro‐N‐(2‐ethyl‐6‐methylphenyl)‐N‐(2‐methoxy‐1‐methylethyl)‐acetamide] were prepared and used to disperse the herbicides into the soil by watering it with the herbicide solutions until saturation. The herbicides were applied once and their initial soil concentration was 66 μg g−1 soil. This moderately toxic concentration was selected in pre‐experiments. The development of herbicide‐treated and control plants was followed by measuring their shoot heights for 5 weeks following the herbicide treatments. At the end of 5‐week‐exposure periods plants were harvested and root and shoot fresh weights were measured. For thiol and GST analyses leaf samples were taken after 1 and 2 weeks of herbicide exposure. In separate experiments leaves were cut from middle leaf positions of 75–80‐d‐old‐poplars and were exposed to acetochlor and metolachlor by putting their petioles into 2.5×10−4 M aqueous solutions of the herbicides. Leaves were incubated for 2 d in a growth chamber under permanent illumination (150 μmol m−2 s−1) at 22 °C. For comparison detached leaves were exposed also to 2.5×10−4 M aqueous solutions of the herbicide acifluorfen (5‐[2‐chloro‐4‐(trifluoromethyl)‐phenoxy]‐2‐nitrobenzoic acid) for 2 d. This herbicide can markedly increase GSH levels and induce GST activities in various plants (Gullner and Dodge, 2000; Pascal et al., 2000). To assess the effects of acifluorfen on the γ‐ECS activity poplar leaves were exposed to 10−4 M acifluorfen solutions for 2 d. All herbicides were purchased from Riedel‐de Haen (Seelze, Germany). Biochemical analyses For thiol and GST analysis, leaves were sampled from each poplar line at upper, middle and lower leaf positions (approximately at the 4–5th, 10–11th and 16–18th leaves, denoted by counting them from the apex) corresponding to developing, mature and senescent leaves. The levels of GSH and its thiol precursors (cysteine and γ‐EC) were determined by reversed‐phased HPLC with spectrofluorometric detection after derivatization with monobromobimane (Strohm et al., 1995). In parallel experiments the foliar amounts of oxidized glutathione (GSSG) were also measured by the above HPLC method after blocking the free thiol group of GSH in the leaf extracts by N‐ethylmaleimide and subsequent reduction of GSSG by dithiothreitol (Strohm et al., 1995). Leaf cell‐free extracts for enzyme activity measurements were prepared as described earlier (Fodor et al., 1997). GST activities were determined spectrophotometrically by measuring the formation of the conjugate reaction product at 340 nm using 1‐chloro‐2,4‐dinitrobenzene (CDNB) as substrate (Mauch and Dudler, 1993). Protein contents were determined by the Bradford method using bovine serum albumin as standard (Bradford, 1976). Thiol levels were measured also in detached leaves exposed to acetochlor, metolachlor or acifluorfen. The activity of γ‐ECS was determined in acifluorfen‐treated and control detached leaves by the reversed‐phased HPLC method (Rüegsegger and Brunold, 1992). Statistics At least four (biometric measurements) or three (biochemical determinations) independent parallel experiments were carried out in each case. The significant differences between mean values were evaluated by Student's t‐test. Differences were considered to be significant at P=0.05. To compare the development of shoots of transgenic and wild‐type poplars the slope values of growth curves were calculated and the homogeneity of slopes was tested by regression analysis with the procedure GLM (software SAS version 6.12). Results Phytotoxic effects of chloroacetanilide herbicides No visible phytotoxic symptoms appeared on the leaves or stems of transgenic or wild‐type poplars after their exposure to acetochlor or metolachlor mixed into the soil for five weeks following the treatments. However, shoot development, measured as shoot height, was considerably retarded in the herbicide‐treated plants as compared to the untreated controls (Fig. 1). The effects of acetochlor and metolachlor on shoot growth did not differ significantly in either poplar line. The growth retardation observed in wild‐type poplars was comparable to that of the transgenic line 6LgI. However, shoot development of the line 11ggs was significantly less inhibited by the herbicides than those of the other two lines (Fig. 1). The effects of the two herbicides were similar. These results indicate the elevated tolerance of line 11ggs against both chloroacetanilide herbicides. Shoot and root fresh weights of transgenic and wild‐type poplar plants were measured at the end of 5‐week experimental periods. Similar to the shoot development data, the shoot fresh weights were markedly reduced by the herbicide. The shoot fresh weights of wild‐type plants were reduced by 45% and 46% after 5 weeks of exposure to acetochlor or metolachlor, respectively (Fig. 2). The shoot fresh weights of both transgenic lines were markedly less reduced by herbicide treatments (by 23–30%) showing that the transgenic lines are more tolerant to chloroacetanilide herbicides than wild‐type plants (Fig. 2). The root fresh weights were more strongly reduced by herbicide treatments than shoot fresh weights. In wild‐type plants this reduction amounted to 71% and 67% after 5 weeks of exposure to acetochlor or metolachlor, respectively (Fig. 3). The root fresh weights also proved the increased herbicide tolerance of poplars overexpressing γ‐ECS. In the lines 11ggs and 6LgI the root fresh weight was reduced only by 54% and 51% after 5 weeks of exposure to acetochlor, respectively. The effects of metolachlor were very similar to those of acetochlor (Fig. 3). Fig. 1. View largeDownload slide Retardation of shoot growth as a consequence of acetochlor and metolachlor treatments (66 μg g−1 soil) of wild type and two transgenic (11ggs and 6LgI) poplar lines. Symbols: (○) control (tap water); (•) acetochlor; (▴) metolachlor. The symbol * shows significant differences between slopes of 11ggs and the other two lines treated by acetochlor or metolachlor at P=5% (n=4). Fig. 1. View largeDownload slide Retardation of shoot growth as a consequence of acetochlor and metolachlor treatments (66 μg g−1 soil) of wild type and two transgenic (11ggs and 6LgI) poplar lines. Symbols: (○) control (tap water); (•) acetochlor; (▴) metolachlor. The symbol * shows significant differences between slopes of 11ggs and the other two lines treated by acetochlor or metolachlor at P=5% (n=4). Fig. 2. View largeDownload slide Reduction of shoot fresh weight brought about by 35 d acetochlor and metolachlor treatments (66 μg g−1 soil) of wild‐type and two transgenic (11ggs and 6LgI) poplar lines. Percentage values are related to the shoot fresh weights of corresponding untreated poplar plants. The symbol * shows significant differences between herbicide‐treated transgenic and wild‐type plants at P=5% (n=4). Fig. 2. View largeDownload slide Reduction of shoot fresh weight brought about by 35 d acetochlor and metolachlor treatments (66 μg g−1 soil) of wild‐type and two transgenic (11ggs and 6LgI) poplar lines. Percentage values are related to the shoot fresh weights of corresponding untreated poplar plants. The symbol * shows significant differences between herbicide‐treated transgenic and wild‐type plants at P=5% (n=4). Fig. 3. View largeDownload slide Reduction of root fresh weight brought about by 35 d acetochlor and metolachlor treatments (66 μg g−1 soil) of wild‐type and two transgenic (11ggs and 6LgI) poplar lines. Percentage values are related to the root fresh weights of corresponding untreated poplar plants. The symbol * shows significant differences between herbicide‐treated transgenic and wild‐type plants at P=5% (n=4). Fig. 3. View largeDownload slide Reduction of root fresh weight brought about by 35 d acetochlor and metolachlor treatments (66 μg g−1 soil) of wild‐type and two transgenic (11ggs and 6LgI) poplar lines. Percentage values are related to the root fresh weights of corresponding untreated poplar plants. The symbol * shows significant differences between herbicide‐treated transgenic and wild‐type plants at P=5% (n=4). Foliar thiol levels Since GSH metabolism plays a crucial role in the detoxification of chloroacetanilide herbicides, foliar levels of GSH and its precursors cysteine and γ‐EC were measured and compared in 75–80 d old transgenic and wild‐type poplar lines at three different leaf positions. In accordance with earlier reports (Noctor et al., 1996, 1998a) the foliar GSH (Fig. 4) and γ‐EC (Fig. 5) levels were significantly higher in the transgenic poplars than in the wild type, which can explain the elevated herbicide tolerance of transgenic poplars. The GSH levels depended only slightly on the leaf position (Fig. 4). The exposure of poplar plants to acetochlor or metolachlor mixed into the soil resulted in a marked increase of foliar GSH content after 2 weeks. Generally, the most significant elevations of GSH level were found in developing upper leaves (Fig. 4). In the leaves of wild‐type poplars only metolachlor treatments led to increased GSH levels. The GSH content of middle and lower leaves did not change considerably and significant increases were observed only in leaves of the acetochlor‐treated 11ggs line (Fig. 4). These results show that the differing herbicide tolerance of transgenic lines and wild type may be partly explained by the enhanced inducibility of foliar GSH levels, particularly in line 11ggs. A similar, but less significant increase of GSH levels was also observed after 1 week of herbicide exposure (data not shown). No significant accumulation of GSSG was found in the leaves of herbicide‐treated poplars (data not shown), showing that herbicide exposure did not lead to significant oxidative stress in the poplar leaves. The foliar amounts of GSSG were in the range of 9–21 nmol g−1 FW (3.8–7.4% of total glutathione). GSSG levels of lower leaves were slightly smaller than those of middle and upper leaves. The foliar γ‐EC levels were also significantly elevated after 2 weeks exposure to acetochlor and metolachlor (Fig. 5). One week exposures resulted in a slight increase of γ‐EC levels only (data not shown). The most substantial increases of γ‐EC content were observed in the upper leaves as observed for GSH. In these leaves only the metolachlor treatments led to significantly increased γ‐EC levels, while in the middle and lower leaves the acetochlor treatments also caused such an effect (Fig. 5). Cysteine levels of transgenic and wild‐type poplar leaves did not differ significantly at any leaf positions (detected levels were in the range of 1.9–5.2 nmol g−1 FW) and were not significantly altered by either herbicide mixed into the soil (data not shown). Fig. 4. View largeDownload slide Changes in the foliar glutathione content of wild‐type and two transgenic (11ggs and 6LgI) poplar lines exposed to acetochlor and metolachlor (66 μg g−1 soil) for 2 weeks. Abbreviations: C, control; A, acetochlor; M, metolachlor. The symbol * shows significant differences between treated and control plants at P=5% (n=3). Fig. 4. View largeDownload slide Changes in the foliar glutathione content of wild‐type and two transgenic (11ggs and 6LgI) poplar lines exposed to acetochlor and metolachlor (66 μg g−1 soil) for 2 weeks. Abbreviations: C, control; A, acetochlor; M, metolachlor. The symbol * shows significant differences between treated and control plants at P=5% (n=3). Fig. 5. View largeDownload slide Changes in the foliar γ‐glutamylcysteine content of wild type and two transgenic (11ggs and 6LgI) poplar lines exposed to acetochlor and metolachlor (66 μg g−1 soil) for 2 weeks. Abbreviations: C, control; A, acetochlor; M, metolachlor. The symbol * shows significant differences between treated and control plants at P=5% (n=3). Fig. 5. View largeDownload slide Changes in the foliar γ‐glutamylcysteine content of wild type and two transgenic (11ggs and 6LgI) poplar lines exposed to acetochlor and metolachlor (66 μg g−1 soil) for 2 weeks. Abbreviations: C, control; A, acetochlor; M, metolachlor. The symbol * shows significant differences between treated and control plants at P=5% (n=3). GST activity Although chloroacetanilide herbicides may react with GSH non‐enzymatically, GST isoenzymes play an important role in the detoxification of these herbicides in plants by catalysing the formation of a conjugate between the pesticide molecules and GSH (Marrs, 1996; Hatton et al., 1996). In order to assess the possible correlations between GST activity and herbicide tolerance, GST activities were measured in acetochlor‐ and metolachlor‐treated transgenic and wild‐type poplars. High levels of GST activities were found in all poplar leaves studied (Fig. 6). The activity depended on the leaf position. Highest activities were detected in developing leaves at upper leaf positions and the activity gradually decreased towards the lower leaves. The GST activities did not differ significantly between transgenic and wild‐type plants. Treatments with acetochlor or metolachlor did not result in any significant increase of GST activity after 1 week of exposure (data not shown). However, after 2 weeks of exposure both herbicides significantly induced GST activities in developing upper leaves, but not in the middle or lower leaves (Fig. 6). The extent of enzyme induction did not differ significantly between the wild type and transgenic lines (Fig. 6). The effects of the two herbicides were similar. The soluble protein contents of the upper leaves did not change significantly as a consequence of herbicide treatments (data not shown). It is interesting to note that the increases of both GSH levels and GST activities were always considerably higher in the upper leaves than in the middle and lower leaves, in which the changes were often not significant. Fig. 6. View largeDownload slide Changes in the foliar glutathione S‐transferase activity of wild type and two transgenic (11ggs and 6LgI) poplar lines exposed to acetochlor and metolachlor (66 μg g−1 soil) for 2 weeks. Abbreviations: C, control; A, acetochlor; M, metolachlor. The symbols * and ** show significant differences between treated and control plants at P=5% and 1%, respectively (n=3). Fig. 6. View largeDownload slide Changes in the foliar glutathione S‐transferase activity of wild type and two transgenic (11ggs and 6LgI) poplar lines exposed to acetochlor and metolachlor (66 μg g−1 soil) for 2 weeks. Abbreviations: C, control; A, acetochlor; M, metolachlor. The symbols * and ** show significant differences between treated and control plants at P=5% and 1%, respectively (n=3). Experiments with detached leaves In a separate set of experiments the effects of chloroacetanilide herbicides on GSH metabolism were compared with the effect of acifluorfen, which is a known inducer of GSH levels in plants (Gullner and Dodge, 2000; Pascal et al., 2000). Detached leaves were exposed to 2.5×10−4 M solutions of acetochlor, acifluorfen and metolachlor for 2 d. Marked increases of cysteine, γ‐EC and GSH levels were observed in acifluorfen‐treated leaves (Fig. 7), but less markedly also by exposure to the chloroacetanilide herbicides. Acifluorfen strongly increased these thiol levels also at 10−4 M concentration (data not shown). Cysteine levels were substantially elevated by acifluorfen, but were not significantly influenced by the other two herbicides (Fig. 7). Massive inductions of γ‐EC levels were observed in the herbicide‐treated transgenic plants, particularly in the 6LgI line, in which all herbicide treatment led to increased γ‐EC levels. The accumulation of γ‐EC caused by acifluorfen was markedly increased when leaves were incubated for 2 d in darkness (data not shown). All herbicide treatments significantly elevated the GSH levels in all poplar lines, but the chloroacetanilide herbicides were less effective than acifluorfen (Fig. 7). Acifluorfen treatments increased the foliar GSSG amounts 2.3–4.9‐fold in leaves of all poplar lines, showing that the herbicide had a marked oxidative effect. In contrast to acifluorfen, the chloroacetanilide herbicides did not influence the GSSG content of the leaves. To investigate the cause of the strong increase of γ‐EC levels by acifluorfen, γ‐ECS activities were measured in detached leaves of transgenic and wild‐type poplar lines that were exposed to acifluorfen for 2 d in constant light. No γ‐ECS activity was detectable in wild‐type leaves, consistent with earlier experiments (Noctor et al., 1996, 1998a). In the leaves of the 11ggs and 6LgI lines the γ‐ECS activities were 172±21 and 290±37 nmol γ‐EC mg protein−1 min−1 (n=3), respectively. Acifluorfen treatments did not alter these activities significantly in either transformant (data not shown). Fig. 7. View largeDownload slide Changes of cysteine, γ‐glutamylcysteine and glutathione contents in detached leaves of wild type and two transgenic (11ggs and 6LgI) poplar lines exposed to 2.5×10−4 M aqueous solutions of acetochlor, acifluorfen and metolachlor for 2 d. Abbreviations: C, control; A, acetochlor; Aci, acifluorfen; M, metolachlor. The symbols *, ** and *** show significant differences between treated and control plants at P=5%, 1% and 0.1%, respectively (n=3). Fig. 7. View largeDownload slide Changes of cysteine, γ‐glutamylcysteine and glutathione contents in detached leaves of wild type and two transgenic (11ggs and 6LgI) poplar lines exposed to 2.5×10−4 M aqueous solutions of acetochlor, acifluorfen and metolachlor for 2 d. Abbreviations: C, control; A, acetochlor; Aci, acifluorfen; M, metolachlor. The symbols *, ** and *** show significant differences between treated and control plants at P=5%, 1% and 0.1%, respectively (n=3). Discussion Since the exact mode of action of chloroacetanilide herbicides is still unknown (Wu et al., 2000), the phytotoxic effects of chloroacetanilide herbicides were characterized by the growth response of poplars. No significant differences were found in growth rates or phenotypic appearance between untreated transgenic poplar lines overexpressing γ‐ECS activity and wild‐type plants (Figs 1–3). Transformation of poplars by the γ‐ECS gene did not alter the foliar GST activity, either (Fig. 6). These results are in accordance with earlier reports which showed that transgenic and wild‐type poplar lines share a similar basic metabolism (Noctor et al., 1996, 1998a). Consistent with these findings, transgenic Indian mustard lines overexpressing γ‐ECS activity did not show any distinguishable difference in visual appearance compared with wild‐type plants (Zhu et al., 1999). Recently, it has been reported that transgenic tobacco plants overexpressing a chloroplast‐targeted γ‐ECS showed light‐intensity dependent chlorotic or necrotic symptoms (Creissen et al., 1999). These authors supposed that these transgenic tobacco plants suffered continuously from oxidative damage due to the failure of the redox‐sensing process in the chloroplasts. It is not known why transformation of tobacco plants with the bacterial γ‐ECS gene led to such differing consequences to those observed with other plant species. Exposure to chloroacetanilide herbicides retarded shoot development and decreased the root and shoot fresh weights of poplar plants (Figs 1–3). Transgenic poplars overexpressing γ‐ECS proved to be significantly more tolerant to the herbicides than wild‐type plants. Chloroacetanilide herbicides are known to be metabolized in tolerant plants through the formation of water‐soluble intermediates which are mainly conjugates with GSH (Jablonkai and Hatzios, 1991; Hatton et al., 1996). A positive correlation between foliar GSH concentration and the tolerance towards these herbicides has been demonstrated in several plant species (Jablonkai and Hatzios, 1993). Therefore it can be supposed that the elevated γ‐EC and GSH level of transgenic poplar plants (Figs 4, 5) resulted in rapid herbicide degradation and, as a consequence, in elevated herbicide tolerance. GSH and γ‐EC levels can be determining factors in the selectivity of chloroacetanilide herbicides between transgenic and wild‐type poplars. In addition, the increased inducibility of foliar contents of these non‐protein thiols by chloroacetanilide herbicides (Figs 4, 5) can also contribute to the elevated herbicide tolerance of transgenic lines. The conjugation of chloroacetanilide herbicides with GSH may proceed either enzymatically, mediated by GST enzymes, or non‐enzymatically. The relative contribution of non‐enzymatic conjugation in the overall formation of herbicidal conjugates with GSH depends on several factors including the concentrations of the reaction partners, and the chemical structure of the herbicides (Jablonkai and Hatzios, 1993). In atrazine‐treated maize high GST activities correlated with herbicide tolerance (Hatton et al., 1996). GST gene expression is inducible by a wide range of endogenous and xenobiotic chemical compounds, including phytohormones, heavy metals, herbicides, and herbicide safeners (Marrs, 1996). Elevated GST levels were also observed in the experiments using transgenic and wild‐type poplar lines after exposures to chloroacetanilide herbicides. Interestingly, GST induction was observed only in developing, upper leaves after chloroacetanilide poisoning (Fig. 6). The most considerable increases of non‐protein thiol levels were observed in developing leaves (Figs 4, 5). The cause of these phenomena is not known. The extent of GST induction did not differ significantly between the more herbicide‐tolerant transgenic and wild‐type poplar lines. According to these results the increased herbicide tolerance of transgenic lines can not be explained by a higher level or inducibility of GST enzymes. However, it is known that GST activities of herbicide‐specific isoenzymes measured with the herbicides as substrates can substantially differ from GST activities measured with the model substrate CDNB used in our studies (Jablonkai and Hatzios, 1993; Hatton et al., 1996). Therefore future determinations of specific GST activities using radioactively labelled chloroacetanilide herbicides as substrates may reveal significant differences between transgenic and wild‐type poplars. The experiments with detached leaves showed that the herbicide acifluorfen, which is a known inducer of foliar GSH levels (Gullner and Dodge, 2000), strongly elevated the foliar cysteine, γ‐EC and GSH levels in poplar leaves (Fig. 7). In particular, acifluorfen dramatically increased the γ‐EC levels in the transgenic poplar lines. In comparison to acifluorfen, the chloroacetanilide herbicides were less effective in increasing these thiol levels, probably because they did not exert any peroxidative effect on poplar leaves, in contrast to acifluorfen. The increase of γ‐EC level by acifluorfen was very strong in leaves incubated in darkness, particularly at the transgenic poplar lines (data not shown). The accumulation of γ‐EC in darkened leaves has been already observed in several plants including poplar and it is known that this phenomenon is caused by the limited availability of glycine in darkness (Buwalda et al., 1990; Noctor and Foyer, 1998). Acifluorfen did not influence significantly the γ‐ECS activities in the transgenic poplar lines. These results suggest that acifluorfen induces the biosynthesis of cysteine, which is a limiting factor of γ‐EC biosynthesis in poplar (Strohm et al., 1995; Noctor et al., 1998a). Little information is available about the phytoremediation of soils contaminated by chloroacetanilide herbicides. The removal of metolachlor and other herbicides from contaminated soils by using the noxious but herbicide‐tolerant weed Kochia scoparia has been investigated (Anderson et al., 1994; Arthur et al., 1999). They reported that the rhizosphere of herbicide‐tolerant plants was an important component in biologically remediating pesticide‐contaminated soils. These studies also showed that a general obstacle in phytoremediation was finding appropriate plant species that can tolerate greater concentrations of pesticides in soils. In the experiments of this study each poplar line showed considerable tolerance towards chloroacetanilide herbicides, probably due to their high GST activities (Fig. 6) and non‐protein thiol levels (Figs 4, 5). Therefore poplars, particularly transgenic lines having elevated herbicide‐tolerance, seem to be a reasonable choice for the removal of herbicides from soils. Untransformed poplars have already been used for the phytoremediation of soils contaminated by the herbicide atrazine, which is also detoxified by the formation of GSH‐conjugates (Burken and Schnoor, 1997). In summary, transgenic poplar plants overexpressing γ‐ECS activity in the cytosol or in the chloroplasts showed increased tolerance to chloroacetanilide herbicides which is probably due to their elevated endogenous γ‐EC and GSH contents and the marked inducibility of foliar amounts of these thiols after herbicide exposure. These results suggest that these transgenic poplar lines are good candidates for the phytoremediation of soils polluted with chloroacetanilide herbicides. However, it seems that further genetic transformations, in particular with genes encoding herbicide‐specific GST isoenzymes, may still improve their detoxification capacities for practical phytoremediation purposes at herbicide contaminated sites. 3 To whom correspondence should be addressed at the Hungarian Academy of Sciences. Fax: +36 1 356 3698. E‐mail: ggull@nki.hu The research was financially supported by UNIDO (Vienna, Austria) and a grant of the Deutscher Akademischer Austauschdienst for G Gullner. References Anderson TA, Kruger EL, Coats JR. 1994. Enhanced degradation of a mixture of three herbicides in the rhizosphere of a herbicide‐tolerant plant. Chemosphere  28, 1551–1557. Google Scholar Arisi A‐CM, Mocquot B, Lagriffoul A, Mench M, Foyer CH, Jouanin L. 2000. 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TI - Enhanced tolerance of transgenic poplar plants overexpressing γ‐glutamylcysteine synthetase towards chloroacetanilide herbicides
JF - Journal of Experimental Botany
DO - 10.1093/jexbot/52.358.971
DA - 2001-05-01
UR - https://www.deepdyve.com/lp/oxford-university-press/enhanced-tolerance-of-transgenic-poplar-plants-overexpressing-Yb2BbIE06w
SP - 971
EP - 979
VL - 52
IS - 358
DP - DeepDyve
ER -