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• W2139860372 abstract "Plant Tau class glutathione transferases (GSTUs) detoxify diphenylether herbicides such as fluorodifen, determining their selectivity in crops and weeds. Using reconstructive PCR, a series of mutant GSTUs were generated from in vitro recombination and mutagenesis of the maize sequences ZmGSTU1 and ZmGSTU2 (with the prefix Zm designating Zea mays L.). A screen of 5000 mutant GSTUs identified seven enzymes with enhanced fluorodifen detoxifying activity. The best performing enhanced fluorodifen detoxifying mutant (EFD) had activity 19-fold higher than the parent enzymes, with a single point mutation conferring this enhancement. Further mutagenesis of this residue generated an EFD with a 29-fold higher catalytic efficiency toward fluorodifen as compared with the parents but with unaltered catalysis toward other substrates. When expressed in Arabidopsis thaliana, the optimized EFD, but not the parent enzymes, conferred enhanced tolerance to fluorodifen. Molecular modeling predicts that the serendipitous mutation giving the improvement in detoxification is due to the removal of an unfavorable interaction together with the introduction of a favorable change in conformation of residues 107–119, which contribute to herbicide binding. Plant Tau class glutathione transferases (GSTUs) detoxify diphenylether herbicides such as fluorodifen, determining their selectivity in crops and weeds. Using reconstructive PCR, a series of mutant GSTUs were generated from in vitro recombination and mutagenesis of the maize sequences ZmGSTU1 and ZmGSTU2 (with the prefix Zm designating Zea mays L.). A screen of 5000 mutant GSTUs identified seven enzymes with enhanced fluorodifen detoxifying activity. The best performing enhanced fluorodifen detoxifying mutant (EFD) had activity 19-fold higher than the parent enzymes, with a single point mutation conferring this enhancement. Further mutagenesis of this residue generated an EFD with a 29-fold higher catalytic efficiency toward fluorodifen as compared with the parents but with unaltered catalysis toward other substrates. When expressed in Arabidopsis thaliana, the optimized EFD, but not the parent enzymes, conferred enhanced tolerance to fluorodifen. Molecular modeling predicts that the serendipitous mutation giving the improvement in detoxification is due to the removal of an unfavorable interaction together with the introduction of a favorable change in conformation of residues 107–119, which contribute to herbicide binding. The relative rate of herbicide detoxification in crops and weeds is a primary determinant of their selectivity (1Owen W.J. Roberts T. Metabolism of Agrochemicals in Plants. John Wiley and Sons Ltd., London2000: 211-258Google Scholar). Crops rapidly metabolize herbicides by oxidative or hydrolytic reactions followed by conjugation with sugars or peptides and vacuolar sequestration of the polar products (1Owen W.J. Roberts T. Metabolism of Agrochemicals in Plants. John Wiley and Sons Ltd., London2000: 211-258Google Scholar). In weeds, these detoxification reactions are slower. Glutathione S-transferases (GSTs) 1The abbreviations used are: GST, glutathione S-transferase; GSTF, Phi class GST; GSTU, Tau class GST; CDNB, 1-chloro-2,4-dinitrobenzene; EFD, enhanced fluorodifen detoxifying mutant; Zm, from Z. mays L. 1The abbreviations used are: GST, glutathione S-transferase; GSTF, Phi class GST; GSTU, Tau class GST; CDNB, 1-chloro-2,4-dinitrobenzene; EFD, enhanced fluorodifen detoxifying mutant; Zm, from Z. mays L. have a well characterized role in determining the metabolism and selectivity of chloroacetanilide, thiocarbamate, and chloro-s-triazine herbicides in maize (2Edwards R. Dixon D.P. Cobb A.H. Kirkwood R.C. Herbicides and Their Mechanisms of Action. Sheffield Academic Press, Sheffield, UK2000: 38-91Google Scholar). GSTs catalyze the conjugation of these herbicides with the tripeptide glutathione (γ-glutamyl-cysteinyl-glycine) to form non-toxic S-glutathionylated products (2Edwards R. Dixon D.P. Cobb A.H. Kirkwood R.C. Herbicides and Their Mechanisms of Action. Sheffield Academic Press, Sheffield, UK2000: 38-91Google Scholar). In other crops, alternative GST activities determine herbicide selectivity. For example, photobleaching diphenylether herbicides such as fluorodifen are rapidly detoxified by GSTs in legumes (see Fig. 1A) but less efficiently in maize (3Frear D.S. Swanson H.R. Pestic. Biochem. Physiol. 1973; 3: 473-482Google Scholar). Species-dependent GST-mediated detoxification can be explained by differences in the expression of the six distinct families of plant GST genes, classified as the Phi, Zeta, Tau, Theta, Lambda, and dehydroascorbate reductase classes (4McGonigle B. Keeler S.J. Lau S.-M.C. Koeppe M.K. O'Keefe D.P. Plant Physiol. 2000; 124: 1105-1120Google Scholar, 5Dixon D.P. Lapthorn A. Edwards R. Genome Biol. 2002; 3Google Scholar, 6Dixon D.P. Davis B.G. Edwards R. J. Biol. Chem. 2002; 277: 30859-30869Google Scholar). The plant-specific Phi and Tau GSTs are primarily responsible for herbicide detoxification, showing class specificity in substrate preference. Phi enzymes (GSTFs) are highly active toward chloroacetanilide and thiocarbamate herbicides, whereas the Tau enzymes (GSTUs) are efficient in detoxifying diphenylethers and aryloxyphenoxypropionates (7Jepson I. Lay V.J. Holt D.C. Bright S.W.J. Greenland A.J. Plant Mol. Biol. 1994; 26: 1855-1866Google Scholar, 8Thom R. Cummins I. Dixon D.P. Edwards R. Cole D.J. Lapthorn A.J. Biochemistry. 2002; 41: 7008-7020Google Scholar). In maize, GSTFs are the major class of expressed GST (7Jepson I. Lay V.J. Holt D.C. Bright S.W.J. Greenland A.J. Plant Mol. Biol. 1994; 26: 1855-1866Google Scholar), whereas in soybean, GSTUs predominate, with this difference accounting for the differential detoxification of different classes of herbicide in the two crops (4McGonigle B. Keeler S.J. Lau S.-M.C. Koeppe M.K. O'Keefe D.P. Plant Physiol. 2000; 124: 1105-1120Google Scholar).We have been interested in enhancing the detoxifying potential of plant GSTs using the dual approach of DNA shuffling and directed mutagenesis. In maize (Zea mays L.), ZmGSTU1 and ZmGSTU2 are the major Tau enzymes expressed, although considerably less abundant than the ZmGSTFs (9Dixon D.P. Cole D.J. Edwards R. Plant Mol. Biol. 1998; 36: 75-87Google Scholar, 10Dixon D.P. Cole D.J. Edwards R. Plant Mol. Biol. 1999; 40: 997-1008Google Scholar). With the intention of enhancing their detoxifying activity toward fluorodifen, we have used reconstructive error-prone PCR (11Stemmer W.P.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10747-10751Google Scholar) to randomly generate mutant GSTUs derived from both ZmGSTU1 and ZmGSTU2. After large scale screening of the mutants from this screen, the most improved GST was then subjected to selective mutagenesis to further increase activity. The resulting optimized catalysts were then used to transform Arabidopsis thaliana and tested for their ability to increase herbicide tolerance in planta.EXPERIMENTAL PROCEDURESGeneration of Mutant GSTs—Clones pGSTU1 and pGSTU2, pBluescript SK– vectors containing cDNA sequences for ZmGSTU1 and ZmGSTU2, respectively, were obtained from cDNA expression library antibody screens as detailed previously (9Dixon D.P. Cole D.J. Edwards R. Plant Mol. Biol. 1998; 36: 75-87Google Scholar, 10Dixon D.P. Cole D.J. Edwards R. Plant Mol. Biol. 1999; 40: 997-1008Google Scholar). In the work describing their original isolation, ZmGSTU1 was termed ZmGSTV and ZmGSTU2 was termed ZmGSTVI, the new designations reflecting a unifying change in nomenclature (5Dixon D.P. Lapthorn A. Edwards R. Genome Biol. 2002; 3Google Scholar). For reconstructive PCR, 10 μg of each of the plasmids pGSTU1 and pGSTU2 was digested with DNase I (0.25–2.0 units) in 50 μlof50mm Tris-HCl, pH 7.5, 1 mm MgCl2 at 22 °C for 10 min with the reaction stopped with 2 μl of 0.5 m EDTA. Reactions producing fragments of mainly 0–500 bp were used for reconstructive PCR (11Stemmer W.P.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10747-10751Google Scholar). Using 20 ng/μl digested DNA, after 50 cycles (94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s + 3 s/cycle), reaction products of 500 bp–20 kb were obtained, and 1 μl of this mixture was used as template for a standard 50-μl PCR reaction using M13 forward and reverse oligonucleotide primers (30 cycles of 94 °C for 30 s, 50 °C for 30 s, 72 °C for 60 s). PCR products of the expected size were recloned into pBluescript.Screening Mutants for GST Activity—Wells of sterile flat-bottomed, 96-well microtiter plates, each containing 200 μl of LB medium supplemented with antibiotics, were inoculated with individual mutant bacterial colonies taken from a master plate and grown for 16 h to stationary phase. The plates were centrifuged, and the growth medium was removed by aspiration prior to resuspending the bacteria in 200 μl of 0.1 m glycine-NaOH, pH 9.5, containing 0.5 mm fluorodifen and 5 mm glutathione. Plates were incubated at 37 °C (2–4 h), and the release of p-nitrophenol was visually monitored. Mutants showing at least a 5-fold faster accumulation of the yellow p-nitrophenol than the parent ZmGSTU clones were selected for further characterization.Analysis of Recombinant Mutant GSTs—GSTUs cloned in either pBluescript or pET plasmids in Escherichia coli were cultured without isopropyl-1-thio-β-d-galactopyranoside induction, and the respective recombinant proteins were purified using S-hexylglutathione-Sepharose (9Dixon D.P. Cole D.J. Edwards R. Plant Mol. Biol. 1998; 36: 75-87Google Scholar, 10Dixon D.P. Cole D.J. Edwards R. Plant Mol. Biol. 1999; 40: 997-1008Google Scholar). Purified GSTs were extensively dialyzed to remove S-hexylglutathione and assayed for activity toward CDNB (1-chloro-2,4-dinitrobenzene) and fluorodifen (12Edwards R. Physiol. Plant. 1996; 98: 594-604Google Scholar), the latter by following the increase in absorbance at 400 nm after incubation at 30 °C in 0.1 m glycine-NaOH buffer, pH 9.5, containing 5 mm glutathione and 50 μm fluorodifen. High pressure liquid chromatography-based assays for activity toward herbicides other than fluorodifen were as described (13Andrews C.J. Skipsey M. Townson J.K. Morris C. Jepson I. Edwards R. Pestic. Sci. 1997; 51: 213-222Google Scholar) except that assays with fomesafen were carried out in glycine-NaOH buffer, pH 9.5. The protein concentration of purified recombinant GSTs was measured based on their calculated UV absorbance at 280 nm (14Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Google Scholar).Site-directed Mutagenesis—The Q115L point mutation in EFD6 (enhanced fluorodifen detoxifying mutant 6), corresponding to the 113th amino acid residue of ZmGSTU2, was subjected to PCR using specific mutagenic primers, together with a primer to the T7 promoter, using pEFD6 as the template. PCR products were subcloned back into pEFD6 to give constructs expressing the mutants EFD6–115E, EFD6–115N, EFD6–115F, EFD6–115A, and EFD6–115Q containing mutations of leucine 115 to glutamic acid, asparagine, phenylalanine, alanine, and glutamine, respectively.Modeling of Fluorodifen Binding to ZmGSTU2—The ZmGSTU2 protein was modeled on the templates of two Tau class enzymes, wheat TaGSTU4 (8Thom R. Cummins I. Dixon D.P. Edwards R. Cole D.J. Lapthorn A.J. Biochemistry. 2002; 41: 7008-7020Google Scholar) and rice OsGSTU1 (Protein Data Bank accession code 1OYJ), showing 39 and 63% sequence identity to ZmGSTU2, respectively, using the web-based utility SWISSMODEL (www.expasy.ch/swissmod/SWISS-MODEL.html). The model obtained was manually analyzed using the graphics program QUANTA (Accelyrs Inc.) for steric clashes and chemical sensibility. The conformation of the side chain of residue Gln-115 resulted in a steric clash with Tyr-170 and therefore was remodeled using water positions from the OsGSTU1 as a guide. A molecular model of fluorodifen was generated using INSIGHTII and CATALYST (Accelyrs Inc.), and a suitable low energy conformation was docked into the active site using QUANTA.Generation and Analysis of Transgenic Plants—GST sequences were subcloned into pRT108 by PCR from the respective pET-vectors and ligated into the NcoI and BamHI sites of the vector (15Töpfer R. Maas C. Höricke-Grandpierre C. Schell J. Steinbiss H.-H. Methods Enzymol. 1993; 217: 66-78Google Scholar). This cassette was then digested with HindIII and ligated into similarly digested pCAMBIA 3300 (CAMBIA, Canberra, Australia). The resulting vectors were transformed into Agrobacterium tumefaciens strain C58C3 and used to transform A. thaliana ecotype Columbia by floral dipping (16Clough S.J. Bent A.F. Plant J. 1998; 16: 735-743Google Scholar). Dipped plants were grown to seed set, and T1 plants were grown for 10 days in the greenhouse (16-h photoperiod; 23 °C day; 18 °C night) in a 4:1 mixture of general purpose compost and silver sand prior to spraying with 0.02% (w/v) glufosinate ammonium (1 ml/35 cm2) to select for transformants. Surviving plants were resprayed after 7 days. T1 lines showing good GST expression were selected for propagation after analyzing extracts from individual rosette leaves by SDS-PAGE and Western blotting using an anti-ZmGSTU1–2 serum (9Dixon D.P. Cole D.J. Edwards R. Plant Mol. Biol. 1998; 36: 75-87Google Scholar). For each line of interest, ∼10 whole T2 seedlings (14 days old) were extracted and assayed for GST activity and immunoreactive polypeptides by Western blotting after normalizing for total protein content. For fluorodifen spray trials, cells containing five 25-day-old T2 seedlings grown in compost, selected previously for the presence of T-DNA insertions by spraying with glufosinate ammonium to kill segregating wild-type plants, were sprayed with 20 μm fluorodifen formulated in 1% (v/v) acetone and 0.01% (v/v) Tween 20 daily for 14 days with each 23-cm2 cell receiving a 1-ml application.RESULTSGeneration and Characterization of Mutant GSTUs by Reconstructive PCR—ZmGSTU1 and ZmGSTU2, showing 72% sequence identity to one another (9Dixon D.P. Cole D.J. Edwards R. Plant Mol. Biol. 1998; 36: 75-87Google Scholar, 10Dixon D.P. Cole D.J. Edwards R. Plant Mol. Biol. 1999; 40: 997-1008Google Scholar), were employed for reconstructive PCR using equal amounts of DNA fragments of pGSTU2 (pBluescript containing cDNA encoding ZmGSTU2) and pGSTU1 (pBluescript containing cDNA encoding ZmGSTU1). Following in vitro recombination, a single product was amplified by PCR from each reassembled template and cloned into pBluescript SK–. A microtiter plate screening procedure was used to assay 5000 mutant colonies for enhanced GST activity toward the herbicide fluorodifen based on the release of colored p-nitrophenol (Fig. 1, A and B). As compared with the parent ZmGSTUs, a significant proportion of mutants showed moderately enhanced activity, largely due to relatively higher levels of GST expression in the bacteria. Analysis of three such clones showed that they were chimeras of the N-terminal portion of ZmGSTU1 with the remaining sequence composed of ZmGSTU2. In each case, the specific activity of the pure enzyme toward fluorodifen was little affected. However, seven mutants were identified that had at least 5-fold higher enzyme activity (as judged visually) than bacteria expressing either ZmGSTU1 or ZmGSTU2. These EFD mutant clones pEFD1–7, expressing the mutant proteins EFD1–7, were sequenced (see supplementary information). Six of the seven EFDs were highly expressed, containing N termini (between 48 and 62 residues) derived from ZmGSTU1 (Fig. 1C). With the exception of EFD6, the remainder of the coding sequence was derived from ZmGSTU2, interrupted by a region, or regions, of varying size derived from ZmGSTU1 (Fig. 1C). In EFD6, there was no ZmGSTU1 insertion in the C-terminal half of the protein, but instead a single missense mutation corresponding to the substitution of glutamine with leucine at residue 115 (equivalent to residue 113 of ZmGSTU2) was identified.The seven EFD mutants were individually subcloned into the pET-11d plasmid, resulting in the expression of a 28-kDa polypeptide in each case as determined by SDS-PAGE. The kinetics of each EFD was then examined after purifying the enzymes by affinity chromatography, with the exception of EFD7, which was not analyzed further due to the instability of its activity. The recombinant GSTs were assayed with fluorodifen and the related diphenylether herbicides fomesafen and acifluorfen (Fig. 1A). All diphenylether substrates were used at relatively low concentrations (50 μm) due to their limited solubility in water. This made it impossible to directly determine the Km values toward these substrates, which were well above the solubility limit in each case. Instead, a value for enzyme catalytic efficiency (kcat/Km) was calculated in each case (Table I). Of the diphenylethers tested, both parent GSTUs and related EFDs showed a marked substrate preference for fluorodifen. The kcat/Km values for fluorodifen were higher in all the EFDs relative to the parent enzymes. This was in contrast to the results obtained with fomesafen and acifluorfen, with the mutant enzymes EFD1–5 showing similar or slightly reduced values for kcat/Km relative to ZmGSTU1, the only parent showing appreciable activity toward both herbicides. EFD6 did show a modest 4-fold increase in kcat/Km toward acifluorfen relative to ZmGSTU1 as compared with a greater than 18-fold enhancement with fluorodifen as substrate. These results suggested that gene shuffling studies had generated a set of mutant enzymes that had selectively improved activities toward fluorodifen but not toward closely related substrate chemistries. Constrained by their limited solubilities, it was not possible to further investigate the effects of gene shuffling on enzyme binding and turnover of the diphenylethers using kinetic analysis. However, it was possible to determine the effect of mutagenesis on substrate binding and turnover of the model substrate CDNB. Mutants EFD1–5 inclusive had apparent Km values toward GSH and CDNB and associated Vmax values that were within the range determined for the parent GSTUs. This was what would be predicted from mutant enzymes that are effectively chimeras of modular blocks of sequence from the two parents. In contrast, EFD6 had increased Km values toward both CDNB and GSH, coupled with a decreased Vmax. Taken together with the singular changes seen with EFD6 in the cleavage of fluorodifen, these results collectively suggested that this mutant had undergone a more radical change in active site topography than the other EFDs. With the exception of the substitution of residue 115, EFD6 was otherwise a simple chimera of the N-terminal portion of ZmGSTU1 and the remaining sequence of ZmGSTU2 (Fig. 1C). Based on the essentially conserved kinetic characteristics of the other chimeric EFDs in which blocks of sequence had been shuffled, it was concluded that the substitution at residue 115 was a key factor determining the novel detoxifying activities of EFD6. Attention was therefore focused on the selective mutagenesis of this residue.Table IKinetic characteristics of purified mutated and wild-type recombinant GSTUs toward the diphenylethers fluorodifen (Flu), acifluorfen (Aci), and Fomesafen (Fom) and the model spectrophotometric substrate CDNB, as well as GSHEnzymekcat/Km (Flu)kcat/Km (Aci)kcat/Km (Fom)kcat/Km (CDNB)Km (CDNB)aApparent values, calculated at 5 mM glutathione.Vmax (CDNB)aApparent values, calculated at 5 mM glutathione.Km (GSH)bApparent values, calculated at 1 mM CDNB.M-1·S-1M-1·S-1M-1·S-1M-1·S-1μMnanokatal/mgmMZmGSTU12301.636.7018,400101011100.56ZmGSTU2400ND0.42300,00011519101.72EFD152900.770.98219,00020622901.95EFD237001.101.26142,0001369300.76EFD375500.960.84343,00019719501.15EFD423600.621.50185,00020417301.18EFD525000.681.70144,00021215001.05EFD642907.609.10920018007604.75EFD6-115Q220ND0.52193,0001068800.92EFD6-115F50404.203.8047,4005348700.74EFD6-115A11,7004.902.60150,00020010600.78a Apparent values, calculated at 5 mM glutathione.b Apparent values, calculated at 1 mM CDNB. Open table in a new tab Directed Mutagenesis of EFD6 —A side effect of the L115Q substitution in EFD6 was that the mutant enzyme was predominantly expressed in the insoluble fraction in E. coli, unlike the other chimeric EFDs analyzed. Again, this was consistent with this substitution having a major effect on protein structure. Rational site-directed mutagenesis was employed to substitute the mutated residue and determine the effect on enzyme activity with the aim of finding a mutant with both high activity toward fluorodifen and more stable expression as a soluble protein. The original mutation replaced glutamine, a mediumsized neutral residue, with leucine, a smaller hydrophobic residue. This residue was therefore mutated to asparagine (neutral but smaller), glutamic acid (acidic, same size), phenylalanine (hydrophobic, large), or alanine (hydrophobic, small), giving the mutants EFD6–115N, EFD6–115E, EFD6–115F, and EFD6–115A, respectively. Residue 115 was also mutagenized back to glutamine to yield EFD6–115Q, effectively producing the unmutated chimera of ZmGSTU1 and ZmGSTU2. Each of these mutants was expressed in E. coli as a LacZ fusion protein. Lysates from bacteria expressing EFD6–115E or EFD6–115N had negligible GST activity with the misfolded recombinant polypeptides precipitated in the inclusion bodies. In contrast, bacteria expressing EFD6–115F, EFD6–115A, and EFD6–115Q gave good yields of soluble GSTs, which were subjected to kinetic analysis (Table I). EFD6–115Q had characteristics similar to ZmGSTU2 toward both diphenylethers and CDNB, confirming that the point mutation in EFD6 affecting residue 115 was primarily responsible for the enhanced fluorodifen cleaving activity. As determined by comparing kcat/Km values, mutation of residue 115 to phenylalanine to produce EFD6–115F gave an enzyme with similar activity toward fluorodifen, reduced activities toward fomesafen and acifluorfen, and a 5-fold increase with CDNB as substrate. The latter change was effected by decreasing the Km toward CDNB rather than increasing Vmax. The most successful attempt at directed mutagenesis was seen with the substitution of residue 115 of EFD6 to alanine. The resulting mutant EFD6–115A had almost a 3-fold increase in kcat/Km toward fluorodifen relative to EFD6. Overall, the combination of DNA shuffling and directed mutagenesis of key residue 115 had caused a 29-fold increase in detoxifying activity toward fluorodifen relative to the most active parent enzyme ZmGSTU2. As compared with EFD6, the alanine substitution also restored a greater kcat/Km value toward CDNB, again as a result of lowering Km toward the substrate, giving an enzyme with essentially similar kinetic characteristics toward CDNB and glutathione to the parent ZmGSTUs, and suggesting that this mutant did not have the structural problems of EFD6. Similarly, the “optimized” mutant enzyme showed only minor differences in kcat/Km toward acifluorfen and fomesafen, herbicides resembling fluorodifen. When the structurally unrelated chloroacetanilide herbicides acetochlor, alachlor, and metolachlor were assayed as GST substrates, EFD6–115A showed specific activities of 112 picokatal/mg of pure protein, 72 picokatal/mg, and 144 picokatal/mg, respectively. These values were very similar to those determined with the purified ZmGSTUs assayed under identical conditions (ZmGSTU1, 160 picokatal/mg of pure protein 68 picokatal/mg, and 192 picokatal/mg, respectively; ZmGSTU2, 215 picokatal/mg of pure protein, 91 picokatal/mg, and 157 picokatal/mg, respectively).Effect of Expression of EFD Mutants on Tolerance to Fluorodifen in Planta—Transgenic Arabidopsis plants were individually engineered to express the parent ZmGSTUs, EFD6, EFD3, and EFD6–115A under the control of the CaMV35S promoter. This combination of EFDs was selected as EFD6–115A represented the optimized detoxifying enzyme generated through a combination of gene shuffling and directed mutagenesis, whereas EFD3 was the most efficient fluorodifen detoxifying enzyme generated by shuffling alone. None of the resulting T1 or T2 transgenics showed any abnormal phenotype under normal growth conditions. T2 plants were then analyzed for the expression of the introduced GSTUs by Western blotting using an antiserum raised to the ZmGSTU1–2 heterodimer and by assaying for fluorodifen cleaving activity (Fig. 2A). The antiserum recognized 28-kDa polypeptides in all GSTU-transformed lines except those expressing ZmGSTU2, which was due to the weak immunoreactivity of this protein with this antibody (9Dixon D.P. Cole D.J. Edwards R. Plant Mol. Biol. 1998; 36: 75-87Google Scholar). When assayed for fluorodifen detoxifying activity (Fig. 2A), the only lines showing enhanced GST activity toward the herbicide in in vitro assays were those transformed with EFD3 and EFD6–115A, the latter showing 19-fold greater specific activity than the controls. It was then of interest to determine whether or not the transgenic expression of the herbicide detoxifying GST activity could confer tolerance to fluorodifen in planta.Fig. 2Analysis of T2 transgenic Arabidopsis. As shown in A, pools of plants derived from each construct were analyzed by SDS-PAGE and Western blotting using an anti-ZmGSTU1–2 serum. Lane M, molecular mass markers (kDa); 1, wild type; 2, vector control (pCAMBIA3300); 3, ZmGSTU1; 4, ZmGSTU2; 5, EFD3; 6, EFD6 (line 1); 7, EFD6 (line 2); 8, EFD6–115A. The GST activities toward fluorodifen determined in duplicate crude extracts are shown as mean values (picokatal/mg total protein). As shown in B, plants transformed with vector alone, ZmGSTU1, ZmGSTU2, or EFD6–115A were treated daily with 20 μm fluorodifen (sprayed) or were left untreated (unsprayed) for 14 days in a glasshouse.View Large Image Figure ViewerDownload (PPT)Fluorodifen was originally developed as a selective photobleaching herbicide for use in crops of peanuts, soybean cotton, and rice rather than in the more sensitive Brassica species (17Shimabukuro R.H. Lamoureux G.L. Swanson H.R. Walsh W.C. Stafford L.E. Frear D.S. Pestic. Biochem. Physiol. 1973; 3: 483-494Google Scholar). The selectivity of fluorodifen is associated with its greater rate of detoxification in tolerant crops as compared with susceptible weeds, principally through GST-mediated metabolism (3Frear D.S. Swanson H.R. Pestic. Biochem. Physiol. 1973; 3: 473-482Google Scholar, 17Shimabukuro R.H. Lamoureux G.L. Swanson H.R. Walsh W.C. Stafford L.E. Frear D.S. Pestic. Biochem. Physiol. 1973; 3: 483-494Google Scholar). It would therefore be anticipated that enhanced expression of a detoxifying GST in a normally susceptible species would give visibly greater tolerance to the photobleaching activity of fluorodifen, which is a characteristic feature of diphenylether herbicides even in tolerant crops (13Andrews C.J. Skipsey M. Townson J.K. Morris C. Jepson I. Edwards R. Pestic. Sci. 1997; 51: 213-222Google Scholar). Arabidopsis plants were found to be highly sensitive to fluorodifen when sprayed as an unformulated leaf drench treatment. Careful adjustment of the treatments showed that a repeated spraying of 25-day-old Arabidopsis plants with 20 μm fluorodifen gave a sublethal but severe photobleaching, which largely arrested growth. T2 generation controls and plants expressing ZmGSTU1, ZmGSTU2, or the optimized mutant EFD6–115A were then treated with the fluorodifen dosing regime, and the plants were visually assessed. After a 14-day treatment, the vector control, ZmGSTU1 and ZmGSTU2-expressing plants, were severely stunted with the foliage showing signs of photobleaching and necrosis (Fig. 2B). Similarly treated plants expressing EFD6–115A were much less damaged, and although showing some photobleaching, had continued to grow at a rate similar to that of unsprayed plants.The recent availability of crystal structures of GSTUs from wheat (8Thom R. Cummins I. Dixon D.P. Edwards R. Cole D.J. Lapthorn A.J. Biochemistry. 2002; 41: 7008-7020Google Scholar) and rice (Protein Data Bank accession number 1OYJ) has permitted molecular modeling of ZmGSTU2 as well as the EFD mutants binding to fluorodifen. EFD1–5 are essentially ZmGSTU2 with two regions of ZmGSTU1 in the N-terminal domain and C-terminal domain, respectively (Fig. 3A). These changes in the N-terminal region were correlated with high levels of GST expression and a lower Km toward glutathione as compared with the other parent ZmGSTU1 (Table I).Fig. 3A, a ribbon representation of the structural model of ZmGSTU2 monomer colored to highlight the changes in a representative EFD mutant enzyme (EFD2). The region derived from ZmGSTU2 is colored green, that from ZmGSTU1 is colored light blue with regions that differ from ZmGSTU2 highlighted in dark blue, and the critical residue 115 is highlighted in red. Glutathione is shown in ball-and-stick representation, and the docked herbicide fluorodifen is represented in stick form, with both colored according to atom type. B, a detail of the active site of the ZmGSTU2 model with fluorodifen represented in Van der Waals spheres. Residues predicted to be important for fluorodifen binding are shown in stick form and colored according to atom type.View Large Image Figure ViewerDownload (PPT)DISCUSSIONThrough a combination of random and directed mutagenesis, we have derived a GST, EFD-115A, with a greatly increased capacity to detoxify fluorodifen and demonstrated that this mutant enzyme can give enhanced protection against the photobleaching injury incurred by this herbicide when expressed in planta. Although forced evolution directed t" @default.
• W2139860372 created "2016-06-24" @default.
• W2139860372 creator A5006389025 @default.
• W2139860372 creator A5010658292 @default.
• W2139860372 creator A5031574561 @default.
• W2139860372 creator A5052481021 @default.
• W2139860372 date "2003-06-01" @default.
• W2139860372 modified "2023-10-13" @default.
• W2139860372 title "Forced Evolution of a Herbicide Detoxifying Glutathione Transferase" @default.
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