With the increasing promotion of simplified rapeseed cultivation in recent years,the development of cultivars with high resistance to herbicides is urgently needed.We previously developed M342,which shows sulfonylurea...With the increasing promotion of simplified rapeseed cultivation in recent years,the development of cultivars with high resistance to herbicides is urgently needed.We previously developed M342,which shows sulfonylurea herbicide resistance,by targeting acetohydroxyacid synthase(AHAS),a key enzyme in branched-chain amino acid synthesis.In the present study,we used a progeny line derived from M342 for an additional round of ethyl methane sulfonate mutagenesis,yielding the novel mutant DS3,which harbored two mutations in AHAS genes and showed high sulfonylurea resistance.One mutation was the substitution Trp574 Leu,as in M342,according to Arabidopsis protein sequencing.The other site was a newly recognized substitution,Pro197 Leu.A KASP marker targeting Pro197 Leu was developed and reliably predicted the response to sulfonylurea herbicides in the F2 population.The combination of Trp574 Leu and Pro197 Leu in DS3 produced a synergistic effect that greatly increased herbicide resistance.Analysis of the protein structures of AHAS1 and AHAS3 in wild-type and single-gene mutant plants revealed three-dimensional protein conformational changes that could account for differences in herbicide resistance characteristics including toxicity tolerance,AHAS enzyme activity,and AHAS gene expression.展开更多
Cyperus difformis L.is a troublesome weed in paddy fields and has attracted attention due to its resistance to acetohydroxyacid synthase(AHAS)inhibitors.It was found that the amino acid mutation in AHAS was the primar...Cyperus difformis L.is a troublesome weed in paddy fields and has attracted attention due to its resistance to acetohydroxyacid synthase(AHAS)inhibitors.It was found that the amino acid mutation in AHAS was the primary cause for the resistance of Cyperus difformis.However,the effect of different mutations on AHAS function is not clear in Cyperus difformis.To confirm the effect of mutations on AHAS function,six biotypes were collected,including Pro197Arg,Pro197Ser,Pro197Leu,Asp376Glu,Trp574Leu and wild type,from Hunan,Anhui,Jiangxi and Jiangsu provinces,China and the function of AHAS was characterized.The AHAS in vitro inhibition assay results indicated that the mutations decreased the sensitivity of AHAS to pyrazosulfuron-ethyl,in which the I_(50)(the half maximal inhibitory concentration)of wild type AHAS was 0.04μmol L^(-1)and Asp376Glu,Pro197Leu,Pro197Arg,Pro197Ser and Trp574Leu mutations were 3.98,11.50,40.38,38.19 and 311.43μmol L^(-1),respectively.In the determination of enzyme kinetics parameters,the Km and the maximum reaction velocity(Vmax)of the wild type were 5.18 mmol L^(-1)and 0.12 nmol mg^(-1)min^(-1),respectively,and the Km values of AHAS with Asp376Glu,Trp574Leu,Pro197Leu and Pro197Ser mutations were 0.38-0.93 times of the wild type.The Km value of the Pro197Arg mutation was 1.14times of the wild type,and the Vmax values of the five mutations were 1.17-3.33-fold compared to the wild type.It was found that the mutations increased the affinity of AHAS to the substrate,except for the Pro197Arg mutation.At a concentration of 0.0032-100 mmol L^(-1)branched-chain amino acids(BCAAs),the sensitivity of the other four mutant AHAS biotypes to feedback inhibition decreased,except for the Pro197Arg mutation.This study elucidated the effect of different mutations on AHAS function in Cyperus difformis and provided ideas for further study of resistance development.展开更多
A novel kind of acetohydroxyacid synthase（AHAS） inhibitors, arylsulfonylaminocarbonyl-2-thiohydantoins, was synthesized based on biorational design. The structures of these compounds were confirmed by IR, MS, NMR, a...A novel kind of acetohydroxyacid synthase（AHAS） inhibitors, arylsulfonylaminocarbonyl-2-thiohydantoins, was synthesized based on biorational design. The structures of these compounds were confirmed by IR, MS, NMR, and elemental analysis. The results of the preliminary activity tests indicate that most of the arylsulfonylamidecarbonyl-5- methyl-2-thiohydantoins have a higher biological activity than those without 5-methyl substituent.展开更多
Acetohydroxyacid synthase(AHAS)is the target enzyme of several classes of herbicides,such as sulfonylureas and imidazolinones.Now many mutant AHASs with herbicide resistance have emerged along with extensive use of he...Acetohydroxyacid synthase(AHAS)is the target enzyme of several classes of herbicides,such as sulfonylureas and imidazolinones.Now many mutant AHASs with herbicide resistance have emerged along with extensive use of herbicides,therefore it is imperative to understand the detailed interaction mechanism and resistance mechanism so as to develop new potent inhibitors for wild-type or resistant AHAS.With the aid of available crystal structures of the Arabidopsis thaliana(At)AHAS-inhibitor complex,molecular dynamics(MD)simulations were used to investigate the interaction and resistance mechanism directly and dynamically at the atomic level.Nanosecond-level MD simulations were performed on six systems consisting of wild-type or W574L mutant AtAHAS in the complex with three sulfonylurea inhibitors,separately,and binding free energy was calculated for each system using the MM-GBSA method.Comprehensive analyses from structural and energetic aspects confirmed the importance of residue W574,and also indicated that W574L mutation might alert the structural charactersistic of the substrate access channel and decrease the binding affinity of inhibitors,which cooperatively weaken the effective channel-blocked effect and finally result in weaker inhibitory effect of inhibitor and corresponding herbicide resistance of W574L mutant.To our knowledge,it is the first report about MD simulations study on the AHAS-related system,which will pave the way to study the interactions between herbicides and wild-type or mutant AHAS dynamically,and decipher the resistance mechanism at the atomic level for better designing new potent anti-resistance herbicides.展开更多
Homologous and heterologous interactions between acetohydroxyacid synthase(AHAS)I andⅢfrom E.coli have been studied by surface plasmon resonance(SPR).The catalytic and regulatory subunits association for AHAS I(KD=1....Homologous and heterologous interactions between acetohydroxyacid synthase(AHAS)I andⅢfrom E.coli have been studied by surface plasmon resonance(SPR).The catalytic and regulatory subunits association for AHAS I(KD=1.13×10-7 M)was stronger than that for AHASⅢ(KD=5.29×10-7 M).A strong heterologous association between regulatory and catalytic subunits and heterologous activation of catalytic subunits were observed.SPR results combined with enzyme kinetics indicate that the reconstituted heterologous enzymes had similar kinetic properties as homologous enzymes,implying that the regulatory subunit of AHAS I could be replaced by the regulatory subunit of AHASⅢand vice versa.This work may be useful to further understandings of the mechanism of regulation of AHAS.展开更多
Bispyribac is a widely used herbicide that targets the acetohydroxyacid synthase （AHAS） enzyme. Mutations in AHAS have caused serious herbicide resistance that threatened the continued use of the herbicide. So far, ...Bispyribac is a widely used herbicide that targets the acetohydroxyacid synthase （AHAS） enzyme. Mutations in AHAS have caused serious herbicide resistance that threatened the continued use of the herbicide. So far, a unified model to decipher herb- icide resistance in molecular level with good prediction is still lacking. In this paper, we have established a new QSAR method to construct a prediction model for AHAS mutation resistance to herbicide Bispyribac. A series of AHAS mutants concerned with the herbicide resistance were constructed, and the inhibitory properties of Bispyribac against these mutants were meas- ured. The 3D-QSAR method has been transformed to process the AHAS mutants and proposed as mutation-dependent biom- acromolecular QSAR （MB-QSAR）. The excellent correlation between experimental and computational data gave the MB-QSAR/CoMFA model （q2 = 0.615, P = 0.921, F2pred = 0.598） and the MB-QSAR/CoMSIA model （q2 = 0.446, r2 = 0.929, r2pred = 0.612）, which showed good prediction for the inhibition properties of Bispyribac against AHAS mutants. Such MB-QSAR models, containing the three-dimensional molecular interaction diagram, not only disclose to us for the first time the detailed three-dimensional information about the structure-resistance relationships, but may also provide further guidance to resistance mutation evolution. Also, the molecular interaction diagram derived from MB-QSAR models may aid the resistance-evading herbicide design.展开更多
Chlorsulfuron is the first commercialized sulfonylurea herbicide, which targets acetohydroxyacid synthase （AHAS）. Mutations in AHAS have caused serious herbicide resistance to chlorsulfuron. Quantitative description...Chlorsulfuron is the first commercialized sulfonylurea herbicide, which targets acetohydroxyacid synthase （AHAS）. Mutations in AHAS have caused serious herbicide resistance to chlorsulfuron. Quantitative description of the herbicide resistance in molecular level will benefit the understanding of the resistance mechanism and aid the design of resistance-evading herbicide. We have recently established a MB-QSAR （Mutation-dependent Biomac- romolecular Quantitative Structure-Activity Relationship） method to conduct the 3D-QSAR study in biomacro- molecules. Herein, based on the herbicide resistance data measured for a series of AHAS mutants against chlorsul- furon, we constructed MB-QSAR models to quantitatively predict the herbicide resistance and interpret the struc- ture resistance relationships for AHAS mutants against chlorsulfuron. Quite well correlations between the experi- mental and the predicted pKi values were achieved for MB-QSAR/CoMFA （q^2=0.705, r^2=0.918, r^2pred=0.635） and MB-QSAR/CoMSIA （q^2=0.558, r^2=0.940, r^2pred=0.527） models, and interpretation of the MB-QSAR models gave chemical intuitive information to guide the resistance-evading herbicide design.展开更多
On the basis of study on the mechanism of action of sulfonylurea herbicides, nine sulfonylurea derivatives of iso-{xazolidinone} were designed and synthesized. The structures of these compounds were confirmed by means...On the basis of study on the mechanism of action of sulfonylurea herbicides, nine sulfonylurea derivatives of iso-{xazolidinone} were designed and synthesized. The structures of these compounds were confirmed by means of IR, MS, NMR and elemental analysis. The results of preliminary active tests indicate that the compounds have some herbicidal activity. The structure-activity relationship was also studied.展开更多
A novel type of aeetohydrexyacid synthase inhibitors, 4-methyl-3-isoxazolidinone derivatives of sulfonylurea, was designed and synthesized. The structures of these compounds were confirmed by using MS, NMR, and elemen...A novel type of aeetohydrexyacid synthase inhibitors, 4-methyl-3-isoxazolidinone derivatives of sulfonylurea, was designed and synthesized. The structures of these compounds were confirmed by using MS, NMR, and elemental analysis. The results of preliminary active tests indicate that the compounds show a herbicidal activity.展开更多
Thirteen novel phenoxysulfonylureas derivatives were synthesized, and their structures were confirmed by MS, NMR and element analysis. The herbicidal activity and structure-activity relationship were also investigated...Thirteen novel phenoxysulfonylureas derivatives were synthesized, and their structures were confirmed by MS, NMR and element analysis. The herbicidal activity and structure-activity relationship were also investigated. The results of preliminary active tests indicate that the compounds show moderate herbicidal activity.展开更多
It remains a great challenge to control weeds in rapeseed fields in China.Breeding herbicide-resistant rapeseed varieties and using corresponding herbicide formulations has become the most economical and effective way...It remains a great challenge to control weeds in rapeseed fields in China.Breeding herbicide-resistant rapeseed varieties and using corresponding herbicide formulations has become the most economical and effective way to control weeds in rapeseed field.Characterization of more herbicide-resistant genetic resources will provide opportunities for breeders to develop rapeseed herbicide-resistant varieties with good agronomic performance.Previously,we obtained the tribenuron methyl(TBM)-resistant mutant K4 from ZS9(Brassica napus L.)through ethyl methyl sulfonate mutagenesis and TBM foliar-spray screening.In this study,the inheritance and molecular characterization of the mutant K4 are carried out.Genetic investigation indicated that the herbicide-resistance of the K4 was controlled by one dominant allele at a single nuclear gene locus.Molecular characterization showed that a single point substitution at position 535 from C to T in BnAHAS3(BnAHAS3^(535T)),which resulted in a mutation at point 179 in BnAHAS3.The K4 showed a certain degree of resistance to TBM,bensulfuron methyl,and monosulfon sodium,which were 50,30,and 5 times that of ZS9,respectively.AHAS enzyme assay,structural analysis of AHAS proteins,affinity detection between TBM and BnAHAS3 by surface plasmon resonance analysis,and the transgenic experiment in Arabidopsis using BnAHAS3^(535T) confirmed that BnAHAS3^(535T) endow the K4 with herbicides resistance.In addition,an allele-specific marker was developed to quickly distinguish the heterozygous and homozygous mutated alleles BnAHAS3^(535T).In conclusion,our research identified and characterized one novel mutative AHAS allele in B.napus and enriched genetic resource for developing herbicide-resistant rapeseed cultivars.展开更多
基金supported by the National Natural Science Foundation of China(31870519,31901503 and 31671731)the National Key Research and Development Program of China(2016YFD0101300 and 2016YFD0100202-10)+1 种基金the China Agriculture Research System(CARS-12)the Natural Science Foundation of Jiangsu Province(BK20190267)。
文摘With the increasing promotion of simplified rapeseed cultivation in recent years,the development of cultivars with high resistance to herbicides is urgently needed.We previously developed M342,which shows sulfonylurea herbicide resistance,by targeting acetohydroxyacid synthase(AHAS),a key enzyme in branched-chain amino acid synthesis.In the present study,we used a progeny line derived from M342 for an additional round of ethyl methane sulfonate mutagenesis,yielding the novel mutant DS3,which harbored two mutations in AHAS genes and showed high sulfonylurea resistance.One mutation was the substitution Trp574 Leu,as in M342,according to Arabidopsis protein sequencing.The other site was a newly recognized substitution,Pro197 Leu.A KASP marker targeting Pro197 Leu was developed and reliably predicted the response to sulfonylurea herbicides in the F2 population.The combination of Trp574 Leu and Pro197 Leu in DS3 produced a synergistic effect that greatly increased herbicide resistance.Analysis of the protein structures of AHAS1 and AHAS3 in wild-type and single-gene mutant plants revealed three-dimensional protein conformational changes that could account for differences in herbicide resistance characteristics including toxicity tolerance,AHAS enzyme activity,and AHAS gene expression.
基金funded by the National Natural Science Foundation of China(31972281)。
文摘Cyperus difformis L.is a troublesome weed in paddy fields and has attracted attention due to its resistance to acetohydroxyacid synthase(AHAS)inhibitors.It was found that the amino acid mutation in AHAS was the primary cause for the resistance of Cyperus difformis.However,the effect of different mutations on AHAS function is not clear in Cyperus difformis.To confirm the effect of mutations on AHAS function,six biotypes were collected,including Pro197Arg,Pro197Ser,Pro197Leu,Asp376Glu,Trp574Leu and wild type,from Hunan,Anhui,Jiangxi and Jiangsu provinces,China and the function of AHAS was characterized.The AHAS in vitro inhibition assay results indicated that the mutations decreased the sensitivity of AHAS to pyrazosulfuron-ethyl,in which the I_(50)(the half maximal inhibitory concentration)of wild type AHAS was 0.04μmol L^(-1)and Asp376Glu,Pro197Leu,Pro197Arg,Pro197Ser and Trp574Leu mutations were 3.98,11.50,40.38,38.19 and 311.43μmol L^(-1),respectively.In the determination of enzyme kinetics parameters,the Km and the maximum reaction velocity(Vmax)of the wild type were 5.18 mmol L^(-1)and 0.12 nmol mg^(-1)min^(-1),respectively,and the Km values of AHAS with Asp376Glu,Trp574Leu,Pro197Leu and Pro197Ser mutations were 0.38-0.93 times of the wild type.The Km value of the Pro197Arg mutation was 1.14times of the wild type,and the Vmax values of the five mutations were 1.17-3.33-fold compared to the wild type.It was found that the mutations increased the affinity of AHAS to the substrate,except for the Pro197Arg mutation.At a concentration of 0.0032-100 mmol L^(-1)branched-chain amino acids(BCAAs),the sensitivity of the other four mutant AHAS biotypes to feedback inhibition decreased,except for the Pro197Arg mutation.This study elucidated the effect of different mutations on AHAS function in Cyperus difformis and provided ideas for further study of resistance development.
文摘A novel kind of acetohydroxyacid synthase（AHAS） inhibitors, arylsulfonylaminocarbonyl-2-thiohydantoins, was synthesized based on biorational design. The structures of these compounds were confirmed by IR, MS, NMR, and elemental analysis. The results of the preliminary activity tests indicate that most of the arylsulfonylamidecarbonyl-5- methyl-2-thiohydantoins have a higher biological activity than those without 5-methyl substituent.
基金supported by the National Natural Science Foundation of China(Grant Nos.20432010,20421202,and 90713011)the National Key Project for Basic Research(Grant Nos.2008DFA30770 and 2010CB126102)Key Project of Ministry of Education,China(Grant No.104189)and Institute of Scientific Computing(ISC)of Nankai University
文摘Acetohydroxyacid synthase(AHAS)is the target enzyme of several classes of herbicides,such as sulfonylureas and imidazolinones.Now many mutant AHASs with herbicide resistance have emerged along with extensive use of herbicides,therefore it is imperative to understand the detailed interaction mechanism and resistance mechanism so as to develop new potent inhibitors for wild-type or resistant AHAS.With the aid of available crystal structures of the Arabidopsis thaliana(At)AHAS-inhibitor complex,molecular dynamics(MD)simulations were used to investigate the interaction and resistance mechanism directly and dynamically at the atomic level.Nanosecond-level MD simulations were performed on six systems consisting of wild-type or W574L mutant AtAHAS in the complex with three sulfonylurea inhibitors,separately,and binding free energy was calculated for each system using the MM-GBSA method.Comprehensive analyses from structural and energetic aspects confirmed the importance of residue W574,and also indicated that W574L mutation might alert the structural charactersistic of the substrate access channel and decrease the binding affinity of inhibitors,which cooperatively weaken the effective channel-blocked effect and finally result in weaker inhibitory effect of inhibitor and corresponding herbicide resistance of W574L mutant.To our knowledge,it is the first report about MD simulations study on the AHAS-related system,which will pave the way to study the interactions between herbicides and wild-type or mutant AHAS dynamically,and decipher the resistance mechanism at the atomic level for better designing new potent anti-resistance herbicides.
基金Supported by the National Key Project for Basic Research of China(Grant No.2010CB126102)National Natural Science Foundation of China(Grant Nos.20572053,20421202&20432010)Ministry of Education of China(Grant No.104189)
文摘Homologous and heterologous interactions between acetohydroxyacid synthase(AHAS)I andⅢfrom E.coli have been studied by surface plasmon resonance(SPR).The catalytic and regulatory subunits association for AHAS I(KD=1.13×10-7 M)was stronger than that for AHASⅢ(KD=5.29×10-7 M).A strong heterologous association between regulatory and catalytic subunits and heterologous activation of catalytic subunits were observed.SPR results combined with enzyme kinetics indicate that the reconstituted heterologous enzymes had similar kinetic properties as homologous enzymes,implying that the regulatory subunit of AHAS I could be replaced by the regulatory subunit of AHASⅢand vice versa.This work may be useful to further understandings of the mechanism of regulation of AHAS.
文摘Bispyribac is a widely used herbicide that targets the acetohydroxyacid synthase （AHAS） enzyme. Mutations in AHAS have caused serious herbicide resistance that threatened the continued use of the herbicide. So far, a unified model to decipher herb- icide resistance in molecular level with good prediction is still lacking. In this paper, we have established a new QSAR method to construct a prediction model for AHAS mutation resistance to herbicide Bispyribac. A series of AHAS mutants concerned with the herbicide resistance were constructed, and the inhibitory properties of Bispyribac against these mutants were meas- ured. The 3D-QSAR method has been transformed to process the AHAS mutants and proposed as mutation-dependent biom- acromolecular QSAR （MB-QSAR）. The excellent correlation between experimental and computational data gave the MB-QSAR/CoMFA model （q2 = 0.615, P = 0.921, F2pred = 0.598） and the MB-QSAR/CoMSIA model （q2 = 0.446, r2 = 0.929, r2pred = 0.612）, which showed good prediction for the inhibition properties of Bispyribac against AHAS mutants. Such MB-QSAR models, containing the three-dimensional molecular interaction diagram, not only disclose to us for the first time the detailed three-dimensional information about the structure-resistance relationships, but may also provide further guidance to resistance mutation evolution. Also, the molecular interaction diagram derived from MB-QSAR models may aid the resistance-evading herbicide design.
基金This work was financially supported by MOST，the National Natural Science Foundation of China
文摘Chlorsulfuron is the first commercialized sulfonylurea herbicide, which targets acetohydroxyacid synthase （AHAS）. Mutations in AHAS have caused serious herbicide resistance to chlorsulfuron. Quantitative description of the herbicide resistance in molecular level will benefit the understanding of the resistance mechanism and aid the design of resistance-evading herbicide. We have recently established a MB-QSAR （Mutation-dependent Biomac- romolecular Quantitative Structure-Activity Relationship） method to conduct the 3D-QSAR study in biomacro- molecules. Herein, based on the herbicide resistance data measured for a series of AHAS mutants against chlorsul- furon, we constructed MB-QSAR models to quantitatively predict the herbicide resistance and interpret the struc- ture resistance relationships for AHAS mutants against chlorsulfuron. Quite well correlations between the experi- mental and the predicted pKi values were achieved for MB-QSAR/CoMFA （q^2=0.705, r^2=0.918, r^2pred=0.635） and MB-QSAR/CoMSIA （q^2=0.558, r^2=0.940, r^2pred=0.527） models, and interpretation of the MB-QSAR models gave chemical intuitive information to guide the resistance-evading herbicide design.
文摘On the basis of study on the mechanism of action of sulfonylurea herbicides, nine sulfonylurea derivatives of iso-{xazolidinone} were designed and synthesized. The structures of these compounds were confirmed by means of IR, MS, NMR and elemental analysis. The results of preliminary active tests indicate that the compounds have some herbicidal activity. The structure-activity relationship was also studied.
文摘A novel type of aeetohydrexyacid synthase inhibitors, 4-methyl-3-isoxazolidinone derivatives of sulfonylurea, was designed and synthesized. The structures of these compounds were confirmed by using MS, NMR, and elemental analysis. The results of preliminary active tests indicate that the compounds show a herbicidal activity.
基金Supported by the National Natural Science Foundation of China(Nos.20802025 and 20082011)
文摘Thirteen novel phenoxysulfonylureas derivatives were synthesized, and their structures were confirmed by MS, NMR and element analysis. The herbicidal activity and structure-activity relationship were also investigated. The results of preliminary active tests indicate that the compounds show moderate herbicidal activity.
基金funded by the Key Research and Development Project in Shaanxi Province of China(grant number 2022NY-153)the National Natural Science Foundation of China(grant number 32172075)+1 种基金the Key Research and Development Projects of Yangling Seed Industry Innovation Center(YLzyyc2021-01)National science and technology innovation 2030-major agricultural bio-breeding major projects(2022ZD0400804).
文摘It remains a great challenge to control weeds in rapeseed fields in China.Breeding herbicide-resistant rapeseed varieties and using corresponding herbicide formulations has become the most economical and effective way to control weeds in rapeseed field.Characterization of more herbicide-resistant genetic resources will provide opportunities for breeders to develop rapeseed herbicide-resistant varieties with good agronomic performance.Previously,we obtained the tribenuron methyl(TBM)-resistant mutant K4 from ZS9(Brassica napus L.)through ethyl methyl sulfonate mutagenesis and TBM foliar-spray screening.In this study,the inheritance and molecular characterization of the mutant K4 are carried out.Genetic investigation indicated that the herbicide-resistance of the K4 was controlled by one dominant allele at a single nuclear gene locus.Molecular characterization showed that a single point substitution at position 535 from C to T in BnAHAS3(BnAHAS3^(535T)),which resulted in a mutation at point 179 in BnAHAS3.The K4 showed a certain degree of resistance to TBM,bensulfuron methyl,and monosulfon sodium,which were 50,30,and 5 times that of ZS9,respectively.AHAS enzyme assay,structural analysis of AHAS proteins,affinity detection between TBM and BnAHAS3 by surface plasmon resonance analysis,and the transgenic experiment in Arabidopsis using BnAHAS3^(535T) confirmed that BnAHAS3^(535T) endow the K4 with herbicides resistance.In addition,an allele-specific marker was developed to quickly distinguish the heterozygous and homozygous mutated alleles BnAHAS3^(535T).In conclusion,our research identified and characterized one novel mutative AHAS allele in B.napus and enriched genetic resource for developing herbicide-resistant rapeseed cultivars.
