Effect of mutations on acetohydroxyacid synthase (AHAS) function in Cyperus difformis L.

Abstract

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 I50 (the half maximal inhibitory concentration) of wild type AHAS was 0.04 μmol L–1 and Asp376Glu, Pro197Leu, Pro197Arg, Pro197Ser and Trp574Leu mutations were 3.98, 11.50, 40.38, 38.19 and 311.43 μmol 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 1.14 times 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.