A Mutation Causing Imidazolinone Resistance Maps

A mutant of Arabidopsis thaliana, two hundred times more resistant to the imidazolinone herbicide imazapyr than wild-type plants, was isolated by direct selection of seedlings from a mutagenized population. Genetic analysis showed that resistance is due to a single dominant nuclear mutation that could not be separated by recombination from a mutation in the CSR1 gene encoding acetohydroxy acid synthase. Acetohydroxy acid synthase activity in extracts isolated from the mutant was 1000-fold more resistant to inhibition by imazapyr than that of the wild type. The resistant enzyme activity cosegregated with whole plant resistance. These data strongly suggest that the mutation is an allele of CSR1 encoding an imazapyr-resistant AHAS. AHAS2 (EC 4.1.3.18), also known as acetolactate synthase, the first enzymatic step common to the biosynthesis of the three branched-chain amino acids, is the site of action of the structurally diverse classes of herbicides: the sulfonylureas and imidazolinones (13, 18). Single dominant mutations resulting in resistance to sulfonylurea compounds in microorganisms (6, 23) and plants (2, 3, 7) have been shown to be due to single base pair substitutions in genes encoding the AHAS catalytic subunit (10, 14, 23). Although the molecular basis for imidazolinone-resistance has not yet been reported, several lines of evidence suggest that mutations causing such a phenotype will also lie within the AHAS gene. First, enzyme kinetics data have shown that the imidazolinones, like the sulfonylureas, inhibit AHAS activity by binding noncompetitively to a common site on the enzyme (17). Second, a number of variant cell lines ofDatura innoxia resistant to sulfonylurea herbicides showed crossresistance to imidazolinone herbicides (16). Finally, mutants of Chlamydomonas reinhardtii resistant to imidazolinones, chlorsulfuron or both map to the same genetic locus (22). We report here the isolation and characterization of an This work was supported in part by grants from the Natural Science and Engineering Research Council of Canada (No. OGP0036718) and a grant from the National Science Foundation (No. PCM8351595). 2 Abbreviations: AHAS. acetohydroxy acid synthase; imr, symbol for a gene encoding imazapyr resistance; chlorsulfuron, (2-chloro-N[(4-methoxy-6-methyl1 ,3,5-triazin-2)aminocarbonyl] benzenesulfonamide): imazapyr. isopropylamine salt of 2-(4-isopropyl-4-methyl5-oxo-2-imidazolin-2-vl) nicotinic acid. imazapyr-resistant mutant of Arabidopsis thaliana and show that resistance is due to a dominant mutation which maps to the AHAS locus. The recent cloning and characterization of the A. thaliana AHAS gene (10, 15) should facilitate molecular analysis of imidazolinone-resistance in this mutant. MATERIALS AND METHODS