IDENTIFICATION AND CHARACTERIZATION OF A CHLORATE-RESISTANT MUTANT OFARABIDOPSIS-THALIANA WITH MUTATIONS IN BOTH NITRATE REDUCTASE STRUCTURAL GENES NIA1 AND NIA2

Mutant plants defective in the assimilation of nitrate can be selected by their resistance to the herbicide chlorate. In Arabidopsis thalian a, mutations at any one of nine distinct loci confer chlorate resistan ce. Only one of the CHL genes, CHL3, has been shown genetically to be a nitrate reductase (NR) structural gene (NIA2) even though two NR gen es (NIA1 and NIA2) have been cloned from the Arabidopsis genome. Plant s in which the NIA2 gene has been deleted retain only 10% of the wild- type shoot NR activity and grow normally with nitrate as the sole nitr ogen source. Using mutagenized seeds from the NIA2 deletion mutant and a modified chlorate selection protocol, we have identified the first mutation in the NIA1 NR structural gene. nia1, nia2 double mutants hav e only 0.5% of wild-type shoot NR activity and display very poor growt h on media with nitrate as the only form of nitrogen. The nia1-1 mutat ion is a single nucleotide substitution that converts an alanine to a threonine in a highly conserved region of the molybdenum cofactor-bind ing domain of the NR protein. These results show that the NIA1 gene en codes a functional NR protein that contributes to the assimilation of nitrate in Arabidopsis.