IDENTIFICATION OF 2 TUNGSTATE-SENSITIVE MOLYBDENUM COFACTOR MUTANTS, CHL2 AND CHL7, OF ARABIDOPSIS-THALIANA

The characterization of mutants that are resistant to the herbicide chlorate has greatly increased our understanding of the structure and function of the genes required for the assimilation of nitrate. Hundreds of chlorate-resistant mutants have been identified in plants, and almost all have been found to be defective in nitrate reduction due to mutations in either nitrate reductase (NR) structural genes or genes required for the synthesis of the NR cofactor molybdenum-pterin (MoCo). The chlorate-resistant mutant of Arabidopsis thaliana, chl2, is also impaired in nitrate reduction, but the defect responsible for this phenotype has yet to be explained. chl2 plants have low levels of NR activity, yet the map position of the chl2 mutation is clearly distinct from that of the two NR structural genes that have been identified in Arabidopsis. In addition, chl2 plants are not thought to be defective in MoCo, as they have near wild-type levels of xanthine dehydrogenase activity, which has been used as a measure of MoCo in other organisms. These results suggest that chl2 may be a NR regulatory mutant. We have examined chl2 plants and have found that they have as much NR (NIA2) mRNA as wild type a variable but often reduced level of NR protein, and one-eighth the NR activity of wild-type plants. It is difficult to explain these results by a simple regulatory model; therefore, we reexamined the MoCo levels in chl2 plants using a sensitive, specific assay for MoCo: complementation of Neurospora MoCo mutant extracts. We found that chl2 has low levels of MoCo -about one-eighth the wild-type level and less than the level in another Arabidopsis MoCo mutant chl6 (B73). To confirm this result we developed a new diagnostic assay for MoCo mutants, growth inhibition by tungstate. Both chl2 and chl6 are sensitive to tungstate at concentrations that have no effect on wild-type plants. The tungstate sensitivity as well as the chlorate resistance, low NR activity and low MoCo levels all cosegregate, indicating that all are due to a single mutation that maps to the chl2 locus, 10 centimorgans from erecta on chromosome 2. We also report on the isolation of a new chlorate-resistant mutant of Arabidopsis, chl7, which is a MoCo mutant with the same phenotypes as chl2 and chl6.