Isolation and Characterization of a Mutant of Arabidopsis thaliana Resistant to -Methyltryptophan

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Abstract
Mutants of Arabidopsis thaliana have been selected for resistance to growth inhibition at the seedling stage by α-methyltryptophan (aMT). One mutant, amt-1 has been characterized in detail. The appearance and growth rate of the mutant in the absence of the inhibitor are similar to wild type, both as plants and callus. However, mutant plant growth is unaffected by 25 micromolar aMT and mutant callus growth by 50 micromolar aMT, concentrations that completely inhibit the growth of wild-type plants and callus, respectively. Tryptophan levels in mutant and wild-type plants are 24.3 ± 2.7 and 4.7 ± 1.2 micrograms per gram fresh weight, respectively, and in the corresponding callus 64.0 ± 2.6 and 31.8 ± 8.4 micrograms per gram fresh weight, respectively. Anthranilate synthase (AS) activity levels in crude extracts from whole plants are 3.09 ± 0.54 nanomoles per milligram protein per hour in amt-1 and 1.32 ± 0.21 nanomoles per milligram protein per hour in wild-type plants. In crude extracts from callus, anthranilate synthase levels are 11.54 ± 2.05 nanomoles per milligram protein per hour and 7.74 ± 1.58 in amt-1 and wild type, respectively. Enzyme extracts are inhibited by l-tryptophan; the concentrations required for 50% inhibition (I50) are 3.9 and 1.9 micromolar for amt-1 and for wild type, respectively. The mutation segregates as a single nuclear allele and shows incomplete dominance. The concomitant increases in both AS activity and its I50 for tryptophan suggest that the mutation amt-1 either resides in one of the AS structural genes or causes increased expression of an AS isoform with an I50 greater than the average for the entire extract.