A Site-directed Mutagenesis Interrogation of the Carboxy-terminal End of Arabidopsis Thaliana Threonine Dehydratase/deaminase Reveals a Synergistic Interaction Between Two Effector-binding Sites and Contributes to the Development of a Novel...

We fused four mutant omr1 alleles, encoding feedback-insensitive forms of Arabidopsis thaliana biosynthetic threonine dehydratase/deaminase (TD), to the CaMV 35S promoter and transformed these constructs into A. thaliana Columbia wild type plants. The mutant TD forms consisted of our previously isolated double mutant, omr1-1 , and three new site-directed mutants, omr1-5 , omr1-7 , and omr1-8 with single point mutations. We employed site-directed mutagenesis to assay the effects of amino acid substitutions in separate regulatory regions within the carboxy-terminal (C-term) allosteric end. TD assays and growth resistance to the isoleucine (Ile) toxic analog -O-methylthreonine (OMT) confirmed the desensitization to feedback inhibition and the viability of these mutant omr1 alleles as selectable markers, respectively. Two of the site-directed mutants, omr1-5 and omr1-7 , appeared to influence one of the two separate Ile-binding sites and had a notable 13-fold and 15-fold increase in free Ile, respectively. The omr1-8 appeared to influence the other Ile-binding site and resulted in a 2-fold increase in free Ile. The transgenic omr1-1 double mutant affecting both Ile-binding sites, however, displayed a 106-fold increase in free Ile revealing a profound synergistic interplay between these separate Ile-binding sites. While all of the four omr1 alleles conferred resistance to elevated concentrations of OMT, the progeny of omr1-1 initial transformants exhibited a bushy phenotype at the rosette stage. On the other hand, progeny of transformants omr1-5 , omr1-7 , and omr1-8 had a normal phenotype, undistinguishable from wild type. Therefore, alleles omr1-5 , omr1-7 , and omr1-8 , proved to be ideal as environmentally-friendly, dominant, selectable markers for plant transformation.