title: 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 selectable marker.
abstract: 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.
authors.affiliation: Department of Biology, Indiana-Purdue University, 2101 East Coliseum Blvd, IN, USA.
authors.forename: Eric L
authors.initials: EL
authors.lastname: Garcia
authors.forename: George S
authors.initials: GS
authors.lastname: Mourad
mesh_terms: D000483:Alleles; D000495:Allosteric Site / Q000235:genetics; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D004351:Drug Resistance; D005819:Genetic Markers / Q000235:genetics; D007532:Isoleucine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D030821:Plants, Genetically Modified; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013913:Threonine Dehydratase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: D029681:Arabidopsis Proteins; D005819:Genetic Markers; D007532:Isoleucine; D013913:Threonine Dehydratase