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PMID: 15604669 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

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.

Plant molecular biology ·Vol. 55 ·No. 1 ·2004-05-00 ·Pages 121-34

Garcia EL, Mourad GS

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.

MeSH Terms
Alleles Allosteric Site/genetics Amino Acid Sequence Arabidopsis/drug effects,enzymology,genetics Arabidopsis Proteins/chemistry,genetics,metabolism Binding Sites/genetics Drug Resistance Genetic Markers/genetics Isoleucine/analogs & derivatives,metabolism,pharmacology Models, Molecular Molecular Sequence Data Mutagenesis, Site-Directed Mutation Plants, Genetically Modified Protein Structure, Secondary Sequence Homology, Amino Acid Threonine Dehydratase/chemistry,genetics,metabolism Transformation, Genetic
Chemicals
Arabidopsis Proteins Genetic Markers Isoleucine Threonine Dehydratase
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Garcia Eric L
Department of Biology, Indiana-Purdue University, 2101 East Coliseum Blvd, IN, USA.
Mourad George S
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Article Info
Journal
Plant molecular biology
Abbr.
Plant Mol Biol
ISSN
0167-4412
DOI
10.1007/s11103-004-0500-z
Published
2004-05-00
Pages
121-34
Language
English
Region
Netherlands
NLM ID
9106343
Subset
IM
External Links
PubMed source DOI: 10.1007/s11103-004-0500-z View journal details
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