L-O-Methylthreonine-Resistant Mutant of Arabidopsis Defective in Isoleucine Feedback Regulation

Abstract

Threonine dehydratase/deaminase (TD), the first enzyme in the isoleucine biosynthetic pathway, is feedback inhibited by isoleucine. By screening M, populations of ethyl methane sulfonatetreated Arabidopsis thaliana Columbia wild-type seeds, we isolated five independent mutants that were resistant to i-O-methylthreonine, an isoleucine structural analog. Crowth in the mutants was 50-to 600-fold more resistant to i-Omethylthreonine than in the wild type. l h e resistance was dueto a single, dominant nuclear gene that was denoted omrl and was mapped to chromosome 3 in CM11 b, the mutant line exhibiting the highest level of resistance. Biochemical characteristics (specific activities, K, , V,,,, and pH optimum) of TD in extracts from the wild type and C M l l b were similar except for the inhibition constant of isoleucine, which was 50-fold higher in C M l l b than in the wild type. Levels of free isoleucine were 20-fold higher in extracts from C M l l b than in extracts from wild type. Therefore, isoleucine feedback insensitivity in G M l l b is due to a mutant form of the TD enzyme encoded by omrl. l h e mutant allele Omrl of the line C M l l b could provide a new selectable marker for plant genetic transformation.TD, the first enzyme in the biosynthetic pathway of Ile, catalyzes the formation of 2-oxobutyrate from Thr in a two-step reaction. The first step is a dehydration of Thr followed by rehydration and liberation of ammonia (Bryan, 1990). AI1 the reactions downstream from TD are catalyzed by enzymes that are shared by the two main branches of the biosynthetic pathway that lead to the production of the branched-chain amino acids Ile, Leu, and Val. In bacteria and yeast, resistance to the Ile structural analog thiaIle was associated with a loss of feedback sensitivity of TD to Ile (Umbarger, 1971;Kielland-Brandt et al., 1979). In Rosa cells, resistance to the Ile structural analog OMT was also associated with a TD that had reduced sensitivity to feedback inhibition by Ile (Strauss et al., 1985). Being in tissue culture and having high ploidy level, it was not possible to determine the genetic basis of feedback insensitivity to Ile in the Rasa variant, the only plant mutant with an Ile-insensitive TD.We are isolating and characterizing mutants of the branched-chain amino acid biosynthetic pathway at the ' This work was supported by research grants from the Natural MATERIALS A N D METHODS Seed MutagenesisAbout 100,000 seeds of Arabidopsis tkaliana (L.) Heynh Columbia wild type were allowed to imbibe (v/v) in an aqueous solution of 0.2% EMS for 18 h at room temperature. The mutagenized seed (M,) was washed severa1 times with distilled water, planted in subpopulations, and allowed to self-fertilize to produce M, seed. The M, seed were harvested from each subpopulation separately, surface sterilized, and planted on agar-solidified medium containing 0.5 mM OMT. In this medium wild-type seedlings were completely bleached and died immediately after germination, whereas resistant mutants were able to germinate and prod...

Evaluation of the Escherichia coli threonine deaminase gene as a selectable marker for plant transformation

“…We have demonstrated that expression of E. coli TD can impart tolerance towards OMT and that the wild-type ilvA can be used to identify transgenic tobacco lines, albeit relatively inefficiently, thus, confirming the hypothesis put forth by Mourad and King (1995). However, further optimizations will be required to allow this selection system to be of any potential value in plant genetic engineering studies.…”

“…Moreover, there was a 50-fold increase in the Ile feedback insensitivity of TD activity in the OMT-tolerant lines compared to that in wild-type plants (Mourad and King 1995). These results lead the authors to speculate that the omr1 allele of A. thaliana may serve as an effective plant selectable marker gene, coupled with OMT, for use in plant genetic engineering studies (Mourad and King 1995). Slater et al (1999) introduced into A. thaliana and Brassica napus either the E. coli wild-type TD gene, ilvA, or a feedback-insensitive mutant ilvA-466.…”

“…One such example is O-methylthreonine (OMT; Rabinovitz et al 1955). As part of a large mutant screen of Arabidopsis lines that were altered in biosynthesis of branched-chain amino acids, Mourad and King (1995) characterized a set of mutants resistant to OMT. The ability of these mutant lines to develop on OMT was attributed to a single nuclear gene, omr1, a mutant allele of TD, which mapped to chromosome 3.…”

“…The OMT-tolerant Arabidopsis lines possessed substantially enhanced intracellular levels of Ile compared to wild-type plants. Moreover, there was a 50-fold increase in the Ile feedback insensitivity of TD activity in the OMT-tolerant lines compared to that in wild-type plants (Mourad and King 1995). These results lead the authors to speculate that the omr1 allele of A. thaliana may serve as an effective plant selectable marker gene, coupled with OMT, for use in plant genetic engineering studies (Mourad and King 1995).…”

“…This study was undertaken to ascertain if the E. coli wild-type ilvA or the ilvA-466 derivative could be utilized as a selectable marker gene in plant genetic engineering studies when coupled with OMT as the selection agent, thus permitting a direct testing of the hypothesis put forth by Mourad and King (1995).…”