a b s t r a c tIn plants, nucleobase biochemistry is highly compartmented relying upon a well-regulated and selective membrane transport system. In Arabidopsis two proteins, AtAzg1 and AtAzg2, show substantial amino acid sequence similarity to the adenine-guanine-hypoxanthine transporter AzgA of Aspergillus nidulans. Analysis of single and double mutant lines harboring T-DNA insertion alleles AtAzg1-1 and AtAzg2-1 reveal a marked resistance to growth in the presence of 8-azaadenine and 8-azaguanine but not to other toxic nucleobase analogues. Conversely, yeast strains expressing AtAzg1 and AtAzg2 gain heightened sensitivity to growth on 8-azaadenine and 8-azaguanine. Radio-labeled purine uptake experiments in yeast and in planta confirm the function of AtAzg1 and AtAzg2 as plant adenine-guanine transporters.
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...
Acetohydroxy acid synthase (AHAS) is an essential enzyme for many organisms as it catalyzes the first step in the biosynthesis of the branched-chain amino acids valine, isoleucine, and leucine. The enzyme is under allosteric control by these amino acids. It is also inhibited by several classes of herbicides, such as the sulfonylureas, imidazolinones and triazolopyrimidines, that are believed to bind to a relic quinone-binding site. In this study, a mutant allele of AHAS3 responsible for sulfonylurea resistance in a Brassica napus cell line was isolated. Sequence analyses predicted a single amino acid change (557 Trp-->Leu) within a conserved region of AHAS. Expression in transgenic plants conferred strong resistance to the three classes of herbicides, revealing a single site essential for the binding of all the herbicide classes. The mutation did not appear to affect feedback inhibition by the branched-chain amino acids in plants.
a b s t r a c tLocus At5g03555 encodes a nucleobase cation symporter 1 (AtNCS1) in the Arabidopsis genome. Arabidopsis insertion mutants, AtNcs1-1 and AtNcs1-3, were used for in planta toxic nucleobase analog growth studies and radio-labeled nucleobase uptake assays to characterize solute transport specificities. These results correlate with similar growth and uptake studies of AtNCS1 expressed in Saccharomyces cerevisiae. Both in planta and heterologous expression studies in yeast revealed a unique solute transport profile for AtNCS1 in moving adenine, guanine and uracil. This is in stark contrast to the canonical transport profiles determined for the well-characterized S. cerevisiae NCS1 proteins FUR4 (uracil transport) or FCY2 (adenine, guanine, and cytosine transport).
We have isolated a triazolopyrimidine-resistant mutant csrl-2, of Arabidopsis thaliana (L.) Heynh. Here, we compare csrl-2 with the previously isolated mutants csrl and csr1-1, and with wild-type Arabidopsis for responses to members of four classes of herbicides, namely, sulfonylureas, triazolopyrimidines, imidazolinones, and pyrimidyl-oxy-benzoates. Two separable herbicide binding sites have been identified previously on the protein of acetolactate synthase (ALS). Here, the mutation giving rise to csrl, originating in a coding sequence towards the 5' end of the ALS gene, and that in csrl-2, affected the inhibitory action on growth and ALS activity of sulfonylurea and triazolopyrimidine herbicides but not that of the imidazolinones or pyrimidyl-oxybenzoates. The other mutation, in csrl-1, originating in a coding sequence towards the 3' end of the ALS gene, affected the inhibitory action of imidazolinones and pyrimidyl-oxy-benzoates but not that of the sulfonylureas or triazolopyrimidines. Additional, stimulatory effects of some of these herbicides on growth of seedlings was unrelated to their effect on their primary target, ALS. The conclusion from these observations is that one of the two previously identified herbicide-binding sites may bind sulfonylureas and triazolopyrimidines while the other may bind imidazolinones and pyrimidyl-oxy-benzoates within a herbicide-binding domain on the ALS enzyme. Such a comparative study using near-isogenic mutants from the same species allows not only the further definition of the domain of herbicide binding on ALS but also could aid investigation of the relationship between herbicide-, substrate-, and allosteric-binding sites on this enzyme.This research was supported by an Operating Grant from the Natural Sciences and Engineering Research Council of Canada to J.K.
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