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.

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).

A fluoroorotic acid (FOA)-resistant mutant of Arabidopsis thaliana was isolated by screening M2 populations of ethyl methane sulphonate (EMS)-mutagenized Columbia seed. FOA resistance was due to a nuclear recessive gene, for1-1, which locates to a 519 kb region in chromosome 5. Assays of key regulatory enzymes in de novo pyrimidine synthesis (uridine monophosphate synthase) and salvage biochemistry (thymidine kinase) confirmed that FOA resistance in for1-1/for1-1 plants was not due to altered enzymatic activities. Uptake studies using radiolabelled purines, pyrimidines, and [14C]FOA reveal that for1-1/for1-1 plants were specifically defective in the uptake of uracil or uracil-like bases. To confirm such specificity, genetic crosses show that FOR1 is a distinct locus from FUR1 which encodes a deoxyuridine nucleoside transporter. In addition, for1-1/for1-1 plants were restored to FOA sensitivity by transformation with the Escherichia coli uracil transporter gene uraA driven by the cauliflower mosaic virus (CaMV) 35S promoter. Molecular mapping studies reveal that FOR1 does not correspond to loci belonging to any of the six known nucleobase transporter families identified in the Arabidopsis genome. Moreover, FOR1 does not appear to regulate the transcript levels of either uracil transporter-encoding loci At2g03590 or At2g03530. The above results strongly suggest that the for1-1 mutant allele affects a transport mechanism that is specific for the uptake of uracil.