Biochemical and genetic characterization of 5-fluoro-2?-deoxyuridine-resistant mutants of Arabidopsis thaliana

Abstract

Abstract. Two independently isolated 5-fluoro-2'-deoxyuridine (FUdR)-resistant mutant lines of Arabidopsis thaliana (L.) Heynh., FUD-1 and FUD-2, were identified by screening M2 populations of ethylmethane-sulfonatemutagenized seeds. The resistance was found to be due to single, recessive, nuclear gene mutations. Genetic complementation tests indicated that these two mutations were in the same gene locus, which was designated furl, and mapped to linkage group four of Arabidopsis. Enzyme assays indicated that the mutants were not defective in thymidine-kinase activity. Greatly reduced concentrations of intracellular 3H were detected in furl/furl plants compared with the wild type after incubation of wild-type and resistant plants in a medium with [3H]FUdR, indicating that either reduced uptake of FUdR or enhanced efflux of FUdR metabolites was the major reason for FUdR-resistance. furl/furl plants also had significantly decreased uptake of thymidine and uridine compared with the wild type but no difference was found in the uptake of adenosine, guanosine, thymine, uracil or amino acids. It is suggested that the transport system affected in the furl/furl mutants is one specific to pyrimidine nucleosides.

The fluorouridine insensitive 1 (fur1) mutant is defective in equilibrative nucleoside transporter 3 (ENT3), and thus represents an important pyrimidine nucleoside uptake system in Arabidopsis thaliana

“…Toxic substrate analogues have proven useful in the identification and characterization of transport‐deficient mutants (Schmidt et al. , 2004; Wu and King, 1994). In the study reported here, we wished to identify whether any of the eight transport proteins within the Arabidopsis ENT family fulfils a dominant function in nucleoside uptake.…”

“…Remarkably, only mutants that were deficient in a functional AtENT3 gene product were able to grow on fluorouridine. In a similar screen the ethylmethane sulfonate (EMS)‐mutagenized fluorouridine insensitive 1 mutant 1 ( fur1‐1 ) was identified, which could also grow on fluorouridine (Wu and King, 1994). We hypothesized that this mutant might be defective in functional AtENT3 protein, and so decided to investigate the molecular nature of the mutation in fur1‐1 .…”

Nucleoside transport and associated metabolism*

“…In a screen for resistance against the toxic uridine analogue 5‐fluorouridine, the fur1‐1 mutant was identified. This mutant is severely restricted in uptake of pyrimidine nucleosides (Wu & King 1994). In a similar screen with Arabidopsis ENT:T‐DNA insertion mutants, it turned out that only the ENT3 mutant was resistant to 5‐fluorouridine (Traub et al.…”

Characterization of the de novo pyrimidine biosynthetic pathway in Arabidopsis thaliana

“…In addition, work in our lab has demonstrated that the pyrimidine free bases orotic acid, uracil and cytosine and each of their fluorinated analogs are all taken up into plant cells . The proteins responsible for this transport have not been identified, but are apparently different from the transporter of Wu and King ( 1994).…”

“…Wu et al, have identified 5-fluoro-2'-deoxyuridine FUdR resistant mutants of Arabidopsis that are deficient in uptake of FUdR from tissue culture media (Wu et al 1994). This protein also transports thymidine and may be similar to thymidine transporters from yeast and other 8 eukaryotes.…”