Characterization of a heavy-ion induced white flower mutant of allotetraploid Nicotiana tabacum

We characterized a white flower mutant of allotetraploid N. tabacum as a DFR-deficient mutant; one copy of DFR has a cultivar-specific frameshift, while the other was deleted by heavy-ion irradiation. In most plants, white-flowered mutants have some kind of deficiency or defect in their anthocyanin biosynthetic pathway. Nicotiana tabacum normally has pink petals, in which cyanidin is the main colored anthocyanidin. When a relevant gene in the cyanidin biosynthetic pathway is mutated, the petals show a white color. Previously, we generated white-flowered mutants of N. tabacum by heavy-ion irradiation, which is accepted as an effective mutagen. In this study, we determined which gene was responsible for the white-flowered phenotype of one of these mutants, cv. Xanthi white flower 1 (xwf1). Southern blot analysis using a DNA fragment of the dihydroflavonol 4-reductase (DFR) gene as a probe showed that the xwf1 mutant lacked signals that were present in wild-type genomic DNAs. Sequence analysis demonstrated that one copy of the DFR gene (NtDFR2) was absent from the genome of the xwf1 mutant. The other copy of the DFR gene (NtDFR1) contained a single-base deletion resulting in a frameshift mutation, which is a spontaneous mutation in cv. Xanthi. Introduction of NtDFR2 cDNA into the petal limbs of xwf1 by particle bombardment resulted in production of the pink-colored cells, whereas introduction of NtDFR1 cDNA did not. These results indicate that xwf1 is a DFR-deficient mutant. One copy of NtDFR1 harbors a spontaneous frameshift mutation, while the other copy of NtDFR2 was deleted by heavy-ion beam irradiation.