A Torenia (Torenia fournieri Lind. ex Fourn.) Novel Mutant 'Flecked' Produces Variegated Flowers by Insertion of a DNA Transposon into an R2R3-MYB Gene

A novel torenia (Torenia fournieri Lind. ex Fourn.) mutant 'flecked', which bears variegated flowers, was obtained from ethyl methanesulfonate-treated M-2 plants. The lower lip of this mutant has small violet spots with a pale violet background, while that of the normal type is solid violet. The mutant trait frequently reverted to a semicircular violet sector or solid violet lower lip. Germinal revertant plants with a solid violet lower lip also frequently occurred in S-1 plants derived from self-pollinated mutant flowers. In the lower lip of the mutant type, anthocyanin concentration was much lower than in the normal type. This was attributed to decreased expression of the genes encoding anthocyanin biosynthesis enzymes, i.e. torenia chalcone synthase (TfCHS), flavanone 3-hydroxylase (TfF3H), dihydroflavonol 4-reductase (TfDFR), anthocyanidin synthase (TfANS), and UDP-glucose 3-O-flavonoid glucosyltransferase (TfUFGT). In the lower lip of the mutant, expression of a gene encoding R2R3-MYB transcription factor (TfMYB1, Torenia fournieri MYB1) was much lower than in the normal type and the revertants; this was caused by insertion of a Enhancer/Suppressor-Mutator (En/Spm)-like transposon (Ttf1, Transposon Torenia fournieri 1) in the 2nd intron of TfMYB1. Furthermore, it was found that the reversion of anthocyanin accumulation in the lower lips correlated to excision of Ttf1 from the TfMYB1. Overexpression of TfMYB1 in torenia caused anthocyanins to accumulate in the purple callus as a result of enhanced expression of the five structural genes mentioned above, demonstrating that TfMYB1 regulates these genes. Therefore, we concluded that a homozygous allele of Ttf1-inserted TfMYB1 caused the mutant phenotype. Ttf1 is a non-autonomous element because Ttf1 does not have the DNA sequence encoding transposase. Based on these results, potential uses of the flecked mutant for torenia breeding and transposon tagging are discussed.