Discovery of a Protein that Enhances Flower Pigmentation Using a Novel Mutation in Japanese Morning Glory

Many mutations exist in the Japanese morning glory. Some of these occurred in the late Edo Period (between 1600 and 1868), when jumping genes or transposons in the Tpn1 family were inserted into other genes. Approximately 800 transposons exist in the genome of the Japanese morning glory, and these transposons still possess the jumping activity. Therefore, frequently, a novel mutation occurs. By using one such novel mutation, a protein that enhances flower pigmentation was discovered.

In many flowers, coloration is by the pigment anthocyanin. Anthocyanin is a typical pigment in plants, and is a type of polyphenol called a flavonoid. The depth of flower color is determined by the amount of anthocyanin contained in the flower; the color is deeper when there is more anthocyanin. Previously, the factors and mechanisms that control the amount of anthocyanin were not sufficiently known.

This study used a new mutation that reduced the anthocyanin content, thus, making flower color pale. This mutation was not artificially produced but naturally appeared from a commercially available strain called Benichidori. The mutation did not directly appear from Benichidori, however, but rather appeared after three mutations had repeatedly occurred (Fig. 1）.

The first mutation changed Benichidori flowers from dark red to having red spots on a white background. This mutation was discovered by a layperson in Toyama Prefecture in 2000. We obtained the seeds of this strain and cultivated them. Subsequently, two mutations occurred. One was that the mutation that had occurred in Toyama Prefecture was reversed to have the original red flower. The other, which made flower color paler, was used in this study. This strain (AK28) with light pink flowers, which was obtained by a series of lucky accidents, was collected by the National BioResource Project (NBRP) Morning Glory as a new resource.

On a light pink flower of AK28, spots in original dark red of Benichidori are observed. When the seeds of AK28 are sown, a reverse mutant whose flower is in dark red, the same as that of Benichidori, can be obtained. This phenotype is often observed in a mutation in which a transposon participates. On the assumption that a transposon in the Tpn1 family of Japanese morning glory had mutated, we searched for the causative gene. In the search, the transposon display method was used, in which a DNA sequence that connects the transposon is amplified using polymerase chain reaction (PCR) (Fig. 2). The principle of this method is that since a transposon is inserted into a mutant, DNA fragments derived from the causative gene can be amplified in the mutant, but the DNA fragments cannot be amplified in wild-type plants or reverse mutants. The causative gene found in this method encoded a protein whose function had been unknown. We investigated the flower pigments and found that the protein facilitated the biosynthesis of not only anthocyanin but also a flavonoid that is a colorless flavonol. Therefore, we named this protein "enhancer of flavonoid production" (EFP). EFP can triple the efficiency of anthocyanin production.
We further investigated the EFP gene in 39 strains with light-colored flowers collected by the NBRP Morning Glory. Of these, 32 possessed the loss-of-function EFP gene. Similar to Japanese morning glory, EFP in Petunia sp. and Torenia fournieri was revealed to increase the efficiency of anthocyanin production.

At present, the mechanism by which EFP increases the efficiency of anthocyanin production is still unknown. We believe that the elucidation of this mechanism will lead us to understand the mechanism for increasing the efficiency of matter production in plants. Similar to the present study, we will be able to find a new mutation and its causative gene using transposons in Japanese morning glory. We have been screening new mutants using transposons and expect to obtain mutants with higher scientific and aesthetic values than ever.