A bHLH protein partially controls proanthocyanidin and phytomelanin pigmentation in the seed coats of morning glory Ipomoea tricolor

In the morning glory Ipomoea tricolor, the ivory-seed (IVS) protein, which bears a basic helix-loop-helix (bHLH) domain homologous to Arabidopsis transparent testa 8 (TT8) and petunia anthocyanin 1 (AN1) that activate various epidermal traits, was reported to regulate anthocyanin pigmentation in flowers and the dark-brown pigmentation in seeds. In this study, additional roles of the Ipomoea tricolor IVS (ItIVS) gene were elucidated and seed pigments of I. tricolor were also analyzed. Microscopic observations showed that trichomes on seed surface were reduced in seeds of the ivs mutant Blue Star (BS), closely resembling ivs mutant seeds of I. purpurea. Different from petunia AN1, the ItIVS gene appears not to be related to expression of the I. tricolor Na (+)/H (+) exchanger (ItNHX1) gene that increases petal pH during flower opening, because the expression profile of ItNHX1 in BS was virtually indistinguishable from that of the wild type Heavenly Blue (HB). The dark-brown pigments of HB were mainly composed of proanthocyanidins and phytomelanins. As phytomelanin content in BS seeds was about 22% of wild type level, and vanillin and rho-dimethylaminocinnamaldehyde (DMACA) treatments for its immature seeds also showed weak positive reactions, it seems that the ItIVS gene partially activates the accumulation of both pigments. Some biosynthetic steps in anthocyanin and proanthocyanidin biosynthesis are thought to be common in Arabidopsis. However, the chalcone synthase D (CHS-D) and the dihydroflavonol 4-reductase B (DFR-B) genes in Ipomoea, which are essential for anthocyanin biosynthesis, were not transcribed in seed coats of HB, and a dfr-b mutant Pearly Gates (PG) still produces proanthocyanidins suggesting that ItIVS may activate different sets of structural genes for anthocyanin biosynthesis in flowers and proanthocyanidin production in seeds. Our results suggest that proanthocyanidin biosynthesis in morning glory is not accomplished by a unique system like it is in Arabidopsis, and more complicated processes are involved.