Ectopic expression of the flavonoid 3′,5′-hydroxylase gene in Ipomoea nil induces the accumulation of delphinidin-based anthocyanins in the petals and inhibits their opening

BACKGROUND: Many commercially important ornamental species, such as rose, carnation, and chrysanthemum, lack blue flower varieties due to the absence of the flavonoid 3',5'-hydroxylase (F3'5'H) gene, which is essential for delphinidin-based anthocyanin (Dps) biosynthesis pathway. Heterologous F3'5'H genes have been introduced into these species to enable Dps accumulation. However, incompatibility between heterologous F3'5'H and the host metabolic background has been reported, and certain gene-host combinations fail to efficiently produce Dps. This study aimed to evaluate whether Ipomoea nil, a model plant for anthocyanin biosynthesis studies, can serve as a platform for functional analysis of F3'5'H. Although I. nil lacks an endogenous F3'5'H gene, it naturally accumulates cyanidin-based blue pigments in its petals. Introducing Dps biosynthesis into this species provides an opportunity to investigate how Dps accumulation alters floral pigmentation. In this study, I. nil was engineered to produce Dps through ectopic expression of F3'5'H genes from four different plant species. RESULT: This study represents the first successful engineering of Dps biosynthesis in the genus Ipomoea. Transgenic I. nil lines expressing F3'5'H from Eustoma grandiflorum, Campanula medium, and Viola × wittrockiana showed high Dps contents, whereas the gene from Gentiana triflora resulted in low content. High Dps content was strongly correlated with a notable physiological phenotype characterized by inhibited petal cell expansion during flowering, thereby preventing the flowers from being fully open. RNA-Seq analysis revealed significant changes in the expression of genes involved in the salicylic acid signaling pathway, anthocyanin transport, oxidative stress response, and senescence-associated processes in petals with inhibited opening. CONCLUSION: This study suggests that Ipomoea nil cv. Violet can serve as a useful system for functional analysis of F3'5'H. In addition, high-level expression of the F3'5'H gene and the resulting high concentration of Dps were correlated with inhibited petal opening in this cultivar. RNA-seq analysis enabled the identification of genes whose expression changes were strongly correlated with the inhibition of petal opening. These findings provide important insights into the mechanisms underlying the inhibition of petal opening caused by altered pigment accumulation.