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 (Dp) biosynthesis. Heterologous F3′5′H genes have been introduced into these species to enable Dp accumulation. This study aimed to investigate the potential of Ipomoea nil , a diploid species with a short life cycle and high transformation efficiency, as a model plant for anthocyanin biosynthesis study. As I. nil naturally lacks a functional F3′5′H gene, this study aimed to engineer Dp production via ectopic expression of F3′5′H genes from four different plant species. Result This study represents the first successful engineering of Dp-based anthocyanins (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 content, whereas the gene from Gentiana triflora resulted in low content. High levels of Dps accumulation were strongly correlated with a notable physiological phenotype wherein petal cell expansion was inhibited during flowering, preventing the flowers from being fully open. To investigate the underlying mechanism of this inhibition, RNA-Seq analysis was conducted on petal tissues to reveal the significant transcriptomic changes associated with the inhibition. Gene Ontology enrichment analysis revealed differential changes in genes involved in salicylic acid signaling pathway, anthocyanin-related transport, oxidative stress response, and senescence-associated processes. These findings suggest that high-level accumulation of metabolites associated with F3′5′H expression may trigger complex stress or defense responses, which could interfere with normal petal 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 content of Dps were correlated with inhibited petal opening in this cultivar. RNA-Seq analysis revealed that inhibited petals showed elevated expression of stress-responsive genes, anthocyanin-related transporters, and oxidative stress-associated and senescence-related genes. These findings provide important insights into the mechanisms underlying the inhibition of petal opening caused by altered pigment accumulation.