Polyacylated Delphinidin Biosynthesis Catalysed by PhF3'5'H-PhBGLU12-PhSCPL2 Determines the Blue Pigmentation in Cineraria (Pericallis hybrida).

The polyacylation modification of anthocyanins constitutes a pivotal step for plants to develop persistent bright blue floral pigmentation, but the biosynthesis and regulatory mechanisms of polyacylated anthocyanin remain poorly understood. In the blue cineraria (Pericallis hybrida), an important ornamental species, a unique anthocyanin structure is characterised by 7- and 3'-polyacylated delphinidin derivatives that generate its distinctive colour. Here, we identified two modification genes PhBGLU12 and PhSCPL2 in the anthocyanin metabolic pathway, which determine the formation of polyacylated delphinidin derivatives in cineraria. RNAi-mediated silencing of these genes resulted in the transgenic lines exhibiting significantly reduced pigmentation, resulting in faded or pale purple colours in ray florets and leaves. Further investigation revealed that co-expression of PhF3'5'H and PhBGLU12 in the leaves of pink cineraria enabled the detection of multiple polyacylated modified delphinidin derivatives accumulating. The MYB activator PhMYB6 was shown to directly activate the transcription of PhF3'5'H and PhBGLU12, thereby promoting polyacylated anthocyanin biosynthesis. Electrophoretic mobility shift assay (EMSA) and dual-luciferase assay demonstrated that PhWRKY44 influences the anthocyanin metabolic pathway by directly activating the expression of PhMYB6. Notably, PhWRKY44 expression was reciprocally regulated by PhMYB6, establishing a mutually reinforcing positive feedback loop. Functional validation demonstrated that silencing PhWRKY44 reduced anthocyanin accumulation in cineraria. These findings reveal an intricate process and regulatory module for the biosynthesis of polyacylated anthocyanins and provide genetic resources for the molecular breeding of blue ornamental varieties.