Functional analysis of the ScAG and ScAGL11 MADS-box transcription factors for anthocyanin biosynthesis and bicolour pattern formation in ray florets.

Cineraria () is an ornamental plant with pure colour and bicolour cultivars, widely used for landscaping. Anthocyanin biosynthesis influences coloration patterns in cineraria. However, how anthocyanins accumulate and distribute in cineraria is poorly understood. This study investigated the molecular mechanisms underlying anthocyanin biosynthesis and bicolour formation in cineraria using pure colour and bicolour cultivars. Transcriptome and gene expression analysis showed that five genes, , , , , and , were inhibited in the white cultivar and colourless regions of bicolour cultivars. In contrast, two MADS-box genes, and , showed significantly higher expression in the colourless regions of bicolour cultivars. ScAG and ScAGL11 were localized in the nucleus and co-expressed with the bicolour trait. Further functional analysis verified that inhibits anthocyanin accumulation in tobacco (). However, virus-induced gene silencing (VIGS) experiments showed that silencing of and increases anthocyanin content in cineraria leaves. Similar results were observed when and were silenced in the cineraria capitulum, accompanied by the smaller size of the colourless region, specifically in the /-silenced plants. The expression of , , and increased in silenced cineraria leaves and capitulum. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation experiments demonstrated that ScAG interacts with ScAGL11. Moreover, directly inhibited the transcription of while inhibited expression by binding to their promoters separately. The findings reported herein indicate that and negatively regulate anthocyanin biosynthesis in cineraria ray florets, and their differential expression in ray florets influences the bicolour pattern appearance.