Fe 3+ ‐Mediated Blue Pigment Formation via Collaborative Regulation of Iron Homeostasis by VIT1 and Ferritin : Insights for Blue Flower Breeding

The bright blue colouration of Centaurea cyanus (cornflower) is attributed to a supramolecular pigment formed via metal ion chelation with accumulated cyanidin (Cy) and co-pigments, yet the underlying genetic regulatory mechanisms remain incompletely understood. Elucidating this mechanism will facilitate the molecular breeding of cyanidin-based blue flowers, thereby enhancing the ornamental value of flowering plants. Initially, in vitro blue pigment reconstruction experiments identified that Fe³⁺ is the primary metal ion enabling the blue transformation of Cy, and the molar ratio of Cy to Fe³⁺ was found to be critical. Genomic analysis identified 11 VIT/VTL genes and four CcFers genes in cornflower. Expression profiling revealed an inverse expression relationship between CcVIT1a and CcFers across various tissues. Notably, CcVIT1a specifically and highly expressed in blue-coloured ray florets. Subcellular localization and yeast mutant complementation assay showed CcVIT1a in tonoplast, whereas the CcFers reside in plastids. This spatial separation suggests their coordinated role in regulating intracellular iron homeostasis. Virus-induced gene silencing of CcVIT1a caused a colour shift from blue to violet, indicating its critical role in blue colouration of cornflower. Concurrently, the expression of the CcFer1a was significantly upregulated, while the other three CcFers were downregulated, further supporting the idea that CcVIT1a and the CcFers collaboratively regulate intracellular iron homeostasis. Transient overexpression of CcVIT1a in chrysanthemum CB (Cy-determined blue flowers) sample 'Dante purple' induced a colour transition from red-purple to violet, accompanied by the downregulation of both CmVIT and CmFer1. These findings imply a similar synergistic mechanism governing iron homeostasis in chrysanthemum. This study elucidates the cooperative roles of VIT1 and Fer in regulating intracellular iron partitioning. We demonstrate that upregulation of CcVIT1a enhances iron sequestration into the vacuole, thereby promoting the cyanidin-based blue colouration. Collectively, our findings provide novel molecular insights and a potential strategy for breeding blue flowers through the manipulation of cyanidin and iron metabolism.