New insight into the structures and formation of anthocyanic vacuolar inclusions in flower petals

BACKGROUND: Although the biosynthetic pathways for anthocyanins and their regulation have been well studied, the mechanism of anthocyanin accumulation in the cell is still poorly understood. Different models have been proposed to explain the transport of anthocyanins from biosynthetic sites to the central vacuole, but cellular and subcellular information is still lacking for reconciliation of different lines of evidence in various anthocyanin sequestration studies. Here, we used light and electron microscopy to investigate the structures and the formation of anthocyanic vacuolar inclusions (AVIs) in lisianthus (Eustoma grandiflorum) petals. RESULTS: AVIs in the epidermal cells of different regions of the petal were investigated. Three different forms of AVIs were observed: vesicle-like, rod-like and irregular shaped. In all cases, EM examinations showed no membrane encompassing the AVI. Instead, the AVI itself consisted of membranous and thread structures throughout. Light and EM microscopy analyses demonstrated that anthocyanins accumulated as vesicle-like bodies in the cytoplasm, which themselves were contained in prevacuolar compartments (PVCs). The vesicle-like bodies seemed to be transported into the central vacuole through the merging of the PVCs and the central vacuole in the epidermal cells. These anthocyanin-containing vesicle-like bodies were subsequently ruptured to form threads in the vacuole. The ultimate irregular AVIs in the cells possessed a very condensed inner and relatively loose outer structure. CONCLUSION: Our results strongly suggest the existence of mass transport for anthocyanins from biosynthetic sites in the cytoplasm to the central vacuole. Anthocyanin-containing PVCs are important intracellular vesicles during the anthocyanin sequestration to the central vacuole and these specific PVCs are likely derived directly from endoplasmic reticulum (ER) in a similar manner to the transport vesicles of vacuolar storage proteins. The membrane-like and thread structures of AVIs point to the involvement of intravacuolar membranes and/or anthocyanin intermolecular association in the central vacuole.