Abstract
Anthocyanins are some of the most widely occurring secondary metabolites in plants, responsible for the orange, red, purple, and blue colors of flowers and fruits and red colors of autumn leaves. These pigments accumulate in vacuoles, and their color is influenced by chemical decorations, vacuolar pH, the presence of copigments, and metal ions. Anthocyanins are usually soluble in the vacuole, but in some plants, they accumulate as discrete sub-vacuolar structures. Studies have distinguished intensely colored intra-vacuolar bodies observed in the cells of highly colored tissues, termed anthocyanic vacuolar inclusions (AVIs), from more globular, membrane-bound anthocyanoplasts. We describe a system in tobacco that adds additional decorations to the basic anthocyanin, cyanidin 3-O-rutinoside, normally formed by this species. Using this system, we have been able to establish which decorations underpin the formation of AVIs, the conditions promoting AVI formation, and, consequently, the mechanism by which they form.