Insight into Color Variation in Hydrangea

Abstract

The mechanism of blue flower color development from anthocyanins is a very interesting subject. In flower petals, colored cells are usually located in the epidermis and/or the second layer of the tissue where anthocyanins are dissolved in vacuoles. Therefore, to understand the mechanism of coloration, manipulation of the colored vacuoles is essential. Hydrangea (Hydrangea macrophylla) is a very unique flower; what we call the flower is not a true flower petal but actually a sepal. The sepal color is well known for readily changing hues. In the early years of the 20^th century it was already known that the more acidic the soil and the higher the Al³⁺ content in sepals, the bluer the septal color became. To date, one anthocyanin component, delphinidin 3-glucoside, and three co-pigment components, 3-O-caffeoyl, 5-O-caffeoyl, and 5-O-p-coumaroylquinic acids, have been identified and it has been reported that all the colors from red through purple to blue developed from these same components. We were interested in this phenomenon and have tried to explain the chemical mechanisms. In this report I will review our recent studies on blue color development and color variation of hydrangeas.
We prepared protoplasts by treatment of blue and red sepals with cellulase and pectinase, and collected colored protoplasts from the reaction mixture. With a combination of microspectrophotometry and direct vacuolar pH (pHv) measurement by microelectrode, we verified that the bluer the cell color, the higher the pHv. Quantitative analysis of organic and inorganic components in colored cells clarified that significant differences were observed in the content of 5-O-acylquinic acids and Al³⁺ between blue and red cells. According to the data obtained, we mixed the components at the measured pHv of blue and red cells, respectively, and could reproduce the same color as those cells.
To clarify the essential structure for the blue complex, we designed and synthesized various unnatural co-pigments and carried out experiments of blue complex using those co-pigments. We determined that the 1-COOH, 1-OH and 5-ester in 5-O-acylquinic acid, and the catechol structure at the B-ring of anthocyanidin chromophore are essential to forming a complex with Al³⁺ to give a stable blue color. Combined with Vis, CD, NMR and MS measurements, the structure of the hydrangea blue-complex was clarified. The blue-complex exists under equilibrium between chelating and non-chelating structures having a hydrophobic interaction between the anthocyanidin chromophore and the cinnamic acyl part of the quinic acid ester.