Single-cell analysis clarifies mosaic color development in purple hydrangea sepal.

Hydrangea sepals exhibit a wide range of colors, from red, through purple, to blue; the purple color is a color mosaic. However, all of these colors are derived from the same components: simple anthocyanins, 3-O-glycosyldelphinidins, three co-pigment components, acylquinic acids and aluminum ions (Al3+ ). We show the color mosaic is a result of graded differences in intravacuolar factors. In order to clarify the mechanisms of mosaic color, we performed single-cell analyses of vacuolar pH, and anthocyanin, co-pigment and Al3+ content. From the sepals, a protoplast mixture of various colors was obtained. The cell color was evaluated by microspectrophotometry and vacuolar pH then was recorded by using a pH microelectrode. The organic and Al3+ contents were quantified by micro-HPLC. We found that the bluer the cell, the greater the ratio of 5-O-acylquinic acids and Al3+ to anthocyanins. Furthermore, reproducing experiments were conducted by mixing the components under various pH condition; all the colors could be reproduced in the various mixing conditions. Based on the above, we provide experimental evidence for cell color variation in hydrangea. Our study demonstrates the expression of phenotypic differences without any direct genomic control.