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 and the purple color is a color mosaic. However, all these colors are derived from the same components: simple anthocyanins, 3-O-glycosyldelphinidins, three co-pigment components, acylquinic acids, and Al3+ . We show the color mosaic is a result of graded differences in intravacuolar factors. • 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 was then 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. Furthemore, 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.