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The original sepal color of  Hydrangea macrophylla  is blue, although it is well known that sepal color easily changes from blue through purple to red. All the colors are due to a unique anthocyanin, 3- O -glucosyldelphinidin, and both aluminum ion (Al 3+ ) and copigments, 5- O -caffeoyl and/or 5- O - p -coumaroylquinic acid are essential for blue coloration. A mixture of 3- O -glucosyldelphinidin, 5- O -acylquinic acid, and Al 3+  in a buffer solution at pH 4 produces a stable blue solution with visible absorption and circular dichroism spectra identical to those of the sepals, then, we named this blue pigment as ‘hydrangea blue-complex’. The hydrangea blue-complex consists of 3- O -glucosyldelphinidin, Al 3+ , and 5- O -acylquinic acid in a ratio 1:1:1 as determined by the electrospray ionization time-of-flight mass spectrometry and nuclear magnetic resonance spectra. To map the distribution of hydrangea blue-complex in sepal tissues, we carried out cryo-time-of-flight secondary ion mass spectrometry analysis. The spectrum of the reproduced hydrangea blue-complex with negative mode-detection gave a molecular ion at  m/z  = 841, which was consistent with the results of ESI-TOF MS. The same molecular ion peak at  m/z  = 841 was detected in freeze-fixed blue sepal-tissue. In sepal tissues, the blue cells were located in the second layer and the mass spectrometry imaging of the ion attributable to hydrangea blue-complex overlapped with the same area of the blue cells. In colorless epidermal cells, atomic ion of Al 3+  was hardly detected and potassium adduct ion of 5- O -caffeoyl and/or 3- O -acylquinic acid were found. This is the first report about the distribution of aluminum, potassium, hydrangea blue-complex, and copigment in sepal tissues and the first evidence that aluminum and hydrangea blue-complex exist in blue sepal cells and are involved in blue coloration.

Direct mapping of hydrangea blue-complex in sepal tissues of Hydrangea macrophylla