Study on the flower color change by measuring vacuolar pH

蓝色牵牛花的花瓣。天蓝色，开花时颜色由紫红色变为浅蓝色。天蓝色花色素苷分布在上、下表皮的液泡中，而在薄壁组织中没有分布。用质子选择性微电极直接测定了活体花瓣细胞的pH值。蓝色开放花的pHv为7.7，紫红色花蕾的pHv为6.7。为了阐明pH升高的机理，以紫红色花蕾、蓝色开放花和紫色褪色花的花瓣为材料，制备了有色原生质体，并提供了液泡膜。通过免疫印迹技术，H^+-ATP酶和H^+-PPase保留在花蕾和开放的花瓣中。相比之下，这些蛋白质在褪色的花瓣中不存在。花蕾和开放花瓣的液泡膜小泡中PPi和ATP依赖的H^+运输活性几乎处于同一水平，而褪色花瓣的液泡膜小泡中则没有PPi和ATP依赖的H ^+运输活性。因此，液泡的pH不是由质子泵控制的，而可能与另一种离子的运输有关。

The petal of blue morning glory, Ipomoea tricolor cv. heavenly blue, changes its color from purplish red to light blue during blooming. The pigment, heavenly blue anthocyanin, was distributed in the vacuoles of upper and lower epidermis and not in parenchyma. We measured pHv of living petal cell directly using proton selective-microelectrode. The pHv of blue open flower was 7.7 while that of purplish red buds was 6.7. This study provides the clear evidence that the color change is caused by the increase of pHv during blooming.To clarify the pH increasing mechanism the colored protoplasts were prepared from the petals of purplish-red buds, blue open flower and purple faded flower, followed by the vacuolar membrane were provided. By immunoblot technique H^+-ATPase and H^+-PPase retained both in the buds and the open flower petals. In contrast those proteins were absent in faded petals. PPi-and ATP-dependent H^+-transport activities in vacuolar membrane vesicles from the buds and the open flower petals were almost in the same level, although no activities in those from the faded flower petals. Therefore the vacuolar pH was not controlled by proton pumps, but might be concerning with another ion transport.