|Budget Amount *help
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2002: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2001: ¥1,700,000 (Direct Cost: ¥1,700,000)
In order to clarify the role of Ca^<2+> in pollen-stigma interaction of Brassicaceae, dynamic movement of Ca^<2+> in pollen grains and papilla cells during pollination process should be examined precisely and with time. Typically, ion movements in a cell have been analyzed by injecting ion sensitive dye into the cell, but the microinjection often causes physical damage to the cell and it is difficult to inject the dye to a pollen grain because pollen grain is covered with hard coats, such as exine layer. The fluorescent protein indicator for Ca^<2+>, "yellow cameleons" (YC) has been developed to combine the advantages of molecular biological targeting and fluorescence readout. In this study, first two kinds of YC3.1 chimeric genes containing Act1 promoter region or SLG promoter region were transformed into Arabidopsis thaliana. Then two kinds of transgenic plants, which expressed YC3.1 in pollen grains or papilla cells, respectively, were obtained. After the pollen grain was pollinated to the papilla cell using micromanipulator, Ca^<2+> dynamics were monitored with time. Before germination, Ca^<2+> accumulated at the site, which the pollen grain contacted to the papilla cell. In the germinated pollen tube, faster movement of Ca^<2+> was observed, compared with that of in vitro germinated pollen tube. On the other hand, Ca^<2+> movement in the papilla cell was slower than that in pollen grains. Next the YC3.1 chimeric genes were transformed into Brassica rapa. The transgenic plant, which expressed YC3.1 in the pollen grains, was obtained. As a result, dynamic movement of Ca^<2+> in the pollen grains was observed only in cross-pollination. Dynamic movement of Ca^<2+> in pollen grains and papilla cells during pollination process could be monitored precisely and with time using transgenic plants, which expressed YC genetically.