| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2006: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2005: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
1. We expressed yellow cameleon 3.6, a Ca^<2+> indicator based on green fluorescent protein (GFP), in the pollen grains and papilla cells of Arabidopsis thaliana and monitored [Ca^<2+>]_cyt dynamics during pollination. In the pollen grain, [Ca^<2+>]_cyt, increased at the potential germination site soon after hydration and remained augmented until germination. As in previous in vitro germination studies, [Ca^<2+>]_cyt oscillations were observed in the tip region of the growing pollen tube, but the oscillation frequency was faster and [Ca^<2+>]_cyt was higher than had been observed in vitro. In the pollinated papilla cell, remarkable increases in [Ca^<2+>]_cyt occurred three times in succession, just under the site of pollen-grain attachment. [Ca^<2+>]_cyt increased first soon after pollen hydration, with a second increase occurring after pollen protrusion. The third and most remarkable [Ca^<2+>]_cyt increase took place when the pollen tube penetrated into the papilla cell wall.
2. We exa
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mined the role of actin, a key cytoskeletal component regulating the transport system for hydration and germination, in the papilla cell during cross-pollination. Using rhodamine-phalloidin staining, we showed that cross-pollination induced actin polymerization. By monitoring transiently expressed GFP-mTalin, we observed the concentration of actin bundles at the cross-pollen attachment site and actin reorganization. We further showed that the coat of cross-pollen is sufficient to mediate this response. The actin-depolymerizing drug cytochalasin D (CD) significantly inhibited pollen hydration and germination during cross-pollination, further emphasizing a role for actin in these processes. Additionally, three-dimensional electron microscopic tomography revealed the close association of the actin cytoskeleton with an apical vacuole network. Cross-pollination led to vacuolar rearrangements toward the site of pollen attachment. Taken together, our data suggest that cross-pollination differentially affect the dynamics of actin cytoskeleton leading to changes in vacuolar structure associated with hydration and germination. Less
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