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Assessing the role of calcium pumps in the gravity response in single-celled spores of Ceratopteris richardii.  S.J. Roux¹, T.J. Bushart¹, A. ul Haque², and D.M. Porterfield^{2 1}Molecular Cell and Developmental Biology, The University of Texas at Austin, and ²Department of Agriculture and Biological Engineering, Purdue University, West Lafayette, IN.

   Previous investigations into how gravity directs the polarity of growth in fern spores of Ceratopteris richardii revealed a key role for a trans-cell calcium current during the period gravity fixes the polarity. This calcium current reorients 180^o when the spores are rotated 180^o. Blocking calcium channels with nifedipine disrupts the calcium flux and disorients the subsequent rhizoid emergence, indicating that calcium uptake is required for gravity to orient the direction of rhizoid emergence. Here we report on the role that calcium-pumping ATPases play in the calcium flux and polarity establishment.

   Spores grown in a fixed orientation were exposed to various concentrations of BHQ, an inhibitor of endomembrane Ca²⁺-ATPases, or Eosin Y, an inhibitor of plasma membrane Ca²⁺-ATPases, and assayed for changes in germination and polarity establishment. Both 100 µM BHQ and 10 µM Eosin Y inhibit 100% of rhizoid emergence if applied continuously.  Eosin Y also depresses the calcium efflux of the spores. However, limited exposure to either drug from 0-24 hrs, the period of polarity fixation, does not disrupt the ability of gravity to orient rhizoid emergence. In a few cases this short-term exposure to BHQ appears to lead to a very small (<5%) but statistically significant increase in downward emerging rhizoids.

   Taken together, the inhibitor data indicate that Ca²⁺ pump activity is required for the initiation of polarized growth, but is not specifically required for gravity to orient the direction of that growth. Because calcium channel activity is required for the gravity response in Ceratopteris, our data are consistent with the hypothesis that the intake of calcium at the bottom of the spore, possibly through stretch-activated calcium channels, is the key event for initiating the gravity response. A model has been developed based on this and other evidence. (Supported by NAG2-1586 and NAG10-295)