[29]

Studies of the Gravity-Directed Calcium Current in Germinating Spores of Ceratopteris richardii through CrACA1 and Microarray Analyses.   T.J. Bushart, M.L. Salmi, and S.J. Roux.  Section of Molecular, Cell, and Developmental Biology, The University of Texas at Austin, Austin, TX.

During the period in which gravity determines their developmental polarity, germinating spores of the fern Ceratopteris richardii exhibit a bottom-to-top calcium current. This current is parallel to the subsequent axis of cell development, is capable of rapid reorientation (<42 s), and its disruption by nifedipine prevents proper orientation. The exit of calcium from the top of the cell is presumed to be achieved by an ATP-dependent pump. Toward this end, we have identified three cDNAs from a Ceratopteris EST library whose sequences predict that they encode Ca2+-ATPase pumps, two of which are predicted to be plasma-membrane localized. As seen by quantitative RT-PCR assays, the RNA expression level of the fully sequenced CrACA1 peaks at the same point that the calcium flux is maximal. Antibodies have been raised to a 93-mer peptide corresponding to the large loop between transmembrane regions 4 and 5 of CrACA1. Initial Western blotting shows the presence of protein through 24 h. Immunolocalization and inhibition studies are underway to further characterize the role of this Ca2+-ATPase in the gravity response. To investigate other elements connected with the calcium flux, our microarray data from a 1g developmental timecourse and a 1g-to-microgravity comparison was examined for significant changes. These analyses reveal six genes with expression changes during the first 48 h of spore development. Only one of these (Accession BE642184) shows an expression profile coincident with the calcium flux profile. This expression profile is altered in microgravity, suggesting that this gene, which putatively encodes a C2 domain protein, may be important in coordinating the Ca2+ signal, specifically in relation to polarized vesicle transport. These investigations are important steps in the identification and characterization of the molecular components that drive and respond to the gravity-directed calcium current.

(Supported by NASA: NAG2-1586, NAG10-295, NNG04G045H)