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USE OF RNAi TO CHARACTERIZE CALCIUM SIGNALLING GENES EXPRESSED IN CERATOPTERIS RICHARDII DURING SPORE GERMINATION.  S.C. Stout, M.L. Salmi, T.J. Bushart, G.B. Clark, S.J. Roux.  Section of Molecular, Cell and Developmental Biology, The University of Texas at Austin.

     Gravity directs polarity development in the single celled spore of Ceratopteris richardii.  The vector of gravity fixes spore polarity during a limited window between 12 and 24 hours after light initiation of spore germination.  Concurrent with this period of graviresponsiveness, an efflux of calcium is found from the top of the spore and a corresponding influx is found along the bottom.  This bottom to top polar calcium current rapidly reorients when the spore is rotated 180є, and treatment with a calcium channel blocker reduces both the calcium current and the spore’s ability to correctly respond to gravity.

     As part of a cDNA microarray project, over 2,800 ESTs were sequenced from a cDNA library.  Several genes involved in calcium signaling and homeostasis are represented in the EST library, including calmodulin, calcium dependent protein kinase (CDPK), calcium-ATPase, and annexin.  Our work focuses on characterizing these genes using the RNAi technique.

     We have cloned and sequenced a full-length calmodulin, which is expressed during the period of gravity fixation.  The expected amino acid sequence has over 90% identity to other higher plant calmodulins, and anti-cauliflower calmodulin antibodies recognize the heterologously expressed protein.

     Spores imbibed with dsRNA corresponding to the calmodulin show delayed germination compared to controls.  However, there is no change in polarity development.  We are testing whether the germination delay can be attributed to an RNAi induced decrease in calmodulin expression, and we are continuing to investigate other genes of interest with this technique.

     (Supported by NASA: NAG10-0295, NAG2-1347, and NGT5-50371)