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The Role of Nitric Oxide and cGMP as Downstream Signals for Transduction of Calcium Dependent Gravisensing.   M. L. Salmi¹, K. E. Morris², S. J. Roux¹, and D. M. Porterfield³, ¹Molecular Cell and Developmental Biology, Univ. of Texas, Austin, TX, ²Depart. of Biological Sciences., Univ. of Missouri-Rolla, Rolla, MO, ³Purdue Univ., Depart. of Agricultural. & Biological  Engineering, Depart. of Horticulture and Landscape Architecture, Bindley Bioscience. Center-Physiological Sensing Facility, West Lafayette, IN

   We have been investigating a role for NO/cGMP signaling in transducing the stimulus of gravity to directed polarization of Ceratopteris richardii fern spores. These cells undergo gravity-directed cell polarity development, that is driven by polar calcium currents. Application of specific NO modulators disrupts the calcium-dependent gravity response that controls cell polarity development in a dose dependent manner. Furthermore the effects of NO donor (Spermine nonoate) exposure were antagonized by application of NO scavengers. Drugs that modulate NO-dependent cGMP levels in the cells (guanylate cyclase inhibitors LY83583 and ODQ; and PDE5 inhibitor Viagra®) disrupt gravity-directed cell polarity in a dose-dependent manner. Viagra® effects were antagonized by application of NO scavengers suggesting a connection between NO and cGMP in the signaling pathway. In order to identify other molecular components of the signaling system, we used preliminary microarray analysis, to determine how gene expression changes in response to Viagra® treatment. This identified approximately 65 genes that could mediate signaling, nuclear regulation, and protein synthesis/folding/degradation in higher plants. Based on the microarray results, we subsequently analyzed gene expression changes using quantitative real-time RT-PCR (Q RT-PCR) for four of the identified genes, which have strong sequence similarity to known signal transduction or stress response genes. In spores treated with Viagra®, these four genes have significant changes in abundance throughout the first 24 h of development not seen in control samples. Similarly expression of these four genes was also evaluated in dibutryly cGMP treated spores, and this revealed significant changes in two of the four genes analyzed.