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Calcium regulation in Tradescantia virginiana: Roles for involvement of inositol 1,4,5-trisphosphate and cyclic ADP-ribose
Fluorescent ratiometric imaging and spectrofluorometric analysis was used to study two signal transduction mechanisms in the stamen hair cells of Tradescantia virginiana. The first study determined the metabolic pathway necessary for the inactivation of Inositol 1,4,5-trisphosphate mediated calcium release from intracellular stores in the living plant cell. Tradescantia stamen hair cells, preloaded with the calcium sensitive ratiometric dye fura-2-dextran, were injected with analogs of Inositol 1,4,5-trisphosphate and cytosolic calcium levels monitored by ratiometric imaging. The injected analogs were selected due to their insensitivity to various kinases and phosphatases for which Inositol 1,4,5-trisphosphate is a substrate. We determined that the 5-phosphatase is the preferred pathway for inactivation of Inositol 1,4,5-trisphosphate in the living plant cell. The second study investigated cyclic ADP-ribose mediated calcium release in the intact plant cell and determined the presence of the metabolic machinery necessary to synthesize cyclic ADP-ribose from its precursor NAD+. Cyclic ADP-ribose was observed to cause calcium release in the stamen hair cells of Tradescantia that were preloaded with the calcium sensitive dye fura-2-dextran. Evidence of cyclic ADP-ribose synthesis was determined using two experimental techniques. Homogenates of the sea urchin Lytechnicus piclus were used as bioassays to detect cyclic ADP-ribose in extracts of Tradescantia stamen hair cells that were incubated with [special characters omitted]NAD+. Cyclic ADP-ribose synthesis was detected from fluorimetric analysis of the homogenate as the calcium sensitive dye fluo-3 was present in the homogenate to detect cyclic ADP-ribose mediated calcium release from sea urchin egg microsomes. We also determined cyclic ADP-ribose synthesis by injection of fura-2-dextran loaded stamen hair cells with [special characters omitted]NAD+ and observing a delayed calcium increase in the cytosol. These results establish the metabolic fate of inositol 1,4,5-trisphosphate in plant cells and demonstrate the biochemical capability for plant cells to synthesize cyclic ADP-ribose to mediate calcium release in plants.
Botany|Cellular biology|Molecular biology
DePass, Anthony Lyndon, "Calcium regulation in Tradescantia virginiana: Roles for involvement of inositol 1,4,5-trisphosphate and cyclic ADP-ribose" (1999). Doctoral Dissertations Available from Proquest. AAI9920594.