9305941 Franceschi In plants, calcium is taken up and transported along with water. The amount taken up in many plants is directly proportional to the amount of calcium in the soil solution. The water evaporates from the surfaces of the plant and, over time, large amounts of calcium can accumulate in various organs, which necessitates high capacity calcium sequestration systems in the organs of such plants. It is hypothesized that calcium oxalate formation has evolved as a mechanism in plants for removing calcium when it is present at levels that can no longer be controlled by the lower-capacity mechanisms common to all cells. The observations that most plant families produce calcium oxalate, that this product can account for up to 85% of the dry weight of some plant species, and that 90% of the calcium in plants can be in this form, support this hypothesis. Calcium binding proteins have been identified that are specific to those cells (crystal idioblasts) that produce calcium oxalate. One protein is cytosolic and shows antigenic similarity to animal calsequestrin. It is hypothesized that this protein is responsible for buffering the activity of the large fluxes of calcium which must be passing through the cytoplasmic compartment during the rapid process of crystal formation. Three other proteins have been found to be associated with the calcium oxalate crystal matrix, and antibodies have been raised to these proteins. These matrix proteins are thought to be responsible for regulating nucleation and/or growth of the crystals. The next general goal of this research is to characterize the structure and function of these unique proteins to further understand how they function in calcium regulation in plants. Specific objectives are to: (1) Characterize of the molecular properties of the matrix proteins by obtaining and analyzing cDNAs for these proteins. (2) Determine the chemical properties of the matrix proteins. The effects of purified matrix proteins on crystal nucleation and growth will be studied in vitro. (3) Resolve the molecular relationship of matrix proteins and crystal matrix. Immunocytochemical techniques will be used to determine the pattern of protein intercalation in the crystal matrix. (4) Characterize the calsequestrin-like protein. Using antibody and cDNA probes to animal calsequestrin, a cDNA will be isolated from a library constructed from Pistia stratiotes. The cDNA will be sequenced and characterized. (5) Determine the subcellular location of the calsequestrin-like protein by immunocytochemistry. %%% Calcium is involved in the control of many physiological processes in plants, but excess calcium can be harmful. Thus, regulation of calcium is critical to normal plant growth, development and productivity. Previous results from this laboratory suggest that formation of calcium oxalate crystals is a mechanism used by plants to sequester excess calcium. This research will examine the mechanisms by which plants form calcium oxalate crystals. The results will contribute to our understanding of how plants regulate their internal calcium levels. ***
