9632027 Franceschi Calcium (Ca) is involved in the control of many physiological processes in plants, and therefore regulation of Ca activity is critical to normal plant growth, development and productivity. Ca is taken up in the root and transported along with water. The water evaporates from the surfaces of the plant, and over time large amounts of Ca can accumulate in various organs, which necessitates high capacity Ca sequestration systems. Ca oxalate formation in plants has evolved as a high capacity mechanism for removing Ca when it is present at levels that can no longer be controlled by the lower capacity mechanisms common to all cells. This idea is supported by observations that most plant families produce Ca oxalate crystals, that this product can account for up to 85% of the dry weight of some plant species, and that 90% of the Ca in plants can be in this form. The goal of this project is to elucidate features of cells called Ca oxalate crystal idioblasts, which are responsible for large scale Ca accumulation. Crystal idioblasts are structurally and biochemically specialized for Ca accumulation and Ca oxalate crystal formation. Four proteins have been identified that play key roles in crystal formation in crystal idioblasts. These are: "matrix protein," a unique vacuolar Ca binding protein; calreticulin, a high capacity Ca binding protein of the endoplasmic reticulum (ER); an ER Ca ATPase (ERCA) which acts as a Ca pump; and oxalate oxidase, which is involved in release of Ca from crystals when free Ca availability becomes limited. In addition, ultrastructural examination shows that crystal idioblasts have abundant ER and Golgi bodies. The aim of the proposed research is to characterize the structure, function and control of developmental expression of these proteins, and thus to gain a better understanding of how they regulate Ca levels in plant tissues and cells. The specific objectives are to: 1, complete the analysis of cDNAs for matrix protein, calreticulin, ERCA an d oxalate oxidase, and produce antibodies to the proteins isolated from a bacterial expression system; 2, purify matrix protein from an expression system and examine its physical-chemical properties in vitro; 3, characterize the temporal and spatial regulations of the proteins and their mRNA transcripts during changing Ca nutrition; 4, determine the pattern of expression of the proteins and their transcripts at the subcellular level from crystal idioblast initiation through maturation; and 5, characterize the formation and modification of the ER and Golgi systems during idioblast development. Molecular biological techniques will be used to accomplish all or parts of the first 3 objectives. Biochemical techniques, Western and Northern blotting, and high resolution in situ hybridization visualization and immunocytochemistry by the light and electron microscopy will be used for objectives 3 and 4. Objective 5 will be completed using laser scanning confocal and standard transmission electron microscopy. %%% A better understanding of the mechanism of Ca oxalate crystal formation is imperative to our understanding of Ca regulation in plants, as well as for the potential manipulation of this process, or selected features of it, by biochemical or molecular genetic means. This project will significantly enhance our understanding of Ca oxalate crystal formation specifically, and will provide important new information on mechanisms of Ca regulation in plants in general. Furthermore, it should be kept in mind that plants are not the only living organisms that manufacture mineral crystalline structures based on calcium salts; for example, the exquisitely precisely-sculpted spicules and mineralized exoskeletons (e.g., oyster shells) of invertebrates, and the bones and teeth of vertebrates, are also examples of this general process of biomineralization. The Ca oxalate crystals of plant crystal idioblasts represent a relatively simple model system for biomineralization, and it is anticipated that the results of this project will provide important new insights into the general area of biomineralization which will be useful not only to biologists interested in how nature manufactures mineralized materials, but also to materials scientists and engineers who are interested in either harnessing or mimicking natural processes to create novel and useful ceramic structural materials. ***
