This project focuses on two important developmental processes occurring during the normal differentiation of tracheary elements (TE), namely, the coordination of secondary cell wall synthesis with the cell's programmed cell death (PCD). Tracheary elements are the cellular corpses that form the water conducting vessels of plant xylem. As TEs develop, they lay down a rigid secondary wall. At the conclusion of this process, death of the developing TE is triggered by its own extracytoplasmic signal. Autolysis of the cytoplasm ensues after release of nascent hydrolases, stored in the vacuole, but the synthesis of these hydrolases begins well before the new wall is completed. Strict coordination of the PCD and autolysis with wall formation is critical because inappropriate timing of cell death would have detrimental effects, not only on the function of the developing corpse, but on the entire developing cell flank (vessel). Recent data from the PI's lab support the following model: prior to cell wall formation, hydrolases axe synthesized and sequestered in the vacuole. During the final stage of secondary wall synthesis, a 40-kD serine protease is secreted and induces the influx of calcium that directly or indirectly activates rupture of the tonoplast and subsequent release of the hydrolases. The proposed project will utilize the well-characterized zinnia cell culture system to address 3 questions about the terminal events of tracheary element differentiation: What is the protease, and how does the signal trigger cell death? The secreted protease will be partially purified in order to obtain internal sequence for cloning the corresponding cDNA. The protease will be expressed in E. coli to analyze its proteolytic activity and in both zinnia roots and arabidopsis plants to test its function. The cleavage specificity will be determined using both the partially purified protease and the recombinant protease and artificial and natural substrates. Arabidopsis homologs of the protease will be identified and the effect of regulated overexpression will be examined. For the latter, an estrogen-inducible system will be used to control expression. The extracellular matrix is recognized as a store of information directing the development of plant and animal cells but how it does this is not understood, especially within a single transduction pathway such as PCD. The proposed model system has several advantages over other systems to now determine the mechanism of signal transduction involved in cell differentiation. Furthermore, the mechanism of regulation of PCD in any context is not well understood and the significance of these results could have impact on many developmental processes from coordination of TE development and wood formation to control of neoplasia. Moreover, this work will address how the extracellular matrix provides signals for cellular development and more specifically, how a novel secreted protease regulates cell death.
