Programmed cell death (PCD) is recognized to be a fundamental process found in all eukaryotes ranging from yeasts, plants and animals. In animal apoptosis, a specialized form of PCD, the core death engine is highly conserved with a family of specialized cysteine proteases called caspases as the determinate executioner. In plants and yeasts, however, canonical caspases are absent from sequenced genomes and instead, a family of distantly related cysteine proteases called metacaspases were identified through structure-based iterative database searches. In this proposed project, the function of selected metacaspase genes in the model plant Arabidopsis (mouse-ear cress) will be studied through detailed genetic, molecular, cell biological and biochemical analyses. Specifically, the investigator aims to uncover some of the upstream regulators and downstream targets of metacaspases. This proposed work will thus provide the foundation for a comprehensive understanding of the regulatory pathways involving metacaspases and cell death regulation in higher plants. Comparison to related studies with animal caspases will provide a better framework for understanding the evolution of PCD in higher eukaryotes. In a broader context, this study should contribute to our understanding of plant responses to biotic and abiotic stresses since PCD is often associated with these conditions, and thus provide important knowledge for future crop improvement. This project will also have broader impact in providing a diversified, interdisciplinary training to postdoctoral researchers. In addition, the investigators will also provide research experience for undergraduate, graduate and high school students through the Douglass Women in Science and George H. Cook Scholar programs of Rutgers University, and the RISE summer research program for high schools.
In the past two decades, programmed cell death (PCD) has been shown to be an important process that is highly conserved in all eukaryotes ranging from fungi, plants and animals. Although plant PCD has been shown to be important for development and stress responses in higher plants, the underlying mechanisms and identity of the critical players that control this process remain elusive. This NSF-funded project focused on the analysis of a specialized family of plant proteases, called metacaspases, that was hypothesized to play critical roles in orchestrating PCD in the model plant Arabidopsis. Using a combination of genetic, molecular and biochemical approaches, our work showed that the most highly expressed metacaspase family member, AtMC4, is involved in mediating cell death activation in response to plant pathogens and oxidative stresses. In addition, we showed that the activation of the latent activity of the AtMC4 protein requires controlled cleavage at a specific site in the pro-enzyme. This autolytic activation was shown to be activated in vivo concomitant with PCD activation, and its suppression by mutagenesis renders AtMC4 inactive as a cell death mediator. Together with two other reports on the genetic study of other members of the metacaspase family in Arabidopsis, our work now clearly demonstrated the in vivo roles of metacaspases in plants as well as established the biochemical foundation for delineating the mechanisms through which these important enzymes are regulated and their downstream protein targets that ultimately leads to PCD. In addition to revealing the importance of metacaspases in regulating plant PCD, our biochemical studies also provided support for the evolutionarily conserved functions and activation mechanisms of these proteases with their animal counterpart caspases. As such, it provided support for the hypothesis that metacaspase may be the more ancestral form of cell death regulators that later diverged and elaborated to give rise to present day caspases in animals. Since plant PCD activation by certain plant pathogens as well as during environmental stresses is responsible for significant crop loss in agriculture worldwide, our study on metacaspases as a key activator of PCD during these situations may provide potential gene targets for crop improvement through molecular breeding or genetic engineering. Lastly, we have trained an undergraduate, Mariya Skvortsova, and a female technician, Yi Zhang, during this project and thus have broadened the participation of underrepresented segments of the workforce in science and technology.
