Dissecting the Regulatory Network of Sphingolipid Biosynthesis in Plants

Sphingolipids are a structurally diverse group of lipids recognized as important components of cellular membranes and regulators of processes during development and in response to environmental stresses. Much progress has been made characterizing the enzymes of the biosynthetic pathway revealing that sphingolipids are essential molecules in plants and that their synthesis and degradation needs to be tightly regulated. Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis and is a primary regulatory point for homeostasis. ORM proteins have been identified as negative regulators of SPT activity, however the mechanistic details of the regulation and other functional roles of these proteins are only beginning to be understood. In this work, we show that ORM1 and ORM2 are essential for life cycle completion in Arabidopsis thaliana. Through the characterization of ORM gene-edited mutants we described that unregulated sphingolipid biosynthesis resulted in ceramide hyperaccumulation, altered organellar structures and increased senescence- and pathogenesis-related gene expression. Furthermore, the study of a structural ORM1 variant provided information about a transmembrane domain involved in the interaction with SPT. In this thesis, we also provide insights into the physiological effects caused by mutations in SPT that induce the production of deoxysphingolipids. Our research demonstrates that plants expressing these mutations showed early senescence and reduced sensitivity to the cell death induced by Fumonisin B1. These findings suggest functional roles of deoxysphingolipids that have not been explored in plants. Finally, we describe a labeling approach to build a sphingolipid kinetic model to study the metabolic flux of sphingolipids during pathogen infection. This study considers a more comprehensive view of the sphingolipid metabolic network that changes dynamically when perturbed. Overall, this study encompasses several aspects of sphingolipid biology in plants. From a directed understanding of the regulatory mechanism and how enzyme variants can lead to the synthesis of atypical sphingolipids to a more comprehensive understanding of the metabolic network. The combination of these approaches will provide important information to understand the regulation of sphingolipids homeostasis