Sphingolipids contribute to protein trafficking and the organization and physical properties of membranes as major structural components of the plasma membrane, tonoplast, and endomembrane system. Sphingolipids also act as dynamic regulators of basic cellular processes, including programmed cell death (PCD), which is triggered by the accumulation of the bioactive sphingolipid metabolites long-chain bases and ceramides. The project is designed to elucidate the regulatory components and their interactions that finely mediate the requirements for sufficient amounts of sphingolipids to support cell viability and growth yet prevent the buildup of sphingolipid metabolites to levels that induce PCD. The project will build upon emerging evidence for post-translational regulation of sphingolipid synthesis through serine palmitoyltransferase (SPT), a heterodimeric enzyme that catalyzes the first step in this pathway. Arabidopsis homologs of new players in the SPT regulatory network will be characterized. These include small subunits of SPT (ssSPT) that stimulate SPT activity and ORMs that provide reversible negative-regulation of SPT activity in response to intracellular sphingolipid levels. Contributions of members of Arabidopsis ssSPT and ORM gene families to the regulation of SPT and sphingolipid synthesis will be determined. Using yeast-based tools, post-translational modifications of Arabidopsis ORMs will be assessed and interactions of SPT and regulatory proteins will be determined. Experiments will also be conducted to identify any additional components of the SPT regulatory system. Results from the yeast-based studies will provide the basis for experiments designed to uncover the in planta transduction of SPT regulation in response to perturbations in sphingolipid homeostasis. These studies will take advantage of Arabidopsis mutants with dysfunctions in sphingolipid biosynthetic regulation and fungal mycotoxins that generate enhanced levels of PCD-inducing long-chain bases. Elucidation of the mechanisms responsible for controlling the synthesis of sphingolipids will not only have implications for crop productivity but will also be of broad general interest since sphingolipids are both essential and highly bioactive in all eukaryotic cells.
Broader Impacts: The project encompasses graduate and undergraduate training and high school educational outreach. The research will be conducted by graduate students at University of Nebraska-Lincoln (UNL) and the Uniformed Services University of the Health Sciences (USUHS). Opportunities for an annual two-to-four week exchange of graduate students between UNL and USUHS will broaden training experiences for student participants. To meet a growing educational need at UNL, a graduate level, team-taught course in lipid biochemistry will be established under the leadership of PI Cahoon and co-PI Stone. This course will include modules on sphingolipid function and metabolism and will broadly engage students with interests ranging from nutrition, biofuels, and lipid signaling. Undergraduate training will be incorporated into the project through the UNL Undergraduate Creative Activities and Research Experiences (UCARE) program that provides a two year opportunity for undergraduate students to develop and conduct independent research projects. Undergraduate training in line with the project goals will also be provided through established NSF REU programs at UNL and science and technology mentorship programs at USUHS, which actively recruit women and underrepresented minority students. The project will also facilitate educational outreach to high school students through lab-based workshops for the annual UNL Women in Science program and by hosting of high school students in the Nebraska EPSCoR-supported Young Nebraska Scientist Initiative, a summer program designed to strengthen the K-12 STEM student pipeline.
