Forward and reverse genetic approaches to understanding sphingolipid metabolism and functions in plants using the model bryophyte Physcomitrella patens

ended

Sphingolipids are essential lipids that are ubiquitous among eukaryotes. Plants produce structurally diverse sphingolipids that are involved in many processes, including maintenance of plasma membrane integrity and microdomain formation, cell growth and division, polar secretion, and programmed cell death (PCD) signalling. They have primarily been investigated in Arabidopsis thaliana, for which an extensive genetic toolkit has been available for decades. Genome sequences and tools for genome editing are now available for a wide variety of species, offering a better understanding of metabolic and functional diversity, and enabling study of evolutionary history and ancestral functions. The bryophyte Physcomitrella patens is an early-diverged land plant and a relatively new model organism. Preliminary work revealed a unique sphingolipid profile for Physcomitrella, and diversification of gene families associated with the biosynthesis of glycosylinositol phosphorylceramides (GIPCs), the most abundant and diverse class of sphingolipids in plants. The precise functions of GIPCs have been challenging to study in Arabidopsis due to non-viable or pleiotropic mutant phenotypes, complex organ structure, and difficulties with extraction and detection of GIPCs. I propose reverse-genetic characterization of GIPC biosynthesis in Physcomitrella, where expansion of gene families and simple morphology will facilitate mutant analysis. Further, I will use Physcomitrella to dissect the connection between sphingolipid metabolism and PCD, which is well-recognized, but mechanistically obscure. I will perform a mutant screen with Physcomitrella protoplasts for resistance to the ceramide synthase inhibitor and PCD trigger Fumonisin B1. The causal mutations will be identified by next-generation mapping and characterized. Altogether, this work offers novel and unique insight into the metabolism and functions of essential and abundant metabolites, and the mechanisms that regulate PCD.