Investigation of Pathways for Complex Sphingolipid Biosynthesis in Arabidopsis thaliana (L.) Heynh

Sphingolipids are essential components of eukaryote membranes. The ceramide backbone of complex sphingolipids is composed of an 18 carbon Long Chain Base (LCB) bound to a 16-26 carbon fatty acid (FA) through an amide linkage. Ceramides are synthesized de novo from a free LCB and fatty acyl coA by ceramide synthase (sphingosine N-acyl transferase, EC 2.3.1.24) which can be inhibited by the fungal mycotoxin Fumonisin B1. Arabidopsis thaliana contains three ceramide synthases denoted LOH1, LOH2, and LOH3 that have previously been hypothesized to have unique substrate preferences that control the final sphingolipid composition, different susceptibilities to Fumonisin, and different influences plant growth/development. This dissertation works to answers to these questions as well as identify novel complex sphingolipid biosynthetic pathways. Through the use of in vitro assays it was found that LOH1 and LOH3 prefer LCBs witth hydroxyls at the C1, C2, and C4 positions (trihydroxy) and C20-24 saturated FA while LOH2 prefers LCBs with hydroxyls at the C1 and C2 positions (dihydroxy) and C16 saturated fatty acids. None of the isoforms were able to use Delta9 desaturated acyl CoAs which are abundant in the final sphingolipid profile. Surprisingly LOH2 showed the highest level of activity with C4 unsaturated LCBs which are not commonly found in leaf. Each isoform was also overexpressed in planta to determine the effects ceramide composition has on plant growth. Overexpression of LOH1 or LOH3 led to an increase in biomass while overexpression of LOH2 resulted in a dwarf phenotype. Both the in vitro assays and in planta overexpression found LOH1 to the most susceptible to FB1 inhibition. In addition to ceramide synthesis a novel Delta8 LCB desaturase from castor bean was identified which required the presence of a Delta4 double bond for activity. The presence of Delta4,8 unsaturated LCBs was found to result in increased glucoscylceramide levels as revealed by LCB feeding experiments and pollen sphingolipid profiling. Therefore, it is hypothesized that the presence of a Delta4 unsaturation targets LCBs through a LOH2-like ceramide synthase for subsequent Delta8 desaturation and glucosylceramide synthesis.