Comparison of Sds-page to automated parallel capillary electrophoresis for enzyme size and purity assessments

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ORM Expression Alters Sphingolipid Homeostasis and Differentially Affects Ceramide Synthase Activity

Sphingolipid synthesis is tightly regulated in eukaryotes. This regulation in plants ensures sufficient sphingolipids to support growth while limiting the accumulation of sphingolipid metabolites that induce programmed cell death. Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis and is considered the primary sphingolipid homeostatic regulatory point. In this report, Arabidopsis (Arabidopsis thaliana) putative SPT regulatory proteins, orosomucoidlike proteins AtORM1 and AtORM2, were found to interact physically with Arabidopsis SPT and to suppress SPT activity when coexpressed with Arabidopsis SPT subunits long-chain base1 (LCB1) and LCB2 and the small subunit of SPT in a yeast (Saccharomyces cerevisiae) SPT-deficient mutant. Consistent with a role in SPT suppression, AtORM1 and AtORM2 overexpression lines displayed increased resistance to the programmed cell death-inducing mycotoxin fumonisin B1, with an accompanying reduced accumulation of LCBs and C16 fatty acid-containing ceramides relative to wild-type plants. Conversely, RNA interference (RNAi) suppression lines of AtORM1 and AtORM2 displayed increased sensitivity to fumonisin B1 and an accompanying strong increase in LCBs and C16 fatty acid-containing ceramides relative to wild-type plants. Overexpression lines also were found to have reduced activity of the class I ceramide synthase that uses C16 fatty acid acyl-coenzyme A and dihydroxy LCB substrates but increased activity of class II ceramide synthases that use very-long-chain fatty acyl-coenzyme A and trihydroxy LCB substrates. RNAi suppression lines, in contrast, displayed increased class I ceramide synthase activity but reduced class II ceramide synthase activity. These findings indicate that ORM mediation of SPT activity differentially regulates functionally distinct ceramide synthase activities as part of a broader sphingolipid homeostatic regulatory network

Investigation of Pathways for Complex Sphingolipid Biosynthesis in \u3ci\u3eArabidopsis thaliana\u3c/i\u3e (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 ω9 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 Δ8 LCB desaturase from castor bean was identified which required the presence of a Δ4 double bond for activity. The presence of Δ4,8 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 Δ4 unsaturation targets LCBs through a LOH2-like ceramide synthase for subsequent Δ8 desaturation and glucosylceramide synthesis. Advisor: Edgar B. Cahoo

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 Δ9 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 Δ8 LCB desaturase from castor bean was identified which required the presence of a Δ4 double bond for activity. The presence of Δ4,8 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 Δ4 unsaturation targets LCBs through a LOH2-like ceramide synthase for subsequent Δ8 desaturation and glucosylceramide synthesis

ORM Expression Alters Sphingolipid Homeostasis and Differentially Affects Ceramide Synthase Activity

Sphingolipid synthesis is tightly regulated in eukaryotes. This regulation in plants ensures sufficient sphingolipids to support growth while limiting the accumulation of sphingolipid metabolites that induce programmed cell death. Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis and is considered the primary sphingolipid homeostatic regulatory point. In this report, Arabidopsis (Arabidopsis thaliana) putative SPT regulatory proteins, orosomucoidlike proteins AtORM1 and AtORM2, were found to interact physically with Arabidopsis SPT and to suppress SPT activity when coexpressed with Arabidopsis SPT subunits long-chain base1 (LCB1) and LCB2 and the small subunit of SPT in a yeast (Saccharomyces cerevisiae) SPT-deficient mutant. Consistent with a role in SPT suppression, AtORM1 and AtORM2 overexpression lines displayed increased resistance to the programmed cell death-inducing mycotoxin fumonisin B1, with an accompanying reduced accumulation of LCBs and C16 fatty acid-containing ceramides relative to wild-type plants. Conversely, RNA interference (RNAi) suppression lines of AtORM1 and AtORM2 displayed increased sensitivity to fumonisin B1 and an accompanying strong increase in LCBs and C16 fatty acid-containing ceramides relative to wild-type plants. Overexpression lines also were found to have reduced activity of the class I ceramide synthase that uses C16 fatty acid acyl-coenzyme A and dihydroxy LCB substrates but increased activity of class II ceramide synthases that use very-long-chain fatty acyl-coenzyme A and trihydroxy LCB substrates. RNAi suppression lines, in contrast, displayed increased class I ceramide synthase activity but reduced class II ceramide synthase activity. These findings indicate that ORM mediation of SPT activity differentially regulates functionally distinct ceramide synthase activities as part of a broader sphingolipid homeostatic regulatory network