Identifying Regulatory Components of Phytosterol Biosynthesis in the Genomics Era
Feldman, Maximilian Jeffrey
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The biosynthesis of sterol molecules is both ubiquitous and essential in the eukaryotic lineage. Despite our advanced knowledge of the structural genes and enzymes involved in phytosterol biosynthesis in Arabidopsis thaliana, our understanding of how this biosynthetic pathway is regulated remains unsatisfactory. This is in stark contrast with to the case in other eukaryotes, where the mechanistic details of pathway regulation are well documented.In the context of this dissertation I utilized a data mining strategy to identify candidate regulatory proteins of sterol biosynthesis and examine their function using reverse genetics. The first study is a detailed characterization of bHLH93, a basic helix-loop-helix protein (bHLH) protein that was identified as a candidate transcriptional regulator of phytosterol metabolism based on the correlation of gene expression vectors in leaf/shoot tissue and the statistical enrichment of DNA motifs in the promoters of sterol biosynthetic genes. bHLH93 is a transcription factor that is localized to the nucleus in plant cells. bhlh93 mutants exhibit pleiotropic phenotypic defects including decreases in bolt length, downward curling of rosette leaves, and accumulation of dark green pigments when grown under continuous, moderately intense light. Metabolically, bhlh93 mutants exhibit dramatic alterations within isoprenoid metabolism (decreases in phytosterols and increases in plastidial isopreniods) and significant perturbations in lipid metabolism. Gene expression changes observed in bhlh93-1/bhlh61-1 double knockouts and a bHLH93 over-expression line suggest that bHLH93 is essential for the light-dependent coordination of development and metabolism in Arabidopsis thaliana. In a second study, I investigated the function of Arabidopsis proteins that contain a putative sterol-sensing domain (SSD). The Arabidopsis genome encodes two highly similar proteins with putative SSDs, AtSSD1 and AtSSD2. Based on sequence comparisons it appears that SSD proteins of plants are most similar to Niemann Pick Type C proteins found in other organisms. Our reverse genetic analysis suggests that AtSSD1 and AtSSD2 are at least partially redundant and essential for both reproductive and vegetative development. Analyses of AtSSD1/atssd1/atssd2/atssd2 double mutant lines suggest that these proteins are likely important in the regulation of fatty acid but not phytosterol metabolism.