Genetic and biochemical characterization of the squalene epoxidase gene family in Arabidopsis thaliana
Rasbery, Jeanne M.
Doctor of Philosophy
As sessile organisms, plants have developed the ability to synthesize an impressive array of metabolites to meet environmental challenges. Terpenes constitute the largest group of plant natural products, yet the biological functions of most of these compounds remain unknown. One class of terpenes, the triterpenoids, are 30-carbon compounds derived from squalene that include hormones, membrane sterols, and antifungal compounds among others. Indeed, more than 100 triterpene skeletons have been identified in plants (Xu et al., 2004). Triterpenoid biosynthesis occurs through a sequence of enzymatic reactions beginning with the conversion of squalene into oxidosqualene by squalene epoxidase (SQE) enzymes. This work focuses on characterizing the squalene epoxidase family in the plant Arabidopsis thaliana. I used biochemical and molecular genetic approaches to characterize the Arabidopsis SQE family. Six putative squalene epoxidase encoding genes were identified in the Arabidopsis genome. Using heterologous expression in yeast and chemical analysis, I demonstrated that three members of the SQE family (SQE1, SQE2, and SQE3) have squalene epoxidase activity. In contrast, the other members of the SQE family (SQE4, SQE5, and SQE6) lacked SQE activity in yeast and may have adopted unique substrate preferences. I used molecular genetic approaches to survey SQE expression in plants and characterize mutations in each of the Arabidopsis SQE genes. SQE1 and SQE3 are expressed in most plant tissues throughout development, whereas expression other SQE genes is more restricted. I demonstrated that SQE1 is essential for normal plant development. sqe1-3 and sqe1-4 mutants are dwarf plants with root, stem, and hypocotyl elongation defects. In addition, sqe1-3 and sqe1-4 mutants produce inviable seed. Chemical analysis demonstrated that sqe1-3 roots and aerial tissues have ∼10-fold increase in squalene, suggesting the triterpene pathway is blocked in this mutant. Together, this work demonstrates that members of the Arabidopsis SQE gene family have unique and overlapping roles in plant growth and development, and suggests that diversity of function at the squalene epoxidase step may contribute to triterpenoid diversity in plants.
Molecular biology; Genetics