Squalene epoxidase converts squalene into oxidosqualene, the precursor of all known angiosperm cyclic triterpenoids, which include membrane sterols, brassinosteroid phytohormones, and non-steroidal triterpenoids. In this work, we have identified six putative Arabidopsis squalene epoxidase (SQE) enzymes and used heterologous expression in yeast to demonstrate that three of these enzymes, SQE1, SQE2, and SQE3, can epoxidize squalene. We isolated and characterized Arabidopsis sqe1 mutants and discovered severe developmental defects, including reduced root and hypocotyl elongation. Adult sqe1-3 and sqe1-4 plants have diminished stature and produce inviable seeds. The sqe1-3 mutant accumulates squalene, consistent with a block in the triterpenoid biosynthetic pathway. Therefore, SQE1 function is necessary for normal plant development, and the five SQE-like genes remaining in this mutant are not fully redundant with SQE1.Plants are estimated to produce more than 500,000 secondary metabolites (1). These compounds have many functions, including attracting pollinators, communicating with neighboring plants, and defending against pathogens and herbivores (2, 3). The importance of secondary metabolites is highlighted by the extensive resources that plants invest in producing these compounds. Although once thought to be metabolically simple, more than 170 secondary metabolites have been identified in Arabidopsis thaliana (reviewed in Ref. 4).Triterpenoids are the 30-carbon subset of terpenoids, the largest class of secondary metabolites. Triterpenoid biosynthesis is diagramed in Fig. 1. Isopentenyl diphosphate and dimethylallyl diphosphate are synthesized from mevalonate and oligomerized to farnesyl diphosphate by farnesyl diphosphate synthase (FPS).5 Farnesyl diphosphate is dimerized to squalene by squalene synthase. Squalene epoxidase (SQE)-mediated oxidation then produces oxidosqualene, which triterpene synthases cyclize to Ͼ80 triterpene skeletons (5, 6). Further metabolism of these compounds produces membrane sterols, brassinosteroid phytohormones, saponins, other defense compounds, cuticular waxes, and numerous triterpenoids that have not been functionally characterized.The yeasts and mammals that have been investigated each encode a single squalene epoxidase. In contrast, several plants have multiple genes predicted to encode squalene epoxidases, a diversity suggesting that this step may be subject to additional or unique regulation in plants. Two Medicago truncatula SQE enzymes have been biochemically characterized (7). The Brassica napus (8), Populus trichocarpa, and Oryza sativa genomes each have multiple predicted SQE enzymes. Despite the likely importance of SQE to plant growth and development, no plant mutants with defects in these enzymes have been reported.In this work, we heterologously expressed the six Arabidopsis putative SQE enzymes in Saccharomyces cerevisiae lacking squalene epoxidase to determine which have squalene epoxidase activity. We isolated Arabidopsis sqe1 loss-of-function mutants and found t...
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