Biosynthesis of methylcinnamate in Ocimum basilicum L.
Methylcinnamate (MC, C10H10O2), a secondary metabolite with a phenylpropanoid (C6-C3) chemical skeleton, is a volatile natural product present in the essential oils of some basil chemotypes (Ocimum basilicum L., Lamiaceae). Sampling methods for the quantitation of MC content in basil in vivo were first compared using variable extraction solvent amounts and procedures, based on a validated GC system. Quantitative analysis of the highly volatile MC in vivo needed to be standardized and a recovery assay was found to be a critical step. The coefficient factor from the recovery experiment can be used to calculate the original volatile content in plant in vivo and minimize variation of experimental results from different research groups and variable sampling methods. ^ We hypothesized that from phenylalanine there were two major steps in the biosynthesis of MC, formation of cinnamic acid and methylation of cinnamic acid to methylcinnamate. Two enzymes, phenylalanine ammonia-lyase (PAL) and S-adenosyl-L-methionine:cinnamate carboxyl methyltransferase (SAM:CCMT), for methylcinnamate biosynthesis, have been purified and characterized by using preparative PAGE, series of chromatographic techniques using the intact glandular trichomes isolated from basil leaves. The PAL exhibited a unique and smaller Mr. (152,000 kD), a homotetramer composed of four 38 kD subunits. The SAM:CCMT is the first purified carboxyl methyltransferase (SAM: CCMT) for secondary metabolites in plants, with a Mr. of 29 kD. ^ The relationships between MC content, PAL and SAM:CCMT activities were examined during basil plant development. Results showed SAM:CCMT activity to be highly correlated with MC content in young basil leaves. This conclusion was also supported by the kinetic data of both PAL and SAM:CCMT. ^ A cDNA library for methylcinnamate basil was constructed by using the basil leaf tissue that exhibited the highest PAL and SAM:CCMT activities during the entire plant development process. ^
Major Professor: James E. Simon, Purdue University.
Agriculture, Food Science and Technology|Biology, Plant Physiology