FUNCTIONAL GENOMICS TO DISSECT TRITERPENE SAPONIN BIOSYNTHESIS IN Medicago truncatula

¹ Plant Biology Division, The Samuel Roberts Noble Foundation, Inc., P.O. Box 2180, Ardmore, OK, 73402

² Departments of Chemistry and Biochemistry and Cell Biology, Rice University, Houston, Texas 77005

Saponins of the model legume Medicago truncatula are triterpene glycosides (Huhman and Sumner, 2002), the aglycones of which are derived from ß-amyrin, an oleanane pentacyclic triterpene, product of the cyclization of 2,3-oxidosqualene. We have proposed a hypothetical biosynthetic pathway for M. truncatula saponins based on previously established biosynthetic principles and phytochemical analysis (Suzuki et al., 2002). The biosynthetic steps following ß-amyrin formation include cytochrome P450-dependent hydroxylation/oxidations and glycosyl transfer reactions. Utilizing the genomic resources of M. truncatula, we have identified and functionally characterized cDNA clones encoding three early enzymes of ß-amyrin formation; namely squalene synthase, squalene epoxidase, and ß-amyrin synthase (ß-AS). Methyl jasmonate (MeJA) strongly induced early saponin pathway genes in M. truncatula cell suspension cultures associated with accumulation of saponins (Suzuki et al., 2002). Up-regulation of saponin biosynthesis in M. truncatula cell suspension culture by MeJA, coupled to the use of DNA array and bioinformatics tools, provides a strategy for molecular dissection of the M. truncatula saponin pathway. 128 putative cytochrome P450s and 164 glycosyltransferases (GTs) from M. truncatula EST libraries were used for gene macroarray analysis which allowed the identification of 38 P450s and 33 GTs up-regulated in response to MeJA. Thirteen of the P450 and 14 of the GT candidates exhibit coordinated expression with ß-AS in at least three M. truncatula EST libraries, and do not show strong expression in libraries where ß-AS is not expressed. Functional characterization of these genes is in progress and will be discussed.
References Huhman, D.V. and Sumner, L.W. (2002) Phytochemistry 59: 347–360. Suzuki, H. et al., (2002) Plant Journal 32: 1033-1048.