Discovering plant secondary metabolite pathways – functional analysis of secondary metabolism related genes

We have designed a novel technology for unravelling the genes involved in the plant secondary metabolism. This technology called Solucel® is based on the genome wide identification and functional analysis of genes involved in the production of phytopharmaceuticals in plant cell cultures. As a model system we used Nicotiana tabacum (BY-2) cell culture in which the nicotine alkaloid production was induced by applying methyl jasmonate as an elicitor (1). Altogether 20 000 transcript tags were visualised. The sequences of the methyl jasmonate modulated (MJM) gene tags were compared to the public databases and 47 of the total 591 MJM gene tags were chosen for further studies. Of particular interest were the genes encoding for protein kinases, signal transducing proteins, transcription factors and other master regulators. Functional analysis of the MJM genes is being performed by using transgenic cell lines of BY-2. Full length cDNAs were constructed and delivered to A. tumefaciens and A. rhizogenes for the establishment of transgenic cell suspension cultures and hairy root cultures, respectively. The transformed cell lines were subjected to metabolite analysis and compared to wild type lines in order to determine the functional properties of the inserted gene construct. In addition, the correlation of the gus-expression and metabolite accumulation in cells carrying promoter-reporter constructs were shown to facilitate the identification of the functional genes. So far, two genes coding for different transcription factors have shown potential to be involved in the regulation of nicotine alkaloid biosynthesis. So-called combinatorial biochemistry approach was applied with the aim of unravelling the possibilities to broaden the chemical diversity of valuable plant derived compounds. The genes derived from tobacco transcriptional profiling were transformed to related or non-related species Hyoscyamus and Catharantus by Agrobacterium transformation, or if required, by using biolistics approaches. Up to date, one gene has shown interesting effect in hairy roots, where besides exceptionally high tropane alkaloid contents, also remarkable changes in secondary metabolite profile was observed, indicating the gene possessing an important role in secondary metabolism. (1) Goossens A, Hakkinen ST, Laakso I, Seppanen-Laakso T, Biondi S, DeSutter V, Lammertyn F, Nuutila AM, Soderlund H, Zabeau M, lnze D, Oksman-Caldentey K-M. Proc Nall Acad Sci 2003; 100: 8595-8600