Towards understanding the biosynthesis of plant secondary metabolites by combining transcriptional profiling with targeted metabolome analysis

Plant secondary metabolites are characterized by an enormous chemical diversity and presently appoximately 100 000 of them have been isolated. They include many high-value pharmaceuticals e.g. anticancer drugs. However, their production is complicated due to the low contents in plants and cell cultures or extremely complex chemical synthesis. Plant metabolic engineering has met only limited success, in sharp contrast to microorganisms, since our knowledge about the biosynthesis of secondary metabolites is still very limited. Genetic maps of biosynthetic pathways are far from complete and the regulation of these pathways is poorly understood. We designed a novel approach using tobacco BY-2 cell culture as a model system, in which a cDNA-AFLP based transcript-profiling technique is linked with targeted metabolic profiling of plant cells to simultaneously identify genes involved in secondary metabolism on a genome-wide scale. From the 20000 transcript tags visualized 591 were jasmonate-modulated with different kinetics. Cluster analysis of expression profiles showed that half of the genes were induced already after 1-4 hours. These genes were of special interest since accumulation of metabolites started after 12 hours of elicitation. No homology to a known sequence was found for 16% of the tags whereas 66% were similar to genes with a known function, and 18% revealed similarity to genes without an allocated function. Functional analysis in transgenic cell lines is focused on genes encoding signal transduction proteins, kinases, phosphatases and transcription factors as well as those with unknown function. Besides identifying several novel genes, we were able to characterize poorly understood branches of secondary metabolite biosynthetic pathways in tobacco, leading to nicotine alkaloids and phenylpropanoids, by using hyphenated analytical tools (GC-, HPLC-MS). The great advantage of this novel technology platform is its universal application to any plant or cell culture of interest without pre-existing gene sequence databases