The plant secondary metabolism - unravelling the metabolite pathways by functional genomics tools

Plants are able to produce chemically diverse secondary metabolites, which have important functions for plants in survival as well as for human use, e.g. as high-value pharmaceuticals, dyes and pesticides. These compounds are often highly complex in their structures and thus their production by chemical synthesis becomes economically unfeasible. Many of the valuable plant-derived pharmaceuticals, e.g. anticancer drugs are still extracted from whole plants. However, the bioproduction of these metabolites in plants are often extremely low and thus plant cell and tissue cultures have been exploited as to overcome the problems associated with the time-consuming cultivation of whole plants. Plant metabolic engineering has met only limited success, in contrast to micro-organisms, since our knowledge about biosynthetic pathways of secondary compounds, and the regulation thereof, are still poorly understood in particular at the genetic level. We designed a novel approach, in which a cDNA-AFLP based transcript-profiling technique is linked with targeted metabolic profiling to simultaneously identify the genes involved in plant secondary metabolism. The model system consisted of tobacco BY-2 cell culture that was treated with methyl jasmonate for the induction of the metabolite production. From the 20000 transcript tags visualised 591 were jasmonate-modulated. Cluster analysis of the expression profiles showed that the half of the genes were induced already after 1-4 hours. Homology searches with the sequences revealed that 58 % of the tags displayed similarity with genes of known functions and 16 % with the gene without allocated function. No homology to a known sequence was found for 26 % of the tags. The accumulation of nicotine alkaloid metabolites started after 12 hours and the metabolites were produced with different kinetics. Functional analysis of these genes is being performed by using transgenic cell lines, with the special interest in genes encoding putative proteins of unknown function and signal transducing proteins. Besides identifying several novel genes, we were able to elucidate the branches of secondary metabolite biosynthesis in tobacco leading to nicotine alkaloids and phenylpropanoids by using hyphenated analytical tools (GC-, HPLC-MS). This powerful novel technology is applicable to any plant or cell culture without pre-excisting knowledge of gene sequences.