A novel technology for the discovery of plant secondary metabolite pathways by integrating transcript and metabolic profiling

Many of the valuable plant secondary metabolites are still isolated from whole plants, since the chemical synthesis of these often multi-chiral compounds is economically unfeasable. Even if plant cell culture systems have been exploited to bridge the problems much encountered with the time-consuming cultivation of whole plants, secondary product yields from cell cultures are often very low. Plant metabolic engineering has met only limited success, 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 technology Solucel®, which is applicable to any plant or cell culture without pre-excisting sequence knowledge of genes studied. Integration of cDNA-AFLP based transcript profiling technique with the targeted metabolic profiling the simultaneous identification of the genes involved in plant secondary metabolism is accomplished. As a model system we used Nicotiana tabacum (BY-2) cell culture in which the nicotine alkaloid production was induced by elicitor. From altogether 20 000 transcript tags visualised, 591 were either induced or repressed by methyl jasmonate. 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. Nicotine alkaloids accumulated in elicited cells following divergent kinetic pattern. For the first time alkaloid anatalline was shown to accumulate in tobacco cell cultures, being inducible by methyl jasmonate. Anatalline was present in two isomeric forms, further characterization of this compound was performed with NMR studies. Functional analysis of the methyl jasmonate modulated 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. Overall 47 genes were chosen for functional testing, of which 22 have now been overexpressed in BY-2 culture. So far, of the genes assayed, 3 have shown to result in an altered metabolite accumulation pattern compared to control cell line and those lines will be subjected for further studies. So-called Combinatorial Biochemistry approach is used with the genes deriving from tobacco transcriptional profiling. Genes are transformed to related species leading to the formation of transgenic hairy roots. Up to date, one gene has shown interesting effect in Hyoscyamus hairy roots, where besides exceptionally high tropane alkaloid contents, also remarkable changes in secondary metabolite profile was observed, indicating an important role in secondary metabolism.