Elucidation of mechanisms required for convergent evolution of secondary metabolite pathways in plants

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Plants synthesise a multitude of secondary metabolites. Variability seems to be essential for the function of these Specialised metabolites in communication with the environment and as an arsenal for chemical defence. Establishment of secondary metabolic pathways is based on the common primary metabolism. Specific enzymes are then involved to generate the secondary metabolites, these are e.g. cytochrome P450 monooxygenases, 2-oxoglutarate dependent dioxygenases, methyltransferases, glycosyltransferases. In the genomes large gene families of these modifying enzymes constitute the Toolbox from which unique enzymes are recruited. Little is known about the principles to choose a member of this arsenal, to shape it for the requirements of a distinct step in biosynthesis and to coordinate the reaction sequence. Isolation and comparison of complete sets of biosynthetic genes for pathways that result from convergent evolution will allow getting insight into these mechanisms. Benzoxazinoid biosynthesis is well suited for such an analysis.Benzoxazinoids are defence-related secondary metabolites found in three orders of the angiosperms: Poales, Ranunculales and Lamiales. The complex biosynthesis is biochemically and on gene level (Bx-genes) completely elucidated in maize. All of the 8 biosynthetic genes are located in a cluster, such a genomic organisation has meanwhile been detected for further defence-related biosynthetic pathways. In the grasses the Bx-genes are monophyletic. We have isolated the first gene of the pathway, Bx1, from Consolida orientalis (Ranunculaceae) and Lamium galeobdolon (Lamiaceae). BX1 is a homolog of the alpha-subunit of tryptophan synthase and has been recruited in each of the three orders independently. Recently we could show for C. orientalis that the specific UDP-glucosyltransferase and beta-glucosidase of the pathway evolved by repeated evolution recruiting members of the respective gene families that are non-orthologous to the monocot Bx-gene lineages. No data for these genes are available for the Lamiales.We propose to isolate all missing benzoxazinois-specific genes of C. orientalis and L. galeobdolon. The comparison of the biosynthetic genes of the three orders will reveal preservation of the sequence of reaction steps, possibly existing restrictions for the recruitment of genes out of the given pool, elucidate whether modifications of recruited genes follow a common pattern and whether the formation of a gene cluster parallels for the establishment of the biosynthetic pathway. Candidate genes will be identified by Transcriptomics and verified by functional characterisation in heterologous systems. To get a further independent proof for the isolation of benzoxazinoid-specific genes we intend to establish Virus Induced Gene Silencing, initially for C. orientalis.