Cell specific piperine biosynthesis in black pepper (Piper nigrum L.) fruits and roots

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Piperine is the pungent principle and the main alkaloid of black pepper (Piper nigrum L.). In this proposal we will characterize remaining steps of the piperine biosynthetic pathway and investigate piperine biosynthesis also at the cellular level. Unknown enzymatic steps of the pathway include the critical chain extension of a presumed phenylpropanoid derived precursor and the complete set of genes required for piperidine biosynthesis. This project is based on differential transcriptome data that were previously generated and already revealed a number of candidate transcripts that were co-expressed with transcripts encoding piperine synthase. a key enzymatic step of the biosynthesis. Transient expression of the complete piperine biosynthesis in Nicotiana benthamiana using the Golden Gate modular cloning system will finally verify the suggested piperine biosynthesis pathway and may enable us to establish the production of potentially biologically or pharmacologically active piperamides. In order to understand piperine biosynthesis at the cellular level, we will perform single cell metabolomics, proteomics, and also transcriptomics of individual piperine accumulating cells of black pepper fruits at two different stages of development versus neighboring cells that do not accumulate piperamides. Identification of these cells is facilitated by the characteristic blue fluorescence of piperine and piperamides observed in cells of the fruit perisperm as well as the root cortex. Piperine accumulating cells of the fruits will be compared with fluorescing root cortex cells that accumulate a distinct blend of piperamides. Fruit and root cells can be isolated by laser capture microdissection. Targeted metabolomics will enable us to identify cell specific metabolites including potential precursors of piperine formation while targeted proteomics will support identification of enzymes and proteins that might be participating in piperamide formation. Data will be completed by analyzing cell specific transcript formation. Two alternative methods are suggested to obtain single cell transcriptome data: laser capture microdissection and isolation of protoplasts, combined with cell sorting by FACS. The single cell approach will not only allow us to obtain a complete picture of piperine biosynthesis at the cellular level but may provide answers to the puzzling question how these cells are able to synthesize and store virtually water insoluble compounds like piperine in molar concentrations.