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

胡椒碱是黑胡椒（Piper nigrum L.）的刺激性成分和主要生物碱。在本提案中，我们将描述胡椒碱生物合成途径的剩余步骤，并在细胞水平上研究胡椒碱生物合成。该途径的未知酶解步骤包括假定的苯丙衍生物前体的关键链延伸和哌啶生物合成所需的全套基因。该项目基于之前生成的差异转录组数据，并且已经揭示了许多与编码胡椒碱合成酶的转录本共表达的候选转录本。生物合成的关键酶促步骤。利用金门模块克隆系统在烟叶中瞬时表达完整的胡椒碱生物合成，将最终验证所提出的胡椒碱生物合成途径，并使我们能够建立具有潜在生物学或药理活性的胡椒酰胺的生产。为了了解胡椒碱在细胞水平上的生物合成，我们将对黑胡椒果实中处于两个不同发育阶段的胡椒碱积累细胞与不积累胡椒胺的邻近细胞进行单细胞代谢组学、蛋白质组学和转录组学研究。在果实外胚层和根皮层的细胞中观察到胡椒碱和胡椒酰胺的特征蓝色荧光，有助于鉴定这些细胞。果实的胡椒碱积累细胞将与荧光根皮质细胞进行比较，这些细胞积累了不同的胡椒酰胺混合物。果实和根细胞可以通过激光捕获显微解剖分离。靶向代谢组学将使我们能够鉴定细胞特异性代谢物，包括胡椒碱形成的潜在前体，而靶向蛋白质组学将支持鉴定可能参与胡椒酰胺形成的酶和蛋白质。数据将通过分析细胞特异性转录物形成来完成。提出了两种获取单细胞转录组数据的方法：激光捕获显微解剖和原生质体分离，结合FACS细胞分选。单细胞方法不仅可以让我们在细胞水平上获得胡椒碱生物合成的完整图像，而且可以为这些细胞如何能够合成和储存摩尔浓度的几乎不溶于水的化合物（如胡椒碱）提供答案。

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.