The terminal enzymatic step in piperine biosynthesis is co‐localized with the product piperine in specialized cells of black pepper (Piper nigrum L.). (20th June 2022)

• Record Type: Journal Article
• Authors: Jäckel, Luise; Schnabel, Arianne; Stellmach, Hagen; Klauß, Ulrike; Matschi, Susanne; Hause, Gerd; Vogt, Thomas
• Is Part Of: Plant journal. Volume 111:Number 3(2022)
• Page Start: 731
• Page End: 747
• Publication Date: 2022-06-20
• Subjects: Piper nigrum -- piperine -- specialized metabolism -- alkaloid -- enzyme purification -- fluorescence microscopy -- laser microdissection; Plant molecular biology -- Periodicals; Plant cells and tissues -- Periodicals; Botany -- Periodicals; 580
• DOI: 10.1111/tpj.15847
• Languages: English
• ISSNs: 0960-7412

Abstract

SUMMARY: Piperine (1‐piperoyl piperidine) is responsible for the pungent perception of dried black pepper ( Piper nigrum ) fruits and essentially contributes to the aromatic properties of this spice in combination with a blend of terpenoids. The final step in piperine biosynthesis involves piperine synthase (PS), which catalyzes the reaction of piperoyl CoA and piperidine to the biologically active and pungent amide. Nevertheless, experimental data on the cellular localization of piperine and the complete biosynthetic pathway are missing. Not only co‐localization of enzymes and products, but also potential transport of piperamides to the sink organs is a possible alternative. This work, which includes purification of the native enzyme, immunolocalization, laser microdissection, fluorescence microscopy, and electron microscopy combined with liquid chromatography electrospray ionization tandem mass spectrometry (LC‐ESI‐MS/MS), provides experimental evidence that piperine and PS are co‐localized in specialized cells of the black pepper fruit perisperm. PS accumulates during early stages of fruit development and its level declines before the fruits are fully mature. The product piperine is co‐localized to PS and can be monitored at the cellular level by its strong bluish fluorescence. Rising piperine levels during fruit maturation are consistent with the increasing numbers of fluorescent cells within the perisperm. Signal intensities of individual laser‐dissected cells when monitored by LC‐ESI‐MS/MS indicate molar concentrations of this alkaloid. Significant levels of piperine and additional piperamides were also detected in cells distributed in the cortex of black pepper roots. In summary, the data provide comprehensive experimental evidence of and insights into cell‐specific biosynthesis and storage of piperidine alkaloids, specific and characteristic for the Piperaceae. By a combination of fluorescence microscopy and LC‐MS/MS analysis we localized the major piperidine alkaloids to specific cells of the fruit perisperm and the root cortex. Immunolocalization of native piperine and piperamide synthases shows that enzymes are co‐localized with high concentrations of products in these idioblasts. Significance Statement: Piperine synthase and piperine localize in specialized cells of the black pepper fruit perisperm. While piperine synthase accumulation drops during fruit ripening, a steady increase in the number of fluorescent piperine‐accumulating idioblasts can be observed during fruit maturation in the perisperm. Laser microdissection and LC‐MS data confirm that piperine levels in these cells rise to molar concentrations. Piperine and other piperamides also accumulate in specialized cells of the root cortex.