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Endocytosis of metals in plants is a growing field of study involving metal uptake from the rhizosphere. Uranium, which is naturally and artificially released into the rhizosphere, is known to be taken up by certain species of plant, such as Nicotiana tabacum, and we hypothesize that endocytosis contributes to the uptake of uranium in tobacco. The endocytic uptake of uranium was investigated in tobacco BY-2 cells using an optimized setup of culture in phosphate-deficient medium. A combination of methods in biochemistry, microscopy and spectroscopy, supplemented by proteomics, were used to study the interaction of uranium and the plant cell. We found that under environmentally relevant uranium concentrations, endocytosis remained active and contributed to 14% of the total uranium bioassociation. Proteomics analyses revealed that uranium induced a change in expression of the clathrin heavy chain variant, signifying a shift in the type of endocytosis taking place. However, the rate of endocytosis remained largely unaltered. Electron microscopy and energy-dispersive X-ray spectroscopy showed an adsorption of uranium to cell surfaces and deposition in vacuoles. Our results demonstrate that endocytosis constitutes a considerable proportion of uranium uptake in BY-2 cells, and that endocytosed uranium is likely targeted to the vacuole for sequestration, providing a physiologically safer route for the plant than uranium transported through the cytosol.Graphical abstract
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Piperine (1-piperoyl piperidine) is responsible for the pungent perception of dried black pepper (Pipernigrum) fruits and essentially contributes to the aromatic properties of this spice in combination with ablend of terpenoids. The final step in piperine biosynthesis involves piperine synthase (PS), which catalyzesthe 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 thesink organs is a possible alternative. This work, which includes purification of the native enzyme, immunolocalization, laser microdissection, fluorescence microscopy, and electron microscopy combinedwith liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), providesexperimental evidence that piperine and PS are co-localized in specialized cells of the black pepper fruit peri-sperm. PS accumulates during early stages of fruit development and its level declines before the fruits arefully mature. The product piperine is co-localized to PS and can be monitored at the cellular level by itsstrong bluish fluorescence. Rising piperine levels during fruit maturation are consistent with the increasingnumbers of fluorescent cells within the perisperm. Signal intensities of individual laser-dissected cells whenmonitored by LC-ESI-MS/MS indicate molar concentrations of this alkaloid. Significant levels of piperineand additional piperamides were also detected in cells distributed in the cortex of black pepper roots. Insummary, the data provide comprehensive experimental evidence of and insights into cell-specific biosyn-thesis and storage of piperidine alkaloids, specific and characteristic for the Piperaceae. By a combination offluorescence microscopy and LC-MS/MS analysis we localized the major piperidine alkaloids to specific cellsof the fruit perisperm and the root cortex. Immunolocalization of native piperine and piperamide synthasesshows that enzymes are co-localized with high concentrations of products in these idioblasts.