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Abstract Manganese (Mn2+) is essential for a diversity of processes, including photosynthetic water splitting and the transfer of glycosyl moieties. Various Golgi-localized glycosyltransferases that mediate cell wall matrix polysaccharide biosynthesis are Mn2+ dependent, but the supply of these enzymes with Mn2+ is not well understood. Here, we show that the BIVALENT CATION TRANSPORTER 3 (BICAT3) localizes specifically to trans-cisternae of the Golgi. In agreement with a role in Mn2+ and Ca2+ homeostasis, BICAT3 rescued yeast (Saccharomyces cerevisiae) mutants defective in their translocation. Arabidopsis (Arabidopsis thaliana) knockout mutants of BICAT3 were sensitive to low Mn2+ and high Ca2+ availability and showed altered accumulation of these cations. Despite reduced cell expansion and leaf size in Mn2+-deficient bicat3 mutants, their photosynthesis was improved, accompanied by an increased Mn content of chloroplasts. Growth defects of bicat3 corresponded with an impaired glycosidic composition of matrix polysaccharides synthesized in the trans-Golgi. In addition to the vegetative growth defects, pollen tube growth of bicat3 was heterogeneously aberrant. This was associated with a severely reduced and similarly heterogeneous pectin deposition and caused diminished seed set and silique length. Double mutant analyses demonstrated that the physiological relevance of BICAT3 is distinct from that of ER-TYPE CA2+-ATPASE 3, a Golgi-localized Mn2+/Ca2+-ATPase. Collectively, BICAT3 is a principal Mn2+ transporter in the trans-Golgi whose activity is critical for specific glycosylation reactions in this organelle and for the allocation of Mn2+ between Golgi apparatus and chloroplasts.
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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.