title: Existence of two parallel mechanisms for glucose uptake in heterotrophic plant cells.
abstract: The implied existence of two mechanisms for glucose uptake into heterotrophic plant cells was investigated using the fluorescent glucose derivative 2-NBDG (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose), two membrane impermeable fluorescent markers (3000 mol. wt. dextran-Texas Red (d-TR) and Alexa-488), hexose carrier and endocytic inhibitors (phloridzin and wortmannin-A, respectively), and fluorescent and confocal microscopy. Both phloridzin and wortmannin-A significantly reduced the uptake of 2-NBDG into sycamore cultured cells, which was confirmed by fluorescent microscopy. Phloridzin prevented 2-NBDG uptake exclusively into the cytosol, whereas the wortmannin-A effect was more general, with 2-NBDG uptake into the vacuole being the more affected. Simultaneous incubation of cells in the membrane-impermeable fluorescent probes Alexa-488 and d-TR for 24 h resulted in co-localization of the labelling in the central vacuole and other endosomal compartments. Cytoplasts, cells devoid of vacuoles, were instrumental in demonstrating the transport of 2-NBDG by separate uptake mechanisms. In cytoplasts incubated simultaneously in 2-NBDG and d-TR for 2 h, a green fluorescent cytosol was indicative of transport of hexoses across the plasmalemma, while the co-localization of 2-NBDG and d-TR in internal vesicles demonstrated transport via an endocytic system. The absence of vesicles when cytoplasts were pre-incubated in wortmannin-A authenticated the endocytic vesicular nature of the co-shared 2-NBDG and d-TR fluorescent structures. In summary, uptake of 2-NBDG occurs by two separate mechanisms: (i) a plasmalemma-bound carrier-mediated system that facilitates 2-NBDG transport into the cytosol, and (ii) an endocytic system that transports most of 2-NBDG directly into the vacuole.
authors.affiliation: University of Florida, Institute of Food and Agricultural Sciences, Horticultural Sciences Department, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850-2299, USA. eje@crec.ifas.ufl.edu
authors.forename: Ed
authors.initials: E
authors.lastname: Etxeberria
authors.forename: Pedro
authors.initials: P
authors.lastname: González
authors.forename: Patricia
authors.initials: P
authors.lastname: Tomlinson
authors.forename: Javier
authors.initials: J
authors.lastname: Pozueta-Romero
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives*; D031002:Acer / Q000166:cytology / Q000378:metabolism*; D001693:Biological Transport, Active; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D003847:Deoxyglucose / Q000031:analogs & derivatives*; D004705:Endocytosis / Q000502:physiology*; D009004:Monosaccharide Transport Proteins / Q000502:physiology*; D010940:Plant Proteins / Q000502:physiology*
chemical_list: D009004:Monosaccharide Transport Proteins; D010940:Plant Proteins; D003847:Deoxyglucose; D009327:4-Chloro-7-nitrobenzofurazan; C098340:2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose