4.7 Article

Existence of two parallel mechanisms for glucose uptake in heterotrophic plant cells

Journal

JOURNAL OF EXPERIMENTAL BOTANY
Volume 56, Issue 417, Pages 1905-1912

Publisher

OXFORD UNIV PRESS
DOI: 10.1093/jxb/eri185

Keywords

cytoplasts; endocytosis; hexose symporter; photoassimilate transport; vacuole

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\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 colocalization 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: (1) a plasmalemma-bound carrier-mediated system that facilitates 2-NBDG transport into the cytosol, and (ii) an enclocytic system that transports most of 2-NBDG directly into the vacuole.

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