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JXB Advance Access published online on May 23, 2005
Journal of Experimental Botany, doi:10.1093/jxb/eri185
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© The Author [2005]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: journals.permissions@oupjournals.org
Received December 24, 2004
Accepted April 7, 2005

RESEARCH PAPER

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

Ed Etxeberria 1*, Pedro González 2, Patricia Tomlinson 3, and Javier Pozueta-Romero 4

1 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; Agrobioteknologia eta Natura Baliabideetako Instituta, Nafarroako Unibertsitate Publikoa and Consejo Superior de Investigaciones Científicas, Mutiloako etorbidea zembaki gabe, 31192 Mutiloabeti, Nafarroa, Spain
2 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
3 Department of Biological Sciences, Berry College, Mt. Berry GA, 30149, USA
4 Agrobioteknologia eta Natura Baliabideetako Instituta, Nafarroako Unibertsitate Publikoa and Consejo Superior de Investigaciones Científicas, Mutiloako etorbidea zembaki gabe, 31192 Mutiloabeti, Nafarroa, Spain

* To whom correspondence should be addressed.
Ed Etxeberria, E-mail: eje{at}crec.ifas.ufl.edu


  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.

Keywords: Cytoplasts; endocytosis; hexose symporter; photoassimilate transport; vacuole.
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