h+-independent glutamine transport in plant root tipsh +独立谷氨酰胺运输在植物根技巧.pdfVIP

+ H -Independent Glutamine Transport in Plant Root Tips 1,2 2 3 1 Huaiyu Yang , Martin Bogner , York-Dieter Stierhof , Uwe Ludewig * 1 Plant Biomass and Nutrition, Institute of Botany, Darmstadt University of Technology, Darmstadt, Germany, 2 Plant Physiology, Center for Molecular Biology of Plants ¨ ¨ ¨ ¨ (ZMBP), University of Tubingen, Tubingen, Germany, 3 Microscopy Unit, Center for Molecular Biology of Plants (ZMBP), University of Tubingen, Tubingen, Germany Abstract Background: Glutamine is one of the primary amino acids in nitrogen assimilation and often the most abundant amino acid in plant roots. To monitor this important metabolite, a novel genetically encoded fluorescent FRET-reporter was constructed and expressed in Arabidopsis thaliana. As a candidate for the glutamine fluxes, the root tip localized, putative amino acid transporter CAT8 was analyzed and heterologously expressed in yeast and oocytes. Principal Findings: Rapid and reversible in vivo fluorescence changes were observed in reporter-expressing root tips upon exposure and removal of glutamine. FRET changes were detected at acid and neutral pH and in the presence of a protonophore, suggesting that part of the glutamine fluxes were independent of the pH. The putative amino acid transporter CAT8 transported glutamine, had a half maximal activity at ,100 mM and the transport was independent of external pH. CAT8 localized not only to the plasma membrane, but additionally to the tonoplast, when tagged with GFP. Ultrastructural analysis confirmed this dual localization and additionally identified CAT8