Visualization of arginine influx into plant cells using a specific FRET-sensor.
Amino acids are not only the building blocks of proteins, but are the metabolic precursors of a variety of primary and secondary metabolites. In order to detect and visualize how plants transport, sense, and store amino acids with sub-cellular specificity, chimeric fluorescent proteins that respond to changes in amino acid concentrations were constructed. The reporter element of these sensors consists of a periplasmic bacterial protein that undergoes large, non-enzymatic conformational changes upon binding of its substrate. The receptor protein was attached to ECFP and an environmentally insensitive YFP derivative at opposite ends. Fluorescence resonance energy transfer changes were specifically observed after addition of arginine and to a lesser extent ornithine. The recombinant sensor showed a concentration-dependent increase in the fluorescence ratio with an apparent in vitro affinity for arginine of approximately 2 mM. A mutation in the binding pocket lowered the affinity and decreased the specificity. When expressed in E. coli, an increase in the fluorescence ratio was specifically detected after exposure to arginine and ornithine. Transient expression of the sensor in plant cell protoplasts and stable expression in Arabidopsis roots revealed specific fluorescence changes upon addition of arginine. The analysis suggests that fluorescent amino acid sensors may be versatile tools for studying the in vivo dynamics of metabolism and compartmentalization in plants.
Bogner
Martin
M
Zentrum für Molekularbiologie der Pflanzen, Pflanzenphysiologie, Universität Tübingen, Auf der Morgenstelle 1, 72076 Tübingen, Germany.
Ludewig
Uwe
U
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D001120:Arginine / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001693:Biological Transport, Active; D002451:Cell Compartmentation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009952:Ornithine / Q000378:metabolism; D018517:Plant Roots / Q000378:metabolism; D030821:Plants, Genetically Modified; D011523:Protoplasts / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D001120:Arginine; D009952:Ornithine