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A Real-Time Fluorogenic Assay for the Visualization of Glycoside Hydrolase Activity in Planta |
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Ibatullin FM, Banasiak A, Baumann MJ, Greffe L, Takahashi J, Mellerowicz EJ, Brumer H, 2009, A Real-Time Fluorogenic Assay for the Visualization of Glycoside Hydrolase Activity in Planta. Plant Physiology, 151: 1741–1750.
There currently exists a diverse array of molecular probes for the in situ localization of polysaccharides, nucleic acids, andproteins in plant cells, including reporter enzyme strategies (e.g. protein-glucuronidase fusions). In contrast, however, there isa paucity of methods for the direct analysis of endogenous glycoside hydrolases and transglycosidases responsible for cell wallremodeling. To exemplify the potential of fluorogenic resorufin glycosides to address this issue, a resorufin b-glycoside of axylogluco-oligosaccharide (XXXG-b-Res) was synthesized as a specific substrate for in planta analysis of XEH activity. Theresorufin aglycone is particularly distinguished for high sensitivity in muro assays due to a low pKa (5.8) and large extinctioncoefficient (« 62,000 M21cm21), long-wavelength fluorescence (excitation 571 nm/emission 585 nm), and high quantum yield(0.74) of the corresponding anion. In vitro analyses demonstrated that XXXG-b-Res is hydrolyzed by the archetypal plant XEH,nasturtium (Tropaeolum majus) NXG1, with classical Michaelis-Menten substrate saturation kinetics and a linear dependence onboth enzyme concentration and incubation time. Further, XEH activity could be visualized in real time by observing thelocalized increase in fluorescence in germinating nasturtium seeds and Arabidopsis (Arabidopsis thaliana) inflorescent stems byconfocal microscopy. Importantly, this new in situ XEH assay provides an essential complement to the in situ xyloglucanendotransglycosylase assay, thus allowing delineation of the disparate activities encoded by xyloglucan endotransglycosylase/hydrolase genes directly in plant tissues. The observation that XXXG-b-Res is also hydrolyzed by diverse microbial XEHsindicates that this substrate, and resorufin glycosides in general, may find broad applicability for the analysis of wallrestructuring by polysaccharide hydrolases during morphogenesis and plant-microbe interactions.
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