Ibatullin FM, Banasiak A, Baumann MJ, Greffe L, Takahashi J, Mellerowicz EJ, Brumer H
A real-time fluorogenic assay for the visualization of glycoside hydrolase activity in plants
Plant Physiology: 2009 151:1741-1750

Abstract
There currently exists a diverse array of molecular probes forthe in situ localization of polysaccharides, nucleic acids,and proteins in plant cells, including reporter enzyme strategies(e.g. protein-glucuronidase fusions). In contrast, however,there is a paucity of methods for the direct analysis of endogenousglycoside hydrolases and transglycosidases responsible for cellwall remodeling. To exemplify the potential of fluorogenic resorufinglycosides to address this issue, a resorufin β-glycosideof a xylogluco-oligosaccharide (XXXG-β-Res) was synthesizedas a specific substrate for in planta analysis of XEH activity.The resorufin aglycone is particularly distinguished for highsensitivity in muro assays due to a low pKa (5.8) and largeextinction coefficient ({epsilon} 62,000 M–1cm–1), long-wavelengthfluorescence (excitation 571 nm/emission 585 nm), and high quantumyield (0.74) of the corresponding anion. In vitro analyses demonstratedthat XXXG-β-Res is hydrolyzed by the archetypal plant XEH,nasturtium (Tropaeolum majus) NXG1, with classical Michaelis-Mentensubstrate saturation kinetics and a linear dependence on bothenzyme concentration and incubation time. Further, XEH activitycould be visualized in real time by observing the localizedincrease in fluorescence in germinating nasturtium seeds andArabidopsis (Arabidopsis thaliana) inflorescent stems by confocalmicroscopy. Importantly, this new in situ XEH assay providesan essential complement to the in situ xyloglucan endotransglycosylaseassay, thus allowing delineation of the disparate activitiesencoded by xyloglucan endotransglycosylase/hydrolase genes directlyin plant tissues. The observation that XXXG-β-Res is alsohydrolyzed by diverse microbial XEHs indicates that this substrate,and resorufin glycosides in general, may find broad applicabilityfor the analysis of wall restructuring by polysaccharide hydrolasesduring morphogenesis and plant-microbe interactions.

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