@ARTICLE{Ibatullin20091741,
author={Ibatullin, F.M. and Banasiak, A. and Baumann, M.J. and Greffe, L. and Takahashi, J. and Mellerowicz, E.J. and Brumer, H.},
title={A real-time fluorogenic assay for the visualization of glycoside hydrolase activity in planta},
journal={Plant Physiology},
year={2009},
volume={151},
number={4},
pages={1741-1750},
doi={10.1104/pp.109.147439},
note={cited By 17},
url={https://www.scopus.com/inward/record.uri?eid=2-s2.0-71049171637&doi=10.1104%2fpp.109.147439&partnerID=40&md5=ab771a8e83aebb30992520d40ba96710},
affiliation={School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Centre, SE-10691 Stockholm, Sweden; Petersburg Nuclear Physics Institute, Russian Academy of Science, Molecular and Radiation Biology Division, Gatchina, St. Petersburg 188300, Russian Federation; Department of Forest Genetics and Plant Physiology, SLU, Umea Plant Science Centre, 90183 Umea, Sweden; Institute of Plant Biology, University of Wroclaw, 50-328 Wroclaw, Poland},
abstract={There currently exists a diverse array of molecular probes for the 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 endogenous glycoside hydrolases and transglycosidases responsible for cell wall remodeling. To exemplify the potential of fluorogenic resorufin glycosides to address this issue, a resorufin β-glycoside of a xylogluco-oligosaccharide (XXXG-β-Res) was synthesized as a specific substrate for in planta analysis of XEH activity. The resorufin aglycone is particularly distinguished for high sensitivity in muro assays due to a low pKa (5.8) and large extinction coefficient (ε 62,000 M-1cm-1), 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-β-Res is hydrolyzed by the archetypal plant XEH, nasturtium (Tropaeolum majus) NXG1, with classical Michaelis-Menten substrate saturation kinetics and a linear dependence on both enzyme concentration and incubation time. Further, XEH activity could be visualized in real time by observing the localized increase in fluorescence in germinating nasturtium seeds and Arabidopsis (Arabidopsis thaliana) inflorescent stems by confocal microscopy. Importantly, this new in situ XEH assay provides an essential complement to the in situ xyloglucan endotransglycosylase assay, thus allowing delineation of the disparate activities encoded by xyloglucan endotransglycosylase/hydrolase genes directly in plant tissues. The observation that XXXG-β-Res is also hydrolyzed by diverse microbial XEHs indicates that this substrate, and resorufin glycosides in general, may find broad applicability for the analysis of wall restructuring by polysaccharide hydrolases during morphogenesis and plant-microbe interactions. © 2009 American Society of Plant Biologists.},
correspondence_address1={Brumer, H.; School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Centre, SE-10691 Stockholm, Sweden; email: harry@biotech.kth.se},
issn={00320889},
coden={PLPHA},
pubmed_id={19783642},
language={English},
abbrev_source_title={Plant Physiol.},
document_type={Article},
source={Scopus},
}

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In contrast, however, there is a paucity of methods for the direct analysis of endogenous glycoside hydrolases and transglycosidases responsible for cell wall remodeling. To exemplify the potential of fluorogenic resorufin glycosides to address this issue, a resorufin β-glycoside of a xylogluco-oligosaccharide (XXXG-β-Res) was synthesized as a specific substrate for in planta analysis of XEH activity. The resorufin aglycone is particularly distinguished for high sensitivity in muro assays due to a low pKa (5.8) and large extinction coefficient (ε 62,000 M-1cm-1), 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-β-Res is hydrolyzed by the archetypal plant XEH, nasturtium (Tropaeolum majus) NXG1, with classical Michaelis-Menten substrate saturation kinetics and a linear dependence on both enzyme concentration and incubation time. Further, XEH activity could be visualized in real time by observing the localized increase in fluorescence in germinating nasturtium seeds and Arabidopsis (Arabidopsis thaliana) inflorescent stems by confocal microscopy. Importantly, this new in situ XEH assay provides an essential complement to the in situ xyloglucan endotransglycosylase assay, thus allowing delineation of the disparate activities encoded by xyloglucan endotransglycosylase/hydrolase genes directly in plant tissues. The observation that XXXG-β-Res is also hydrolyzed by diverse microbial XEHs indicates that this substrate, and resorufin glycosides in general, may find broad applicability for the analysis of wall restructuring by polysaccharide hydrolases during morphogenesis and plant-microbe interactions. © 2009 American Society of Plant Biologists.","correspondence_address1":"Brumer, H.; School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Centre, SE-10691 Stockholm, Sweden; email: harry@biotech.kth.se","issn":"00320889","coden":"PLPHA","pubmed_id":"19783642","language":"English","abbrev_source_title":"Plant Physiol.","document_type":"Article","source":"Scopus","bibtex":"@ARTICLE{Ibatullin20091741,\r\nauthor={Ibatullin, F.M. and Banasiak, A. and Baumann, M.J. and Greffe, L. and Takahashi, J. and Mellerowicz, E.J. and Brumer, H.},\r\ntitle={A real-time fluorogenic assay for the visualization of glycoside hydrolase activity in planta},\r\njournal={Plant Physiology},\r\nyear={2009},\r\nvolume={151},\r\nnumber={4},\r\npages={1741-1750},\r\ndoi={10.1104/pp.109.147439},\r\nnote={cited By 17},\r\nurl={https://www.scopus.com/inward/record.uri?eid=2-s2.0-71049171637&doi=10.1104%2fpp.109.147439&partnerID=40&md5=ab771a8e83aebb30992520d40ba96710},\r\naffiliation={School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Centre, SE-10691 Stockholm, Sweden; Petersburg Nuclear Physics Institute, Russian Academy of Science, Molecular and Radiation Biology Division, Gatchina, St. Petersburg 188300, Russian Federation; Department of Forest Genetics and Plant Physiology, SLU, Umea Plant Science Centre, 90183 Umea, Sweden; Institute of Plant Biology, University of Wroclaw, 50-328 Wroclaw, Poland},\r\nabstract={There currently exists a diverse array of molecular probes for the in situ localization of polysaccharides, nucleic acids, and proteins in plant cells, including reporter enzyme strategies (e.g. protein-glucuronidase fusions). 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Further, XEH activity could be visualized in real time by observing the localized increase in fluorescence in germinating nasturtium seeds and Arabidopsis (Arabidopsis thaliana) inflorescent stems by confocal microscopy. Importantly, this new in situ XEH assay provides an essential complement to the in situ xyloglucan endotransglycosylase assay, thus allowing delineation of the disparate activities encoded by xyloglucan endotransglycosylase/hydrolase genes directly in plant tissues. 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