A Real-Time Fluorogenic Assay for the Visualization of Glycoside Hydrolase Activity in Planta

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A Real-Time Fluorogenic Assay for the Visualization of Glycoside Hydrolase Activity in Planta

Ibatullin, Farid M. (author): KTH,Skolan för bioteknologi (BIO)

Banasiak, Alicja (author)

Baumann, Martin J. (author): KTH,Skolan för bioteknologi (BIO)

Greffe, Lionel (author): KTH,Skolan för bioteknologi (BIO)

Takahashi Schmidt, Junko (author): Swedish University of Agricultural Sciences,Sveriges lantbruksuniversitet,Institutionen för skoglig genetik och växtfysiologi,Department of Forest Genetics and Plant Physiology

Mellerowicz, Ewa (author): Swedish University of Agricultural Sciences,Sveriges lantbruksuniversitet,Institutionen för skoglig genetik och växtfysiologi,Department of Forest Genetics and Plant Physiology

Brumer, Harry (author): KTH,Glykovetenskap

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(creator_code:org_t)

2009-09-25
2009
English.
In: Plant Physiology. - : Oxford University Press (OUP). - 0032-0889 .- 1532-2548. ; 151:4, s. 1741-1750

Related links:: http://www.plantphys...; show more...; https://urn.kb.se/re...; https://doi.org/10.1...; https://res.slu.se/i...; show less...

Journal article (peer-reviewed)

Abstract Subject headings

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 beta-glycoside of a xylogluco-oligosaccharide (XXXG-beta-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 pK(a) (5.8) and large extinction coefficient (epsilon 62,000 M-1 cm(-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-beta-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-beta-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.

Subject headings

LANTBRUKSVETENSKAPER -- Lantbruksvetenskap, skogsbruk och fiske -- Skogsvetenskap (hsv//swe)
AGRICULTURAL SCIENCES -- Agriculture, Forestry and Fisheries -- Forest Science (hsv//eng)

Keyword

xyloglucan endo-transglycosylase
transfer-catalyzed synthesis
single-enzyme molecules
beta-d-glucopyranosides
cell-wall metabolism
structural-analysis
in-vivo
polysaccharide degradation
ph
regulation
oligosaccharides

Publication and Content Type

ref (subject category)
art (subject category)

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By the author/editor: Ibatullin, Farid ...; Banasiak, Alicja; Baumann, Martin ...; Greffe, Lionel; Takahashi Schmid ...; Mellerowicz, Ewa; show more...; Brumer, Harry; show less...

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AGRICULTURAL SCIENCES: AGRICULTURAL SCI ...; and Agriculture Fore ...; and Forest Science

Articles in the publication: Plant Physiology

By the university: Royal Institute of Technology; Swedish University of Agricultural Sciences

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