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

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 pK_a (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.The development and application of molecular probes for the localization of biomolecules in planta continues to have a profound impact on the field of plant physiology. A number of elegant techniques have been devised for the detection of nucleic acids, polypeptides, and polysaccharides in situ, including DNA/RNA hybridization (Jin and Lloyd, 1997), reporter protein fusions (Jefferson et al., 1987; Ehrhardt, 2003; Chapman et al., 2005; Stewart, 2005; Berg and Beachy, 2008; Nelson et al., 2008), immunohistochemical methods (Walker et al., 2001; Chapman et al., 2005), applications of natural carbohydrate-binding proteins (Knox, 2008), and direct spectroscopy (Vicente et al., 2007). While there now exists a considerable toolbox to identify the location to which biomolecules are directed in the cell, elucidation of specific biochemical function at the site of localization often remains challenging.Presently, there is a growing interest in the roles of glycoside hydrolases (GHs) and transglycosylases in plant cell wall biogenesis, remodeling, and degradation (Minic and Jouanin, 2006; Vicente et al., 2007; Gilbert et al., 2008; Lopez-Casado et al., 2008). A technical limitation of many studies, however, is that enzyme activities can only be measured for crude whole-tissue extracts, or purified or recombinant enzymes, and thus cannot be directly correlated with the high-resolution in situ localization of other biomacromolecules. As such, the in situ analysis of GH activities responsible for the degradation of plant cell wall polysaccharides has received comparatively little attention, primarily due to a paucity of convenient assay methods (Vicente et al., 2007). Some notable exceptions include the use of commercially available X (5-bromo-4-chloro-3-indolyl glycoside) substrates for the detection of exoglycosidase activity (Monroe et al., 1999; Chantarangsee et al., 2007; Macquet et al., 2007; Wen et al., 2008). Likewise, transglycosylase activity has been visualized in higher plant and yeast cell walls using sulforhodamine-oligosaccharide acceptor substrates (Vissenberg et al., 2000; Bourquin et al., 2002; Nishikubo et al., 2007; Cabib et al., 2008). Both types are examples of end point, or stopped, assays, in which precipitated indigoid dyes or incorporated fluorescent oligosaccharide conjugate, respectively, are observed after a terminal incubation time.In this study, we have developed the use of resorufin glycosides as substrates for the real-time, continuous observation of GH activity in situ (Fig. 1). Enzymatic hydrolysis of such substrates releases the resorufin aglycone, which is distinguished by a low pK_a value (5.8) and a large extinction coefficient (ε 62,000 m⁻¹cm⁻¹), long-wavelength fluorescence (excitation/emission maxima, 571 nm/585 nm), and high quantum yield (0.74) of the resorufinyl anion (Bueno et al., 2002). The pK_a value and spectral properties make resorufin glycosides particularly suitable for high sensitivity in muro enzyme activity assays due to significant ionization of resorufin at typical apoplastic pH values (Felle, 2005). To highlight the potential of this class of substrates in cell wall morphological studies, we have chemically synthesized a xylogluco-oligosaccharide (XGO) resorufin β-glycoside (XXXG-β-Res; Fig. 1 1]; XGO nomenclature according to Fry et al., 1993) and demonstrated its use for the real-time imaging of xyloglucan endohydrolase (XEH) activity in plant tissues from nasturtium (Tropaeolum majus) and Arabidopsis (Arabidopsis thaliana) by confocal fluorescence microscopy.Open in a separate windowFigure 1.Use of resorufin glycosides as fluorogenic substrates for glycosidases. R = saccharide or hydrogen; 1] and 2], substrates for determination of (xylo)glucanase activity. Oligosaccharide nomenclature is according to Fry et al. (1993). See online article for color version of this figure.]