Glycoside Hydrolase Activities in Cell Walls of Sclerenchyma Cells in the Inflorescence Stems of Arabidopsis thaliana Visualized in Situ

Techniques for in situ localization of gene products provide indispensable information for understanding biological function. In the case of enzymes, biological function is directly related to activity, and therefore, knowledge of activity patterns is central to understanding the molecular controls of plant development. We have previously developed a novel type of fluorogenic substrate for revealing glycoside hydrolase activity in planta, based on resorufin β-glycosides Here, we explore a wider range of such substrates to visualize glycoside hydrolase activities in Arabidopsis inflorescence stems in real time, especially highlighting distinct distribution patterns of these activities in the secondary cell walls of sclerenchyma cells. The results demonstrate that β-1,4-glucosidase, β-1,4-glucanase and β-1,4-galactosidase activities accompany secondary wall deposition. In contrast, xyloglucanase activity follows a different pattern, with the highest signal observed in mature cells, concentrated in the middle lamella. These data further the understanding of the process of cell wall deposition and function in sclerenchymatic tissues of plants.Science, Faculty ofOther UBCNon UBCChemistry, Department ofReviewedFacult

A Real-Time Fluorogenic Assay for the Visualization of Glycoside Hydrolase Activity in Planta1[C][OA]

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

Dispersion of the Thermodynamically Immiscible Polypropylene and Ethylene—Propylene Triple Synthetic Rubber Polymer Blends Using Supercritical SEDS Process: Effect of Operating Parameters

In this paper, we present the results of dispersion of thermodynamically immiscible polypropylene (PP) and ethylene-propylene triple synthetic rubber (EPTSR) polymer blends using the Solution-Enhanced Dispersion by Supercritical Fluid (SEDS) technique at operation conditions in the pressure range of (8 to 25) MPa and at temperatures t = 40 °C and 60 °C. The kinetics of crystallization and phase transformation in polymer blends obtained by conventional method (melt blending) and by mixing in the SEDS process have been studied using the DSC technique. The effects of the SEDS operation process on the physical—chemical (melting temperature, heat of fusion) and mechanical (microparticle size) characteristics of the SEDS-produced polymer blends were studied

Sequencing, biochemical characterization, crystal structure and molecular dynamics of cellobiohydrolase Cel7A from Geotrichum candidum

The ascomycete Geotrichum candidum is a versatile and efficient decay fungus that is involved, for example, in biodeterioration of compact discs; notably, the 3C strain was previously shown to degrade filter paper and cotton more efficiently than several industrial enzyme preparations. Glycoside hydrolase (GH) family 7 cellobiohydrolases (CBHs) are the primary constituents of industrial cellulase cocktails employed in biomass conversion, and feature tunnel-enclosed active sites that enable processive hydrolytic cleavage of cellulose chains. Understanding the structure-function relationships defining the activity and stability of GH7 CBHs is thus of keen interest. Accordingly, we report the comprehensive characterization of the GH7 CBH secreted by G. candidum (GcaCel7A). The bimodular cellulase consists of a family 1 cellulose-binding module (CBM) and linker connected to a GH7 catalytic domain that shares 64% sequence identity with the archetypal industrial GH7 CBH of Hypocrea jecorina (HjeCel7A). GcaCel7A shows activity on Avicel cellulose similar to HjeCel7A, with less product inhibition, but has a lower temperature optimum (50 °C versus 60-65 °C, respectively). Five crystal structures, with and without bound thio-oligosaccharides, show conformational diversity of tunnel-enclosing loops, including a form with partial tunnel collapse at subsite -4 not reported previously in GH7. Also, the first O-glycosylation site in a GH7 crystal structure is reported - on a loop where the glycan probably influences loop contacts across the active site and interactions with the cellulose surface. The GcaCel7A structures indicate higher loop flexibility than HjeCel7A, in accordance with sequence modifications. However, GcaCel7A retains small fluctuations in molecular simulations, suggesting high processivity and low endo-initiation probability, similar to HjeCel7A. Database Structural data are available in the Protein Data Bank under the accession numbers 5AMP, 4ZZV, 4ZZW, 4ZZT, and 4ZZU. The Geotrichum candidum GH family 7 cellobiohydrolase nucleotide sequence is available in GenBank under accession number KJ958925. Enzymes Glycoside hydrolase family 7 reducing end acting cellobiohydrolase We report the characterization of the GH7 CBH secreted by ascomycete G. candidum (GcaCel7A). X-ray data revealed the first O-glycosylation in a GH7 crystal structure on a loop where the glycan influences loop contacts and interactions with the cellulose surface. Even though GcaCel7A structures indicate higher loop flexibility than H. jecorina Cel7A, molecular simulations suggest high processivity and low endo-initiation probability similar to HjeCel7A.</p

Characterization and three-dimensional structures of two distinct bacterial xyloglucanases from families GH5 and GH12

The plant cell wall is a complex material in which the cellulose microfibrils are embedded within a mesh of other polysaccharides, some of which are loosely termed "hemicellulose." One such hemicellulose is xyloglucan, which displays a beta-1,4-linked D-glucose backbone substituted with xylose, galactose, and occasionally fucose moieties. Both xyloglucan and the enzymes responsible for its modification and degradation are finding increasing prominence, reflecting both the drive for enzymatic biomass conversion, their role in detergent applications, and the utility of modified xyloglucans for cellulose fiber modification. Here we present the enzymatic characterization and three-dimensional structures in ligand free and xyloglucan- oligosaccharide complexed forms of two distinct xyloglucanases from glycoside hydrolase families GH5 and GH12. The enzymes, Paenibacillus pabuli XG5 and Bacillus licheniformis XG12, both display open active center grooves grafted upon their respective (beta/alpha)(8) and beta-jelly roll folds, in which the side chain decorations of xyloglucan may be accommodated. For the beta-jelly roll enzyme topology of GH12, binding of xylosyl and pendant galactosyl moieties is tolerated, but the enzymeis similarly competent in the degradation of unbranched glucans. In the case of the (beta/alpha)(8) GH5 enzyme, kinetically productive interactions are made with both xylose and galactose substituents, as reflected in both a high specific activity on xyloglucan and the kinetics of a series of aryl glycosides. The differential strategies for the accommodation of the side chains of xyloglucan presumably facilitate the action of these microbial hydrolases in milieus where diverse and differently substituted substrates may be encountered.</p