School of Biotechnology, Royal Institute of Technology, AlbaNova University Centre, SE-10691 Stockholm, Sweden.
Plant Physiol. 2009 Dec;151(4):1741-50. doi: 10.1104/pp.109.147439. Epub 2009 Sep 25.
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.
目前，有多种用于原位定位植物细胞中多糖、核酸和蛋白质的分子探针，包括报告酶策略（例如，蛋白-葡糖醛酸酶融合）。然而，用于直接分析负责细胞壁重塑的内源性糖苷水解酶和转糖苷酶的方法却很少。为了说明荧光素糖苷在解决这一问题上的潜力，我们合成了木糖葡糖醛酸寡糖的荧光素 β-糖苷（XXXG-β-Res），作为植物内分析 XEH 活性的特定底物。由于低 pK(a)（5.8）和大消光系数（epsilon 62,000 M(-1) cm(-1)）、长波长荧光（激发 571nm/发射 585nm）以及相应阴离子的高量子产率（0.74），荧光素糖苷在壁内测定中具有特别高的灵敏度。体外分析表明，XXXG-β-Res 被典型的植物 XEH、nasturtium（Tropaeolum majus）NXG1 水解，具有经典的米氏-门登哈根底物饱和动力学和对酶浓度和孵育时间的线性依赖性。此外，通过共聚焦显微镜观察发芽 nasturtium 种子和拟南芥（Arabidopsis thaliana）花序茎中荧光的局部增加，可以实时观察到 XEH 活性。重要的是，这种新的原位 XEH 测定为原位木葡聚糖内切转糖苷酶测定提供了重要补充，从而可以直接在植物组织中描绘木葡聚糖内切转糖苷酶/水解酶基因编码的不同活性。XXXG-β-Res 也被多种微生物 XEH 水解的观察表明，这种底物以及一般的荧光素糖苷可能在形态发生和植物-微生物相互作用过程中分析多糖水解酶对细胞壁重构的分析中具有广泛的适用性。
