目的:成熟果实的软化涉及细胞壁果胶通过三种类型的反应部分解聚:酶水解,酶消除(裂解酶催化)和非酶氧化断裂。两种已知的裂解酶活性是果胶酸裂解酶和鼠李糖半乳糖醛酸裂解酶(RGL),可能分别引起果胶的同半乳糖醛酸和鼠李糖半乳糖醛酸-I(RG-I)结构域的中链裂解。然而,尚未测试RGL是否在体内表现出作用的重要生物学问题。
方法:我们开发了一种用于特异性和灵敏地检测体内RGL产品的方法,基于Driselase对细胞壁的消化和RGL作用的特征性不饱和指纹产物(四糖)的检测。
结果:在模型实验中,已通过商业RGL在体外部分切割的马铃薯RG-I被Driselase消化,释放不饱和四糖(\'ΔUA-Rha-GalA-Rha\'),作为RGL行动的诊断。这种高酸性指纹化合物是从单糖(半乳糖醛酸,半乳糖,鼠李糖等.)通过在pH2下电泳,然后通过薄层色谱(TLC)与ΔUA-GalA(果胶酸裂解酶作用的指纹图谱)分离。'ΔUA-Rha-GalA-Rha'通过质谱和酸水解确认为4-脱氧-β-1-苏-己-4-烯吡喃糖醛酸基-(1→2)-1-鼠李糖基-(1→4)-d-半乳糖醛酸基-(1→2)-1-鼠李糖。driselase消化来自不同成熟水果的细胞壁[日期,沙棘,蔓越莓,紫杉(紫杉),芒果,李子,黑莓,苹果,梨和草莓]产生相同的指纹化合物,证明RGL在收获前已在这些果实中体内起作用。来自成熟日期的消化物中的“指纹”:(半乳糖醛酸酯鼠李糖)比例约为1:72(mol/mol),表明内源性RG-I中〜1.4%的骨架Rha→GalA键已被体内RGL作用裂解。
结论:结果首次证明了RGL,以前从水果基因表达的研究中得知,蛋白质组学研究和体外酶活性,在软水果的壁中表现出酶作用,因此可能有助于水果软化。
OBJECTIVE: The softening of ripening fruit involves partial depolymerization of cell-wall pectin by three types of reaction: enzymic hydrolysis, enzymic elimination (lyase-catalysed) and non-enzymic oxidative scission. Two known lyase activities are pectate lyase and rhamnogalacturonan lyase (RGL), potentially causing mid-chain cleavage of homogalacturonan and rhamnogalacturonan-I (RG-I) domains of pectin respectively. However, the important biological question of whether RGL exhibits action in vivo had not been tested.
METHODS: We developed a method for specifically and sensitively detecting in-vivo RGL products, based on Driselase digestion of cell walls and detection of a characteristic unsaturated \'fingerprint\' product (tetrasaccharide) of RGL action.
RESULTS: In model experiments, potato RG-I that had been partially cleaved in vitro by commercial RGL was digested by Driselase, releasing an unsaturated tetrasaccharide (\'ΔUA-Rha-GalA-Rha\'), taken as diagnostic of RGL action. This highly acidic fingerprint compound was separated from monosaccharides (galacturonate, galactose, rhamnose, etc.) by electrophoresis at pH 2, then separated from ΔUA-GalA (the fingerprint of pectate lyase action) by thin-layer chromatography. The \'ΔUA-Rha-GalA-Rha\' was confirmed as 4-deoxy-β-l-threo-hex-4-enopyranuronosyl-(1→2)-l-rhamnosyl-(1→4)-d-galacturonosyl-(1→2)-l-rhamnose by mass spectrometry and acid hydrolysis. Driselase digestion of cell walls from diverse ripe fruits [date, sea buckthorn, cranberry, yew (arils), mango, plum, blackberry, apple, pear and strawberry] yielded the same fingerprint compound, demonstrating that RGL had been acting in vivo in these fruits prior to harvest. The \'fingerprint\' : (galacturonate + rhamnose) ratio in digests from ripe dates was approximately 1 : 72 (mol/mol), indicating that ~1.4 % of the backbone Rha→GalA bonds in endogenous RG-I had been cleaved by in-vivo RGL action.
CONCLUSIONS: The results provide the first demonstration that RGL, previously known from studies of fruit gene expression, proteomic studies and in-vitro enzyme activity, exhibits enzyme action in the walls of soft fruits and may thus be proposed to contribute to fruit softening.