Fruit softening: evidence for rhamnogalacturonan lyase action in vivo in ripe fruit cell walls
Fruit softening: evidence for rhamnogalacturonan lyase action in vivo in ripe fruit cell walls
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DOI：
10.1093/aob/mcad197
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发表时间：
2024-01-05
期刊：
影响因子：
4.2
通讯作者：
Fry，Stephen C.
中科院分区：
文献类型：
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作者：
Al-Hinai，Thurayya Z. S.;Mackay，C. Logan;Fry，Stephen C.
背景与目的成熟果实的软化过程包括三种反应：酶解、酶消除(裂解酶催化)和非酶氧化裂解。已知的两种裂解酶活性是果胶裂解酶和鼠李半乳糖醛酸裂解酶(RGL)，它们可能分别导致果胶的高半乳糖醛酸和鼠李半乳糖醛酸-I(RG-I)结构域的中链断裂。然而，RGL是否显示活体肌动蛋白这一重要的生物学问题还没有得到测试。方法我们建立了一种特异和灵敏地检测活体RGL产物的方法，该方法基于Driselase对Rgl活性的消化和对Rg1活性的特征不饱和指纹产物(四糖)的检测。关键结果在模型实验中，商业Rg1体外部分切割的马铃薯RG-I被Driselase消化，释放出一种不饱和的四糖(‘ΔUA-Rha-Gala-Rha’)，作为Rg1活性的诊断。这种高度酸性的指纹图谱化合物是从单糖(半乳糖醛酸、半乳糖、鼠李糖等)中分离出来的。经pH 2电泳法分离，再用薄层层析法从果胶裂解酶指纹图谱(ΔUA-Gala)中分离得到。经质谱和酸解鉴定为4-deoxy-β-l-threo-hex-4-enopyranuronosyl-(1→2)-l-rhamnosyl-(1→4)-d-galacturonosyl-(1→2)-l-rhamnose，确定为ΔUA-Rha-Gala-Rha。Driselase对不同成熟水果[枣、沙棘、越橘、红豆杉、芒果、李子、黑莓、苹果、梨和草莓]的细胞壁进行Driselase消化，得到相同的指纹图谱，表明Rg1在采收前一直在这些水果中发挥作用。成熟枣消化液中的“指纹”：(半乳糖醛酸 + 鼠李糖)比例约为1：72，表明内源RG-I中约1.4%的主链→-GAL键已被RGL活体切割。结论本研究首次证明Rg1在软果果壁中具有酶作用，可能有助于果实软化。
Background and AimsThe 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 actionin vivohad not been tested.MethodsWe developed a method for specifically and sensitively detectingin-vivoRGL products, based on Driselase digestion of cell walls and detection of a characteristic unsaturated ‘fingerprint’ product (tetrasaccharide) of RGL action.Key ResultsIn model experiments, potato RG-I that had been partially cleavedin vitroby 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 actingin vivoin 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 byin-vivoRGL action.ConclusionsThe results provide the first demonstration that RGL, previously known from studies of fruit gene expression, proteomic studies andin-vitroenzyme activity, exhibits enzyme action in the walls of soft fruits and may thus be proposed to contribute to fruit softening.