Fruit softening: evidence for pectate lyase action in vivo in date (Phoenix dactylifera) and rosaceous fruit cell walls.
Fruit softening: evidence for pectate lyase action in vivo in date (Phoenix dactylifera) and rosaceous fruit cell walls.
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DOI：
10.1093/aob/mcab072
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发表时间：
2021-09-07
期刊：
影响因子：
4.2
通讯作者：
Fry SC
中科院分区：
文献类型：
--
作者：
Al Hinai TZS;Vreeburg RAM;Mackay CL;Murray L;Sadler IH;Fry SC
在果实发育过程中发生的程序性软化需要细胞壁多糖，特别是果胶的断裂。提出的机制包括壁酶或羟基自由基的作用。在水果提取物中发现的酶活性包括果胶酸裂解酶（PL）和内切多聚半乳糖醛酸酶（EPG），其在体外分别通过β-消除和水解在中链切割去酯化的同型半乳糖醛酸聚糖。然而，PL是否在体内表现出作用的重要生物学问题尚未得到测试。我们开发了一种特异性和灵敏度检测体内PL产物的方法，基于Driselase消化细胞壁多糖和检测PL作用的特征不饱和产物。在模型体外实验中，用Driselase完全消化已被商业PL部分裂解的果胶同型半乳糖醛酸聚糖，释放不饱和二糖（“Δ UA-GalA”），作为PL作用的诊断。用电泳法从饱和寡聚半乳糖醛酸（EPG）产物中分离出ΔUA-GalA，然后用薄层层析法将ΔUA-GalA与高级同系物分离。经核磁共振谱分析，ΔUA-GalA为4-脱氧-β-l-苏型-己-4-烯吡喃糖基-（1→4）-d-半乳糖醛酸。Driselase消化来自枣（Phoenix dactylifera）、梨（Pyrus communis）、花楸（Sorbus aucuparia）和苹果（Malus pumila）的成熟果实的细胞壁产生ΔUA-GalA，表明PL在收获前已经在这些果实中体内起作用。通过负模式质谱法（包括碰撞诱导解离（CID）裂解）验证了日期衍生的ΔUA-GalA。成熟枣中的ΔUA-GalA：GalA比值约为1：20（mol mol-1），表明成熟枣中的ΔUA-GalA：GalA比值约为1：20（mol-1）。内源性同型半乳糖醛酸聚糖中5%的键已被体内PL作用裂解。结果提供了第一个证明，PL，以前已知的水果基因表达，蛋白质组学研究和体外酶活性的研究，表现出酶的作用，在软水果的墙壁，因此可能会提出有助于水果软化。
The programmed softening occurring during fruit development requires scission of cell wall polysaccharides, especially pectin. Proposed mechanisms include the action of wall enzymes or hydroxyl radicals. Enzyme activities found in fruit extracts include pectate lyase (PL) and endo-polygalacturonase (EPG), which, in vitro, cleave de-esterified homogalacturonan in mid-chain by β-elimination and hydrolysis, respectively. However, the important biological question of whether PL exhibits action in vivo had not been tested. We developed a method for specifically and sensitively detecting in-vivo PL products, based on Driselase digestion of cell wall polysaccharides and detection of the characteristic unsaturated product of PL action. In model in-vitro experiments, pectic homogalacturonan that had been partially cleaved by commercial PL was digested to completion with Driselase, releasing an unsaturated disaccharide (‘ΔUA–GalA’), taken as diagnostic of PL action. ΔUA–GalA was separated from saturated oligogalacturonides (EPG products) by electrophoresis, then subjected to thin-layer chromatography (TLC), resolving ΔUA–GalA from higher homologues. The ΔUA–GalA was confirmed as 4-deoxy-β-l-threo-hex-4-enopyranuronosyl-(1→4)-d-galacturonic acid by NMR spectroscopy. Driselase digestion of cell walls from ripe fruits of date (Phoenix dactylifera), pear (Pyrus communis), rowan (Sorbus aucuparia) and apple (Malus pumila) yielded ΔUA–GalA, demonstrating that PL had been acting in vivo in these fruits prior to harvest. Date-derived ΔUA–GalA was verified by negative-mode mass spectrometry, including collision-induced dissociation (CID) fragmentation. The ΔUA–GalA:GalA ratio from ripe dates was roughly 1:20 (mol mol–1), indicating that approx. 5 % of the bonds in endogenous homogalacturonan had been cleaved by in-vivo PL action. The results provide the first demonstration that PL, 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.
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