Abstract
Background and AimsThe 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.MethodsWe 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.Key ResultsIn 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.ConclusionsThe 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.
Highlights
Fruit softening in generalThe programmed softening that occurs during the ripening of many fruit species requires cell wall loosening and a reduction in cell–cell adhesion as a result of dissolution of the pectin-rich middle lamella (Jarvis et al, 2003; Brummell, 2006)
Key Results In model in-vitro experiments, pectic homogalacturonan that had been partially cleaved by commercial pectate lyase (PL) was digested to completion with Driselase, releasing an unsaturated disaccharide (‘ΔUA–galacturonic acid (GalA)’), taken as diagnostic of PL action
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
Summary
Fruit softening in generalThe programmed softening that occurs during the ripening of many fruit species requires cell wall loosening and a reduction in cell–cell adhesion as a result of dissolution of the pectin-rich middle lamella (Jarvis et al, 2003; Brummell, 2006). Characteristic modifications include solubilization and depolymerization of pectin, loss of neutral sugars from pectic side chains, cell wall swelling and disassembly of the xyloglucan–cellulose network (Paniagua et al, 2017) These modifications are partly due to non-enzymic reactions with reactive oxygen species (especially the hydroxyl radical, ·OH; Dumville and Fry, 2000; Airianah et al, 2016) or expansins (Brummell et al, 1999), and partly the result of wall-modifying enzymes secreted into the apoplast during ripening. These enzymes act by cleaving polysaccharides, resulting in mechanical weakening. The link by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited
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