Fruit softening: evidence for pectate lyase action in vivo in date (Phoenix dactylifera) and rosaceous fruit cell walls.
Ann Bot
; 128(5): 511-525, 2021 09 07.
Article
in En
| MEDLINE
| ID: mdl-34111288
ABSTRACT
BACKGROUND AND AIMS:
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 ß-elimination and hydrolysis, respectively. However, the important biological question of whether PL exhibits action in vivo had not been tested.METHODS:
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. KEYRESULTS:
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-ß-l-threo-hex-4-enopyranuronosyl-(1â4)-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-GalAGalA ratio from ripe dates was roughly 120 (mol mol-1), indicating that approx. 5 % of the bonds in endogenous homogalacturonan had been cleaved by in-vivo PL action.CONCLUSIONS:
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.Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Phoeniceae
/
Fruit
Type of study:
Prognostic_studies
Language:
En
Journal:
Ann Bot
Year:
2021
Document type:
Article
Affiliation country:
United kingdom