Fruit softening: evidence for pectate lyase action in vivo in date (Phoenix dactylifera) and rosaceous fruit cell walls.

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 β-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.

Key results

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 CID fragmentation. The ΔUA-GalA : GalA ratio from ripe dates was roughly 1:20 (mol/mol), indicating that ~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.

SUBMITTER: Al Hinai TZS 

PROVIDER: S-EPMC8422893 | biostudies-literature |

REPOSITORIES: biostudies-literature