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

Thurayya Al Hinai, Robert A. M. Vreeburg, Logan Mackay, Lorna Murray, Ian Sadler, Stephen C. Fry

Research output: Contribution to journalArticlepeer-review

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 beta-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 (‘ UA–GalA’), taken as diagnostic of PL action. UA–GalA was separated from saturated oligogalacturonides (EPG products) by electrophoresis, then subjected to thin-layer chromatography (TLC), resolving UA–GalA from higher homologues. The UA–GalA was confirmed as 4-deoxy--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 UA–GalA, demonstrating that PL had been acting in vivo in these fruits prior to harvest. Date-derived UA–GalA was verified by negative-mode mass spectrometry, including CID fragmentation.

The UA–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.
Original languageEnglish
Pages (from-to)511–525
Number of pages15
JournalAnnals of Botany
Volume128
Issue number5
Early online date10 Jun 2021
DOIs
Publication statusPublished - 8 Oct 2021

Keywords

  • cell wall
  • driselase
  • fruit softening
  • high voltage paper electrophoresis
  • homogalacturonan
  • pectate lyase
  • date (Phoenix dactylifera)
  • pear (Pyrus communis)
  • rowan (Sorbus aucuparia)
  • apple (Malus pumila)

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