Fruit Softening: Evidence for Rhamnogalacturonan Lyase Action in Vivo in Ripe Fruit Cell Walls

Overview

Journal Ann Bot

Publisher Oxford University Press

Specialty Biology

Date 2024 Jan 5

PMID 38180460

Authors

Thurayya Z S Al-Hinai

C Logan Mackay

Stephen C Fry

Affiliations

Soon will be listed here.

Abstract

Background And Aims: The softening of ripening fruit involves partial depolymerization of cell-wall pectin by three types of reaction: enzymic hydrolysis, enzymic elimination (lyase-catalysed) and non-enzymic oxidative scission. Two known lyase activities are pectate lyase and rhamnogalacturonan lyase (RGL), potentially causing mid-chain cleavage of homogalacturonan and rhamnogalacturonan-I (RG-I) domains of pectin respectively. However, the important biological question of whether RGL exhibits action in vivo had not been tested.

Methods: We developed a method for specifically and sensitively detecting in-vivo RGL products, based on Driselase digestion of cell walls and detection of a characteristic unsaturated 'fingerprint' product (tetrasaccharide) of RGL action.

Key Results: In model experiments, potato RG-I that had been partially cleaved in vitro by commercial RGL was digested by Driselase, releasing an unsaturated tetrasaccharide ('ΔUA-Rha-GalA-Rha'), taken as diagnostic of RGL action. This highly acidic fingerprint compound was separated from monosaccharides (galacturonate, galactose, rhamnose, etc.) by electrophoresis at pH 2, then separated from ΔUA-GalA (the fingerprint of pectate lyase action) by thin-layer chromatography. The 'ΔUA-Rha-GalA-Rha' was confirmed as 4-deoxy-β-l-threo-hex-4-enopyranuronosyl-(1→2)-l-rhamnosyl-(1→4)-d-galacturonosyl-(1→2)-l-rhamnose by mass spectrometry and acid hydrolysis. Driselase digestion of cell walls from diverse ripe fruits [date, sea buckthorn, cranberry, yew (arils), mango, plum, blackberry, apple, pear and strawberry] yielded the same fingerprint compound, demonstrating that RGL had been acting in vivo in these fruits prior to harvest. The 'fingerprint' : (galacturonate + rhamnose) ratio in digests from ripe dates was approximately 1 : 72 (mol/mol), indicating that ~1.4 % of the backbone Rha→GalA bonds in endogenous RG-I had been cleaved by in-vivo RGL action.

Conclusions: The results provide the first demonstration that RGL, 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.

References

Seymour G . Pectate lyase action in vivo and fruit softening. A commentary on: 'Fruit softening: evidence for pectate lyase action in vivo in date (Phoenix dactylifera) and rosaceous fruit cell walls'. Ann Bot. 2021; 128(5):i-ii. PMC: 8422885. DOI: 10.1093/aob/mcab096. View

Brummell D, Dal Cin V, Lurie S, Crisosto C, Labavitch J . Cell wall metabolism during the development of chilling injury in cold-stored peach fruit: association of mealiness with arrested disassembly of cell wall pectins. J Exp Bot. 2004; 55(405):2041-52. DOI: 10.1093/jxb/erh228. View

Miedes E, Lorences E . Xyloglucan endotransglucosylase/hydrolases (XTHs) during tomato fruit growth and ripening. J Plant Physiol. 2008; 166(5):489-98. DOI: 10.1016/j.jplph.2008.07.003. View

Kobayashi M, Matoh T, Azuma J . Two Chains of Rhamnogalacturonan II Are Cross-Linked by Borate-Diol Ester Bonds in Higher Plant Cell Walls. Plant Physiol. 1996; 110(3):1017-1020. PMC: 157802. DOI: 10.1104/pp.110.3.1017. View

Azadi P, ONeill M, Bergmann C, Darvill A, Albersheim P . The backbone of the pectic polysaccharide rhamnogalacturonan I is cleaved by an endohydrolase and an endolyase. Glycobiology. 1995; 5(8):783-9. DOI: 10.1093/glycob/5.8.783. View

Mohnen D . Pectin structure and biosynthesis. Curr Opin Plant Biol. 2008; 11(3):266-77. DOI: 10.1016/j.pbi.2008.03.006. View

Molina-Hidalgo F, Franco A, Villatoro C, Medina-Puche L, Mercado J, Hidalgo M . The strawberry (Fragariaxananassa) fruit-specific rhamnogalacturonate lyase 1 (FaRGLyase1) gene encodes an enzyme involved in the degradation of cell-wall middle lamellae. J Exp Bot. 2013; 64(6):1471-83. DOI: 10.1093/jxb/ers386. View

Uluisik S, Seymour G . Pectate lyases: Their role in plants and importance in fruit ripening. Food Chem. 2019; 309:125559. DOI: 10.1016/j.foodchem.2019.125559. View

Yang L, Huang W, Xiong F, Xian Z, Su D, Ren M . Silencing of SlPL, which encodes a pectate lyase in tomato, confers enhanced fruit firmness, prolonged shelf-life and reduced susceptibility to grey mould. Plant Biotechnol J. 2017; 15(12):1544-1555. PMC: 5698048. DOI: 10.1111/pbi.12737. View

10.

Al Hinai T, Vreeburg R, Mackay C, Murray L, Sadler I, Fry S . Fruit softening: evidence for pectate lyase action in vivo in date (Phoenix dactylifera) and rosaceous fruit cell walls. Ann Bot. 2021; 128(5):511-525. PMC: 8422893. DOI: 10.1093/aob/mcab072. View

11.

Begum R, Fry S . Arabinogalactan-Proteins as Boron-Acting Enzymes, Cross-Linking the Rhamnogalacturonan-II Domains of Pectin. Plants (Basel). 2023; 12(23). PMC: 10707938. DOI: 10.3390/plants12233921. View

12.

Mutter M, Colquhoun I, Schols H, Beldman G, Voragen A . Rhamnogalacturonase B from Aspergillus aculeatus is a rhamnogalacturonan alpha-L-rhamnopyranosyl-(1-->4)-alpha-D-galactopyranosyluronide lyase. Plant Physiol. 1996; 110(1):73-7. PMC: 157695. DOI: 10.1104/pp.110.1.73. View

13.

Zhang W, Zhao S, Zhang L, Xing Y, Jia W . Changes in the cell wall during fruit development and ripening in Fragaria vesca. Plant Physiol Biochem. 2020; 154:54-65. DOI: 10.1016/j.plaphy.2020.05.028. View

14.

Wang D, Yeats T, Uluisik S, Rose J, Seymour G . Fruit Softening: Revisiting the Role of Pectin. Trends Plant Sci. 2018; 23(4):302-310. DOI: 10.1016/j.tplants.2018.01.006. View

15.

Wang W, Wang Y, Yi H, Liu Y, Zhang G, Zhang L . Biochemical Characterization of Two Rhamnogalacturonan Lyases From ATCC 8483 With Preference for RG-I Substrates. Front Microbiol. 2022; 12:799875. PMC: 8787155. DOI: 10.3389/fmicb.2021.799875. View

16.

Marin-Rodriguez M, Smith D, Manning K, Orchard J, Seymour G . Pectate lyase gene expression and enzyme activity in ripening banana fruit. Plant Mol Biol. 2003; 51(6):851-7. DOI: 10.1023/a:1023057202847. View

17.

Perrone P, Hewage C, Thomson A, Bailey K, Sadler I, Fry S . Patterns of methyl and O-acetyl esterification in spinach pectins: new complexity. Phytochemistry. 2002; 60(1):67-77. DOI: 10.1016/s0031-9422(02)00039-0. View

18.

Fry S . High-Voltage Paper Electrophoresis (HVPE). Methods Mol Biol. 2020; 2149:1-31. DOI: 10.1007/978-1-0716-0621-6_1. View

19.

Ishii T . O-acetylated oligosaccharides from pectins of potato tuber cell walls. Plant Physiol. 1997; 113(4):1265-72. PMC: 158249. DOI: 10.1104/pp.113.4.1265. View

20.

Schuchmann M, von Sonntag C . The effect of oxygen on the OH-radical-induced scission of the glycosidic linkage of cellobiose. Int J Radiat Biol Relat Stud Phys Chem Med. 1978; 34(4):397-400. DOI: 10.1080/09553007814551051. View