Fruit softening: evidence for rhamnogalacturonan lyase action in vivo in ripe fruit cell walls

Background and aims The softening of ripening fruit involves partial depolymerisation 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 (‘UA-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 UA–GalA (the fingerprint of pectate lyase action) by thin-layer chromatography (TLC). The ‘UA-Rha-GalA-Rha’ was confirmed as 4-deoxy--L-threo-hex-4-enopyranuronosyl-(12)-L-rhamnosyl-(14)-D-galacturonosyl-(12)-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 RhaGalA 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. <br/