Apoplastic hydroxyl radicals and their in-vivo action on textural polysaccharides in ripening fruit tissues

Our principal goal is to update the current picture of the mechanism of tissue softening during fruit ripening. Specifically, we wish to test critically the proposed contribution of apoplastic hydroxyl radicals (•OH) to the scission of structural polysaccharides in fruits. Hydroxyl radicals have been proposed to contribute, in synergy with hydrolases, transglycosylases and expansins, to fruit softening. If such a contribution is confirmed, it will be of great importance, and interest, to take this into account in understanding the mechanism of fruit softening, and therefore in assessing the opportunities for the successful commercial control of fruit softening.
To achieve our goal, we will exploit several major fruits — and especially pears (in view of initial successful experiments with these pomes) but also others — to work towards the following objectives:
• To demonstrate unequivocally the presence and identity, in fruits, of polysaccharides that have been subjected, in the recent past, to natural attack by endogenous hydroxyl radicals. This work will make use of a novel radio¬chemical ‘finger¬printing’ method recently developed in this laboratory.
• To refine the finger¬printing method, by instrumental approaches (NMR and MS), and especially to identify two highly characteristic but unidentified products with very high electrophoretic mobility.
• To demonstrate the on-going production of hydroxyl radicals in the fruit apoplast. This work will make use of another novel method, also recently developed in this laboratory, involving the use of a 3H-labelled •OH-‘trap’ (reporter molecule) infiltrated into the apoplast of living fruit.
• To use non-toxic hydroxyl radical scavengers to provide more definitive evidence that the ‘trapping’ method accurately reports •OH production.
• To determine whether apoplastic •OH action coincides with episodes of breakdown and/or solubilisation of specific structural polysaccharides, during ripening and over-ripening, in several diverse fruits.
• To begin a study of the effect of non-toxic hydroxyl radical scavengers on the process of fruit softening — a possible indication of the functional role of •OH in ripening.

The attractive ‘softening’ that occurs during the ripening of fruits is mainly achieved by natural changes in fruit cell wall properties. Many cell wall enzymes have been studied in relation to the softening process, with various degrees of success in different fruits. This indicates that fruit softening is not always achieved in one specific way, but by mechanisms that can differ between different fruit species. In addition to changes in cell walls caused by enzymes, a non-enzymatic cleaving mechanism has been suggested, based on the ability of hydroxyl radicals (•OH) to attack polysaccharides.



All the ingredients to produce hydroxyl radicals are present in the cell walls of living plant cells, and hydroxyl radicals have indeed been detected in plant cells, although it has not been unequivocally demonstrated that they are in the ‘apoplast’, i.e. the solution which permeates the cell walls. In addition, no proof had been given for the attack of hydroxyl radicals on cell wall polysaccharides. To find a method to test the natural occurrence of such attack, and to apply it to ripening fruit to see whether hydroxyl radical attack in polysaccharides is involved in fruit softening, were the main objectives of this project.



When a hydroxyl radical attacks a cell wall polysaccharide, it will cause recognisable chemical changes. The approach used in this project is to detect these changes was by ‘labelling’ them with either a radioactive or a fluorescent compound. We have used the method with success on pure polysaccharides, and were also able to detect an indicative (‘fingerprint’) compound in the cell wall polysaccharides of ripe banana fruit. Given that this is a novel technique, the promising results have to be approached with care, and we are currently performing all the controls necessary to validate the positive results.

The project showed that it is possible to 'fingerprint' plant cell walls, e.g. those of ripening fruits, to determine whether their constituent polysaccharides have been naturally attacked by hydroxyl radicals. Hydroxyl radicals are widely regarded as detrimental to life, but may in fact play important roles in natural situations where structural polymers, such as those of cell walls, are being actively degraded. We showed by model experiments using purified polysaccharides that it is possible to recognise hydroxy-attached molecules, and that the strategy can be readily applied across to the cell walls of fruits and vegetables. The results increase out understanding of the 'nuts and bolts' of cell wall loosening in agriculturally important situations such as germination and fruit ripening.