Inhibition of Polygalacturonase in Tomato Pericarp Tissue by Lysophosphatidylethanolamine: Implications in Fruit Shelf-life

Abstract

The pericarp tissue of red mature tomato (Lycopersicon esculentum cv. Gagliano) was used to exctract polygalacturonase (PG) enzyme. The technique for assaying PG activity involves measurement of released reducing groups that were linked together in pectin. Since the crude extract of PG from pericarp will contain considerable reducing groups, we found that repeated washings of the cell wall pulp removed much of the sugars and thus minimized the background absorbance without loss of PG activity. There is an inherent perplexity concerning the selection of blank for PG assay. This is because (i) the enzyme extract contains both the substrate (pectin) and product (free reducing groups) involved in the reaction; (ii) the color development with cyanoacetamide requires heating for 10 min. Thus, even though the reaction is terminated with borate buffer (pH 9.0) the breakdown of pectin continues chemically by heat; (iii) the absorbance from both pectin and enzyme together at zero time termination was always lower than the sum of absorbances from pectin alone and enzyme alone. This suggests that when together in the same tube, the enzyme appears to protect the pectin from physical breakdown during the period of 10 min. boil needed to develop color using the cyanoacetamide. Thus, the most appropriate blank is processing separately the solutions of enzyme alone and substrate pectin alone for color development and then adding the two absorbances. Using this improved assay we found that lysophosphatidylethanolamine (LPE) inhibited tomato PG activity. This inhibition appears to depend on the ripening stage of the fruit. Our results suggest that LPE is able to impart firmness to tomato fruit by reducing the PG activity, which in turn could protect the pectin/middle lamellae from enzymic breakdown. The effects of LPE on PG activity are distinct from those of Triton X-100 and lysophosphatidylcholine.