Phospholipase Dα Activity in Mesocarp Tissues from Fruit of Cucumis melo L. Reticulatus and Inodorus Cultivars

In muskmelon (Cucumis melo L.) fruit, phospholipase Dα [PLDα (EC 3.1.4.4)] activity is associated with mesocarp tissue softening and plasma membrane degradation during senescence. PLDα activity is regulated by a number of factors, most notably [Ca²⁺], as well as pH, substrate lipid composition, and reaction time and temperature; however, no enzyme assay has been established specifically for C. melo mesocarp tissues. This study determined optimal conditions to assay total PLDα activity in homogenates of lyophilized muskmelon mesocarp tissues, which were as follows: incubation in medium buffered at pH 5.7 including 20 mM Ca²⁺ for 30 min at 37 °C. The assay was utilized to measure PLDα activity in hypodermal and middle mesocarp tissues from fruit of four cultivars representing netted (‘Cruiser’ and ‘Nitro’) and hybrid honey dew (‘Honey Yellow’ and ‘Morning Ice’) types of muskmelon. Tissue samples were excised from fruit and frozen in liquid N₂ on day 0 after overnight transport, after 5 d storage at 4 °C, and after 5 d at 4 °C plus 3 d at 21 °C. At the end of this 8-d regime, all fruit were still in excellent condition. Generally, PLDα specific activity (units/min/mg protein) was high on day 0 in both mesocarp tissues from all four cultivars, and there was a substantial increase in activity after 5 d at 4 °C, except for hypodermal mesocarp from ‘Morning Ice’ and ‘Nitro’, which showed no change. Transfer of fruit to 21 °C for 3 d after 5 d at 4 °C resulted in a marked decline in PLDα activity in both mesocarp tissues from ‘Cruiser’ melons, whereas the decrease was more modest in mesocarp from ‘Honey Yellow’ and ‘Nitro’, and activity increased in middle and hypodermal mesocarp from ‘Morning Ice’. Optimization of the PLDα enzyme assay for muskmelon mesocarp tissues, in addition to the observed differences among fruit of the four cultivars, establishes a framework for further investigation of the role of PLDα activity in postharvest fruit softening, senescence, and loss of quality.