Cloning of phospholipase Dα and lipoxygenase genes CmPLDα1 and CmLOX1 and their expression in fruit, floral, and vegetative tissues of 'honey brew' hybrid honeydew melon

Abstract

Increases in phospholipase D [PLD (EC 3.1.4.4)] and lipoxygenase [LOX (EC 1.13.11.12)] activities are thought to play a critical role in senescence of mesocarp tissues in netted and nonnetted muskmelon (Cucumis melo L.) fruits. We have cloned and characterized two full-length cDNAs, CmPLDα1 and CmLOX1, encoding PLDα and LOX proteins in honeydew melon (C. melo Inodorus Group cv. Honey Brew). Relative levels of expression of the corresponding genes were determined by semi-quantitative RT-PCR in developing and mature fruit mesocarp tissues [20-60 d after pollination (DAP)], as well as in roots, leaves, and stems from 4-week-old and flowers from 6- to 7-week-old plants. The coding regions of CmPLDα1 and CmLOX1 cDNAs are, respectively, 2427 and 2634 nucleotides long, encoding proteins 808 and 877 amino acids in length. CmPLDα1 is very similar to PLDα genes from castor bean (Ricinis communis L.), cowpea (Vigna unguiculata L.), strawberry (Fragaria xananassa Duch.) and tomato (Lycopersicon esculentum Mill.) (77% nucleotide identity), and is the first PLD gene cloned from a cucurbit species. CmLOX1 has 94% nucleotide identity to a cucumber (Cucumis sativus L.) LOX gene expressed in roots and 80% identity to cucumber cotyledon lipid body LOX. In general, transcript of CmPLDα1 was much more abundant than that of CmLOX1, but relative levels of transcript in the various organs and tissues were similar for the two genes. Expression was highest in roots, flowers, and fruit mesocarp tissues. CmPLDα1 expression in fruit was essentially constitutive throughout development, although maximum levels occurred at 50 and 55 DAP, respectively, in middle and hypodermal mesocarp. CmLOX1 expression was generally higher in middle than in hypodermal mesocarp with maximum transcript levels occurring at 55 and 50 DAP, respectively. Overall, the patterns of expression of CmPLDα1 and CmLOX1 are consistent with a model in which their encoded enzymes act in tandem to promote or accelerate senescence in fruit mesocarp tissues.