Investigation of Volatile Ester Biosynthesis in Melon Fruit (Cucumis melo L., botanical group reticulatus)

Melon (Cucumis melo) is widely cultivated in the United States, and the aroma of the fruit is an important aspect for consumer appreciation of quality. Aroma is a complex mixture of many different volatile compounds, including fruity-- and sweet--scented volatile esters. Two of the major ester components of melon aroma, 2--methylpropyl acetate and 2--methylbutyl acetate, are derived from the branched--chain amino acids (BCAAs) valine and isoleucine, respectively. Aldehydes derived from BCAAs are known to be reduced to alcohols by alcohol dehydrogenases, and alcohol acyltransferase enzymes catalyze the esterification reaction to form volatile esters. How BCAAs are converted to their aldehyde derivatives remains unclear. It was suggested that the BCAAs are converted to ketoacids by transamination and a ketoacid decarboxylase catalyzes the subsequent reaction to yield aldehydes. Two candidate decarboxylases (PDC1 and PDC2) putatively involved in the pathway were identified and characterized. Recombinant CmPDC1 enzyme could carry out the efficient decarboxylation of pyruvate and also had significant activity toward a range of straight-- and branched--chain alpha--keto acid substrates. On the other hand, CmPDC2 had very low or no activity toward all alpha--keto acids tested. RNAi--mediated silencing of CmPDC1 and/or CmPDC2 expression in melon showed that CmPDC1 is involved in acetaldehyde, propanal and pentanal production, while CmPDC2 does not contribute significantly to volatile biosynthesis in melon fruit. Importantly, our results also demonstrate that neither of these decarboxylases is involved in BCAA--derived aldehyde formation in melon. We therefore hypothesized and tested the occurrence of an alternative Co--enzyme A--dependent biochemical pathway involving BCAA--derived carboxylic acids as intermediates. Precursor enrichment studies demonstrated that 2--methylpropanoic acid was an intermediate in 2--methylpropyl acetate biosynthesis in melon. Functional analysis of candidate Acyl--CoA Lyase 1 (CmACH1) showed it had activity toward both 2--methylpropanoyl--CoA and palmitoyl--CoA to produce 2--methylpropanoic acid and palmitic acid in vitro. Agrobacterium--mediated transient silencing of CmACH1 resulted in a decrease in 2--methylpropyl acetate production, suggesting that CmACH1 is involved in 2--methylpropyl acetate biosynthesis. During the course of this work, a rapid Agrobacterium --mediated transient silencing method was developed, enabling over 70% reduction in target gene expression within four days after inoculation.