ASHS 2025 Conference

Citramalate synthase is proposed to be the first committed step in the formation of branched chain esters in apples based on detailed biochemical, pharmacological, isotopic, and molecular data. However, the function of the full citramalate pathway, from the formation of citramalate to the formation of the branched chain esters containing 2-methylbutyl and 2-methylbutanoate moieties has not been tested using a gain of function model. In this work, we report on the transient transformation of ripening banana fruit with functional and non-functional alleles of MdCMS (MdCMS_1 and MdCMS_2, respectively) and report on the compounds induced by the addition of this gene. Banana do not produce appreciable levels of citramalate and make little to no 2-methylbutyl and 2-methylbutanoate containing esters. Following the transformation event, citramalate accumulated in banana transformed with MdCMS_1, but not MdCMS-2. Similarly, banana with the functional form of MdCMS produced modest levels of 2-methylbutyl acetate and 2-methylbutyl butanoate esters. Fruit transformed with MdCMS_1 also accumulated isoleucine as a by-product of the enhanced pathway throughput. Finally, there was a marked enhancement of propyl esters, likely a function of an increase in alpha-ketobutyrate, which is an intermediate in the citramalate pathway between citramalic acid and 2-methylbutyl and 2-methylebutnoate esters. The data provide the last necessary evidence for the conclusively documenting function of citramalate synthase as a functional pathway in apple useful for the production of aroma active branched-chain esters.