Apple and banana fruits produce anteiso- and iso-branched-chain esters from newly synthesized precursors

Inhibitors of acetohydroxyacid synthase, the common enzyme of branched-chain amino acid biosynthesis, were applied to ripening apple (Malus xdomestica Borkh.), banana (Musa spp.), and flowering quince (Chaenomeles xsuperba) fruits to discern the contribution of newly synthesized precursors to branched-chain ester formation. After treatment, anteiso- and iso-branched-chain volatiles (i.e., those related to isoleucine, and valine and leucine, respectively) were observed to universally decrease in content. Fruits recovered production following exogenous feeding of branched-chain -ketoacids. Furthermore, apple and banana fruits were capable of metabolizing all three branched-chain -ketoacids to esters. Among free amino acids, only the branched-chain amino acids with correspondingly reduced branched-chain esters had a lesser concentration following treatment with inhibitor. Our results ultimately reject the hypothesis that anteiso- and iso-branched-chain esters are derived from preexisting amino acids and instead support the hypothesis that these esters are the product of de novo precursor biosynthesis. The novel use of these inhibitors also allowed for further investigation of branched-chain volatile biosynthesis, the citramalate synthase pathway, and the importance of precursor availability in fruits. Notably, in Valery banana fruit, ethyl acetate and butyl acetate were found to be dependent on acetohydroxyacid synthase activity for production whereas 1-methylbutyl acetate and 1-methylbutyl butanoate (sec-branched-chain esters) were not. Inhibitor usage on apples also allowed for a sensory study that found that humans can discern the absence of 2-methylbutyl and 2-methylbutanoate esters in apple fruit. Additionally, a population genetics analysis found that there is selection pressure against apples that lack these esters.