Changes in Free Amino Acid Content in 'Jonagold' Apple Fruit as Related to Branched-chain Ester Production, Ripening, and Senescence

The relationship among the free amino acid content, the expression of genes related to branched-chain amino acid metabolism {branched-chain aminotransferase [BCAT], alpha-keto acid decarboxylase [pyruvate decarboxylase (PDC)], and threonine deaminase [TD]}, and the production of branched-chain (BC) esters during ripening and senescence in 'Jonagold' apple fruit (Malus Xdomestica) was studied. Eighteen amino acids were measured by liquid chromatography coupled with tandem mass spectrometry. The content for all amino acids changed with developmental stage and some shared similar patterns of accumulation/diminution. The pattern for isoleucine differed from all other amino acids, increasing more than 20-fold during the ripening process. The onset of the increase was concomitant with the onset of increasing ethylene and BC ester production and the content remained elevated even during senescence. The elevated isoleucine levels are consistent with an increase in the flux through the pathway leading to the formation and degradation of the isoleucine precursor alpha-keto-beta-methylvalerate, which is used for production of BC esters containing 2-methylbutanol and 2-methylbutanoate moieties. Unexpectedly, the content of threonine, the amino acid from which isoleucine is thought to be derived in plants, did not change in concert with isoleucine, but rather declined somewhat after ripening was well underway. Patterns in the expression of some, but not all, of the putative BCAT and PDC genes appeared to reflect the rise and fall in ester formation; however, the expression of putative TD genes did not change during ripening. The patterns in gene expression and amino acid content are interpreted to suggest that the synthesis of alpha-keto-beta-methylvalerate and isoleucine during apple ripening may depend on an as yet uncharacterized pathway that bypasses threonine, similar to the citramalate pathway found in some bacteria.