Evaluation of Isoleucine and Citramalic Acid Content Using High and Low Branched-Chain Ester Producing Lines From Geneva Malus Core Collection

Ripening apple fruit from 10 lines (Malus xdomestica and Malus xhybrid) from the Malus core collection of the germplasm repository at the New York Agricultural Experiment Station in Geneva, NY, were selected for this study. These lines were selected to study the biological difference between high and low branched-chain (BC) ester-producing lines. High BC lines had a significant isoleucine increase, about a 4- to 27-fold change during ripening. However, in all low BC lines, isoleucine decreased or remain unchanged. Citramalic acid, which is formed from the condensation of pyruvate and 2-acetyl-CoA by citramalate synthase (CIM) and linked to a newly described pathway for the biosynthesis of BC esters in apple, increased in content about 1.3- to 2.9-fold in low BC lines and 15- to 165-fold in high BC lines. The accumulation of isoleucine and an increase in citramalic acid in these apple lines suggest that the hypothesized citramalate pathway may activate upon ripening, thereby supplying the BC ester precursor, α-keto-β-methylvalerate, which is also the immediate precursor to isoleucine. In addition, a ripening-related increase in 2-ethylmalate was detected.  2-Ethylmalate can be synthesized from the condensation of pyruvate and α-ketobutyrate via the action of CIM. The expression of MdCIM increased during the ripening stage for all lines and did not differ between low and high BC lines. The observation of high MdCIM expression for all lines can be explained from our previously characterized two CIM proteins, MdCIM1, and MdCIM2.  MdCIM1 is active, but MdCIM2 is not. It is possible that the MdCIM1 predominated in high BC lines, whereas MdCIM2 predominated in low BC lines.