Citramalate synthase yields a biosynthetic pathway for isoleucine and straight- and branched-chain ester formation in ripening apple fruit

A plant pathway that initiates with the formation of citramalate from pyruvate and acetyl-CoA by citramalate synthase (CMS) is shown to contribute to the synthesis of alpha-ketoacids and important odor-active esters in apple (Malus x domestica) fruit. Microarray screening led to the discovery of a gene with high amino acid similarity to 2-isopropylmalate synthase (IPMS). However, functional analysis of recombinant protein revealed its substrate preference differed substantially from IPMS and was more typical of CMS. MdCMS also lacked the regulatory region present in MdIPMS and was not sensitive to feedback inhibition. C-13-acetate feeding of apple tissue labeled citramalate and alpha-ketoacids in a manner consistent with the presence of the citramalate pathway, labeling both straight- and branched-chain esters. Analysis of genomic DNA (gDNA) revealed the presence of two nearly identical alleles in Jonagold fruit (MdCMS_1 and MdCMS_2), differing by two nonsynonymous single-nucleotide polymorphisms (SNPs). The mature proteins differed only at amino acid 387, possessing either glutamine(387) (MdCMS_1) or glutamate(387) (MdCMS_2). Glutamate(387) was associated with near complete loss of activity. MdCMS expression was fruit-specific, increasing severalfold during ripening. The translated protein product was detected in ripe fruit. Transient expression of MdCMS_1 in Nicotiana benthamiana induced the accumulation of high levels of citramalate, whereas MdCMS_2 did not. Domesticated apple lines with MdCMS isozymes containing only glutamate(387) produced a very low proportion of 2-methylbutanol and 2-methylbutanoate (2MB) and 1-propanol and propanoate (PROP) esters. The citramalate pathway, previously only described in microorganisms, is shown to function in ripening apple and contribute to isoleucine and 2MB and PROP ester biosynthesis without feedback regulation.

Automated summary

The citramalate pathway, previously described in microorganisms, is shown to be functional in ripening apples, contributing to the synthesis of isoleucine and 2MB and PROP esters without feedback regulation.