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

Abstract

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 α-ketoacids and important odor-active esters in apple (Malus × 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. 13C-acetate feeding of apple tissue labeled citramalate and α-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 glutamine387 (MdCMS_1) or glutamate387 (MdCMS_2). Glutamate387 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 glutamate387 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.

Emission Patterns of Esters and Their Precursors Throughout Ripening and Senescence in ‘Redchief Delicious’ Apple Fruit and Implications Regarding Biosynthesis and Aroma Perception

“…The slide was then placed into a horizontally oriented 30-mL glass vial sealed with a cap housing a valved septum (Mininert valve 20/400 mm; Sigma-Aldrich). The samples were incubated for 5 or 21 h at 22 C (Sugimoto et al, 2021).…”

“…The incorporation of 13 C into headspace volatiles was analyzed by GC/MS as described (Sugimoto et al, 2021).…”

“…The generation of isotopologs (mass isotopomers) of headspace volatiles following feeding by 1-[ 13 C]-or 2-[ 13 C] or 1,2-[ 13 C 2 ] acetic acid were quantified as previously described (Sugimoto et al, 2021). The selected fragment ions (m/z), their formula, and the portion of the target molecule contributing the base fragment ion are provided (Supplemental Table 2).…”

“…In apple, the carbon chain-length of the alcohol portion of the ester usually varies between 1-and 6-carbons and the acid portion has a typical chain-length from 2-to 8-carbons and both the alkyl and alkanoate moieties can be straight or branched. The branched-chain esters are derived from the pathways associated with branched-chain amino acid biosynthesis via 1-carbon (1-C) fatty acid biosynthesis (FAB) (Kroumova and Wagner, 2003;Sugimoto et al, 2011Sugimoto et al, , 2015Sugimoto et al, , 2021 and degradation (Nursten, 1970;P erez et al, 2002;Tressl and Drawert, 1973;Wyllie et al, 1995). Straight-chain fatty acids are believed to be derived from fatty acid metabolism via degradative (Bartley et al, 1985;Brackmann et al, 1993;Fellman et al, 2000;Nursten, 1970;Rowan et al, 1996Rowan et al, , 1999 and synthetic Beaudry, 2013, Contreras et al, 2015;Kroumova and Wagner, 2003;Schiller et al, 2015;Sugimoto et al, 2021;Tan and Bangerth, 2001) processes.…”

“…In addition, we wanted to determine if production rates of individual esters were in agreement with available kinetic analyses of MdAAT1 and use these data to infer whether changes in AAT substrate specificity take place during apple fruit ripening, which would indicate multiple AAT isozymes impact apple aroma. Further, we wished to determine whether esters were synthesized in 'Redchief Delicious' apples via the newly described citramalate pathway, which uses 1-C FAB, and/ or via the relatively more fully characterized 2-C FAB pathway (Kroumova and Wagner, 2003;Risso et al, 2008;Sugimoto et al, 2011Sugimoto et al, , 2015Sugimoto et al, , 2021Xu et al, 2004) by evaluating the incorporation of acetic acid as a native compound and as 1-, 2-, and 1,2-[ 13 C 2 ] isotopologs (Sugimoto et al, 2021).…”