Advanced studies of apple (Malus domestica Borkh) development, physiology, and biochemistry have been hampered by the lack of appropriate genomics tools. One exception is the recent acquisition of extensive expressed sequence tag (EST) data. The entire available EST dataset for apple resulted from the efforts of at least 20 contributors and was derived from more than 70 cDNA libraries representing diverse transcriptional profiles from a variety of organs, fruit parts, developmental stages, biotic and abiotic stresses, and from at least nine cultivars. We analyzed apple EST sequences available in public databanks using statistical algorithms to identify those apple genes that are likely to be highly expressed in fruit, expressed uniquely or preferentially in fruit, and/or temporally or spatially regulated during fruit growth and development. We applied these results to the analysis of biochemical pathways involved in biosynthesis of precursors for volatile esters and identified a subset of apple genes that may participate in generating flavor and aroma components found in mature fruit.Cultivated apple (Malus domestica Borkh) is among the most diverse and ubiquitously cultivated fruit species. Apple is a member of the Rosaceae family, which includes many commercial fruit (e

The relationship among the free amino acid content, the expression of genes related to branched-chain amino acid metabolism {branched-chain aminotransferase [BCAT], α-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 ×domestica) 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 α-keto-β-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 α-keto-β-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.

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.

The volatile ester and alcohol profiles of ripening apple fruit from 184 germplasm lines in the USDA Malus Germplasm Repository at the New York Agricultural Experiment Station in Geneva, NY, USA, were evaluated. Cluster analysis suggested biochemical relationships exist between several ester classes. A strong linkage was revealed between 2-methylbutanoate, propanoate, and butanoate esters, suggesting the influence of the recently proposed "citramalic acid pathway" in apple fruit. Those lines with a high content of esters formed from 2-methylbutan-1-ol and 2-methylbutanoate (2MB) relative to straight-chain (SC) esters (high 2MB/SC ratio) exhibited a marked increase in isoleucine and citramalic acid during ripening, but those lines with a low content did not. Thus, the data were consistent with the existence of the hypothesized citramalic acid pathway and suggest that the Geneva Malus Germplasm Repository, appropriately used, could be helpful in expanding our understanding of mechanisms for fruit volatile synthesis and other aspects of secondary metabolism.

No abstract