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Plants control the quantities of methyl salicylate through a variety of biosynthetic pathways, including the methylation of salicylic acid to form methyl salicylate and subsequent glycosylation to prevent methyl salicylate emission. Here, we identified a subclade of tomato methyl esterases, SALICYLIC ACID METHYL ESTERASE1-4, responsible for demethylation of methyl salicylate to form salicylic acid in fruits. This family was identified by proximity to a highly significant methyl salicylate genome-wide association study locus on chromosome 2. Genetic mapping studies in a biparental population confirmed a major methyl salicylate locus on chromosome 2. Fruits from SlMES1 knockout lines emitted significantly (P\u2009&amp;lt;\u20090,05, t test) higher amounts of methyl salicylate than wild-type fruits. Double and triple mutants of SlMES2, SlMES3, and SlMES4 emitted even more methyl salicylate than SlMES1 single knockouts\u2014but not at statistically distinguishable levels\u2014compared to the single mutant. Heterologously expressed SlMES1 and SlMES3 acted on methyl salicylate in vitro, with SlMES1 having a higher affinity for methyl salicylate than SlMES3. The SlMES locus has undergone major rearrangement, as demonstrated by genome structure analysis in the parents of the biparental population. Analysis of accessions that produce high or low levels of methyl salicylate showed that SlMES1 and SlMES3 genes expressed the highest in the low methyl salicylate lines. None of the MES genes were appreciably expressed in the high methyl salicylate-producing lines. We concluded that the SlMES gene family encodes tomato methyl esterases that convert methyl salicylate to salicylic acid in ripe tomato fruit. Their ability to decrease methyl salicylate levels by conversion to salicylic acid is an attractive breeding target to lower the level of a negative contributor to flavor.<\/jats:p>","DOI":"10.1093\/plphys\/kiac509","type":"journal-article","created":{"date-parts":[[2022,10,31]],"date-time":"2022-10-31T11:49:06Z","timestamp":1667216946000},"page":"110-124","source":"Crossref","is-referenced-by-count":25,"title":["A family of methyl esterases converts methyl salicylate to salicylic acid in ripening tomato fruit"],"prefix":"10.1093","volume":"191","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1889-4885","authenticated-orcid":false,"given":"Elizabeth M","family":"Frick","sequence":"first","affiliation":[{"name":"Horticultural Sciences, University of Florida , Gainesville, Florida 32611, 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