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Background: Although many of the volatile constituents of flavor and aroma in citrus have been identified, the knowledge of molecular mechanisms and regulation of volatile production are very limited. Our aim was to understand mechanisms of flavor volatile production and regulation in mandarin fruit. Result: Fruits of two mandarin hybrids, Temple and Murcott with contrasting volatile and non- volatile profiles, were collected at three developmental stages. A combination of methods, including the isobaric tags for relative and absolute quantification (iTRAQ), quantitative real-time polymerase chain reaction, gas chromatography, and high-performance liquid chromatography, was used to identify proteins, measure gene expression levels, volatiles, sugars, organic acids and carotenoids. Two thirds of differentially expressed proteins were identified in the pathways of glycolysis, citric acid cycle, amino acid, sugar and starch metabolism. An enzyme encoding valencene synthase gene (Cstps1) was more abundant in Temple than in Murcott. Valencene accounted for 9.4% of total volatile content in Temple, whereas no valencene was detected in Murcott fruit. Murcott expression of Cstps1 is severely reduced. Conclusion: We showed that the diversion of valencene and other sesquiterpenes into the terpenoid pathway together with high production of apocarotenoid volatiles might have resulted in the lower concentration of carotenoids in Temple fruit. [ABSTRACT FROM AUTHOR]