The CsDof1.8-CsLIPOXYGENASE09 module regulates C9 aroma production in cucumber

Nine-carbon aldehydes and their relative alcohols (C9 aromas) are the main aroma compounds of cucumber (Cucumis sativus L.) fruits and provide a unique cucumber-like note. However, the key regulators of C9 aroma accumulation in cucumber fruit are poorly characterized. Based on C9 aroma dynamic analysis and transcriptome analysis during fruit development of two different cucumber inbred lines, Q16 and Q24, Lipoxygenase09 (CsLOX09) was identified as a candidate gene for C9 aroma accumulation. Additionally, Q24 with higher CsLOX09 expression accumulated more C9 aromas than Q16. To verify the function of CsLOX09, Cslox09 homozygote knockout lines were created. C9 aroma content decreased by 80.79% to 99.16% in these mutants compared to the wild type. To further explore the reasons for the difference in CsLOX09 expression between Q16 and Q24 fruits, a co-expression network was constructed by integrating the C9 aroma-associated metabolism and transcriptomic data. Eighteen candidate transcription factors were highly correlated with the expression of CsLOX09. DNA binding with One Finger 1.8 (CsDof1.8) was confirmed to bind directly to the A/TAAAG motif of the CsLOX09 promoter through dual-luciferase, yeast one-hybrid, chromatin immunoprecipitation-qPCR and electrophoretic mobility shift assays. Furthermore, C9 aroma content and CsLOX09 expression were significantly increased in the CsDof1.8 overexpression lines. Overall, these data elucidate the metabolic regulation of C9 aromas in cucumber and provide a foundation for facilitating the regulation of flavor in cucumber breeding.