Integration of transcriptomics and metabolomics reveals the regulation of (E)-β-ionone synthesis by the CsKHZ1-CsCCD4 module in Chinese orchid (Cymbidium sinense)

Cymbidium sinense is a renowned ornamental orchid species appreciated for its captivating floral fragrance. To investigate the molecular mechanisms underlying floral scent formation, we performed an integrated analysis of transcriptomics and metabolomics across five developmental stages and three floral tissues. Metabolomic profiling revealed that ( E )-β-ionone is the predominant volatile compound, with its levels fluctuating significantly during flower development and correlating with fragrance intensity. Notably, the sepals and petals were identified as the primary sources of floral scent, with their emission increasing as the flowers bloomed. Transcriptomic analysis revealed 1272 differentially expressed genes during the period of significant floral scent enhancement, with most genes, including CsCCD4 , enriched in metabolic pathways and secondary metabolite biosynthesis. Weighted gene co-expression network analysis (WGCNA) identified CsCCD4 as a central hub gene, closely associated with ( E )-β-ionone biosynthetic pathways. Functional validation through stable transformation in tobacco and transient expression in Phalaenopsis petals confirmed that CsCCD4 catalyzes ( E )-β-ionone production, promoting floral fragrance accumulation. Additionally, yeast one-hybrid screening, dual luciferase, electrophoretic mobility shift, and protoplast assays showed that CsKHZ1 , a zinc finger transcription factor, binds the CsCCD4 promoter and activates transcription, reported here for the first time. These findings suggest that the CsKHZ1 - CsCCD4 module regulates ( E )-β-ionone accumulation and enhances floral fragrance in C. sinense . This study provides a deeper understanding of the molecular networks governing floral scent formation and offers valuable genetic resources for future breeding programs aimed at enhancing fragrance traits in orchids. Moreover, the identification of key regulatory genes holds potential for biotechnological applications in the production of high-value volatiles. • Metabolomic analysis revealed ( E )-β-ionone as the main floral scent component in Cymbidium sinense . • Transcriptomic analysis identified CsCCD4 as a key differentially expressed gene positively correlated with ( E )-β-ionone accumulation. • WGCNA identified CsCCD4 as a hub gene within an ( E )-β-ionone-related gene co-expression network. • Heterologous expression of CsCCD4 in Nicotiana alata and Phalaenopsis significantly increased ( E )-β-ionone production. • Promoter assays provided the first evidence that CsKHZ1 binds the CsCCD4 promoter and activates transcription.