The regulatory landscape of β-caryophyllene biosynthesis in pak choi

β-Caryophyllene is a key volatile sesquiterpene involved in plant defense and contributes to the characteristic aroma of pak choi (Brassica campestris). This study aimed to elucidate the regulatory landscape of β-caryophyllene biosynthesis in pak choi to understand the genetic and molecular mechanisms controlling the production of this volatile sesquiterpene. Among 61 germplasm accessions of pak choi, β-caryophyllene was detected in only 11 accessions. Genetic analysis revealed that β-caryophyllene production is controlled by a single dominant gene. Fine mapping and gene sequencing identified the candidate gene B. campestris terpene synthases 21 (BcTPSa21), which encodes a β-caryophyllene synthase. Functional validation of BcTPSa21 through transient expression of BcTPSa21 in Nicotiana benthamiana leaves and enzyme activity assays in vitro confirmed its role in β-caryophyllene biosynthesis. A single nucleotide polymorphism (SNP) (C-T) in the promoter region of BcTPSa21 was found to affect the binding of the transcription factor BcMYC2, thereby influencing gene expression. Additionally, BcDIVARICATA (an R-R-type MYB TF BcDIV) was identified as a negative regulator of β-caryophyllene synthesis. The molecular experiments showed that abscisic acid participates in the biosynthesis of β-caryophyllene via the B. campestris pyrabactin resistance 1-like (BcPYL6)-BcDIVARICATA-BcMYC2 module. RNA-seq analysis suggested that under temperature stress, the transcription of BcTPSa21 and the biosynthesis of β-caryophyllene were the collective result of multilevel regulation. These findings provide comprehensive insights into the regulatory mechanisms governing β-caryophyllene biosynthesis in pak choi, identifying key factors and regulatory modules involved and offering a foundation for enhancing the flavor quality of pak choi through targeted genetic interventions.