6-benzyladenine, a cytokinin, promotes the accumulation of essential oil, flavonoids, and phenolics in Santalum album heartwood by interacting with other hormones

Santalum album L. is an economically important species that can produce highly valued essential oil (EO) consisting mainly of santalols in its aromatic heartwood. Elicitors stimulate the production of EO, but the underlying mechanism remains unknown. In this study, the regulatory role and ability of a cytokinin (6-benzyladenine, BA) to promote the accumulation of secondary metabolites, was investigated. The levels of EO and total flavonoids increased 2.3- and 1.5-fold, respectively one year after BA treatment. The top significantly enriched Kyoto Encyclopedia of Genes and Genomes pathways of upregulated differentially expressed genes (DEGs) and differentially accumulated metabolites contained ‘terpenoid biosynthesis’, ‘phenylpropanoid biosynthesis’, and ‘flavonoids biosynthesis’, displaying similar profiles to those detected in natural heartwood from 10-year-old trees. Prominently, the content of santalols was increased by approximately 90-fold by BA relative to the control and was associated with the contents of flavonoids and phenolics. BA also enhanced the contents of cytokinins, an auxin (IAA), gibberellins, jasmonic acid and an ethylene precursor while DEGs associated with the biosynthesis and signaling of these hormones were significantly upregulated. Dual-LUC assays confirmed that identified SaMYC2a-2c and three SaERFs activated SaSSY and SaCYP736A167 expression. Moreover, exogenous BA regulated EO biosynthesis in sandalwood suspension cells but could not directly activate the expression of these two genes in these cells. We infer from these findings that exogenously applied BA positively participated in the biosynthesis of sandalwood EO by interacting with other hormones. These findings also reveal details of the regulatory mechanism underlying the cytokinin-induced production of secondary metabolites, which determine the fragrance and color of S. album heartwood. • An understanding of essential oil formation in Santalum album heartwood was sought. • Exogenous BA promoted the accumulation of secondary metabolites in stems. • BA activated cambial differentiation and led to heartwood formation. • BA also regulates essential oil biosynthesis in sandalwood suspension cells. • Cytokinin positively influenced EO biosynthesis by interacting with other hormones.