CRISPR/Cas9-mediated targeted mutagenesis of bZIP2 in Salvia miltiorrhiza leads to promoted phenolic acid biosynthesis

Phenolic acids comprise the major bioactive compounds in Salvia miltiorrhiza, which can be increased by abscisic acid (ABA) elicitation. However, the underlying regulation mechanism is poorly understood. Here, the ABA-induced transcriptome revealed that bZIP2 was significantly induced, which expressed highly in roots. Bioinformatic analysis revealed that bZIP2 belong to the subgroup A and contained a bZIP domain together with four conservative regions (C1, C2, C3, and C4). Phenolic acid content was increased in bZIP2 knock-out lines medicated by CRISPR/Cas9 technology. In contrast, overexpressing lines produced less phenolic acids by downregulating the PAL gene. Biochemistry assays showed that bZIP2 binds to the ABRE2 element in the promoter region of the PAL gene and suppresses its expression. Besides, bZIP2 physically interacted with SnRK2.3, SnRK2.4, SnRK2.6, and SnRK2.10. Overall, these results demonstrated that bZIP2 is a negative regulator in phenolic acid biosynthesis, which provides new insights for the metabolic engineering of phenolic acids.