Melatonin activates the ZjERF34-ZjMYB1 regulatory pathway to promote flavonoid biosynthesis and salt tolerance in jujube (Ziziphus jujuba Mill.)

Flavonoids are essential secondary metabolites that contribute to fruit nutritional quality and stress adaptation. Jujube (Ziziphus jujuba Mill.) accumulates abundant flavonoids; however, the regulatory mechanisms by which melatonin (MT) modulates flavonoid biosynthesis remain poorly understood.

In this study, exogenous MT treatment markedly promoted flavonoid accumulation in jujube leaves and triggered dynamic changes in endogenous MT levels, implying a regulatory function of MT in this process. Transcriptomic and correlation analyses identified ZjMYB1 as a key MT-responsive regulator strongly associated with flavonoid accumulation. Functional characterization revealed that silencing ZjMYB1 significantly decreased flavonoid content and downregulated genes in the phenylpropanoid pathway, whereas its overexpression enhanced flavonoid accumulation in jujube and heterologous systems. ZjMYB1 directly activated the key structural genes ZjF3H and ZjFLS by binding to MYB cis-elements in their promoters. Furthermore, the MT-responsive transcription factor ZjERF34 was identified as an upstream regulator of ZjMYB1. ZjERF34 directly targets the ZjMYB1 promoter to enhance its transcription, as demonstrated by Y1H, GUS staining, and dual-luciferase assays, establishing a hierarchical ERF–MYB regulatory network. In addition, ZjMYB1 overexpression significantly enhanced salt tolerance, correlating with increased flavonoid accumulation and upregulation of stress-responsive genes.

Collectively, our findings reveal a MT–ZjERF34–ZjMYB1–ZjF3H/FLS regulatory module that links hormonal signaling with flavonoid biosynthesis and abiotic stress responses, providing new insights and potential molecular targets for improving fruit quality and stress resilience in horticultural crops.