Overexpression of MhERF1 promotes flavonoid accumulation in hairy roots of the Tibetan medicinal plant Mirabilis himalaica.

Mirabilis himalaica, a valuable traditional Tibetan medicinal plant, is an important natural source of bioactive flavonoids. Due to excessive harvesting, this species has become endangered, highlighting the need to enhance the production of its medicinal metabolites through molecular breeding and metabolic engineering. In this study, analysis of previously generated transcriptomic and metabolomic datasets identified MhERF1 as a candidate transcription factor gene associated with flavonoid accumulation. The MhERF1 coding sequence was cloned, and Agrobacterium rhizogenes-mediated hairy-root transformation framework was adapted to evaluate the function of MhERF1 overexpression in M. himalaica. Metabolomic profiling showed that overexpression of MhERF1 enhanced flavonoid accumulation in hairy roots, with 421 flavonoid compounds detected and 138 differential flavonoid metabolites identified, of which 107 were upregulated, predominantly enriched in the "flavonoid biosynthesis" pathway. Transcriptomic analysis identified 6,467 differentially expressed genes (DEGs), including significant upregulation of key structural genes such as CHS and C3'H. Integrated analysis indicated that MhERF1 upregulated the expression of key structural genes involved in the flavonoid pathway and formed a putative co-expression network with other transcription factors, such as MYB and C2H2. These findings support MhERF1 as a candidate positive regulator of flavonoid metabolism and provide a basis for further functional validation and metabolic-engineering studies in M. himalaica.