Molecular insights into DaERF108-mediated regulation on asperosaponin VI biosynthesis under cold tolerance in...

Plants frequently modulate their hormonal signaling pathways in response to stress, thereby regulating the synthesis of secondary metabolites and adapting to fluctuations in their surroundings. The APETALA2/ethylene-responsive factor (AP2/ERF) domain transcription factors are important in regulating abiotic stress tolerance. The accumulation of asperosaponin VI in the root was significantly enhanced under low temperature stress, which exhibited a correlation with the AP2/ERF family. However, the involvement of AP2/ERF in regulating asperosaponin VI biosynthesis under cold stress remains ambiguous. Under cold stress conditions below 10 °C, we observed the accumulation of asperosaponin VI and an increase in jasmonic acid (JA) levels. This response was attributed to the activation of the JA synthesis pathway induced by low temperatures. Additionally, a comprehensive analysis of the full-length transcriptome of Dipsacus asper identified a total of 80 DaAP2/ERF transcription factors, which exhibited significant homology with Arabidopsis thaliana and Citrus ERFs based on phylogenetic analysis. Furthermore, qRT-PCR analysis demonstrated that both cold stress and methyl jasmonate (MeJA) induction upregulated DaERF108 expression. The expression of DaERF108 is notably upregulated in the leaves and during the early stages of growth and development of D. asper, while subcellular localization analysis confirmed its presence in the nucleus. The overexpression of DaERF108 significantly enhanced the accumulation of oleanolic acid, a precursor of asperosaponin VI, and activated the triterpenoid biosynthesis pathway in Arabidopsis roots. Additionally, the overexpression of DaERF108 induced the activation of the terpenoid synthesis pathway under cold stress conditions. Notably, there was a positive correlation between DaERF108 expression and genes involved in asperosaponin VI biosynthesis, particularly with 3-hydroxy-3-methylglutaryl coenzyme A synthase (DaHMGS). The interaction between DaERF108 and the GCC-box element in the DaHMGS promoter was demonstrated by LUC and Y1H assays, leading to enhanced activity. These findings suggest that DaERF108 specifically binds to the G-box element, thereby regulating DaHMGS gene expression, activating the JA signaling pathway, and promoting asperosaponin VI biosynthesis in response to cold stress.