Integrated miRNA-mRNA-degradome analysis unveils the key regulatory mechanism of ginseng under temperature-water stress.

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conclusionMYB and bHLH are the key transcription factors to coordinate the balance between antioxidant system and secondary metabolism under combined temperature-water stress. Low temperature and drought severely impede medicinal plant growth and secondary metabolite accumulation. Identifying key regulatory genes or modules and clarifying their molecular mechanisms is critical for advancing the ginseng industry. Using ginseng hairy roots, we established a composite stress system combining 5 °C with 10-30% polyethylene glycol. We assessed effects on ginseng hairy roots growth, ginsenoside content, and stress-related physiological responses, and performed miRNA, transcriptome, and degradome sequencing analysis. Low-temperature and mild-drought (LTD) treatment significantly promoted ginsenoside accumulation, peaking on day 8, and enhanced antioxidant enzyme activity, indicating robust stress tolerance. Co-analysis identified four miRNAs, mtr-MIR159a-p3_2ss16GT17CT_1, mtr-MIR159a-p3_2ss16GT17CT_2, ptc-miR319a_L + 1R-1, and stu-miR482c_2ss12AT18GA that target MYB and bHLH transcription factor family and exhibit negative expression correlations. These miRNAs likely act as central regulators of ginsenoside biosynthesis under combined temperature-water stress, providing mechanistic insights into how ecological factors modulate ginsenoside production.