Title: Physiological and transcriptome responses of Panax ginseng roots and functional characterization of PgNRT1 under different nitrogen supply status

Nitrogen (N) is an essential macronutrient that determines the growth and medicinal quality of Panax ginseng. Optimizing N application methods and improving N use efficiency hold great significance for the cultivation of ginseng. The present study aimed to systematically explore the regulatory mechanisms and key molecular pathways of ginseng growth with conventional nitrate N supply (NN), double nitrate N supply (DNN), conventional ammonium N supply (AN), and N deficiency supply (ND). We quantified N content and N metabolism-related enzyme activities in 2-year-old ginseng under different N treatments, identified key genes via transcriptome analysis, and validated PgNRT1 function through heterologous expression in Arabidopsis thaliana and Solanum tuberosum (potato). The results showed that DNN supply significantly promoted the development of ginseng lateral roots, enhanced plant photosynthetic capacity, increased nitrate accumulation in plants, and activated the activities of N metabolism enzymes, including nitrate reductase (NR), glutamine synthetase (GS), and glutamate synthase (GOGAT). Transcriptomic analysis revealed that DNN supply specifically upregulated nitrogen metabolism genes (encoding NR, GS, GOGAT). DNN supply significantly promoted root elongation in the PgNRT1-overexpressing Arabidopsis lines and rescued the inhibition of root elongation without N supply, and increased tuber fresh weight in the PgNRT1-transgenic potato lines. In conclusion, DNN supply promotes ginseng growth primarily through enhancing N metabolic processes, and the nitrate transporter gene PgNRT1 was identified as a key regulator which mediated the nitrate-induced growth promotion in ginseng.