The AhDREB transgene expression activates NtP5CS and NtSUSY, promoting osmotic adjustment in transgenic tobacco under salt stress

Soil salinity is a major environmental constraint that limits the growth and productivity of peanut (Arachis hypogaea L.), a legume adapted to mildly acidic soils but highly sensitive to saline-alkaline conditions. Dehydration-Responsive Element Binding (DREB) transcription factors are key regulators of plant responses to abiotic stresses. In this study, the AhDREB gene from peanut was introduced into tobacco (Nicotiana tabacum) to examine its functional role under salt stress. The transgenic lines (L32.2 and L37.2) exhibited strong induction of AhDREB expression upon exposure to 150 and 250 mM NaCl, with transcript levels increasing up to 2.34-fold compared with untreated controls (P < 0.001). Quantitative RT-PCR analysis revealed that AhDREB enhanced the transcription of two osmolyte-related genes, NtP5CS and NtSUSY. Under saline conditions, the expression of these genes was 1.20-1.89-fold higher in transgenic lines than in wild-type (WT) plants and 4.74-7.66-fold higher than in non-stress conditions (P < 0.001). Consistently, both lines accumulated greater amounts of proline and soluble sugars, showing 2.09-2.30-fold and 2.40-4.70-fold increases, respectively, compared with the WT. Relative to non-stress conditions, proline and sugar contents increased by 3.59-5.47 fold and 3.75-7.65-fold, respectively. Line L37.2 accumulated higher proline levels, whereas L32.2 exhibited greater sugar content, indicating distinct osmolyte regulation patterns. Overall, the AhDREB gene enhances salt tolerance in tobacco by transcriptionally activating osmolyte biosynthetic pathways and improving cellular osmotic adjustment, providing molecular evidence for its potential application in developing stress-tolerant peanut cultivars.