Integrated physiological and transcriptomic analysis reveals novel key genes underlying cold tolerance in Nicotiana tabacum

Cold stress represents one of the most prevalent abiotic stresses that severely limit the growth, productivity, and geographical distribution of crops. As a thermophilic economic crop, tobacco (Nicotiana tabacum) is particularly vulnerable to chilling injury, often resulting in reduced survival rates and significant yield losses. To investigate the molecular basis of cold tolerance, we conducted comparative analyses of contrasting tobacco varieties: cold-resistant Gui tobacco 40 (GZ40) and cold-sensitive Tai tobacco 8 (TY8). Physiological assessments showed that GZ40 exhibited superior cold tolerance, as evidenced by milder wilting, faster post-stress recovery, and enhanced antioxidant defenses. Specifically, after 96 h of cold stress, SOD activity in GZ40 was 65.5% higher than TY8, along with 19.1% lower electrolyte leakage and 71.1% lower proline accumulation relative to TY8. Comparative transcriptomic profiling revealed 20,566 DEGs between EG and EGA and the ET versus ETA comparison identified 16,382 DEGs. Transcriptome profiling of these DEGs identified significant enrichment in cold responsive pathways including proteasome function, photosynthesis, and carbohydrate metabolism. Through weighted gene co-expression network analysis, we identified two key modules, midnight blue and orange, showing distinct expression patterns between varieties. Further analysis of hub genes within these modules highlighted several promising candidates: phospholipase Dβ1 (PLD) involved in membrane remodeling and signal transduction, pentatricopeptide repeat proteins (PPR) regulating organellar gene expression, and translocase of chloroplast 120 (TOC120) ensuring the stability of photosynthetic apparatus. These findings provide novel insights into non-canonical cold resistance mechanisms in tobacco, identifying potential genetic targets for molecular breeding approaches to enhance cold tolerance in tobacco.