Multiple omics insights into the regulatory networks of long-term chilled stress-induced early flowering and reduced yield in tobacco (Nicotiana tabacum cv. KRK26)

Long-term cold stress during early spring transplantation induced early flowering in KRK26, a physiological response closely associated with reduced yield and quality of the flue-cured tobacco. A two-year field experiment was conducted across three locations and three transplanting periods to evaluate the agronomic traits of tobacco plants, the appearance quality and sensory quality of the flue-cured tobacco. The results showed that the early-transplanted plants were significantly inhibited in their growth and development as well as in the quality of the tobacco leaves due to the impact of low-temperature stress. To further explore the molecular regulatory mechanism of cold-induced early flowering on the quality of tobacco plants, a positioning experiment and greenhouse simulation experiment were carried out in 2024. The results showed that the early-transplanted plants experienced a long period of low-temperature exposure (<10°C), exhibited significant dwarfing, early flowering, and a significant reduction in the first-class leaf yield compared to those transplanted later. Long-term cold stress disrupted redox homeostasis, as evidenced by decreased glutathione levels and reduced activities of key antioxidant enzymes (POD and MDHAR), while increasing levels of oxidized ascorbate derivatives (DHA and MDHA). Transcriptomic analyses revealed downregulation of cytoplasmic translation processes, accompanied by activation of circadian rhythm and gibberellin homeostasis pathways. Importantly, WGBS identified methylation difference (predominantly CpG/CHG contexts) within regulatory regions of key genes, despite unchanged global methylation levels. This targeted hypomethylation resulted in transcriptional activation of key flowering regulators, including the age-pathway genes NtSPL5a and NtAP1La . In conclusion, long-term cold stress triggers early flowering and yield loss in KRK26 primarily through hypomethylation-mediated epigenetic activation of expression of key genes related to flowering. This process is further compounded by transcriptomic reprogramming and disruption of the ROS system imbalance. These findings provide a molecular foundation for developing strategies to mitigate cold-induced yield reduction in tobacco cultivation. • Long-term cold stress during early spring transplantation causes early flowering in KRK26, reducing yield and quality. • Cold stress disrupts redox homeostasis and increases oxidative damage by suppressing antioxidant enzyme activities. • Transcriptomic changes included downregulation of translation processes and activation of circadian and gibberellin pathways. • Epigenetic hypomethylation activates flowering-related genes NtSPL5a and NtAP1La, promoting early flowering.