Abstract

Cold stress is a major environmental challenge that affects tobacco ( Nicotiana tabacum L.) growth and yield potential. However, the precise mechanisms underlying the tobacco plant's response remain largely unclear. In this study, we identified a cold-induced CCCH-type zinc finger protein, NtZFP66L1, as an important positive regulator of cold tolerance in tobacco. Overexpressing NtZFP66L1 improved cold resistance, while knocking it out made plants more sensitive to cold. NtZFP66L1 enhances cold tolerance by targeting NtPP2C-37 and NtGLK1 ; knocking out NtPP2C-37 increased cold tolerance, while loss of NtGLK1 decreased cold resistance. In plants overexpressing NtZFP66L1 , abscisic acid (ABA) biosynthesis-related genes, and ABA content were upregulated under both normal and cold stress conditions. Conversely, their expression and content were decreased in knockout lines. Notably, applying external ABA did not rescue the cold-sensitive phenotype of zfp66l1 mutants. These results suggest that NtZFP66L1 mainly influences low-temperature responses through the ABA signaling pathway. Overall, our findings show that NtZFP66L1 acts as a positive regulator in cold stress by both suppressing the NtPP2C-37 -involved ABA signaling pathway and activating NtGLK1 expression. This research offers valuable insights for genetically improving tobacco cultivars to enhance their ability to adapt to cold stress.