NtLYK5 negatively regulates drought resistance in tobacco through H₂O₂-mediated leaf stomatal opening and closing

WGCNA mined the unknown gene NtLYK5, and VIGS and RNA-seq analyses suggested that NtLYK5 mediates the negative regulation of hydrogen peroxide production for drought resistance. Drought during the seedling stage of tobacco (Nicotiana tabacum), a water-sensitive and economically important crop, has serious adverse effects on transplant survival and tobacco plant growth. In this study, we conducted transcriptome sequencing on drought-tolerant and drought-sensitive recombinant inbred lines (RILs) from the F7 generation of the cross "NC82 × Bina No. 1." Using weighted gene co-expression network analysis (WGCNA), BLAST alignment, bioinformatics analysis, and qRT-PCR, we identified a key candidate gene NtLYK5, which is highly associated with drought resistance but previously had an unknown function. Virus-induced gene silencing to suppress NtLYK5 expression in tobacco enhanced drought resistance, reduced hydrogen peroxide (H₂O₂) levels, and increased catalase (CAT) activity. Further transcriptomic analysis of drought-tolerant and drought-sensitive lines revealed differentially expressed genes (DEGs) associated with NtLYK5-induced drought resistance, which were significantly enriched in the MAPK signaling pathway. Under drought stress, H₂O₂ acts as a signaling molecule affecting the expression of DEGs such as ANP1, which induces H₂O₂ production and cell death, thereby preventing the stomata from closing properly. These results suggest that NtLYK5 is a key gene that negatively regulates drought tolerance in tobacco seedlings by modulating H₂O₂-induced stomatal movement.