OsERF101-OsACO1 module acts a negative regulator of salinity resistance in rice by modulating ethylene-associated ROS...

Soil salinity severely constrains rice productivity worldwide. As an essential phytohormone, ethylene participates in the developmental processes and stress adaptation of plants; however, the regulatory mechanisms linking ethylene biosynthesis to salt tolerance in rice remain incompletely understood. Here, we demonstrate that the ethylene biosynthesis gene OsACO1 and the AP2/ERF transcription factor OsERF101 negatively regulate salinity tolerance in seedlings. The CRISPR/Cas9-mediated knockout mutants, osaco1 and oserf101, exhibited significantly enhanced survival under 150 mM NaCl treatment, along with reduced reactive oxygen species (ROS) accumulation and enhanced antioxidative enzymatic activity. Transcriptome profiling revealed that salt-treated osaco1 seedlings displayed extensive transcriptional reprogramming, including upregulation of genes involved in ion homeostasis, ROS detoxification, hormone signaling (JA/ABA/ET), transcriptional regulation, and stress defense. A salt-responsive module associated with the osaco1 genotype was detected using weighted gene co-expression network analysis. Promoter motif enrichment and intersection analyses highlighted AP2/ERF transcription factors as key regulatory candidates. The direct interaction between OsERF101 and the GCC-box element within the OsACO1 promoter and the transcriptional activation of OsACO1 expression were confirmed through yeast one-hybrid, dual-luciferase reporter, and electrophoretic mobility shift assays. Collectively, our results support a model in which OsERF101 positively regulates OsACO1 expression, thereby modulating ethylene-associated ROS homeostasis and hormone crosstalk to fine-tune salt stress responses. Our findings elucidate the mechanisms underlying the ethylene-driven regulation of salinity resistance in rice and identify potential targets for molecular breeding of salinity-tolerant cultivars.