Integrated physiological and transcriptome analysis identifies EgNAC9 as a master regulator of cold tolerance in oil palm.

Oil palm (Elaeis guineensis Jacq.) is the highest-yielding oil crop globally, however, its geographical distribution is severely constrained by pronounced sensitivity to low temperatures, which disrupt physiological homeostasis and impair agronomic performance. Cold exposure precipitates a rapid suppression of photosynthetic capacity, reflected by declines in stomatal conductance, intercellular CO₂ concentration, and pigment stoichiometry, indicative of early photoprotective modulation. To elucidate the molecular determinants underlying cold acclimation in this tropical perennial, we employed an integrative multi-omics strategy encompassing physiological measurements, high-resolution transcriptomics, and weighted gene co-expression network analysis (WGCNA). Acute cold stress elicited transient induction of antioxidant enzymes (SOD, CAT) and proline accumulation, whereas prolonged exposure resulted in excessive ROS accumulation and oxidative cytotoxicity. Network topology analysis identified EgNAC9 as a high-connectivity hub transcription factor strongly co-expressed with ROS-scavenging pathways. Functional characterization in Arabidopsis thaliana demonstrated that EgNAC9 overexpression confers enhanced cold tolerance through augmented root system vigor, optimized stomatal regulation, and attenuation of oxidative damage. Mechanistic assays further revealed that EgNAC9 directly binds to and transcriptionally activates the EgCAT1 promoter, constituting a cold-responsive regulatory module that reinforces antioxidant capacity. Collectively, these findings delineate EgNAC9 as a key regulatory node orchestrating ROS homeostasis and cold stress resilience in oil palm and provide a mechanistic foundation for molecular breeding of cold-adapted cultivars suitable for marginal and subtropical environments.