Objective This study aimed to investigate the function and potential regulatory mechanisms of the EgrNAC13, a stress responsive NAC (SNAC) gene, in the abiotic stress responses of Eucalyptus, particularly under cold and salinity conditions. The findings are expected to provide valuable genetic resources and a theoretical foundation for improving abiotic stress tolerance in Eucalyptus through molecular marker-assisted breeding.

Method Multiple sequence alignment and phylogenetic analyses of EgrNAC13 and its homologs from different plant species were conducted to characterize its protein structure and evolutionary relationships. A EgrNAC13 overexpression vector, driven by 35S, was constructed and transformed into Arabidopsis thaliana. Based on transgene expression analysis of EgrNAC13 in transgenic lines, two overexpression lines were selected for cold and salt stress treatments, and their stress responsive phenotypes were evaluated. In addition, the potential regulatory mechanisms of EgrNAC13 were preliminarily explored by measuring superoxide dismutase (SOD) and peroxidase (POD) activities, as well as by analyzing the expression patterns of key cold responsive genes including AtCBF1、AtCBF2和AtRD29A by qRT-PCR at different time points during cold treatment.

Result Phylogenetic analysis revealed clear evolutionary divergence of EgrNAC13 between monocotyledonous and dicotyledonous plants, as well as between woody and herbaceous species. The protein exhibited high amino acid sequence conservation among Myrtaceae species. The NAM domain sequences of EgrNAC13 homologous proteins from different species are highly similar, indicating functional conservation among these proteins. Transgenic Arabidopsis lines overexpressing EgrNAC13 displayed significantly enhanced tolerance to both cold and salinity stress. Following exposure to −6 °C, the survival rates of EgrNAC13-OE1 and EgrNAC13-OE5 reached 81.6% and 85.1%, respectively, which were significantly higher than that of the wild type (32.3%). Under both cold and salt stress conditions, SOD activity was significantly higher in the overexpression lines than in the wild type. POD activity, however, was significantly elevated only under salt stress. This indicate that EgrNAC13 can improve plant stress tolerance to low temperature and high salt stress by enhancing the scavenging capacity of reactive oxygen species. During cold treatment, the expression levels of AtCBF1 and AtCBF2 in the overexpression lines were significantly upregulated at 3 h and 12 h of 4 °C treatment, respectively. In contrast, AtRD29A expression was consistently and significantly induced in the overexpression lines under both normal and cold treatment conditions. Therefore, EgrNAC13 mediates plant responses to low temperature stress mainly by regulating the CBF pathway.

Conclusion EgrNAC13, a highly conserved NAC transcription factor in Myrtaceae species, significantly enhances cold and salt tolerance in transgenic Arabidopsis by increasing antioxidant enzyme activities, particularly SOD and POD. Moreover, EgrNAC13 may mediate stress responses mainly through CBF-dependent pathways. These findings provide a foundation for further elucidation of the stress resistance functions and molecular mechanisms of EgrNAC13 in Eucalyptus and offer promising genetic resources for stress tolerant breeding programs.