NtSAP9 confers freezing tolerance in Nicotiana tabacum plants

Abiotic stresses, such as extreme temperatures, drought, and salinity, significantly affect plant growth and productivity. Among these, cold stress is particularly detrimental, impairing cellular processes and leading to reduced crop yields. In recent years, stress-associated proteins (SAPs) containing A20 and AN1 zinc-finger domains have emerged as crucial regulators in plant stress responses. However, the functions of SAPs in tobacco plants remain unclear. Here, we isolated Nicotiana tabacum SAP9 (NtSAP9), whose expression was induced by cold treatment, based on RNA-sequences data. Knock down of NtSAP9 expression reduced freezing tolerance, while overexpression conferred freezing tolerance in transgenic tobacco plants, as indicated by relative electrolytic leakage and photosystem II photochemical efficiency. Untargeted metabolomics via liquid chromatography-tandem mass spectrometry revealed distinct metabolic profiles between WT and NtSAP9-overexpressing tobacco plants under normal and low temperature conditions. Upregulation of amino acids like D-Glutamine, DL-Glutamine, and O-Acetyl-L-serine suggests NtSAP9 enhances cold tolerance. Further expression analysis by quantitative real-time PCR indicated that NtSAP9 participates in cold stress response possibly through amino acid synthesis-related genes expression, such as glutamine synthetase and glutamate dehydrogenase. These findings improve our understanding of SAP proteins in tobacco's response to cold stress.