Interactive effects of cadmium stress and freeze–thaw cycles on Secale cereale L.: Protective role of exogenous betaine

Cadmium (Cd) and the freeze-thaw cycle (FTC) are serious concerns worldwide, and plants at high altitudes are particularly vulnerable to these stresses. This study aims to evaluate the role of Betaine (B) in increasing Secale cereale L. tolerance to FTC and Cd stress. In this study, Secale cereale L . was chosen as a model to investigate the effect of B application on seedlings exposed to FTC and Cd stress. Photosynthetic and non-photosynthetic parameters were measured. An integrated biomarker response (IBR) model and molecular docking were also used for a comprehensive assessment. The results showed that Cd and FTC significantly elevated malondialdehyde (MDA), hydrogen peroxide (H 2 O 2 ), ascorbate peroxidase (APX), glutamate decarboxylase (GAD), nonprotein thiol (NPT), phytochelatins (PCs), and reduced glutathione (GSH) activities. Combined stress resulted in significant increases of 9.42%, 10.7%, and 4.6% in GAD, H2O2, and NPT, respectively, and increases of 2.5% and 16.1% in PCs and MDA activities, respectively. FTC and Cd adversely affected photosynthetic parameters, causing decreases of 19.4%, 58.2%, and 56.3% in water use efficiency (WUE), transpiration rate (Tr), and photosynthetic rate (Pn), respectively, while intercellular CO₂ concentration (Ci) increased by 61.7%. With exogenous B application, antioxidant enzyme activities were elevated, while MDA, H 2 O 2 , and relative conductivity (RC) were reduced. These results suggest that B is a capable agent for mitigating abiotic stress in cold-exposed and Cd-contaminated environments. The outcomes provide essential insights for future research on ecology, environmental pollution, and management strategies to enhance plant resilience. • Freeze–thaw cycles and cadmium pose combined ecological stress to rye seedlings. • Dual stress elevates oxidative damage and disrupts photosynthetic performance. • Betaine mitigates Cd–FTC toxicity by strengthening antioxidant defenses.