Heterologous Expression of triticale PROLYL AMINOPEPTIDASE (TsPAP1) Enhances Copper Stress Tolerance in Arabidopsis thaliana by Strengthening the Enzymatic Antioxidant Defense System
Heavy metal pollution has become a major environmental challenge limiting agricultural productivity worldwide. Copper (Cu), although an essential micronutrient, becomes phytotoxic at elevated concentrations, primarily by inducing oxidative stress. Among the most widespread metabolic adjustments triggered by abiotic stress is the accumulation of proline, which is a compatible osmolyte that stabilizes proteins and membranes and helps maintain cellular redox homeostasis. This paper demonstrates that the heterologous expression of TsPAP1, a triticale gene encoding a prolyl aminopeptidase, enhances proline accumulation and confers increased Cu tolerance in Arabidopsis thaliana. Under Cu stress, transgenic lines maintained superior physiological performance relative to the wild type (WT), as evidenced by the reduced biomass loss and lower accumulation of malondialdehyde and reactive oxygen species. Cu exposure activated antioxidant defenses; however, the induction of catalase (CAT), class III peroxidases (POD), ascorbate peroxidase (APX), and glutathione reductase (GR) activities was more pronounced in transgenic lines. Transcriptomic analysis revealed a higher expression of genes encoding antioxidant isoforms localized to chloroplasts (sAPX, CSD2), the cytosol (APX1), peroxisomes (CAT1), and the apoplast (Prx02, Prx51), indicating targeted reinforcement of the multi-compartmental redox defense system. Together, these findings identify TsPAP1 as a potential regulator of proline-dependent redox homeostasis that contributes to enhanced Cu tolerance through the coordinated activation of antioxidant networks. The results further suggest that PAP-dependent peptide turnover contributes to proline-mediated stress adaptation, linking peptide metabolism with antioxidant regulation and highlighting TsPAP1 as a promising target for engineering heavy metal-resilient crops.