Molecular Trojan Effect of Microplastic Diethyl Phthalate Drives Multiscale Stress Vortex through Interfacial Engineering in Cold Agroecosystems during Freeze–Thaw Cycles

Global climate change exacerbates the synergistic effects of freeze–thaw (FT) cycles and emerging pollutants in cold-region ecosystems. To elucidate their multidimensional stress mechanisms, this study integrated a “seed-to-seed” full-life-cycle soil cultivation experiment (120 days), physio-ecological assays, molecular dynamics (MD) simulations, and multiomics technologies to systematically analyze the cascading damage mechanisms in rye induced by the combined stress of FT, microplastics (MPs), and diethyl phthalate (DEP). Long-term experiments demonstrated that MPs + DEP copollution led to approximately 27.5% reduction in spike length, over 36% decrease in 1000-grain weight, and an 18–23 d delay in flowering time; these indicators worsened further with the superposition of FT, indicating significant inhibition of reproductive growth. At the physiological mechanism level, DEP competitively inhibited ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity, impeding carbon assimilation; MPs induced thylakoid membrane lipid peroxidation, disrupting the electron transport chain; and FT exacerbated chloroplast ultrastructural damage, collectively causing a 41.1% decrease in the photosynthetic rate (Pn), a 65.8% reduction in stomatal conductance (Gs), and a 140% increase in the malondialdehyde (MDA) content. MD simulations revealed that FT enhanced the binding stability of nonspecific lipid-transfer protein (nsLTP) with DEP, promoting the upward translocation of pollutants, with the highest DEP residue in grains reaching 0.306 ± 0.038 mg/kg, posing a potential food safety risk. Metabolomic analysis indicated that MPs activated genes promoting cell wall fibrosis defense, whereas DEP inhibited lipoxygenase, leading to lipid accumulation, with Mg2+ loss and S accumulation exacerbating the oxidative damage cascade. The endophytic microbiome facilitated cooperative pollutant degradation via the Pseudomonas acidovorax module, achieving partial ecological compensation. This study reveals a “stress compensation–metabolic imbalance–oxidative damage” vicious cycle mechanism, which advances our understanding of composite pollution risks in high-latitude farmland and the synergistic effects of climate change and pollutants.