The Micro-battlefield under climate change: the domino effect of freeze-thaw cycles on Tributyl phosphate accumulation in grains via nutritional, metabolomic, and microbial profiling.

Under the context of climate change, the risks posed by emerging combined pollution to food security, particularly the accumulation and toxicity of contaminants in the edible parts of crops, remain poorly understood. This study investigated the individual and combined effects of freeze-thaw cycles (FTC) and the flame retardant tributyl phosphate (TBP) on rye growth, physiology, rhizosphere microbiome, quality, and grain accumulation through short-term hydroponic and long-term soil cultivation experiments. The results showed that combined FTC and TBP stress exerted a significant synergistic toxic effect on rye, including inhibited plant growth and photosynthesis, aggravated oxidative damage, and disruption of the rhizosphere microbial community structure (e.g., significantly increased abundance of Acidovorax). Long-term combined stress delayed heading, reduced thousand-kernel weight, and led to TBP accumulation in grains (up to 0.25 mg/kg), corresponding to a grain bioaccumulation factor of 0.50. Metabolomic analysis revealed that differentially accumulated metabolites under combined stress were mainly enriched in pathways such as purine metabolism, leading to significant reprogramming of primary metabolites like amino acids and monosaccharides. Nutritional quality analysis showed that combined stress increased crude protein (7.7%) and fiber components but decreased nitrogen-free extract, altered mineral absorption patterns, and likely interfered with cell wall metabolism and nutrient accumulation via molecular interactions between TBP and target proteins such as pectin methylesterase. Transcriptomics further indicated that combined stress specifically regulated pathways including fatty acid biosynthesis and ascorbate metabolism. In summary, FTC exacerbates TBP accumulation and toxicity in rye grains by disturbing the rhizosphere microecology and interfering with key metabolic and nutritional pathways, ultimately affecting yield and quality. This study provides important insights for assessing the migration risk of pollutants along the food chain and highlights the direct implications for dietary exposure assessment of organophosphate esters in cereals, thereby supporting food safety evaluation under climate change.