Objectives Foxtail millet (Setaria italica L. P. Beauv) exhibits strong tolerance to nutrient-poor soils. Exploring the changes in root morphology and physiological metabolism in response to low phosphorus (P) stress in foxtail millet is crucial for understanding its mechanisms of low-P tolerance.

Methods Hydroponic experiments were conducted using low-P tolerant genotype B254 and low P-sensitive genotype B171. Two treatments were set up: normal and low-P Hoagland nutrient solutions with KH₂PO₄ concentrations of 0.25 mmol/L and 0.0025 mmol/L, respectively. At 7 and 14 days after treatment in the nutrient solutions, samples were collected for analysis of agronomic traits, root morphology, P content, and acid phosphatase (ACP) activity. Metabolites in leaves and roots were analyzed using LC-MS/MS technology, with VIP>1, |log₂(FC)|≥1, and P<0.05 as criteria for screening differential metabolites. The main metabolic pathways of these differential metabolites were analyzed using the KEGG metabolic pathway database.

Results Under low-P conditions, the plant height of foxtail millet significantly decreased by 32.8%, and root P content can be decreased by 75.84%. However, the total root length, root surface area of the tolerant genotype B254 significantly increased by 33.7% and 59.5%, respectively, demonstrating strong low-P tolerance. Meanwhile, chlorophyll, anthocyanin, and ACP activity also significantly increased under low-P conditions, the ACP activity in the leaves and roots of the B254 germplasm significantly increased by 91.83% and 22.91%, respectively, and this increase was more pronounced in B254. Correlation analysis showed that the correlation between plant height, root development, and physiological adaptive traits was significantly enhanced under low-P conditions. Metabolomics analysis revealed significant differences in metabolite types between the two cultivars under low-P stress. Specifically, B171 accumulated large amounts of flavonoids, terpenes, and lipids in both leaves and roots, while the differential metabolites in B254 were mainly amino acids and lipids, especially the accumulation of lysophosphatidic acid, a precursor for the synthesis of special ester phospholipids. KEGG metabolic pathway analysis found that low-P stress significantly enriched purine metabolism in foxtail millet leaves and phenylalanine metabolism in roots.

Conclusions Low P stress can induce foxtail millet germplasm with low phosphorus tolerant to enhance acid phosphatase activity, increase anthocyanin content, and promote the synthesis of metabolites such as flavonoids, amino acids, and lipids. Notably, the accumulation of phospholipid precursors such as lysophosphatidic acid plays a crucial role. The differential metabolites were primarily observed in purine metabolism and phenylalanine metabolism pathways.