Aluminum (Al) toxicity and low phosphorus availability (LP) are the top two co-existing edaphic constraints limiting agriculture productivity in acid soils. Plants have evolved versatile mechanisms to cope with the two stresses alone or simultaneously. However, the specific and common molecular mechanisms, especially those involving flavonoids and carbohydrate metabolism, remain unclear. Laboratory studies were conducted on two wheat genotypes—Fielder (Al-tolerant and P-efficient) and Ardito (Al-sensitive and P-inefficient)—exposed to 50 μM Al and 2 μM Pi (LP) in hydroponic solutions. After 4 days of stress, wheat roots were analyzed using transcriptomics and targeted metabolomics techniques. In Fielder, a total of 2296 differentially expressed genes (DEGs) were identified under Al stress, with 1535 upregulated and 761 downregulated, and 3029 DEGs were identified under LP stress, with 1591 upregulated and 1438 downregulated. Similarly, 4404 DEGs were identified in Ardito under Al stress, with 3191 upregulated and 1213 downregulated, and 1430 DEGs were identified under LP stress, with 1176 upregulated and 254 downregulated. GO annotation analysis results showed that 4079 DEGs were annotated to the metabolic processes term. These DEGs were significantly enriched in the phenylpropanoid, flavonoid, flavone and flavonol biosynthesis, and carbohydrate metabolism pathways by performing the KEGG enrichment analysis. The targeted metabolome analysis detected 19 flavonoids and 15 carbohydrate components in Fielder and Ardito under Al and LP stresses. In Fielder, more responsive genes and metabolites were involved in flavonoid metabolism under LP than Al stress, whereas the opposite trend was observed in Ardito. In the carbohydrate metabolism pathway, the gene and metabolite expression levels were higher in Fielder than in Ardito. The combined transcriptome and metabolome analysis revealed differences in flavonoid- and carbohydrate-related genes and metabolites between Fielder and Ardito under Al and LP stresses, which may contribute to Fielder’s higher resistance to Al and LP. The results of this study lay a foundation for pyramiding genes and breeding multi-resistant varieties.
