A Pathway‐Based Metabolomics Approach Reveals Metabolic Strategies and Reprogramming in Drought‐Tolerant Soybean Genotypes

ABSTRACT Soybean ( Glycine max ) is a globally important crop, yet its productivity is highly susceptible to drought stress, particularly in rainfed systems. To better understand the biochemical basis of drought tolerance, this study employed pathway‐based metabolomics to characterize differentially expressed metabolites between drought‐tolerant and susceptible soybean genotypes and to identify underlying mechanisms. Metabolite profiles of leaves collected from both watered and drought‐stressed conditions were analyzed using liquid chromatography–mass spectrometry. Discriminant metabolites were identified through multivariate statistical analysis and pathway mapping. Principal component analysis revealed distinct metabolic variation primarily under drought stress, indicating that drought‐tolerant genotypes engage in active metabolic reprogramming in response to water deficit. Overall, the metabolic responses of drought‐tolerant genotypes were less pronounced than those of susceptible ones, suggesting a more selectively managed allocation of metabolic resources in the tolerant genotypes. Pathway analysis indicated that tolerant genotypes selectively enhanced specific primary and secondary metabolic processes, including central carbon metabolism, shikimate pathway–associated metabolites, and specific amino acid pools, while also displaying divergent allocation within phenylpropanoid (secondary metabolism) and branched‐chain amino acid pathways (primary metabolism). Both tolerant and susceptible cultivars exhibited shared drought responses, including hormonal activation, lipid remodeling, accumulation of phenylpropanoid intermediates, and osmoprotective amino acids. This study demonstrates that drought adaptation arises from the interplay between conserved biochemical adjustments and genotype‐specific reprogramming in primary and secondary metabolism, providing metabolite‐level insights that can guide future large‐scale field studies aimed at selecting genotypes for drought tolerance using biomarkers.