Abstract
Background: Drought stress is a primary environmental constraint limiting crop growth and productivity. Current drought-related plant research predominantly focuses on whole-leaf analyses, neglecting the spatial heterogeneity of metabolites within leaf tissues. Methods: This study combined transcriptomic and metabolomic approaches to investigate spatially distinct metabolic responses in marginal versus central regions of Medicago lupulina L. leaves under PEG-simulated drought. Results: Findings demonstrated that TCA cycle metabolites exhibited relative stability between leaf margins and centers under drought conditions, suggesting preserved core metabolic functionality in central tissues to sustain stress tolerance. Additionally, shikimic acid displayed a significantly reduced regional gradient in stressed tissues (PEG Margin vs. PEG Center) compared to controls. Phenylalanine, tryptophan, liquiritigenin, isoliquiritigenin, coproporphyrin III, and coproporphyrinogen III itself exhibited significantly increased internal gradient differences in stressed groups compared to control groups. The coordinated upregulation of key biosynthetic genes (e.g., TAT, AST, FNS II) in both the marginal and central regions of stressed leaves indicates a metabolic shift toward the biosynthesis of downstream defensive flavonoids. These metabolites and genes accumulated preferentially in margin regions of stressed leaves, indicative of localized activation of defense-associated metabolic pathways. Conclusions: This study reveals a spatially partitioned metabolic response to drought stress in M. lupulina leaves, where defensive metabolism is preferentially enhanced at the leaf margins while core metabolic homeostasis is maintained. These findings provide new spatial insights into plant drought acclimation and identify potential targets for improving crop resilience through the fine-tuning of local metabolism.