Abstract
Symbiotic nitrogen fixation in legumes is highly sensitive to soil salinity, yet the spatially resolved molecular mechanisms governing nodule dysfunction remain poorly understood. In this study, we integrated spatial transcriptomics (10x Visium) and matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to map the micro-environmental landscape of transcriptomic and metabolic alterations in indeterminate root nodules of Medicago truncatula exposed to acute salt stress (150 mM NaCl). High-resolution spatial profiling resolved distinct zonation-dependent responses across the developmental continuum of the nodule (Zones I–IV and the nodule cortex). Salt stress triggered a rapid spatial metabolic reprogramming characterized by a severe depletion of critical dicarboxylates—specifically malate and succinate—within the symbiotic nitrogen-fixing Zone III, directly correlating with localized down-regulation of host sucrose synthase (MtSUCS1) and bacteroid dicarboxylate transporter genes. Concurrently, salt stress induced localized accumulation of protective osmolytes, including proline and pinitol, predominantly restricted to the non-infected cortical parenchyma and Zone II. Spatial transcriptomics further uncovered an unexpected micro-localized activation of AP2/ERF transcription factors and glutathione S-transferase pathways in the outer cortex, alongside premature transcriptional signatures of senescence in Zone III. These integrated spatial multi-omics findings demonstrate that salinity impairs nitrogen fixation through localized carbon starvation and cell-type-specific oxidative stress rather than systemic metabolic shutdown. Our work offers high-resolution insights into legume-rhizobium symbioses under abiotic stress, highlighting cell-layer-specific targets for engineering salt-tolerant leguminous crops.
