Multi‐Generational High Nitrogen Application Inhibits Seed Germination in Wheat: Insights Into Metabolic Dynamics and Microbial Interactions

Excessive nitrogen (N) application can affect soil health and crop performance, yet the multi-generational effects of prolonged high-N exposure on wheat seed germination remain unclear. We profiled seed metabolomes and endophytic bacterial communities across seven consecutive wheat generations (F1-F7) under normal-N and high-N regimes, and assessed germination of F7 seeds. Spatial metabolomics, spatial transcriptomics and 16S rRNA sequencing were used to resolve embryo- and endosperm-specific responses. Seven generations of high-N treatment delayed progeny seed germination and decreased the germination index by 12.3%, whereas the vigor index was not significantly affected. Spatial omics revealed strong embryo-endosperm heterogeneity and a high-N-associated shift in endosperm metabolism, including a 73.9% decrease in endosperm L-aspartic acid. Compartment-resolved microbiome analysis showed enrichment of Bacillus under high-N exposure. Functional assays showed that L-aspartic acid promoted germination/early outgrowth, whereas Bacillus grew with L-aspartic acid as the sole C/N source and reduced germination under L-aspartic acid-supplemented conditions. These results support a model in which multi-generational high N reduces wheat seed germination through endosperm amino-acid depletion and altered endophytic bacterial assembly.