Cultivar-specific spatial metabolic differentiation across rice grain microregions at the wax-ripening stage

Pigmented rice is rich in anthocyanins, flavonoids, phenolic acids, and other specialized metabolites. However, their spatial distribution within grain microregions remains insufficiently characterized. Here, untargeted LC-MS/MS and AFADESI-MSI were integrated to characterize global and microregional metabolic profiles in black-grained YZ8 and white-grained YC2332 at the wax-ripening stage, using three biological replicates per cultivar. AFADESI-MSI comparisons were based on biological-replicate-level ROI abundances, and metabolite assignments were treated as putative because authentic standards were not used for structural confirmation. The two cultivars were clearly separated at the global metabolite level and exhibited pronounced microregional heterogeneity. YZ8 showed relatively high ion signals of anthocyanin-, flavonoid-, and phenolic acid-related features in the outer layer, whereas YC2332 showed relatively high signals of sterol-, quinone-, and glycerophospholipid-related features. The outer layer, endosperm, and embryo displayed distinct metabolic characteristics within each cultivar. These results reveal cultivar-specific spatial metabolic patterns that require validation across broader rice germplasm.