Shrunken2 (Sh2) and Brittle2 (Bt2) play a crucial role in the developmental processes of the aleurone layers in maize.

The aleurone layer, located on the outermost region of the endosperm, is rich in lipids, proteins, and minerals and can substantially enhance grain nutritional value with increased thickness. ADP-glucose pyrophosphorylase is a critical rate-limiting enzyme in starch biosynthesis, with Shrunken2 (Sh2) and Brittle2 (Bt2) encoding its large and small subunits, respectively. Nevertheless, the influence of these genes on maize (Zea mays) aleurone layer development remains poorly understood. We conducted genetic analysis, map-based cloning, and allelic validation using the Huajian1F (HJ1F) paternal line containing multi-aleurone layers, and identified that Sh2 regulates aleurone layer thickness in HJ1F. Additional studies demonstrated that the bt2 mutant displayed the phenotype of aleurone layer thickening. Both sh2 and bt2 mutants exhibited a substantial increase in the contents of grain oil, total protein, and mineral elements. RNA sequencing analysis indicated that differentially expressed genes were enriched in fatty acid and amino acid metabolic pathways during aleurone layer formation. ZmDof3 (DNA-binding one zinc finger protein 3) and NKD2 (NAKED ENDOSPERM2), which participate in aleurone layer thickening in maize, showed significant differential expression during the aleurone layer development of sh2 and bt2 mutants. Yeast one-hybrid assays, β-galactosidase activity assays, and the luciferase reporter system confirmed that ZmDof3 and NKD2 regulate the expression of Sh2 and Bt2. This study has established that Sh2 and Bt2 control starch biosynthesis and play a pivotal role in maize aleurone layer development.