Temporal dynamics of protein phosphorylation and S-nitrosylation direct maize seed germination programs

Seed germination directly influences seedling establishment and ultimately crop yield. While hormonal and transcriptional regulations of germination are well characterized, the role of post-translational modifications (PTMs) remains largely unexplored. In this resource article, we developed a phosphate-affinity tag-switch (PAT-switch) proteomics workflow enabling simultaneous, site-specific profiling of phosphorylation and S-nitrosylation during maize seed germination. Using this strategy, we identified over 5921 S-nitrosylation sites-the most comprehensive S-nitrosoproteome reported in maize to date-and 15 947 phosphorylation sites. Quantitative analysis revealed that phosphorylation predominantly occurs early in germination, activating primary metabolism and regulatory pathways. In contrast, S-nitrosylation is more prominent in later stages, likely fine-tuning metabolism and redox balance. Integrating these PTM datasets uncovered extensive and dynamic interactions that modulate cell programs required for seedling establishment, particularly among enzymes and signaling hubs in glycolysis and hormone pathways. Notably, we identified a conserved SnRK1α kinase, in which adjacent S-nitrosylation at Cys¹⁷² and phosphorylation at Thr¹⁷⁰ reciprocally modulates kinase activity via local structural effects. Specifically, kinase activity assays and western blotting demonstrated that NO-mediated S-nitrosylation reduces the efficiency of Thr¹⁷⁰ phosphorylation. Molecular dynamics simulations further revealed that S-nitrosylation of Cys¹⁷² stabilized the activation loop in a conformation that restricts Thr¹⁷⁰ accessibility. Collectively, our results highlight the importance of the PTM landscape and inter-PTM interactions in mediating developmental transitions during maize seed germination, providing novel insights and resources that may lead to crop improvements via manipulating PTM networks.