Comparative Transcriptomic Analysis of High- and Low-Protein Wheat Lines Reveals Differential Nitrogen Responses at the Seedling Stage

Nitrogen (N) availability is a critical determinant of grain yield and protein quality in wheat (Triticum aestivum L.). To elucidate the molecular mechanisms underlying nitrogen response associated with nitrogen use efficiency (NUE), a comparative transcriptomic analysis of high grain protein content (HP) and low grain protein content (LP) wheat lines during N resupply at the seedling stage is conducted in this study, with sampling conducted at T1 (one day after treatment) and T3 (three days after treatment). Our results reveal that the HP line exhibits an early-responsive and well-coordinated metabolic pattern, whereas the LP line shows a distinct temporal response characterized by delayed adjustments. Integrated GSEA and KEGG analyses demonstrated that the HP line prioritized protein processing in the endoplasmic reticulum and diterpenoid biosynthesis, potentially associated with enhanced protein quality control and early signaling efficacy. This allows the HP line to synchronize its N assimilation machinery with the transient peak of N availability at T1 and establishes a robust foundation for protein accumulation. Conversely, the LP line redirected its metabolic resources toward glutathione metabolism and flavonoid biosynthesis to mitigate N-induced oxidative instability. This metabolic shift increases the energetic usage required for antioxidant defense and subsequently deviates resources away from productive N assimilation. These divergent metabolic landscapes were orchestrated by a hierarchical network of transcription factors (TFs). In leaves, the MYB and NAC families showed a more disciplined and immediate increase in the HP line, whereas the LP line demonstrated a delayed peak at T3. In root tissues, while Dof and NAC families were rapidly induced and concluded in the HP line, the LP line exhibited a sluggish sensing-to-response mechanism with prolonged or specific late-stage activation at T3. These results indicate that the capacity for rapid metabolic synchronization and disciplined transcriptomic mobilization is a key physiological indicator of high-protein potential in wheat. This insight provides essential molecular targets for breeding programs aimed at improving NUE and grain quality.