Characterization of Grain Protein Accumulation Across Different Spikelet Positions and the Mechanisms Underlying Their Responses to Different Nitrogen Fertilization Practices

Understanding protein accumulation responses to the nitrogen (N) fertilizer at different spikelet positions and physiological mechanisms is essential for high-quality, high-yield wheat. We investigated two cultivars, strong-gluten KX3302 and medium-gluten ZM27, under four N regimes: LN-C, LN-S, HN-C, and HN-S. Parameters analyzed included grain weight (GW), grain protein content (GPC), protein fractions, sedimentation value (SV), N metabolism enzymes, endogenous phytohormones, and glutenin gene expression across spikelet positions. Middle/basal grains exceeded apical grains in GW, GPC, α/β-gliadin, HMW-GS, and LMW-GS; γ-gliadin and SV were higher apically/middle. Superior grains (SGs) surpassed inferior grains (IGs) in GW, GPC, fractions, and beneficial phytohormones. N metabolism enzymes and HMW-GS expression peaked midspikelet and in SGs. HN-S enhanced GW, GPC, SV, and fractions versus LN-C (especially SGs), but GW and HMW-GS increased more in IGs, reducing disparities. Stage-optimized high N elevates protein accumulation and quality by regulating hormones, enzymes, and genes, while boosting GW/SV and reducing positional differences.