Differential regulation of starch structure-function relationships by pre- and post-anthesis nitrogen in hard red spring wheat.

Nitrogen (N) fertilization regulates carbon allocation during grain filling, but its effects on starch hierarchy and functionality in Hard Red Spring (HRS) wheat remain unclear. This study examined four N rates (0-168 kg N ha-1), including post-anthesis N, across three HRS cultivars, four locations and two growing seasons. Increasing N reduced total starch content (64.53-62.45%) and starch damage (5.08-4.62%) (p < 0.05), while increasing amylopectin content (73.66-77.32%), amylopectin molecular weight (5.76-8.12 × 106 Da), relative crystallinity, short-range molecular order, and starch branching. These changes increased gelatinization temperatures and enthalpy and improved hydration properties. Pre-anthesis N (N112) treatment produced the strongest changes in starch composition, crystallinity, and thermal behavior, whereas the N168 split treatment, which included post-anthesis N, was associated primarily with additional changes in branching and hydration. Although cultivar responses varied, structure-function relationships were consistent across environments. Nitrogen timing therefore regulates starch organization across structural levels and offers a mechanistic approach to modifying starch functionality through agronomic management.