Natural variation in ZmNRT2.5 modulates husk leaf width and promotes seed protein content in maize

The husk leaf of maize (Zea mays) encases the ear as a modified leaf and plays pivotal roles in protecting the ear from pathogen infection, translocating nutrition for grains and warranting grain yield. However, the natural genetic basis for variation in husk leaf width remains largely unexplored. Here, we performed a genome-wide association study for maize husk leaf width and identified a 3-bp InDel (insertion/deletion) in the coding region of the nitrate transporter gene ZmNRT2.5. This polymorphism altered the interaction strength of ZmNRT2.5 with another transporter, ZmNPF5, thereby contributing to variation in husk leaf width. We also isolated loss-of-function mutants in ZmNRT2.5, which exhibited a substantial decrease in husk leaf width relative to their controls. We demonstrate that ZmNRT2.5 facilitates the transport of nitrate from husk leaves to maize kernels in plants grown under low-nitrogen conditions, contributing to the accumulation of proteins in maize seeds. Together, our findings uncovered a key gene controlling maize husk leaf width and nitrate transport from husk leaves to kernels. Identification of the ZmNRT2.5 loci offers direct targets for improving the protein content of maize seeds via molecular-assisted maize breeding.