Roots are essential organs for wheat to absorb water and mineral nutrients. Exploring key genes that control root morphology and deciphering the underlying regulatory mechanisms are of great significance for genetic improvement of root architecture and nitrogen-use efficiency (NUE) in wheat.

Epigenetic regulations play a crucial role in root development, especially in the adaption to dynamic environmental cues. N6-methyladenine (m6A), one of the most important RNA modifications in eukaryotes, is catalyzed by methyltransferase complexes, recognized by N6-methyladenine-binding proteins, and removed by demethylases. To date, there have been no reports of m6A demethylase in the regulation of nitrogen-dependent root development in wheat.

In our study, a stable major QTL (qMrl-7B) associated with the maximum root length was finely located in the 0.26 Mb interval on chromosome 7B of Kenong 9204. Based on expression analysis and sequence alignment, the candidate gene of qMrl-7B was identified as TaRL-7B, which encodes an m6A demethylase and positively regulates root length.

Loss of function of TaRL-7B resulted in reduced seedling root length and plant height, while its overexpression enhanced these traits, particularly under nitrogen-deficient conditions. Significantly, TaRL-7B expression was positively correlated with tiller number, grain width and grain weight, indicating its great potential for improving crop yield.

These results proved that the m6A demethylase synergistically regulates root length, tiller number and yield potential, providing elite gene resources for improving wheat root system and breeding high-yielding varieties under various nitrogen regimes.