Abstract: Foxtail millet (Setaria italica), a vital drought-resistant crop, plays a significant role in ensuring food and nutritional security. However, its drought resistance mechanism is not fully understood. N⁶-methyladenosine (m⁶A) modification of RNA, a prevalent epi-transcriptomic modification in eukaryotes, provides a binding site for m⁶A readers and affects plant growth and stress responses by regulating RNA metabolism. In this study, we unveiled that the YT521-B homology (YTH) family gene SiYTH1 positively regulated the drought tolerance of foxtail millet. Notably, the siyth1 mutant exhibited reduced stomatal closure and augmented accumulation of excessive H₂O₂ under drought stress. Further investigations demonstrated that SiYTH1 positively regulated the transcripts harboring m⁶A modification related to stomatal closure and reactive oxygen species (ROS) scavenging under drought stress. SiYTH1 was uniformly distributed in the cytoplasm of SiYTH1-GFP transgenic foxtail millet. It formed dynamic liquid-like SiYTH1 cytosol condensates in response to drought stress. Moreover, the cytoplasmic protein SiYTH1 was identified as a distinct m⁶A reader, facilitating the stabilization of its directly bound SiARDP and ROS scavenging-related transcripts under drought stress. Furthermore, natural variation analysis revealed SiYTH1^AGTG as the dominant allele responsible for drought tolerance in foxtail millet. Collectively, this study provides novel insights into the intricate mechanism of m⁶A reader-mediated drought tolerance and presents a valuable genetic resource for improving drought tolerance in foxtail millet breeding.

Key words: drought stress, mRNA stability, N⁶-methyladenosine (m⁶A) reader, phase separation, SiYTH1, Setaria italica