OsNAC20 and OsNAC26 are the key regulators in rice grain filling.

Grain filling is a complex trait governed by multiple physiological processes. Although numerous transcription factors (TFs) have been shown to regulate storage substance synthesis, the central regulators that coordinate these diverse pathways remain largely unknown. OsNAC20 and OsNAC26 function as direct regulators of storage substance synthesis. Here, we detected that OsNAC20 and OsNAC26 regulate grain filling beyond this role. Combining DNA affinity purification sequencing analysis (DAP-seq) and RNA sequencing (RNA-seq), we found that OsNAC20/26 regulate storage substance biosynthesis by activating bZIP58, FLO2, FLO11, VPE1, and GS1;3, affect cellular hormone metabolic process by activating YUC11 and YUC9, influence hormone-mediated signaling by activating PYL8 and PYL9, and modulate transmembrane transport by activating YSL2 and SWEET11. Meanwhile, OsNAC20/26 interacted with bZIP58, RPBF, and SAPK3, thereby enhancing their transactivation on genes associated with starch and storage protein synthesis. In addition, it was detected that OsNAC20/26 could be induced by abscisic acid (ABA). Overall, this study reveals a grain-filling regulatory network surrounding OsNAC20 and OsNAC26, providing an important theoretical basis for in-depth analysis of the molecular mechanism of rice grain filling.