<scp>OsRF2b</scp> interacting with <scp>OsbZIP61</scp> modulates nitrogen use efficiency and grain yield via heterodimers in rice

Nitrogen (N) is critical for crop growth, and enhancing nitrogen use efficiency (NUE) is essential for achieving high grain yields and quality in crop production. In this study, we identified two transcription factors OsRF2b and OsbZIP61, which localize in the nucleus and function as negative regulators of NUE and grain yield in rice. OsRF2b responds to nitrogen supplies, and overexpression of OsRF2b resulted in growth retardation, reduced nitrogen content, and lower NUE in transgenic lines, leading to decreased grain yield. In contrast, the rf2b mutant exhibited increased plant height, grain size, and NUE, which contribute to improved grain yield. Furthermore, RT-qPCR results showed that OsRF2b overexpression significantly downregulated OsNRT1.1B and OsNRT2.1/2.2 expression, while the OsRF2b mutation significantly upregulated these genes. Additionally, yeast one-hybrid, EMSA, and transient expression assays demonstrated that OsRF2b directly binds to the promoter region of OsNRT1.1B and suppresses its expression. Moreover, bimolecular fluorescence complementation (BiFC) and luciferase complementation imaging (LCI) assays indicated that OsRF2b interacts with OsbZIP61, and OsbZIP61 overexpression also decreased rice NUE and grain yield. In conclusion, we identified the bZIP transcription factor OsRF2b, which interacts with OsbZIP61 and acts as negative regulators of NUE and grain yield by forming heterodimers and directly binding to the promoter of OsNRT1.1B and suppressing its expression. These findings provide new insights into the mechanisms underlying two bZIPs involved in NUE and grain yield, which offer a genetic basis for rice breeding in the future.