Summary: 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.

Improving plant nitrogen use efficiency (NUE) is important for many crops, especially in hybrid breeding. Reducing nitrogen inputs is key for achieving sustainable rice production and decreasing environmental problems. Here, we analyzed the transcriptomic and physiological changes in two indica restorer lines (Nanhui511 and Minghui23) under high (HN) and low nitrogen (LN) conditions. Nanhui511 (NH511) was more sensitive to different nitrogen supplies and exhibited higher nitrogen uptake and NUE under HN conditions by increasing lateral root and tiller numbers in the seedling and maturation stages, respectively. It also exhibited a lower survival rate than that of MH23 when planted in chlorate-containing hydroponic solution, indicating its high nitrogen uptake ability under different nitrogen supply conditions. When transferred from LN to HN conditions, NH511 had 2456 differentially expressed genes, whereas MH23 had only 266. Furthermore, genes related to nitrogen utilization showed differential expression in NH511 under HN conditions, while the opposite was observed in MH23. These results will help decrease certain limitations of hybrid rice breeding and provide novel data for high-NUE hybrid rice cultivation.

Rice productivity relies heavily on nitrogen fertilization, and improving nitrogen use efficiency (NUE) is important for hybrid rice breeding. Reducing nitrogen inputs is the key to achieving sustainable rice production and reducing environmental problems. Here, we analyzed the genome-wide transcriptomic changes in microRNAs (miRNAs) in the indica rice restorer cultivar Nanhui 511 (NH511) under high (HN) and low nitrogen (LN) conditions. The results showed that NH511 is sensitive to nitrogen supplies and HN conditions promoted the growth its lateral roots at the seedling stage. Furthermore, we identified 483 known miRNAs and 128 novel miRNAs by small RNA sequencing in response to nitrogen in NH511. We also detected 100 differentially expressed genes (DEGs), including 75 upregulated and 25 downregulated DEGs, under HN conditions. Among these DEGs, 43 miRNAs that exhibited a 2-fold change in their expression were identified in response to HN conditions, including 28 upregulated and 15 downregulated genes. Additionally, some differentially expressed miRNAs were further validated by qPCR analysis, which showed that miR443, miR1861b, and miR166k-3p were upregulated, whereas miR395v and miR444b.1 were downregulated under HN conditions. Moreover, the degradomes of possible target genes for miR166k-3p and miR444b.1 and expression variations were analyzed by qPCR at different time points under HN conditions. Our findings revealed comprehensive expression profiles of miRNAs responsive to HN treatments in an indica rice restorer cultivar, which advances our understanding of the regulation of nitrogen signaling mediated by miRNAs and provides novel data for high-NUE hybrid rice cultivation.