Rice transcription factors OsNIGT2/3 regulate nitrogen acquisition by repressing OsNRTs and OsAMTs under high-nitrogen conditions

Abstract

Introduction: Nitrogen is a crucial nutrient for crop growth, yet its utilization efficiency is generally low, leading to resource waste and serious environmental problems. Future agricultural sustainability requires improved crop NUE. In this study, we investigated the functions of the rice transcription factors OsNIGT2 and OsNIGT3 in nitrate uptake under high-nitrogen conditions.

Methods: Hydroponic experiments and field tests were conducted to investigate the effects of OsNIGT2 and OsNIGT3 on physiological phenotypes and nitrogen use efficiency. Transcriptome analysis was used to explore the genome-wide transcriptional landscape of the two genes in response to nitrate availability. DNA affinity purification sequencing (DAP-seq) was employed to identify genomic sites bound by OsNIGT2, subsequently, yeast one-hybrid and transient expression assays verified the regulatory effects of OsNIGT2 and OsNIGT3 on key genes.

Results: The double mutants of OsNIGT2 and OsNIGT3 presented significantly greater dry weights, nitrogen contents, total amino acid contents, and nitrate levels than the WT, whereas the single mutants presented no significant differences. These findings indicate the functional redundancy of OsNIGT2 and OsNIGT3 in regulating nitrogen uptake and assimilation. OsNIGT2 and OsNIGT3 act as transcriptional repressors, negatively regulating the expression of nitrogen absorption-related genes. Furthermore, DAP-seq identified potential targets bound by OsNIGT2, highlighting its role in the expression of several nitrogen and phosphorus utilization-related genes. Additionally, field tests shown that OsNIGT2 and OsNIGT3 knockout promotes both grain yield and NUE. This study provides potential genetic targets for improving yield and NUE in rice and other crops, laying a significant foundation for future crop improvement.

Keywords: NUE; OsAMTs; OsNIGT2; OsNIGT3; OsNRTs; rice.

Figure 1

OsNIGT2 and OsNIGT3 are transcriptional repressors that are strongly induced by nitrate. (A) Heatmap of G2-like transcription factors expression under NH₄NO₃ treatment, the number represents the processing time (hour). (B) Nitrate-specific induction of OsNIGT2 and OsNIGT3 expression. (C) A phylogenetic tree of the G2-like transcription factor subfamily constructed by ClustalW alignment and the maximum likelihood method. (D) Time-course analyses of OsNIGT2 and OsNIGT3 expression levels in response to nitrate. (E) The OsNIGT2-GFP and OsNIGT3-GFP signals colocalize with the cell nucleus marker OsWRKY29 (fused with RFP), bars = 10 μm. (F) OsNIGT2 and OsNIGT3 transcriptional repression assay. Asterisks indicate significant differences of LUC/REN ratio between the Empty and OsNIGT2/NIGT3 according to two-tailed Student’s t tests: **,P<0.01.

Figure 2

Spatial expression patterns of OsNIGT2 and OsNIGT3. (A) Spatial patterns of OsNIGT2 expression as detected by GUS staining in OsNIGT2pro:GUS. (B) Spatial patterns of OsNIGT3 expression as detected by GUS staining in OsNIGT3pro:GUS. Two-week-old seedlings were incubated with various compounds (4 mM KCl, KH₂PO₄, NH₄Cl, KNO₃, and NH₄NO₃) for 3 hours before GUS staining. Bar = 200 mm.

Figure 3

OsNIGT2 and OsNIGT3 knockout increased N accumulation in hydroponic culture. (A) Growth morphology of Nipponbare (WT), nigt2, nigt3, and nigt2/3 (two lines) grown in hydroponic culture. (B, C) Dry weights of shoots and roots. (D, E) Total N concentrations in shoots and roots. (F, G) Total amino acid concentrations in leaves and roots. (H, I) NO₃ ^¯ concentrations in leaves and roots. Con, concentration; DW, dry weight. Asterisks indicate significant differences between the WT and nigt2 or nigt3 single mutants or double nigt2/3 mutant according to two-tailed Student’s t tests: *, P < 0.05; **, P < 0.01. Detailed data were shown in Supplementary Table S4 .

Figure 4

Chlorate sensitivity assay in Nipponbare (WT) and nigt2/3 double mutants. (A) Growth morphology of Nipponbare (WT) nigt2/3 double mutants grown in hydroponic culture. (B) Chlorate sensitivity was calculated from the mortality rate of plants poisoned with chlorate. Asterisks indicate significant differences of death rate between the WT and double nigt2/3 mutant according to two-tailed Student’s t tests: **,P<0.01.

Figure 5

Expression profiling analysis of Nipponbare and nigt2/3-ko1 double mutant seedlings under different nitrate concentrations. (A) Number of upregulated and downregulated genes in ko vs. WT under LN (0.2 mM KNO₃) and HN (4 mM KNO₃) conditions. (B) Venn diagram analysis of the common and specific genes unique to and shared between the different pairwise comparisons. (C) Heatmap of nitrogen uptake and assimilation genes under LN and HN conditions. (D) Gene Ontology (GO) classification of DEGs from nigt2/3-ko1 vs. WT under LN conditions. (E) GO classification of DEGs from nigt2/3-ko1 vs. WT plants under HN conditions.

Figure 6

Genome-wide binding profiles of OsNIGT2 from the DAP-seq analysis. (A) Distribution of OsNIGT2-binding regions in the rice genome. Promoter region, -2 kb to the transcription start site (TSS); terminator, +2 kb to the transcription termination site (TTS); intergenic region, 2 kb upstream of the TSS or 2 kb downstream of the TTS. (B) OsNIGT2-binding motif identified by MEME-ChIP in 400 bp flanking sequences around the genic peak summits and density plot of this motif around peak summits. (C) Y1H assay of OsNIGT2 and OsNIGT3 interactions with potential promoters. NI2, pGADT7-NIGT2; NI3, pGADT7-NIGT3; wt-pro represents the original promoter sequence; mu-pro represents a mutation in the B-box of the promoter (GAATATTC → tAcgATTC or GAATC → tAcgC).

Figure 7

Dual luciferase reporter assay of the interaction of OsNIGT2 and OsNIGT3 with potential promoters. Asterisks indicate significant differences of LUC/REN ratio between the Empty and OsNIGT2/NIGT3 according to two-tailed Student’s t tests: **, P< 0.01.

Figure 8

OsNIGT2 and OsNIGT3 knockout promotes grain yield and NUE. (A) Growth phenotype of wild-type (WT) and nigt2/3-ko1 plants at maturing stage in the HN field; (B–E) Plant height (B), Tiller number per 5 plants (C), Grain yield per 5 plants (D), Actual yield (E) of WT and mutants under LN and HN conditions; (F) Agronomic NUE of WT and mutants. Values are the means ± SD (four replications), asterisks indicate significant differences between the WT and single nigt2, nigt3, and dual nigt2/3 mutants by two-tailed Student’s t-test: *, P < 0.05; **, P < 0.01.