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A genetic module boosts grain yield and nitrogen use efficiency by improving nitrate transport in maize

Nature Genetics volume 58, pages 618–629 (2026)Cite this article

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A Publisher Correction to this article was published on 23 March 2026

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Abstract

Although nitrogen fertilizer use has boosted crop yields, excessive application diminishes crop nitrogen use efficiency (NUE) and causes environmental problems. Therefore, increasing crop NUE is urgently needed for agricultural sustainability. Through a genome-wide association study, we identified a locus, NCR1 (Nitrate Concentration Regulator 1), that correlates with nitrate concentrations in maize root xylem. NCR1 encodes a MYB transcription factor that positively regulates the transcription of nitrate transporter NRT2.3 expressed predominantly in root xylem parenchyma cells. The NCR1–NRT2.3 transcription module responds to external nitrogen and controls nitrate translocation from roots to shoots. The superior NCR1−In allele with a 123-bp promoter deletion has decreased in frequency as nitrogen fertilizer use in China has increased. Overexpression of NCR1 or NRT2.3, or introgression of NCR1−In, increases grain yield and nitrogen content in the shoot and seed. This study uncovers a crucial genetic module for improving grain yield and NUE in maize.

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Fig. 1: GWAS identified NCR1, which associates with root-to-shoot NO3− transport.
Fig. 2: NCR1 promotes root-to-shoot nitrate transport.
Fig. 3: NRT2.3 mediates nitrate loading into the root xylem.
Fig. 4: NCR1 binds directly to the NRT2.3 promoter and regulates NRT2.3 expression positively.
Fig. 5: A Harbinger-like TE in the NRT2.3 promoter was selected during maize domestication.
Fig. 6: NCR1−In allele has been lost gradually during modern maize breeding in China.
Fig. 7: Yield traits and nitrogen content of NCR1 OE and NRT2.3 OE hybrids in field test.
Fig. 8: Model for the NCR1–NRT2.3 module involved in root-to-shoot nitrate transport in maize.

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Data availability

The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information. A reporting summary for this article is available in Supplementary Information. The B73 reference genomic sequences are collected from the MaizeGDB (https://www.maizegdb.org/). RNA-sequencing data and CUT&Tag data can be found in the National Center for Biotechnology Information under accession number PRJNA1117020. Source data are provided with this paper.

Code availability

No custom code was generated. All code used to analyze the sequence data is publicly available in the SAMtools section of GitHub63 (https://github.com/samtools/samtools).

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Acknowledgements

We thank the High-performance Computing Platform of China Agricultural University for its support of large-scale computation. We acknowledge Beijing PARATERA Tech CO., Ltd (https://paratera.com/) for providing HPC resources that have contributed to the research results reported within this study. The transgenic seeds of maize were created by the Center for Crop Functional Genomics and Molecular Breeding of China Agricultural University. This work was supported by grants from the National Key Research and Development Program of China (2021YFF1000500), National Natural Science Foundation of China (32025004, 32425041, 32302660), STI2030-Major Projects (2023ZD0406704, 2023ZD04069, 2023ZD04071), China Postdoctoral Science Foundation (2021M703535), Pinduoduo-China Agricultural University Research Fund (PC2023A01004) and Beijing Outstanding University Discipline Program.

Author information

Author notes

  1. These authors contributed equally: Meiling Zhang, Ziqi Wu.

Authors and Affiliations

  1. State Key Laboratory of Maize Bio-breeding, National Maize Improvement Center, Department of Plant Genetics and Breeding, China Agricultural University, Beijing, China

    Meiling Zhang, Liangliang Huang, Xiaomeng Shen, Ziwei Luo, Bo Yang, Chao Bian, Haiming Zhao, Jian Chen, Weibin Song & Jinsheng Lai

  2. State Key Laboratory of Plant Environmental Resilience (SKLPER), College of Biological Sciences, China Agricultural University, Beijing, China

    Ziqi Wu, Kangqi Wang, Yingying Hu, Burebiyanmu Wubulikasimu, Yizhou Qin, Junzheng Fu, Xiqing Wang, Feng Qin & Yi Wang

  3. Frontiers Science Center for Molecular Design Breeding (MOE), China Agricultural University, Beijing, China

    Feng Qin, Chao Bian, Haiming Zhao, Jian Chen, Weibin Song, Yi Wang & Jinsheng Lai

  4. Center for Crop Functional Genomics and Molecular Breeding, China Agricultural University, Beijing, China

    Xiaoming Zhao, Xiqing Wang, Feng Qin, Chao Bian, Haiming Zhao, Jian Chen, Weibin Song, Yi Wang & Jinsheng Lai

  5. Sanya Institute, China Agricultural University, Hainan, China

    Feng Qin, Chao Bian, Haiming Zhao, Weibin Song, Yi Wang & Jinsheng Lai

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  1. Meiling Zhang
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Contributions

J.L. and Y.W. directed the research and supervised the project. M.Z. and Z.W. performed most of the experiments. M.Z., C.B., Y.W. and J.L. wrote the manuscript. Z.W., M.Z. and Y.Q. performed the field tests in Beijing, Sanya and Gongzhuling. M.Z., Z.W. and J.F. collected and analyzed the field data. L.H. and X.S. provided the sequencing data of inbred lines. K.W., Y.H., B.W., Y.Q., J.F., Z.L. and B.Y. contributed to the seedlings culture in greenhouse and sample collection for GWAS. X.Z., X.W., F.Q., H.Z., J.C. and W.S. contributed to the generation and collection of biological materials and participated in project discussions.

Corresponding authors

Correspondence to Yi Wang or Jinsheng Lai.

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Competing interests

Two patent applications related to this work have been submitted by J.L., Y.W., M.Z., Z.W., W.S., H.Z., J.C. and C.B. (application nos. 202411076358.2 (PCT/CN2025/108660) and 202411077906.3 (PCT/CN2025/108631)). The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Identification and molecular characterization of NCR1.

(a) Phylogenetic tree of 181 maize inbred lines based on UPGMA. Among these lines, 104 accessions are sourced from China, 35 accessions from the United States, 42 accessions from other countries around the globe. Using a Bayesian approach and phylogenetic tree method, eight distinct subpopulations were identified and validated, comprising 19 Tropical, 19 Lancaster, 16 Reid, 12 TSPT, 11 Iodent, 11 X-group, 10 P-group, 9 A632, and 74 mixed individuals. (b) Population structure of 181 inbred lines assessed by STRUCTURE. Eight ancestral populations were identified, represented by distinct colors: tropical (red), X-group (blue), Iodent (green), TPST (brown), P-group (purple), Reid (orange), A632 (cyan), and Lancaster (pink). Each vertical line represents an inbred line; colored segment height represents membership percentage in each ancestral population. (c) Genome-wide linkage disequilibrium analysis of 181 maize inbred lines. The values 1.6 kb and 28 kb at the red dashed lines represent the distance between molecular markers when r2 decreases to 0.2 or 0.15, respectively. (d) NO3− concentration in root xylem sap under LN condition. (e) Distribution of NO3− concentration in xylem sap under LN and HN conditions. (f) Local Manhattan plot over the 127.75-127.85 Mb region on Chr 3. The SNPs exceeding the threshold are labeled red dots and located on or near the NCR1 gene. (g) Gene structure of NCR1 and mutation sites of ncr1 mutant lines. Black boxes represent exons, and lines represent introns. The sgRNA target site and the PAM motif are indicated. A single base-pair insertion (A) in ncr1−1 is shown in blue. The deleted sequences in ncr1−2 (17 bp) and ncr1−3 (14 bp) are shown using blue dashes. (h) Sequence difference between the NCR1−In and NCR1+In promoters. The NCR1−In promoter contains an E-box motif. (i) Subcellular localization of NCR1-GFP protein in maize mesophyll cell protoplasts. The nucleus was visualized using DAPI staining. (j) Transcript levels of NCR1 in the roots of NCR1 OE lines. Data are mean ± SE. n represents numbers of biologically independent samples. Two-sided Student’s t-test was used to determine P values.

Source data

Extended Data Fig. 2 RNA-seq analysis of NCR1-regulated genes in root.

(a) Volcano plots showing the differentially expressed genes (DEGs) in root regulated by NCR1 in response to LN treatment. Red and blue dots represent upregulated and downregulated genes, respectively (absolute fold change >1.5, P < 0.05). Three biological replicates were performed. (b) Hierarchical clustering and heatmap of 1,298 NCR1-regulated DEGs and key GO terms. DEGs show segregation into nine co-expressed clusters. GO enrichment analysis of heatmap DEG clusters is shown in Supplementary Table 7. (c) Venn diagram showing the overlap between NCR1-regulated genes under LN conditions and LN-responsive genes. (d) A heat map of DEGs involved in nitrate absorption and nitrogen metabolism. The color key (blue to red) represents gene expression (FPKM) as fold changes. (e) Distributions of NCR1 binding sites at the NRT1.1B, NiR1, and GS1-1 genes shown as Integrated Genome Browser windows. Notable peaks were calculated by MACS2. The bottom track indicates genes with transcription direction on the chromosome. Blue bars indicate NCR1 binding peaks in the promoter regions. (f) Transcript levels of NR4, NiR1, and GS1-1 genes in the NCR1−In (n = 14 accessions) and NCR1+In (n = 21 accessions) maize inbred lines after HN and LN treatment for 15 days. Statistical significance was determined by a two-sided t-test. The horizontal bars of boxes represent minima, 25th percentiles, medians, 75th percentiles, and maxima.

Source data

Extended Data Fig. 3 NRT2.3 expression pattern and NO3− transport analyses.

(a) NRT2.3 expression levels in different tissues (n = 3 biologically independent samples). (b) GUS staining showing transcriptional change of NRT2.3 in response to nitrogen supply using ProNRT2.3:GUS plants. Longitudinal sections (a and b) and cross sections (c to f) of the primary root are shown. (c) Subcellular localization of NRT2.3-GFP in tobacco (Nicotiana benthamiana) leaves. The AtCBL1n-OFP and AtCBL2-mCherry were used as the PM and tonoplast markers, respectively. (d) Nitrate uptake assay in Xenopus oocytes. Oocytes expressing different cRNA combinations were incubated in the solution containing 0.25 or 5 mM K15NO3 at pH 5.5 or 7.5 for 6 h. Then, the 15N content in oocytes was measured. Data are mean ± SE. Each point represents one replicate from one oocyte. n represents numbers of biologically independent samples. Different letters indicate significant differences (P < 0.05) based on one-way ANOVA (Tukey’s test). (e) Mutation sites in two nrt2.3 mutant lines. (f) Transcript levels of NRT2.3 in the roots of NRT2.3 OE lines. Data are mean ± SE. n represents numbers of biologically independent samples. Different letters indicate significant differences (P < 0.05) based on one-way ANOVA (Tukey’s test). (g) to (i) 15N content (g), shoot/root 15N ratio (h), and total N content (i) in the WT and nrt2.3 mutants. The seedlings were cultured in 0.4 mM 15NO3− solution for 1 day. Then, the 15N content in the shoot and root was measured, respectively. Data are mean ± SE. Shoot and root samples from four seedlings were mixed as one biological replicate for each material. n represents numbers of biologically independent samples. Different letters indicate significant difference (P < 0.05) based on the one-way ANOVA (Tukey’s test). (j) Positions of P1 to P4 fragments in the NRT2.3 promoter. MYB core and MRE cis-elements are shown. Gray lines represent MYB core (CNGTTR). Red lines indicate MRE (AACCaaa).

Source data

Extended Data Fig. 4 NRT2.3 is genetically epistatic to NCR1.

(a) Phenotype comparison among WT, ncr1-1, nrt2.3-1 single mutant, and ncr1-1 nrt2.3-1 double mutant after LN and HN treatment for 14 days. (b) Mutation sites of NCR1 and NRT2.3 in the double mutant. (c) to (e) Shoot dry weight (c), NO3− concentration in xylem sap (d), and shoot nitrogen content (e) of different plants after LN and HN treatment for 14 days. Data are mean ± SE. Each point represents one biological replicate from one seedling. n represents numbers of biologically independent samples. Different letters indicate significant difference (P < 0.05) based on the one-way ANOVA (Tukey’s test). (f) Phenotype comparison among WT, ncr1-1, NRT2.3 OE-2, and ncr1-1 NRT2.3 OE-2 plants after LN and HN treatment for 15 days. (g) Mutation sites of NCR1 in the ncr1-1 NRT2.3 OE-2 plants. (h) to (j) NRT2.3 transcript level (h) (n = 3 biologically independent samples), NO3− concentration in xylem sap (i), and shoot nitrogen content (j) of different plants after LN and HN treatment for 15 days. Data are mean ± SE. Each point in (h) represents one biological replicate from four seedlings. Each point in (i) and (j) represents one biological replicate from one seedling. n represents numbers of biologically independent samples. Different letters indicate significant difference (P < 0.05) based on the one-way ANOVA (Tukey’s test).

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Extended Data Fig. 5 Domestication analysis of NRT2.1, NRT2.2, and NRT2.3.

(a) XP-CLR values between maize and parviglumis. The region (70-71 Mb) in Chr 5 containing the NRT2.3 gene (red) is shown. (b) and (c) Nucleotide diversity (π) of NRT2.1 (b) and NRT2.2 (c) genes among teosinte, maize landrace, and maize inbred line. Nucleotide diversity (π) values were calculated using a 1000-bp sliding window and a 100-bp step. (d) Sequence comparison of NRT2.3 genes from 11 teosinte varieties, 7 landraces, and 8 maize inbred lines. The green box indicates the position of the 536-bp Harbinger-like TE.

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Extended Data Fig. 6 Ear phenotype and yield traits of ncr1 and nrt2.3 inbred lines in field test.

Ear phenotype (a and b) and kernel weight per ear (c and d) of ncr1 and nrt2.3 inbred lines under two nitrogen conditions in the field test in Sanya, China, in 2023. The data in (c) and (d) were obtained from at least 38 plants for each line. Data are mean ± SE. Different letters indicate significant difference (P < 0.05) based on the one-way ANOVA (Tukey’s test).

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Extended Data Fig. 7 Ear phenotype, yield traits, and nitrogen content of NCR1 OE and NRT2.3 OE inbred lines in field test.

Ear phenotype (a and b), kernel weight per ear (c), NUE (d), leaf nitrogen content (e), and stem nitrogen content (f) of NCR1 OE and NRT2.3 OE inbred lines under two nitrogen conditions in the field test in Sanya, China, in 2023. Data are mean ± SE. The data in (c) were obtained from 60 plants for each line. The bars within violin plots represent 25th percentiles, medians, and 75th percentiles. The data in (d) were derived from at least 3 plots; each line contained 39 plants in one plot. The ear leaves and ear stems were collected for nitrogen content measurement. n represents numbers of biologically independent samples. The horizontal bars of boxes in (e) and (f) represent minima, 25th percentiles, medians, 75th percentiles, and maxima. Different letters indicate significant difference (P < 0.05) based on the one-way ANOVA (Tukey’s test).

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Extended Data Fig. 8 Yield traits of NCR1 OE and NRT2.3 OE hybrid lines in the field test in Gongzhuling.

Ear weight (a), ear length (b), kernel number per row (c), kernel row number (d), ear diameter (e), and 100-kernel weight (f) of NCR1 OE and NRT2.3 OE hybrids under two nitrogen conditions in field test in Gongzhuling, China, 2023. Data are mean ± SE. The data in (a) were obtained from 60 plants for each line. In (b) to (f), n represents numbers of biologically independent samples. The horizontal bars of boxes in (a) and (f) represent minima, 25th percentiles, medians, 75th percentiles, and maxima. Different letters indicate significant difference (P < 0.05) based on the one-way ANOVA (Tukey’s test).

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Extended Data Fig. 9 Yield traits of NCR1 OE and NRT2.3 OE hybrid lines in the field test in Beijing and Sanya.

Kernel yield per plot (a and e), NUE (b and f), ear weight (c and g), and ear length (d and h) of NCR1 OE and NRT2.3 OE hybrids under two nitrogen conditions in the field test. The data from (a) to (d) were obtained from the field test in Beijing, China, in 2023. The data from (e) to (h) were obtained from the field test in Sanya, China, in 2023. Data are mean ± SE. The data in (a), (b), (e), and (f) were derived from 3 plots. The data in (c) and (g) were obtained from 60 plants for each line. The bars within violin plots represent 25th percentiles, medians, and 75th percentiles. The data in (d) and (h) were obtained from 15 plants for each line. Different letters indicate significant difference (P < 0.05) based on the one-way ANOVA (Tukey’s test).

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Extended Data Fig. 10 NCR1−In frequency analyses.

(a) NCR1−In frequencies in different maize populations. (b) Pearson correlation analysis of NCR1−In frequencies and nitrogen application levels for maize in 11 countries. The nitrogen application levels for maize production in various countries were obtained from the International Fertilizer Association (IFA, https://www.fertilizer.org/). P values are determined by the two-sided Pearson correlation coefficient analysis. (c) The NCR1−In frequencies of maize inbred lines from 11 countries used in (b).

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Supplementary information

Reporting Summary (download PDF )

Supplementary Tables 1–10 (download XLS )

Table 1: The 181 maize inbred lines used in GWAS. Table 2: NCR1 resequencing data of 155 maize inbred lines. Table 3: DEGs in WT after LN treatment. Table 4: DEGs showing opposite changes in the ncr1 mutant and NCR1 OE plants under LN conditions. Table 5: DEGs showing opposite changes in the ncr1 mutant and NCR1 OE plants under HN conditions. Table 6: NRT2.3 genotypes in teosinte, landrace, and maize inbred lines. Table 7: GO enrichment analysis of heatmap DEG clusters. Table 8: NCR1 genotypes in the elite inbred lines in China. Table 9: NCR1 genotypes in two different groups of maize inbred lines in the United States. Table 10: Primer sequences used in this study.

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Zhang, M., Wu, Z., Huang, L. et al. A genetic module boosts grain yield and nitrogen use efficiency by improving nitrate transport in maize. Nat Genet 58, 618–629 (2026). https://doi.org/10.1038/s41588-026-02532-y

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