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Ferredoxin-mediated mechanism for efficient nitrogen utilization in maize

Nature Plants volume 11, pages 643–659 (2025)Cite this article

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Abstract

Nitrogen (N) is an essential macronutrient for plant development and, ultimately, yield. Identifying the genetic components and mechanisms underlying N use efficiency in maize (Zea mays L.) is thus of great importance. Nitrate (NO3−) is the preferred inorganic N source in maize. Here we performed a genome-wide association study of shoot NO3− accumulation in maize seedlings grown under low-NO3− conditions, identifying the ferredoxin family gene ZmFd4 as a major contributor to this trait. ZmFd4 interacts and co-localizes with nitrite reductases (ZmNiRs) in chloroplasts to promote their enzymatic activity. Furthermore, ZmFd4 forms a high-affinity heterodimer with its closest paralogue, ZmFd9, in a NO3−-sensitive manner. Although ZmFd4 exerts similar biochemical functions as ZmFd9, ZmFd4 and ZmFd9 interaction limits their ability to associate with ZmNiRs and stimulate their activity. Knockout lines for ZmFd4 with decreased NO3− contents exhibit more efficient NO3− assimilation, and field experiments show consistently improved N utilization and grain yield under N-deficient conditions. Our work thus provides molecular and mechanistic insights into the natural variation in N utilization, instrumental for genetic improvement of yield in maize and, potentially, in other crops.

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Fig. 1: ZmNCRG1 is associated with natural variation in shoot NO3− contents under low-NO3− conditions.
Fig. 2: ZmNCRG1 encodes the ferredoxin ZmFd4.
Fig. 3: InDel194 is strongly associated with ZmFd4 transcript levels and shoot NO3− contents.
Fig. 4: ZmFd4 interacts with ZmNiR1 and regulates NiR activity.
Fig. 5: ZmFds associate with ZmNiR1 and ZmFd4 associates with ZmFd9.
Fig. 6: ZmFd4 and ZmFd9 compete with each other.
Fig. 7: Evaluation of yield performance of Zmfd4 knockout mutants.
Fig. 8: Conceptual model of ZmFd-mediated NO3− assimilation and maize grain yield in response to low N availability.

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Data availability

Gene sequences and amino acid sequences used in this Article were collected from Maize GDB (maize; https://www.maizegdb.org/), Gramene (rice; http://ensembl.gramene.org/index.html) and TAIR10 (Arabidopsis; https://www.arabidopsis.org/). All data supporting the findings of this study are available within the main text or the supplementary information files. The reporting summary for this article is presented as a supplementary information file. Source data are provided with this paper.

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Acknowledgements

We thank X. Yang for providing published inbred lines and helping with data analysis; and S. Huang, C. Jiang, G. Bi, C. Liu and S. Zhang for helpful discussions. The transgenic maize lines were generated 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 to J.Z.), the National Natural Science Foundation of China (32170265 and 32441022 to J.Z.), the Chinese Universities Scientific Fund (2024TC084 to J.Z.), the Pinduoduo-China Agricultural University Research Fund (PC2024B01005 to J.Z.), the Hainan Provincial Natural Science Foundation of China (323CXTD379 to J.Z.), and the Central Guidance on Local Science and Technology Development Fund of Shanxi Province (YDZJSX2024D040 to C.T. and J.Z.).

Author information

Author notes

  1. These authors contributed equally: Guannan Jia, Guojingwei Chen, Zhaoheng Zhang.

Authors and Affiliations

  1. State Key Laboratory of Plant Environmental Resilience, College of Biological Sciences, Center for Crop Functional Genomics and Molecular Breeding, Frontiers Science Center for Molecular Design Breeding (MOE), China Agricultural University, Beijing, China

    Guannan Jia, Guojingwei Chen, Zhaoheng Zhang, Yaping Wang, Kaina Zhang, Xiaoyun Zhao, Xiaoming Zhao, Zhen Li, Yan Guo, Zhizhong Gong & Jing Zhang

  2. Shanxi Key Laboratory of Sorghum Genetic and Germplasm Innovation, Sorghum Research Institute, Shanxi Agricultural University, Jinzhong, China

    Chenghua Tian

  3. Department of Neurology of The First Affiliated Hospital of USTC, MOE Key Laboratory for Membraneless Organelles and Cellular Dynamics, Hefei National Research Center for Physical Sciences at the Microscale, Center for Advanced Interdisciplinary Science and Biomedicine of IHM, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China

    Jie Luo & Linfeng Sun

  4. State Key Laboratory of Forage Breeding-by-Design and Utilization and Key Laboratory of photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China

    Wenqiang Yang

  5. Institute of Science and Technology Austria (ISTA), Klosterneuburg, Austria

    Jiří Friml

  6. College of Life Sciences, Institute of Life Science and Green Development, Hebei University, Baoding, China

    Zhizhong Gong

  7. Sanya Institute of China Agricultural University, Sanya, China

    Jing Zhang

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  1. Guannan Jia
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Contributions

J.Z. designed the study. G.J., G.C. and Z.Z. performed most of the experiments. C.T. helped with field testing. Y.W. performed subcellular ___location analysis. J.L. performed ITC binding measurement. K.Z. performed the stable nitrogen isotope analysis with an isotope-ratio mass spectrometer. Xiaoyun Zhao performed LC–MS/MS analysis. Xiaoming Zhao performed the maize transformation. G.J., G.C., Z.Z., C.T., Y.W., J.L., Z.L., L.S., W.Y., Y.G., J.F., Z.G. and J.Z. analysed the data. G.J., J.F., Z.G. and J.Z. wrote the paper.

Corresponding author

Correspondence to Jing Zhang.

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Nature Plants thanks Chengcai Chu, Xiangdong Fu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 ZmNCRG1 confers natural variation in shoot NO3− contents under low-NO3− conditions.

a, Ranked distribution of shoot NO3− contents across 340 maize inbred lines. All inbred lines were grown under 0.05 mM NO3− conditions for 15 d, and the shoot tissues were collected for NO3− content measurements. b, Local Manhattan plot showing the GWAS results in the region of ZmNCRG1 for shoot NO3− contents from seedlings grown under 0.05 mM NO3− conditions. An 800-bp genomic region (Chr2: 59.7228–59.7236 Mb) is shown. The horizontal dashed line represents the Bonferroni-adjusted significance threshold (−log10(P) = 5). Two adjacent SNPs (Chr2_59723461 and Chr2_59723508) showing significant association with shoot NO3− contents are highlighted in red. The locations of the leading SNPs (Chr2_59723461 and Chr2_59723508) and the structure of ZmNCRG1 (Zm00001d003797) are shown. Blue box represents the coding region.

Extended Data Fig. 2 Mutation of ZmFd4 does not affect fresh or dry weight and root-to-shoot NO3− distribution.

a, Genomic structure of the ZmFd4 locus and sequence analysis of ZmFd4 in three knockout mutants generated via CRISPR/Cas9. Blue box represents the coding region. The target sequences are shown in blue font, and the protospacer adjacent motif (PAM) is in orange font. Zmfd4-c1 harbors two 1-bp insertions (after 150 and 208 bp downstream of the ATG translation start codon, respectively); Zmfd4-c2 carries a complex mutation pattern with an 18-bp deletion (134–151 bp) and a 1-bp insertion (after position 208 bp); and Zmfd4-c3 harbors a 58-bp deletion (151–208 bp). b, c, Fresh weight (b) and dry weight (c) of the wild type and Zmfd4 mutants grown under 4 mM NO3− (NN) or 0.05 mM NO3− (LN) conditions. Data are shown as means ± s.d. (b, n = 21 plants; c, n = 19 plants). d, NO3− contents analyzed in the roots of 15-d-old wild-type and Zmfd4 seedlings grown under 4 or 0.05 mM NO3− conditions. Data are shown as means ± s.d. (n = 3 biologically independent samples). e, Shoot/root 15NO3− concentration ratio of the wild type and Zmfd4 mutants grown under 4 or 0.05 mM NO3− conditions. Plants were exposed to 15NO3− for 24 h. Data are shown as means ± s.d. (n = 6 biologically independent samples).

Extended Data Fig. 3 Genotyping of ZmFd4 in NIL plants generated by introgressing ZmFd4 allele from inbred line CIMBL144 into Dan340.

Genotyping the BC4F2 segregating plants carrying a homozygous ZmFd4CIMBL144 or ZmFd4Dan340 allele or heterozygous ZmFd4CIMBL144/ZmFd4Dan340.

Source data

Extended Data Fig. 4 Chloroplast-localized ZmFd4 interacts with ZmNiR2 and does not affect photosynthetic capacity.

a, Subcellular localization of ZmFd4. Confocal images show the overlap of the ZmFd4-GFP signal with chlorophyll autofluorescence in maize protoplasts. Scale bars, 10 μm. b, Y2H assay showing that ZmFd4 interacts with ZmNiR2. c, In vitro pull-down assay showing that MBP-ZmNiR2 purified from E. coli can pull down GST-ZmFd4. d, LCI assay showing that ZmFd4 interacts with ZmNiR2 in N. benthamiana leaves. e, Co-IP assay showing that ZmFd4 associates with ZmNiR2 in maize protoplasts. f, g, Colocalization of ZmNiR1 (f) and ZmNiR2 (g) with chloroplast-localized ZmFd4 in N. benthamiana leaves. Scale bars, 10 μm. h–j, Chlorophyll fluorescence parameters of 15-d-old seedlings grown under 4 or 0.05 mM NO3− conditions: Fv/Fm (h), Y(II) (i), and Y(I) (j). Data are shown as means ± s.d. (h, i, n = 14 independent assays; j, n = 20 independent assays). k, P700 redox kinetics triggered by far-red illumination in 15-d-old seedlings grown under 4 or 0.05 mM NO3− conditions. l, Total C contents in the shoots of wild type and Zmfd4 mutants. Data are shown as means ± s.d. (n = 6 biologically independent samples). m, C/N content ratio of the wild type and Zmfd4 mutants. Data are shown as means ± s.d. (n = 6 biologically independent samples). n, Relative transcript levels of ZmNiRs in wild type and Zmfd4 mutants, as determined by RT–qPCR. Total RNA was isolated from shoots of 15-d-old seedlings grown under 0.05 mM NO3− conditions. Data are shown as means ± s.d. (n = 6 biologically independent samples). o, Shoot NO2− contents of wild-type and Zmfd4 plants grown under 0.05 mM NO3− conditions for 15 d. Data are shown as means ± s.d. (n = 3 biologically independent samples). A two-sided t-test was used to determine statistical significance.

Source data

Extended Data Fig. 5 ZmFds interact with ZmNiR2 and regulate NiR activity.

a, Y2H assay showing that ZmFd1, ZmFd2, ZmFd7, and ZmFd9 interact with ZmNiR2. b, In vitro pull-down assay showing that MBP-ZmNiR2 purified from E. coli can pull down GST-ZmFd1, GST-ZmFd2, GST-ZmFd4, GST-ZmFd7, and GST-ZmFd9. c, LCI assay showing that ZmFd1, ZmFd2, ZmFd7, and ZmFd9 interact with ZmNiR2 in N. benthamiana leaves. d, Co-IP assay showing that ZmFd9 associates with ZmNiR2 in maize protoplasts. e, Ability of recombinant His-ZmFds to stimulate NiR activity based on the consumption of NO2− by ferredoxin-dependent NiR in wild-type leaf extracts. Data are shown as means ± s.d. (n = 4 biologically independent samples). f, Immunoblot assay for the indicated fusion proteins in the LCI assays related to Fig. 5j, using an antibody recognizing full-length firefly LUC that reacts with N- (upper bands) and C-terminal (lower bands) firefly LUC fragments.

Source data

Extended Data Fig. 6 ZmFd4 and ZmFd9 compete with each other.

a, In vitro pull-down assay showing that ZmFd9 diminishes the interaction between ZmFd4 and ZmNiR2, and ZmFd4 also interferes with the interaction between ZmFd9 and ZmNiR2. Numbers below the lanes indicate the relative band intensities normalized to loading controls. b, Co-IP assay showing that ZmFd9 inhibits the association of ZmFd4 with ZmNiR2, and ZmFd4 weakens the association of ZmFd9 with ZmNiR2. Numbers below the lanes indicate the relative band intensities normalized to loading controls. c, LCI assay showing that ZmFd9 inhibits the interaction of ZmFd4 with ZmNiR2 (left image), and ZmFd4 also represses the interaction of ZmFd9 with ZmNiR2 (right image) in N. benthamiana leaves. Application of 4 mM NO3− attenuates the competition of ZmFd4 and ZmFd9 for ZmNiR2. Quantification of relative LUC activity is shown on the left and right graphs. Data are shown as means ± s.d. (n = 3 independent assays). Different lowercase letters indicate significant differences (P < 0.05; one-way ANOVA with Fisher’s LSD test). d, e, Immunoblot analyses of the indicated fusion proteins in the LCI assays related to Fig. 6c (d) and Extended Data Fig. 6c (e). An antibody recognizing full-length firefly LUC reacts with N- (lower bands) and C-terminal (upper bands) firefly LUC fragments. f, Effect of co-incubating ZmFd4 and ZmFd9 on the activity of ZmNiRs, as analyzed in an in vitro system. The concentration of recombinant ZmFds and ZmNiRs used for enzymatic assays was 12 µM. Data are shown as means ± s.d. (left graph: n = 4 biologically independent samples; right graph: n = 6 biologically independent samples). Different lowercase letters indicate significant differences (P < 0.05; one-way ANOVA with Fisher’s LSD test). g, Effect of ZmFd9 on NiR activity, as analyzed in a semi-in vitro system employing enzyme extracts derived from the wild type and Zmfd4 mutant, along with recombinant GST-ZmFd9. The concentration of ZmFd9 used for enzymatic assays was 12 µM. Data are shown as means ± s.d. (n = 6 biologically independent samples). Different lowercase letters indicate significant differences (P < 0.05; one-way ANOVA with Fisher’s LSD test). h, Redox potential of recombinant ZmFd4 and ZmFd9. Cyclic voltammograms were measured in 50 μM solutions of ZmFds versus a standard calomel electrode.

Source data

Extended Data Fig. 7 Characteristic analysis of different transgenic maize lines.

a, Genomic structure of the ZmFd9 locus and sequence analysis of ZmFd9 in three knockout mutants generated via CRISPR/Cas9. Blue box represents the coding region. The target sequences are in blue font, and the PAM is in orange font. Zmfd9-c1 harbors two 1-bp insertions (after 45 and 104 bp downstream of the ATG translation start codon, respectively); Zmfd9-c2 carries a 74-bp deletion (42–115 bp); and Zmfd9-c3 harbors two 1-bp insertions (after 42 and 103 bp, respectively). All three mutant alleles are predicted to produce truncated ZmFd9 proteins. b, Representative photographs of wild-type and Zmfd9 plants grown under 4 or 0.05 mM NO3− conditions for 15 d. Scale bars, 10 cm. c–e, Relative ZmNiR transcript levels (c), NiR activity (d), and shoot NO3− contents (e) in wild-type and Zmfd9 plants grown under 0.05 mM NO3− conditions. Data are shown as means ± s.d. (c, d, n = 6 biologically independent samples; e, n = 3 biologically independent samples). A two-sided t-test was used to determine statistical significance. f, Effect of ZmFd4 on NiR activity, as analyzed in a semi-in vitro system employing enzyme extracts derived from the wild type and Zmfd9 mutant, along with recombinant GST-ZmFd4. The concentration of ZmFd4 used for enzymatic assays was 12 µM. Data are shown as means ± s.d. (n = 4 biologically independent samples). Different lowercase letters indicate significant differences (P < 0.05; one-way ANOVA with Fisher’s LSD test). g, Genomic structure of the ZmFd4 and ZmFd9 loci and sequence analysis of the target sites in two double knockout mutant lines generated via CRISPR/Cas9. Blue boxes represent the coding regions. The target sequences are in blue font, and the PAM is in orange font. Zmfd4 Zmfd9-c1 harbors a 52-bp deletion (bases 441–492) in ZmFd4, and a C-to-T substitution (at 423 bp; synonymous mutation) and a replacement of a 9-bp fragment (439–447 bp) with a 10-bp sequence in ZmFd9. Zmfd4 Zmfd9-c2 carries a 2-bp deletion (after 446 bp) in ZmFd4 and a 1-bp insertion (after 445 bp) in ZmFd9. Both lines are predicted to produce frameshift proteins of ZmFd4 and ZmFd9. h–j, Relative ZmNiRs transcript levels (h), NiR activity (i), and shoot NO3− contents (j) in wild-type and Zmfd4 Zmfd9 plants grown under 0.05 mM NO3− conditions. Data are shown as means ± s.d. (n = 3 biologically independent samples). A two-sided t-test was used to determine statistical significance. k, Representative photographs of wild-type and Zmfd4 Zmfd9 plants grown under 4 or 0.05 mM NO3− conditions for 14 d. Scale bars, 10 cm. l, Relative ZmNiR2 transcript levels in wild-type and ZmNiR2 overexpression plants grown under 4 mM NO3− conditions. Data are shown as means ± s.d. (n = 3 biologically independent samples). A two-sided t-test was used to determine statistical significance. m, o, NiR activity (m) and shoot NO3− contents (o) in wild-type and ZmNiR2-OE plants grown under 0.05 mM NO3− conditions. Data are shown as means ± s.d. (m, n = 6 biologically independent samples; o, n = 3 biologically independent samples). A two-sided t-test was used to determine statistical significance. n, Representative photographs of wild-type and ZmNiR2-OE plants grown under 4 or 0.05 mM NO3− conditions for 16 d. Scale bars, 10 cm.

Extended Data Fig. 8 Field tests of agronomic traits of Zmfd4 knockouts.

a–l, Plant height (a), ear leaf length (b), ear leaf width (c), days to heading (d), days to anthesis (e), days to silking (f), anthesis-silking interval (g), ear length (h), ear diameter (i), kernel length (j), kernel width (k), and kernel thickness (l) of the wild type and Zmfd4 mutants grown under normal- (NN) or low-N (LN) field conditions. Data are shown as means ± s.d. (a–i, n = 10, 9, 10, 10, 9, 10 plants for columns in order from left to right; j–l, n = 5 biologically independent samples).

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Supplementary Information

Supplementary Tables 1–4.

Reporting Summary

Supplementary Data

Supplementary Datasets 1–9.

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Source Data Extended Data Fig. 3

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Jia, G., Chen, G., Zhang, Z. et al. Ferredoxin-mediated mechanism for efficient nitrogen utilization in maize. Nat. Plants 11, 643–659 (2025). https://doi.org/10.1038/s41477-025-01934-w

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