Abstract

Nitrogen is an essential macronutrient for plant growth and basic metabolic processes. The application of nitrogen-containing fertilizer increases yield, which has been a substantial factor in the green revolution1. Ecologically, however, excessive application of fertilizer has disastrous effects such as eutrophication2. A better understanding of how plants regulate nitrogen metabolism is critical to increase plant yield and reduce fertilizer overuse. Here we present a transcriptional regulatory network and twenty-one transcription factors that regulate the architecture of root and shoot systems in response to changes in nitrogen availability. Genetic perturbation of a subset of these transcription factors revealed coordinate transcriptional regulation of enzymes involved in nitrogen metabolism. Transcriptional regulators in the network are transcriptionally modified by feedback via genetic perturbation of nitrogen metabolism. The network, genes and gene-regulatory modules identified here will prove critical to increasing agricultural productivity.

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Data availability

RNA sequencing data that support the findings of this study have been deposited in NCBI with the primary accession code GSE107988. Supplementary Tables, R code and Cytoscape files can be found at: https://www.bradylab.org/resources/ or https://github.com/agaudinier/Gaudinier2018.

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Acknowledgements

We thank N. M. Crawford for chl1-5 seeds, P. J. Etchells for wox14-1 and lbd4-1 seeds, and E. E. Sparks and P. N. Benfey for erf107-1, abf4-2, eel-1, vip1-1 and erf070 seeds. Some seed stocks were obtained from the Arabidopsis Biological Resource Center (ABRC) at Ohio State University. We thank E. A. Ainsworth and S. B. Gray for help with chlorophyll and protein assays, K. Kajala for help with RNA-seq libraries and E. M. McGinnis for help with root measurements. We thank K. Dehesh for discussions. This research was funded by DuPont Pioneer. A.G. was also supported by the Elsie Taylor Stocking Memorial Fellowship, the Katherine Esau Graduate Summer Fellowship and the University of California, Davis Dissertation Year Fellowship. J.R.-M. was supported by a UC-MEXUS CONACYT PhD Fellowship. D.J.K., M.T. and S.M.B. acknowledge funding from NSF-MCB-1330337. S.M.B. was partially funded by an HHMI Faculty Scholar Fellowship.

Reviewer information

Nature thanks M. Bennett, C. Hodgman and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Affiliations

  1. Department of Plant Biology and Genome Center, University of California, Davis, Davis, CA, USA

    • Allison Gaudinier
    • , Joel Rodriguez-Medina
    • , Anne-Maarit Bågman
    • , Jessica Foret
    • , Michelle Tang
    •  & Siobhan M. Brady
  2. Cold Spring Harbor Laboratory, Cold Spring Harbor, Cold Spring Harbor, NY, USA

    • Lifang Zhang
    • , Andrew Olson
    • , Christophe Liseron-Monfils
    •  & Doreen Ware
  3. DuPont Pioneer, Johnston, IA, USA

    • Shane Abbitt
    • , Bo Shen
    •  & Mary J. Frank
  4. Department of Plant Sciences, University of California, Davis, Davis, CA, USA

    • Michelle Tang
    • , Baohua Li
    • , Daniel E. Runcie
    •  & Daniel J. Kliebenstein
  5. DynaMo Center of Excellence, University of Copenhagen, Frederiksberg C, Denmark

    • Daniel J. Kliebenstein
  6. US Department of Agriculture, Agricultural Research Service, Ithaca, NY, USA

    • Doreen Ware

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Contributions

S.M.B., B.S. and D.W. conceived the project. A.G., L.Z., J.F., S.A. and M.T. cloned promoters. A.G. and S.M.B. designed experiments and A.G., D.J.K. and S.M.B. contributed to data analysis experimental design. A.G., J.F., A.-M.B., M.T. and B.L. performed enhanced yeast one-hybrid screens. A.G. genotyped plants. A.G., A.-M.B. and M.T. performed plant phenotyping. A.G., J.R.-M., A.O. and C.L.-M. performed bioinformatics. A.G. performed transcription factor–target correlation analysis. C.L.-M. and A.O. performed NeCorr analysis. J.R.-M. performed network analysis (enrichment tests), analysis of RNA sequencing data, clustering and network-metabolic analysis. A.G., S.M.B., D.E.R., D.J.K., B.S., D.W. and M.J.F. provided discussion, experimental design and analysis suggestions. S.M.B. and A.G. wrote the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Siobhan M. Brady.

Extended data figures and tables

  1. Extended Data Fig. 1 Combinatorial interactions between transcription factors and promoters of genes associated with nitrogen metabolism, signalling and nitrogen-associated processes.

    Rectangles, promoters; ovals, transcription factors; diamonds, genes represented as both promoters and transcription factors. Nitrogen-associated biological processes are indicated by promoter colour. A grey line indicates a transcription factor–promoter interaction. Light green, nitrogen transporter; yellow, organ growth; dark green, nitrate assimilation; light purple, nitrogen signalling; light blue, nitrogen-linked; orange, carbon metabolism; red, ethylene; dark blue, auxin; teal, carbon transporter; dark purple, amino acid metabolism; pink, transcription factors linked to nitrogen.

  2. Extended Data Fig. 2 Genes in the YNM regulated by hormone signalling.

    The YNM. Genes coloured in each panel are regulated by the CPK–NLP7 signalling cascade or indicated hormone. P value indicates significance for enrichment in the network using a two-sided Fisher’s exact test. a, Genes regulated by the CPK–NLP7 signalling cascade (cyan). b, Genes regulated by abscisic acid (purple). c, Genes regulated by ethylene (red). d, Genes regulated by methyl jasmonate (orange). e, Genes regulated by auxin (dark blue). f, Genes regulated by cytokinin (light blue). g, Genes regulated by brassinosteroid (green). h, Genes regulated by gibberellic acid (pink). Gene lists used for enrichment tests can be found in Supplementary Table 4.

  3. Extended Data Fig. 3 Wild-type root growth.

    RSA for wild-type (Col-0) nine-day-old seedlings in both limiting (1 mM) and sufficient (10 mM) KNO3 conditions. ag, Traits measured were primary root length (a), number of lateral roots (b), total lateral root length (c), average lateral root length (d), total root length (e), lateral root density (f) and the ratio of lateral root length contributing to the total root length (g). Box plots are centred at the data median and mark from the 25th to the 75th percentile. Individual measurements are plotted as black dots. n = 209 1 mM KNO3, n = 201 10 mM KNO3, P values were calculated using two-way ANOVAs.

  4. Extended Data Fig. 4 Principal component analysis of all wild-type root traits.

    Dark blue, roots grown on 10 mM KNO3; light blue, roots grown on 1 mM KNO3. a, PC1 captures 69% of the variation and PC2 captures 19% of the variation. b, PC2 plotted with PC3 captures 9% of the variation. c, PC1 plotted with PC3 (n = 209 1 mM KNO3, n = 201 10 mM KNO3).

  5. Extended Data Fig. 5 YNM sub-network involved in nitrogen-associated influence on RSA.

    a, The YNM. Blue, genes associated with root length (Supplementary Table 10); yellow, genes associated with lateral root development29; green, genes associated with root length and lateral root development. Heavy black borders denote genes with a mutant root phenotype from this study. b, Sub-network of YNM with genes associated with RSA, and their first neighbour connections.

  6. Extended Data Fig. 6 Nitrogen, carbon and carbon:nitrogen ratio in transcription-factor mutants.

    a, Percentage of natural abundance of 15N in total shoot tissue. b, Percentage of natural abundance of 13C in total shoot tissue. c, Ratio of natural abundance of 13C to 15N. *P < 0.05 using a two-way ANOVA; exact n and P values for the analysis can be found in Supplementary Table 10. Box plots are centred at the data median and mark from the 25th to the 75th percentile. Individual measurements are plotted as black dots.

  7. Extended Data Fig. 7 Chlorophyll levels across transcription-factor mutants.

    a, Chlorophyll levels measured by chlorophyll content index. b, Total chlorophyll levels measured by ethanol extraction. *P < 0.05 using a two-way ANOVA; exact n and P values for the analysis can be found in Supplementary Table 10. Box plots are centred at the data median and mark from the 25th to the 75th percentile. Individual measurements are plotted as black dots.

  8. Extended Data Fig. 8 Clustering of nitrogen-responsive genes in the root, in transcription-factor mutants.

    The expression in the root of genes responsive to nitrogen availability (Supplementary Table 15) was analysed in the mutant background of each transcription factor, and clustered using dominant pattern identification. Gene expression in each mutant background was expressed as the log2(fold change) of the expression of a given gene in 1 mM nitrate relative to 10 mM nitrate, and relative to its expression in wild type (log2(fold change) in 1 mM nitrate relative to 10 mM nitrate. Colours on the y axis indicate each respective cluster or module. Gene names are indicated on the far right.

  9. Extended Data Fig. 9 Clusters of YNM genes in mutants of enzymes involved in nitrogen metabolism and their transcriptional regulators.

    a, Clusters of genes significantly differentially expressed in the microarray analysis of nitrogen-metabolism mutants and nitrogen transcriptional regulator mutants. b, Clusters overlaid on the YNM.

  10. Extended Data Fig. 10 Differentially expressed genes in the YNM in mutants of enzymes involved in nitrogen metabolism, and their transcriptional regulators.

    The YNM. Genes are coloured by the number of mutant datasets in which they are found to be differentially expressed (white = 0, dark purple = 10).

Supplementary information

  1. Supplementary Information

    This file contains a guide for Supplementary Tables 1-16.

  2. Reporting Summary

  3. Supplementary Data 1

    Root and shoot phenotype boxplots. Boxplots display results from phenotyping analysis for all T-DNA mutant lines tested. Boxplots are centered at the data median and mark from the 25th to the 75th percentile. Individual measurements are plotted as black dots. Biological replicates (n) and significance results from two-way ANOVA is recorded for each measurement for each genotype in Supplemental Table 13. * = p-value < 0.05.

  4. Supplementary Data 2

    ANOVA tables from all phenotyping analyses. Biological replicates (n) are recorded for each measurement for each genotype in Supplemental Table 13.

  5. Supplementary Tables

    This zipped file contains Supplementary Tables 1-16 – see Supplementary Information document for full table legends.

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