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A feedback regulatory loop by MAPK–CCA1 engages auxin signalling to stimulate root foraging for nitrate

Nature Plants (2026)Cite this article

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Abstract

In eucaryotes, mitogen-activated protein kinase (MAPK) cascades are evolutionarily conserved signalling modules crucial for growth regulation and stress tolerance. However, the regulatory role of MAPK in nutrient sensing by plants remains largely unclear. Here we uncovered MEKK14 and its paralogue MEKK13 determine lateral root elongation via enhanced cell division and expansion. We further fine-mapped a naturally occurring histidine-to-glutamine substitution in MEKK14 that weakens protein kinase activity and attenuates lateral root growth and response to nitrate (NO3−). We further demonstrate that NO3− transcriptionally upregulates MEKK13/14 depending on NLP7 to activate a MKK3–MPK1/2/7/14 signalling module. Downstream of this signalling cascade, the core oscillator of the circadian clock CCA1 is phosphorylated and stabilized to feedback induce MEKK13/14 expression and to activate auxin signalling-dependent lateral root foraging for NO3−. Our findings reveal a positive-feedback phosphorylation–transcriptional regulatory loop in root NO3− foraging, extending the regulatory function of MAPK signalling in the nutrient sensing.

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Fig. 1: GWAS maps MEKK14 as determinant for LR length.
Fig. 2: His to Gln substitution at position 172 of MEKK14 weakens kinase activity and decreases LR elongation.
Fig. 3: MEKK13/14 determine LR elongation under NO3−.
Fig. 4: The MKK3–MPK1/2/7/14 module stimulates LR elongation under NO3−.
Fig. 5: Group C MPKs phosphorylate CCA1 to stimulate LR elongation under NO3−.
Fig. 6: Phosphorylation stabilizes CCA1 to feedback activate MEKK13/14 expression.
Fig. 7: A MAPK–CCA1 cascade stimulates LR elongation via auxin signalling.

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Data availability

Data are available within the Article and the Supplementary Information. Additional datasets are available from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

We thank J. Fuge and A. Bieber (Leibniz Institute of Plant Genetics and Crop Plant Research, Gatersleben) for excellent technical assistance; Z. Ding (Shandong University, China) and Y. Xiang (Chinese Academy of Agricultural Sciences) for sharing mpk1,2,14 and mpk7 mutants. This work was supported by grants to Z.J. by Biological Breeding-National Science and Technology Major Project (2023ZD04072), National Natural Science Foundation of China (32370270), Disciplinary Breakthroughs Project of Ministry of Education (JYB2025XDXIM702), Chinese Universities Scientific Fund (2023RC052, 1031-00114341, 2025RC027) and Pinduoduo-China Agricultural University Research Fund (PC2024B01009).

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Author notes

  1. These authors contributed equally: Xiaofei Zhang, Shanmin Zhou.

Authors and Affiliations

  1. State Key Laboratory of Nutrient Use and Management, College of Resources and Environmental Sciences, National Academy of Agriculture Green Development, China Agricultural University, Beijing, China

    Xiaofei Zhang, Shanmin Zhou, Lixing Yuan, Fusuo Zhang & Zhongtao Jia

  2. Molecular Plant Nutrition, Department of Physiology & Cell Biology, Leibniz-Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany

    Jingyi Guo, Ricardo F. H. Giehl & Nicolaus von Wirén

  3. State Key Laboratory of Crop Stress Adaptation and Improvement, School of Life Sciences, Henan University, Kaifeng, China

    Xiaodong Xu

  4. Future Food Beacon and School of Biosciences, University of Nottingham, Nottingham, UK

    Malcolm J. Bennett

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  1. Xiaofei Zhang
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Contributions

Z.J., R.F.H.G. and N.v.W. conceived the project and designed the experiments. X.Z., S.Z. and Z.J. performed experiments; J.G. genotyped the mpk mutants; L.Y. and F.Z. contributed to helpful discussions; X.X. provided critical plant materials. Z.J., R.F.H.G. and N.v.W. wrote the manuscript with the support of X.X., L.Y. and M.J.B.

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Correspondence to Nicolaus von Wirén or Zhongtao Jia.

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Extended data

Extended Data Fig. 1 His to Gln substitution at position 172 does not impact the interaction of MEKK14 proteoforms with MKK3.

Y2H assay showing that MEKK14172H and MEKK14172Q interact similarly with MKK3. SD/-Leu-Trp, synthetic defined (SD) medium lacking Leu and Trp; SD/-Leu-Trp-His-Ade, SD medium lacking Leu, Trp, His and Ade.

Source data

Extended Data Fig. 2 Expression pattern of MEKK14 and MEKK13.

a-b Overview of promoter::GUS activity of MEKK14 (a) and MEKK13 (b) in 10-day-old light-grown seedlings. c-d Promoter activity of MEKK14 (c) and MEKK13 (d) in primary root tips. e Expression pattern of MEKK13 in early-stage LR primordia (i), LR primordia close to emergence (ii) and emerged LR (iii). 10-day-old seedlings grown on 11.4 mM N were sampled and subjected to GUS staining for 1 hour. Scale bars, 1 cm in a-b and 100 μm in c-e.

Source data

Extended Data Fig. 3 MEKK14 allelic variants determine phenotypic variation of NO3−-dependent LR growth.

a-c Average LR length of accessions from 172H haplotype (Col-0, Sha, Yo-0, Ty-1 and Rmx-A02) and 172Q haplotype (Ra-0, Uod-1, Ren-1, Pog-0, Van-0, Sg-1, Per-1, Wa-0, Ws-2 and Est-0) plants grown under 2 mM KCl (a) or KNO3 (b) and appearance of plants from representative genotypes (c). Symbols with crossbars represent means ± s.e.m. Individual symbols represent averaged LR length of respective accessions assessed from 6-10 independent plants. In a and b, n = 5 and 10 for 172H and 172Q haplogroup, respectively. d-e Appearance of plants (d), and average LR length (e) of wild types (Col-0, Sha, Ra-0 and Uod-1) and mekk14 crispr mutant plants grown on either 2 mM KCl or KNO3. Symbols with crossbars represent means ± s.e.m. Number of plants analyzed in KCl and KNO3: n = 16/14 (Col-0), 16/14 (cr2_Col-0), 16/14 (cr7_Col-0), 16/15 (Sha), 15/13 (cr7_Sha), 15/15 (cr10_Sha),16/14 (Ra-0), 16/16 (cr1_Ra-0), 16/16 (cr2_Ra-0), 16/15 (Uod-1), 15/16 (cr8_ Uod-1), 16/15 (cr11_Col-0), respectively. Scale bar, 1 cm. Different letters indicate significant differences at P < 0.01 according to one-way ANOVA and post-hoc Tukey’s test.

Source data

Extended Data Fig. 4 Loss-of-function of MKK3 reduces meristem size and cell length under NO3−.

a-b. Representative confocal images of root meristems (a) and mature cells (b) of Col-0 and mkk3 LRs grown on 2 mM KCl or KNO3. Red lines in a and arrowheads in b indicate meristem sizes (a) and the boundaries between two consecutive mature cortical cells (b). Scale bars, 100 μm. c-d Length of the meristems (c) and cortical cells (d) of LRs from Col-0 and mkk3 plants. Number of roots analyzed in KCl/KNO3: n = 12/12, 12/12 and 12/12 in e and 72/72, 72/72 and 72/72 in f for Col-0, mkk3-1 and mkk3-2, respectively. Blue symbols with crossbars represent mean ±s.e.m. Different letters indicate significant differences at P < 0.001 according to one-way ANOVA and post-hoc Tukey’s test.

Source data

Extended Data Fig. 5 MPK1, 2, and 14 single mutants exhibit inhibited lateral root elongation.

a-b Appearance of plants (a) and average LR length (b) of wild-type (Col-0), mpk1-1, mpk1-2, mpk14-1, mpk14-2 and mpk2-2 mutant plants grown on 2 mM KCl or KNO3. Scale bars, 1 cm. Number of roots analyzed in KCl/KNO3: n = 20/20, 19/19, 19/20,19/19, 20/20 and 19/20 for Col-0, mpk1-1, mpk1-2, mpk14-1, mpk14-2 and mpk2-2, respectively. c and e. Representative confocal images of root meristems (c) and mature cells (e) of Col-0 and mpk LRs. Red lines in c and arrowheads in e indicate meristem sizes (c) and the boundaries between two consecutive mature cortical cells (e). Scale bars, 100 μm. d and f. Meristem size (d) and cortical cell length (f) of LRs from Col-0 and mkks plants. Number of roots or cells analyzed in KCl/KNO3: n = 12/12, 12/12 and 12/12 in d and 72/72, 72/72, 72/72 and 72/72 in f for Col-0, mkk3-1 and mkk3-2, respectively. Blue symbols with crossbars represent mean ± s.e.m. P values related to differences between respective T-DNA KO and wild-type Col-0 were calculated according to Welch’s t-test.

Source data

Extended Data Fig. 6 Multiple deletion lines of MPK1/2/7/14 have decreased meristem size and cell length under NO3−.

a-b. Representative confocal images of root meristems (a) and mature cells (b) of Col-0, tko and qko (#1 and #2) LRs grown on 2 mM KCl or KNO3. Red lines in a and arrowheads in b indicate meristem sizes (a) and the boundaries between two consecutive mature cortical cells (b). c-d Meristem size (c) and cortical cell length (d) of LRs from Col-0, tko and qko (#1 and #2) plants. Number of LR analyzed in KCl/ KNO3: n = 13/12 (Col-0), 13/12 (tko), 15/10 (qko-#1) and 15/13 (qko-#2) in c; n = 72/76 (Col-0), 65/64 (tko), 70/60 (qko-#1), 66/63 (qko-#2) in d, respectively. Blue symbols with crossbars represent means ± s.e.m. Different letters indicate significant differences at P < 0.05 according to one-way ANOVA and post-hoc Tukey’s test.

Source data

Extended Data Fig. 7 NO3− activates a MEKK14-MKK3-MPK1/2/7/14 phospho-relay to promote LR growth.

a MEKK13/14- and MKK3-dependent activation of MPK1/2/7/14 phosphorylation by NO3−. 7-day-old seedlings precultured on high N were subjected to 4 days N starvation and then exposed to 10 mM KNO3. Recombinant GST-tagged MPK1/2/7/14 protein was incubated with total protein extracts from wild-type (Col-0), mekk13/14 and mkk3 seedlings for 1-4 h of nitrate treatment. Abundance of GST-MPK1/2/7/14 and phosphorylated GST-MPK1/2/7/14 proteins was assessed with Coomassie Brilliant Blue staining (CBB) and anti-thiophosphate ester-specific antibody (anti-TPE). Actin was used as internal control for equal protein loading. The band intensity of phosphorylated MPK was quantified with Image J (version 1.53e) and normalized to Actin with time 0 in Col-0 samples set to 1.0. b-c Appearance of plants (b), and average LR length (c) of wild-type (Col-0), mekk13/14 and two representative T3 transgenic lines expressing GFP-tagged MKK3DD under MEKK14 promoter grown under 2 mM KCl and KNO3. Number of plants analyzed in KCl/KNO3: n = 16/16, 16/15, 16/15 and 15/15 for Col-0, mekk14, MKK3DD-#1 and MKK3DD-#2, respectively. d-e Appearance of plants (d), and average LR length (e) of wild-type (Col-0), mkk3-2 and two representative T3 transgenic lines expressing GFP-tagged MPKsDD under MKK3 promoter grown under 2 mM KCl and KNO3. Number of plants analyzed in KCl/KNO3: n = 16/16, 16/16, 16/16, 15/15, 15/16, 16/15, 16/16, 16/15, 16/14 and 16/16 for Col-0, mkk3-2, MPK1DD-#1 and MPK1DD-#2, MPK2DD-#1, MPK2DD-#2, MPK7DD-#1, MPK7DD-#2, MPK14DD-#1 and MPK14DD-#2, respectively. Scale bars, 1 cm. Different letters indicate significant differences at P < 0.01 according to one-way ANOVA and post-hoc Tukey’s test.

Source data

Extended Data Fig. 8 CCA1 interacts with MPK1/2/7/14 in yeast two-hybrid and split luciferase complementation imaging assays.

a-d Y2H assay showing CCA1 interaction with MPK1 (a), MPK2 (b), MPK7 (c) and MPK14 (d). Co-transformation of CCA1-BD with empty AD or AD-MPK1/2/7/14, or of AD-MPK1/2/7/14 with empty BD vector. AD and BD vectors served as negative controls. SD/-Leu-Trp, synthetic defined (SD) medium lacking Leu and Trp; SD/-Leu-Trp-His-Ade, SD medium lacking Leu, Trp, His, and Ade. e-h Split-LUC complementation imaging assay in N. benthamiana testing interaction between CCA1 and MPK1 (e), MPK2 (f), MPK7 (g) and MPK14 (h). Indicated constructs were transiently expressed in N. benthamiana by Agrobacterium-mediated transformation, and the GUS (β-glucuronidase) protein served as negative controls. nLUC, N-terminal fragment of firefly luciferase; cLUC, C-terminal fragment of firefly luciferase.

Source data

Extended Data Fig. 9 Exogenous auxin supply restores the LR growth response to NO3−.

a-b Appearance of plants (a) and average LR length (b) of wild-type (Col-0) and cca1-1 mutant plants grown on 2 mM KCl or KNO3 supplied with or without 20 nM IAA. c-d Appearance of plants (c) and average LR length (d) of wild-type (Col-0), mekk13/14, mkk3-2 and qko-#1 mutant plants grown on 2 mM KCl or KNO3 supplied with or without 20 nM IAA. Scale bars, 1 cm. Symbols with crossbars represent means ± s.e.m. n = 17 and 16 for Col-0 and cca1-1 under respective N and auxin treatment in b; n = 16 for respective treatment and genotype in d. Different letters indicate significant differences at P < 0.001 according to one-way ANOVA and post-hoc Tukey’s test.

Source data

Extended Data Fig. 10 External auxin supply to LRs in a split-root system restores the LR growth response of cca1-1 to NO3− without penalizing primary root growth.

a Schematic representation of the spilt-agar experiment with auxin treatment. Col-0 and cca1-1 mutant were firstly precultured on 1/2 MS medium identical to sufficient N (11.4 mM) treatment but with N supplied exclusively as 1 mM Gln. After 6 days, seedlings of homogenous size from each genotype were transferred to either 2 mM KCl or KNO3 for 5 days enabling LR outgrowth and phenotypic characterization shown in figure panel c and d. Then, these seedlings were transferred to split-agar plates, so that only the branching zone containing emerged LRs was exposed to 10 or 20 nM IAA. After 3 days, roots were scanned and subjected to quantification of the length of individual LRs and primary root. b-d Appearance of plants (b), average LR length (c) and primary root length (d) of wild-type (Col-0) and cca1-1 mutant plants grown under either 2 mM KCl or KNO3 in the presence or absence of IAA in the split-agar plates for 3 days. Only LRs grown in the auxin-supplied compartments were considered for quantification. Scale bars, 1 cm. Number of roots analyzed given in KCl/KNO3 in c and d: n = 9/8 and 8/8 for Col-0 and cca1-1 in panel DAT5; 9/8 and 9/8 for Col-0 and cca1-1 in panel mock; 8/8 and 8/8 for Col-0 and cca1-1 in panel 10 nM IAA; 8/8 and 8/8 for Col-0 and cca1-1 in panel 20 nM IAA. Symbols with crossbars represent mean ± s.e.m. Different letters indicate significant differences at P < 0.01 according to one-way ANOVA and post-hoc Tukey’s test.

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Zhang, X., Zhou, S., Guo, J. et al. A feedback regulatory loop by MAPK–CCA1 engages auxin signalling to stimulate root foraging for nitrate. Nat. Plants (2026). https://doi.org/10.1038/s41477-026-02225-8

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