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A type 2C protein phosphatase activates high-affinity nitrate uptake by dephosphorylating NRT2.1

Abstract

The nitrate transporter NRT2.1, which plays a central role in high-affinity nitrate uptake in roots, is activated at the post-translational level in response to nitrogen (N) starvation1,2. However, the critical enzymes required for the post-translational activation of NRT2.1 remain to be identified. Here, we show that a type 2C protein phosphatase, designated CEPD-induced phosphatase (CEPH), activates high-affinity nitrate uptake by directly dephosphorylating Ser501 of NRT2.1, a residue that functions as a negative phospho-switch in Arabidopsis2. CEPH is predominantly expressed in epidermal and cortex cells in roots and is upregulated by N starvation via a CEPDL2/CEPD1/2-mediated long-distance signalling from shoots3,4. The loss of CEPH leads to marked decreases in high-affinity nitrate uptake, tissue nitrate content and plant biomass. Collectively, our results identify CEPH as a crucial enzyme in the N-starvation-dependent activation of NRT2.1 and provide molecular and mechanistic insights into how plants regulate high-affinity nitrate uptake at the post-translational level in response to the N environment.

Data availability

The microarray data have been deposited in the NCBI Gene Expression Omnibus under accession number GSE160649. The raw MS data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository under accession number PXD022343. The Arabidopsis lines generated in this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank H. Fukuda (Agilent Technologies) for technical advice regarding phosphopeptide enrichment. This research was supported by a Grant-in-Aid for Scientific Research (S) (no. 18H05274 to Y.M.), a Grant-in-Aid for Transformative Research Areas (A) (no. 20H05907 to Y.M.) and a Grant-in-Aid for JSPS Fellows (no. 20J20049 to Y.O.) from the Japan Society for the Promotion of Science.

Author information

Affiliations

  1. Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan

    Yuri Ohkubo & Yoshikatsu Matsubayashi

  2. Institute of Transformative Bio-Molecules, Nagoya University, Nagoya, Japan

    Keiko Kuwata

Contributions

Y.M. and Y.O. conceived the project and designed the experiments. Y.O. performed all of the biological experiments. K.K. performed the phosphopeptide enrichment and nano LC–MS/MS analyses. Y.M. and Y.O. interpreted the results and wrote the manuscript.

Corresponding author

Correspondence to
Yoshikatsu Matsubayashi.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Plants thanks Brent Kaiser and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 At4g32950 is an Arabidopsis PP2C subfamily E protein.

a, A combined phylogenetic tree of PP2C subfamily E proteins from Arabidopsis (At), potato (St), Medicago truncatula (Mt), rice (Os), maize (Zm) and moss Physcomitrella patens (Pp). At4g32950 was classified into a distinct clade (shaded) that includes members from potato, Medicago, rice and maize, but not mosses. Numbers represent bootstrap values from 1,000 replicates. b, Expression stability of two different reference genes, EF-1α and TUA4, between WT and cepd1,2 cepdl2 triple mutant plants. TUA4 and EF-1α expression level in WT and cepd1,2 cepdl2 was quantified by RT-qPCR using EF-1α and TUA4 as reference genes, respectively. c, Expression of NRT2.1 in WT and cepd1,2 cepdl2 triple mutant plants. Data are presented as the mean ± SD, and each dot represents a biological replicate. P-values are 2.76 × 10−2 for 3 mM and 1.61 × 10−4 for 1 mM. For (b) and (c), statistical significance is determined by two-tailed non-paired Student’s t test (*P < 0.05, ‘ns’ indicates not significant). Source data

Extended Data Fig. 2 Phenotypic analysis of ceph-1 mutant plants.

a, Fresh weight of 14-day-old shoots of WT, ceph-1 mutant, and ceph-1 plants complemented with CEPH grown on 1 mM NO3 medium. b, Fresh weight of 14-day-old shoots of WT, ceph-1 mutant, and ceph-1 plants complemented with CEPH-GFP grown on 1 mM NO3 medium. c, Root phenotypes of 10-day-old WT and ceph-1 mutant plants grown on 1 mM NO3 medium. Scale bar = 1 cm. d, Primary root length of 10- or 14-day-old WT and ceph-1 mutant plants grown on 1 mM NO3 medium. e, Ratio of total lateral root (LR) length to primary root (PR) length of 10- or 14-day-old WT and ceph-1 mutant plants grown on 1 mM NO3 medium. P-values are 2.23 × 10−2 for 10 d and 4.01 × 10−3 for 14 d. f, Phenotypes of 14-day-old WT and ceph-1 mutant plants grown under N-replete conditions (10 mM NH4+, 10 mM NO3). Scale bar = 5 mm. g, Fresh weight of the shoots and roots described in (f). (h) HATS and calculated LATS NO3 uptake activities of 14-day-old WT and ceph-1 mutant plants grown on 1 mM NO3 medium. P-values are 6.13 × 10−5 for HATS and 1.32 × 10−3 for LATS. i, Expression of nitrate transporter genes in WT and ceph-1 mutant plants. j, Expression of CEPD1/2 and CEPDL2 in WT and ceph-1 mutant plants. P-value is 3.81 × 10−3 for CEPDL2. k, Induction level of CEPH in XVE-CEPH plants 6 h after estradiol treatment. P-value is 3.65 × 10−3. Data are presented as the mean ± SD, and each dot represents a biological replicate. For (a) and (b), different letters indicate statistically significant differences (P < 0.05, one-way ANOVA with post-hoc Tukey’s test). For (d), (e) and (g)-(k), statistical significance is determined by two-tailed non-paired Student’s t test (*P < 0.05, ‘ns’ indicates not significant). Source data

Extended Data Fig. 3 Venn diagram showing overlap between data sets.

a, Venn diagram for numbers of phosphopeptide ion peaks identified in the Lys-C–digested samples and overlap between the forward and reciprocal analyses. b, Venn diagram for numbers of phosphopeptide ion peaks identified in the trypsin-digested samples and overlap between the forward and reciprocal analyses.

Extended Data Fig. 4 Quantitative phosphoproteomic analysis of the CEPH substrate.

a, Mass spectra of the NRT2.1 G478 nonphosphorylated peptide (position 478-493 in NRT2.1 protein) derived from reciprocally 14N- and 15N-labeled plants, showing comparable NRT2.1 protein abundance in WT and ceph-1 plants. b, Mass spectra of the NRT2.1 T521 phosphopeptide (position 516-530 in NRT2.1) derived from reciprocally labeled plants, showing comparable phosphorylation levels in WT and ceph-1 plants. (c) Dephosphorylation of the NRT2.1 S28 phosphopeptide with CEPH-GFP detected by LC-MS in the selected ion monitoring mode at m/z 831.9.

Extended Data Fig. 5 NRT2.1 S501 phosphorylation acts as a negative phospho-switch that represses HATS activity.

a, Phenotypes of 14-day-old WT, nrt2.1-2.2 mutant, nrt2.1-2.2 complemented with NRT2.1[S501A] and nrt2.1-2.2 complemented with NRT2.1[S501D] grown on 1 mM NO3 medium. Scale bar = 2 mm. b, Fresh weight of 14-day-old shoots of the plants shown in (a). c, Root HATS activity of the plants shown in (a). d, Mass spectra of the NRT2.1 G478 nonphosphorylated peptide (position 478-493 in NRT2.1 protein) and ACT8 A21 peptide (position 21-30 in ACT8 protein) derived from WT and NRT2.1[S501D] plants. Decrease in ACT8 protein abundance in S501D plants suggests the decrease in overall protein expression level is due to a nitrate uptake defect in S501D plants. Considering this, protein abundance of NRT2.1 was not significantly affected by the S501D mutation. e, Root HATS activity of 14-day-old nrt2.1-2.2 mutant plants complemented with NRT2.1[S501A], ceph-1 mutant, ceph-1 mutant expressing NRT2.1[S501A] and ceph-1 mutant expressing NRT2.1[S501D]. The plotted S501A/nrt2.1-2.2 data is the same as that shown in Extended Data Fig. 5c, as the experiments were done concurrently. f, Mass spectra of the NRT2.1 G478 nonphosphorylated peptide (position 478-493 in NRT2.1 protein) derived from reciprocally 14N- and 15N-labeled plants, showing comparable NRT2.1 protein abundance in mock and estradiol-treated XVE-CEPH plants. Data are presented as the mean ± SD, and each dot represents a biological replicate. For (b), (c) and (e), different letters indicate statistically significant differences (P < 0.05, one-way ANOVA with post-hoc Tukey’s test). Source data

Supplementary information

Supplementary Table 1

List of the top ten CEPDL2/CEPD1/2-regulated genes in roots. The table includes the GeneChip signal intensity, signal intensity ratio (log2(FC)), z score and gene description. The results are sorted by the z score of CEPDL2ox plants.

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Ohkubo, Y., Kuwata, K. & Matsubayashi, Y. A type 2C protein phosphatase activates high-affinity nitrate uptake by dephosphorylating NRT2.1.
Nat. Plants (2021). https://doi.org/10.1038/s41477-021-00870-9

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