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Discovery of nitrate–CPK–NLP signalling in central nutrient–growth networks

Abstract

Nutrient signalling integrates and coordinates gene expression, metabolism and growth. However, its primary molecular mechanisms remain incompletely understood in plants and animals. Here we report unique Ca2+ signalling triggered by nitrate with live imaging of an ultrasensitive biosensor in Arabidopsis leaves and roots. A nitrate-sensitized and targeted functional genomic screen identifies subgroup III Ca2+-sensor protein kinases (CPKs) as master regulators that orchestrate primary nitrate responses. A chemical switch with the engineered mutant CPK10(M141G) circumvents embryo lethality and enables conditional analyses of cpk10 cpk30 cpk32 triple mutants to define comprehensive nitrate-associated regulatory and developmental programs. Nitrate-coupled CPK signalling phosphorylates conserved NIN-LIKE PROTEIN (NLP) transcription factors to specify the reprogramming of gene sets for downstream transcription factors, transporters, nitrogen assimilation, carbon/nitrogen metabolism, redox, signalling, hormones and proliferation. Conditional cpk10 cpk30 cpk32 and nlp7 mutants similarly impair nitrate-stimulated system-wide shoot growth and root establishment. The nutrient-coupled Ca2+ signalling network integrates transcriptome and cellular metabolism with shoot–root coordination and developmental plasticity in shaping organ biomass and architecture.

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Figure 1: Nitrate triggers unique Ca2+-CPK signalling.
Figure 2: Chemical genetic analyses of CPK10, CPK30 and CPK32.
Figure 3: CPK10, CPK30 and CPK32 control primary nitrate-responsive transcriptome.
Figure 4: Dynamic Nitrate–CPK signalling orchestrates development of shoots and roots.
Figure 5: The CPK–NLP signalling connection.
Figure 6: Nitrate–CPK–NLP signalling is crucial in nutrient–growth networks.

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Acknowledgements

We thank T. Asai for the pNLP7-NLP7-GFP and pNLP7-NLP7(S205A)-GFP constructs, X. Y. Liu and R. Q. Ye for the pUBQ10-NLS-TdTomato construct, X. C. Zhang and M. R. Knight for the aequorin transgenic line, J. Bush for management of the plant facilities, Q. Hall for advice on embryo analysis, ABRC, NASC and the Salk Institute for T-DNA insertion lines, K. Holton and H. Lee for advice on statistics analyses, and L. Shi, J. Bush and A. Diener for comments. K.S. thanks F. Rutaganira for help with characterization of 3MBiP. The Research is supported by the NIH, NSF and WJC Special Project (PJ009106) RDA-Korea to J.S., and by CREST-JPMJCR15O5, JST and JSPS-KAKENHI grants 25252014/26221103 to S.Y. and 15H05616 to M.K., and the NSFC grant 31670246 to K.L.

Author information

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Authors

Contributions

K.L., Y.N., J.S. and S.Y. conceived and initiated the project, and designed the experiments, K.L., Y.N., M.K., Y.W., H.D., H.S.C., L.L., M.B. and J.S. performed experiments. M.M., Y.N. and H.S.C. performed bioinformatics. S.M. and T.I. conducted LC–MS/MS analysis. C.Z. and K.S. provided 3MBiP and suggestions. J.S., K.L., Y.N. and S.Y. wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Kun-hsiang Liu, Shuichi Yanagisawa or Jen Sheen.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks H. Sakakibara and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Nitrate promotes plant development and induces Ca2+ signatures in leaves and roots.

a, Nitrate promotes shoot and root development. Plants were germinated without an exogenous nitrogen source for 4 days and then transferred to the plates supplemented with different concentrations of KNO3, NH4Cl or glutamine for 7 days. Scale bars, 1 cm. The experiments were repeated twice with 10 seedlings for each treatment with consistent results. b, c, Distinct Ca2+ signatures induced by nitrate and flg22 in aequorin transgenic plants. Arabidopsis transgenic seedlings constitutively expressing the Ca2+ reporter protein apoaequorin were grown in liquid medium containing 2.5 mM ammonium succinate as the sole nitrogen source for 6 days. Aequorin was reconstituted with 10 μM coelenterazine overnight in the dark. The results are presented as relative light units (RLU) in response to 10 mM KCl or KNO3 (b) or to 100 nM flg22 or water (c) at intervals of 1 s. Error bars, ± s.e.m., n = 10 seedlings. The experiments were repeated three times with similar results. The RLU value is cut-off at 3,500. d, NRT1.1 is highly expressed in shoots and mesophyll protoplasts. The signal counts of the genes in roots and shoots were derived from previously published microarray data4. The signal counts of the genes for mesophyll protoplasts were derived from previously published microarray data60. LHCB2.2 serves as a leaf-specific expression control. The control gene UBQ10 is constitutively and highly expressed in roots, shoots and mesophyll protoplasts. Error bars, s.d., n = 3 biological replicates from mesophyll protoplasts. e, Nitrate induction of the endogenous NIR gene as a primary nitrate-responsive marker gene in seedlings and mesophyll protoplasts. NIR expression was quantified by RT–qPCR analysis. Arabidopsis seedlings or mesophyll protoplasts were treated with 10 mM KCl or KNO3 for 2 h. Error bars, s.d., n = 3 biological replicates. f, g, Time-lapse images of nitrate-stimulated Ca2+ signalling in roots of intact transgenic GCaMP6 plants. The entire time-lapse recording of Ca2+ signals stimulated by 10 mM KCl or KNO3 in the root tip (f) or the elongated region (g) is shown in Supplementary Videos 3 and 4. Seedlings were grown on basal medium without nitrogen for 4 days and then stimulated by KCl or KNO3. Scale bars, 10 μm. The experiments were repeated three times with 10 seedlings for each treatment with consistent results. Source Data for d and e can be found in the Supplementary Information.

Source data

Extended Data Figure 2 Calcium mediates the nitrate response in seedlings and mesophyll protoplasts.

a, Ca2+ channel blockers diminish primary nitrate-responsive transcription. RT–qPCR analyses with 7-day-old seedlings. 0.5 mM KNO3, 15 min. Error bars, s.d., n = 3 biological replicates. *P < 0.05, **P < 0.0001 (two-way ANOVA with Tukey’s multiple comparisons test). b, An antagonist of Ca2+ sensors (W7) inhibits primary nitrate-responsive transcription. Error bars, s.d., n = 3 biological replicates. *P < 0.05, **P < 0.0001 (two-way ANOVA with Tukey’s multiple comparisons test). c, Nitrate stimulates putative endogenous CPKs in an in-gel kinase assay. 10 mM KNO3, 10 min. d, Time-course analysis of NIR-LUC activity in response to nitrate induction. Mesophyll protoplasts co-transfected with NIR-LUC and UBQ10-GUS (as the internal control) were incubated in WI buffer for 4 h and then induced by 0.5 mM KCl or KNO3 for 0.5, 1, 2 and 3 h. The fold change is calculated relative to the value of KCl treatment at each time point. Error bars, s.d., n = 3 biological replicates. e, Nitrate-specific induction of NIR-LUC expression. Transfected mesophyll protoplasts were incubated in WI buffer for 4 h and then induced by 0.5 mM KCl or different nitrogen sources for 2 h. Error bars, s.d., n = 3 biological replicates. f, Sensitive regulation of NIR-LUC by nitrate. Transfected mesophyll protoplasts were incubated in WI buffer for 4 h and then induced by different concentration of KCl or KNO3 for 2 h. Error bars, s.d., n = 3 biological replicates. g, Relation tree of Arabidopsis CPK proteins. The relation tree was generated by ClustalX and Treeview algorithms using the complete protein sequences of CPKs. The subgroup III CPKs that enhanced NIR-LUC activity by more than two-fold are highlighted. Genes encoding CPK14 and CPK24 are not expressed in mesophyll cells. Source Data for a, b, df can be found in the Supplementary Information.

Source data

Extended Data Figure 3 Analyses of single and double cpk and icpk mutants in subgroup III CPKs.

a, b, The cpk T-DNA insertion lines. All cpk mutants were isolated and confirmed by PCR analysis of genomic DNA using gene-specific primers and a T-DNA left-border primer. Lines represent introns or promoters, whereas dark and light grey boxes represent exons and untranslated regions, respectively. Arrows represent primers used for genotyping (see Supplementary Table 4). c, RT–PCR analysis of CPK transcripts in cpk mutants. TUB4 is the housekeeping control gene. d, Analyses of nitrate-responsive marker gene expression in cpk mutants. Seedlings (7-day-old) were induced by 0.5 mM KCl or KNO3 for 15 min. Relative expression of nitrate-responsive marker genes was analysed by RT–qPCR and normalized to the expression of UBQ10. The expression level is calculated relative to the value of wild-type seedlings treated with KCl. Error bars, s.d., n = 3 biological replicates. e, Single and double cpk mutants lack an overt phenotype. Plants were germinated and grown on the ammonium succinate medium for 3 days and then transferred to basal medium plates supplemented with 5 mM KNO3 for 6 days. To analyse the chemical analogue-sensitive mutants, wild-type and icpk10,30 seedlings were transferred to basal medium plates supplemented with 5 mM KNO3 and 1 μM 3MBiP for 6 days, and 3MBiP was reapplied every 2 days after transfer. Scale bar, 1 cm. Images are representative of 10 seedlings. f, g, The average fresh weight of 9-day-old single and double cpk mutants. Error bars, s.d., n = 12 seedlings. h, The average fresh weight of 9-day-old double cpk mutants and icpk supplemented with or without 3MBiP. Error bars, s.d., n = 12 seedlings. i, j, Primary nitrate-responsive gene expression is reduced in cpk double mutants. RT–qPCR analyses with 7-day-old seedlings. 0.5 mM KNO3, 15 min. Error bars, s.d., n = 3 seedlings. *P < 0.05, **P < 0.0001 (two-way ANOVA with Tukey’s multiple comparisons test). Source Data for d, i and j can be found in the Supplementary Information.

Source data

Extended Data Figure 4 RNA-seq and qRT–PCR data analyses and functional classification.

Biological triplicate RNA-seq experiments were performed and analysed with DESeq2. a, Nitrate–CPK-downregulated genes. Dark grey, nitrate–CPK target genes (q ≤ 0.05). b, Classification of nitrate–CPK-downregulated genes. The MapMan functional categories for nitrate-downregulated genes are presented. c, Enriched functional categories of nitrate-upregulated genes. d, Enriched functional categories of nitrate-downregulated genes. The fold enrichment is calculated as follows: (number of classified_input_set/number of total_input_set)/(number of classified_reference_set/number of total_reference_set). The P value is calculated in Excel using a hypergeometric distribution test. The categories were sorted by fold enrichment with a P ≤ 0.05 cut-off. e, Nitrate–CPK target genes regulate nitrogen transport and metabolism. f, RT–qPCR analyses of nitrate–CPK target genes in eight functional classes in seedlings. 10 mM KNO3, 15 min. Error bars, s.d., n = 3 biological replicates. NIA1/2, NIR, NRT2.1/2.2, G6PD2/3, GLN, GDH3, UPM1, FD3 and FNR1/2 genes were regulated by the CPK10, CPK30 and CPK32 protein kinases1,3,4,5,12,13,16,28. Transcription factor genes NLP3, HRS1 and TGA4 were primary nitrate–CPK target genes3,12,13,15,20. g, The fold changes of expression levels of nitrate-upregulated genes in wild-type and icpk seedlings listed in f. The table provided the Source Data for the histograms presented in f. n = 3 biological replicates.

Extended Data Figure 5 Primary nitrate-upregulated genes are present in diverse experimental systems.

Venn diagrams (http://www.cmbi.ru.nl/cdd/biovenn/) were used to present the comparison and overlaps between the list of primary nitrate-upregulated genes defined in this study and the nitrate-upregulated genes at 20 min defined by previously published gene sets4,10,13. Red, 394 nitrate-upregulated genes identified in this study with a log2 ≥ 1 and q ≤ 0.05 cut-off; light red, 992 nitrate-upregulated genes in this study with a q ≤ 0.05 cut-off; dark blue, 338 nitrate-upregulated genes from ref. 4 with a log2 ≥ 1 cut-off for both biological duplicate datasets; green, 366 nitrate-upregulated genes from the ref. 10 dataset with a log2 ≥ 1 cut-off; light blue, 227 nitrate-upregulated genes from ref. 13 with a log2 ≥ 1 cut-off. Gene numbers in each group and the percentage of overlapped nitrate-upregulated genes in previously published datasets are shown.

Extended Data Figure 6 Quantitative analyses of root growth phenotype in wild-type and icpk seedlings in response to ammonium and nitrate.

a, The icpk mutant displays defects in nitrate-stimulated lateral root establishment. Wild-type and icpk mutant seedlings were germinated and grown on ammonium succinate medium for 3 days, and then transferred to a plate supplemented with 5 mM KNO3, 2.5 mM ammonium succinate, 5 mM KCl or 5 mM glutamine in the presence of 1 μM 3MBiP for 5 days. Scale bars, 1 cm. Images are representative of 6 seedlings. b, Primary root (PR) length was similar in 8-day-old wild-type and icpk seedlings. Error bars, s.d., n = 16 seedlings. c, Lateral root primordium (LRP) density decreased significantly in 8-day-old icpk seedlings in response to nitrate. Error bars, s.d., n = 15 seedlings. *P < 0.05 (Student’s t-test). d, Lateral root (LR) length was markedly reduced in icpk seedlings in the presence of nitrate. Error bars, s.d., n = 10 seedlings. *P < 0.05 (Student’s t-test). e, The development of lateral roots is severely retarded in icpk. The developmental stages of the third lateral root in 6-day-old wild-type and icpk seedlings induced by nitrate for 3 days are shown. Scale bars, 100 μm. Images are representative of 6 seedlings. f, Time-course analyses of icpk defects in nitrate-specific lateral root development stages I–VII41. Em, emerged primordia. Error bars, s.e.m., n = 16 seedlings. g, Chi-square test of wild-type and icpk lateral root development. Wild-type and icpk seedlings were compared on two categories, early lateral root development stages before emergence (stage I–VII) and afterwards (Em + LR). The low P value indicates the high level of association between the genotype and development stages.

Extended Data Figure 7 Nitrate-induced NLP phosphorylation.

a, Nitrate-induced mobility shift of MYC-tagged NLP6. Transgenic seedlings expressing MYC-tagged NLP6 were grown in liquid medium containing 0.5 mM ammonium succinate as a sole nitrogen source for 4 days and then treated with 10 mM KCl or KNO3 for indicated periods. Immunoblot analysis was carried out with proteins extracted from the seedlings using anti-MYC and anti-histone H3 (HIS) antibodies. b, Effect of alkaline phosphatase treatment on mobility shift of MYC-tagged NLP6. Proteins from seedlings treated with 10 mM KNO3 for 0 or 30 min were subjected to CIP treatment. The experiments were repeated twice with consistent results. c, An antagonist of Ca2+ sensors (W7) diminished nitrate-triggered phosphorylation of NLP7. d, Phosphorylation of histone by CPK10, CPK30 and CPK32 is Ca2+-dependent. e, Alignment of the amino acid sequences around the conserved CPK phosphorylation site in all NLPs from A. thaliana and L. japonicus (Lj). Conserved amino acid residues are indicated by black boxes. The CPK phosphorylation motif indicated by an underline was identified by multiple web-based bioinformatics tools and literature analysis61,62,63,64,65,66 with a candidate serine (Ser205 in NLP7) that is uniquely conserved in nine Arabidopsis NLPs and four orthologous L. japonicus NLPs (outlined in red), but not in L. japonicus NIN. LjNIN, a variant of NLP, which evolved specifically for symbiotic nitrogen fixation in legumes, lacks a CPK phosphorylation site. f, Ser205 in NLP7 is the phosphorylation site for CPK10, CPK30 and CPK32. g, Nitrate-induced mobility shift was abolished for NLP7(S205A). h, Overexpression of NLP7 and CPK10ac showed similar synergism with nitrate for NIR-LUC activation in protoplast transient assays. NLP7 or CPK10ac alone was not effective to enhance NIR-LUC expression without nitrate. CPK10(KM) lacking kinase activity and NLP7(S205A) lacking the CPK10, CPK30 and CPK32 phosphorylation site served as negative controls. NLP7 or CPK10ac protein expression was detected by immunoblot analyses before dividing protoplasts equally, and treated with 0.5 mM KCl or KNO3 for 2 h. UBQ10-GUS is a transient expression control. Error bars, s.d., n = 5 biological replicates.

Extended Data Figure 8 The CPK phosphorylation residue Ser205 is required for NLP7 nuclear retention triggered by nitrate at the plant root tip.

a, CPK–GFPs are not processed in response to nitrate. Proteins from CPK–GFP-transfected protoplasts were analysed by immunoblots with an anti-GFP antibody. b, Confocal image of NLP7–GFP or NLP7(S205A)–GFP in transgenic nlp7-1 complementation plants in response to nitrate. GFP images recorded at 0 or 8 min after 10 mM KNO3 induction are shown. Scale bars, 100 μm. The experiments were repeated 3 times with 10 seedlings for each line with consistent results.

Extended Data Figure 9 Nitrate enhancement of proliferation in the lateral root primordia.

a, b, Ser205 is crucial for NLP7-mediated transcriptional activation of target genes with diverse functions. Genome-wide transcriptional profiling by RNA-seq was performed with mesophyll protoplasts expressing NLP7–HA or NLP7(S205A)–HA or the control plasmid for 4.5 h. Red, NLP7 target genes identified by both ChIP–chip13 and DNA affinity purification sequencing (DAP-seq)67; black, ChIP–chip only; grey, DAP-seq only. c, Normalized HTSeq read counts of NLP7-activated genes (listed in a and b) from RNA-seq experiments. Normalized read counts of NLP7-activated genes calculated as the original HTseq counts divided by the normalization factors were extracted after DESeq2 analysis.

Extended Data Figure 10 Complementation analyses of nlp7-1 with NLP7–GFP or NLP7(S205A)–GFP in transgenic Arabidopsis plants.

a, The shoot fresh weight of wild-type, nlp7-1, NLP7-GFP/nlp7-1 and NLP7(S205A)–GFP/nlp7-1 shoots. Error bars, s.e.m., n = 10 seedlings. **P ≤ 0.0001 versus wild-type control (one-way ANOVA with Tukey’s multiple comparisons test). Plants were grown on 25 mM KNO3 medium for 21 days. Data from three independent complement lines are presented. b, The root fresh weight of 11-day-old wild-type, nlp7-1, NLP7–GFP/nlp7-1 and NLP7(S205A)–GFP/nlp7-1. Seedlings were germinated on the ammonium succinate medium for 3 days and then transferred to the plates supplemented with 5 mM KNO3 for 8 days. Error bars, s.e.m., n = 10 seedlings. **P ≤ 0.0001 versus wild-type control (one-way ANOVA with Tukey’s multiple comparisons test).

Supplementary information

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Nitrate stimulates dynamic Ca2+ increase in mesophyll protoplasts expressing GCaMP6

Mesophyll protoplasts expressing GCaMP6 were treated with 10 mM KNO3. The time-lapse video shows the representative GFP fluorescence signal changes in response to nitrate treatment using the Leica DM5000B microscope. The images are representative of 10 protoplasts. The images were acquired every two sec for 6 min and then made into a stack and converted to a video (7 frames/sec, 1 sec in the video equals to 12 sec in the recording). (AVI 3506 kb)

Nitrate stimulates dynamic Ca2+ increase in transgenic cotyledons expressing GCaMP6

The cotyledon of a GCaMP6 transgenic seedling was treated with 10 mM KNO3. The time-lapse video shows the representative GFP fluorescence signal changes in response to nitrate treatment using the Leica TCS NT SP1confocal microscope. The images are representative of 10 seedlings. The images were acquired every 10 sec for 8 min and then made into a stack and converted to a video (7 frames/sec, 1 sec in the video equals to 1 min in the recording). (AVI 4013 kb)

Nitrate stimulates dynamic Ca2+ increase in the transgenic root tip expressing GCaMP6

The root tip of a GCaMP6 transgenic seedling was treated with 10 mM KNO3. The time-lapse video shows the representative GFP fluorescence signal changes in response to nitrate treatment using the Leica TCS NT SP1confocal microscope. The images are representative of 10 seedlings. The images were acquired every 10 sec for 5 min and then made into a stack and converted to a video (7 frames/sec, 1 sec in the video equals to 1 minute in the recording). (AVI 346 kb)

Nitrate stimulates dynamic Ca2+ increase in the transgenic root elongated region expressing GCaMP6

The root elongated region of a GCaMP6 transgenic seedling was treated with 10 mM KNO3. The time-lapse video shows the representative GFP fluorescence signal changes in response to nitrate treatment using the Leica TCS NT SP1 confocal microscope. The images are representative of 10 seedlings. The images were acquired every 10 sec for 5 min and then made into a stack and converted to a video (7 frames/sec, 1 sec in the video equals to 1 minute in the recording). (AVI 1498 kb)

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Liu, Kh., Niu, Y., Konishi, M. et al. Discovery of nitrate–CPK–NLP signalling in central nutrient–growth networks. Nature 545, 311–316 (2017). https://doi.org/10.1038/nature22077

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