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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Gene Expression Omnibus
Nunes-Nesi, A., Fernie, A. R. & Stitt, M. Metabolic and signaling aspects underpinning the regulation of plant carbon nitrogen interactions. Mol. Plant 3, 973–996 (2010)
Kiba, T. & Krapp, A. Plant nitrogen acquisition under low availability: regulation of uptake and root architecture. Plant Cell Physiol. 57, 707–714 (2016)
Konishi, M. & Yanagisawa, S. Emergence of a new step towards understanding the molecular mechanisms underlying nitrate-regulated gene expression. J. Exp. Bot. 65, 5589–5600 (2014)
Wang, R., Okamoto, M., Xing, X. & Crawford, N. M. Microarray analysis of the nitrate response in Arabidopsis roots and shoots reveals over 1,000 rapidly responding genes and new linkages to glucose, trehalose-6-phosphate, iron, and sulfate metabolism. Plant Physiol. 132, 556–567 (2003)
Scheible, W. R. et al. Genome-wide reprogramming of primary and secondary metabolism, protein synthesis, cellular growth processes, and the regulatory infrastructure of Arabidopsis in response to nitrogen. Plant Physiol. 136, 2483–2499 (2004)
Bouguyon, E. et al. Multiple mechanisms of nitrate sensing by Arabidopsis nitrate transceptor NRT1.1. Nature Plants 1, 15015 (2015)
Wang, R., Xing, X., Wang, Y., Tran, A. & Crawford, N. M. A genetic screen for nitrate regulatory mutants captures the nitrate transporter gene NRT1.1. Plant Physiol. 151, 472–478 (2009)
Wang, R. et al. Genomic analysis of the nitrate response using a nitrate reductase-null mutant of Arabidopsis. Plant Physiol. 136, 2512–2522 (2004)
Castaings, L. et al. The nodule inception-like protein 7 modulates nitrate sensing and metabolism in Arabidopsis. Plant J. 57, 426–435 (2009)
Krouk, G., Mirowski, P., LeCun, Y., Shasha, D. E. & Coruzzi, G. M. Predictive network modeling of the high-resolution dynamic plant transcriptome in response to nitrate. Genome Biol. 11, R123 (2010)
Liu, K. H., McCormack, M. & Sheen, J. Targeted parallel sequencing of large genetically-defined genomic regions for identifying mutations in Arabidopsis. Plant Methods 8, 12 (2012)
Konishi, M. & Yanagisawa, S. Arabidopsis NIN-like transcription factors have a central role in nitrate signalling. Nat. Commun. 4, 1617 (2013)
Marchive, C. et al. Nuclear retention of the transcription factor NLP7 orchestrates the early response to nitrate in plants. Nat. Commun. 4, 1713 (2013)
Guan, P. et al. Nitrate foraging by Arabidopsis roots is mediated by the transcription factor TCP20 through the systemic signaling pathway. Proc. Natl Acad. Sci. USA 111, 15267–15272 (2014)
Alvarez, J. M. et al. Systems approach identifies TGA1 and TGA4 transcription factors as important regulatory components of the nitrate response of Arabidopsis thaliana roots. Plant J. 80, 1–13 (2014)
Obertello, M., Shrivastava, S., Katari, M. S. & Coruzzi, G. M. Cross-species network analysis uncovers conserved nitrogen-regulated network modules in rice. Plant Physiol. 168, 1830–1843 (2015)
Hu, H. C., Wang, Y. Y. & Tsay, Y. F. AtCIPK8, a CBL-interacting protein kinase, regulates the low-affinity phase of the primary nitrate response. Plant J. 57, 264–278 (2009)
Ho, C. H., Lin, S. H., Hu, H. C. & Tsay, Y. F. CHL1 functions as a nitrate sensor in plants. Cell 138, 1184–1194 (2009)
Léran, S. et al. Nitrate sensing and uptake in Arabidopsis are enhanced by ABI2, a phosphatase inactivated by the stress hormone abscisic acid. Sci. Signal. 8, ra43 (2015)
Medici, A. et al. AtNIGT1/HRS1 integrates nitrate and phosphate signals at the Arabidopsis root tip. Nat. Commun. 6, 6274 (2015)
Vidal, E. A., Álvarez, J. M., Moyano, T. C. & Gutiérrez, R. A. Transcriptional networks in the nitrate response of Arabidopsis thaliana. Curr. Opin. Plant Biol. 27, 125–132 (2015)
Knight, H., Trewavas, A. J. & Knight, M. R. Cold calcium signaling in Arabidopsis involves two cellular pools and a change in calcium signature after acclimation. Plant Cell 8, 489–503 (1996)
Boudsocq, M. et al. Differential innate immune signalling via Ca2+ sensor protein kinases. Nature 464, 418–422 (2010)
Boudsocq, M. & Sheen, J. CDPKs in immune and stress signaling. Trends Plant Sci. 18, 30–40 (2013)
Reddy, A. S., Ali, G. S., Celesnik, H. & Day, I. S. Coping with stresses: roles of calcium- and calcium/calmodulin-regulated gene expression. Plant Cell 23, 2010–2032 (2011)
Simeunovic, A., Mair, A., Wurzinger, B. & Teige, M. Know where your clients are: subcellular localization and targets of calcium-dependent protein kinases. J. Exp. Bot. 67, 3855–3872 (2016)
Ebert, D. H. & Greenberg, M. E. Activity-dependent neuronal signalling and autism spectrum disorder. Nature 493, 327–337 (2013)
Sakakibara, H., Kobayashi, K., Deji, A. & Sugiyama, T. Partial characterization of the signaling pathway for the nitrate-dependent expression of genes for nitrogen-assimilatory enzymes using detached maize leaves. Plant Cell Physiol. 38, 837–843 (1997)
Riveras, E. et al. The calcium ion is a second messenger in the nitrate signaling pathway of Arabidopsis. Plant Physiol. 169, 1397–1404 (2015)
Forde, B. G. Glutamate signalling in roots. J. Exp. Bot. 65, 779–787 (2014)
Giehl, R. F., Gruber, B. D. & von Wirén, N. It’s time to make changes: modulation of root system architecture by nutrient signals. J. Exp. Bot. 65, 769–778 (2014)
Xiong, Y. et al. Glucose-TOR signalling reprograms the transcriptome and activates meristems. Nature 496, 181–186 (2013)
Chen, T. W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013)
Yuan, F. et al. OSCA1 mediates osmotic-stress-evoked Ca2+ increases vital for osmosensing in Arabidopsis. Nature 514, 367–371 (2014)
Charpentier, M. et al. Nuclear-localized cyclic nucleotide-gated channels mediate symbiotic calcium oscillations. Science 352, 1102–1105 (2016)
Brandt, B. et al. Calcium specificity signaling mechanisms in abscisic acid signal transduction in Arabidopsis guard cells. eLife 4, e03599 (2015)
Gan, Y., Bernreiter, A., Filleur, S., Abram, B. & Forde, B. G. Overexpressing the ANR1 MADS-box gene in transgenic plants provides new insights into its role in the nitrate regulation of root development. Plant Cell Physiol. 53, 1003–1016 (2012)
Liu, Y. et al. Structural basis for selective inhibition of Src family kinases by PP1. Chem. Biol. 6, 671–678 (1999)
Zhang, C. et al. Structure-guided inhibitor design expands the scope of analog-sensitive kinase technology. ACS Chem. Biol. 8, 1931–1938 (2013)
Kiba, T., Takei, K., Kojima, M. & Sakakibara, H. Side-chain modification of cytokinins controls shoot growth in Arabidopsis. Dev. Cell 27, 452–461 (2013)
Malamy, J. E. & Benfey, P. N. Organization and cell differentiation in lateral roots of Arabidopsis thaliana. Development 124, 33–44 (1997)
Sheen, J. Master regulators in plant glucose signaling networks. J. Plant Biol. 57, 67–79 (2014)
Efeyan, A., Comb, W. C. & Sabatini, D. M. Nutrient-sensing mechanisms and pathways. Nature 517, 302–310 (2015)
Rebsamen, M. et al. SLC38A9 is a component of the lysosomal amino acid sensing machinery that controls mTORC1. Nature 519, 477–481 (2015)
Yoo, S. D., Cho, Y. H. & Sheen, J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat. Protocols 2, 1565–1572 (2007)
Xiang, C., Han, P., Lutziger, I., Wang, K. & Oliver, D. J. A mini binary vector series for plant transformation. Plant Mol. Biol. 40, 711–717 (1999)
Clough, S. J. & Bent, A. F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998)
Guo, J. et al. Involvement of Arabidopsis RACK1 in protein translation and its regulation by abscisic acid. Plant Physiol. 155, 370–383 (2011)
Kato, Y., Konishi, M., Shigyo, M., Yoneyama, T. & Yanagisawa, S. Characterization of plant eukaryotic translation initiation factor 6 (eIF6) genes: The essential role in embryogenesis and their differential expression in Arabidopsis and rice. Biochem. Biophys. Res. Commun. 397, 673–678 (2010)
Alonso, J. M. et al. Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301, 653–657 (2003)
Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013)
Anders, S., Pyl, P. T. & Huber, W. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015)
Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014)
Van Verk, M. C., Hickman, R., Pieterse, C. M. & Van Wees, S. C. RNA-Seq: revelation of the messengers. Trends Plant Sci. 18, 175–179 (2013)
de Hoon, M. J., Imoto, S., Nolan, J. & Miyano, S. Open source clustering software. Bioinformatics 20, 1453–1454 (2004)
Saldanha, A. J. Java Treeview–extensible visualization of microarray data. Bioinformatics 20, 3246–3248 (2004)
Axelos, M., Curic, C., Mazzolini, L., Bardet, C. & Lescure, N. A protocol for transient gene expression in Arabidopsis thaliana protoplasts isolated from cell suspension cultures. Plant Physiol. Biochem. 30, 123–128 (1992)
Aki, T., Shigyo, M., Nakano, R., Yoneyama, T. & Yanagisawa, S. Nano scale proteomics revealed the presence of regulatory proteins including three FT-Like proteins in phloem and xylem saps from rice. Plant Cell Physiol. 49, 767–790 (2008)
Aki, T. & Yanagisawa, S. Application of rice nuclear proteome analysis to the identification of evolutionarily conserved and glucose-responsive nuclear proteins. J. Proteome Res. 8, 3912–3924 (2009)
Baena-González, E., Rolland, F., Thevelein, J. M. & Sheen, J. A central integrator of transcription networks in plant stress and energy signalling. Nature 448, 938–942 (2007)
Cheng, S. H., Willmann, M. R., Chen, H. C. & Sheen, J. Calcium signaling through protein kinases. The Arabidopsis calcium-dependent protein kinase gene family. Plant Physiol. 129, 469–485 (2002)
Curran, A. et al. Calcium-dependent protein kinases from Arabidopsis show substrate specificity differences in an analysis of 103 substrates. Front. Plant Sci. 2, 36 (2011)
Song, C. et al. Systematic analysis of protein phosphorylation networks from phosphoproteomic data. Mol. Cell. Proteomics 11, 1070–1083 (2012)
Yaffe, M. B. et al. A motif-based profile scanning approach for genome-wide prediction of signaling pathways. Nat. Biotechnol. 19, 348–353 (2001)
Soyano, T., Shimoda, Y. & Hayashi, M. NODULE INCEPTION antagonistically regulates gene expression with nitrate in Lotus japonicus. Plant Cell Physiol. 56, 368–376 (2015)
Suzuki, W., Konishi, M. & Yanagisawa, S. The evolutionary events necessary for the emergence of symbiotic nitrogen fixation in legumes may involve a loss of nitrate responsiveness of the NIN transcription factor. Plant Signal. Behav. 8, e25975 (2013)
O’Malley, R. C. et al. Cistrome and epicistrome features shape the regulatory DNA landscape. Cell 165, 1280–1292 (2016)
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.
The authors declare no competing financial interests.
Reviewer Information Nature thanks H. Sakakibara 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 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.
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, d–f can be found in the Supplementary Information.
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.
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.
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.
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).
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This file contains Supplementary Table 2 (XLSX 83 kb)
This file contains Tables 3-6. (PDF 229 kb)
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)
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)
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)
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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