Abstract
Nitrate (NO3−) is the primary source of nitrogen preferred by most arable crops, including wheat. The pioneering experiment on primary nitrate response (PNR) was carried out three decades ago. Since then, much research has been carried out to understand the NO3− signaling. Nitrate is sensed by the dual affinity NO3− transceptor NPF6.3, which further relays the information to a master regulator NIN-like protein 7 (NLP7) through calcium-dependent protein kinases (CPK10, CPK30, CPK32), highlighting the importance of calcium ion (Ca2+) as one of the important secondary messengers in relaying the NO3− signaling in Arabidopsis. In a previous study, we found that Ca2+ regulates nitrogen starvation response in wheat. In this study, 10 days old NO3−-starved wheat seedlings were exposed to various treatments. Our study on time course changes in expression of PNR sentinel genes; NPF6.1, NPF6.2, NRT2.1, NRT2.3, NR, and NIR in wheat manifest the highest level of expression at 30 min after NO3− exposure. The use of Ca2+ chelator EGTA confirmed the involvement of Ca2+ in the regulation of transcription of NPFs and NRTs as well the NO3− uptake. We also observed the NO3− dose-dependent and tissue-specific regulation of nitrate reductase activity involving Ca2+ as a mediator. The participation of Ca2+ in the PNR and NO3− signaling in wheat is confirmed by pharmacological analysis, physiological evidences, and protoplast-based Ca2+ localization.
Similar content being viewed by others
Data availability
All data generated or analysed during this study are included in this published article [and its supplementary information files].
References
Adavi SB, Sathee L (2019) Elevated CO2-induced production of nitric oxide differentially modulates nitrate assimilation and root growth of wheat seedlings in a nitrate dose-dependent manner. Protoplasma 256:147–159
Adavi SB, Sathee L (2021) Elevated CO2 differentially regulates root nitrate transporter kinetics in a genotype and nitrate dose-dependent manner. Plant Sci 305:110807
Adavi SB, Sathee L (2021) Influence of calcium on nitrate starvation response of bread wheat. Plant Physiol Reports. https://doi.org/10.1007/s40502-021-00626-9
Adavi SB, Sathee L (2021) Elevated CO2 alters tissue balance of nitrogen metabolism and downregulates nitrogen assimilation and signalling gene expression in wheat seedlings receiving high nitrate supply. Protoplasma 258:219–233
Bender KW, Zielinski RE, Huber SC (2018) Revisiting paradigms of Ca2+ signaling protein kinase regulation in plants. Biochem J 475:207–223
Brooks MD, Cirrone J, Pasquino AV, Alvarez JM, Swift J, Mittal S, Juang C-L, Varala K, Gutiérrez RA, Krouk G et al (2019) Network walking charts transcriptional dynamics of nitrogen signaling by integrating validated and predicted genome-wide interactions. Nat Commun 10:1–13
Buchner P, Hawkesford MJ (2014) Complex phylogeny and gene expression patterns of members of the NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family (NPF) in wheat. J Exp Bot 65(19):5697–5710
CH Ho Slhhyt (2009) CHL1 functions as a nitrate sensor in plants. Cell 138:1184–1194
Crawford NM, Forde BG (2002) Molecular and developmental biology of inorganic nitrogen nutrition. Arab Book Am Soc Plant Biol 11:1–25
Davidson EA (2009) The contribution of manure and fertilizer nitrogen to atmospheric nitrous oxide since 1860. Nat Geosci 2:659–662
Deng M, Moureaux T, Caboche M (1989) Tungstate, a molybdate analog inactivating nitrate reductase, deregulates the expression of the nitrate reductase structural gene. Plant Physiol 91:304–309
Douglas P, Pigaglio E, Ferrer A, Halford NG, MacKintosh C (1997) Three spinach leaf nitrate reductase-3-hydroxy-3-methylglutaryl-CoA reductase kinases that are regulated by reversible phosphorylation and/or Ca2+ ions. Biochem J 325:101–109
Downes MT (1978) An improved hydrazine reduction method for the automated determination of low nitrate levels in freshwater. Water Res 12:673–675
Dubey RS, Srivastava RK, Pessarakli M (2021) Physiological mechanisms of nitrogen absorption and assimilation in plants under stressful conditions. Handbook of plant and crop physiology. CRC Press, pp. 579–616
Engelsberger WR, Schulze WX (2012) Nitrate and ammonium lead to distinct global dynamic phosphorylation patterns when resupplied to nitrogen-starved Arabidopsis seedlings. Plant J 69:978–995
Evans HJ, Nason A (1953) Pyridine nucleotide-nitrate reductase from extracts of higher plants. Plant Physiol 28:233–254
Girin T, Lejay L, Wirth J, Widiez T, Palenchar PM, Nazoa P, Touraine B, Gojon A, Lepetit M (2007) Identification of a 150 bp cis-acting element of the AtNRT2.1 promoter involved in the regulation of gene expression by the N and C status of the plant. Plant, Cell Environ 30:1366–1380
Gowri G, Kenis JD, Ingemarsson B, Redinbaugh MG, Campbell WH (1992) nitrate reductase transcript is expressed in the primary response of maize to environmental nitrate. Plant Mol Biol 18:55–64
Gutiérrez RA, Gifford ML, Poultney C, Wang R, Shasha DE, Coruzzi GM, Crawford NM (2007) Insights into the genomic nitrate response using genetics and the Sungear Software System. J Exp Bot 58:2359–2367
Herschman HR (1991) Primary response genes induced by growth factors and tumor promoters. Annu Rev Biochem 60:281–319
Hirel B, Tétu T, Lea PJ, Dubois F (2011) Improving nitrogen use efficiency in crops for sustainable agriculture. Sustainability 3:1452–1485
Hu H-C, Wang Y-Y, Tsay Y-F (2009) AtCIPK8, a CBL-interacting protein kinase, regulates the low-affinity phase of the primary nitrate response. Plant J 57:264–278
Jia X, Zhang X, Qu J, Han R (2016) Optimization conditions of wheat mesophyll protoplast isolation. Agric Sci 7(12):850–858
Kaiser WM, Weiner H, Huber SC (1999) Nitrate reductase in higher plants: a case study for transduction of environmental stimuli into control of catalytic activity. Physiol Plant 105:384–389
Klepper L, Flesher D, Hageman RH (1971) Generation of reduced nicotinamide adenine dinucleotide for nitrate reduction in green leaves. Plant Physiol 48:580–590
Konishi M, Yanagisawa S (2013) Arabidopsis NIN-like transcription factors have a central role in nitrate signalling. Nat Commun 4:1–9
Krapp A, Fraisier V, Scheible W-R, Quesada A, Gojon A, Stitt M, Caboche M, Daniel-Vedele F (1998) Expression studies of Nrt 2: 1Np, a putative high-affinity nitrate transporter: evidence for its role in nitrate uptake. Plant J 14:723–731
Krouk G (2017) Nitrate signalling: calcium bridges the nitrate gap. Nat Plants 3:1–2
Krouk G, Crawford NM, Coruzzi GM, Tsay Y-F (2010a) Nitrate signaling: adaptation to fluctuating environments. Curr Opin Plant Biol 13:265–272
Krouk G, Lacombe B, Bielach A, Perrine-Walker F, Malinska K, Mounier E, Hoyerova K, Tillard P, Leon S, Ljung K, Zazimalova E, Benkova E, Nacry P, Gojon A (2010b) Nitrate-regulated auxin transport by NRT1.1 defines a mechanism for nutrient sensing in plants. Dev Cell 18:927–937
Krouk G, Mirowski P, LeCun Y, Shasha DE, Coruzzi GM (2010c) Predictive network modeling of the high-resolution dynamic plant transcriptome in response to nitrate. Genome Biol 11(12):R123. https://doi.org/10.1186/gb-2010-11-12-r123
Lambeck IC, Fischer-Schrader K, Niks D, Roeper J, Chi J-C, Hille R, Schwarz G (2012) Molecular mechanism of 14-3-3 protein-mediated inhibition of plant nitrate reductase. J Biol Chem 287:4562–4571
Lejay L, Tillard P, Lepetit M, Olive FD, Filleur S, Daniel-Vedele F, Gojon A (1999) Molecular and functional regulation of two NO3–uptake systems by N-and C-status of Arabidopsis plants. Plant J 18:509–519
Liu K, Niu Y, Konishi M, Wu Y, Du H, Sun Chung H, Li L, Boudsocq M, McCormack M, Maekawa S et al (2017a) Discovery of nitrate–CPK–NLP signalling in central nutrient–growth networks. Nature 545:311–316
Liu KH, Niu Y, Konishi M, Wu Y, Du H, Sun Chung H, Li L, Boudsocq M, McCormack M, Maekawa S, Ishida T, Zhang C, Shokat K, Yanagisawa S, Sheen J (2017b) Discovery of nitrate-CPK-NLP signalling in central nutrient-growth networks. Nature 545:311–316
Liu KH, Diener A, Lin Z, Liu C, Sheen J (2020) Primary nitrate responses mediated by calcium signalling and diverse protein phosphorylation. J Exp Bot 71:4428–4441
Liu KH, Liu M, Lin Z, Wang ZF, Chen B, Liu C, Guo A, Konishi M, Yanagisawa S, Wagner G, Sheen J (2022) NIN-like protein 7 transcription factor is a plant nitrate sensor. Science 377(6613):1419–1425
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2- $Δ$$Δ$CT method. Methods 25:402–408
Marchive C, Roudier F, Castaings L, Bréhaut V, Blondet E, Colot V, Meyer C, Krapp A (2013) Nuclear retention of the transcription factor NLP7 orchestrates the early response to nitrate in plants. Nat Commun 4:1–9
Medici A, Krouk G (2014) The primary nitrate response: a multifaceted signalling pathway. J Exp Bot 65:5567–5576
Migocka M, Warzybok A, Papierniak A, Kłobus G (2013) NO3−/H+ antiport in the tonoplast of cucumber root cells is stimulated by nitrate supply: evidence for a reversible nitrate-induced phosphorylation of vacuolar NO3−/H+ antiport. PLoS ONE 8:e73972
Mu X, Luo J (2019) Evolutionary analyses of NIN-like proteins in plants and their roles in nitrate signaling. Cell Mol Life Sci 76:3753–3764
Muños S, Cazettes C, Fizames C, Gaymard F, Tillard P, Lepetit M, Lejay L, Gojon A (2004) Transcript profiling in the chl1-5 mutant of arabidopsis reveals a role of the nitrate transporter NRT1.1 in the regulation of another nitrate transporter, NRT2.1 W inside a box sign. Plant Cell 16:2433–2447
Nair TVR, Abrol YP (1973) Nitrate reductase activity in developing wheat ears. Experientia 29:1480–1481
Owen AG, Jones DL (2001) Competition for amino acids between wheat roots and rhizosphere microorganisms and the role of amino acids in plant N acquisition. Soil Biol Biochem 33:651–657
Pouteau S, Cherel I, Vaucheret H, Caboche M (1989) Nitrate reductase mRNA regulation in Nicotiana plumbaginifolia nitrate reductase-deficient mutants. Plant Cell 1:1111–1120
Riveras E, Alvarez JM, Vidal EA, Oses C, Vega A, Gutiérrez RA (2015) The calcium ion is a second messenger in the nitrate signaling pathway of Arabidopsis. Plant Physiol 169:1397–1404
Rubin G, Tohge T, Matsuda F, Saito K, Scheible W-R (2009) Members of the LBD family of transcription factors repress anthocyanin synthesis and affect additional nitrogen responses in Arabidopsis. Plant Cell 21:3567–3584
Ruffel S, Krouk G, Ristova D, Shasha D, Birnbaum KD, Coruzzi GM (2011) Nitrogen economics of root foraging: transitive closure of the nitrate–cytokinin relay and distinct systemic signaling for N supply vs. demand. Proc Natl Acad Sci 108:18524–18529
Sakakibara H, Kobayashi K, Deji A, Sugiyama T (1997) 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
Sander L, Jensen PE, Back LF, Stummann BM, Henningsen KW (1995) Structure and expression of a nitrite reductase gene from bean (Phaseolus vulgaris) and promoter analysis in transgenic tobacco. Plant Mol Biol 27:165–177
Sane PV, Kumar N, Baijal M, Singh KK, Kochhar VK (1987) Activation of nitrate reductase by calcium and calmodulin. Phytochemistry 26:1289–1291
Sathee L, Krishna GK, Adavi SB, Jha SK, Jain V (2021) Role of protein phosphatases in the regulation of nitrogen nutrition in plants. Physiol Mol Biol Plants 27:1–12
Sato T, Maekawa S, Konishi M, Yoshioka N, Sasaki Y, Maeda H, Ishida T, Kato Y, Yamaguchi J, Yanagisawa S (2017) Direct transcriptional activation of BT genes by NLP transcription factors is a key component of the nitrate response in Arabidopsis. Biochem Biophys Res Commun 483:380–386
Stitt M (1999) Nitrate regulation of metabolism and growth. Curr Opin Plant Biol 2:178–186
Sueyoshi K, Mitsuyama T, Sugimoto T, Kleinhofs A, Warner RL, Oji Y (1999) Effects of inhibitors for signaling components on the expression of the genes for nitrate reductase and nitrite reductase in excised barley leaves. Soil Sci Plant Nutr 45:1015–1019
Vidal EA, Moyano TC, Canales J, Gutiérrez RA (2014) Nitrogen control of developmental phase transitions in Arabidopsis thaliana. J Exp Bot 65:5611–5618
Vidmar JJ, Zhuo D, Siddiqi MY, Schjoerring JK, Touraine B, Glass ADM (2000) Regulation of high-affinity nitrate transporter genes and high-affinity nitrate influx by nitrogen pools in roots of barley. Plant Physiol 123:307–318
Walch-Liu PIA, Ivanov II, Filleur S, Gan Y, Remans T, Forde BG (2006) Nitrogen regulation of root branching. Ann Bot 97:875–881
Wang Y-H, Garvin DF, Kochian LV (2001) Nitrate-induced genes in tomato roots. Array analysis reveals novel genes that may play a role in nitrogen nutrition. Plant Physiol 127:345–359
Wang R, Okamoto M, Xing X, Crawford NM (2003) 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
Wang R, Tischner R, Gutiérrez RA, Hoffman M, Xing X, Chen M, Coruzzi G, Crawford NM (2004) Genomic analysis of the nitrate response using a nitrate reductase-null mutant of arabidopsis. Plant Physiol 136:2512–2522
Wang Y-Y, Hsu P-K, Tsay Y-F (2012) Uptake, allocation and signaling of nitrate. Trends Plant Sci 17:458–467
Wang Y-Y, Cheng Y-H, Chen K-E, Tsay Y-F (2018) Nitrate transport, signaling, and use efficiency. Annu Rev Plant Biol 69:85–122
Wang X, Feng C, Tian LL, Hou C, Tian W, Hu B, Zhang Q, Ren Z, Niu Q, Song J, Kong D, Liu L, He Y, Ma L, Chu C, Luan S, Li L (2021) A transceptor–channel complex couples nitrate sensing to calcium signaling in Arabidopsis. Mol Plant 14:774–786
Yuan S, Zhang Z-W, Zheng C, Zhao Z-Y, Wang Y, Feng L-Y, Niu G, Wang C-Q, Wang J-H, Feng H et al (2016) Arabidopsis cryptochrome 1 functions in nitrogen regulation of flowering. Proc Natl Acad Sci 113:7661–7666
Zhao L, Zhang W, Yang Y, Li Z, Li N, Qi S, Crawford NM, Wang Y (2018) The Arabidopsis NLP7 gene regulates nitrate signaling via NRT1. 1-dependent pathway in the presence of ammonium. Sci Rep 8:1–13
Acknowledgements
The authors thank the ICAR-Indian Agricultural Research Institute for funding and providing the necessary facilities. SAB acknowledges DST-INSPIRE for the junior research fellowship support received during the study.
Author information
Authors and Affiliations
Contributions
SAB conducted the experiments with the help of LS. SAB prepared the first draft. LS conceived the idea and finalized the draft.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no competing interests.
Additional information
Handling Editor: Néstor Carrillo
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Adavi, S.B., Sathee, L. Calcium regulates primary nitrate response associated gene transcription in a time- and dose-dependent manner. Protoplasma 261, 257–269 (2024). https://doi.org/10.1007/s00709-023-01893-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00709-023-01893-z