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Emerging Players in the Nitrate Signaling Pathway

2017; Elsevier BV; Volume: 10; Issue: 8 Linguagem: Inglês

10.1016/j.molp.2017.07.006

ISSN

1674-2052

Autores

Grace Armijo, Rodrigo A. Gutiérrez,

Tópico(s)

Pancreatic function and diabetes

Resumo

Nitrogen (N)-based fertilizers are routinely used to increase agricultural productivity for both food and non-food uses of crops. Unfortunately, excess N fertilizers escape to the environment, leading to detrimental effects on the ecosystem and human health. Understanding how plants sense and respond to different N nutrients or metabolites to regulate metabolism, physiology, growth, and development is essential for sustained yields while reducing agriculture's environmental and economic costs. Several recent publications report important components of the nitrate signaling pathway, and the role of Ca2+ as a second messenger downstream of nitrate perception in roots and shoots is now clear. Moreover, using a combination of reverse genetics and chemical inhibitors, three key Ca2+-sensor protein kinases (CPKs) have been linked to nitrate transcriptional control by NLP7. This emerging nitrate signaling pathway integrates nitrate perception, second messengers to early signaling events, and transcriptional control, leading to metabolic, physiological, and developmental changes. The only nitrate sensor proposed thus far is the dual-affinity nitrate transporter NRT1.1. NRT1.1 triggers nitrate responses and is required for normal expression of nitrate-responsive genes. Moreover, studies of protein mutations that uncouple transport and signaling activities provide evidence that both processes are performed independently by the same protein. The mutant allele chl1-9, where a leucine is replaced by proline at position 492, displays a nitrate uptake defect but shows a typical biphasic primary nitrate response for NRT2.1 (Ho et al., 2009Ho C.H. Lin S.H. Hu H.C. Tsay Y.F. CHL1 functions as a nitrate sensor in plants.Cell. 2009; 138: 1184-1194Abstract Full Text Full Text PDF PubMed Scopus (893) Google Scholar). In other assays, however, chl1-9 and chl1-5 (deletion mutant of NRT1.1) were similar in the long-term repression of NRT2.1 expression and lateral root growth without nitrate (Bouguyon et al., 2015Bouguyon E. Brun F. Meynard D. Kubes M. Pervent M. Leran S. Lacombe B. Krouk G. Guiderdoni E. Zazimalova E. et al.Multiple mechanisms of nitrate sensing by Arabidopsis nitrate transceptor NRT1.1.Nat. Plants. 2015; 1: 15015Crossref PubMed Scopus (205) Google Scholar). It was proposed that NRT1.1 functions as a “transceptor,” with dual transport and sensing functions, which is critical for activating signal transduction cascades in response to the nitrate signal. However, it is also possible that additional sensing mechanisms exits for either external or internal nitrate with distinct or overlapping signaling functions associated with NRT1.1. NRT1.1 displays either high or low affinity for nitrate transport, depending on the phosphorylation status of a threonine 101 residue. The phosphorylated form of NRT1.1 is a high-affinity transporter, whereas the non-phosphorylated form behaves as a low-affinity nitrate transporter (Liu and Tsay, 2003Liu K.H. Tsay Y.F. Switching between the two action modes of the dual-affinity nitrate transporter CHL1 by phosphorylation.EMBO J. 2003; 22: 1005-1013Crossref PubMed Scopus (374) Google Scholar). The protein complex CIPK23-CBL9 (CIPK, CBL-Interacting Protein Kinase; CBL, Calcineurin-B like Protein) has been implicated in the dual-affinity transition changes of NRT1.1 through phosphorylation (Noguero and Lacombe, 2016Noguero M. Lacombe B. Transporters involved in root nitrate uptake and sensing by Arabidopsis.Front. Plant Sci. 2016; 7: 1391Crossref PubMed Scopus (55) Google Scholar). More recently, ABI2 (ABA-insensitive 2), a protein phosphatase 2C family member, and the calcium sensor CBL1 were identified as additional components that modulate NRT1.1 transport function, NRT2.1 expression, and root developmental responses to nitrate (Leran et al., 2015Leran S. Edel K.H. Pervent M. Hashimoto K. Corratge-Faillie C. Offenborn J.N. Tillard P. Gojon A. Kudla J. Lacombe B. Nitrate sensing and uptake in Arabidopsis are enhanced by ABI2, a phosphatase inactivated by the stress hormone abscisic acid.Sci. Signal. 2015; 8: ra43Crossref PubMed Scopus (140) Google Scholar). NRT1.1 is capable of triggering independent signaling pathways in response to nitrate in Arabidopsis roots. Different NRT1.1 mutant alleles exhibit distinct responses to nitrate at the transcriptome level as well as for repression of lateral root development (Bouguyon et al., 2015Bouguyon E. Brun F. Meynard D. Kubes M. Pervent M. Leran S. Lacombe B. Krouk G. Guiderdoni E. Zazimalova E. et al.Multiple mechanisms of nitrate sensing by Arabidopsis nitrate transceptor NRT1.1.Nat. Plants. 2015; 1: 15015Crossref PubMed Scopus (205) Google Scholar). For instance, the phosphorylated form of NRT1.1 is responsible for the NRT1.1-dependent regulation of lateral root development (Bouguyon et al., 2015Bouguyon E. Brun F. Meynard D. Kubes M. Pervent M. Leran S. Lacombe B. Krouk G. Guiderdoni E. Zazimalova E. et al.Multiple mechanisms of nitrate sensing by Arabidopsis nitrate transceptor NRT1.1.Nat. Plants. 2015; 1: 15015Crossref PubMed Scopus (205) Google Scholar). Furthermore, mutations affect NRT1.1-mediated auxin transport (P492L and T101A substitution) as well as NRT1.1-dependent regulation of lateral root primordia. It was suggested that P492L substitution suppresses both nitrate transport by NRT1.1 and auxin transport, in which the phosphorylated form of NRT1.1 would be predominantly active (Bouguyon et al., 2015Bouguyon E. Brun F. Meynard D. Kubes M. Pervent M. Leran S. Lacombe B. Krouk G. Guiderdoni E. Zazimalova E. et al.Multiple mechanisms of nitrate sensing by Arabidopsis nitrate transceptor NRT1.1.Nat. Plants. 2015; 1: 15015Crossref PubMed Scopus (205) Google Scholar). The chl1-9 allele shows altered NRT1.1 subcellular localization to the plasma membrane, displaying predominantly intracellular localization, which could explain its transport-defective phenotype. On the other hand, NRT1.1-dependent repression of NRT2.1 requires the phosphorylated form of NRT1.1 (Bouguyon et al., 2015Bouguyon E. Brun F. Meynard D. Kubes M. Pervent M. Leran S. Lacombe B. Krouk G. Guiderdoni E. Zazimalova E. et al.Multiple mechanisms of nitrate sensing by Arabidopsis nitrate transceptor NRT1.1.Nat. Plants. 2015; 1: 15015Crossref PubMed Scopus (205) Google Scholar). At least four different modes of NRT1.1-dependent signaling were suggested (Bouguyon et al., 2015Bouguyon E. Brun F. Meynard D. Kubes M. Pervent M. Leran S. Lacombe B. Krouk G. Guiderdoni E. Zazimalova E. et al.Multiple mechanisms of nitrate sensing by Arabidopsis nitrate transceptor NRT1.1.Nat. Plants. 2015; 1: 15015Crossref PubMed Scopus (205) Google Scholar). Short-term induction of NRT2.1 at high nitrate is negatively affected by T101D, but not by P492L and T101A substitutions. Long-term regulation of NRT2.1 expression by high nitrogen and the repression of lateral root emergence in the absence of nitrate show the opposite pattern, with no effect of T101D substitution, and loss of signaling resulting from P492L or T101A mutations. Genes related to the extracellular matrix, secreted proteins, and oxidation-reduction reactions are under NRT1.1-dependent regulation that is suppressed by both T101A and T101D mutations, but not by P492L, indicating a yet uncharacterized signaling role of NRT1.1 (Bouguyon et al., 2015Bouguyon E. Brun F. Meynard D. Kubes M. Pervent M. Leran S. Lacombe B. Krouk G. Guiderdoni E. Zazimalova E. et al.Multiple mechanisms of nitrate sensing by Arabidopsis nitrate transceptor NRT1.1.Nat. Plants. 2015; 1: 15015Crossref PubMed Scopus (205) Google Scholar). Early studies in Zea mays (maize) and Hordeum vulgare (barley) leaves found that EGTA or the calcium channel blocker LaCl3 are able to repress nitrate induction of primary response genes such as nitrate reductase (NR) and nitrite reductase (NiR), suggesting that calcium may be important for nitrate responses (reviewed in Undurraga et al., 2017Undurraga S.F. Ibarra-Henriquez C. Fredes I. Alvarez J.M. Gutierrez R.A. Nitrate signaling and early responses in Arabidopsis roots.J. Exp. Bot. 2017; 68: 2541-2551Crossref PubMed Scopus (57) Google Scholar). More recently, using a cytosolic aequorin calcium reporter, it was shown that nitrate treatment induces a rapid increase in cytoplasmic Ca2+ levels in roots that required NRT1.1 (Riveras et al., 2015Riveras E. Alvarez J.M. Vidal E.A. Oses C. Vega A. Gutierrez R.A. The calcium ion is a second messenger in the nitrate signaling pathway of Arabidopsis.Plant Physiol. 2015; 169: 1397-1404Crossref PubMed Scopus (140) Google Scholar). In addition, pharmacological inhibition of PLC activity blocked the induction of cytoplasmic Ca2+ levels after nitrate treatments and affected expression of nitrate-responsive genes (Riveras et al., 2015Riveras E. Alvarez J.M. Vidal E.A. Oses C. Vega A. Gutierrez R.A. The calcium ion is a second messenger in the nitrate signaling pathway of Arabidopsis.Plant Physiol. 2015; 169: 1397-1404Crossref PubMed Scopus (140) Google Scholar), indicating that phospholipase C (PLC) activity is also implicated in nitrate signaling. Treatment with LaCl3 or the PLC inhibitor U73122 significantly reduced nitrate induction of NRT2.1, NRT3.1, NiR, and TGACG SEQUENCE-SPECIFIC BINDING PROTEIN1 (TGA1) genes (Riveras et al., 2015Riveras E. Alvarez J.M. Vidal E.A. Oses C. Vega A. Gutierrez R.A. The calcium ion is a second messenger in the nitrate signaling pathway of Arabidopsis.Plant Physiol. 2015; 169: 1397-1404Crossref PubMed Scopus (140) Google Scholar). In another recent study, using the GCaMP6 calcium biosensor, Liu et al., 2017Liu K.H. Niu Y. Konishi M. Wu Y. Du H. Sun Chung H. Li L. Boudsocq M. McCormack M. Maekawa S. et al.Discovery of nitrate-CPK-NLP signalling in central nutrient-growth networks.Nature. 2017; 545: 311-316Crossref PubMed Scopus (287) Google Scholar investigated live Ca2+ signaling in mesophyll protoplasts of leaf cells and found that nitrate specifically stimulates Ca2+ signature in the nucleus and cytosol. Although results from these two studies showed different kinetics for nitrate-induced calcium concentration changes, the importance of calcium as a second messenger in nitrate signaling is now clear. Discrepancies may be due to different experimental conditions or methodologies utilized, which in the second study allowed for dynamic and subcellular imaging of Ca2+ signaling triggered by nitrate, which was not previously possible (Liu et al., 2017Liu K.H. Niu Y. Konishi M. Wu Y. Du H. Sun Chung H. Li L. Boudsocq M. McCormack M. Maekawa S. et al.Discovery of nitrate-CPK-NLP signalling in central nutrient-growth networks.Nature. 2017; 545: 311-316Crossref PubMed Scopus (287) Google Scholar). In addition, activation of nitrate-responsive marker genes NiR, G6PD3 G6PD3, and FNR2 was also compromised in seedlings treated with blockers of plasma membrane Ca2+ channels, gadolinium (Gd3+) and lanthanum (La3+), or calmodulin inhibitor W7 (Liu et al., 2017Liu K.H. Niu Y. Konishi M. Wu Y. Du H. Sun Chung H. Li L. Boudsocq M. McCormack M. Maekawa S. et al.Discovery of nitrate-CPK-NLP signalling in central nutrient-growth networks.Nature. 2017; 545: 311-316Crossref PubMed Scopus (287) Google Scholar). These two independent studies clearly show that Ca2+ has a crucial role in nitrate signaling in plants. Interestingly, not all nitrate-responsive genes depend on this signaling pathway. Induction of AUXIN SIGNALING F-BOX3 (AFB3) by nitrate, part of the AFB3/miR393 regulatory module, was not affected by calcium channel blocker and PLC inhibitor treatments (Riveras et al., 2015Riveras E. Alvarez J.M. Vidal E.A. Oses C. Vega A. Gutierrez R.A. The calcium ion is a second messenger in the nitrate signaling pathway of Arabidopsis.Plant Physiol. 2015; 169: 1397-1404Crossref PubMed Scopus (140) Google Scholar), suggesting the existence of Ca2+-dependent and -independent pathways downstream of NRT1.1. One of the direct consequences of Ca2+ increase is the change in protein phosphorylation status, controlling the activity of key components of the nitrate signaling pathway and other targets. The recent report by Liu et al., 2017Liu K.H. Niu Y. Konishi M. Wu Y. Du H. Sun Chung H. Li L. Boudsocq M. McCormack M. Maekawa S. et al.Discovery of nitrate-CPK-NLP signalling in central nutrient-growth networks.Nature. 2017; 545: 311-316Crossref PubMed Scopus (287) Google Scholar made a significant step toward closing the gap between Ca2+ and the first layer of transcriptional regulators. Using in-gel kinase assays, they detected an enhanced activity of endogenous Ca2+-sensor protein kinases (CPKs) in response to nitrate. In addition, using a luciferase (LUC) reporter gene NIR-LUC, which exhibits a physiological nitrate response in transgenic Arabidopsis plants, they identified a subgroup III of constitutively active CPKs as most effective for synergistically activating the NIR-LUC reporter. Three CPKs, CPK10, CPK30, and CPK32, were proposed as key regulators of primary nitrate responses, connecting the influx of calcium and phosphorylation of target proteins as orchestrators of the response that would active transcription factor targets to coordinate the primary nitrate response (Liu et al., 2017Liu K.H. Niu Y. Konishi M. Wu Y. Du H. Sun Chung H. Li L. Boudsocq M. McCormack M. Maekawa S. et al.Discovery of nitrate-CPK-NLP signalling in central nutrient-growth networks.Nature. 2017; 545: 311-316Crossref PubMed Scopus (287) Google Scholar). Nitrate-coupled CPK signaling phosphorylates transcription factors for regulating the expression of downstream genes that affects nitrogen assimilation, carbon/nitrogen metabolism, and proliferation, among others (Liu et al., 2017Liu K.H. Niu Y. Konishi M. Wu Y. Du H. Sun Chung H. Li L. Boudsocq M. McCormack M. Maekawa S. et al.Discovery of nitrate-CPK-NLP signalling in central nutrient-growth networks.Nature. 2017; 545: 311-316Crossref PubMed Scopus (287) Google Scholar). The NIN-LIKE PROTEIN (NLP) family of transcription factors were identified as nitrate-responsive cis-element (NRE)-binding proteins, which function as transcriptional activators in the nitrate-regulated expression of Arabidopsis genes (Konishi and Yanagisawa, 2013Konishi M. Yanagisawa S. Arabidopsis NIN-like transcription factors have a central role in nitrate signalling.Nat. Commun. 2013; 4: 1617Crossref PubMed Scopus (254) Google Scholar, Marchive et al., 2013Marchive C. Roudier F. Castaings L. Brehaut V. Blondet E. Colot V. Meyer C. Krapp A. Nuclear retention of the transcription factor NLP7 orchestrates the early response to nitrate in plants.Nat. Commun. 2013; 4: 1713Crossref PubMed Scopus (308) Google Scholar). A putative DNA-binding domain, RWP-RK, present in the nine Arabidopsis NLPs was shown to bind NRE in yeast, suggesting that all NLPs may bind this element (Konishi and Yanagisawa, 2013Konishi M. Yanagisawa S. Arabidopsis NIN-like transcription factors have a central role in nitrate signalling.Nat. Commun. 2013; 4: 1617Crossref PubMed Scopus (254) Google Scholar). Furthermore, the N-terminal region of NLP6 was shown to function as a nitrate-responsive domain (Konishi and Yanagisawa, 2013Konishi M. Yanagisawa S. Arabidopsis NIN-like transcription factors have a central role in nitrate signalling.Nat. Commun. 2013; 4: 1617Crossref PubMed Scopus (254) Google Scholar). Alignment of nine Arabidopsis NLPs protein sequences plus four orthologous sequences from Lotus japonicus, revealed a conserved residue Ser205, which was proposed as a target of CPK phosphorylation (Liu et al., 2017Liu K.H. Niu Y. Konishi M. Wu Y. Du H. Sun Chung H. Li L. Boudsocq M. McCormack M. Maekawa S. et al.Discovery of nitrate-CPK-NLP signalling in central nutrient-growth networks.Nature. 2017; 545: 311-316Crossref PubMed Scopus (287) Google Scholar). Interestingly, NLP7 was shown to be phosphorylated by CPK10, and interaction of both proteins was also shown in the nucleus of protoplasts in the presence of nitrate (Liu et al., 2017Liu K.H. Niu Y. Konishi M. Wu Y. Du H. Sun Chung H. Li L. Boudsocq M. McCormack M. Maekawa S. et al.Discovery of nitrate-CPK-NLP signalling in central nutrient-growth networks.Nature. 2017; 545: 311-316Crossref PubMed Scopus (287) Google Scholar). NLP7 is a regulator of early nitrate-dependent gene expression changes, and can translocate to the nucleus in response to nitrate treatments (Marchive et al., 2013Marchive C. Roudier F. Castaings L. Brehaut V. Blondet E. Colot V. Meyer C. Krapp A. Nuclear retention of the transcription factor NLP7 orchestrates the early response to nitrate in plants.Nat. Commun. 2013; 4: 1713Crossref PubMed Scopus (308) Google Scholar). In addition to NLP7, others transcription factors known to participate in the regulation of gene expression in response to nitrogen nutrient/metabolite treatments include Arabidopsis Nitrate Regulated 1 (ANR1), LOB Domain Containing proteins (LBD37/38/39), Squamosa Promoter Binding Protein-Like 9 (SPL9), Basic Leucine-Zipper 1 (bZIP1), NAC Domain Containing Protein 4 (NAC4), TGA1/TGA4, Teosinte Branched1/Cycloidea/Proliferating Cell Factor 20 (TCP20), and Nitrate Regulatory Gene 2 (NRG2). However, interaction with cognate promoter targets has been experimentally verified only for TGA1, NLP6/7, bZIP1, and TCP20 (reviewed in Undurraga et al., 2017Undurraga S.F. Ibarra-Henriquez C. Fredes I. Alvarez J.M. Gutierrez R.A. Nitrate signaling and early responses in Arabidopsis roots.J. Exp. Bot. 2017; 68: 2541-2551Crossref PubMed Scopus (57) Google Scholar). TCP20 was found to play a critical role in nitrate-induced transcriptional changes and systemic signaling since it induces expression of NRT1.1 only under low nitrate conditions, while TGA1 and TGA4 regulate expression of genes in response to high nitrate treatments and are involved in nitrate transport and metabolic functions, particularly controlling the expression of two high-affinity nitrate transporter genes NRT2.1 and NRT2.2 by binding directly to their promoters (O'Brien et al., 2016O'Brien J.A. Vega A. Bouguyon E. Krouk G. Gojon A. Coruzzi G. Gutierrez R.A. Nitrate transport, sensing, and responses in plants.Mol. Plant. 2016; 9: 837-856Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar). Furthermore, upregulation of TGA1 expression is calcium-dependent (Riveras et al., 2015Riveras E. Alvarez J.M. Vidal E.A. Oses C. Vega A. Gutierrez R.A. The calcium ion is a second messenger in the nitrate signaling pathway of Arabidopsis.Plant Physiol. 2015; 169: 1397-1404Crossref PubMed Scopus (140) Google Scholar). Interestingly, TGA1 could be phosphorylated by mitogen-activated protein kinases, which regulate many essential cellular processes in response to a stimulus (Popescu et al., 2009Popescu S.C. Popescu G.V. Bachan S. Zhang Z. Gerstein M. Snyder M. Dinesh-Kumar S.P. MAPK target networks in Arabidopsis thaliana revealed using functional protein microarrays.Genes Dev. 2009; 23: 80-92Crossref PubMed Scopus (389) Google Scholar). Recently, CIPK23–TGA1 nuclear interaction was experimentally demonstrated (Yazaki et al., 2016Yazaki J. Galli M. Kim A.Y. Nito K. Aleman F. Chang K.N. Carvunis A.-R. Quan R. Nguyen H. Song L. et al.Mapping transcription factor interactome networks using HaloTag protein arrays.Proc. Natl. Acad. Sci. USA. 2016; 113: E4238-E4247Crossref PubMed Scopus (47) Google Scholar). These results suggest that phosphorylation in response to nitrate treatments may be also important for TGA1 activation. Determining the mechanism of activation for TGA1 and other nitrate regulatory factors is certainly an area of great interest. In summary, these recent discoveries provide new insights for nitrate signal transduction and activation of transcriptional networks in response to N nutrient, as illustrated in the Figure 1. While the understanding of the nitrate signaling pathway has been advanced, many important questions remain to be answered. For instance, how does nitrate perception elicit an increase in cytosolic Ca2+ level? What is the biochemical role of NRT1.1 in this process? Which and how are PLCs implicated in nitrate signaling? How many nitrate-sensing mechanisms exist? Are these signaling pathways conserved across land plants? Unraveling the molecular basis of nitrate sensing and regulation by answering these questions would be an important step toward developing better strategies to increase nitrogen use efficiency to avoid detrimental environmental impact while sustaining crop yields. Research in our group is funded by grants from Fondo de Desarrollo de Areas Prioritarias (FONDAP) Center for Genome Regulation (15090007), Millennium Nucleus Center for Plant Systems and Synthetic Biology (NC130030), Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT) 1141097 to R.A.G. and Department of Energy DE-FOA-0001207 to G.C.

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