Nitrate in 2020: Thirty Years from Transport to Signaling Networks.

Nitrogen (N) is an essential macronutrient for plants and a major limiting factor for plant growth and crop production. Nitrate is the main source of N available to plants in agricultural soils and in many natural environments. Sustaining agricultural productivity is of paramount importance in the current scenario of increasing world population, diversification of crop uses, and climate change. Plant productivity for major crops around the world, however, is still supported by excess application of N-rich fertilizers with detrimental economic and environmental impacts. Thus, understanding how plants regulate nitrate uptake and metabolism is key for developing new crops with enhanced N use efficiency and to cope with future world food demands. The study of plant responses to nitrate has gained considerable interest over the last 30 years. This review provides an overview of key findings in nitrate research, spanning biochemistry, molecular genetics, genomics, and systems biology. We discuss how we have reached our current view of nitrate transport, local and systemic nitrate sensing/signaling, and the regulatory networks underlying nitrate-controlled outputs in plants. We hope this summary will serve not only as a timeline and information repository but also as a baseline to define outstanding questions for future research.

PHB3 regulates lateral root primordia formation via NO-mediated degradation of AUXIN/INDOLE-3-ACETIC ACID proteins.

We have previously shown that Arabidopsis thaliana Prohibitin 3 (PHB3) controls auxin-stimulated lateral root (LR) formation; however, the underlying molecular mechanism is unknown. Here, we demonstrate that PHB3 regulates lateral root (LR) development mainly through influencing lateral root primordia (LRP) initiation, via affecting nitric oxide (NO) accumulation. The reduced LRP in phb3 mutant was largely rescued by treatment with a NO donor. The decreased NO accumulation in phb3 caused a lower expression of GATA TRANSCRIPTION FACTOR 23 (GATA23) and LATERAL ORGAN BOUNDARIES DOMAIN 16 (LBD16) through inhibiting the degradation of INDOLE-3-ACETIC ACID INDUCIBLE 14/28 (IAA14/28). Overexpression of either GATA23 or LBD16 in phb3 mutant background recovered the reduced density of LRP. These results indicate that PHB3 regulates LRP initiation via NO-mediated auxin signalling, by modulating the degradation of IAA14/28.

Interacting TCP and NLP transcription factors control plant responses to nitrate availability.

Plants have evolved adaptive strategies that involve transcriptional networks to cope with and survive environmental challenges. Key transcriptional regulators that mediate responses to environmental fluctuations in nitrate have been identified; however, little is known about how these regulators interact to orchestrate nitrogen (N) responses and cell-cycle regulation. Here we report that teosinte branched1/cycloidea/proliferating cell factor1-20 (TCP20) and NIN-like protein (NLP) transcription factors NLP6 and NLP7, which act as activators of nitrate assimilatory genes, bind to adjacent sites in the upstream promoter region of the nitrate reductase gene, NIA1, and physically interact under continuous nitrate and N-starvation conditions. Regions of these proteins necessary for these interactions were found to include the type I/II Phox and Bem1p (PB1) domains of NLP6&7, a protein-interaction module conserved in animals for nutrient signaling, and the histidine- and glutamine-rich domain of TCP20, which is conserved across plant species. Under N starvation, TCP20-NLP6&7 heterodimers accumulate in the nucleus, and this coincides with TCP20 and NLP6&7-dependent up-regulation of nitrate assimilation and signaling genes and down-regulation of the G2/M cell-cycle marker gene, CYCB1;1 TCP20 and NLP6&7 also support root meristem growth under N starvation. These findings provide insights into how plants coordinate responses to nitrate availability, linking nitrate assimilation and signaling with cell-cycle progression.

The long noncoding RNA T5120 regulates nitrate response and assimilation in Arabidopsis.

Long noncoding RNAs (lncRNAs) are crucial regulators in many plant biological processes. However, it remains unknown whether lncRNAs can respond to nitrate or function in nitrate regulation. We detected 695 lncRNAs, 480 known and 215 novel, in Arabidopsis seedling roots; six showed altered expression in response to nitrate treatment, among which T5120 showed the highest induction. Overexpression of T5120 in Arabidopsis promoted the response to nitrate, enhanced nitrate assimilation and improved biomass and root development. Biochemical and molecular analyses revealed that NLP7, a master nitrate regulatory transcription factor, directly bound to the nitrate-responsive cis-element (NRE)-like motif of the T5120 promoter and activated T5120 transcription. In addition, T5120 partially restored the nitrate signalling and assimilation phenotypes of nlp7 mutant, suggesting that T5120 is involved in NLP7-mediated nitrate regulation. Interestingly, the expression of T5120 was regulated by the nitrate sensor NRT1.1. Therefore, T5120 is modulated by NLP7 and NRT1.1 to regulate nitrate signalling. Our work reveals a new regulatory mechanism in which lncRNA T5120 functions in nitrate regulation, providing new insights into the nitrate signalling network. Importantly, lncRNA T5120 can promote nitrate assimilation and plant growth to improve nitrogen use efficiency.

The Arabidopsis NRG2 Protein Mediates Nitrate Signaling and Interacts with and Regulates Key Nitrate Regulators.

We show that NITRATE REGULATORY GENE2 (NRG2), which we identified using forward genetics, mediates nitrate signaling in Arabidopsis thaliana. A mutation in NRG2 disrupted the induction of nitrate-responsive genes after nitrate treatment by an ammonium-independent mechanism. The nitrate content in roots was lower in the mutants than in the wild type, which may have resulted from reduced expression of NRT1.1 (also called NPF6.3, encoding a nitrate transporter/receptor) and upregulation of NRT1.8 (also called NPF7.2, encoding a xylem nitrate transporter). Genetic and molecular data suggest that NRG2 functions upstream of NRT1.1 in nitrate signaling. Furthermore, NRG2 directly interacts with the nitrate regulator NLP7 in the nucleus, but nuclear retention of NLP7 in response to nitrate is not dependent on NRG2. Transcriptomic analysis revealed that genes involved in four nitrogen-related clusters including nitrate transport and response to nitrate were differentially expressed in the nrg2 mutants. A nitrogen compound transport cluster containing some members of the NRT/PTR family was regulated by both NRG2 and NRT1.1, while no nitrogen-related clusters showed regulation by both NRG2 and NLP7. Thus, NRG2 plays a key role in nitrate regulation in part through modulating NRT1.1 expression and may function with NLP7 via their physical interaction.

Molecular Regulation of Nitrate Responses in Plants.

Nitrogen is an essential macronutrient that affects plant growth and development. Improving the nitrogen use efficiency of crops is of great importance for the economic and environmental sustainability of agriculture. Nitrate (NO3-) is a major form of nitrogen absorbed by most crops and also serves as a vital signaling molecule. Research has identified key molecular components in nitrate signaling mainly by employing forward and reverse genetics as well as systems biology. In this review, we focus on advances in the characterization of genes involved in primary nitrate responses as well as the long-term effects of nitrate, especially in terms of how nitrate regulates root development.

The Arabidopsis CPSF30-L gene plays an essential role in nitrate signaling and regulates the nitrate transceptor gene NRT1.1.

Plants have evolved sophisticated mechanisms to adapt to fluctuating environmental nitrogen availability. However, more underlying genes regulating the response to nitrate have yet to be characterized. We report here the identification of a nitrate regulatory mutant whose mutation mapped to the Cleavage and Polyadenylation Specificity Factor 30 gene (CPSF30-L). In the mutant, induction of nitrate-responsive genes was inhibited independent of the ammonium conditions and was restored by expression of the wild-type 65 kDa encoded by CPSF30-L. Molecular and genetic evidence suggests that CPSF30-L works upstream of NRT1.1 and independently of NLP7 in response to nitrate. Analysis of the 3'-UTR of NRT1.1 showed that the pattern of polyadenylation sites was altered in the cpsf30 mutant. Transcriptome analysis revealed that four nitrogen-related clusters were enriched in the differentially expressed genes of the cpsf30 mutant. Nitrate uptake was decreased in the mutant along with reduced expression of the nitrate transporter/sensor gene NRT1.1, while nitrate reduction and amino acid content were enhanced in roots along with increased expression of several nitrate assimilatory genes. These findings indicate that the 65 kDa protein encoded by CPSF30-L mediates nitrate signaling in part by regulating NRT1.1 expression, thus adding an important component to the nitrate signaling network.

Nitrate foraging by Arabidopsis roots is mediated by the transcription factor TCP20 through the systemic signaling pathway.

To compete for nutrients in diverse soil microenvironments, plants proliferate lateral roots preferentially in nutrient-rich zones. For nitrate, root foraging involves local and systemic signaling; however, little is known about the genes that function in the systemic signaling pathway. By using nitrate enhancer DNA to screen a library of Arabidopsis transcription factors in the yeast one-hybrid system, the transcription factor gene TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1-20 (TCP20) was identified. TCP20, which belongs to an ancient, plant-specific gene family that regulates shoot, flower, and embryo development, was implicated in nitrate signaling by its ability to bind DNA in more than 100 nitrate-regulated genes. Analysis of insertion mutants of TCP20 showed that they had normal primary and lateral root growth on homogenous nitrate media but were impaired in preferential lateral root growth (root foraging) on heterogeneous media in split-root plates. Inhibition of preferential lateral root growth was still evident in the mutants even when ammonium was uniformly present in the media, indicating that the TCP20 response was to nitrate. Comparison of tcp20 mutants with those of nlp7 mutants, which are defective in local control of root growth but not in the root-foraging response, indicated that TCP20 function is independent of and distinct from NLP7 function. Further analysis showed that tcp20 mutants lack systemic control of root growth regardless of the local nitrate concentrations. These results indicate that TCP20 plays a key role in the systemic signaling pathway that directs nitrate foraging by Arabidopsis roots.

The Arabidopsis Prohibitin Gene PHB3 Functions in Nitric Oxide-Mediated Responses and in Hydrogen Peroxide-Induced Nitric Oxide Accumulation.

To discover genes involved in nitric oxide (NO) metabolism, a genetic screen was employed to identify mutants defective in NO accumulation after treatment with the physiological inducer hydrogen peroxide. In wild-type Arabidopsis thaliana plants, NO levels increase eightfold in roots after H(2)O(2) treatment for 30 min. A mutant defective in H(2)O(2)-induced NO accumulation was identified, and the corresponding mutation was mapped to the prohibitin gene PHB3, converting the highly conserved Gly-37 to an Asp in the protein's SPFH domain. This point mutant and a T-DNA insertion mutant were examined for other NO-related phenotypes. Both mutants were defective in abscisic acid-induced NO accumulation and stomatal closure and in auxin-induced lateral root formation. Both mutants were less sensitive to salt stress, showing no increase in NO accumulation and less inhibition of primary root growth in response to NaCl treatment. In addition, light-induced NO accumulation was dramatically reduced in cotyledons. We found no evidence for impaired H(2)O(2) metabolism or signaling in the mutants as H(2)O(2) levels and H(2)O(2)-induced gene expression were unaffected by the mutations. These findings identify a component of the NO homeostasis system in plants and expand the function of prohibitin genes to include regulation of NO accumulation and NO-mediated responses.

A molecular framework underlying low-nitrogen-induced early leaf senescence in Arabidopsis thaliana.

Nitrogen (N) deficiency causes early leaf senescence, resulting in accelerated whole-plant maturation and severely reduced crop yield. However, the molecular mechanisms underlying N-deficiency-induced early leaf senescence remain unclear, even in the model species Arabidopsis thaliana. In this study, we identified Growth, Development and Splicing 1 (GDS1), a previously reported transcription factor, as a new regulator of nitrate (NO3-) signaling by a yeast-one-hybrid screen using a NO3- enhancer fragment from the promoter of NRT2.1. We showed that GDS1 promotes NO3- signaling, absorption and assimilation by affecting the expression of multiple NO3- regulatory genes, including Nitrate Regulatory Gene2 (NRG2). Interestingly, we observed that gds1 mutants show early leaf senescence as well as reduced NO3- content and N uptake under N-deficient conditions. Further analyses indicated that GDS1 binds to the promoters of several senescence-related genes, including Phytochrome-Interacting Transcription Factors 4 and 5 (PIF4 and PIF5) and represses their expression. Interestingly, we found that N deficiency decreases GDS1 protein accumulation, and GDS1 could interact with Anaphase Promoting Complex Subunit 10 (APC10). Genetic and biochemical experiments demonstrated that Anaphase Promoting Complex or Cyclosome (APC/C) promotes the ubiquitination and degradation of GDS1 under N deficiency, resulting in loss of PIF4 and PIF5 repression and consequent early leaf senescence. Furthermore, we discovered that overexpression of GDS1 could delay leaf senescence and improve seed yield and N-use efficiency (NUE) in Arabidopsis. In summary, our study uncovers a molecular framework illustrating a new mechanism underlying low-N-induced early leaf senescence and provides potential targets for genetic improvement of crop varieties with increased yield and NUE.

Multiple regulatory elements in the Arabidopsis NIA1 promoter act synergistically to form a nitrate enhancer.

To accommodate fluctuating nutrient levels in the soil, plants modulate their metabolism and root development via signaling mechanisms that rapidly reprogram the plant transcriptome. In the case of nitrate, over 1,000 genes are induced or repressed within minutes of nitrate exposure. To identify cis-regulatory elements that mediate these responses, an enhancer screen was performed in transgenic Arabidopsis (Arabidopsis thaliana) plants. A 1.8-kb promoter fragment from the nitrate reductase gene NIA1 was identified that acts as a nitrate enhancer when fused to a 35S minimal promoter. Enhancer activity was localized to a 180-bp fragment, and this activity could be enhanced by the addition of a 131-bp fragment from the nitrite reductase promoter. A promoter construct containing the 180- and 131-bp fragments was also induced by nitrite and repressed by ammonium, indicating that it was responsive to multiple nitrogen signals. To identify specific regulatory elements within the 180-bp NIA1 fragment, a transient expression system using agroinfiltration of Nicotiana benthamiana was developed. Deletion analysis identified three elements corresponding to predicted binding motifs for homeodomain/E-box, Myb, and Alfin1 transcription factors. A fully active promoter showing nitrate and nitrite enhancer activity equivalent to that of the wild-type 180-bp fragment could be built from these three elements if the spacing between the homeodomain/E-box and Myb-Alfin1 sites was equivalent to that of the native promoter. These findings were validated in transgenic Arabidopsis plants and identify a cis-regulatory module containing three elements that comprise a nitrate enhancer in the NIA1 promoter.

The Arabidopsis NLP7 gene regulates nitrate signaling via NRT1.1-dependent pathway in the presence of ammonium.

Nitrate is not only an important nutrient but also a signaling molecule for plants. A few of key molecular components involved in primary nitrate responses have been identified mainly by forward and reverse genetics as well as systems biology, however, many underlining mechanisms of nitrate regulation remain unclear. In this study, we show that the expression of NRT1.1, which encodes a nitrate sensor and transporter (also known as CHL1 and NPF6.3), is modulated by NIN-like protein 7 (NLP7). Genetic and molecular analyses indicate that NLP7 works upstream of NRT1.1 in nitrate regulation when NH4+ is present, while in absence of NH4+, it functions in nitrate signaling independently of NRT1.1. Ectopic expression of NRT1.1 in nlp7 resulted in partial or complete restoration of nitrate signaling (expression from nitrate-regulated promoter NRP), nitrate content and nitrate reductase activity in the transgenic lines. Transcriptome analysis revealed that four nitrogen-related clusters including amino acid synthesis-related genes and members of NRT1/PTR family were modulated by both NLP7 and NRT1.1. In addition, ChIP and EMSA assays results indicated that NLP7 may bind to specific regions of the NRT1.1 promoter. Thus, NLP7 acts as an important factor in nitrate signaling via regulating NRT1.1 under NH4+ conditions.

FIP1 Plays an Important Role in Nitrate Signaling and Regulates CIPK8 and CIPK23 Expression in Arabidopsis.

Unraveling the molecular mechanisms of nitrate regulation and deciphering the underlying genetic network is vital for elucidating nitrate uptake and utilization in plants. Such knowledge could lead to the improvement of nitrogen-use efficiency in agriculture. Here, we report that the FIP1 gene (factor interacting with poly(A) polymerase 1) plays an important role in nitrate signaling in Arabidopsis thaliana. FIP1 encodes a putative core component of the polyadenylation factor complex. We found that FIP1 interacts with the cleavage and polyadenylation specificity factor 30-L (CPSF30-L), which is also an essential player in nitrate signaling. The induction of nitrate-responsive genes following nitrate treatment was inhibited in the fip1 mutant. The nitrate content was also reduced in fip1 seedlings due to their decreased nitrate uptake activity. Furthermore, the nitrate content was higher in the roots but lower in the roots of fip1, which may result from the downregulation of NRT1.8 and the upregulation of the nitrate assimilation genes. In addition, qPCR analyses revealed that FIP1 negatively regulated the expression of CIPK8 and CIPK23, two protein kinases involved in nitrate signaling. In the fip1 mutant, the increased expression of CIPK23 may affect nitrate uptake, resulting in its lower nitrate content. Genetic and molecular evidence suggests that FIP1 and CPSF30-L function in the same nitrate-signaling pathway, with FIP1 mediating signaling through its interaction with CPSF30-L and its regulation of CIPK8 and CIPK23. Analysis of the 3'-UTR of NRT1.1 showed that the pattern of polyadenylation sites was altered in the fip1 mutant. These findings add a novel component to the nitrate regulation network and enhance our understanding of the underlying mechanisms for nitrate signaling.

Interference with the citrulline-based nitric oxide synthase assay by argininosuccinate lyase activity in Arabidopsis extracts.

There are many reports of an arginine-dependent nitric oxide synthase activity in plants; however, the gene(s) or protein(s) responsible for this activity have yet to be convincingly identified. To measure nitric oxide synthase activity, many studies have relied on a citrulline-based assay that measures the formation of L-citrulline from L-arginine using ion exchange chromatography. In this article, we report that when such assays are used with protein extracts from Arabidopsis, an arginine-dependent activity was observed, but it produced a product other than citrulline. TLC analysis identified the product as argininosuccinate. The reaction was stimulated by fumarate (> 500 microM), implicating the urea cycle enzyme argininosuccinate lyase (EC 4.3.2.1), which reversibly converts arginine and fumarate to argininosuccinate. These results indicate that caution is needed when using standard citrulline-based assays to measure nitric oxide synthase activity in plant extracts, and highlight the importance of verifying the identity of the product as citrulline.

New insights into nitric oxide metabolism and regulatory functions.

Nitric oxide (NO) has been intensively studied to elucidate the role of this enigmatic signaling molecule in plant development, metabolism and disease responses. Many studies using pharmacological and biochemical tools have demonstrated that NO functions in hormone responses, programmed cell death, defense gene induction and signal transduction pathways. NO originates from two sources in plants: nitrite and arginine. Recent studies using mutants and transgenic plants have confirmed these key findings and have gone further to identify (i) a new mechanism to modulate NO bioactivity involving hemoglobin, (ii) a gene involved in arginine-dependent NO synthesis, and (iii) a novel function for NO signaling in flowering. These findings continue to elucidate the expanding role of NO in plant biology.

Overexpression of the Maize ZmNLP6 and ZmNLP8 Can Complement the Arabidopsis Nitrate Regulatory Mutant nlp7 by Restoring Nitrate Signaling and Assimilation.

Nitrate is a key nutrient that affects maize growth and yield, and much has yet to be learned about nitrate regulatory genes and mechanisms in maize. Here, we identified nine ZmNLP genes in maize and analyzed the functions of two ZmNLP members in nitrate signaling. qPCR results revealed a broad pattern of expression for ZmNLP genes in different stages and organs with the highest levels of transcript expression of ZmNLP6 and ZmNLP8. When ZmNLP6 and ZmNLP8 were overexpressed in the Arabidopsis nitrate regulatory gene mutant nlp7-4, nitrate assimilation and induction of nitrate-responsive genes in the transgenic plants were recovered to WT levels, indicating that ZmNLP6 and ZmNLP8 can replace the essential roles of the master nitrate regulatory gene AtNLP7 in nitrate signaling and metabolism. ZmNLP6 and ZmNLP8 are localized in the nucleus and can bind candidate nitrate-responsive cis-elements in vitro. The biomass and yield of transgenic Arabidopsis lines overexpressing ZmNLP6 and ZmNLP8 showed significant increase compared with WT and nlp7-4 mutant line in low nitrate conditions. Thus, ZmNLP6 and ZmNLP8 regulate nitrate signaling in transgenic Arabidopsis plants and may be potential candidates for improving nitrogen use efficiency of maize.

Expression of the Arabidopsis transposable element Tag1 is targeted to developing gametophytes.

The Arabidopsis transposon Tag1 undergoes late excision during vegetative and germinal development in plants containing 35S-Tag1-GUS constructs. To determine if transcriptional regulation can account for the developmental control of Tag1 excision, the transcriptional activity of Tag1 promoter-GUS fusion constructs of various lengths was examined in transgenic plants. All constructs showed expression in the reproductive organs of developing flowers but no expression in leaves. Expression was restricted to developing gametophytes in both male and female lineages. Quantitative RT-PCR analysis confirmed that Tag1 expression predominates in the reproductive organs of flower buds. These results are consistent with late germinal excision of Tag1, but they cannot explain the vegetative excision activity of Tag1 observed with 35S-Tag1-GUS constructs. To resolve this issue, Tag1 excision was reexamined using elements with no adjacent 35S promoter sequences. Tag1 excision in this context is restricted to germinal events with no detectable vegetative excision. If a 35S enhancer sequence is placed next to Tag1, vegetative excision is restored. These results indicate that the intrinsic activity of Tag1 is restricted to germinal excision due to targeted expression of the Tag1 transposase to developing gametophytes and that this activity is altered by the presence of adjacent enhancers or promoters.

AtNIGT1/HRS1 integrates nitrate and phosphate signals at the Arabidopsis root tip.

Nitrogen and phosphorus are among the most widely used fertilizers worldwide. Nitrate (NO3(-)) and phosphate (PO4(3-)) are also signalling molecules whose respective transduction pathways are being intensively studied. However, plants are continuously challenged with combined nutritional deficiencies, yet very little is known about how these signalling pathways are integrated. Here we report the identification of a highly NO3(-)-inducible NRT1.1-controlled GARP transcription factor, HRS1, document its genome-wide transcriptional targets, and validate its cis-regulatory elements. We demonstrate that this transcription factor and a close homologue repress the primary root growth in response to P deficiency conditions, but only when NO3(-) is present. This system defines a molecular logic gate integrating P and N signals. We propose that NO3(-) and P signalling converge via double transcriptional and post-transcriptional control of the same protein, HRS1.

A framework integrating plant growth with hormones and nutrients.

It is well known that nutrient availability controls plant development. Moreover, plant development is finely tuned by a myriad of hormonal signals. Thus, it is not surprising to see increasing evidence of coordination between nutritional and hormonal signaling. In this opinion article, we discuss how nitrogen signals control the hormonal status of plants and how hormonal signals interplay with nitrogen nutrition. We further expand the discussion to include other nutrient-hormone pairs. We propose that nutrition and growth are linked by a multi-level, feed-forward cycle that regulates plant growth, development and metabolism via dedicated signaling pathways that mediate nutrient and hormonal regulation. We believe this model will provide a useful concept for past and future research in this field.

Nitrate signaling: adaptation to fluctuating environments.

Nitrate (NO(3)(-)) is a key nutrient as well as a signaling molecule that impacts both metabolism and development of plants. Understanding the complexity of the regulatory networks that control nitrate uptake, metabolism, and associated responses has the potential to provide solutions that address the major issues of nitrate pollution and toxicity that threaten agricultural and ecological sustainability and human health. Recently, major advances have been made in cataloguing the nitrate transcriptome and in identifying key components that mediate nitrate signaling. In this perspective, we describe the genes involved in nitrate regulation and how they influence nitrate transport and assimilation, and we discuss the role of systems biology approaches in elucidating the gene networks involved in NO(3)(-) signaling adaptation to fluctuating environments.