Nitrate Response Research Articles

Differential phytotoxic responses of silver nitrate (AgNO3) and silver nanoparticle (AgNps) in Cucumis sativus L.

The present study was undertaken to evaluate the effects of silver nitrate (AgNO3) and biosynthesized silver nanoparticle (AgNps) on Cucumis sativus L seedlings. Results indicated that both the forms of silver significantly reduced the growth which may be accompanied due to increased accumulation of silver in plants (4708.2±108.75mg/kg). Both the treatments showed steep reduction (> in AgNO3 treatments) in photosynthetic performance, total chlorophyll, carotenoids and total protein content and significantly (P<0.05) increased oxidative stress (MDA, H2O2, SOR; i.e. > in AgNO3 treatment). The histochemical observations (NBT & DAB) of oxidative stress markers (H2O2 and O2−) were also in accordance with their total estimation of H2O2 and O2− in both the treatments. Chlorophyll florescence parameters were also significantly (P<0.05) influenced by AgNO3 and AgNps treatments and showed remarkable modifications.Though, both the treatments (AgNO3 and AgNps) showed anatomical impacts on the root cortical cells, however, degeneration of cortical cells and disintegration of endodermis in AgNO3 treatments were more prominent. Taking impacts of the both forms of silver together, present study suggests that the AgNO3 is more toxic than AgNPs while potential risks of both forms is critical on the growth and development of Cucumis seedlings. We suggest further studies to explore the underlying mechanisms and to understand the effective levels of both forms of silver to be used in sustaining agricultural productions.

Discovery of nitrate-CPK-NLP signalling in central nutrient-growth networks.

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.

Nitrate signaling and early responses in Arabidopsis roots.

Nitrogen (N) is an essential macronutrient that impacts many aspects of plant physiology, growth, and development. Besides its nutritional role, N nutrient and metabolites act as signaling molecules that regulate the expression of a wide range of genes and biological processes. In this review, we describe recent advances in the understanding of components of the nitrate signaling pathway. Recent evidence posits that in one nitrate signaling pathway, nitrate sensed by NRT1.1 activates a phospholipase C activity that is necessary for increased cytosolic calcium levels. The nitrate-elicited calcium increase presumably activates calcium sensors, kinases, or phosphatases, resulting in changes in expression of primary nitrate response genes. Consistent with this model, nitrate treatments elicit proteome-wide changes in phosphorylation patterns in a wide range of proteins, including transporters, metabolic enzymes, kinases, phosphatases, and other regulatory proteins. Identifying and characterizing the function of the different players involved in this and other nitrate signaling pathways and their functional relationships is the next step to understand N responses in plants.

The Effect of Betaine on Nitrate and Cardiovascular Response to Exercise

The Effect of Betaine on Nitrate and Cardiovascular Response to Exercise

Direct transcriptional activation of BT genes by NLP transcription factors is a key component of the nitrate response in Arabidopsis

Nitrate modulates growth and development, functioning as a nutrient signal in plants. Although many changes in physiological processes in response to nitrate have been well characterized as nitrate responses, the molecular mechanisms underlying the nitrate response are not yet fully understood. Here, we show that NLP transcription factors, which are key regulators of the nitrate response, directly activate the nitrate-inducible expression of BT1 and BT2 encoding putative scaffold proteins with a plant-specific domain structure in Arabidopsis. Interestingly, the 35S promoter-driven expression of BT2 partially rescued growth inhibition caused by reductions in NLP activity in Arabidopsis. Furthermore, simultaneous disruption of BT1 and BT2 affected nitrate-dependent lateral root development. These results suggest that direct activation of BT1 and BT2 by NLP transcriptional activators is a key component of the molecular mechanism underlying the nitrate response in Arabidopsis.

Early nitrogen-deprivation responses in Arabidopsis roots reveal distinct differences on transcriptome and (phospho-) proteome levels between nitrate and ammonium nutrition.

Plant roots acquire nitrogen predominantly as ammonium and nitrate, which besides serving as nutrients, also have signaling roles. Re-addition of nitrate to starved plants rapidly re-programs the metabolism and gene expression, but the earliest responses to nitrogen deprivation are unknown. Here, the early transcriptional and (phospho)proteomic responses of roots to nitrate or ammonium deprivation were analyzed. The rapid transcriptional repression of known nitrate-induced genes proceeded the tissue NO3- concentration drop, with the transcription factor genes LBD37/38 and HRS1/HHO1 among those with earliest significant change. Similar rapid transcriptional repression occurred in loss-of-function mutants of the nitrate response factor NLP7 and some transcripts were stabilized by nitrate. In contrast, an early transcriptional response to ammonium deprivation was almost completely absent. However, ammonium deprivation induced a rapid and transient perturbation of the proteome and a differential phosphorylation pattern in proteins involved in adjusting the pH and cation homeostasis, plasma membrane H+ , NH4+ , K+ and water fluxes. Fewer differential phosphorylation patterns in transporters, kinases and other proteins occurred with nitrate deprivation. The deprivation responses were not just opposite to the re-supply responses, but identified NO3- deprivation-induced mRNA decay and signaling candidates potentially reporting the external nitrate status to the cell.

Integration of Hormonal and Nutritional Cues Orchestrates Progressive Corolla Opening.

Flower opening is essential for pollination and thus successful sexual reproduction; however, the underlying mechanisms of its timing control remain largely elusive. We identify a unique cucumber (Cucumis sativus) line '6457' that produces normal ovaries when nutrients are under-supplied, and super ovaries (87%) with delayed corolla opening when nutrients are oversupplied. Corolla opening in both normal and super ovaries is divided into four distinct phases, namely the green bud, green-yellow bud, yellow bud, and flowering stages, along with progressive color transition, cytological tuning, and differential expression of 14,282 genes. In the super ovary, cell division and cell expansion persisted for a significantly longer period of time; the expressions of genes related to photosynthesis, protein degradation, and signaling kinases were dramatically up-regulated, whereas the activities of most transcription factors and stress-related genes were significantly down-regulated; concentrations of cytokinins (CKs) and gibberellins were higher in accordance with reduced cytokinin conjugation and degradation and increased expression of gibberellin biosynthesis genes. Exogenous CK application was sufficient for the genesis of super ovaries, suggesting a decisive role of CKs in controlling the timing of corolla opening. Furthermore, 194 out of 11,127 differentially expressed genes identified in pairwise comparisons, including critical developmental, signaling, and cytological regulators, contained all three types of cis-elements for CK, nitrate, and phosphorus responses in their promoter regions, indicating that the integration of hormone modulation and nutritional regulation orchestrated the precise control of corolla opening in cucumber. Our findings provide a valuable framework for dissecting the regulatory pathways for flower opening in plants.

Dielectric response of potassium nitrate in a restricted geometry

The dielectric properties of nanocomposite materials (NCM) on basis of porous glasses with KNO3 embedded into the pores have been studied in the temperature interval 293 K–570 K and in the frequency diapason 0.1–107 Hz. The three types of porous glasses with average pore diameter 320 (PG320), 46 (PG46) and 7 (PG7) nm have been used for production NCM from melted KNO3. It is shown that dielectric permittivity demonstrates the essential growth in the high-temperature phase for all types of NCM. The temperature of the ferroelectric phase transition decreases at a reduction of average pore diameters and the temperature interval of stability of ferroelectric phase essentially enlarges. In case of NCM on basis of PG7 there is no any anomalies corresponding to the phase transition in this NCM and on cooling up to room temperature ferroelectric phase remains stable. From analysis of dielectric response, the relaxation process responsible for the ferroelectric phase transition has been detected and the temperature dependencies of process parameters have been determined.

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.

Chloride Sources and Losses in Two Tile-Drained Agricultural Watersheds.

Chloride is a relatively unreactive plant nutrient that has long been used as a biogeochemical tracer but also can be a pollutant causing aquatic biology impacts when concentrations are high, typically from rock salt applications used for deicing roads. Chloride inputs to watersheds are most often from atmospheric deposition, road salt, or agricultural fertilizer, although studies on agricultural watersheds with large fertilizer inputs are few. We used long-term (21 and 17 yr) chloride water quality data in two rivers of east-central Illinois to better understand chloride biogeochemistry in two agricultural watersheds (Embarras and Kaskaskia), the former with a larger urban land use and both with extensive tile drainage. During our sampling period, the average chloride concentration was 23.7 and 20.9 mg L in the Embarras and Kaskaskia Rivers, respectively. Annual fluxes of chloride were 72.5 and 61.2 kg ha yr in the Embarras and Kaskaskia watersheds, respectively. In both watersheds, fertilizer chloride was the dominant input (∼49 kg ha yr), with road salt likely the other major source (23.2 and 7.2 kg ha yr for the Embarras and Kaskaskia watersheds, respectively). Combining our monitoring data with earlier published data on the Embarras River showed an increase in chloride concentrations as potash use increased in Illinois during the 1960s and 1970s with a lag of about 2 to 6 yr to changes in potash inputs based on a multiple-regression model. In these agricultural watersheds, riverine chloride responds relatively quickly to potash fertilization as a result of tile-drainage.

The nitrate inducible NAC transcription factor TaNAC2-5A controls nitrate response and increases wheat yield

The nitrate inducible NAC transcription factor TaNAC2-5A controls nitrate response and increases wheat yield

The changing trend in nitrate concentrations in major aquifers due to historical nitrate loading from agricultural land across England and Wales from 1925 to 2150

Nitrate is necessary for agricultural productivity, but can cause considerable problems if released into aquatic systems. Agricultural land is the major source of nitrates in UK groundwater. Due to the long time-lag in the groundwater system, it could take decades for leached nitrate from the soil to discharge into freshwaters. However, this nitrate time-lag has rarely been considered in environmental water management. Against this background, this paper presents an approach to modelling groundwater nitrate at the national scale, to simulate the impacts of historical nitrate loading from agricultural land on the evolution of groundwater nitrate concentrations. An additional process-based component was constructed for the saturated zone of significant aquifers in England and Wales. This uses a simple flow model which requires modelled recharge values, together with published aquifer properties and thickness data. A spatially distributed and temporally variable nitrate input function was also introduced. The sensitivity of parameters was analysed using Monte Carlo simulations. The model was calibrated using national nitrate monitoring data. Time series of annual average nitrate concentrations along with annual spatially distributed nitrate concentration maps from 1925 to 2150 were generated for 28 selected aquifer zones. The results show that 16 aquifer zones have an increasing trend in nitrate concentration, while average nitrate concentrations in the remaining 12 are declining. The results are also indicative of the trend in the flux of groundwater nitrate entering rivers through baseflow. The model thus enables the magnitude and timescale of groundwater nitrate response to be factored into source apportionment tools and to be taken into account alongside current planning of land-management options for reducing nitrate losses.

The Nitrate-Inducible NAC Transcription Factor TaNAC2-5A Controls Nitrate Response and Increases Wheat Yield.

Nitrate is a major nitrogen resource for cereal crops; thus, understanding nitrate signaling in cereal crops is valuable for engineering crops with improved nitrogen use efficiency. Although several regulators have been identified in nitrate sensing and signaling in Arabidopsis (Arabidopsis thaliana), the equivalent information in cereals is missing. Here, we isolated a nitrate-inducible and cereal-specific NAM, ATAF, and CUC (NAC) transcription factor, TaNAC2-5A, from wheat (Triticum aestivum). A chromatin immunoprecipitation assay showed that TaNAC2-5A could directly bind to the promoter regions of the genes encoding nitrate transporter and glutamine synthetase. Overexpression of TaNAC2-5A in wheat enhanced root growth and nitrate influx rate and, hence, increased the root's ability to acquire nitrogen. Furthermore, we found that TaNAC2-5A-overexpressing transgenic wheat lines had higher grain yield and higher nitrogen accumulation in aerial parts and allocated more nitrogen in grains in a field experiment. These results suggest that TaNAC2-5A is involved in nitrate signaling and show that it is an exciting gene resource for breeding crops with more efficient use of fertilizer.

G-protein α-subunit (GPA1) regulates stress, nitrate and phosphate response, flavonoid biosynthesis, fruit/seed development and substantially shares GCR1 regulation in A. thaliana.

Heterotrimeric G-proteins are implicated in several plant processes, but the mechanisms of signal-response coupling and the roles of G-protein coupled receptors in general and GCR1 in particular, remain poorly understood. We isolated a knock-out mutant of the Arabidopsis G-protein α subunit (gpa1-5) and analysed its transcriptome to understand the genomewide role of GPA1 and compared it with that of our similar analysis of a GCR1 mutant (Chakraborty et al. 2015, PLoS ONE 10(2):e0117819). We found 394 GPA1-regulated genes spanning 79 biological processes, including biotic and abiotic stresses, development, flavonoid biosynthesis, transcription factors, transporters and nitrate/phosphate responses. Many of them are either unknown or unclaimed explicitly in other published gpa1 mutant transcriptome analyses. A comparison of all known GPA1-regulated genes (including the above 394) with 350 GCR1-regulated genes revealed 114 common genes. This can be best explained by GCR1-GPA1 coupling, or by convergence of their independent signaling pathways. Though the common genes in our GPA1 and GCR1 mutant datasets constitute only 26% of the GPA1-regulated and 30% of the GCR1-responsive genes, they belong to nearly half of all the processes affected in both the mutants. Thus, GCR1 and GPA1 regulate not only some common genes, but also different genes belonging to the same processes to achieve similar outcomes. Overall, we validate some known and report many hitherto unknown roles of GPA1 in plants, including agronomically important ones such as biotic stress and nutrient response, and also provide compelling genetic evidence to revisit the role of GCR1 in G-protein signalling.

The Calcium Ion Is a Second Messenger in the Nitrate Signaling Pathway of Arabidopsis.

Understanding how plants sense and respond to changes in nitrogen availability is the first step toward developing strategies for biotechnological applications, such as improvement of nitrogen use efficiency. However, components involved in nitrogen signaling pathways remain poorly characterized. Calcium is a second messenger in signal transduction pathways in plants, and it has been indirectly implicated in nitrate responses. Using aequorin reporter plants, we show that nitrate treatments transiently increase cytoplasmic Ca(2+) concentration. We found that nitrate also induces cytoplasmic concentration of inositol 1,4,5-trisphosphate. Increases in inositol 1,4,5-trisphosphate and cytoplasmic Ca(2+) levels in response to nitrate treatments were blocked by U73122, a pharmacological inhibitor of phospholipase C, but not by the nonfunctional phospholipase C inhibitor analog U73343. In addition, increase in cytoplasmic Ca(2+) levels in response to nitrate treatments was abolished in mutants of the nitrate transceptor NITRATE TRANSPORTER1.1/Arabidopsis (Arabidopsis thaliana) NITRATE TRANSPORTER1 PEPTIDE TRANSPORTER FAMILY6.3. Gene expression of nitrate-responsive genes was severely affected by pretreatments with Ca(2+) channel blockers or phospholipase C inhibitors. These results indicate that Ca(2+) acts as a second messenger in the nitrate signaling pathway of Arabidopsis. Our results suggest a model where NRT1.1/AtNPF6.3 and a phospholipase C activity mediate the increase of Ca(2+) in response to nitrate required for changes in expression of prototypical nitrate-responsive genes.

Sensitivity of tropical tropospheric composition to lightning NOx production as determined by replay simulations with GEOS‐5

AbstractThe sensitivity of tropical tropospheric composition to the source strength of nitrogen oxides (NOx) produced by lightning (LNOx) is analyzed for September through November 2007 using the NASA GEOS‐5 model constrained by MERRA fields, with full GMI stratospheric‐tropospheric chemistry and an LNOx algorithm that is appropriate for use in a climate modeling setting; satellite retrievals from OMI, TES, and OMI/MLS; and in situ measurements from SHADOZ ozonesondes. Global mean LNOx production rates of 0 to 492 mol NO flash−1 and the subsequent responses of NOx, ozone (O3), hydroxyl radical (OH), nitric acid (HNO3), peroxyacetyl nitrate (PAN), and NOy (NOx + HNO3 + PAN) are investigated. The radiative implications associated with LNOx‐induced changes in tropospheric O3 are assessed. Increasing the LNOx production rate by a factor of 4 (from 123 to 492 mol flash−1) leads to tropical upper tropospheric enhancements of greater than 100% in NOx, OH, HNO3, and PAN. This increase in LNOx production also leads to O3 enhancements of up to 60%, which subsequently yields a factor‐of‐three increase in the mean net radiative flux at the tropopause. An LNOx source of 246 mol flash−1 agrees reasonably well with measurements, with an approximate factor‐of‐two uncertainty due to the short length of the study, inconsistencies in the observational data sets, and systematic biases in modeled LNOx production. Further research into the regional dependencies of lightning flash rates and LNOx production per flash, along with improvements in satellite retrievals, should help resolve the discrepancies that currently exist between the model and observations.

Two short sequences in OsNAR2.1 promoter are necessary for fully activating the nitrate induced gene expression in rice roots.

Nitrate is an essential nitrogen source and serves as a signal to control growth and gene expression in plants. In rice, OsNAR2.1 is an essential partner of multiple OsNRT2 nitrate transporters for nitrate uptake over low and high concentration range. Previously, we have reported that −311 bp upstream fragment from the translational start site in the promoter of OsNAR2.1 gene is the nitrate responsive region. To identify the cis-acting DNA elements necessary for nitrate induced gene expression, we detected the expression of beta-glucuronidase (GUS) reporter in the transgenic rice driven by the OsNAR2.1 promoter with different lengths and site mutations of the 311 bp region. We found that −129 to −1 bp region is necessary for the nitrate-induced full activation of OsNAR2.1. Besides, the site mutations showed that the 20 bp fragment between −191 and −172 bp contains an enhancer binding site necessary to fully drive the OsNAR2.1 expression. Part of the 20 bp fragment is commonly presented in the sequences of different promoters of both the nitrate induced NAR2 genes and nitrite reductase NIR1 genes from various higher plants. These findings thus reveal the presence of conserved cis-acting element for mediating nitrate responses in plants.

Phytosiderophores revisited: 2′-deoxymugineic acid-mediated iron uptake triggers nitrogen assimilation in rice (Oryza sativa L.) seedlings

Poaceae plants release phytosiderophores into the rhizosphere in order to chelate iron (Fe), which often exists in insoluble forms especially under high pH conditions. The impact of phytosiderophore treatment at the physiological and molecular levels in vivo remains largely elusive, although the biosynthesis of phytosiderophores and the transport of phytosiderophore-metal complexes have been well studied. We recently showed that the application of 30 μM of the chemically synthesized phytosiderophore 2′-deoxymugineic acid (DMA) was sufficient for apparent full recovery of otherwise considerably reduced growth of hydroponic rice seedlings at high pH. Moreover, unexpected induction of high-affinity nitrate transporter gene expression as well as nitrate reductase activity indicates that the nitrate response is linked to Fe homeostasis. These data shed light on the biological relevance of DMA not simply as a Fe chelator, but also as a trigger that promotes plant growth by reinforcing nitrate assimilation.

Metabolic and co-expression network-based analyses associated with nitrate response in rice.

BackgroundUnderstanding gene expression and metabolic re-programming that occur in response to limiting nitrogen (N) conditions in crop plants is crucial for the ongoing progress towards the development of varieties with improved nitrogen use efficiency (NUE). To unravel new details on the molecular and metabolic responses to N availability in a major food crop, we conducted analyses on a weighted gene co-expression network and metabolic profile data obtained from leaves and roots of rice plants adapted to sufficient and limiting N as well as after shifting them to limiting (reduction) and sufficient (induction) N conditions.ResultsA gene co-expression network representing clusters of rice genes with similar expression patterns across four nitrogen conditions and two tissue types was generated. The resulting 18 clusters were analyzed for enrichment of significant gene ontology (GO) terms. Four clusters exhibited significant correlation with limiting and reducing nitrate treatments. Among the identified enriched GO terms, those related to nucleoside/nucleotide, purine and ATP binding, defense response, sugar/carbohydrate binding, protein kinase activities, cell-death and cell wall enzymatic activity are enriched. Although a subset of functional categories are more broadly associated with the response of rice organs to limiting N and N reduction, our analyses suggest that N reduction elicits a response distinguishable from that to adaptation to limiting N, particularly in leaves. This observation is further supported by metabolic profiling which shows that several compounds in leaves change proportionally to the nitrate level (i.e. higher in sufficient N vs. limiting N) and respond with even higher levels when the nitrate level is reduced. Notably, these compounds are directly involved in N assimilation, transport, and storage (glutamine, asparagine, glutamate and allantoin) and extend to most amino acids. Based on these data, we hypothesize that plants respond by rapidly mobilizing stored vacuolar nitrate when N deficit is perceived, and that the response likely involves phosphorylation signal cascades and transcriptional regulation.ConclusionsThe co-expression network analysis and metabolic profiling performed in rice pinpoint the relevance of signal transduction components and regulation of N mobilization in response to limiting N conditions and deepen our understanding of N responses and N use in crops.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1056) contains supplementary material, which is available to authorized users.

NODULE INCEPTION antagonistically regulates gene expression with nitrate in Lotus japonicus.

Legumes produce root nodules as symbiotic organs where nitrogen-fixing bacteria are accommodated. Lotus japonicus NODULE INCEPTION (NIN) is an essential factor that specifically and positively regulates nodulation processes, and has evolved from a member of the NIN-like proteins, of which Arabidopsis homologs target nitrate-responsive elements (NREs), and activate gene expression in response to nitrate. It is therefore assumed that the NIN-mediated transcriptional network overlaps with those regulated by NLPs, because of their common DNA-binding RWP-RK domains. However, nodulation is inhibited in the presence of nitrate, and involvement of NIN in nitrate responses has remained largely unknown. Here we determined a consensus of NIN-binding nucleotide sequences (NBSs) by in vitro experiments, and revealed that the sequence pattern was very similar to those of NREs. Chromatin immunoprecitiation (ChIP)-PCR analyses showed that NIN targeted NREs in L. japonicus nitrate-inducible gene promoters, including LjNIR1, LjNRT2.1 and LjNRT2.2. Affinities of NIN binding to the NREs were comparable with that to NBS-yB1a, an NBS on the symbiotic LjNF-YB1 promoter, indicating that NREs are potential targets of NIN. However, rhizobial infection did not activate LjNIR1, LjNRT2.1 and LjNRT2.2. NIN ectopic expression interfered with nitrate-dependent activation of these genes. Nitrate treatment followed by NIN activation down-regulated expression of symbiotic NIN target genes. Our results showed that NIN and nitrate antagonistically regulate expression of genes that are activated by nitrate and NIN, respectively. We propose that this antagonistic relationship prevents inappropriate activation of genes in response to nitrate and rhizobial infection.