Nitrate Signaling Research Articles

An autoinhibitory mechanism controls RNA-binding activity of the nitrate-sensing protein NasR.

The ANTAR domain harnesses RNA‐binding activity to promote transcription attenuation. Although several ANTAR proteins have been analyzed by high‐resolution structural analyses, the residues involved in RNA‐recognition and transcription attenuation have not been identified. Nor is it clear how signal‐responsive domains are allosterically coupled with ANTAR domains for control of gene expression. Herein, we examined the sequence conservation of ANTAR domains to find residues that may associate with RNA. We subjected the corresponding positions of Klebsiella oxytoca NasR to site‐directed alanine substitutions and measured RNA‐binding activity. This revealed a functionally important patch of residues that forms amino acid pairing interactions with residues from NasR’s nitrate‐sensing NIT domain. We hypothesize these amino acid pairing interactions are part of an autoinhibitory mechanism that holds the structure in an “off” state in the absence of nitrate signal. Indeed, mutational disruption of these interactions resulted in constitutively active proteins, freed from autoinhibition and no longer influenced by nitrate. Moreover, sequence analyses suggested the autoinhibitory mechanism has been evolutionarily maintained by NasR proteins. These data reveal a molecular mechanism for how NasR couples its nitrate signal to RNA‐binding activity, and generally show how signal‐responsive domains of one‐component regulatory proteins have evolved to exert control over RNA‐binding ANTAR domains.

Nitrate Uptake, Transport and Signaling Regulation Pathways

Nitrate Uptake, Transport and Signaling Regulation Pathways

DCAPS markers developed for nitrate transporter genes TaNRT2L12s associating with 1 000-grain weight in wheat

Nitrate transporters (NRTs) are regulators of nitrate assimilation and transport. The genome sequences of TaNRT2L12-A, -B and -D were cloned from wheat (Triticum aestivum L.), and polymorphisms were analyzed by sequencing. TaNRT2L12-D in a germplasm population was highly conserved. However, 38 single nucleotide polymorphisms (SNPs) in TaNRT2L12-A coding region and 11 SNPs in TaNRT2L12-B coding region were detected. Two derived cleaved amplified polymorphic sequences (dCAPS) markers A-CSNP1 and A-CSNP2 were developed for TaNRT2L12-A based on SNP-351 and SNP-729, and three haplotypes were identified in the germplasm population. B-CSNP1 and B-CSNP2 were developed for TaNRT2L12-B based on SNP-237 and SNP-1227, and three haplotypes were detected in the germplasm population. Association analyses between the markers and agronomic traits in 30 environments and phenotypic comparisons revealed that A-CSNP2-A is a superior allele of shorter plant height (PH), length of penultimate internode (LPI) and peduncle length (PL), B-CSNP2-G is a superior allele of higher grain number per spike (GNS). Hap-6B-1 containing both superior alleles B-CSNP1-C and B-CSNP2-A is a superior haplotype of 1 000-grain weight (TGW). Expression analysis showed that TaNRT2L12-B is mainly expressed in the root base and regulated by nitrate. Therefore, TaNRT2L12 may be involved in nitrate transport and signaling to regulate TGW in wheat. The superior alleles and dCAPS markers of TaNRT2L12-A/B are beneficial to genetic improvement and germplasm enhancement with molecular markers-assisted selection.

Autoregulation of nodulation pathway is dispensable for nitrate-induced control of rhizobial infection

ABSTRACTLegumes possess the autoregulation of nodulation (AON) pathway which is responsible for maintaining optimal root nodule number. In Lotus japonicus, AON comprises the CLE-HAR1-TML module, which plays an essential role in transmitting signals via root-to-shoot-to-root long-distance signaling. In addition to AON’s principal role of negatively regulating nodule number, a recent study revealed another in the systemic control of rhizobial infection. Nitrate also negatively regulates the pleiotropic phases of legume-Rhizobium symbioses, including rhizobial infection and nodule number. Nitrate signaling has recently been shown to use AON components such as CLE-RS2 and HAR1 to control nodule number. Here we consider the role of a loss-of-function mutation in CLE-RS1, -RS2 and TML in rhizobial infection in relation to nitrate. Our results agree with previous findings and support the hypothesis that AON is required for the control of rhizobial infection but not for its nitrate-induced control. Furthermore, we confirm that the tml mutants exhibit nitrate sensitivity that differs from that of cle-rs2 and har1. Hence, while the nitrate-induced control mechanism of nodule number uses AON components, an unknown pathway specific to nitrate may exist downstream of HAR1, acting in parallel with the HAR1> TML pathway.

Regulation of expression of genes associated with nitrate response by osmotic stress and combined osmotic and nitrogen deficiency stress in bread wheat (Triticum aestivum L.)

In drought prone areas, often farmers use less nitrogen, and thus the crop is subjected to combined stress (low N + osmotic stress). Since understanding the regulation of genes involved in nitrate signalling, uptake and assimilation under water-deficit (osmotic stress) is important for improving yield under the combined stress environments, this study analysed the regulation of genes coding for N responses under low N, osmotic stress (OS) and combined stress conditions in seedlings of a wheat. The results revealed that HD2967, a mega wheat variety, was more tolerant to short-term N starvation, OS and combined stress as compared with C306, a drought tolerant check. Interestingly, it was found that low N stress can also lead to accumulation of ABA in wheat seedlings. Real-time RT-qPCR analysis revealed that in addition to low N stress, OS also regulated expression of nitrate signalling genes (TaCIPK8, TaCIPK23, TaNLP4, TaSPL9, TabHLH1 and TaNAC4), HATS gene TaNRT2.1, LATS genes (TaNRT6.5 and TaNPF7.1), nitrate and nitrite assimilation genes and ammonium assimilation genes at least in one tissue of one of the genotypes. Combined stress was found to have significant interaction in regulation genes for nitrate signalling, uptake and assimilation. TabZIP1 and TaPIMP1 TF were identified as new players in low N response in wheat. Thus, osmotic stress and combined stress modulates the genes for N responses, and genotypic variation exists for this in wheat. The common expression pattern of N response genes found under low N and OS may probably regulated, at least in part, by ABA-dependent pathway, as ABA accumulation was induced by both OS and low N stresses. Functional analysis of the osmotic stress regulated genes coding for N response will help enhance tolerance of wheat to combined stress conditions.

OsNAR2.2 plays a vital role in the root growth and development by promoting nitrate uptake and signaling in rice

Plants in soil faces great fluctuations of external mineral nutrient availability, and they have developed sophisticated nutrient sensing systems to regulate their physiological responses to prevent nutrient deficiency. However, complete knowledge of the regulatory system is required to maximize inorganic nitrogen (N) uptake and utilization. In this study, we report a partner protein for high-affinity nitrate transport, OsNAR2.2. OsNAR2.2 was involved in the root growth in a nitrate-dependent manner in rice, and this process was closely associated with auxin. Expression analysis showed that OsNAR2.2 responded to nitrate and various plant hormone signals. Knockdown of OsNAR2.2 by T-DNA insertion not only significantly repressed the primary root elongation, but also severely reduced the number of lateral root and adventitious root. Further research indicated that the size of meristematic zone and epidermal cell length of mature zone in the primary root tip were remarkably reduced, and the formation of lateral root primordial was constrained in osnar2.2 mutant. Interestingly, the repression of root growth in osnar2.2 mutant was observed when NO3− but not NH4+ was used as N source in the medium. The NO3− content in osnar2.2 root was significantly reduced under NO3− conditions, in comparison with that of wild type. Meanwhile, the free IAA accumulation as well as the expression of auxin biosynthesis and transport genes was altered in osnar2.2 root, suggesting there might be a crosslink between the nitrate and auxin signaling. Together, OsNAR2.2 plays a vital role in rice root growth and development in a nitrate-dependent manner, which might be associated with auxin signaling.

The NMN Module Conducts Nodule Number Orchestra.

Legumes control nodule number through nodulation and autoregulation of nodulation (AON) pathways. Nodule Inception (NIN) is essential for rhizobial infection and nodule organogenesis in legumes. The GmNINa-miR172c-NNC1 (NMN) module, which consists of two positive regulators, GmNINa and miR172c, and a suppressor, NNC1, integrates both pathways. GmNINa activates miR172c to downregulate NNC1, leading to nodulation, while NNC1 inhibits miR172c expression, forming a negative feedback loop. GmNINa and NNC1 interact with each other and antagonistically fine-tune GmRIC1/RIC2 expression, turning AON on and off. Conversely, activation of AON inhibits GmNINa and miR172c expression, thereby reducing their inhibitory effects on NNC1 to attenuate both nodulation signaling and AON. The NMN module functions not only as an “accelerator” of the nodulation signal to promote nodulation but also as a “brake” on the signal by activating AON to orchestrate nodule number.

Nitrate Signaling in Plants: Mechanisms of Implementation

The review deals with the mechanisms of nitrate signaling covering its sensing by transceptors located in the plasma membrane, intracellular signal transduction, and multiplication by a cascade of intermediates as well as regulation of expression of target genes and transduction between the organs involving major phytohormones with consideration for spatial separateness of their biosyntheses in the whole plant. The contribution of signaling into C/N balance of the organism based on key reactions of carbohydrate and nitrogen metabolism are discussed.

NRT1.1s in plants: functions beyond nitrate transport.

Arabidopsis AtNRT1.1 (CHL1/AtNPF6.3) is the first nitrate transporter identified in plants and was initially found to play a role in nitrate uptake and transport. AtNRT1.1 also displays auxin transport activity and mediates nitrate-modulated root development, suggesting that it has transport capacity for multiple substrates. Subsequent work revealed that AtNRT1.1 can respond to environmental nitrate fluctuations by altering its nitrate transport activity, modulated by phosphorylation, leading to the critical finding that AtNRT1.1 acts as a transceptor for nitrate sensing. Recent studies have revealed how OsNRT1.1B, the functional homologue of AtNRT1.1 in rice, mediates nitrate signal transduction from the plasma membrane to the nucleus, and how OsNRT1.1B integrates the nitrate and phosphate signaling networks. OsNRT1.1B has also been shown to be involved in regulating the root microbiota to facilitate organic nitrogen mineralization in soil, thus mediating plant–microbe interactions. Furthermore, the divergent functions of OsNRT1.1A and OsNRT1.1B in regulating nitrogen use in rice suggest that the function of NRT1.1 is still far from fully understood. In this review, we focus on the most recent progress on the molecular mechanisms of NRT1.1s in plants, with the aim of providing an up-to-date view of the versatile functions of NRT1.1 in nitrogen utilization in plants.

Nitrate regulation of lateral root and root hair development in plants.

Nitrogen (N) is one of the most important macronutrients for plant growth and development. However, the concentration and distribution of N varies in soil due to a variety of environmental factors. In response, higher plants have evolved a developmentally flexible root system to efficiently take up N under N-limited conditions. Over the past decade, significant progress has been made in understanding this form of plant ‘root-foraging’ behavior, which is controlled by both a local and a long-distance systemic nitrate signaling pathway. In this review, we focus on the key components of nitrate perception, signaling, and transduction and its role in lateral root development. We also highlight recent findings on the molecular mechanisms of the nitrate systemic signaling pathway, including small signaling peptides involved in long-distance shoot–root communication. Furthermore, we summarize the transcription factor networks responsible for nitrate-dependent lateral root and root hair development.

Downstream evolution of wastewater treatment plant nutrient signals using high‐temporal monitoring

AbstractWastewater treatment plants are major point‐sources of nutrients to streams globally, but the impact on receiving streams is not always clear. Previous research has shown mixed responses in receiving streams, with some showing no net retention through in‐stream processing for large distances below plants and some showing high rates of processing and retention. This study focuses on Sandy Run, a small, suburban stream in Montgomery County, PA, that receives effluent from two plants, where effluent makes up an estimated 50% of outlet discharge at baseflow. Two sites were monitored in late summer baseflow using high‐temporal loggers to evaluate nitrate and phosphate retention with distance below the plants. Effluent quantity was monitored immediately below the effluent outfalls using specific conductivity as a conservative signal of solute fluctuations throughout the day. A site 1 km downstream showed diel nitrate changes, but despite moderate gross primary productivity and ecosystem respiration rates, there was little net retention of nutrients and the diel nitrate signal can be attributed to advection and dispersion of variable upstream effluent. A site 5.4 km below the plant showed a diel nitrate signal as well, but baseflow daily hysteresis plots of nitrate and specific conductivity showed the effluent and nitrate peaks did not coincide. Instead, the effluent input signal was seen overnight, but there was in‐stream removal and release processes during the day. Over the distance to this site, the stream was metabolizing some of the high nutrient loads, although gross primary productivity and ecosystem respiration rates were lower. It is important to understand subdaily changes in nutrient processing to fully quantify the impacts of effluent on small streams at different scales. Furthermore, looking at the diel signal without considering conservative transport would overestimate in‐stream processing.

Nitrate Induction of Primary Root Growth Requires Cytokinin Signaling in Arabidopsis thaliana.

Nitrate can act as a potent signal to control growth and development in plants. In this study, we show that nitrate is able to stimulate primary root growth via increased meristem activity and cytokinin signaling. Cytokinin perception and biosynthesis mutants displayed shorter roots as compared with wild-type plants when grown with nitrate as the only nitrogen source. Histological analysis of the root tip revealed decreased cell division and elongation in the cytokinin receptor double mutant ahk2/ahk4 as compared with wild-type plants under a sufficient nitrate regime. Interestingly, a nitrate-dependent root growth arrest was observed between days 5 and 6 after sowing. Wild-type plants were able to recover from this growth arrest, while cytokinin signaling or biosynthesis mutants were not. Transcriptome analysis revealed significant changes in gene expression after, but not before, this transition in contrasting genotypes and nitrate regimes. We identified genes involved in both cell division and elongation as potentially important for primary root growth in response to nitrate. Our results provide evidence linking nitrate and cytokinin signaling for the control of primary root growth in Arabidopsis thaliana.

A wheat transcription factor positively sets seed vigour by regulating the grain nitrate signal.

Summary Seed vigour and early establishment are important factors determining the yield of crops. A wheat nitrate‐inducible NAC transcription factor, TaNAC2, plays a critical role in promoting crop growth and nitrogen use efficiency (NUE), and now its role in seed vigour is revealed. A TaNAC2 regulated gene was identified that is a NRT2‐type nitrate transporter TaNRT2.5 with a key role in seed vigour.Overexpressing TaNAC2‐5A increases grain nitrate concentration and seed vigour by directly binding to the promoter of TaNRT2.5‐3B and positively regulating its expression. TaNRT2.5 is expressed in developing grain, particularly the embryo and husk.In Xenopus oocyte assays TaNRT2.5 requires a partner protein TaNAR2.1 to give nitrate transport activity, and the transporter locates to the tonoplast in a tobacco leaf transient expression system. Furthermore, in the root TaNRT2.5 and TaNRT2.1 function in post‐anthesis acquisition of soil nitrate. Overexpression of TaNRT2.5‐3B increases seed vigour, grain nitrate concentration and yield, whereas RNA interference of TaNRT2.5 has the opposite effects. The TaNAC2‐NRT2.5 module has a key role in regulating grain nitrate accumulation and seed vigour. Both genes can potentially be used to improve grain yield and NUE in wheat.

The calcium sensor OsCBL1 modulates nitrate signaling to regulate seedling growth in rice.

Nitrate signaling integrates and coordinates gene expression and plant growth; however, the underlying molecular mechanisms involved remain poorly understood. Our previous study revealed that rice calcineurin B-like protein 1 (OsCBL1) modulates lateral root elongation by affecting auxin biosynthesis. Here, we report that OsCBL1 also modulates nitrate signaling to regulate rice seedlings growth. Compared with wild-type seedlings, seedlings of OsCBL1-knockdown (OsCBL1-KD) plants showed a suppressed growth phenotype, which included reduced root and shoot fresh weights and shorter radicles, crown roots, and lateral roots, when grown in nitrogen-free conditions. Although the growth defects of OsCBL1-KD plants could be partially rescued by the addition of nitrate to the growth conditions, the nitrate uptake capability of the OsCBL1-KD plants did not differ from that of wild-type plants as assessed via nitrate content and 15NO3− influx experiments. The nitrate-regulated expression of nitrate signal sentinel genes (OsNRT2.1 and OsNRT2.2) was affected in the OsCBL1-KD plants under both long- and short-term nitrate treatments. Overall, our results showed a novel role for OsCBL1 in the regulation of nitrate signaling and nitrate-mediated rice growth.

Nitrate inhibits primary root growth by reducing accumulation of reactive oxygen species in the root tip in Medicago truncatula

In Medicago truncatula, nitrate, acting as a signal perceived by NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER FAMILY 6.8 (MtNPF6.8), inhibits primary root growth through a reduction of root cell elongation. Since reactive oxygen species (ROS) produced and converted in root tip (O2•− → H2O2 → •OH) have been reported to control cell elongation, the impact of nitrate on the distribution of these ROS in the primary root of M. truncatula was analyzed. We found that nitrate reduced the content of O2•−, H2O2 and •OH in the root tip of three wild type genotypes sensitive to nitrate (R108, DZA, A17), inhibition of root growth and O2•− accumulation being highly correlated. Nitrate also modified the capacity of R108 root tip to produce or remove ROS. The ROS content decrease observed in R108 in response to nitrate is linked to changes in peroxidase activity (EC1.11.1.7) with an increase in peroxidative activity that scavenge H2O2 and a decrease in hydroxylic activity that converts H2O2 into •OH. These changes impair the accumulation of H2O2 and then the accumulation of •OH, the species responsible for cell wall loosening and cell elongation. Accordingly, nitrate inhibitory effect was abolished by externally added H2O2 or mimicked by KI, an H2O2 scavenger. In contrast, nitrate has no effect on ROS production or removal capacities in npf6.8-2, a knockdown line insensitive to nitrate, affected in the nitrate transporter MtNPF6.8 (in R108 background) by RNAi. Altogether, our data show that ROS are mediators acting downstream of MtNPF6.8 in the nitrate signaling pathway.

Feedforward Control of Plant Nitrate Transporter NRT1.1 Biphasic Adaptive Activity

Defective nitrate signaling in plants causes disorder in nitrogen metabolism, and it negatively affects nitrate transport systems, which toggle between high- and low-affinity modes in variable soil nitrate conditions. Recent discovery of a plasma membrane nitrate transceptor protein NRT1.1—a transporter cum sensor—provides a clue on this toggling mechanism. However, the general mechanistic description still remains poorly understood. Here, we illustrate adaptive responses and regulation of NRT1.1-mediated nitrate signaling in a wide range of extracellular nitrate concentrations. The results show that the homodimeric structure of NRT1.1 and its dimeric switch play an important role in eliciting specific cytosolic calcium waves sensed by the calcineurin-B-like calcium sensor CBL9, which activates the kinase CIPK23, in low nitrate concentration that is, however, impeded in high nitrate concentration. Nitrate binding at the high-affinity unit initiates NRT1.1 dimer decoupling and priming of the Thr101 site for phosphorylation by CIPK23. This phosphorylation stabilizes the NRT1.1 monomeric state, acting as a high-affinity nitrate transceptor. However, nitrate binding in both monomers, retaining the unmodified NRT1.1 state through dimerization, attenuates CIPK23 activity and thereby maintains the low-affinity mode of nitrate signaling and transport. This phosphorylation-led modulation of NRT1.1 activity shows bistable behavior controlled by an incoherent feedforward loop, which integrates nitrate-induced positive and negative regulatory effects on CIPK23. These results, therefore, advance our molecular understanding of adaptation in fluctuating nutrient availability and are a way forward for improving plant nitrogen use efficiency.

Methods for Measuring Nitrate Reductase, Nitrite Levels, and Nitric Oxide from Plant Tissues.

Nitrogen (N) is one of the most important nutrients which exist in both inorganic and organic forms. Plants assimilate inorganic form of N [nitrate (NO3-), nitrite (NO2-) or ammonium (NH4+)] and incorporate into amino acids. The metabolism of N involves a series of events such as sensing, uptake, and assimilation. The initial stage is sensing, triggered by nitrate or ammonium signals initiating signal transduction processes in N metabolism. The assimilation pathway initiates with NO3-/NH4+ transport to roots via specific high and low affinity (HATs and LATs) nitrate transporters or directly via ammonium transporters (AMTs). In cytosol the NO3- is reduced to NO2- by cytosolic nitrate reductase (NR) and the produced NO2- is further reduced to NH4+ by nitrite reductase (NiR) in plastids. NR has capability to reduce NO2- to nitric oxide (NO) under specific conditions such as hypoxia, low pH, and pathogen infection. The produced NO acts as a signal for wide range of processes such as plant growth development and stress. Here, we provide methods to measure NR activity, NO2- levels, and NO production in plant tissues.

Phosphorylation-Mediated Dynamics of Nitrate Transceptor NRT1.1 Regulate Auxin Flux and Nitrate Signaling in Lateral Root Growth.

The dual-affinity nitrate transceptor NITRATE TRANSPORTER1.1 (NRT1.1) has two modes of transport and signaling, governed by Thr-101 (T101) phosphorylation. NRT1.1 regulates lateral root (LR) development by modulating nitrate-dependent basipetal auxin export and nitrate-mediated signal transduction. Here, using the Arabidopsis (Arabidopsis thaliana) NRT1.1T101D phosphomimetic and NRT1.1T101A nonphosphorylatable mutants, we found that the phosphorylation state of NRT1.1 plays a key role in NRT1.1 function during LR development. Single-particle tracking revealed that phosphorylation affected NRT1.1 spatiotemporal dynamics. The phosphomimetic NRT1.1T101D form showed fast lateral mobility and membrane partitioning that facilitated auxin flux under low-nitrate conditions. By contrast, nonphosphorylatable NRT1.1T101A showed low lateral mobility and oligomerized at the plasma membrane (PM), where it induced endocytosis via the clathrin-mediated endocytosis and microdomain-mediated endocytosis pathways under high-nitrate conditions. These behaviors promoted LR development by suppressing NRT1.1-controlled auxin transport on the PM and stimulating Ca2+-ARABIDOPSIS NITRATE REGULATED1 signaling from the endosome.

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.

Evolutionary analyses of NIN-like proteins in plants and their roles in nitrate signaling.

Nitrogen (N) is one of the most important essential macro-elements for plant growth and development, and nitrate represents the most abundant inorganic form of N in soils. The nitrate uptake and assimilation processes are finely tuned according to the available nitrate in the surroundings as well as by the internal finely coordinated signaling pathways. The NIN-like proteins (NLPs) harbor both RWP-RK, and Phox and Bem1 (PB1) domains, and they belong to the well-characterized plant-specific RWP-RK transcription factor gene family. NLPs are known to be involved in the nitrate signaling pathway by activating downstream target genes, and thus they are implicated in the primary nitrate response in the nucleus via their RWP-RK domains. The PB1 domain is a ubiquitous protein-protein interaction domain and it comprises another regulatory layer for NLPs via the protein interactions within NLPs or with other essential components. Recently, Ca2+-Ca2+ sensor protein kinase-NLP signaling cascades have been identified and they allow NLPs to have central roles in mediating the nitrate signaling pathway. NLPs play essential roles in many aspects of plant growth and development via the finely tuned nitrate signaling pathway. Furthermore, recent studies have highlighted the emerging roles played by NLPs in the N starvation response, nodule formation in legumes, N and P interactions, and root cap release in higher plants. In this review, we consider recent advances in the identification, evolution, molecular characteristics, and functions of the NLP gene family in plant growth and development.