# Nitrate Sensing and Signaling in Plants: Comparative Insights and Nutritional Interactions

Sandrine Ruffel, Jorge del Rosario, Benoît Lacombe, Hatem Rouached,
Rodrigo A Gutiérrez, Gloria M Coruzzi, Gabriel Krouk

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Sandrine Ruffel, Jorge del Rosario, Benoît Lacombe, Hatem Rouached, Rodrigo A Gutiérrez, et al. Nitrate Sensing and Signaling in Plants: Comparative Insights and Nutritional Interactions. Annual Review of Plant Biology, 2025, 76 (1), pp.25-52. 10.1146/annurev-arplant-083123-053039. hal-05076570

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*Annual Review of Plant Biology*

# Nitrate Sensing and Signaling
in Plants: Comparative Insights
and Nutritional Interactions

Sandrine Ruffel,<fnref n="1" />,* Jorge Del Rosario,<fnref n="2" />,*,
Benoît Lacombe,<fnref n="1" /> Hatem Rouached,<fnref n="3" />

Benoît Lacombe,<fnref n="1" /> Hatem Rouached,<fnref n="3" />

Rodrigo A. Gutiérrez,<fnref n="2" /> Gloria M. Coruzzi,<fnref n="4" />

and Gabriel Krouk<fnref n="1" />

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Annu. Rev. Plant Biol. 2025. 76:25–52

The *Annual Review of Plant Biology* is online at
plant.annualreviews.org

https://doi.org/10.1146/annurev-arplant-083123-053039

Copyright © 2025 by the author(s). This work is
licensed under a Creative Commons Attribution 4.0
International License, which permits unrestricted
use, distribution, and reproduction in any medium,
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See credit lines of images or other third-party
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## Keywords

nitrate, primary nitrate response, nitrogen starvation response, nitrate sensing, plants, bacteria, fungi, algae, nitrogen use efficiency, phosphorus, potassium, systemic signaling, carbon signaling

## Abstract

Plant nitrogen nutrition is an essential and energy-costly component of terrestrial food chains. Understanding nitrate sensing in plants can lead to improved crop yields and nutrient use efficiency, directly impacting food security and agricultural sustainability. Herein, we review and present a comprehensive framework for understanding nitrate sensing in plants, integrating molecular, genetic, and physiological aspects. We begin by detailing the primary nitrate response and nitrate starvation response, which are central to the plant's ability to sense and respond to nitrate availability. We then explore the intricate interactions between nitrate signaling and other nutritional pathways such as those for carbon, phosphorus, potassium, and sulfur assimilation and reactive oxygen species (ROS) handling, and how it unfolds in long-distance systemic communication between roots and shoots.

<footnote n="1">¹IPSiM, Université de Montpellier, CNRS, INRAE, Montpellier, France;
email: gkrouk@gmail.com</footnote>

<footnote n="2">²Facultad de Ciencias Biológicas, Institute for Integrative Biology (iBio), Center for Genome Regulation (CRG), Institute of Ecology and Biodiversity (IEB), Pontificia Universidad Católica de Chile, Santiago, Chile</footnote>

<footnote n="3">³Department of Plant, Soil, and Microbial Sciences, The Plant Resilience Institute, Michigan State University, East Lansing, Michigan, USA</footnote>

<footnote n="4">⁴Department of Biology, Center for Genomics and Systems Biology, New York University, New York, NY, USA</footnote>

<footnote>*These authors contributed equally to this article</footnote>
Finally, evolutionary insights are provided by comparing nitrate-sensing mechanisms across different plant species as well as Bacteria, Archaea, Chlorophyta, Charophyta (algae), and Fungi, revealing how these mechanisms may have evolved in diverse ecological niches. This review not only provides a framework to project our present and future understanding of plant nitrate and nitrogen nutrition but also offers potential strategies for improving nutrient use efficiency in crops through genetic and biotechnological interventions.

## Contents

<table><tr><td>1. INTRODUCTION</td><td>26</td></tr><tr><td>2. NITRATE SENSING AND RELATED SIGNALING CASCADES IN PLANTS</td><td>27</td></tr><tr><td>2.1. Definition of the Primary Nitrate Response</td><td>27</td></tr><tr><td>2.2. The Primary Nitrate Response: The Molecular Backbone (NRT1.1, CPKs, and NLP7)</td><td>28</td></tr><tr><td>2.3. NLP7: From a Pioneer Central Regulator to a Nitrate Sensor</td><td>28</td></tr><tr><td>2.4. N Dose Response to Nitrate</td><td>30</td></tr><tr><td>2.5. Nitrogen Starvation Response</td><td>31</td></tr><tr><td>2.6. Nitrogen Starvation Response/Primary Nitrate Response Interrelationships</td><td>31</td></tr><tr><td>2.7. Signaling Interactions</td><td>32</td></tr><tr><td>3. COMPARISON OF CORE SENSING AND SIGNALING MECHANISMS</td><td>39</td></tr><tr><td>3.1. Bacterial Sensor Systems</td><td>39</td></tr><tr><td>3.2. Archaea Sensor System</td><td>41</td></tr><tr><td>3.3. Fungal Sensor System</td><td>41</td></tr><tr><td>3.4. Viridiplantae Sensor Systems</td><td>41</td></tr><tr><td>3.5. Comparison of the Nitrate-Sensing Systems</td><td>42</td></tr><tr><td>4. CONCLUSIONS AND GENERAL REMARKS</td><td>43</td></tr></table>

## 1. INTRODUCTION

The plant kingdom exhibits a unique proficiency in utilizing nitrate ($NO_3^−$) as a nitrogen (N) source, and it has been hypothesized to be an important byproduct of the soil microbial activity (15). Conversely, plants can use reduced forms of N such as ammonium ($NH_4^+$), amino acids, peptides, and urea, for example, which are less energetically costly to assimilate but are not extremely abundant in soils because they are substrates of microbial nutrition (15). This competition between soil microbes and plants is likely why nitrate serves as the primary source of inorganic N for plants in aerobic soil conditions (15, 31). Due to its mobility, which is partly influenced by its own negative charge and the negative charge of clay, the concentration of $NO_3^−$ ions in the soil solution can vary significantly, by as much as four orders of magnitude (31, 148). In this context, plants evolved several adaptive strategies to mitigate the effect of these fluctuations (117, 146, 159), in particular, by developing sensory functions linked to molecular signaling pathways that are the focal point of this review. The discovery of these sensory functions highlights the fact that nitrate is much more than an N source; it is also a very potent signaling molecule for plants as well as for other organisms.
Nitrate is known to control almost every aspect of plant physiology and development. This ranges from its very well-described action on (a) root and shoot development, in particular, through intimate mechanisms that involve changes in hormonal fluxes in meristems (1, 76, 79, 81, 83); (b) transpiration via stomatal opening (54); (c) flowering transition (51, 130); (d) germination (2); (e) shoot branching (33); and (f) shoot development through the control of ploidy (109). Numerous recent reviews have been published, elegantly detailing these mechanisms and their associated molecular networks (117, 146, 159, 163).

Thus, we decided to focus this review on establishing a specific framework that defines the core molecular pathways controlling and delineating the nitrate response, namely the primary nitrate response (PNR), and its counterpart, the nitrogen starvation response (NSR) (Section 2). In Section 3 we focus on how these central signaling pathways interact with other nutrition-related pathways and unfold in systemic relationships, and in Section 4 we explain the evolutionary understanding of the nitrate-sensing mechanisms found in plants and how they compare with those in other branches of life. Taken together, we believe this offers a framework to comprehend current knowledge on nitrate signaling organization and to position future discoveries in this area.

# 2. NITRATE SENSING AND RELATED SIGNALING CASCADES IN PLANTS

## 2.1. Definition of the Primary Nitrate Response

Nitrate provision profoundly influences gene expression in plants, playing a crucial role in their acclimation to fluctuating nutritional environments. Depending on the conditions before or during nitrate application, this treatment can alter the expression of up to 10% of the genes in *Arabidopsis thaliana*. The initial molecular responses to nitrate are referred to as the PNR. The PNR describes the rapid and specific response of plants to nitrate, but this definition has evolved over time starting from the 1990s.

At least three different, nonexclusive definitions of the PNR exist in the literature, with the particular challenge being the definition of the term primary. The concept of the PNR was initially introduced in 1992 by Gowri et al. (50). It was described as the accumulation of maize nitrate reductase (NR) messenger RNA (mRNA) in the presence of a protein synthesis inhibitor. The authors observed that the protein synthesis inhibitor did not affect the transcriptional regulation of the *NR* gene when nitrate was present, leading them to suggest that the nitrate-sensing mechanism is constitutively expressed or at least long-lived. Consequently, this response can be considered primary, as it occurs without the need for new protein synthesis.

Secondly, the term primary could be associated with the fact that the response triggered by nitrate application is due to nitrate itself rather than a product derived from its assimilation (such as $NO_2^-$, $NH_4^+$, amino acids). The genesis of the PNR concept can thus also be traced back to research in the late 1990s, when scientists began to investigate the unique role of nitrate in plant signaling and metabolism. The seminal work by Crawford & Glass (31) first proposed nitrate's dual function in plants, highlighting its importance in both nutrition and signaling. This distinction can be identified in two ways. One method involves studying the gene expression response to nitrate in an NR-null mutant (160), a deletion of the major *nitrate reductase (NIA)2* gene, and an insertion in the *NIA1* gene. NR is the initial enzyme in the assimilation pathway, converting $NO_3^-$ to $NO_2^-$.
If changes in gene expression are observed following nitrate treatment in this genetic background (an NR-null mutant), they can be attributed to nitrate itself and not a product of its assimilation.

The third approach to defining the term primary involves using time as a discriminating factor. The genome-wide dynamics of the PNR have been extensively studied in *Arabidopsis*, revealing rapid gene expression changes in response to nitrate application (80, 158, 161). These studies

NR: nitrate reductase

NIA: *nitrate reductase*
gene
NIR: nitrite reductase

**G6PDH:** glucose
6-phosphate
dehydrogenase

**HRS1:** hypersensitive to
low P₁-elicited primary
root shortening 1

**HHO:** hypersensitive
to low P₁-elicited
primary root
shortening homologs
(also known as NIGTs)

**CIPK:** calcineurin
B-like interacting
protein kinase

**NIN:** nodule
inception

**TCP20:** TEOSINTE
BRANCHED 1,
CYCLOIDEA, PCF1
transcription factor
family 20

**NLP:** NIN-like
protein

observed gene regulation as early as 3–5 min after treatment. Specifically, they identified at least two waves of gene expression (80) with different dynamics in roots and shoots (158). The rapid response timescale (within minutes) is likely incompatible with the effects of potential assimilation products. Additionally, nitrate-responsive genes such as *nitrite reductase* (NIR) respond to nanomolar concentrations of nitrate (161), further indicating that these responses are likely due to nitrate itself and not its assimilation products.

In conclusion, the PNR can be defined using at least three nonexclusive criteria, encompassing kinetic, genetic, and pharmacological contexts (fully discussed in 105).

## 2.2. The Primary Nitrate Response: The Molecular Backbone (NRT1.1, CPKs, and NLP7)

The PNR is activated when nitrate-depleted or N-depleted plants are supplied with nitrate (Figure 1a), triggering a rapid response within minutes (62, 80, 161). Sentinel genes of the PNR, such as *NIA1*, *NIR1*, and *glucose 6-phosphate dehydrogenase* (G6PDH), are swiftly regulated by nitrate (80, 158). Transcription factor genes such as *hypersensitive to low P₁-elicited primary root shortening 1* (*HRS1*) and *HRS1 homolog 1* (*HHO1*) are also sentinel genes but play a crucial role in the control of the NSR (see Section 2.6).

The PNR is the most thoroughly studied and understood N-related signaling pathway, with several key molecular players identified. These include the nitrate transceptor NRT1.1 (aka CHL1/NPF6.3). NRT1.1 was first identified as a dual-affinity nitrate transporter. The gene was cloned as the corresponding mutants (*chl1*) are chlorate (a toxic analog of nitrate) resistant (93, 155). Numerous observations were made showing that the transport activity of NRT1.1 and different phenotypes related to development (129) or gene regulation (82, 112) could be decorrelated. Definitive proof of the role of NRT1.1 as a nitrate sensor was published in 2009, when a mutant was isolated that genetically decoupled the transport and the sensing activity of the protein (62). Outside of the PNR context, NRT1.1 also displays a nitrate-sensing function that involves its auxin transport activity. This mechanism controls lateral root development in response to nitrate (79) but falls out of the scope of this review as it has so far not been related to the PNR or NSR. Key research has highlighted the importance of nitrate-triggered calcium signaling as a central link between the NRT1.1 transceptor and the nuclear events that regulate the PNR (77, 92, 132). Thus, the molecular backbone also includes a range of kinases and phosphatases such as calcineurin B-like interacting protein kinase 8 (CIPK8) (66), CIPK23 (62), CPK10, CIPK30, CIPK32 (92), and ABI2 (88). Additionally, several transcription factors play crucial roles in the PNR, including nodule inception (NIN)-like protein 6 (NLP6)/NLP7 (20, 162) (details in Section 2.3), TGA1 (3), NRG2 (167), TCP20 (TEOSINTE BRANCHED 1, CYCLOIDEA, PCF1 transcription factor family 20) (52), and SPL9 (80). We propose that the NRT1.1/kinases/NLP axis can be considered a molecular backbone of the PNR (77) (Figure 1).

## 2.3. NLP7: From a Pioneer Central Regulator to a Nitrate Sensor

Through the use of genetics, NLP7 was established as central to the PNR. At the same time, it was identified through reverse genetics, according to homology with the *Chlamydomonas* gene *NIT2* (20) (see Section 3) and a genetic screen for nitrate response (162). In both cases, the *nlp7* mutants display PNRs with reduced magnitude. More recently, this master transcription factor (4, 20, 74, 100, 162) was shown to also be a nitrate sensor (91). Once nitrate is transported or sensed by NRT1.1, a rapid wave of $Ca^{2+}$ causes the activation of group III calcium-sensor protein kinases (CPKs) in seconds, which in turn phosphorylates NLP7 to retain it in the nucleus, activating early nitrate-response genes within minutes (92, 100) (Figure 1). Its action is related
<figure><img src="image_4.png" /><figcaption>Figure 1</figcaption></figure>

Relationships between the three core nutritional responses: (a) the PNR, (b) the NSR, and (c) the PSR. (Top) The graphs illustrate the dynamics and sentinel genes used to evaluate these responses. (Bottom) The diagrams depict the different molecular relationships among the elements of these three pathways. Orange arrows indicate the influence of one pathway on various elements where a formal causal gene has not yet been identified but regulation has been established (or vice versa for PHO2). Genes shown in red are considered effectors of the pathway, triggering the appropriate metabolic or physiological response (e.g., NRT2.4 and NRT2.5 trigger the very high-affinity nitrate response upon NSR activation). Sensed ions/elements as well as their sensors are shown in purple. Abbreviations: CBL7, calcineurin B-like 7; CPK, calcium-sensor protein kinase; GDH3, glutamate dehydrogenase 3; HHO, HRS1 homolog; HRS1, hypersensitive to low *P*<sub>i</sub>-elicited primary root shortening 1; mRNA, messenger RNA; N, nitrogen; NIGT, nitrate-induced GARP transcription factor; NIR, nitrite reductase; NLA, NITROGEN LIMITATION ADAPTION; NLP, NIN-like protein; NO<sub>3</sub><sup>-</sup>, nitrate; NRT, nitrate transporter; NSR, nitrogen starvation response; P, phosphorus; PHO, phosphate; PHR, phosphate response; PHT1, phosphate transporter 1; PNR, primary nitrate response; PSR, phosphate starvation response; ROS, reactive oxygen species.

to the transcription factor subfamily, as NLP7 has been found to interact with NLP6, and both
are retained in the nucleus in response to nitrate (27). Another level of nitrate regulation has
been uncovered, as NLP7 has been shown to bind nitrate and act as an intracellular nitrate sen-
sor using the genetically encoded split mCitrine-NLP7 nitrate biosensor (sCiNiS) (91). The role
of NLP7 in mediating the dynamics of the PNR comes from time-series studies of the NLP7
mode of action following nuclear import (4). Specifically, these studies implicate NLP7 as a puta-
tive pioneer transcription factor acting in a hit-and-run mode of transcription. The hit-and-run
model supported for NLP7 (4), first described for another master transcription factor, the general
N-response bZIP1, follows the hypothesis that a transcription factor trigger/pioneer can form a stable transcriptional complex (the hit), allowing transcription to continue even after the initiat-ing transcription factor is no longer bound (the run) (4, 38, 121, 141). Similar results implicated a hit-and-run mechanism for NLP7 to initiate a cascade of early nitrate-responsive downstream transcription factors in the PNR (4). Time-series chromatin immunoprecipitation-sequencing (ChIP-seq) experiments conducted in a plant cell-based transcription factor perturbation assay called TARGET (9) showed that NLP7 were transiently bound to early nitrate-response genes, and 4-thiouracil-labeling of nascent mRNA confirmed the active transcription of these hit-and-run targets. The transient binding of NLP7 to these early targets was confirmed by DamID-seq methylation. DamID-seq uses a fusion protein of DNA adenine methyltransferase (Dam) to detect transcription factor-DNA-binding events by leaving a stable methylation mark at the adenine base in GAmeTC sequences within 1 kb from transcription factor-DNA-binding sites (4). Overall, the multiple levels of NLP7 regulation highlight the important role of NLP7 in the PNR to ensure fast and broad acclimation by the plant to fluctuating nitrate levels (Figure 1).

## 2.4. N Dose Response to Nitrate

Nitrate sensing enables plants to reprogram their genome-wide expression and alter metabolism and development. But as mentioned above (Section 2.1), the concentration of this ion can also vary dramatically by orders of magnitude (31). Thus, the magnitude of the response (the peak at which the PNR sentinel genes respond to nitrate provision) is also a matter of research. In an initial observation of the magnitude of such a peak, Hu et al. (66) demonstrated that the transcriptional response of several sentinel genes belonging to the PNR follows a biphasic curve reminiscent of high-affinity and low-affinity transport activity. They also found that this response is regulated by CIPK8 (66). To explore the mechanism underlying plant response to N dose, Swift et al. (151) exposed *Arabidopsis* seedlings to a matrix of N doses (ammonium nitrate) and monitored genome-wide expression changes as a function of time. Mathematical modeling of this RNA-sequencing data revealed that the dynamics of transcriptomic responses to N dose indeed mirrors simple enzyme kinetics described by Michaelis & Menten in 1913 (68). According to the Michaelis-Menten kinetics model, changing enzyme abundance will impact the maximum rate of reaction possible ($V_{max}$). Because transcription factors establish the rates at which transcription takes place, they can be directly analogized to the catalytic enzymes in the Michaelis-Menten model.

A gene whose expression significantly fit the Michaelis-Menten model enabled Swift et al. (151) to estimate the maximum rate of transcript change, known as $V_{max}$, as well as $K_m$, the dose of N at which half of $V_{max}$ was achieved. Using these parameters, the Michaelis-Menten kinetic model could explain the expression of 30% of N dose-responsive genes. The Michaelis-Menten model predicts the rate at which a catalyst can convert a substrate to its product. By analogy, transcription factors act in part as the catalyst that drives rates of transcript change. To test this, they investigated and demonstrated that altering transcription factor abundance in planta could impact the maximum rate at which transcript abundance changed ($V_{max}$) in responses to N dose and focused on TGA1, a master gene in the N response (3). Specifically, the overexpression of TGA1 leads to an increase in the maximum rate of transcript change in response to N dose ($V_{max}$), as predicted by the Michaelis-Menten model (151). To confirm that TGA1 sits downstream of the NRT1.1 nitrate transceptor, genes whose expression is dependent on NRT1.1 (16) were overlapped with genes regulated by TGA1 (17), as well as genes whose expression is modeled by Michaelis-Menten kinetics (151). The significant overlap between these gene lists may suggest that NRT1.1 and TGA1 are in the same pathway (Figure 1) that controls rates of transcriptional change in response to nitrate dose. Importantly, the impact of changing the level of TGA1 (overexpression or mutant
analysis of a TGA1/double mutant) affected the kinetics of the N-dose response at the molecular level and led to an acceleration in N dose-dependent plant growth rates (151). It is worth noting here that we independently refer to N or nitrate dose in this paragraph, as it has not been properly studied, and it is not yet possible to rule out that part of the observed signal may contain not only nitrate-related signals but potentially also signals from other N sources, as defined by Ristova et al. (131). However, the involvement of NRT1.1 in this pathway clearly indicates that nitrate is a component.

## 2.5. Nitrogen Starvation Response

The NSR is another critical signaling pathway that becomes active in plants when there is a prolonged (days) absence of N (and consequently of nitrate) in the environment, indicating a deficiency of N (Figure 1b). This response involves several molecular events and sentinel genes, such as those encoding the very high-affinity nitrate transporters NRT2.4 and NRT2.5, which are induced to capture the remaining traces of nitrate from the soil (72, 73, 99, 137). Additionally, *glutamate dehydrogenase 3 (GDH3)* is thought to be involved in N recycling under these conditions (137). Despite its importance, the NSR remains less understood at the molecular level compared to the PNR. Research has shown that the calcineurin B-like 7 (CBL7) calcium sensor affects the expression of *NRT2.4* and *NRT2.5* within the NSR context (98). The microRNA miR169 also plays a role in regulating NFYA transcription factors in response to N starvation, significantly impacting the transcription of *NRT2.1* and *NRT1.1* (169). However, the direct involvement of NFYA-encoding genes in the NSR has not been conclusively demonstrated. Overexpression of LBD37, LBD38, and LBD39 transcription factors influences anthocyanin production in N-deprived plants and potentially regulates nitrate transporters (133), including *NRT2.5*, although no direct regulatory targets for these transcription factors have been identified. More recently, three studies have identified GARP transcription factors (138), namely HRS1/HHOs [aka nitrate-induced GARP transcription factors (NIGTs)] as transcriptional repressors of *NRT2.4* and *NRT2.5* (73, 99, 137). Very interesting phenotypes emerged from these studies: A quadruple mutant for the subfamily of HHOs displays a twofold increase in its capacity to transport nitrate in the high-affinity range that is correlated to a better growth on N-containing media (137). Phenotypes of the quadruple *hbo* mutant are reminiscent of those that have been described for the *bt1/bt2* mutant, which may be involved in the NSR pathway, but this hypothesis has not been formally tested. Indeed, Araus et al. (6) determined that the *bt2/bt1* mutant overexpresses *NRT2* genes in low N conditions that improve N use efficiency. Taken together, this body of information supports defining the NSR as the counterpart to the PNR, as it represents a regulatory mode that signals nitrate and N hunger to the plant (Figure 1).

## 2.6. Nitrogen Starvation Response/Primary Nitrate Response Interrelationships

On one hand, the PNR triggers the regulation of many genes (up to 10% of the genome) (160). It is interesting to note that the vast majority of sentinel genes being strongly and robustly regulated by the PNR are central to the regulation of the NSR. These include *LBD37*, *LBD38*, *LBD39*, *HRS1*, *HHO1*, *HHO2*, *HHO3*, and *broad-complex*, *tramtrack*, and *bric-à-brac* (*BT*)1/*BT2*, as nicely demonstrated in a meta-analysis of several transcriptomes (19) (Figure 1). This makes perfect sense, as when nitrate is present (i.e., the PNR is activated), it triggers a nitrate satiety signal intended to shut down the NSR (73, 137). Here, we could ask if the reverse relationship is also true: Are the central regulators of the PNR under the control of NSR sentinels? To our knowledge, this has not been properly studied yet. However, the central regulators and sensors, namely NRT1.1

**NIGT:**
nitrate-induced GARP
transcription factor
(also known as HHO)

**BT:** broad-complex,
tramtrack, and
bric-à-brac
C: carbon

OPPP: oxidative
pentose phosphate
pathway

and NLP7, are not known to be strongly regulated by the NSR. However, one particularly strong
candidate could be PHO2. Indeed, it has been shown that PHO2 is induced by N starvation and
that the mutant displays defects in the PNR response (107). In conclusion, the reciprocal control
of the PNR by NSR marker genes is not yet obvious, although it might be expected, and it requires
further research to be formally established and may also involve other nutrient-related signaling
pathways as discussed in Section 2.7.

**2.7. Signaling Interactions**

This core regulatory module, made of the interaction of the PNR and the NSR (Figure 1), is not strictly isolated and is under the strong influence of other signaling pathways. In the following sections, we review the current knowledge of the external influence on the nitrate sensing pathways and how they are integrated between roots and shoots.

**2.7.1. Carbon interaction.** N transport and assimilation, especially when nitrate is their main source, require large amounts of energy (ATP), reducing power (NADPH), and C skeleton (2-oxoglutarate from the TCA cycle). All of these molecules are synthesized in the carbon (C) metabolic pathways tightly linking N and C metabolism through what are likely intricate regulatory networks (29). Evidence for C influence on the regulation of nitrate utilization was reported multiple times in the 1950s. As such, the physiological analysis of root nitrate uptake (influx and efflux) in relation to nitrate accumulation, translocation, and metabolism has been shown to vary during day/night cycles, mediated by the availability of photosynthesates transported to the roots (34, 35, 124). Accordingly, the genetic characterization of the nitrate transport system in *Arabidopsis* allowed researchers to show that the expressions of *NRT2.1* and *NRT1.1* are diurnally regulated, reaching a peak at the end of the day, and are maintained and induced by sucrose provision in the dark (86), similarly to what was previously shown for NR activity and expression (26).

The catalytic activity of the glycolytic enzyme hexokinase (HXK) is required for the glucose-mediated regulation of *NRT2.1* and *NRT1.1* (85). In particular, glucose 6-phosphate or products of its metabolism through the oxidative pentose phosphate pathway (OPPP) or an enzyme belonging to this pathway are involved (32, 87). However, the regulation of *NRT2.1* by the redox status (discussed in Section 2.7.4) may also suggest that the reducing power produced by the OPPP, namely the NADPH, could be also be part of the C-signaling branch (12, 21).

The sugar-dependent regulation of nitrate transport and assimilation is likely not plugged into
the PNR-related regulatory network, as suggested by the normal *NRT2.1* response to nitrate in
the hexokinase mutant *gin2-1* (32). Consistently, in a transcriptomic analysis of root responses
combining different N and light/C supplies, none of the known nitrate regulators of *NRT2.1*
(e.g., *TGA1*) displayed a light- or C-dependent expression (134).

Alternatively, the strategy to coordinate the expression of the final targets of the PNR (i.e., *NRT2.1*) with C metabolism may rely on the recruitment of different transcription factors, some of which are known to be involved in sugar/light signaling. In a cell-based system, bZIP1 was shown to be a master hit-and-run regulator (described in Section 2.3) hitting transiently a set of targets, including *NRT2.1* and *NLP3*, to propagate the nitrate signal (38, 121). Initially, the transcription factor bZIP1, acting downstream of HXK, had been shown to be repressed by exogenous sugar, resulting in a derepression of seedling growth (69). Actually, bZIP1, similar to other members of the C/S bZIP transcription factors, acts downstream of SnRK1 in a low-energy signaling pathway, positively controlling the expression of genes involved in branched-chain amino acid catabolism (57, 116, 125). Therefore, it would be interesting to understand whether bZIP1 controls independently the propagation of two seemingly opposite signals (i.e., the starvation signal stopping
growth versus the nitrate signal stimulating it) or instead corresponds to a general nutrient signal transducer allowing catalysis of the transcription of a large gene set.

The transcriptomic analysis of root responses combining N and C supplies was precisely designed to specifically identify molecular mechanisms regulating the expression of *NRT2* genes in response to N and C signal interaction (134). Ruffel et al. (134) isolated three transcription factors, never previously found in nitrogen-related transcriptomic data sets, based on their expression profiles depending on N and light provision. One of them is the transcription factor bHLH093, which partly controls the induction of *NRT2.1* and *NRT2.4* by light in combination with N deprivation or low nitrate supply (134). A new signaling cascade downstream of the transcription factor NLP7, described in Section 2.3, as a key activator of the nitrate-responsive gene regulatory network, has been characterized for its role in integrating nitrate and high light conditions to protect the photosynthesis machinery. In this pathway, NLP7 directly controls the transcription level of the two homeodomain-leucine zipper class I transcription factors, *HOMEBOX PROTEIN 52* (*HB52*) and *HB54*, whose high level of expression depends also on light intensity. The main target of HB52 and HB54 is *VAR2*, a gene encoding the FtsH2 subunit of the chloroplast FtsH protease, involved in the quality control of photosynthesis proteins (7). Recently, it has been shown that NLP7 is able to interact in vivo with NLP2. Interestingly, NLP2 also plays a role in the early nitrate response gene regulatory network but with a specific regulatory role in the expression of genes involved in glycolysis in nitrate supply conditions (44). Altogether, these findings illustrate how various types of branching in the PNR can coexist to integrate combinations of signals and nutrients.

### 2.7.2. Phosphorus interaction.
The homeostatic interaction between N and phosphorus (P) significantly influences plant responses to deficiencies in either nutrient (36, 60, 78). This influence is evident in how N or P availability regulates the expression of numerous N- or P-related genes (107). It is noteworthy that the choice of N source, such as nitrate rather than ammonium, is a potent modulator of phosphate starvation-response (PSR) gene expression (156). In *Arabidopsis*, the N and P signaling interplay is further evident in the NIGT1/HHO pathway, which inhibits the expression of P-related genes, including *SPX* genes, in an N-dependent manner (73, 99, 156) (Figure 1). Additionally, a member of the *SPX* gene family (144), *NITROGEN LIMITATION ADAPTATION (NLA)*, plays a critical role in regulating P homeostasis (122) (Figure 1). Initially identified for its involvement in N limitation responses, NLA encodes an E3 ligase that specifically requires UBC24 for polyubiquitylation (122). NLA associates specifically with the high-affinity $P_i$ transporter PHT1;4, and their interaction at the plasma membrane likely adjusts $P_i$ uptake activity (122). Consistently, genetic inactivation of NLA leads to P overaccumulation (122). NLA activity is dependent on PHOSPHATE 2 (PHO2) (122), and mutations in PHO2 result in $P_i$ overaccumulation in shoots (8, 10), thereby phenocopying the *nla* mutants. *PHO2* encodes a ubiquitin-conjugating E2 enzyme variant that operates downstream of the PHOSPHATE RESPONSE 1 (PHR1) transcription factor (10) (Figure 1) and regulates the abundance of the PHO1 protein, thereby influencing $P_i$ uptake and translocation (94). This elucidates the mechanism underlying the $P_i$ overaccumulation phenotype in *pho2* mutants in the presence of $P_i$ in the growth media. While PHO2 was originally identified for its role in $P_i$ homeostasis, it has become increasingly recognized for its role in N/P crosstalk. PHO2 modulates late phases of the PNR via mutual feedback control of NRT1.1, and *pho2* mutants demonstrate near-total abolishment of N control over the PSR (107) (Figure 1).

Integrative regulation of N and P homeostasis is conserved between dicotyledonous and monocotyledonous plant species, although the mechanisms may differ (64, 107). For instance, while the *Arabidopsis* PHR1 protein turnover increases under N starvation (107), the rice OsPHR3

SPX: SYG1, PHO81, and XPR1

NLA: NITROGEN LIMITATION ADAPTATION

UBC24: ubiquitin-conjugating enzyme 24
expression increases with N supplementation (150). Research in rice has revealed additional regulatory layers by revealing the interaction between the nitrate sensor NRT1.1B and the Pᵢ signaling repressor OsSPX4. Nitrate perception enhances their interaction, leading to the ubiquitylation and degradation of OsSPX4 through the recruitment of the E3 ubiquitin ligase OsNBIP1 by OsNRT1.1B. This degradation enables OsPHR2 to translocate into the nucleus and commence the transcription of PSR target genes (64). This complexity of N and P interactions in plants underscores the need for further research to better understand these mechanisms. This is particularly relevant in the light of shortcomings in strategies aimed at improving N and/or P homeostasis, raising questions about the need to untangle plant responses to N from responses to P and vice versa (36). One example highlighting this need is the inhibitory effect of P deficiency on root growth when nitrate availability is sufficient. Early studies have demonstrated that the highly nitrate-inducible GARP transcription factor, HRS1, and its close homolog, HHO1, repress primary root growth in response to P-deficiency conditions, but only in the presence of nitrate (106). This suggests that plants respond to P deficiency only in the presence of N in the environment. Moreover, research in *Arabidopsis* has revealed a surprising prioritization of stress response between N and P deficiencies. While P deficiency triggers extensive gene expression reprogramming, concurrent N and P deficiency results in a reduced transcriptional response, indicating a higher priority for N (107). Therefore, future studies should delve deeper into the molecular mechanisms integrating N and P signaling. By designing strategies to decouple the influence of N on P-deficiency responses and vice versa, we can potentially enhance plant resilience to N and P limitations.

### 2.7.3. Sulfur interaction.
One of the critical roles of N in cell function is its involvement in amino acid biosynthesis, including the synthesis of sulfur (S)-containing amino acids such as cysteine and methionine (164). While N metabolism provides the essential amino groups and precursor molecules, such as serine, for cysteine synthesis, S metabolism supplies the requisite sulfide to complete the process (164). This highlights the crucial coordination between N and S homeostasis in plants to meet their metabolic demands. Plants maintain N content in their tissues through tightly regulated mechanisms to prevent both excessive accumulation and deficiency of these essential nutrients. For instance, S availability influences N uptake, as shown by studies on the transcriptional regulation of N uptake transporters under S deficiency (37). These studies reveal that transcription factors regulating S-deficiency responses also affect N-related genes. For example, in *Arabidopsis*, SULFUR LIMITATION 1 [SLIM1, also known as ETHYLENE-INSENSITIVE3-LIKE 3 (EIL3)], a key regulator of S metabolism, significantly regulates N transporters (104). Mutations in *SLIM1* reduce the expression of nitrate transporters such as *NRT2.5* and upregulate *NRT1.7* and *NTR1.9* in *Arabidopsis* (37). Additionally, overexpression of *SLIM1* under the 35S promoter (35S::*SLIM1* lines) results in significantly reduced nitrate levels within a month after sowing, with further decreases as the plants mature (5). These findings strongly indicate that overexpressing the SLIM1 transcription factor accelerates developmental senescence by reducing N accumulation (5). Interestingly, GLUTAMINE SYNTHETASE 2 (GS2), an enzyme essential for N assimilation and metabolism, is consistently downregulated during senescence (59). However, it was observed to be upregulated in the *slim1* mutant (37). This finding suggests that SLIM1 negatively regulates GS2 during plant senescence, highlighting its critical role in coordinating S and N signaling pathways. This genetic regulation enables plants to adapt their metabolic demands to different developmental stages (37, 104). While significant progress has been made in understanding N and S metabolism and their interconnections, much remains to be learned about the precise role of SLIM1 in this interplay, as well as the mechanisms of N and S sensing and signaling crosstalk in plants. This knowledge is crucial for developing
strategies to improve the efficiency of N and S use and enhance crop tolerance to deficiencies in
these essential nutrients.

**2.7.4. Reactive oxygen species interaction.** Reactive oxygen species (ROS) are byproducts of aerobic metabolism that can cause damage to cells, but they are now recognized as key regulators and signaling molecules (108). It was shown that under N starvation, ROS levels increase in *Arabidopsis* roots (147). More recent studies in *Arabidopsis* revealed that ROS play a crucial role in NSR signal transduction (137) (Figure 1). N deprivation triggers the accumulation of hydrogen peroxide (H₂O₂ at least), a versatile signaling modulator. Inhibiting H₂O₂ accumulation with agents such as potassium iodide and diphenyleneiodonium almost totally abolishes the NSR (137). Interestingly, HHOs have been shown to repress ROS production, creating a parallel way to control the NSR via controlling redox status (137) (Figure 1). Other studies have highlighted the role of ROS in N-related signaling; however, they are beyond the scope of this review, as they do not directly pertain to the core signaling pathways, specifically the PNR and NSR.

**2.7.5. Potassium interaction.** The relationship between nitrate and potassium (K+) is much less documented, in particular at the molecular level that could help to decipher whether nitrate sensing is influenced by K+ availability. Concerning physiology, in most plant species, the uptake rates of K+ and nitrate from the soil seem positively correlated. This effect can be explained by the improved charge balance during nutrient uptake and long-distance transport and by the K+-induced activation of the enzymes involved in nitrate assimilation. For instance, plants grown in the presence of nitrate take up and accumulate more K+ than when grown with NH₄⁺. However, little is known about the direct influences of one ion on the transport of the other (30). We can, however, hypothesize that such a connection exists, as K+ is the main cation in the xylem sap and thus the most probable counterion to nitrate transported to aerial parts. In leaves, nitrate is stored inside vacuoles, again using K+ as a counterion, where both ions contribute to osmotic adjustment (11). So it is not absurd to hypothesize that K+ availability may have an influence on nitrate signaling. The influence of K starvation over N transcriptomic response was noted by Kellermeier et al. (71); they identified genes being under the combinatorial control of both nutrients. However, none of the sentinel genes known to mark the PNR nor the NSR are included. It is important to note that Kellermeier et al. (71) provided the combination of nutritional deprivations at the same time. Maybe K+ deprivation preceding nitrate treatment would change the dynamics of the PNR, but this has to be experimentally tested.

Interestingly, a set of experiments have started to shed light on a potential N/K crosstalk mechanism. The key component identified to date is the transporter NRT1.5/NPF7.3, initially identified as a nitrate efflux transporter and later as a potassium transporter. NRT1.5/NPF7.3 can affect K+ translocation (from roots to shoots) under low-nitrate conditions (39). NRT1.5 is also involved in xylem K+ and nitrate transport under K+ limitation (89). Later, myeloblastosis transcription factor 59 (MYB59) was shown to regulate *NRT1.5* expression and balance K+/nitrate transport (42). Under sufficient conditions, MYB59 binds to the *NRT1.5* promoter, enhancing nutrient (K+ and nitrate) translocation. Conversely, under deficient conditions, MYB59 is down-regulated, reducing *NRT1.5* transcript levels. This highlights MYB59's role in coregulating K+ and nitrate transport in the xylem and suggests that it could be a key factor in signaling crosstalk (42). However, this will need experimental investigation.

Finally, it is tempting to hypothesize that CIPK23 may play a role in the relationship between K+ and nitrate uptake. Indeed, CIPK23 was first identified to regulate AKT1, a main K+ channel (166). Later, CIPK23 was also shown to directly regulate the transport activity of NRT1.1 (an important nitrate transporter and sensor) (62). By integrating signals related to K+ and nitrate availability, CIPK23 helps plants adjust their uptake strategies. Further research on CIPK23's

MYB59:
myeloblastosis
transcription factor 59
specific mechanisms can deepen our understanding of how nitrate and K⁺ transport and sensing are actively coordinated. It is worth noting, however, that CIPK23 is also important in the control of iron (43) and ammonium (149) homeostasis, which make the interrelationships between nutrients even more intricate, but this falls out of the scope of the present article. Whether these physiological interactions are related to the control of nitrate- and K⁺-sensing systems is still largely unknown and will probably inspire exciting avenues of research.

### 2.7.6. Long-distance nitrate signaling.
If the PNR has been shown as being mainly cell autonomous, intricate root→shoot→root long-distance mechanisms are at play to coordinate nitrate acquisition and foraging. The idea that shoots may exert control over the root nitrate acquisition system was presented early in a series of seminal works on barley by Drew & Saker (40) and Drew et al. (41) in the 1970s. Using a variant of the split-root system, they showed that lateral root growth and nitrate uptake were increased specifically in the part of the main root axis locally supplied with nitrate compared to plants homogeneously supplied with the same nitrate concentration. This compensatory response to partial N deficiency, resulting in maintaining a shoot:root ratio similar to plants uniformly supplied with nitrate, led the authors to conclude that the production of dry matter by the shoots exerted a constraint on the root activity (40). Then, the main hypothesis was that the systemic regulation of the nitrate uptake system is related to the N-demand/satiety status of the whole plant relying on the shoot-to-root carbohydrate supply and/or relayed by the products of nitrate assimilation, namely $NH_4^+$, circulating amino acids, or even peptides (67, 86, 111). However, in split-root experiments designed to modulate the N status downward and leading to enhanced nitrate uptake in the nitrate-supplied root, the amino acid contents in phloem were not decreased (153).

In line with the systemic regulation of nitrate uptake, the expression of the nitrate transporter *NRT2.1*, in addition to being rapidly regulated by local nitrate provision through the PNR, is regulated by N status (47, 86). In subsequent studies, the transcriptional regulation of *NRT2.1* became a prime target to decipher the molecular mechanisms associated with the systemic regulation of the nitrate acquisition system, leading to the identification of a small region of 150 bp in its promoter responsible for systemic regulation (49). Using the split-root experimental setup, transcriptomic analysis of *Arabidopsis* roots under different N conditions (homogeneous nitrate supply, heterogeneous nitrate supply, or homogeneous N deprivation) reveals that systemic regulation of the transporter is rapidly established within a few hours. This systemic regulation occurs even without nitrate assimilation, as shown in a nitrate reductase-null mutant. Additionally, several other genes related to nitrate transport and assimilation, such as *NRT3.1*, *NIR*, and *G6PDH*, exhibit similar systemic regulation (135, 137, 138) (**Figure 2**).

Combining this experimental framework with genomic and genetic approaches, the molecular basis of the nitrate-related long-distance regulatory network is now better understood, even allowing one to start considering the evolutive trajectory of the long-distance signaling network regulating the diversity of the N acquisition system (e.g., $N_2$ fixation through symbiotic interaction) (48, 135) (**Figure 2**). In heterogeneous nitrate medium, two main root-to-shoot signals have been found to be involved in the systemic regulation of the nitrate transporter *NRT2.1* by N status. The first one corresponds to the *trans*-zeatin (*tZ*) form of cytokinin synthetized from the nitrate-supplied roots. Indeed, impairing the root-to-shoot translocation of *tZ* abolishes the molecular and physiological response to heterogeneity (128) (**Figure 2**). In fully nitrate-supplied plants in which the expression of *NRT2.1* is reduced compared to plants exposed to nitrate heterogeneity, *tZs* are also translocated to shoots but with a higher level of accumulation since the whole root system produces cytokinins upon nitrate stimulation. Therefore, it has been proposed that the regulation of *NRT2.1* and other coregulated genes in roots depends on the level of *tZ*
<figure><img src="image_5.png" /><figcaption>Figure 2</figcaption></figure>

Model of an N systemic signaling network regulating root transport and development in NO₃⁻ heterogeneous conditions. Whenever a root system is exposed to a heterogeneous nutrient condition, as illustrated here, the perception of the local environment is determinant. Local NO₃⁻ supply, likely through the PNR, triggers the biosynthesis of CK and, in particular, the *tZ* form. The root local N deprivation induces the synthesis of CEPs, with the corresponding genes being transcriptionally regulated by the cooperation between the transcription factors TCP20 and HBI1. Hormones and peptides are translocated to the shoots via the xylem flow (*double blue arrows*). Within the aerial part, these root signals are integrated with the nutrient status through mechanisms that are still unknown (possibly molecular interaction or dynamic localization). The root response to heterogeneity, especially the stimulation of root development and NO₃⁻ transport in the NO₃⁻-rich patch, is then modulated by likely multiple shoot-to-root signals emerging from the integration of multiple signals in the shoots (*orange and brown arrows*). Among them, CEPD proteins target in roots the expression of a phosphatase, namely CEPH, which dephosphorylates NRT2.1, activating NO₃⁻ transport. HY5 and miR2111 may play a role in the shoot control of roots. The connection of these signals to local NO₃⁻ signaling and TCP20 activity in the transcriptional control of NRT2.1, for instance, and root development remains to be explored. Abbreviations: C, carbon; CEP, C-terminally encoded peptides; CEPD, CEP downstream; CEPR, CEP receptor; CK, cytokinin; *NIA*, *nitrate reductase*; N, nitrogen; *NIR*, *nitrite reductase*; NO₃⁻, nitrate; PNR, primary nitrate response; *tZ*, *trans-zeatin*.

accumulation in shoots (128). The second root-to-shoot signals, regulating *NRT2.1* at a systemic level, are 15-amino acid C-terminally encoded peptides (CEPs) whose expressions are induced in N-deprived roots. They are translocated to the shoots where they are perceived by two leucine-rich repeat receptor kinases (LRR-RKs) renamed CEP receptor 1 (CEPR1) and CEPR2 (152).

**CEP:** C-terminally encoded peptide

**LRR-RK:** leucine-rich repeat receptor kinase
**CEPD**: CEP downstream (peptide family)

**HY5**: long hypocotyl 5

The perception of CEPs in shoots triggers the expression of polypeptides of around 100 amino acids belonging to a glutaredoxin family and renamed CEP downstream 1 (CEPD1) and CEPD2. CEPDs are translocated from the shoots to the roots leading specifically to *NRT2.1* induction in nitrate supply roots (119) (Figure 2). An additional polypeptide, belonging to the same family and named CEPD-Like 2 (CEPDL2), has been found upregulated by a lower N status in shoots. CEPDL2 functions also as a shoot-to-root mobile signal stimulating nitrate uptake and transport inside the plant, together with CEPD1 and CEPD2 (120). The shoot CEPDL2/CEPD1/CEPD2 signals mediate in roots the upregulation of the phosphatase, CEPH, which is involved in the dephosphorylation of NRT2.1 at Ser501, leading to the activation of nitrate transport (118). It is still unclear how *tZ* (nitrate side) and CEP (–N side) coexist and interact in the shoots (Figure 2). However, given that a high level of *CEPDL* expression in shoots required *tZ* in shoots (120), one could speculate the following model in which the systemic N demand does not strictly rely on the –N-deprived roots: Nitrate transport regulation in heterogeneous nitrate conditions would depend on the integration at the shoot level of these two root signals mediated by different local environments. The level of *tZ* originating from roots and accumulated in shoots would transduce a signal for the presence and the level of nitrate available for the whole root system, sustain the expression level of the *CEPDL2*, and then enhance the activity of the CEP-mediated pathway to regulate nitrate uptake according to the N status (Figure 2).

The transcription factor TCP20 is a central regulator in the N-demand systemic signaling pathway. In yeast one-hybrid assays, TCP20 binds to the promoter of the nitrate-responsive genes *NRT2.1*, *NIA1*, and *NRT1.1*. Moreover, in vitro, TCP20 binds the 150-bp fragment in the *NRT2.1* promoter previously identified as sufficient for systemic regulation (53), suggesting that this transcription factor may act downstream in the N-systemic demand pathway. However, TCP20 interacts with the transcription factor HBI1 in N-deprivation conditions to directly promote the expression of CEPs (28), positioning this transcription factor upstream in the N-systemic demand pathway. In accordance with a regulatory role in systemic signaling, *NRT2.1* regulation as well as the root developmental response to nitrate heterogeneity are impaired in *tcp20* mutants (28, 53). Altogether, one could suggest that TCP20 acts locally in roots, transducing N deprivation to trigger systemic N-demand signaling and priming the functional targets required for N foraging.

Two additional shoot-to-root regulators are interesting to mention because of their role in systemically controlling root nitrate acquisition and development in *Arabidopsis* and because they illustrate the diversity of signals used for interorgan communication. The first one is the bZIP transcription factor HY5. In shoots, HY5 enhances the expression of genes related to photosynthesis, the biosynthesis of trehalose 6-phosphate, and sucrose efflux transporters *SWEET11* and *SWEET12* (24, 25, 154). Upon illumination, HY5 is translocated from shoots to roots and directly upregulates its own transcription as well as that of *NRT2.1*, allowing it to coordinate light response with root growth (25). It is noteworthy that HY5 also displays a root-autonomous role in repressing the transcription of NRT1.1 in high ambient temperature. Thermodependency phenotypes driven by this local regulatory module correspond to root elongation and a decrease in P content (84). Interestingly, in the transcriptomic analysis of roots from plants experiencing different N status, HY5 belongs to the population of genes regulated by the N systemic and local environment (136), suggesting that one branch of the systemic N-demand signaling indeed involves rapid modification of C metabolism. The second branch is AtMIR2111, which targets, in roots, the accumulation of *HOMOLOG OF LEGUME TML (HOLT)*. In legumes, this regulatory module is central in the regulation of nodulation; MIR2111 positively regulates the symbiotic infection. In *Arabidopsis*, overexpression of this microRNA as well as the mutation of the target gene, *HOLT*, leads to a reduction of lateral root initiation
at low and moderate nitrate concentrations, impairing plants' ability to forage for nutrients (145).

Plant adaptation to nitrate heterogeneity largely relies on a long-distance signaling network, allowing plants to rebalance the activity of all organs evolving in different local environments. Multiple branches, involving a diversity of signals (e.g., hormones, peptides, proteins, and RNA), have been identified and in the future will need to be unified in a single and dynamic systemic model.

# 3. COMPARISON OF CORE SENSING AND SIGNALING MECHANISMS

Nitrate-signaling mechanisms were first discovered in other kingdoms. However, it is unclear whether nitrate signaling in plants is related to other nitrate-sensing mechanisms across life domains. Below, we summarize the knowledge related to nitrate sensing in various kingdoms and provide a comparative analysis with nitrate sensing in plants, providing a first hint into understanding their evolutionary paths.

## 3.1. Bacterial Sensor Systems

The nitrate-binding domain in NLP7 has been described as homologous to the GAF domain in NreA from *Staphylococcus carnosus*, a part of the NreABC system (91). NreA binds nitrate within its GAF domain, representing the first instance of this domain binding nitrate (113). The binding mechanism of NreA is distinct from other nitrate-binding proteins, such as NarX or NrtA. NreA binds nitrate in a monomeric pocket, unlike the multimeric interfaces seen in other proteins (113). This nitrate-sensing mechanism involves the activation of NreB histidine kinase by the NreA-NO₃⁻ complex, followed by the phosphorylation of NreC-P, which then binds target promoter regions (157) (**Figure 3b**). In addition to this mechanism, bacteria exhibit considerable diversity in nitrate-sensing systems. For instance, in *Escherichia coli*, NarQ and NarX function as nitrate- and nitrite-sensing histidine kinases (NSHKs). In *Mycobacterium tuberculosis*, NarS, related to NarXL, accomplishes a similar function (55). Other nitrate-sensing proteins such as McpN from *Pseudomonas aeruginosa*, NasR from *Klebsiella oxytoca*, and NreA from *S. carnosus* have also been described. Interestingly, despite their differences, McpN, NarQ, and NarX exhibit a similar nitrate-binding structure, suggesting a conserved function (56).

NarX and NarQ histidine kinases (which are NSHKs) belong to two-component systems featuring domains such as HAMP, GAF, or PAS and a sensor region known as the P-box, which is oriented toward the periplasmic region (157). NSHKs are classified into three groups (55): The first lacks HAMP and GAF domains and corresponds to the Bacteroidetes; the second includes NarQ-like proteins from Gammaproteobacteria with HAMP- and GAF-like domains; and the third consists of NarX-like proteins, encompassing all other NSHKs. Nevertheless, the nitrate-sensing function in all these proteins is not related to their GAF domains. As a two-component system, in the first step, NarX phosphorylates NarL, or NarQ phosphorylates NarP in response to nitrate or nitrite, then binds the target promoter region of *narG*, *napF*, or *nrfA* genes (**Figure 3a**). Interestingly, although NarQ may phosphorylate both NarL and NarP, NarX interacts mainly with NarL, demonstrating a cross-regulation between these two sensor systems (115).

Other nitrate-sensing mechanisms have been described in *P. aeruginosa*, where nitrate chemotaxis is facilitated by the PilJ-type McpN-ligand-binding domain (LBD), a member of the methyl-chemotaxis proteins with low similarity to NarX/NarQ histidine kinases, and is not regulated by this two-component system (101). Unlike NarX/NarQ, McpN specifically binds nitrate and controls chemotaxis proteins from the Che pathway. Interestingly, the nitrate-binding residues of the MCP domain from McpN align with NarX nitrate-binding sites, indicating a
a Gram negative

<figure><img src="image_6.png" /></figure>

Bacteria/Archaea

b Gram positive

<figure><img src="image_7.png" /></figure>

c Archaea

<figure><img src="image_8.png" /></figure>

h

<figure><img src="image_9.png" /></figure>

e Chlorophyte

<figure><img src="image_10.png" /></figure>

f Plant

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

g

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

(Caption appears on following page)
<caption>**Figure 3** (Figure appears on preceding page)</caption>

(a–f) Representation of the main components of nitrate-sensing systems in different organisms across the domains of life. The example shown of Gram-negative bacteria could be *Escherichia coli* or *Dickeya dadantii*; the example shown of Gram-positive bacteria is *Staphylococcus carnosus*. (g) Comparison of nitrate-binding domains in land plant NLP phylogeny. (h) Comparison of nitrate-binding domains in closest land plant AtNRT1.1/AtNPF6.3 phylogeny. The phylogeny indicates identity level, and red asterisks correspond to nitrate-interacting residues. Abbreviations: aa, amino acid; Ac, *Adiantum capillus-veneris*; At, *Arabidopsis thaliana*; Atri, *Amborella tricbopoda*; Cb, *Chara braunii*; Mp, *Marchantia polymorpha*; NLP, NIN-like protein; NO₃⁻, nitrate; Os, *Oryza sativa*; P, phosphorus; Pp, *Physcomitrium patens*; Ps, *Picea sitchensis*; Sm, *Selaginella moellendorffii*; Tc, *Taxus chinensis*.

similar nitrate-sensing mechanism between them and potentially a common evolutionary history. Other bacterial sensors include NIT proteins, which regulate gene expression, cellular motility, and enzyme activity in response to nitrate changes. NasR from *K. oxytoca*, the first-described chemosensor with a NIT domain oriented to the N-terminal region, is responsible for its sensor activity (Figure 3a). This domain does not resemble NarX or NarQ proteins but shares structural features with them. Additionally, NasR contains an ANTAR domain, and other NIT proteins may feature HAMP, GAF, and PAS domains.

## 3.2. Archaea Sensor System

In contrast to well-described nitrate-sensing mechanisms in bacteria, mechanisms of nitrate perception in archaeal systems are largely unknown. Archaea species possess many histidine kinase transmembrane receptors with GAF or PAS domains coded in their genomes, associated with other domains such as CHASE4 or CHASE6, indicating their possible function as sensory proteins (171). Nevertheless, despite the presence of two-component systems in Archaea, they lack the main classes of bacterial response regulators, including the NarL system (46). On the other hand, archaeal species that respire nitrate use a membrane-localized nitrate reductase, with an active site oriented toward the outside of the cell and other domains oriented toward the intracellular space (Figure 3c). However, it remains unclear whether this protein can sense nitrate or whether other nitrate-sensing systems exist in Archaea (103).

## 3.3. Fungal Sensor System

NLPs belong to the RWP-RK group, whose members play a crucial role in regulating nitrogen metabolism in plants but are absent in fungi. This absence may indicate a gene loss after divergence from the plant group and a lack of nitrate sensor activity mediated by NLPs (142). The transcription factors NirA and AreA are described as the main nitrate-sensing components in *Aspergillus nidulans*. NirA accumulates in the nucleus and requires the transcriptional regulator AreA (Figure 3d). AreA belongs to the GATA factors, which are present in other Eukarya such as metazoans and plants (143), and contains a conserved DNA-binding region with a Cys4 zinc finger (14, 97). During nitrate starvation, AreA mediates chromatin remodeling, allowing NirA to bind the promoters of genes such as nitrate reductase and nitrite reductase (13). Additionally, AreA and NirA are directly involved in the regulation of the nitrate transporter NrtA under nitrate and nitrate-limiting conditions (95). Related systems have been described in other Ascomycota and Basidiomycota (97, 126, 127, 170).

## 3.4. Viridiplantae Sensor Systems

In Viridiplantae, RWK-RP family proteins play diverse roles in physiological processes such as gamete sexual determination, root nodulation, and nitrate-inducible gene expression, highlighting their versatility and near-exclusivity to this group. Evolutionary studies indicate that members of the RDK family evolved independently in Chlorophyta and Streptophyta, marking an important
evolutionary event (139). In this context, NLPs have evolved from a specific copy from the Charophyta group, as it is present only in *Chara braunii*. On the other hand, Chlorophyta contain NIT2 proteins that function similarly to NLPs in land plants (110). Notably, *Chlamydomonas reinhardtii's* NIT2 protein was the first to require intracellular nitrate to activate *NIA1*, demonstrating its role as a nitrate sensor, similar to what has been described for plant NLPs (18) (*Figure 3ef*). Phylogenetic analysis places the NIT2 protein at the base of the NLP group, indicating an early divergence during land plant evolution. However, NIT2 does not contain the GAF or PB1 domains.

The NLP family has expanded considerably in land plants due to whole-genome duplications, as observed in *Brassica rapa* (23), *Helianthus annuus*, *Populus trichocarpa*, and *Gossypium hirsutum* (110). This significant gene expansion is evident in phylogenetic analyses, where NLPs are typically categorized into three subgroups. However, these analyses usually exclude basal land plants, ferns, and gymnosperms. It is important to highlight the differences from NIN proteins that have diverged from a particular NLP group and show variations in their N-terminal conformation, which may explain the functional losses compared to NLPs (22, 90).

Oomycetes, formerly considered part of the fungi group, are now classified as members of the Stramenopila clade, which are related to golden-brown algae, diatoms, and brown algae. Genome analysis proved that species from *Phytophthora*, *Pythium*, *Plasmopara*, and *Saprolegnia*, all considered pathogens, present many copies of RWP-RK proteins in their genomes. However, the closest proteins to the NLP group do not present GAF-like or PB1 domains (168). Thus, even though they resemble NIT2 proteins, the function of these proteins within the Stramenopila clade remains unknown.

NPF family members likely originated in Chlorophyta and are versatile transporters of a variety of molecules, including nitrate, auxin, glucosinolates, and gibberellin (70). Chlorophyta and Charophyta transport nitrate via NPF, NRT2, and NAR1 transporters. However, unlike land plants, where the NPF family is typically large, Chlorophyta and Charophyta have a low number of NPFs or none (45). Instead, they possess different copies of NRT2 and NAR1 (61, 140). This information suggests that the transport functions of NPF proteins originated in Chlorophyta, and as more complex organs developed, new NPF subfamily members in plants acquired neofunctionalization (96, 140) (*Figure 3f*). In NRT1.1, amino acids His356, Thr360, Tyr388, and Glu476 are described as main residues that interact with nitrate, and His356 residue is particularly important for the interaction, corresponding to a feature that distinguishes it from other NPF6 subfamily members (62, 123). OsNRT1.1B (OsNPF6.5) is considered a homolog of NRT1.1 in rice (*Oryza sativa*), playing an important role in nitrate uptake, transport, and sensing. *Figure 3a* represents the variability among the closest NRT1.1 nitrate-binding regions from Charophyta to angiosperm NPFs. It shows that His356 is particular to both NRT1.1 and OsNRT1.1B, which could explain their specific function as nitrate sensors. Nevertheless, other residues involved in nitrate interactions do not appear to be highly conserved within this specific NPF6 subgroup. Remarkably, OsNRT1.1B appears to result from directional selection in the *indica* variety of rice (65). Despite the conserved transport functions of NPF proteins, the sensor function has yet to be explored in other land plant lineages and seems to be absent in Chlorophyta and Charophyta NPFs (114). Notably, despite the conserved transport function, detailed analysis of conserved motifs and transmembrane regions related to their function has shown considerable variation among land plants. This indicates the presence of unknown transport function mechanisms (96).

## 3.5. Comparison of the Nitrate-Sensing Systems

Recently, it was demonstrated that the NLP7 GAF-like domain, located in the N-terminal region of the protein, is responsible for the central role of nuclear export under nitrate deficiency and
has a transcriptional activator function (165). Further investigation on the NLP7 GAF-like domain demonstrated that it contains a nitrate-binding domain similar to the NreA nitrate-binding pocket. Surprisingly, compared to what happens in the NPF6 subgroup, the amino acids that interact with nitrate in NLP7 are highly conserved in other NLPs, from basal land plants to angiosperms (91). A small representation of NLP's nitrate-binding domain from Charophyta to angiosperms allows us to compare interacting nitrate residues and predict the possibility of similar functions among them (Figure 3b). GAF domains are described as ubiquitous motifs in all domains of life, being part of phosphodiesterases, adenylyl cyclases, and other signaling and sensory proteins, with a similar structure to PAS domains (63, 102). However, more studies are needed to determine if these structural similarities correspond to an ancestral origin of these protein groups and if nitrate-sensing functions have appeared recently or have been lost in other plant lineages. In this sense, not all proteins that present domains related to nitrate-sensing activity may play this role, as it is observed in the NIT domain, which seems to be present in other proteins but without a nitrate-binding function (101).

In addition to these characteristics, the PB1 domain's role in NLP-NLP and NLP-TCP20 interactions in *Arabidopsis* adds another layer of functional complexity during the nitrate-signaling process that has not yet been explored in other land plants (75).

Related to NPF nitrate sensor activity, even though the POT domain is distributed in Bacteria and Eukarya, there is no evidence that NRT1.1 sensor activity may be present in members of other NPF subfamilies. Despite analyses demonstrating that the ExxER/K motif and residues responsible for TMH4-TMH10 salt bridges are conserved in NPF from Charophyta to land plants, conserved regions containing nitrate-binding residues and the evaluation of sensor activity remain unknown (96).

Finally, the diversity of nitrate-sensing systems from bacteria to land plants is a testament to the complexity of this field. Even if some proteins have similar structural organization or activity, no clear evidence indicates a common ancestor of nitrate sensor systems. Environmental and other conditions may have regulated the origin of each system across the domains, especially in land plants, but profound new evolutionary analyses are needed to clarify their origin.

## 4. CONCLUSIONS AND GENERAL REMARKS

Here, we propose a comprehensive framework as a blueprint on how nitrate-sensing mechanisms are intricately linked with overall nutritional interactions within plants, ensuring efficient nutrient uptake and utilization. Central to this model are the PNR and the NSR (Figure 1), which collectively modulate the plant's capacity to sense and respond to nitrate availability in the soil.

Nitrate sensing is demonstrated to be a multifaceted process, deeply intertwined with broader metabolic networks, including interactions with other essential nutrients such as C, P, K, S, and ROS, that also unfolds in systemic root-to-shoot communications (Figure 2).

Furthermore, this framework provides evolutionary insights into nitrate sensing and signaling by discussing the conservation and divergence of key molecular players across different plant species in the light of other nitrate-sensing mechanisms in other branches of life (e.g., Bacteria, Chlorophyta, Charophyta, and Fungi) (Figure 3). It explores how these mechanisms have evolved to adapt to varying environmental conditions and nutrient availability, offering a broader perspective on the evolutionary biology of nutrient sensing in plants.

By elucidating the complex network of interactions that govern nitrate sensing and signaling, this framework not only enhances our understanding of plant nutrition but also has opened new avenues for improving nutrient use efficiency in crops through genetic and biotechnological interventions (58, 65, 137). This integrative approach provides a robust foundation for future research
aimed at optimizing plant growth and productivity in diverse environmental conditions and to
face climate change challenges.

## SUMMARY POINTS

1. The primary nitrate response (PNR) is activated during the transition from nitrogen-deficient (–N) to nitrate-sufficient (+N) conditions, whereas the nitrogen starvation response (NSR) operates from nitrogen sufficiency (+N) to nitrogen deficiency (–N).

2. The PNR serves as the core nitrate-signaling pathway, integrating the functions of both known nitrate sensors NRT1.1 (a nitrate transceptor located at the membrane) and NLP7 (a transcription factor).

3. The PNR is closely intertwined with the NSR, with many sentinel genes and regulatory elements from the PNR influencing and coordinating the activation of the NSR under low-nitrate conditions, ensuring transitions between nitrate availability and deficiency.

4. The NSR/PNR module is deeply interconnected with various other signaling pathways, including those for phosphorus (P), carbon (C), reactive oxygen species (ROS), potassium (K), and sulfur (S). These interactions enable plants to coordinate nitrogen sensing with broader nutrient and stress responses.

5. Nitrate sensing occurs in different plant organs and is coordinated through robust long-distance communication involving cytokinins (CKs), peptides (e.g., CEP and CEPD), and long noncoding RNAs (lncRNAs).

6. The evolutionary origins and adaptations of nitrate sensing proteins, such as NRT1.1 and NLP7, remain to be fully elucidated. Further research is needed to understand how this signaling module evolved across different plant lineages and its connections to nitrate sensing systems in other life domains.

7. Manipulating key elements of nitrate sensing, such as NLPs, NRT1.1, and regulators such as HRS1 and HHO proteins, has demonstrated significant potential for improving nitrogen use efficiency (NUE). These advances offer promising avenues for biotechnological applications to enhance agricultural sustainability and demonstrate how fundamental research is the foundation of innovation and rarely the other way around.

## DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review.

## ACKNOWLEDGMENTS

Our work is supported by the CNRS (IRP-CoopNet2) to G.K., S.R., and G.M.C. and by the University of Montpellier (I-SITE MUSE project AI3P) to G.K. This work was also publicly funded through ANR (the French National Research Agency) under the Investissements d'avenir program (reference ANR-16-IDEX-0006) and the PRC programs FatRoot ANR-21-CE13-0019-03 and DeepPep ANR-23-CE20-0020-01 to G.K., S.R., and H.R. Work in G.M.C.'s lab is supported by National Institutes of Health grant RO1-GM1211753. Work in R.A.G.'s lab is supported by the ANID-Millennium Science Initiative Program-Millennium Institute for Integrative Biology
(ICN17_022), Center for Genome Regulation (ICN2021_044), and ANID-FONDECYT 1220594. Collaboration between H.R., B.L., G.K., and S.R. is supported by the International Associated Lab (IAL In-Combi) granted by INRAE for use by IPSiM and Michigan State University's Plant Resilience Institute. The US National Science Foundation grant MCB-2334561 supports H.R.'s lab.

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