Nitrate Signaling, Functions, and Regulation of Root System Architecture: Insights from Arabidopsis thaliana

Root system architecture (RSA) is required for the acquisition of water and mineral nutrients from the soil. One of the essential nutrients, nitrate (NO3−), is sensed and transported by nitrate transporters NRT1.1 and NRT2.1 in the plants. Nitrate transporter 1.1 (NRT1.1) is a dual-affinity nitrate transporter phosphorylated at the T101 residue by calcineurin B-like interacting protein kinase (CIPKs); it also regulates the expression of other key nitrate assimilatory genes. The differential phosphorylation (phosphorylation and dephosphorylation) strategies and underlying Ca2+ signaling mechanism of NRT1.1 stimulate lateral root growth by activating the auxin transport activity and Ca2+-ANR1 signaling at the plasma membrane and the endosomes, respectively. NO3− additionally functions as a signal molecule that forms a signaling system, which consists of a vast array of transcription factors that control root system architecture that either stimulate or inhibit lateral and primary root development in response to localized and high nitrate (NO3−), respectively. This review elucidates the so-far identified nitrate transporters, nitrate sensing, signal transduction, and the key roles of nitrate transporters and its downstream transcriptional regulatory network in the primary and lateral root development in Arabidopsis thaliana under stress conditions.


Introduction
Nitrogen significantly influences plant growth and development. Plants adopt numerous strategies to modulate the uptake capacity of their roots to cope with spatial and temporal fluctuations in N availability [1]. In plants, the root architecture adjusts to these environmental fluctuations [2,3] and synchronizes the NO 3 − supply and demand inside the plants by the coordination of the systemic signal required to deal with root NO 3 − acquisition [4].
The regulatory pattern of root NO 3 − uptake simplifies the root transport system in two ways; The first is the rapid uptake after the NO 3 − provision, which requires de novo protein synthesis [5,6], and the other is the root NO 3 − efflux, strongly upregulated by N deficiency or low availability and downregulated by high nitrate supply [7,8]. An important hypothesis arising from the recently in chl1-5 mutants. To study the function of CIPK8, the two independent T-DNA insertion mutants (cipk8-1 and cipk8-2) were isolated and a reduction in the cipk8 mutant was apparent via the induction of nitrate-responsive genes NRT1.1, NRT2.1, NIA1, and NiR. This then clearly demonstrated that CIPK8 functions as a positive regulator of the primary NO 3 − response in the low-affinity system [25].
In the fip1 mutant, the increased expression of CIPK23 may affect NO 3 − uptake and subsequently reduce NO 3 − content. Molecular genetics suggest that FIP1 and CPSF30-L operate similar NO 3 − signaling pathways. FIP1-induced NO 3 − signaling interacts with CPSF30-L and is regulated by CIPK8 and CIPK23 [34,35]. The role of the subgroup III protein kinases (CPKs) CPK10, -30, -32 in NO 3 − regulated root growth was examined [15]. The NO 3 − -induced LR primordial density was reduced and LR elongation was significantly hindered in icpk [15], thus associating the inhibition of nitrate-CPK-stimulated genes with transcription, metabolism, and transport activities [15]. The activity of the CPKs can be enriched within 10 min in response to nitrate. These CPKs have been distinguished as the primary regulators that coordinate the essential NO 3 − response [15] and modulate various essential cell and metabolic functions instantly triggered by NO 3 − [36,37].
CPK10 and CPK30 have also been shown to be associated with the abscisic acid (ABA) responsiveness of the mesophyll protoplasts, which is a promising avenue of research on the coregulation of NO 3 − and ABA pathways. Both have been speculated to contribute to the regulation of the root growth and gene expression [37]. For instance, ABI2 (ABA-insensitive 2) phosphatase is a fundamental component of the ABA sensing system [38]. Besides the CIPK23-CBL9 complex functioning in the dual affinity transition changes of NRT1.1, ABI2 and CBL1 also interact with phosphorylated CIPK23, which is recognized as an additional segment of this regulation process. NO 3 − sensitivity instigates a rapid increase in the cytoplasmic Ca 2+ level downstream of NRT1.1 in a PLC-dependent manner [28]. In short, nitrate-mediated CPK signaling phosphorylates transcription factors to regulate the expression of downstream genes that affect nitrogen assimilation, carbon/nitrogen metabolism, and proliferation [15]. However, it is possible that additional NO 3 − sensors and NRT1.1-independent pathways could be involved in the Ca 2+ influx and other signaling measures [28,39] (Figure 1). An increase in Ca 2+ initiates a change in the protein phosphorylation status while controlling the movement of the key component of the NO 3 − signaling pathway. CPK10, 30, and 32 work as regulators Figure 1. Summary of early responses in nitrate signaling and assimilation. NO3 − signaling pathway switches its affinity via phosphorylation (modified from Undurraga [41]). Nitrate-responsive genes are depicted in light green, transcription factors in purple, and microRNAs in grey. For clarity purposes, the cell nucleus is shown. (A) phosphatidylinositol-specific (PI-PLC) and Ca 2+ -dependent pathways. At Low NO3 − condition, protein kinases CBL1/9-CIPK23 complex phosphorylates NRT1. 1 and changes it into a high-affinity transporter, which activates PLC and results in calcium influx (Ca 2+ acts as a second messenger). This cascade mediates changes in the expression of transcription factors (TGA1/4 *) and genes involved in nitrate transport (NRT2.1, NRT2.2, and NRT3.1) and nitrate assimilation (NIA1 and NiR). (B) Nonphosphorylated form of NRT1.1-induced signaling. Nitrateinduced Ca 2+ -ANR1 signaling that promotes lateral root (LR) initiation is assumed to be a nonphosphorylated form of NRT1.1 signaling after the supply of nitrate in limited-nitrate conditions. (C) PI-PLC and Ca 2+ -independent pathways. Conversely, AFB3 is regulated by nitrate in a phospholipase C (PLC)-and calcium-independent manner. ABF3 modulates the expression of NAC4 and OBP4 with subsequent effects on root remodeling. Finally, nitrate assimilation produces organic N, which induces miR393 and represses miR167 (grey) and regulates the abundance of AFB3 and ARF8, respectively. * TGA1 and TGA4 are redundant regulatory factors that mediate nitrate responses in Arabidopsis roots. However, the interaction between TGA4 and the PLC-calcium pathway has not been experimentally validated.
Ca 2+ sensor proteins perceive changes in the (Ca 2+ )cyt and subsequently transduce downstream signaling cascades to stimulate alteration of enzymatic activity, cytoskeleton orientation, phosphorylation, and gene expression [42,43]. This was further confirmed by the pretreatment of seedlings with phospholipase C inhibitors or Ca 2+ channel blockers, which severely affected NO3 − - Figure 1. Summary of early responses in nitrate signaling and assimilation. NO 3 − signaling pathway switches its affinity via phosphorylation (modified from Undurraga [41]). Nitrate-responsive genes are depicted in light green, transcription factors in purple, and microRNAs in grey. For clarity purposes, the cell nucleus is shown. Phosphatidylinositol-specific (PI-PLC) and Ca 2+ -dependent pathways. At Low NO 3 − condition, protein kinases CBL1/9-CIPK23 complex phosphorylates NRT1.1 and changes it into a high-affinity transporter, which activates PLC and results in calcium influx (Ca 2+ acts as a second messenger). This cascade mediates changes in the expression of transcription factors (TGA1/4 *) and genes involved in nitrate transport (NRT2.1, NRT2.2, and NRT3.1) and nitrate assimilation (NIA1 and NiR). Nonphosphorylated form of NRT1.1-induced signaling. Nitrate-induced Ca 2+ -ANR1 signaling that promotes lateral root (LR) initiation is assumed to be a nonphosphorylated form of NRT1.1 signaling after the supply of nitrate in limited-nitrate conditions. (C) PI-PLC and Ca 2+ -independent pathways. Conversely, AFB3 is regulated by nitrate in a phospholipase C (PLC)-and calcium-independent manner. ABF3 modulates the expression of NAC4 and OBP4 with subsequent effects on root remodeling. Finally, nitrate assimilation produces organic N, which induces miR393 and represses miR167 (grey) and regulates the abundance of AFB3 and ARF8, respectively. * TGA1 and TGA4 are redundant regulatory factors that mediate nitrate responses in Arabidopsis roots. However, the interaction between TGA4 and the PLC-calcium pathway has not been experimentally validated. Ca 2+ sensor proteins perceive changes in the (Ca 2+ ) cyt and subsequently transduce downstream signaling cascades to stimulate alteration of enzymatic activity, cytoskeleton orientation, phosphorylation, and gene expression [42,43]. This was further confirmed by the pretreatment of seedlings with phospholipase C inhibitors or Ca 2+ channel blockers, which severely affected NO 3 − -responsive gene expression in Arabidopsis, indicating the function of Ca 2 as a secondary messenger in NO 3 − signaling pathways. A model was therefore suggested, where the (Ca 2+ ) cyt level increases by NRT1.1 and phospholipase C activity in response to NO 3 − , which is required for changes in the prototypical NO 3 − -responsive gene expression [16]. Taken together, both NRT1.1 and phospholipase activity are mandatory for NO 3 − -mediated increase in cytoplasmic Ca 2+ levels and IP3 ( Figure 1) [16]. PLC enzymes are membrane-associated, resulting in the remodeling of lipid membranes by the breakdown of phospholipids and the subsequent production of multiple secondary messengers [16]. In plants, two classes of PLCs exist, and they are distinguished based on their substrate specificity. One is phosphatidylinositol-specific (PI-PLC) and the other is non-specific (NPC). Plant NPCs share homology with bacterial PLCs. NPCs can incline either phosphatidylcholine-specific phospholipase C (PC-PLC), phosphatidylethanolamine (PE-PLC), or phosphatidylserine (PS-PLC). However, PI-PLC is the most considered class of PLC, which hydrolyzes phosphatidylinositol 4, 5-bisphosphate (PIP2) from the plasma membrane to create IP3 and diacylglycerol (DAG) [44]. The nitrate signaling and phosphatidylinositol-specific PI-PLC links were found in Arabidopsis. Nitrate triggers Ca 2+ and inositol 1, 4, 5-triphosphate (IP3), which were not witnessed in the plant's pretreatment with PLC inhibitor U73122. For instance, the NRT1.1 mutants, chl1 and chl9, revealed that this was an NRT1.1-based response. The associated rise in IP3 after NO 3 − treatment also suggested that the activity of phospholipase C (PLC) was associated with this signaling pathway [16].

Nitrate-Induced Ca 2+ and PI-PLC-Dependent Signalling
Phosphatidylinositol-specific phospholipase C (PI-PLC) is the major part of nitrate signaling and transport, modulated by the phosphorylation/dephosphorylating process. Both plasma membrane and tonoplast nitrate transport activity are regulated by phosphorylation [27,29]. In Arabidopsis, Ca 2+ has a definite role in plant signal transduction and is also significant for the NO 3 − -mediated signaling of gene expression. As stated earlier, NO 3 − treatment rapidly increased the cytoplasmic Ca 2+ level in the roots [27,29] ( Figure 1) and nitrate is absorbed in the root cell by plasma-membrane-localized nitrate transporter families, NRT1 and NRT2 [22]. NRT1.1/CHL1 is a low-affinity transporter that switches to a high-affinity transport system when NRT1.1 is phosphorylated at the threonine residue 101(T101) by protein kinase CBL1/9-CIPK23 [9]. The protein complex CIPK23-CBL9 (CBL-interacting protein kinase (CIPK); calcineurin-B like protein (CBL)) and CIPK8 have been implicated in the dual-affinity transition changes of NRT1.1 through phosphorylation [33]. More recent studies have revealed that a protein phosphatase 2C (PP2C) family member, ABI2 (ABA-insensitive 2), and the calcium sensor CBL1 were distinguished as supplementary constituents that modulate NRT1.1 transport functions and NRT2.1 expression in root growth NO 3 − responses [38] (Figure 1).
Hence, the phosphorylation activates a weak upregulation of high-affinity nitrate transporter NRT2.1 [14], and subsequently induces NRT1.1, NRT2.1, NRT2.2, and NRT2.4 under nitrate-starved seedlings after nitrate supply, while upregulating all the nitrate assimilatory genes [27,50]. CPK phosphorylates the NLP TFs, particularly NLP7, which interact with CPK20 in the nucleolus under NO 3 − availability. Besides NPL7, more TFs, such as TCP20, also contribute to the NO 3 − -induced transcriptional changes and systemic signaling. In contrast, TGA1/4 controls the genes which participate in the PNR, transport, metabolic, and developmental processes [28]. Under limited-nitrate conditions, the NRT1.1 is, therefore, phosphorylated at the T101 in order to stimulate NRT1.1 association with membrane microdomains at the plasma membrane (PM). When nitrate supply is increased, the nonphosphorylated NRT1.1 shows oligomerization and low structural mobility at the PM, thereby initiating rapid inducible endocytosis. These activities could promote LR growth by switching NRT1.1-auxin transport activity on the PM and stimulating Ca 2+ -ANR1 signaling from the endosomes (discussed in detail in Section 3.2.1, nonphosphorylated nitrate signaling) [51].  [14]. In contrast, T101Dexpressing transgenic plants that mimic phosphorylated NRT1.1/CHL1 displayed only high-affinity NO 3 − uptake activity and are activated only at a high-affinity primary NO 3 − response. This suggests that T101D can only bind NO 3 − with a high-affinity uptake system [14]. Subsequently, it could be possible that binding sites with low affinity could be blocked by T101 phosphorylation [14] ( Figure 2).

Differential Phosphorylation
Genes 2020, 11, x FOR PEER REVIEW 6 of 24
However, after the point mutation at the plasma membrane, the mode of NRT1.1T101 phosphorylation may be different in both NO3 − uptake and signaling. Transgenic plants of T101A, which mimic the NRT1.1/CHL1 dephosphorylation, exhibits only low-affinity NO3 − uptake, but can also sense NO3 − at high-affinity range, with the high-affinity for NO3 − being comparatively less than the wild-type (WT) [14]. These properties propose that WT NRT1.1 and the T101A mutant may have two NO3 − -binding sites; high affinity and low affinity. It is worth noting that only the low-affinity binding site of the T101A mutant can be transported over the plasma membrane (PM). Unlike NO3 − uptake, NO3 − binding to both sites of T101A mutants could trigger the NO3 − response. This could justify the reason why the CHL1T101A mutant still exhibits a biphasic primary response [14]. In contrast, T101D-expressing transgenic plants that mimic phosphorylated NRT1.1/CHL1 displayed only high-affinity NO3 − uptake activity and are activated only at a high-affinity primary NO3 − response. This suggests that T101D can only bind NO3 − with a high-affinity uptake system [14]. Subsequently, it could be possible that binding sites with low affinity could be blocked by T101 phosphorylation [14] (Figure 2).   The two NO 3 − binding sites depicted here ( Figure 2) have two adaptations of a single binding site.
Taken together, these findings suggest that at the low level of NO 3 − sensing, T101 phosphorylation keeps the PNR, whereas, for uptake and substrate-binding, T101 phosphorylation may repress the low-affinity NO 3 − binding and is then required to use the high-affinity transport system [14].
Under both HN and LN conditions, T101A seedlings exhibited a transient increase in (Ca 2+ ) cyt [51], while T101D seedlings displayed a decrease in [Ca 2+ ] cyt . Concomitantly with [Ca 2+ ] cyt accumulation, HN-stimulated expression of ANR1 in LRs is sensed in T101A, but not in T101D. In the light of these findings, it is suggested that a nonphosphorylated form of NRT1.1 could activate the Ca 2+ -CPKs-NLPs signaling pathway to induce the expression of ANR1, and subsequently control LR elongation [51]. It was analyzed that intracellular transport of T101A and T101D in LR cells showed that differential phosphorylation of NRT1.1 enhanced the implementation of NRT1.1-stimulated signal transduction in LR growth [51]. Phosphorylated NRT1.1 takes up the sparingly accessible NO 3 − from the soil at high affinity and induces the NRT2.1 expression to a lower extent compared to the low-affinity state [52] (Figure 2). Under high NO 3 conditions, NRT1.1-induced auxin transport is inhibited, and shortly after NO 3 − -treatment, the dual affinity modes of the NRT1.1 are regulated at Thr-101(T101) phosphorylation [52]. As mentioned earlier, under low NO 3 − conditions, phosphorylation at T101 stimulates NRT1.1 association with a functional membrane microdomain at PM [51], confirming the NRT1.1-mediated auxin flux, and subsequently repressing their growth by reducing the LRP auxin level. With an increased NO 3 − level, nonphosphorylated NRT1.1 shows oligomerization and low lateral mobility at the PM and rapid inducible endocytosis. This activity may stimulate LR development by supporting NRT1.1-auxin transport activity on the PM to induce Ca 2+ -ANR1-signaling from the endosome [51]. Further studies have shown that seedlings of T101A had much higher LR density than that of T101D when grown under low NO 3 − conditions (0.2 mM), whereas in high NO 3 − conditions (1 mM), no significant difference was observed in the LR density of the mutants compared to WT plants [51]. These findings confirm that that T101A and more nonphosphorylated WT NRT1.1 promote LR growth in LN by suppressing basipetal auxin transport, and subsequently accumulating auxin in the LR tips [51].

Nitrate-Induced Ca 2+ and PI-PLC-Independent Signaling
Ca 2+ and PI-PLC are not affected by the expression of NO 3 − responsive auxin signaling F-Box3 (AFB3) protein, indicating that beyond Ca 2+ and PI-PLC, there is a PI-PLC-independent pathway that controls the regulation of the nitrate-sensitive genes [16,55] (Figure 1). Hence, NRT1.1 toggles within the phosphorylation status of a critical threonine residue from low-to high-affinity states. This residue is amongst the second and third transmembrane helices of NRT1.1 located in the intracellular side [14,25].
In Arabidopsis root, Ca 2+ and PI-PLC-independent miR393/AFB3 regulatory modules are recognized as nitrate responsive genes, which assimilate nitrate and auxin signaling [56]. Nitrate induced LRs are dependent on miR167, and its target auxin-responsive factor ARF8 mRNA [57] plays a distinctive role in regulating several genes connected via a network to promote the stimulation of LR initiation and inhibition of elongated roots in response to N [57] (Figure 1). This earlier identified regulatory module, controlled by miR393 microRNA and the AFB3 auxin receptor, stimulates LRs in response to external and internal NO 3 − applications [51,58]. AFB3 is induced by NO 3 − and repressed by miR393, whereas nitrate reduction and assimilation produced N metabolites, which induces miR393 [59] (Figure 1). Furthermore, AFB3 coregulates NAC4 and OBP4, and this coregulation is confirmed by using the green fluorescence protein (GFP)-expressing lines after 2 h, in response to nitrate. AFB3, activated in the pericycle, indicated that the AFB3-NAC4-OBF4 complex might build a regulatory module that controls LR growth in a NO 3 − -dependent manner [56].
Nitrate-stimulated AFB3 induced in the root might be a specific signaling network of Aux/IAA and ARF factors to modulate NAC4 activation and LR growth. The abundant Aux/IAA-ARF modules chronologically generate new LRs and control LR development in Arabidopsis. The lateral root basal meristem (the zone between meristem and elongation) depends on IAA28 and ARF proteins, which include transcription factors ARF5, ARF6, ARF7, ARF8, and ARF19 [13,53]. In plant RSA, the LR initiation and emergence of the AFB3 overexpression line and the afb3 mutant line have emerging roles compared to wild-types and display increased growth of LRs under nitrate-sufficient conditions. Additional findings revealed that the transcription factor NAC4, which functions downstream of AFB3, might be involved in two dependent pathways of RSA regulation [52,58]. Following AFB3, NAC4 acts downstream in the pericycle cell to alter LR density in nitrate treatments [9,51].

Auxin Response Network
Auxin signaling is primarily passed over by transcriptional pathways for morphogenesis and developmental processes, which include TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX (TIR1/AFB) proteins, AUXIN/INDLOE-3-ACETIC ACID (AUX/IAA) transcriptional co-regulators and AUXIN RESPONSE FACTOR (ARF) transcription factors [60][61][62]. At low levels of auxin concentration, members of the transcriptional inhibitor family AUXIN/IAA-INDUCIBLE (AUX/IAA) interact with the DNA-binding protein of ARF [56,57], while the ARF proteins function to detect the auxin-response promoter elements (AuxREs) in various auxin-regulated genes to activate or suppress their expression [63,64]. AUX/IAA protein inhibits the ARF function either by passively inhibiting ARF proteins from their target promoters [65] or by binding ARF with the corepressor TOPLESS (TPL) for inactivation of the chromatin and silencing of ARF target genes [56,59,66]. An increase in auxin concentration by an auxin-induced module of the coreceptor complex consists of F-box protein from the TRANSPORT INHIBITOR RESPONSE 1 (TIR1)/AUXIN SIGNALING F-BOX PROTEIN (AFB) family and is an Aux/IAA member [60,67,68]. TIR1/ABFs, a subunit of nuclear S-PHASE KINASE ASSOCIATED PROTEIN 1-CULLIN-F-BOX PROTEIN (SCF)-type E3 ubiquitin-protein ligases (SCFTIR/AFB), stimulate the recognition of substrates. The auxin response is initiated by connecting hormones to the TIR1/AFB receptor. The auxin receptor is part of the SCFTIR1/AFB ubiquitin ligase complex [69,70]. Binding of auxin to its receptor TIR1/AFB activates the information and breakdown of the polyubiquitination of the Aux/IAA inhibitor, which subsequently releases the inhibition of ARF transcription factors, which induce the transcription of auxin-responsive genes [71,72]. This represents the pivot of auxin signaling.
In a simpler form, auxin-initiated AUX/IAA removal relieves ARF inhibition and activates the transcription of primary genes. Remarkably, the auxin response network is enough to reconstitute the AuxRE-dependent activation of reporter genes in yeast [73]. Hence, in Arabidopsis root, a miR393/AFB3 regulatory module is recognized as nitrate-responsive, which assimilates nitrate and auxin signaling to promote root growth [56]. Only those pathways discussed in the present review are depicted. The green arrows indicate systemic transport and assimilation, the black arrows indicate positive signaling as a stimulatory effect, red lines indicate negative signaling as an inhibitory effect, the orange lines depict the unknown positive and negative signaling pathways, and dotted lines represent the unconfirmed nitrate-mediated signaling pathways. The low nitrate and severely low nitrate conditions have been reported to have a stimulatory and inhibitory effect on LR development, respectively, while high NO3 − supply has an inhibitory effect on LR growth [24] (see text for further information). External NO3 regulates primary root growth in Arabidopsis. The receptor for the external glutamate signal is shown Only those pathways discussed in the present review are depicted. The green arrows indicate systemic transport and assimilation, the black arrows indicate positive signaling as a stimulatory effect, red lines indicate negative signaling as an inhibitory effect, the orange lines depict the unknown positive and negative signaling pathways, and dotted lines represent the unconfirmed nitrate-mediated signaling pathways. The low nitrate and severely low nitrate conditions have been reported to have a stimulatory and inhibitory effect on LR development, respectively, while high NO 3 − supply has an inhibitory effect on LR growth [24] (see text for further information). External NO 3 regulates primary root growth in Arabidopsis. The receptor for the external glutamate signal is shown as a glutamate-gated Ca 2+ channel because these are known to be activated at root tips [79]. However, its specific role in this signaling pathway is unconfirmed (see text for further information).
To this end, this response is glutamate-specific in Arabidopsis since an ongoing study of the impact of 17 other proteinogenic amino acids on the architecture of the roots found none that could produce its distinctive effect on root architecture [77]. By using a chemical genetic approach, the MEKK1 MAP kinase gene has since been investigated as part of the glutamate signaling pathways in PR tips [80]. MEKK1 functions mainly as a distinctive immune system and its expression was demonstrated to be profoundly receptive to a variety of abiotic factors [81]. Nitrate exhibits a strong signal to stimulate the primary root development by enhancing the activity of the meristem and cytokinin signaling. Cytokinin sensing and biosynthesis mutants showed shorter roots compared with wild-type when subjected to NO 3 − treatments, especially when NO 3 − is the primary source [82]. Histological studies of the root tip revealed reduced cell division and elongation in the cytokinin receptor double mutant ahk2/ahk4 (histidine kinase) compared with WT plants under adequate NO 3 − supply. It is worth noting that as NO 3 − -mediated restriction in the root growth was observed between 5 and 6 days after planting, the WT plants had the potential to recover from the growth-restricted condition, whereas cytokinin signaling or biosynthesis mutants were most certainly not capable of recovering [82].
In addition, the transcriptomic analysis indicated that genes associated with both cell division and elongation are possibly significant for PR development in response to NO 3 − , thereby indicating the interaction between nitrate and cytokinin signals in regulating PR development in Arabidopsis [82].

Effects on Lateral Root Growth and Development
The growth of lateral roots is strongly affected by the concentration of N in the growth environment. For instance, in low NO 3 − soil, patches of high NO 3 − have a localized stimulatory impact on LR development, which varies in different plant species [2,74], whereas under high NO 3 − conditions (with no restricted growth), LR development is repressed [83]. Further studies also revealed that NO 3 − plays a prominent role in regulating LRs. Generally, low NO 3 − has a dual effect on the LRs, such as stimulatory as well as inhibitory effects, whereas high NO 3 − supply only exhibits an inhibitory effect on LR growth and development of LRs [4]. In other words, there are two clear morphological adaptations. Under N-deficient conditions, the LRs are significantly stimulated; however, when exposed to more severe N deficiency, the entire LR length reduces and LR formation disappears [13]. This is initiated by the signaling impact of NO 3 − itself, rather than downstream metabolites [2].

Stimulatory Effect of Low Nitrate on LR Growth
The low NO 3 − -stimulated Arabidopsis LR development depends on the role of the auxin biosynthetic gene TAR2 (tryptophan aminotransferase related 2; Figure 3), which is expressed in the pericycle and vasculature of developed roots close to the root tip and is stimulated under low-nitrogen conditions. In WT plants, the low NO 3 − restored auxin accumulation in the primordial of the nonemerged LRs, with an additional three cell layers and LR emergence. On the other hand, these low N-stimulated auxin accumulation and root developmental responses were disrupted in tar2 null mutants [4,51]. Subsequently, TAR2 is required for restructuring the root architecture in response to low N conditions. Another nitrate responsive gene, BBX16 (bobby sox homolog), belongs to the constans-like zinc finger family.  might act as a key factor in this signaling pathway [4]. It was thus demonstrated that OsNRT2.1 could be involved in the nitrate-dependent pathway of root elongation by regulating auxin transport to the roots under low NO 3 − conditions [90]. Apart from the aforementioned pathways comprising both transcriptional factors and hormonal signals, nitric oxides (NOs) have been accounted for as a significant NO 3 − -mediated signal which regulates RSA in plants [79,82]. In rice, NO produced by NR could enhance the inadequate production of N by developing LR initiation under partial NO 3 − availability [91,92]. To this end, LRs are significantly stimulated by mild NO 3 − deficiency. Different molecular players are involved in the regulation of different stages of plant growth and development.

Inhibitory Effects of Severely Low Nitrate on LR Growth
Earlier studies have found that the impact of NO 3 − was related to the ability of the localized NO 3 − supply to stimulate LR elongation [23,83]. Experimental estimation of using a limited, rather than uniform, NO 3 − treatment initiates the specific effects of the external NO 3 − on LR development, and this can be observed under conditions where the systemic effects, due to changes in the N status of the plant, can be limited to a greater extent [2,4]. Under severe N deficiency, both LR formation and length are repressed in plants [93]. An additional system, downstream of CLV1 feedback, regulates the transcript level of the N-responsive CLE genes in the roots for fine-tuning of the signal amplitude [4,89]. In other words, CLEs-CLV acts as a regulatory module in NO 3 − signaling pathways, and it also antagonistically controls the growth of LRs under limited N conditions [4,94]. Similarly, one member of the CEP (C-TERMINALLY ENCODED PEPTIDE) gene family has been shown to arrest root growth [95]. The analysis of OE-lines of several CEP genes demonstrates their distinctive function. It was reported that CEPs have an antagonistic effect on LR growth while initiating a delay in PR and LR growth [95].
Another mechanism of the systemic inhibition of LR growth is associated with the inhibition of LRs in response to NO 3 − . Limited NO 3 − supply significantly increases abscisic acid (ABA) accumulation, as this ABA accumulation inactivates its coreceptor ABI2 (ABA-insensitive 2) and protein phosphate 2C (PP2C) [96] (Figure 3). The ABI2 then co-interacts with Ca 2+ -sensor subunit CBL1 and the kinases (CBL1-CIPK23) complexes, with their substrate being NRT1. conditions decreased the entire root system, whereas, when plants are subjected to low NO 3 − concentrations (10 µM), the PR part exposed to high NO 3 − triggered the local induction of LR elongation [2,83]. However, the global inhibitory effect of NO 3 − appeared to be as a result of prolonged exposure of plants to ample NO 3 − supply. The LR elongation under this condition was also suppressed in the areas of the root system that were subjected to the state of low NO 3 − conditions [2,97].
As reported earlier, the AFB3 receptor gene is strongly induced by NO 3 − , and the LR initiation is specifically diminished in afb3 mutants [59]. Research on the nitrate reductase (NR)-null mutants has revealed that NO 3 − itself was the main stimulator of AFB3. AFB3 expression feedback is regulated by nitrate-assimilatory products, such as miR393, a micro RNA that targets AFB3 transcript for degradation. This pathway has further confirmed the findings that nitrate (NO 3 − ) induced NAC4 and OBP4 transcription factors, functioning downstream of AFB3. Taken together with the results obtained from nac4 mutants, the afb3 mutant displays an apparent reduction in LR growth in response to NO 3 − [56].
Recent studies have demonstrated that high-affinity NO 3 − transporter AtNRT2.1 may be involved in the inhibition of LR initiation at high C: N ratios [98]. Also, the involvement of the ABA affecting LR growth, in response to NO 3 − , might be connected to the recently identified ABA receptor [98]. Nitrate reductase (NR)-lacking mutants display sensitivity to this systemic inhibitory effect, indicating that NO 3 − concentration in the tissue of plant cells may function in inhibitory signal induction. Thus, this model defines root branching, as modulated by inhibitory signals via internal N status and external NO 3 − supply [83].
Furthermore, ABA, which is associated with the systematic inhibitory effect of high NO 3 − on LR growth, might be connected with the recently identified ABA receptor FLOWERING CONTROL LOCUS A (FCA). In addition, root architecture response to the recently identified external L-glutamate conceivably provides a significant tool for studying biological functions of plant glutamate receptors and amino acid signaling [98]. It was also reported that FCA possibly acts as a receptor for ABA. The loss of function mutant fca displays low sensitivity to the inhibitory effect of ABA on LRs, indicating that FCA might be a constituent in signaling transduction pathways associated with high NO 3 − ABA-mediated inhibition of LRs [4,99,100].
It has been genetically proven that inhibition by ABA and NO 3 − is mediated by the same signaling mechanism. For instance, the LABI (lateral roots ABA-insensitive) is characterized based on the LR production affinity when exposed to 0.5 µM, which is less sensitive to the high NO 3 − -induced LR inhibition [4] (Figure 3). The identification of LABI genes could give indepth information about the signaling mechanism underlying this inhibition [98]. Interestingly, all the mutants produced shorter primary roots phenotypes, which indicated that LR development could be intrinsically correlated with PR growth. It was reported that the presence of the PR meristem is required for high NO 3 − and ABA-induced inhibition; however, this inhibition could be eliminated by the removal of the PRs [4].
Furthermore, root architecture response to glutamate may give an essential experimental framework to study glutamate signaling in plants and to elucidate the possible roles of the glutamate receptor [98]. Recent studies have shown that high NO 3 − supply (30 mM) stimulated ABA accumulation in the emerging root tips by discharging it from the inactive stores via ER-localized β-GLUCOSIDASE1 (BG1) to regulate root development. This information provides a system for NO 3 − -induced root development via the regulation of ABA accumulation in the root tips. It was hypothesized that there is a close association between ABA and NO 3 − signaling to coregulate LR growth [81]. A recent study has also shown that myb29-1 mutants increased the LR length, LR density, and total length under adequate NO 3 − supply in a genotype-dependent manner [48] (Table 1).

Coordinated Regulation of Nitrate and Other Messengers on RSA
Root foraging for NO 3 − involves both local and systemic signaling. NO 3 − -auxin-CK regulation could also be a key constituent of N systemic signaling, which coordinates nutritional requirements among various organs at different growth stages [101,102].

Nitrate-Mediated Auxin Allocation
A systemic regulation that includes the inhibition of auxin translocation from the shoot to root suppresses LR initiation and development and subsequently affects NO 3 − use efficiency in plants [103].
In such a situation, growing Arabidopsis thaliana on a nitrate medium was observed to have reduced auxin contents in the roots, while increasing the auxin content in the shoots. These findings have demonstrated that high NO 3 − inhibits the translocation of auxin from the shoots to the roots [78].
In addition, nitric oxide (NO) was found to be a key nitrate-related signal that regulates plant RSA and the signaling cascade of lateral root formation induced by auxin [104]. It can be deduced from the previous observation that a decrease in NO 3 − provision tends to promote auxin translocation from shoot to root. The high NO 3 − -inhibited root growth is a consequence of condensed cell elongation, and also probably due to the changes in meristematic length. Higher NO 3 − supply diminished the IAA concentration in the phloem exudates. The NO 3 − -induced inhibited root growth was closely associated with the reduction of auxin in the roots, especially in the regions close to the root tips. The regrowth of PRs by external NAA and IAA under high NO 3 − levels confirms that this inhibitory effect via high NO 3 − might be partially associated with the reduced IAA level in the roots [42].
However, the effect of NO 3 − on root growth could be complicated by the fact that high NO 3 − concentration (50 mM) triggers complete inhibition of LR development [105]. It has been experimentally confirmed that these responses are linked to an auxin transport inhibitor. To this end, the local supply of nitrate reduced the transport of auxin from shoot to root, and this subsequently resulted in decreased root auxin concentration to a level more appropriate for lateral root growth. However, for the stimulation of LRs, a change in the root auxin concentration only is not adequate. Regardless of these models, few ideas concerning the transcriptional gene regulatory system are known [106]. Furthermore, under available nitrate conditions, the auxin level in the root decreased compared to low NO 3 − conditions, and nitrate application seemed to inhibit auxin transport from shoot to root.
In many cases, the external IAA partially lowers the stimulatory effect of localized nitrate. High nitrate supply reduces the IAA concentration in the phloem exudates; thus, suppression of root growth by high nitrate is mainly dependent on the reduction of IAA levels in the roots, specifically in the root tip region. It could be deduced that the inhibitory impact of high nitrate concentration on the restricted root growth may be associated with the decline in auxin content in the roots [42]. The currently accessible information leading to a potential connection between nitrate and auxin accumulation influences the rate of auxin biosynthesis, transport, and allocation of auxin from root to shoot [107].

Nitrate-Mediated Cytokinin Allocation
Cytokinin (CK) affects intercellular auxin transport by regulating the expression of numerous auxin transport components, and thus balances the auxin distribution to regulate the size of root meristem [108]. Findings have also shown that the NO 3 − -CK shoot-root dependent system exhibits the NO 3 − demands of the whole plant, which affects root growth in NO 3 − rich patches of the soil [109].
Since CK could be widely distributed throughout the entire plant cell, CK-induced root-shoot coordination is a proposed model of systemic signaling for nutritional status [110]. CK activity could be closely associated with NO 3 − accessibility. Apart from the downstream metabolites of NO 3 − , NO 3 − has been known to initiate rapid de novo CK synthesis and accumulation in Arabidopsis roots [111]. The CK biosynthesis occurs in different parts of the plant tissue, where the adenosine phosphate-isopentenyltransferase (IPT) is expressed. IPTs are the primary enzymes that mainly influence the rate of CK biosynthesis, such as the prenylation of adenosine 5 phosphates and ATP and ADP at the N 6 -terminal with dimethyl diphosphate (DMAPP) [112].
In Arabidopsis, IPT3 is regulated in a NO 3 − -dependent manner. are known to be transcriptionally activated by CK and its disruption influences the basal expression of a significant number of CK-regulated genes, including type-A ARRs. CRFs are involved in promoting plant growth and leaf senescence [113]. The close regulation of the CYP735A2 and IPT3 by NO 3 − could be a major factor shaping NO 3 − -dependent spatio-temporal CK distribution in plants, and also regulating root system architecture in response to several abiotic stresses [114]. In short, nitrate and two hormonal mediators, CK and its antagonistic partner, auxin, act in synergy to modulate CK biosynthesis for root development.

Role of NO 3 − Transporters in Mitigating Plant Stress
Nitrate transporters are ultimately responsible for the absorption of NO 3 − from the soil and translocation of NO 3 − to various aerial parts of the plant [115]. NO 3 − transporter NRT1.1 acts as a positive growth regulator of vegetative and reproductive organs [116]. Studies have shown that AtNRT1.1/AtNPF6.3/CHL1 might be involved in the tolerance of the plant to proton toxicity; further studies on chl1 mutants, however, have revealed a reduced proton tolerance when compared with WT [117]. Moreover, the accumulation of sodium (Na + ) in the plant was found to be defective on npf6. 3 [120].
AtNPF3.1 transported ABA and GA (gibberellic acid) in vitro [124]. The interaction between NO 3 −and NRT-mediated NO 3 − uptakes on exposure to Pb in Arabidopsis via NRT-related mutants [125] demonstrates a new strategy for plant tolerance to lead (Pb) contamination [125]. Under low NO 3 − conditions, an NRT2 member, AtNRT2.1, contributes to iHATS (inducible high-affinity transport system) and plays a crucial role in the RSA, while AtNRT2.4 contributes to plant biomass production. AtNRT2.5 also stimulates mature plants under NO 3 − -deficient conditions [126].
ATNRT2.6 expression is induced after phytopathogenic bacterium inoculation. Hence, plants with low NRT2.6 expression show lower tolerance to pathogenic attacks [127]. Interestingly, there is a correlation between NRT2.6 expression and reactive oxygen species (ROS) accumulation in response to E. amylovora infection and treatments with the redox-active herbicide methyl viologen. This indicates a probable link between NRT2.6 activity and the production of ROS response to biotic and abiotic stresses [127].
In the chloride channel family (CLC), AtCLC accumulates anions in the vacuole when stomata are open, and also facilitates anion release during stomatal closure in response to stress hormones like abscisic acid (ABA) [128]. In addition to the NO 3 transporter, the NO 3 − -associated transcription factor, phloem-mobile CEPD-like 2 (CEPDL2)-polypeptide contributes to NO 3 − acquisition, along with CEPD1 and CEPD2, which mediate root N status, and the loss of each of these three proteins severely impair N homeostasis in the plants. A similar study showed that shoots of the CEPDL2/CEPD1/2 genotype characterize a high-affinity NO 3 − uptake duration in the roots, thereby indicating a systematic regulation of root N acquisition [84]. ANTHOCYANIN PIGMENT1 (PAP1) and its homolog PAP2/MYB90 were strongly stimulated by NO 3 − [129]. Recent research has demonstrated that three LBDs regulate anthocyanin synthesis via repression of PAP1 and PAP2. MYB and bHLH (basic helix-loop-helix) proteins form complexes with TTG1 (TRANSPARENT TESTA GLABRA1) WD40-repeat protein in Arabidopsis to modulate several other epidermal gene expressions such as anthocyanin regulation, proanthocyanin, and mucilage biosynthesis in the seed coat or trichome and root hair organogenesis [49].

Conclusions
RSA response of the plant to NO 3 − accessibility represents a prominent model to study developmental plasticity; however, the underlying mechanism remains highly obscure [130]. One of the most important discoveries in the past few years has been the involvement of NO 3 − transporters NRT1.1 and NRT2.1 in early response signaling, and their effects on the morphological adaptation of the plant RSA. Despite their roles as transporters and in signaling response, NRT1.1 cannot fully explain the complete mechanism of the NO 3 − responses observed in plants [43]. However, some findings have supported the previous speculation that NO 3 − transporters could act as early NO 3 − sensors [98]. This provides critical insights into understanding the ability to sense NO 3 − as well as other nutrients [52].
In this review, we have summarized in depth the characterization of the nitrate transporters NRT1.1 and NRT2.1 in Arabidopsis (Figure 1), delivering clues on how NO 3 − is sensed, taken up, and mobilized, and their modification by phosphorylation at the T101 residue has also been well demonstrated. In addition, the influences of physiological growth on RSA under low and high NO 3 − conditions, and the underlying molecular players, including TFs and N metabolites, are hypothesized and are associated with the transcriptional control of significant NO 3 − -responsive genes, which include NIA1, NIA2, NiR, NR, NRT2.1, -2.2, -2.4, -2.5, and NRT3.1. However, the fact is that different TFs, NLP7, TGA1/4, and TCP20, can regulate the expression of the same target gene, NRT2.1 (Figure 1). These TFs co-interact in response to NO 3 − to regulate root growth.
Despite the development of multiple NO 3 − signaling pathways regulating RSA and the characterization of primary Ca 2+ -induced responses elucidated in the present review, many important inquires on how PLCs are implicated in nitrate signaling and the specificity of the protein kinases that switch the different constituents of PLCs are yet to be answered. Moreover, the speculated nonphosphorylated form of the NRT1.1-signaling Ca 2+ -CPKs-NLPs pathway has received trivial experimental attention. Additionally, PLC-and Ca 2+ -independent nitrate signaling pathways have another component, as evidenced by AFB3 expression and its downstream TFs, which lead to the possibility that there might be another second messenger involved in nitrate responses. There are more nitrate regulatory modules in existence, with no clues about their signaling pathways and components; however, they are the fundamental contributors controlling LR development. Hence, functional identification and characterization of the various players associated with this and other NO 3 − signaling pathways and their possible functions in the root architecture of Arabidopsis is the next step to try and comprehend the NO 3 − responses that will facilitate crop genetics improvement.

Acknowledgments:
The authors would like to thank Anelia Marais for the critical reading of the manuscript.

Conflicts of Interest:
The authors declared that they have no conflict of interest. Regulator of V-ATPase in vacuolar membrane protein 2