Auxin has been proposed as a long-range signal from shoot to root mediating root development in response to nitrate (Forde and Lorenzo, 2001) and there are strong connections between auxin and nitrate signaling, which could cooperatively regulate LR development (Zhang et al., 1999; Gutiérrez et al., 2007; Tian et al., 2008; Krouk et al., 2010; Mounier et al., 2014). A positive effect of mild N deprivation on LR formation in Arabidopsis required an auxin biosynthesis gene TAR2 (tryptophan aminotransferase related 2). TAR2 can convert L-Trp to indole-3-pyruvic acid (IPyA), which is the first step in the IPyA pathway branching from a Trp-dependent auxin pathway (Tao et al., 2008; Won et al., 2011; Zhao, 2012). Under low NO3– supply, the expression of TAR2 was up-regulated, resulting in an increase in IAA levels in the developing LRs. However, the tar2-c null mutants had much shorter total LR length and fewer visible LR numbers, indicating that mutation of TAR2 impaired the LR formation (Ma et al., 2014). Therefore, low NO3–-stimulated LR emergence depended on root-synthesized auxin in a TAR2-dependent manner. However, the components acting upstream of TAR2 remain unclear.

Another example of the stimulatory effect of low nitrate on LR development was mediated by an Arabidopsis AGL17-clade MADs-box gene AGL21. The expression of AGL21 was induced by N deprivation and auxin. The study found that AGL21-overexpressing plants produced more visible and longer LRs, while agl21 mutants showed a reduction on LR elongation under N-restricted conditions. Moreover, auxin biosynthesis genes YUC5, YUC8, and TAR3 were significantly up-regulated in AGL21-overexpressing plants and down-regulated in agl21 mutants (Yu L. et al., 2014). These findings suggested that AGL21 positively regulated LR development by enhancing local auxin biosynthesis in LR primordia and LRs.

In addition, a previous study on rice revealed that knockdown of OsNAR2.1, the complementary partener of NRT2.1, inhibited LR formation under low NO3– concentrations, by reducing the expression levels of PINs in roots (Huang et al., 2015). This evidence demonstrated that NAR2.1 played a positive role in regulating LRs development by affecting auxin polar transport under low NO3– conditions. As the author mentioned, the effects of NAR2.1 on LR formation were most likely by a combination of NO3– uptake and NO3– signaling. NAR2.1 has no NO3– transport function itself, therefore, the role of NRT2.1 may be indispensable in this signaling pathway, which has not been determined.

Apart from the above-mentioned pathways including transcription factors and hormonal signals, more recently, nitric oxide (NO) has been reported to be a key nitrate-related signal to regulate the root system architecture in plants (Chen et al., 2010; Meng et al., 2012; Trevisan et al., 2014). In rice, NO generated by the nitrate reductase (NR) could improve the N acquisition capacity by increasing LR initiation under partial nitrate nutrition (Sun et al., 2015). However, a study in maize supported that NO played a key role in early nitrate perception and could induce a more elevated primary root elongation after low nitrate resupply (Manoli et al., 2014). Therefore, the role of NO in the nitrate signaling pathway needs further evaluation.

In a word, nitrate signaling pathways have a stimulatory effect on LR development under mild N deficiency. The molecular players involved in the pathways regulate different stages of LR development through affecting not only auxin biosynthesis but its transport. Moreover, we speculate that nitrate signaling components act upstream of auxin on regulating LR development.

Under severe N deficiency, both formation and length of LRs were inhibited in plants (Gruber et al., 2013). Further studies indicated that they are involved in CLE [CLV3/ENDOSPERM SURROUNDING REGION (ESR)]-related peptides and the CLAVATA1 (CLV1) lecuine-rich repeat receptor-like kinase regulatory module. CLEs and its receptors CLVs control meristem functions and were required for maintenance of shoot apical meristem, respectively, in plants (Clark et al., 1997; Brand et al., 2000; Kondo et al., 2006; Mitchum et al., 2008; Ogawa et al., 2008; Stahl et al., 2009). In addition, CLE genes (CLE1, 3, 4 and 7) were up-regulated in root pericycle cells when plants were under limited nitrate conditions (<100 μM). What’s more, the CLE-overexpressing plants had significantly inhibited outgrowth of LRP and their emergence, while the clv1 mutants exhibited an increased LR growth. Moreover, overexpression of CLE3 inhibited LR growth in wild-type plants, but not in clv1 mutants. The up-regulation of CLE2, -3, -4, and -7 in clv1 mutants suggested the amplitude of the CLE peptide was feedback-regulated by CLV1 (Araya et al., 2014a,b). In short, CLEs-CLV acted as a regulatory module in the nitrate signaling pathway and negatively regulated lateral root development under N deficiency condition. In addition, C-TERMINALLY ENCODED PEPTIDE (CEP) hormones, 15-amino-acid peptides, are proved to be the negative regulators of root development and growth in plants (Ohyama et al., 2008; Delay et al., 2013; Tabata et al., 2014). A study in Medicago truncatula found that the expression of MtCEP1 was up-regulated by N starvation and overexpressing MtCEP1 caused the inhibition on LR formation, indicating that MtCEP1 peptide could negatively modulate LR formation under low NO3– concentrations. The further RNA-seq analysis revealed that transcription factors WRKY, bZIP, MYB and homologs of LOB29, SUPERROOT2, etc., may act downstream of MtCEP1 (Imin et al., 2013).

Another mechanism for systemic inhibition of LR development in response to N deficiency was identified. Low nitrate triggered a significant increase in ABA accumulation, and ABA accumulation would inactivate its coreceptor ABI2 (ABA-insensitive 2), a protein phosphatase 2C (PP2C) (Joshi-Saha et al., 2011). ABI2 then interacted with and dephosphorylated the Ca²⁺-sensor subunit CBL1 and the kinase CIPK23 (CBL1-CIPK23) complex, whose substrate was NRT1.1. Moreover, CIPK23 could interact with NRT1.1 in the plasma membrane and phosphorylate NRT1.1, which inhibited the activity of NRT1.1 at low nitrate concentrations (Ho et al., 2009). A recent study reconfirmed the above-mentioned speculative pathway. Mutation of ABI2 leads to the activation of CBL1-CIPK23 complex, then results in reduced root NO3– uptake by repressing the transport activity of NRT1.1 under nitrate deficiency (Léran et al., 2015). As ABA could inhibit LR formation by increasing endogenous ABA biosynthesis in plants (Guo et al., 2009) and the downstream components involved in this pathway are still unclear. Therefore, it is ambiguous whether the negative influence on LR development involving ABA under N deficiency is due to the impaired NO3– signaling effects or ABA physiological function.

In addition, there are some evidences that nitrate transporters with a sensing role are involved in nitrate signaling pathway. NRT1.1 acts as a NO3– transceptor, with a dual transporter/sensor function in signaling pathway (Remans et al., 2006; Ho et al., 2009; Gojon et al., 2011). However, NRT1.1 could control the growth of LRP under the absence of NO3– or low NO3– condition (Bouguyon et al., 2016). While under N-limited condition, AtNRT2.1 might act as NO3– sensor or signaling component repressing LR initiation, which was independent of its NO3– uptake activity (Little et al., 2005; Remans et al., 2006). Finally, although the definite mechanism is still uncertain, the negative effect of NRT1.1/NRT2.1 on LR development may represent a distinct systemic pathway under low nitrate conditions.

In all, although acting antagonistically and having opposite effects, these systemic pathways coexist to regulate LR development in response to low N. Which pathway taking action depends on the degree of N deficiency that the plants suffered from or their specific environment conditions.

Auxin biosynthesis, polar transport, and signal transduction are key players in regulation of LR development (Casimiro et al., 2001; De Smet et al., 2006; Laskowski et al., 2008; Rubio et al., 2009; Krouk et al., 2011; De Smet, 2012). Therefore, the involvement of auxin in the high nitrate concentration-induced inhibitory effect on LR development is of no surprise. Auxin receptor AFB3 was strongly induced by high nitrate concentration, but was significantly repressed by NO3– metabolites via the feedback regulation by miR393 (Vidal et al., 2010). miR393/AFB3 was identified as a unique module to regulate LR initiation in Arabidopsis. The pathway was extended by the founding that nitrate-induction of transcription factors NAC4 and OBP4 required the existence of AFB3; Similar to the afb3 mutant, a nac4 mutant was less sensitive to nitrate-stimulated LR initiation. Meanwhile, the expression of OBP4 was significantly reduced in nac4 mutants (Vidal et al., 2013). Therefore, it can be speculated that AFB3 acts upstream of NAC4 and OBP4 to regulate the density of LR. A recent study has extended this pathway in the other direction, nitrate-regulation of AFB3 and NAC4 is dependent on the NO3– transport function of NRT1.1, but not its sensing function (Vidal et al., 2014). Except for an auxin signaling pathway, high nitrate regulates LR development also through affecting auxin levels in roots. For instance, when external nitrate concentration was greater than 10 mM, the elongation of LRs in maize was inhibited, which was due to a reduction of auxin translocation from shoot to root in phloem. Therefore, a reduced auxin level in roots resulted in the inhibition of LR growth (Tian et al., 2008).

At high concentrations, nitrate inhibited LR outgrowth through ABA signaling (Signora et al., 2001; Vidal et al., 2010). ABA insensitive mutants abi4-1, abi4-2 and abi5-1 were less sensitive to the inhibitory effect of high nitrate on LR; and ABA synthesis mutant abas produced a significant increase in LR growth, suggesting that the inhibitory effect caused by high nitrate on LR growth was required for both ABA accumulation and signaling (Signora et al., 2001). In addition, genetic analysis revealed that ABA signaling acted downstream of NO3– perception in the regulation of root architecture (Signora et al., 2001). However, a new study demonstrates that high NO3– supply (30 mM) stimulates ABA accumulation in growing roots tips by releasing it from inactive stores via ER-localized β-GLUCOSIDASE1 (BG1), thereby regulating root growth (Ondzighi-Assoume et al., 2016). These data provide a mechanism for NO3–-regulated root growth via the regulation of ABA accumulation in the root tip. In conclusion, we can speculate that there is a close interaction between ABA and nitrate signaling, which could jointly regulate LR formation.

Many evidences proved that ethylene inhibited LR development in plants by altering auxin transport and there was crosstalk between ethylene and auxin in regulating LR formation (Ivanchenko et al., 2008; Negi et al., 2008; Lewis et al., 2011). However, the crosstalk between ethylene and nitrate signaling regulating LR development is rarely reported. A previous study found that ethylene was involved in nitrate-dependent LR growth and branching in Arabidopsis. High nitrate induced a rapid burst of ethylene in immature LRs. The ethylene burst then upregulated and downregulated the expression of AtNRT1.1 and AtNRT2.1 in roots, respectively, resulting in the inhibition of immature LR growth in Arabidopsis. In addition, the etr1-3 and ein2-1 mutants were insensitive to high nitrate concentrations, suggesting that ethylene signaling genes ETR1 and EIN2 might involve in this regulatory pathway (Tian et al., 2009). As there is no related study exploring the players downstream of NRT1.1 and NRT2.1, the mechanism of LR inhibition by ethylene under high nitrate condition becomes obscure. However, IAA and ACC (the precursor of ethylene) could inhibit root elongation synergistically (Stepanova et al., 2007; Swarup et al., 2007). The auxin transport or biosynthesis mediated by ethylene may be the possible explanation under this situation.