Insights on Phytohormonal Crosstalk in Plant Response to Nitrogen Stress: A Focus on Plant Root Growth and Development

Nitrogen (N) is a vital mineral component that can restrict the growth and development of plants if supplied inappropriately. In order to benefit their growth and development, plants have complex physiological and structural responses to changes in their nitrogen supply. As higher plants have multiple organs with varying functions and nutritional requirements, they coordinate their responses at the whole-plant level based on local and long-distance signaling pathways. It has been suggested that phytohormones are signaling substances in such pathways. The nitrogen signaling pathway is closely associated with phytohormones such as auxin (AUX), abscisic acid (ABA), cytokinins (CKs), ethylene (ETH), brassinosteroid (BR), strigolactones (SLs), jasmonic acid (JA), and salicylic acid (SA). Recent research has shed light on how nitrogen and phytohormones interact to modulate physiology and morphology. This review provides a summary of the research on how phytohormone signaling affects root system architecture (RSA) in response to nitrogen availability. Overall, this review contributes to identifying recent developments in the interaction between phytohormones and N, as well as serving as a foundation for further study.


Introduction
Enhancing crop yields and reducing environmental risks simultaneously is a massive challenge in sustainable agricultural development. In the past four decades, agricultural food production has doubled worldwide, and nitrogen (N) fertilizer use has increased seven-fold. Plants require N as a major macronutrient, and its availability has been recognized for years as a critical factor in crop production and food security [1,2]. The overuse of N fertilizers results in a 25-50% decrease in plant N uptake efficiency and a 60-70% decrease in plant nitrogen use efficiency (NUE) [3,4]. Furthermore, the overuse of N fertilizers not only negatively influences human health but also boosts agricultural production costs and entails environmental risks, such as water resource contamination and soil salinization [5].
Enhancing NUE in plants is crucial to enhancing yields and quality, reducing nutrient input costs, and improving soil, water, and air quality [6]. In addition to reducing fertilizer input costs, higher NUE by plants can reduce nutrient losses and increase crop production. The goal of improving crop NUE is to understand the whole system, from the macro level (agroecosystem) to the molecular level [7]. Thus, improving crop yields and reducing environmental risks requires a better understanding of how plants improve NUE. N, an essential macronutrient for plants, is absorbed in two ways from the soil: inorganically, such as nitrate (NO 3 − ) or ammonium (NH 4 + ), or organically, mainly as free The lateral root primordium (LRP) is initiated and organized by CKs, most likely by disrupting the auxin gradient and inhibiting its formation. CK has been shown to regulate the endocytic recycling of the auxin efflux carrier PIN1 during lateral root (LR) development by redirecting it for vacuolar lytic degradation [54][55][56]. Aside from monitoring nitrate responses from root to shoot, CK acts as long-distance messengers. A significant amount of CK is produced when nitrate is applied, and as a result, that hormone may pass through the vascular bundles [57,58]. This leads to the accumulation of CK when nitrate increases the expression of CK biosynthesis genes CYP735A and IPT3 [48,59]. As a result of the nitrate treatment, seven genes in the CK pathway are also activated [60][61][62].
Nitrate feeding is proposed to increase the synthesis of isopentenyl (iP)-type CK in root phloem after IPT3 is activated. CYP735A converts this type of CK into trans-zeatin (tZ), which facilitates leaf expansion and modulates gene expression changes in the shoot when exposed to high levels of nitrates ( Figure 1). Aside from its proposed role as a long-distance signal, CK can also monitor local nitrogen (organ level) availability [63]. CK inhibits the accumulation of nitrate and ammonia transporters in nitrate-provided Arabidopsis plants' roots. This mechanism may reflect a negative regulatory mechanism, which decreases nitrate consumption in non-limited conditions [64,65]. tween CK and nitrogen [49,50]. In Plantago major, exogenous CK treatment can partially resolve growth-limiting results caused by low nitrogen supply [51,52]. A similar association has been recorded in Arabidopsis thaliana (A. thaliana) [53]. The lateral root primordium (LRP) is initiated and organized by CKs, most likely by disrupting the auxin gradient and inhibiting its formation. CK has been shown to regulate the endocytic recycling of the auxin efflux carrier PIN1 during lateral root (LR) development by redirecting it for vacuolar lytic degradation [54][55][56]. Aside from monitoring nitrate responses from root to shoot, CK acts as long-distance messengers. A significant amount of CK is produced when nitrate is applied, and as a result, that hormone may pass through the vascular bundles [57,58]. This leads to the accumulation of CK when nitrate increases the expression of CK biosynthesis genes CYP735A and IPT3 [48,59]. As a result of the nitrate treatment, seven genes in the CK pathway are also activated [60][61][62].
Nitrate feeding is proposed to increase the synthesis of isopentenyl (iP)-type CK in root phloem after IPT3 is activated. CYP735A converts this type of CK into trans-zeatin (tZ), which facilitates leaf expansion and modulates gene expression changes in the shoot when exposed to high levels of nitrates ( Figure 1). Aside from its proposed role as a long-distance signal, CK can also monitor local nitrogen (organ level) availability [63]. CK inhibits the accumulation of nitrate and ammonia transporters in nitrate-provided Arabidopsis plants' roots. This mechanism may reflect a negative regulatory mechanism, which decreases nitrate consumption in non-limited conditions [64,65]. Recent studies have revealed that when roots are exposed to various nitrate sources, C-TERMINALLY ENCODED PEPTIDE 1 (CEP1), CEP RECEPTOR (CEPR), and CEP DOWNSTREAMS (CEPDs) play key roles in regulating NRT2.1 (Figure 1) [67]. CEPDL2 can transport from the phloem to the cortex cells when NRT2.1 is expressed after CEP1/2 and CEPDL2 have moved from shoot to root through the phloem [68].
In response to changes in the light environment, it has been documented that ELONGATED HYPCOTYL 5 (HY5) translocates from the shoot to the root through the phloem and stimulates NRT2.1 expression [69]. CKs may play a role in controlling global Recent studies have revealed that when roots are exposed to various nitrate sources, C-TERMINALLY ENCODED PEPTIDE 1 (CEP1), CEP RECEPTOR (CEPR), and CEP DOWN-STREAMS (CEPDs) play key roles in regulating NRT2.1 (Figure 1) [67]. CEPDL2 can transport from the phloem to the cortex cells when NRT2.1 is expressed after CEP1/2 and CEPDL2 have moved from shoot to root through the phloem [68].
In response to changes in the light environment, it has been documented that ELON-GATED HYPCOTYL 5 (HY5) translocates from the shoot to the root through the phloem and stimulates NRT2.1 expression [69]. CKs may play a role in controlling global nutrient acquisition, while others (CEPD/CEPDL and HY5) may play more specialized roles in regulating nitrate uptake [70].
A model was proposed for CK's function as a root-to-shoot nitrate signal [71]. The ARABIDOPSIS HIS KINASE (AHKs, CK receptors), specifically (AHK3 and AHK5), have been identified to play critical roles in root and shoot growth ( Figure 2) [72]. Specific roles have been identified for each CK receptor. Notably, AHK3 and AHK5 play key roles in regulating the cell differentiation zone of the root meristem, root hair, and root elongation [73,74]. Table 1 summarizes a detailed summary of CK-related genes and their implications in RSA plasticity in response to various forms and concentrations of N.

Abscisic Acid (ABA) Modulates RSA in Response to Nitrogen Stress
Abscisic acid (ABA) is often referred to as a stress hormone but is actually a messenger that interprets both biotic and abiotic signals associated with the environment [81]. At the same time, it has been found that several plant species link ABA levels with nitrogen status. Although there is considerable evidence of a correlation between ABA levels and nitrogen status in several plant species, the relationship between both is not generally consistent [30,65,[82][83][84][85]. For instance, there is no statistically significant difference in ABA levels of A. thaliana between high-nitrogen (HN) and low-nitrogen (LN) seedlings [86,87]. Whether changes in ABA content are important to nitrogen signaling is still uncertain, but it is becoming evident that ABA is involved in nitrogen signaling. Several studies indicate that ABA is involved in lateral root growth in response to a high nitrate supply in A. thaliana [88,89]. Mutants from A. thaliana with impaired ABA synthesis showed a decreased inhibition of lateral root formation after applying nitrate, suggesting that ABA signaling, at least partially, underpins the nitrate-induced root branching repression [90]. A typical plant response to ABA is the suppression of lateral root initiation [91].
Another group of A. thaliana mutants showing ABA-insensitive lateral root initiation (LABI mutants) has shown decreased sensitivity to nitrate resupply, supporting the idea of specific regulatory elements for ABA nitrate signaling [88]. Identifying LABI genes will be a milestone in understanding the mechanisms behind this inhibition effect.
Furthermore, the rapid increase in ABA levels after nitrate treatment of barley roots may indicate that ABA directly controls the plant's response to high nitrate levels. However, it may also be used to adapt to sudden changes in nitrate availability [92,93]. Further evidence for a correlation between ABA and nitrogen signaling was found in a recent study in a Medicago truncatula lateral-root-organ-defective (LATD) mutant [94]. The LATD mutant exhibits severe abnormalities in root meristem maintenance and development, which are rectified by exogenous ABA treatment [95,96]. Notably, the LATD mutant has a nitrate-insensitive primary root development, and the LATD gene encodes a transporter belonging to the NRT1 (PTR) family [97,98]. Cytokinin stimulates, and auxin and ABA inhibit the expression of the LATD gene in root tips ( Figure 3) [13].
senger that interprets both biotic and abiotic signals associated with the environment [81]. At the same time, it has been found that several plant species link ABA levels with nitrogen status. Although there is considerable evidence of a correlation between ABA levels and nitrogen status in several plant species, the relationship between both is not generally consistent [30,65,[82][83][84][85]. For instance, there is no statistically significant difference in ABA levels of A. thaliana between high-nitrogen (HN) and low-nitrogen (LN) seedlings [86,87]. Whether changes in ABA content are important to nitrogen signaling is still uncertain, but it is becoming evident that ABA is involved in nitrogen signaling. Several studies indicate that ABA is involved in lateral root growth in response to a high nitrate supply in A. thaliana [88,89]. Mutants from A. thaliana with impaired ABA synthesis showed a decreased inhibition of lateral root formation after applying nitrate, suggesting that ABA signaling, at least partially, underpins the nitrate-induced root branching repression [90]. A typical plant response to ABA is the suppression of lateral root initiation [91].
Another group of A. thaliana mutants showing ABA-insensitive lateral root initiation (LABI mutants) has shown decreased sensitivity to nitrate resupply, supporting the idea of specific regulatory elements for ABA nitrate signaling [88]. Identifying LABI genes will be a milestone in understanding the mechanisms behind this inhibition effect.
Furthermore, the rapid increase in ABA levels after nitrate treatment of barley roots may indicate that ABA directly controls the plant's response to high nitrate levels. However, it may also be used to adapt to sudden changes in nitrate availability [92,93]. Further evidence for a correlation between ABA and nitrogen signaling was found in a recent study in a Medicago truncatula lateral-root-organ-defective (LATD) mutant [94]. The LATD mutant exhibits severe abnormalities in root meristem maintenance and development, which are rectified by exogenous ABA treatment [95,96]. Notably, the LATD mutant has a nitrate-insensitive primary root development, and the LATD gene encodes a transporter belonging to the NRT1 (PTR) family [97,98]. Cytokinin stimulates, and auxin and ABA inhibit the expression of the LATD gene in root tips (Figure 3) [13].
. In primary and lateral roots, LATD may regulate the activation of the meristems through a nitrate-ABA signaling pathway due to its homology to nitrate transporters [36]. This evidence also suggests a similar transition plays the same role in nodule formation. Given that LATD is expressed in both lateral roots and nodules, controlling LATD might be extremely important for maintaining a balance between lateral root and nodule development ( Figure 3). LATD may perform a sensing function, possibly in response to nitrate or ABA, or it may be part of a sensing system. Future biological experiments should assist us in understanding how LATD works.
ABA also influences LR growth in the presence of localized NO 3 − availability. It was documented that mutants lacking ABA biosynthesis (aba1-1, 2-3, 2-4, and 3-2) as well as those lacking ABA insensitivity (abi4-1, 4-2, and 5-1) exhibited longer LR as a result of localized NO 3 − supply than wild type (WT) (Figure 2) [99], suggesting that ABA acts as a negative regulator to modulate LR elongation. A summary of all the genes involved in N uptake and transport that are regulated by ABA is shown in Table 2. Table 2. List of genes and their roles associated with N stress and ABA in Arabidopsis and wheat.

Gene Name
Gene ID Functions References

AtPYL2 At2g26040
Involved in ABA signaling and PR and LR growth in response to NO 3 AtSnRK2.

Auxin (AUX) Modulates RSA in Response to Nitrogen Supply
Auxins are a group of important phytohormones that modulate plant development and morphology to environmental conditions and are vital under nitrogen stress [23,104,105]. Auxin accumulation is dependent on nitrogen sources [35,103]. Nitrogen can modulate auxin signals in both A. thaliana and rice [106,107]. Auxin signaling is upregulated in response to nitrogen starvation, as shown by the DR5:: GUS reporting method and reverse genetic approaches [20,49]. Auxin signaling is involved in primary and lateral root development in Arabidopsis in response to nitrate [108][109][110]. It has been widely thought that nitrogen signals are transmitted from shoot to root because auxin is transported basipetally and promotes lateral root growth [111,112]. As a result of high doses of nitrate being applied to maize, root growth is reduced, and less auxin is produced in the roots [113,114]. More important findings come from multiple transcriptome studies, indicating that the nitrate treatment impact genes are involved in auxin transport [115][116][117].
Similarly, a switch from a high-nitrate to a low-nitrate medium showed that auxin, in the roots, increased following lateral root growth in A. thaliana. Furthermore, plants grown in a medium with LN levels produced lower levels of auxins in their shoots than in a medium with HN [118,119]. Additionally, Arabidopsis seedlings cultivated under LN conditions produced greater root auxin levels than those grown under HN conditions, which indicates that dicot and monocot plants share a similar mechanism for controlling root auxin levels according to the nitrogen level of the plant [120]. However, it is critical to investigate this idea further because auxins do not reduce lateral root development suppression in plants growing in high-nitrate environments, implying that these hormones do not particularly control nitrate signaling. However, other signals may still be necessary to alter nitrate inhibitor effects [88].
A recent study in understanding auxin action suggests that the driving force of auxinregulated growth and development is the concentration gradient and the differential sensitivity of various cell types, apart from the auxin levels in tissue. Cell-to-cell polar transport establishes the auxin gradient, and the differential sensitivity is achieved by modulating signaling components [121,122]. Recent research indicates that nitrogen signaling is regulated by the same or similar mechanisms [10,82,123,124]. It is now evident that auxins play a crucial role in regulating root system architecture at various stages, such as biosynthesis, signaling, transport, and auxin distribution.
Multiple studies have shown that auxin biosynthesis, transport, and accumulation are affected by different nitrogen regimes in maize [113], Arabidopsis [110], soybean [125], and rice [126,127]. Several important auxin-related regulatory modules that respond to nitrogen availability in Arabidopsis have been identified, including TAR2, a gene involved in auxin biosynthesis, auxin transporters such as PIN1, PIN2, PIN4, and PIN7, as well as molecular components that control their subcellular distribution [128]. As an N-responsive gene in the pericycle, AUXIN RESPONSE FACTOR 8 (ARF8) was identified as involved in auxin signaling ( Figure 2) [129]. The ratio between the initiation of LRs and their emergence is controlled by ARF8 and its associated microRNA167s [130]. The nitrate transceptor, NRT1.1, is another mechanism that contributes to nitrogen-auxin interplay underpinning root system adaptation [131]. In NO 3 − -rich patches of soil, roots colonize, and this adaptation causes AXR4, a gene initially implicated in auxin signaling, to be activated ( Figure 2) [132]. In addition, AXR4 was found to target AUX1 to the plasma membrane, indicating it plays an important role in auxin transport [133]. However, AXR4's potential as a nitrate carrier or sensor remains to be seen. More recently, RSA has been controlled by a unique N-regulatory network based on miR393/AFB3 [134].
It has been demonstrated that NRT1.1/CHL1, a nitrate sensor and dual-affinity nitrate transporter, contributes to the nitrate-induced expression of AtIPT3 ( Figure 2) [131]. Auxin transport by NRT1.1 is thought to explain the discovery that a chl1 mutant accumulates auxin in LRP [135], which suggests that nitrate and auxin signaling are interconnected. Table 3 summarizes all genes and their functions involved in AUX-regulated N uptake and transport.

Ethylene (ETH) Modulates RSA in Response to Nitrogen Stress
Ethylene is a master regulator of root growth and development due to its involvement in both indole acetic acid (IAA) trafficking and partitioning along the primary root and root growth [143]. Ethylene boosts IAA production by triggering tryptophan aminotransferase (TAA1) and tryptophan aminotransferase-related (TAR1 and TAR2) enzymes, which have similar roles in ethylene responses [144]. It has been demonstrated that nitrogen consumption reduces leaf longevity primarily by altering the levels of ethylene in leaves. Perhaps a high N concentration increases the activity of enzymes involved in ethylene biosynthesis, accounting for the decrease in leaf N and leaf longevity caused by cobalt-chloride-induced reduction in ethylene biosynthesis [145].
Several researchers have explored the short-term (≤24 h) ethylene response to changes in external nitrate availability through the expression of nitrate transporter (NRT) genes [146,147]. Seedlings were grown in a low-nitrate (0.1 mM) concentration solution for 5 days before being transferred to a high external concentration (10 mM) solution for 6-24 h and analyzed for NO 3 − /ethylene interactions [145]. Another study examined this interplay by growing seedlings in high-nitrate environments (10 mM) for 1 week before transferring them to environments with low external nitrate levels (0.2 mM) for 24 h [148]. Following a burst of ethylene production (0.5-1 h) from both stresses in the roots, the ethylene concentration gradually decreased. Moreover, the exogenous application of the ethylene synthesis precursors AMINOCYCLOPROPANE CARBOXYLIC ACID SYNTHASE (ACC) and AMINOETHOXYVINYLGLYCINE (AVG) at low and high nitrate concentrations led to the upregulation and downregulation of nitrate transporters (AtNRT1.1 and AtNRT2.1) [145]. However, etr1-3 and ein2-1 mutants showed no sensitivity to high nitrate concentrations [145], concluding that ethylene biosynthesis and signaling are necessary for regulating both AtNRT genes ( Figure 4A).    A high level of nitrate may modulate the actions of ethylene on nitrate transporters and metabolism [150]. It was documented that the expression of AtNRT2.1 rather than AtNRT1.1 was associated with ethylene biosynthesis and signaling responses in seedlings transferred from high to low nitrate concentrations [148]. In particular, a study of the expression of AtNRT2.1 in wild-type and mutant forms of the ctr1-1, ein3-1, and eil-1 genes revealed that ethylene repressed AtNRT2.1 and nitrate acquisition through one of the ethylene signaling cascade's components [148]. Therefore, a feedback loop under nitrate deficiency is linked to ethylene biosynthesis and signaling and AtNRT2.1 expression ( Figure 4B). Consequently, the results show that ethylene biosynthesis and signaling play a role in the short-term responses to nitrate deficiency and excess via fine-tuning the expression of the AtNRT2.1 and AtNRT1.1 genes. This study does not demonstrate how ethylene can affect nitrate uptake by altering the root system's long-term morphology (Figure 2).

(A)
An interesting study reveals that ethylene may serve as a signal for plant-to-plant communication in rice under high-density stress conditions when ethylene production is linked to genes involved in ethylene homeostasis [151]. Researchers found that the amount of nitrogen (N) in the soil can affect the growth of ethylene-dependent rice plants. They also found that the development of the plants was hampered in high-density situations when N was either limited (3 mM NO 3 − ) or sufficient (10 mM NO 3 − ). In Brassica juncea plants, N availability impacts photosynthesis, stomatal conductance, and growth and influences ethylene's evolution [152,153]. According to a field study, ethephon (an ethylene-releasing chemical) improved ethylene production and photosynthesis of plants grown with N levels of 40 and 80 mg kg −1 [152]. Table 4 lists all of the genes and their functions that are involved with ETH-regulated N uptake and transport.
Many plant species produce strigolactones as plant hormones [164]. Despite being primarily produced by roots, strigolactones are also synthesized in other parts of plants [165]. Furthermore, it has been reported that strigolactones' biosynthesis is impacted by N, Pi, and S deficiencies [166,167]. SL biosynthesis can increase with the depletion of these nutrients, but distinct catalytic steps have been observed. The expression levels of MAX3 and MAX4 are consistently increased by N and Pi deficiency in A. thaliana, similar to Oryza sativa D17 and D10 [166,168].
Low levels of N have been demonstrated to trigger an undisclosed mechanism that reduces the concentrations of jasmonic acid (JA) and jasmonic acid-isoleucine (JA-Ile) in A. thaliana roots [169][170][171].
There is currently limited understanding of how the availability of nutrients affects the production of salicylic acid (SA) in the roots. Two recent studies have demonstrated that roots that lack both N and Pi accumulate more SA [172,173]. Despite this, little is known about the underlying mechanism. The gene functions associated with N uptake and transport regulated by BR, SA, and SL are summarized in Table 5.

Conclusions and Prospects
This review comprehensively explains the molecular mechanisms of phytohormones in nitrogen signaling. Phytohormones (CK, ABA, AUX, ETH, BR, SL, JA, and SA) are the key players that regulate several signaling pathways in response to nitrogen stress and modulate root system architecture ( Figure 2). Nitrogen and phytohormones signify a considerable research gap for each defined effect on root growth and development. Researchers have reported an interaction between auxin and other phytohormones (such as CK, AUX, ABA, etc.) that regulates RSA [182][183][184][185]. However, it is uncertain if interactions between multiple phytohormones have a role in response to N availability.
Furthermore, it is worth exploring how phytohormone signals are influenced by N availability through crosstalk between key regulatory elements. It is a fascinating hypothesis, but many questions remain unanswered. What is the process for assessing and translating nutritional status into phytohormone signals? The process of sensing and translating takes place in which cell, tissue, or organ? Which nutrient signals are the most important? What is the effective way of transmitting phytohormones and nutrients to plant target sites? Where and how are the phytohormonal signal and nutrient-specific signals interpreted and incorporated to deliver a proper nutrient response? In conjunction with system biology approaches, genomic research possesses a great perspective to identify the most critical players in interconnected regulatory networks.
It is most advantageous to apply advanced biotechnology approaches, including genomewide association studies, omics, and bioinformatics, to decipher the genetic response of RSA to N signals and pinpoint valuable natural alleles. In addition, the identified favorable alleles can be utilized to improve N utilization efficiency using CRISPR/Cas9 technology in crops by determining root tissue/cell-specific expression and allele-specific modification. A clear understanding of genetic regulation of nitrogen use efficiency (NUE) and breeding crops with high yields using low nitrogen fertilizer inputs will be essential in the future with the use of advanced biotechnologies and accumulated basic research findings.