Among all nutrients, nitrogen (N) is quantitatively the most important essential mineral element for plants and thus its availability a critical determinant in agricultural plant production. The accessibility of N for plant roots varies considerably through space and time, due to soil heterogeneity, anthropogenic N inputs, water flows, microbial activity, and other factors that dislocate or convert N forms (Bloom, 2015). Plants acquire N mostly in the form of ammonium (NH₄ ⁺) and nitrate (NO₃ ^-), with the latter being the major form in most plants grown on crop soils (Miller et al., 2007). As plants have adapted to fluctuating NO₃ ^- concentrations and external pH levels, they evolved sophisticated transport systems that enable efficient NO₃ ^- uptake coupled with protons (H⁺), ensuring N acquisition even when NO₃ ^- availability is limited in acidic conditions (Tsai and Schmidt, 2021). At micromolar substrate concentrations, root uptake is mainly mediated by high-affinity NRT2-type transporters (Bouguyon et al., 2012), whereas at millimolar concentrations the high-affinity transporters become repressed and low-affinity transporters take over. Here, the transceptor NRT1.1/NPF6.3/CHL1 (hereby NRT1.1) plays a central role as it confers most of the low-affinity NO₃ ^- transport capacity (Krouk et al., 2010).

NRT1.1 also mediates NO₃ ^- signaling and triggers the downstream transcriptional upregulation of a plethora of NO₃ ^–inducible genes (Wang et al., 2009; Bouguyon et al., 2015). Moreover, NRT1.1 is extensively involved in cellular and physiological processes of plant growth and development. These include NO₃ ^- root-to-shoot translocation, shoot transpiration, auxin transport, seed dormancy relief, and enhanced tolerance to adverse environmental conditions such as H⁺ excess, sodium, cadmium, zinc, lead, or NH₄ ⁺ toxicity, as well as iron or phosphorus (Pi) deficiency (Fang et al., 2021). Although specific topics associated with the NO₃ ^- transport and sensing functions of NRT1.1 have been discussed in multiple reviews, only a few studies have revealed its signaling function in the absence of NO₃ ^- (Wang et al., 2009; Jian et al., 2018; Hachiya et al., 2011, 2024).

With regard to NH₄ ⁺ uptake systems, high-affinity transporters of the AMT family appear being of major importance in plant roots, because NH₄ ⁺ concentrations in the majority of crop soils usually range between 20 and 200 μM (Lark et al., 2004). Out of the five AMT proteins expressed in Arabidopsis roots, AMT1.1, AMT1.2, AMT1.3, and AMT1.5, are responsible for high-affinity NH₄ ⁺ uptake in N-deficient plants (Loqué et al., 2006; Yuan et al., 2007). Between 60 and 70% of this transport capacity is mediated by AMT1.1 and AMT1.3, while AMT1.2 with its lower affinity confers another approx. 20% and mainly retrieves NH₄ ⁺ from the apoplastic transport route (Duan et al., 2018). As concluded from the remaining uptake capacity in the quadruple knock-out line qko (amt1.1, 1.2, 1.3, 2.1), AMT1.5 confers less than 10% of the overall high-affinity NH₄ ⁺ uptake capacity, probably at fairly high affinity (estimated K_m ~5 µM (Yuan et al., 2007)). On the other hand, under certain conditions, NH₄ ⁺ concentrations can exceed those of NO₃ ^- and be the main source of N, such as in acidic or paddy soils, which can cause toxicity and arrested plant growth (Xu et al., 2012). Unlike H⁺ cotransport during NO₃ ^- uptake, only transport activities of AMT1.1 in wheat and common bean are pH-dependent, exhibiting an acid-stimulated regulatory mode (Søegaard, 2009; Ortiz-Ramirez et al., 2011), whereas the transport activities in Arabidopsis, tomato, and rice are pH-independent when expressed in oocytes.

Although the environmental challenge urges to reduce the input of NO₃ ^–based fertilizers (Howarth, 2008), NH₄ ⁺ as the main source of N is problematic, and is partly overcome in agricultural practice by the coating of urea fertilizers with urease inhibitors to generate a slow and continuous release of NH₄ ⁺. For this reason, the application of both N form in plant species-specific ratios has proven beneficial for crops (Britto and Kronzucker, 2013). However, the reason for NO₃ ^- and NH₄ ⁺ positive synergy in plant growth and development is not fully understood. Furthermore, while the molecular mechanisms by which plants mediate and modulate the activity of their uptake systems for NO₃ ^- and NH₄ ⁺ have been well characterized, it has remained unclear to what extent these transport systems interact to ensure optimal N uptake and growth. In this review, the interdependency of the AMT1s and NRT1.1 during N uptake balance is discussed. Emphasis is placed on how NRT1.1 signaling, both NO₃ ^–dependent and independent, affects NH₄ ⁺ tolerance, alongside the underestimated role of AMT-dependent NH₄ ⁺ uptake in nutrient homeostasis and cellular pH regulation.

Hachiya and Noguchi (2011), and later Jian et al. (2018), pointed out that a NO₃ ^–independent function of NRT1.1 might exist in Arabidopsis, as functional disruption of NRT1.1 in the so-called chl1–5 mutant confers increased tolerance to sole NH₄ ⁺ nutrition. The main reason for this phenomenon remains unclear. The reduction of shoot transpiration in the chl1–5 mutant (Guo et al., 2003) was excluded as the cause of the observed NH₄ ⁺ tolerance, since no significant differences in the stomatal conductance were found with respect to the wild-type under sole NH₄ ⁺ nutrition (Jian et al., 2018).

There is also some divergent findings regarding the possible transcriptional and post-transcriptional repression of AMTs in the chl1–5 as for its NH₄ ⁺ tolerance. On one hand, Hachiya et al. (2011, 2024) did not observe differences in AMT transcript levels between wild-type Col-0 and chl1–5 under sole NH₄ ⁺ nutrition. Similarly, Muños et al. (2004) did not find differences in the ¹⁵NH₄ ⁺ uptake capacity under same conditions, leading to the conclusion that NH₄ ⁺ tolerance is unlikely to result from AMT repression or lower NH₄ ⁺ uptake. On the contrary, Jian et al. (2018) observed a significant downregulation of AMT genes in the mutant (specifically of AMT1.1, 1.3 and 1.5), together with a decline in the ¹⁵NH₄ ⁺ uptake under sole NH₄ ⁺ nutrition. Likewise, they observed a lower accumulation of free NH₄ ⁺, which the authors attributed to decreased NH₄ ⁺ uptake combined with the induction of glutamine synthetase activity in the roots. They suggested that the enhanced primary assimilation of NH₄ ⁺ in chl1–5 plays a crucial role in its NH₄ ⁺ tolerance. The later study by Hachiya et al. (2021) demonstrated that glutamine synthetase activity in roots attenuates NH₄ ⁺ toxicity, whereas in shoot causes acidic stress that arrests cell growth in Arabidopsis.

Another process that appears to contribute to mitigating the NH₄ ⁺ toxicity in chl1–5 is the promotion of glucosinolate (GSL) biosynthesis. According to the microarray analysis of Wang et al. (2009), several genes relevant to the aliphatic GSL biosynthetic pathway were induced in chl1–5 mutant in the absence of NO₃ ^-. Recently, Hachiya et al. (2024) confirmed that the biosynthesis of GSLs and their positive regulator genes MYB28 and MYB29 were indeed significantly induced in chl1–5 in the absence of NO₃ ^-. Since GSLs are N-rich secondary metabolites whose synthesis increases under NH₄ ⁺ nutrition (Marino et al., 2016; Coleto et al., 2017), it is plausible that NH₄ ⁺ tolerance of chl1–5 could be due in part to the promotion of NH₄ ⁺ assimilation into these compounds rather than into Gln, a H⁺ generating process.

Taken together, despite some discrepancies in AMT expression and/or activity, the results suggest that the NH₄ ⁺ tolerance observed in chl1–5 mutants is more closely linked to reduced NH₄ ⁺ accumulation in the shoot. However, more studies are needed to elucidate how GSLs, other genes or hormones are involved in the NO₃ ^–independent signaling of NRT1.1 during NH₄ ⁺ nutrition.

An additional detail is that the NH₄ ⁺-tolerance of chl1–5 in the absence of NO₃ ^- was more pronounced at lower pH (Hachiya et al., 2011), so there might be a pH-dependent effect that could ‘amplify’ NRT1.1-dependent signaling. Indeed, excess NH₄ ⁺ uptake mediated by AMTs leads to considerable acidification of the apoplastic pH, which causes ionic imbalances during nutrient uptake and an array of constraints to plant fitness in the long-term. Decreasing the medium pH from 6.5 to 5.5 increased the expression and activity of NRT1.1 irrespective of the presence of NO₃ ^-, indicating that pH alone can trigger NRT1.1 readiness in anticipation of reduced levels of available NO₃ ^- (Tsay et al., 1993; Ye et al., 2021). Although it has not yet been demonstrated which form of NRT1.1 exists predominantly under low pH/high NH₄ ⁺ conditions, it is well known that CBL-INTERACTING PROTEIN KINASE 23 (CIPK23) stabilizes the NRT1.1 monomeric state by phosphorylation, enabling NRT1.1 to act as a high-affinity NO₃ ^- transceptor (Ho et al., 2009; Rashid et al., 2020). Since CIPK23 is also induced by low pH and excess NH₄ ⁺ (see next section) (Straub et al., 2017; Ye et al., 2021), and post-translationally de-repressed by the protein phosphatase ABA-INSENSITIVE 1 and 2 (Ganz et al., 2022; Léran et al., 2015), CIPK23 likely enhances phosphorylation of AMT1 to prevent NH₄ ⁺ uptake dependent toxicity, while NRT1.1 to stimulate NO₃ ^- once available. In addition to CIPK23, the kinase CIPK15 also represses the activity of AMT1.1 in Arabidopsis through phosphorylation under high ammonium conditions (Chen et al., 2020). Also, the phosphatases ABI1 and ABI2 inactivate CIPK15 via dephosphorylation, although there is currently no evidence that they also target NRTs. So far, one could speculate whether might be a ‘competition’ between NRT1.1 and AMTs as common targets for post-translational modifiers (e.g., CIPK23, ABI1, ABI2). This competition could potentially reduce the capacity of those kinases and phosphatases to control their activity, thereby influencing N uptake.

In the field, NH₄ ⁺ accumulation and low pH often arise simultaneously, because nitrification is generally inhibited under low pH condition. However, strict NH₄ ⁺ conditions are unlikely in most crop soils, and even the presence of a low concentration of NO₃ ^- improves NH₄ ⁺ tolerance (Garnica et al., 2010; Hachiya et al., 2012; Zheng et al., 2015; Fang et al., 2016; Du et al., 2021). Thus, under most soil conditions, the growth and tolerance of defected NRT1.1 mutants to NH₄ ⁺/low pH are reduced due to their restricted NO₃ ^-/H⁺ uptake and primary NO₃ ^- response (Fang et al., 2016). At the gene expression level, NRT1.1 is also induced by low external pH that achieve a maximum at pH 5.0 in Arabidopsis (Ye et al., 2021). A low pH is compensated by more efficient NO₃ ^- uptake by NRT1.1, whose protonated H356 residue is essential for increasing the NO₃ ^- and H⁺ cotransport (Sun et al., 2014). Therefore, NRT1.1 is essential to avoid further H⁺ rhizotoxicity, and in turn the associated ionic imbalances such as excessive root accumulation of Fe²⁺ and Mn²⁺, or lower phosphorous (Pi) availability (Kochian et al., 2004).

In the context of NH₄ ⁺ nutrition, apoplastic acidification is promoted under excessive NH₄ ⁺ nutrition. The AMT-dependent NH₄ ⁺ uptake contributes to depolarizing the electrical membrane potential, which in turn enhances net H⁺ efflux, primarily mediated by AHA2 (Meier et al., 2020). Therefore, the induction of NRT1.1 during NH₄ ⁺-promoted apoplastic acidification is plausible for plants to ensure a correct charge balance, which this is in what lies partially the NO₃ ^–dependent alleviation of NH₄ ⁺ toxicity when sufficient external NO₃ ^- is present. Under low external NO₃ ^- conditions, NRT1.1 is also involved in regulating the apoplastic pH through modulation of AHA2 via the co-receptor kinase QSK1 (Zhu et al., 2024). NRT1.1 interacts with QSK1, prompting QSK1 to phosphorylate AHA2 at the S899 site, which inhibits AHA2 activity and reduces the H⁺ efflux into the apoplast. This mechanism decreases external acidification and is associated with the repression of lateral root (LR) growth under unfavorable low NO₃ ^- conditions. This functional connection between NRT1.1 and AHA2 extends to the antagonistic role of AMTs, as NH₄ ⁺ activates AMT1.3 signaling, promoting LR emergence and higher-order LR branching (Meier et al., 2020). While the role of auxin in NO₃ ^–mediated LR elongation is well established, further research is needed to determine whether AMTs and auxin also contribute to regulating the NRT1.1-QSK1-AHA2 module, particularly under varying N ratios and concentrations.

On top of NRT1.1 function, additional players have been added during adaptation of Arabidopsis to NH₄ ⁺ nutrition and low pH, such as the S-type channel SLAH3 (Zheng et al., 2015; Xiao et al., 2022), and the C2H2-type zinc finger transcription factor SENSITIVE TO PROTON RHIZOTOXICITY 1 (STOP1) (Ye et al., 2021). SLAH3 form a functional unit with NRT1.1 and exports chloride and NO₃ ^- from the cytosol, whereas STOP1 plays a central role in the induction of NRT1.1 and CIPK23 as well as in the excretion of organic acids to solubilize external Pi. Hence, SLAH3 and STOP1 link external NH₄ ⁺ with the reported efficient of NO₃ ^- and Pi acquisition, respectively (Tian et al., 2021; Ye et al., 2021). Considering that CIPK23 also phosphorylates NRT1.1, the potassium transporter AKT1, and the metal transporter IRT1, STOP1 appears to be behind in ensuring the nutrient balance and ion homeostasis during excessive NH₄ ⁺ nutrition.

Lastly, there are regulatory elements influenced under co-provision of NH₄ ⁺, altering the NRT1.1-dependent NO₃ ^- response, such as NIN LIKE PROTEIN 7 (NLP7) or the POLYADENYLATION FACTOR SUBUNIT 3 (CPSF30). Zhao et al. (2018) revealed that NLP7 acts as a positive regulatory factor upstream of NRT1.1 when NH₄ ⁺ is present, modulating the NO₃ ^- signaling function of NRT1.1. Similarly, the presence of NH₄ ⁺ prompts CPSF30 to act upstream of NRT1.1 in NO₃ ^- signaling without affecting the CPSF30 expression (Li et al., 2017).

Although the mechanisms underlying the NO₃ ^– and NH₄ ⁺-dependent sensing function of NRT1.1 remain unclear, it could all respond to a constant fine adjustment in the intracellular pH. In fact, NH₄ ⁺ and NO₃ ^- account for about 70% of the cations and anions taken up by plants, so they can exert a strong influence on intracellular pH despite the strong buffering capacity of the cytosol. The Figure 1 reveals a complex picture, involving for the first time an interconnection between NRTs and AMTs, whose contribution to regulating pH-dependent mechanisms of N uptake will depend on the balance between nitrate- and ammonium-based nutrition, and vice versa. In the case of NO₃ ^- nutrition, H⁺ consumption during NO₃ ^- reduction to nitrite and NH₄ ⁺ increases cytosolic pH, and this declines plasma membrane (PM) H⁺-ATPase activity and the consequent respiratory expense. Therefore, cotransport of H⁺ together with a cytosolic pH increase during NO₃ ^- uptake and reduction contributes to overall plant cell alkalinization. On the other hand, the decrease in pH driven in part by the AMTs-mediated NH₄ ⁺ uptake enrich the transcription of key players that activates not only the expression of NRT1.1, but also earlier genes involved in the acquisition of other nutrients whose availability or acquisition is particularly sensitive to external pH changes. For instance, Pi uptake is also an anion/H⁺ cotransport process and the second most important macronutrient for plants, aside from its close relationship with Fe availability and homeostasis. While, under low Pi conditions, the repression of NRT2s by NIGTs might be part of an adaptive response to reduce energy consumption and maintain the ionic balance, AMTs are upregulated to promote the NH₄ ⁺ uptake and subsequent H⁺ release. For this reason, the AMT1 family is considered to play a role in local Pi signaling, and potentially increases Pi solubility in the medium (Paz-Ares et al., 2022). The discovery of NRT1.1 and AMT-dependent regulation of N and P-related genes may lead to a deeper understanding of the strategies employed by plants to adapt to changing nutrient environments.

To date, attempts to enhance N use efficiency in crops through modulation of AMT gene expression have achieved limited success, likely due to NH₄ ⁺ sensitivity. This phenomenon represents a bottleneck in improving NH₄ ⁺-based nutrition, as NH₄ ⁺ when supplied as the predominant N form proves highly deleterious for most crops (Britto and Kronzucker, 2002). Notably, NH₄ ⁺ sensitivity exhibits substantial interspecies and intraspecies variability among cultivars, reflecting an intrinsic ecophysiological adaptation (Rivero-Marcos et al., 2024), where the role of AMTs in NH₄ ⁺ acquisition and signaling/adaptation may also vary substantially across plant systems.

In contrast, NO₃ ^- transporters have been more thoroughly characterized, owing partly to their broader functional diversity across plant species (Ruffel et al., 2025). This disparity is not surprising given the predominance of NO₃ ^- as the primary inorganic N form in aerobic soils - a consequence of microbial competition for reduced N compounds, agricultural tillage practices, and other edaphic and climate factors (Mao et al., 2025). The nitrate transporter NRT1.1 has evolved dual functionality as a NO₃ ^- sensor, like the TF NLP7, and the overexpression of these components enhances growth and N use efficiency in several species (e.g., AtNRT1.1, Sakuraba et al., 2021; AtNLP7, Yu et al., 2016; OsNRT1.1A, Hu et al, 2015; OsNRT1.1B, Wang et al., 2018).

A key unresolved question concerns the synergistic growth response observed when NO₃ ^- and NH₄ ⁺ are co-supplied. Remarkably, even micromolar NO₃ ^- concentrations (≤100 μM) - considered non-nutritional – to NH₄ ⁺-fed plants can significantly alleviate NH₄ ⁺ toxicity and stimulate growth in some NH₄ ⁺-preferring species (Garnica et al., 2009; Yan et al., 2023). This strongly implicates the primary NO₃ ^- response (PNR) pathway in growth optimization, and likely includes downstream AMT-related components. Consequently, classical PNR components like NRT1.1, but also potentially AMTs - which may serve dual roles as NH₄ ⁺ transporters and sensors (Pflüger et al., 2024) - represent promising targets for crop improvement strategies. Future research should investigate kinases and phosphatases regulated by NO₃ ^- signaling that may modulate AMT activity, and potential physical interactions between NRT1.1 and AMTs, which is plausible given the known ‘promiscuity’ among N transporters and key proteins regulating N uptake (Zhu et al., 2024).

Here, it has been shown how AMTs and NRTs sustain the vital uptake of N, thus affecting the N use efficiency of the plant and cellular pH homeostasis. While AMT-dependent NH₄ ⁺ uptake largely contributes to apoplast and rhizosphere acidification, NO₃ ^- and other nutrient uptake such as Pi becomes more efficient through pH-dependent induction of TFs like STOP1. NRT1.1 has the most essential contribution not only in NO₃ ^- acquisition and signaling, but also in modulating NH₄ ⁺ response in the absence of NO₃ ^-. Therefore, understanding the mechanisms by which plants regulate N acquisition in dependence or not of NRT1.1, and evaluating the contribution of AMTs to changes in internal and external pH, is critical for improving N use efficiency and nutrient uptake across diverse species and crop systems. These insights indicate that conventional approaches examining the responses to NO₃ ^- and NH₄ ⁺ separately, or after N deprivation, are no longer sufficient. Field-relevant advances will require integrated approaches using both N forms and corresponding mutants, capitalizing on their synergistic potential to achieve growth optimization and yield improvement under reduced N inputs.