Ammonium Phytotoxicity and Tolerance: An Insight into Ammonium Nutrition to Improve Crop Productivity

: Ammonium sensitivity is considered a globally stressful condition that affects overall crop productivity. The major toxic symptom associated with ammonium nutrition is growth retardation, which has been associated with a high energy cost for maintaining ion, pH, and hormone homeostasis and, eventually, the NH 3 /NH 4 + level in plant tissues. While certain species/genotypes exhibit extreme sensitivity to ammonium, other species/genotypes prefer ammonium to nitrate as a form of nitrogen. Some of the key tolerance mechanisms used by the plant to deal with NH 4+ toxicity include an enhanced activity of an alternative oxidase pathway in mitochondria, greater NH 4+ assimilation plus the retention of the minimum level of NH 4+ in leaves, and/or poor response to extrinsic acidiﬁcation or pH drop. Except for toxicity, ammonium can be considered as an energy-efﬁcient nutrition in comparison to nitrate since it is already in a reduced form for use in amino acid metabolism. Through effective manipulation of the NH 4+ /NO 3 − ratio, ammonium nutrition can be used to increase productivity, quality, and resistance to various biotic and abiotic stresses of crops. This review highlights recent advancements in ammonium toxicity and tolerance mechanisms, possible strategies to improve ammonium tolerance, and omics-based understanding of nitrogen use efﬁciency (NUE) in plants.


Introduction
Nitrogen (N) holds a prominent position in the metabolic system of plants, as it is part of amino acids, which are the building blocks of proteins and enzymes. Among all the mineral elements, N is regarded as one of the vital macronutrients for all living tissues in plants, including metabolism, resource distribution, growth, and development [1]. Nitrogen uptake is necessary for plant growth as it is crucial for the production of coenzymes, proteins, and photosynthetic processes. Although dinitrogen gas makes up about 78% of the atmosphere, plants cannot use it precisely. About 65% of the available N for the biosphere is produced via the biological N fixation process, which converts atmospheric N 2 to ammonium. Ammonium (NH 4 + ) and nitrate (NO 3 − ) are the two forms of accessible N that plants can take up from the soil and are assimilated into amino acids, either in the roots or shoots, depending on the plant species. In many plant species, the majority of the nitrate taken up by the roots is transferred to the shoot, where it is further assimilated into amino acids [2]. However, in a few species such as peas, amino acid synthesis primarily occurs in the roots, before being transported by the xylem to mature source leaves, where the amino-N is used in a variety of metabolic activities or loaded into the phloem to provide N to developing sinks, such as fruit and seeds [3]. Therefore, the main factors affecting soil nitrogen input for plant nutrition and agricultural yield are the decomposition of organic

Nitrogen Metabolism and Ammonium Assimilation
N is a nutrient that plants can obtain from a variety of compounds, viz., nitrate, urea, ammonium, and amino acids and use for a variety of metabolic processes, including the synthesis of nucleic acids and proteins, while also as signaling and storage molecules. An essential physiological mechanism for the growth and development of plants is the assimilation of nitrogen into carbon skeletons. Under aerobic conditions, NO 3 − acts as the main source of N with rapid mobility and storability in vacuoles; however, its reduction to NH 4 + is required for the synthesis of many organic compounds and proteins. All inorganic N is first converted to ammonium since it is the only reduced N form that plants can employ to assimilate into N-carrying amino acids [8]. A two-stage process is involved to convert nitrate into NH 4 + ; in the first step, nitrate reductase converts NO 3 − into nitrite in the cytoplasm, and in the second step, nitrite is turned into NH 4 + by nitrite reductase in the plastids [11]. Then, ammonia is assimilated into glutamine (Gln) and glutamate (Glu) through the glutamate synthase/glutamine-2-oxoglutarate amino transferase (GS/GOGAT) cycle [12,13] (Figure 1). The enzyme, glutamine synthetase (GS) has a vital role in the assim-ilation of NH 4 + into glutamine, while it is also a rate-limiting step in the NH 4 + assimilation. Additionally, GOGAT and GS have two isoforms and their location has been identified as being tissue specific. For instance, NADH-GOGAT (NADH-dependent glutamate synthase) and GS1 are implicated in nitrogen assimilation in the roots, while Fd-GOGAT (reduced ferredoxin-dependent glutamate synthase) and GS2 are primarily involved in nitrogen assimilation in leaves [13]. Among five GS1 genes in Arabidopsis (GLN1;1-GLN1;5), GLN1;2 is the main isoenzyme responsible for GS1 activity in the roots. The gln1;2 mutant exhibits poor GS activity, excessive ammonium buildup, and decreased plant growth when supplied with ammonia, indicating that GLN1;2 is crucial for both assimilation of ammonia and detoxification in roots [14]. NADH-GOGAT serves as the primary isoform in the roots of Arabidopsis and exogenous ammonium supply greatly induces the expression of this isoform. Under the conditions of ammonium availability, the nadh-gogat mutant exhibits lower glutamate formation and biomass buildup [15]. However, as endogenous NH 4 + concentration rises, a different route regulated by the mitochondrial NAD(H)-dependent glutamate dehydrogenase (GDH) helps to lower this internal NH 4 + concentration (Figure 1). The GDH enzyme catalyzes the reversible amination of 2-oxoglutarate (2-OG) to form glutamate, using ammonium as a substrate. Therefore, the GDH enzyme is theoretically able to either assimilate or release ammonium. Evidence suggests that under normal conditions, GDH operates in the direction of Glu deamination to form ammonium [16]. However, when ammonium concentration increases above a certain threshold, particularly under stress conditions [17], or when plants are grown under sole NH 4 + N source, the GDH activity is greatly induced to lower the internal concentration of NH 4 + [18]. The ability of GDH to release N from amino acids results in the creation of a keto-acid, 2-OG, and NH 3 , which can, then, be independently recycled for use in respiration and the synthesis of amides, respectively. The C-skeletons generated by other metabolic pathways, including respiration and photosynthesis, can also be used to integrate ammonia into the amino acids, glutamine, and glutamate. However, ammonium is thought to be toxic to plants at higher concentrations, as it can result in proton extrusion, which is linked to ammonium uptake, cytosolic pH alterations, and the dissociation of photophosphorylation in plants. The transport of nitrate occurs through a nitrate transporter (NRT) followed by its conversion into nitrite by nitrate reductase (NR) in the cytoplasm and its subsequent reduction into ammonium ions by nitrite reductase (NiR) in the chloroplast. The nitrate transporters act as H + /NO3 ̶ symporters that depend on the proton-motive force produced by the plasma membrane H + -ATPases. According to the hypothesis postulated by Coskun et al. [19], ammonium (NH4 + ) is taken up as the nonelectrogenic flux of NH3 through the plasma membrane, mediated by aquaporins (AQP). Ammonium is also transported through high-affinity ammonium transporters (AMT) and to some extent through the non-specific cation channels (NSCC) or K + channels. The ammonium ion that enters the cell directly goes to the GS/GOGAT cycle, where NH4 + is assimilated into glutamine (Gln) by glutamine synthetase and glutamate by glutamate synthase (GOGAT) in the chloroplast. Glutamine can also be converted into asparagine (Asn) via asparagine synthetase (ASN). The enzyme Figure 1. Mechanism of uptake and assimilation of nitrate and ammonium ions in plants. The transport of nitrate occurs through a nitrate transporter (NRT) followed by its conversion into nitrite by nitrate reductase (NR) in the cytoplasm and its subsequent reduction into ammonium ions by nitrite reductase (NiR) in the chloroplast. The nitrate transporters act as H + /NO 3 − symporters that depend on the proton-motive force produced by the plasma membrane H + -ATPases. According to the hypothesis postulated by Coskun et al. [19], ammonium (NH 4 + ) is taken up as the non-electrogenic flux of NH 3 through the plasma membrane, mediated by aquaporins (AQP). Ammonium is also transported through high-affinity ammonium transporters (AMT) and to some extent through the nonspecific cation channels (NSCC) or K + channels. The ammonium ion that enters the cell directly goes to the GS/GOGAT cycle, where NH 4 + is assimilated into glutamine (Gln) by glutamine synthetase and glutamate by glutamate synthase (GOGAT) in the chloroplast. Glutamine can also be converted into asparagine (Asn) via asparagine synthetase (ASN). The enzyme glutamate dehydrogenase (GDH) yields ammonium and 2-oxoglutarate (2-OG), a product that links nitrogen metabolism with photorespiration, which involves three cell organelles, such as chloroplast, peroxisome, and mitochondria. The photorespiratory product, glyoxylate from chloroplast is converted into glycine by the action of glutamate: glyoxylate aminotransferase (GGT) in peroxisome, while two molecules of glycine are converted into one molecule of serine by the combined action of glycine decarboxylase (GDC) and serine hydroxymethyltransferase (SHMT) in mitochondria.

Signs of Ammonium-Induced Toxicity
Ammonium toxicity often develops, when a plant is exposed to high levels of ammonium in the environment, or alternatively, when plant cells spontaneously overproduce ammonium due to enhanced proteolytic activity as a consequence of environmental stress. Widely varying documented NH 4 + toxicity symptoms typically manifest at external NH 4 + values over 0.1 to 0.5 mmol L −1 [20]. The primary phenotypic indicators of ammonium toxicity in plants are restricted root growth and chlorosis of the leaves [10]. Ammonium toxicity modifies the biochemical and physiological makeup of the plant, affecting its intracellular pH, osmotic balance, metabolism of phytohormones and polyamines, and ability to absorb nutrients ( Figure 2). Ammonium toxicity can also cause a reduction in the amount of carbon availability, damage to the chloroplast's ultrastructure, an increase in proton efflux, ineffective transmembrane ammonium cycling, suppression of the enzyme GDP-mannose pyrophosphorylase, as well as oxidative stress [17,19]. Ammonium toxicity is also accompanied by lower levels of chlorophyll a and b and carotenoids, a decline in photosynthetic rates, and a rise in the production of ethylene [19,21]. In cucumber, higher levels of ammonium were found to be toxic, causing toxicity symptoms such as suppression of growth, chlorosis, curling of leaves, and low tissue accumulation of calcium and magnesium, while the addition of lesser amounts of NH 4 + to NO 3 − has drastically improved the growth rate [22]. Increased ammonium levels have negatively impacted the uptake of cations, such as Ca, K, Mg, and Si, in papaya, while also reducing growth [23]. Excess ammonium has resulted in calcium deficit, restricted N uptake, altered N/Ca ratio as well as physiological changes in tomato plants [24]. Recently, the interactive role of ammonium nutrition with iron (Fe) homeostasis has been demonstrated in the model plant Brachypodium distachyon [25]. It was shown that plants that had been fed with ammonium showed heightened susceptibility to iron deficiency, indicating that ammonium nutrition resulted in decreased Fe uptake and root-to-shoot transfer. Thus, in order to better comprehend and enhance ammonium use efficiency and tolerance in plants, future studies are required to explain the mechanisms driving the interaction between Fe and NH 4 + , particularly regarding the mechanism leading to Fe deficiency under ammonium stress.
Root development is more strongly impacted by excess NH 4 + than any other plant organ. Higher NO 3 − concentrations promote lateral root growth, while higher NH 4 + concentrations result in stunted roots with slow growth rates. Overabundance of NH 4 + primarily shortens primary roots by preventing cell expansion and division in the meristems. Under these circumstances, GDP-mannose pyrophosphorylase (GMPase)-mediated NH 4 + efflux increases in the meristematic zone, leading to a higher NH 4 + influx in mature root cells [26]. Investigations in Lotus japonicus, employing split-root systems have revealed that root-specific NH 4 + -induced reactions are regulated by NH 4 + transporters that are more closely related to NH 4 + sensing than its assimilation. In both L. japonicus and Arabidopsis, AMT1;3 mediates the short-root phenotype induced by NH 4 + nutrition [27,28]. Ammonium suppresses root gravitropism in a similar way as it inhibits primary root growth. This phenomenon is linked to a considerable delay in the asymmetrical auxin distribution in the elongation zone of the root and a significant reduction in the expression of PIN2, the auxin exporter. ARG1 (Altered Response to Gravity 1) acts as an antagonist to PIN2 to re-establish root gravitropism through PIN3-mediated lateral auxin distribution and AUX1-induced shootward auxin reflux at the root apex under ammonium input [29]. The defective root gravitropism is greatly influenced by the antagonistic interaction between PIN2 and ARG1 in maintaining the asymmetric auxin fluxes under ammonium supply. Thus, PIN2 and ARG1 are the key targets of NH 4 + -induced impairment of root gravitropism.

Postulated Theories of Ammonium Toxicity and Tolerance
The ability of a plant to tolerate ammonium largely depends on its capacity to assimilate ammonium. Shoots are typically the most affected area of the plant to NH4 + feeding, after roots. The early signs of NH4 + toxicity arise at the root level with a significant alteration of the root system architecture (RSA), as roots are the prime NH4 + sensor [30] ( Figure  2). The exogenous NH4 + eventually reaches the leaves once the root system's storage capacity has been exhausted. The harmful effects of NH4 + on leaves are mainly associated with its impact on photosynthesis and its ability to cause oxidative stress. For instance, a higher concentration of NH4 + (5 mM) has affected photosynthesis by binding NH3 in the oxygen-evolving complex (OEC) of photosystem II in the cyanobacterium, Synechocystis [31]. In A. thaliana, NH4 + nutrition did not alter the photosynthetic efficiency but raised the amounts of mitochondrial reactive oxygen species (ROS) in the leaf [32]. In line with this, few other studies have confirmed that NH4 + nutrition caused enhanced lipid peroxidation and glutathione-ascorbate cycle enzyme activity, or alterations in the redox state, implying higher ROS production [33,34]. The tolerance mechanisms used by plants to deal with NH4 + toxicity have not been well described because the primary causes of NH4 + toxicity

Postulated Theories of Ammonium Toxicity and Tolerance
The ability of a plant to tolerate ammonium largely depends on its capacity to assimilate ammonium. Shoots are typically the most affected area of the plant to NH 4 + feeding, after roots. The early signs of NH 4 + toxicity arise at the root level with a significant alteration of the root system architecture (RSA), as roots are the prime NH 4 + sensor [30] (Figure 2). The exogenous NH 4 + eventually reaches the leaves once the root system's storage capacity has been exhausted. The harmful effects of NH 4 + on leaves are mainly associated with its impact on photosynthesis and its ability to cause oxidative stress. For instance, a higher concentration of NH 4 + (5 mM) has affected photosynthesis by binding NH 3 in the oxygen-evolving complex (OEC) of photosystem II in the cyanobacterium, Synechocystis [31]. In A. thaliana, NH 4 + nutrition did not alter the photosynthetic efficiency but raised the amounts of mitochondrial reactive oxygen species (ROS) in the leaf [32]. In line with this, few other studies have confirmed that NH 4 + nutrition caused enhanced lipid peroxidation and glutathione-ascorbate cycle enzyme activity, or alterations in the redox state, implying higher ROS production [33,34]. The tolerance mechanisms used by plants to deal with NH 4 + toxicity have not been well described because the primary causes of NH 4 + toxicity have not been determined clearly. According to the classical hypothesis of ammonium toxicity, some of the key traits contributing to NH 4 + tolerance in plants include enhanced alternative oxidase pathway activity in mitochondria, greater NH 4 + assimilation and retention of low levels of NH 4 + in leaves, and/or poor sensitivity to extrinsic pH acidification [35]. Therefore, several factors, including root carbon metabolism and the capacity to maintain high respiration rates, the ability to limit NH 4 + accumulation inside tissues, and tolerance to acidification of the root zone affect a plant's overall ability to withstand high NH 4 +  (2) the aquaporins (AQP) that aid in the non-electrogenic NH 3 flow through the plasma membrane at high NH 4 + concentrations [37]; (3) the non-specific cation channels (NSCC) and K + specific channels that transport NH 4 + ions in competition with K + , which are similar in size and surface charge density ( Figure 1) [38]. One of the main causes of NH 4 + toxicity is the impact of NH 4 + nutrition on reducing the concentrations of cations (K + , Ca 2+ , and Mg 2+ ) in plant tissue. The harmful effects of NH 4 + nutrition can, however, be reduced by raising K + concentrations in the root media. K + can lessen the toxicity of NH 4 + by enhancing the incorporation of NH 4 + into organic N-compounds via the activation of enzymes such as glutamate dehydrogenase, glutamine synthetase, etc., as well as by limiting the absorption of NH 4 + by low-affinity transporters [39]. K + concentrations in the root medium have the potential to obstruct the transport of NH 3 by specifically regulating the activity of AQP and the plant's water balance [40].

Signaling Responses Involved in Ammonium Sensing and Tolerance
Effective nutrient harvesting by plant roots requires a quick and sensitive assessment of nutrient availability in the environment. Nutrient transporters located at the plasma membrane are the key players in sensing nutrients in the external environment. For example, NRT1.1/NPF6.3 in Arabidopsis and Mep2 in yeast have been recognized as the major transporters of nitrate and ammonium, respectively, across the plasma membrane [41]. Numerous factors are engaged in the crosstalk signaling that controls plant growth and development under the conditions of NH 4 + sensitivity and tolerance.

AMT-Mediated Extracellular Ammonium Sensing Responses
The extracellular sensing and transportation of ammonium are mediated through the phospho-dependent allosteric mechanism of activating the C-terminal domain of AMT1;1 in Arabidopsis [41,42]. The cytosolic C-terminus of each monomer of the trimeric AMT1 complex connects to the pore region of the adjacent subunit to transactivate the ammonium transport. The trans-inactivation of both the individual monomer as well as the entire trimeric complex is facilitated by the phosphorylation of threonine residue in the C-terminus of any monomer that leads to the relieving of contact between the C-terminus and the conducting pore. The critical threonine residue T460 in the C-terminal domain of AMT1;1 is dephosphorylated in conditions of low ammonium availability, whereas conditions of high ammonium supply result in phosphorylation and deactivation of the AMT1;1 trimer. Moreover, it has been demonstrated that glutamine, a product of ammonium assim-ilation, and methionine sulfoximine (MSX), the glutamine synthetase inhibitor, are unable to induce T460 phosphorylation, suggesting that extracellular ammonium alone can specifically trigger the phospho-dependent inactivation of AMT1;1. Thus, in order to prevent excessive ammonium absorption, it has been postulated that AMT1;1 itself functions as a transceptor, whereby it attracts a kinase from the cytosol following extracellular ammonium binding, and confers either direct or indirect phosphorylation of AMT1;1. Intriguingly, trans-inactivation by C-terminal phosphorylation extends to heterotrimeric AMT1 complexes in addition to homotrimeric ones, as demonstrated for AMT1;1, AMT1;2 [43], and AMT1;3 [44]. This greatly increases the potential for regulating the exchange of signals between AMT isoforms via protein-protein interactions. In furtherance, AMT1;3 plays a crucial role in ammonium-mediated lateral root branching. Therefore, it appears that AMT1;1 and AMT1;3 are significant players in various signaling pathways, and their colocalization and physical interaction enable the coordination of both ammonium sensing and transport in response to the external availability of ammonium in the rhizosphere ( Figure 3). Surprisingly as AMT1;3 seems to resist the C-terminal phosphorylation and inactivation caused by ammonium, an alternate feedback mechanism is activated in response to increased ammonium supply [45]. Following ammonium addition, AMT1;3 proteins aggregate and are subsequently removed from the plasma membrane through the endocytic pathway, as demonstrated in the glutamine synthetase mutant gln1;2. Hence this alternative shutoff mechanism helps to prevent ammonium toxicity in root cells. the exchange of signals between AMT isoforms via protein-protein interactions. In furtherance, AMT1;3 plays a crucial role in ammonium-mediated lateral root branching.
Therefore, it appears that AMT1;1 and AMT1;3 are significant players in various signaling pathways, and their colocalization and physical interaction enable the coordination of both ammonium sensing and transport in response to the external availability of ammonium in the rhizosphere (Figure 3). Surprisingly as AMT1;3 seems to resist the C-terminal phosphorylation and inactivation caused by ammonium, an alternate feedback mechanism is activated in response to increased ammonium supply [45]. Following ammonium addition, AMT1;3 proteins aggregate and are subsequently removed from the plasma membrane through the endocytic pathway, as demonstrated in the glutamine synthetase mutant gln1;2. Hence this alternative shutoff mechanism helps to prevent ammonium toxicity in root cells. In low ammonium conditions, the crucial threonine residue T460 in the C-terminal domain of AMT1;1 is dephosphorylated allowing the ammonium transport pores to open and allows for ammonium uptake. AMT1;3 sends a signal to encourage lateral root development when roots are exposed to localized ammonium, which leads to ammonium scavenging. AMT1;1 is rendered inactive by phosphorylation of its C-terminal domain in response to increased ammonium availability. Elevated cytosolic ammonium levels cause AMT1;3 to be clustered and internalized into the cytoplasm by the endocytic pathway, which further stops the uptake of ammonia. The tonoplast-localized receptor-like kinase CAP1 facilitates the compartmentation of ammonium into vacuoles mediated through aquaporins (AQP), which protects cells from ammonium toxicity. In low ammonium conditions, the crucial threonine residue T460 in the C-terminal domain of AMT1;1 is dephosphorylated allowing the ammonium transport pores to open and allows for ammonium uptake. AMT1;3 sends a signal to encourage lateral root development when roots are exposed to localized ammonium, which leads to ammonium scavenging. AMT1;1 is rendered inactive by phosphorylation of its C-terminal domain in response to increased ammonium availability. Elevated cytosolic ammonium levels cause AMT1;3 to be clustered and internalized into the cytoplasm by the endocytic pathway, which further stops the uptake of ammonia. The tonoplast-localized receptor-like kinase CAP1 facilitates the compartmentation of ammonium into vacuoles mediated through aquaporins (AQP), which protects cells from ammonium toxicity.

CAP1-Mediated Intracellular Ammonium Sensing Responses
In addition to ammonium assimilation, alternative mechanisms are also in operation to provide tolerance to ammonium toxicity. It has been shown in Arabidopsis that CAP1, a receptor-like kinase, mediates the compartmentation of ammonium in vacuoles under conditions of high intracellular ammonium [46] (Figure 3). The tonoplast localized unique regulator of the NH 4 + homeostasis signal, CAP1 has been found to regulate the polar development of root hairs by maintaining tip-focused cytoplasmic Ca 2+ gradients as well as ROS gradients. A CAP1 knockout mutant (cap1-1), exhibited an increased concentration of cytoplasmic NH 4 + . In addition, on Murashige and Skoog media, the root hair growth of cap1-1 was suppressed, while NH 4 + depletion restored the Ca 2+ gradient required for normal growth. The comparatively alkaline cytosolic pH of cap1-1 root hairs and the reduced net NH 4 + inflow across the vacuolar membrane suggested that CAP1 mutation promoted NH 4 + accumulation in the cytoplasm. Moreover, ectopic expression of CAP1 in the yeast mutant npr1 accomplished the missing function of NPR1, a crucial component of the ammonium-sensing pathway, and restored the phosphorylation as well as the ammonium transport activity of Mep2. This suggests that CAP1 may also function in plants as a regulatory component in intracellular ammonium-sensing events [46]. Autophosphorylation of CAP1 upon exposure to ammonium has been confirmed in Arabidopsis through a phosphoproteomics approach [46,47]. Moreover, the location of CAP1 in the tonoplast suggests that CAP1 may phosphorylate vacuolar ammonium transporters. The aquaporins, TIP2;1 and TIP2;3 that are located in the tonoplast and involved in ammonium compartmentation might be the potential putative targets for CAP1. However, so far only the transcriptional upregulation of the ammonia-conducting aquaporin TIP2;3 has been evidenced in ammonium treated cap1-1 mutants [48]. Thus, the transcriptional regulators responsible for amplifying the ammonium-sensing event and orchestrating the subsequent ammonium responses are still unclear. Hence, future studies that examine the local integration of external and internal ammonium-dependent signals to control AMT-dependent ammonium transport are of utmost importance.

Nitrate, Auxins, NO, and Polyamines-Mediated Signaling of NH 4 + Tolerance
The alleviating role of NO 3 − in ammonium toxicity has been mediated through the crosstalk signaling among NO 3 − , NRT1.1/NPF6.3, NO 3 − transporter, and auxin. Since NO 3 − induces the expression of the NRT1.1/NPF6.3 transporter gene, plants lacking NO 3 − in the root medium may experience disruptions in this signaling pathway-for example, short and less branched roots in the case of NH 4 + and urea-fed Medicago truncatula, respectively [49]. The auxin-responsive reporter DR5:GUS-reporter has been used in a mutant strain of Arabidopsis to study the function of auxin signaling in the growth of primary roots fed with NH 4 + . The study showed that the response of the reporter to auxins under NH 4 + nutrition was dramatically reduced, whereas exogenous auxin-only partially restored root growth [50]. It was determined that NRT1.1/NPF6.3 had a NO 3 --independent function since NRT1.1/NPF6.3 defective mutants had a better tolerance for high concentrations of NH 4 + and NO 3 − , which when administered did not increase this tolerance towards NH 4 + . In NO 3 − -mediated reduction of ammonium stress, NO 3 − efflux channels, such as SLAH3, also played a significant role [51]. In the absence of NO 3 − , SLAH3-defective mutants are hypersensitive to high NH 4 + concentrations as well as low pH. Plants lack the essential enzyme, carbamoyl phosphate synthase (CPS)-type I of animals; nevertheless, they appear to possess a functional CPS-type II that utilizes glutamine as the NH 3 source for carbamoyl-P synthesis. As a result, plants are unable to produce urea directly from NH 4 + , which would lower the internal NH 4 + concentration under stress. The plant urea cycle comprises two phases: the biosynthetic phase, which produces arginine, and the catabolic phase, where arginine can be broken down by arginase to produce urea and ornithine or act as a substrate for arginine decarboxylase to make agmatine. Either agmatine or ornithine can further produce putrescine, spermidine, and other polyamines. Arginine has been proposed as an intermediate in NO synthesis and links the urea cycle with NH 4 + tolerance. It has been implied that nitric oxide (NO) derived from NO 3 through nitrate reductase also plays a profound role in auxin-signaling by acting downstream of auxin. This was confirmed by arginase-negative mutants of Arabidopsis, through which increased NO builds up and effluxes, while promoting the development of lateral and adventitious roots [52]. The root tips, developing lateral roots, and hypocotyls of these mutants consistently displayed enhanced auxin signaling. However, the source of NO in plants fed with NH 4 + or urea as the only N nutrition remains elusive. It was also found that the exogenous application of polyamines such as spermidine and spermine has led to the increased synthesis of NO in Arabidopsis [53]. It has been hypothesized that an excess of ammonium resulting either from NH 4 + nutrition or from polyamine metabolism may operate as a feedback inhibitor of polyamine deamination, thereby reducing polyamine metabolism. Further, metabolic studies in peas and Arabidopsis have revealed that ammonium supplementation caused arginine accumulation, presumably indicating impaired polyamine production [44,54].

Ameliorated Crop Quality and Productivity by Ammonium Nutrition
In agricultural settings, the two most common types of inorganic nitrogen taken by the roots of higher plants are ammonium (NH 4 + ) and nitrate (NO 3 − ). In the rhizosphere environment, the absorption of nitrate nitrogen is associated with the inorganic cation uptake and the release of OH − , leading to an alkaline rhizosphere, while ammonium nitrogen absorption takes place by the uptake of inorganic anion and the release of H + , causing an acidic rhizosphere. An overabundance of free NH 4 + in the cytosol is likely the primary source of ammonium toxicity and plants employ a number of rational ways to manage NH 4 + levels, including absorption into organic compounds, storage of NH 4 in the vacuole, and outflow of NH 4 to the apoplast and rhizosphere. Despite ammonium stress affecting almost all plant species, there is considerable intra-and interspecific variation in response to ammonium feeding. While some species/genotypes exhibit severe sensitivity to ammonium when growing, other species/genotypes exhibit ammonium preference. The influence of ammonium and nitrate nutrition varied among selected bedding plants, such as Salvia splendens, marigold, Ageratum houstonianum, and Petunia hybrida [55]. Salvia and marigold were highly sensitive to NH 4 + , while ageratum, being tolerant to NH 4 + , was more sensitive to NO 3 − . The study also revealed that NH 4 + -fed plants contained more anions, such as more Cland fewer cations than NO 3 − -fed plants. NH 4 + nutrition has significantly enhanced the chlorophyll contents of ageratum and petunia, while NO 3 − nutrition increased the chlorophyll contents of Salvia [56]. Upon investigating the effect of NH 4 + and NO 3 − in the presence and absence of Cl − , the uptake of NO 3 was suppressed by NH 4 + , while the uptake of NH 4 + was unaffected by NO 3 − and the uptake of Clwas counteracted by both NH 4 + and NO 3 − [57]. In Arabidopsis, ammonium as a predominant N source negatively impacted biomass production [58,59], while in another study, ammonium nutrition was found to be beneficial in the improvement of glucosinolate metabolism [60]. Ammonium as a sole N source has improved the grain quality in wheat by increasing the grain reserve proteins, such as gliadins and glutenins [61]. The effects of N nutrition in the form of NH 4 + and NO 3 have been extensively studied in many plant species. However, the requirement of N sources varied among species. For example, NO 3 − nutrition positively regulated the growth of wheat [62], sugar beet [63], tobacco [64], beans [65], and canola [66], whereas NH 4 + nutrition was found suitable for the growth of larch and pine [67], tomato [68], and rice [69]. Nevertheless, employing NO 3 − as the exclusive source of nitrogen causes N leaching and nitrate buildup in plants [70,71]. For the majority of species, a combination of ammonium and nitrate is recommended as an excellent nutrient recipe than using nitrate or ammonium alone. Numerous studies have achieved high productivity and quality in many plant species by combining NH 4 + and NO 3 in a particular ratio (Table 1). For instance, optimum concentrations of NH 4 + and NO 3 have greatly influenced the growth and flowering of Petunia hybrida, grown in rockwool and peat-lite media. The highest shoot dry weight was obtained when equal concentrations of NH 4 + and NO 3 − (9 me. L −1 each) were added to the peat-lite media. Flowering was delayed at increased concentrations of both forms of nitrogen; however, increased NO 3 highly affected flowering [72]. The japonica rice cultivars exhibited a high ammonium preference along with a partial requirement of nitrate (NH 4 + :NO 3 − ,75:25) for efficient photosynthetic activity, higher NUE and faster biomass accumulation [73]. In the case of cabbage, an equal ratio of ammonium and nitrate has greatly improved the seedling fresh weight, root length, and contents of phosphate (H 2 PO 4 -), and mineral nutrients (K, Ca, and Mg), while also significantly reducing the nitrate content in leaves [74]. Upon investigating the effect of various NH 4 + :NO 3 − proportions in strawberries, the ideal NH 4 + : NO 3 − ratio for superior fruit development and quality in the semi-hydroponic fertigation system was found to be 29:71 [75]. In Capsicum annuum L, the ratio of ammonium to nitrate at 25:75 drastically improved root growth, and the accumulations of nutrient elements (N, P, and K) accumulation and dry matter [76]. The treatment also improved the fruit quality by raising vitamin C, contents of total phenols, flavonoids, soluble sugar and protein, dry matter, and pungency levels. The ammonium tolerance was further substantiated by greater levels of the enzymes, GS and GOGAT as well as higher relative expression of the genes encoding these enzymes. Similarly, the NH 4 + :NO 3 − ratio of 25:75 has been shown to enhance N uptake and assimilation in flowering Chinese cabbage, mostly via controlling the pH of the nutrient solution [77]. It has been well established that NH 4 + uptake and assimilation are associated with proton release processes leading to the acidification or pH drop of the rhizosphere. Thus, the interaction in the decrease in pH and K + uptake in NH 4 + tolerance was investigated in hydroponically cultured ageratum, salvia, cabbage, and lettuce plants at three NH 4 + :NO 3 − ratios (0:100, 50:50, and 100:0) [78]. The plants, such as ageratum and lettuce, were found to be more tolerant to NH 4 + feeding, which exhibited a rapid decrease of solution pH as well as improved K + influx when compared to NH 4 + sensitive, salvia, and cabbage. In another study, the effect of NH 4 + and NO 3 − ratios (0:100, 50:50, and 100:0) on growth, photosynthesis, nitrogen, and carbohydrate concentrations alongside nitrogen assimilation enzymes were evaluated among NH 4 + sensitive (cabbage) and tolerant (lettuce) species. The study reinforced that both plants benefitted from equal ratios of NH 4 + and NO 3 − (50:50) with enhanced growth and quality. Moreover, in contrast to cabbage, the GS and GDH activities in lettuce were boosted in response to an increase in the level of external NH 4 + leading to reduced NH 4 + accumulation [79]. Thus, the co-provision of NH 4 + with NO 3 triggers a synergistic growth response that can outpace the maximum growth rates on either nitrogen source alone. For all of the above studies indicated, it is significant to note that the optimal ratio of NH 4 + :NO 3 is also influenced by the total N concentration and the source of NH 4 + -N. The requirement of total N concentration varied among plant species, ranging between 5.0 and 15 mM L -1 , and in all the trials, the total N content was maintained constant, while the ratios of NH 4 + to NO 3 were altered (Table 1). Additionally, the source of NH 4 + -N plays a vital role in determining the plant responses to nutrition, osmotic balance, stress, and overall plant development. Ammonium sulfate (AS) is one of the most widely used N fertilizers, which comprises 21% N and 60% sulfur (S). It is a great source of S for crops that demand huge supplies and offers a balanced supply of both N and S. In terms of nutrition, N and S play very close relationships with one another, particularly because both nutrients are necessary for the synthesis of chlorophyll and are components of amino acids. Sulfur is a structural element found in amino acids, protein disulfide bonds, cofactors, and vitamins. S deficiency impacts the biomass, general morphology, yield, and nutritional value of the plants [80]. Hence, the addition of a N source in the form of AS, also meets the S requirements of plants, promoting overall growth.
AS may have certain potential agronomic and environmental advantages over other N fertilizers, such as urea and ammonium nitrate (AN), including (1) minimal to no N loss via NH 3 volatilization upon surface application to acid or neutral soils, (2) a better source of N for saline soils as it lessens the detrimental effects of NaCl on plant growth and for saline-sodic calcareous soils, as it enhances soil structure, (3) minimal NO 3 -N leaching from AS than AN, (4) improved N efficiency due to less denitrification than AN and reduced emission of greenhouse gases (NO and N 2 O), and (5) the formation of more acidic root rhizosphere by preferred absorption of NH 4 -N of AS over NO 3 -N of AN, which may boost soil phosphorus (P) and micronutrient availability [81]. Although AN has the highest proportion of N (33-34%), the use of AN is not recommended for flooded rice soils that are either rainfed or irrigated mainly due to the significant N losses from the denitrification of nitrate. Therefore, AS becomes the preferred choice of fertilizer for rice growers [81]. On the other hand, other N fertilizers, such as ammonium chloride, improved the yield of ratoon in sugarcane during the first cycle but decreased the yield in subsequent cycles. As sugarcane is moderately sensitive to soil salinity, the decrease in yield could be attributed to the saline effect caused by the residual Clfrom NH 4 Cl - [82]. Similarly, the application of NH 4 Clhas provoked the accumulation of cadmium in wheat due to the increased Clconcentration in the soil [83]. Hence, rationale utilization of the optimum concentration of NH 4 Cl -, especially in saline-sensitive crops is highly crucial, as Clappears to be the preferable counter ion to NH 4 + , the increased Clions exacerbate the symptoms of salinity stress, in addition to ammonium toxicity.
Ammonium fertilizers are ineffective in cold climates and irrigated systems due to the significantly decreased activity of nitrifying microorganisms. Since NH 4 + is a cation, it greatly adheres to most soils, especially to clays and organic matter; thus, there is minimal leaching off with water. Hence, this causes more ammonium toxicity in irrigated systems where nitrification is greatly inhibited. Therefore, management of appropriate NH 4 + -N source and concentration in irrigated fields is of prime requirement. Recently, several studies have examined the interaction effect between the NH 4 + -N forms and irrigation systems in cultivated fields. For example, AS application has drastically improved the lint yield in a drip-irrigated, moderately saline cotton field by reducing rhizosphere soil pH and salinity as well as improving P nutrition [84]. In another study, AN fertilization at 120% crop evapotranspiration (ETc), and drip irrigation conditions significantly improved the water use efficiency, chlorophyll, tuber quality, and yield of potatoes compared to AS and urea fertilization [85]. Eggplant is sensitive to NH 4 + nutrition in solution culture, while it has responded positively to an appropriate ratio (50:50) of AN fertilization with 75% irrigation level through improved vegetative growth, water use efficiency, and yield compared to AS fertilization [86].
Although nitrate fertilizers are less likely to be hazardous to plants than ammonium fertilizers, they contribute to significant N loss through NO 3 leaching and higher NO emissions. Inhibitors of soil nitrification, including dicyandiamide (DCD), have been suggested as an efficient way to lessen soil N loss and N 2 O emissions. It has been extensively demonstrated that using nitrification inhibitors in conjunction with ammonium fertilizers in agricultural fields can reduce the environmental impact of nitrogen fertilizers [87]. However, the maintenance of N in the NH 4 + form in soil by such compounds also raises concerns for crops that are sensitive to ammonium [88]. Hence, future research is needed to determine how nitrification inhibitors and the increased NH 4 + -N concentrations in soil affect crop growth and yields.

Ameliorated Crop Resilience to Biotic and Abiotic Stresses by Ammonium Nutrition
Ammonium stress can have beneficial effects on crop resilience, to biotic and abiotic stresses, and generate high-quality products, although it may have a modest detrimental influence on productivity in agricultural contexts. The synthesis of most defense-related secondary metabolites depends on nitrogen metabolism, which also serves as a key step in the production of nitric oxide (NO), whose function in plant-pathogen interactions has been extensively investigated [110]. Hence, nitrogen metabolism closely relates to plant immunity. Although plants that have been fed nitrate have generally shown enhanced resistance to pathogenic attacks [111,112], few species, particularly tomatoes, have shown notable resistance to pathogens when fed with ammonium. Ammonium-mediated repression of the virulence of Fusarium oxysporum has been demonstrated in tomatoes, mainly by inhibiting the penetration of virulence components through cellophane [113]. Another study demonstrated that the mild toxicity generated by ammonium stress has led to the buildup of H 2 O 2 in tomato plants, which served as a signal to activate systemic acquired acclimation (SAA), and thereby provided resistance to the infections caused by Pseudomonas syringae [114]. Similarly, ammonium-mediated alterations in carbon and nitrogen metabolisms have been reported to increase the resistance against P. syringae in tomatoes [115]. In rice, the overexpression of the ammonium transporter AMT1;2 improved NUE and resistance to sheath blight disease caused by Rhizoctonia solani, further indicating that ammonium uptake and assimilation are necessary for this defense [116].
In furtherance, it has been recognized that an optimum nitrate-to-ammonium ratio can enhance protein synthesis, decrease the lipid peroxidation rate, and boost the plant's ability to withstand various abiotic stresses. Ammonium feeding may cause a multitude of reactions, many of which are defense responses that are typical of various abiotic stress conditions. The significance of ammonium in alleviating heavy metal toxicity has been proven through some of the studies. The positive effect of NH 4 NO 3 in improving manganese tolerance has been reported in Trifolium subterraneum L. genotypes [117]. Ammonium nutrition has been found to increase the aluminum (Al) tolerance in soybean [118], rice [119], and Lespedeza bicolor [120]. The primary symptom of Al toxicity is the inhibition of root elongation. Ammonium nutrition, however, dramatically increased root length and reduced Al buildup in roots under Al toxicity. The major mechanism of Al tolerance in the above-mentioned species was the restricted Al absorption, as a result of competition between positively charged Al + and NH 4 + for binding sites in the apoplast of root cell walls. Ammonium has been shown to have a strong mitigating effect on cadmium (Cd) toxicity by increasing antioxidant activity and glutathione-ascorbate cycle efficiency in rice [121]. The higher Cd detoxification effect of NH 4 + compared to has been demonstrated in the hyperaccumulator, Solanum nigrum L [122]. Ammonium-mediated Cd toxicity in S. nigrum has been affected through decreased absorption and accumulation of Cd via downregulation of Cd transport-related genes, increased immobilization of Cd in the cell wall of roots by binding to pectin and cell wall components, and through the upregulation of expansin, (SnExp), a gene responsible for metal tolerance. In contrast to the report on the impact of NH 4 + in worsening iron deficiency symptoms of the model plant, B. distachyon [25], the alleviating effects of NH 4 NO 3 and NO in iron deficiency has been documented in pears [123]. The study showed that NH 4 NO 3 significantly reduced the chlorosis of leaves caused by iron deficiency, mostly through promoting the activity of ferric chelate reductase and nitrogen assimilation enzymes. Ammonium has been found to improve the salinity tolerance of plants such as citrus [124], Sorghum bicolor [125], maize, [126], and tomato [127]. Ammonium has ameliorated the polyethylene glycol (PEG)-induced water stress tolerance in rice through improved regulation of NH 4 + uptake, assimilation, and aquaporin activity [128,129]. An appropriate ratio of NH 4 + : NO 3 -(10:90) has effectively mitigated the low light intensity stress in Brassica pekinensis by modulating the expression of ammonium assimilatory enzymes, such as GS and NR [105]. In cucumber, the greater flux rate of NH 4 + into vascular bundles of the lateral vein, midrib, and shoot tip, due to the increased conversion of NO 3 to NH 4 + , has substantially aided in overcoming low-temperature stress, as an energy conservatory mechanism [130]. An equal ratio of ammonium and nitrate (50:50) was found to improve the biomass, photosynthetic efficiency, protein content, and decrease lipid peroxidation in baby lettuce under heat stress conditions [131].

Omics Investigations in Comprehending NUE
Numerous key regulatory genes, such as transporters, reductase, synthase, glutamate dehydrogenase, and aminotransferase are involved in the various processes of nitrogen metabolism, including the uptake, translocation, assimilation, and remobilization. Until recently several "omics" studies have focused primarily on rapidly altering gene expression and metabolites in plants under various sources of nitrogen. For instance, a comparative transcriptomic analysis between the cultivated and wild lettuce revealed the tolerance mechanisms exhibited by wild under nitrogen starvation conditions [132]. The study evidenced an enhanced expression of nitrate transporter genes (NRT2.1, NRT2.4, and NRT2.5) and glutamate synthase genes (GLN1 and GLN2) in wild lettuce compared to the cultivated one, suggesting high-efficiency N uptake under low nitrogen conditions. Hence, the study gave insights into improving the NUE of cultivated lettuce by the introgression of novel alleles from the wild species in the future. In perennial ryegrass, transcriptome profiling under various nitrogen levels has identified four crucially enriched pathways, such as the photosynthesis-antenna protein, carotenoid biosynthesis, steroid biosynthesis, and C5branched dibasic acid metabolism, particularly at excessive nitrogen stress conditions [133]. Transcriptomic analysis was used to examine the processes of ammonium toxicity and tolerance in an aquatic macrophyte Myriophyllum aquaticum under situations of high NH 4 + stress [134]. The study evidenced the transcriptional upregulation of chlorophyll a/b binding proteins genes contributing to high NH 4 + resistance in the leaves. Moreover, the upregulation of nitrogen metabolism genes, along with the upregulation of catalase genes aided in combating NH 4 + stress, by means of counteracting the reactive oxygen species. A comparative transcriptome analysis between high NUE and low NUE cultivars of Brassica juncea showed that under low nitrate treatments, the high NUE cultivar had an upregulation of genes involved in N absorption, remobilization, and assimilation [135]. Furthermore, the co-expression network analysis has identified a number of nitrate regulatory modules that may be involved in the control of nitrate reductase activity, phenylpropanoid biosynthesis, and carbon:nitrogen interaction in B. juncea. The activation of phenylpropanoid and ROS scavenging pathways to counteract NH 4 + toxicity has also been confirmed by the transcriptional responses of the common duckweed Lemna minor under ammonium toxicity [136]. In Camelia sinensis, NRT, AMT, AQP, GS, and GOGAT have been identified as key differentially expressed genes (DEGs) regulating ammonium uptake and assimilation [137]. Analysis of the transcriptome of Salicornia neei, a halophyte grown in saline water containing 3 mM ammonium has led to the precise identification of the ammonium detoxification mechanism, mainly through the upregulation of glutamine synthetase and glutamate synthase genes involved in ammonium homeostasis [138].
As N is necessary for the synthesis of a wide range of metabolites, such as amino acids, proteins, lipids, nucleic acids, and chlorophyll, then, measuring metabolite levels using metabolomic techniques can reveal fundamental details about how biological processes react to physiological or environmental changes brought on by N status [139]. A clear discrimination of metabolic profiles of tea leaves and roots in response to N deficiency was observed by untargeted metabolomics using a GC-TOF/MS method [140]. Surprisingly, N deprivation elevated the concentrations of major phenylpropanoids, organic acids, and amino acids in roots, while in leaves there was a significant downregulation of amino acids. Thus, the differential metabolite accumulation in tea leaves and roots corresponds to their quality and stress response, respectively, under N deficiency conditions. An integrative metabolomic response of multiple species in response to ammonium nutrition showed that the accumulation of the essential amino acids varied depending on the species; however, the synthesis of Gln and/or asparagine (Asn) served as a more precise biomarker of ammonium nutrition [141]. The high NUE in maize fed with a mixture of urea and ammonium has been demonstrated with an integrated transcriptomics and metabolomics study [142]. In addition to a rapid elevation of the transcripts of nitrogen assimilation genes, including ZmGLN1;2, ZmGLN1;5, ZmAMT1.1a, ZmGOT1, and ZmGOT3, the combination of urea and ammonium also caused a significant buildup of amino acids, including tyrosine (Tyr), arginine (Arg), phenylalanine (Phe), and methionine (Met), in shoots. Quantitative proteomics analysis was carried out to investigate the ammonium-mediated growth inhibition of tomato roots. Even though the proteins (K4BPV5 and K4D9J3) corresponding to ammonium assimilation were upregulated, there was limited assimilation of ammonium leading to root growth inhibition. The reason for the higher accumulation of ammonium leading to toxicity could be explained by the downregulation of a protein, Q5NE21 corresponding to carbonic anhydrase, a key player in C metabolism [143].

Strategies to Overcome Ammonium Toxicity
The vast understanding of ammonium toxicity through extensive studies has led to the development of several strategies for overcoming ammonium toxicity, both at the laboratory and agricultural field levels ( Figure 2). In laboratory conditions, ammonium toxicity can be reduced by increasing CO 2 concentrations, light intensities, and exogenous organic or inorganic carbon supplies [22,144,145]. The regulation of the pH of the nutrient solution has also been suggested as an efficient way to manage the symptoms of ammonium toxicity [146]. The beneficial role of silicon (Si) in mitigating ammonium toxicity and other abiotic stresses has been extensively studied [147,148]. Silicon application has effectively reversed the adverse effects of ammonium toxicity and enhanced the growth parameters, photosynthesis, and NUE of many plant species ( Table 2). The ability of potassium and nitrate to counteract the harmful effects of ammonium on plants suggests that the strategic use of mineral fertilizers, including K + and NO 3 -, in the field could maximize nitrogen utilization and increase the safety of agricultural plants. It is a recommended practice to fertilize soils with ammonium and nitrate, in the form of NH 4 NO 3 to balance the effects of NH 4 + as NO 3 absorption helps in the alkalinization of the rhizosphere. However, the development of genetically modified (GM) plants is the potential strategy for achieving stable ammonium tolerance. In such cases, it may be beneficial to overexpress the transporters or enzymes involved in the assimilation, detoxification, and compartmentation of ammonia, whereas silencing the genes responsible for ammonia uptake and translocation may result in nitrogen deficiency or other detrimental effects as it can block crucial metabolic pathways [149]. The role of hormones, such as ABA that are involved in AMOS1/EGY1-dependent retrograde signaling of ammonium stress and auxins at elevated concentrations have been reported to alleviate ammonium toxicity [26,50]. Table 2: alleviation of ammonium toxicity using silicon supplementation in various plant species. Table 2. Alleviation of ammonium toxicity using silicon supplementation in various plant species.

PLANT SPECIES Mode of Si Application in the Cultivation System
Form and Concentration of Si Used

Mitigating Effects of Si under Ammonium Toxicity Reference
Zea mays L.
In the nutrient solution of the hydroponics system 10 mmol L −1 K 2 SiO 3 Increased N accumulation, shoot, and root dry mass production [150] Lycopersicon esculentum Mill.
In the nutrient solution of the hydroponics system 1 mmol L −1 monosilicic acid Improved N and Si use efficiency, increased leaf area, root area, dry biomass, total chlorophyll, carotenoids, and reduced oxidative stress [22] Cucumis sativus L. var.

Hokushin and Tsubasa
In the nutrient solution of the hydroponics system 0, 1, and 10 mmol L −1 K 2 SiO 3 Greater dry mass, N accumulation, green color index (GCI), and increased nitrate reductase activity [151] Brassica oleracea var. Botrytis cv Barcelona and Brassica oleracea var. Italica) cv BRO 68 In the nutrient solution of the hydroponics system 2 mmol L −1 silicon using stabilized sodium and potassium silicate (114.9 g L −1 Si and 18.9 g L −1 K 2 O).
In the nutrient solution of the vermiculate cultivation 2 mmol L −1 K 2 SiO 3 Greater stem and root diameter, root length, dry matter, leaf green color index (GCI), increased accumulation of ions (N, Si, K, Ca, and Mg), improved NUE, and reduced electrolyte leakage [153] Beta vulgaris L.
In the nutrient solution of the vermiculate cultivation in growth chamber 28.5 g L −1 H 4 SiO 4 Improved photosynthesis, stomatal conductance, transpiration, dry mass production, and accumulation of N and Si in roots [154] Raphanus sativus L.
In the nutrient solution of the hydroponics system 2 mmol L −1 K 2 SiO 3 Improved photosynthesis, transpiration, stomatal conductance, water-use efficiency, and higher total dry biomass [155] Eucalyptus urophylla x Eucalyptus grandis Fertigation of Si in vermiculate cultivation 2 mmol L −1 K 2 SiO 3 Increased dry mass production and improved the photosynthetic apparatus by decreasing fluorescence, and improving the quantum efficiency of photosystem II [156] Brassica campestris L. ssp. pekinensis In the nutrient solution of the hydroponics system 0.0 and 1.0 me·L −1 K 2 SiO 3 Improved growth parameters, photosynthesis, stomatal conductance, cation accumulation (Ca, Mg, K, and Si) antioxidant enzyme activities, and N assimilation enzyme activities [157] Salvia splendens 'Vista Red' In the nutrient solution of the hydroponics substrate system 0.0 and 1.0 me·L −1 K 2 SiO 3 Improved growth parameters, photosynthesis, stomatal conductance, cation accumulation (Ca, Mg, K, and Si) antioxidant enzyme activities, and N assimilation enzyme activities as well as reduced accumulation of ROS, and malondialdehyde (MDA) [158] Brassica lee ssp. namai Ssamchoo 'Chunssamhwang51' In the nutrient solution of the hydroponics system 0.0 and 10.7 me·L −1 K 2 SiO 3 Increased shoot fresh weight, chlorophyll content, leaf area, vitamin C, and antioxidant enzyme activities [108]

Conclusions and Future Perspectives
Ammonium assimilation into plant metabolites requires limited energy than nitrate assimilation, as it is already in reduced form. Thus, ammonium nutrition can be considered an energy-saving option that can be readily utilized by plants. However, the toxic effect of ammonium demands the optimization of its use to minimize its detrimental effects. Utilizing high-efficiency N fertilizers that contain both NH 4 + and NO 3 can significantly aid in managing ammonium toxicity issues and increase plant NUE in agricultural fields. To achieve high plant productivity at the laboratory level, different concentrations, and combinations of NH 4 + and NO 3 must be investigated, because the ideal ratio of NH 4 + to NO 3 probably varies for different species. Future research is highly needed to confirm the positive effects of ammonium in increasing plant quality, productivity, and resilience to biotic and abiotic stressors and their mechanisms behind. Moreover, the combined use of physiological and multi-omics studies will allow the exploration of the molecular intricacies of ammonium toxicity and tolerance in the future.