Integrated comparative transcriptome and physiological analysis reveals the metabolic responses underlying genotype variations in NH4+ tolerance

Several mechanisms have been proposed to explain NH₄⁺ toxicity. However, the core information about the biochemical regulation of plants in response to NH₄⁺ toxicity is still lacking. In this study, the tissue NH₄⁺ concentration is an important factor contributing to variations in plant growth even under nitrate nutrition and NH₄⁺ tolerance under ammonium nutrition. Furthermore, NH₄⁺ led to the reprogramming of the transcriptional profile, as genes related to trehalose-6-phosphate and zeatin biosynthesis were downregulated, whereas genes related to nitrogen metabolism, camalexin, stilbenoid and phenylpropanoid biosynthesis were upregulated. Further analysis revealed that a large number of genes, which enriched in phenylpropanoid and stilbenoid biosynthesis, were uniquely upregulated in the NH₄⁺- tolerant ecotype Or-1. These results suggested that the NH₄⁺-tolerant ecotype showed a more intense response to NH₄⁺ by activating defense processes and pathways. Importantly, the tolerant ecotype had a higher ¹⁵NH₄⁺ uptake and nitrogen utilization efficiency, but lower NH₄⁺, indicating the tolerant ecotype maintained a low NH₄⁺ level, mainly by promoting NH₄⁺ assimilation rather than inhibiting NH₄⁺ uptake. The carbon and nitrogen metabolism analysis revealed that the tolerant ecotype had a stronger carbon skeleton production capacity with higher levels of hexokinase, pyruvate kinase, and glutamate dehydrogenase activity to assimilate free NH₄⁺, Taken together, the results revealed the core mechanisms utilized by plants in response to NH₄⁺, which are consequently of ecological and agricultural importance.

Introduction

Nitrogen (N) is an essential plant macronutrient that is required for growth and is thus important in agricultural production. NH₄⁺-based fertilizers contain commonly used N forms synthesized via the Haber–Bosch process (Halvorson et al., 2014). While NH₄⁺ is the preferred form of N for plants at low concentrations, it is toxic to plants at high concentrations (von Wirén et al., 2000; Britto and Kronzucker, 2002). As NH₄⁺ escapes little with water in soil and elevated CO₂ reduces nitrate reductions in C3 species, a “more NH₄⁺ solution” was considered to mitigate nitrogen pollution and improve crop yields (Rubio-Asensio and Bloom, 2016; Subbarao and Searchinger, 2021). However, excess NH₄⁺/NH₃ atmospheric depositions have attracted attention in recent decades as they caused environmental problems related to species richness and composition (Stevens et al., 2004; Clark and Tilman, 2008; Duprè et al., 2010). The mechanisms underlying NH₄⁺ toxicity in plants are consequently of ecological and agricultural importance.

NH₄⁺ toxicity causes a retardation in plant growth, shortened roots, reduced root gravitropism, and leaf chlorosis (Britto and Kronzucker, 2002; Li et al., 2014). Several mechanisms have been proposed to explain NH₄⁺ toxicity, including the depletion of organic acids (Hachiya et al., 2010), deficiency of cations (Hoopen et al., 2010), futile transmembrane NH₄⁺ cycling (Britto et al., 2001; Li et al., 2022), photodamage to photosystem II (Dai et al., 2014), reactive oxygen and nitrogen species (RONS) induced oxidative stress (Liu et al., 2022), and the disruption of hormonal homeostasis (Li et al., 2012). The reactions of NH₄⁺ conjugation to glutamic acid and the synthesis of glutamic acid are critical for the detoxification of NH₄⁺, and are catalyzed by glutamine synthase (GS), glutamate synthase (GOGAT), and glutamate dehydrogenase (GDH) (Masclaux-Daubresse et al., 2006). Studies have shown that GS activity is upregulated by high external NH₄⁺ levels and that NH₄⁺-tolerant plants have higher GS activity and lower levels of NH₄⁺ accumulation in their tissues (Cruz et al., 2006; Guan et al., 2016). In addition, NH₄⁺ uptake is tightly controlled through the NH₄⁺-dependent inhibition of AMT1 by the phosphorylation of Thr-460 (Straub et al., 2017).

In recent years, numerous genetic loci controlling NH₄⁺ toxicity have been identified by screening for mutants. The Arabidopsis mutant hsn1-1 and its alelic mutant vtc1 are hypersensitive to NH₄⁺ because of point mutations in the gene encoding GDP-mannose pyrophosphorylase (GMPase). Defects in protein N-glycosylation of hsn1-1 have been linked to the root hypersensitivity phenotype (Qin et al., 2008). Based on leaf hypersensitivity to NH₄⁺, ammonium overly sensitive 1 (amos1) and amos2 were identified. amos1 was found to be an allelic mutation of EGY1 encoding a plastid metalloprotease. AMOS1/EGY1 mediated plastid retrograde signaling regulates NH₄⁺-responsive genes to maintain chloroplast functionality (Li et al., 2012). An ammonium tolerance mutant (amot1) was identified as allelic to EIN3, which positively regulates ROS production and induces oxidative stress under NH₄⁺ stress (Li et al., 2019a). Moreover, other genetic loci that control root development and gravitropism have been identified in Arabidopsis, including auxin resistant 1 (aux1), tiny root hair 1 (trh1), dolichol phosphate mannose synthase1 (dpms1), and gravitropism sensitive to ammonium 1 (gsa1) (Li et al., 2014).

Although the numerous genetic loci were found to associate with the toxicity of NH₄⁺ nutrition, the function studies of individual genes cannot fully reflect the natural responses of plants to NH₄⁺ toxicity. The tolerance of plants to NH₄⁺ depends on the species and variety. For example, some rice cultivars have adapted to NH₄⁺, while Arabidopsis thaliana and the Brassicaceae family are sensitive to NH₄⁺ (Esteban et al., 2016; Li et al., 2021). In this study, we explore the cellular threshold of NH₄⁺ concentration by investigated the A. thaliana natural accessions. More importantly, the core information about the biochemical regulation of plants in response to NH₄⁺ toxicity was identified by comparative assessments of the sensitive and tolerant species.

Materials and methods

Plant material and culture conditions

A natural accessions of Arabidopsis thaliana were used in this study (Table S1). Surface-sterilized seeds were sown onto nutritional soil in a greenhouse (300 μmol photons m^-2 s^-1, 16 h photoperiod, 22°C) for 7 d. Then, eight uniformly growing plants of each ecotype were grown hydroponically with 4 L of nutritional media per pot, containing 1 mM Ca(NO₃)₂ or 1 mM (NH₄)₂SO₄. The other essential nutrients in the two different nitrogen nutrient solutions were the same as our previous study (Chen et al., 2021), specially 1.25 mM KCl, 0.625 mM KH₂PO₄, 0.5 mM MgSO₄, 25 μM Fe-EDTA, 17.5 μM H₃BO₃, 3.5 μM MnCl₂, 0.25 μM ZnSO₄, 0.05 μM NaMoO₄, and 0.125 μM CuSO₄. It is of note that the concentration of Ca²⁺ in the solution was uniformly set to 1.5 mM, and The pH was buffered to 6.0 using 2.5 mM MES. The thaliana natural accessions was grown under the NH₄⁺ and NO₃^-.

The seedlings were first grown in 1 mM Ca(NO₃)₂ nutrient solution for 5 d and then transferred to nitrogen-free nutrient solution for 3 d. Subsequently, the thaliana natural accessions were grown under the NH₄⁺ and NO₃^- treatments for 8 days to investigate the NH₄⁺ concentration and fresh weight. The culture solution was refreshed every 4 days.

Measurement of the NH₄⁺ and total nitrogen concentrations

NH₄⁺ was extracted with deionized water using fresh rosette leaves. The supernatant was used to determine the NH₄⁺ concentration after the samples were centrifuged at 12,000 × g. Briefly, 20 μL supernatant was mixed with 0.5 mL phenol solution (10 g/L Phenol, 100 mg/L sodium nitrosoferricyanide) and 5 mL sodium hypochlorite alkaline solution (Sodium hydroxide 5 g, sodium hydrogen phosphate 3.53 g, sodium phosphate 15.9 g, sodium hypochlorite solution (ω= 5.25%) 5 mL, dissolved in 500 ml deionized water). The NH₄⁺ concentrations were measured colorimetrically using phenol hypochlorite (Berthelot reaction) colorimetry at 630 nm, and (NH₄)₂SO₄ was used as a standard.

For total nitrogen (TN) measurement, the plants were sampled and then dried at 105°C for half an hour and further dried at 65°C until a constant weight. The samples were weighed 0.100g using a thousand balance, packed in 150 mL narrow-mouth triangular flasks, boiled with H₂SO₄-H₂O₂ until transparent and clear. The solution was transferred to a 50 mL volumetric flask and made up to volume. The solution was then filtered and analyzed using a continuous-flow analyzer AA3 (Autoanalyzer 3; SEAL, Germany). NUtE (Nitrogen utilization efficiency) was calculated as dry shoot biomass/shoot N.

Enzyme activity

Fresh samples (100 mg) were homogenized in 3 mL of 50 mM Tris-HCl buffer (pH 8.0) containing 2 mM Mg²⁺, 2 mM DTT, and 0.4 M sucrose. After centrifuged at 10000 × g and 4°C for 10 min, the supernatant was then used to determine the activity of glutamine synthase. The activities of GS were measured according to the reference (Chen et al., 2018). Briefly, 0.5 mL supernatant was mixed with 1.6 mL reagent^①, which is composed of 100 mM Tris-HCl (pH 7.4), 80 mM MgSO4, 20 mM glutamate, 20 mM cysteic acid, 2 mM EGTA and 80 mM hydroxyl-amine hydrochloride, and 0.7 mL reagent^②, which is composed of 40 mM ATP. The 3 mL reaction mixture was incubated for 30 min at 37°C and was terminated by adding 1 mL FeCl₃ reagent (88 mM FeCl₃, 670 mM HCl and 200 mM TCA). After 10 min, the mixture was centrifuged at 4000 × g for 10 min, and the absorption value of supernatanta solution was determined at 540 nm wavelength.

NADH-dependent glutamate dehydrogenase was measured according to the reference (Groat and Vance, 1981). Briefly, fresh samples (100 mg) were homogenized in 3 mL of 50 mM Tris-HCl buffer (pH 8.0). 50 μL of the supernatant was mixed with the 950 μL reagent, which contained 100 μM NADH, 2.5 mM 2-oxoglutarate (2-OG), 200 mM NH₄Cl. The enzyme activity was defined as the reduction of absorbance due to NADH at 340 nm. The assay of enzyme activity was performed according the instruction of commercial kit (NADH-GDH kit, BC1465, Solarbio).

Pyruvate (Pyr) concentrations were measured according to the reference with some modifications (Kachmar and Boyer, 1953). Pyr reacts with 2, 4-dinitrophenylhydrazine to form pyruvate-2, 4-dinitrophenylhydrazone, which could be determined by calorimetry. Briefly, 0.1 g Fresh samples (100 mg) were homogenized in 1 mL trichloroacetic acid (8%) and placed on ice for 3 min. After centrifuged at 8000 × g and 4°C for 10 min, 75 μL the supernatant was added to the enzyme label plate and added 25 μL 2, 4-dinitrophenylhydrazine (0.05%), to react 2 min. Finally, 125 μL 1.5 M NaOH was added, and the absorption value of the tube was determined at 520 nm wavelength. The assay was performed using the Micro Pyruvate Assay Kit (BC2205; Solarbio).

Pyruvate kinase (PK) activity was measured according to the reference (Lepper et al., 2010). The assay was performed according to the instructions of the Pyruvate Kinase Assay Kit (BC0540; Solarbio). Briefly, 0.1 g Fresh samples (100 mg) were homogenized in 3 mL extracting solution, which contained 100 mM Tris-HCl (pH 7.5), 10 mM β-mercaptoethanol, 12.5% (v/v) glycerine, l mM EDTA-Na₂, 10 mM MgCl₂, and 1% (m/v) PVP-40. After centrifuged at 8000 × g and 4°C for 10 min, 0.1 mL of the supernatant was mixed with 0.9 mL reagent, which contained 100 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 0.16 mM NADH, 75 mM KCl, 5.0 mM ADP, 7.0 units L-lactate dehydrogenase (LDH), and 1.0 mM phosphoenolpyruvate (PEP). The absorption values at 340 nm wavelength were immediately recorded at 20s and 140s.

Hexose kinase (HXK) activity was measured according to the reference (Pancera et al., 2006). The assay was performed according to the instructions of the Hexose Kinase Assay Kit (BC0740; Solarbio). Briefly, 0.1 g Fresh samples (100 mg) were homogenized in 1 mL extracting solution. After centrifuged at 8000 × g and 4°C for 10 min, 10 μL the supernatant was added to the enzyme label plate, followed by 10 μL G-6-PDH solution (0.12 g/L) and 180 μL reagent, which 5 μl of a 2.0 mM NADP⁺ solution, 15 μl of a 0.1 M ATP solution, 50 μl of a 1 M glucose solution, and 110 μL of a 100 mM Tris–HCl buffer (pH 7.5). The absorption values at 340 nm wavelength were immediately recorded at 20s and 320s.

¹⁵N-NH₄⁺ isotope tracing

The objective of ¹⁵N-labeling experiment after 3d of N starvation is investigate the transport capacity of both genotypes. Seedlings were first grown in 1 mM Ca(NO₃)₂ nutrient solution for 5 d, and then transferred to a nitrogen-free nutrient solution for 3 d. Subsequently, the plants were transplanted into 1 mM (¹⁵NH₄)₂SO₄ with a ¹⁵N abundance of 5%. Samples were taken at 3, 6, and 24 h to determine the ¹⁵N content.

Comparative transcriptome analysis

The seedlings were grown in a NO₃^- nutrient solution for 5 d, and then transferred to a nitrogen-free nutrient solution for 3 d to deplete the stored NO₃^-. Subsequently, the plants were transferred to 1 mM Ca(NO₃)₂ or 1 mM (NH₄)₂SO₄ for 1 d. The total RNA was then extracted from the roots of the NH₄⁺-tolerant and -sensitive ecotypes. Three biological replicates were used for each ecotype under each treatment. The sequencing library was generated using the NEBNext Ultra™ RNA Library Prep Kit from Illumina (New York, NEB, USA), followed by sequencing on an Illumina HiSeq 2500 platform (San Diego, CA, USA). Gene expression levels were calculated using the FPKM method (Fragments Per Kilobase per Million mapped reads. Differentially expressed genes (DEGs) were defined as genes with |log₂ ^{(fold change)} | >1 and a false discovery rate (FDR) < 0.05. The comparative transcriptome between NH₄⁺ and NO₃^- was identified through comparisons of the FPKM values for each gene between NH₄⁺ and NO₃^-. The comparative transcriptome between the two genotypes was identified through comparisons of the FPKM values for each gene between Or-1 and the Rak-2. GO enrichment analysis was performed by the agriGo program (http://bioinfo.cau.edu.cn/agriGO/). The significantly enriched GO terms were defined with corrected P < 0.05.

Statistical analysis

Graphical values represent the mean ± SD. Statistical analyses were conducted using two-tailed Student’s t-tests or one-way analysis of variance (ANOVA). Least significant difference (LSD) tests for different treatments were used for multiple comparisons with P < 0.05. The significance level was set at P < 0.05 (*) and P < 0.01 (**).

Results

The tissue NH₄⁺ concentration was negatively linked to Arabidopsis growth

A panel of A. thaliana natural accessions was grown under identical concentrations of NH₄⁺ and NO₃^-. The growth of each ecotype was significantly inhibited by NH₄⁺ (Figures 1A, B). Conversely, nitrogen concentration was significantly higher in plants grown under NH₄⁺ than under NO₃^- conditions (Figure 1C). However, the free NH₄⁺ concentration in the shoots was significantly higher than that under NO₃^-, and its concentration varied widely among ecotypes (Figure 1D). The two-segment linear model accurately simulated the relationship between fresh weight and tissue NH₄⁺ concentration under different nitrogen sources (Figure 1E, Table S1). The fresh weight sharply decreased with the tissue NH₄⁺ concentration (<50 μg/g) under NO₃^- conditions, and the pace of decline slowed when the NH₄⁺ concentration was > 50 g/g under NH₄⁺ conditions (Figure 1E). Our data revealed that the tissue NH₄⁺ concentration was negatively linked to Arabidopsis growth, even under NO₃^- culture conditions.

Figure 1

Figure 1 The tissue NH₄⁺ concentration is negatively linked to the growth of Arabidopsis. After 8 d cultivation with NO₃^- or NH₄⁺ nutrition, the physiological parameters including the fresh weight, rosette length, nitrogen concentration and free NH₄⁺ were investigated. (A) The fresh weight, and (B) rosette length under nitrate or NH₄⁺ nutrition. (C) Total nitrogen concentration, and (D) free NH₄⁺ concentration in the plants. (E) The correlation analysis of free NH₄⁺ with fresh weight. Dashed lines indicated two linear models. Pearson R² values are given when p <.05. Student’s t test (**p <.01) was used to analyze statistical significance. For fresh weight and rosette length, results of each ecotype are means of eight biological replicates, and for TN and free NH₄⁺ concentration, results of each ecotype are means of three biological replicates.

Sensitivity to NH₄⁺ between Or-1 and the Rak-2

We comprehensively considered the biomass under ammonium nitrogen and the biomass under normal conditions, and excluded the ecotypes with poor growth under nitrate nitrogen. Additionally, the Or-1 and Rak-2 showed similar growth period. Thus, ecotypes Or-1 and Rak-2 were selected for further investigation. Or-1 grew better under both NO₃^- and NH₄⁺ nutrition (Figure 2A), which was also supported by its biomass (Figure 2B). With NO₃^- nutrition, the biomass of Rak-2 was 18.4% smaller than that of Or-1. However, the difference in biomass between the two ecotypes was 53% under NH₄⁺ nutrition (Figure 2B). The free NH₄⁺ concentration in Rak-2 was significantly higher than that in Or-1 under both nitrate and NH₄⁺ nutrition conditions (Figure 2C). Thus, Or-1 was characterized as an NH₄⁺-tolerant ecotype with high biomass and low NH₄⁺ concentration, while Rak-2 was characterized as an NH₄⁺ -sensitive ecotype.

Figure 2

Figure 2 Different sensitivity to NH₄⁺ nutrition between the Or-1 and the Rak-2. (A) Photos of the two ecotypes under NO₃^- or NH₄⁺ nutrition. (B) The dry weight of whole plants, and (C) free NH₄⁺ concentration of the two ecotypes under NO₃^- or NH₄⁺ nutrition. For dry weight, results of each ecotype are means ± SD of eight biological replicates, and for free NH₄⁺ concentration, results of each ecotype are means ± SD of three biological replicates. **p < .01.

The response of the transcriptome to NH₄⁺ was analyzed to explore the underlying molecular mechanisms. In Or-1, 1016 genes were upregulated and 687 genes were downregulated by NH4+ (Figure S1A). In Rak-2 cells, 483 and 637 genes were upregulated and downregulated by NH4+, respectively (Figure S1B). NO₃^–responsive genes (e.g., NRT2.1, NIA1, G6PD3, and FNR2) were activated by NO₃^- in both ecotypes (Figure S1). Based on the fold change and FDR of the differentially expressed genes (DEGs), the genes (CLE4, PMEI4, FAR3, HHO1) were strongly activated by NO₃^-, and the genes (ALMT1, CCX1, ALD1, PBS3, DUR3) were strongly activated by NH₄⁺ (Figure S1).

Core biological processes and pathways involved in the responses to NH₄⁺

The DEGs in the two ecotypes were used to analyze the core biological processes and pathways in response to NH₄⁺. The Gene Ontology (GO) enrichment analysis indicated that genes involved in hydrogen peroxide transport, camalexin biosynthesis, trehalose biosynthesis, and glutamate dehydrogenase activity were the most enriched terms regulated by NH₄⁺ (Figure 3A). The genes (PIP2.1, PIP2.4, TIP1.1, TIP1.2), annotated to transport H₂O and H₂O₂, respectively, were repressed by NH₄⁺ (Figure 3B). However, the genes (NAC042, CYP71B15, AtCYP71B2, MPK9, WRKY33, PAD4) that participate in camalexin biosynthesis, were activated by NH₄⁺ (Figure 3B). The trehalose-6-phosphate synthesis genes (TPS8, TPS 10) were repressed by NH₄⁺, while the trehalose-6-phosphate phosphatase genes (TPPD, TPPG) were activated (Figure 3B). The results suggest that NH₄⁺ represses the process of water transport and trehalose-6-phosphate synthesis but induces camalexin synthesis.

Figure 3

Figure 3 Overview of the transcriptome and the DEGs that were highly enriched in the core biological process and pathways in response to NH₄⁺. (A) Gene ontology (GO) enrichment analysis of the DEGs under NH₄⁺ stress. (B) The expression profiling of the genes involving in camalexin biosynthesis, H₂O₂ transmembrane transport and trehalose biosynthesis. (C) The enrichment pathways of the down-regulated genes by NH₄⁺. (D) The enrichment pathways of the up-regulated genes by NH₄⁺. (E) The effects of NH₄⁺ nutrition on the pathway of nitrogen assimilation. (F) The effects of NH₄⁺ nutrition on the pathway of zeatin biosynthesis. The red lines indicated the process was facilitated and the blue lines indicated the process was inhibited by NH₄⁺.

By analyzing the up- or downregulated gene sets, the KEGG results showed that the most enriched pathways were activated or repressed by NH₄⁺. The zeatin biosynthesis pathway was the most enriched process and was repressed by NH₄⁺ (Figure 3C). The isopentenyl-transferase gene (IPT) and the cytochrome P450 monooxygenase CYP735A encodes the rate-limiting enzyme in CK synthesis. Surprisingly, the transcript levels of IPT3, 5, 7 with NH₄⁺ nutrients were 6%, 39%, and 22%, respectively, of those under NO₃^- nutrients, and the transcript levels of CYP735A1, 2 under NH₄⁺ nutrients were only 10% and 15%, respectively, of those under NO₃^- nutrient conditions (Figure 3E; Figure S2). However, the CK oxidase genes (CKX1, 5, 6) under NH₄⁺ nutrient were 1.6 and 3.1 times higher than that under nitrate nutrient. These results support the conclusion that NH₄⁺ represses CK synthesis but reduced the level of active CK.

The most enriched pathway activated by NH₄⁺ was stilbenoid biosynthesis, followed by phenylalanine biosynthesis, glutathione metabolism, phenylpropanoid biosynthesis, and glutamate metabolism (Figure 3D). Nitrogen metabolism was simultaneously enriched in the upregulated and downregulated pathways, as the nitrate transporters (NRT1.1, NRT2.1, NRT3.1) and reduction genes (NIA1, NiR1) were strongly repressed by NH₄⁺, and NH₄⁺ assimilation genes (GLN1.4, GLN1.3, GDH3, GDH2, GDH1) were highly activated by NH₄⁺ (Figure 3E).

Stilbenoid, phenylpropanoid, and sucrose metabolism enriched in the tolerant ecotype were up-regulated by NH₄⁺

To explore the molecular mechanism of NH₄⁺ sensitivity in Or-1 and Rak-2, we compared the transcriptomes of the NH₄⁺-sensitive ecotype Rak-2 and the NH₄⁺-tolerant ecotype Or-1, under NH₄⁺ nutrient conditions. Overall, there were 2460 DEGs between the two ecotypes (Figure 4A; Figure S3). Among them, the expression of 1291 genes were higher in the NH₄⁺-tolerant ecotype Or-1, which was more than the number of genes with higher expression in the NH₄⁺-sensitive ecotype Rak-2 (Figure 4A). The functions of these DEGs were predicted using GO enrichment analysis. Some enriched GO terms between the two genotypes overlapped with those that responded to NH₄⁺, such as camalexin biosynthesis, water channel activity, and nitrilase activity (Figure 4B; Figure 3A).

Figure 4

Figure 4 Transcriptional characterization of the DEGs between the NH₄⁺-tolerant ecotype and the NH₄⁺-sensitive ecotype. (A) The expression profiling of DGEs between the NH₄⁺-tolerant ecotype and the NH₄⁺-sensitive ecotype. (B) GO enrichment analysis of the DGEs. (C) The enrichment pathways of the DGEs with higher expression in the NH₄⁺-tolerant ecotype Or-1. (D) The enrichment pathways of the DGEs with higher expression in the NH₄⁺-sensitive ecotype Rak-2. The circle size indicates the number of DEGs, and the rich factor indicates the degree of enrichment of the KEGG pathways.

By analyzing the higher expression genes in the Rak-2 compared with the Or-1, The most enriched pathways were found to be thiamine metabolism, pentose phosphate pathway (Figure 4D). Analysis of genes with higher expression in Or-1 than in Rak-2 under NH₄⁺-rich conditions. The most enriched pathways included stilbenoid, phenylpropanoid, glutathione, and sucrose metabolism (Figure 4C). Importantly, these pathways enriched in the Or-1 were up-regulated by NH₄⁺ (Figure S4).

The NH₄⁺-tolerant ecotype Or-1 exhibits a more intensive response to NH₄⁺ by activating defense processes and pathways

As shown in the VENN diagram, 148 DEGs between the two ecotypes were simultaneously responsive to NH₄⁺ (Figure 5A). In addition, 391 DEGs between the two ecotypes specifically responded to NH₄⁺ in the tolerant ecotype Or-1 (Figure 5A). Similarly, 194 DEGs between the two ecotypes specifically responded to NH₄⁺ in the sensitive ecotype Rak-2 (Figure 5A). The core 148 genes were classified into upregulated and downregulated genes following NH₄⁺ treatment (Figures 5B, C; Table S2). Among the core 148 genes, a higher proportion of genes were upregulated by NH₄⁺, especially Or-1 (Figure 5C). The cytochrome P450 genes CYP86A4, CYP71B22, CYP706A2, and CYP709B3 were strongly induced by NH₄⁺ and showed higher Or-1 expression (Figure 5C). The transcription factors WRKY55, WRKY41, bZIP8, and ZAT8 were more strongly induced in the NH₄⁺-tolerant ecotype Or-1 than that in the NH₄⁺-sensitive ecotype Rak-2 (Figure 5C).

Figure 5

Figure 5 Analysis of the common and specific NH₄⁺-responsive genes between the two ecotypes. (A) The VENN diagram of the DEGs. (B) The commonly down-regulated DEGs between the two ecotypes. (C) The commonly down-regulated DEGs between the two ecotypes. (D) The DEGs between the two ecotypes specifically responded to NH₄⁺ only in one ecotype. (E) The expression profiling of the DEGs specifically responded to NH₄⁺ in the sensitive ecotype Rak-2. (F) The expression profiling of the DEGs specifically responded to NH₄⁺ in the tolerant ecotype Or-1. (G) GO enrichment analysis of the DGEs specifically responded to NH₄⁺ in the tolerant ecotype Or-1. (H) The enrichment pathways of the DGEs specifically responded to NH₄⁺ in the tolerant ecotype Or-1.

Surprisingly, 194 DEGs between the two ecotypes that specifically responded to NH₄⁺ in Rak-2 were mostly downregulated by NH₄⁺ (Figures 5D, E; Table S2). Conversely, 391 DEGs between the two ecotypes specifically responded to NH₄⁺ in Or-1 plants (Figures 5D, F; Table S2). These genes were mostly upregulated by NH₄⁺ and showed higher expression levels in Or-1 cells (Figures 5D, F). These results suggest that a large number of genes were uniquely upregulated in the NH₄⁺- tolerant ecotype Or-1. The function of these unique NH₄⁺-responsive genes in the Or-1 were predicted using GO enrichment analysis and KEGG pathways. The most highly enriched biological processes were defense responses, including responses to ABA, water deprivation, chitin, wounding, and oxidative stress (Figure 5G). Similarly, the KEGG pathway analysis indicated that nitrogen metabolism, phenylpropanoid biosynthesis, glutathione metabolism, and plant hormone signal transduction were the most highly enriched pathways (Figure 5H). Collectively, these results suggest that the NH₄⁺-tolerant ecotype Or-1 exhibits a more intensive response to NH₄⁺ by activating defense processes and pathways, including phenylpropanoid biosynthesis, nitrogen metabolism, and glutathione metabolism.

The tolerant ecotype maintained a low NH₄⁺ level, mainly by promoting NH₄⁺ assimilation rather than inhibiting NH₄⁺ uptake

As the NH₄⁺-sensitive ecotype, Rak-2 accumulated more NH₄⁺ than the NH₄⁺-tolerant ecotype Or-1 (Figure 2C), their uptake and assimilation of NH₄⁺ were investigated. Both the concentration and accumulation of ¹⁵N were significantly higher in the NH₄⁺-tolerant ecotype Or-1 (Figures 6A, B). The higher nitrogen utilization efficiency (NUtE) in the tolerant ecotype indicated the higher efficiency of the conversion of shoot N into shoot biomass (Figure 6C). The expression of AMT2 and AMT1.2 were also higher in the NH₄⁺-tolerant ecotype (Figure 6D), which supports the results for ¹⁵NH₄⁺ assimilation. Although the GS activity of both ecotypes increased with increasing NH₄⁺ concentration, no difference was observed between the two (Figure 6E). The expression of Gln1s was significantly induced by NH₄⁺, and only the expression of Gln1.4 slightly higher in the NH₄⁺ -sensitive ecotype (Figure 6D). However, GDH activity was strongly induced by NH₄⁺ nutrition and was significantly higher in the tolerant ecotype when compared with that in the sensitive ecotype (Figure 6F). Accordingly, the expression of GDH1 and GDH2 was more significantly induced in the roots of the tolerant ecotype than in those of the sensitive ecotype (Figure 6D).

Figure 6

Figure 6 Analysis of NH₄⁺ assimilation capacity between the two varieties. (A, B) The short-term of ¹⁵NH₄⁺ uptake for 3 h, 6 h and 24 h. (C) The nitrogen utilization efficiency (NUtE) between the two ecotypes under NH₄⁺ nutrition. NUtE is calculated by biomass/nitrogen. (D) Differential expression profiling of the NH₄⁺ uptake and assimilation genes, including the NH₄⁺ transporter genes AMTs, glutamine synthesis genes GLN and glutamate dehydrogenase genes GDH. (E) The activity of glutamine synthesis in the roots of two ecotypes. (F) The activity of GDH in the roots of two ecotypes. (G) Differential expression profiling of the carbohydrate metabolism genes, including the cell wall-bound invertase gene CWINV1, hexokinase gene HXK2 and pyruvate kinase gene PK4. (H) The activity of hexokinase in the roots of two ecotypes. (I) The content of pyruvate in the roots of two ecotypes. (J) The schematic diagram of carbon and nitrogen metabolism. The enzymes and metabolite highlighted in red font represent higher activity or content in the root of tolerant ecotype. Results are means ± SD of three biological replicates. *p < .05 and **p < .01.

The vital nodes of C and N metabolism were also investigated. The expression of hexokinase gene (HXK2) and pyruvate kinase gene (PK4) were much higher in the roots of the tolerant ecotype than the sensitive ecotype with NH₄⁺ nutrition (Figure 6G). These results are consistent with the enzymatic activities of HXK and PK (Figure 6H; Figure S5). Importantly, pyruvate content, which is essential for N metabolism, was reduced by NH₄⁺ nutrition, whereas its content was significantly higher in the roots of the tolerant ecotype than in the sensitive ecotype under NH₄⁺ nutrition (Figure 6I). These results indicated that the tolerant ecotype maintained a low NH₄⁺ level, mainly by promoting NH₄⁺ assimilation rather than inhibiting NH₄⁺ uptake.

Discussion

The tissue NH₄⁺ concentration is an important factor contributing to variations in plant growth and NH₄⁺ tolerance

NH₄⁺ is toxic to plants, even at micromolar concentrations (von Wirén et al., 2000). Many hypotheses have been proposed to explain NH₄⁺ toxicity, such as the depletion of organic acids, deficiency of cations, futile transmembrane NH₄⁺ cycling and so on (Li et al., 2014). Previous studies have explored the medium NH₄⁺ concentration toxic to different crops (Britto and Kronzucker, 2002). A previous study has expounded the critical role of NH₄⁺ in Arabidopsis natural variability in NH₄⁺ tolerance (Sarasketa et al., 2014). In this study, we found the two-segment linear model accurately simulated the relationship between fresh weight and tissue NH₄⁺ concentration under different nitrogen sources (Figure 1E). When the tissue NH₄⁺ concentration was below 50 μg/g under nitrate conditions, the fresh weight rapidly decreased with the tissue NH₄⁺ concentration. Whereafter, the pace of declines slowed, when the tissue NH₄⁺ concentration was over 50 μg/g under NH₄⁺ conditions, (Figure 1E). This phenomenon possibly enlightened that low concentrations of NH₄⁺ do not cause obvious symptoms of NH₄⁺ toxicity, such as leaf chlorosis, but affect plant growth in an imperceptible way. However, the visible symptoms of NH₄⁺ toxicity appeared over 50 μg/g under NH₄⁺ conditions. A previous study revealed that the shoot K⁺ was positively correlated with the growth and NH₄⁺ tolerance (Chen et al., 2021), and this correlation may be indirectly caused by the inhibition of K⁺ transport. Thus, the tissue NH₄⁺ concentration is an important factor contributing to variations in plant growth and NH₄⁺ tolerance.

Plants deal with NH₄⁺ overload by using different strategies, including NH₄⁺ transporters and NH₄⁺ assimilation regulation. Inactivation of AMT1;1 and AMT1;2 by interaction with CIPK23 is important for NH₄⁺ uptake regulation; Thus, the Loss of CIPK23 increases root NH₄⁺ uptake and confers hypersensitivity to NH₄⁺ (Straub et al., 2017). In the present study, the NH₄⁺-tolerant ecotype Or-1 accumulated more ¹⁵N-labeled NH₄⁺ (Figures 6A, B), thus the repression of AMT transporter cannot explain the tolerance mechanism of Or-1. Instead, the NH₄⁺-tolerant ecotype displayed higher expression of AMT2 and AMT1.2 than the NH₄⁺ -sensitive ecotype Rak-2, and its expression were induced by NH₄⁺ (Figure 6D). Recently study found the expression of ZmAMT1s were induced by NH₄⁺ nutrition and discovered glutamine rather than NH₄⁺ regulated ZmAMT1s (Hui et al., 2022). The higher content of assimilated ¹⁵N-labeled H₄⁺ and NUtE indicated that the tolerant ecotype Or-1 had a stronger NH₄⁺ assimilation capacity, thus induced the expression of AMTs through a positive feedback by the NH₄⁺ assimilate - glutamine.

The tolerant ecotype had a stronger NH₄⁺ assimilation capacity to alleviating NH₄⁺ toxicity

In plants, NH₄⁺ assimilation generally occurs via the GS/GOGAT cycle. Cytosolic (GS1) and chloroplast (GS2) isoforms play opposing roles in NH₄⁺ stress. Root Gln1.2 alleviates NH₄⁺ toxicity, whereas shoot Gln2 aggravates NH₄⁺ toxicity in a pH-dependent manner (Hachiya et al., 2021). Indeed, the expression of Gln1s was significantly induced by NH₄⁺, whereas the expression of Gln2 was repressed (Figure 6D), suggesting an interesting regulation of GS to adapt NH₄⁺ stress in plants. Although the GS activity of both ecotypes increased with increasing NH₄⁺ concentration, no difference was observed between the two ecotypes (Figure 6E). Another study found no correlation between GS activity and shoot biomass in Arabidopsis under NH₄⁺ nutrition (Sarasketa et al., 2014). Thus, variations in GS activity may not be crucial for NH₄⁺ tolerance in different Arabidopsis ecotypes. GDH can incorporate NH₄⁺ independently of the GS/GOGAT cycle and plays a critical role in the detoxification of NH₄⁺ in stress conditions (Xian et al., 2020). Although the capacity of GDH to synthesize Glu in vivo has not been clearly demonstrated, heterologous expression of fungal GDH in plants could alleviates NH₄⁺ toxicity and improve nitrogen assimilation (Tang et al., 2018; Yan et al., 2021). In this study, GDH activity and its encoding genes in both ecotypes were induced by NH₄⁺ nutrition, and the activity was much higher in the tolerant ecotype than in the sensitive ecotype (Figure 6F). Moreover, GDH1 and GDH2 were more strongly induced in the roots of the tolerant ecotype than in those of the sensitive ecotype (Figure 6D). Overall, these results possibly indicate the critical role of GDH in Arabidopsis natural variation in NH₄⁺ tolerance.

The sucrose metabolism pathway was enriched in the tolerant ecotype, indicating that this process was more active in the NH₄⁺-tolerant ecotype (Figure 4C; Figure S4). Hexokinase (HXK) and Pyruvate kinase (PK) function crucial roles in Glycolysis and TCA cycle, providing 2-OG and energy for N metabolism (Stitt, 1999). In the present work, the expression of HXK2 and PK4 were much higher in the root of tolerant ecotype than the sensitive ecotype under NH₄⁺ nutrition (Figure 6G). These results are consistent with the enzymatic activities of HXK and PK (Figure 6H; Figure S5). Importantly, the crucial carbon metabolite pyruvate was reduced by NH₄⁺ nutrition, whereas its content was significantly higher in the roots of the tolerant ecotype than in the sensitive ecotype under NH₄⁺ nutrition (Figure 6I). These results suggest that the tolerant ecotype had a stronger carbon skeleton (2-OG) production capacity for NH₄⁺ assimilation (Figure 6J).

Coordination of carbon (C) and nitrogen (N) metabolism is essential for plant growth and stress tolerance (Reguera et al., 2013; Liang et al., 2020). The higher nitrogen utilization efficiency (NUtE) in the tolerant ecotype indicated the higher efficiency of the conversion of shoot N into shoot biomass (Figure 6C). The NUtE was similar among the accessions with different nitrogen use efficiency and uniformly decreased with high N supply, but the accessions differed in their NUtE under N restriction (Menz et al., 2018). The nitrogen was not limited and the concentration was even higher under NH₄⁺ nutrition than that under nitrate nutrition. Thus, the higher NUtE in the tolerant ecotype may suggest better-coordinated C/N metabolism compared with the sensitive ecotype under NH₄⁺ stress.

The tolerant ecotype displayed stronger defense responses by activating phenylpropanoids and the derived stilbenoids

NH₄⁺ induces ROS formation, leading to oxidative damage in plants (Podgorska et al., 2013; Liu et al., 2022). Here, stilbenoid biosynthesis, phenylpropanoid biosynthesis, and glutathione metabolism pathways were commonly activated by NH₄⁺ in all ecotypes (Figure 3D). Stilbenoids, which are hydroxylated derivatives of stilbene belonging to the phenylpropanoid family, have been shown to have a wide spectrum of biological functions, such as antioxidant and antimicrobial activities (Dong and Lin, 2021). Many other secondary metabolites, such as anthocyanins and flavonols, share a common origin in the phenylpropanoid biosynthetic pathway and functions as an ROS scavenger induced by abiotic stress (Dong and Lin, 2021). Treatment with flavonoids, such as naringenin, reduces oxidative damage under Cd stress in rice (Chen et al., 2022). Moreover, phenylpropanoid metabolism is associated with the reinforcement of cell walls under NH₄⁺ nutrition, which was considered as an important NH₄⁺ tolerance mechanism (Royo et al., 2019; Yang et al., 2022). Importantly, the NH₄⁺-tolerant ecotype Or-1 showed a more intense response to NH₄⁺ by activating pathways, including phenylpropanoid and stilbenoid biosynthesis (Figure 4C). Therefore, the biosynthesis of phenylpropanoids and stilbenoids derived from NH₄⁺ may play a positive role in the defense response to NH₄⁺ toxicity, and this thus warrants further investigation.

The tolerant ecotype was more responsive to NH₄⁺ stress than the sensitive ecotype. These genes were highly enriched in defense responses, including their responses to ABA, water deprivation, chitin, wounding, and oxidative stress (Figure 5G). ABA signaling plays a key role in oxidative stress responses and is associated with the induction of antioxidant defense systems (Yang et al., 2015; Li et al., 2019b). Interestingly, the NH₄⁺- tolerant ecotype has stronger ABA responses and phenylpropanoid metabolism for ROS scavenging (Figures 5G, H). Recently, a research investigated the genetic variation underlying differential ammonium and nitrate responses in Arabidopsis thaliana, and found the preferring ammonium or nitrate, appeared to be generated by combinations of loci rather than a few large-effect loci, which most are specific to a developmental or defense trait under specific nitrogen source (Katz et al., 2022). In our results, we also found only few genes were simultaneously responsive to NH₄⁺ between the two genotypes (Figure 5A). This suggested that ammonium tolerance genotypes are a combination of multiple mini-effect genes.

Conclusion

In this study, the tissue content of NH₄⁺ was found the main cause for NH₄⁺ toxicity, and the two-segment linear model accurately simulated the relationship between fresh weight and tissue NH₄⁺ concentration under different nitrogen sources. we revealed that the tolerant ecotype maintained a low NH₄⁺ level, mainly by promoting NH₄⁺ assimilation rather than inhibiting NH₄⁺ uptake. The carbon and nitrogen metabolism analysis revealed that the tolerant ecotype had a stronger carbon skeleton (2-OG) production capacity with higher levels of hexokinase (HXK), pyruvate kinase (PK), and GDH activity to assimilate free NH₄⁺. Furthermore, the core information about the biochemical regulation of plants in response to NH₄⁺ toxicity was identified. The most enriched pathways included nitrogen metabolism, camalexin, stilbenoid and phenylpropanoid biosynthesis were upregulated by NH₄⁺. Interestingly, a large number of genes, which enriched in phenylpropanoid and stilbenoid biosynthesis, were uniquely upregulated in the NH₄⁺- tolerant ecotype. These results suggested that the NH₄⁺-tolerant ecotype showed a more intense response to NH₄⁺ by activating defense processes and pathways.

Data availability statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: Gene Expression Omnibus, GSE243624.

Author contributions

HC: Conceptualization, Data curation, Formal analysis, Writing – original draft. WL: Data curation, Formal analysis, Methodology, Writing – original draft. WZ: Investigation, Software, Validation, Writing – review & editing. JZ: Data curation, Formal analysis, Project administration, Writing – review & editing. QZ: Writing – review & editing. ZZ: Resources, Supervision, Writing – review & editing.

Funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This study was financially supported in part by the National Natural Science Foundation of China (Grant No. 32172669; U21A20236); the Scientific Research Fund of Hunan Provincial Education Department (Grant No. 22A0157).

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2023.1286174/full#supplementary-material

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