Bicarbonate-Dependent Detoxification by Mitigating Ammonium-Induced Hypoxic Stress in Triticum aestivum Root

Simple Summary Ammonium (NH4+) is usually toxic to plant growth when used as the sole or dominant N source. Exploring the underlying molecular mechanisms of NH4+ toxicity and how to minimize NH4+ toxicity may greatly benefit crop productivity. In this study, the underlying mechanism of NH4+ toxicity and bicarbonate (HCO3−)-dependent alleviation in wheat was investigated. Comprehensive transcriptomic and physiological analyses suggested that NH4+ nutrition alone stimulated fermentation and glycolysis, promoted the activity of alternative respiratory pathways, suppressed TCA cycle pathways, and reduced ATP synthesis; adding HCO3− relieved the toxic effects of NH4+ nutrition. Our results reveal the importance of C and N interactions for alleviating NH4+ toxicity, likely by mitigating root hypoxic stress. As the first report on the hypoxic stress triggered by NH4+ treatment, this study provides novel insights into the mechanisms of NH4+ toxicity and its alleviation, which may present potential solutions for improving the nitrogen use efficiency in wheat. Abstract Ammonium (NH4+) toxicity is ubiquitous in plants. To investigate the underlying mechanisms of this toxicity and bicarbonate (HCO3−)-dependent alleviation, wheat plants were hydroponically cultivated in half-strength Hoagland nutrient solution containing 7.5 mM NO3− (CK), 7.5 mM NH4+ (SA), or 7.5 mM NH4+ + 3 mM HCO3− (AC). Transcriptomic analysis revealed that compared to CK, SA treatment at 48 h significantly upregulated the expression of genes encoding fermentation enzymes (pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), and lactate dehydrogenase (LDH)) and oxygen consumption enzymes (respiratory burst oxidase homologs, dioxygenases, and alternative oxidases), downregulated the expression of genes encoding oxygen transporters (PIP-type aquaporins, non-symbiotic hemoglobins), and those involved in energy metabolism, including tricarboxylic acid (TCA) cycle enzymes and ATP synthases, but upregulated the glycolytic enzymes in the roots and downregulated the expression of genes involved in the cell cycle and elongation. The physiological assay showed that SA treatment significantly increased PDC, ADH, and LDH activity by 36.69%, 43.66%, and 61.60%, respectively; root ethanol concentration by 62.95%; and lactate efflux by 23.20%, and significantly decreased the concentrations of pyruvate and most TCA cycle intermediates, the complex V activity, ATP content, and ATP/ADP ratio. As a consequence, SA significantly inhibited root growth. AC treatment reversed the changes caused by SA and alleviated the inhibition of root growth. In conclusion, NH4+ treatment alone may cause hypoxic stress in the roots, inhibit energy generation, suppress cell division and elongation, and ultimately inhibit root growth, and adding HCO3− remarkably alleviates the NH4+-induced inhibitory effects on root growth largely by attenuating the hypoxic stress.

Simple Summary: Ammonium (NH 4 + ) is usually toxic to plant growth when used as the sole or dominant N source.Exploring the underlying molecular mechanisms of NH 4 + toxicity and how to minimize NH 4 + toxicity may greatly benefit crop productivity.In this study, the underlying mechanism of NH 4 + toxicity and bicarbonate (HCO 3 − )-dependent alleviation in wheat was investigated.
Comprehensive transcriptomic and physiological analyses suggested that NH 4 + nutrition alone stimulated fermentation and glycolysis, promoted the activity of alternative respiratory pathways, suppressed TCA cycle pathways, and reduced ATP synthesis; adding HCO 3 − relieved the toxic effects of NH 4 + nutrition.Our results reveal the importance of C and N interactions for alleviating NH 4 + toxicity, likely by mitigating root hypoxic stress.As the first report on the hypoxic stress triggered by NH 4 + treatment, this study provides novel insights into the mechanisms of NH 4 + toxicity and its alleviation, which may present potential solutions for improving the nitrogen use efficiency in wheat.
Abstract: Ammonium (NH 4 + ) toxicity is ubiquitous in plants.To investigate the underlying mechanisms of this toxicity and bicarbonate (HCO 3 − )-dependent alleviation, wheat plants were hydroponically cultivated in half-strength Hoagland nutrient solution containing 7.5 mM NO 3 − (CK), 7.5 mM NH 4 + (SA), or 7.5 mM NH 4 + + 3 mM HCO 3 − (AC).Transcriptomic analysis revealed that compared to CK, SA treatment at 48 h significantly upregulated the expression of genes encoding fermentation enzymes (pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), and lactate dehydrogenase (LDH)) and oxygen consumption enzymes (respiratory burst oxidase homologs, dioxygenases, and alternative oxidases), downregulated the expression of genes encoding oxygen transporters (PIP-type aquaporins, non-symbiotic hemoglobins), and those involved in energy metabolism, including tricarboxylic acid (TCA) cycle enzymes and ATP synthases, but upregulated the glycolytic enzymes in the roots and downregulated the expression of genes involved in the cell cycle and elongation.
The physiological assay showed that SA treatment significantly increased PDC, ADH, and LDH activity by 36.69%,43.66%, and 61.60%, respectively; root ethanol concentration by 62.95%; and lactate efflux by 23.20%, and significantly decreased the concentrations of pyruvate and most TCA cycle intermediates, the complex V activity, ATP content, and ATP/ADP ratio.As a consequence, SA significantly inhibited root growth.AC treatment reversed the changes caused by SA and alleviated the inhibition of root growth.In conclusion, NH 4 + treatment alone may cause hypoxic stress in

Introduction
Nitrogen (N) is one of most essential nutrients for plant growth and development and is involved in the biosynthesis of proteins, nucleic acids, chlorophyll, and several hormones [1][2][3].Therefore, appropriate N fertilizer use is critical in increasing crop yield and improving the quality of agricultural products.Ammonium (NH 4 + ) and nitrate (NO 3 − ) are the two main N sources for plants [4][5][6].NH 4 + can be more directly assimilated by plant cells, while NO 3 − uptake and reduction (i.e., conversion of NO 3 − to NH 4 + via the actions of nitrate reductase and nitrite reductase) consume large amounts of ATP and reducing equivalents, and the resulting NH 4 + is then used by plants [5,7,8].Therefore, it is widely acknowledged that NH 4 + is the preferred N source with respect to energy cost.To increase crop yields, farmers tend to apply excessive N fertilizers; however, only 30-40% of the fertilizer is estimated to be taken up by plant roots [8,9].Moreover, excess N fertilizer application often suppresses crop growth, decreases kernel yield, and causes environmental pollution [10].In particular, when using NH 4 + as the sole N source, most plants exhibit severe growth retardation, leaf chlorosis, a lower net photosynthetic rate, and other toxic symptoms, commonly referred to as NH 4 + syndrome [11,12].Therefore, optimizing N fertilization can improve nitrogen use efficiency (NUE), reduce the cost of N inputs, and decrease environmental pollution [13].
Many studies have been conducted to elucidate the underlying mechanisms by which exogenous substances alleviate NH 4 + toxicity.It was found that elevated potassium (K + ) reduced futile NH 4 + cycling on the plasma membrane in rice (Oryza sativa L.) [23], and decreased vacuolar H + -ATPase activity and inhibited NH 4 + accumulation in Arabidopsis thaliana roots [24].Low levels of NO 3 − attenuate NH 4 + toxicity by upregulating ACLA-3 (encoding ATP-citrate lyase A-3) and increasing the production of several key metabolites in the tricarboxylic acid (TCA) cycle in Panax notoginseng [25] and wheat [7].SnRK1.1 allows SLAH3-mediated NO 3 − efflux by phosphorylating the C-terminal of SLAH3 at site S601, thereby alleviating high-NH 4 + /low-pH stress in Arabidopsis thaliana [26].Exogenous α-ketoglutarate (KGA), a key C skeleton for N assimilation, alleviates NH 4 + stress in tomato (Solanum lycopersicum L.) [27].Silicon (Si) alleviates NH 4 + toxicity by accelerating NH 4 + assimilation via the actions of glutamine synthetase, glutamate synthase, and glutamate dehydrogenase in cabbage (Brassica campestris L.) [28] or increasing the shoot cytokinin content in tomato [29].
When used as the dominant N source, NH 4 + stimulates respiratory O 2 consumption in Arabidopsis, barley (Hordeum vulgare L.), wheat, and maize (Zea mays L.) to meet the needs of ATP, resulting in higher carbon dioxide (CO 2 ) evolution [30][31][32].Therefore, the reduced NH 4 + toxicity after the exogenous addition of CO 3 2− [33], HCO 3 − [34], or CO 2 [35] may be explained by improved carbohydrate accumulation, balanced C and N metabolism, and a greater ability to cope with the depletion of organic acids [25].Notably, these changes are involved in the TCA cycle, where intermediates, mainly KGA, can be used as C skeletons for NH 4 + assimilation [36,37].Wheat is one of the most important food crops, and about one third of the global population currently consumes wheat [38].Improving NUE is important to improve grain yield and processing quality and reduce the cost of wheat production [39].In this study, comprehensive transcriptomic and physiological analyses were conducted to investigate + toxicity and its HCO 3 − -dependent alleviation in wheat.Our results show that NH 4 + nutrition alone stimulated fermentation and glycolysis, promoted the activity of alternative respiratory pathways, suppressed TCA cycle pathways, and reduced ATP synthesis; adding HCO 3 − relieved the toxic effects of NH 4 + nutrition.Our results reveal the importance of C and N interactions for alleviating NH 4 + toxicity, likely by mitigating root hypoxic stress.

Plant Material and Growth Conditions
Seeds of wheat (cultivar Jimai 22) were surface-sterilized using 70% ethanol for 45 s and washed 5 times with distilled water.The sterilized seeds were then germinated on moist filter paper placed inside Petri dishes at 23 • C.After 3 days, the uniform-sized seedlings were transferred to black plastic pots with dimensions of 10 cm × 8 cm × 5 cm (length, width, height) containing distilled water and grown in a growth chamber at 25 • C/21 • C (day/night) under 14 h/10 h (light/dark) for 5 days under the following conditions: light intensity 450 µmol m −2 s −1 and humidity 70 ± 5%.Each pot contained 15 plants and the distilled water was renewed every 2 days.
In our preliminary experiment, we found that the roots of 8-day-old seedlings grew best in half-strength Hoagland nutrient solution containing 7.5 mM NO 3 − (Table S1), and 7.5 mM NH 4 + showed significant inhibition of root growth compared with 7.5 mM NO 3 − .An experiment using gradient concentrations showed that 3 mM HCO 3 − significantly improved the root growth of wheat seedlings fed 7.5 mM NH 4 + .Therefore, the following experiment was conducted using 8-day-old seedlings that were fixed on polystyrene plates and hydroponically cultured in half-strength Hoagland nutrient solution containing 7.5 mM NO 3 − (CK, applied as KNO 3 and Ca(NO 3 ) 2 ), 7.5 mM NH 4 + (sole ammonium (SA), applied as 7.5 mM NH 4 Cl), or 7.5 mM NH 4 + + 3 mM HCO 3 − (ammonium and bicarbonate (AC), applied as 7.5 mM NH 4 Cl and 3 mM KHCO 3 ).The solutions were renewed every 2 days.Each treatment was repeated in triplicate.Potassium in the nutrient solution was balanced by the addition of K 2 SO 4 .

Measurement of Plant Fresh Weight (FW)
Wheat seedlings were collected at 24, 48, 72, and 96 h after treatment and separated into shoots and roots, which were dried with absorbent paper and immediately weighed.Three biologically independent experiments, each with three replicates, were conducted to calculate the net increase in FW.

RNA Extraction and Detection
Fresh roots (approximately 0.1 g) were fully ground in a mortar with 1 mL RLT and 100 µL PLANTaid at room temperature.The homogenate was transferred to a centrifuge tube, vigorously shaken for 15 s, and centrifuged at 13,000 rpm for 5 min.Then, 450 µL of supernatant was transferred to a new centrifuge tube, and the volume of half absolute ethanol was added, blown, and mixed.The mixture was added to an adsorption column for RA (the adsorption column was placed in the collection tube) and centrifuged at 13,000 rpm for 60 s, and the waste liquid was abandoned.Total RNA extracted from roots underwent RNA-Seq analysis at Novogene (Beijing, China).Three biological replicates of each sample were used for RNA-Seq analysis.RNA quality and quantity were checked using a spectrophotometer (NanoDrop ND-1000 UV-Vis spectrophotometer; Nanodrop Technologies, Wilmington, DE, USA).The integrity of the final RNA samples was checked by denaturing gel electrophoresis on 1.4% (w/v) formaldehyde agarose gels, and the concentration was determined photometrically (NanoDrop).Purified RNA was treated with a Turbo DNase-free kit.cRNA synthesis and labeling, array hybridization, and scanning were performed at imaGenes GmbH (Berlin, Germany).

Library Construction and Quality Inspection
There are 2 ways to construct a library: ordinary NEB construction and chain-specific construction.The NEBNext ® Ultra™ RNA Library Prep Kit from Illumina ® was used to build the library.After RNA library construction, initial quantification was characterized on 1% agarose gels and examined using the NanoPhotometer ® spectrophotometer (Implen, Westlake Village, CA, USA).RNA concentrations were measured using a Qubit ® RNA Assay Kit in a Qubit ® 2.0 Fluorimeter (Life Technologies, Carlsbad, CA, USA).The RNA integrity number was analyzed by accurate detection of RNA integrity and library insert size using an Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA).After the insert size was determined, effective library concentrations were accurately quantified by qRT-PCR (above 2 nM) to ensure quality.

Sequencing
After inspection, libraries were pooled and sequenced against the effective concentration and target data volume.Four fluorescently labeled dNTP, DNA polymerase, and adapter primers were amplified to the sequenced flow cells.When the sequencing cluster extended the complementary strand, each fluorescently labeled dNTP was released.The sequencer captured the fluorescence signal and the light signal was converted to the sequencing peak through the computer software to obtain the sequence information of the fragments to be measured.

Data Quality Control
The image data of sequencing fragments measured using a high-throughput sequencer were converted into sequence data (reads) by CASAVA bases, which mainly contained the sequence information of the sequencing fragments and the corresponding quality information.The sequencing error rate distribution was checked and Q20, Q30, and GC contents were determined, and a small number of reads with low sequencing quality were filtered out of the raw data to obtain clean reads with high quality for subsequent analysis (Table S2).The clean reads were quickly and accurately aligned to the reference genome using HISAT2-2.1.0software to obtain the mapping information of reads on the reference genome.

Analysis of Total Differentially Expressed Genes (DEGs)
After quantifying gene expression, we performed a statistical analysis of their expression data to screen the samples for genes with significantly different expression levels in different states.The original read count was first standardized (normalized) and mainly corrected for sequencing depth.The statistical model then calculated the hypothesis testing probability (P adj ) and performed multiple-hypothesis test correction to obtain the false discovery rate (FDR, commonly notated as P adj ).Finally, the number of DEGs for each comparative combination was counted and screened to analyze the expression of target genes.

GO and KEGG Enrichment Analysis of DEGs
Gene Ontology (GO) proteome annotation and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database were used to annotate pathways, and they were derived from the online DAVID Bioinformatics tools (https://david.ncifcrf.gov/home.jsp;accessed on 28 March 2023).First, identified gene IDs were converted to Entrez gene IDs and then mapped to GO IDs by gene ID.GO is a comprehensive database describing gene function, divided into three parts: biological process (BP), cellular component (CC), and molecular function (MF).Correction for multiple hypothesis tests was carried out by using standard false discovery rate control methods.In the following discussion, GO functional enrichment was determined with P adj < 0.05 as the threshold for significance.KEGG is a comprehensive database that integrates information on genomic, chemical, and systematic functions.KEGG pathway enrichment analysis of DEGs was performed with P adj < 0.05 as the threshold for significance.

Enzymatic Assays
Fresh root tissue (approximately 1.0 g) was added with 1.6 mL of pre-cooled phosphate buffer (1 mM AsA, 3 mM β-mercaptoethanol, 0.5 mM PMSF, 2% PVP, 1 mM EDTA, pH 7.8).The mixture was ground with liquid nitrogen, the extract was centrifuged at 4 • C at 12,000× g for 20 min, and the supernatant was used for the determination of enzyme activity.Complex V, pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), lactate dehydrogenase (LDH), pyruvate kinase (PK), and pyruvate dehydrogenase complex (PDHC) activity was determined using commercial chemical detection kits (Comin, Suzhou Comin Biotechnology Co., Ltd., Suzhou, China) according to the instructions provided by the manufacturer.Absorbance measurement was performed using a 96-well microplate reader (Rayto RT-6100, Rayto Company, Shenzhen, China), and the corresponding calculation formula was used to calculate enzyme activity.

Measurement of ATP and ADP Content
Fresh roots (approximately 1.0 g) were homogenized using a mortar and pestle with 5 mL of extracting solution (96% ethanol, 0.1 M EDTA, pH 7) at 78 • C.This homogenate was heated in a boiling water bath for 1 min and filled with nitrogen for 10 min, and then the supernatant was diluted with available Tris-EDTA.The ATP and ADP content was measured on ice by chemiluminescent analysis using commercial chemical detection kits (Comin, Suzhou Comin Biotechnology Co., Ltd., Suzhou, China) according to the manufacturer's instructions.

Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)
qRT-PCR was conducted using TaqPro Universal SYBR qPCR Mastermix (Q712-02, Vazyme, Nanjing, China) according to the manufacturer's instructions.Three independent biological repetitions were performed.qRT-PCR was performed under the following conditions: 95 • C for 3 min, followed by 40 cycles of 95 • C for 10 s, 58 • C for 30 s, and 72 • C for 30 s.The glyceraldehyde 3-phosphate dehydrogenase gene was used as the reference gene.The obtained Cq values were used as the original data to calculate the relative expression levels of DEGs via the 2 −∆∆cq method.

Statistical Analysis
All data analyses were conducted with Data Processing System (DPS) statistical software (DPS 10.05, Hangzhou, Zhejiang, China), and the least-significant-different (LSD) test (p ≤ 0.05) was used to compare significant differences between treatments.

Plant Growth
No significant difference in the net increase in shoot FW of the wheat seedlings was observed between CK, AC, and SA treatments at 24, 48, 72, and 96 h (Figure 1a,c).At 24 h, no significant difference in the net increase in root FW was observed between CK, SA, and AC treatments (Figure 1b).However, the net increase in root biomass of the SA-treated wheat seedlings was significantly decreased at 48, 72, and 96 h compared to CK, while the net increase under AC treatment was increased compared to SA (Figure 1b,c).These results indicate that NH 4 + treatment suppresses root growth and the addition of HCO 3 − partially restores root growth.

Plant Growth
No significant difference in the net increase in shoot FW of the wheat seedlings was observed between CK, AC, and SA treatments at 24, 48, 72, and 96 h (Figure 1a,c).At 24 h, no significant difference in the net increase in root FW was observed between CK, SA, and AC treatments (Figure 1b).However, the net increase in root biomass of the SA-treated wheat seedlings was significantly decreased at 48, 72, and 96 h compared to CK, while the net increase under AC treatment was increased compared to SA (Figure 1b,c).These results indicate that NH4 + treatment suppresses root growth and the addition of HCO3 − partially restores root growth

DEGs under Different N Treatments
After filtering out low-quality reads and adapter sequences, a total of 1.38 billion clean reads were obtained with a Q30-based percentage of 91.16% and an average GC content above 55.4%.Combining the GC content and Q30, we believed that the sequencing results were highly accurate and relatively reliable for further experimental analysis.
We performed differential significance analysis of DEGs from the roots of three plant groups (CK, SA, and AC) and conducted paired comparisons between wheat roots subjected to different N treatments.Our screening criteria for significance of DEGs were |log2 (fold change)| ≥ 1 and p ≤ 0.05.In the comparison between SA and CK, a total of 97,506 DEGs were identified, of which 5738 DEGs were upregulated and 6449 DEGs were downregulated.In AC vs. CK, 96,653 DEGs were identified, including 369 upregulated and 81 downregulated DEGs.In AC vs. SA, 97,383 DEGs were identified, with 10,507 upregulated and 9692 downregulated DEGs (Figure 2).These data suggest that the roots strongly respond to NH4 + stress and that the HCO3 − -dependent alleviation of NH4 + stress is extremely complex.

DEGs under Different N Treatments
After filtering out low-quality reads and adapter sequences, a total of 1.38 billion clean reads were obtained with a Q30-based percentage of 91.16% and an average GC content above 55.4%.Combining the GC content and Q30, we believed that the sequencing results were highly accurate and relatively reliable for further experimental analysis.
We performed differential significance analysis of DEGs from the roots of three plant groups (CK, SA, and AC) and conducted paired comparisons between wheat roots subjected to different N treatments.Our screening criteria for significance of DEGs were |log 2 (fold change)| ≥ 1 and p ≤ 0.05.In the comparison between SA and CK, a total of 97,506 DEGs were identified, of which 5738 DEGs were upregulated and 6449 DEGs were downregulated.In AC vs. CK, 96,653 DEGs were identified, including 369 upregulated and 81 downregulated DEGs.In AC vs. SA, 97,383 DEGs were identified, with 10,507 upregulated and 9692 downregulated DEGs (Figure 2).These data suggest that the roots strongly respond to NH 4 + stress and that the HCO 3 − -dependent alleviation of NH 4 + stress is extremely complex.

Expression of Fermentation Genes and Concentration of Fermentation Products
The expression of fermentation transcripts, PDC, ADH, LDH, alanine aminotransferase (AlaAT), and NAD-dependent formate dehydrogenase (FDH) was upregulated under SA treatment at 48 h compared with CK.However, under AC treatment, the expression of these DEGs was significantly downregulated compared with SA treatment (Figure 3).

Expression of Fermentation Genes and Concentration of Fermentation Products
The expression of fermentation transcripts, PDC, ADH, LDH, alanine aminotransferase (AlaAT), and NAD-dependent formate dehydrogenase (FDH) was upregulated under SA treatment at 48 h compared with CK.However, under AC treatment, the expression of these DEGs was significantly downregulated compared with SA treatment (Figure 3).
To further verify whether high NH 4 + causes hypoxic stress in wheat roots, the contents of fermentation products were determined.The results show that NH 4 + alone caused a significant accumulation of ethanol (62.95% increase compared to CK) in the roots (Figure 3).Although the root LA content was decreased in wheat plants under SA treatment, the LA content in the nutrient solution was increased by 23.20%, suggesting that a larger amount of LA was released from the root to apoplast and then to the medium.However, root ethanol accumulation and LA efflux rate were decreased by 39.36% and 10.44%, respectively, after the application of HCO 3 − (Figure 3).These results suggest that NH 4 + alone may induce low O 2 stress in the root cells, leading to alcohol and lactate fermentation; the HCO 3 − -dependent alleviation of NH 4 + stress may be associated with the relief of low O 2 stress and the lower accumulation of fermentation products.To further verify whether high NH4 + causes hypoxic stress in wheat roots, the contents of fermentation products were determined.The results show that NH4 + alone caused a significant accumulation of ethanol (62.95% increase compared to CK) in the roots (Figure 3).Although the root LA content was decreased in wheat plants under SA treatment, the LA content in the nutrient solution was increased by 23.20%, suggesting that a larger amount of LA was released from the root to apoplast and then to the medium.However, root ethanol accumulation and LA efflux rate were decreased by 39.36% and 10.44%, respectively, after the application of HCO3 − (Figure 3).These results suggest that NH4 + alone may induce low O2 stress in the root cells, leading to alcohol and lactate fermentation; the HCO3 − -dependent alleviation of NH4 + stress may be associated with the relief of low O2 stress and the lower accumulation of fermentation products.

Expression of Genes Involved in Hypoxic Stress
To further verify whether SA treatment would induce hypoxic stress in terms of root growth, 11 classes of hypoxia-inducible genes were collected from previous publications (Table 1).Among these genes, hypoxia-inducible factor (HIF), burst oxidase homologs (Rbohs), internal and external alternative NADH dehydrogenase, alternative oxidases (AOXs), nodulin intrinsic proteins (NIPs), aspartate aminotransferase (AspAT), AlaAT, the pivotal enzymes of γ-aminobutyric acid (GABA shunt (glutamate decarboxylase (GAD), GABA transaminase (GABA-T), and succinate-semialdehyde dehydrogenase (SSADH)), ethylene biosynthesis, mitochondrial dicarboxylate carrier, mitochondrial arginine carrier, SLAH3, allene oxide synthase, and nudix hydrolase were significantly upregulated under NH 4 + alone and downregulated after the application of HCO 3 − .On the contrary, prolyl 4-hydroxylases (PHDs), plasma membrane intrinsic proteins (PIPs), non-symbiotic hemoglobins (PGBs), DNA methylation, and chromatin structure regulatory mechanisms were significantly downregulated under NH 4 + treatment and upregulated after the addition of HCO 3 − .The differential expression of these genes under SA is highly consistent with what was previously reported in plants under hypoxic stress (Table 1; for NIPs, see Figure 3), further suggesting that NH 4 + treatment alone may induce hypoxic stress in wheat roots.Table 1.List of genes related to hypoxic stress in wheat roots.

Gene ID Gene Expression
lated after the addition of HCO3 − .The differential expression of these genes under SA highly consistent with what was previously reported in plants under hypoxic stress (T ble 1; for NIPs, see Figure 3), further suggesting that NH4 + treatment alone may indu hypoxic stress in wheat roots.

Gene ID Gene Expression
lated after the addition of HCO3 − .The differential expression of these genes under SA highly consistent with what was previously reported in plants under hypoxic stress (T ble 1; for NIPs, see Figure 3), further suggesting that NH4 + treatment alone may indu hypoxic stress in wheat roots.

Expression of DEGs Involved in O 2 Transport or Consumption Processes
To explore the possible causes of SA-induced hypoxic stress, we analyzed the transcript abundance of genes involved in O 2 transport or consumption processes.The results show that the expressions of 14 genes encoding PIP-type AQPs were significantly downregulated and those of 5 genes encoding Rbohs were significantly upregulated under SA treatment (Table 1), and after the addition of HCO 3 − , the PIP AQPs were significantly upregulated and Rbohs genes were significantly downregulated (Table 1).

Glycolysis, Pyruvate Metabolism, TCA Cycle, Fermentation, Shikimate Pathway, and GABA Shunt
The expression levels of DEGs involved in glycolysis, including phosphofructokinase (PFK), orthologs to phosphoglycerate kinase (PGK), and enolase (ENO), were generally upregulated in roots under SA treatment compared to CK, except for hexokinase (HK) (Figure 5).After the addition of HCO3 − , the expression of DEGs encoding glycolytic enzymes, including PFK, PGK, and ENO, was downregulated, while HK was upregulated (Figure 5).These results indicate that the flux of glycolysis may be increased under NH4 + treatment and downregulated with the addition of HCO3 − .3.6.Glycolysis, Pyruvate Metabolism, TCA Cycle, Fermentation, Shikimate Pathway, and GABA Shunt The expression levels of DEGs involved in glycolysis, including phosphofructokinase (PFK), orthologs to phosphoglycerate kinase (PGK), and enolase (ENO), were generally upregulated in roots under SA treatment compared to CK, except for hexokinase (HK) (Figure 5).After the addition of HCO 3 − , the expression of DEGs encoding glycolytic enzymes, including PFK, PGK, and ENO, was downregulated, while HK was upregulated (Figure 5).These results indicate that the flux of glycolysis may be increased under NH 4 + treatment and downregulated with the addition of HCO 3 − .As shown in Figure 5, SA enhanced the transcript levels of DEGs encoding phosphoenolpyruvate carboxylase (PEPC), phosphoenolpyruvate carboxylase kinase (PEPCK), and NADP-malic enzyme (ME) for anaplerotic routes associated with the TCA cycle, while AC decreased the transcript levels of these genes.In the pyruvate metabolism and TCA cycle pathways, the expression of DEGs encoding PK, PDHC, malate dehydrogenase (MDH), and citrate synthase (CS) was downregulated under NH4 + treatment.After the addition of HCO3 − , the expression of DEGs encoding PK, PDHC, MDH, and CS was upregulated (Figure 5).Briefly, these data indicate that the capacity of the TCA cycle may be suppressed in roots when wheat plants are exposed to NH4 + alone and may be promoted when HCO3 − is added.
The physiological assay showed that the activity of PDC, ADH, and LDH in roots was significantly increased by 36.69%,43.66%, and 61.60%, respectively, under SA treatment and decreased by 19.69%, 13.52%, and 32.57% after the addition of HCO3 − , whereas the activity of PK and pyruvate dehydrogenase (PDH) in roots decreased by 11.29% and 11.15%, respectively, under NH4 + treatment and increased by 4.37% and  As shown in Figure 5, SA enhanced the transcript levels of DEGs encoding phosphoenolpyruvate carboxylase (PEPC), phosphoenolpyruvate carboxylase kinase (PEPCK), and NADP-malic enzyme (ME) for anaplerotic routes associated with the TCA cycle, while AC decreased the transcript levels of these genes.In the pyruvate metabolism and TCA cycle pathways, the expression of DEGs encoding PK, PDHC, malate dehydrogenase (MDH), and citrate synthase (CS) was downregulated under NH 4 + treatment.After the addition of HCO 3 − , the expression of DEGs encoding PK, PDHC, MDH, and CS was upregulated (Figure 5).Briefly, these data indicate that the capacity of the TCA cycle may be suppressed in roots when wheat plants are exposed to NH 4 + alone and may be promoted when HCO 3 − is added.The physiological assay showed that the activity of PDC, ADH, and LDH in roots was significantly increased by 36.69%,43.66%, and 61.60%, respectively, under SA treatment and decreased by 19.69%, 13.52%, and 32.57% after the addition of HCO 3 − , whereas the activity of PK and pyruvate dehydrogenase (PDH) in roots decreased by 11.29% and 11.15%, respectively, under NH 4 + treatment and increased by 4.37% and 93.17% after the addition of HCO 3 − (Table 2), which was highly consistent with the RNA-Seq results (Figure 5).The concentrations of Ala, formate, aromatic amino acids (Trp, Tyr, and Phe), and GABA increased significantly in the roots of wheat plants under SA treatment compared with CK.The addition of HCO 3 − led to the decreased synthesis of these amino acids and formate.The concentrations of TCA cycle intermediates, including Pyr, CA, KGA, succinate, fumarate, malate, and OAA, were significantly decreased in the roots when subjected to SA treatment, while under AC treatment, the concentrations were increased (Table 3).

ATP Synthesis
All DEGs encoding ATP synthases were downregulated under SA treatment and upregulated when HCO 3 − was added (Figure 6a).Accordingly, the activity of complex V significantly decreased in the roots under SA compared with CK and significantly increased under AC compared with SA (Figure 6b).As a consequence, root ATP content decreased and ADP content increased, resulting in a lower ATP/ADP ratio under SA treatment compared with CK (Figure 6c,d).Adding HCO 3 − led to a significant increase in ATP content and decrease in ADP content, resulting in a higher ATP/ADP ratio compared with SA (Figure 6c,d).

DEGs Involved in Cell Division and Elongation
A total of 72 DEGs, including 40 genes encoding mitosis-related proteins, 26 genes associated with cell division, and 6 genes involved in cell elongationin the roots, were downregulated by SA treatmentand upregulated by AC treatment (Table 4).Taken together, these data suggest that NH4 + alone can suppress the rate of cell division in meristems and cell elongation in the elongation zone, thereby decreasing root growth, while adding HCO3 − promotes the cell cycle rate and restores root growth.

DEGs Involved in Cell Division and Elongation
A total of 72 DEGs, including 40 genes encoding mitosis-related proteins, 26 genes associated with cell division, and 6 genes involved in cell elongationin the roots, were downregulated by SA treatmentand upregulated by AC treatment (Table 4).Taken together, these data suggest that NH 4 + alone can suppress the rate of cell division in meristems and cell elongation in the elongation zone, thereby decreasing root growth, while adding HCO 3 − promotes the cell cycle rate and restores root growth.KEGG analysis revealed that in SA vs. CK, the significantly enriched pathways included DNA replication; amino acid biosynthesis; alanine, aspartate, and glutamate metabolism; amino sugar and nucleotide sugar metabolism; nitrogen metabolism; glycolysis/gluconeogenesis; starch and sucrose metabolism; carbon metabolism; and the citrate cycle (TCA cycle) (Figure 8a).In AC vs. SA, the pathways were mainly clustered in DNA replication; amino acid biosynthesis; carbon metabolism; glycolysis/gluconeogenesis; the pentose phosphate pathway; nitrogen metabolism; alanine, aspartate, and glutamate metabolism; amino sugar and nucleotide sugar metabolism; nucleotide metabolism; the citrate cycle (TCA cycle); beta-alanine metabolism; and pyruvate metabolism (Figure 8b).These results indicate that an array of physiological processes in wheat roots are affected by NH4 + stress.KEGG analysis revealed that in SA vs. CK, the significantly enriched pathways included DNA replication; amino acid biosynthesis; alanine, aspartate, and glutamate metabolism; amino sugar and nucleotide sugar metabolism; nitrogen metabolism; glycolysis/gluconeogenesis; starch and sucrose metabolism; carbon metabolism; and the citrate cycle (TCA cycle) (Figure 8a).In AC vs. SA, the pathways were mainly clustered in DNA replication; amino acid biosynthesis; carbon metabolism; glycolysis/gluconeogenesis; the pentose phosphate pathway; nitrogen metabolism; alanine, aspartate, and glutamate metabolism; amino sugar and nucleotide sugar metabolism; nucleotide metabolism; the citrate cycle (TCA cycle); beta-alanine metabolism; and pyruvate metabolism (Figure 8b).These results indicate that an array of physiological processes in wheat roots are affected by NH 4 + stress.

Validation of Hub Genes by qRT-PCR
Sixteen hub genes were selected to further verify the reliability of the RNA-Seq data by determining the RNA expression levels.The qRT-PCR results showed that the 7 hub genes, including ADH, 1-aminocyclopropane-1-carboxylate synthase (ACS), and AOXs, were significantly upregulated, and the 9 hub genes, associated with PIP, PDHC, DNA, and chromatin metabolic processes and mitosis, were significantly downregulated in SA vs. CK, while in AC vs. SA, these 16 DEGs showed the reverse expression trend.The qRT-PCR results were highly consistent with those of RNA-Seq analysis (Figure 9).

Validation of Hub Genes by qRT-PCR
Sixteen hub genes were selected to further verify the reliability of the RNA-Seq data by determining the RNA expression levels.The qRT-PCR results showed that the 7 hub genes, including ADH, 1-aminocyclopropane-1-carboxylate synthase (ACS), and AOXs, were significantly upregulated, and the 9 hub genes, associated with PIP, PDHC, DNA, and chromatin metabolic processes and mitosis, were significantly downregulated in SA vs. CK, while in AC vs. SA, these 16 DEGs showed the reverse expression trend.The qRT-PCR results were highly consistent with those of RNA-Seq analysis (Figure 9).

HCO3 − Alleviates the Inhibition of Root Growth under NH4 + Treatment Alone
Increasing evidence has demonstrated that NH4 + as a dominant N source inhibits root growth in Arabidopsis thaliana, wheat, rice, and other plants [6,7,14,66], suggesting that roots are highly sensitive to NH4 + [25].In the present study, we found that NH4 + treatment alone inhibited root growth and adding HCO3 − attenuated the inhibitory effects of NH4 + (Figure 1a-c)

Fermentation Is Stimulated by NH4 + and Mitigated after Addition of HCO3 −
The underlying mechanisms of NH4 + toxicity remain largely unknown [7,21,26].In the present study, we found an increased transcript abundance of DEGs encoding PDC, ADH, and LDH; an increased activity of PDC, ADH, and LDH; and subsequent ethanol and LA accumulation under NH4 + treatment, while the addition of HCO3 − significantly attenuated these changes (Figures 3 and 9, Table 2).The increased rate of LA efflux under NH4 + treatment may be due to the higher expression of NIPs (Figure 3), as observed in Arabidopsis and other plants under hypoxic conditions [67,68].These results are highly consistent with the findings reported in Glycine max [69].Considering that PDC, ADH, and LDH are reliable markers of fermentative processes launched by hypoxic stress [49, 51,60], it would be reasonable to assume that NH4 + alone can induce hypoxia in the roots and exogenous HCO3 − can attenuate this stress.This assumption corroborates the finding that the plant response to NH4 + may overlap with the response to low O2 stress [32].Increasing evidence has demonstrated that NH 4 + as a dominant N source inhibits root growth in Arabidopsis thaliana, wheat, rice, and other plants [6,7,14,66], suggesting that roots are highly sensitive to NH 4 + [25].In the present study, we found that NH 4 + treatment alone inhibited root growth and adding HCO 3 − attenuated the inhibitory effects of NH 4 + (Figure 1a-c).

−
The underlying mechanisms of NH 4 + toxicity remain largely unknown [7,21,26].In the present study, we found an increased transcript abundance of DEGs encoding PDC, ADH, and LDH; an increased activity of PDC, ADH, and LDH; and subsequent ethanol and LA accumulation under NH 4 + treatment, while the addition of HCO 3 − significantly attenuated these changes (Figures 3 and 9, Table 2).The increased rate of LA efflux under NH 4 + treatment may be due to the higher expression of NIPs (Figure 3), as observed in Arabidopsis and other plants under hypoxic conditions [67,68].These results are highly consistent with the findings reported in Glycine max [69].Considering that PDC, ADH, and LDH are reliable markers of fermentative processes launched by hypoxic stress [49, 51,60], it would be reasonable to assume that NH 4 + alone can induce hypoxia in the roots and exogenous HCO 3 − can attenuate this stress.This assumption corroborates the finding that the plant response to NH + treatment.Rbohs are a family of plasma-membrane-bound enzymes that transfer electrons from cytosolic NADPH/NADH to apoplastic O 2 with the production of reactive oxygen species, and thus play various roles in defense response and morphogenetic processes [12,43].In this study, Rbohs genes were significantly upregulated in roots subjected to NH 4 + and significantly downregulated after the application of HCO 3 − (Table 1).These data suggest that the higher expression of Rbohs genes under NH 4 + treatment dampens O 2 uptake and contributes to cellular O 2 depletion (Figure 10).

Differential Expression of Hypoxia Response Genes Indicates That NH4 + Induces Cellular O2 Deprivation and HCO3 − Alleviates This Stress
Previous studies have reported that numerous hypoxia-inducible factors, such as HIF, Rbohs, internal and external alternative NADH dehydrogenases, AOXs, NIPs, AlaAT, and ethylene biosynthesis, were significantly induced by hypoxic stress, while PIPs, PGB, PHDs, DNA methylation, and chromatin structure regulatory mechanisms were significantly downregulated.In our study, we observed that the transcriptomic response of core hypoxia-inducible genes to NH4 + treatment was highly consistent with what was observed in earlier studies on hypoxia-stressed plants (Table 1, Figure 3), further supporting the notion that NH4 + treatment may induce hypoxic stress in wheat roots.The HCO3 − -dependent alleviation of NH4 + toxicity may be associated with the attenuated hypoxic stress.

O2 Uptake, Transport, and Consumption May Be Associated with Cellular O2 Availability
It is interesting to explore how hypoxic conditions are established in wheat roots under NH4 + treatment.Rbohs are a family of plasma-membrane-bound enzymes that transfer electrons from cytosolic NADPH/NADH to apoplastic O2 with the production of reactive oxygen species, and thus play various roles in defense response and morphogenetic processes [12,43].In this study, Rbohs genes were significantly upregulated in roots subjected to NH4 + and significantly downregulated after the application of HCO3 − (Table 1).These data suggest that the higher expression of Rbohs genes under NH4 + treatment dampens O2 uptake and contributes to cellular O2 depletion (Figure 10).Plant AQPs are localized in cell membranes to transport water molecules, O 2 , and CO 2 , and are involved in the hypoxia response [72][73][74].Five subfamilies of AQPs, including the PIPs and NIPs, have been categorized in higher plants [75].In particular, the overexpression of PIP1;3 has been observed to improve the rate of root O 2 utilization and respiration, and to promote plant growth under hypoxic stress by mediating glycolysis, pyruvate metabolism, and the TCA cycle in the roots of canola (Brassica napus) [76].In the present study, we found that NH 4 + treatment reduced the transcript levels of genes encoding PIP-type AQPs, while the addition of HCO 3 − led to a significantly upregulated expression (Table 1, Figure 9).Based on these results, it is conceivable that the downregulated expression of PIP-type AQPs at least partially contributes to the low O 2 stress in roots under NH 4 + treatment (Figure 10).
Dioxygenases catalyze the incorporation of one or two O 2 atoms into target organic substrates in various metabolic reactions, including DNA replication, RNA modification, and histone demethylation [41,77].Therefore, high dioxygenase activity would induce O 2 overconsumption [41].In this study, we observed that most genes encoding dioxygenases, including ACO1, non-heme dioxygenases, gibberellin 2-beta-dioxygenases, and 9-cis-epoxycarotenoid dioxygenases, were upregulated by NH 4 + treatment (Figure 4).So, we speculate that the higher upregulated expression of dioxygenases would promote the incorporation of molecular O 2 into various substrates and decrease free O 2 availability, thus leading to cellular hypoxia in the roots of plants fed NH 4 + alone (Figure 10).AOX is one of the terminal oxidases of the plant mitochondrial ETC [46,78].AOX has a non-proton motive characteristic and delivers electrons from ubiquinone to O 2 to generate H 2 O by bypassing two sites of H + pumping in complexes III and IV of the cytochrome pathway, which dramatically reduces ATP generation [44,46,78].Our results show that the transcript abundance of four AOX genes was increased in the roots of plants fed only NH 4 + , while HCO 3 − supplementation led to a significantly decreased expression of these genes (Table 1, Figure 9).The upregulation of AOX genes under NH 4 + treatment may greatly increase O 2 consumption without ATP production, which may be associated with hypoxic stress in root cells (Figure 10).
PGBs have an extremely high affinity for O 2 and extremely slow O 2 dissociation properties [79] and can serve as terminal electron acceptors in hypoxic root tissue [80,81].Under hypoxic conditions, the expression of Pgb1.1 and Pgb1.2 was upregulated, thus mitigating the inhibitory effect of O 2 deprivation on root growth in maize [43].Fe is involved in the biosynthesis of heme molecules [79], and its deficiency leads to physiological hypoxia [82].Mugineic acid (MA) is involved in Fe translocation in plant tissues as an Fe chelator, and its biosynthesis requires a precursor, nicotianamine (NA, formed from S-adenosyl methionine via nicotianamine synthase (NAS)) in graminaceous plants [83] and 2 ′ -deoxymugineic-acid 2 ′ -dioxygenases [84].Our results show that all genes encoding PGBs, NAS, 2 ′ -deoxymugineic-acid 2 ′ -dioxygenases, and Fe 2+ transport proteins in the roots were significantly downregulated under NH 4 + treatment and were upregulated after HCO 3 − application (Table 1).Considering that Fe is a cofactor of PGBs, and PGBs function as O 2 carriers and potential terminal electron acceptors in hypoxic root tissue, it is tempting to speculate that intracellular O 2 availability and respiratory use are reduced in the roots of wheat plants fed NH 4 + and increased after the addition of HCO 3 − (Figure 10).It has been well documented that O 2 is used as a terminal electron acceptor in ETC [46].During aerobic respiration, about 36 moles of ATPs are produced [85].However, under hypoxic conditions, ATP synthesis from oxidative phosphorylation is reduced [41]; thus, plant cells will rely on other metabolic pathways, such as glycolysis, leading to reduced ATP generation (2 moles of ATPs per mole of glucose) [52,85].This metabolic switch may be regulated by many factors, such as hypoxia-inducible factor 1 (HIF-1) [85].In the present study, we observed that DEGs encoding PFK, PGK, and ENO were upregulated under NH 4 + treatment and downregulated after HCO 3 − was added (Figure 5).In short, NH 4 + treatment simulates glycolysis, presumably because of the lower O 2 availability in the root cells, while the addition of HCO 3 − may mitigate the hypoxic stress and improve the glycolytic pathway.

Supplementing with HCO 3
− Ameliorates NH 4 + -Repressed TCA Cycle It is well known that the TCA cycle provides essential C skeletons for the assimilation of NH 4 + into amino acids [34], while producing energy [5].Therefore, the functions of the TCA cycle may be important in the alleviation of NH 4 + toxicity.In the present study, we found that the DEGs encoding PEPC and PEPCK were upregulated under NH 4 + treatment and downregulated after HCO 3 − was added (Figure 5).PEPC catalyzes the carboxylation of PEP in the presence of HCO 3 − to form OAA, and PEPCK catalyzes the decarboxylation of OAA to PEP in the gluconeogenesis pathway [86].Therefore, the higher expression of PEPC and PEPCK under NH 4 + conditions could lead to futile cycling between PEP and OAA in the cytoplasm, resulting in a lower accumulation of OAA in the roots (Table 3), which decreases the C anaplerosis in the TCA cycle.
In addition, PEP can also be converted to chorismate through the shikimate pathway, producing aromatic amino acids Trp, Tyr, and Phe [87].In this study, we observed that under NH 4 + conditions, the expression of all AroL, AroC, and PheA genes encoding the shikimate pathway enzymes was significantly upregulated and the concentrations of Trp, Tyr, and Phe were increased, which in turn significantly reduced the concentration of Pyr (glycolytic terminal intermediate) under NH 4 + treatment (Tables 1 and 3).The application of HCO 3 − led to increased Pyr concentration (Table 3).Ala and formate are synthesized via AlaAT and FDH, respectively, using Pyr as a precursor [88,89].AlaAT and FDH expression was induced and Ala and formate accumulated in roots under NH 4 + treatment, and these were repressed by the addition of HCO 3 − (Figure 3), leading to more ions in the NH 4 + -treated plants (Table 3).Furthermore, the expression levels and activity of PK and PDHC decreased with NH 4 + treatment and increased after the addition of HCO 3 − (Figures 5 and 9, Table 2).These changes were greatly attenuated after the addition of HCO 3 − .Based on these results, it can be reasonably hypothesized that NH 4 + nutrition alone suppresses the flux of Pyr in the TCA cycle and adding HCO 3 − mitigates this suppression.The irreversible α-decarboxylation of glutamate catalyzed by GAD in plant tissues synthesizes GABA, a bypassing step in the TCA cycle known as GABA shunt [90].In our study, the expression of GDA, GABA-T, and SSADH genes and the GABA content were significantly upregulated in roots under NH 4 + treatment, while after HCO 3 − addition, the expression was downregulated and GABA content was decreased (Tables 1 and 3).Furthermore, we observed that the expression of most TCA cycle enzymes and the concentrations of key intermediates were reduced under NH 4 + treatment and increased after HCO 3 − was applied (Figure 5, Table 3).Based on these results, we propose that NH 4 + treatment alone reduces the overall capacity of the TCA cycle, likely due to the suppressed flow of C and enhanced GABA shunt.It was encouraging to find that the addition of HCO 3 − mitigated the suppressed TCA cycle activity.This finding is consistent with findings reported in wheat [53] and rice [91] and findings reported by Cramer and Lewis [30] and Bialczyk et al. [34].

ATP Biosynthesis Is Inhibited by NH 4
+ and Promoted after Addition of HCO 3

−
As discussed above, the functions of alternative NAD(P)H:ubiquinone oxidoreductases and AOXs are not coupled to proton translocation, and thus inhibit ATP production [46,92].In the present study, we found that the expression of alternative NAD(P)H:ubiquinone oxidoreductases and AOXs was upregulated in roots under NH 4 + treatment and downregulated after the addition of HCO 3 − (Table 1, Figure 9), while the transcription level of PGBs was reduced under NH 4 + alone and increased after the addition of HCO 3 − (Table 1).In agreement with this, we observed that the downregulated ATP synthase expression, reduced ETC complex V activity, and lower ATP content under NH 4 + treatment were largely mitigated after the addition of HCO 3 − (Figure 6a-c).Considering that hypoxic stress induces the fermentation and glycolysis pathways, suppresses the TCA cycle, and limits energy generation [85,93], we conclude that NH 4 + -induced changes in the roots may be due to low O 2 stress, and that adding HCO 3 − may promote O 2 transport to the mitochondria and oxidative phosphorylation with PGBs as electron acceptors, ultimately improving ATP synthesis in the roots.Cell division and cell elongation are the principal processes that determine root growth, and they occur in spatially distinct developmental zones [94].The precise regulation of chromatin structure in the nucleus is closely related to the transcriptional reprogramming associated with cell proliferation in root apical meristems and growth and development in Arabidopsis and rice [95].In this study, we found that the expression of all genes related to DNA and chromatin metabolic processes, cell division, and cell elongation was downregulated in roots exposed to NH 4 + and upregulated after the addition of HCO 3 − (Tables 1 and 4, Figure 9).Hence, we propose that cell cycle arrest and elongation inhibition may directly account for root growth inhibition under NH 4 + treatment and that applying HCO 3 − attenuates the inhibitory effects of this treatment.
4.9.Ethylene Signaling Is Involved in Regulating NH 4 + Toxicity and its Alleviation by HCO 3

−
Numerous studies have shown that ethylene contributes to the expression of core hypoxia genes and hypoxia acclimation by enhancing the production and stabilization of Group VII Ethylene Response Factors (ERFVIIs) [66,96,97].ERFVIIs facilitate the induction of genes involved in fermentation, glycolysis, energy maintenance, and C metabolism under hypoxic stress in Arabidopsis [98], rice [99], and tobacco [44].In the present study, transcriptome-wide analysis showed that the expression of genes encoding ACSs, ACOs, and two pivotal enzymes in ethylene synthesis was upregulated under NH 4 + treatment but downregulated after HCO 3 − addition (Table 1, Figure 9).It seems reasonable to assume that ethylene synthesis is increased and is involved in NH 4 + toxicity as a signaling agent, and that adding HCO 3 − negatively regulates ethylene signal transduction, thus improving root growth (Figure 10).

Conclusions
Treatment with NH 4 + alone led to significantly restrained wheat root growth and induced the expression of hypoxia-response core genes, indicating that hypoxia may be the primary cause of NH 4 + toxicity in root cells.Hypoxic stress appears to be associated with the upregulation of Rbohs, dioxygenases, and AOXs and the downregulation of AQPs.As a consequence, the capacity of the TCA cycle is reduced and the production of ATP is inhibited, eventually dampening the root cell cycle, elongation, and growth.Compared with NH 4 + nutrition alone, the addition of HCO 3 − significantly improved hypoxia-related metabolic processes, boosted ATP generation, promoted root cell division and elongation, and ultimately enhanced root growth in wheat seedlings.Ethylene signaling may be involved in NH 4 + toxicity and HCO 3 − -dependent detoxification.As the first report on hypoxic stress triggered by NH 4 + treatment, this study provides novel insights into the mechanisms of NH 4 + toxicity and its alleviation, further providing a valuable molecular basis for studying how to improve NUE.In particular, based on the data reported here, we can strongly recommend splitting the proportion of N fertilizer for wheat production in the field.Alternatively, C-containing fertilizers such as ammonium bicarbonate and urea should be used as the preferred N source.Retaining the C-rich residue of previous crops is also a practical strategy when using NH 4 + fertilizers as the dominant N source.These practices could minimize NH 4 + toxicity and increase nutrient use efficiency and wheat grain yield.

Supplementary Materials:
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/biology13020101/s1,Table S1: Fresh weight of wheat seedlings grown in different strengths of Hoagland nutrient solution for 48 h; Table S2: Quality assessment of RNA-Seq sequencing results.

Figure 6 .
Figure 6.Effects of N treatments on ATP synthesis in roots of wheat seedlings: (a) expression o DEGs encoding ATP synthases; (b) activity of root complex V; (c) ATP and ADP content; (d) ratio of ATP to ADP.Values represent mean ± SD from three independent biological replicates.Differen lowercase letters above columns indicate significant differences at p < 0.05.Wheat seedlings were treated with 7.5 mM NO3 − (CK), 7.5 mM NH4 + (SA), or 7.5 mM NH4 + + 3 mM HCO3 − (AC).

Figure 6 .
Figure 6.Effects of N treatments on ATP synthesis in roots of wheat seedlings: (a) expression of DEGs encoding ATP synthases; (b) activity of root complex V; (c) ATP and ADP content; (d) ratio of ATP to ADP.Values represent mean ± SD from three independent biological replicates.Different lowercase letters above columns indicate significant differences at p < 0.05.Wheat seedlings were treated with 7.5 mM NO 3 − (CK), 7.5 mM NH 4 + (SA), or 7.5 mM NH 4 + + 3 mM HCO 3 − (AC).

Figure 7 .
Figure 7. GO analysis of DEGs under different N treatments: (a) SA vs. CK; (b) AC vs. SA.X-axis indicates −Log10 (p-value), and Y-axis is enriched GO terms.

Figure 7 .
Figure 7. GO analysis of DEGs under different N treatments: (a) SA vs. CK; (b) AC vs. SA.X-axis indicates −Log 10 (p-value), and Y-axis is enriched GO terms.

Figure 8 .
Figure 8. KEGG analysis of DEGs under different N treatments: (a) SA vs. CK; (b) AC vs. SA.X-axis is enrichment score, Y-axis is KEGG pathway; size of dots represents number of genes annotated to KEGG pathway; and red to green in color bar indicate significance (high to low, respectively) of enrichment.

Figure 8 .
Figure 8. KEGG analysis of DEGs under different N treatments: (a) SA vs. CK; (b) AC vs. SA.X-axis is enrichment score, Y-axis is KEGG pathway; size of dots represents number of genes annotated to KEGG pathway; and red to green in color bar indicate significance (high to low, respectively) of enrichment.

3 −
Alleviates the Inhibition of Root Growth under NH 4 + Treatment Alone

Figure 10 .
Figure10.Schematic model of NH4 + toxicity and HCO3 − -dependent alleviation in roots of wheat seedlings.Under NH4 + treatment, significantly upregulated Rbohs consume large amounts of apoplastic O2,downregulated PIP-type AQPs decrease O2 uptake and transport from the ambient environment to the cell, and upregulated dioxygenases and AOXs increase intracellular O2 consumption, thus reducing cellular O2 availability.O2 deprivation then suppresses oxidative phosphorylation and ATP production, in turn activating the ethylene-enhanced ERFVII pool and stim-

Figure 10 .
Figure 10.Schematic model of NH 4 + toxicity and HCO 3 − -dependent alleviation in roots of wheat seedlings.Under NH 4 + treatment, significantly upregulated Rbohs consume large amounts of apoplastic O 2 , downregulated PIP-type AQPs decrease O 2 uptake and transport from the ambient environment to the cell, and upregulated dioxygenases and AOXs increase intracellular O 2 consumption, thus reducing cellular O 2 availability.O 2 deprivation then suppresses oxidative phosphorylation and ATP production, in turn activating the ethylene-enhanced ERFVII pool and stimulating hypoxic metabolism, such as alcoholic and lactic fermentation, and regulates the expression of hypoxia-like responsive genes encoding PDC, ADH, LDH, and PGBs [70].O 2 -binding PGBs may function as terminal electron acceptor electron transport chains (ETCs), thus downregulated expression of PGBs under NH 4 + may reduce electron transport inETC and then ATP generation.Higher ethylene production further consumes more molecular O 2 [71].Conversely, adding HCO 3 − greatly ameliorates the negative

Author
Contributions: X.L. and L.K. conceived and designed the study and wrote the manuscript.Y.Z. and C.T. participated in the experiments.X.L. and H.L. analyzed the transcriptome data and constructed the figures.C.T. performed the physiological determination.H.X. and S.F. modified the manuscript.All authors have read and agreed to the published version of the manuscript.

Table 1 .
List of genes related to hypoxic stress in wheat roots.

Table 1 .
List of genes related to hypoxic stress in wheat roots.
− (AC).Numbers in color scale bar indicate log 2 (FC) in gene expression.

Table 2 .
Fermentation and Pyr metabolism enzyme activity.

Table 3 .
Effects of N treatments on concentrations of metabolites from amino acid metabolism, Pyr metabolism, TCA cycle, and GABA shunt in roots.

Table 4 .
Differentially expressed genes related to cell division and cell elongation in wheat roots under different N treatments.
[32]ay overlap with the response to low O 2 stress[32].Previous studies have reported that numerous hypoxia-inducible factors, such as HIF, Rbohs, internal and external alternative NADH dehydrogenases, AOXs, NIPs, AlaAT, and biosynthesis, were significantly induced by hypoxic stress, while PIPs, PGB, PHDs, DNA methylation, and chromatin structure regulatory mechanisms were significantly downregulated.In our study, we observed that the transcriptomic response of core hypoxiainducible genes to NH 4 + treatment was highly consistent with what was observed in earlier studies on hypoxia-stressed plants (Table1, Figure3), further supporting the notion Uptake, Transport, and Consumption May Be Associated with Cellular O 2 Availability It is interesting to explore how hypoxic conditions are established in wheat roots under NH 4 ethylene 4 + alone on these processes.Two juxtaposed arrows indicate differential expression of genes in SA vs. CK and AC vs. SA.The question mark in the figure indicates that this process remains to be elucidated.Red and blue arrows indicate increased and decreased transcript abundance, respectively.ACO, 1-aminocyclopropane-1-carboxylate oxidase; ACS, 1-aminocyclopropane-1-carboxylate synthase; ADH, alcohol dehydrogenase; AOX, alternative oxidase; AQP, aquaporins; ERFVII, Group VII Ethylene Response Factor; LDH, lactate dehydrogenase; PDC, pyruvate decarboxylase; PGB, nonsymbiotic hemoglobin; Rbohs, respiratory burst oxidase homolog.