# Excessive assimilation of ammonium by plastidic glutamine synthetase is a major cause of ammonium toxicity in Arabidopsis thaliana

Takushi Hachiya, Jun Inaba, Mayumi Wakazaki, Mayuko Sato, Kiminori

Toyooka, Atsuko Miyagi, Maki Kawai-Yamada, Takatoshi Kiba, Alain Gojon,

Hitoshi Sakakibara

► To cite this version:

Takushi Hachiya, Jun Inaba, Mayumi Wakazaki, Mayuko Sato, Kiminori Toyooka, et al.. Excessive assimilation of ammonium by plastidic glutamine synthetase is a major cause of ammonium toxicity in Arabidopsis thaliana. 2019. hal-02788815

HAL Id: hal-02788815

https://hal.inrae.fr/hal-02788815

Preprint submitted on 5 Jun 2020

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
1 **Title:** Excessive assimilation of ammonium by plastidic glutamine synthetase is a major

2 cause of ammonium toxicity in *Arabidopsis thaliana*

3 **Authors and affiliations:** Takushi Hachiya<sup>a,b,c</sup>, Jun Inaba<sup>d</sup>, Mayumi Wakazaki<sup>d</sup>, Mayuko

4 Sato<sup>d</sup>, Kiminori Toyooka<sup>d</sup>, Atsuko Miyagi<sup>e</sup>, Maki Kawai-Yamada<sup>e</sup>, Takatoshi Kiba<sup>b,d</sup>,

5 Alain Gojon<sup>f</sup>, and Hitoshi Sakakibara<sup>b,d</sup>

6 <sup>a</sup>Department of Molecular and Functional Genomics, Interdisciplinary Center for Science

7 Research, Shimane University, Matsue 690-8504, Japan, <sup>b</sup>Department of Biological

8 Mechanisms and Functions, Graduate School of Bioagricultural Sciences, Nagoya

9 University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan, <sup>c</sup>Institute for

10 Advanced Research, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8602,

11 Japan, <sup>d</sup>RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro-cho, Tsurumi-

12 ku, Yokohama, Kanagawa 230-0045, Japan, <sup>e</sup>Graduate School of Science and

13 Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama-city, Saitama

14 338-8570, Japan, <sup>f</sup>Biochimie et Physiologie Moléculaire des Plantes,

15 CNRS/INRA/SupAgro-M/Montpellier University, Montpellier 34060, France.

16 **Corresponding Author:** Takushi Hachiya (http://orcid.org/0000-0002-9239-6529)

17 Department of Molecular and Functional Genomics, Center for Interdisciplinary Center

18 for Science Research, Shimane University, Matsue 690-8504, Japan

19 E-mail; takushi.hachiya@life.shimane-u.ac.jp, Tel; +81-852-32-6288

20 **Keywords:** Acidic stress, Ammonium toxicity, Glutamine synthetase

21

22
23 **Abstract**

Plants use nitrate and ammonium in the soil as their main nitrogen sources. Recently, ammonium has attracted attention due to evidence suggesting that, in C₃ species, an elevated CO₂ environment inhibits nitrate assimilation. However, high concentrations of ammonium as the sole nitrogen source for plants causes impaired growth, i.e. ammonium toxicity. Although ammonium toxicity has been studied for a long time, the primary cause remains to be elucidated. Here, we show that ammonium assimilation in plastids rather than ammonium accumulation is a primary cause for toxicity. Our genetic screen of ammonium-tolerant Arabidopsis lines with enhanced shoot growth identified plastidic GLUTAMINE SYNTHETASE 2 (GLN2) as the causal gene. Our reciprocal grafting of wild-type and GLN2 or GLN1;2-deficient lines suggested that shoot GLN2 activity results in ammonium toxicity, whilst root GLN1;2 activity prevents it. With exposure to toxic levels of ammonium, the shoot GLN2 reaction produced an abundance of protons within cells, thereby elevating shoot acidity and stimulating expression of acidic stress- responsive genes. Application of an alkaline ammonia solution to the toxic ammonium medium efficiently alleviated the ammonium toxicity with a concomitant reduction in shoot acidity. Consequently, we conclude that a primary cause of ammonium toxicity is acidic stress in the shoot. This fundamental insight provides a framework for enhanced understanding of ammonium toxicity in plants.

42

43

44
## 45 Introduction

Nitrate and ammonium are the main sources of nitrogen (N) for most plants.

Recent studies suggest that elevated CO₂ inhibits nitrate reduction in C₃ species, such as wheat and *Arabidopsis*, whereas ammonium utilization does not decrease (1). One estimate predicts that only a 1% drop in nitrogen use efficiency could increase worldwide cultivation costs for crops by about $1 billion annually (2). Therefore, increasing ammonium use by crops is an important goal for agriculture as CO₂ levels rise in the world; however, millimolar concentrations of ammonium as the sole N source causes growth suppression and chlorosis in plants, compared with nitrate (3, 4, 5). This phenomenon is widely known as ammonium toxicity, but the primary cause of impaired growth remains to be identified.

Plants grown in high ammonium conditions show several distinct characteristics from those grown in nitrate (3, 4, 5). These toxic symptoms have evoked several hypotheses about the toxic causes, including futile transmembrane ammonium cycling, deficiencies in inorganic cations and organic acids, impaired hormonal homeostasis, disordered pH regulation, and the uncoupling of photophosphorylation; however, some of the symptoms are not directly associated with growth suppression by ammonium toxicity (6), making it difficult to determine the toxic cause. Several efforts have isolated ammonium-sensitive mutants in *Arabidopsis thaliana* and determined their causative genes (4, 5). *GMP1* is a causal gene whose deficiency causes stunted growth of primary roots under high ammonium conditions (7). Given that *GMP1* is crucial for synthesizing GDP-mannose as a substrate for N-glycosylation, lack of N-glycoproteins
67 could be involved in ammonium hypersensitivity. In accordance with this hypothesis, the
68 ammonium-dependent inhibition of primary root growth was shown to be partly
69 attenuated by the lack of a GDP-mannose pyrophosphohydrolase that hydrolyses GDP-
70 mannose to mannose 1-phosphate and GMP (8). In another study, a genetic screen
71 focusing on severely chlorotic Arabidopsis leaves identified AMOS1, a gene encoding a
72 plastid metalloprotease, as a factor for improving ammonium tolerance (9).
73 Transcriptome analysis revealed that an AMOS1-dependent mechanism regulates more
74 than half of the transcriptional changes triggered by toxic levels of ammonium. On the
75 other hand, recent studies found that ammonium toxicity was partly alleviated by
76 deficiencies in EIN2 and EIN3, regulators of ethylene responses, or by the application of
77 ethylene biosynthesis and action inhibitors (10, 11). This suggests that ammonium
78 toxicity would be mediated via the ethylene signaling pathway.

79 The above-described genetic studies have succeeded in determining molecular
80 components closely associated with ammonium toxicity. Nevertheless, the initial event
81 that triggers ammonium toxicity remains to be identified and characterized. To address
82 this question, we screened ammonium-insensitive Arabidopsis lines that were expected
83 to attenuate toxicity and isolated ami2. Interestingly, the defect in ami2 was
84 downregulation of the GLUTAMINE SYNTHETASE 2 (GLN2) gene encoding an
85 ammonium assimilatory enzyme. We identified that in the presence of toxic levels of
86 ammonium, large levels of proton production, due to excessive primary assimilation of
87 ammonium by GLN2, aggravate the acidic burden and lead to plant toxicity.
88

88
## 89 Results

### 90 A Genetic Screen Isolated an Ammonium-Insensitive Mutant.
To find ammonium-insensitive lines, a gain-of-function population of the Arabidopsis FOX (full-length cDNA overexpressing) lines (12) was used. An apparent ammonium-insensitive mutant was identified that shows enhanced growth of cotyledons that are greener than wild-type (Col) when grown on 10 mM ammonium as the sole N source; the mutant was named ammonium-insensitive 2 (*ami2*) (Fig. 1A). The fresh weights of *ami2* 11-d-old shoots were approximately double those of Col when grown on ammonium (Fig. 1B). In contrast, in media containing 10 mM nitrate or 5 mM ammonium plus 5 mM nitrate, the shoot fresh weights of *ami2* were less than those of Col. In media containing 10 mM ammonium, the percentage increase in fresh weight of *ami2* relative to Col was much larger for shoots (by ca. 110%) than for roots (by ca. 50%) (Fig. 1C). The greater shoot growth in *ami2* was reduced in media with lower concentrations of ammonium (0.4, 2 mM), in which the shoot growth of Col was greater than that when grown on media containing 10 mM ammonium (Fig. 1D). Moreover, nitrate addition in the presence of 10 mM ammonium attenuated the deficiency in shoot growth more effectively in Col than in *ami2*, decreasing the growth difference in a concentration-dependent manner (*SI Appendix*, Fig. S1 A and B). A time-course analysis of shoot growth revealed that increased ammonium tolerance of the *ami2* plants compared to Col was significant as soon as 5 d after culture initiation (*SI Appendix*, Fig. S1C). These results indicate that ammonium tolerance in *ami2* is manifested specifically under harsh ammonium conditions.
To corroborate this enhanced ammonium tolerance in *ami2*, we performed microarray experiments and compared the expression of genes responsive to toxic levels of ammonium (9) between the Col and *ami2* shoots growing in media containing 10 mM ammonium (Fig. 1E and *Datasets*, Table S1). The transcript levels of ammonium-inducible genes were significantly reduced in *ami2* shoots compared with Col shoots, whereas those of ammonium-repressive genes showed the opposite trend. A reverse transcription-quantitative PCR (RT-qPCR) analysis confirmed that expression of *MIOX2* and *PDH2*, two representative ammonium-inducible genes, was more upregulated in the presence of ammonium than in nitrate-containing media in Col, but not in *ami2* (*SI Appendix*, Fig. S2A). The expression of a house-keeping gene *TIP41* was less changed (*SI Appendix*, Fig. S2B). Collectively, these results indicate that ammonium toxicity is attenuated in *ami2* shoots.

**GLUTAMINE SYNTHETASE 2 is a Major Causative Gene for Ammonium Toxicity.**

Next, to identify the causative gene in *ami2*, we recovered the transgene in a vector using specific primers and sequenced the construct. The gene was identified as *GLUTAMINE SYNTHETASE 2 (GLN2)*, the sole plastidic isoform in *A. thaliana* (Fig. 2A and *SI Appendix*, Fig. S2C). Because the transgene was driven by the cauliflower mosaic virus *35S* promoter, we expected that overexpression of *GLN2* would enhance ammonium tolerance; however, in media containing 10 mM ammonium, the transcript levels of *GLN2* in *ami2* shoots were downregulated to about 5% of those in Col (Fig. 2B). In contrast, among the major cytosolic *GLUTAMINE SYNTHETASE* genes (*GLN1s*), *GLN1*;1 was
132 upregulated in *ami2* shoots, but *GLN1;2* and *GLN1;3* were slightly downregulated (SI Appendix, Fig. S2D). Also, an immunoblot analysis using anti-GLN antibodies (13) 133 confirmed that the protein levels of GLN2 were remarkably lower in *ami2* shoots 134 compared with Col, whereas the signal intensities corresponding to GLN1s were 135 comparable between the mutant and wild type (Fig. 2C). These findings suggested that 136 overexpression of *GLN2* cDNA would result in a co-suppression event (14), which would 137 make it difficult to test for phenotypic complementation by introducing the *GLN2* 138 transgene. To ensure that reduced expression of *GLN2* enhances ammonium tolerance, 139 we obtained another *GLN2*-deficient line having a T-DNA insertion at the 3'-UTR region 140 of *GLN2* (SALK_051953, designated as *gln2*, SI Appendix, Fig. S2C). As expected, *gln2* 141 phenocopied *ami2* in terms of the reduced *GLN2* and GLN2 expression (Fig. 2 B and C), 142 the enhanced ammonium tolerance (Fig. 2 D and E), and the lowered induction of 143 ammonium-inducible genes when grown on ammonium (SI Appendix, Fig. S2A). Thus, 144 we concluded that *GLN2* is a causative gene for ammonium toxicity. 145

146

147 **Shoot GLN2 Causes Ammonium Toxicity; Root GLN1;2 Attenuates Ammonium Toxicity.** Previous studies had reported that mutants deficient in *AtGLN1;2* were 148 hypersensitive to millimolar concentrations of ammonium (15-17). We also confirmed 149 the ammonium hypersensitivity of *gln1;2-1* and *gln1;2-2* (SI Appendix, Fig. S3 A and B). 150 *GLN2* and *GLN1;2*, therefore, have opposite effects on ammonium toxicity. To discover 151 how GLN2 and GLN1;2 are involved in the toxicity, we evaluated the distribution of 152 *GLN2* and *GLN1;2* expression between shoots and roots in Col plants. In the presence of 153
154 ammonium or nitrate, the steady-state levels of *GLN2* expression were consistently higher
155 in the shoots than in the roots, whereas expression of *GLN1*;2 was much higher in the
156 roots (Fig. 3 *A* and *B* and *SI Appendix*, Fig. S4 *A* and *B*), implying that both shoot GLN2
157 and root GLN1;2 could affect ammonium toxicity. To support this hypothesis, we
158 performed a growth analysis using reciprocally-grafted plants between Col and *ami2* (Fig.
159 3C and *SI Appendix*, Fig. S4C) and between Col and *gln1*;2-1 (Fig. 3D and *SI Appendix*,
160 Fig. S4D). Prior to the analysis, we confirmed that shoot expression of *GLN2* was lower
161 in the *ami2*-derived shoots irrespective of root-genotype (*SI Appendix*, Fig. S5), because
162 *GLN2* mRNA is suggested to be root-to-shoot mobile (18). Only when the scion was
163 derived from *ami2* was shoot growth significantly enhanced in the presence of 10 mM
164 ammonium (Fig. 3C). On the other hand, deficiency in root *GLN1*;2 content was
165 sufficient to decrease shoot growth in ammonium (Fig. 3D). Further, we observed that in
166 ammonium-grown plants, the total enzymatic activities of GLNs were significantly
167 reduced by ca. 30-40% in 5-d-old shoots of *ami2* and *gln2* and by ca. 40-60% in 5-d-old
168 roots of *gln1*;2-1 and *gln1*;2-2 compared with Col (*SI Appendix*, Fig. S6 *A* and *B*).
169 Additionally, partially compensatory inductions of other GLNs were found in the mutants
170 (*SI Appendix*, Fig. S6 *C* and *D*). Our findings demonstrate that although shoot GLN2
171 causes ammonium toxicity in the shoot, root GLN1;2 attenuates ammonium toxicity.

172

173 Decreased GLN2 Activity Reduces the Conversion of Ammonium to Amino Acids
174 in Shoots. It is generally held that ammonium *per se* is a toxic compound (19). On the
175 other hand, a deficiency in *GLN2* content should lead to ammonium accumulation in the
176 shoot. Our determination of shoot ammonium content revealed that *ami2* and *gln2* shoots grown on 10 mM ammonium both accumulated more than 100 µmol g⁻¹ fresh weight of ammonium (Fig. 4A), albeit the two mutants accumulated more fresh weight than Col (SI Appendix, Fig. S7). This result indicates that ammonium assimilation by GLN2, rather than ammonium accumulation, triggers ammonium toxicity in the shoot.

181 An ample supply of ammonium increases the concentrations of amino acids compared with nitrate supply alone (6, 20). In particular, the molar ratios of Gln to Glu are elevated at higher ammonium levels, suggesting that Gln synthesis by glutamine synthetase (GLN) overflows glutamate synthase (GOGAT) capacity. Our hierarchical cluster analysis of amino acid content in shoots clearly demonstrated that the type of N source, i.e. 10 mM ammonium or nitrate, was the strongest determinant for plant amino acid composition (Fig. 4B and SI Appendix, S8A). In this analysis, Col and the GLN2-deficient lines categorized into separate clusters depending on the N source. The molar ratio of Gln to Glu (Fig. 4C), total amino acid-N content per amino acid (SI Appendix, Fig. S8B), total amino acid-N content per fresh weight (SI Appendix, Fig. S8C), and the molar ratios of N to C in total amino acids (SI Appendix, Fig. S8D) were consistently larger in ammonium-grown shoots than nitrate-grown shoots, and this large ammonium-N input was partly but significantly attenuated by GLN2 deficiency. These findings suggest that the GLN2 reaction leads to excessive incorporation of ammonium-N into amino acids in shoots when toxic levels of ammonium are present.
**Ammonium Assimilation by GLN2 Causes Acidic Stress.** The amino acid profiles suggested that metabolic imbalances due to excessive ammonium assimilation by GLN2 could be a cause of ammonium toxicity. We have previously demonstrated that nitrate addition at adequate concentrations mitigates ammonium toxicity without reducing amino acid accumulation (6). Therefore, a phenomenon triggered by some GLN2-mediated process other than amino acid accumulation should be a cause of ammonium toxicity.

Notably, the GLN reaction is a proton-producing process (21). The stoichiometry of this reaction is two protons per each glutamine produced, one proton of which is derived from ATP hydrolysis and the other is from deprotonation of $NH_4^+$. Conversely, the subsequent ferredoxin-dependent glutamate synthase (Fd-GOGAT) reaction consumes two protons per one glutamine incorporated. Given that the molar ratio of Gln to Glu was about 10 in the ammonium condition but close to 1 in the nitrate condition (Fig. 4C), proton production in the ammonium condition could proceed beyond its consumption. Strikingly, a previous study found that 43% of ammonium-inducible genes correspond to acidic stress-inducible genes in Arabidopsis roots (22, 23). Thus, we hypothesized that excessive ammonium assimilation by GLN2 causes acidic stress to the plants growing in ammonium.

We re-surveyed our microarray data by focusing on previously identified acidic stress-responsive genes (23) (Fig. 5A and *Datasets*, Table S2). All acidic stress-inducible genes were entirely downregulated in *ami2* shoots compared with Col, whereas the acidic stress-repressive genes showed the opposite trend. The transcript levels of *ALMT1*, a typical acidic stress-inducible gene, were determined in the shoots of Col and *ami2* plants
219 incubated in 10 mM ammonium or nitrate with or without methionine sulfoximine (MSX),
220 an inhibitor of the GLN reaction (Fig. 5B). ALMT1 expression was much higher in the
221 ammonium-treated Col shoots than the nitrate-treated samples. This ammonium-
222 dependent induction was significantly diminished in the ami2 shoots and was mimicked
223 by MSX treatment. Also, other proton-inducible genes such as GABA-T, GAD1, GDH2,
224 PGIP1, and PGIP2 (24) were ammonium-inducible, and their inductions were suppressed
225 or attenuated by GLN2 deficiency (SI Appendix, Fig. S9A). These results support our
226 hypothesis associating ammonium assimilation with acidic stress. Moreover, in Col and
227 ami2 reciprocally-grafted plants growing in the ammonium condition, ALMT1 expression
228 was significantly lower in the ami2-derived shoots than the Col-derived shoots (SI
229 Appendix, Fig. S9B), indicating that shoot GLN2 locally causes acidic stress to the shoot.
230 Furthermore, ALMT1 expression was analyzed using grafted plants between Col and a
231 mutant lacking the STOP1 transcription factor (stop1-KO) that induces ALMT1 to
232 respond to acidic stress (24) (SI Appendix, Fig. S9C). In the stop1-KO-derived shoots, the
233 ammonium-dependent induction of ALMT1 disappeared, reconfirming the notion that
234 acidic stress occurs in plants growing in ammonium.

235 It is widely accepted that the reduction from nitrate to ammonium consumes a
236 proton, suggesting that nitrate reduction could attenuate acidic stress caused by excess
237 ammonium and might explain why nitrate addition alleviates ammonium toxicity. To
238 verify this hypothesis, we analyzed shoot expression of ALMT1 using grafted plants
239 between Col and the NITRATE REDUCTASE-null mutant (designated as NR-null) (25)
240 (SI Appendix, Fig. S9D). Addition of 2.5 mM nitrate diminished the ammonium-
241 dependent *ALMT1* induction in the Col-derived shoots but not in the NR-null-derived
242 shoots, thereby supporting the above hypothesis.

243 To obtain direct evidence for ammonium-dependent proton production, we
244 measured the proton concentrations of water extracts from the Col and *ami2* shoots
245 incubated in media containing 10 mM ammonium or nitrate with or without MSX (Fig.
246 5C). The ammonium-treated Col shoots contained the highest concentrations of protons;
247 proton content was significantly decreased by *GLN2* deficiency and by MSX treatment
248 to levels comparable to those in nitrate-treated shoots. A similar trend was observed
249 among the Col, *ami2*, and *gln2* shoots grown on ammonium- or nitrate-containing media
250 (*SI Appendix*, Fig. S9E).

251 The presence of ammonium in cultures generally acidifies the external media
252 (22). Thus, we quantified the proton efflux from the Col and *ami2* shoots incubated in
253 media containing 10 mM ammonium or nitrate with or without MSX (Fig. 5D).
254 Incubation of the Col shoots in the presence of ammonium strongly acidified the external
255 media, which was alleviated by *GLN2* deficiency and by MSX treatment. A similar
256 tendency was observed by qualitative measurements with a pH indicator of proton
257 effluxes from mesophyll cells where GLN2 is predominantly expressed (*SI Appendix*, Fig.
258 S9F). Thus, we conclude that ammonium assimilation by GLN2 without nitrate increases
259 shoot acidity.

260

261 **Ammonium Toxicity is Closely Associated with Acidic Stress.** If acidic stress rather
262 than ammonium accumulation has a dominant effect on ammonium toxicity, an
263 application of alkaline ammonia should reduce the toxicity. Given that the GLN2 reaction
264 is a primary cause of increased acidic stress, an elevation in medium pH may increase the
265 shoot growth of Col more effectively than that of the GLN2-deficient mutants. As
266 expected, addition of a 25% ammonia solution to media containing 10 mM ammonium
267 elevated the pH from 5.7 to 6.7 and significantly improved shoot growth with a
268 concomitant decrease in acidity (Fig. 6A). Fresh weights of Col shoots grown at pH 6.7
269 increased by ca. 180% compared with those grown at pH 5.7, whereas fresh weights of
270 *ami2* and *gln2* shoots only increased by ca. 30% and 60%, respectively (Fig. 6B). In
271 addition, the acid-sensitive STOP1-deficient mutants had slightly but significantly lower
272 shoot growth when grown in 10 mM ammonium (Fig. 6C and SI Appendix, Fig. S9G),
273 although their acid-hypersensitivity has been described only in roots to date (24, 26).
274 Moreover, the NR-null-derived shoots that lack a proton-consuming nitrate reduction
275 capacity failed to attenuate ammonium toxicity by nitrate addition (Fig. 6D). Collectively,
276 our results lead to the conclusion that acidic stress is one of the primary causes of
277 ammonium toxicity.

278

279 **GLN2 Causes Ammonium Toxicity Independently of NRT1.1.** We have already
280 reported that Arabidopsis NITRATE TRANSPORTER 1.1 (NRT1.1), acting as a nitrate
281 transceptor, also aggravates ammonium toxicity (27). This result was recently confirmed
282 by another group (11). Thus, we investigated whether NRT1.1 and GLN2 increase the
283 sensitivity to ammonium through a common mechanism. A RT-qPCR analysis revealed
284 that deficiency of either *NRT1.1* or *GLN2* did not downregulate the expression of the
other gene (*SI Appendix*, Fig. S10A), and that *GLN2* expression was almost 3-times higher in *nrt1.1* than in Col. Therefore, the enhanced ammonium tolerance of *nrt1.1* cannot be explained by reduced *GLN2* expression as in *gln2*. Moreover, a homozygous double mutant of *NRT1.1* and *GLN2* showed slightly but significantly larger shoot fresh weight, leaf number, shoot diameter, and chlorophyll content compared with any of the single mutants (*SI Appendix*, Fig. S10 *B-D*). These findings suggest that *NRT1.1* and *GLN2* are implicated in ammonium-sensitivity independently.

292

293

294

295

296

297

298

299

300

301

302

303

304

305

306
307 **Discussion**

308 Although ammonium is a toxic compound for plant growth, our results demonstrate that
309 ammonium assimilation by shoot GLN2 rather than ammonium accumulation is a major
310 cause of ammonium toxicity (Fig. 2 and Fig. 4). In plants growing in toxic levels of
311 ammonium as a sole N source, assimilation of ammonium by GLN2 would occur largely
312 due to bypassing nitrate reduction as the rate-limiting step for N assimilation. The
313 resultant increase in the ratio of Gln to Glu content (Fig. 4C) corresponds to the
314 preferential enhancement of the proton-producing GLN reaction over the proton-
315 consuming GOGAT reaction. This metabolic imbalance exerted by the GLN2 reaction
316 leads to the production of large amounts of protons in shoot cells that stimulate proton
317 effluxes to the apoplasm; however, the volume of the shoot apoplasm is probably too
318 small to accommodate such a large proton efflux. Thus, in the presence of toxic levels of
319 ammonium, the GLN2 reaction causes acidic stress inside and outside the cells and
320 triggers acidic stress responses that modulate gene expression (Fig. 5 and SI Appendix,
321 Fig. S9 A-F). Given that the wild-type when grown at a higher pH phenocopies the
322 ammonium-insensitive lines at lower pH, the acidic stress-sensitive mutants show
323 ammonium-hypersensitivity, and proton-consuming nitrate reduction alleviates
324 ammonium toxicity (Fig. 6 and SI Appendix, Fig. S9G), we conclude that acidic stress is
325 one of the primary causes for ammonium toxicity. In this framework, upregulation of
326 Arabidopsis NIA1 and NIA2 genes encoding nitrate reductase (NR) (23, 24) and activation
327 of spinach NR (28) responding to acidic stress are understandable regulatory responses
328 in the context of maintaining cellular pH homeostasis.
The present study does not address how ammonium-dependent acidification
triggers growth deficiency at the cellular and subcellular scales. The chloroplastic
localization of GLN2 indicates that proton production must occur within chloroplasts in
the elevated ammonium condition. A previous study reported abnormal chloroplast
membrane structure including swollen compartments at late stages of ammonium toxicity
(29); however, we did not find any similar structural changes in the shoots of ammonium-
grown plants (SI Appendix, Fig. S11A), where the intermediates of the Calvin-Benson
cycle were not depleted compared with nitrate-grown shoots (SI Appendix, Fig. S11B).
These observations do not support a deficiency in chloroplast function as a primary cause
of ammonium toxicity. Apoplastic pH in sunflower leaves and cytosolic pH in carrot cell
suspensions decrease after application of millimolar levels of ammonium (30, 31). The
ammonium-inducible genes whose expression is downregulated by GLN2 deficiency,
PGIP1 and PGIP2 (SI Appendix, Fig. S9A), contribute to cell wall stabilization under
acidic stress (26), implying apoplastic acidification as a target of ammonium toxicity. On
the other hand, in the presence of toxic levels of ammonium, the GABA shunt-related
genes (SI Appendix, Fig. S9A) and oxygen uptake rates (32) are induced as biochemical
pH-stats (33) that may represent an intracellular acidic burden. Given that changes in pH
environments influence a wide spectrum of physiological processes, elucidating the
relationship between ammonium-dependent acidification and growth deficiency awaits
future study.

At the whole-plant scale, our grafting work demonstrated that root GLN1;2
activity attenuates ammonium toxicity in the shoots, whilst shoot GLN2 activity causes
351 the condition (Fig. 3). Considering that GLN1;2 is the ammonium-inducible low-affinity
352 enzyme expressed in the epidermis and cortex of roots, and its deficiency elevates
353 ammonium levels in xylem sap when ammonium is supplied (17), root GLN1;2 could act
354 as a barrier to prevent the shoot-to-root transport of ammonium, thus avoiding ammonium
355 assimilation by shoot GLN2. In oilseed rape plants, replacing 3 mM nitrate in a nutrient
356 solution with 10 mM ammonium increased the ammonium levels in xylem sap linearly
357 with time, attaining concentrations greater than 5 mM (34), which could indicate breaking
358 through the barrier. On the other hand, we did not determine whether shoot GLN1
359 isozymes attenuate or deteriorate the toxicity. With ammonium nitrate nutrition,
360 Arabidopsis shoot GLN1;2 activity promotes shoot growth (35). We observed a larger
361 protein signal corresponding to shoot GLN1s when plants received ammonium rather
362 than nitrate nutrition (SI Appendix, Fig. S12 A and B), implying a barrier function of
363 GLN1 in the shoot. Further grafting work using several combinations of multiple mutants
364 on GLN1s are required to confirm this hypothesis.

365 The present study demonstrated that *GLN2* and *NRT1.1* reduce ammonium
366 tolerance via separate mechanisms when plants experience high ammonium conditions
367 (*SI Appendix*, Fig. S10). On the other hand, these genes are nitrate-inducible genes that
368 are crucial for plant adaptation to nitrate-dominant environments (36, 37). This
369 observation suggests that the adaptive traits to nitrate and ammonium could be exclusive,
370 and therefore, breeding elevated CO₂-adapted crops in terms of their mode of N utilization,
371 i.e. ammonium-tolerant crops, might sacrifice their adaptability to nitrate.
372
373 **Materials and Methods**

374 Detailed information on plant materials, their growth conditions, isolation of ammonium-

375 insensitive lines, expression analyses for mRNAs and proteins, the grafting procedure,

376 the activity assay, metabolite analysis by mass spectrometry, physiological analyses,

377 TEM observations, and statistical analyses is provided in SI Appendix, SI Materials and

378 *Methods*.

379

380

381

382

383

384

385

386

387

388

389

390

391

392

393

394
395 **ACKNOWLEDGMENTS.** Seeds of the *A thaliana* NR-null mutant were kindly 396 provided by Prof. Nigel M. Crawford, Division of Biological Sciences, University of 397 California, San Diego. This work was supported by the Building of Consortia for the 398 Development of Human Resources in Science and Technology, by the Japan Society for 399 the Promotion of Science KAKENHI Grant No. JP17K15237, by the Inamori Foundation, 400 by the Agropolis Foundation No. 1502-405, and by a Grant-in-Aid for Young Scientists 401 from Shimane University.

402

403

404

405

406

407

408

409

410

411

412

413

414

415

416
417 References

418 1. Rubio-Asensio JS, Bloom AJ (2016) Inorganic nitrogen form: a major player in wheat and Arabidopsis responses to elevated CO2. *J Exp Bot* 68: 2611-2625.

420 2. Kant S, Bi YM, Rothstein SJ (2011) Understanding plant response to nitrogen limitation for the improvement of crop nitrogen use efficiency. *J Exp Bot* 62: 1499-1509.

423 3. Britto DT, Kronzucker HJ (2002) NH<sub>4</sub><sup>+</sup> toxicity in higher plants: a critical review. *J Plant Physiol* 159: 567-584.

425 4. Li B, Li G, Kronzucker HJ, Baluška F, Shi W (2014) Ammonium stress in *Arabidopsis* signaling, genetic loci, and physiological targets. *Trends Plant Sci* 19: 107-114.

428 5. Esteban R, Ariz I, Cruz C, Moran JF (2016) Mechanisms of ammonium toxicity and the quest for tolerance. *Plant Sci* 248: 92-101.

430 6. Hachiya, T et al. (2012) Nitrate addition alleviates ammonium toxicity without lessening ammonium accumulation, organic acid depletion and inorganic cation depletion in Arabidopsis thaliana shoots. *Plant Cell Physiol* 53: 577-591.

433 7. Qin C, et al. (2008) GDP-mannose pyrophosphorylase is a genetic determinant of ammonium sensitivity in *Arabidopsis thaliana*. *Proc Natl Acad Sci USA* 105: 18308-18313.

436 8. Tanaka H, et al. (2015) Identi cation and characterization of *Arabidopsis* AtNUDX9 as a GDP-d-mannose pyrophosphohydrolase: its involvement in root growth inhibition in response to ammonium. *J Exp Bot* 66: 5797-5808.
439 9. Li B, et al. (2012) Arabidopsis plastid metalloprotease AMOS1/EGY1 integrates with
440 ABA signaling to regulate global gene expression in response to ammonium stress.

441 *Plant Physiol* 160: 2040-2051.

442 10. Li G, et al. (2019) The Arabidopsis *AMOT1/EIN3* gene plays a important role in the
443 amelioration of ammonium toxicity. *J Exp Bot* 70: 1375-1388

444 11. Jian S, et al. (2018) NRT1.1-related $NH_4^+$ toxicity is associated with a disturbed
445 balance between $NH_4^+$ uptake and assimilation. *Plant Physiol* 178: 1473-1488.

446 12. Ichikawa T, et al. (2006) The FOX hunting system: an alternative gain-of-function
447 gene hunting technique. *Plant J* 48 : 974-985.

448 13. Sakakibara H, Kawabata S, Hase T, Sugiyama T (1992) Differential effects of nitrate
449 and light on the expression of glutamine synthetases and ferredoxin-dependent
450 glutamate synthase in maize. *Plant Cell Physiol* 33: 1193-1198.

451 14. Cheng XF, Wang ZY (2005) Overexpression of *COL9*, a *CONSTANS-LIKE* gene,
452 delays flowering by reducing expression of *CO* and *FT* in *Arabidopsis thaliana*. *Plant
453 J* 43: 758-768.

454 15. Lothier J, et al. (2011) The cytosolic glutamine synthetase GLN1;2 plays a role in the
455 control of plant growth and ammonium homeostasis in *Arabidopsis* rosettes when
456 nitrate supply is not limiting. *J Exp Bot* 62: 1375-1390.

457 16. Guan M, Møller IS, Schjøerring JK (2015) Two cytosolic glutamine synthetase
458 isoforms play specific roles for seed germination and seed yield structure in
459 *Arabidopsis*. *J Exp Bot* 66: 203-212.
460 17. Konishi N, et al. (2017) Contributions of two cytosolic glutamine synthetase isozymes
to ammonium assimilation in *Arabidopsis* roots. *J Exp Bot* 68: 613-625.

462 18. Thieme CJ, et al. (2015) Endogenous *Arabidopsis* messenger RNAs transported to distant tissues. *Nat Plants* 1: 15025.

464 19. Bittsánszky A, Pilinszky K, Gyulai G, Komives T (2015) Overcoming ammonium toxicity. *Plant Sci* 231: 184-190.

466 20. Sato S, Soga T, Nishioka T, Tomita M (2004) Simultaneous determination of the main
metabolites in rice leaves using capillary electrophoresis mass spectrometry and
capillary electrophoresis diode array detection. *Plant J* 40: 151-163.

469 21. Britto DT, Kronzucker HJ (2005) Nitrogen acquisition, PEP carboxylase, and cellular
pH homeostasis: new views on old paradigms. *Plant Cell Environ* 28: 1396-1409.

471 22. Patterson K, et al. (2010) Distinct signalling pathways and transcriptome response
472 signatures differentiate ammonium- and nitrate-supplied plants. *Plant Cell Environ*
473 33: 1486-1501.

474 23. Lager I, et al. (2010) Changes in external pH rapidly alter plant gene expression and
475 modulate auxin and elicitor responses. *Plant Cell Environ* 33: 1513-1528.

476 24. Sawaki Y, et al. (2009) STOP1 regulates multiple genes that protect Arabidopsis from
477 proton and aluminum toxicities. *Plant Physiol* 150: 281-294.

478 25. Wang R, Xing X, Crawford N (2007) Nitrite acts as a transcriptome signal at
479 micromolar concentrations in Arabidopsis roots. *Plant Physiol* 145: 1735-1745.

480 26. Kobayashi Y, et al. (2014) STOP2 activates transcription of several genes for Al- and
481 Low pH-tolerance that are regulated by STOP1 in *Arabidopsis*. *Mol Plant* 7: 311-322.
27. Hachiya T, et al. (2011) Evidence for a nitrate-independent function of the nitrate sensor NRT1.1 in Arabidopsis thaliana. *J Plant Res* 124: 425-430.

28. Kaiser WM, Brendle-Behnisch E (1995) Acid-base-modulation of nitrate reductase in leaf tissues. *Planta* 196: 1-6.

29. Puritch GS, Barker AV (1967) Structure and function of tomato leaf chloroplasts during ammonium toxicity. *Plant Physiol* 42: 1229-1238.

30. Carroll AD, et al. (1994) Ammonium assimilation and the role of γ-aminobutyric acid in pH homeostasis in carrot cell suspensions. *Plant Physiol* 106: 513-520.

31. Hoffmann B, Plänker R, Mengel K (1992) Measurement of pH in the apoplast of sunflower leaves by means of fluorescence. *Physiol Plant* 84: 146-153.

32. Hachiya T, et al. (2010) Ammonium-dependent respiratory increase is dependent on the cytochrome pathway in *Arabidopsis thaliana* shoots. *Plant Cell Environ* 33: 1888-1897.

33. Sakano K (1998) Revision of Biochemical pH-Stat: Involvement of Alternative Pathway Metabolisms. *Plant Cell Physiol* 39: 467-473.

34. Schjoerring JK, Husted S, Mäck G, Mattsson M (2002) The regulation of ammonium translocation in plants. *J Exp Bot* 53: 883-890.

35. Guan M, Schjoerring JK (2016) Peering into the separate roles of root and shoot cytosolic glutamine synthetase 1;2 by use of grafting experiments in Arabidopsis. *Plant Signal Behav* 11: e1245253.

36. Hachiya T, Sakakibara H (2017) Interactions between nitrate and ammonium in their uptake, allocation, assimilation, and signaling in plants. *J Exp Bot* 68: 2501-2512.
504 37. Marche C, et al. (2013) Nuclear retention of the transcription factor NLP7

505 orchestrates the early response to nitrate in plants. *Nat Commun* 4: 1713.

506

507

508

509

510

511

512

513

514

515

516

517

518

519

520

521

522

523

524

525
526 **Figure Legends**

527 **Fig. 1** Enhanced shoot growth of *ami2* in the presence of 10 mM ammonium. (A) A representative photograph of shoots from the wild-type (Col) and *ami2* grown on media containing 10 mM ammonium for 11 d. The scale bar represents 5 mm. (B) Fresh weights (FW) of shoots from Col and *ami2* grown on media containing 10 mM ammonium (mean ± SD; n = 10), 5 mM ammonium nitrate (mean ± SD; n = 5), or 10 mM nitrate (mean ± SD; n = 5) for 11 d. (C) FW of shoots and roots from Col and *ami2* grown on media containing 10 mM ammonium for 11 d (mean ± SD; n = 26). (D) FW of shoots grown on media containing 0.4, 2, or 10 mM ammonium for 11 d (mean ± SD, n = 5). (E) Box plots of the differences in the expression of the ammonium stress-responsive genes between the Col and *ami2* shoots 3 d after transfer to media containing 10 mM ammonium. The gene list was obtained from (9) (For further details, see *Datasets*, Table S1). Two independent experiments (Exp1 and Exp2) were performed. Nine shoots from three plates constituted a single biological replicate. An individual box plot shows the median (heavy vertical line), the 25th to 75th percentiles (right and left sides of the box), the 10th to 90th percentiles (whiskers), and the mean (closed circle). (B-D) Six shoots from one plate constituted a single biological replicate. (B, E) Welch's *t*-test was run at $\alpha = 0.05$; *p* < 0.05. (C, D) Tukey-Kramer's multiple comparison test was conducted at a significance level of *P* < 0.05 only when a one-way ANOVA was significant at *P* < 0.05. Different letters denote significant differences.
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547 **Fig. 2** Downregulation of *GLN2* enhances ammonium tolerance. (A) Genomic PCR using 548 *GLN2*-specific primers. g and c denote the PCR fragments derived from genomic DNA 549 and cDNA sequences corresponding to *GLN2*, respectively. (B) Relative transcript levels 550 of *GLN2* in the shoots of Col, *ami2*, and *gln2* 3 d after transfer to media containing 10 551 mM ammonium or 10 mM nitrate (mean ± SD; n = 3). Six shoots from two plates 552 constituted a single biological replicate. (C) Immunodetection of GLN1s and GLN2 553 isoproteins using specific antisera raised against maize GLN following SDS-PAGE and 554 immunoblotting of total proteins from the shoots of Col, *ami2*, and *gln2* 5 d after transfer 555 to media containing 10 mM ammonium or 10 mM nitrate. LSU denotes large subunits of 556 RuBisCO. (D) A representative photograph of shoots from Col, *ami2*, and *gln2* 7 d after 557 transfer to media containing 10 mM ammonium or 10 mM nitrate. The scale bar 558 represents 10 mm. (E) FW of shoots from Col, *ami2*, and *gln2* 7 d after transfer to media 559 containing 10 mM ammonium (mean ± SD; n = 8) or 10 mM nitrate (mean ± SD; n = 5). 560 Mean values of three shoots from one plate constituted a single biological replicate. (B, 561 E) Tukey-Kramer's multiple comparison test was conducted at a significance level of *P* 562 < 0.05 only when a one-way ANOVA was significant at *P* < 0.05. Different letters denote 563 significant differences. 564

565 **Fig. 3** Shoot GLN2 causes ammonium toxicity, whilst root GLN1;2 attenuates 566 ammonium toxicity. (A) Relative transcript levels of *GLN2* in the shoots and roots of Col 567 grown on media containing 10 mM ammonium for 5, 8, or 11 d (mean ± SD; n = 3). (B) 568 Relative transcript levels of *GLN1;2* in the shoots and roots of Col grown on media
containing 10 mM ammonium for 5, 8, or 11 d (mean ± SD; n = 3). (A, B) Twelve shoots and roots from one plate constituted a single biological replicate. Welch's t-test was run at α = 0.05; *p* < 0.05. (C) FW of shoots from reciprocally-grafted plants between Col (C) and *ami2* (a) 7 d after transfer to media containing 10 mM ammonium (mean ± SD; n = 8). (D) FW of shoots from reciprocally-grafted plants between Col (C) and *gln1.2-1* 7 d after transfer to media containing 10 mM ammonium (mean ± SD; n = 10). (C, D) One shoot from one plate constituted a single biological replicate. Tukey-Kramer's multiple comparison test was conducted at a significance level of *P* < 0.05 only when a one-way ANOVA was significant at *P* < 0.05. Different letters denote significant differences. Representative photograph of shoots 7 d after transfer to media containing 10 mM ammonium are shown. The scale bar represents 10 mm.

**Fig. 4** Decreased activity of GLN2 reduces the conversion of ammonium to amino acids in shoots. (A) The shoot ammonium content of Col, *ami2*, and *gln2* 5 d after transfer to media containing 10 mM ammonium or 10 mM nitrate (mean ± SD; n = 3). Three shoots from one plate constituted a single biological replicate. (B) Hierarchical clustering of the shoot amino acid content of Col (C), *ami2* (a), and *gln2* (g) 5 d after transfer to media containing 10 mM ammonium or 10 mM nitrate. The color spectrum from yellow to blue corresponds to the relative content of each amino acid. (C) The molar ratio of Gln to Glu in the shoots of Col, *ami2*, and *gln2* 5 d after transfer to media containing 10 mM ammonium or 10 mM nitrate (mean ± SE; n = 3). (A, C) Tukey-Kramer's multiple comparison test was conducted at a significance level of *P* < 0.05 only when a one-way
ANOVA was significant at *P* < 0.05. Different letters denote significant differences. (B, C) Six shoots from two plates constituted a single biological replicate. Three biological replicates were sampled separately three times.

**Fig. 5** Ammonium assimilation by GLN2 causes acidic stress. (A) Box plots of the differences in expression of the acidic stress-responsive genes between the Col and *ami2* shoots 3 d after transfer to media containing 10 mM ammonium. The gene list was obtained from (23) (For further details, see *Datasets*, Table S2). Two independent experiments (Exp1 and Exp2) were performed. Nine shoots from three plates constituted a single biological replicate. An individual box plot shows the median (heavy vertical line), the 25th to 75th percentiles (right and left sides of the box), the 10th to 90th percentiles (whiskers), and the mean (closed circle). Welch's *t*-test was run at α = 0.05; \* *p* < 0.05.

(B) Effects of MSX treatment on the relative transcript level of *ALMT1* in the Col and *ami2* shoots 3 d after transfer to media containing 10 mM ammonium or 10 mM nitrate. The transcript levels were evaluated both by RT-qPCR (mean ± SD; n = 3) and semi-quantitative RT-PCR with agarose gel electrophoresis. *ACTIN2* (*ACT2*) was the internal standard. Three shoots from one plate constituted a single biological replicate. (C) Effects of MSX treatment on proton concentrations in water extracts from the Col and *ami2* shoots 3 d after transfer to media containing 10 mM ammonium or 10 mM nitrate (mean ± SD; n = 3). Three shoots from one plate constituted a single biological replicate. (D) Effects of MSX treatment on proton efflux rates from the Col and *ami2* shoots 3 d after transfer to media containing 10 mM ammonium or 10 mM nitrate (mean ± SE; n = 3).
Three shoots from one plate constituted a single biological replicate. (B-D) Tukey-Kramer's multiple comparison test was conducted at a significance level of *P* < 0.05 only when a one-way ANOVA was significant at *P* < 0.05. Different letters denote significant differences.

**Fig. 6** Ammonium toxicity is closely linked with acidic stress. (A) Effects of NH₃ application on shoot FW and proton concentrations in water extracts of Col grown on media containing 10 mM ammonium or 10 mM nitrate for 5 d (mean ± SD; n = 3). Thirty-seven shoots from one plate constituted a single biological replicate. The pH was adjusted to pH 5.7 with 1N KOH; subsequently, 25% (v/v) ammonia was added to adjust the pH from 5.7 to 6.7. A representative photograph of 11-d-old shoots grown on 10 mM ammonium (12 plants per plate) is shown. (B) Effects of intermediate pH on the FW of shoots from Col, *ami2*, and *gln2* grown on media containing 10 mM ammonium for 11 d (mean ± SD; n = 6). Three shoots from one plate constituted a single biological replicate. The pH was adjusted to pH 5.7 with 1N KOH; subsequently, 1N NaOH was used to adjust the pH from 5.7 to 6.7 to maintain the potassium concentration constant among all samples. A representative photograph of 11-d-old shoots is shown. (C) FW of shoots from Col, *stop1-KO* (*stop1-k*), and the *stop1* mutant (*stop1-m*) grown on media containing 10 mM ammonium (mean ± SD; n = 20) or 10 mM nitrate (mean ± SD; n = 5) for 11 d. Six shoots from one plate constituted a single biological replicate. Welch's *t*-test was run at *α* = 0.05; \* *p* < 0.05. NS denotes not significant. (D) FW of shoots from plants grafted between Col (C) and the NR-null mutant (*nr*) 7 d after transfer to media containing 10
635 mM ammonium ($NH_4^+$) or 2.5 mM nitrate and 10 mM ammonium ($NH_4^+ NO_3^-$)

636 conditions (mean ± SD; n = 3). One shoot from one plate constituted a single biological

637 replicate. A representative photograph of shoots 7 d after transfer to media is shown. (A,

638 *B*, *D*) Tukey-Kramer's multiple comparison test was conducted at a significance level of

639 *P* < 0.05 only when a one-way ANOVA was significant at *P* < 0.05. Different letters

640 denote significant differences. The scale bar represents 10 mm.
<figure><img src="image_2.png" /><figcaption>Figure 1</figcaption></figure>

**Fig. 1** Enhanced shoot growth of *ami2* in the presence of 10 mM ammonium. (A) A representative photograph of shoots from the wild-type (Col) and *ami2* grown on media containing 10 mM ammonium for 11 d. The scale bar represents 5 mm. (B) Fresh weights (FW) of shoots from Col and *ami2* grown on media containing 10 mM ammonium (mean ± SD; n = 10), 5 mM ammonium nitrate (mean ± SD; n = 5), or 10 mM nitrate (mean ± SD; n = 5) for 11 d. (C) FW of shoots and roots from Col and *ami2* grown on media containing 10 mM ammonium for 11 d (mean ± SD; n = 26). (D) FW of shoots grown on media containing 0.4, 2, or 10 mM ammonium for 11 d (mean ± SD, n = 5). (E) Box plots of the differences in the expression of the ammonium stress-responsive genes between the Col and *ami2* shoots 3 d after transfer to media containing 10 mM ammonium. The gene list was obtained from (9) (For further details, see *Datasets*, Table S1). Two independent experiments (Exp1 and Exp2) were performed. Nine shoots from three plates constituted a single biological replicate. An individual box plot shows the median (heavy vertical line), the 25th to 75th percentiles (right and left sides of the box), the 10th to 90th percentiles (whiskers), and the mean (closed circle). (B-D) Six shoots from one plate constituted a single biological replicate. (B, E) Welch's *t*-test was run at $\alpha = 0.05$; \*p < 0.05. (C, D) Tukey-Kramer's multiple comparison test was conducted at a significance level of *P* < 0.05 only when a one-way ANOVA was significant at *P* < 0.05. Different letters denote significant differences.
<figure><img src="image_3.png" /><figcaption>Figure 2</figcaption></figure>

**Fig. 2** Downregulation of *GLN2* enhances ammonium tolerance. (A) Genomic PCR using *GLN2*-specific primers. g and c denote the PCR fragments derived from genomic DNA and cDNA sequences corresponding to *GLN2*, respectively. (B) Relative transcript levels of *GLN2* in the shoots of Col, *ami2*, and *gln2* 3 d after transfer to media containing 10 mM ammonium or 10 mM nitrate (mean ± SD; n = 3). Six shoots from two plates constituted a single biological replicate. (C) Immunodetection of GLN1s and GLN2 isoproteins using specific antisera raised against maize GLN following SDS-PAGE and immunoblotting of total proteins from the shoots of Col, *ami2*, and *gln2* 5 d after transfer to media containing 10 mM ammonium or 10 mM nitrate. LSU denotes large subunits of RuBisCO. (D) A representative photograph of shoots from Col, *ami2*, and *gln2* 7 d after transfer to media containing 10 mM ammonium or 10 mM nitrate. The scale bar represents 10 mm. (E) FW of shoots from Col, *ami2*, and *gln2* 7 d after transfer to media containing 10 mM ammonium (mean ± SD; n = 8) or 10 mM nitrate (mean ± SD; n = 5). Mean values of three shoots from one plate constituted a single biological replicate. (B, E) Tukey-Kramer's multiple comparison test was conducted at a significance level of *P* < 0.05 only when a one-way ANOVA was significant at *P* < 0.05. Different letters denote significant differences.
<figure><img src="image_4.png" /><figcaption>Figure 3</figcaption></figure>

**Fig. 3** Shoot GLN2 causes ammonium toxicity, whilst root GLN1;2 attenuates ammonium toxicity. (A) Relative transcript levels of *GLN2* in the shoots and roots of Col grown on media containing 10 mM ammonium for 5, 8, or 11 d (mean ± SD; n = 3). (B) Relative transcript levels of *GLN1;2* in the shoots and roots of Col grown on media containing 10 mM ammonium for 5, 8, or 11 d (mean ± SD; n = 3). (A, B) Twelve shoots and roots from one plate constituted a single biological replicate. Welch's *t*-test was run at $\alpha = 0.05$; *p* < 0.05. (C) FW of shoots from reciprocally-grafted plants between Col (C) and *ami2* (a) 7 d after transfer to media containing 10 mM ammonium (mean ± SD; n = 8). (D) FW of shoots from reciprocally-grafted plants between Col (C) and *gln1.2-1* 7 d after transfer to media containing 10 mM ammonium (mean ± SD; n = 10). (C, D) One shoot from one plate constituted a single biological replicate. Tukey-Kramer's multiple comparison test was conducted at a significance level of *P* < 0.05 only when a one-way ANOVA was significant at *P* < 0.05. Different letters denote significant differences. Representative photograph of shoots 7 d after transfer to media containing 10 mM ammonium are shown. The scale bar represents 10 mm.
<figure><img src="image_5.png" /><figcaption>Figure 4</figcaption></figure>

**Fig. 4** Decreased activity of GLN2 reduces the conversion of ammonium to amino acids in shoots. (A) The shoot ammonium content of Col, *ami2*, and *gln2* 5 d after transfer to media containing 10 mM ammonium or 10 mM nitrate (mean ± SD; n = 3). Three shoots from one plate constituted a single biological replicate. (B) Hierarchical clustering of the shoot amino acid content of Col (C), *ami2* (a), and *gln2* (g) 5 d after transfer to media containing 10 mM ammonium or 10 mM nitrate. The color spectrum from yellow to blue corresponds to the relative content of each amino acid. (C) The molar ratio of Gln to Glu in the shoots of Col, *ami2*, and *gln2* 5 d after transfer to media containing 10 mM ammonium or 10 mM nitrate (mean ± SE; n = 3). (A, C) Tukey-Kramer's multiple comparison test was conducted at a significance level of *P* < 0.05 only when a one-way ANOVA was significant at *P* < 0.05. Different letters denote significant differences. (B, C) Six shoots from two plates constituted a single biological replicate. Three biological replicates were sampled separately three times.
<figure><img src="image_6.png" /><figcaption>**Figure 5**</figcaption></figure>

**Fig. 5** Ammonium assimilation by GLN2 causes acidic stress. (A) Box plots of the differences in expression of the acidic stress-responsive genes between the Col and *ami2* shoots 3 d after transfer to media containing 10 mM ammonium. The gene list was obtained from (21) (For further details, see *Datasets*, Table S2). Two independent experiments (Exp1 and Exp2) were performed. Nine shoots from three plates constituted a single biological replicate. An individual box plot shows the median (heavy vertical line), the 25th to 75th percentiles (right and left sides of the box), the 10th to 90th percentiles (whiskers), and the mean (closed circle). Welch's *t*-test was run at $\alpha = 0.05$; \*p < 0.05. (B) Effects of MSX treatment on the relative transcript level of *ALMT1* in the Col and *ami2* shoots 3 d after transfer to media containing 10 mM ammonium or 10 mM nitrate. The transcript levels were evaluated both by RT-qPCR (mean ± SD; n = 3) and semi-quantitative RT-PCR with agarose gel electrophoresis. *ACTIN2* (*ACT2*) was the internal standard. Three shoots from one plate constituted a single biological replicate. (C) Effects of MSX treatment on proton concentrations in water extracts from the Col and *ami2* shoots 3 d after transfer to media containing 10 mM ammonium or 10 mM nitrate (mean ± SD; n = 3). Three shoots from one plate constituted a single biological replicate. (D) Effects of MSX treatment on proton efflux rates from the Col and *ami2* shoots 3 d after transfer to media containing 10 mM ammonium or 10 mM nitrate (mean ± SE; n = 3). Three shoots from one plate constituted a single biological replicate. (B-D) Tukey-Kramer's multiple comparison test was conducted at a significance level of *P* < 0.05 only when a one-way ANOVA was significant at *P* < 0.05. Different letters denote significant differences.
<figure><img src="image_7.png" /><figcaption>Figure 6</figcaption></figure>

Fig. 6 Ammonium toxicity is closely linked with acidic stress. (A) Effects of NH₃ application on shoot FW and proton concentrations in water extracts of Col grown on media containing 10 mM ammonium or 10 mM nitrate for 5 d (mean ± SD; n = 3). Thirty-seven shoots from one plate constituted a single biological replicate. The pH was adjusted to pH 5.7 with 1N KOH; subsequently, 25% (v/v) ammonia was added to adjust the pH from 5.7 to 6.7. A representative photograph of 11-d-old shoots grown on 10 mM ammonium (12 plants per plate) is shown. (B) Effects of intermediate pH on the FW of shoots from Col, *ami2*, and *gln2* grown on media containing 10 mM ammonium for 11 d (mean ± SD; n = 6). Three shoots from one plate constituted a single biological replicate. The pH was adjusted to pH 5.7 with 1N KOH; subsequently, 1N NaOH was used to adjust the pH from 5.7 to 6.7 to maintain the potassium concentration constant among all samples. A representative photograph of 11-d-old shoots is shown. (C) FW of shoots from Col, *stop1-KO* (*stop1*-k), and the *stop1* mutant (*stop1-m*) grown on media containing 10 mM ammonium (mean ± SD; n = 20) or 10 mM nitrate (mean ± SD; n = 5) for 11 d. Six shoots from one plate constituted a single biological replicate. Welch's *t*-test was run at α = 0.05; \*p < 0.05. NS denotes not significant. (D) FW of shoots from plants grafted between Col (C) and the NR-null mutant (*nr*) 7 d after transfer to media containing 10 mM ammonium (NH₄⁺) or 2.5 mM nitrate and 10 mM ammonium (NH₄⁺NO₃⁻) conditions (mean ± SD; n = 3). One shoot from one plate constituted a single biological replicate. A representative photograph of shoots 7 d after transfer to media is shown. (A, B, D) Tukey-Kramer's multiple comparison test was conducted at a significance level of *P* < 0.05 only when a one-way ANOVA was significant at *P* < 0.05. Different letters denote significant differences. The scale bar represents 10 mm.