Plastid-localized amino acid metabolism coordinates rice ammonium tolerance and nitrogen use efficiency

Ammonium toxicity affecting plant metabolism and development is a worldwide problem impeding crop production. Remarkably, rice (Oryza sativa L.) favours ammonium as its major nitrogen source in paddy fields. We set up a forward-genetic screen to decipher the molecular mechanisms conferring rice ammonium tolerance and identified rohan showing root hypersensitivity to ammonium due to a missense mutation in an argininosuccinate lyase (ASL)-encoding gene. ASL localizes to plastids and its expression is induced by ammonium. ASL alleviates ammonium-inhibited root elongation by converting the excessive glutamine to arginine. Consequently, arginine leads to auxin accumulation in the root meristem, thereby stimulating root elongation under high ammonium. Furthermore, we identified natural variation in the ASL allele between japonica and indica subspecies explaining their different root sensitivity towards ammonium. Finally, we show that ASL expression positively correlates with root ammonium tolerance and that nitrogen use efficiency and yield can be improved through a gain-of-function approach. This study identifies argininesuccinate lyase, a key member of the urea cycle for arginine biosynthesis, as an important molecular component of rice root tolerance to ammonium and so as a potential lead to improve nitrogen use efficiency and yield.

Ammonium toxicity affecting plant metabolism and development is a worldwide problem impeding crop production.Remarkably, rice (Oryza sativa L.) favours ammonium as its major nitrogen source in paddy fields.We set up a forward-genetic screen to decipher the molecular mechanisms conferring rice ammonium tolerance and identified rohan showing root hypersensitivity to ammonium due to a missense mutation in a n a rg in in os uc cinate lyase (ASL)-encoding gene.ASL localizes to plastids and its expression is induced by ammonium.ASL alleviates ammonium-inhibited root elongation by converting the excessive glutamine to arginine.Consequently, arginine leads to auxin accumulation in the root meristem, thereby stimulating root elongation under high ammonium.Furthermore, we identified natural variation in the ASL allele between japonica and indica subspecies explaining their different root sensitivity towards a m m on ium.F i n a l ly, we show that ASL expression positively correlates with root ammonium tolerance and that nitrogen use efficiency and yield can be improved through a gain-of-function approach.
Nitrogen (N) is one of the essential nutrients for plant growth and crop yield because its metabolism in vivo leads to the production of diverse organic compounds, such as proteins, nucleic acids, chlorophyll and plant hormones.Nitrate (NO 3 − ) and ammonium (NH 4 + ) are two primary inorganic N sources for plants.Nitrate is reduced to NH 4 + by the sequential action of nitrate reductase and nitrite reductase, whereas NH 4  + can be incorporated directly into amino acids via the glutamine synthetase (GS)/glutamine-2-oxoglutarate aminotransferase (GOGAT) pathway 1 .However, excessive NH 4 + leads to severe suppression of plant growth and yield, and becomes a serious risk for agricultural and ecological systems 2,3 .Thus improving plant tolerance to NH 4 + is key to enhanced NH 4  + utilization and improved nitrogen use efficiency (NUE).Symptoms of NH 4 + toxicity in plants have been suggested to be a consequence of repressed protein N-glycosylation 4,5 , chloroplast development [6][7][8] , hormone metabolism 6,9,10 , glucosinolate metabolism and Fe homeostasis 11 .The primary cause of NH 4  + toxicity has been explained by futile transmembrane NH 3 cycling 12,13 , feedback regulation on NH 4 + assimilation 14 and low pH/acidic stress to plants resulting from NH 4 + flux Article https://doi.org/10.1038/s41477-023-01494-xnitrogen (rohan).The rohan root response to NH 4 + was not altered by supplying P, K or Fe (Extended Data Fig. 1c,d); rohan also exhibited root hypersensitivity to external Gln, a N storage form in vivo 27 , rather than other nutrients, indicating that the rohan root response is specific for high N (Extended Data Fig. 1e,f).Furthermore, the rohan root phenotype was consistently observed in a paddy field supplied with low N (75 kg ha −1 urea) and high N (350 kg ha −1 urea) (Fig. 1e,f).
Root elongation is determined by meristem cell division activity and subsequent cell elongation.Compared with the N-free condition, NH 4  + treatment caused a reduction in meristem length and cortical cell number in WT, and this inhibitory effect on meristem division was enhanced in rohan (Fig. 1c,d).By contrast, cortical cell elongation was not affected in rohan either for the N-free condition or NH 4  + supply (Extended Data Fig. 1h,i).We further determined the root meristem activity of rohan by employing a 5-ethynyl-2'-deoxyuridine (EdU)-based DNA synthesis phase (S-phase) assay 28 .NH 4  + treatment led to a weaker EdU signal in the root meristems of both WT and rohan compared with under the N-free condition.Nevertheless, the root meristem of rohan showed less EdU signal than that of WT under the high NH 4  + condition (Fig. 1c,d).These results indicated that the root phenotype of rohan resulted from reduced meristem activity under a high NH 4  + supply.Next, we investigated whether the hypersensitive rohan root response to NH 4 + toxicity was caused by NH 4 + accumulation and the herewith associated in vivo acidic stress.Transcription analysis showed that the respective expression levels of AMT1.1, AMT1.2, AMT1.3 and AMT2.1 in the roots were similar between WT and rohan (Extended Data Fig. 2a).Consistent with this, the rohan mutation had a minor effect on root NH 4  + uptake, and also showed attenuated root proton influx and acidification compared with the WT (Fig. 1g and Extended Data Fig. 2b,c).In addition, rohan exhibited lower sensitivity in root elongation to low pH compared with WT under either N-free or high NH 4  + conditions (Extended Data Fig. 2d,e).These results therefore suggest that the root response of rohan to NH 4 + is most probably not a consequence of NH 4 + uptake and/or acidic stress.

ROHAN encodes an N-responsive ASL
To identify the mutation causing the root hypersensitivity of rohan to NH 4 + , we performed a MutMap analysis using an F 2 segregating population of rohan backcrossed with the parental line.We found that the rohan mutation was inherited as a single recessive mutation by observing the root phenotype of the F 1 progeny and self-pollinated F 2 population.Seedlings of the F 1 progeny showed a parent-like root sensitivity to NH 4  + treatment, and F 2 progeny phenotypically segregated in an almost 3:1 ratio for a respective NH 4  + sensitive and hypersensitive root phenotype (χ 2 = 0.58; P < 0.05) (Extended Data Fig. 3a,b).Detailed bulked genomic sequencing and MutMap analysis on the F 2 progeny identified a non-synonymous single base change (C to T) 1,706 bp downstream of the start codon of Os03g05500 (Fig. 2a and Extended Data Fig. 3c).The gene encodes an ASL that is predicted to act in the urea cycle and catalyses the conversion of argininosuccinate into Arg and fumarate 26,29 .We further employed a widely targeted metabolomics analysis and detected a massive accumulation of argininosuccinic acid and citrulline, but reduced concentrations of arginine-related compounds in the rohan (Fig. 2d,e and Supplementary Table 1), confirming its function in converting argininosuccinic acid into Arg.
ASL gene is highly conserved within the plant kingdom (Extended Data Fig. 4a).In rice, ASL contains seven exons, and the mutation in rohan results in a Pro211Leu (P211L) substitution at the third exon (Fig. 2a).Pro211 is highly conserved among plant species (Extended Data Fig. 4c).By structural modelling the ASL protein based on the ASL crystal structure in Bacillus coli, we found Pro211 at the hinge region linking protein domains (Fig. 2h and Extended Data Fig. 4c).To confirm that the P211L mutation is responsible for ASL function and root response to NH 4 + , we performed genetic complementation by transforming rohan with a VENUS-fused ASL WT protein driven by its and assimilation 7,8 .Recent studies have also revealed that NH 4 + toxicity is coupled with NH 4 + uptake and l-glutamine (Gln) synthesis by reducing apoplast pH and provoking severe acidic stress in Arabidopsis 15,16 .In Arabidopsis mutants showing reduced NH 4 + uptake (for example, the ammonium transporters (AMTs) quadruple mutant) or Gln synthesis (for example, gln2-1), the toxic effects of NH 4 + on seedling growth are largely relieved.NH 4 + toxicity can also be alleviated in mutants of NRT1.1, which activate nitrate-independent signalling to regulate NH 4 + uptake and assimilation 17,18 , or by a nitrate efflux channel SLAH3, which increases external nitrate levels to buffer the rhizosphere pH 19 .Together, these results highlight that NH 4 + /NH 3 fluxes and assimilation are critical to NH 4 + toxicity in Arabidopsis.Rice is considered as an NH 4 + -tolerant plant because NH 4 + is the primary N source for rice in the paddy field, where the nitrification process is low 20 .AMT-mediated NH 4 + uptake has a predominant role in promoting rice shoot growth and yield 21 , but negatively regulates the root system architecture, in particular by reducing rice root elongation and gravitropism 22,23 .Several studies have suggested that the inhibitory effect of NH 4 + on rice root elongation is probably due to its impact on the biosynthesis of plant hormones such as auxin, ethylene and brassinosteroids, which are critical for sustaining root development 16,24,25 .Our recent work also showed that root acidification resulting from NH 4 + uptake triggered asymmetric auxin distribution in the root cap, leading to the loss of root gravitropism under high NH 4 + (ref.16).However, root elongation inhibited by NH 4 + could not be fully mitigated by buffering the rhizosphere pH or providing an external nitrate supply, suggesting a different impact of NH 4 + in root development between Arabidopsis and rice.
To decipher the molecular machinery that allows rice to circumvent NH 4 + toxicity on root growth, we employed a forward-genetic approach and identified ROOT HYPERSENSITIVE TO AMMONIUM NITROGEN (ROHAN) as a key regulator of NH 4 + tolerance in rice.ROHAN encodes a plastid-localized argininosuccinate lyase (ASL), which was previously shown to regulate l-arginine (Arg) biosynthesis and root elongation 26 .Under high NH 4 + , ASL enhances the conversion of excessive Gln to Arg, thereby alleviating NH 4 + inhibition of root elongation.In addition, we show that a missense substitution in the ASL coding sequence in a particular rice cultivar can confer root tolerance to high NH 4

+
. Finally, we demonstrate that ASL not only helps rice to cope with NH 4 + toxicity, but also promotes rice yield and NUE under low and high N supplies, and represents an important target for rice molecular breeding of NH 4 + tolerance and NUE.

Rice rohan displays root growth hypersensitivity to ammonium
To uncover the genetic control of rice root responses to NH 4 + , an ethyl methylsulfonate-mutagenized population (10,000 mutant lines) was generated from a local elite japonica cultivar Wuyungeng7 (hereafter designated as the wild-type (WT)).We then screened for mutants showing altered root length when grown in hydroponic solutions supplied with 2.5 mM NH 4 + , a concentration that inhibits seminal root (SR) elongation 16 .Because NH 4 + uptake results in proton release 16 , 2-morpholinoethanesulfonic acid monohydrate was supplemented to exclude root phenotypes caused by lowering pH.We identified one mutant displaying severely inhibited SR elongation when compared with the WT under high NH 4 + supply (74% shorter than the WT SR length); in the absence of N, however, the mutant SR length was reduced less compared with the high NH 4 + condition (36% decrease) (Fig. 1a,b).We further observed that the highest sensitivity of SR elongation to NH 4 + in the mutant occurred at 2.5 mM NH 4 + , and that SR elongation was less sensitive to varying NO 3 − concentrations (Extended Data Fig. 1a).Moreover, a time-course experiment showed that the inhibitory effect of NH 4 + on SR elongation in the mutant was more pronounced after 3 days of treatment than after 1 day (Extended Data Fig. 1b).Because of the striking inhibition of root growth under NH 4 + supply, we named the mutant root hypersensitive to ammonium Article https://doi.org/10.1038/s41477-023-01494-xnative promoter (proASL:ASL-VENUS rohan).More than 24 individual lines were obtained, and lines with ASL-VENUS expression in the root showed root sensitivity to NH 4 + identical to that of the WT (Fig. 2b,c).In addition, CRISPR-Cas9 knockout lines of ASL mimicked the root phenotype of rohan under the high NH 4 + condition (Fig. 2b,c and Extended Data Fig. 3d).Thus, ASL is responsible for the rohan root phenotype, whereas its function is dependent on the Pro211 residue.
By generating a transcriptional reporter line of ASL in the WT background, we observed strong expression of proASL:GUS in the root meristem and the stele of the elongation zone, which is in agreement with an earlier report by Xia et al. 26 .Furthermore, proASL:GUS was also found to be expressed in lateral root primordia and emerged lateral roots (Extended Data Fig. 4b), indicating the critical role of ASL in regulating rice root development.In the complemented lines, the ASL-VENUS signal was also highly detected at the root meristem and localized to plastids at the cellular level, confirming the earlier reported localization in onion epidermal cells 26 (Fig. 2f).The Pro211Leu (P211L) substitution did not affect the subcellular localization of ASL (Fig. 2g) and suggested the Pro211 residue is key to its protein function in plastids.
We next investigated whether ASL expression was regulated by external N supply.Quantitative polymerase chain reaction with reverse   The experiment was repeated three times with similar results.Scale bar, 1 mm.

Article
https://doi.org/10.1038/s41477-023-01494-xtranscription (RT-qPCR) analysis showed that ASL expression in roots was gradually increased by NH 4 + and Gln over 24 h, whereas it was less induced by NO 3 − treatment (Fig. 2i).This was further confirmed by the expression analysis of proASL:GUS, showing strong induction of ASL expression in the root meristem by NH 4 + after 24 h (Fig. 2j).The induction of ASL expression by NH 4 + was suppressed in either the quadruple mutants of AMTs (qko) 30 or in the mutants of Glutamine Synthetase (gs1;1), which are defective in Gln synthesis (Extended Data Fig. 5).Taken together, these data demonstrate that ASL is a N-responsive gene that encodes a plasmid-localized protein regulating root development.

Defect in Gln to Arg conversion causes root hypersensitivity to NH 4 +
ASL has been shown to play a role in amino acid metabolism by catalysing the last step of Arg biosynthesis 26 .Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of differential metabolites also suggested that Arg biosynthesis was strongly attenuated in rohan (Extended data Fig. 6).Furthermore, Arg produced by ASL is believed to be required for normal root elongation in rice 26 , suggesting a possible link between ASL function in plastids and root tolerance to NH 4 + .To address the latter, we measured the content of 20 free amino acids in roots of rohan and WT under the N-free condition and a high NH 4 + supply.NH 4 + treatment increased the overall content of amino acids, especially Asn and Gln in both WT and rohan roots (Extended Data Fig. 7a).Remarkably, upon high NH 4 + supply, more Gln and free NH 4 + accumulated in roots of the rohan and asl CR 4 mutants than in the WT.The opposite trend was observed for the Arg content, which was reduced more in the mutants than in the WT roots (Fig. 3a).In rice, NH 4 + is assimilated to Gln depending on glutamine synthase (GS) activity in the cytosol (Supplementary Fig. 1) 31,32 , and Gln is subsequently converted to Arg by the urea cycle in the plastid (Fig. 3b) 33,34 .Thus, the increased Gln and NH 4 + in rohan roots might be due to the perturbation of Arg synthesis.In the complementing line of rohan, the contents of Gln, NH 4 + and Arg in the root were all recovered to the WT levels (Fig. 3a and Extended Data Fig. 7a), confirming that ASL acts on the conversion of NH 4 + /Gln to Arg.We next questioned whether the observed metabolic defect was associated with rohan root sensitivity to NH 4 + by testing the effects of Gln and Arg application on root growth.We observed substantial inhibition of SR meristem cell division and elongation at 0.3 mM Gln in the WT.In rohan, however, a Gln concentration as low as 0.03 mM caused substantial inhibition of SR meristem cell division and elongation (Fig. 3c,d,g,h and Extended Data Fig. 7d).This suggested that root elongation of rohan is hypersensitive to Gln, mimicking its response to NH 4 + .Interestingly, addition of l-methionine sulfoximine (MSO), a competitive inhibitor of GS 35 , restored SR elongation in both the WT and rohan under high NH 4 + supply (Fig. 3c,d and Extended Data Fig. 7d).Meanwhile, qko and gs1;1 mutants both showed resistance to NH 4 + treatment towards SR elongation (Fig. 3e,f), indicating that Gln accumulation is a major factor responsible for NH 4 + toxicity to root elongation in rice.
Intriguingly, arrested SR meristem cell division and elongation in rohan under high NH 4 + and Gln could be rescued by supplying Arg, with the optimum concentration being 0.3 mM, whereas this had a minor effect on SR meristem cell division and elongation in the WT (Fig. 3g,h and Extended Data Fig. 7b,c).However, external Arg supplies could not rescue NH 4 + -and Gln-inhibited SR elongation in the WT (Extended Data Fig. 7b,c), suggesting that endogenous Arg, produced by ASL, is required for root elongation under high NH 4 + .Thus, we concluded that the biological function of ASL in converting excessive NH 4 + /Gln into Arg in vivo, enables alleviation of NH 4 + -induced inhibition of rice root elongation.

ASL promotes N metabolism and photosynthesis under a high NH 4 + supply
We next aimed to reveal the mechanism underlying ASL-mediated root tolerance to NH 4 + by performing RNA-sequencing (RNA-seq) analysis of root meristems in rohan and WT under a N-free condition and an NH 4 + supply.In agreement with the hypersensitive root phenotypes of rohan under high NH 4 + , a more diverged transcriptional pattern between rohan and the WT under high NH 4 + was observed compared with the N-free condition (Extended Data Fig. 8a).
To specify the molecular pathways downstream of ASL, we analysed differentially expressed genes (DEGs) in the WT and rohan under NH 4 + and N-free conditions.NH 4 + treatment led to a higher number of DEGs in rohan compared with WT after 1 day of treatment; the number of DEGs in rohan was increased mainly after 3 days of treatment (Extended Data Fig. 8b).Gene set enrichment analysis (GSEA) of these DEGs based on Gene Ontology and KEGG suggested that the N metabolism pathway was strongly affected in rohan (Extended Data Fig. 8c), consistent with its altered amino acid metabolism.Interestingly, genes associated with ammonium assimilation and Gln synthesis, including three GS1 members and GDH1, were all found to be downregulated in rohan under NH 4 + treatment after 3 days (Extended Data Fig. 8d,e), which might result from feedback inhibition by the accumulated Gln in rohan.Interestingly, several cation proton exchanger and ATPase encoding genes, for example CHX13, CHX15, CAX1 and OVP1, had induced expression under the high NH 4 + condition; their expressions were suppressed in rohan (Extended Data Fig. 9), which accounts for the reduced proton flux in rohan.
In addition, photosynthesis pathway genes were also enriched in rohan under high NH 4 + (Extended Data Fig. 8b).Increasing the N supply can promote photosynthesis in rice 36,37 .Consistent with this, a high NH 4 + supply induced the photosynthetic rate, transpiration rate, stomatal conductance and photosystem II (PSII) in WT seedlings, but severely repressed them in rohan, correlating with retarded shoot growth in rohan under NH 4 + supply (Supplementary Fig. 2).Photosynthesis and shoot growth deficiency in rohan recovered in the complementation line or with an external Arg supply (Supplementary Fig. 2b).These results show the importance of ASL for efficient amino acid metabolism in photosynthesis.

The rohan root response to NH 4 + is mediated by auxin metabolism
Interestingly, GSEA of DEGs revealed that hormone metabolism was also affected in rohan under high NH 4 + (Extended Data Fig. 10a).Among Asterisks indicate significant differences (two-sided Student's t-test).Gln and Arg were used at 0.3 mM.In a and f, letters denote significant differences (P < 0.05, by one-way ANOVA followed by Duncan's test).In c and e, the white dotted line indicates the position of the root tip when the seedlings were transferred to the media supplied with the indicated chemicals.In the boxplots in d and h, the centre line is the median, the box indicates upper/lower quartiles and whiskers denote minimum/maximum (n is the number of individual seedlings treated).
In c, e and g, similar results were obtained in three independent experiments.

Article
https://doi.org/10.1038/s41477-023-01494-xplant hormones, auxin plays a crucial role in regulating root elongation and responses to N sources and photosynthesis 38,39 .The expression of several genes involved in auxin conjugation (GH3s) and auxin signalling (ARF2, IAA6, IAA14, and small auxin-up RNAs (SAURs)) was found to be downregulated in rohan under high NH 4 + (Fig. 4a).GH3s and SAURs are auxin-inducible genes 40 , thus changes in their expression indicated altered endogenous auxin levels in rohan.On measuring endogenous indole-3-acetic acid (IAA) content, we detected a substantial reduction in the root tip of rohan compared with that of the WT under either high NH 4 + or Gln supply, whereas IAA was only slightly reduced in rohan under N deficiency (Extended Data Fig. 10b).The IAA content in rohan root tip was recovered by its complementation or exogenous Arg (Extended Data Fig. 10b), showing the importance of ASL for endogenous auxin metabolism in the root tip.
To reveal spatial changes in auxin levels in root tips, we introduced DR5rev:3xVENUS, a sensitive auxin reporter 41 , into rohan by crossing.The DR5 signal was strong in the root epidermis and stele of both the WT and rohan in the N-free condition.Under high NH 4 + , WT roots had a lower DR5 signal in the stele but an increased signal in the epidermis, whereas the DR5 signal almost disappeared from the root stele and epidermis of rohan.Interestingly, exogenous Arg elevated DR5 expression in the root stele and epidermis of rohan (Fig. 4b,c and Extended Data Fig. 10c), corresponding to its effect in restoring SR elongation in rohan.Remarkably, Arg induced DR5 expression in the root epidermis of the WT under high NH 4 + (Fig. 4b,c).Because Arg failed to rescue root elongation in NH 4 + -treated WT, these results indicate that changes in auxin levels in the root meristem stele are correlated with the ASL-mediated SR response to NH 4 + .Regulation of root meristem activity and root elongation in response to N sources is mediated by auxin efflux carrier PIN-FORMED (PIN)-dependent polar auxin transport 42 .We also found that several genes encoding PIN were transcriptionally repressed in rohan under high NH 4 + (Fig. 4a), as confirmed by RT-qPCR (Extend Data Fig. 10e).We further validated the role of PINs in mediating the rohan root response to NH 4 + by using two well-characterized potent polar auxin transport inhibitors, 1-N-naphthylphthalamic acid (NPA) and 2-[4-(diethylamino)-2-hydroxybenzoyl]benzoic acid (BUM) 43,44 , to bypass the functional redundancy of PIN proteins.Under high NH 4 + supply, rohan showed less sensitivity to NPA and BUM than the WT regarding SR elongation (Fig. 4d,e and Extended Data Fig. 10d).Following inhibition of SR elongation by NPA, NPA treatment dismissed DR5 expression in the root stele of the WT upon NH 4 + treatment, resembling DR5 expression in the root stele of rohan (Fig. 4b,c).Moreover, exogenous IAA failed to trigger DR5 expression in stele and SR elongation in rohan, suggesting the complete suppression of PIN activity in rohan (Fig. 4b,c and Supplementary Fig. 3).
PIN1 expression abundances were largely repressed in rohan under high NH 4 + (Fig. 4a), and PIN1 paralogue genes are highly expressed in rice root stele and mediate shoot-to-root auxin transport and auxin accumulation 45,46 .We further generated a CRISPR-Cas9 knockout mutant of PIN1a and PIN1b (Extended Data Fig. 10f).Remarkably, pin1apin1b mutants had a lower IAA content in root tip (Extended Data Fig. 10g), and showed shorter root meristems and SRs, mimicking the rohan root phenotype (Fig. 4g, i), confirming the requirement of PIN-mediated polar auxin transport for the ASL-dependent effect on SR elongation.Together, our data demonstrated a critical role of auxin metabolism and polar auxin transport underlying ASL-controlled root tolerance to NH 4 + .

ASL allele confers rice root tolerance to NH 4 + toxicity
Because of the important role of ASL in NH 4 + root tolerance, we further investigated its genetic variation in rice germplasm.Using nucleotide diversity (π) and fixation index (Fst) analysis, we found evidence for decreased nucleotide diversity of ASL in the japonica and indica subpopulations compared with the wild rice population (Oryza rufipogon), whereas regions around the ASL locus showed differentiation between subspecies japonica and indica, indicative of possible selective pressure during domestication (Fig. 5a,b).Detailed analysis showed 37 single nucleotide polymorphism (SNP) variants, including 35 introns, 1 missense and 1 synonymous within the ASL gene.We further focused on the missense SNP (Chr310847318, c.3972A > G), which showed apparent indica (94.91%) to japonica (96.76%) differentiation and led to a Lys470Arg substitution (Fig. 5d,e).The allele frequency of the SNP from japonica, indica I, indica II, indica III, aus and aromas subpopulations were calculated and are shown on the geographic map (Fig. 5c).
To validate the function of the SNP, we randomly selected a total of 100 SNP A -variant and SNP G -variant accessions to evaluate their root sensitivity to high NH 4 + .Our results revealed a higher root sensitivity to NH 4 + in SNP G -variant accessions than in SNP A -variant accessions (Fig. 5f and Supplementary Fig. 4a).Furthermore, ASL expression in SNP G -variant accessions was less induced than in SNP A -variant accessions by high NH 4 + (Fig. 5g).These results suggest that the SNP variation of ASL might be associated with transcriptional responses and SR elongation to NH 4 + .To experimentally validate this possibility, we further conducted allelic complementation by transforming SNP G -variant (proASL:ASL K470R , with only one missense SNP difference from the japonica cultivar Wuyungeng7 genome) into the rohan.Unlike the SNP A -variant, which could restore SR elongation (Fig. 2b,c), independent SNP G -variant lines failed to rescue the SR elongation under high NH 4 + supply (Fig. 5h,i), demonstrating that the natural ASL variants cause the divergence in root tolerance to NH 4 + .

ASL expression determines rice tolerance to NH 4 + and NUE
Because ASL expression is correlated with root tolerance to NH 4 + , we further generated overexpression lines of ASL (UBIL:ASL) in a rohan background.Two independent UBIL:ASL lines, which had higher ASL expression (Supplementary Fig. 4b), showed longer SR than the WT with different increasing NH 4 + concentrations (Fig. 6a,b).By contrast, rohan displayed lower expression of ASL in roots and shorter SRs than the WT (Supplementary Fig. 4b).These results underline once again the importance of ASL expression for root tolerance to NH 4 + .We further planted mutant and overexpression lines of ASL in a paddy field supplied with low (75 kg ha −1 ), moderate (150 kg ha −1 ) and high nitrogen (350 kg ha −1 ).Compared with the WT, ASL overexpression led to notable increases in N content, biomass, tiller number, grain yield, grain proteins and NUE under all N conditions, which were all reduced in rohan (Fig. 6c,d and Supplementary Fig. 5).Collectively, these results show that ASL expression positively correlates with root tolerance to high NH 4 + and NUE.

Discussion
Improving plant tolerance to NH 4 + thus represents a promising strategy to improve crop NUE and yield.Although previous studies in rice have shown that reduced ammonium uptake or Gln synthesis     (Fig. 6e).Importantly, induction of ASL expression not only promotes rice root tolerance to NH 4 + but also increases the NUE and yield under low and high N supplies in the paddy field.Therefore, our results suggest that the ASL-mediated Gln to Arg conversion coordinates root tolerance to NH 4 + toxicity and NUE.Amino acids are a primary storage form of N and exert diverse functions in plant development and defence 47 .ASL is one of the rate-limiting enzymes of the urea cycle and is utilized in Arg production 26,29 .Using metabolomics analysis combined with analysis of the amino acid content, ASL was demonstrated to act specifically in the conversion pathway of Gln to Arg (Fig. 3), indicating a critical role for the urea cycle in Gln metabolism in plastids.In Arabidopsis, the elevated apoplast acidification resulting from Gln synthesis has been suggested to cause NH 4 + toxicity in the shoot 15 .Gln had a similar inhibitory effect on root growth in rice, and rohan roots show hypersensitivity to an external Gln supply, which can be suppressed when Gln synthesis is attenuated (Fig. 3).These results demonstrate that Gln accumulation is a primary cause underlying NH 4 + toxicity in rice root development under pH-buffered conditions.However, rohan roots exhibited reduced proton influx, root acidification and insensitivity to low pH (Extended Data Fig. 2), arguing that ASL-mediated root tolerance to NH 4 + is linked to metabolic detoxication rather than root acidification.
The regulation of N in plant root development is mediated by phytohormones such as auxin, ethylene and brassinosteroids 16,25,48 .Auxin is a prominent regulator of root meristem activity and root elongation, and also interacts with other hormones to regulate root development 49 .Our transcriptome data revealed a robust transcriptional regulation of ASL in auxin signalling pathways (Fig. 4).We demonstrated that ASL plays a role in maintaining high auxin levels in root stele, which is required for root elongation under high NH 4 + .Auxin accumulation in root meristem is mediated by auxin efflux carrier PIN-dependent polar auxin transport 50 .We further identified PIN1 as a downstream signalling component of ASL.PIN1 activity in the stele is required for shoot-to-root auxin transport and SR elongation in rice 45,46,51 .ASL can induce PIN1 expression to facilitate tissue-specific auxin accumulation at the root stele to stimulate root elongation (Fig. 4).In Arabidopsis, high NH 4 + was also found to affect PIN activity to adjust root growth 42 .Therefore, our study suggests a molecular link between the N metabolic pathway and the phytohormone signal in regulating rice root tolerance to NH 4 + .Further investigations are needed to uncover the mechanism underlying the transcriptional regulation of ASL and NH 4 + in auxin homeostasis.ASL is highly conserved in various plant species.We identified a natural variant of ASL (SNP A ) in rice that increases root tolerance to high external NH 4 + .This tolerance allele, SNP A , is mainly distributed in japonica cultivars but is absent from indica cultivars, correlating with the higher root tolerance of japonica cultivars compared with indica cultivars (Fig. 5).Because ASL expression is highly elevated by high NH 4 + in japonica cultivars in contrast to indica cultivars, the elite allele of ROHAN might determine root tolerance to NH 4 + toxicity by regulating its expression in response to external NH 4 + .We further confirmed that ASL overexpression enhanced root tolerance to NH 4 + and increased rice yield and NUE (Fig. 6), which might be because of its promotive effects on photosynthesis (Supplementary Fig. 2).
In summary, we identified ASL as a critical regulator of NH 4 + tolerance and NUE in rice.Our study reveals a molecular link between amino acid metabolism and the root response to an external N supply, and suggests that polar auxin transport-mediated auxin accumulation in the root meristem is required for ASL function.Because rice varieties harbouring the elite variant of ASL display a higher root tolerance to NH 4 + without penalty in plant growth and yield, ASL may represent a potential candidate target for genetic editing or marker-assisted breeding to develop rice varieties tolerant to NH 4 + toxicity and help to increase rice NUE and yield under large quantities of urea-based fertilizer.The identification of ASL therefore represents an important element to set up strategies to cope with the excessive use of N fertilizer, which has resulted in several environmental issues, such as surface water eutrophication, groundwater pollution, greenhouse gas emission and soil acidification.

Plant materials
Rice (Oryza sativa cv.Wuyungeng7) seeds were mutagenized by treating them with 1.0% ethyl methylsulfonate (Sigma-Aldrich, catalogue no.62-50-0) for 12 h 52 .M 2 seeds obtained from self-pollinated M 1 plants were used to screen NH 4 + -sensitive mutants based on root elongation at 2.5 mM NH 4 + .In brief, seeds from 10,000 M 2 lines were bulked, and 100 seeds were sown on a mesh in a 500-ml volume cup.Young seedlings were subjected to ammonium treatment 3 days after germination with a hydroponic culture containing 2.5 mM ammonium.The root elongation of M 3 progeny was finally assessed in the same manner to confirm the NH 4 + -sensitive phenotype of the candidate mutant rohan.The rice DR5rev:3xVENUS-N7 transgenic reporter line has been described previously 41 .

Root phenotype analysis
Rice seedling roots were imaged at a resolution of 400 dots per inch using an EPSON Expression 11000XL scanner.Seedling roots were scanned before and after treatments.We then measured the SR length using Fiji image analysis software (http://fiji.sc/).SR elongation was calculated as the difference between SR lengths after and before treatments.

Root tissue acidification assay
Roots from WT, rohan and rohan-C1 (complementary line of the rohan) seedlings, treated with or without 2.5 mM NH 4 + for 6 days, were harvested, frozen immediately with liquid N 2 and ground to a powder.Two volumes of H 2 O were added to the frozen powder followed by centrifugation at 10,000g at room temperature for 10 min.The supernatant was measured with a 0.15 mM concentration of the pH indicator bromocresol purple (Sigma-Aldrich, catalogue no.115-42-2) 15,53 .The supernatant media was proportional to the volume of media in which assays were performed.Media pH was adjusted with KOH, and media standards of known pH (4.0-6.5) were prepared in the same way.

Genetic mapping of the rohan mutation
To identify the causal mutation, an F 2 population was generated by backcrossing rohan with the parent Wuyungeng7 and this was then used for genetic mapping via a modified MutMap analysis 54 .We then performed whole-genome resequencing of two F 2 segregant bulks: 20 individuals showing the parent phenotype and 20 displaying the rohan NH 4 + -sensitive phenotype.Next, DNA was isolated from leaves of each individual using the DNeasy Plant Mini Kit (Qiagen); DNA bulk was created by mixing individual DNAs equally to reduce sequencing bias.Sequencing libraries with an average insert size of 350-500 bp were constructed using the Illumina DNA Prep Kit and sequenced with PE150 (Paired-end, 150 bp) mode using Illumina NovaSeq 6000 platform (Annoroad Gene Technology).Raw FASTQ data were trimmed using Fastp v.0.20 (ref.55), with sequencing adaptors, low-quality bases and short reads (<40 bp).Cleaned data were then aligned to the rice reference genome (IRGSP1.0,https://rapdb.dna.affrc.go.jp/) using BWA v.0.7.17 (ref.56).PCR duplication was removed by Sambamba v.0.8. 1 (ref.57).SNP calling was performed using Gene Analysis Toolkit v.4.2.1 (ref.58), and all variants were scored by Euclidean distance (ED) and fitted using a sliding window approach 54 .ED 4 was then calculated by raising ED to the fourth power to decrease noise.Candidate causal mutations with significance at the 95% confidence interval were finally identified.

RNA-seq analysis
Root tip segments (~1 cm) from 20 WT and rohan seedlings, treated with or without 2.5 mM NH 4 + for 1 and 3 days, were dissected from the SRs and collected with three independent biological repeats for RNA-seq analysis.Total RNA was extracted using TRIzol reagent and digested with RNase-free DNase (Qiagen) according to the manufacturer's instructions.RNA was then purified and concentrated using a RNeasy column (TaKaRa).RNA integrity numbers were assessed using an Agilent 2100 bioanalyzer (Agilent Technologies).RNA quantification was done using a Qubit 4 fluorometer (Thermo Fisher Scientific).Purification of poly-A messenger RNA and construction of complementary DNA were accomplished with a TruSeq RNA Library Preparation Kit (Illumina).RNA-seq was finally performed using an Illumina NovaSeq 6000 platform with PE150 (Paired-end, 150 bp) (Annoroad Gene Technology).
RNA-seq datasets were analysed following a custom protocol 59 .Raw data were also cleaned using Fastp v.0.20 55 .Cleaned reads were aligned to the rice reference genome using STAR v.2.7.8a with a splicing-aware method and two-pass mode 60 .The aligned reads were assembled separately into transcripts for each sample with the reference annotation-based transcript assembly algorithm and generated an updated transcript annotation with a gene transfer format (GTF)-formatted file using StringTie v.2.1.3 (ref.61).Finally, the expression level of genes was quantified and normalized with the above-updated GTF file using HTSeq, respectively 62 .Only the genes with a fragments per kilobase of transcript per million fragments mapped >1 in at least six samples were used for downstream gene expression analysis.DESeq2 was used to perform pairwise comparisons between conditional samples to identify DEGs with the updated GTF file 63 .In our study, genes were considered differentially expressed according to the following criteria: log 2 (fold change) (log 2 (FC)) ≥1 and adjusted P < 0.05.
Venn diagrams and heatmaps were plotted from the DEGs using custom R scripts.Enrichment analysis based on Gene Ontology and KEGG databases was carried out using PlantGSAD 64 .The Benjamini-Yekutieli method was used for P-value adjustment.In addition, cluster-Profiler v.3.14, a GSEA method 65 , was further employed to determine whether a previously defined set of genes such as nitrogen metabolism and plant hormone signal transduction shows concordant differences in response to NH 4 + treatment between the mutant and the WT.

Metabolite profiling
Fresh roots (1.5 g) from WT and rohan seedlings, treated with or without 2.5 mM NH 4 + for 4 days, were harvested and freeze-dried for metabolite profiling with three independent biological repeats.The lyophilized tissues were ground using a mixer mill (MM 400; Retsch) and 100 mg of powder was extracted overnight at 4 °C with 1.0 ml of 70% aqueous methanol, pure methanol for water and lipid-solubility metabolites, respectively.Following centrifugation at 10,000g for 10 min, all the supernatants were pooled and filtered with a membrane.Subsequently, an ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry system was used to analyse the widely targeted metabolomics analysis at Metware Biotechnology.Quantification of metabolites was carried out using a scheduled multiple reaction monitoring method according to previous studies 66 .
Orthogonal partial least squares-discriminant analysis (OPLS-DA) was performed to detect sample correlation using the R package Meta-boAnalystR.Differential metabolites analysis was determined by variable importance in projection (≥1), absolute log 2 (FC) (|log 2 (FC)| ≥ 1) and false discovery rate <0.05.Variable importance in projection values were extracted from the OPLS-DA result, which also contains score plots and permutation plots, and was generated using the R package MetaboAnalystR.The data was log transformed (log 2 ) and meant centring before OPLS-DA.To avoid overfitting, a permutation test (200 permutations) was performed.

Allelic variation and local adaption analysis
For allelic variation analysis of ASL, SNP datasets of 446 accessions of common wild rice (Oryza rufipogon) and the 3,000 Rice Genomes Project were obtained from a previous study 67,68 .The nucleotide diversity (π) and Fst statistics were calculated and fitted using the sibling window method (window size: 2 kb; step size: 1 kb) for wild rice and rice subpopulations using VCFtools v.0.1.17(ref.69).Functional effects of variants from ASL and its surrounding regions (with a 2 kb flanking sequence on both sides) were predicted by SNPeff v. 4.3 (ref.70).The allele frequency of missense SNP (Chr3:10847318) from six rice Article https://doi.org/10.1038/s41477-023-01494-xsubgroups (japonica, indica I, indica II, indica III, aromatic and aus) of the 3,000 varieties were calculated and integrated into a geographical map according to their subpopulation and origin information from the Rice SNP-Seek Database (https://snp-seek.irri.org).Moreover, SR length and expression abundance corresponding to different alleles were also measured from a selection of 50 japonica and 50 indica varieties.

Plastid construction and plant transformation
The Gateway system (Invitrogen) was employed to generate most genetic constructs.For transcriptional fusions, ~2,000 promoter fragments upstream of the start codon were first amplified by PCR from genomic DNA and cloned into pDONRP41R using BP Clonase™ II, whereas the ASL open reading frame with or without a stop codon was amplified from genomic DNA and cloned into pDONR221 or pDON-R2F3R, respectively.Subsequently, the vectors were introduced into different Gateway expression vectors by using LR clonase TM enzyme.The Gateway expression vectors pMK7SNFM14GW, pHb7m34GW and pHb7m24GW were used for the promoter-GUS (β-glucuronidase) fusion, the complementation of the mutant and generating the overexpression lines, respectively.

Histochemical analysis and microscopy
For GUS assays, roots from proASL:GUS transgenic rice seedlings, treated with or without 2.5 mM NH 4 + for 1 day, were collected and soaked in 100 mM Tris/NaCl buffer (pH 7).After incubation at 37 °C for 30 min, root tissues were soaked in 100 mM Tris/NaCl buffer (pH 7.0) containing 2 mM X-gluc (Warbio, catalogue no.114162-64-0) and 2 mM ferricyanide, and kept at 37 °C for 10 h.Roots were imaged with a Leica DM2500 microscope (Leica Microsystems).For anatomical sections, GUS-stained samples were fixed overnight and embedded following a published protocol 72 .
DR5rev.VENUS-N7 expression in the root tip of rice was observed with a Leica SP8 laser-scanning microscope equipped with a white laser and hybrid laser detectors.Before confocal imaging, the root tips of DR5rev:3xVENUS-N7 transgenic rice seedlings grown under different treatments were cleaned using a modified ClearSee method 73 .Root tissues were first fixed with 4% paraformaldehyde (Sigma-Aldrich, catalogue no.30525-89-4) in PBS buffer under vacuum for 4 h at room temperature, washed twice with PBS, and then transferred to ClearSee solution 73 for 6 days at room temperature.To investigate the subcellular of ASL, the root tips of proASL:ASL:VENUS transgenic rice seedlings were also cleaned using the above ClearSee method, and then stained with calcofluor white (CW) (0.1%, in ClearSee solution; Yuanye Bio, catalogue no.4404-43-7) for 1 h.The root tips were further washed with ClearSee solution before confocal imaging.Rice root meristems were imaged using modified pseudo-Schiff propidium iodide (mPS-PI) staining to visualize the cell organization of rice SR tips 16 .

EdU staining
EdU staining was performed using an EdU kit (Click-iT EdU Alexa Fluor 488 HCS assay; Invitrogen, catalogue no.C10350), according to the manufacturer's instructions.Roots of 4-day-old seedlings were immersed in 20 μM EdU solutions for 2 h, and fixed for 30 min in 3.7% formaldehyde solution diluted with phosphate buffer (pH 7.2, 0.1% Triton X-100), followed by 30 min of incubation with EdU detection cocktail.An Olympus MXV10 microscope with a green fluorescent protein channel was used to capture the images.

RNA extraction and real-time qPCR analysis
Root tip segments (~1 cm) from the WT and rohan seedlings, treated with or without 2.5 mM NH 4 + for the indicated times, were dissected from the SR and harvested for real-time qPCR analysis.For each treatment or genotype, at least 20 individual roots were sampled, and three independent biological replicates were performed.Total RNA was isolated using the plant RNA purification reagent (Invitrogen).cDNA was synthesized from 1 μg of RNA using the Advantage RT-for-PCR Kit (TaKaRa) according to the manufacturer's instructions and was diluted 20 times for subsequent qPCR.Real-time PCR was performed in a real-time PCR machine (LightCycler 480, Roche Diagnostics) in accordance with the manufacturer's manual in a reaction mixture of 10 μl of TB Green Fast qPCR mix (CellAmp Direct TB Green RT-qPCR Kit).OsActin and OsUBQ10 were used as housekeeping genes, and three biological replicates were analysed.Primer sequences are listed in the Supplementary Table 2, together with all the other primers used in this study.

Amino acid analysis
After 4 days of N-free and NH 4 + treatments, the roots of WT and rohan plants were collected in liquid nitrogen, ground and mixed manually.Samples were then transferred to 5-ml tubes, frozen with liquid nitrogen, and transferred to the lyophilizer (BILON, FD-2C) for 3 days.Lyophilized root tissues (~3 mg) were used for extraction with 80% methanol, followed by incubation at 70 °C for 15 min under shaking (600 r.p.m.).Following centrifugation at 10,000g for 15 min at room temperature, the supernatants were collected and the pellet was re-extracted with 20% methanol as described above.All supernatants were collected in a new pellet and dried in a Termovap sample concentrator (MD200-1; Allsheng).The dried pellets were dissolved in 500 μl of ultrapure water.Then, 500 μl of 4% 5-sulfosalicylic acid dihydrate was added to redissolve the pellets, followed by centrifugation at 12,000g for 15 min at room temperature.The supernatants were collected and filtered using a 0.22 μm water filter.Samples were analysed using a LA8080 automatic amino acid analyser (Hitachi).All steps were performed according to the manufacturer's instructions.The total amino acid content was calculated as the sum of the content of each amino acid in the plant.

Photosynthesis parameters and seeds protein analysis
Simultaneous measurement of photosynthesis parameters was performed on light-adapted leaves using a LI-6,800 infrared gas analysis Article https://doi.org/10.1038/s41477-023-01494-xsystem.Before the measurements, leaves of 21-day-old rice seedlings were placed in a greenhouse at a photosynthetic photon flux density of 1,500 μmol m −2 s −1 with an ambient CO 2 concentration of 400 μmol mol −1 .After equilibration to a steady state, 0.8 s saturating pulses of light (~8,000 μmol m −2 s −1 ) were applied to measure maximum fluorescence (F m ′) and steady-state fluorescence (F s ); gas-exchange parameters were recorded.The Φ PSII (photosystem II quantum yield) was calculated as: Φ PSII = (F m ′ − F s )/ F m ′.Gas-exchange parameters include net photosynthesis rate, stomatal conductance and transpiration rate.The crude protein content of the seeds was detected using a near infrared spectroscope (DA7250).

Growth conditions and agronomic trait measurement in paddy field
Three-week-old rice seedlings were transplanted to the paddy field in June and left there until the harvest stage in October.The paddy field (yellow-brown soil type) was located in a subtropical area, specifically in Baima of Nanjing (119° 18′ E, 31° 61′ N) under long-day conditions (13.5 h day) in summer.Rice seedlings were planted at a distance of 20 cm, and 7 seedlings × 7 rows were planted under 75, 150 and 350 kg ha −1 N. N fertilizers (urea) were applied to the field on the day before transplanting, tillering and flowering, respectively, with 50%, 25% and 25% of the total nitrogen fertilizer applied.
To measure agronomic traits, such as tiller numbers and yield per plant, ten plants were randomly selected from each genotype under different N conditions.A soil column with an area of 30 × 30 cm 2 and a depth of 45 cm surrounding the selected plants was excavated to determine the root phenotype under soil conditions.After removing the soil, clods covering the roots and clay adhering to the root surface was gently washed away with water.Following removal of the soil, the roots were measured and photographed.

Total tissue N content analysis
Ten individual rice seedlings of WT (O.sativa cv.Wuyungeng7), rohan and transgenic ASL-OE plants were harvested at ripping stage from the field and heated at 105 °C for 30 min.Dry weight was recorded as biomass values.Total N content in plants was measured according to the Kjedhal method 75 and determined using a continuous flow analyser (SEAL AA3).Total N content was estimated as the sum of the N contents of all plant's parts.NUE was calculated by grain yield per N supplied.

IAA content analysis
Roots from WT, rohan, rohan-C1 and pin1apin1b seedlings were harvested after the indicated treatments, and immediately frozen in liquid nitrogen for IAA content analysis in accordance with a published procedure 76 .Before chromatographic measurement, the dehydrated precipitate (100 mg) was resuspended with 200 μl of 80% methanol.For chromatographic separations, ultrahigh performance liquid chromatography (UHPLC) (Acquity UPLC-I-Class, Waters) equipped with a reverse-phase column (BEH-C18 column, 2.1 × 50 mm, 1.7 μm; Waters) was used as the stationary phase, and formic acid (FA) in water (0.1%, v/v, buffer A) and methanol (buffer B) were employed as the mobile phase.Auxin extracts were subjected to a tandem quadrupole mass spectrometer (Xevo TQ-S micro; Waters), for IAA quantification equipped with an electrospray ionization source.Data acquisition and processing were performed using Masslynx software (v.4.1;Waters).Mass transitions were monitored as follows: m/z 176.07-130.00;and external correction method based on IAA standards was used for relative IAA measurement.

Statistics and reproducibility
The experiments performed in this study were repeated at least three times, and similar results were obtained.All the results were analysed and plotted in GraphPad Prism v.9 (www.graphpad.com/features).SPSS software was used for statistical analysis.Significant differences between the two sets of data were determined by Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001), whereas differences among more than two sets of data were analysed with one-way analysis of variance (ANOVA) followed by Duncan's multiple comparisons (P < 0.05).The sample size used to derive the statistics is indicated in the figure legends or in the figures.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Fig. 1 |
Fig. 1 | rohan root growth is hypersensitive to NH 4 + .a,b, Root phenotype (a) and SR length (b) of WT and rohan seedlings treated with or without 2.5 mM NH 4 + for 15 days.The white dotted line indicates the position of the root tip when the seedlings were transferred to media supplied with or without NH 4 + .Scale bar, 1 cm.Data represent the mean ± s.d.c, Confocal images (upper) and EdU staining (lower) of WT and rohan root meristem under N-free and NH 4 + treatments for 4 days.Red and yellow arrowheads indicate stem cells and the start of the transition zone, respectively.White arrowheads indicate the most shootward root tip EdU staining position.Scale bar, 100 μm.d, Quantification of Article https://doi.org/10.1038/s41477-023-01494-x

PFumarateFig. 2 |
Fig. 2 | ROHAN encodes a plastid-localized ASL.a, Location of the rohan mutation site (red arrowhead) in ASL.UTR, untranslated region.b,c, Root phenotype (b) and SR length (c) of the indicated seedlings treated with 2.5 mM NH 4 + for 6 days.rohan-C1 and rohan-C2 are two independent complementary lines of rohan, and asl CR 4 and asl CR 23 are two independent CRISPR lines.The white dotted line indicates the position of the root tip when the seedlings were transferred to media supplied with NH 4 + .Scale bar, 1 cm.Data represent the mean ± s.d.(numbers in columns represent the number of individual seedlings treated), and the letters denote significant differences (P < 0.05, by one-way ANOVA followed by Duncan's test).d,e, Schematic representation of the urea cycle pathway (d) and relative intensity (e) of argininosuccinic acid, citrulline, N-feruloylputrescine and N-feruloylagmatine between WT and rohan seedlings based on widely targeted metabolomic analysis.Data represent the mean ± s.d. of three biological replicates, and asterisks indicate significant differences

Fig. 3 | 4 +/
Fig. 3 | rohan is defective in the conversion of NH 4 + /Gln to Arg. a, Contents of Gln, Arg and free NH 4 + in the roots of indicated seedlings treated with or without NH 4 + for 6 days.Data represent the mean ± s.d. of three biological replicates.DW, dry weight.b, Proposed working model for the regulation of ASL in Gln and Arg metabolism.c,d, Root phenotype (c) and SR length (d) of WT and rohan seedlings treated with 0.3 mM Gln, 10 μM MSO and 0.3 mM Arg, under the indicated N conditions for 6 days.Scale bar, 1 cm.Asterisks indicate significant differences in relation to the WT or rohan under 0 μM chemical treatments (two-sided Student's t-test, **P < 0.01, ****P < 0.0001).e,f, Root phenotype (e) and SR length (f) of WT (O.sativa cv.Nipponbare), gs1;1 GS1 mutant and qko-1 quadruple mutant of AMT seedlings treated with or without 2.5 mM NH 4 + for 6 days.Scale bar, 1 cm.Data represent the mean ± s.d.(numbers in columns represent the number Articlehttps://doi.org/10.1038/s41477-023-01494-x

Fig. 4 |
Fig. 4 | The root response of rohan to NH 4 + is mediated by auxin.a, Heatmap of expression Z-scores of DEGs that are relevant to auxin signal transduction and homeostasis.b,c, Expression pattern (b) and quantification of DR5rev:3xVENUS-N7 signal (c) in the root stele and epidermis of WT and rohan seedlings under the indicated treatments for 2 days.The white and green arrowheads indicate stele (St) and epidermis (Ep), respectively; 2.5 mM NH 4 + , 0.3 mM Gln, 0.3 mM Arg, 10 nM IAA and 10 nM NPA were used.Scale bar, 100 μm.d,e, Root phenotype (d) and relative SR length (e) of WT and rohan seedlings treated with 100 nM NPA or BUM in the presence of 2.5 mM NH 4 + for 6 days.Scale bar, 1 cm.f,g, Root phenotype of WT and pin1apin1b seedlings treated with 2.5 mM NH 4 + (f) and relative SR length (g) of WT and pin1apin1b seedlings grown under N-free conditions or 2.5 mM NH 4 + for 6 days.Scale bar, 1 cm.Data represent

+
Article https://doi.org/10.1038/s41477-023-01494-xcould considerably alleviate NH 4 + -inhibition of root growth, they also reported retarded shoot growth, indicating an antagonistic interplay between NH 4+ tolerance and NUE30,31 .Here, we identified ASL, a gene encoding a plastid-localized ASL, essential in determining the NH 4 + tolerance of rice roots.Our study revealed that NH 4 + toxicity impeding rice root growth results from the accumulation of NH 4 + /Gln in roots.ASL is transcriptionally induced in the root meristem by NH 4 + and facilitates the conversion of over-accumulated NH 4

Fig. 5 |
Fig. 5 | Genetic variation in ASL is associated with its regulatory role in root response to NH 4 + .a, Nucleotide diversity (π) of ASL gene and flanking regions between different rice subspecies.b, Mean Fst value of ASL gene and flanking regions between different rice subspecies.c, Geographic distribution of the SNP(Chr3:10847318) allele frequency in different rice varieties based on the 3,000 Rice Genome database.d, Distribution of the SNP(Chr3:10847318) in japonica and indica rice subspecies based on the 3,000 Rice Genome Project database.e, Amino acid change caused by the SNP mutant (A < -G).f, Comparison of relative SR length (+N/−N) between japonica and indica subspecies seedlings grown under 2.5 mM NH 4 + (+N) or N-free (−N) condition for 6 days.The thick dashed line indicates the median, the thin dashed lines indicate the 25th and 75th percentiles (n = 50 cultivars for either japonica or indica subspecies, ≥10 seedlings per cultivar) and the asterisk indicates a significant difference (two-sided Student's t-test).g, Comparison of relative

Fig. 6 | 4 +.
Fig. 6 | ASL expression determines rice tolerance to NH 4 + toxicity and NUE.a,b, Root phenotype (a) and relative SR length (b) of the WT and two independents transgenic ASL overexpression lines treated with varying concentrations of NH 4 + for 6 days.The white dotted line indicates the position of the root tip when the seedlings were transferred to media supplemented with NH 4 + .Scale bar, 1 cm. in the boxplot, the centre line is the median, the box indicates the upper/lower quartiles and whiskers denote minimum/maximum (n is the number of individual seedlings treated).Asterisks indicate significant differences relative to WT under each concentration of NH 4 + (two-sided Student's t-test).c, Gross morphology (upper) and total grains per plant (lower) of WT, Photosynthesis

Extended Data Fig. 1 | 4 +,Extended Data Fig. 7 | 4 +/Extended Data Fig. 9 |NH 4 +-
The seminal root growth of rohan is hypersensitive to external NH 4 + .a, Relative seminal roots (SR) of wild-type (WT) and rohan seedlings treated with varying concentrations of NH 4 + and NO 3 − for 6 days.b, Relative SR length of WT and rohan plants treated without or with 2.5 mM NH 4 + over one day to five days.c,d, Root phenotype (c) and SR length (d) of WT and rohan seedlings grown in modified Kimura B solution supplied with or without indicated nutrient elements.Scale bars, 1 cm.Data represent the means ± s.d.(numbers in columns represent the number of individual seedlings treated).e,f, Root (e) and SR length (f) of WT and rohan seedlings grown in H 2 O supplied with or without indicated nutrients.the concentrations of chemicals were used as follows: 0.3 mM Gln, 1.25 mM (NH 4 ) 2 SO 4 , 0.3 mM NaH 2 PO 4 , 0.65 mM K 2 SO 4 , 1 mM CaCl 2 , 1 mM MgSO 4 , 20 μM EDTA-Fe, and 0.5 mM Na 2 SiO 3 .Scale bars, 1 cm.Data represent the means ± s.d.(numbers in columns represent the number of individual seedlings treated).h,i, Confocal images (h) and cortex cell length (i) in root maturation zone of WT and rohan seedlings treated with or without NH 4 + for 4 days.Ep, epidermis.Ex, exodermis.Sc, Sclerenchyma.Co, cortex.Scale bars, 100 μm.Data represent the means ± s.d.(numbers in columns represent the number of individual seedlings treated).a,b, Boxplot, median (centerline), upper/lower quartiles (box), minimum/maximum (whiskers) (n indicates the number of individual seedlings treated), and the asterisks indicate significant differences between WT and rohan under each concentration of NH 4 + or NO 3 − (a) or each day (b) (two-sided student's t-test, **** P < 0.0001).c,e, The yellow dotted lines indicate the positions of root tip when the seedings were transferred to media supplied with or without indicated nutrients.f,i, The asterisks indicate significant differences (two-sided student's t-test); ns, not significant.Extended Data Fig. 2 | ASL-mediated seminal root elongation is independent of NH 4 + uptake and low pH.a, Relative expression level of the ammonium transporters AMT1.1, AMT1.2, AMT1.3 and AMT2.1 in the root tips of wild-type (WT) and rohan seedlings treated with or without 2.5 mM NH 4 + for 4 days.Data represent the means ± s.e. of three biological replicates, and the asterisks indicate significant differences (two-sided student's t-test).b, Quantification of the net NH 4 + and proton fluxes in the root tip of WT and rohan seedlings treated with or without 2.5 mM NH Data represent the means ± s.d.(n = 3 individual seedlings of each line), and the asterisks indicate significant differences between WT and rohan at each tested position along the root (two-sided student's t-test, **** P < 0.0001).c, BCP staining of grinded root tissues showing the root acidification of WT, rohan and rohan-C1 seedlings grown under either N-free or 2.5 mM NH 4 + condition (three biological replicates).d,e, Root phenotype (d) and seminal roots (SR) length (e) of WT and rohan seedlings treated with or without 2.5 mM NH 4 + , under the conditions of pH 5.5 and pH 4.0.The white dotted line indicates the position of the root tips when the seedlings were transferred to N-free or NH 4 + media with different pHs.Scale bar, 1 cm.Data represent the means ± s.d.(numbers in columns represent the number of individual seedlings treated).The asterisks indicate significant differences between pH 5.5 and pH 4.0 treatments (two-sided student's t-test).Extended Data Fig. 3 | Map-based cloning of ASL gene.a, Root phenotypes of the wild-type (WT), rohan, and the F 1 progeny of the rohan x wild-type backcross treated with 2.5 mM NH 4 + for 6 days.12 independent F1 progeny seedlings showed identical root phenotype.Scale bar, 1 cm.b, Segregation statistic of the wild-type and rohan root phenotypes in the backcross F 2 progeny.c, Euclidean distance association analysis of the ASL candidate interval in the rice genome.ED 4 was calculated based on the SNP and InDel frequency.The red arrow indicates the chromosomal location of the ASL gene.ED, Euclidean Distance.d, Sketch map of mutation sites in the CRISPR knockout lines of ASL.Red triangles indicate the mutation target sites.Extended Data Fig. 6 | Metabolomic analysis of wild-type and rohan.a, Principal component analysis (PCA) between wild-type (WT) and rohan.b, Metabolites abundant ranking profile based on log2 (Fold change) and p-value between wild-type and rohan.c, Distribution of differential metabolites.Red represents up-regulating in rohan while blue represents down-regulating.d, KEGG classification of differential metabolites between WT and rohan.e, KEGG enrichment analysis of differential metabolites between WT and rohan.ASL acts on the conversion of NH Gln to Arg. a, Contents of free amino acids in the roots of wild-type (WT), rohan, and rohan-C1 treated with or without 2.5 mM NH 4 + for 6 days.rohan-C1 is a complementary line of the rohan.Data represent the means ± s.d. of four biological repeats, and the asterisks indicate significant differences relative to WT (two-sided student's t-test), ND, not detected.b,c, Root phenotype (b) and relative seminal roots (SR) length (c) of WT and rohan seedlings grown under indicated treatments for 6 days.The white dotted line indicates the position of the root tip when the seedlings were transferred to media supplied with indicated chemicals.Scale bars, 1 cm.Data represent the means ± s.d.(numbers in columns represent the number of individual seedlings treated), and the letters denote significant differences (P < 0.05, by one-way ANOVA followed by Duncan's test).d, Relative seminal roots (SR) length of WT and rohan seedlings treated with varying concentrations of Gln, MSO, and Arg, under the indicated N condition for 6 days.Boxplot, median (centerline), upper/lower quartiles (box), minimum/ maximum (whiskers) (n indicates the number of individual seedlings treated), and the asterisks indicate significant differences relative to WT or rohan under control treatment (0 mM Gln, MSO, or Arg) (two-sided student's t-test, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001).Similar results were obtained in three independent experiments.The induced expression of proton flux-associated genes is suppressed in rohan.Relative expression levels of indicated proton flux-associated genes in the root tips of wild-type (WT) and rohan seedlings treated with or without 2.5 mM NH 4 + for 3 days.Data represent the means ± s.e. of three biological repeats, and the asterisks indicate significant differences (two-sided student's t-test).