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

doi:10.21203/rs.3.rs-1474062/v1

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

https://doi.org/10.21203/rs.3.rs-1474062/v1

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published 21 Aug, 2023

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Ammonium toxicity affecting plant metabolism and development is a worldwide problem impeding crop production. Remarkably, rice (Oryza sativa L) favors 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 the rohan mutant showing root hyper-sensitivity to ammonium due to a missense mutation in an arginine-succinate lyase-encoding gene. ROHAN localizes to plastids while its expression is induced by ammonium. ROHAN alleviates ammonium-inhibited root elongation by converting the excessive glutamine into 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 ROHAN allele between Japonica and Indica subspecies explaining their different root sensitivity towards ammonium. Finally, we show that ROHAN expression positively correlates with root ammonium tolerance and that nitrogen use efficiency and yield can be improved through a gain-of-function approach.

Ammonium toxicity

Nitrogen use efficiency

Root elongation

ROHAN

Gln

Arg

Auxin

Oryza sativa L.

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 (NO₃^-) and ammonium (NH₄⁺) are two primary inorganic N sources for plants. Nitrate is reduced into NH₄⁺ by ferredoxin-dependent nitrite reductases, consuming NADH or NADPH as reductants, while NH₄⁺ can be directly incorporated into amino acids via the GS-GOGAT pathway¹. Thus NH₄⁺ is considered a more efficient and economical N fertilizer than NO₃^- to promote crop yield². However, excessive NH₄⁺ causes severe toxic effects on plant shoot and root growth³. Because the intensive use of NH₄⁺-based fertilizers in the past decades has resulted in dangerous accumulation of NH₄⁺in agricultural soils⁴, improving plant tolerance to NH₄⁺ is key to the enhanced utilization of NH₄⁺ and improve N use efficiency (NUE).

The mechanism underlying plant NH₄⁺ tolerance have been extensively investigated in Arabidopsis through genetic approaches, and NH₄⁺ toxicity is suggested to be a consequence of repressed metabolic processes³, protein glycosylation⁵, chloroplast development⁶, hormone metabolism^6–8, glucosinolate metabolism and Fe homeostasis⁹. Recent studies have also revealed that NH₄⁺ toxicity is coupled with NH₄⁺ uptake and glutamine (Gln) accumulation by reducing root apoplast pH and provoking severe acidic stress to Arabidopsis^10,11. In Arabidopsis mutants showing reduced NH₄⁺ uptake (for instance, the ammonium transporters AMTs quadruple mutant) or Gln synthesis (e.g. gln2-1), the toxic effects of NH₄⁺ on seedling growth are vastly relieved. The NH₄⁺ toxicity can also be alleviated when the NRT1.1-mediated nitrate influx is reduced or the SLAH3-mediated nitrate efflux is enhanced, which both increase external nitrate levels to buffer the rhizosphere pH^12,13. Together, these results suggest that root acidification is the primary cause of NH₄⁺ toxicity in Arabidopsis.

Rice is considered as a NH₄⁺ tolerant species since NH₄⁺ is the primary N source for rice in the paddy field, where the nitrification process is low¹⁴. AMT-mediated NH₄⁺ uptake plays a predominant role in promoting rice shoot growth and yield¹⁵ but negatively regulates root system architecture, in particular by reducing rice root elongation and gravitropism^16,17. Several studies suggested that the inhibitory effect of NH₄⁺ 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^11,18,19. Our recent work also showed that root acidification resulting from NH₄⁺ uptake triggered the asymmetric auxin distribution in the root cap, leading to the loss of root gravitropism under high NH₄⁺¹¹. However, root elongation inhibited by NH₄⁺could not be fully mitigated by buffering the rhizosphere pH or by supplying external nitrate supply, thus suggesting a different impact of NH₄⁺ in root development between Arabidopsis and rice.

In order to decipher the molecular machinery allowing rice to circumvent NH₄⁺ toxicity on root growth, we employed a forward genetic approach and identified ROOT HYPERSENSITIVE TO AMMONIUM NITROGEN (ROHAN) as a key regulator of NH₄⁺ tolerance in rice. We further show that ROHAN encodes a plastid-localized argininosuccinate lyase which metabolizes excessive Gln into arginine (Arg), by doing so, alleviating NH₄⁺-inhibition of root elongation. Additionally, we show that a substitution in the ROHAN coding sequence present of a particular rice cultivar can confer root tolerance to high NH₄⁺. Finally, we demonstrate that ROHAN not only help to cope with NH4⁺ toxicity but also promotes rice yield and NUE under low and high N supplies, which represents an important target for rice molecular breeding of NH₄⁺ tolerance and NUE.

Rice rohan mutants display root growth hypersensitivity to ammonium

To discover the genetic control of rice root responses to NH₄⁺, an EMS-mutagenized population (10,000 mutant lines) was generated starting from a local elite Japonica cultivar Wuyungeng 7 (hereafter designated as the wild-type). We then screened for mutants showing altered root length when grown in hydroponic solutions supplied with 2.5 mM NH₄⁺, a concentration that is known to cause inhibition of seminal root (SR) elongation¹¹. Because NH₄⁺ uptake results in proton release¹¹, MES was supplemented in order to exclude root phenotypes caused by lowering pH. We identified one mutant displaying severely inhibited SR elongation when compared with the wild-type under high NH₄⁺ supply (73.9% shorter than the wild-type SR length). In absence of N however, the mutant SR length was slightly shorter than the wild-type (35.9% decrease) (Fig. 1a and b). We further observed that the highest sensitivity of SR elongation to NH₄⁺ in the mutant occurred at 2.5 mM NH₄⁺, and that SR elongation was less sensitive to varying NO₃^- concentrations (Extended Data Fig. 1a). A time-course experiment moreover showed that the inhibitory effect of NH₄⁺ on SR elongation in the mutant was more pronounced after three days of treatment than one day (Extended Data Fig. 1b). Furthermore, the root phenotype of this mutant was consistently observed in the paddy field supplied with low N (75 kg/ha urea) and high N supply (350 kg/ha urea) (Fig. 1e and f). Because of the striking inhibition of root growth under ammonium supply, we named the mutant root hypersensitive to ammonium nitrogen (rohan).

Root elongation is determined by the meristem cell division activity and the subsequent cell elongation. Thus, we investigated the root meristem development in rohan under different N conditions. Compared to the N-free control condition, NH₄⁺ treatment caused a significant reduction of both meristem length and cortical cell number in wild-type, and this inhibitory effect on meristem division was enhanced in rohan (Fig. 1c and d). In contrast, the cortical cell elongation was not affected in rohan neither for N-free or NH₄⁺ supply (Extended Data Fig. 1c and d). We further determined the root meristem activity of the mutant rohan by employing an EdU-based S-phase (DNA synthesis phase) assay²⁰. NH₄⁺ treatment led to weaker EdU signal in the root tips of both the wild-type and rohan than under N-free condition. Nevertheless, root meristems of rohan showed less EdU signal than wild-type under high NH₄⁺ supply (Fig. 1c and d). These results indicated that root phenotype of rohan resulted from reduced meristem activity under high NH₄⁺ supply.

Next, we investigated whether the hypersensitive rohan root response to NH₄⁺ toxicity was caused by NH₄⁺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 wild-type and rohan (Extended Data Fig. 2a). Consistently, the rohan mutation had a minor effect on NH₄⁺ uptake of roots and it also showed a lower root proton influx than the wild-type (Fig. 1g, and Extended Data Fig. 2b). Additionally, rohan exhibited an equal reduction in root elongation under low pH treatment as wild-type (Extended Data Fig. 2c and d). These results therefore suggested that the root response of rohan to NH₄⁺ was most likely not a consequence of NH₄⁺ uptake and acidic stress.

ROHAN encodes an N-responsive and plasmid-localized argininosuccinate lyase

To identify the mutation causing the root hypersensitivity of rohan to ammonium, we performed a MutMap analysis using an F₂ 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 F₁ progeny and self-pollinated F₂ population. The seedlings of the F₁ progeny showed a parent-like root sensitivity to NH₄⁺ treatment, and F₂ progeny phenotypically segregated in nearly 3:1 ratio for a respective NH₄⁺ sensitive and hypersensitive root phenotype (χ2 = 0.58; P < 0.05) (Extended Data Fig. 3a and b). Detailed bulked genomic sequencing and MutMap analysis on the F₂ progeny identified a non-synonymous single base change (C to T) at position 1706-bp downstream of the start codon of Os03g05500 (Fig. 2a and Extended Data Fig. 3c). The gene encodes an argininosuccinate lyase (ASL), which is known to catalyzes the conversion of argininosuccinate into arginine and fumarate²¹. ROHAN/ASL gene is highly conserved within the plant kingdom (Extended Data Fig. 4a).

In rice, ROHAN/ASL contains seven exons, and the mutation in rohan results in a Pro211Leu (P211L) substitution located at the third exon (Fig. 2a). Pro211 is highly conserved among plant species (Extended Data Fig. 4c). By structural modelling the ROHAN/ASL protein based on the ASL crystal structure in Bacillus coli, we found the Pro211 at the hinge region linking protein domains (Fig. 2f and Extended Data Fig. 4c). To confirm that the P211L mutation is responsible for ROHAN function and root response to NH₄⁺, we further performed genetic complementation by transforming rohan mutants with VENUS-fused ROHAN/ASL wild-type protein driven by its native promoter (proROHAN:ROHAN-VENUS rohan). Over 24 individual lines were obtained, and lines with ROHAN-VENUS expression in the root showed identical root sensitivity to NH₄⁺ as the wild-type (Fig. 2b and c). Additionally, knock-out lines of ROHAN/ASL1, generated by CRISPR-cas9, mimicked the root phenotype of rohan under high NH₄⁺ supply (Fig. 2b and c). Thus, ASL is responsible for rohan root phenotype while its function is dependent on the Pro211 residue.

By generating a transcriptional reporter line of ROHAN in the wild-type background, we observed a strong expression of proROHAN:GUS in the root meristem and the stele of the elongation zone. proROHAN:GUS was also found to be expressed in lateral root primordia and emerged lateral roots (Extended Data Fig. 4b), indicating a critical role of ROHAN in regulating rice root development. In the complemented lines, the ROHAN-VENUS signal was also highly detected at the root meristem and localized to plastids at the cellular level (Fig. 2d). The Pro211Leu (P211L) did not affect the subcellular localization of ROHAN (Fig. 2e) and suggested the Pro211 residue is key to its protein function in plastids.

We next investigated whether ROHAN expression was regulated by external NH₄⁺ supply. qRT-PCR analysis showed that ROHAN expression in roots gradually increased by NH₄⁺ application over 24h, while it was less induced by NO₃^- treatment (Fig. 2g). This was further confirmed by the expression analysis of proROHAN:GUS, showing a strong induction of ROHAN expression in the root meristem by NH₄⁺ after 24h (Fig. 2h). All together, these data demonstrate that ROHAN is a NH₄⁺ responsive gene that encodes a plasmid-localized protein regulating root development.

Metabolic defect of NH₄⁺/Gln conversion to Arg in rohan causes its root hypersensitivity to ammonium

The ROHAN encoded ASL has previously been shown to play a role in amino acid (AA) metabolism by catalyzing the last step of Arg biosynthesis. Furthermore, Arg produced by ASL is believed to be required for normal root elongation in rice²² suggesting a possible link between ROHAN function in plastids and root tolerance to NH₄⁺. To address the latter, we measured the content of 20 free AAs in roots of rohan and wild-type under N-free condition and high NH₄⁺ supply. NH₄⁺ treatment increased the overall content of AAs, especially Asn and Gln both in wild-type and rohan roots (Extended Data Fig. 5a). Remarkably, upon high NH₄⁺supply, more Gln and free NH₄⁺ accumulated in roots of the rohan and rohan^CR4 mutants than in the wild-type. The opposite trend was observed for the Arg content, being more reduced in the mutants than in the wild-type roots (Fig. 3a). NH₄⁺ is assimilated to Gln by Glutamine synthetase (GS), and Gln is subsequently converted to Arg by the Urea cycle (Fig. 3b)^23,24. Thus, the increased Gln and NH₄⁺ in rohan roots might be due to the perturbation of Arg synthesis. In the complementing line of rohan, the contents of Gln, NH₄⁺ and Arg in the root were all recovered to the wild-type levels (Fig. 3a and Extended Data Fig. 5a), confirming that ROHAN acts on the conversion of NH₄⁺/Gln to Arg.

Next, we questioned if the observed metabolic defect was associated with rohan root sensitivity to NH₄⁺ by testing the effects of Gln and Arg application on root growth. We observed a substantial inhibition of SR meristem cell division and elongation at 0.3 mM Gln treatment in the wild-type. In rohan however, Gln concentration as low as 0.03 mM Gln already caused significant inhibition of SR meristem cell division and elongation (Fig. 3c, d, h, i and Extended Data Fig. 5g). This suggested that root elongation of rohan is hypersensitive to Gln, mimicking its response to NH₄⁺. Interestingly, the addition of methionine sulfoximine (MSO), a potent inhibitor of GS²⁵, restored SR elongation in both the wild-type and rohan under high NH₄⁺ supply (Fig. 3c and d). We further used CRISPR-Cas9 to generate quadruple mutants of AMTs (qko)²⁶ and a gs1;1 single mutant, which are defective in Gln synthesis (Extended Data Fig. 5b and c)²⁷. Both qko and gs1;1 mutants showed resistance to NH₄⁺ treatment towards SR elongation (Fig. 3e and f) indicating that Gln accumulation is a major factor responsible for NH₄⁺ toxicity to root elongation in rice.

Intriguingly, the arrested SR meristem cell division and elongation in rohan under high NH₄⁺ could be rescued by the supply of Arg, with the optimum concentration at 0.3 mM, while it had a minor effect on SR meristem cell division and elongation in the wild-type (Fig. 3d, h, i, and Extended Data Fig. 5d). However, external Arg supplies could not rescue the NH₄⁺- and Gln-inhibited SR elongation in the wild-type (Extended Data Fig. 5e and f), suggesting that endogenous Arg, produced by ROHAN, is required for root elongation under high NH₄⁺. Thus, we concluded that the biological function of ROHAN in converting excessive NH₄⁺/Gln to Arg in vivo, enables the alleviation of NH₄⁺ inhibited rice root elongation.

ROHAN promotes nitrogen metabolism and photosynthesis under high ammonium supply

We next aimed to unveil the mechanism underlying ROHAN-mediated root tolerance to NH₄⁺ by performing RNA-seq analysis of root meristems in rohan and wild-type under N-free condition and NH₄⁺ supply. In agreement with the hypersensitive root phenotypes of rohan mutants under high NH₄⁺, a more diverged transcriptional pattern between rohan mutants and the wild-type under high NH₄⁺ compared to N-free was observed (Extended Data Fig. 6a).

To specify the molecular pathways downstream of ROHAN, we analyzed differentially expressed genes in the wild-type and rohan under NH₄⁺ and N-free conditions. NH₄⁺ treatment led to a higher number of differentially expressed genes in rohan than wild-type after 1d treatment, and the number of differentially expressed genes in rohan was increased mainly after 3 days of treatment (Extended Data Fig. 6b). Gene set enrichment analysis of these differentially expressed genes based on GO and KEGG suggested that the N metabolism pathway was significantly affected in rohan (Extended Data Fig. 6c), 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 significantly down-regulated in rohan under NH₄⁺ treatment (Extended Data Fig. 6d and e), which might result from a feedback inhibition by the accumulated Gln in rohan.

In addition, photosynthesis pathway genes were also significantly enriched in rohan under high NH₄⁺ (Extended Data Fig. 6b). Increasing N supply can promote photosynthesis in rice^28,29. Consistently, high NH₄⁺ supply significantly induced photosynthetic rate, transpiration rate, stomatal conductance and PSII in wild-type seedlings, severely repressed in rohan, correlating with retarded shoot growth in rohan mutants under NH₄⁺ supply (Extended Data Fig.7b). The photosynthesis and shoot growth deficiency in rohan was recovered in the complementation line or by external Arg supply (Extended Data Fig.7a). These results show the importance of ROHAN for efficient AA metabolism in photosynthesis.

The rohan root response to NH₄⁺ is mediated by auxin metabolism

Interestingly, gene set enrichment analysis of differentially expressed genes revealed that hormone metabolism was also affected in rohan under high NH₄⁺ (Extended Data Fig. 8a). Among plant hormones, auxin plays a crucial role in regulating root elongation and responses to N sources and photosynthesis^30,31. The expression of several genes involved in auxin conjugation (i.e., GH3s) and auxin signaling (i.e., ARF2, IAA6, IAA14, and small auxin-up RNAs, SAURs) was found to be down-regulated in rohan under high NH₄⁺ (Fig. 4a). GH3s and SAURs are auxin-inducible genes³², thus the changes in their expression indicated altered endogenous auxin levels in rohan. By measuring endogenous IAA content, we indeed detected a significant reduction of IAA content in the root tip of rohan as compared to that of in the wild-type under high NH₄⁺supply, while it was only slightly reduced in rohan under N deficiency (Extended Data Fig. 8b). In the complementation line of rohan, IAA content in the root tip was fully recovered (Extended Data Fig. 8b), showing the importance of ROHAN 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 reporter³³, into rohan mutants by crossing. The DR5 signal was strong in the root epidermis and stele of both the wild-type and rohan in N-free condition. Under high NH₄⁺ treatment, wild-type roots had a lower DR5 signal in the stele but an increased signal in the epidermis, whereas it almost disappeared from the root stele and epidermis of rohan. Interestingly, exogenous Arg elevated DR5 expression in the root stele and epidermis of rohan mutants (Fig. 4b, c and Extended Data Fig. 8c), corresponding to its effect in restoring SR elongation in rohan mutants. These results indicate that the changes of auxin levels in the root tip are correlated with the ROHAN-mediated SR elongation in response to NH₄⁺.

Regulation of the root meristem activity and root elongation in response to N sources is mediated by auxin efflux carrier PIN-dependent polar auxin transport (PAT)³⁴. Accordingly, we also found that several PIN encoding genes were transcriptionally repressed in rohan mutants under high NH₄⁺ (Fig. 4a). We further validated the role of PINs in mediating rohan root response to NH₄⁺ by using two well-characterized potent polar auxin transport inhibitors, 1-N-naphthylphthalamic acid (NPA) and BUM^35,36, in order to bypass the functional redundancy of PIN proteins. Under high NH₄⁺ supply, rohan mutants showed higher sensitivity to NPA and BUM with respect to SR elongation, as this was completely arrested at low concentration of NPA and BUM (Fig. 4d and e). Following inhibition of SR elongation by NPA, NPA treatment resulted in a higher accumulation of DR5 expression in the root epidermis of the wild-type upon NH₄⁺ treatment (N-free), while this induction was blocked in rohan. Moreover, exogenous IAA failed to trigger DR5 expression and root elongation in rohan mutants, suggesting a complete suppression of PIN activity in rohan mutants (Fig. 4b and Extended Data Fig. 8c). We further generated a CRISPR-Cas9 knock-out mutant of PIN1a and PIN1b, whose expression abundances were significantly repressed in rohan mutants under high NH₄⁺. The pin1apin1b mutants showed shorter root meristems and SRs, mimicking the rohan mutant root phenotype (Fig. 4g and i), confirming the requirement of PIN-mediated polar auxin transport for the ROHAN-dependent effect on SR elongation. Altogether, our data demonstrated a critical role of auxin metabolism and polar auxin transport underlying ROHAN-controlled root tolerance to ammonium stress.

ROHAN allele confers rice root tolerance to NH₄⁺ toxicity

Because of the significant role of ROHAN in NH₄⁺ root tolerance, we further investigated its genetic variation in rice germplasm. By nucleotide diversity (π) and Fst analysis, we found evidence for decreased nucleotide diversity of ROHAN in the japonica and indica subpopulations as compared to the wild rice population (Oryza rufipogon), while regions around the ROHAN locus showed significant differentiation between subspecies japonica and indica, indicative for a possible result of selective pressure during domestication (Fig. 5a and b). Detailed analysis showed 37 SNP variants, including 35 introns, one missense, and one synonymous within the ROHAN gene. We further focused on the missense SNP (Chr3:10847318, c.3972A>G), which showed apparent Indica (94.91%)-Japonica (96.76%) differentiation and led to a Lys470Arg substitution (Fig. 5d and e). The allele frequency of the SNP from japonica, indica I, indica II, indica III, aus, and aromas subpopulations were calculated and shown on the geographic map (Fig. 5c).

To validate the function of the SNP, we randomly selected in total 100 SNP^A-variant and SNP^G-variant accessions to evaluate their root sensitivity to high NH₄⁺. Our results revealed a higher root sensitivity to NH₄⁺ in SNP^G-variant accessions than SNP^A-variant accessions (Fig. 5f). Furthermore, ROHAN expression in SNP^G-variant accessions was less induced than SNP^A-variant accessions by high NH₄⁺ (Fig. 5g). These results suggest that the SNP variation of ROHAN might be associated with transcriptional responses and SR elongation to NH₄⁺. To experimentally validate this possibility, we further conducted allelic complementation by transforming SNP^G-variant (proROHAN:ROHAN^K470R, with only one missense SNP difference from the Japonica cultivar Wuyungeng 7 genome) into the rohan. Unlike the SNP^A-variant, which could restore SR elongation (Fig. 2b and c), while independent SNP^G-variant lines failed to rescue the SR elongation under high NH₄⁺ supply (Fig. 5h and i), demonstrating that the natural ROHAN variants cause the divergence of the root tolerance to NH₄⁺.

ROHAN expression determines the rice tolerance to NH₄⁺ toxicity and nitrogen use efficiency

Since ROHAN expression is correlated with root tolerance to NH₄⁺, we further generated overexpression lines of ROHAN (UBIL:ROHAN) in a rohan background. Two independent UBIL:ROHAN lines, which had higher ROHAN expression (Extended Data Fig. 9b), showed longer SR than the wild-type upon different increasing NH₄⁺ concentrations (2.5mM ~ 10mM) (Fig. 6a and b). In contrast, rohan displayed lower expression of ROHAN in roots and shorter SRs than wild-type (Extended Data Fig. 9b). These results underline once again the importance of ROHAN expression for root tolerance to NH₄⁺.

We further planted the mutant and overexpression lines of ROHAN in the paddy field supplied with low (75 kg/ha), moderate (150 kg/ha), and high nitrogen (350 kg/ha). Compared to the wild-type, ROHAN overexpression led to a significant increase in tiller number, grain yield, grain proteins and nitrogen use efficiency under all N conditions, whereas rohan mutants showed lower tiller number, yield, grain proteins and NUE (Fig. 6c and d). Collectively, these results show that ROHAN expression positively correlates with root tolerance to high NH₄⁺ and NUE.

Ammonium nitrogen can cause severe toxic effects on various plant physiological and developmental processes. In particular, NH₄⁺ represses growth of the root system, which in turn leads to reduced nutrient and water uptake and therefore dramatically decreases crop yield. Improving plant tolerance to NH₄⁺ thus represents a promising strategy to improve crop nitrogen use efficiency and yield. Although previous studies in rice have shown that reduced ammonium uptake or Gln synthesis could considerably alleviate the NH₄⁺-inhibition of root growth, they also reported on retarded shoot growth, indicating an antagonistic interplay between NH₄⁺ tolerance and NUE^26,37. Here, we identified ROHAN, a gene encoding a plastid-localized ASL, essential in determining the NH₄⁺ tolerance of rice roots. Our study revealed that NH₄⁺ toxicity impeding rice root growth results from the accumulation of NH₄⁺/Gln in roots. ROHAN/ASL is transcriptionally induced in the root meristem by NH₄⁺ and facilitates the conversion of over-accumulated NH₄⁺/Gln into Arg, which is essential for auxin homeostasis and root elongation under high NH₄⁺(Fig. 6e). Significantly, induction of ROHAN expression not only promotes rice root tolerance to NH₄⁺ but also increases the NUE and yield under low and high N supplies in the paddy field. Therefore, our results suggest that the ROHAN-mediated Gln to Arg conversion coordinates root tolerance to NH₄⁺ toxicity and NUE.

Amino acids are a primary storage form of N and exert diverse functions in plant development and defense³⁸. NH₄⁺ is assimilated via the GS-GOOAT pathway into Gln and further converted into other amino acids. The ROHAN encoded ASL is one of the rate-limiting enzymes of the urea cycle and is utilized in the production of arginine²¹. ROHAN shares the same plastidial localization with GS1;1¹⁰ (Fig. 2), a critical enzyme for Gln synthesis. By analyzing the amino acid content, ROHAN was found to act specifically in the conversion pathway of Gln to Arg (Fig. 3), indicating a critical role of the urea cycle in Gln metabolism in plastids. In Arabidopsis, the elevated Gln content and root apoplast acidification by high NH₄⁺ supply has been suggested as the primary cause of NH₄⁺ toxicity¹⁰. Consistently, Gln had a similar inhibitory effect on root growth in rice, and rohan mutant roots show hypersensitive to external Gln supply, which can be suppressed when Gln synthesis is attenuated (Fig. 3). These results demonstrate that NH₄⁺-induced accumulation of endogenous Gln is a common mechanism underlying NH₄⁺ toxicity in rice root development. However, rohan mutant roots exhibited normal sensitivity to low pH (Extended Data Fig. 2), arguing that ROHAN-mediated root tolerance to NH₄⁺ is unlikely linked to root acidification.

The regulation of N in plant root development is mediated by phytohormones such as auxin, ethylene, and brassinosteroid^11,39,40. Auxin is a prominent regulator of root meristem activity and root elongation, and also interacts with other hormones to regulate root development⁴¹. Our transcriptome data revealed a robust transcriptional regulation of ROHAN in auxin signaling pathways (Fig. 4). We demonstrated that ROHAN plays a role in maintaining high auxin levels and spatial distribution at the root tip, required for root elongation under high NH₄⁺. Local auxin accumulation is mediated by auxin efflux carrier PIN-dependent polar auxin transport. We further identified PIN1 as a downstream signaling component of ROHAN. PIN1 activity is required for rice root meristem activity and SR elongation. PIN1 is transcriptionally activated by ROHAN to facilitate the tissue-specific auxin accumulation at the root tip to stimulate root elongation. In Arabidopsis, high ammonium was also found to affect PIN activity.to adjust root growth³⁴. Therefore, our study suggests a molecular link between the N metabolic pathway and the phytohormone signal in regulating rice root tolerance to ammonium. Further investigations will be needed to uncover the mechanism underlying the transcriptional regulation of ROHAN and NH₄⁺ in auxin homeostasis.

ROHAN is highly conserved in various plant species. In this study, we further identified a natural variant of ROHAN (SNP^A) in rice that increases root tolerance to high external NH₄⁺. 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 as compared to Indica cultivars (Fig. 5). As ROHAN expression is highly elevated by high NH₄⁺ in Japonica cultivars in contrast to Indica cultivars, the elite allele of ROHAN might determine root tolerance to NH₄⁺ toxicity through regulating its expression in response to external NH₄⁺ supply.

In summary, we identified ROHAN as a critical regulator of NH₄⁺ tolerance and NUE in rice. Our study reveals a molecular link between amino acid metabolism and root response to external N supply and suggests that polar auxin transport-mediated local auxin accumulation in the root meristem is required for ROHAN function. As rice varieties harboring the elite variant of ROHAN display a higher root tolerance to NH₄⁺ without penalty in plant growth and yield, ROHAN may represent a potential candidate target for genetic editing or marker-assisted breeding to develop crop varieties tolerant to ammonium toxicity and help to increase rice NUE and yield under the large quantities of urea-based fertilizer. The identification of ROHAN therefore represents an important element to set up strategies to cope with the excessive use of N fertilizer that has resulted in several environmental issues, such as surface water eutrophication, groundwater pollution, greenhouse gas emission and soil acidification.

Acknowledgements

We thank Hongye Qu, Xiaoli Dai and Kaiyun Qian for their technical help with the ¹⁵N uptake assays and confocal imaging. This work was supported by China National Key Program for Research and Development (2021YFF1000403), National Natural Science Foundation (No. 32072658 and 31822047), Key Research and Development Program of Jiangsu Province (BE2020339), the Fundamental Research Funds for the Central Universities (KJYQ201903 and KYT201802), and Innovative Research Team Development Plan of the Ministry of Education of China (No. IRT_17R56) and the Research Foundation – Flanders (FWO): Bilateral Scientific Cooperation fund with China (NSFC) (No. G002817N).Y.M.X is supported by grant from the Chinese Scholarship Council (CSC, 201806850032).

Author contributions

W.X. and T.B. directed the experiments. Y.M.X and Y.D.L. performed most of the experiments and analysis. L.T.J., L.L.Z, M.Z and L.L. helped with vector construction. Y.H.L helped with root section. M.J.T and M.W. helped the IAA content analysis. W.C.Q. helped the phenotyping analysis. H.D.G., and PM.P. helped the imaging. S.Y.L. helped the amino acid content analysis. All authors discussed the results and contributed to the finalization of the manuscript.

Conflict of interest statement

All authors state no conflict of interest concerning this manuscript.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL databases under the following accession numbers: ROHAN (Os03g0305500), AMT1;1 (Os04g0509600), AMT1;2 (Os02g0620500), AMT1;3 (Os02g0620600), AMT2;1 (Os05g0468700), GS1;1 (Os02g0735200), PIN1a (Os06g0232300), PIN1b (Os02g0743400).

Plant materials

Rice (Orzya sativa cv. Wuyungeng7) seeds were mutagenized by treating them with 1.0% ethyl methylsulfonate (EMS) for 12 h as previously described⁴². M₂ seeds obtained from self-pollinated M₁ plants were used for the screening of NH₄⁺-sensitive mutants based on root elongation at 2.5 mM ammonium. In brief, seeds from 10,000 M₂ lines were bulked, and 100 seeds were sown on a mesh in a 500ml 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 M₃ progeny was finally assessed in the same manner to confirm the NH₄⁺-sensitive phenotype of the candidate mutant rohan. The rice DR5rev:3xVENUS-N7 transgenic reporter line was previously described ³³.

Plant growth conditions

Wild-type, mutants and transgenic rice seeds were surface-sterilized with 70% (v/v) ethanol for 2 min, followed by 30% (v/v) bleach containing 0.01% Tween 80 for 30 min. After 5 times washing with sterilized water, rice seeds were germinated at 37 ℃ for 3 days. Germinated rice seedlings were first grown in water for another 3 days in a growth chamber under a photoperiod of 14 h light (~ 200 μmol•m^-2•sec^-1 light density and ~ 60% humidity) and 10 h dark at 28 ℃. Subsequently, the rice seedlings with around 2 cm seminal roots were transferred to a hydroponic medium with modified Kimura B solution (500 ml volume for each cup with 10 seedlings, pH 5.5, 0.25g MES) for different treatments according to the previous study¹¹. For N-free treatment, nitrogen sources (NH₄)₂SO₄and KNO₃ were replaced with K₂SO₄ at a concentration of 1.25 mM. For NO₃ ^-treatment, (NH₄)₂SO₄ was replaced with 2.5 mM KNO₃. The rice seedlings were treated for 6 days unless otherwise noted, and the hydroponic culture was refreshed every 2 days.

Root phenotype analysis

The rice seedling roots were imaged with a 400dpi resolution by an EPSON Expression 11000XL scanner. Seedling roots were scanned before and after treatments. We then measured the seminal roots (SR) length using Fiji image analysis software (http://fiji.sc/). The SR elongation was finally calculated as the difference between the SR lengths after and before treatments.

Genetic mapping of the rohan mutation

To identify the causal mutation, a F₂population was generated by backcrossing rohan mutant with the parent Wuyungeng 7 then used for genetic mapping through a modified MutMap analysis⁴³. We then performed whole genome re-sequencing of two F₂ segregant bulks: 20 individuals showing the parent phenotype and 20 others displaying the rohan NH₄⁺-sensitive phenotype. Next, DNA was isolated from leaves for each individual using the DNeasy Plant Mini Kit (Qiagen) as previously described⁴², and ultimately each DNA bulk was created by mixing equally individual DNA in order to reduce sequencing bias. Sequencing libraries with an average insert size of approximately 350–500 bp were constructed using the Illumina DNA Prep Kit and sequenced with PE150 mode using Illumina NovaSeq 6000 platform (Zhejiang Annoroad Biotechnology Co., Ltd., China). Raw FASTQ data were trimmed using Fastp v0.20⁴⁴, with sequencing adapters, 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 v0.7.17⁴⁵. PCR duplication was removed by Sambamba v0.8.1⁴⁶. SNP calling was performed using GATK v4.2.1⁴⁷, and all variants were scored by ED (Euclidean distance) and fitted by sliding window approach⁴³. ED⁴ 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. Data availability of the raw Whole-genome sequencing (WGS) datasets, including parents and bulked DNA pools, have been deposited on NCBI BioProject (https://www.ncbi.nlm.nih.gov/bioproject) under the accession number PRJNA808438

RNA-seq analysis

Roots from the wild-type and rohan mutant seedlings, treated with or without high NH₄⁺ for 1 d and 3 d, were collected with three independent biological repeats for RNA-seq analysis. Total RNA was extracted using TRIzol reagent and digested with RNase-free DNase (Qiagen, Germany) according to the manufacturer’s instructions. RNA was then purified and concentrated using an RNeasy column (Takara, Japan). RNA integrity numbers (RINs) were assessed by an Agilent 2100 bioanalyzer (Agilent Technologies, United States). The quantification of RNA was done using a Qubit 4 Fluorometer (Thermo Fisher Scientific Company, Germany). Purification of the poly-A mRNA and construction of the cDNA realized with the TruSeq RNA Library Preparation Kit (Illumina, United States). RNA sequencing was finally performed using the Illumina NovaSeq 6000 platform with PE150 (Zhejiang Annoroad Biotechnology Co., China).

RNA-seq datasets were then analyzed following a custom protocol previously published⁴⁸. Raw data were also cleaned using Fastp v0.20⁴⁴. Cleaned reads were aligned to the rice reference genome using STAR v2.7.8a with a splicing-aware method and two-pass mode⁴⁹. The aligned reads were separately assembled into transcripts for each sample with the reference annotation-based transcript (RABT) assembly algorithm and generated an updated transcript annotation with GTF-formatted file using StringTie v2.1.3⁵⁰. Finally, the expression level of genes was quantified and normalized with the above-updated GTF file using HTSeq, respectively⁵¹. Only the genes with an FPKM (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 differentially expressed (DEG) genes with the updated GTF file⁵². In our study, genes were considered as differentially expressed according to the following criteria: Log2 (Fold change) ≥ 1 and the adjusted p-value < 0.05. Raw RNA-Seq datasets were also available on NCBI BioProject under PRJNA808101.

Venn diagrams and heatmaps were plotted from the differentially expressed genes using custom R scripts. Enrichment analysis based on GO and KEGG databases was carried out using PlantGSAD⁵³. The Benjamini–Yekutieli method was used for P-value adjustment. Besides, clusterProfiler v3.14, a gene set enrichment analysis (GSEA) method⁵⁴, was further employed to determine if a prior defined set of genes such as nitrogen metabolism and plant hormone signal transduction shows concordant differences in response to NH₄⁺ treatment between the mutant and the wild-type.

Allelic variation and local adaption analysis

For allelic variation analysis of ROHAN, SNP datasets of 446 accessions of common wild rice (Oryza rufipogon) and 3K Rice Genomes Project were obtained from a previous study^55,56. The nucleotide diversity (Pi) and Fst statistics were calculated and fitted with the sibling window method (window size:2 kb; step size:1 kb) for wild rice and rice subpopulations using VCFtools v.0.1.17⁵⁷. Functional effects of variants from ROHAN and its surrounding regions (with 2 Kb flanking sequence on both sides) were predicted by SNPeff v4.3⁵⁸. Allele frequency of missense SNP (Chr3:10847318) from six rice subgroups (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, Carlsbad, CA, USA) was employed to generate most genetic constructs. For transcriptional fusions, first, ~2k promoter fragments upstream of the start codon were amplified by PCR from genomic DNA and cloned into pdonrp41R by Bp reactions. Second, the ROHAN open reading frame with or without stop codon was amplified from genomic DNA and was cloned into pDONR221 or pDONR2F3R, respectively. Subsequently, the vectors were introduced into different expression vectors by LR reactions. The expression vectors pmk7snfm14GW, pHb7m34GW and pHb7m24GW were respectively used for the promoter-GUS fusion, the complementation of the mutant and generating the overexpression lines.

To generate pCRISPR-OsROHAN, pCRISPR-OsGS1;1 and pCRISPR-OsPIN1a, pCRISPR-OsPIN1b gene-editing constructs, the corresponding target sequences were blasted by CRISPR-GE (http://skl.scau.edu.cn/home/), and cloned into guide RNA (sgRNA) expression cassettes by overlapping PCR, the fragments were subsequentially cloned into the BsaI site of the pYLCRISPR/Cas9-MH vector.

All constructs were verified by DNA sequencing analysis. The primers used are listed in the Supplementary Table S1. pUBIL:ROHAN, pROHAN:ROHAN1:VENUS and pROHAN:VENUS;ROHAN were transformed into the rohan mutant. pYLCRISPR/Cas9-MH-ROHAN, proROHAN:GUS and pCRISPR-OsPIN1a, pCRISPR-OsPIN1b were transformed into the wild-type. pYLCRISPR/Cas9-MH-OsGS1;1 was transformed into Nipponbare. All plasmids were transformed into rice plants using Agrobacterium-mediated transformation⁵⁹.

Histochemical analysis and microscopy

GUS assays were done as previously described⁶⁰. Roots were imaged with a Leica DM2500 microscope (Leica Microsystems, Wetzlar, Germany). For the anatomical sections, GUS-stained samples were fixed overnight and embedded following a published protocol⁶¹.

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. The root tips of rice DR5rev:3xVENUS-N7 transgenic rice seedlings grown under different treatments were cleaned with a modified ClearSee method preceding confocal imaging as previously described¹¹. Rice root meristems were imaged using a modified mPS-PI staining to clear and visualize the cell organization of rice SR tips¹¹.

RNA extraction and quantitative real-time PCR analysis

Total RNA was isolated using the plant RNA purification reagent (Invitrogen). cDNA was synthesized from 1 µg of RNA with the Advantage® RT-for-PCR Kit (TaKaRa) according to the manufacturer’s instructions and was diluted 20 times for subsequent quantitative (q)PCR. Real-time PCR was done in Real-time PCR machine (LightCycler 480, Roche Diagnostics) according to the manufacturer’s manuals in a reaction mixture of 10 µL of TB Green Fast qPCR mix (CellAmp™ Direct TB Green® RT-qPCR Kit). The OsActin was selected as a housekeeping gene, and three biological replicates were analyzed. Primer sequences are listed in the Supplementary Table S1, together with all the other primers in this study.

Subcellular localization of ROHAN

To investigate the subcellular localization of ROHAN, the pROHAN:ROHAN:VENUS and pROHAN::ROHAN^P211L::VENUS constructs were transformed into rice shoot protoplasts using polyethylene glycol (PEG)-mediated transformation⁶². Protoplasts isolated from rice and pROHAN:ROHAN:VENUS transgenic seedlings were observed using a confocal laser (Leica LSP8). YFP fluorescence was observed at 525 nm for emission and 514 nm for excitation.

EDU staining

EDU staining was performed using an EdU kit (C10350, Click-iT EdU Alexa Fluor 488 HCS assay; Invitrogen), accordingly to the manufacturer’s instructions. Roots of 4-days-old seedlings were immersed in 20 µM EDU solutions for 2 h and fixed for 30 min in 3.7% formaldehyde solution in phosphate buffer (pH 7.2) with 0.1% Tritonx-100, followed by 30 min of incubation with EDU detection cocktail. An Olympus MXV10 microscope with GFP channel was used to capture the images.

Amino acid analysis

Wild-type and rohan plants were grown under different treatments for 4 days. Roots were collected for amino acid extraction as previously described⁶³. Roots were collected in liquid nitrogen, then grinded and mixed manually. Samples were then transferred to 5 ml tubes, frozen with liquid nitrogen, and transferred to the lyophilizer (BILON, FD-2C, Shanghai, China) for 3 days. Lyophilized root tissues (~ 3 mg) were used for extraction with 80% methanol followed by incubation at 70℃ for 15 mins and shaking (600 rpm). Following centrifugation at 10,000 g for 15 min at room temperature, the supernatants were collected, 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, China). The dried pellets were dissolved in 500 µL ultrapure water. 500 µL 4% 5-Sulfosalicylic acid dihydrate was added to re-dissolve followed by centrifugation at 12,000g for 15min at room temperature. The supernatants were collected and filtered by 0.22 µm water filter. The samples were analyzed with a LA8080 automatic amino acid analyzer (Hitachi, Tokyo, Japan). All steps were performed accordingly to the manufacturer’s instructions.

Photosynthesis parameter and seeds protein analysis

Simultaneous measurements of photosynthesis parameters were performed on light-adapted leaves with a LI-6,800 infrared gas analysis system. Prior to the measurements, the leaves were placed in a greenhouse at a photosynthetic photon flux density of 1,500 μmol·m^-2s^-1 with an ambient CO₂ concentration of 400 μmol/mol. After equilibration to a steady state, 0.8 s saturating pulses of light (~8,000 μmol·m^-2s^-1) were applied to measure the maximum (F_m') and steady-state fluorescence (F_s); gas exchange parameters were recorded. The Φ_PSII was calculated as: Φ_PSII= (F_m'- F_s)/ F_m'. Gas exchange parameters include net photosynthesis rate, stomatal conductance and transpiration rate. The seeds crude protein content was detected with a Near Infrared Spectroscope (NIRS) (DA7250, China).

IAA content analysis

Germinated rice seedlings (3-day-old) were transferred to nutrient solution containing NH₄⁺ or not. After 4 days of treatment, the roots were harvested and immediately frozen in liquid nitrogen. Auxin extraction procedure was conducted as described in⁶⁴. Before chromatographic measurement, the dehydrated precipitate was re-suspended with 200 µL 80% MeOH. For chromatographic separations, UHPLC (Acquity UPLC-I-Class™, Waters, Milford, MA, USA) equipped with a reverse phase column (Waters BEH-C18 column, 2.1 × 50 mm, 1.7 μm) as the stationary phase was used, and 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, Waters Xevo TQ-S micro, for IAA quantification (Waters, Milford, MA, USA) equipped with an electrospray ionization (ESI) source. Data acquisition and processing were performed using Masslynx software (version 4.1, Waters, Manchester, UK). The mass transitions were monitored as follows: m/z 176.07-130.00, external correction method based on IAA standards was used for relative IAA measurement.

Statistical analysis

The experiments performed in this study were repeated at least three times, and all the results were presented as the mean ± SD. SPSS software was used for statistical analysis. The significant difference between the two sets of data was determined by Student’s t-test, whereas the difference among more than two sets of data was analyzed with one-way ANOVA followed by Duncan’s multiple comparisons.

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

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