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

Nature Plants volume 9, pages 1514–1529 (2023)Cite this article

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Abstract

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.

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Fig. 1: rohan root growth is hypersensitive to NH4+.
Fig. 2: ROHAN encodes a plastid-localized ASL.
Fig. 3: rohan is defective in the conversion of NH4+/Gln to Arg.
Fig. 4: The root response of rohan to NH4+ is mediated by auxin.
Fig. 5: Genetic variation in ASL is associated with its regulatory role in root response to NH4+.
Fig. 6: ASL expression determines rice tolerance to NH4+ toxicity and NUE.

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Data availability

The raw whole-genome sequencing (WGS) datasets and RNA-seq datasets have been deposited on NCBI BioProject (https://www.ncbi.nlm.nih.gov/bioproject) under the accession numbers PRJNA808438 and PRJNA808101. The datasets of widely targeted metabolomics are deposited in the OMIX, China Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences with accession number OMIX004476 (https://ngdc.cncb.ac.cn/omix), and are also available in Supplementary Table 1. Source data are provided with this paper. For any datasets unavailable through the links above, they can be requested from the corresponding author.

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Acknowledgements

We thank H. Qu, X. Dai and K. Qian for their technical help with the 15N uptake assays and confocal imaging. This work was supported by China National Key Programme for Research and Development (grant no. 2021YFF1000403), the Project of Sanya Yazhou Bay Science and Technology City (grant no. SCKJ-JYRC-2022-21), National Natural Science Foundation (grant nos 32072658 and 31822047), Key Research and Development Programme of Jiangsu Province (grant no. BE2020339), Jiangsu Seed Industry Revitalization Project (grant no. JBGS [2021] 011) and the Research Foundation – Flanders: Bilateral Scientific Cooperation fund with China (grant no. G002817N). Y.X. is supported by a grant from the Chinese Scholarship Council (grant no. 201806850032).

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Author notes

  1. These authors contributed equally: Yuanming Xie, Yuanda Lv.

Authors and Affiliations

  1. State Key Laboratory of Crop Genetics & Germplasm Enhancement and Utilization and MOA Key Laboratory of Plant Nutrition and Fertilization in Lower-Middle Reaches of the Yangtze River, Sanya Institute of Nanjing Agricultural University, Nanjing Agricultural University, Nanjing, China

    Yuanming Xie, Letian Jia, Lulu Zheng, Yonghui Li, Ming Zhu, Long Luo, Shaoyan Lin, Le Luo, Guohua Xu & Wei Xuan

  2. Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium

    Yuanming Xie, Hugues De Gernier, Pierre-Mathieu Pélissier, Hans Motte & Tom Beeckman

  3. VIB-UGent Center for Plant Systems Biology, Ghent, Belgium

    Yuanming Xie, Hugues De Gernier, Pierre-Mathieu Pélissier, Hans Motte & Tom Beeckman

  4. Excellence and Innovation Center, Jiangsu Academy of Agricultural Sciences, Nanjing, China

    Yuanda Lv & Weicong Qi

  5. Department of Plant Pathology, Nanjing Agricultural University, Nanjing, China

    Mengjun Tian & Ming Wang

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Contributions

W.X. and T.B. directed the experiments. Y.X. and Y. Lv performed most of the experiments and analysis. L.J., L.Z., M.Z. and Long Luo helped with vector construction. Y. Li helped with root section. M.T and M.W. helped the indole-3-acetic acid content analysis. W.Q. helped the phenotyping analysis. H.D.G. P.-M.P. and H.M. helped the imaging. S.L. and Le Luo helped the amino acid content analysis. All authors discussed the results and contributed to the finalization of the manuscript.

Corresponding authors

Correspondence to Tom Beeckman or Wei Xuan.

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Nature Plants thanks Takushi Hachiya and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 The seminal root growth of rohan is hypersensitive to external NH4+.

a, Relative seminal roots (SR) of wild-type (WT) and rohan seedlings treated with varying concentrations of NH4+ and NO3−for 6 days. b, Relative SR length of WT and rohan plants treated without or with 2.5 mM NH4+ 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 phenotype (e) and SR length (f) of WT and rohan seedlings grown in H2O supplied with or without indicated nutrients. the concentrations of chemicals were used as follows: 0.3 mM Gln, 1.25 mM (NH4)2SO4, 0.3 mM NaH2PO4, 0.65 mM K2SO4, 1 mM CaCl2, 1 mM MgSO4, 20 μM EDTA-Fe, and 0.5 mM Na2SiO3. 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 NH4+ 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 NH4+ or NO3− (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.

Source data

Extended Data Fig. 2 ASL-mediated seminal root elongation is independent of NH4+ 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 NH4+ 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 NH4+ and proton fluxes in the root tip of WT and rohan seedlings treated with or without 2.5 mM NH4+, 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 NH4+ 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 NH4+, 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 NH4+ 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).

Source data

Extended Data Fig. 3 Map-based cloning of ASL gene.

a, Root phenotypes of the wild-type (WT), rohan, and the F1 progeny of the rohan x wild-type backcross treated with 2.5 mM NH4+ 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 F2 progeny. c, Euclidean distance association analysis of the ASL candidate interval in the rice genome. ED4 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. 4 Phylogenetic and protein sequence analysis of ASL.

a, Phylogenetic tree of ASL established with MEGA 7.0 software by Neighbor-jointing method with 1000 bootstrapping trials. The numbers on the branch indicate bootstrapping values. Escherichia coli (Ec), Triticum aestivum (TAE), Oryza sativa (Os), Medicago truncatula (Mt), Selaginella moellendorffii (Sm), Arabidopsis thaliana (At), Sorghum bicolor (Sb), Zea mays (Zm), Hordeum vulgare (HV), Brachypodium distachyon (Bd). b, Expression pattern of proASL:GUS in the root tissues of wild-type (Oryza sativa cv. Wuyungeng7) seedlings. LR, lateral root; LRP, lateral root primordium; DZ, differentiation zone; MZ, meristem zone. Similar results were obtained from root sections of three independent seedlings. Scale bars, 100 μm. T-LRP, transverse section of lateral root primordium. T-DZ, transverse section of root differentiation zone. T-MZ, transverse section of root meristem zone. Scale bars, 100 μm. c, Alignment of ASL amino acid sequence in different plant species using ESPript 3 software. Red arrowhead indicates the location of mutation site. Accession numbers for aligned sequences: EcASL, WP_109536061.1; AtASL, NP_196653.1; OsASL, XP_015632553.1; BdASL, XP_010228716.1; HvASL, BAJ96216.1; SmASL, EFJ24721.1; MtASL, XP_003602904.2; TaeASL, XP_044394196.1.

Extended Data Fig. 5 The effects of NH4+ on the Gln content in gs1;1 mutants, and ASL expression in gs1;1 and qko mutants.

a, Sketch map of mutation sites in the CRISPR knockout lines of Glutamine Synthestase (GS1;1). b, Quantification of Gln content in the root tips of wild-type (Oryza sativa cv. Nipponbare) and two gs1;1 mutant lines treated with or without 2.5 mM NH4+ for 6 days. Data represent the means ± s.d. of three biological repeats, and the asterisks indicate significant differences relative to wild-type (two-sided student’s t-test). ns, not significant. c, Relative expression level of ASL at the root tip of wild-type, gs1;1-1, gs1;1-2, qko-1 and qko-2 seedlings under treatments with or without 2.5 mM NH4+ for 3 days. Data represent the means ± s.e. of three biological replicates, and asterisks indicate significant differences relative to N-free (two-sided student’s t-test). Similar results were obtained in three independent experiments.

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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.

Extended Data Fig. 7 ASL acts on the conversion of NH4+/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 NH4+ 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.

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Extended Data Fig. 8 ASL regulates the nitrogen metabolism pathway at the transcriptional level.

a, Venn diagram showing the number of differentially expressed genes affected by 2.5 mM NH4+ treatment in the root tips of wild-type (WT) and rohan. b, Gene set enrichment analysis of differentially expressed genes based on GO and KEGG databases between the wild-type and rohan. c, GSEA results of nitrogen metabolism related gene sets in rohan comparing with WT under one-day and three -day treatment of 2.5 mM NH4+, Permutation test, two-side. d, Heatmap of Z-score values from expression abundance of genes associated with nitrogen metabolism. Each treatment is a column, while each row is a gene. The colour ranged from red for positive Z-score values to blue for negative values. e, Relative expression level of indicated genes in the root tips of WT and rohan seedlings treated with or without 2.5 mM NH4+ 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).

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Extended Data Fig. 9 The NH4+-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 NH4+ 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).

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Extended Data Fig. 10 rohan root response to NH4+ is mediated by auxin metabolism.

a, GSEA results of plant hormone signal transduction related gene sets in rohan comparing with wild-type (WT) under one-day and three-day treatment of high NH4+, Permutation test, two-side. b, Quantification of the free IAA content in the roots of WT, rohan and rohan-C1 (complementary line of the rohan) seedlings treated with or without 2.5 mM NH4+, 0.3 mM Gln, 0.3 mM Arg and their combination for 6 days. Data represent the means ± s.d. of 5 biological replicates,and the asterisks indicate significant differences relative to WT (two-sided student’s t-test). ND, not detected. c, Expression pattern of DR5rev:3xVENUS-N7 in the root tips of WT and rohan seedlings grown under N-free condition for 48 h (related to Fig. 4d). The white and green arrowheads indicate St (stele) and Ep (epidermis), respectively. Similar results were obtained in three independent experiments. Scale bar, 100 μm. d, Relative SR length of seminal roots (SR) length of WT and rohan seedlings treated with NPA or BUM, under N-free condition for 6 days (related to Fig. 4g). 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 BUM or NPA (two-sided student’s t-test). e, Relative expression levels of PINs at the root tip of WT and rohan seedlings under 2.5 mM NH4+ treatments for 3 days. Data represent the means ± s.e. of three biological replicates, and the asterisks indicate significant differences (two-sided student’s t-test). f, Sketch map of mutation of CRISPR knockout lines of PIN1a and PIN1b. Red triangles indicate the mutation target sites. g, Quantification of free IAA content in the root of WT and pin1apin1b seedlings treated with or without 2.5 mM NH4+ for 6 days. Data represent the means ± s.d. of 5 biological replicates, and the asterisks indicate significant differences (two-sided student’s t-test).

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Supplementary information

Supplementary Information

Supplementary Figs 1–5.

Reporting Summary

Supplementary Tables

Supplementary Table 1 Metabolite profiling of wild-type and rohan seedlings. Supplementary Table 2 Sequences of primers used in this study.

Supplementary Data

Table 1 Statistical source data for Supplementary Fig. 2. Table 2 Statistical source data for Supplementary Fig. 3. Table 3 Statistical source data for Supplementary Fig. 4. Table 4 Statistical source data for Supplementary Fig. 5.

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Xie, Y., Lv, Y., Jia, L. et al. Plastid-localized amino acid metabolism coordinates rice ammonium tolerance and nitrogen use efficiency. Nat. Plants 9, 1514–1529 (2023). https://doi.org/10.1038/s41477-023-01494-x

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