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Transfer cells mediate nitrate uptake to control root nodule symbiosis

Abstract

Root nodule symbiosis enables nitrogen fixation in legumes and, therefore, improves crop production for sustainable agriculture1,2. Environmental nitrate levels affect nodulation and nitrogen fixation, but the mechanisms by which legume plants modulate nitrate uptake to regulate nodule symbiosis remain unclear1. Here, we identify a member of the Medicago truncatula nitrate peptide family (NPF), NPF7.6, which is expressed specifically in the nodule vasculature. NPF7.6 localizes to the plasma membrane of nodule transfer cells (NTCs), where it functions as a high-affinity nitrate transporter. Transfer cells show characteristic wall ingrowths that enhance the capacity for membrane transport at the apoplasmic–symplasmic interface between the vasculature and surrounding tissues3. Importantly, knockout of NPF7.6 using CRISPR–Cas9 resulted in developmental defects of the nodule vasculature, with excessive expansion of NTC plasma membranes. npf7.6 nodules showed severely compromised nitrate responsiveness caused by an attenuated ability to transport nitrate. Moreover, npf7.6 nodules exhibited disturbed nitric oxide homeostasis and a notable decrease in nitrogenase activity. Our findings indicate that NPF7.6 has been co-opted into a regulatory role in nodulation, functioning in nitrate uptake through NTCs to fine-tune nodule symbiosis in response to fluctuating environmental nitrate status. These observations will inform efforts to optimize nitrogen fixation in legume crops.

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Fig. 1: NPF7.6 encodes a high-affinity nitrate transporter that is specifically expressed in NTCs.
Fig. 2: Knockout of NPF7.6 reduces the nitrate responsiveness during nodule development.
Fig. 3: NPF7.6 is required for nodule vasculature development and plasma membrane development of NTCs.
Fig. 4: NPF7.6 functions in nitrate uptake through NTCs to regulate nodule symbiosis.

Data availability

The original RNA-seq data have been deposited at Genome Sequence Archive (https://bigd.big.ac.cn/gsa/) and can be accessed through the GSA accession number CRA001389. The data for the current study are available within the paper and the Supplementary Information. Source Data for Figs. 1, 2 and 4 are provided with the paper. Raw data or materials generated during this study are available on reasonable request.

References

  1. Ferguson, B. J. et al. Legume nodulation: the host controls the party. Plant Cell Environ. 42, 41–45 (2018).

    PubMed  Google Scholar 

  2. Olivares, J., Bedmar, E. J. & Sanjuan, J. Biological nitrogen fixation in the context of global change. Mol. Plant Microbe Interact. 26, 486–494 (2013).

    CAS  PubMed  Google Scholar 

  3. Offler, C. E., McCurdy, D. W., Patrick, J. W. & Talbot, M. J. Transfer cells: cells specialized for a special purpose. Annu. Rev. Plant Biol. 54, 431–454 (2003).

    CAS  PubMed  Google Scholar 

  4. Griesmann, M. et al. Phylogenomics reveals multiple losses of nitrogen-fixing root nodule symbiosis. Science 361, aat1743 (2018).

    Google Scholar 

  5. Martin, F. M., Uroz, S. & Barker, D. G. Ancestral alliances: plant mutualistic symbioses with fungi and bacteria. Science 356, aad4501 (2017).

    Google Scholar 

  6. Canfield, D. E., Glazer, A. N. & Falkowski, P. G. The evolution and future of Earth’s nitrogen cycle. Science 330, 192–196 (2010).

    CAS  PubMed  Google Scholar 

  7. Barbulova, A., Rogato, A., D’Apuzzo, E., Omrane, S. & Chiurazzi, M. Differential effects of combined N sources on early steps of the Nod factor-dependent transduction pathway in Lotus japonicus. Mol. Plant Microbe Interact. 20, 994–1003 (2007).

    CAS  PubMed  Google Scholar 

  8. van Noorden, G. E. et al. Molecular signals controlling the inhibition of nodulation by nitrate in Medicago truncatula. Int. J. Mol. Sci. 17, 1060 (2016).

    PubMed Central  Google Scholar 

  9. Nanjareddy, K. et al. Nitrate regulates rhizobial and mycorrhizal symbiosis in common bean (Phaseolus vulgaris L.). J. Integr. Plant Biol. 56, 281–298 (2014).

    CAS  PubMed  Google Scholar 

  10. Nishida, H. & Suzaki, T. Nitrate-mediated control of root nodule symbiosis. Curr. Opin. Plant Biol. 44, 129–136 (2018).

    CAS  PubMed  Google Scholar 

  11. Fred, E. B. & Graul, E. J. The effect of soluble nitrogenous salts on nodule formation. J. Am. Soc. Agron. 8, 316–328 (1916).

    CAS  Google Scholar 

  12. Streeter, J. & Wong, P. P. Inhibition of legume nodule formation and N2 fixation by nitrate. Criti. Rev. Plant Sci. 7, 1–23 (1988).

    CAS  Google Scholar 

  13. Xu, G., Fan, X. & Miller, A. J. Plant nitrogen assimilation and use efficiency. Annu. Rev. Plant Biol. 63, 153–182 (2012).

    CAS  PubMed  Google Scholar 

  14. Corratge-Faillie, C. & Lacombe, B. Substrate (un)specificity of Arabidopsis NRT1/PTR FAMILY (NPF) proteins. J. Exp. Bot. 68, 3107–3113 (2017).

    CAS  PubMed  Google Scholar 

  15. Aubry, E., Dinant, S., Vilaine, F., Bellini, C. & Le Hir, R. Lateral transport of organic and inorganic solutes. Plants 8, 20 (2019).

    CAS  PubMed Central  Google Scholar 

  16. Hsu, P. K. & Tsay, Y. F. Two phloem nitrate transporters, NRT1.11 and NRT1.12, are important for redistributing xylem-borne nitrate to enhance plant growth. Plant Physiol. 163, 844–856 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. McCurdy, D. W. & Hueros, G. Transfer cells. Front. Plant Sci. 5, 672 (2014).

    PubMed  PubMed Central  Google Scholar 

  18. Gama, T. S., Aguiar-Dias, A. C. & Demarco, D. Transfer cells in trichomatous nectary in Adenocalymma magnificum (Bignoniaceae). Acad. Bras. Cienc. 88, 527–537 (2016).

    CAS  Google Scholar 

  19. Sosso, D. et al. Seed filling in domesticated maize and rice depends on SWEET-mediated hexose transport. Nat. Genet. 47, 1489–1493 (2015).

    CAS  PubMed  Google Scholar 

  20. Bohlmann, H. & Sobczak, M. The plant cell wall in the feeding sites of cyst nematodes. Front. Plant Sci. 5, 89 (2014).

    PubMed  PubMed Central  Google Scholar 

  21. Yamaji, N., Sasaki, A., Xia, J. X., Yokosho, K. & Ma, J. F. A node-based switch for preferential distribution of manganese in rice. Nat. Commun. 4, 2442 (2013).

    PubMed  Google Scholar 

  22. Pate, J. S., Gunning, B. E. S. & Briarty, L. G. Ultrastructure and functioning of the transport system of the leguminous root nodule. Planta 85, 11–34 (1969).

    CAS  PubMed  Google Scholar 

  23. Briarty, L. G. Repeating particles associated with membranes of transfer cells. Planta 113, 373–377 (1973).

    CAS  PubMed  Google Scholar 

  24. Sutter, J. U. et al. Abscisic acid triggers the endocytosis of the Arabidopsis KAT1 K+ channel and its recycling to the plasma membrane. Curr. Biol. 17, 1396–1402 (2007).

    CAS  PubMed  Google Scholar 

  25. Wang, L., Xue, Y. Q., Xing, J. J., Song, K. & Lin, J. X. Exploring the spatiotemporal organization of membrane proteins in living plant. Cells Ann. Rev. Plant Biol. 69, 525–551 (2018).

    CAS  Google Scholar 

  26. Demir, F. et al. Arabidopsis nanodomain-delimited ABA signaling pathway regulates the anion channel SLAH3. Proc. Natl Acad. Sci. USA 110, 8296–8301 (2013).

    CAS  PubMed  Google Scholar 

  27. Wang, L. L. et al. CRISPR/Cas9 knockout of leghemoglobin genes in Lotus japonicus uncovers their synergistic roles in symbiotic nitrogen fixation. N. Phytol. 224, 818–832 (2019).

    CAS  Google Scholar 

  28. Berger, A., Guinand, S., Boscari, A., Puppo, A. & Brouquisse, R. Medicago truncatula Phytoglobin 1.1 controls symbiotic nodulation and nitrogen fixation via the regulation of nitric oxide concentration. N. Phytol. (in the press).

  29. Ott, T. et al. Symbiotic leghemoglobins are crucial for nitrogen fixation in legume root nodules but not for general plant growth and development. Curr. Biol. 15, 531–535 (2005).

    CAS  PubMed  Google Scholar 

  30. Appleby, C. A. Leghemoglobin and rhizobium respiration. Ann. Rev. Plant Physiol. 35, 443–478 (1984).

    CAS  Google Scholar 

  31. Berger, A. et al. Pathways of nitric oxide metabolism and operation of phytoglobins in legume nodules: missing links and future directions. Plant Cell Environ. 41, 2057–2068 (2018).

  32. Berger, A., Boscari, A., Frendo, P. & Brouquisse, R. Nitric oxide signaling, metabolism and toxicity in nitrogen-fixing symbiosis. J. Exp. Bot. 70, 4505–4520 (2019).

    CAS  PubMed  Google Scholar 

  33. Mathieu, C., Moreau, S., Frendo, P., Puppo, A. & Davies, M. J. Direct detection of radicals in intact soybean nodules: presence of nitric oxide-leghemoglobin complexes. Free Radic. Biol. Med. 24, 1242–1249 (1998).

    CAS  PubMed  Google Scholar 

  34. Sanchez, C. et al. Production of nitric oxide and nitrosylleghemoglobin complexes in soybean nodules in response to flooding. Mol. Plant Microbe Interact. 23, 702–711 (2010).

    CAS  PubMed  Google Scholar 

  35. Lin, J. S. et al. NIN interacts with NLPs to mediate nitrate inhibition of nodulation in Medicago truncatula. Nat. Plants 4, 942–952 (2018).

    CAS  PubMed  Google Scholar 

  36. Nishida, H. et al. A NIN-LIKE PROTEIN mediates nitrate-induced control of root nodule symbiosis in Lotus japonicus. Nat. Commun. 9, 499 (2018).

    PubMed  PubMed Central  Google Scholar 

  37. Li, Y. G. et al. Disruption of the rice nitrate transporter OsNPF2.2 hinders root-to-shoot nitrate transport and vascular development. Sci. Rep. 5, 9635 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Leran, S. et al. A unified nomenclature of NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family members in plants. Trends Plant Sci. 19, 5–9 (2014).

    CAS  PubMed  Google Scholar 

  39. Nour-Eldin, H. H. et al. NRT/PTR transporters are essential for translocation of glucosinolate defence compounds to seeds. Nature 488, 531–534 (2012).

    CAS  PubMed  Google Scholar 

  40. Pellizzaro, A., Alibert, B., Planchet, E., Limami, A. M. & Morere-Le Paven, M. C. Nitrate transporters: an overview in legumes. Planta 246, 585–595 (2017).

    CAS  PubMed  Google Scholar 

  41. Krouk, G. et al. Nitrate-regulated auxin transport by NRT1.1 defines a mechanism for nutrient sensing in plants. Dev. Cell 18, 927–937 (2010).

    CAS  PubMed  Google Scholar 

  42. Yan, L. et al. High-efficiency genome editing in Arabidopsis using YAO Promoter-Driven CRISPR/Cas9 system. Mol. Plant 8, 1820–1823 (2015).

    CAS  PubMed  Google Scholar 

  43. Limpens, E. et al. RNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula. J. Exp. Bot. 55, 983–992 (2004).

    CAS  PubMed  Google Scholar 

  44. Wang, C. et al. NODULES WITH ACTIVATED DEFENSE 1 is required for maintenance of rhizobial endosymbiosis in Medicago truncatula. N. Phytol. 212, 176–191 (2016).

    CAS  Google Scholar 

  45. Zhang, X. et al. The host actin cytoskeleton channels rhizobia release and facilitates symbiosome accommodation during nodulation in Medicago truncatula. N. Phytol. 221, 1049–1059 (2018).

    Google Scholar 

  46. Rupp, R. A., Snider, L. & Weintraub, H. Xenopus embryos regulate the nuclear localization of XMyoD. Genes Dev. 8, 1311–1323 (1994).

    CAS  PubMed  Google Scholar 

  47. Wang, W. et al. Expression of the nitrate transporter gene OsNRT1.1A/OsNPF6.3 confers high yield and early maturation in rice. Plant Cell 30, 638–651 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank Y. Wang for providing M. truncatula R108 seeds and the pCambia1391z expression vector; X. Li and X. Tan for helping with sample preparation and taking SEM images; Y. Feng for 3D reconstruction; Y. Li and D. Chen for assistance with the nitrogenase activity assay; L. Su and Y. Wu for providing technical assistance with imaging; and Y. Xue, Y. Wang, C. Chu, B. Hu and W. Wang for discussion. This study was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (grant no. XDB27040210), National Transgenic Major Program (grant no. 2019ZX08010-004), National Science Fund for Distinguished Young Scholars (grant no. 31925003) and by grants from the State Key Laboratory of Plant Genomics.

Author information

Authors and Affiliations

Authors

Contributions

Q.W. designed the project, performed most of the experiments and wrote the manuscript. Y.H. constructed the expression vectors and conducted the screening of transgenic plants. Z.R. prepared and injected the Xenopus oocytes. J.R. cultivated the transgenic plants. X.Z., C.Z., J.T. and Y.Y. provided essential technical assistance. J.S. and G.F.G. analysed the data. L.L. supervised the 15N-uptake assay and analysed the data. Z.K. conceived the project, interpreted the data and revised the article.

Corresponding author

Correspondence to Zhaosheng Kong.

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The authors declare no competing interests.

Additional information

Peer review information: Nature Plants thanks Mingyong Zhang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–13 and the associated legends, and legends for Supplementary Videos 1–5.

Reporting Summary

Supplementary Tables 1–6.

Supplementary Table 7.

Supplementary Video 1

Subcellular localization of pNPF7.6::NPF7.6-GFP.

Supplementary Video 2

The ultrastructure and 3D organization of xylem parenchyma transfer cells.

Supplementary Video 3

3D reconstruction of xylem parenchyma transfer cells in control R108.

Supplementary Video 4

The ultrastructure and 3D organization of wall ingrowth in control R108.

Supplementary Video 5

The ultrastructure and 3D organization of wall ingrowth in npf7.6 mutant.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 4

Statistical source data.

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Wang, Q., Huang, Y., Ren, Z. et al. Transfer cells mediate nitrate uptake to control root nodule symbiosis. Nat. Plants 6, 800–808 (2020). https://doi.org/10.1038/s41477-020-0683-6

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