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Transfer cells mediate nitrate uptake to control root nodule symbiosis

Nature Plants volume 6, pages 800–808 (2020)Cite this article

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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.

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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.

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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.

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Authors and Affiliations

  1. State Key Laboratory of Plant Genomics, Institute of Microbiology, Innovative Academy of Seed Design, Chinese Academy of Sciences, Beijing, China

    Qi Wang, Yige Huang, Xiaxia Zhang, Jing Ren, Chen Zhang, Juan Tian, Yanjun Yu & Zhaosheng Kong

  2. College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China

    Yige Huang, Xiaxia Zhang, Jing Ren, Chen Zhang & Zhaosheng Kong

  3. College of Life Sciences, Capital Normal University, Beijing, China

    Zhijie Ren & Legong Li

  4. Chinese Academy of Sciences Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China

    Jiaqi Su & George F. Gao

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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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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.

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Statistical source data.

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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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