Plant Molecular Biology

, Volume 100, Issue 3, pp 265–283 | Cite as

Quantitative phosphoproteomic analyses provide evidence for extensive phosphorylation of regulatory proteins in the rhizobia–legume symbiosis

  • Zaibao Zhang
  • Danxia Ke
  • Menghui Hu
  • Chi Zhang
  • Lijun Deng
  • Yuting Li
  • Jiuli Li
  • Hai Zhao
  • Lin Cheng
  • Lei WangEmail author
  • Hongyu YuanEmail author


Key message

Symbiotic nitrogen fixation in root nodules of grain legumes is essential for high yielding. Protein phosphorylation/dephosphorylation plays important role in root nodule development. Differences in the phosphoproteomes may either be developmental specific and related to nitrogen fixation activity. An iTRAQ-based quantitative phosphoproteomic analyses during nodule development enables identification of specific phosphorylation signaling in the Lotus–rhizobia symbiosis.


During evolution, legumes (Fabaceae) have evolved a symbiotic relationship with rhizobia, which fix atmospheric nitrogen and produce ammonia that host plants can then absorb. Root nodule development depends on the activation of protein phosphorylation-mediated signal transduction cascades. To investigate possible molecular mechanisms of protein modulation during nodule development, we used iTRAQ-based quantitative proteomic analyses to identify root phosphoproteins during rhizobial colonization and infection of Lotus japonicus. 1154 phosphoproteins with 2957 high-confidence phosphorylation sites were identified. Gene ontology enrichment analysis of functional groups of these genes revealed that the biological processes mediated by these proteins included cellular processes, signal transduction, and transporter activity. Quantitative data highlighted the dynamics of protein phosphorylation during nodule development and, based on regulatory trends, seven groups were identified. RNA splicing and brassinosteroid (BR) signaling pathways were extensively affected by phosphorylation, and most Ser/Arg-rich (SR) proteins were multiply phosphorylated. In addition, many proposed kinase-substrate pairs were predicted, and in these MAPK6 substrates were found to be highly enriched. This study offers insights into the regulatory processes underlying nodule development, provides an accessible resource cataloging the phosphorylation status of thousands of Lotus proteins during nodule development, and develops our understanding of post-translational regulatory mechanisms in the Lotus–rhizobia symbiosis.


Nodule Lotus japonicus Rhizobia Mass spectrometry Phosphoproteomics 



This work was supported by Natural Science Foundation of Henan Provincial Science and Technology (Grant No. 182300410063), key scientific research projects of Henan higher education institutions (Grant No. 18A180031), National Natural Science Foundation of China (Grant No. 31400213), Funding scheme for young core teachers of Xinyang Normal University (2015, 2016), Nanhu Scholars Program for Young Scholars of XYNU and the foundation and frontier technology research of Henan Province (Grant No. 162300410257).

Author contributions

ZZ, HY and LW designed the research; ZZ and DK performed data analysis and wrote the manuscript; MH, CZ, LD, YL, JL, LC and HZ performed protein extraction and MS analysis.

Supplementary material

11103_2019_857_MOESM1_ESM.pdf (542 kb)
Supplementary Figure S1 The homolog protein analysis of maize SPS protein (ZmSPS) based on amino acid sequence. One red star indicate phosphoprotein and two red stars indicate differential phosphoprotein. (PDF 542 KB)
11103_2019_857_MOESM2_ESM.pdf (307 kb)
Supplementary Figure S2 Distribution of differentially regulated phosphoproteins according to their predicted functions. Significantly overrepresented functional categories (p < 0.01) are marked with a red star. The grey bar represents total protein and the red bar represents rhizobium-responsive phosphoproteins. (PDF 307 KB)
11103_2019_857_MOESM3_ESM.pdf (148 kb)
Supplementary Figure S3 Phylogenetic classification (A) and sequence alignment of LjRBOHs (B). The phosphorylated sites are marked with a red star. (PDF 147 KB)
11103_2019_857_MOESM4_ESM.pdf (374 kb)
Supplementary Figure S4 Sequence alignments of phosphorylated aquaporins. The phosphorylated sites are marked with a red star. (PDF 374 KB)
11103_2019_857_MOESM5_ESM.xlsx (1019 kb)
Supplementary Table S1 Identified phosphopeptides in phosphoproteomic analyses. (XLSX 1019 KB)
11103_2019_857_MOESM6_ESM.xlsx (663 kb)
Supplementary Table S2 Comparative analysis of phosphopeptides. (XLSX 663 KB)
11103_2019_857_MOESM7_ESM.xlsx (220 kb)
Supplementary Table S3 Overview of differentially accumulated phosphopeptides. Sheet 1, 1: Overview of identified M. loti phosphoproteins; 2: Identified differentially accumulating phosphopeptides of SR (Ser/Arg-rich) proteins; 3: The differentially accumulating phosphosites in spliceosome proteins; 4: The microtubule-associated proteins 65; 5: Differentially accumulating phosphopeptides of transporters; 6: One phosphopeptide of cullin (Lj1g3v4916290); 7: 5 MAPKs, 4 MAPKKKs, and 1 LjSYMRK; 8: The detailed summary of the phosphorylated peptides with a regulatory trend; 9: One sucrose phosphate synthase; 10: Comparative analysis of differentially accumulating phosphopeptides of protein kinases; 11: One respiratory burst oxidase; 12: Comparative analysis of differentially accumulating phosphopeptides of GTPases; 13: Differentially accumulating phosphatases identified in phosphoproteome; 14: Comparative analysis of differentially accumulating transcription factors identified in phosphoproteome; 15: List of predicated kinase to substrate relationships in phosphoproteome; 16: List of predicated protein kinase / phosphatase in the root during rhizobial inoculation. Sheet 2, 1: The differentially accumulating phosphopeptides of containing ......TP.....; 2: The differentially accumulating phosphopeptides of containing ......SD.....; 3: The differentially accumulating phosphopeptides of containing ......SS.....; 4: The differentially accumulating phosphopeptides of containing ......SP...... (XLSX 219 KB)
11103_2019_857_MOESM8_ESM.xlsx (78 kb)
Supplementary Table S4 Tables showing differentially phosphorylated proteins over different time intervals. Sheet 1: Differentially phosphorylated proteins between T0 and T5h. Sheet 2: Differentially phosphorylated proteins between T0 and T3d. Sheet 3: Differentially phosphorylated proteins between T0 and T7d. (XLSX 77 KB)


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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Zaibao Zhang
    • 1
    • 2
  • Danxia Ke
    • 1
    • 2
  • Menghui Hu
    • 2
  • Chi Zhang
    • 2
  • Lijun Deng
    • 2
  • Yuting Li
    • 2
  • Jiuli Li
    • 2
  • Hai Zhao
    • 2
  • Lin Cheng
    • 2
  • Lei Wang
    • 1
    • 2
    Email author
  • Hongyu Yuan
    • 1
    • 2
    Email author
  1. 1.Henan Key Laboratory of Tea Plant BiologyXinyang Normal UniversityXinyangChina
  2. 2.College of Life ScienceXinyang Normal UniversityXinyangChina

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