Tree Genetics & Genomes

, 15:10 | Cite as

Phosphoproteomic changes in root cells of Poncirus trifoliata (L.) Raf. induced by Rhizophagus intraradices inoculation

  • Fuxi Bai
  • Fang Song
  • Zijun Zheng
  • Huimin Yu
  • Xiuxin Deng
  • Shunyuan Xiao
  • Zhiyong PanEmail author
Original Article
Part of the following topical collections:
  1. Disease Resistance


Arbuscular mycorrhiza (AM) is a widespread endosymbiosis between terrestrial plants and AM fungi belonging to sub-phylum Glomeromycotina (Mycologia 108: 1028–1046, 2016). To date, many plant genes involved in establishing the AM symbiosis have been identified. Yet, the precise mechanisms governing the early signaling process are still not well understood. In this study, we employed the isotope tags for relative and absolute quantification (iTRAQ) and LC–MS/MS analysis to investigate the phosphoproteomic changes in the root cells of Poncirus trifoliata (L.) Raf. (a common citrus rootstock) during the establishment of the AM symbiosis. A total of 1920 unique phosphopeptides derived from 1016 phosphoproteins were identified, which collectively contained 2308 phosphorylation sites. Motif-X analysis of all the detected phosphopeptides showed that 25 phosphoserine motifs and 4 phosphothreonine motifs were overrepresented in phosphorylation processes. Among the phosphoserine motifs, [SDXE] and [PXSP] showed the highest fold increase upon colonization by Rhizophagus intraradices, suggesting that they may play potential roles in AM symbiosis. At 1 week post-inoculation of R. intraradices, the phosphorylation levels of 65 phosphopeptides were significantly changed (p value < 0.05 and fold change > 1.35), with 39 upregulated and 26 downregulated, implying that at least some of the 65 phosphoproteins may be involved in the signal transduction during early events of the AM symbiosis. This study provides a comprehensive phosphoproteomic analysis in a citrus rootstock and the phosphoproteomic changes should shed light onto the signaling mechanisms involved in the establishment of AM symbiosis.


Phosphoproteome Arbuscular mycorrhizal symbiosis Rhizophagus intraradices Poncirus 



We would like to thank Yunliu Zeng (Huazhong Agricultural University) for critical discussion and suggestions and Chengquan Yang (North West Agriculture and Forestry University) for his technical assistance with bioinformatic analysis and manuscript revision.

Author contributions

ZY. P., XX. D., and SY. X. conceived and designed the experiments. FX. B., F. S., ZJ. Z., and HM. Y. prepared the materials and performed the experiments, FX. B. and HM. Y. collected, analyzed, and deposited the data. FX. B. proofread the final draft and revised the manuscript. All authors have read and approved the manuscript.


This work was funded by the National Key Research and Development Program of China (Grant Number 2017YFD0202001), National Natural Science Foundation of China (No. 31521092), and the Fundamental Research Funds for the Central Universities (Grant Number 2662018JC039).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Data archiving statement

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (Deutsch et al. 2017) via the PRIDE (Vizcaino et al. 2016) partner repository with the dataset identifier PXD007840.

Supplementary material

11295_2019_1317_MOESM1_ESM.xlsx (380 kb)
Table S1 List of identified phosphorylation sites and changes in their phosphorylation levels upon Rhizophagus intraradices inoculation in Poncirus. a Complete list of total identified phosphorylation sites and changes in their phosphorylation levels upon R. intraradices inoculation in Poncirus. b Phosphopeptides identified in this experiment with p value < 0.05. (XLSX 380 kb)
11295_2019_1317_MOESM2_ESM.xlsx (276 kb)
Table S2 Mercator functional annotation of phosphoprotein sequence data. a Mercator functional annotation of total phosphoprotein sequence data. b Mercator functional annotation of significant changed phosphoprotein sequence data. (XLSX 275 kb)
11295_2019_1317_MOESM3_ESM.xlsx (119 kb)
Table S3 The blast result of total phosphoproteins searching for the P3DB database. a The blast result of total phosphoproteins searching for the P3DB database. b Novel phosphoproteins identified in this experiment. (XLSX 119 kb)
11295_2019_1317_MOESM4_ESM.xlsx (43 kb)
Table S4 The blast results of the Poncirus phosphoproteins (p value < 0.05) against the Medicago and soybean genome. The proteins that labeled with green were significantly changed in Medicago or soybean phosphoproteome and they were the best hit or the e-value was zero. The proteins that labeled with gray were significantly changed in Medicago or soybean phosphoproteome, but they were not the best hit and the e-value was not zero. (XLSX 43 kb)
11295_2019_1317_MOESM5_ESM.xlsx (13 kb)
Table S5 The pathway enrichment analysis of the significantly changed phosphoproteins by using KOBAS3.0 (XLSX 12 kb)
11295_2019_1317_MOESM6_ESM.pdf (7.5 mb)
Fig. S1 Ink stained roots of Poncirus at different time point post-Rhizophagus intraradices inoculation. a Ink-stained roots of AM group after 1 weeks. b Ink-stained root of AM group 2 weeks post-inoculation. c Ink-stained root of AM group 8 weeks post-inoculation. Arbuscular(ab), vesicle(ve), and hyphae(hp) were shown. Scale bars, 200 μm. (PDF 7711 kb)
11295_2019_1317_Fig3_ESM.png (1.5 mb)
Fig. S2

Phosphorylation motif logos that enriched in Rhizophagus intraradices responsive phosphoproteome. Motif-X analysis was performed with a 15 amino acid window, an occurrence of 20, a significance of 10–5, and the C. sinensis protein database was used as the background database to normalize the score against a random distribution of amino acids (PNG 1488 kb)

11295_2019_1317_MOESM7_ESM.eps (4.3 mb)
High resolution image (EPS 4426 kb)


  1. Amanchy R, Periaswamy B, Mathivanan S, Reddy R, Tattikota SG, Pandey A (2007) A curated compendium of phosphorylation motifs. Nat Biotechnol 25:285–286. CrossRefPubMedGoogle Scholar
  2. Anderson JC, Bartels S, Besteiro MAG, Shahollari B, Ulm R, Peck SC (2011) Arabidopsis MAP kinase phosphatase 1 (AtMKP1) negatively regulates MPK6-mediated PAMP responses and resistance against bacteria. Plant J 67:258–268. CrossRefPubMedGoogle Scholar
  3. Bartels S, Anderson JC, Besteiro MAG, Carreri A, Hirt H, Buchala A, Metraux JP, Peck SC, Ulm R (2009) Map kinase phosphatase1 and protein tyrosine phosphatase1 are repressors of salicylic acid synthesis and SNC1-mediated responses in arabidopsis. Plant Cell 21:2884–2897. CrossRefPubMedPubMedCentralGoogle Scholar
  4. Beausoleil SA, Villen J, Gerber SA, Rush J, Gygi SP (2006) A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat Biotechnol 24:1285–1292. CrossRefPubMedGoogle Scholar
  5. Bergmann DC, Lukowitz W, Somerville CR (2004) Stomatal development and pattern controlled by a MAPKK kinase. Science 304:1494–1497. CrossRefPubMedGoogle Scholar
  6. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL (2009) BLAST plus : architecture and applications. BMC Bioinformatics 10:Artn 421. CrossRefGoogle Scholar
  7. Chou MF, Schwartz D (2011) Biological sequence motif discovery using motif-x Current protocols in bioinformatics:13.15. 11–13.15. 24Google Scholar
  8. Deguchi Y, Banba M, Shimoda Y, Chechetka SA, Suzuri R, Okusako Y, Ooki Y, Toyokura K, Suzuki A, Uchiumi T, Higashi S, Abe M, Kouchi H, Izui K, Hata S (2007) Transcriptome profiling of Lotus japonicus roots during arbuscular mycorrhiza development and comparison with that of nodulation. DNA Res 14:117–133. CrossRefPubMedPubMedCentralGoogle Scholar
  9. Den Herder G, Yoshida S, Antolin-Llovera M, Ried MK, Parniske M (2012) Lotus japonicus E3 ligase seven in absentia4 destabilizes the symbiosis receptor-like kinase SYMRK and negatively regulates rhizobial infection. Plant Cell 24:1691–1707. CrossRefGoogle Scholar
  10. Genre A, Chabaud M, Balzergue C, Puech-Pages V, Novero M, Rey T, Fournier J, Rochange S, Becard G, Bonfante P, Barker DG (2013) Short-chain chitin oligomers from arbuscular mycorrhizal fungi trigger nuclear Ca2+ spiking in Medicago truncatula roots and their production is enhanced by strigolactone. New Phytol 198:179–189. CrossRefPubMedGoogle Scholar
  11. Gnad F, Ren S, Cox J, Olsen JV, Macek B, Oroshi M, Mann M (2007) PHOSIDA (phosphorylation site database): management, structural and evolutionary investigation, and prediction of phosphosites. Genome Biol 8:R250. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Grimsrud PA, den Os D, Wenger CD, Swaney DL, Schwartz D, Sussman MR, Ane JM, Coon JJ (2010) Large-scale phosphoprotein analysis in Medicago truncatula roots provides insight into in vivo kinase activity in legumes. Plant Physiol 152:19–28. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Humphrey SJ, James DE, Mann M (2015) Protein phosphorylation: a major switch mechanism for metabolic regulation. Trends Endocrinol Metab 26:676–687. CrossRefPubMedGoogle Scholar
  14. Jayaraman D, Richards AL, Westphall MS, Coon JJ, Ane JM (2017) Identification of the phosphorylation targets of symbiotic receptor-like kinases using a high-throughput multiplexed assay for kinase specificity. Plant J 90:1196–1207. CrossRefPubMedPubMedCentralGoogle Scholar
  15. Jiang Y, Wang W, Xie Q, Liu N, Liu L, Wang D, Zhang X, Yang C, Chen X, Tang D, Wang E (2017) Plants transfer lipids to sustain colonization by mutualistic mycorrhizal and parasitic fungi. Science 356:1172–1175. CrossRefPubMedGoogle Scholar
  16. Jin Y, Liu H, Luo D, Yu N, Dong W, Wang C, Zhang X, Dai H, Yang J, Wang E (2016) DELLA proteins are common components of symbiotic rhizobial and mycorrhizal signalling pathways. Nat Commun 7:12433. CrossRefPubMedPubMedCentralGoogle Scholar
  17. Larsen MR, Thingholm TE, Jensen ON, Roepstorff P, Jorgensen TJD (2005) Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol Cell Proteomics 4:873–886. CrossRefPubMedGoogle Scholar
  18. Liu JY, Blaylock LA, Endre G, Cho J, Town CD, VandenBosch KA, Harrison MJ (2003) Transcript profiling coupled with spatial expression analyses reveals genes involved in distinct developmental stages of an arbuscular mycorrhizal symbiosis. Plant Cell 15:2106–2123. CrossRefPubMedPubMedCentralGoogle Scholar
  19. Lohse M, Nagel A, Herter T, May P, Schroda M, Zrenner R, Tohge T, Fernie AR, Stitt M, Usadel B (2014) Mercator: a fast and simple web server for genome scale functional annotation of plant sequence data. Plant Cell Environ 37:1250–1258. CrossRefPubMedGoogle Scholar
  20. Luginbuehl LH, Menard GN, Kurup S, Van Erp H, Radhakrishnan GV, Breakspear A, Oldroyd GED, Eastmond PJ (2017) Fatty acids in arbuscular mycorrhizal fungi are synthesized by the host plant. Science 356:1175–1178. CrossRefPubMedGoogle Scholar
  21. Maillet F, Poinsot V, Andre O, Puech-Pages V, Haouy A, Gueunier M, Cromer L, Giraudet D, Formey D, Niebel A, Martinez EA, Driguez H, Becard G, Denarie J (2011) Fungal lipochitooligosaccharide symbiotic signals in arbuscular mycorrhiza. Nature 469:58–63. CrossRefPubMedGoogle Scholar
  22. Manthey K, Krajinski F, Hohnjec N, Firnhaber C, Puhler A, Perlick AM, Kuster H (2004) Transcriptome profiling in root nodules and arbuscular mycorrhiza identifies a collection of novel genes induced during Medicago truncatula root endosymbioses. Mol Plant-Microbe Interact 17:1063–1077. CrossRefPubMedGoogle Scholar
  23. Maurel C, Verdoucq L, Luu DT, Santoni V (2008) Plant aquaporins: membrane channels with multiple integrated functions. Annu Rev Plant Biol 59:595–624. CrossRefPubMedGoogle Scholar
  24. Mbengue M, Camut S, de Carvalho-Niebel F, Deslandes L, Froidure S, Klaus-Heisen D, Moreau S, Rivas S, Timmers T, Herve C, Cullimore J, Lefebvre B (2010) The Medicago truncatula E3 ubiquitin ligase PUB1 interacts with the LYK3 symbiotic receptor and negatively regulates infection and nodulation. Plant Cell 22:3474–3488. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Medina MJH, Gagnon H, Piche Y, Ocampo JA, Garrido JMG, Vierheilig H (2003) Root colonization by arbuscular mycorrhizal fungi is affected by the salicylic acid content of the plant. Plant Sci 164:993–998. CrossRefGoogle Scholar
  26. Miyata K, Kozaki T, Kouzai Y, Ozawa K, Ishii K, Asamizu E, Okabe Y, Umehara Y, Miyamoto A, Kobae Y, Akiyama K, Kaku H, Nishizawa Y, Shibuya N, Nakagawa T (2014) The bifunctional plant receptor, OsCERK1, regulates both chitin-triggered immunity and arbuscular mycorrhizal symbiosis in rice. Plant Cell Physiol 55:1864–1872. CrossRefPubMedGoogle Scholar
  27. Nadal M, Paszkowski U (2013) Polyphony in the rhizosphere: presymbiotic communication in arbuscular mycorrhizal symbiosis. Curr Opin Plant Biol 16:473–479. CrossRefPubMedGoogle Scholar
  28. Nakagami H, Sugiyama N, Mochida K, Daudi A, Yoshida Y, Toyoda T, Tomita M, Ishihama Y, Shirasu K (2010) Large-scale comparative phosphoproteomics identifies conserved phosphorylation sites in plants. Plant Physiol 153:1161–1174. CrossRefPubMedPubMedCentralGoogle Scholar
  29. Oldroyd GED (2013) Speak, friend, and enter: signalling systems that promote beneficial symbiotic associations in plants. Nat Rev Microbiol 11:252–263. CrossRefPubMedGoogle Scholar
  30. Parniske M (2008) Arbuscular mycorrhiza: the mother of plant root endosymbioses. Nat Rev Microbiol 6:763–775. CrossRefPubMedGoogle Scholar
  31. Pruitt KD, Tatusova T, Maglott DR (2007) NCBI reference sequences (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res 35:D61–D65.
  32. Remy W, Taylor TN, Hass H, Kerp H (1994) Four hundred-million-year-old vesicular arbuscular mycorrhizae. Proc Natl Acad Sci U S A 91:11841–11843CrossRefGoogle Scholar
  33. Rose CM, Venkateshwaran M, Volkening JD, Grimsrud PA, Maeda J, Bailey DJ, Park K, Howes-Podoll M, den Os D, Yeun LH, Westphall MS, Sussman MR, Ane JM, Coon JJ (2012) Rapid phosphoproteomic and transcriptomic changes in the rhizobia-legume symbiosis. Mol Cell Proteomics 11:724–744. CrossRefPubMedPubMedCentralGoogle Scholar
  34. Ross PL, Huang YLN, Marchese JN, Williamson B, Parker K, Hattan S, Khainovski N, Pillai S, Dey S, Daniels S, Purkayastha S, Juhasz P, Martin S, Bartlet-Jones M, He F, Jacobson A, Pappin DJ (2004) Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics 3:1154–1169. CrossRefPubMedGoogle Scholar
  35. Salih E (2005) Phosphoproteomics by mass spectrometry and classical protein chemistry approaches. Mass Spectrom Rev 24:828–846. CrossRefPubMedGoogle Scholar
  36. Sandberg A, Lindell G, Kallstrom BN, Branca RM, Danielsson KG, Dahlberg M, Larson B, Forshed J, Lehtio J (2012) Tumor proteomics by multivariate analysis on individual pathway data for characterization of vulvar cancer phenotypes. Mol Cell Proteomics 11:ARTN M112.016998. CrossRefGoogle Scholar
  37. Schmutz J, Cannon SB, Schlueter J, Ma JX, Mitros T, Nelson W, Hyten DL, Song QJ, Thelen JJ, Cheng JL, Xu D, Hellsten U, May GD, Yu Y, Sakurai T, Umezawa T, Bhattacharyya MK, Sandhu D, Valliyodan B, Lindquist E, Peto M, Grant D, Shu S, Goodstein D, Barry K, Futrell-Griggs M, Abernathy B, du J, Tian Z, Zhu L, Gill N, Joshi T, Libault M, Sethuraman A, Zhang XC, Shinozaki K, Nguyen HT, Wing RA, Cregan P, Specht J, Grimwood J, Rokhsar D, Stacey G, Shoemaker RC, Jackson SA (2010) Genome sequence of the palaeopolyploid soybean. Nature 463:178–183. CrossRefPubMedGoogle Scholar
  38. Serna-Sanz A, Parniske M, Peck SC (2011) Phosphoproteome analysis of Lotus japonicus roots reveals shared and distinct components of symbiosis and defense. Mol Plant-Microbe Interact 24:932–937. CrossRefPubMedGoogle Scholar
  39. Shu B, Xia R-X, Wang P (2012) Differential regulation of Pht1 phosphate transporters from trifoliate orange (Poncirus trifoliata L. Raf) seedlings. Sci Hortic 146:115–123. CrossRefGoogle Scholar
  40. Smith SE, Read DJ (2010) Mycorrhizal symbiosis. Academic press, Cambridge, MAGoogle Scholar
  41. Spatafora JW, Chang Y, Benny GL, Lazarus K, Smith ME, Berbee ML, Bonito G, Corradi N, Grigoriev I, Gryganskyi A, James TY, O'Donnell K, Roberson RW, Taylor TN, Uehling J, Vilgalys R, White MM, Stajich JE (2016) A phylum-level phylogenetic classification of zygomycete fungi based on genome-scale data. Mycologia 108:1028–1046. CrossRefPubMedPubMedCentralGoogle Scholar
  42. Stacey G, McAlvin CB, Kim SY, Olivares J, Soto MJ (2006) Effects of endogenous salicylic acid on nodulation in the model legumes Lotus japonicus and Medicago truncatula. Plant Physiol 141:1473–1481. CrossRefPubMedPubMedCentralGoogle Scholar
  43. Sugiyama N, Nakagami H, Mochida K, Daudi A, Tomita M, Shirasu K, Ishihama Y (2008) Large-scale phosphorylation mapping reveals the extent of tyrosine phosphorylation in arabidopsis. Mol Syst Biol 4:ARTN 193. CrossRefGoogle Scholar
  44. Tran HNN, Brechenmacher L, Aldrich JT, Clauss TR, Gritsenko MA, Hixson KK, Libault M, Tanaka K, Yang F, Yao QM, Pasa-Tolic L, Xu D, Nguyen HT, Stacey G (2012) Quantitative phosphoproteomic analysis of soybean root hairs inoculated with Bradyrhizobium japonicum. Mol Cell Proteomics 11:1140–1155. CrossRefGoogle Scholar
  45. Van Ness LK, Jayaraman D, Maeda J, Barrett-Wilt GA, Sussman MR, Ane JM (2016) Mass spectrometric-based selected reaction monitoring of protein phosphorylation during symbiotic signaling in the model legume, Medicago truncatula. PLoS One 11:e0155460. CrossRefPubMedPubMedCentralGoogle Scholar
  46. Vierheilig H, Coughlan AP, Wyss U, Piche Y (1998) Ink and vinegar, a simple staining technique for arbuscular-mycorrhizal fungi. Appl Environ Microbiol 64:5004–5007PubMedPubMedCentralGoogle Scholar
  47. Wingenter K, Schulz A, Wormit A, Wic S, Trentmann O, Hoermiller II, Heyer AG, Marten I, Hedrich R, Neuhaus HE (2010) Increased activity of the vacuolar monosaccharide transporter TMT1 alters cellular sugar partitioning, sugar signaling, and seed yield in Arabidopsis. Plant Physiol 154:665–677. CrossRefPubMedPubMedCentralGoogle Scholar
  48. Wingenter K, Trentmann O, Winschuh I, Hormiller II, Heyer AG, Reinders J, Schulz A, Geiger D, Hedrich R, Neuhaus HE (2011) A member of the mitogen-activated protein 3-kinase family is involved in the regulation of plant vacuolar glucose uptake. Plant J 68:890–900. CrossRefPubMedGoogle Scholar
  49. Wisniewski JR, Zougman A, Nagaraj N, Mann M (2009) Universal sample preparation method for proteome analysis. Nat Methods 6:359–U360. CrossRefPubMedGoogle Scholar
  50. Xu Q, Chen LL, Ruan XA, Chen DJ, Zhu AD, Chen CL, Bertrand D, Jiao WB, Hao BH, Lyon MP, Chen JJ, Gao S, Xing F, Lan H, Chang JW, Ge XH, Lei Y, Hu Q, Miao Y, Wang L, Xiao S, Biswas MK, Zeng W, Guo F, Cao H, Yang X, Xu XW, Cheng YJ, Xu J, Liu JH, Luo OJ, Tang Z, Guo WW, Kuang H, Zhang HY, Roose ML, Nagarajan N, Deng XX, Ruan Y (2013) The draft genome of sweet orange (Citrus sinensis). Nat Genet 45:59–U92. CrossRefPubMedGoogle Scholar
  51. Young ND, Debelle F, Oldroyd GED, Geurts R, Cannon SB, Udvardi MK, Benedito VA, Mayer KFX, Gouzy J, Schoof H, Van de Peer Y, Proost S, Cook DR, Meyers BC, Spannagl M, Cheung F, De Mita S, Krishnakumar V, Gundlach H, Zhou SG et al (2011) The Medicago genome provides insight into the evolution of rhizobial symbioses. Nature 480:520–524. CrossRefPubMedPubMedCentralGoogle Scholar
  52. Yuan SL, Zhu H, Gou HL, Fu WW, Liu LJ, Chen T, Ke DX, Kang H, Xie Q, Hong ZL, Zhang ZM (2012) A ubiquitin ligase of symbiosis receptor kinase involved in nodule organogenesis. Plant Physiol 160:106–117. CrossRefPubMedPubMedCentralGoogle Scholar
  53. Zhang XW, Dong WT, Sun JH, Feng F, Deng YW, He ZH, Oldroyd GED, Wang ET (2015) The receptor kinase CERK1 has dual functions in symbiosis and immunity signalling. Plant J 81:258–267. CrossRefPubMedGoogle Scholar
  54. Zhang ZB, Liu YN, Huang H, Gao MH, Wu D, Kong Q, Zhang YL (2017) The NLR protein SUMM2 senses the disruption of an immune signaling MAP kinase cascade via CRCK3. EMBO Rep 18:292–302. CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Key Laboratory of Horticultural Plant Biology (Ministry of Education), Key Laboratory of Horticultural Crop Biology and Genetic Improvement (Central Region, Ministry of Agriculture), College of Horticulture and Forestry SciencesHuazhong Agricultural UniversityWuhan CityPeople’s Republic of China
  2. 2.Institute for Bioscience and Biotechnology Research & Department of Plant Sciences and Landscape ArchitectureUniversity of Maryland, College ParkRockvilleUSA

Personalised recommendations