Abstract
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.
Abstract
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.
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Amor BB, Shaw SL, Oldroyd GE, Maillet F, Penmetsa RV, Cook D, Long SR, Denarie J, Gough C (2003) The NFP locus of Medicago truncatula controls an early step of Nod factor signal transduction upstream of a rapid calcium flux and root hair deformation. Plant J Cell Mol Biol 34(4):495–506
Antolin-Llovera M, Ried MK, Binder A, Parniske M (2012) Receptor kinase signaling pathways in plant-microbe interactions. Ann Rev Phytopathol 50:451–473. https://doi.org/10.1146/annurev-phyto-081211-173002
Barbazuk WB, Fu Y, McGinnis KM (2008) Genome-wide analyses of alternative splicing in plants: opportunities and challenges. Genome Res 18(9):1381–1392. https://doi.org/10.1101/gr.053678.106
Borisov AY, Madsen LH, Tsyganov VE, Umehara Y, Voroshilova VA, Batagov AO, Sandal N, Mortensen A, Schauser L, Ellis N, Tikhonovich IA, Stougaard J (2003) The Sym35 gene required for root nodule development in pea is an ortholog of Nin from Lotus japonicus. Plant Physiol 131(3):1009–1017. https://doi.org/10.1104/pp.102.016071
Boscari A, Del Giudice J, Ferrarini A, Venturini L, Zaffini AL, Delledonne M, Puppo A (2013) Expression dynamics of the Medicago truncatula transcriptome during the symbiotic interaction with Sinorhizobium meliloti: which role for nitric oxide? Plant Physiol 161(1):425–439. https://doi.org/10.1104/pp.112.208538
Campanoni P, Blatt MR (2007) Membrane trafficking and polar growth in root hairs and pollen tubes. J Exp Bot 58(1):65–74. https://doi.org/10.1093/jxb/erl059
Cazalla D, Zhu J, Manche L, Huber E, Krainer AR, Caceres JF (2002) Nuclear export and retention signals in the RS domain of SR proteins. Mol Cell Biol 22(19):6871–6882
Clarke VC, Loughlin PC, Day DA, Smith PM (2014) Transport processes of the legume symbiosome membrane. Front Plant Sci 5:699. https://doi.org/10.3389/fpls.2014.00699
Dalla Via V, Traubenik S, Rivero C, Aguilar OM, Zanetti ME, Blanco FA (2017) The monomeric GTPase RabA2 is required for progression and maintenance of membrane integrity of infection threads during root nodule symbiosis. Plant Mol Biol 93(6):549–562. https://doi.org/10.1007/s11103-016-0581-5
Dam S, Dyrlund TF, Ussatjuk A, Jochimsen B, Nielsen K, Goffard N, Ventosa M, Lorentzen A, Gupta V, Andersen SU, Enghild JJ, Ronson CW, Roepstorff P, Stougaard J (2014) Proteome reference maps of the Lotus japonicus nodule and root. Proteomics 14(2–3):230–240. https://doi.org/10.1002/pmic.201300353
de la Fuente van Bentem S, Anrather D, Roitinger E, Djamei A, Hufnagl T, Barta A, Csaszar E, Dohnal I, Lecourieux D, Hirt H (2006) Phosphoproteomics reveals extensive in vivo phosphorylation of Arabidopsis proteins involved in RNA metabolism. Nucleic Acids Res 34(11):3267–3278. https://doi.org/10.1093/nar/gkl429
Desbrosses G, Kopka C, Ott T, Udvardi MK (2004) Lotus japonicus LjKUP is induced late during nodule development and encodes a potassium transporter of the plasma membrane. Mol Plant Microbe Interact 17(7):789–797. https://doi.org/10.1094/mpmi.2004.17.7.789
Downie JA (2014) Legume nodulation. Curr Biol: CB 24 (5):R184–R190. https://doi.org/10.1016/j.cub.2014.01.028
Eichhorn H, Klinghammer M, Becht P, Tenhaken R (2006) Isolation of a novel ABC-transporter gene from soybean induced by salicylic acid. J Exp Bot 57(10):2193–2201. https://doi.org/10.1093/jxb/erj179
Endre G, Kereszt A, Kevei Z, Mihacea S, Kalo P, Kiss GB (2002) A receptor kinase gene regulating symbiotic nodule development. Nature 417(6892):962–966. https://doi.org/10.1038/nature00842
Fedorova E, Thomson R, Whitehead LF, Maudoux O, Udvardi MK, Day DA (1999) Localization of H(+)-ATPases in soybean root nodules. Planta 209(1):25–32. https://doi.org/10.1007/s004250050603
Ferguson BJ, Indrasumunar A, Hayashi S, Lin MH, Lin YH, Reid DE, Gresshoff PM (2010) Molecular analysis of legume nodule development and autoregulation. J Integr Plant Biol 52(1):61–76. https://doi.org/10.1111/j.1744-7909.2010.00899.x
Fremin C, Guegan JP, Plutoni C, Mahaffey J, Philips MR, Emery G, Meloche S (2016) ERK1/2-induced phosphorylation of R-Ras GTPases stimulates their oncogenic potential. Oncogene 35(43):5692–5698. https://doi.org/10.1038/onc.2016.122
Gargantini PR, Gonzalez-Rizzo S, Chinchilla D, Raices M, Giammaria V, Ulloa RM, Frugier F, Crespi MD (2006) A CDPK isoform participates in the regulation of nodule number in Medicago truncatula. Plant J Cell Mol Biol 48(6):843–856. https://doi.org/10.1111/j.1365-313X.2006.02910.x
Gavrin A, Jansen V, Ivanov S, Bisseling T, Fedorova E (2015) ARP2/3-mediated actin nucleation associated with symbiosome membrane is essential for the development of symbiosomes in infected cells of Medicago truncatula root nodules. Mol Plant Microbe Interact 28(5):605–614. https://doi.org/10.1094/mpmi-12-14-0402-r
Gebril S, Seger M, Villanueva FM, Ortega JL, Bagga S, Sengupta-Gopalan C (2015) Transgenic alfalfa (Medicago sativa) with increased sucrose phosphate synthase activity shows enhanced growth when grown under N2-fixing conditions. Planta 242(4):1009–1024. https://doi.org/10.1007/s00425-015-2342-0
Graham PH, Vance CP (2003) Legumes: importance and constraints to greater use. Plant Physiol 131(3):872–877. https://doi.org/10.1104/pp.017004
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(1):19–28. https://doi.org/10.1104/pp.109.149625
Guo H, Li L, Aluru M, Aluru S, Yin Y (2013) Mechanisms and networks for brassinosteroid regulated gene expression. Curr Opin Plant Biol 16(5):545–553. https://doi.org/10.1016/j.pbi.2013.08.002
Hu X, Li N, Wu L, Li C, Li C, Zhang L, Liu T, Wang W (2015) Quantitative iTRAQ-based proteomic analysis of phosphoproteins and ABA-regulated phosphoproteins in maize leaves under osmotic stress. Sci Rep 5:15626. https://doi.org/10.1038/srep15626
Huang JZ, Huber SC (2001) Phosphorylation of synthetic peptides by a CDPK and plant SNF1-related protein kinase. Influence of proline and basic amino acid residues at selected positions. Plant Cell Physiol 42(10):1079–1087
Huang JZ, Hardin SC, Huber SC (2001) Identification of a novel phosphorylation motif for CDPKs: phosphorylation of synthetic peptides lacking basic residues at P-3/P-4. Arch Biochem Biophys 393(1):61–66. https://doi.org/10.1006/abbi.2001.2476
Ivashuta S, Liu J, Liu J, Lohar DP, Haridas S, Bucciarelli B, VandenBosch KA, Vance CP, Harrison MJ, Gantt JS (2005) RNA interference identifies a calcium-dependent protein kinase involved in Medicago truncatula root development. Plant Cell 17(11):2911–2921. https://doi.org/10.1105/tpc.105.035394
Jang JC (2016) Arginine-rich motif-tandem CCCH zinc finger proteins in plant stress responses and post-transcriptional regulation of gene expression. Plant Sci 252:118–124. https://doi.org/10.1016/j.plantsci.2016.06.014
Kalo P, Gleason C, Edwards A, Marsh J, Mitra RM, Hirsch S, Jakab J, Sims S, Long SR, Rogers J, Kiss GB, Downie JA, Oldroyd GE (2005) Nodulation signaling in legumes requires NSP2, a member of the GRAS family of transcriptional regulators. Science 308(5729):1786–1789. https://doi.org/10.1126/science.1110951
Kapranov P, Jensen TJ, Poulsen C, de Bruijn FJ, Szczyglowski K (1999) A protein phosphatase 2C gene, LjNPP2C1, from Lotus japonicus induced during root nodule development. Proc Natl Acad Sci USA 96(4):1738–1743
Ke D, Fang Q, Chen C, Zhu H, Chen T, Chang X, Yuan S, Kang H, Ma L, Hong Z, Zhang Z (2012) The small GTPase ROP6 interacts with NFR5 and is involved in nodule formation in Lotus japonicus. Plant Physiol 159(1):131–143. https://doi.org/10.1104/pp.112.197269
Khan M, Rozhon W, Bigeard J, Pflieger D, Husar S, Pitzschke A, Teige M, Jonak C, Hirt H, Poppenberger B (2013) Brassinosteroid-regulated GSK3/Shaggy-like kinases phosphorylate mitogen-activated protein (MAP) kinase kinases, which control stomata development in Arabidopsis thaliana. J Biol Chem 288(11):7519–7527. https://doi.org/10.1074/jbc.M112.384453
Kiirika LM, Bergmann HF, Schikowsky C, Wimmer D, Korte J, Schmitz U, Niehaus K, Colditz F (2012) Silencing of the Rac1 GTPase MtROP9 in Medicago truncatula stimulates early mycorrhizal and oomycete root colonizations but negatively affects rhizobial infection. Plant Physiol 159(1):501–516. https://doi.org/10.1104/pp.112.193706
Kobayashi M, Ohura I, Kawakita K, Yokota N, Fujiwara M, Shimamoto K, Doke N, Yoshioka H (2007) Calcium-dependent protein kinases regulate the production of reactive oxygen species by potato NADPH oxidase. Plant Cell 19(3):1065–1080. https://doi.org/10.1105/tpc.106.048884
Konert G, Rahikainen M, Trotta A, Durian G, Salojarvi J, Khorobrykh S, Tyystjarvi E, Kangasjarvi S (2015) Subunits B’gamma and B’zeta of protein phosphatase 2A regulate photo-oxidative stress responses and growth in Arabidopsis thaliana. Plant Cell Environ 38(12):2641–2651. https://doi.org/10.1111/pce.12575
Kouchi H, Imaizumi-Anraku H, Hayashi M, Hakoyama T, Nakagawa T, Umehara Y, Suganuma N, Kawaguchi M (2010) How many peas in a pod? Legume genes responsible for mutualistic symbioses underground. Plant Cell Physiol 51(9):1381–1397. https://doi.org/10.1093/pcp/pcq107
Larsen MR, Thingholm TE, Jensen ON, Roepstorff P, Jorgensen TJ (2005) Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol Cell Proteom: MCP 4(7):873–886. https://doi.org/10.1074/mcp.T500007-MCP200
Lee M, Lee K, Lee J, Noh EW, Lee Y (2005) AtPDR12 contributes to lead resistance in Arabidopsis. Plant Physiol 138(2):827–836. https://doi.org/10.1104/pp.104.058107
Levy J, Bres C, Geurts R, Chalhoub B, Kulikova O, Duc G, Journet EP, Ane JM, Lauber E, Bisseling T, Denarie J, Rosenberg C, Debelle F (2004) A putative Ca2+ and calmodulin-dependent protein kinase required for bacterial and fungal symbioses. Science 303(5662):1361–1364. https://doi.org/10.1126/science.1093038
Limpens E, Franken C, Smit P, Willemse J, Bisseling T, Geurts R (2003) LysM domain receptor kinases regulating rhizobial Nod factor-induced infection. Science 302(5645):630–633. https://doi.org/10.1126/science.1090074
Liu N, Ni Z, Zhang H, Chen Q, Gao W, Cai Y, Li M, Sun G, Qu YY (2018) The gene encoding subunit A of the vacuolar H(+)-ATPase from cotton plays an important role in conferring tolerance to water deficit. Front Plant Sci 9:758. https://doi.org/10.3389/fpls.2018.00758
Ma B, Reynolds CM, Raetz CR (2008) Periplasmic orientation of nascent lipid A in the inner membrane of an Escherichia coli LptA mutant. Proc Natl Acad Sci USA 105(37):13823–13828. https://doi.org/10.1073/pnas.0807028105
Ma Q, Wu M, Pei W, Li H, Li X, Zhang J, Yu J, Yu S (2014) Quantitative phosphoproteomic profiling of fiber differentiation and initiation in a fiberless mutant of cotton. BMC Genom 15:466. https://doi.org/10.1186/1471-2164-15-466
Madsen EB, Madsen LH, Radutoiu S, Olbryt M, Rakwalska M, Szczyglowski K, Sato S, Kaneko T, Tabata S, Sandal N, Stougaard J (2003) A receptor kinase gene of the LysM type is involved in legume perception of rhizobial signals. Nature 425(6958):637–640. https://doi.org/10.1038/nature02045
Madsen EB, Antolin-Llovera M, Grossmann C, Ye J, Vieweg S, Broghammer A, Krusell L, Radutoiu S, Jensen ON, Stougaard J, Parniske M (2011) Autophosphorylation is essential for the in vivo function of the Lotus japonicus Nod factor receptor 1 and receptor-mediated signalling in cooperation with Nod factor receptor 5. Plant J Cell Mol Biol 65(3):404–417. https://doi.org/10.1111/j.1365-313X.2010.04431.x
Majerus PW, Kisseleva MV, Norris FA (1999) The role of phosphatases in inositol signaling reactions. J Biol Chem 274(16):10669–10672
Marx H, Minogue CE, Jayaraman D, Richards AL, Kwiecien NW, Siahpirani AF, Rajasekar S, Maeda J, Garcia K, Del Valle-Echevarria AR, Volkening JD, Westphall MS, Roy S, Sussman MR, Ane JM, Coon JJ (2016) A proteomic atlas of the legume Medicago truncatula and its nitrogen-fixing endosymbiont Sinorhizobium meliloti. Nat Biotechnol 34(11):1198–1205. https://doi.org/10.1038/nbt.3681
Messinese E, Mun JH, Yeun LH, Jayaraman D, Rouge P, Barre A, Lougnon G, Schornack S, Bono JJ, Cook DR, Ane JM (2007) A novel nuclear protein interacts with the symbiotic DMI3 calcium- and calmodulin-dependent protein kinase of Medicago truncatula. Mol Plant-Microbe Interact MPMI 20(8):912–921. https://doi.org/10.1094/mpmi-20-8-0912
Middleton PH, Jakab J, Penmetsa RV, Starker CG, Doll J, Kalo P, Prabhu R, Marsh JF, Mitra RM, Kereszt A, Dudas B, VandenBosch K, Long SR, Cook DR, Kiss GB, Oldroyd GE (2007) An ERF transcription factor in Medicago truncatula that is essential for Nod factor signal transduction. Plant Cell 19(4):1221–1234. https://doi.org/10.1105/tpc.106.048264
Miller JB, Pratap A, Miyahara A, Zhou L, Bornemann S, Morris RJ, Oldroyd GE (2013) Calcium/calmodulin-dependent protein kinase is negatively and positively regulated by calcium, providing a mechanism for decoding calcium responses during symbiosis signaling. Plant Cell 25(12):5053–5066. https://doi.org/10.1105/tpc.113.116921
Misteli T, Caceres JF, Clement JQ, Krainer AR, Wilkinson MF, Spector DL (1998) Serine phosphorylation of SR proteins is required for their recruitment to sites of transcription in vivo. J Cell Biol 143(2):297–307
Mohd-Radzman NA, Laffont C, Ivanovici A, Patel N, Reid D, Stougaard J, Frugier F, Imin N, Djordjevic MA (2016) Different pathways act downstream of the CEP peptide receptor CRA2 to regulate lateral root and nodule development. Plant Physiol 171(4):2536–2548. https://doi.org/10.1104/pp.16.00113
Ngo JC, Chakrabarti S, Ding JH, Velazquez-Dones A, Nolen B, Aubol BE, Adams JA, Fu XD, Ghosh G (2005) Interplay between SRPK and Clk/Sty kinases in phosphorylation of the splicing factor ASF/SF2 is regulated by a docking motif in ASF/SF2. Mol Cell 20(1):77–89. https://doi.org/10.1016/j.molcel.2005.08.025
Nguyen TH, Brechenmacher L, Aldrich JT, Clauss TR, Gritsenko MA, Hixson KK, Libault M, Tanaka K, Yang F, Yao Q, Pasa-Tolic L, Xu D, Nguyen HT, Stacey G (2012) Quantitative phosphoproteomic analysis of soybean root hairs inoculated with Bradyrhizobium japonicum. Mol Cell Proteom: MCP 11(11):1140–1155. https://doi.org/10.1074/mcp.M112.018028
Oldroyd GE, Downie JA (2008) Coordinating nodule morphogenesis with rhizobial infection in legumes. Ann Rev Plant Biol 59:519–546. https://doi.org/10.1146/annurev.arplant.59.032607.092839
Paumi CM, Chuk M, Chevelev I, Stagljar I, Michaelis S (2008) Negative regulation of the yeast ABC transporter Ycf1p by phosphorylation within its N-terminal extension. J Biol Chem 283(40):27079–27088. https://doi.org/10.1074/jbc.M802569200
Popp C, Ott T (2011) Regulation of signal transduction and bacterial infection during root nodule symbiosis. Curr Opin Plant Biol 14(4):458–467. https://doi.org/10.1016/j.pbi.2011.03.016
Qing D, Yang Z, Li M, Wong WS, Guo G, Liu S, Guo H, Li N (2016) Quantitative and functional phosphoproteomic analysis reveals that ethylene regulates water transport via the C-terminal phosphorylation of aquaporin PIP2;1 in Arabidopsis. Mol Plant 9(1):158–174. https://doi.org/10.1016/j.molp.2015.10.001
Radutoiu S, Madsen LH, Madsen EB, Felle HH, Umehara Y, Gronlund M, Sato S, Nakamura Y, Tabata S, Sandal N, Stougaard J (2003) Plant recognition of symbiotic bacteria requires two LysM receptor-like kinases. Nature 425(6958):585–592. https://doi.org/10.1038/nature02039
Ragel P, Rodenas R, Garcia-Martin E, Andres Z, Villalta I, Nieves-Cordones M, Rivero RM, Martinez V, Pardo JM, Quintero FJ, Rubio F (2015) The CBL-interacting protein kinase CIPK23 regulates HAK5-mediated high-affinity K+ uptake in Arabidopsis roots. Plant Physiol 169(4):2863–2873. https://doi.org/10.1104/pp.15.01401
Reddy ASN (2004) Plant serine/arginine-rich proteins and their role in pre-mRNA splicing. Trends Plant Sci 9(11):541–547. https://doi.org/10.1016/j.tplants.2004.09.007
Rival P, de Billy F, Bono JJ, Gough C, Rosenberg C, Bensmihen S (2012) Epidermal and cortical roles of NFP and DMI3 in coordinating early steps of nodulation in Medicago truncatula. Development 139(18):3383–3391. https://doi.org/10.1242/dev.081620
Rivero C, Traubenik S, Zanetti ME, Blanco FA (2017) Small GTPases in plant biotic interactions. Small GTPases. https://doi.org/10.1080/21541248.2017.1333557
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 Proteom: MCP 11(9):724–744. https://doi.org/10.1074/mcp.M112.019208
Schauser L, Roussis A, Stiller J, Stougaard J (1999) A plant regulator controlling development of symbiotic root nodules. Nature 402(6758):191–195. https://doi.org/10.1038/46058
Schwartz D, Gygi SP (2005) An iterative statistical approach to the identification of protein phosphorylation motifs from large-scale data sets. Nat Biotechnol 23(11):1391–1398. https://doi.org/10.1038/nbt1146
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: MPMI 24(8):932–937. https://doi.org/10.1094/mpmi-09-10-0222
Singh S, Katzer K, Lambert J, Cerri M, Parniske M (2014) CYCLOPS, a DNA-binding transcriptional activator, orchestrates symbiotic root nodule development. Cell Host Microbe 15(2):139–152. https://doi.org/10.1016/j.chom.2014.01.011
Sjovall-Larsen S, Alexandersson E, Johansson I, Karlsson M, Johanson U, Kjellbom P (2006) Purification and characterization of two protein kinases acting on the aquaporin SoPIP2;1. Biochim Biophys Acta 1758(8):1157–1164. https://doi.org/10.1016/j.bbamem.2006.06.002
Smertenko AP, Chang HY, Sonobe S, Fenyk SI, Weingartner M, Bogre L, Hussey PJ (2006) Control of the AtMAP65-1 interaction with microtubules through the cell cycle. J Cell Sci 119(Pt 15):3227–3237. https://doi.org/10.1242/jcs.03051
Smit P, Raedts J, Portyanko V, Debelle F, Gough C, Bisseling T, Geurts R (2005) NSP1 of the GRAS protein family is essential for rhizobial Nod factor-induced transcription. Science 308(5729):1789–1791. https://doi.org/10.1126/science.1111025
Stolarczyk EI, Reiling CJ, Paumi CM (2011) Regulation of ABC transporter function via phosphorylation by protein kinases. Curr Pharm Biotechnol 12(4):621–635
Stracke S, Kistner C, Yoshida S, Mulder L, Sato S, Kaneko T, Tabata S, Sandal N, Stougaard J, Szczyglowski K, Parniske M (2002) A plant receptor-like kinase required for both bacterial and fungal symbiosis. Nature 417(6892):959–962. https://doi.org/10.1038/nature00841
Suzaki T, Yoro E, Kawaguchi M (2015) Leguminous plants: inventors of root nodules to accommodate symbiotic bacteria. Int Rev Cell Mol Biol 316:111–158. https://doi.org/10.1016/bs.ircmb.2015.01.004
Thingholm TE, Jorgensen TJ, Jensen ON, Larsen MR (2006) Highly selective enrichment of phosphorylated peptides using titanium dioxide. Nat Protoc 1(4):1929–1935. https://doi.org/10.1038/nprot.2006.185
Tillemans V, Leponce I, Rausin G, Dispa L, Motte P (2006) Insights into nuclear organization in plants as revealed by the dynamic distribution of Arabidopsis SR splicing factors. Plant Cell 18(11):3218–3234. https://doi.org/10.1105/tpc.106.044529
Valdés-López O, Sussman MR (2015) Leveraging large-scale approaches to dissect the rhizobia–legume symbiosis. In: Biological nitrogen fixation. Wiley, Hoboken, pp 799–806. https://doi.org/10.1002/9781119053095.ch79
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(5):e0155460. https://doi.org/10.1371/journal.pone.0155460
von Arnim AG (2001) A hitchhiker’s guide to the proteasome. Science’s STKE: signal transduction knowledge environment 2001 (97):pe2. https://doi.org/10.1126/stke.2001.97.pe2
Wada S, Tanabe K, Yamazaki A, Niimi M, Uehara Y, Niimi K, Lamping E, Cannon RD, Monk BC (2005) Phosphorylation of candida glabrata ATP-binding cassette transporter Cdr1p regulates drug efflux activity and ATPase stability. J Biol Chem 280(1):94–103. https://doi.org/10.1074/jbc.M408252200
Wang L, Liang W, Xing J, Tan F, Chen Y, Huang L, Cheng CL, Chen W (2013) Dynamics of chloroplast proteome in salt-stressed mangrove Kandelia candel (L.) Druce. J Proteome Res 12(11):5124–5136. https://doi.org/10.1021/pr4006469
Wisniewski JR, Zougman A, Nagaraj N, Mann M (2009) Universal sample preparation method for proteome analysis. Nat Methods 6(5):359–362. https://doi.org/10.1038/nmeth.1322
Yano K, Yoshida S, Muller J, Singh S, Banba M, Vickers K, Markmann K, White C, Schuller B, Sato S, Asamizu E, Tabata S, Murooka Y, Perry J, Wang TL, Kawaguchi M, Imaizumi-Anraku H, Hayashi M, Parniske M (2008) CYCLOPS, a mediator of symbiotic intracellular accommodation. Proc Natl Acad Sci USA 105(51):20540–20545. https://doi.org/10.1073/pnas.0806858105
Ye J, Zhang X, Young C, Zhao X, Hao Q, Cheng L, Jensen ON (2010) Optimized IMAC-IMAC protocol for phosphopeptide recovery from complex biological samples. J Proteome Res 9(7):3561–3573. https://doi.org/10.1021/pr100075x
Ye J, Zhang Z, Long H, Zhang Z, Hong Y, Zhang X, You C, Liang W, Ma H, Lu P (2015) Proteomic and phosphoproteomic analyses reveal extensive phosphorylation of regulatory proteins in developing rice anthers. Plant J 84(3):527–544. https://doi.org/10.1111/tpj.13019
Ye J, Zhang Z, You C, Zhang X, Lu J, Ma H (2016) Abundant protein phosphorylation potentially regulates Arabidopsis anther development. J Exp Bot 67(17):4993–5008. https://doi.org/10.1093/jxb/erw293
Yun CY, Velazquez-Dones AL, Lyman SK, Fu XD (2003) Phosphorylation-dependent and -independent nuclear import of RS domain-containing splicing factors and regulators. J Biol Chem 278(20):18050–18055. https://doi.org/10.1074/jbc.M211714200
Zulawski M, Braginets R, Schulze WX (2013) PhosPhAt goes kinases--searchable protein kinase target information in the plant phosphorylation site database PhosPhAt. Nucleic Acids Res 41(D1):D1176–D1184. https://doi.org/10.1093/nar/gks1081
Acknowledgements
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).
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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.
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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)
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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)
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Supplementary Figure S3 Phylogenetic classification (A) and sequence alignment of LjRBOHs (B). The phosphorylated sites are marked with a red star. (PDF 147 KB)
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Supplementary Figure S4 Sequence alignments of phosphorylated aquaporins. The phosphorylated sites are marked with a red star. (PDF 374 KB)
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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)
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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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Zhang, Z., Ke, D., Hu, M. et al. Quantitative phosphoproteomic analyses provide evidence for extensive phosphorylation of regulatory proteins in the rhizobia–legume symbiosis. Plant Mol Biol 100, 265–283 (2019). https://doi.org/10.1007/s11103-019-00857-3
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DOI: https://doi.org/10.1007/s11103-019-00857-3