Abstract
Key message
An affinity-based chemical proteomic technique enabled direct identification of BAP-interacting proteins in wheat, including the well-known cytokinin-binder, cytokinin-binding protein 1.
Abstract
In this work, we show the development of a chemical proteomic technique for the identification of proteins binding to natural aromatic cytokinins (CKs). 6-benzylaminopurine (BAP) and documented CK-binder, wheat germ-allocated cytokinin-binding protein 1 (CBP-1), were suggested as an ideal proof-of concept affinity pair. Therefore, wheat grains were chosen as a model plant material. The BAP affinity beads were prepared by the immobilization of synthesized BAP-derived ligand to a commercial, pre-activated resin and used to isolate target proteins. The proteomic analysis of complex plant extracts is often complicated by the presence of highly abundant background proteins; in this case, the omnipresent alpha-amylase inhibitors (AAIs). To cope with this problem, we included SDS–PAGE, in-gel trypsin digestion and fraction pooling prior to shotgun analysis, which brought about an obvious drop in the signals belonging to the obstructing proteins. This was accompanied by a sharp increase in the number of identified BAP targets in comparison to a conventional in-solution digestion approach. To distinguish specific CK-binding proteins from those having a general affinity for nucleotide-like compounds, competitive pull-downs with natural nucleotides and free BAP were included in every affinity experiment. By this approach, we were able to identify a group of BAP-interacting proteins, which were subsequently found to be related to biological processes affected by CKs. Moreover, the selected affinity enrichment strategy was verified by the detection of the aforementioned CK-interacting protein, CBP-1. We propose that the developed method represents a promising tool for appealing research of as yet unknown CK molecular partners in plants.
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Abbreviations
- AAI:
-
Alpha-amylase inhibitor
- BAP:
-
6-Benzylaminopurine
- C2-AHA-BAP:
-
2-(6-Aminohexyl)-6-benzyl-9H-purine-2,6-diamine
- CBP-1:
-
Cytokinin-binding protein 1
- CK:
-
Cytokinin
- DMSO:
-
Dimethylsulfoxide
- EDTA:
-
Ethylenediaminetetraacetic acid
- FDR:
-
False discovery rate
- GO:
-
Gene ontology
References
Argueso CT, Ferreira FJ, Kieber JJ (2009) Environmental perception avenues: the interaction of cytokinin and environmental response pathways. Plant Cell Environ 32:1147–1160
Bantscheff M, Hopf C, Savitski MM, Dittmann A, Grandi P, Michno AM, Schlegl J, Abraham Y, Becher I, Bergamini G, Boesche M, Delling M, Dümpelfeld B, Eberhard D, Huthmacher C, Mathieson T, Poeckel D, Reader V, Strunk K, Sweetman G, Kruse U, Neubauer G, Ramsden NG, Drewes G (2011) Chemoproteomics profiling of HDAC inhibitors reveals selective targeting of HDAC complexes. Nat Biotechnol 29:255–265
Breitkopf SB, Oppermann FS, Kéri G, Grammel M, Daub H (2010) Proteomics analysis of cellular imatinib targets and their candidate downstream effectors. J Proteome Res 9:6033–6043
Brenner WG, Ramireddy E, Heyl A, Schmülling T (2012) Gene regulation by cytokinin in Arabidopsis. Front Plant Sci 3:8
Brinegar AC, Stevens A, Fox JE (1985) Biosynthesis and degradation of a wheat embryo cytokinin-binding protein during embryogenesis and germination. Plant Physiol 79:706–710
Candiano G, Bruschi M, Musante L, Santucci L, Ghiggeri GM, Carnemolla B, Orecchia P, Zardi L, Righetti PG (2004) Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis 25:1327–1333
Cerny M, Dycka F, Bobalova J, Brzobohaty B (2011) Early cytokinin response proteins and phosphoproteins of Arabidopsis thaliana identified by proteome and phosphoproteome profiling. J Exp Bot 62:921–937
Cerny M, Novak J, Habanova H, Cerna H, Brzobohaty B (2016) Role of the proteome in phytohormonal signaling. Biochim Biophys Acta 1864:1003–10015
Chamrad I, Rix U, Stulakov A, Gridling M, Parapatics K, Muller AC, Altiok C, Colinge J, Superti-Furga G, Haura EB, Bennet KL (2013) A minimalized chemical proteomic approach for target profiling of clinical kinase inhibitors in tumor biopsies. J Proteome Res 12:4005–4017
Chamrad I, Simersky R, Beresova L, Strnad M, Sebela M, Lenobel R (2014) Proteomic identification of a candidate sequence of wheat cytokinin-binding protein 1. J Plant Growth Regul 33:896–902
Childs KL, Hamilton JP, Zhu W, Ly E, Cheung F, Wu H, Rabinowicz PD, Town CD, Buell CR, Chan AP (2007) The TIGR plant transcript assemblies database. Nucleic Acids Res 35(Database issue):D846–D851
Dadvar P, O’Flaherty M, Scholten A, Rumpel K, Heck AJR (2009) A chemical proteomics based enrichment technique targeting the interactome of the PDE5 inhibitor PF-450124. Mol Biosyst 5:472–482
Dupont FM, Vensel WH, Tanaka CK, Hurkman WJ, Altenbach SB (2011) Deciphering the complexities of the wheat flour proteome using quantitative two-dimensional electrophoresis, three proteases and tandem mass spectrometry. Proteome Sci 9:10
Erion JL, Fox JE (1981) Purification and properties of a protein which binds cytokinin-active 6-substituted purines. Plant Physiol 67:156–162
Fernbach NV, Planyavsky M, Müller A, Breitwieser FP, Colinge J, Rix U, Bennett KL (2009) Acid elution and one-dimensional shotgun analysis on an Orbitrap mass spectrometer: an application to drug affinity chromatography. J Proteome Res 8:4753–4765
Franco OL, Rigden DJ, Melo FR, Grossi-De-Sá MF (2002) Plant alpha-amylase inhibitors and their interaction with insect alpha-amylases. Eur J Biochem 269:397–412
Gan S, Amasino RM (1995) Inhibition of leaf senescence by autoregulated production of cytokinin. Science 270:1986–1988
Heintz D, Erxleben A, High AA, Wutz V, Reski R, Van Dorsselaer A, Sarnighausen E (2006) Rapid alteration of the phosphoproteome in the moss Physcomitrella patens after cytokinin treatment. J Proteome Res 5:2283–2293
Jeffery DA, Bogyo M (2003) Chemical proteomics and its application to drug discovery. Curr Opin Plant Biol 14:87–95
Kaminek M, Trckova M, Fox JE, Gaudinova A (2003) Comparison of cytokinin-binding proteins from wheat and oat grains. Physiol Plant 117:453–458
Kravets VS, Kolesnikov YS, Kretynin SV, Getman IA, Romanov GA (2010) Rapid activation of specific phospholipase(s) D by cytokinin in Amaranthus assay system. Physiol Plant 138:249–255
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Li J, Rix U, Fang B, Bai Y, Edwards A, Colinge J, Bennett KL, Gao J, Song L, Eschrich S, Superti-Furga G, Koomen J, Haura EB (2010) A chemical and phosphoproteomic characterization of dasatinib action in lung cancer. Nat Chem Biol 6:291–299
Marhavy P, Bielach A, Abas L, Abuzeineh A, Duclercq J, Tanaka H, Parezova M, Petrasek J, Friml J, Kleine-Vehn J, Benkova E (2011) 0 Cytokinin modulates endocytic trafficking of PIN1 auxin efflux carrier to control plant organogenesis. Dev Cell 21:796–804
McCarthy FM, Wang N, Magee GB, Nanduri B, Lawrence ML, Camon EB, Barrell DG, Hill DP, Dolan ME, Williams WP, Luthe DS, Bridges SM, Burgess SC (2006) AgBase: a functional genomics resource for agriculture. BMC Genomics 7:229
O’Brien JA, Benková E (2013) Cytokinin cross-talking during biotic and abiotic stress responses. Front Plant Sci 4:451
Olsen JV, de Godoy LMF, Li G, Macek B, Mortensen P, Pesch R, Makarov A, Lange O, Horning S, Mann M (2005) Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap. Mol Cell Proteomics 4:2010–2021
Raijmakers R, Dadvar P, Pelletier S, Gouw J, Rumpel K, Heck AJR (2010) Target profiling of a small library of phosphodiesterase 5 (PDE5) inhibitors using chemical proteomics. ChemMedChem 5:1927–1936
Raines T, Shanks C, Cheng CY, McPherson D, Argueso CT, Kim HJ, Franco-Zorrilla JM, López-Vidriero I, Solano R, Vaňková R, Schaller GE, Kieber JJ (2016) The cytokinin response factors modulate root and shoot growth and promote leaf senescence in Arabidopsis. Plant J 85:134–147
Rappsilber J, Mann M, Ishihama Y (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2:1896–1906
Rix U, Superti-Furga G (2009) Target profiling of small molecules by chemical proteomics. Nat Chem Biol 5:616–624
Scholten A, Poh KM, van Veen TAB, van Breukelen B, Vos MA, Heck AJR (2006) Analysis of the cGMP/cAMP interactome using a chemical proteomics approach in mammalian heart tissue validates sphingosine kinase type 1-interacting protein as a genuine and highly abundant AKAP. J Proteome Res 5:1435–1447
Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1:2856–2860
Tanaka M, Takei K, Kojima M, Sakakibara H, Mori H (2006) Auxin controls local cytokinin biosynthesis in the nodal stem in apical dominance. Plant J 45:1028–1036
Vescovi M, Riefler M, Gessuti M, Novák O, Schmülling T, Lo Schiavo F (2012) Programmed cell death induced by high levels of cytokinin in Arabidopsis cultured cells is mediated by the cytokinin receptor CRE1/AHK4. J Exp Bot 63:2825–2832
Werner T, Schmülling T (2009) Cytokinin action in plant development. Curr Opin Plant Biol 12:527–538
Werner T, Holst K, Pörs Y, Guivarc’h A, Mustroph A, Chriqui D, Grimm B, Schmülling T (2008) Cytokinin deficiency causes distinct changes of sink and source parameters in tobacco shoots and roots. J Exp Bot 59:2659–2672
Zwack PJ, Rashotte AM (2015) Interactions between cytokinin signalling and abiotic stress responses. J Exp Bot 66:4863–4871
Acknowledgements
We thank David A. Morris for helpful discussions, advices, careful reading and critical remarks on the manuscript. We are grateful to Petr Galuszka and David Kopečný for their help with the TIGR database. This work was supported by the Grant LO1204 from the National Program of Sustainability I by the Ministry of Education, Youth and Sports, Czech Republic.
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Communicated by Vincent Thijs.
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Simerský, R., Chamrád, I., Kania, J. et al. Chemical proteomic analysis of 6-benzylaminopurine molecular partners in wheat grains. Plant Cell Rep 36, 1561–1570 (2017). https://doi.org/10.1007/s00299-017-2174-4
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DOI: https://doi.org/10.1007/s00299-017-2174-4