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Analysis of anti-BZR1 antibody reveals the roles BES1 in maintaining the BZR1 levels in Arabidopsis

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Abstract

The transcription factor brassinazole-resistant 1 (BZR1) is a key component and positive regulator of the brassinosteroid (BR) signaling pathway. In this study, we report the cloning, expression, and purification of BZR1 for preparation of rabbit antibody against BZR1. The full-length coding sequence of BZR1 was cloned into the pET28a cloning vector and expressed in Escherichia coli to produce recombinant His-tagged BZR1. Using affinity chromatography with Ni-NTA beads under denaturing conditions, we were able to purify an approximately 38-kD recombinant His-BZR1 fusion protein, which was used to raise antiserum against BZR1. The antibody titer and sensitivity were evaluated by indirect ELISA and immunoblot. Furthermore, the antibody was used to detect protein from extracts derived from plant tissues, and was able to detect three major forms of BZR1 in Arabidopsis plants as well as in other three monocot species rice, Setaria viridis and sorghum. The produced anti-BZR1 antibody will be instrumental in determining the molecular mechanisms underlying BZR1 activity. Thus, we believe that our antibody represents a powerful tool for analyzing the role of BZR1 in plant physiology and characterizing the crosstalk between proteins in signaling cascades involving BZR1 in dicot and monocot plants.

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References

  • Bai MY, Shang JX, Oh E, Fan M, Bai Y, Zentella R, Sun TP, Wang ZY (2012) Brassinosteroid, gibberellin and phytochrome im**e on a common transcription module in Arabidopsis. Nat Cell Biol 14:810–817

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Bai MY, Zhang LY, Gampala SS, Zhu SW, Song WY, Chong K, Wang ZY (2007) Functions of OsBZR1 and 14-3-3 proteins in brassinosteroid signaling in rice. Proc Natl Acad Sci USA 104: 13839–13844

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Cheon J, Fujioka S, Dilkes BP, Choe S (2013) Brassinosteroids regulate plant growth through distinct signaling pathways in Selaginella and Arabidopsis. PLoS One 8:e81938

    Article  PubMed Central  PubMed  Google Scholar 

  • Choe S, Schmitz RJ, Fujioka S, Takatsuto S, Lee MO, Yoshida S, Feldmann KA, Tax FE (2002) Arabidopsis brassinosteroid-insensitive dwarf12 mutants are semidominant and defective in a glycogen synthase kinase 3b-like kinase. Plant Physiol 130: 1506–1515

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Chung Y, Choe S (2013) The regulation of brassinosteroid biosynthesis in Arabidopsis. Crit Rev Plant Sci 32:396–410

    Article  Google Scholar 

  • Frey A, Di Canzio J, Zurakowski D (1998) A statistically defined endpoint titer determination method for immunoassays. J Immunol Methods 221:35–41

    Article  CAS  PubMed  Google Scholar 

  • He JX, Gendron JM, Sun Y, Gampala SS, Gendron N, Sun CQ, Wang ZY (2005) BZR1 is a transcriptional repressor with dual roles in brassinosteroid homeostasis and growth responses. Science 307: 1634–1638

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • He JX, Gendron JM, Yang Y, Li J, Wang ZY (2002) The GSK3-like kinase BIN2 phosphorylates and destabilizes BZR1, a positive regulator of the brassinosteroid signaling pathway in Arabidopsis. Proc Natl Acad Sci USA 99:10185–10190

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • He JX, Gendron JM, Yang Y, Li J, Wang ZY (2002) The GSK3-like kinase BIN2 phosphorylates and destabilizes BZR1, a positive regulator of the brassinosteroid signaling pathway in Arabidopsis. Proc Natl Acad Sci USA 99:10185–10190

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Kim B, Fujioka S, Kwon M, Jeon J, Choe S (2013) Arabidopsis Brassinosteroid-overproducing gulliver3-D/dwarf4-D mutants exhibit altered responses to Jasmonic acid and pathogen. Plant Cell Rep 32:1139–1149

    Article  CAS  PubMed  Google Scholar 

  • Kim B, Jeong YJ, Corvalan C, Fujioka S, Cho S, Park T, Choe S (2014) Darkness and gulliver2/phyB mutation decrease the abundance of phosphorylated BZR1 to activate brassinosteroid signaling in Arabidopsis. Plant J 77:1737–747

    Article  Google Scholar 

  • Kim B, Kim G, Fujioka S, Takatsuto S, Choe S (2012) Overexpression of 3beta-hydroxysteroid dehydrogenases/C-4 decarboxylases causes growth defects possibly due to abnormal auxin transport in arabidopsis. Mol Cells 34: 77–84

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Kim B, Kwon M, Jeon J, Schulz B, Corvalán C, Jeong YJ, Choe S (2014) The Arabidopsis gulliver2/phyB mutant exhibits reduced sensitivity to brassinazole. J Plant Biol 57:20–27

    Article  CAS  Google Scholar 

  • Kim TW, Guan S, Sun Y, Deng Z, Tang W, Shang JX, Burlingame AL, Wang ZY (2009) Brassinosteroid signal transduction from cell-surface receptor kinases to nuclear transcription factors. Nat Cell Biol 11:1254–1260

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Kim TW, Wang ZY (2010) Brassinosteroid signal transduction from receptor kinases to transcription factors. Annu Rev Plant Biol 61:681–704

    Article  CAS  PubMed  Google Scholar 

  • Kleber-Janke T, Becker WM (2000) Use of modified BL21(DE3) Escherichia coli cells for high-level expression of recombinant peanut allergens affected by poor codon usage. Protein Expr Purif 19:419–424

    Article  CAS  PubMed  Google Scholar 

  • Li J, Nam KH (2002) Regulation of brassinosteroid signaling by a GSK3/SHAGGY-like kinase. Science 295:1299–1301

    CAS  PubMed  Google Scholar 

  • Li L, Deng XW (2005) It runs in the family: regulation of brassinosteroid signaling by the BZR1-BES1 class of transcription factors. Trends Plant Sci 10:266–268

    Article  CAS  PubMed  Google Scholar 

  • Maharjan PM, Choe S (2011) High temperature stimulates DWARF4 (DWF4) expression to increase hypocotyl elongation in Arabidopsis. J Plant Biol 54:425–429

    Article  CAS  Google Scholar 

  • Mora-Garcia S, Vert G, Yin Y, Cano-Delgado A, Cheong H, Chory J (2004) Nuclear protein phosphatases with Kelch-repeat domains modulate the response to brassinosteroids in Arabidopsis. Genes Dev 18:448–460

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Oh E, Zhu JY, Wang ZY (2012) Interaction between BZR1 and PIF4 integrates brassinosteroid and environmental responses. Nat Cell Biol 14:802–809

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Ryu H, Kim K, Cho H, Park J, Choe S, Hwang I (2007) Nucleocytoplasmic shuttling of BZR1mediated by phosphorylation is essential in Arabidopsis brassinosteroid signaling. Plant Cell 17:2749–2762

    Article  Google Scholar 

  • Schenk PM, Baumann S, Mattes R, Steinbiss HH (1995) Improved high-level expression system for eukaryotic genes in Escherichia coli using T7 RNA polymerase and rare ArgtRNAs. Biotechniques 19:196–198, 200

    CAS  PubMed  Google Scholar 

  • Sun Y, Fan XY, Cao DM, Tang W, He K, Zhu JY, He JX, Bai MY, Zhu S, Oh E, Patil S, Kim TW, Ji H, Wong WH, Rhee SY, Wang ZY (2010) Integration of brassinosteroid signal transduction with the transcription network for plant growth regulation in Arabidopsis. Dev Cell 19:765–777

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Tang W, Yuan M, Wang R, Yang Y, Wang C, Oses-Prieto JA, Kim TW, Zhou HW, Deng Z, Gampala SS, Gendron JM, Jonassen EM, Lillo C, DeLong A, Burlingame AL, Sun Y, Wang ZY (2011) PP2A activates brassinosteroid-responsive gene expression and plant growth by dephosphorylating BZR1. Nat Cell Biol 13: 124–131

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Vert G, Nemhauser J, Geldner N, Hong F, Chory J (2005) Molecular mechanisms of steroid hormone signaling in plants. Annu Rev Cell Dev Biol 21:177–201

    Article  CAS  PubMed  Google Scholar 

  • Wang ZY, Nakano T, Gendron J, He J, Chen M, Vafeados D, Yang Y, Fujioka S, Yoshida S, Asami T, Chory J (2002) Nuclear-localized BZR1 mediates brassinosteroid-induced growth and feedback suppression of brassinosteroid biosynthesis. Dev Cell 2:505–513

    Article  CAS  PubMed  Google Scholar 

  • Yang Y, Dai L, **a H, Zhu K, Liu H, Chen K (2013) Protein profile of rice (Oryza sativa) seeds. Genet Mol Biol 36:87–92

    Article  PubMed Central  PubMed  Google Scholar 

  • Yin Y, Vafeados D, Tao Y, Yoshida S, Asami T, Chory J (2005) A new class of transcription factors mediates brassinosteroid-regulated gene expression in Arabidopsis. Cell 120:249–259

    Article  CAS  PubMed  Google Scholar 

  • Yin Y, Wang ZY, Mora-Garcia S, Li J, Yoshida S, Asami T, Chory J (2002) BES1 accumulates in the nucleus in response to brassinosteroids to regulate gene expression and promote stem elongation. Cell 109:181–191

    Article  CAS  PubMed  Google Scholar 

  • Yu X, Li L, Guo M, Chory J, Yin Y (2008) Modulation of brassinosteroidregulated gene expression by Jumonji domain-containing proteins ELF6 and REF6 in Arabidopsis. Proc Natl Acad Sci USA 105:7618–7623

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Yu X, Li L, Zola J, Aluru M, Ye H, Foudree A, Guo H, Anderson S, Aluru S, Liu P, Rodermel S, Yin Y (2011) A brassinosteroid transcriptional network revealed by genome-wide identification of BESI target genes in Arabidopsis thaliana. Plant J 65:634–646

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. School of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul, 151-747, Korea

    Yu Jeong Jeong, Claudia Corvalán & Sunghwa Choe

  2. Convergence Research Center for Functional Plant Products, Advanced Institutes of Convergence Technology, 864-1, Iuidong, Yeongtong-gu, Suwon-si, Gyeonggi-do, 443-270, Korea

    Soon Il Kwon & Sunghwa Choe

  3. Plant Genomics and Breeding Institute, Seoul National University, Seoul, 151-921, Korea

    Sunghwa Choe

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  1. Yu Jeong Jeong
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  2. Claudia Corvalán
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  3. Soon Il Kwon
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  4. Sunghwa Choe
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Correspondence to Sunghwa Choe.

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Jeong, Y.J., Corvalán, C., Kwon, S.I. et al. Analysis of anti-BZR1 antibody reveals the roles BES1 in maintaining the BZR1 levels in Arabidopsis. J. Plant Biol. 58, 87–95 (2015). https://doi.org/10.1007/s12374-014-0289-5

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  • DOI: https://doi.org/10.1007/s12374-014-0289-5

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