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Plant Gene Regulatory Networks pp 83–103Cite as

Analysis of a Plant Transcriptional Regulatory Network Using Transient Expression Systems

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1629))

Abstract

In plant biology, transient expression systems have become valuable approaches used routinely to rapidly study protein expression, subcellular localization, protein-protein interactions, and transcriptional activity prior to in vivo studies. When studying transcriptional regulation, luciferase reporter assays offer a sensitive readout for assaying promoter behavior in response to different regulators or environmental contexts and to confirm and assess the functional relevance of predicted binding sites in target promoters. This chapter aims to provide detailed methods for using luciferase reporter system as a rapid, efficient, and versatile assay to analyze transcriptional regulation of target genes by transcriptional regulators. We describe a series of optimized transient expression systems consisting of Arabidopsis thaliana protoplasts, infiltrated Nicotiana benthamiana leaves, and human HeLa cells to study the transcriptional regulations of two well-characterized transcriptional regulators SCARECROW (SCR) and SHORT-ROOT (SHR) on one of their targets, CYCLIN D6 (CYCD6).

Here, we illustrate similarities and differences in outcomes when using different systems. The plant-based systems revealed that the SCR–SHR complex enhances CYCD6 transcription, while analysis in HeLa cells showed that the complex is not sufficient to strongly induce CYCD6 transcription, suggesting that additional, plant-specific regulators are required for full activation. These results highlight the importance of the system and suggest that including heterologous systems, such as HeLa cells, can provide a more comprehensive analysis of a complex gene regulatory network.

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References

  1. Pajoro A, Madrigal P, Muiño JM et al (2014) Dynamics of chromatin accessibility and gene regulation by MADS-domain transcription factors in flower development. Genome Biol 15:R41

    Article  PubMed  PubMed Central  Google Scholar 

  2. Franco-Zorrilla JM, López-Vidriero I, Carrasco JL et al (2014) DNA-binding specificities of plant transcription factors and their potential to define target genes. Proc Natl Acad Sci U S A 111:2367–2372

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Ogawa N, Biggin MD (2012) High-throughput SELEX determination of DNA sequences bound by transcription factors in vitro. In: Deplancke B, Gheldof N (eds) Gene regulatory networks. Humana Press, Totowa, NJ, pp 51–63

    Chapter  Google Scholar 

  4. Cheng C, Gao X, Feng B et al (2013) Plant immune response to pathogens differs with changing temperatures. Nat Commun 4:2530

    PubMed  PubMed Central  Google Scholar 

  5. Cruz-Ramírez A, Díaz-Triviño S, Blilou I et al (2012) A bistable circuit involving SCARECROW-RETINOBLASTOMA integrates cues to inform asymmetric stem cell division. Cell 150:1002–1015

    Article  PubMed  PubMed Central  Google Scholar 

  6. Long Y, Smet W, Cruz-Ramírez A et al (2015) Arabidopsis BIRD zinc finger proteins jointly stabilize tissue boundaries by confining the cell fate regulator SHORT-ROOT and contributing to fate specification. Plant Cell 27:1185–1199

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Pauklin S, Vallier L (2013) The cell-cycle state of stem cells determines cell fate propensity. Cell 155:135–147

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Yoshida H, Hirano K, Sato T et al (2014) DELLA protein functions as a transcriptional activator through the DNA binding of the indeterminate domain family proteins. Proc Natl Acad Sci U S A 111:7861–7866

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Ar B, Je T, Jf H (1988) Optimized use of the firefly luciferase assay as a reporter gene in mammalian cell lines. Biotechniques 7:1116–1122

    Google Scholar 

  10. Ow DW, Wet JRD, Helinski DR et al (1986) Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants. Science 234:856–859

    Article  CAS  PubMed  Google Scholar 

  11. Bhaumik S, Gambhir SS (2002) Optical imaging of Renilla luciferase reporter gene expression in living mice. Proc Natl Acad Sci 99:377–382

    Article  CAS  PubMed  Google Scholar 

  12. Williams TM, Burlein JE, Ogden S et al (1989) Advantages of firefly luciferase as a reporter gene: application to the interleukin-2 gene promoter. Anal Biochem 176:28–32

    Article  CAS  PubMed  Google Scholar 

  13. Wood KV (1994) Luciferase assay method. http://www.google.com/patents/US5283179

  14. Bioluminescent Reporter Gene Assays Protocols and Applications Guide. https://nld.promega.com/~/media/files/resources/paguide/letter/chap8.pdf?la=en

    Google Scholar 

  15. Brunoud G, Wells DM, Oliva M et al (2012) A novel sensor to map auxin response and distribution at high spatio-temporal resolution. Nature 482:103–106

    Article  CAS  PubMed  Google Scholar 

  16. Liao C-Y, Smet W, Brunoud G et al (2015) Reporters for sensitive and quantitative measurement of auxin response. Nat Methods 12:207–210

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Alcaraz-Pérez F, Mulero V, Cayuela ML (2008) Application of the dual-luciferase reporter assay to the analysis of promoter activity in Zebrafish embryos. BMC Biotechnol 8:81

    Article  PubMed  PubMed Central  Google Scholar 

  18. Konishi M, Yanagisawa S (2013) Arabidopsis NIN-like transcription factors have a central role in nitrate signalling. Nat Commun 4:1617

    Article  PubMed  Google Scholar 

  19. Muller B, Sheen J (2008) Cytokinin and auxin interaction in root stem-cell specification during early embryogenesis. Nature 453:1094–1097

    Article  PubMed  PubMed Central  Google Scholar 

  20. Helariutta Y, Fukaki H, Wysocka-Diller J et al (2000) The SHORT-ROOT gene controls radial patterning of the Arabidopsis root through radial signaling. Cell 101:555–567

    Article  CAS  PubMed  Google Scholar 

  21. Sabatini S, Heidstra R, Wildwater M et al (2003) SCARECROW is involved in positioning the stem cell niche in the Arabidopsis root meristem. Genes Dev 17:354–358

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Scheres B, Wolkenfelt H, Willemsen V et al (1994) Embryonic origin of the Arabidopsis primary root and root meristem initials. Development 120:2475–2487

    CAS  Google Scholar 

  23. Cui H, Levesque MP, Vernoux T et al (2007) An evolutionarily conserved mechanism delimiting SHR movement defines a single layer of endodermis in plants. Science 316:421–425

    Article  CAS  PubMed  Google Scholar 

  24. Sozzani R, Cui H, Moreno-Risueno MA et al (2010) Spatiotemporal regulation of cell-cycle genes by SHORTROOT links patterning and growth. Nature 466:128–132

    Google Scholar 

  25. Moreno-Risueno MA, Sozzani R, Yardımcı GG et al (2015) Transcriptional control of tissue formation throughout root development. Science 350:426–430

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Levesque MP, Vernoux T, Busch W et al (2006) Whole-genome analysis of the SHORT-ROOT developmental pathway in arabidopsis. PLoS Biol 4:e143

    Google Scholar 

  27. Long Y, Goedhart J, Schneijderberg M et al (2015) SCARECROW-LIKE23 and SCARECROW jointly specify endodermal cell fate but distinctly control SHORT-ROOT movement. Plant J 84:773–784

    Article  CAS  PubMed  Google Scholar 

  28. Welch D, Hassan H, Blilou I et al (2007) Arabidopsis JACKDAW and MAGPIE zinc finger proteins delimit asymmetric cell division and stabilize tissue boundaries by restricting SHORT-ROOT action. Genes Dev 21:2196–2204

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Cui H, Kong D, Liu X et al (2014) SCARECROW, SCR-LIKE 23 and SHORT-ROOT control bundle sheath cell fate and function in Arabidopsis thaliana. Plant J 78:319–327

    Article  CAS  PubMed  Google Scholar 

  30. Doelling JH, Pikaard CS (1993) Transient expression in Arabidopsis thaliana protoplasts derived from rapidly established cell suspension cultures. Plant Cell Rep 12:241–244

    Google Scholar 

  31. Rensink WA, Lee Y, Liu J et al (2005) Comparative analyses of six solanaceous transcriptomes reveal a high degree of sequence conservation and species-specific transcripts. BMC Genomics 6:124

    Google Scholar 

  32. Nakagawa T, Kurose T, Hino T et al (2007) Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. J Biosci Bioeng 104:34–41

    Google Scholar 

  33. He TC, Chan TA, Vogelstein B et al (1999) PPARdelta is an APC-regulated target of nonsteroidal anti-inflammatory drugs. Cell 99:335–345

    Google Scholar 

  34. Goedhart J et al (2010) Bright cyan fluorescent protein variants identified by fluorescence lifetime screening. Nat Methods 7(2):137–139

    Google Scholar 

  35. Yoo S-D, Cho Y-H, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2:1565–1572

    Google Scholar 

  36. Liu L, Zhang Y, Tang S et al (2010) An efficient system to detect protein ubiquitination by agroinfiltration in Nicotiana benthamiana. Plant J 61:893–903

    Google Scholar 

  37. Ma L, Lukasik E, Gawehns F et al (2012) The use of agroinfiltration for transient expression of plant resistance and fungal effector proteins in nicotiana benthamiana leaves. In: Bolton MD, Thomma BPHJ (eds) Plant fungal pathogens. Humana Press, Totowa, NJ, pp 61–74

    Google Scholar 

  38. FuGENE® 6 Transfection Reagent Protocol. https://nld.promega.com/resources/protocols/technical-manuals/101/fugene-6-transfection-reagent-protocol/

    Google Scholar 

  39. GloMax® 96 Microplate Luminometer for Luminescence Assays. https://nld.promega.com/products/fluorometers-luminometers-multimode-readers/luminometers/glomax-96-microplate-luminometer/

    Google Scholar 

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Acknowledgments

We are grateful to Dr Erin Spark, Dr Wenkun Zhou, and Jorge Zamora Zaragoza for their helpful comments on this chapter.

pEGB-35S:Renilla:Tnos vector for constitutive rLUC expression was kindly provided by Prof. Smeekens Laboratory, Utrecht University, the Netherlands. pGreen vectors containing fLUC for promoters assays and constitutive rLUC were kindly provided by Dr. Rumyana Karlova, Wageningen University and Research Centre, the Netherlands. Plasmid for constitutive rLUC expression p-CMV-Renilla and mammalian protocol was kindly provided by Dr. Isabel Sanchez Perez Department of Biochemistry; School of Medicine; Biomedical Research Institute of Madrid CSIC/UAM; Madrid, Spain.

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Authors and Affiliations

  1. Plant Developmental Biology, Wageningen University and Research Centre, Droevendaalsesteeg 1, Wageningen, 6708PB, The Netherlands

    Sara Díaz-Triviño, Ben Scheres & Ikram Blilou

  2. Laboratoire de Reproduction et Développement des Plantes, Ecole Normale Supérieure de Lyon, 46 Allée d’Italie, 69364, Lyon Cedex 07, France

    Yuchen Long

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  1. Sara Díaz-Triviño
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  2. Yuchen Long
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  3. Ben Scheres
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  4. Ikram Blilou
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Correspondence to Ikram Blilou .

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Editors and Affiliations

  1. Department for Plant Cell and Molecular Biology, Institute for Biology, Humboldt-Universität zu Berlin, Berlin, Germany

    Kerstin Kaufmann

  2. Institute for Biochemistry and Biology, University of Potsdam, Potsdam, Germany

    Bernd Mueller-Roeber

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Díaz-Triviño, S., Long, Y., Scheres, B., Blilou, I. (2017). Analysis of a Plant Transcriptional Regulatory Network Using Transient Expression Systems. In: Kaufmann, K., Mueller-Roeber, B. (eds) Plant Gene Regulatory Networks. Methods in Molecular Biology, vol 1629. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7125-1_7

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  • DOI: https://doi.org/10.1007/978-1-4939-7125-1_7

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7124-4

  • Online ISBN: 978-1-4939-7125-1

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