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Plant Transcription Factors pp 61–79Cite as

Generation of Inducible Transgenic Lines of Arabidopsis Transcription Factors Regulated by MicroRNAs

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1830))

Abstract

Transcription factors play key regulatory roles in all the life processes across kingdoms. In plants, the genome of a typical model species such as Arabidopsis thaliana encodes over 1500 transcription factors that regulate the expression dynamics of all the genes in time and space. Therefore, studying their function by analyzing the loss and gain-of-function lines is of prime importance in basic plant biology and its agricultural application. However, the current approach of knocking out genes often causes embryonic lethal phenotype, while inactivating one or two members of a redundant gene family yields little phenotypic changes, thereby making the functional analysis a technically challenging task. In such cases, inducible knock-down or overexpression of transcription factors appears to be a more effective approach. Restricting the transcription factors in the cytoplasm by fusing them with animal glucocorticoid/estrogen receptors (GR/ER) and then re-localizing them to the nucleus by external application of animal hormone analogues has been a useful method of gene function analysis in the model plants. In this chapter, we describe the recent advancements in the GR and ER expression systems and their use in analyzing the function of transcription factors in Arabidopsis.

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References

  1. Riechmann JL, Heard J, Martin G, Reuber L, Jiang C-J, Keddie J, Adam L, Pineda O, Ratcliffe OJ, Samaha RR, Creelman R, Pilgrim M, Broun P, Zhang JZ, Ghandehari D, Sherman BK, Yu G-L (2000) Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes. Science 290:2105–2110

    Article  CAS  PubMed  Google Scholar 

  2. Vaquerizas JM, Kummerfeld SK, Teichmann SA, Luscombe NM (2009) A census of human transcription factors: function, expression and evolution. Nat Rev Genet 10:252–263

    Article  CAS  PubMed  Google Scholar 

  3. Shiu S-H, Shih M-C, Li W-H (2005) Transcription factor families have much higher expansion rates in plants than in animals. Plant Physiol 139:18–26

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Charoensawan V, Wilson D, Teichmann SA (2010) Genomic repertoires of DNA-binding transcription factors across the tree of life. Nucleic Acids Res 38:7364–7377

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Ezer D, Shepherd SJ, Brestovitsky A, Dickinson P, Cortijo S, Charoensawan V, Box MS, Biswas S, Jaeger K, Wigge PA (2017) The G-box transcriptional regulatory code in Arabidopsis. Plant Physiol 175:628–640

    PubMed  PubMed Central  CAS  Google Scholar 

  6. Odell JT, Nagy F, Chua N-H (1985) Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313:810–812

    Article  CAS  PubMed  Google Scholar 

  7. Cornejo M-J, Luth D, Blankenship KM, Anderson OD, Blechl AE (1993) Activity of a maize ubiquitin promoter in transgenic rice. Plant Mol Biol 23:567–581

    Article  CAS  PubMed  Google Scholar 

  8. Bowling SA (1997) The cpr5 mutant of arabidopsis expresses both NPR1-dependent and NPR1-independent resistance. Plant Cell 9:1573–1584

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Moore I, Samalova M, Kurup S (2006) Transactivated and chemically inducible gene expression in plants. Plant J 45:651–683

    Article  CAS  PubMed  Google Scholar 

  10. Lee GH, Sohn SH, Park EY, Park YD (2012) Aberrant promoter methylation occurred from multicopy transgene and SU(VAR)3-9 homolog 9 (SUVH9) gene in transgenic Nicotiana benthamiana. Funct Plant Biol 39:764–773

    Article  CAS  Google Scholar 

  11. Kanno T, Huettel B, Mette MF, Aufsatz W, Jaligot E, Daxinger L, Kreil DP, Matzke M, Matzke AJM (2005) Atypical RNA polymerase subunits required for RNA-directed DNA methylation. Nat Genet 37:761–765

    Article  CAS  PubMed  Google Scholar 

  12. Biłas R, Szafran K, Hnatuszko-Konka K, Kononowicz AK (2016) Cis-regulatory elements used to control gene expression in plants. Plant Cell Tissue Organ Cult 127:269–287

    Article  CAS  Google Scholar 

  13. Dutt M, Dhekney SA, Soriano L, Kandel R, Grosser JW (2014) Temporal and spatial control of gene expression in horticultural crops. Hortic Res 1:14047

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Potenza C, Aleman L, Sengupta-Gopalan C (2004) Targeting transgene expression in research, agricultural, and environmental applications: promoters used in plant transformation. Vitr Cell Dev Biol 40:1–22

    Article  CAS  Google Scholar 

  15. Corrado G, Karali M (2009) Inducible gene expression systems and plant biotechnology. Biotechnol Adv 27:733–743

    Article  PubMed  Google Scholar 

  16. Sharma AK, Sharma MK (2009) Plants as bioreactors: recent developments and emerging opportunities. Biotechnol Adv 27:811–832

    Article  CAS  PubMed  Google Scholar 

  17. Junker BH, Chu C, Sonnewald U, Willmitzer L, Fernie AR (2003) In plants the alc gene expression system responds more rapidly following induction with acetaldehyde than with ethanol. FEBS Lett 535:136–140

    Article  CAS  PubMed  Google Scholar 

  18. Caddick MX, Greenland AJ, Jepson I, Krause KP, Qu N, Riddell KV, Salter MG, Schuch W, Sonnewald U, Tomsett AB (1998) An ethanol inducible gene switch for plants used to manipulate carbon metabolism. Nat Biotechnol 16:177–180

    Article  CAS  PubMed  Google Scholar 

  19. Salter MG, Paine JA, Riddell KV, Jepson L, Greenland AJ, Caddick MX, Brian Tomsett A (1998) Characterisation of the ethanol-inducible alc gene expression system for transgenic plants. Plant J 16:127–132

    Article  CAS  Google Scholar 

  20. Roslan HA, Salter MG, Wood CD, White MRH, Croft KP, Robson F, Coupland G, Doonan J, Laufs P, Brian Tomsett A, Caddick MX (2001) Characterization of the ethanol-inducible alc gene-expression system in Arabidopsis thaliana. Plant J 28:225–235

    Article  CAS  PubMed  Google Scholar 

  21. Sweetman JP, Chu C, Qu N, Greenland AJ, Sonnewald U, Jepson I (2002) Ethanol vapor is an efficient inducer of the alc gene expression system in model and crop plant species. Plant Physiol 129:943–948

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Garoosi GA, Salter MG, Caddick MX, Tomsett AB (2005) Characterization of the ethanol-inducible alc gene expression system in tomato. J Exp Bot 56:1635–1642

    Article  CAS  PubMed  Google Scholar 

  23. Picard D (1993) Steroid-binding domains for regulating the functions of heterologous proteins in cis. Trends Cell Biol 3:278–280

    Article  CAS  PubMed  Google Scholar 

  24. Picard D, Khursheed B, Garabedian MJ, Fortin MG, Lindquist S, Yamamoto KR (1990) Reduced levels of hsp90 compromise steroid receptor action in vivo. Nature 348:166–168

    Article  CAS  PubMed  Google Scholar 

  25. Schena M, Lloyd AM, Davis RW (1991) A steroid-inducible gene expression system for plant cells. Proc Natl Acad Sci U S A 88:10421–10425

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Günl M, Liew EF, David K, Putterill J (2009) Analysis of a post-translational steroid induction system for GIGANTEA in Arabidopsis. BMC Plant Biol 9:141

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Aoyama T, Chua NH (1997) A glucocorticoid-mediated transcriptional induction system in transgenic plants. Plant J 11:605–612

    Article  CAS  PubMed  Google Scholar 

  28. Moore I, Gälweiler L, Grosskopf D, Schell J, Palme K (1998) A transcription activation system for regulated gene expression in transgenic plants. Proc Natl Acad Sci U S A 95:376–381

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Kang HG, Fang Y, Singh KB (1999) A glucocorticoid-inducible transcription system causes severe growth defects in Arabidopsis and induces defense-related genes. Plant J 20:127–133

    Article  CAS  PubMed  Google Scholar 

  30. Andersen SU, Cvitanich C, Hougaard BK, Roussis A, Grønlund M, Jensen DB, Frøkjær LA, Jensen EØ (2003) The glucocorticoid-inducible GVG system causes severe growth defects in both root and shoot of the model legume Lotus japonicus. Mol Plant-Microbe Interact 16:1069–1076

    Article  CAS  PubMed  Google Scholar 

  31. Amirsadeghi S, McDonald AE, Vanlerberghe GC (2007) A glucocorticoid-inducible gene expression system can cause growth defects in tobacco. Planta 226:453–463

    Article  CAS  PubMed  Google Scholar 

  32. Zuo J, Niu Q-W, Chua N-H (2000) An estrogen-based transactivator XVE mediates highly inducible gene expression in transgenic plants. Plant J 24:265–273

    Article  CAS  PubMed  Google Scholar 

  33. Samalova M, Brzobohaty B, Moore I (2005) pOp6/LhGR: a stringently regulated and highly responsive dexamethasone-inducible gene expression system for tobacco. Plant J 41:919–935

    Article  CAS  PubMed  Google Scholar 

  34. Craft J, Samalova M, Baroux C, Townley H, Martinez A, Jepson I, Tsiantis M, Moore I (2005) New pOp/LhG4 vectors for stringent glucocorticoid-dependent transgene expression in Arabidopsis. Plant J 41:899–918

    Article  CAS  PubMed  Google Scholar 

  35. Mueller-Fahrnow A, Egner U (1999) Ligand-binding domain of estrogen receptors. Curr Opin Biotechnol 10:550–556

    Article  CAS  PubMed  Google Scholar 

  36. Högger P, Rohdewald P (1994) Binding kinetics of fluticasone propionate to the human glucocorticoid receptor. Steroids 59:597–602

    Article  PubMed  Google Scholar 

  37. Bruce W, Folkerts O, Garnaat C, Crasta O, Roth B, Bowen B (2000) Expression profiling of the maize flavonoid pathway genes controlled by estradiol-inducible transcription factors CRC and P. Plant Cell 12:65–80

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Okuzaki A, Konagaya K, Nanasato Y, Tsuda M, Tabei Y (2011) Estrogen-inducible GFP expression patterns in rice (Oryza sativa L.). Plant Cell Rep 30:529–538

    Article  CAS  PubMed  Google Scholar 

  39. Wallrath LL, Lu Q, Granok H, Elgin SCR (1994) Architectural variations of inducible eukaryotic promoters: preset and remodeling chromatin structures. BioEssays 16:165–170

    Article  CAS  PubMed  Google Scholar 

  40. Ainley WM, Key JL (1990) Development of a heat shock inducible expression cassette for plants: characterization of parameters for its use in transient expression assays. Plant Mol Biol 14:949–967

    Article  CAS  PubMed  Google Scholar 

  41. Shinmyo A, Yoshida K, Kasai T (1996) Construction of gene expression system in cultured tobacco cells. Ann N Y Acad Sci 782:97–106

    Article  CAS  PubMed  Google Scholar 

  42. Lee K-T, Chen S-C, Chiang B-L, Yamakawa T (2007) Heat-inducible production of beta-glucuronidase in tobacco hairy root cultures. Appl Microbiol Biotechnol 73:1047–1053

    Article  CAS  PubMed  Google Scholar 

  43. Freeman J, Sparks CA, West J, Shewry PR, Jones HD (2011) Temporal and spatial control of transgene expression using a heat-inducible promoter in transgenic wheat. Plant Biotechnol J 9:788–796

    Article  CAS  PubMed  Google Scholar 

  44. Masclaux F, Galaud JP (2011) Heat-inducible RNAi for gene functional analysis in plants. Methods Mol Biol 744:37–55

    Article  CAS  PubMed  Google Scholar 

  45. Lawrence RJ, Pikaard CS (2003) Transgene-induced RNA interference: a strategy for overcoming gene redundancy in polyploids to generate loss-of-function mutations. Plant J 36:114–121

    Article  CAS  PubMed  Google Scholar 

  46. Guo HS, Fei JF, Xie Q, Chua NH (2003) A chemical-regulated inducible RNAi system in plants. Plant J 34:383–392

    Article  CAS  PubMed  Google Scholar 

  47. Reddy GV, Meyerowitz EM (2005) Stem-cell homeostasis and growth dynamics can be uncoupled in the Arabidopsis shoot apex. Science 310:663–667

    Article  Google Scholar 

  48. Chen S, Hofius D, Sonnewald U, Bornke F (2003) Temporal and spatial control of gene silencing in transgenic plants by inducible expression of double-stranded RNA. Plant J 36:731–740

    Article  CAS  PubMed  Google Scholar 

  49. Wielopolska A, Townley H, Moore I, Waterhouse P, Helliwell C (2005) A high-throughput inducible RNAi vector for plants. Plant Biotechnol J 3:583–590

    Article  CAS  PubMed  Google Scholar 

  50. Liu S, Yoder JI (2016) Chemical induction of hairpin RNAi molecules to silence vital genes in plant roots. Sci Rep 6:37711

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Schwab R (2006) Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell 18:1121–1133

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Hirano T, Matsuzawa T, Takegawa K, Sato MH (2011) Loss-of-function and gain-of-function mutations in FAB1A/B impair endomembrane homeostasis, conferring pleiotropic developmental abnormalities in Arabidopsis. Plant Physiol 155:797–807

    Article  CAS  PubMed  Google Scholar 

  53. Bartel DP (2004) MicroRNAs. Cell 116:281–297

    Article  CAS  Google Scholar 

  54. Zhang B, Pan X, Cobb GP, Anderson TA (2006) Plant microRNA: a small regulatory molecule with big impact. Dev Biol 289:3–16

    Article  CAS  PubMed  Google Scholar 

  55. Kozomara A, Griffiths-Jones S (2014) MiRBase: annotating high confidence microRNAs using deep sequencing data. Nucleic Acids Res 42(Database issue):D68–D73. https://doi.org/10.1093/nar/gkt1181

    Article  PubMed  CAS  Google Scholar 

  56. Weigel D, Ahn JH, Blázquez MA, Borevitz JO, Christensen SK, Fankhauser C, Ferrándiz C, Kardailsky I, Malancharuvil EJ, Neff MM, Nguyen JT, Sato S, Wang Z-Y, Xia Y, Dixon RA, Harrison MJ, Lamb CJ, Yanofsky MF, Chory J (2000) Activation tagging in arabidopsis. Plant Physiol 122:1003–1014

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Palatnik JF, Allen E, Wu X, Schommer C, Schwab R, Carrington JC, Weigel D (2003) Control of leaf morphogenesis by microRNAs. Nature 425:257–263

    Article  CAS  PubMed  Google Scholar 

  58. Aukerman MJ, Sakai H (2003) Regulation of flowering time and floral organ identity by a microRNA and its APETALA2 -like target genes. Plant Cell 15:2730–2741

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Williams L, Grigg SP, Xie M, Christensen S, Fletcher JC (2005) Regulation of Arabidopsis shoot apical meristem and lateral organ formation by microRNA miR166g and its AtHD-ZIP target genes. Development 132:3657–3668

    Article  CAS  PubMed  Google Scholar 

  60. Ori N, Cohen AR, Etzioni A, Brand A, Yanai O, Shleizer S, Menda N, Amsellem Z, Efroni I, Pekker I, Alvarez JP, Blum E, Zamir D, Eshed Y (2007) Regulation of LANCEOLATE by miR319 is required for compound-leaf development in tomato. Nat Genet 39:787–791

    Article  CAS  PubMed  Google Scholar 

  61. Schommer C, Palatnik JF, Aggarwal P, Chételat A, Cubas P, Farmer EE, Nath U, Weigel D (2008) Control of jasmonate biosynthesis and senescence by miR319 targets. PLoS Biol 6:1991–2001

    Article  CAS  Google Scholar 

  62. Mallory AC, Bartel DP, Bartel B (2005) MicroRNA-directed regulation of Arabidopsis AUXIN RESPONSE FACTOR17 is essential for proper development and modulates expression of early auxin response genes. Plant Cell 17:1360–1375

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Nag A, King S, Jack T (2009) miR319a targeting of TCP4 is critical for petal growth and development in Arabidopsis. Proc Natl Acad Sci U S A 106:22534–22539

    Article  PubMed  PubMed Central  Google Scholar 

  64. Challa KR, Aggarwal P, Nath U (2016) Activation of YUCCA5 by the transcription factor TCP4 integrates developmental and environmental signals to promote hypocotyl elongation in Arabidopsis. Plant Cell 28:2117–2130

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Helliwell C, Waterhouse P (2003) Constructs and methods for high-throughput gene silencing in plants. Methods 30:289–295

    Article  CAS  PubMed  Google Scholar 

  66. Clough SJ, Bent AF (1998) Floral dip: a simplified method for agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743

    Article  CAS  PubMed  Google Scholar 

  67. Yamaguchi N, Winter CM, Wellmer F, Wagner D (2015) Identification of direct targets of plant transcription factors using the GR fusion technique. Methods Mol Biol 1284:123–38

    Google Scholar 

  68. Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51–59

    Article  CAS  PubMed  Google Scholar 

  69. Simon R, Igeno MI, Coupland G (1996) Activation of floral meristem identity genes in Arabidopsis. Nature 384:59–62

    Article  CAS  PubMed  Google Scholar 

  70. Ito T, Wellmer F, Yu H, Das P, Ito N, Alves-Ferreira M, Riechmann JL, Meyerowitz EM (2004) The homeotic protein AGAMOUS controls microsporogenesis by regulation of SPOROCYTELESS. Nature 430:356–360

    Article  CAS  PubMed  Google Scholar 

  71. Sun B, Looi L-S, Guo S, He Z, Gan E-S, Huang J, Xu Y, Wee W-Y, Ito T (2014) Timing mechanism dependent on cell division is invoked by Polycomb eviction in plant stem cells. Science 343:1248559

    Article  CAS  PubMed  Google Scholar 

  72. Sablowski RWM, Meyerowitz EM (1998) A homolog of NO APICAL MERISTEM is an immediate target of the floral homeotic genes APETALA3/PISTILLATA. Cell 92:93–103

    Article  CAS  PubMed  Google Scholar 

  73. Morohashi K, Grotewold E (2009) A systems approach reveals regulatory circuitry for Arabidopsis trichome initiation by the GL3 and GL1 selectors. PLoS Genet 5(2):e1000396. https://doi.org/10.1371/journal.pgen.1000396

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  74. Wagner D, Sablowski RWM (2001) Glucocorticoid fusions for transcription factor. In: Weigel D, Glazebrook J (eds) Arabidopsis-a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  75. Bent A (2006) Arabidopsis thaliana floral dip transformation method. Methods Mol Biol 343:87–103

    PubMed  CAS  Google Scholar 

  76. Brand L, Hörler M, Nüesch E, Vassalli S, Barrell P, Yang W, Jefferson RA, Grossniklaus U, Curtis MD (2006) A versatile and reliable two-component system for tissue-specific gene induction in Arabidopsis. Plant Physiol 141:1194–1204

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Borghi L (2010) Inducible gene expression systems for plants. Methods Mol Biol 655:65–75

    Google Scholar 

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Acknowledgments

We thank Nam Hai Chua (Rockefeller University, USA) for pER8 vector and P. Waterhouse (CSIRO, Australia) for pHANNIBAL and pART27 vectors. PA was supported by fellowship from CSIR, Govt. of India; KRC, MR and PS were supported by fellowships from MHRD, Govt. of India. Authors thank DST-FIST, UGC Centre for Advanced Study and DBT-IISc Partnership Program for the funding and infrastructure support. Pooja Aggarwal and Krishna Reddy Challa contributed equally to this work.

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  1. Pooja Aggarwal and Krishna Reddy Challa contributed equally to this work.

Authors and Affiliations

  1. Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India

    Pooja Aggarwal, Krishna Reddy Challa, Monalisha Rath, Preethi Sunkara & Utpal Nath

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  1. Pooja Aggarwal
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  2. Krishna Reddy Challa
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  3. Monalisha Rath
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  4. Preethi Sunkara
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  5. Utpal Nath
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Correspondence to Utpal Nath .

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Editors and Affiliations

  1. Plant Stem Cell Regulation and Floral Patterning Laboratory, Nara Institute of Science and Technology, Ikoma, Nara, Japan

    Nobutoshi Yamaguchi

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Aggarwal, P., Challa, K.R., Rath, M., Sunkara, P., Nath, U. (2018). Generation of Inducible Transgenic Lines of Arabidopsis Transcription Factors Regulated by MicroRNAs. In: Yamaguchi, N. (eds) Plant Transcription Factors. Methods in Molecular Biology, vol 1830. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8657-6_4

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  • DOI: https://doi.org/10.1007/978-1-4939-8657-6_4

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