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Real-Time Genetic Manipulations of the Cytokinin Pathway: A Tool for Laboratory and Field Studies

  • Martin Schäfer
  • Stefan MeldauEmail author
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Part of the Methods in Molecular Biology book series (MIMB, volume 1569)

Abstract

Although many established tools for cytokinin (CK) pathway manipulations are well suitable for the analysis of molecular interactions, their use on a whole plant scale is often limited by the induction of severe developmental defects. To circumvent this problem, different methods were developed that allow for a more precise manipulation of the CK pathway. Here we present one of these systems, the pOp6/LhGR system for chemically inducible gene expression. This system allows regulation on a spatial, temporal, and quantitative scale and therefore provides a superior tool for analyzing the role of CKs in the interactions of plants with their environment. The pOp6/LhGR system was tested for RNAi-mediated gene silencing and heterologous gene expression and was successfully used for CK pathway manipulations in different model organisms (Arabidopsis thaliana, Nicotiana tabaccum, Nicotiana attenuata, Citrus sinensis × C. trifoliate). Here we describe specific aspects of the screening procedure and present an experimental setup that can not only be used in the laboratory but is also applicable under field conditions.

Key words

Cytokinins Isopentenyl transferase pOp6 LhGR Dexamethasone Stress response Ecology Fieldwork 

References

  1. 1.
    Kudo T, Kiba T, Sakakibara H (2010) Metabolism and long-distance translocation of cytokinins. J Integr Plant Biol 52:53–60CrossRefPubMedGoogle Scholar
  2. 2.
    Werner T, Nehnevajova E, Köllmer I, Novak O, Strnad M, Krämer U, Schmülling T (2010) Root-specific reduction of cytokinin causes enhanced root growth, drought tolerance, and leaf mineral enrichment in Arabidopsis and tobacco. Plant Cell 22:3905–3920CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Riefler M, Novak O, Strnad M, Schmülling T (2006) Arabidopsis cytokinin receptor mutants reveal functions in shoot growth, leaf senescence, seed size, germination, root development, and cytokinin metabolism. Plant Cell Online 18:40–54CrossRefGoogle Scholar
  4. 4.
    Argueso CT, Ferreira FJ, Epple P, To JPC, Hutchison CE, Schaller GE, Dangl JL, Kieber JJ (2012) Two-component elements mediate interactions between cytokinin and salicylic acid in plant immunity. PLoS Genet 8:e1002448CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Novák J, Pavlů J, Novák O, Nožková-Hlaváčková V, Špundová M, Hlavinka J, Koukalová Š, Skalák J, Černý M, Brzobohatý B (2013) High cytokinin levels induce a hypersensitive-like response in tobacco. Ann Bot 112:41–55CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Gan SS, Amasino RM (1995) Inhibition of leaf senescence by autoregulated production of cytokinin. Science 270:1986–1988CrossRefPubMedGoogle Scholar
  7. 7.
    Qin H, Gu Q, Zhang J, Sun L, Kuppu S, Zhang Y, Burow M, Payton P, Blumwald E, Zhang H (2011) Regulated expression of an isopentenyltransferase gene (IPT) in peanut significantly improves drought tolerance and increases yield under field conditions. Plant Cell Physiol 52:1904–1914CrossRefPubMedGoogle Scholar
  8. 8.
    Camargo SR, Cancado GM, Ulian EC, Menossi M (2007) Identification of genes responsive to the application of ethanol on sugarcane leaves. Plant Cell Rep 26:2119–2128CrossRefPubMedGoogle Scholar
  9. 9.
    Guo HS, Fei JF, Xie Q, Chua NH (2003) A chemical-regulated inducible RNAi system in plants. Plant J 34:383–392CrossRefPubMedGoogle Scholar
  10. 10.
    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 41:899–918CrossRefPubMedGoogle Scholar
  11. 11.
    Samalova M, Brzobohaty B, Moore I (2005) pOp6/LhGR: a stringently regulated and highly responsive dexamethasone-inducible gene expression system for tobacco. Plant J 41:919–935CrossRefPubMedGoogle Scholar
  12. 12.
    Schäfer M, Brütting C, Gase K, Reichelt M, Baldwin I, Meldau S (2013) “Real time” genetic manipulation: a new tool for ecological field studies. Plant J 76:506–518CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Wielopolska A, Townley H, Moore I, Waterhouse P, Helliwell C (2005) A high-throughput inducible RNAi vector for plants. Plant Biotechnol J 3:583–590CrossRefPubMedGoogle Scholar
  14. 14.
    Rossignol P, Orbović V, Irish VF (2014) A dexamethasone-inducible gene expression system is active in Citrus plants. Sci Hortic 172:47–53CrossRefGoogle Scholar
  15. 15.
    Miller JS, Nguyen T, Stanley-Samuelson DW (1994) Eicosanoids mediate insect nodulation responses to bacterial infections. Proc Natl Acad Sci U S A 91:12418–12422CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Walton CH (1959) Clinical experience with dexamethasone. Can Med Assoc J 81:724–726PubMedPubMedCentralGoogle Scholar
  17. 17.
    Corrado G, Karali M (2009) Inducible gene expression systems and plant biotechnology. Biotechnol Adv 27:733–743CrossRefPubMedGoogle Scholar
  18. 18.
    Moore I, Samalova M, Kurup S (2006) Transactivated and chemically inducible gene expression in plants. Plant J 45:651–683CrossRefPubMedGoogle Scholar
  19. 19.
    Gase K, Weinhold A, Bozorov T, Schuck S, Baldwin IT (2011) Efficient screening of transgenic plant lines for ecological research. Mol Ecol Resour 11:890–902CrossRefPubMedGoogle Scholar
  20. 20.
    Velten J, Cakir C, Youn E, Chen J, Cazzonelli CI (2012) Transgene silencing and transgene-derived siRNA production in tobacco plants homozygous for an introduced AtMYB90 construct. PLoS One 7:e30141CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Moore I, Galweiler 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 95:376–381CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Baldwin I (1996) Chap. 49. In: Städler E, Rowell-Rahier M, Bauer R (eds) Proceedings of the 9th international symposium on insect-plant relationships, vol 53. Methyl jasmonate-induced nicotine production in Nicotiana attenuata: Inducing defenses in the field without wounding. Springer, Netherlands, pp 213–220Google Scholar
  23. 23.
    Kallenbach M, Bonaventure G, Gilardoni PA, Wissgott A, Baldwin IT (2012) Empoasca leafhoppers attack wild tobacco plants in a jasmonate-dependent manner and identify jasmonate mutants in natural populations. Proc Natl Acad Sci U S A 109:E1548–E1557CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Kessler A, Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature. Science 291:2141–2144CrossRefPubMedGoogle Scholar
  25. 25.
    Meldau S, Baldwin IT, Wu J (2011) SGT1 regulates wounding- and herbivory-induced jasmonic acid accumulation and Nicotiana attenuata’s resistance to the specialist lepidopteran herbivore Manduca sexta. New Phytol 189:1143–1156CrossRefPubMedGoogle Scholar
  26. 26.
    Heidekamp F, Dirkse WG, Hille J, van Ormondt H (1983) Nucleotide sequence of the Agrobacterium tumefaciens octopine Ti plasmid-encoded tmr gene. Nucleic Acids Res 11:6211–6223CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Bohner S, Gatz C (2001) Characterisation of novel target promoters for the dexamethasone-inducible/tetracycline-repressible regulator TGV using luciferase and isopentenyl transferase as sensitive reporter genes. Mol Gen Genet 264:860–870CrossRefPubMedGoogle Scholar
  28. 28.
    Medford JI, Horgan R, El-Sawi Z, Klee HJ (1989) Alterations of endogenous cytokinins in transgenic plants using a chimeric isopentenyl transferase gene. Plant Cell 1:403–413CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Ueda N, Kojima M, Suzuki K, Sakakibara H (2012) Agrobacterium tumefaciens tumor morphology root plastid localization and preferential usage of hydroxylated prenyl donor is important for efficient gall formation. Plant Physiol 159:1064–1072CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Aoyama T, Chua N-H (1997) A glucocorticoid-mediated transcriptional induction system in transgenic plants. Plant J 11:605–612CrossRefPubMedGoogle Scholar
  31. 31.
    Schäfer M, Meza-Canales ID, Brütting C, Baldwin IT, Meldau S (2015) Cytokinin concentrations and CHASE-DOMAIN CONTAINING HIS KINASE 2 (NaCHK2)- and NaCHK3-mediated perception modulate herbivory-induced defense signaling and defenses in Nicotiana attenuata. New Phytol 207(3):645–658CrossRefPubMedGoogle Scholar
  32. 32.
    Kallenbach M, Alagna F, Baldwin IT, Bonaventure G (2010) Nicotiana attenuata SIPK, WIPK, NPR1, and fatty acid-amino acid conjugates participate in the induction of jasmonic acid biosynthesis by affecting early enzymatic steps in the pathway. Plant Physiol 152:96–106CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Weinhold A, Kallenbach M, Baldwin IT (2013) Progressive 35S promoter methylation increases rapidly during vegetative development in transgenic Nicotiana attenuata plants. BMC Plant Biol 13:99CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Robison MM, Smid MPL, Wolyn DJ (2006) Organic solvents for the glucocorticoid inducer dexamethasone: are they toxic and unnecessary in hydroponic systems? Can J Bot 84:1013–1018CrossRefGoogle Scholar
  35. 35.
    Williams AC, Barry BW (2012) Penetration enhancers. Adv Drug Deliv Rev 64(Suppl):128–137CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of Molecular EcologyMax-Planck-Institute for Chemical EcologyJenaGermany
  2. 2.Research & DevelopmentKWS SAAT SEEinbeckGermany

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