Plant Molecular Biology

, Volume 59, Issue 4, pp 631–645 | Cite as

Identification of a Novel Cis-Element Exhibiting Cytokinin-Dependent Protein Binding in Vitro in the 5′-region of NADPH-Protochlorophyllide Oxidoreductase Gene in Cucumber

  • Naoki Fusada
  • Tatsuru MasudaEmail author
  • Hirofumi Kuroda
  • Hiroshi Shimada
  • Hiroyuki Ohta
  • Ken-ichiro Takamiya


Cytokinins and light activate the transcription of the cucumber NADPH-protochlorophyllide reductase (POR) gene. We have previously reported that 2.3 kb of the 5′-region of this gene contains a cis-element that is responsive to cytokinin. In this study, to identify the cytokinin-responsive cis-element corresponding to chlorophyll biosynthesis and chloroplast development, we performed transient expression assays in etiolated cucumber cotyledons. A 5′-deletional analysis indicated that a 411-bp fragment (−451 to −40 bp) contained at least one of the cis-elements related to cytokinin-responsiveness. Gel mobility shift assays also detected cytokinin-enhanced binding in this region. DNase I footprinting analysis, using a 150-bp fragment (−490 to −340 bp) as the probe, identified the cytokinin-enhanced protected sequence as 5′-ATATTAGTGATAT-3′. More detailed gel mobility shift and competition analyses identified 5′-TATTAG-3′ as the sequence critical for cytokinin-enhanced binding. Mutations in the identified sequence in the transient expression assay caused a reduced but retained cytokinin-responsiveness, as well as low reporter activity of untreated control. These results suggest that the identified sequence is a novel cis-element exhibiting cytokinin-dependent protein binding in vitro, which may function effectively when interacting with other cytokinin-related elements. The effects of this element on the chloroplast development are discussed in relation to other cytokinin-related elements.


chlorophyll cucumber cytokinin DNA-binding proteins NADPH-protochlorophyllide oxidoreductase promoter 



Arabidopsis response regulator








firefly luciferase


monogalactosyldiacylglycerol synthase






NADPH-protochlorophyllide reductase


rennila luciferase


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  1. Aoyama, T., Oka, A. 2003Cytokinin signal transduction in plant cellsJ. Plant Res.116221231CrossRefPubMedGoogle Scholar
  2. Apel, K., Santel, H.J., Redlinger, T.E., Falk, H. 1980The protochlorophyllide holochrome of barley (Hordeum vulgare L.). Isolation and characterization of the NADPH:protochlorophyllide oxidoreductaseEur. J. Biochem.111251258PubMedGoogle Scholar
  3. Beevers, L., Lovers, B., Pearson, J.A., Wareing, P.F. 1970Phytochrome and hormonal control of expansion and greening of etiolated wheat leavesPlanta90286294CrossRefGoogle Scholar
  4. Bensadoun, A. and Weinstein, D. 1976. Assay of proteins in the presence of interfering materials. Anal. Biochem. 70: 241–250Google Scholar
  5. Binns, A.N. 1994Cytokinin accumulation and action: biochemical, genetic, and molecular approachesAnnu. Rev. Plant Physiol. Plant Mol. Biol.45173196CrossRefGoogle Scholar
  6. Chory, J., Reinecke, D., Sim, S., Washburn, T., Brenner, M. 1994A role for cytokinins in de-etiolation in Arabidopsis (det mutants have an altered response to cytokinins)Plant Physiol.104339347PubMedGoogle Scholar
  7. D’Agostino, I.B., Deruere, J., Kieber, J.J. 2000Characterization of the response of the Arabidopsis response regulator gene family to cytokininPlant Physiol.12417061717CrossRefPubMedGoogle Scholar
  8. Fletcher, R.A., McCullagh, D. 1971Benzyladenine as a regulator of chlorophyll synthesis in cucumber cotyledonsCan. J. Bot.4921972201Google Scholar
  9. Frick, G., Su, Q., Apel, K., Armstrong, G.A. 2003An Arabidopsis porB porC double mutant lacking light-dependent NADPH:protochlorophyllide oxidoreductases B and C is highly chlorophyll-deficient and developmentally arrestedPlant J.35141153CrossRefPubMedGoogle Scholar
  10. Fusada, N., Masuda, T., Kuroda, H., Shiraishi, T., Shimada, H., Ohta, H., Takamiya, K. 2000NADPH-protochlorophyllide oxidoreductase in cucumber is encoded by a single gene and its expression is transcriptionally enhanced by illuminationPhotosynth. Res.64147154CrossRefPubMedGoogle Scholar
  11. Griffiths, W.T. 1974Protochlorophyll and protochlorophyllide as precursors for chlorophyll synthesis in vitroFEBS Lett.49196200CrossRefPubMedGoogle Scholar
  12. Heyl, A., Schmulling, T. 2003Cytokinin signal perception and transductionCurr. Opin. Plant Biol.6480488CrossRefPubMedGoogle Scholar
  13. Hosoda, K., Imamura, A., Katoh, E., Hatta, T., Tachiki, M., Yamada, H., Mizuno, T., Yamazaki, T. 2002Molecular structure of the GARP family of plant Myb-related DNA binding motifs of the Arabidopsis response regulatorsPlant Cell1420152029CrossRefPubMedGoogle Scholar
  14. Howell, S.H., Lall, S., Che, P. 2003Cytokinins and shoot developmentTrends Plant Sci.8453459CrossRefPubMedGoogle Scholar
  15. Hutchison, C.E., Kieber, J.J. 2002Cytokinin signaling in ArabidopsisPlant Cell14S47S59CrossRefPubMedGoogle Scholar
  16. Imamura, A., Kiba, T., Tajima, Y., Yamashino, T., Mizuno, T. 2003In vivo and in vitro characterization of the ARR11 response regulator implicated in the His-to-Asp phosphorelay signal transduction in Arabidopsis thalianaPlant Cell Physiol.44122131CrossRefPubMedGoogle Scholar
  17. Jin, G., Davey, M.C., Ertl, J.R., Chen, R., Yu, Z.T., Daniel, S.G., Becker, W.M., Chen, C.M. 1998Interaction of DNA-binding proteins with the 5′-flanking region of a cytokinin-responsive cucumber hydroxypyruvate reductase genePlant Mol. Biol.38713723CrossRefPubMedGoogle Scholar
  18. Kakimoto, T. 2003Perception and signal transduction of cytokininsAnnu. Rev. Plant. Biol.54605627CrossRefPubMedGoogle Scholar
  19. Knypl, J.S. 1969Inhibition of chlorophyll synthesis by growth retardants and coumarin, and its reversal by potassiumNature22410251026Google Scholar
  20. Kobayashi, K, Awai, K, Takamiya, K, Ohta, H. 2004Arabidopsis type B monogalactosyldiacylglycerol synthase genes are expressed during pollen tube growth and induced by phosphate starvationPlant Physiol.134640648CrossRefPubMedGoogle Scholar
  21. Kuroda, H., Masuda, T., Ohta, H., Shioi, Y., Takamiya, K. 1995Light-enhanced gene expression of NADPH-protochlorophyllide oxidoreductase in cucumberBiochem. Biophys. Res. Commun.210310316CrossRefPubMedGoogle Scholar
  22. Kuroda, H., Masuda, T., Ohta, H., Shioi, Y., Takamiya, K. 1996Effects of light, developmental age and phytohormones on the expression of the encoding NADPH-Protochlorophyllide oxidoreductase in Cucumis sativusPlant Physiol. Biochem.341722Google Scholar
  23. Kuroda, H., Masuda, T., Fusada, N., Ohta, H., Takamiya, K. 2000Expression of NADPH-protochlorophyllide oxidoreductase gene in fully green leaves of cucumberPlant Cell Physiol.41226229PubMedGoogle Scholar
  24. Kuroda, H., Masuda, T., Fusada, N., Ohta, H., Takamiya, K. 2001Cytokinin-induced transcriptional activation of NADPH-protochlorophyllide oxidoredutase gene in cucumberJ. Plant Res.11417Google Scholar
  25. Kusnetsov, V., Herrmann, R.G., Kulaeva, O.N., Oelmuller, R. 1998Cytokinin stimulates and abscisic acid inhibits greening of etiolated Lupinus luteus cotyledons by affecting the expression of the light-sensitive protochlorophyllide oxidoreductaseMol. Gen. Genet.2592128CrossRefPubMedGoogle Scholar
  26. Kusnetsov, V., Landsberger, M., Meurer, J., Oelmuller, R. 1999The assembly of the CAAT-box binding complex at a photosynthesis gene promoter is regulated by light, cytokinin, and the stage of the plastidsJ. Biol. Chem.2743600936014CrossRefPubMedGoogle Scholar
  27. Lebedev, N., Timko, P.M. 1998Protochlorophyllide photoreductionPhotosynth. Res.58523CrossRefGoogle Scholar
  28. Longo, G.P., Bracale, M., Rossi, G., Longo, C.P. 1990Benzyladenine induces the appearance of LHCP-mRNA and of the relevant protein in dark-grown excised watermelon cotyledonsPlant Mol. Biol.14569573CrossRefPubMedGoogle Scholar
  29. Masuda, T., Ohta, H., Shioi, Y., Tsuji, H., Takamiya, K. 1995Stimulation of glutamyl-tRNA reductase activity by benzyladenine in greening cucumber cotyledonsPlant Cell Physiol.3612371243Google Scholar
  30. Masuda, T., Fusada, N., Oosawa, N., Takamatsu, K., Yamamoto, Y.Y., Ohto, M., Nakamura, K., Goto, K., Shibata, D., Shirano, Y., Hayashi, H., Kato, T., Tabata, S., Shimada, H., Ohta, H., Takamiya, K. 2003Functional analysis of isoforms of NADPH: protochlorophyllide oxidoreductase (POR), PORB and PORC, in Arabidopsis thalianaPlant Cell Physiol.44963974CrossRefPubMedGoogle Scholar
  31. Masuda, T., Takamiya, K. 2004Novel insights into the enzymology, regulation and physiological functions of light-dependent protochlorophyllide oxidoreductase in angiospermsPhotosynth. Res.81129CrossRefGoogle Scholar
  32. Mikami, K., Nakayama, T., Kawata, T., Tabata, T., Iwabuchi, M. 1989Specific interaction of nuclear HBP-1 with the conserved hexameric sequence ACGTCA in the regulatory region of wheat histone genesPlant Cell Physiol.30107119Google Scholar
  33. Mok, D.W., Mok, M.C. 1996Cytokinin metabolism and actionAnnu. Rev. Plant Physiol. Plant Mol. Biol.5289118CrossRefGoogle Scholar
  34. Mok, D.W., Mok, M.C. 2001Cytokinin metabolism and actionAnnu. Rev. Plant Physiol. Plant Mol. Biol5289118CrossRefPubMedGoogle Scholar
  35. Natesan, S., Gilman, M. 1995YY1 facilitates the association of serum response factor with the c-fos serum response elementMol. Cell. Biol.1559755982PubMedGoogle Scholar
  36. Oka, A., Sakai, H., Iwakoshi, S. 2002His–Asp phosphorelay signal transduction in higher plants: receptors and response regulators for cytokinin signaling in Arabidopsis thalianaGenes Genet. Syst.77383391CrossRefPubMedGoogle Scholar
  37. Oosawa, N., Masuda, T., Awai, K., Fusada, N., Shimada, H., Ohta, H., Takamiya, K. 2000Identification and light-induced expression of a novel gene of NADPH-protochlorophyllide oxidoreductase isoform in Arabidopsis thalianaFEBS Lett.474133136CrossRefPubMedGoogle Scholar
  38. Sakai, H., Honma, T., Aoyama, T., Sato, S., Kato, T., Tabata, S., Oka, A. 2001ARR1, a transcription factor for genes immediately responsive to cytokininsScience29415191521CrossRefPubMedGoogle Scholar
  39. Seyedi, M., Selstam, E., Timko, M.P., Sundqvist, C. 2001The cytokinin 2-isopentenyladenine causes partial reversion to skotomorphogenesis and induces formation of prolamellar bodies and protochlorophyllide657 in the lip1 mutant of peaPhysiol. Plant.112261272CrossRefPubMedGoogle Scholar
  40. Sheen, J. 2002Phosphorelay and transcription control in cytokinin signal transductionScience29616501652CrossRefPubMedGoogle Scholar
  41. Su, Q., Frick, G., Armstrong, G., Apel, K. 2001POR C of Arabidopsis thaliana: a third light- and NADPH-dependent protochlorophyllide oxidoreductase that is differentially regulated by lightPlant Mol. Biol.47805813CrossRefPubMedGoogle Scholar
  42. Wingender, E., Chen, X., Fricke, E., Geffers, R., Hehl, R., Liebich, I., Krull, M., Matys, V., Michael, H., Ohnhauser, R., Pruss, M., Schacherer, F., Thiele, S., Urbach, S. 2001The TRANSFAC system on gene expression regulationNucleic Acids Res.29281283CrossRefPubMedGoogle Scholar
  43. Yamaryo, Y., Kanai, D., Awai, K., Masuda, T., Shimada, H., Takamiya, K., Ohta, H. 2000Transcriptional regulation by light and phytohormones of the MGD gene in cucumberBiochem. Soc. Trans.28738740CrossRefPubMedGoogle Scholar
  44. Yamaryo, Y., Kanai, D., Awai, K., Shimojima, M., Masuda, T., Shimada, H., Takamiya, K., Ohta, H. 2003Light and cytokinin play a co-operative role in MGDG synthesis in greening cucumber cotyledonsPlant Cell Physiol.44844855CrossRefPubMedGoogle Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  • Naoki Fusada
    • 1
    • 2
  • Tatsuru Masuda
    • 2
    • 3
    Email author
  • Hirofumi Kuroda
    • 1
    • 4
  • Hiroshi Shimada
    • 1
  • Hiroyuki Ohta
    • 1
  • Ken-ichiro Takamiya
    • 1
  1. 1.Graduate School of Bioscience and BiotechnologyTokyo Institute of TechnologyYokohamaJapan
  2. 2.ATM System ProjectERATO, Japan Science and Technology AgencyYokohamaJapan
  3. 3.Graduate School of Arts and SciencesUniversity of TokyoMeguro-ku TokyoJapan
  4. 4.Plant Function Exploration Team, Plant Functional Genomics Research GroupGenomic Sciences Center, RIKEN Yokohama InstituteJapan

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