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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
Article

Abstract

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.

Keywords

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

Abbreviations

ARR

Arabidopsis response regulator

BA

benzyladenine

Chl

chlorophyll

Chlide

chlorophyllide

LUC

firefly luciferase

MGD

monogalactosyldiacylglycerol synthase

MGDG

monogalactosyldiacylglycerol

Pchlide

protochlorophyllide

POR

NADPH-protochlorophyllide reductase

RLUC

rennila luciferase

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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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