Identification of a novel cis-element exhibiting cytokinin-dependent protein binding in vitro in the 5'-region of NADPH-protochlorophyllide oxidoreductase gene in cucumber.
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.
Fusada
Naoki
N
Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259, Nagatsuta, Midori-ku, Yokohama, 226-8501, Japan.
Masuda
Tatsuru
T
Kuroda
Hirofumi
H
Shimada
Hiroshi
H
Ohta
Hiroyuki
H
Takamiya
Ken-Ichiro
K
D001483:Base Sequence; D001667:Binding, Competitive; D018553:Cucumis sativus / Q000201:enzymology* / Q000235:genetics*; D003583:Cytokinins / Q000494:pharmacology*; D018983:DNA Footprinting; D003850:Deoxyribonuclease I / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D009687:Nuclear Proteins / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding / Q000187:drug effects; D017384:Sequence Deletion / Q000235:genetics
D003583:Cytokinins; D009687:Nuclear Proteins; D044925:Oxidoreductases Acting on CH-CH Group Donors; C023886:protochlorophyllide reductase; D003850:Deoxyribonuclease I