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.