Interaction of DNA-binding proteins with the 5 '-flanking region of a cytokinin-responsive cucumber hydroxypyruvate reductase gene

Transcription of the cucumber hpr-A gene is responsive to cytokinin and lig ht. To investigate the molecular basis for transcriptional regulation by cy tokinin, we have identified DNA sequences and proteins that may be involved in the regulation of hpr-A gene expression. Transient expression assays in etiolated cucumber cotyledons indicate that the 315 bp fragment (-382 to - 67) contains sequences necessary for cytokinin responsiveness of the lucife rase reporter gene. Band shift assays detected cytokinin-enhanced and -redu ced protein binding sites in a 97 bp fragment (-382 to -285) upstream of th e hpr-A gene. DNase I footprinting identified two protein-protected sites, a 15 bp sequence, 5'-AAATGACGAAAATGC-3', that contains an as-1 TGACG motif found in other plant promoters, and a 13 bp sequence, 5'-AAGATTGATTGAG-3', of unknown function. Two-dimensional band shift analysis of the cytokinin-r esponsive DNA protein complex revealed the presence of six DNA protein inte ractions. Band shift assays showed that cytokinin and light have different effects on the interaction of nuclear proteins to the 97 bp fragment of the hpr-A gene. These data suggest that cytokinin and light do not share ident ical signal transduction pathways in regulating hpr-A gene expression.