| Homeodomain (HD) proteins play an important role in cell fate determination and body plan definition by binding to regulatory regions of key target genes. Using the yeast two-hybrid system and a potato KNOX HD protein, POTH1, as bait, seven distinct interacting proteins have been identified from a potato (Solanum tuberosum L.) stolon library. All seven cDNAs belong to the BEL1-like family of transcription factors. Through deletion mutation analysis, we identified a protein-binding domain present in the carboxy-end of the KNOX domain of POTH1. The protein-binding domain in the BEL protein is located in the amino-terminal half of the 120-residue conserved region. BELs showed differential patterns of mRNA accumulation in various potato organs. The level of one of the BELs, StBEL-5, mRNA increased under short-day photoperiods in both leaves and stolons. Similar to overexpression lines of POTH1, transgenic lines that overexpressed StBEL-5 exhibited enhanced tuber formation under noninductive conditions and decreased mRNA levels of ga20ox1, a gene that encodes a key enzyme in the GA biosynthetic pathway.; Gel-shift assays demonstrated that StBEL5 and POTH1 bind to the regulatory region of ga20ox1 from potato. In tandem, StBEL5 and POTH1 had a greater binding affinity for the ga20ox1 promoter than either alone. The StBEL5-POTH1 heterodimer bound specifically to a composite site (T/A)GA(C/G)(T/A)(T/A)GAC containing two TGAC cores. Using a transcription assay with a GUS marker, StBEL5 and POTH1 alone suppressed the activity of the ga20ox1 promoter by more than 50%, in tandem, 80%. Dominant negative constructs containing amino-terminal protein binding domains of StBEL5 or POTH1 blocked the repression activity of StBEL5 or POTH1, respectively. The mutated ga20ox1 promoter that could be bound by StBEL5 or POTH1 individually but not by the StBEL5-POTH1 heterodimer also abolished the repression activity of StBEL5, POTH1, and the StBEL5-POTH1 heterodimer. These results suggest that StBEL5 and POTH1 promote tuberization by working as a tandem complex to repress ga20ox1 transcription. |
