Functional dissection of a rice high-pI alpha-amylase gene promoter

Deletion analysis has previously shown that a 260 bp fragment, located between positions -230 and +29 of the 5' end of a rice high-pI alpha--amylase gene, OSamy-c, is required for gibberellic acid (GA3)-dependent transcriptional activation. We have since established a quantitative transient assay based on expression of a luciferase reporter gene in rice aleurone cells and continued to characterize the OSamy-c promoter for GA3-dependent regulatory sequences. Using this method, we have shown that the DNA sequence between -158 and -46 (sequence I) is sufficient to confer GA3-responsive activation on OSamy-c. We have also shown that this sequence is capable of directing GA3-dependent expression from a heterologous minimal promoter. Our results also showed that sequence I confers GA3 regulatory control in an orientation-dependent manner and interacts with two further upstream DNA sequences, II and III, in a combination which mildly enhances the level of the GA3 response exhibited by sequence I. Thus, we propose that sequence I confers the fundamental GA3-responsive character on OSamy-c, and that regulatory proteins that bind sequences II and III interact with each other and with regulatory proteins that bind sequence I, effectively to modulate the GA3 response.