The metallothionein-like gene from buckwheat: Structural and functional analysis of the promoter region

The buckwheat ( Fagopyrum esculentum Moench) metallothionein- like gene was cloned and its 59 regulatory region was examined. Computer analysis of this region using overlapping data from three different databases predicted the existence of regulatory sequences that could be involved in responses to different hormonal and external stimuli ( ERE, heat shock-HSE, light and stress-GT-1, I-box, GATA, G-box, metal-MRE) as well as the presence of putative binding sites for plant-specific transcription factors (Dof1, NtBBF1, Athb-1). Further investigation included analysis of the interactions of distal and proximal parts of the defined regulatory region and purified Dof1 Delta C domain of maize Dof1 and the HD-Zip-1 domain of Arabidopsis thaliana Athb-1 transcription factors as well as with buckwheat leaf nuclear extract. The identity of putative Dof1- and Athb-1-binding sites was confirmed in the proximal region. More important, we found that some proteins from buckwheat leaf nuclear extract compete for the Dof1- binding sites, indicating the presence of a similar protein type in that extract. We also showed that buckwheat leaf nuclear extract itself interacted with both proximal and distal promoter regions. Interaction with the proximal region resulted in the formation of a single HMW complex, while five separate complexes with the distal region were detected, indicating interaction with predicted G- and I-boxes, which are probably involved in light- and/or stress-regulated MT3 gene expression. The promoter ability of the cloned buckwheat MT gene regulatory region was characterized by measuring the activity of the GUS reporter gene in the leaves of transgenic tobacco. Buckwheat MT3 promoter activity was also analyzed for its response to stress produced by either hydrogen peroxide or UV treatment. We found that both treatments increased GUS activity.