Abstract
The beta-phaseolin (phas) gene, which encodes one of the major seed storage proteins of P. vulgaris, is tightly regulated at the transcription level resulting in strict tissue-specific and spatial expression during embryonic development. The phas proximal promoter contains a complex arrangement of core promoter elements including three TATA boxes as well as several putative initiator elements. To delineate the respective contributions of the core promoter elements to transcription initiation we have performed site-directed mutagenesis of the phas promoter. In vivo expression studies were performed on transgenic Arabidopsis harboring phas promoter mutants driving expression of the beta-glucuronidase (gus) reporter gene. Quantitative assessment of GUS activity in seeds bearing the promoter mutants indicated that both sequence and spacing of the TATA elements influenced the efficiency of transcription. Substitution, insertion or deletion mutations had no effect on histochemical staining patterns indicating that strict spacing requirements are not essential for correct spatial expression of phas during embryogenesis. Further evaluation of the phas promoter by in vitro transcription analysis revealed the presence of multiple TATA-dependent transcription initiation start sites. The distance between TATA elements and transcription start sites was maintained in insertion and deletion mutants through the creation of novel initiation sites, indicating that positioning of the TATA elements rather than DNA sequence was the primary determinant of start site location. We conclude that, while dispensable for proper spatial distribution, the complex architecture of the phas promoter is required to ensure high levels of accurate phas transcription initiation in the developing embryo.
Highlights
The proximal promoter region of a gene contains core promoter elements that determine the basal transcription activity of the gene and typically direct the positioning of the transcription initiation start site
Substitution mutations were made in individual TATA elements (Fig. 1) and the ability of debilitated promoters to drive expression of the reporter gene was assessed in seeds and embryos of stably-transformed Arabidopsis plants
In agreement with the in vivo analysis, both TATA1 and TATA2 were found to contribute to phas promoter activity. These results further suggested that TATA1 mediates initiation at Ϫ12 while TATA2 is principally responsible for directing initiation at the primary transcription start site situated at ϩ1
Summary
Initiator; DPE, downstream promoter element; phas, -phaseolin; TBP, TATA-binding protein; hTBP, human TATA-binding protein; UAS, upstream activating sequence. If any, of having more than one TBP binding site has not been well documented, recent analysis of the tubulin promoter in soybean indicates that the two TATA boxes present in this promoter function additively to direct transcription in seedlings. These two TATA boxes are differentially sensitive to light conditions [8], suggesting that, in the case of tubulin, the multiple TATA boxes play a role in directing high levels of transcription and provide a mechanism for titrating gene activity in response to altered environmental conditions. We conclude that the complex architecture of the TATA region of the phas promoter is not essential for maintaining spatially regulated expression of phas during embryogenesis, but is required to ensure high levels of phas message production during development
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