5′ deletion analysis of the potato starch phosphorylase gene: an upstream sequence defines distal regulatory elements and a proximal organ-dependent promoter

Abstract

The expression of the starch phosphorylase gene is regulated through the control of transcription initiation as well as by another mechanism which could be mRNA stability. The −1862 to +26 region of the gene expressed the β-glucuronidase (GUS) reporter gene in transgenic potato plants grown in vitro, at normal levels in both stems and microtubers, and also in leaves but at reduced levels. This pattern of expression is in agreement with the nuclear transcription data. Regions of the phosphorylase promoter involved in transcriptional regulation of the gene were analysed by 5′-deletion analysis in transgenic plants. The deletion analysis allowed the characterization of positive and possibly negative cis-acting elements that influence the level of activity of the phosphorylase promoter. Deletion of the region between −550 and −324 led to a large reduction in GUS activity in transformed plants, indicating that a major positive element lies in this region. The proximal region of the promoter (−164 to +26) was observed to be sufficient for preferential expression of the reporter gene in stems and tubers. An electrophoretic mobility shift assay using nuclear extracts from potato tubers, revealed the binding of nuclear proteins to the −550 to −324 region. This promoter region is very rich in dA dT base pairs, a characteristic shared by many plant gene regulatory elements. Nuclear proteins extracted from potato tubers were also shown to bind to a double-stranded oligonucleotide corresponding to an A T -rich region (−396 to −376) of the phosphorylase promoter. Therefore, functional assays and protein-DNA interactions indicate that the −550 to −324 region enhances the level of transcription of the promoter and that organ-dependent regulatory elements are present in the proximal region of the promoter.