5'-DELETION ANALYSIS OF THE POTATO STARCH PHOSPHORYLASE GENE - AN UPSTREAM SEQUENCE DEFINES DISTAL REGULATORY ELEMENTS AND A PROXIMAL ORGAN-DEPENDENT PROMOTER

The expression of the starch phosphorylase gene is regulated through t he control of transcription initiation as well as by another mechanism which could be mRNA stability. The -1862 to +26 region of the gene ex pressed the beta-glucuronidase (GUS) reporter gene in transgenic potat o plants grown in vitro, at normal levels in both stems and microtuber s, and also in leaves but at reduced levels. This pattern of expressio n is in agreement with the nuclear transcription data. Regions of the phosphorylase promoter involved in transcriptional regulation of the g ene were analysed by 5'-deletion analysis in transgenic plants. The de letion analysis allowed the characterization of positive and possibly negative cis-acting elements that influence the level of activity of t he phosphorylase promoter. Deletion of the region between -550 and -32 4 led to a large reduction in GUS activity in transformed plants, indi cating that a major positive element lies in this region. The proximal region of the promoter (-164 to +26) was observed to be sufficient fo r preferential expression of the reporter gene in stems and tubers. An electrophoretic mobility shift assay using nuclear extracts from pota to tubers, revealed the binding of nuclear proteins to the -550 to -32 4 region. This promoter region is very rich in dA/dT base pairs, a cha racteristic shared by many plant gene regulatory elements. Nuclear pro teins extracted from potato tubers were also shown to bind to a double -stranded oligonucleotide corresponding to an AVT-rich region (-396 to -376) of the phosphorylase promoter. Therefore, functional assays and protein-DNA interactions indicate that the -550 to -324 region enhanc es the level of transcription of the promoter and that organ-dependent regulatory elements are present in the proximal region of the promote r.