Molecular analysis of the 5′-upstream region of a gibberellin-inducible cytosolic glutamine synthetase gene (GS1b) expressed in pine vascular tissue

The promoter region of the cytosolic glutamine synthetase GS1b gene was isolated from the conifer Pinus sylvestris L. (Scots pine). The 1,171-bp stretch of sequence lying immediately upstream of the transcriptional start site was sufficient to drive the expression of a reporter gene in a manner consistent with the expression pattern of the native GS1b gene. Computer analysis of putative cis elements in this promoter region revealed the presence of an AT box, an AC motif similar to those found in other genes expressed in the vascular tissue, and a gibberellin (GA)-responsive element. Consistent with the latter finding, GS1b gene expression was induced by exogenously supplied gibberellic acid (GA(3)) in germinating pine embryos and pine seedlings. In order to examine if the putative GA-response element found in the GS1b promoter could function in the regulation of GS1b expression, a series of deletions of the upstream gene region were fused to the uidA reporter gene, and transient expression analyzed either in untreated or in GA(3)-treated pine (Pinus pinaster Ait.) protoplasts. Deletion analysis revealed that sequences containing the GA-responsive element, located between -1005 and -724 bp were essential for the increased promoter activity observed in response to GA(3). Furthermore, electrophoretic-mobility-shift assays showed that pine nuclear proteins bind to a 22-bp sequence that contains the GA-response element, located between -768 and -747 bp relative to the transcription start site.