Interaction of cis-acting elements in the expression of a gene encoding cytosolic glutamine synthetase in pine seedlings

Abstract

Glutamine synthetase (GS) genes, GS1a and GS1b, in pine (Pinus sylvestris L.) are differentially regulated in tissue specificity and during seedling development. To gain insight into the regulatory mechanisms controlling their expression, we have analysed the 5′-flanking sequences of the gene GS1a using a transient expression system in pine protoplasts. Structural analysis of this region revealed the presence of putative regulatory elements including two AT-rich elements and a poly CT consensus sequence. A series of 5′- and 3′-deletions of the untranslated region covering the three putative elements, −800 to −626, −626 to −427 and +118 to +177 were analysed to demonstrate the functional implications of these elements in gene regulation. An electrophoretic mobility-shift assay showed that nuclear proteins prepared from pine cotyledons interact with both AT-rich regions (−800 to −427). Interestingly, no protein binding was detected when the untranslated region (+118 to +177) was included, even if deletion of that region suppressed promoter activity in the transient expression experiments conducted. However, simultaneous deletion of both types of cis elements, A/T and CT, resulted in a recovery of promoter activity of 50%. These results suggest a key regulatory role of the CT box by the interaction with A/T stretches in the distal part of the promoter and possibly with the proximal region (−427 to −1).