Differences in structure of adventitious roots in Salix clones with contrasting characteristics of cadmium accumulation and sensitivity

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).