Functional analysis of two bean nitrate reductase promoters in transgenic tobacco

Transcription of nitrate reductase (NR) genes is regulated by nitrate as well as a variety of other environmental and developmental factors. Two NR genes have previously been characterized in bean (Phaseolus vulgaris). Both genes are nitrate- and light-inducible and their mRNA expression level displays a circadian rhythm. The two genes, NR1 and NR2, show differences in developmental and tissue-specific expression. In order to deliniate elements in the NR1 and NR2 promoters that are involved in these expression patterns, eight 5' deleted Versions of the NR1 promoter and one fragment of the NR2 promoter were cloned in front of the GUS reporter gene and transformed into tobacco. Approximately 50% of the regenerated transformants expressed detectable levels of GUS activity. For the eight NR1 promoter deletion constructs, differences in promoter strength were observed indicating both positive and negative elements in the promoter. For both the NR1 and the NR2 promoter-GUS constructs no significant nitrate regulation could be detected in the transgenic plants. A possible explanation for this is that elements needed for proper nitrate regulation of the NR1 and NR2 genes are located outside the analysed 3 500 bp and 1 400 bp promoter regions, respectively. A diurnal rhythm regulation similar to that seen for the endogenous nitrate reductase in bean plants, could be found for the GUS mRNA expression in several of the transformants containing the NR1 and NR2 promoter-GUS constructs.