A 330 BP REGION OF THE SPINACH NITRITE REDUCTASE GENE PROMOTER DIRECTS NITRATE-INDUCIBLE TISSUE-SPECIFIC EXPRESSION IN TRANSGENIC TOBACCO

Nitrite reductase is an enzyme in the nitrate assimilatory pathway whose expression is induced upon the addition of nitrate. Furthermore, it is known to be located in chloroplasts in leaves and plastids in roots. A 3.1 kb 5' upstream region of the spinach nitrite reductase (NiR) gene promoter was shown previously to confer nitrate inducibility on the beta-glucuronidase (GUS) reporter gene expression in both the leaves and the roots of transgenic tobacco plants. In the present study, this 3.1 kb promoter fragment as well as a series of promoter deletion constructs, fused to a GUS gene, were utilized to delineate the region of NiR promoter involved in nitrate regulation of NiR expression by studying the cellular localization of NiR-GUS expression as well as its regulation by nitrate. In plants carrying the longest promoter fragment (-3100 from the transcription start site) and promoter sequences progressively deleted to -330 bp, the expression of GUS was markedly increased in the presence of nitrate, and this expression was found to occur in mesophyll cells in leaves and in the vascular tissues of stem and roots. When nitrate was added to NiR-GUS plants grown in the absence of nitrate, significant levels of GUS activity could be seen in the roots after 2 h and in the leaves after 6 h. Further 5' deletion of the promoter to -200 bp abolished the nitrate induction of GUS expression, indicating that the 130 bp region of the nitrite reductase promoter located between -330 and -200 is required for full nitrate-inducible tissue-specific expression.