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 who se expression is induced upon the addition of nitrate. Furthermore, it is known to be located in chloroplasts in leaves and plastids in root s. A 3.1 kb 5' upstream region of the spinach nitrite reductase (NiR) gene promoter was shown previously to confer nitrate inducibility on t he beta-glucuronidase (GUS) reporter gene expression in both the leave s and the roots of transgenic tobacco plants. In the present study, th is 3.1 kb promoter fragment as well as a series of promoter deletion c onstructs, fused to a GUS gene, were utilized to delineate the region of NiR promoter involved in nitrate regulation of NiR expression by st udying the cellular localization of NiR-GUS expression as well as its regulation by nitrate. In plants carrying the longest promoter fragmen t (-3100 from the transcription start site) and promoter sequences pro gressively deleted to -330 bp, the expression of GUS was markedly incr eased in the presence of nitrate, and this expression was found to occ ur in mesophyll cells in leaves and in the vascular tissues of stem an d 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 r oots 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 t issue-specific expression.