A 330 bp region of the spinach nitrite reductase gene promoter directs nitrate‐inducible tissue‐specific expression in transgenic tobacco

Abstract

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 β‐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.

5[prime] Proximal Regions of Arabidopsis Nitrate Reductase Genes Direct Nitrate-Induced Transcription in Transgenic Tobacco

“…This problem can also be overcom? by genninating T2 seeds directly on ammonium medium a s described by Rastogi et al (1993) in analysis of nitrate induction on NiR. Whether the differences between our resultj and those of Vaucheret et al (1992) result from differences in the reporter gene, plant species, and/or the experimental protocol used for nitrate induction remains unclear.…”

“…It is generally agreed that NiR genes are expressed at a higher leve1 than NR genes (Layzell, 1990). Deletion analysis of the spinach NiR promoter revealed that a 330-bp 5' flanking region is sufficient in achieving high levels of expression in the presence of nitrate (Rastogi et al, 1993).…”

Expression of NADH-Specific and NAD(P)H-Bispecific Nitrate Reductase Genes in Response to Nitrate in Barley

“…Presumably eis-acting elements and truns-acting inducer protein(s) are required for NR induction. A 130-bp region of the spinach NiR promoter located between -330 and -200 is essential for induction of NiR gene expression in transgenic tobacco plants (Rastogi et al, 1993). However, this region of the NiR gene has no discernible conservation with 5' upstream DNA sequences of NR genes (Rastogi et al, 1993).…”

Light Regulation of Nitrate uptake, Assimilation and Metabolism

“…Promoter analysis of bean (Sander et al 1995), spinach (Rastogi 1993, Neininger et al 1994, Sivasankar et al 1998 tobacco (Dorbe et al 1998) and birch (Warning and Hachtel 2000) have revealed cis-acting elements involved in nitrate induction of NII transcription. Promoter analysis was performed by linking the promoters, or promoter fragments, to a reporter gene, and introducing this into tobacco, or in one case (Dorbe et al 1998) Arabidopsis.…”