Structure and expression of a nitrite reductase gene from bean (Phaseolus vulgaris) and promoter analysis in transgenic tobacco

Abstract

A structural gene encoding nitrite reductase (NiR) in bean (Phaseolus vulgaris) has been cloned and sequenced. The NiR gene is present as a single copy encoding a protein of 582 amino acids. The bean NiR protein is synthesized as a precursor with an amino-terminal transit peptide (TP) consisting of 18 amino acid residues. The bean NiR transit peptide shows similarity to the TPs of other known plant NiRs. The NiR gene is expressed in trifoliate leaves and in roots of 20-day old bean plants where transcript accumulation is nitrate-inducible. Gene expression occurs in a circadian rhythm and induced by light in leaves of dark-adapted plants. A particular 100 bp sequence is present in the promoter and in the first intron of the NiR gene. Several copies of this 100 bp sequence are present in the bean genome. Comparisons between the promoter of the bean NiR gene and of two bean nitrate reductase genes (NR1 and NR2) show a limited number of conserved motifs, although the genes are presumed to be co-regulated. Comparisons are also made between the bean NiR promoter and the spinach NiR promoter. Transformation of tobacco plants with the bean NiR promoter fused to the GUS reporter gene (beta-glucuronidase) shows that the bean NiR promoter is nitrate-regulated and that the presence of the 100 bp sequence influences the level of GUS activity. NiR-coding sequences are not required for nitrate regulation but have a quantitative effect on the measured GUS activity.

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.…”

Coordination of Plant Metabolism and Development by the Circadian Clock.

“…In this pathway, nitrate (NO,-) is transported into roots cells, where it is reduced by nitrate reductase (NR) to nitrite (NO,-), which is then reduced to NH4+ by nitrite reductase (NiR; Sander et al, 1995). NH4+ can then be transported throughout the plant mainly in the form of asparagine (Lea and Miflin, 1980), which is produced by a glutamine-dependent asparagine synthetase (AS; Lam et al, 1994).…”

“…NH4+ can then be transported throughout the plant mainly in the form of asparagine (Lea and Miflin, 1980), which is produced by a glutamine-dependent asparagine synthetase (AS; Lam et al, 1994). Interestingly, NR and NiR have maximal expression and activity during the light phase, and their gene expression is circadian clock regulated in bean (NiR; Sander et al, 1995), tobacco (NR; Deng et al, 1990), and Arabidopsis (NR; Pilgrim et al, 1993). By contrast, AS mRNA levels peak during the night phase in pea (Tsai and Coruzzi, 1990) and Arabidopsis (Lam et al, 1994), although the accumulation of this enzyme does not appear to be clock regulated (Tsai and Coruzzi, 1990).…”

“…The differences in lighydark (LD) phase expression of NR, NiR, and AS may be a function of the energy requirements for the corresponding enzyme reactions (Lam et al, 1994;Sander et al, 1995). For example, NR and NiR enzymes require large amounts of reducing power, which is readily available when the plants are photosynthesizing (Pilgrim et al, 1993;Sander et al, 1995); hence, the clock coordinates the maximal expression of these genes during the light phase.…”

Structure–function relationship of assimilatory nitrite reductases from the leaf and root of tobacco based on high‐resolution structures

“…Overall, about 84 residues were variable when Nii3 and Nii4 were compared. Nii3 and Nii4 in which the transit peptides located at the N-termini were removed (18 residues) 27 were used in this study. Nii3 and Nii4 are 569 and 566 residues in length and have estimated molecular weights of 63.7 and 63.5 kDa, respectively.…”