Regulation Of Nitrate Reductase Gene Expression Research Articles

Nitrate reductase enzymes in alga Chattonella subsalsa are regulated by environmental cues at the translational and post-translational levels

Nitrate reductase (NR) catalyzes the rate-limiting step in nitrate assimilation. Plant and algal NRs have a highly conserved domain architecture but differ in regulation. In plants, NR activity is regulated by reversible phosphorylation and subsequent binding of 14-3-3 proteins at a conserved serine residue. Algal NRs typically lack 14-3-3 binding motifs, which have only recently been identified in a few algal species. Previous research indicates that the alga, Chattonella subsalsa, possesses a novel NR, NR2-2/2HbN (NR2), which incorporates a 2/2 hemoglobin domain. A second NR (NR3) in C. subsalsa lacks the cytochrome b5 (heme-Fe) domain but includes a putative binding motif for 14-3-3 proteins. The expression of NR2 and NR3 genes indicates that NR2 transcript abundance was regulated by light, nitrogen source, and temperature, while NR3 transcript levels were only regulated by light. Here, we measured total NR activity in C. subsalsa and the potential for regulation of NR activity by putative 14-3-3 binding proteins. Results indicate that NR activity in C. subsalsa was regulated by light, nitrogen source, and temperature at the translational level. NR activity was also regulated by endogenous rhythm and temperature at the post-translational level, supporting the hypothesis that NR3 is regulated by 14-3-3 binding proteins. Together with a previous report describing the regulation of NR gene expression in C. subsalsa, results suggest that C. subsalsa responds to environmental conditions by differential regulation of NRs at transcriptional, translational, and post-translational levels. This flexibility may provide a competitive advantage for this species in the environment. To date, this is the first report which provides evidence for the potential post-translational regulation of NR by 14-3-3 proteins in algal species and suggests that regulatory mechanisms for NR activity may be shared between plants and some algal species.

The Regulatory Region Controlling the Nitrate-Responsive Expression of a Nitrate Reductase Gene, NIA1, in Arabidopsis

Nitrate reductase (NR) is the enzyme that catalyzes the first step of nitrate assimilation. It is well known that the expression of NR genes is rapidly induced in various plants by nitrate. Previously, the activity of a tobacco NR gene promoter was reported to be high in tobacco plants grown on medium containing ammonium as the sole nitrogen source, but low in tobacco plants grown on nitrate-containing medium. This cast some doubt on the role of the NR gene promoter in the nitrate-inducible expression of this gene. Furthermore, in previous studies, transformation with genomic fragments containing NR loci restored the reduced NR activity in NR mutants to a limited extent, suggesting a complex regulation of NR gene expression. Here, we show that although the 1.9 kb promoter of an NR gene in Arabidopsis, NIA1, is not activated by nitrate, the expression of a GUS (β-glucuronidase) reporter gene inserted between the 5'- and 3'-flanking sequences of the NIA1 coding region is strongly induced by nitrate. When the 3'-flanking sequence was fused downstream of the GUS gene under the control of the 35S minimal promoter, its expression was also strongly induced by nitrate. Furthermore, dissection analysis of the 3'-flanking region revealed that the sequence downstream of the transcriptional terminator rather than the 3'-untranslated region plays a role in nitrate-inducible expression, indicating a requirement for the 3'-flanking sequence for the nitrate-inducible transcription of NIA1. We also show that the 2.7 kb promoter sequence of NIA2, another NR gene of Arabidopsis, cannot direct nitrate-inducible expression.

The regulation of the nitrate assimilation and amino acid biosynthetic studied in transformed tobacco plants with reduced Fd-GOGAT activity

Tobacco (Nicotiana tabacum) plants expressing a partial ferredoxin-dependent glutamine a-oxoglutarate aminotransferase (Fd-GOGAT) cDNA in the antisense orientation under the control of the 35S promoter, were used to study the metabolism of amino acids, 2-oxoglutarate and ammonium following the transition from CO2 enrichment (where photorespiration is inhibited) to air (where photorespiration is a major process of ammonium production in leaves). In the Fd-GOGAT antisense plants, the photorespiration-dependent increases in foliar ammonium, glutamine, a-KG and total amino acids were proportional to the decrease in Fd-GOGAT activity. The transgenic plants were used to examine the roles of ammonium, glutamine (Gln) and a-KG in the regulation of nitrate reductase (NR: EC 1.6.6.1) gene expression. In air, the simultaneous accumulation of Gln and a-KG was accompanied by an increase in the steady state level of NR transcripts. We conclude that the negative effect of Gln on NR transcript abundance is offset by high a-KG. It is therefore likely that the relative amounts of a-KG and Gln are more important in controlling NR gene transcription than the concentration of either metabolite alone. In addition we found that several pathways of amino acid biosynthesis were modified when NH4+ and Gln accumulate in leaves and that this accumulation was not due to increased proteolytic activity. The accumulation of a-KG, Gln and NH4+ in the leaves of the transformed plants with low Fd-GOGAT activity may initiate pathways of signal transduction leading to the simultaneous modulation of several pathways of nitrate assimilation and amino acid biosynthetsis.

Light Regulation of Nitrate Reductase Gene Expression in Maize Involves a G-Protein

This paper reports three lines of evidence to demonstrate the presence of heterotrimeric G-proteins in maize and their involvement in the regulation of nitrate reductase gene expression by light: (1) Southern blot analysis of maize genomic DNA using a human Ha-ras cDNA probe revealed specific bands indicating the presence of G-protein (α subunit) gene(s) in maize. Northern blot analysis of maize total RNA using the same probe revealed that the putative Gα gene(s) is transcriptionally active. (2) Western blots containing purified plasma membrane proteins from maize leaves showed specific binding of γ [35S]-labeled GTP in a red light-dependent manner, indicating the involvement of G-proteins in mediating the light signal. The size of the putative Gα gene product (∼45 kDa) indicates that it may be a heterotrimeric G-protein. (3) Cholera toxin mimicked the effect of red light to enhance the transcript levels of nitrate reductase (NR), indicating that G-proteins may mediate light regulation of NR gene expression.

A chlorate-resistant mutant defective in the regulation of nitrate reductase gene expression in Arabidopsis defines a new HY locus.

Light acts both directly as a signal and indirectly through photosynthesis to regulate the expression of genes encoding nitrate reductase (NR). Here, we report the isolation and characterization of a novel chlorate-resistant mutant that is defective in the regulation of NR gene expression. The response of NR2, but not NR1 or the gene encoding nitrate reductase (NiR), to light signals was impaired in this Arabidopsis mutant, designated cr88. In addition to NR2, the light regulation of the genes encoding the chlorophyll a/b binding protein (CAB) and the small subunit of ribulose bisphosphate carboxylase (RBCS) was also impaired in this mutant. These results suggest that the pathway through which light regulates the expression of NR2, CAB, and RBCS genes is different from those that regulate the expression of NR1 and NiR. An examination of the deetiolation process under different light spectrum showed that cr88 is defective in red light-mediated deetiolation. Complementation tests with various long hypocotyl (hy) mutants indicated that CR88 identifies a new HY locus. The possible functions of CR88 are discussed.

Effects of constitutive expression of nitrate reductase in transgenic Nicotiana plumbaginifolia L. in response to varying nitrogen supply

Transformed Nicotiana plumbaginifolia plants with constitutive expression of nitrate reductase (NR) activity were grown at different levels of nitrogen nutrition. The gradients in foliar NO 3 − content and maximum extractable NR activity observed with leaf order on the shoot, from base to apex, were much decreased as a result of N-deficiency in both the transformed plants and wild type controls grown under identical conditions. Constitutive expression of NR did not influence the foliar protein and chlorophyll contents under any circumstances. A reciprocal relationship between the observed maximal extractable NR activity of the leaves and their NO 3 − content was observed in plants grown in nitrogen replete conditions at low irradiance (170 μmol photons·m−2 ·s−1). This relationship disappeared at higher irradiance (450 μmol photons·m−2·S−1) because the maximal extractable NR activity in the leaves of the wild type plants in these conditions increased to a level that was similar to, or greater than that found in constitutive NR-expressors. Much more NO 3 − accumulated in the leaves of plants grown at 450 μmol photons·m−2·s−1 than in those grown at 170 μmol photons·m−2·s−1 in N-replete conditions. The foliar NO 3 − level and maximal NR activity decreased with the imposition of N-deficiency in all plant types such that after prolonged exposure to nitrogen depletion very little NO 3 − was found in the leaves and NR activity had decreased to almost zero. The activity of NR decreased under conditions of nitrogen deficiency. This regulation is multifactoral since there is no regulation of NR gene expression by NO 3 − in the constitutive NR-expressors. We conclude that the NR protein is specifically targetted for destruction under nitrogen deficiency. Consequently, constitutive expression of NR activity does not benefit the plant in terms of increased biomass production in conditions of limiting nitrogen.

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

Nitrate reductase (NR) is the first enzyme in nitrate assimilation, a critical process for plant survival. The regulation of NR gene expression is complex, involving both internal and external factors. Of these, nitrate induction of NR gene expression has been studied most extensively and is well conserved among bacteria, fungi, and higher plants. We are interested in understanding the mechanism of nitrate induction of higher plant NR genes. Here we describe promoter analyses of the 5' flanking regions of the Arabidopsis NR genes, NR1 and NR2, with respect to nitrate induction of gene expression. To facilitate these analyses, a nitrate induction procedure using T1 transgenic tobacco plants was established. Approximately 1.5-kb 5' flanking regions of the two Arabidopsis NR genes (NR1 and NR2) were fused to a reporter gene and its expression in transgenic plants was analyzed. Deletion analyses of these regions show that 238- and 188-bp 5' flanking regions of the NR1 and NR2, respectively, contain sequences responsive to nitrate induction.

Constitutive expression of nitrate reductase allows normal growth and development of Nicotiana plumbaginifolia plants.

A nitrate reductase (NR) deficient mutant of Nicotiana plumbaginifolia totally impaired in the production of NR transcript and protein was restored for NR activity by transformation with a chimaeric NR gene. This gene was composed of a full-length tobacco NR cDNA fused to the CaMV 35S promoter and to termination signals from the tobacco NR gene. The transgenic plants we obtained were viable and fertile and expressed from one-fifth to three times the wild-type NR activity in their leaves. The analysis of chimeric NR gene expression in these plants showed, by comparison with wild-type plants, that the regulation of NR gene expression by light, nitrate and circadian rhythm takes place at the transcriptional level. However, unlike nitrate, light was required for the accumulation of NR protein in transgenic plants, suggesting that NR expression is also controlled at the translational and/or post-translational level.