Expression of Leaf Nitrate Reductase Genes from Tomato and Tobacco in Relation to Light-Dark Regimes and Nitrate Supply

Abstract

The influence of light-dark cycles and nitrate supply on nitrate reductase (NR) mRNA levels was studied in two plant species, tobacco (Nicotiana tabacum) and tomato (Lycopersicon esculentum) using specific NR DNA probes. In the same series of experiments, changes in the levels of NR protein (NRP) by enzyme-linked immunosorbent assay and changes in the level of NADH-nitrate reductase activity (NRA) were also followed. During a light-dark cycle, it was found that in both tomato and tobacco, NR mRNA accumulation increased rapidly during the dark period and reached a maximum at the beginning of the day, while NRP reached a peak 2 and 4 hours after mRNA peaked, for tomato and tobacco, respectively. At the end of the day, the amount of mRNA was decreased by a factor of at least 100 compared to sunrise in both species. These results demonstrate that light is involved, although probably not directly, in the regulation of the NR gene expression at the mRNA level. The peak of NRA in tobacco coincided with the peak in NR mRNA accumulation (i.e. sunrise), whereas in tomato the peak of NRA was approximately 5 to 6 hours after sunrise. There is no obvious correlation between NRP and NRA levels during the day. In nitrogen starvation experiments, a rapid decrease of NRP and NRA was detected, while NR mRNA levels were not significantly altered. Upon nitrate replenishment, nitrogen-starved plants accumulated NR mRNA rapidly. These results suggest that the availability of nitrogen affects the expression of NR activity at the transcriptional as well as at the post-transcriptional levels.clones coding for the NR apoprotein (3,4,8) has allowed the study of gene transcription.The influence of light on NRA has been described in a variety of higher plants (for a review see Ref. 10). For many species, extractable NRA rapidly increases when etiolated, nitrate-grown seedlings are placed in white light. By immunological methods, NRA induction has been correlated with de novo synthesis of NRP in barley (21) and maize (17). In this paper, using specific probes for tobacco (3) and tomato (to be published), we show that during a 24 h cycle, the de novo synthesis of NRP during the day follows increases in pools of NR mRNA during the last part of the night.Since the early experiments ofTang and Wu (23) of the apoprotein in response to nitrate was correlated with an increase in the mRNA level. We have studied the levels of NR mRNA, NRP and NRA during nitrate starvation/induction experiments using tobacco and tomato. MATERIALS AND METHODSHigher plants, algae, and some bacteria assimilate nitrogen from their environment primarily by the nitrate reducing pathway. The first step of this route is catalyzed by the enzyme NR' (EC 1.6.6.1.), which reduces the nitrate to nitrite. Nitrate reduction is considered the controlling step in the assimilation of nitrate and has been studied extensively in bacteria, fungi, and higher plants (28). In plants, the enzyme is an homodimer carrying three cofactors, namely FAD, Cyt b557, and the molybdenum cofacto...

Induction of Nitrate Assimilatory Enzymes in the Tree Betula pendula

“…The decline in NR mRNA after peak accumulation in leaves and roots argues for a negative feedback control of NR mRNA synthesis and/or degradation in birch. Probably this reflects a general mechanism of NR regulation in plants, because similar changes of NR mRNA pools following the induction by nitrate were also found in herbaceous species (11,18). Although feedback regulation of NR by products of nitrate assimilation has been reported for fungi (21), the stimuli involved in plants are not known.…”

“…Upon transfer of ammonium-grown seedlings to a nitrate-containing medium, NR activities dramatically increased in leaves and roots of illuminated plants. This induction strictly depends on the presence of light, thus resembling the situation found for NADH-specific NR in herbaceous species (11,18,23 (11). This could not be tested in birch due to the lack of NR-specific antibodies.…”

Diurnal Rhythmicity in the Pattern of mRNAs in the Leaves of Sinapis alba

“…On the other hand, several recent studies have demonstrated diurnal or circadian rhythmicity in the level of specific mRNAs (8,9,13,18,21,25,28 (6). Gels were prepared for fluorography using Amplify (Amersham, UK) and exposed to preflashed Kodak X-Omat AR film, 48 Leaves of S. alba were harvested and their mRNA analyzed at the beginning (0 h), middle (4 h), and end of the day (8 h) as well as three times during the following night (12,16, and 20 h).…”

Atividade das enzimas redutase do nitrato e glutamina sintetase em cafeeiro arábica.

“…Segundo Galangau et al (1988) e Vincentz et al (1993) a atividade da enzima RN nas folhas e raízes pode ser induzida pela presença do substrato…”

“…A luz e o teor de nitrato são fatores importantes na indução e manutenção da atividade da enzima RN nos tecidos vegetais (Hageman & Flesher, 1960;Beevers & Hageman, 1969;Galangau et al, 1988;Vincentz et al, 1993). De acordo com diversos autores a atividade da RN nas folhas de várias espécies, aumenta durante as horas de luz e decresce no escuro (Harper & Hageman, 1972;Lewis et al, 1982;Hipkin et al, 1984).…”