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...

Messenger RNAs encoding the nitrate reductase apoenzyme from tobacco can be translated in a cell-free system. Poly(A)+ mRNA fractions from the 23-32 S area of a sucrose gradient were used to build a cDNA library in the expression vector gt11 with an efficiency of cloning of approximately 10(4) recombinants/ng mRNA. Recombinant clones were screened with a rabbit polyclonal antibody directed against the corn nitrate reductase, which cross reacts specifically with the nitrate reductases from dicotyledons. Among 240000 recombinant plaques, eight clones were isolated containing inserts of sizes ranging from 1.6 kb to 2.1 kb and sharing sequence homologies. Seven of these clones contained a common internal 1.6 kb EcoRI fragment. The identity of these clones was confirmed as follows. A fusion protein of 170 kDa inducible by IPTG and recognized by the rabbit nitrate reductase antibody was expressed by a lysogen derived from one of the recombinants. The antibodies binding the fused protein were eluted and shown to be inhibitory to the catalytic activity of tobacco nitrate reductase. Two monoclonal antibodies directed against nitrate reductase were also able to bind the hybrid protein. The 1.6 kb EcoRI fragment was sequenced by the method of Sanger. The open reading frame corresponding to a translational fusion with the -galactosidase coding sequence of the vector shared strong homology at the amino acid level with the heme-binding domain of proteins of the cytochrome b5 superfamily and with human erythrocyte cytochrome b5 reductase. When the 1.6 kb EcoRI fragment was used as a probe for Northern blot experiments a signal corresponding to a 3.5 kb RNA was detected in tobacco and in Nicotiana plumbaginifolia mRNA preparations but no cross-hybridization with corn mRNAs was detected. The probe hybridized with low copy number sequences in genomic blots of tobacco DNA.

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