Tungstate, a Molybdate Analog Inactivating Nitrate Reductase, Deregulates the Expression of the Nitrate Reductase Structural Gene

Abstract

Nitrate reductase (NR, EC 1.6.6.1) from higher plants is a homodimeric enzyme carrying a molybdenum cofactor at the catalytic site. Tungsten can be substituted for molybdenum in the cofactor structure, resulting in an inactive enzyme. When nitratefed Nicotiana tabacum plants were grown on a nutrient solution in which tungstate was substituted for molybdate, NR activity in the leaves decreased to a very low level within 24 hours while NR protein accumulated progressively to a level severalfold higher than the control after 6 days. NR mRNA level in molybdategrown plants exhibited a considerable day-night fluctuation. However, when plants were treated with tungstate, NR mRNA level remained very high. NR activity and protein increased over a 24-hour period when nitrate was added back to N-starved molybdate-grown plants. NR mRNA level increased markedly during the first 2 hours and then decreased. In the presence of tungstate, however, the induction of NR activity by nitrate was totally abolished while high levels of NR protein and mRNA were both induced, and the high level of NR mRNA was maintained over a 10-hour period. These results suggest that the substitution of tungsten for molybdenum in NR complex leads to an overexpression of the NR structural gene. Possible mechanisms involved in this deregulation are discussed.In higher plants, the regulation of NR' has been studied extensively under various physiological conditions. A number of factors such as light, mineral nutrition, growth regulators and environmental stress have been shown to affect its activity (21). The precise mechanisms by which these factors modulate NR expression are, at present, not well understood. Molecular tools such as NR monoclonal antibodies (8) and a NR cDNA probe (3) have been obtained in our laboratory to study the regulation of the NR gene expression in tobacco.Higher plant NADH-NR (EC 1.6.6.1) is a nuclear geneencoded homodimer, having a subunit mol wt of 105,000 to 115,000 and containing flavin adenine dinucleotide, hemeFe, and molybdenum cofactor as prosthetic groups (4). Molybdenum is directly implicated in the electron transfer for nitrate reduction. Tungsten, a metal classified with Cr, Mo, and U in the Mendeleieff table, can compete with molybdenum for incorporation into the enzyme complex and results in enzyme inactivation (15,17). In this work, we show that ' Abbreviations: NR, nitrate reductase; rbcS, ribulose bisphosphate carboxylase small subunit. the substitution of tungstate for molybdate in the nutrient solution enhances strongly the expression ofthe NR structural gene. MATERIALS AND METHODS Plant Material and Experimental ConditionsTobacco seeds (Nicotiana tabacum cv Xanthi) were germinated and grown on a sand-peat mixture (1/1, v/v) in the greenhouse. A nitrate and ammonium-containing nutrient solution (10) was applied daily. After 1 month, the seedlings were transferred to a controlled culture room for experimentation under the following conditions: 85% RH, 16 h photoperiod (180 ,umol* m-2 s', fluorescent lam...

Nitrate Control of Root Hydraulic Properties in Plants: Translating Local Information to Whole Plant Response

“…To elucidate the signaling path responsible for the observed changes in root hydraulic conductance, we pretreated tomato and cucumber plants with a molybdenum-free medium containing tungstate, a well-known inhibitor of nitrate assimilation (Deng et al, 1989). The treatment resulted in strong inhibition of nitrate reductase (NR), effectively removing any downstream products of nitrate assimilation (Table II).…”

A Nitrate-Induced frq-Less Oscillator in Neurospora crassa

“…Figure 1A,B shows the observed NRA rhythm of bd and frq 9 mutants in continuous darkness (DD). Because N. crassa NR is photoactivated like plant NR (Ninnemann, 1997;Roldán and Butler, 1980) and because plant NRA rhythms are generally light induced (Deng et al, 1989;Lillo, 1984Lillo, , 1993Lillo and Ruoff, 1989;McClung and Kay, 1994), we tested whether the Neurospora NRA rhythm may also occur under continuous light (LL) conditions. LL does not abolish the NRA rhythm, as the results in Figure 1C,D show, in contrast to Neurospora's sporulation rhythm and FRQ oscillations (Collett et al, 2002).…”

Tungsten in biological systems

“…Thus, Azotobacter chroococcum grown in the presence of 99Mo yields active radiolabelled NR, but in the presence of J8SW inactive radiolabelled NR is produced [198]. On the other hand, the ability to grow in the presence of W can be used as a diagnostic tool for the identification of regulatory NR mutants, and W has been used to monitor the expression of NR in plants [203,204,328] as well as in bacteria [105,193].…”