Metabolite control of squash nitrate reductase

Langendorfer, Randall L.; Watters, Michelle T.; John, Sabu

doi:10.1016/0168-9452(88)90077-5

Cited by 17 publications

(14 citation statements)

References 15 publications

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“…Exogenous supplies of amino acids reduced NR activity in tobacco cell culture (1 1) and in corn roots (18), and decreased both NR activity and NR protein level in squash cotyledons (16). Therefore, it is possible that the expression of higher plant NR is also regulated in response to the status of reduced nitrogen in the tissues.…”

Section: Discussionmentioning

confidence: 99%

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

Deng¹,

Moureaux²,

Caboche³

1989

Plant Physiol.

139

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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...

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Section: Discussionmentioning

confidence: 99%

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

Deng¹,

Moureaux²,

Caboche³

1989

Plant Physiol.

139

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“…Gln, Glu, and Asp) are assumed to exert, in normal conditions, negative feedback on NR expression at the transcriptional level, although the exact signaling mechanism is so far not clear (Langendorfer et al, 1988;Deng et al, 1991;Sivasankar and Oaks, 1995;Scheible et al, 2000;Stitt et al, 2002). In C1 plants, circadian rhythmicity of the NR mRNA driven by the 35S promoter is lost, which indicates that this fluctuation is the result of transcriptional control in wild-type plants (Vincentz and Caboche, 1991).…”

mentioning

confidence: 99%

Posttranslational Regulation of Nitrate Reductase Strongly Affects the Levels of Free Amino Acids and Nitrate, whereas Transcriptional Regulation Has Only Minor Influence

et al. 2006

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Diurnal variations in nitrate reductase (NR) activity and nitrogen metabolites were examined in wild-type Nicotiana plumbaginifolia and transformants with various degrees of NR deregulation. In the C1 line, NR was only deregulated at the transcriptional level by placing the NR gene under the control of the cauliflower mosaic virus 35S RNA promoter. In the Del8 and S521D lines, NR was additionally deregulated at the posttranslational level either by a deletion mutation in the N-terminal domain or by a mutation of the regulatory phosphorylation site (serine-521). Posttranslational regulation was essential for pronounced diurnal variations in NR activity. Low nitrate content was related to deregulation of NR, whereas the level of total free amino acids was much higher in plants with fully deregulated NR. Abolishing transcriptional and posttranslational regulation (S521D plants) resulted in an increase of glutamine and asparagine by a factor of 9 and 14, respectively, compared with wild type, whereas abolishing transcriptional regulation (C1 plants) only resulted in increases of glutamine and asparagine by factors ,2. Among the minor amino acids, isoleucine and threonine, in particular, showed enhanced levels in S521D. Nitrate uptake rates were the same in S521D and wild type as determined with 15 N feeding. Deregulation of NR appears to set the level of certain amino acids, whereas diurnal variations were still determined by light/dark. Generally, deregulation of NR at the transcriptional level did not have much influence on metabolite levels, but additional deregulation at the posttranslational level resulted in profound changes of nitrogen metabolite levels.

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“…Generally, a negative correlation has been observed between root amide levels and net nitrogen uptake, as well as NR protein and activity (Langendorfer et al, 1988;Shiraishi et al, 1992). Similarly, extemal applications of Glu inhibited NOS-uptake when given before induction (Breteler and Amozis, 1985) and have been observed to cause a more severe reduction in Nosuptake than similar pretreatments using Gln (Muller and Touraine, 1992).…”

Section: The Effects Of Gln and Clumentioning

confidence: 99%

“…Extemal applications of amides and amino acids have been used previously to affect the inorganic nitrogen assimilatory pathway (Oaks et al, 1977;Breteler and Amozis, 1985;Langendorfer et al, 1988;Lee et al, 1992;Muller and Touraine, 1992;Shiraishi et al, 1992). Generally, a negative correlation has been observed between root amide levels and net nitrogen uptake, as well as NR protein and activity (Langendorfer et al, 1988;Shiraishi et al, 1992).…”

Section: The Effects Of Gln and Clumentioning

confidence: 99%

Investigation of the Apparent Induction of Nitrate Uptake in Barley (Hordeum vulgare L.) Using NO3--Selective Microelectrodes (Modulation of Coarse Regulation of NO3- Uptake by Exogenous Application of Downstream Metabolites in the NO3- Assimilatory Pathway)

Henriksen

Spanswick

1993

Plant Physiol.

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Metabolite control of squash nitrate reductase

Cited by 17 publications

References 15 publications

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

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

Posttranslational Regulation of Nitrate Reductase Strongly Affects the Levels of Free Amino Acids and Nitrate, whereas Transcriptional Regulation Has Only Minor Influence

Investigation of the Apparent Induction of Nitrate Uptake in Barley (Hordeum vulgare L.) Using NO3--Selective Microelectrodes (Modulation of Coarse Regulation of NO3- Uptake by Exogenous Application of Downstream Metabolites in the NO3- Assimilatory Pathway)

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