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CELL BIOLOGY AND SIGNAL TRANSDUCTION

Posttranslational Regulation of Nitrate Reductase Strongly Affects the Levels of Free Amino Acids and Nitrate, whereas Transcriptional Regulation Has Only Minor Influence^1,[OA]

Faculty of Science and Technology, University of Stavanger, N–4036 Stavanger, Norway (U.S.L., C.L.); and Unité de Nutrition Azotée des Plantes, Institut National de la Recherche Agronomique, F–78026 Versailles cedex, France (M.-T.L., I.Q., C.M.)

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 ¹⁵N 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.

The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) are: Christian Meyer (meyer{at}versailles.inra.fr) and Cathrine Lillo (cathrine.lillo{at}uis.no).

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Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.074633.

^* Corresponding author; e-mail cathrine.lillo{at}uis.no; fax 47–5183–1750.

Received November 23, 2005; returned for revision January 7, 2006; accepted January 9, 2006.

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