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Published on February 3, 2006; 10.1104/pp.105.074633

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Received November 23, 2005
Returned for revision December 13, 2005
Accepted January 9, 2006

Post-translational regulation of nitrate reductase strongly affects the levels of free amino acids and nitrate, whereas transcriptional regulation has only minor influence

Unni S Lea , Marie-Thérèse Leydecker , Isabelle Quilleré , Christian Meyer , and Cathrine Lillo *

University of Stavanger, Faculty of Science and Technology, N-4036 Stavanger, Norway
Unité de Nutrition Azotée des Plantes INRA, F-78026 Versailles Cedex, France

* Corresponding author; email: Cathrine.lillo{at}uis.no.

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 CaMV 35S RNA promoter. In Del8 and S521D lines NR was additionally deregulated at the post-translational level by a deletion mutation in the N-terminal domain or by mutation of the regulatory phosphorylation site (Ser 521), respectively. Post-translational 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 post-translational regulation (S521D plants) resulted in an increase of Gln and Asn by a factor of 9 and 14, respectively, compared with WT, whereas abolishing transcriptional regulation (C1 plants) only, resulted in increases of Gln and Asn by factors less than 2. Among the minor amino acids especially Ile and Thr showed enhanced levels in S521D. Nitrate uptake rates were the same in S521D and WT 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/darkness. Generally, deregulation of NR at the transcriptional level did not have much influence on metabolite levels, but additional deregulation at the post-translational level resulted in profound changes of N-metabolite levels.




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