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

Abstract

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.

Otimização da análise da atividade da redutase do nitrato e sua caracterização em folhas de cana-de-açúcar

“…Resultados semelhantes foram encontrados em folhas de plantas do tipo C3, que também apresentaram maiores reduções de nitrato em alta luminosidade (Lea et al, 2006;Kolton et al, 2012), diferentemente de folhas de plantas do tipo CAM, como o abacaxizeiro [Ananas comosus (L.) Merr. ], em que essa enzima mostrou-se mais ativa no início da manhã (Freschi et al, 2009).…”

“…Nas junções entre os grupos prostéticos da enzima (resíduo de serina), pode ocorrer a fosforilação, seguida da ligação da proteína 14-3-3, a qual controla rapidamente e de forma reversível a atividade da RN em reposta à luminosidade ou à escuridão. Assim, no escuro, a proteína se liga ao fosfato do resíduo de serina e inativa a enzima, e, se as plantas forem expostas à luz novamente, ocorre o desligamento da proteína 14-3-3 e a enzima torna-se ativa outra vez (Crawford et al, 2000;Souza et al, 2002;Lea et al, 2006).…”

Assimilação metabólica de nitrogênio em plântulas de seringueira cultivadas com nitrato ou amônio

“…A atividade in vitro da RN presente nas raízes aumentou nas plantas cultivadas com nitrato (Figura 2), o que era esperado e evidencia a indução da enzima pelo substrato (Lea et al, 2006), já que nas plantas cultivadas na presença de amônio e na ausência de N a atividade reduziu.…”

Protein Phosphatase 2A B55 and A Regulatory Subunits Interact with Nitrate Reductase and Are Essential for Nitrate Reductase Activation

“…At both levels signals from the chloroplasts are involved in initiating an increase in NR activity levels, but these signals and their processing are still unknown (Jonassen et al, 2008;Lillo, 2008;Krouk et al, 2010;Nunes-Nesi et al, 2010). At the posttranslational level NR is rapidly activated by dephosphorylation, and active photosynthesis is necessary for this activation (Kaiser and Brendle-Behnisch, 1991;Huber et al, 1994;Douglas et al, 1995;Bachmann et al, 1996;Campbell, 1999;Lea et al, 2006). The phosphorylation status of NR, or any other protein, is the result of the action of both kinases and phosphatases.…”