ALBERT

Notes: Diurnal changes of transcript levels for key enzymes in nitrate and organic acid metabolism and the accompanying changes of enzyme activities and metabolite levels were investigated in nitrogen-sufficient wild-type tobacco, in transfomants with decreased expression of nitrate reductase, and in nitrate-deficient wild-type tobacco. (i) In nitrogen-sufficient wild-type plants, transcript levels for nitrate reductase (NR, EC 1.6.6.1), nitrite reductase (NIR, EC 1.7.7.1) and phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) were high at the end of the night and decreased markedly during the light period. The levels of these three transcripts were increased and the diurnal changes were damped in genotypes with decreased expression of nitrate reductase. The levels of these transcripts were very low in nitrate-limited wild-type plants, except for a small rise after irrigation with 0·2 mM nitrate. (ii) The levels of the transcripts for cytosolic pyruvate kinase (PK, EC 2.7.1.40), mitochondrial citrate synthase (CS, EC 4.1.3.7) and NADP-isocitrate dehydrogenase (NADP-ICDH, EC 1.1.1.42) were highest at the end of the light period and beginning of the night. These three transcripts increase and the diurnal changes were damped in genotypes with decreased expression of NR. (iii) The diurnal changes of transcript levels were accompanied by changes in the activities of the encoded enzymes. The activities of NR and PEPC were highest in the early part of the light period, whereas the activities of PK and NADP-ICDH were highest later in the light period and during the first part of the night and CS activity was highest at the end of the night. Activity of PEPC, PK, CS and NADP-ICDH increased and the diurnal changes were damped in genotypes with low expression of NR. Activity of all four enzymes decreased in nitrate-limited wild-type plants. (iv) In the light, malate accumulated, citrate decreased, and about 30% of the assimilated nitrate accumulated temporarily as glutamine, ammonium, glycine and serine. These changes were reversed during the night. (v) It is proposed that the diurnal changes of expression facilitate preferential synthesis of malate to act as a counter-anion for pH regulation during the first part of the light period when NR activity is high, and preferential synthesis of 2-oxoglutarate to act as a nitrogen acceptor later in the day when large amounts of nitrogen have accumulated in ammonium, glutamine and other amino acids including glycine in the photorespiration pathway, and NR activity has been decreased.

Notes: Nitrate assimilation in leaves requires synthesis of malate to counteract alkalinization, and synthesis of 2-oxoglutarate to act as an acceptor in the GOGAT pathway. We have investigated whether malate or 2-oxoglutarate regulate nitrate reductase (NIA, EC 1·6.6·1) expression. (i) Diurnal changes of NIA expression and organic acid levels were compared in tobacco leaves. The NIA transcript rose during the night and decreased during the day, and NIA activity rose to a maximum during the first 4 h of the light period and fell during the second part of the light period. Malate accumulated to high levels during the light period and decreased during the night. The 2-oxoglutarate increased by 40% at the beginning and decreased towards the end of the light period. The glutamine : 2-oxoglutarate ratio was steady during the first part of the light period and increased markedly during the second part of the light period. The diurnal changes of the NIA transcript level were inversely correlated to the diurnal changes of malate, and unrelated to the changes of 2-oxoglutarate or the glutamine : 2-oxoglutarate ratio. The decrease of NIA activity in the second part of the light period correlated with an increase of the glutamine : 2-oxoglutarate ratio. (ii) Leaves were detached 4 h into the light period and supplied with malate or 2-oxoglutarate via the petiole, to investigate their impact on the gradual decrease of the NIA transcript and NIA activity during the second part of the light period. Physiologically relevant changes of malate led to a further decrease of the NIA transcript level and a 27–60% decrease of NIA activity. A large increase of 2-oxoglutarate stabilized the NIA transcript level but had only slight effects on NIA activity. (iii) Plants were darkened for 16–24 h to reduce the NIA transcript level and NIA activity to low levels, and leaves were then detached and supplied with malate or 2-oxoglutarate for 4 h in the light to investigate their impact on the light-induction of NIA. The increase of the NIA transcript and NIA activity was antagonized by malate, and slightly accelerated by 2-oxoglutarate. (iv) Plants were placed in the dark for 60 h to reduce NIA activity to the limit of detection, and leaf discs were then incubated in the dark on sucrose to achieve a photosynthesis-independent increase of NIA activity. This was strongly inhibited by malate. (v) It is concluded that malate inhibits NIA expression, affecting both the NIA transcript level and NIA activity. Although the results are consistent with a role for 2-oxoglutarate in the regulation of NIA expression, the impact is less marked and no endogenous changes of 2-oxoglutarate were found that are likely to have a significant effect on NIA expression.