ALBERT

Notes: The effect of elevated [CO2] on biomass, nitrate, ammonium, amino acids, protein, nitrate reductase activity, carbohydrates, photosynthesis, the activities of Rubisco and six other Calvin cycle enzymes, and transcripts for Rubisco small subunit, Rubisco activase, chlorophyll a binding protein, NADP-glyceraldehyde-3-phosphate dehydrogenase, aldolase, transketolase, plastid fructose-1,6-bisphosphatase and ADP-glucose pyrophosphorylase was investigated in tobacco growing on 2, 6 and 20 m M nitrate and 1, 3 and 10 m M ammonium nitate. (i) The growth stimulation in elevated [CO2] was attenuated in intermediate and abolished in low nitrogen. (ii) Elevated [CO2] led to a decline of nitrate, ammonium, amino acids especially glutamine, and protein in low nitrogen and a dramatic decrease in intermediate nitrogen, but not in high nitrogen. (iii) Elevated [CO2] led to a decrease of nitrate reductase activity in low, intermediate and high ammonium nitrate and in intermediate nitrate, but not in high nitrate. (iii) At low nitrogen, starch increased relative to sugars. Elevated [CO2] exaggerated this shift. ADP-glucose pyrophosphorylase transcript increased in low nitrogen, and in elevated [CO2]. (iv) In high nitrogen, sugars rose in elevated [CO2], but there was no acclimation of photosynthetic rate, only a small decrease of Rubisco and no decrease of other Calvin cycle enzymes, and no decrease of the corresponding transcripts. In lower nitrogen, there was a marked acclimation of photosynthetic rate and a general decrease of Calvin cycle enzymes, even though sugar levels did not increase. The decreased activities were due to a general decrease of leaf protein. The corresponding transcripts did not decrease except at very low nitrogen. (v) It is concluded that many of the effects of elevated [CO2] on nitrate metabolism, photosynthate allocation, photosynthetic acclimation and growth are due to a shift in nitrogen status.

Notes: To assess how diurnal changes of nitrate reductase (NIA) expression in leaves interact with upstream and downstream processes during nitrate utilization, nitrate uptake, and nitrate and ammonium metabolism were investigated at several times during the diurnal cycle in wild-type tobacco. Plants were grown hydroponically on 2 mM nitrate to exclude possible complications due to changes in the external availability of nitrate, and to allow nitrate uptake to be measured in the growth conditions. (a) In leaves, the NIA transcript decreases during the day and recovers at night, and NIA activity increases three-fold during the first part and declines during the second part of the light period. Nitrate decreases during the day and recovers at night, ammonium, glutamine, glycine and serine increase during the day and decrease at night, and 2-oxoglutarate increases three-fold after illumination and decreases during the last part of the light period. The amplitudes of the diurnal changes are similar to or larger than in tobacco grown on high nitrate in sand. The transcript for plastid glutamine synthetase (GLN2) is low at the end of the night and increases during the day, and glutamine synthetase activity increases to a peak at the end of the day and decreases at night. (b) In the roots, transcript levels for the high affinity nitrate transporter (NRT2) increase in the day and decrease at night. Nitrate uptake is about 40% higher during the day than at night. (c) Comparison of the diurnal changes of the leaf metabolite pools with the rate of nitrate uptake allows diurnal changes in fluxes to be estimated. During the first part of the light, the rate of nitrate assimilation is about two-fold higher than the rate of nitrate uptake, and also exceeds the rate at which reduced nitrogen is metabolized in the GOGAT pathway. The resulting decrease of leaf nitrate and accumulation of nitrogen in intermediates of ammonium metabolism and photorespiration represent about 40 and 15%, respectively, of the total nitrate that enters the plant in 24 h. Later in the diurnal cycle as NIA expression and activity decline, this imbalance is reversed. NRT2 expression and nitrate uptake remain relatively high, and nitrate taken up during the night is used to replenish the leaf nitrate pool. Increased GLN2 expression in leaves during the second part of the light period allows continued assimilation of ammonium released during photorespiration and remobilization of the reduced nitrogen that accumulated earlier in the diurnal cycle.