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: The influence of elevated [CO2] on the uptake and assimilation of nitrate and ammonium was investigated by growing tobacco plants in hydroponic culture with 2 mm nitrate or 1 mm ammonium nitrate and ambient or 800 p.p.m. [CO2]. Leaves and roots were harvested at several times during the diurnal cycle to investigate the levels of the transcripts for a high-affinity nitrate transporter (NRT2), nitrate reductase (NIA), cytosolic and plastidic glutamine synthetase (GLN1, GLN2), the activity of NIA and glutamine synthetase, the rate of 15N-nitrate and 15N-ammonium uptake, and the levels of nitrate, ammonium, amino acids, 2-oxoglutarate and carbohydrates. (i) In source leaves of plants growing on 2 mm nitrate in ambient [CO2], NIA transcript is high at the end of the night and NIA activity increases three-fold after illumination. The rate of nitrate reduction during the first part of the light period is two-fold higher than the rate of nitrate uptake and exceeds the rate of ammonium metabolism in the glutamate: oxoglutarate aminotransferase (GOGAT) pathway, resulting in a rapid decrease of nitrate and the accumulation of ammonium, glutamine and the photorespiratory intermediates glycine and serine. This imbalance is reversed later in the diurnal cycle. The level of the NIA transcript falls dramatically after illumination, and NIA activity and the rate of nitrate reduction decline during the second part of the light period and are low at night. NRT2 transcript increases during the day and remains high for the first part of the night and nitrate uptake remains high in the second part of the light period and decreases by only 30% at night. The nitrate absorbed at night is used to replenish the leaf nitrate pool. GLN2 transcript and glutamine synthetase activity rise to a maximum at the end of the day and decline only gradually after darkening, and ammonium and amino acids decrease during the night. (ii) In plants growing on ammonium nitrate, about 30% of the nitrogen is derived from ammonium. More ammonium accumulates in leaves during the day, and glutamine synthetase activity and glutamine levels remain high through the night. There is a corresponding 30% inhibition of nitrate uptake, a decrease of the absolute nitrate level, and a 15–30% decrease of NIA activity in the leaves and roots. The diurnal changes of leaf nitrate and the absolute level and diurnal changes of the NIA transcript are, however, similar to those in nitrate-grown plants. (iii) Plants growing on nitrate adjust to elevated [CO2] by a coordinate change in the diurnal regulation of NRT2 and NIA, which allows maximum rates of nitrate uptake and maximum NIA activity to be maintained for a larger part of the 24 h diurnal cycle. In contrast, tobacco growing on ammonium nitrate adjusts by selectively increasing the rate of ammonium uptake, and decreasing the expression of NRT2 and NIA and the rate of nitrate assimilation. In both conditions, the overall rate of inorganic nitrogen utilization is increased in elevated [CO2] due to higher rates of uptake and assimilation at the end of the day and during the night, and amino acids are maintained at levels that are comparable to or even higher than in ambient [CO2]. (iv) Comparison of the diurnal changes of transcripts, enzyme activities and metabolite pools across the four growth conditions reveals that these complex diurnal changes are due to transcriptional and post-transcriptional mechanisms, which act several steps and are triggered by various signals depending on the condition and organ. The results indicate that nitrate and ammonium uptake and root NIA activity may be regulated by the sugar supply, that ammonium uptake and assimilation inhibit nitrate uptake and root NIA activity, that the balance between the influx and utilization of nitrate plays a key role in the diurnal changes of the NIA transcript in leaves, that changes of glutamine do not play a key role in transcriptional regulation of NIA in leaves but instead inhibit NIA activity via uncharacterized post-transcriptional or post-translational mechanisms, and that high ammonium acts via uncharacterized post-transcriptional or post-translational mechanisms to stabilize glutamine synthetase activity during the night.

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.