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Notes: These experiments use Nia30(145), a tobacco nia1nia2 double null mutant transformed with a NIA2 construct, to define when sugar supply plays the dominating role in the regulation of nitrate reductase (NIA) expression. The null alleles of Nia30(145) are transcribed and translated to produce non-functional NIA transcript and NIA protein, providing an endogenous reporter system to track NIA expression at the transcript and protein level. The re-introduced NIA2 construct is expressed at low efficiency, providing a background in which the response to changes in sugar status is not complicated by simultaneous changes in the rate of nitrate assimilation and the levels of nitrate and glutamine. In an alternating light–dark regime, Nia30(145) contained high levels of nitrate and low levels of glutamine and other amino acids. This drives constitutive overexpression of NIA. After transfer of Nia30(145) to continuous darkness, nitrate remains high and glutamine low, but the NIA transcript level and NIA protein decreased significantly within 24 h and were undetectable from 48 h onwards. The decrease of the NIA transcript level was fully reversed and the decrease of NIA protein was partly reversed when leaves were detached from the pre-darkened plants and supplied with sucrose in the dark. The decrease was not reversed by nitrate or cytokinin. The NIA transcript disappeared when the leaf sugar content fell below 4 μmol hexose equivalents g−1 FW, and recovered when sugars rose above 8 μmol hexose equivalents g−1 FW. It is concluded that low sugar represses NIA, completely overriding signals derived from nitrate and nitrogen metabolism.

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.

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: 
AGPase, ADP glucose pyrophosphorylase
GS, glutamine synthetase
GOGAT, glutamate : oxoglutarate amino transferase
NADP-ICDH, NADP-dependent isocitrate dehydrogenase
NR, nitrate reductase
OPPP, oxidative pentose phosphate pathway
3PGA, glycerate-3-phosphate
PEPCase, phosphoenolpyruvate carboxylase
Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase
SPS, sucrose phosphate-synthase

This review first summarizes the numerous studies that have described the interaction between the nitrogen supply and the response of photosynthesis, metabolism and growth to elevated [CO2]. The initial stimulation of photosynthesis in elevated [CO2] is often followed by a decline of photosynthesis, that is typically accompanied by a decrease of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), an accumulation of carbohydrate especially starch, and a decrease of the nitrogen concentration in the plant. These changes are particularly marked when the nitrogen supply is low, whereas when the nitrogen supply is adequate there is no acclimation of photosynthesis, no major decrease in the internal concentration of nitrogen or the levels of nitrogen metabolites, and growth is stimulated markedly. Second, emerging evidence is discussed that signals derived from nitrate and nitrogen metabolites such as glutamine act to regulate the expression of genes involved in nitrate and ammonium uptake and assimilation, organic acid synthesis and starch accumulation, to modulate the sugar-mediated repression of the expression of genes involved in photosynthesis, and to modulate whole plant events including shoot–root allocation, root architecture and flowering. Third, increased rates of growth in elevated [CO2] will require higher rates of inorganic nitrogen uptake and assimilation. Recent evidence is discussed that an increased supply of sugars can increase the rates of nitrate and ammonium uptake and assimilation, the synthesis of organic acid acceptors, and the synthesis of amino acids. Fourth, interpretation of experiments in elevated [CO2] requires that the nitrogen status of the plants is monitored. The suitability of different criteria to assess the plant nitrogen status is critically discussed. Finally the review returns to experiments with elevated [CO2] and discusses the following topics: is, and if so how, are nitrate and ammonium uptake and metabolism stimulated in elevated [CO2], and does the result depend on the nitrogen supply? Is acclimation of photosynthesis the result of sugar-mediated repression of gene expression, end-product feedback of photosynthesis, nitrogen-induced senescence, or ontogenetic drift? Is the accumulation of starch a passive response to increased carbohydrate formation, or is it triggered by changes in the nutrient status? How do changes in sugar production and inorganic nitrogen assimilation interact in different conditions and at different stages of the life history to determine the response of whole plant growth and allocation to elevated [CO2]?

Notes: Tobacco seedlings were grown in nutrient agar at a range of ammonium nitrate concentrations either without added sucrose, or with 100 mol m–3 sucrose. In the absence of added sucrose, nitrogen-limited plants had increased levels of glucose, fructose and sucrose, decreased chlorophyll, decreased protein, and decreased Rubisco activity, but the level of the transcript for the small subunit of Rubisco (RbcS) did not decrease compared with nitrogen-sufficient plants. When sucrose was added to nitrogen-sufficient seedlings, there was an increase of sucrose, glucose and fructose in the leaves, growth was increased, and the chlorophyll and protein content, Rubisco activity, and the RbcS transcript level did not change. When sucrose was added to nitrogen-limited seedlings, there was a further increase of sucrose, glucose and fructose, growth was not increased, and there was a further decrease of chlorophyll, protein and Rubisco activity, and a marked decrease of the RbcS transcript level. To check that the decrease of the RbcS transcript level was not an indirect effect due to changes of nitrogen metabolites after adding sugars, glucose was added to Chenopodium cells in the presence and absence of glutamine or azaserine. Changes of glutamine that suffice to increase and decrease the level of the transcript for nitrate reductase (Nia) do not affect the RbcS transcript concentration, and glucose addition still led to a decrease of the RbcS transcript level when the internal glutamine concentration was high. Tobacco seedlings were also grown in nutrient agar at a range of phosphate concentrations either without added sucrose, or with 100 mol m–3 sucrose. Phosphate-limited seedlings did not show a decrease of chlorophyll, protein, Rubisco activity, or the level of the RbcS transcript, compared with phosphate-sufficient seedlings. The addition of sucrose to phosphate-limited plants led to a similar increase of sugars to that seen after adding sucrose to nitrogen-limited seedlings, but did not alter chlorophyll, protein, Rubisco activity, or the level of the RbcS transcript. The addition of sucrose to phosphate-limited plants led to a slight increase of the level of the transcript for nitrate reductase (Nia), increased nitrate reductase activity, and a marked increase of the amino acid content. Phosphate limitation led to an increased level of the transcript for the regulatory subunit of ADP glucose pyrophosphorylase (AgpS2), and this response was strengthened when sucrose was added. The regulation of AgpS2 expression by phosphate and sucrose was further investigated by feeding sucrose and phosphate to detached source leaves via the transpiration stream. The level of the AgpS2 transcript decreased after feeding phosphate and increased after feeding sucrose, and the effect of sucrose was antagonised by phosphate. It is concluded that the response to sugar signalling is modulated by nitrogen and phosphate in a gene-specific manner. The significance of these results for understanding the visual phenotype of nitrogen- and phosphate-limited plants, and the response of photosynthesis and starch synthesis to the plant nutrient status is discussed.

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.

Notes: Wild-type and antisense rbcS tobacco (Nicotiana tabacum) plants were grown in a glasshouse in midsummer in Portugal with an irradiance of 1500–2000 μmol m−2s−1 and daytime temperatures of 30–35 °C. The Rubisco content of the transformants was lower by 35, 80 and over 90% than that of the wild-type. Gas exchange was measured over three separate days. There was a near-linear relation between Rubisco content and photosynthetic rate during the period of high irradiance, allowing a flux control coefficient of 0.83–0.89 to be estimated. The relation deviated slightly from linearity, because the internal CO2 concentration (c;) was higher in the transformants than in the wild-type (190 and 275 μmol mol−1 in plants with 35 and 80% less Rubisco, respectively, compared with 175 μmol mol−1 for wild-type), compensating to some extent for the decreased Rubisco content. This increase in ci occurred because the stomatal conductance (g) remained unaltered or was even higher in plants with decreased Rubisco, despite the lower rate of CO2 assimilation. As a consequence, water use efficiency declined. The decreased rate of photosynthesis was not accompanied by a stoichiometric decrease in apparent growth rate. These results are discussed in relation to earlier studies of the plant set in growth cabinets. It is concluded that tobacco can adjust over a wide range of growth conditions to avoid a onesided limitation by Rubisco, but that in extreme environmental conditions this capacity to adapt is exhausted.

Notes: The carbohydrate content of photosynthetic cells or tissues was increased by feeding glucose to autotrophic Chenopodium rubrum cell suspension cultures, by feeding glucose via the transpiration stream to detached spinach leaves, and by expressing yeast-derived invertase in the apoplast of tobacco leaves. Extracts were prepared, and plastidic and cytosolic isoenzymes were separated by electrophoresis and assayed by in situ activity staining. In all three systems, compared to control treatments, accumulation of carbohydrate led to decreased activity of plastid starch phosphorylase and phosphoglucose mutase, but not of the corresponding cytosolic isoenzymes. It led to increased activity of cytosolic 6-hosphogluconate dehydrogenase but not of the plastid isoenzyme. The activities of cytosolic and plastidic aldolase, triose-phosphate isomerase, and phosphoglucose isomerase were unaltered. The transcript and activity of nitrate reductase and pyrophosphate:fructose-6-phosphate phosphotransferase (both cytosolic enzymes) increased. The transcript levels for the S-gene of ADP-glucose pyrophosphorylase (a plastid enzyme) increased, but the overall enzyme activity decreased slightly. It is concluded that high carbohydrate leads to a selective change in the enzyme complement of the plastid, retaining enzymes which are required for glycolysis and losing enzymes which are needed for photosynthesis or starch mobilization.

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.