Notes: Abstract: Metabolism of [U-13C5]glutamine was studied in primary cultures of cerebral cortical astrocytes in the presence or absence of extracellular glutamate. Perchloric acid extracts of the cells as well as redissolved lyophilized media were subjected to nuclear magnetic resonance and mass spectrometry to identify 13C-labeled metabolites. Label from glutamine was found in glutamate and to a lesser extent in lactate and alanine. In the presence of unlabeled glutamate, label was also observed in aspartate. It could be clearly demonstrated that some [U-13C5]glutamine is metabolized through the tricarboxylic acid cycle, although to a much smaller extent than previously shown for [U-13C5]glutamate. Lactate formation from tricarboxylic acid cycle intermediates has previously been demonstrated. It has, however, not been demonstrated that pyruvate, formed from glutamate or glutamine, may reenter the tricarboxylic acid cycle after conversion to acetyl-CoA. The present work demonstrates that this pathway is active, because [4,5-13C2]glutamate was observed in astrocytes incubated with [U-13C5]glutamine in the additional presence of unlabeled glutamate. Furthermore, using mass spectrometry, mono-labeled alanine, glutamate, and glutamine were detected. This isotopomer could be derived via the action of pyruvate carboxylase using 13CO2 produced within the mitochondria or from labeled intermediates that had stayed in the tricarboxylic acid cycle for more than one turn.

Notes: Abstract: Primary cultures of cerebral cortical astrocytes were incubated with [U-13C]glutamate (0.5 mM) in modified Dulbecco's medium for 2 h. Perchloric acid (PCA) extracts of the cells as well as redissolved lyophilized media were subjected to NMR spectroscopy to identify 13C-labeled metabolites. NMR spectra of the PCA extracts exhibited distinct multiplets for glutamate, aspartate, glutamine, and malate. The culture medium showed peaks for a multitude of compounds released from the astrocytes, among which lactate, glutamine, alanine, and citrate were readily identifiable. For the first time incorporation of label into lactate from glutamate was clearly demonstrated by doublet formation in the C-3 position and two doublets in the C-2 position of lactate. This labeling pattern can only occur by incorporation from glutamate, because natural abundance will only produce singlets in proton-decoupled 13C spectra. Glutamine, released into the medium, was labeled uniformly to a large extent, but the C-3 position not only showed the expected apparent triplet but also a doublet due to 13C incorporation into the C-4 position of glutamine. The doublet accounted for 11% of the total label in the glutamine synthesized and released within the incubation period. The corresponding labeling pattern of [13C]glutamate in the PCA extracts showed that 19% of the glutamate contained 12C. Labeling of lactate, citrate, malate, and aspartate as well as incorporation of 12C into uniformly labeled glutamate and glutamine could only arise via the tricarboxylic acid cycle. The relative amount of glutamate metabolized via this route is at least 70% as calculated from the areas of the C-3 resonances of these compounds. Only a maximum of 30% was converted to glutamine directly.

Notes: Antibodies raised against a peptide fragment (residues 60–456) of potato sucrose phosphate synthase (SPS) were used to investigate whether potato plants contain multiple forms of SPS. When a partially purified preparation of SPS from cold-stored potato tubers was separated on 5% polyacrylamide gel electrophoresis (PAGE), four immunopositive bands were found with estimated molecular weights of 125, 127, 135 and 145 kDa. These bands were also found in rapidly prepared extracts and were termed SPS-1a, SPS-1b, SPS-2 and SPS-3, respectively. Direct evidence that SPS-1a and SPS-1b represent active SPS was provided by the finding that both are greatly reduced in plants expressing an antisense sequence derived from the potato leaf SPS gene. SPS-2 was not decreased in the antisense plants, indicating that it has a significantly different sequence. Evidence that SPS-2 represents active SPS was obtained by showing that the amount of SPS-1a and SPS-1b protein remaining in the leaves and tubers of antisense potato plants was too low to account for the remaining SPS activity. The four immunopositive SPS forms had different tissue distributions. SPS-1a was the major form in all tissues except petals, sepals and stamens. SPS-1b and SPS-2 were absent in very young growing tissues but were present as minor forms in source leaves and sprouting tubers. The SPS-1b level was especially high in petals and sepals, and the SPS-2 level was especially high in the stamens. SPS-3 was only detected in very young tissues. The four forms also showed different responses to low temperature. Transfer of tubers to 4°C led to a specific and reversible increase of SPS-1b during the next 4 d. The appearance of SPS-1b correlated with a change in the kinetic properties of SPS that has recently been shown (Hill et al. 1996) to play a key role in triggering the accumulation of sugars in cold-stored tubers. The appearance of SPS-1b protein at low temperature was accompanied by an increase of SPS transcript. Incubation of tuber slices with calyculin A and okadaic acid to alter the phosphorylation state of SPS did not lead to appearance or disappearance of SPS-1b. It is concluded that potato plants contain several forms of SPS that have different functions in growing and mature tissues, in flower parts, and in acclimation to low temperature.

Notes: Higher rates of nitrate assimilation are required to support faster growth in enhanced carbon dioxide. To investigate how this is achieved, tobacco plants were grown on high nitrate and high light in ambient and enhanced (700 μmol mol–1) carbon dioxide. Surprisingly, enhanced carbon dioxide did not increase leaf nitrate reductase (NR) activity in the middle of the photoperiod. Possible reasons for this anomalous result were investigated. (a) Measurements of biomass, nitrate, amino acids and glutamine in plants fertilized once and twice daily with 12 mol m–3 nitrate showed that enhanced carbon dioxide did not lead to a nitrate limitation in these plants. (b) Enhanced carbon dioxide modified the diurnal regulation of NR activity in source leaves. The transcript for nia declined during the light period in a similar manner in ambient and enhanced carbon dioxide. The decline of the transcript correlated with a decrease of nitrate in the leaf, and was temporarily reversed after re-irrigating with nitrate in the second part of the photoperiod. The decline of the transcript was not correlated with changes of sugars or glutamine. NR activity and protein decline in the second part of the photoperiod, and NR is inactivated in the dark in ambient carbon dioxide. The decline of NR activity was smaller and dark inactivation was partially reversed in enhanced carbon dioxide, indicating that post-transcriptional or post-translational regulation of NR has been modified. The increased activation and stability of NR in enhanced carbon dioxide was correlated with higher sugars and lower glutamine in the leaves. (c) Enhanced carbon dioxide led to increased levels of the minor amino acids in leaves. (d) Enhanced carbon dioxide led to a large decrease of glycine and a small decrease of serine in leaves of mature plants. The glycine:serine ratio decreased in source leaves of older plants and seedlings. The consequences of a lower rate of photorespiration for the levels of glutamine and the regulation of nitrogen metabolism are discussed. (e) Enhanced carbon dioxide also modified the diurnal regulation of NR in roots. The nia transcript increased after nitrate fertilization in the early and the second part of the photoperiod. The response of the transcript was not accentuated in enhanced carbon dioxide. NR activity declined slightly during the photoperiod in ambient carbon dioxide, whereas it increased 2-fold in enhanced carbon dioxide. The increase of root NR activity in enhanced carbon dioxide was preceded by a transient increase of sugars, and was followed by a decline of sugars, a faster decrease of nitrate than in ambient carbon dioxide, and an increase of nitrite in the roots. (f) To interpret the physiological significance of these changes in nitrate metabolism, they were compared with the current growth rate of the plants. (g) In 4–5-week-old plants, the current rate of growth was similar in ambient and enhanced carbon dioxide (≈ 0·4 g–1 d–1). Enhanced carbon dioxide only led to small changes of NR activity, nitrate decreased, and overall amino acids were not significantly increased. (h) Young seedlings had a high growth rate (0·5 g–1 d–1) in ambient carbon dioxide, that was increased by another 20% in enhanced carbon dioxide. Enhanced carbon dioxide led to larger increases of NR activity and NR activation, a 2–3-fold increase of glutamine, a 50% increase of glutamate, and a 2–3-fold increase in minor amino acids. It also led to a higher nitrate level. It is argued that enhanced carbon dioxide leads to a very effective stimulation of nitrate uptake, nitrate assimilation and amino acid synthesis in seedlings. This will play an important role in allowing faster growth rates in enhanced carbon dioxide at this stage.

Notes: As reported in a previous paper [Lerchl et al. (1995) Plant Cell, 7, 259–270], expression of Escherichia coli inorganic pyrophosphatase in the cytosol under the control of the phloem-specific rolC promoter from, Agobacterium rhizogenes results in decreased growth of transgenic tobacco plants. In this paper we investigate the effect of the phloem-specific expression of pyrophosphatase on phloem metabolism, and on plant growth and allocation. A small decrease in the hexose phosphate/UDP-glucose ratio, the ATP/ADP ratio and the respiration rate in the midribs of the transformants provides evidence Hint mobilization of sucrose via pyrophosphate-dependent reactions is necessary for phloem energy metabolism. The source leaves of the transformants had higher levels of carbohydrates and amino acids and a much higher glutamine/glutamate ratio than the wild type, showing that export was inhibited and that the growth inhibition was not due to a lack of photoas-similates or organic nitrogen in the leaves. The accumulation of photoassimilates was paralleled by a decrease in photosynthesis, chlorophyll content and ribulose bisphosphate carboxylase/oxygenase (Rubisco) activity, a small increase in hexose phosphates and triose phosphates and a decrease in glycerate 3-phosphate in the source leaves. There was a decrease of soluble sugars and amino acids in sink leaves of the transformants. In sink leaves amino acids decreased more than carbohydrates and a decrease in the glutamine/ glutamate ratio was observed. This was accompanied by a large decrease of nitrate. Sugars and amino acids were also reduced in the root tips of the transformants. The carbohydrate /amino acid ratio decreased 5-fold in the root tips, indicating a particularly smile shortage of carbohydrates. Relatively high levels of sugars and amino acids in the basal regions of the root and the increase in sugars in the midrib indicate that there is also increased leakage of assimilates out of the phloem during long-distance transport. Metabolism is required to maintain phloem function along the transport route, as well as for the initial step of loading. The transformants showed decreased stem and root growth. The growth inhibition was largest in conditions allowing rapid growth of the wild type (high light and nitrogen supply).

Notes: Since 1980, the use of transgenic plants in modern plant science has become a powerful tool to study whole plant physiology. In this review, we try to summarize the data obtained in the field of photoassimilate partitioning. Attempts to study sink-source interactions concern factors which might limit sink strength and source capacity. Transgenic plants have been used to manipulate the sucrose to starch ratio in order to produce plants with higher sucrose levels in their source leaves. Alterations in partitioning were achieved by manipulating Calvin cycle enzymes, transport proteins and sucrose biosynthetic enzymes. The ability of sink tissues to attract photoassimilates has been altered by either increasing or decreasing sucrose hydrolytic activities. The increase of sucrose hydrolysis was achieved by creating transgenic potato plants with tuber specific yeast-derived invertase. Decreased sucrose utilization was achieved by antisense inhibition of sucrose synthase in potato tubers.

Notes: Acclimation of plants to an increase in atmospheric carbon dioxide concentration is a well described phenomenon. It is characterized by an increase in leaf carbohydrates and a degradation of ribulose 1, 5-bisphosphate carboxylase protein (Rubisco) leading in the long term to a lower rate of CO2 assimilation than expected from the kinetic constants of Rubisco. This article summarizes studies with transgenic plants grown in elevated pCO2 which are modified in their capacity of CO2 fixation, of sucrose and starch synthesis, of triosephosphate and sucrose transport and of sink metabolism of sucrose. These studies show that a feedback accumulation of carbohydrates in leaves play only a minor role in acclimation, because leaf starch synthesis functions as an efficient buffer for photoassimilates. There is some evidence that in elevated pCO2, plants grow faster and senescence is induced earlier.

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: Transfer of potato tubers to low temperature leads after 2–4 d to a stimulation of sucrose synthesis, a decline of hexose-phosphates and a change in the kinetic properties, and the appearance of a new form of sucrose phosphate synthase (SPS). Antisense and co-suppression transformants with a 70–80% reduction in SPS expression have been used to analyse the contribution of SPS to the control of cold sweetening. The rate of sucrose synthesis in cold-stored tubers was investigated by measuring the accumulation of sugars, by injecting labelled glucose of high specific activity into intact tubers, and by providing 50 mol m–3 labelled glucose to fresh tuber slices from cold-stored tubers. A 70–80% decrease of SPS expression resulted in a reproducible but non-proportional (10–40%) decrease of soluble sugars in cold-stored tubers, and a non-proportional (about 25%) inhibition of label incorporation into sucrose, increased labelling of respiratory intermediates and carbon dioxide, and increased labelling of glucans. The maximum activity of SPS is 50-fold higher than the net rate of sugar accumulation in wild-type tubers, and decreased expression of SPS in the transformants was partly compensated for increased levels of hexose-phosphates. It is concluded that SPS expression per se does not control sugar synthesis. Rather, a comparison of the in vitro properties of SPS with the estimated in vivo concentrations of effectors shows that SPS is strongly substrate limited in vivo. Alterations in the kinetic properties of SPS, such as occur in response to low temperature, will provide a more effective way to stimulate sucrose synthesis than changes of SPS expression.