ALBERT

Notes: Starch is of great importance both as a carbon storage reserve in plants and as a biotechnologically important product. The potato tuber is an attractive model system for the study of starch metabolism, because it is a relatively homogenous tissue in which conversion of sucrose to starch represents the dominant metabolic flux. All the major genes of the potato tuber sucrose to starch pathway have been cloned in recent years, allowing the generation of a suite of antisense transgenic lines to be produced in which the activity of each individual enzyme in the pathway is progressively decreased. Investigations of these plants have provided a complete picture of the distribution of control in this important pathway. Sucrose synthase, UGPase, hexokinase, cytosolic phosphoglucomutase, plastidial phosphoglucomutase, the amyloplastidial adenylate translocator, AGPase, starch synthase and starch branching enzyme have flux control coefficients (FCCs) of 0.10, approximating 0.00, approximating 0.00, 0.15, 0.23, 0.98, 0.35, 0.12 and approximating 0.00 for starch accumulation. These results show that the majority of the control on starch accumulation in potato tubers resides in the transfer of adenylate between the cytosol and the amyloplast, with a minor contribution being made by the first two steps of the plastidial starch synthesis pathway (the reactions catalysed by plastidial phosphoglucomutase and AGPase). This contrasts with leaves, in which the majority of the control has been found to reside in the reactions catalysed by plastidial phosphoglucomutase and AGPase. In leaves, ATP for starch synthesis is generated within the plastid via photophosphorylation. Several studies have attempted to increase the rate of starch synthesis by overexpressing pathway enzymes in tubers. The results of these studies and the role of other ATP producers in the starch synthetic process are reviewed. In the same time period methods of non-aqueous fractionation have been adapted to potato tuber tissue in order to ascertain subcellular metabolite levels. Results obtained from these studies allow the calculation of mass action ratios of the constitutive enzymes of the sucrose to starch transition. When taken together with the known regulatory properties of these enzymes the combination of broad control analysis studies and assessment of the mass action ratios of the respective enzymes allows a comprehensive description of this important metabolic network. Some illustrative examples of how this network responds to environmental change are presented. Finally implications of this whole pathway evaluation for more general studies of plant metabolic pathways and networks are discussed.

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 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: 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: The onset of sugar accumulation in cold-stored potato tubers coincides with an activation of sucrose phosphate synthase (SPS) and the appearance of a new form of amylase (Hill et al. 1996; Nielsen et al. 1997). To provide more evidence that these changes are involved in the regulation of cold-induced sugar accumulation we have compared the temperature dependence of sugar accumulation with the temperature dependence of changes in SPS and amylase activity. To do this, we investigated: sugars and metabolites; SPS activity, protein and transcript; invertase activity; and amylolytic activity and amylase forms, during the first 10 d after transferring tubers from room temperature to 3, 5, 7, 9 and 11 °C. After 10 d there was negligible accumulation of sugars at 11, 9 and 7 °C, and a large accumulation at 5 and 3 °C. Activation of SPS was assayed by comparing activity in an assay with limiting substrates and phosphate and an assay with saturating substrate concentrations. The activation state increased slightly at 7 °C, 3- to 4-fold at 5 °C and 5- to 6-fold at 3 °C. The cold-induced change in the kinetic properties of SPS was accompanied by the appearance of a new form of SPS with a slightly higher apparent molecular weight, and by an increase of the SPS transcript. The changes in SPS protein and transcript showed the same temperature dependence as the changes in the kinetic properties. Total starch hydrolysing enzyme activity was unaltered at 7°C, increased at 5°C and increased further (up to 7- to 8-fold) at 3 °C. The increase in amylolytic activity correlated with the appearance of a new amylase band on zymograms. Acid invertase activity showed a similar increase at 3, 5, and 7°C, and it did not correlate with the total sugar accumulation. The cold-induced accumulation of sugar can be reversed by transferring tubers back to warmer temperatures. We compared the decline of sugar levels with the changes of SPS, amylolytic activity and metabolites at various times (up to 14 d) after transfer of tubers from a 4 °C storage (for 14 d) back to 20°C. SPS activation state is reversed and the cold-induced form of SPS virtually disappears during the first 2–4 d at 20 °C. The cold-induced amylase activity also vanishes within 2–4 d.

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.