Increased levels of adenine nucleotides modify the interaction between starch synthesis and respiration when adenine is supplied to discs from growing potato tubers

To investigate the importance of the overall size of the total adenine nucleotide pool for the regulation of primary metabolism in growing potato tubers, freshly cut discs were provided with zero or 2 mM adenine in the presence of 1 or 100 mM [U-¹⁴C]glucose or 100 mM [U-¹⁴C]sucrose in the presence and absence of 20 mM orthophosphate (Pi). Adenine led to a 150–250% increase of the total adenine nucleotide pool, which included an increase of ADP, a larger increase of ATP and an increase of the ATP:ADP ratio. There was a 50–100% increase of ADP-glucose (ADPGlc), and starch synthesis was stimulated. Respiratory oxygen uptake was stimulated, and the levels of glycerate-3-phosphate, phosphoenolpyruvate and α-ketoglutarate decreased. The response to adenine was not modified by Pi. It is proposed that increased ATP stimulates ADPGlc pyrophosphorylase, leading to a higher rate of starch synthesis. The impact on starch synthesis is constrained, however, because increased ADP can lead to a stimulation of respiration and decline of glycerate-3-phosphate, which will inhibit ADPGlc pyrophosphorylase. The quantitative impact depends on the conditions. In the presence of 1 mM glucose, the levels of phosphorylated intermediates and the rate of starch synthesis were low. Adenine led to a relatively large stimulation of respiration, but only a small stimulation of starch synthesis. In the presence of 100 mM glucose, discs contained high levels of phosphorylated intermediates, low ATP:ADP ratios (<3) and low rates of starch synthesis (<20% of the metabolised glucose). Adenine led to marked increase of ATP and 2- to 4-fold stimulation of starch synthesis. Discs incubated with 100 mM sucrose already had high ATP:ADP ratios (>8) and high rates of starch synthesis (>50% of the metabolised sucrose). Adenine led to a further increase, but the stimulation was less marked than in high glucose. These results have implications for the function of nucleotide cofactors in segregating sucrose mobilisation and respiration, and the need for energy conservation during sugar-starch conversions. Received: 9 February 2000 / Accepted: 9 June 2000     

Contribution of adenosine 5′-diphosphoglucose pyrophosphorylase to the control of starch synthesis is decreased by water stress in growing potato tubers

Water stress stimulates sucrose synthesis and inhibits starch and cell-wall synthesis in tissue slices of growing potato (Solanum tuberosum L. cv. Desirée) tubers. Based on the analysis of fluxes and metabolites, Geigenberger et al. (1997, Planta 201: 502–518) proposed that water deficits up to −0.72 MPa stimulate sucrose synthesis, leading to decreased starch synthesis as a result of the resulting decline of phosphorylated metabolite levels, whereas more-severe water deficits directly inhibit the use of ADP-glucose. Potato plants with decreased expression of adenosine 5′-diphosphoglucose pyrophosphorylase (AGPase) have been used to test the prediction that the contribution of AGPase to the control of starch synthesis should decrease in severely water-stressed tuber material. Freshly cut slices from wild-type and antisense tubers were incubated at a range of mannitol concentrations (20, 300 and 500 mM) and the metabolism of [¹⁴C]glucose was analysed. A 86–97% reduction of AGPase activity led to a major but non-stoichiometric inhibition of starch accumulation in intact growing tubers attached to the plant (40–85%), and an inhibition of starch synthesis in non-stressed tuber slices incubated in 20 mM mannitol (60–80%). The inhibition of starch synthesis was accompanied by a 2- to 8-fold increase in the levels of sugars in intact tubers and a 2- to 3-fold stimulation of sucrose synthesis in tuber slices, whereas respiration and cell-wall synthesis were not significantly affected. The strong impact of AGPase on carbon partitioning in non-stressed tubers and tuber slices was retained in slices subjected to moderate water deficit (300 mM mannitol, corresponding to −0.72 MPa). In discs incubated in 500 mM mannitol (corresponding to −1.2 MPa) this response was modified. A 80–97% reduction of AGPase resulted in only a 0–40% inhibition of starch synthesis. Further, the water stress-induced stimulation of sucrose synthesis was abolished in the transformants. The results provide direct evidence that the contribution of AGPase to the control of starch synthesis can be modified by environmental factors, leading to a lower degree of control during severe water deficits. There was also a dramatic decrease in the labelling of cell-wall components in wild-type tuber slices incubated with 300 or 500 mM mannitol. The water stress-induced inhibition of cell-wall synthesis occurred independently of AGPase expression and the accompanying changes in starch and sucrose metabolism, indicating a direct inhibition of cell-wall synthesis in response to water stress. Received: 24 February 1999 / Accepted: 28 May 1999     

Metabolism in slices from growing potato tubers responds differently to addition of sucrose and glucose

The short-term changes in metabolism that occurred after adding glucose or sucrose to freshly cut discs from growing potato (Solanum tuberosum L.) tubers were investigated. (i) When glucose was supplied, there was a marked increase in glycolytic metabolites, and respiration was stimulated. When sucrose was supplied, amounts of glycolytic metabolites including hexose phosphates and 3-phosphoglycerate (3PGA) were similar to or lower than in control discs incubated without sugars, and respiration did not rise initially above that in control discs. This different response to sucrose and glucose was found across the concentration range 5–200 mM. A larger proportion of the metabolised ¹⁴C was converted to starch when [¹⁴C] sucrose was supplied than when [¹⁴C] glucose was supplied. The different effect on metabolite levels, respiration and starch synthesis was largest after 20–30 min, and decreased in longer incubations. (ii) When 5 or 25 mM sucrose was added in the presence of [¹⁴C] glucose, it led to a decrease in hexose phosphates and 3PGA, and a small increase in the rate of starch synthesis compared to discs incubated with glucose in the absence of sucrose. These differences were seen in a 30-min pulse and a 2-h pulse. Whereas ADP-glucose levels after adding sucrose resembled those in control discs, glucose led to a decrease in ADP-glucose. This decrease did not occur when 5 or 25 mM sucrose was added with the glucose. (iii) To check the relevance of these experiments for intact tubers, water or 100 mM mannitol, sucrose or glucose were supplied through the stolon to intact tubers for 24 h. A 0.2 mM solution of [¹⁴C] glucose was then introduced into the tubers, and its metabolism investigated during the next 30 min. Labelling of starch was increased after preincubation with sucrose, and significantly inhibited after preincubation with glucose. (iv) It is concluded that glucose and sucrose have different effects on tuber metabolism. Whereas glucose leads to a preferential stimulation of respiration, sucrose preferentially stimulates starch synthesis via a novel mechanism that allows stimulation of ADP-glucose pyrophosphorylase even though the levels of hexose phosphates and the allosteric activator 3PGA decrease. Received: 9 October 1997 / Accepted: 3 February 1998     

Orotate leads to a specific increase in uridine nucleotide levels and a stimulation of sucrose degradation and starch synthesis in discs from growing potato tubers

Freshly cut discs from growing potato tubers were incubated for 3 h with 10 mM orotate or 10 mM uridine. Control discs incubated without precursors showed a 30–40% decrease of uridine nucleotides, but not of adenine nucleotides. Orotate- and uridine-feeding led to a 1.5- to 2-fold increase in the levels of uridine nucleotides compared with control discs, and a 15–30% increase compared with the original values in intact tubers, but did not alter the levels of adenine nucleotides. Between 70–80% of the uridine nucleotides were present as UDPglucose, 15–25% as UTP, and 2–3% as UDP. The increase of uridine nucleotides involved a similar relative increase of UDPglucose, UTP and UDP. It was accompanied by a slight stimulation of the rate of [¹⁴C]sucrose uptake, a 2-fold stimulation of the rate at which the [¹⁴C]sucrose was subsequently metabolised, a small increase in the levels of hexose phosphates, glycerate-3-phospate and ADPglucose, and a 30% shift in the allocation of the metabolised label in favour of starch synthesis, resulting in a 2.4-fold stimulation of the rate of starch synthesis. Orotate led to a similar increase of uridine nucleotide levels in the presence of [¹⁴C]glucose, but did not significantly alter the rate of glucose uptake and metabolism to starch, nor did it increase the rate of sucrose resynthesis. The levels of uridine nucleotides were high in tubers on 6 to 10-week-old potato plants, and declined in tubers on 12 to 15-week-old plants. Comparison with the effect of the uridine nucleotide level in discs shows that the high levels of uridine nucleotides in tubers on young plants will play an important role in determining the rate at which sucrose can be converted to starch, and that the level of uridine nucleotides is probably co-limiting for sucrose-starch conversions in tubers on older plants. Received: 25 September 1998 / Accepted: 29 December 1998     

Hexose metabolism in discs excised from developing potato (Solanum tuberosum L.) tubers

Metabolism of radiolabelled hexoses by discs excised from developing potato (Solanum tuberosum L.) tubers was been investigated in the presence of acid invertase to prevent accumulation of labelled sucrose in the bathing medium (Viola, 1996, Planta 198: 179–185). When the discs were incubated with either [U-¹⁴C]glucose or [U-¹⁴C]fructose without unlabelled hexoses, the unidirectional rate of sucrose synthesis was insignificant compared with that of sucrose breakdown. The inclusion of unlabelled fructose in the medium induced a dramatic increase in the unidirectional rate of sucroses synthesis in the tuber discs. Indeed, the decline in the sucrose content observed when discs were incubated without exogenous sugars could be completely prevented by including 300 mM fructose in the bathing medium. On the other hand, the inclusion of unlabelled glucose in the medium did not significantly affect the relative incorporation of [U-¹⁴C]glucose to starch, sucrose or glycolytic products. Substantial differences in the intramolecular distribution of ¹³C enrichment in the hexosyl moieties of sucrose were observed when the discs were incubated with either [2-¹³C]fructose or [2-¹³C]glucose. The pattern of ¹³C enrichment distribution in sucrose suggested that incoming glucose was converted into sucrose via the sucrose-phosphate synthase pathway whilst fructose was incorporated directly into sucrose via sucrose synthase. Quantitative estimations of metabolic fluxes in vivo in the discs were also provided. The apparent maximal rate of glucose phosphorylation was close to the extractable maximum catalytic activity of glucokinase. On the other hand, the apparent maximal rate of fructose phosphorylation was much lower than the maximum catalytic activity of fructokinase, suggesting that the activity of the enzyme (unlike that of glucokinase) was regulated in vivo. Although in the discs incubated with or without fructose the rates of starch synthesis or glycolysis were similar, the relative partitioning of metabolic intermediates into sucrose was much higher in discs incubated with fructose (0.6% and 32.6%, respectively). It is hypothesised that the equilibrium of the reaction catalysed by sucrose synthase in vivo is affected in discs incubated with fructose as a result of the accumulation of the sugar in the tissue. This results in the onset of sucrose cycling. Incubation with glucose enhanced all metabolic fluxes. In particular, the net rate of starch synthesis increased from 2.0 mol · hexose · g FW^–1 · h^–1 in the absence of exogenous glucose to 3.7 mol · hexose · g FW^–1 · h^–1 in the presence of 300 mM glucose. These data are taken as an indication that the regulation of fructokinase in vivo may represent a limiting factor in the utilisation of sucrose for biosynthetic processes in developing potato tubers.Abbreviations ADPGlc adenosine 5-diphosphoglucose - Glc6P glucose-6-phosphate - hexose-P hexose phosphate - NMR nuclear magnetic resonance - UDPGlc uridine 5-diphosphoglucose Many thanks to L. Sommerville for skillfull assistance and to J. Crawford and J. Liu for useful discussions on flux analysis. The research was funded by the Scottish Office Agriculture and Fisheries Department.     

The contribution of adenosine 5′-diphosphoglucose pyrophosphorylase to the control of starch synthesis in potato tubers

The aim of this work was to investigate the extent to which starch synthesis in potato (Solanum tuberosum L.) tubers is controlled by the activity of ADPglucose pyrophosphorylase (EC 2.7.7.27; AGPase). In order to do this, fluxes of carbohydrate metabolism were measured in tubers that had reduced AGPase activity as a result of the expression of a cDNA encoding the B subunit in the antisense orientation. Reduction in AGPase activity led to a reduction in starch accumulation, and an increase in sucrose accumulation. The control coefficient of AGPase on starch accumulation in intact plants was estimated to be around 0.3. The fluxes of carbohydrate metabolism were measured in tuber discs from wild-type and transgenic plants by investigating the metabolism of [U-¹⁴C]glucose. In tuber discs, the control coefficient of AGPase over starch synthesis was estimated as 0.55, while the control coefficient of the enzyme over sucrose synthesis was −0.47. The values obtained suggest that AGPase activity exerts appreciable control over tuber metabolism in potato. Received: 24 February 1999 / Accepted: 8 April 1999     

Tuber-specific expression of a yeast invertase and a bacterial glucokinase in potato leads to an activation of sucrose phosphate synthase and the creation of a sucrose futile cycle

Fluxes were investigated in growing tubers from wild-type potato (Solanum tuberosum L. cv. Desiree) and from transformants expressing a yeast invertase in the cytosol under the control of the tuber-specific patatin promoter either alone (EC 3.2.1.26; U-IN2-30) or in combination with a Zymomonas mobilis glucokinase (EC 2.7.1.2; GK3-38) by supplying radiolabelled [¹⁴C]sucrose, [¹⁴C]glucose or [¹⁴C]fructose to tuber discs for a 90-min pulse and subsequent chase incubations of 4 and 12 h, and by supplying [¹⁴C]fructose for 2 h and 4 h to intact tubers attached to the mother plant. Contrary to the expectation that this novel route for sucrose degradation would promote starch synthesis, the starch content decreased in the transgenic lines. Labelling kinetics did not reveal whether this was due to changes in the fluxes into or out of starch. However, they demonstrated that glycolysis is enhanced in the transgenic lines in comparison to the wild type. There was also a significant stimulation of sucrose synthesis, leading to a rapid cycle of sucrose degradation and resynthesis. The labelling pattern indicated that sucrose phosphate synthase (SPS; EC 2.4.1.14) was responsible for the enhanced recycling of label into sucrose. In agreement, there was a 4-fold and 6-fold increase in the activation status of SPS in U-IN2-30 and GK3-38, respectively, and experiments with protein phosphatase inhibitors indicated that this activation involves enhanced dephosphorylation of SPS. It is proposed that this activation of SPS is promoted by the elevated glucose 6-phosphate levels in the transgenic tubers. These results indicate the pitfalls of metabolic engineering without a full appreciation of the metabolic system and regulatory circuits present in the tissue under investigation. Received: 21 July 1998 / Accepted: 5 December 1998     

Osmotic regulation of starch synthesis in potato tubers?

Using potato (Solanum tuberosum L.) tuber discs incubated in a range of mannitol concentrations it has been demonstrated that both sucrose uptake and the conversion of sucrose to starch are sensitive to the osmotic environment of the storage cells. Starch synthesis was optimised at 300 mM but declined sharply at both lower and higher osmotic concentrations. The decline in starch synthesis on either side of optimum was not proportional to the change in mannitol concentration, indicating different inhibitory mechanisms under low and high osmotica. The fraction of the total sucrose converted to starch i.e. the partitioning between sucrose and starch, was also influenced by osmotic environment. The amount of soluble material taken up by the storage cells, but not converted to starch, was maintained under mannitol concentrations (300–400 mM) which inhibited starch synthesis, indicating that sucrose uptake continued during declining starch synthesis. At mannitol concentrations above 400 mM, sucrose uptake was greatly enhanced but no significant change in starch synthesis occurred.     

Cold hardiness in Entomobrya nivalis (Collembola, Entomobryidae): annual cycle of polyols and antifreeze proteins, and antifreeze triggering by temperature and photoperiod

In the Swiss Prealps Entomobrya nivalis hibernates in an inactive state, hidden under bark flakes on spruce. For freeze avoidance it relies on thermal hysteresis proteins (THPs) and polyols (mainly ribitol, with small amounts arabitol and threitol). Polyols are present only during the inactive state, THPs additionally protect during the transition phase in spring and autumn, when animals are still active but frosts may occur. Peak values were recorded in February/March for THPs (3.5 °C hysteresis between melting and freezing point) and for polyols (26 μg mg⁻¹ FW; hemolymph osmolality 680 mosmol l⁻¹). E. nivalis is able to control its hemolymph osmolality independently of body water content. Mean osmolality in summer was 350– 440 mosmol l⁻¹, in winter it was elevated to 650 mosmol l⁻¹, due to a synthesis mainly of ribitol. Body water content varied between 1.8 and 3.3 mg H₂O mg⁻¹ DW, depending on humidity conditions. Experiments on triggering of antifreeze synthesis showed the action of temperature and photoperiod as cues, but there was also evidence for an endogenous rhythm. No clear correlation between antifreeze concentration and supercooling ability could be established, suggesting that gut content or other parameters also play an inportant role. Accepted: 18 November 1995     

Decreased expression of sucrose phosphate synthase strongly inhibits the water stress-induced synthesis of sucrose in growing potato tubers

Water stress stimulates sucrose synthesis and inhibits starch synthesis in wild-type tubers. Antisense and co-suppression potato transformants with decreased expression of sucrose–phosphate synthase (SPS) have been used to analyse the importance of SPS for the regulation of this water-stress induced change in partitioning. (i) In the absence of water stress, a 70–80% decrease in SPS activity led to a 30–50% inhibition of sucrose synthesis and a slight (10–20%) increase of starch synthesis in tuber discs in short-term labelling experiments with low concentrations of labelled glucose. Similar changes were seen in short-term labelling experiments with intact tubers attached to well-watered plants. Provided plants were grown with ample light and water, transformant tubers had a slightly lower water and sucrose content and a similar or even marginally higher starch content than wild-type tubers. (ii) When wild-type tuber slices were incubated with labelled glucose in the presence of mannitol to generate a moderate water deficit (between –0.12 and –0.72 MPa), there was a marked stimulation of sucrose synthesis and inhibition of starch synthesis. A similar stimulation was seen in labelling experiments with wild-type tubers that were attached to water-stressed wild-type plants. These changes were almost completely suppressed in transformants with a 70–80% reduction of SPS activity. (iii) Decreased irrigation led to an increase in the fraction of the dry-matter allocated to tubers in wild-type plants. This shift in allocation was prevented in transformants with reduced expression of SPS. (iv) The results show that operation of SPS and the sucrose cycle in growing potato tubers may lead to a marginal decrease in starch accumulation in non-stressed plants. However, SPS becomes a crucial factor in water-stressed plants because it is required for adaptive changes in tuber metabolism and whole plant allocation.     

Effects of 3 days of carbohydrate supplementation on muscle glycogen content and utilisation during a 1-h cycling performance

This study compared the effects of supplementing the normal diets of six trained cyclists [maximal oxygen uptake O_2max) 4.5 (0.36)l · min⁻¹; values are mean (SD)] with additional carbohydrate (CHO) on muscle glycogen utilisation during a 1-h cycle time-trial (TT). Using a randomised crossover design, subjects consumed either their normal diet (NORM) for 3 days, which consisted of 426 (137) g · day⁻¹ CHO [5.9 (1.4) g · kg⁻¹ body mass (BM)], or additional CHO (SUPP) to increase their intake to 661 (76) g · day⁻¹ [9.3 (0.7) g · kg⁻¹ BM]. The SUPP diet elevated muscle glycogen content from 459 (83) to 565 (62) mmol · kg⁻¹ dry weight (d.w.) (P < 0.05). However, despite the increased pre-exercise muscle glycogen stores, there was no difference in the distance cycled during the TT [40.41 (1.44) vs 40.18 (1.76) km for NORM and SUPP, respectively]. With NORM, muscle glycogen declined from 459 (83) to 175 (64) mmol · kg⁻¹ d.w., whereas with SUPP the corresponding values were 565 (62) and 292 (113) mmol · kg⁻¹ d.w. Accordingly, both muscle glycogen utilisation [277 (64) vs 273 (114) mmol · kg⁻¹ d.w.] and total CHO oxidation [169 (20) vs 165 (30) g · h⁻¹ for NORM and SUPP, respectively] were similar. Neither were there any differences in plasma glucose or lactate concentrations during the two experimental trials. Plasma glucose concentration averaged 5.5 (0.5) and 5.6 (0.6) mmol · l⁻¹, while plasma lactate concentration averaged 4.4 (1.9) and 4.4 (2.3) mmol · l⁻¹ for NORM and SUPP, respectively. The results of this study show that when well-trained subjects increase the CHO content of their diet for 3 days from 6 to 9 g · kg⁻¹ BM there is only a modest increase in muscle glycogen content. Since supplementary CHO did not improve TT performance, we conclude that additional CHO provides no benefit to performance for athletes who compete in intense, continuous events lasting 1 h. Furthermore, the substantial muscle CHO reserves observed at the termination of exercise indicate that whole-muscle glycogen depletion does not determine fatigue at this exercise intensity and duration. Accepted: 25 November 1996     

Glucose-induced inactivation of isocitrate lyase in Aspergillus nidulans

     The existence of a second mechanism of catabolite control of isocitrate lyase of Aspergillus nidulans, in addition to the carbon catabolite repression phenomenon recently reported was analysed. Isocitrate lyase was rapidly and specifically inactivated by glucose. The inactivation was irreversible at all stages in the presence of cycloheximide, showing that reactivation depends on de novo protein synthesis. In addition, analysis of glucose-induced inactivation of isocitrate lyase in a creA ^d -30 strain showed that the creA gene is not involved in this process. Received: 13 May 1994 / Accepted 12 August 1994     

Blood flow in the carotid artery during breath-holding in relation to diving bradycardia

The present study investigated the mechanism of diving bradycardia. A group of 14 healthy untrained male subjects were examined during breath-holding either out of the water (30–33°C), in head-out immersion, or in whole-body submersion (27–29°C) in a diving pool. Blood velocity, blood volume flow in the carotid artery, diastolic blood pressure and electrocardiogram were measured and recorded during the experiments. The peak blood velocity increased by 13.6% (P < 0.01) and R-wave amplitude increased by 57.1% (P < 0.005) when the subjects entered water from air. End-diastolic blood velocity in the carotid artery increased significantly during breath-holding, e.g. increased from 0.20 (SD 0.02) m · s⁻¹ at rest to 0.33 (SD 0.04) m · s⁻¹ (P < 0.001) at 50.0 s in breath-hold submersion to a 2.0-m depth. Blood volume flow in the carotid artery increased by 26.6% (P < 0.05) at 30 s and 36.6% (P < 0.001) at 40 s in breath-hold submersion to a 2.0-m depth. Diastolic blood pressure increased by 15.4% (P < 0.01) at 60 s during breath-holding in head-out immersion. Blood volume flow, and diastolic blood pressure increased significantly more and faster during breath-holding in submersion than out of the water. There was a good negative correlation with the heart rate: the root mean square correlation coefficient r was 0.73 (P < 0.001). It was concluded that an increased accumulation of blood in the aorta and arteries at end-diastole and decreased venous return, caused by an increase in systemic peripheral resistance during breath-holding, underlies diving bradycardia. Accepted: 22 November 1996     

Stem respiration of ponderosa pines grown in contrasting climates: implications for global climate change

We examined the effects of climate and allocation patterns on stem respiration in ponderosa pine (Pinus ponderosa) growing on identical substrate in the cool, moist Sierra Nevada mountains and the warm, dry, Great Basin Desert. These environments are representative of current climatic conditions and those predicted to accompany a doubling of atmospheric CO₂, respectively, throughout the range of many western north American conifers. A previous study found that trees growing in the desert allocate proportionally more biomass to sapwood and less to leaf area than montane trees. We tested the hypothesis that respiration rates of sapwood are lower in desert trees than in montane trees due to reduced stem maintenance respiration (physiological acclimation) or reduced construction cost of stem tissue (structural acclimation). Maintenance respiration per unit sapwood volume at 15°C did not differ between populations (desert: 6.39 ± 1.14 SE μmol m⁻³ s⁻¹, montane: 6.54 ± 1.13 SE μmol m⁻³ s⁻¹, P = 0.71) and declined with increasing stem diameter (P = 0.001). The temperature coefficient of respiration (Q ₁₀) varied seasonally within both environments (P = 0.05). Construction cost of stem sapwood was the same in both environments (desert: 1.46 ± 0.009 SE g glucose g⁻¹ sapwood, montane: 1.48 ± 0.009 SE glucose g⁻¹ sapwood, P = 0.14). Annual construction respiration calculated from construction cost, percent carbon and relative growth rate was greater in montane populations due to higher growth rates. These data provide no evidence of respiratory acclimation by desert trees. Estimated yearly stem maintenance respiration was greater in large desert trees than in large montane trees because of higher temperatures in the desert and because of increased allocation of biomass to sapwood. By analogy, these data suggest that under predicted increases in temperature and aridity, potential increases in aboveground carbon gain due to enhanced photosynthetic rates may be partially offset by increases in maintenance respiration in large trees growing in CO₂-enriched atmospheres. Received: 4 November 1996 / Accepted: 23 January 1997     

Energetics during nursing and early postweaning fasting in hooded seal (Cystophora cristata ) pups from the Gulf of St Lawrence, Canada

In this study we measured growth and milk intake and calculated energy intake and its allocation into metabolism and stored tissue for hooded seal (Cystophora cristata) pups. In addition, we measured mass loss, change in body composition and metabolic rate during the first days of the postweaning fast. The mean body mass of the hooded seal pups (n = 5) at the start of the experiments, when they were new-born, was 24.3 ± 1.3 kg (SD). They gained an average of 5.9 ± 1.1. kg · day⁻¹ of which 19% was water, 76% fat and 5% protein. This corresponds to an average daily energy deposition of 179.8 ± 16.0 MJ. The pups were weaned at an average body mass of 42.5 ± 1.0 kg 3.1 days after the experiment was initiated. During the first days of the postweaning fast the pups lost an average of 1.3 ± 0.5␣kg of body mass daily, of which 56% was water, 16% fat and 28% protein. During the nursing period the average daily water influx for the pups was 124.6 ± 25.8 ml · kg⁻¹. The average CO₂ production during this period was 1.10 ± 0.20 ml · g⁻¹ · h⁻¹, which corresponds to a field metabolic rate of 714 ± 130 kJ ·  kg⁻¹ · day⁻¹, or 5.8 ± 1.1 times the predicted basal metabolic rate according to Kleiber (1975). During the postweaning fast the average daily water influx was reduced to 16.1 ± 6.6 ml · kg⁻¹. The average CO₂ production in␣this period was 0.58 ± 0.17 ml · g⁻¹ · h⁻¹ which corresponds to a field metabolic rate of 375 ± 108 kJ · kg⁻¹ · day⁻¹ or 3.2 ± 0.9 times the predicted basal metabolic rate. Average values for milk composition were 33.5% water, 58.6% fat and 6.2% protein. The pups drank an average of 10.4 ± 1.8␣kg of milk daily, which represents an energy intake of 248.9 ± 39.1 MJ · day⁻¹. The pups were able to store 73.2 ± 7.7% of this energy as body tissue. Accepted: 15 August 1996     

Cold induced vasodilatation and cardiovascular responses in humans during cold water immersion of various upper limb areas

To study the physiological responses induced by immersing in cold water various areas of the upper limb, 20 subjects immersed either the index finger (T1), hand (T2) or forearm and hand (T3) for 30 min in 5°C water followed by a 15-min recovery period. Skin temperature of the index finger, skin blood flow (Q_sk) measured by laser Doppler flowmetry, as well as heart rate (HR) and mean arterial blood pressure (ˉBP_a) were all monitored during the test. Cutaneous vascular conductance (CVC) was calculated as Q_sk / ˉBP_a. Cold induced vasodilatation (CIVD) indices were calculated from index finger skin temperature and CVC time courses. The results showed that no differences in temperature, CVC or cardiovascular changes were observed between T2 and T3. During T1, CIVD appeared earlier compared to T2 and T3 [5.90 (SEM 0.32) min in T1 vs 7.95 (SEM 0.86) min in T2 and 9.26 (SEM 0.78) min in T3, P < 0.01]. The HR was unchanged in T1 whereas it increased significantly at the beginning of T2 and T3 [+13 (SEM 2) beats · min⁻¹ in T2 and +15 (SEM 3) beats · min⁻¹ in T3, P < 0.01] and then decreased at the end of the immersion [−12 (SEM 3) beats · min⁻¹ in T2, and −15 (SEM 3) beats · min⁻¹ in T3, P < 0.01]. Moreover, ˉBP_aincreased at the beginning of T1 but was lower than in T2 and T3 [+9.3 (SEM 2.5) mmHg in T1, P < 0.05;  +20.6 (SEM 2.6) mmHg and 26.5 (SEM 2.8) mmHg in T2 and T3, respectively, P < 0.01]. The rewarming during recovery was faster and higher in T1 compared to T2 and T3. These results showed that general and local physiological responses observed during an upper limb cold water test differed according to the area immersed. Index finger cooling led to earlier and faster CIVD without significant cardiovascular changes, whereas hand or forearm immersion led to a delayed and slower CIVD with a bradycardia at the end of the test. Accepted: 26 November 1996     

The relationship between the rate of starch synthesis, the adenosine 5′-diphosphoglucose concentration and the amylose content of starch in developing pea embryos

Mutations that reduced the rate of starch synthesis in pea (Pisum sativum L.) embryos through effects on enzymes on the pathway from sucrose to adenosine 5′-diphosphoglucose (ADPglucose) also led to a reduction in the amylose content of the starch of developing embryos. Evidence is presented that this relationship between rate of synthesis and the composition of starch is due to the fact that amylopectin-synthesising isoforms of starch synthase have higher affinities for ADPglucose than the amylose-synthesising isoform. First, developing mutant embryos (rb, rug3 and rug4 mutants) displayed both reduced amylose contents in their starches and reduced ADPglucose contents relative to wild-type embryos. Second, incubation of detached, wild-type embryos for 6 h at high and low glucose concentrations resulted in differences in both ADPglucose content and the relative rates of amylose and amylopectin synthesis. At 0.25 M glucose both ADPglucose content and the proportion of synthesised starch that was amylose were about twice as great as at 25 μM glucose. Third, S _0.5 values for soluble (amylopectin-synthesising) starch synthases in developing embryos were several-fold lower than that for granule-bound (amylose synthesising) starch synthase. Estimates of the expected amylose contents of the starch of the mutant embryos, based on the reduction in their ADPglucose contents and on the S _0.5 values of the starch synthases, were very similar to the measured amylose contents. The implications of these results for the determination of starch composition are discussed. Received: 6 February 1999 / Accepted: 22 May 1999     

Control of carbon partitioning and photosynthesis by the triose phosphate/phosphate translocator in transgenic tobacco plants (Nicotiana tabacum). II. Assessment of control coefficients of the triose phosphate/phosphate translocator

 Transgenic tobacco (Nicotiana tabacum L.) plants with decreased and increased transport capacities of the chloroplast triose phosphate/phosphate translocator (TPT) were used to study the control the TPT exerts on the flux of starch and sucrose biosynthesis, as well as CO₂ assimilation, respiration and photosynthetic electron transport. For this purpose, tobacco lines with an antisense repression of the endogenous TPT (αTPT) and tobacco lines overexpressing a TPT gene from Flaveria trinervia (FtTPT) were used. In ambient CO₂, there was no or little effect of altered TPT transport activities on either rates of photosynthetic electron transport and/or CO₂ assimilation. However, in elevated CO₂ (1500 μl · l⁻¹) and low O₂ (2%) the TPT exerted strong control on the rate of CO₂ assimilation (control coefficient for the wild type; C^JA _TPT=0.30) in saturating light. Similarly, the incorporation of ¹⁴C into starch in high CO₂ was increased in tobacco plants with decreased TPT activity, but was reduced in plants overexpressing the TPT from F. trinervia. Thus, the TPT exerted negative control on the rate of starch biosynthesis with a C^JStarch _TPT=−0.19 in the wild type estimated from a hyperbolic curve fitted to the data points. This was less than the positive control strength on the rate of sucrose biosynthesis (C^JSuc _TPT=0.35 in the wild type). Theoretically, the positive control exerted on sucrose biosynthesis should be numerically identical to the negative control on starch biosynthesis unless additional metabolic pathways are affected. The rate of dark respiration showed some correlation with the TPT activity in that it increased in FtTPT overexpressors, but decreased in αTPT plants with an apparent control coefficient of C^JRes _TPT=0.24. If the control on sucrose biosynthesis is referred to as “gain of carbon” (positive control) and the control on starch biosynthesis as well as dark respiration as a “loss of carbon” (negative control) for sucrose biosynthesis and subsequent export, the sum of the control coefficients on dark respiration and starch biosynthesis would be numerically similar to the control coefficient on the rate of sucrose biosynthesis. There was also some control on the rate of photosynthetic electron transport, but only at high light and in elevated CO₂ combined with low O₂. The control coefficient for the rate of photosynthetic electron transport was C^JETR _TPT=0.16 in the wild type. Control coefficients were also calculated for plants with elevated and lowered TPT activity. Furthermore, the extent to which starch degradation/glucose utilisation compensates for the lack of triose phosphate export was assessed. The TPT also exerted control on metabolite contents in air. Received: 26 March 1999 / Accepted: 21 August 1999     

Short latency vestibular evoked potentials in the Japanese quail (Coturnix coturnix japonica)

Short-latency vestibular-evoked potentials to pulsed linear acceleration were characterized in the quail. Responses occurred within 8 ms following the onset of stimuli and were composed of a series of positive and negative peaks. The latencies and amplitudes of the first four peaks were quantitatively characterized. Mean latencies at 1.0 g ms⁻¹ ranged from 1265 ± 208 μs (P1, N = 18) to 4802 ± 441 μs (N4, N = 13). Amplitudes ranged from 3.72 ± 1.51 μV (P1/N1, N = 18) to 1.49 ± 0.77 μV (P3/N3, N = 16). Latency-intensity (LI) slopes ranged from −38.7 ± 7.3 μs dB⁻¹ (P1, N = 18) to −71.6 ± 21.9 μs dB⁻¹ (N3, N = 15) and amplitude-intensity (AI) slopes ranged from 0.20 ± 0.08 μV dB⁻¹ (P1/N1, N = 18) to 0.07 ± 0.04 μV dB⁻¹ (P3/N3, N = 11). The mean response threshold across all animals was −21.83 ± 3.34 dB re: 1.0 g ms⁻¹ (N = 18). Responses remained after cochlear extirpation showing that they could not depend critically on cochlear activity. Responses were eliminated by destruction of the vestibular end organs, thus showing that responses depended critically and specifically on the vestibular system. The results demonstrate that the responses are vestibular and the findings provide a scientific basis for using vestibular responses to evaluate vestibular function through ontogeny and senescence in the quail. Accepted: 18 January 1997