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.     

Generation of rhythmic and arrhythmic behaviour of Crassulacean acid metabolism in Kalanchoë daigremontiana under continuous light by varying the irradiance or temperature: Measurements in vivo and model simulations

Leaves of Kalanchoë daigremontiana Hamet et Perr. at a photon flux density (PFD) above 220 mol·m^–2s^–1 (400–700 nm) or at leaf temperatures above 27.0 °C showed a rapid loss of rhythmicity, and a more or less pronounced damping-out of the endogenous circadian rhythm of CO₂ exchange under continuous illumination. This rhythm was reinitiated after reduction of the PFD by 90–120 mol·m^–2·s^–1 or reduction of leaf temperature by 3.5–11.0 °C under otherwise unchanged external conditions. The reduction in the magnitude of the external control parameter of the Crassulacean acid metabolism (CAM) rhythm (i.e. PFD or leaf temperature) set the phase of the new rhythm. The maxima of CO₂ uptake occurred about 5, 28, 51, 75 h after the reduction. Simulations with a CAM model under comparable conditions showed a similar behaviour. The influence of temperature on the endogenous CAM rhythm observed in K. daigremontiana in vivo could be simulated by incorporating into the model temperature-dependent switch modes for passive efflux of malate from the vacuole to the cytoplasm. Thus, the model indicates that tonoplast function plays an important role in regulation of the endogenous CAM rhythm in K. daigremontiana.Abbreviations CAM Crassulacean acid metabolism - PAR photosynthetically active radiation - PFD photon flux density This work was supported by a grant to F.B. and U.L. from Teilprojekt B5 in the Sonderforschungsbereich 199 of the Deutsche Forschungsgemeinschaft (Bonn, Germany) and by a grant to T. E. E. G. from the Sudienstiftung des deutschen Volkes (Bonn, Germany). Erika Ball is thanked for processing of time-course data for the analysis of Fourier spectra.     

Anaerobic metabolism in the leech (Hirudo medicinalis L.): direct and indirect calorimetry during severe hypoxia

Anaerobic metabolism in the limnic annelid Hirudo medicinalis L. was investigated by direct and indirect calorimetry. During long-term severe hypoxia, the rate of heat dissipation was reduced up to 13% of the aerobic rate. At the same time, the rate of ATP turnover was reduced to about 30% of the aerobic rate, indicating that metabolic depression is an important mechanism to ensure survival of the leech during environmental anaerobiosis. Heat dissipation during hypoxia was monitored under two experimental conditions, favouring either concomitant hypocapnia (continuous N₂ bubbling) or hypercapnia (self-induced hypoxia). The reduction in heat dissipation during hypocapnic hypoxia was less pronounced than during hypercapnic hypoxia, indicating that the different experimental conditions may influence anaerobic metabolism and the extent of metabolic depression. Biochemical analysis of known anaerobic substrates and endproducts provided the basis for indirect calorimetry during self-induced hypoxia. From changes in metabolites, the expected heat dissipation was calculated for initial (0–8 h) and long-term severe hypoxia (8–72 h). During the initial period, the calculated heat dissipation fully accounted for direct calorimetric determination. During long-term hypoxia, only 71% of the measured heat production could be explained from biochemical analysis of metabolites. Therefore, an additional unknown endproduct cannot be excluded, especially when anaerobic ammonia production and analysis of the carbohydrate balance are considered.Abbreviations APW artificial pond water - HPLC high-performance liquid chromatography - fw fresh weight - HP heat production - HD heat dissipation - MR metabolic rate     

Approaching the multifaceted nature of energy metabolism: inactivation of the cytosolic creatine kinases via homologous recombination in mouse embryonic stem cells

To study the physiological role of the creatine kinase/phosphocreatine (CK/PCr) system in cells and tissues with a high and fluctuating energy demand we have concentrated on the site-directed inactivation of the B- and M-CK genes encoding the cytosolic CK protein subunits. In our approach we used homologous recombination in mouse embryonic stem (ES) cells from strain 129/Sv. Using targeting constructs based on strain 129/Sv isogenic DNA we managed to ablate the essential exons of the B-CK and M-CK genes at reasonably high frequencies. ES clones with fully disrupted B-CK and two types of M-CK gene mutations, a null (M-CK^–) and leaky (M-CK¹) mutation, were used to generate chimaeric mutant mice via injection in strain C57BL/6 derived blastocysts. Chimaeras with the B-CK null mutation have no overt abnormalities but failed to transmit the mutation to their offspring. For the M-CK^– and M-CK¹ mutations successful transmission was achieved and heterozygous and homozygous mutant mice were bred. Animals deficient in MM-CK are phenotypically normal but lack muscular burst activity. Fluxes through the CK reaction in skeletal muscle are highly impaired and fast fibres show adaptation in cellular architecture and storage of glycogen. Mice homozygous for the leaky M-CK allele, which have 3-fold reduced MM-CK activity, show normal fast fibres but CK fluxes and burst activity are still not restored to wildtype levels.     

Phosphorylation of glucose by a guanosine-5′-triphosphate (GTP)-dependent glucokinase in Fibrobacter succinogenes subsp. succinogenes S85

Cell extracts of Fibrobacter succinogenes subsp. succinogenes S85 phosphorylated glucose with a GTP-dependent glucokinase. The enzyme showed little activity with ATP (12% of that with GTP). Of other phosphate donors tested, only dGTP and ITP gave high glucokinase activities. Dialyzed extracts required Mg⁺² and K⁺ for maximal activity. In potassium phosphate buffer, glucokinase showed maximum activity at pH 7.5 with glucose-6-phosphate dehydrogenase as the coupling enzyme. In this assay, glucokinase was active with glucose (100%), 2-deoxy-d-glucose (40%), and mannose (20%). Partially purified glucokinase had a molecular weight of 82,000 and a pl of 4.82. Double-reciprocal plots of substrate concentration versus velocity were linear and the enzyme had apparent K_m values of 55 M for glucose and 72 M for GTP. Dialyzed cell extracts of Fibrobacter intestinalis C1A also contained a GTP-dependent glucokinase that showed little activity with ATP. Potassium also stimulated the activity of this enzyme. These results suggest that this unusual glucokinase may be characteristic of the genus Fibrobacter.Abbreviations CHES cyclohexylaminoethanesulfonic acid - GK glucokinase - PEP phosphoenolpyruvate Published with the approval of the Director of the North Dakota Agricultural Experiment Station as journal article no. 2186     

Physiology, thermoregulation and bipedalism

It has long been recognized that the bipedal posture reduces the surface area of the body exposed to the sun. In recent years, a theory has been developed by Wheeler that bipedalism evolved in the ancestor of the Hominidae in order to help relieve thermal stress on the animals in open equatorial environments. Bipedalism was said to afford a distinct adaptive advantage over quadrupedalism by permitting hominids to remain active in the open throughout the day. The heat load of the hypothetical hominid comprises the external environment as modelled by Wheeler and the animal's internal environment (i.e., the internal heat generated by its metabolic and locomotor activities, and its evaporative and respirative cooling capacities). When these factors are integrated in the calculation of the animal's thermal budget, the putative advantage of the bipedal over the quadrupedal posture is considerably reduced. The simulations conducted in this study suggest that the increased time afforded to early hominids in the open by bipedalism was relatively short and, therefore, of little or no adaptive significance. These results suggest that thermoregulatory considerations cannot be implicated as a first cause in the evolution of bipedalism in the hominid ancestor.     

A protein involved in co-ordinated regulation of inorganic carbon and glucose metabolism in the facultative photoautotrophic cyanobacteriumSynechocystis PCC6803

The involvement of a gene ofSynechocystis PCC6803,icfG, in the co-ordinated regulation of inorganic carbon and glucose metabolism, was established. TheicfG gene codes for a 72 kDa protein, which shows no homology with those registered in data libraries. Expression oficfG required glucose, the actual inducer probably being glucose-6-phosphate, and was independent of light and of the external inorganic carbon concentration. Mutants carrying an inactivated copy oficfG were constructed. Their growth characteristics were identical to those of the wild type under all regimes except in limiting inorganic carbon with glucose being present either before or after the transfer to the limiting conditions. These conditions completely prevented growth, both in the light and in the dark. The inhibition could be relieved by several intermediates of the tricarboxylic acid cycle. Assays of various enzymic activities related to inorganic carbon uptake and to its assimilationvia either the Calvin cycle or phosphoenolpyruvate carboxylase did not reveal the level of action of IcfG. Possible models include a blockage of the assimilation of both carbon sources in the absence of IcfG, or the inhibition of Ci incorporation route(s) essential under limiting inorganic carbon conditions, even when glucose is present, and even in the dark.     

The biosynthesis of rosmarinic acid in suspension cultures of Coleus blumei

Suspension cultures of Coleus blumei accumulate very high amounts of rosmarinic acid, an ester of caffeic acid and 3,4-dihydroxyphenyllactate, in medium with elevated sucrose concentrations. Since the synthesis of this high level of rosmarinic acid occurs in only five days of the culture period, the activities of the enzymes involved in the biosynthesis are very high. Therefore all the enzymes necessary for the formation of rosmarinic acid from the precursors phenylalanine and tyrosine could be isolated from cell cultures of Coleus blumei: phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, hydroxycinnamoyl:CoA ligase, tyrosine aminotransferase, hydroxyphenylpyruvate reductase, rosmarinic acid synthase and two microsomal 3- and 3-hydroxylases. The main characteristics of these enzymes of the proposed biosynthetic pathway of rosmarinic acid will be described.Abbreviations DHPL 3,4-dihydroxyphenyllactate - DHPP 3,4-dihydroxyphenylpyruvate - pHPL 4-hydroxyphenyllactate - pHPP 4-hydroxyphenylpyruvate - RA rosmarinic acid     

Uptake,accumulation and metabolism of auxins in tobacco leaf protoplasts

Uptake and metabolism of exogenous naphthalene-1-acetic acid (NAA) and indole-3-acetic acid (IAA) have been studied in tobacco (Nicotiana tabacum L. cv. Xanthi) mesophyll protoplasts. Both auxins entered protoplasts by diffusion under the action of the transmembrane pH gradient without any detectable participation of an influx carrier. Molecules were accumulated by an anion-trapping mechanism and most of them were metabolized within hours, essentially as glucose-ester and amino-acid conjugates. Protoplasts were equipped with a functional auxin-efflux carrier as evidenced by the inhibitory effect of naphthylphtalamic acid on IAA efflux. Basically, similar mechanisms of NAA and IAA uptake occurred in protoplasts. However, the two auxins differed in their levels of accumulation, due to different membrane-transport characteristics, and the nature of the metabolites produced. This shows the need to estimate the accumulation and the metabolism of auxins when analyzing their effects in a given cell system. The internal auxin concentration could be modulated by changing the transmembrane pH gradient, giving an interesting perspective for discriminating between the effects of intra- and extracellular auxin on physiological processes.Abbreviations BA benzoic acid - C_i/C_e accumulation ratio of auxin - IAAasp N-[3-indolylacetyl]-dl-aspartic acid - NAA naphthalene-1-acetic acid - NAAasp N-[1-naphthylacetyl]-l-aspartic acid - NPA N-1-naphthylphthalamic acid The authors thank Dr. M. Caboche (I.N.R.A, Versailles, France) for his generous gifts of some amide derivatives of 1-NAA, Mr. P. Varennes and Dr. B. Das (I.C.S.N., C.N.R.S., Gif-sur-Yvette, France) for recording and interpreting the mass spectra of NAA glucose ester, and Prof. P. Manigault (Institut des Sciences Végétales, Gif-sur-Yvette) for microscopy measurements of protoplast dimensions. This work was supported by funds from the C.N.R.S, I.N.R.A, and E.E.C.