Sul Controllo Della Glicolisi Nel Lievito

Abstract

On the control of carbohydrate utilization in yeast. — The results of a previous investigation showed that in higher plants the stimulating action of 2,4 dinitrophenol (DNP) on oxygen uptake and glycolysis is accompained by a fall of the level of reducing sugars, due to an increase of their respiratory utilization, and thus — according to every evidence — of the rate of hexose phosphate synthesis.

In the present work, the occurrence of a similar phenomenon in yeast (where the inhibiting effect of DNP on glucose uptake is not so much marked as in higher plant tissue) was investigated.

Here again DNP, at a 10^-4M concentration, induced a rapid decrease of the disaccaride trehalose and of glycogen, such as to account for the increased rate of respiration and of fermentation. The ratio between the contributions to CO₂ of Carbons 1 and respectively 6 of glucose was not significantly changed by DNP, which suggests that at least part of the DNP induced increase of glycolysis was mediated by the Embden Meyerhof pathway, and thus that a larger amount of fructose diphosphate was formed in the presence of the uncoupler.

In other experiments the effects of DNP on the dissimilation of C¹⁴ labeled glucose, glycerol and pyruvate to CO₂ and ethanol, and on the incorporation of the radioactive isotope into various fractions, 15 minutes after feeding the labeled substrates, was investigated. It was found that:

1) Glucose and glycerol uptake is not markedly inhibited by DNP at the concentration employed (^10–4M).

2) In the absence of DNP, a considerable portion of the radioactivity fed as glucose or glycerol and taken up by the yeast cells is recovered in the glycogen and trehalose fractions. (35% of the glucose, and 22% of the glycerol taken up). This is also observed for carbons 2 and 3, but not for carbon 1 of pyruvate. This indicates a reversibility of the glycolitic processes comprehended in the region between phospho-enol pyruvate andpolysac-carides; while the pyruvate kinase reaction appears to represent a sharp barrier at the « lower » end of glycolysis.

3) DNP almost completely inhibited the incorporation of C¹⁴ from glucose and glycerol into glycogen and trehalose, although it increased the rate of its dissimilation to CO₂ and ethanol. The total amount of glucose and glycerol transformed in the various metabolites (and thus — according to every evidence — phosphorylated) was somewhat lowered and proteins synthesis severely depressed. These effects are interpreted as due to the uncoupling action of DNP at the mitochondrial level, and to the consequent general decrease of the ATP and UTP levels required for protein and for polysaccharide synthesis.     

Reazioni Metaboliche All'auxina - VII Variazioni a Carico Di Metaboliti Della Glicolisi

Abstract

Metabolic responses to auxin. VII. Effects on glycolysis. — The detection of changes the of level of respiratory substrates could provide an indication on the site of action of auxin. The effects of indoleacetic acid on the contents in several glycolysis intermediates in isolated segments from the growing part of the third internode of etiolated pea seadlings were thus investigated.

Hexose phosphates, triose phosphates, phospho-enol-pyruvate, pyruvate and ethanol were determined enzymatically in the neutralized perchloric extracts from groups of segments incubated for 3 hours in the presence or in the absence of 5 × 10 ^?5 M indoleacetic acid. No significant difference appeared between the results on the perchloric extracts and those obtained after fractionation with barium and alcohol, according to Umbreit et al.

Acetaldehyde was measured as by Klotz. Pyruvate was determined also as a phenyl-hydrazone, according to Cavallini and Frontali; the same method was used for the determination of α-ketoglutarare and of an unknown compound, showing a chromatographic behavior similar to that of an uronic acid.

Auxin induced a modest but significant increase of hexose-6-phosphates (16%) and of fructose di-phosphate (38%). For glucose-1-phosphate and triose phosphates — present at very low concentration — no significant changes could be detected, α-ketoglutarate level appeared unchanged. The compound behaving as an uronic acid increased, in the auxin treated segments, by more than 100%.

A very definite response to auxin appeared at the level of pyruvate. acetaldehyde and alcohol. Pyruvate quite reproducibly increased by 60%, and acetaldehyde production was more than doubled. In contrast, alcohol decreased by about 40%.

These results are discussed in relation to the previous data on the effects of auxin on the oxidative metabolism, and, particularly, to the findings of an increased rate of CO-sensitive respiration and of the rise of the ATP, TPNH and red. glutathione levels in the auxin treated pea internode segments.     

Metabolite labelling reveals hierarchies in Clostridium acetobutylicum that selectively channel carbons from sugar mixtures towards biofuel precursors

Clostridial fermentation of cellulose and hemicellulose relies on the cellular physiology controlling the metabolism of the cellulosic hexose sugar (glucose) with respect to the hemicellulosic pentose sugars (xylose and arabinose) and the hemicellulosic hexose sugars (galactose and mannose). Here, liquid chromatography–mass spectrometry and stable isotope tracers in Clostridium acetobutylicum were applied to investigate the metabolic hierarchy of glucose relative to the different hemicellulosic sugars towards two important biofuel precursors, acetyl‐coenzyme A and butyryl‐coenzyme A. The findings revealed constitutive metabolic hierarchies in C. acetobutylicum that facilitate (i) selective investment of hemicellulosic pentoses towards ribonucleotide biosynthesis without substantial investment into biofuel production and (ii) selective contribution of hemicellulosic hexoses through the glycolytic pathway towards biofuel precursors. Long‐term isotopic enrichment demonstrated incorporation of both pentose sugars into pentose‐phosphates and ribonucleotides in the presence of glucose. Kinetic labelling data, however, showed that xylose was not routed towards the biofuel precursors but there was minor contribution from arabinose. Glucose hierarchy over the hemicellulosic hexoses was substrate‐dependent. Kinetic labelling of hexose‐phosphates and triose‐phosphates indicated that mannose was assimilated but not galactose. Labelling of both biofuel precursors confirmed this metabolic preference. These results highlight important metabolic considerations in the accounting of clostridial mixed‐sugar utilization.     

Metabolism of C-labeled photosynthate and distribution of enzymes of glucose metabolism in soybean nodules

The metabolism of translocated photosynthate by soybean (Glycine max L. Merr.) nodules was investigated by ¹⁴CO₂-labeling studies and analysis of nodule enzymes. Plants were exposed to ¹⁴CO₂ for 30 minutes, followed by ¹²CO₂ for up to 5 hours. The largest amount of radioactivity in nodules was recovered in neutral sugars at all sampling times. The organic acid fraction of the cytosol was labeled rapidly. Although cyclitols and malonate were found in high concentrations in the nodules, they accumulated less than 10% of the radioactivity in the neutral and acidic fractions, respectively. Phosphate esters were found to contain very low levels of total label, which prohibited analysis of the radioactivity in individual compounds. The whole nodule-labeling patterns suggested the utilization of photosynthate for the generation of organic acids (principally malate) and amino acids (principally glutamate).

The radioactivity in bacteroids as a percentage of total nodule label increased slightly with time, while the percentage in the cytosol fraction declined. The labeling patterns for the cytosol were essentially the same as whole nodule-labeling patterns, and they suggest a degradation of carbohydrates for the production of organic acids and amino acids. When it was found that most of the radioactivity in bacteroids was in sugars, the enzymes of glucose metabolism were surveyed. Bacteroids from nodules formed by Rhizobium japonicum strain 110 or strain 138 lacked activity for phosphofructokinase and NADP-dependent 6-phosphogluconate dehydrogenase, key enzymes of glycolysis and the oxidative pentose-phosphate pathways. Enzymes of the glycolytic and pentose phosphate pathways were found in the cytosol fraction.

In three experiments, bacteroids contained about 10 to 30% of the total radioactivity in nodules 2 to 5 hours after pulse-labeling of plants, and 60 to 65% of the radioactivity in bacteroids was in the neutral sugar fraction at all sampling times. This strongly suggests some absorption and metabolism of sugars by bacteroids in spite of the lack of key enzymes. Bacteroids did possess enzymes for the formation of hexose phosphates from glucose or fructose. Radioactivity in α,α-trehalose in bacteroids increased until, after 5 hours, trehalose was a major labeled compound in bacteroids. Thus, trehalose synthesis may be a major fate of sugars entering bacteroids.

     

Measurement of Metabolites Associated with Nonaqueously Isolated Starch Granules from Immature Zea mays L. Endosperm

Starch granules with associated metabolites were isolated from immature Zea mays L. endosperm by a nonaqueous procedure using glycerol and 3-chloro-1,2-propanediol. The soluble extract of the granule preparation contained varying amounts of neutral sugars, inorganic phosphate, hexose and triose phosphates, organic acids, adenosine and uridine nucleotides, sugar nucleotides, and amino acids. Based on the metabolites present and on information about translocators in chloroplast membranes, which function in transferring metabolites from the chloroplast stroma into the cytoplasm, it is suggested that sucrose is degraded in the cytoplasm, via glycolysis, to triose phosphates which cross the amyloplast membrane by means of a phosphate translocator. It is further postulated that hexose phosphates and sugars are produced from the triose phosphates in the amyloplast stroma by gluconeogenesis with starch being formed from glucose 1-phosphate via pyrophosphorylase and starch synthase enzymes. The glucose 1-phosphate to inorganic phosphate ratio in the granule preparation was such that starch synthesis by phosphorylase is highly unlikely in maize endosperm.     

Sul trasporto dei glucidi nelle piante superiori: I. — Aspetti biochimici

Abstract

CARBOHYDRATE TRANSLOCATION IN HIGHER PLANTS. I. - BIOCHEMICAL ASPECTS. — The concentration of soluble sugars and of hexose phosphates and the activity of several enzymes involved in hexose activation and polysaccaride synthesis have been investigated, separately, in the phloematic tissue and in the medullar parenchyma of Cucurbita Pepo internodes.

In the phloematic tissue (including sieve tubes, companion cells and phloematic parenchyma) the concentration of free hexoses appeared of about 50% lower, and that of glucose-6-P and of sucrose of about 100% higher then in the medullar parenchyma. Consistent amounts of raffinose were found only in the phloematic tissue. Paper chromatograms of the sieve tube exudate showed the presence of raffinose and sucrose in a ratio close to unity, and no appreciable amounts of free hexoses.

Determination of enzyme activity on preparations obtained from homogenates from the two types of tissue by repeated ammonium sulfate precipitation showed in the phloematic tissue a high activity of the enzymes hexokinase, UDP-kinase, UDPG-pyrophosphorylase and inorganic pyrophosphatase. The presence in the same tissue of galactosekinase, UDP-Gal-pyrophosphorylase and UDPG-epimerase was also ascertained.

On a protein basis, the activity of UDPG-pyrophosphorylase, inorganic pyrophosphatase and hexokinase appared about 3 times higher in the phloematic tissue than in the parenchyma; while this difference between the two tissues was not so marked for phosphofructokinase, and very small for other enzymes such as ATP-ase and phosphomono-esterase.

These results suggest that the very high activity, in the phloem cells neighbouring the sieve tubes, of the enzyme system catalyzing oligopolysaccaride synthesis could be an important component of the mechanism involved in the accumulation of oligopolysaccarides in the sieve tubes, and thus in sugar translocation. A scheme is proposed according to which the ATP and UTP energy would be utilized by the phloem cells to reach and to maintein a concentration of soluble sugars consistently higher than that prevailing in the contiguous tissues.     

Starch Biosynthesis in Developing Wheat Grain : Evidence against the Direct Involvement of Triose Phosphates in the Metabolic Pathway

We have used ¹³C-labeled sugars and nuclear magnetic resonance (NMR) spectrometry to study the metabolic pathway of starch biosynthesis in developing wheat grain (Triticum aestivum cv Mardler). Our aim was to examine the extent of redistribution of ¹³C between carbons atoms 1 and 6 of [1-¹³C] or [6-¹³C]glucose (or fructose) incorporated into starch, and hence provide evidence for or against the involvement of triose phosphates in the metabolic pathway. Starch synthesis in the endosperm tissue was studied in two experimental systems. First, the ¹³C sugars were supplied to isolated endosperm tissue incubated in vitro, and second the ¹³C sugars were supplied in vivo to the intact plant. The ¹³C starch produced by the endosperm tissue of the grain was isolated and enzymically degraded to glucose using amyloglucosidase, and the distribution of ¹³C in all glucosyl carbons was quantified by ¹³C-NMR spectrometry. In all of the experiments, irrespective of the incubation time or incubation conditions, there was a similar pattern of partial (between 15 and 20%) redistribution of label between carbons 1 and 6 of glucose recovered from starch. There was no detectable increase over background ¹³C incidence in carbons 2 to 5. Within each experiment, the same pattern of partial redistribution of label was found in the glucosyl and fructosyl moieties of sucrose extracted from the tissue. Since it is unlikely that sucrose is present in the amyloplast, we suggest that the observed redistribution of label occurred in the cytosolic compartment of the endosperm cells and that both sucrose and starch are synthesized from a common pool of intermediates, such as hexose phosphate. We suggest that redistribution of label occurs via a cytosolic pathway cycle involving conversion of hexose phosphate to triose phosphate, interconversion of triose phosphate by triose phosphate isomerase, and resynthesis of hexose phosphate in the cytosol. A further round of triose phosphate interconversion in the amyloplast could not be detected. These data seriously weaken the argument for the selective uptake of triose phosphates by the amyloplast as part of the pathway of starch biosynthesis from sucrose in plant storage tissues. Instead, we suggest that a hexose phosphate such as glucose 1-phosphate, glucose 6-phosphate, or fructose 6-phosphate is the most likely candidate for entry into the amyloplast. A pathway of starch biosynthesis is presented, which is consistent with our data and with the current information on the intracellular distribution of enzymes in plant storage tissues.     

Kinetin and carbohydrate metabolism in chinese cabbage

The effects of kinetin on starch and sugar levels and on ¹⁴CO₂ and ³²P-orthophosphate labeling patterns of floated Chinese cabbage (Brassica pekinensis) leaf discs were investigated. Kinetin caused gross starch degradation. Neutral sugars were depressed by 30 to 40% in leaf tissue treated with kinetin for 24 hours. ¹⁴CO₂ labeling of leaf discs pretreated with kinetin for 24 hours showed increased radioactivity in chloroform-soluble material and most sugar phosphates, and a 35 to 40% decrease in radioactivity in the neutral sugars, glucose, sucrose, and fructose. Incorporation into ATP was increased by 40% by kinetin. ³²P-Orthophosphate uptake was inhibited 30% by kinetin. When corrected for uptake, kinetin stimulated incorporation into chloroform-soluble material but had little effect on other cell fractions. These results indicate that kinetin mobilizes starch reserves and increases the flow of sugars required for the synthesis of lipids and structural materials in floated discs.     

Alcuni Aspetti Della Variabilità Del Legno Secondario Di Un Esemplare Di Prunus Persica Stokes

Abstract

Some aspects of the variability in wood structure of a specimen of PRUNUS PERSICA Stokes. — The secondary xylem of a young stem of Prunus persica Stokes has been investigated under three main headings: 1) vessel and fibre length; 2) intrusive fibre growth; 3) relative amount of libriform fibres, fibretracheids and tracheids.

Vessel members and fibres have been reckoned from single rings and from the whole wood body.

The relation between the above mentioned dimensions and the position within the wood body and the width of the growth layers has been calculated.

No remarkable variation has been found along the axis of the stem; on the contrary there is a decrease in both fibres and vessels length along the radius of the stem from inside outwards. Such behaviour having never been recorded before, the possible causes are suggested.

A positive correlation has been found between the width of the growth layers and 1) vessel members and fibres length, 2) fibre intrusive growth, 3) percentage of libriform fibres. In addition a relative correlation between percentage of libriform fibres and age of the cambium has been put in evidence.     

H<Subscript>2</Subscript> synthesis from pentoses and biomass in <Emphasis Type="Italic">Thermotoga</Emphasis> spp.

We have investigated H₂ production on glucose, xylose, arabinose, and glycerol in Thermotoga maritima and T. neapolitana. Both species metabolised all sugars with hydrogen yields of 2.7–3.8 mol mol⁻¹ sugar. Both pentoses were at least comparable to glucose with respect to their qualities as substrates for hydrogen production, while glycerol was not metabolised by either species. Glycerol was also not metabolised by T. elfii. We also demonstrated that T. neapolitana can use wet oxidised wheat straws, in which most sugars are stored in glycoside polymers, for growth and efficient hydrogen production, while glucose, xylose and arabinose are consumed in parallel.     

Mikrobielle Verwertung von Methanol

Hexose phosphate synthetase activity was found in cell-free extracts of methanol-grown Candida boidinii. Incubation of this crude extract with ¹⁴C-formaldehyde and D-ribose-5-phosphate leads to incorporation of radioactivity into fructose-and glucose phosphates. Cells grown on glucose lack the hexose phosphate synthetase activity. No hydroxypyruvate reductase activity, the key enzyme of the serine pathway was found. These results indicate that during growth of C. boidinii on methanol, cell constituents are made by a sugar phosphate pathway similar in concept, if not in absolute molecular detail, to the ribose phosphate cycle in C₁-metabolizing bacteria.     

Biosynthesis of cell-wall polysaccharides in cultured carrot cells

Biosynthesis of the cell wall in carrot cells (Daucus carota L.) cultured in a synthetic liquid medium was studied by measuring the incorporation of radioactive glucose and myo-inositol (MI). When the cells were fed with [¹⁴C]glucose in the presence of 0.01% MI, the label soon appeared in the neutral sugars in the cell wall but little radioactivity was found in the uronic-acid residues even after a prolonged incubation. On the other hand, radioactivity derived from [³H]MI was found to be distributed among uronic acids and pentoses but not in the hexose residues in the wall. The data indicate that MI is an important intermediate for the synthesis of acidic sugars in the wall of cultured carrot cells.Abbreviation MI myo-inositol     

Uptake and metabolism of carbohydrates by epidermal tissue

Isolated epidermis of Commelina communis L. and Tulipa gesneriana L. assimilated ¹⁴CO₂ into malic acid and its metabolites but not into sugars or their phosphates; epidermis could not reduce CO₂ by photosynthesis and therefore must be heterotrophic (Raschke and Dittrich, 1977). If, however, isolated epidermis of Commelina communis was placed on prelabelled mesophyll (obtained by an exposure to ¹⁴CO₂ for 10 min), radioactive sugars appeared in the epidermis, most likely by transfer from the mesophyll. Of the radioactivity in the epidermis, 60% was in sucrose, glucose, fructose, 3-phosphoglyceric acid and sugar phosphates. During a 10-min exposure to ¹⁴CO₂, epidermis in situ incorporated 16 times more radioactivity than isolated epidermal strips. Isolated epidermis of Commelina communis and Tulipa gesneriana took up ¹⁴C-labelled glucose-1-phosphate (without dephosphorylation), glucose, sucrose and maltose. These substances were transformed into other sugars and, simultaneously, into malic acid. Carbons-1 through-3 of malic acid in guard cells can thus be derived from sugars. Radioactivity appeared also in the hydrolysate of the ethanol-insoluble residue and in compounds of the tricarboxylic-acid cycle, including their transamination products. The hydrolysate contained glucose as the only radioactive compound. Radioactivity in the hydrolysate was therefore considered an indication of starch. Starch formation in the epidermis began within 5 min of exposure to glucose-1-phosphate. Autoradiograms of epidermal sections were blackened above the guard cells. Formation of starch from radioactive sugars therefore occurred predominantly in these cells. Epidermis of tulip consistently incorporated more ¹⁴C into malic and aspartic acids than that of Commelina communis (e.g. after a 4-h exposure to [¹⁴C]glucose in the dark, epidermis, with open stomata, of tulip contained 31% of its radioactivity in malate and aspartate, that of Commelina communis only 2%). The results of our experiments allow a merger of the old observations on the involvement of starch metabolism in stomatal movement with the more recent recognition of ion transfer and acid metabolism as causes of stomatal opening and closing.Abbreviation G-1-P glucose-1-phosphate     

Characterization of a regulatory mutant of fructose 1,6-bisphosphatase in Saccharomyces carlsbergensis.

Summary The fdp mutation has been localized on the genome of Saccharomyces carlsbergensis, on chromosome II, between lys2 and tyr1, at a map distance of 31 centimorgan from lys2.Since the fdp mutant does not grow on glucose, fructose, mannose and sucrose, hexose transport and a number of enzymes of carbon metabolism were tested, but no significant differences could be found between the wild type and the mutant. Only the regulatory properties of glycogen synthetase are changed in the mutant, but it is doubtfull whether this can explain its phenotype.The disorganization of carbon metabolism of the mutant upon addition of glucose to the medium was analyzed in more detail. The most prominent feature observed until now is the accumulation of free glucose and hexose phosphates in the cell. This result indicates that somehow the feedback control between hexose transport and metabolism is impaired. Hexose phosphates are known to be toxic to many cells, including yeast. Therefore, accumulation of hexose phosphates in the presence of glucose in the medium, can explain the absence of growth on this carbon source.     

Improved strains of recombinant Escherichia coli for ethanol production from sugar mixtures

Hemicellulose hydrolysates of agricultural residues often contain mixtures of hexose and pentose sugars. Ethanologenic Escherichia coli that have been previously investigated preferentially ferment hexose sugars. In some cases, xylose fermentation was slow or incomplete. The purpose of this study was to develop improved ethanologenic E. coli strains for the fermentation of pentoses in sugar mixtures. Using fosfomycin as a selective agent, glucose-negative mutants of E. coli KO11 (containing chromosomally integrated genes encoding the ethanol pathway from Zymomonas mobilis) were isolated that were unable to ferment sugars transported by the phosphoenolpyruvate-dependent phosphotransferase system. These strains (SL31 and SL142) retained the ability to ferment sugars with independent transport systems such as arabinose and xylose and were used to ferment pentose sugars to ethanol selectively in the presence of high concentrations of glucose. Additional fosfomycin-resistant mutants were isolated that were superior to strain KO11 for ethanol production from hexose and pentose sugars. These hyperproductive strains (SL28 and SL40) retained the ability to metabolize all sugars tested, completed fermentations more rapidly, and achieved higher ethanol yields than the parent. Both SL28 and SL40 produced 60 gl^–1 ethanol from 120 gl^–1 xylose in 60 h, 20% more ethanol than KO11 under identical conditions. Further studies illustrated the feasibility of sequential fermentation. A mixture of hexose and pentose sugars was fermented with near theoretical yield by SL40 in the first step followed by a second fermentation in which yeast and glucose were added. Such a two-step approach can combine the attributes of ethanologenic E. coli for pentoses with the high ethanol tolerance of conventional yeasts in a single vessel.     

The role of the hexose transporter in the chloroplasts of Arabidopsis thaliana L.

The metabolism of wild-type Arabidopsis thaliana L. and its mutant TC265 were compared in order to reveal the role of the chloroplast glucose transporter. Plants were grown in a 12-h photoperiod. From 20 to 40 days after germination, starch per gram fresh weight of shoot in the mutant was four times that in the wild type. The extent of this difference did not alter during this period. Stereological analysis showed that the chloroplasts in the mutant were larger than those in the wild type; the thylakoids appeared to be distorted by the high starch content. [U-¹⁴C]Glucose and [U-¹⁴C]glycerol were supplied, separately, to excised leaves in the dark. [U-¹⁴C]Glucose was a good precursor of sucrose in the wild type and mutant; [U-¹⁴C]glycerol was a poor precursor of sucrose in both. The distribution of ¹⁴C in the wild type was used to calculate that the net flux was from hexose monophosphates to triose phosphates, not vice versa. During the first 4 h of the night the sugar content (75% sucrose, 20% glucose) of the leaves of the mutant dropped sharply, and at all times during the night it was less than that of the wild-type leaves. This drop in sugar coincided with a decrease in the rate of respiration. The growth rate of the mutant was less than that of the wild type. Addition of sucrose restored the rate of respiration at night and increased the rate of growth. It is argued that a major function of the glucose transporter in Arabidopsis chloroplasts is export of the products of starch breakdown that are destined for sucrose synthesis at night.We thank Professor C.R. Somerville for his generous gift of seed of the Arabidopsis mutant TC265. We are also grateful to Mr B. Chapman for assistance with the preparation of the sections for electron microscopy. R.N.T. thanks the Science and Engineering Research Council for a studentship.     

Studies on the low temperature induced biogenesis of glycerol by adult Protophormia terranovae

Physiological and biochemical changes accompanying cold stress in the diapausing adult arctic blowfly, Protophormia terranovae, have been observed. In the laboratory, this insect survives prolonged periods at temperatures in the range of ?1°C to +4°C. Concentrations of free glycerol in excess of 10% of fresh body weight have been measured and the rate of its synthesis is greater at +1°C to +4°C than at ?1°C to 0°C. Under these conditions Protophormia also undergoes significant weight loss (up to 58% over 39 days) presumably in part due to dehydration. Its respiration rate decreases as expected when first shifted from 20°C to 0°C but the rate declines an additional 70% after exposure to 0°C for 24 hr. This lowest rate, which is then maintained, when considered with the initial faster one suggests positive thermal modulation is coupled to inverse thermal compensation during cold stress. This was not observed with nondiapausing Protophormia.Increments in free glycerol are accompanied by decreases in the insect's total glycogen reserves but upon rewarming, they return to pre-cold stress levels. While pre-stress glycogen stores are insufficient to provide for most of the free glycerol which accumulates, ingested carbohydrate present in the crop provides sufficient quantities. Studies with [¹⁴C] glucose indicate it is also metabolically active at low temperature.Neutral glyceride glycerol cannot contribute to net synthesis of free glycerol in significant amounts since the steady state concentrations present in pre-cold stressed insects decrease only slightly during cold stress. Furthermore, the specific radioactivity of acyl glyceride glycerol labelled in vivo with 2-[³H] glycerol before cold stress, remains unchanged during hibernation indicating that acyl glycerides are not turning over glycerol units produced by catabolism of hexose. The results of these studies argue that carbohydrate and not lipid glycerol is the source of the free glycerol which accumulates in Protophormia at low temperatures. The relationship of the above results to possible mechanisms which should permit glycerol accumulation under aerobic conditions are discussed.     

Metabolic processes and carbon nutrient exchanges between host and pathogen sustain the disease development during sunflower infection by <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

Interactions between the necrotrophic fungus Sclerotinia sclerotiorum and one of its hosts, Helianthus annuus L., were analyzed during fungal colonization of plant tissues. Metabolomic analysis, based on ¹³C- and ³¹P-NMR spectroscopy, was used to draw up the profiles of soluble metabolites of the two partners before interaction, and to trace the fate of metabolites specific of each partner during colonization. In sunflower cotyledons, the main soluble carbohydrates were glucose, fructose, sucrose and glutamate. In S. sclerotiorum extracts, glucose, trehalose and mannitol were the predominant soluble carbon stores. During infection, a decline in sugars and amino acids was observed in the plant and fungus total content. Sucrose and fructose, initially present almost exclusively in plant, were reduced by 85%. We used a biochemical approach to correlate the disappearance of sucrose with the expression and the activity of fungal invertase. The expression of two hexose transporters, Sshxt1 and Sshxt2, was enhanced during infection. A database search for hexose transporters homologues in the S. sclerotiorum genome revealed a multigenic sugar transport system. Furthermore, the composition of the pool of reserve sugars and polyols during infection was investigated. Whereas mannitol was produced in vitro and accumulated in planta, glycerol was exclusively produced in infected tissues and increased during colonization. The hypothesis that the induction of glycerol synthesis in S. sclerotiorum exerts a positive effect on osmotic protection of fungal cells and favors fungal growth in plant tissues is discussed. Taken together, our data revealed the importance of carbon–nutrient exchanges during the necrotrophic pathogenesis of S. sclerotiorum.