Metabolic correlates to glycerol biosynthesis in a freeze-avoiding insect,Epiblema scudderiana

Summary The course of glycerol biosynthesis, initiated by exposure to –4°C, was monitored in larvae of the goldenrod gall moth,Epiblema scudderiana, and accompanying changes in the levels of intermediates of glycolysis, adenylates, glycogen, glucose, fructose-2,6-bisphosphate, and fermentative end products were characterized. Production of cryoprotectant was initiated within 6 h after a switch from +16° to –4°C, with halfmaximal levels reached in 30 h and maximal content, 450–500 mol/g wet weight, achieved after 4 days. Changes in the levels of intermediates of the synthetic pathway within 2 h at –4°C indicated that the regulatory sites involved glycogen phosphorylase, phosphofructokinase, and glycerol-3-phosphatase. A rapid increase in fructose-2,6-bisphosphate, an activator of phosphofructokinase and inhibitor of fructose-1,6-bisphosphatase, appeared to have a role in maintaining flux in the direction of glycerol biosynthesis. Analysis of metabolite changes as glycerol production slowed suggested that the inhibitory restriction of the regulatory enzymes was slightly out of phase. Inhibition at the glycerol-3-phosphatase locus apparently occurred first and resulted in a build-up of glycolytic intermediates and an overflow accumulation of glucose. Glucose inhibition of phosphorylase, stimulating the conversion of the activea to the inactiveb forms, appears to be the mechanism that shuts off phosphorylase function, counteracting the effects of low temperature that are the basis of the initial enzyme activation. Equivalent experiments carried out under a nitrogen gas atmosphere suggested that the metabolic make-up of the larvae in autumn is one that obligately routes carbohydrate flux through the hexose monophosphate shunt. The consequence of this is that fermentative ATP production during anoxia is linked to the accumulation of large amounts of glycerol as the only means of maintaining redox balance.Abbreviations G6P glucose-6-phosphate - F6P fructose-6-phosphate - F1, 6P fructose-1,6-bisphosphate - F2,6P ₂ fructose-2,6-bisphosphate - G3P grycerol-3-phosphate - DHAP dinydroxyacetonephosphate - GAP glyceraldehyde-3-phosphate - PEP phosphoenolpyruvate - PFK phosphofructokinase - FBPase fructose-1,6-bisphosphatase - PK pyruvate kinase     

Reduced-activity mutants of phosphoglucose isomerase in the cytosol and chloroplast of Clarkia xantiana

(i) We have studied the influence of reduced phosphoglucose-isomerase (PGI) activity on photosynthetic carbon metabolism in mutants of Clarkia xantiana Gray (Onagraceae). The mutants had reduced plastid (75% or 50% of wildtype) or reduced cytosolic (64%, 36% or 18% of wildtype) PGI activity. (ii) Reduced plastid PGI had no significant effect on metabolism in low light. In high light, starch synthesis decreased by 50%. There was no corresponding increase of sucrose synthesis. Instead glycerate-3-phosphate, ribulose-1,5-bisphosphate, reduction of Q_A (the acceptor for photosystem II) and energy-dependent chlorophyll-fluorescence quenching increased, and O₂ evolution was inhibited by 25%. (iii) Decreased cytosolic PGI led to lower rates of sucrose synthesis, increased fructose-2,6-bisphosphate, glycerate-3-phosphate and ribulose-1,5-bisphosphate, and a stimulation of starch synthesis, but without a significant inhibition of O₂ evolution. Partitioning was most affected in low light, while the metabolite levels changed more at saturating irradiances. (iv) These results provide decisive evidence that fructose-2,6-bisphosphate can mediate a feedback inhibition of sucrose synthesis in response to accumulating hexose phosphates. They also provide evidence that the ensuing stimulation of starch synthesis is due to activation of ADP-glucose pyrophosphorylase by a rising glycerate-3-phosphate: inorganic phosphate ratio, and that this can occur without any loss of photosynthetic rate. However the effectiveness of these mechanisms varies, depending on the conditions. (v) These results are analysed using the approach of Kacser and Burns (1973, Trends Biochem. Sci. 7, 1149–1161) to provide estimates for the elasticities and flux-control coefficient of the cytosolic fructose-1,6-bisphosphatase, and to estimate the gain in the fructose-2,6-bisphosphate regulator cycle during feedback inhibition of sucrose synthesis.Abbreviations and symbols Chl chlorophyll - Fru6P fructose-6-phosphate - Frul,6bisP fructose-1,6-bisphosphate - Fru-1,6Pase fructose-1,6-bisphosphatase - Fru2,6bisP fructose-2,6-bisphosphate - Fru2,6Pase fructose-2,6-bisphosphatase - Glc6P glucose-6-phosphate - PGI phosphoglucose isomerase - Pi inorganic phosphate - Q_A acceptor for photosystem II - Ru1,5bisP ributose-1,5-bisphosphate - SPS sucrose-phosphate synthase     

Time-course phosphorylation of glucose by Saccharomyces carlsbergensis following alterations in cell membrane permeability

Summary The correlation between release of sugar phosphates and the increase of membrane permeability was assessed in Saccharomyces carlsbergensis cells. The highest level of fructose-1,6-diphosphate,FDP, (35–40 mg/ml) was reached after 6h incubation at 35°C (65–70% permeabilized cells) while it was less than 1 mg/ml after 22 h incubation at 15 °C (only 10% permeabilized cells). Assessment of enzymatic activity of hexokinase (HK) phosphofructokinase (PFK) and aldolase (AL) during fermentation showed a higher leakage of both kinases in permeabilized cells than in intact ones.     

Fructose-1,6-bisphosphatase from<Emphasis Type="Italic"> Corynebacterium glutamicum</Emphasis>: expression and deletion of the<Emphasis Type="Italic"> fbp</Emphasis> gene and biochemical characterization of the enzyme

The class II fructose-1,6-bisphosphatase gene of Corynebacterium glutamicum, fbp, was cloned and expressed with a N-terminal His-tag in Escherichia coli. Purified, His-tagged fructose-1,6-bisphosphatase from C. glutamicum was shown to be tetrameric, with a molecular mass of about 140 kDa for the homotetramer. The enzyme displayed Michaelis-Menten kinetics for the substrate fructose 1,6-bisphosphate with a K_m value of about 14 µM and a V_max of about 5.4 µmol min^–1 mg^–1 and k_cat of about 3.2 s^–1. Fructose-1,6-bisphosphatase activity was dependent on the divalent cations Mg²⁺ or Mn²⁺ and was inhibited by the monovalent cation Li⁺ with an inhibition constant of 140 µM. Fructose 6-phosphate, glycerol 3-phosphate, ribulose 1,5-bisphosphate and myo-inositol-monophosphate were not significant substrates of fructose-1,6-bisphosphatase from C. glutamicum. The enzymatic activity was inhibited by AMP and phosphoenolpyruvate and to a lesser extent by phosphate, fructose 6-phosphate, fructose 2,6-bisphosphate, and UDP. Fructose-1,6-bisphosphatase activities and protein levels varied little with respect to the carbon source. Deletion of the chromosomal fbp gene led to the absence of any detectable fructose-1,6-bisphosphatase activity in crude extracts of C. glutamicum WTfbp and to an inability of this strain to grow on the carbon sources acetate, citrate, glutamate, and lactate. Thus, fbp is essential for growth on gluconeogenic carbon sources and likely codes for the only fructose-1,6-bisphosphatase in C. glutamicum.     

Shoot Regeneration from Immature Cotyledons of Cicer Arietinum

Shoot regeneration was achieved from immature cotyledons of five chickpea (Cicer arietinum L.) genotypes: C235, ICC4971, ICC11531, ICC12257 and ICC12873. The cotyledons cultured on Murashige and Skoog (MS) medium supplemented with 3 or 5 mg dm^–3 zeatin with or without 0.04 mg dm^–3 indole acetic acid (IAA) showed formation of cotyledon like structures (CLS) at their proximal ends. Subsequently, shoot regeneration took place in some of the CLS forming explants. CLS were also formed in cotyledons cultured on MS + 0.2 – 1 mg dm^–3 thidiazuron (TDZ); direct shoot regeneration was observed in cotyledons cultured on 1 mg dm^–3 TDZ. The shoot buds elongated on media containing indole butyric acid (IBA), benzylaminopurine (BAP) and gibberellic acid (GA₃). Complete plantlets were obtained by rooting of shoots following pulse treatment with 200 mg dm^–3 IBA for 5 min and culture on growth regulator free half-strength MS medium.     

Cold-hardening results in increased activity of enzymes involved in carbon metabolism in leaves of winter rye (Secale cereale L.)

Light- and CO₂-saturated photosynthesis of nonhardened rye (Secale cereale L. cv. Musketeer) was reduced from 18.10 to 7.17 mol O₂·m^–2·s^–1 when leaves were transferred from 20 to 5°C for 30 min. Following cold-hardening at 5°C for ten weeks, photosynthesis recovered to 15.05 mol O₂·m^–2·s^–1,comparable to the nonhardened rate at 20°C. Recovery of photosynthesis was associated with increases in the total activity and activation of enzymes of the photosynthetic carbon-reduction cycle and of sucrose synthesis. The total hexose-phosphate pool increase by 30% and 120% for nonhardened and cold-hardened leaves respectively when measured at 5°C. The large increase in esterified phosphate in coldhardened leaves occurred without a limitation in inorganic phosphate supply. In contrast, the much smaller increase in esterified phosphate in nonhardened leaves was associated with an inhibition of ribulose-1,5-bisphosphate carboxylase/oxygenase and sucrose-phosphate synthase activation. It is suggested that the large increases in hexose phosphates in cold-hardened leaves compensates for the higher substrate threshold concentrations needed for enzyme activation at low temperatures. High substrate concentrations could also compensate for the kinetic limitations imposed by product inhibition from the accumulation of sucrose at 5°C. Nonhardened leaves appear to be unable to compensate in this fashion due to an inadequate supply of inorganic phosphate.Abbreviations DHAP dihydroxyacetone phosphate - Fru6P fructose-6-phosphate - Fru 1,6BP fructose-1,6-bisphosphate - Fru1,6BPase fructose-1,6-bisphosphatase - Glc6P glucose-6-phosphate - PGA 3-phosphoglycerate - PPFD photosynthetic photon flux density - CH cold-hardened rye grown at 5°C - NH nonhardened rye grown at 24°C - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - SPS sucrose-phosphate synthase - UDPGlc uridine 5-diphosphoglucose This work was supported by operating grants from the Swedish Natural Sciences Research Council to G.Ö. and P.G.     

Transgenic tobacco plants with strongly decreased expression of pyrophosphate: Fructose-6-phosphate 1-phosphotransferase do not differ significantly from wild type in photosynthate partitioning,plant growth or their ability to cope with limiting phosphate,limiting nitrogen and suboptimal temperatures

Transformation of tobacco with the potato gene encoding the subunit of pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) in the antisense orientation under the control of the constitutive CaMV 35S promoter, followed by selfing and crossing of the transformants, generated a line of tobacco (5–37) with up to an 85% reduction in PFP activity in the shoot. Transformants containing a sense construct (4-40-91) contained only 1–3% of wild-type PFP, presumably due to co-suppression. Rates of photosynthesis and partitioning between sucrose and starch in source leaves were identical in 4-40-91 transformants and the wild type. In the dark in sink leaves of 4-40-91 transformants, levels of hexose phosphates were up to 50% higher, glycerate-3-phosphate 30% lower and fructose-2,6-bisphosphate threefold higher than in the wild type; inorganic pyrophosphate, pyruvate and the ATP/ADP ratio were unaltered. Low -PFP and wild-type plants did not differ significantly in their rate of growth at 25° C and 200 mol quanta · m^–2 · s^–1 on full nutrient medium. Growth on limiting phosphate and limiting nitrogen was inhibited identically in the wild type and transformants, and transformants adjusted their shoot/root ratio in an identical manner to the wild type. Differences in fructose-2,6-bisphosphate and glycolytic metabolites between the wild type and transformants were no larger in these suboptimal nutrient conditions, than in optimal conditions. Growth of the wild type and 4-40-91 transformants was inhibited identically at 12° C compared to 25° C. Differences in fructose-2,6-bisphosphate were smaller when the genotypes were compared at 12° C than at 25° C. We conclude that PFP does not play an essential role in photosynthate partitioning in source leaves. During respiratory metabolism in sink leaves it catalyzes a net glycolytic flux, as in potato tubers. However, tobacco seedlings are able to compensate for a large decrease in expression of PFP without loss of growth, or the ability to cope with suboptimal phosphate, nitrogen or temperature.Abbreviations F2,6BP fructose-2,6-bisphosphate - F6P fructose-6-phosphate - G6P glucose-6-phosphate - PFK phosphofructokinase - PFP pyrophosphate-dependent fructose-6-phosphate 1-phosphotransferase - 3-PGA glycerate-3-phosphate - PPi inorganic pyrophosphate - PEP phosphoenolpyruvate This work was supported by the Bundesministerium für Forschung and Technologie (M.S, U.S.) and the Canadian Research Council (S.C., D.D). M.P. was supported by a Royal Society Fellowship.     

Effects of Pluronic F-68 on shoot regeneration from cultured jute cotyledons and on growth of transformed roots

The effects have been studied of the non-ionic surfactant, Pluronic F-68, on the growth in culture of jute (Corchorus capsularis L.) cotyledons with attached petioles, cotyledon explants and transformed roots. Supplementation of culture medium with 0.001–0.5% (w/v) of either commercial grade Pluronic F-68 or a purified fraction prepared by passage through silica gel, stimulated shoot production from the petioles of C. capsularis var. D154 and C134 cotyledons. This effect was most marked in C134, because of the failure of control cotyledons to produce shoots in the absence of Pluronic. Plants regenerated from Pluronic-treated cotyledons were morphologically normal. Growth of transformed roots of C. capsularis var. D154 was stimulated in medium supplemented with commercial grade or purified Pluronic F-68, with maximum increases in both fresh and dry weights with 0.1% (w/v) of the surfactant. Roots cultured in the presence of Pluronic F-68 could be maintained without sub-culture for up to 70 days, whereas roots cultured in the absence of Pluronic required subculture every 7 days, to prevent necrosis. Transformed roots also produced callus in the presence of 0.001–1.0% (w/v) of either commercial grade or purified Pluronic. The biotechnological implications of these results are discussed in relation to the potential value of non-ionic surfactants as growth-stimulating additives to plant culture media.Abbreviations NAA -naphthaleneacetic acid - BA 6-benzyladenine - IAA indole-3-acetic acid - MS Murashige & Skoog (1962)     

Liver metabolism in cold hypoxia: a comparison of energy metabolism and glycolysis in cold-sensitive and cold-resistant mammals

The effects of cold hypoxia were examined during a time-course at 2 °C on levels of glycolytic metabolites: glycogen, glucose, glucose-1-phosphate, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, phosphoenolpyruvate, pyruvate, lactate and energetics (ATP, ADP, AMP) of livers from rats and columbian ground squirrels. Responses of adenylate pools reflected the energy imbalance created during cold hypoxia in both rat and ground squirrel liver within minutes of organ isolation. In rat, ATP levels and energy charge values for freshly isolated livers were 2.54 mol·g^-1 and 0.70, respectively. Within 5 min of cold hypoxia, ATP levels had dropped well below control values and by 8 h storage, ATP, AMP, and energy charge values were 0.21 mol·g^-1, 2.01 mol·g^-1, and 0.17, respectively. In columbian ground squirrels the patterns of rapid ATP depletion and AMP accumulation were similar to those found in rat. In rat liver, enzymatic regulatory control of glycolysis appeared to be extremely sensitive to the decline in cellular energy levels. After 8 h cold hypoxia levels of fructose-6-phosphate decreased and fructose-1,6-bisphosphate increased, thus reflecting an activation of glycolysis at the regulatory step catalysed by phospho-fructokinase fructose-1,6-bisphosphatase. Despite an initial increase in flux through glycolysis over the first 2 min (lactate levels increased 3.7 mol·g^-1), further flux through the pathway was not permitted even though glycolysis was activated at the phosphofructokinase/fructose-1,6-bisphosphatase locus at 8 h, since supplies of phosphorylated substrate glucose-1-phosphate or glucose-6-phosphate remained low throughout the duration of the 24-h period. Conversely, livers of Columbian ground squirrels exhibited no activation or inactivation of two key glycolytic regulatory loci, phosphofructokinase/fructose-1,6-bisphosphatase and pyruvate kinase/phosphoenolpyruvate carboxykinase and pyruvate carboxylase. Although previous studies have shown similar allosteric sensitivities to adenylates to rat liver phospho-fructokinase, there was no evidence of an activation of the pathway as a result of decreasing high energy adenylate, ATP or increasing AMP levels. The lack of any apparent regulatory control of glycosis during cold hypoxia may be related to hibernator-specific metabolic adaptations that are key to the survival of hypothermia during natural bouts of hibernation.Abbreviations DHAP dihydroxyacetonephosphate - EC energy charge - F1,6P₂ fructose-1,6-bisphosphate - F2,6P₂ fructose-2,6-bisphosphate - F6P fructose-6-phosphate - FBP fructose-1,6-bisphosphatase - G1P glucose-1-phosphate - G6P glucose-6-phosphate - GAP glyceraldehyde-3-phosphate - GAPDH glyceraldehyde-3-phosphate dehydrogenase - L/R lactobionate/raffinose-based solution - MR metabolic rate - PDH pyruvate dehydrogenase - PEP phosphoenolpyruvate - PEPCK & PC phosphoenolpyruvate carboxykinase and pyruvate carboxylase - PFK phosphofructokinase; PK, pyruvate kinase - Q ₁₀ the effect of a 10 °C drop in temperature on reaction rates (generally, Q ₁₀=2–3) - TA total adenylates - UW solution University of Wisconsin solution (L/R-based)     

Carnitine acetyltransferase in pea cotyledon mitochondria

Purified pea cotyledon mitochondria did not oxidise acetyl-CoA in the presence of carnitine. However, acetylcarnitine was oxidised. It was concluded that acetylcarnitine passed through the mitochondrial membrane barrier but acetyl-CoA did not. Only a sensitive radioactive assay detected carnitine acetyltransferase in intact mitochondrion or intact mitoplast preparations. When the mitochondria or mitoplasts were burst, acetyl-CoA substrate was available to the matrix carnitine acetyltransferase and a high activity of the enzyme was measured. The inner mitochondrial membrane is there-fore the membrane barrier to acetyl-CoA but acetylcarnitine is suggested to be transported through this membrane via an integral carnitine: acylcarnitine translocator. Evidence is presented to indicate that when the cotyledons from 48-h-grown peas are oxidising pyruvate, acetylcarnitine formed in the mitochondrial matrix by the action of matrix carnitine acetyltransferase may be transported to extra-mitochondrial sites via the membrane translocator.     

Activities of glycolytic enzymes in rapidly proliferating and differentiated C6 glioma cells

Comparisons of glycolytic enzymes between rapidly proliferating and Bt2 cAMP-induced differentiated C6 glioma cells have been made. Rapidly proliferating cells had higher concentrations of glucose-6-phosphate, fructose-6-phosphate and fructose-1,6-bisphosphate compared to morphologically differentiated cells. Under maximally activating conditions, the specific activity and Vmax of hexokinase and phosphofructokinase enzymes were reduced by approximately 3- and 28-fold, respectively, in differentiated cells, without any change in Km values. These results suggest that hexokinase and phosphofructokinase occupy special control positions and the rate of glycolysis is correlated with cellular proliferation of C6 glioma cells.     

In vitro plantlet regeneration from cotyledons of the tree-legume Leucaena leucocephala

Two plant regeneration methods applicable to Leucaenaleucocephala were developed. In the first method, involvingorganogenesis via callus formation, cotyledon, hypocotyl and root segments wereinitiated on MS medium containing different concentrations ofN⁶-benzyladenine (BA), 2,4-dichlorophenoxyacetic acid (2,4-D), andnaphthaleneacetic acid (NAA). Both compact (type I) and friable (type II) calliwere obtained from the cotyledon and hypocotyl explants treated with differentconcentrations of the growth regulators. Shoots were generated only from thefriable calli formed from the cotyledon explants. The calli formed from thehypocotyl explants did not generate shoots and the root explants died withoutforming callus. Cotyledon explants from 3–4 day old seedlings showedmaximum callus induction compared to those from older seedlings. In a secondmethod involving direct organogenesis, excised cotyledons were cultured on 1/2MS medium containing 10–35 mg l^–1N⁶-benzyladenine (BA) for 7–14 days. Transfer of thecotyledonsto regeneration medium containing low BA resulted in callus formation andsubsequent shoot regeneration from the base of the excised cotyledon explants,with up to 100% frequency. Regenerated shoots rooted best on a basal mediumcontaining no growth regulators.     

In vitro plantlet regeneration from cotyledon, hypocotyl and root explants of hybrid seed geranium

In vitro plantlet regeneration systems for the seed geranium (Pelargonium x hortorum Bailey) using cotyledon, hypocotyl and root explants were optimized by studying the influence of seedling age, growth regulators and excision orientation on organogenesis. Indole-3-acetic acid combined with zeatin yielded the highest rate of shoot production on cotyledon explants (0.2–2 shoots per explant). More shoots were produced on explants cut from the most basal region of cotyledons from 2 to 4-day-old seedlings than from older seedlings or more distal cut sites. Hypocotyl explants produced the highest number of shoots, up to 40 shoots per explant, on indole-3-acetic acid (2.8–5.6 mM) + zeatin (4.6 mM) or thidiazuron (4.5 mM). Maximum shoot formation (0.3–1.4 shoots per explant) on root explants occurred when they were cultured on medium containing zeatin. Regenerated shoots rooted best on a basal medium containing no growth regulators. There were substantial differences among cultivars in shoot formation from each of the explant systems.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - TDZ thidiazuron     

Isolation and cell regeneration of protoplasts from sugar pine (Pinus lambertiana)

Protoplasts were isolated at high yields from actively growing callus and cell suspensions of cotyledons and needles of mature trees. The best protoplast growth response was obtained from cell suspensions of cotyledon and needle callus. Lower protoplast yields were obtained directly from young needles of flushing buds on explants from mature shoots (30-year-old trees) growing in vitro. In all cases, the first divisions, promoted by dimethyl sulfoxide, were observed in 10–45% of the protoplasts by 7–10 days. After 25–30 days, colonies of 8–10 cells were established. Browning of protoplast-derived cell cultures was observed within 40–45 days (cotyledons) and 20–25 days (mature tree sources).Abbreviations BA N⁶-Benzyladenine - DCR Douglas-fir cotyledon revised medium - 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethyl sulfoxide - FDA Fluorescein diacetate - Mes 2-(N-morpholino) ethanesulfonic acid - NAA -naphthaleneacetic acid     

Secondary somatic embryogenesis and plant regeneration in cassava

Somatic embryos isolated from mature seed-derived cotyledon cultures of cassava (Mannihot esculenta Crantz) underwent direct secondary somatic embryogenesis or plant development under appropriate incubation conditions. Isolated somatic embryos were subjected to a two-stage culture procedure similar to that which induced their development on cotyledon explants. This involved incubation for 24–30 days on Murashige and Skoog basal medium supplemented with 2–8 mgl^-1 2,4-dichlorophenoxyacetic acid (2,4-D) (Stage I medium) before transfer to medium supplemented with 0.01 mgl^-1 2,4-D and 0.1 mgl^-1 6-benzylamino purine (BAP) (Stage II medium). Under these conditions, secondary somatic embryos developed directly from the cotyledons and shoot-tip region of primary somatic embryos by a developmental process morphologically very similar to that occurring on zygotic cotyledon explants. Apical shoot extension and adventitious root formation occurred when somatic embryos were isolated from parental cultures and incubated on Stage II medium. Somatic embryo-derived plants growing in greenhouse conditions appeared morphologically normal when compared with non-regenerated plants.     

Studies of cotyledon protoplast cultures from Brassica napus,B. campestris and B. oleracea. I: Cell wall regeneration and cell division

Protoplasts isolated from cotyledons of a number of cultivars of Brassica napus, B. campestris and B. oleracea were cultured in different media to study the characteristics of cell wall regeneration and cell division at early stages of culture. Time course analysis using Calcolfluor White staining indicated that cell wall regeneration began in some protoplasts 2–4 h following isolation in all cultivars. 30–70% of cultured cotyledon protoplasts exhibited cell wall regeneration at 24 h and about 60–90% at 72 h after the initiation of culture. Results also indicated that a low percentage (0.4–5.4%) of cultured cotyledon protoplasts entered their first cell division one day after initial culture in all twelve cultivars. The percentage of dividing cells increased linearly up to 40% from 1 to 7 day, indicating that cotyledon protoplasts of Brassica had a high capacity for cell division. Factors that influence the level of cell wall regeneration and cell division during cotyledon protoplast culture have been investigated in this study. Cotyledons from seedlings germinated in a dark/dim light regime provided a satisfactory tissue source for protoplast isolation and culture for all Brassica cultivars used. The percentages of protoplasts exhibiting cell wall regeneration and division were significantly influenced by cultivar and species examined, with protoplasts from all five cultivars of B. campestris showing much lower rates of cell wall regeneration than those of B. napus and B. oleracea over 24–120 h, and with the levels of cell division in B. napus cultivars being much higher than those in B. campestris and B. oleracea over 1–9 days. The capacity of cell wall regeneration and cell division in cotyledon protoplast culture of the Brassica species appears under strong genetic control. Cell wall regeneration in protoplast culture was not affected by the culture medium used. In contrast, the composition of the culture medium played an important role in determining the level of cell division, and the interaction between medium type and cultivars was very significant.Abbreviations BA benzylaminopurine - CPW Composition of Protoplast Washing-solution - CW Calcolfluor White - EDTA ethylenediamine-tetraacetic acid - KT Kinetin - Md MS modified Murashige and Skoog medium - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - PAR photosynthetically active radiation - SDS sodium dodecyl sulfate     

Adenosine stimulates anabolic metabolism in developing castor bean (Ricinus communis L.) cotyledons

In previous experiments it was shown that Castor-bean (Ricinus communis) endosperm releases carbohydrates, amino acids and nucleoside derivatives, which are subsequently imported into the developing cotyledons (Kombrink and Beevers in Plant Physiol 73:370-376, 1983). To investigate the importance of the most prominent nucleoside adenosine for the metabolism of growing Ricinus seedlings, we supplied adenosine to cotyledons of 5-days-old seedlings after removal of the endosperm. This treatment led to a 16% increase in freshweight of intact seedlings within 16 h, compared to controls. Using detached cotyledons, we followed uptake of radiolabelled adenosine and identified 40% of label in solubles (mostly ATP and ADP), 46% incorporation in RNA and 2.5% in DNA, indicating a highly active salvage pathway. About 7% of freshly imported adenosine entered the phloem, which indicates a major function of adenosine for cotyledon metabolism. Import and conversion of adenosine improved the energy content of cotyledons as revealed by a substantially increased ATP/ADP ratio. This effect was accompanied by slight increases in respiratory activity, decreased levels of hexose phosphates and increased levels of fructose-1,6-bisphosphate and triose phosphates. These alterations indicate a stimulation of glycolytic flux by activation of phosphofructokinase, and accordingly we determined a higher activity of this enzyme. Furthermore the rate of [(14)C]-sucrose driven starch biosynthesis in developing castor-bean is significantly increased by feeding of adenosine. In conclusion, our data indicate that adenosine imported from mobilizing endosperm into developing castor-bean cotyledons fulfils an important function as it promotes anabolic reactions in this rapidly developing tissue.     

Metabolism of carbohydrate and lipid reserves in germinated cotton seeds

Utilization of reserve lipid and carbohydrates during germination (0–12 h) and postgerminative growth (12–48 h) was studied in cotton (Gossypium hirsutum L.) seedlings. Raffinose and stachyose were utilized during the germination period and early growth; mobilization was associated with -galactosidase (EC 3.2.1.22) activity. Results from pulse-chase experiments with [³H]raffinose supplied exogenously to 4-h soaked seeds indicated that raffinose-derived catabolites contributed to the coincident increase in cotyledon sucrose and starch, and to the small increase in axis dry weight. Starch appears to be an alternative sink for end products of hydrolysis of reserve carbohydrates prior to the onset of rapid axis growth and cotyledon expansion. Mobilization of neutral lipid commenced at about 16 h after soaking, concomitant with development of key glyoxylate-cycle and other gluconeogenesis-related enzyme activities. Axis dry weight increased three-fold between 24 and 48 h. Results from pulse-chase (3 h, 16 h) experiments in which [2-¹⁴C]acetate was supplied to cotyledons of intact 22-h-old seedlings showed that acetate-derived metabolites were not transported exclusively to the axes, but were partitioned between axes and cotyledons. Only 27% of total incorporated radioactivity was recovered in axes following the chase, 18% was evolved as CO₂, and the rest was recovered in water-soluble substances (20%) and polymers (31%) within the cotyledons. Of the polymers, 55% of the activity was in polysaccharides (Starch, pectic substances, hemicellulose, cellulose), 25% in protein, and 20% in unidentified neutral and acidic compounds. Considering these data, the amount of lipid mobilized, and various routes by which supplied [2-¹⁴C]acetate could be metabolized, it appears that lipidderived compounds contribute only 25–40% of axis dry-weight gain. Lipid-derived substances retained in the cotyledons likely are utilized for expansion and differentiation of the cotyledons into photosynthetic organs.     

Purification and properties of pea (Pisum sativum L.) thioredoxin f,a plant thioredoxin with unique features in the activation of chloroplast fructose-1,6-bisphosphatase

Thioredoxin (Td) f from pea (Pisum sativum L.) leaves was purified by a simple method, which provided a high yield of homogeneous Td f. Purified Td f had an isoelectric point of 5.4 and a relative molecular mass (M_r) of 12 kilodaltons (kDa) when determined by filtration through Superose 12, but an M_r of 15.8 kDa when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein remained fully active for several months when conserved frozen at — 20° C. The pea protein was able to activate fructose1,6-bisphosphatase (FBPase; EC 3.1.3.11), but in contrast to other higher-plant Td f proteins, was not functional in the modulation of NADP⁺-malate dehydrogenase activity. In spite of the absence of immunological cross-reactions of pea and spinach Td f proteins with the corresponding antibodies, pea Td f activated not only the homologous FBPase, but also the spinach enzyme. The saturation curves for pea FBPase, either with fructose-1,6-bisphosphate in the presence of different concentrations of homologous Td f, or with pea Td f in the presence of excess substrate, showed sigmoid kinetics; this can be explained on the basis of a random distribution of fructose-1,6-bisphosphate, and of the oxidized and reduced forms of the activator, among the four Td f- and substrate-binding sites of this tetrameric enzyme. From the saturation curves of pea and spinach Td f proteins against pea FBPase, a 4:1 stoichiometry was determined for the Td f-enzyme binding. This is in contrast to the 2:1 stoichiometry found for the spinach FBPase. The UV spectrum of pea Td f had a maximum at 277 nm, which shifted to 281 nm after reduction with dithiothreitol (s at 280 nm for 15.8-kDa M_r = 6324 M^–1 · cm^–1). The fluorescence emission spectrum after 280-nm excitation had a maximum at 334 nm, related to tyrosine residues; after denaturation with guanidine isothiocyanate an additional maximum appeared at 350 nm, which is concerned with tryptophan groups. Neither the native nor the denatured form showed a significant increase in fluorescence after reduction by dithiothreitol, which means that the tyrosine and tryptophan groups in the reduced Td f are similarly exposed. Pea Td f appears to have one cysteine residue more than the three cysteines earlier described for spinach and Scenedesmus Td f proteins.Abbreviations DDT dithiothreitol - ELISA enzyme-linked immunosorbent assay - FBPase fructose- 1,6-bisphosphatase - kDa kilodalton - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - Td thioredoxin The authors are grateful to Mrs. Francisca Castro and Mr. Narciso Algaba for skilful technical assistance. This work was supported by grant PB87-0431 of Dirección General de Investigación Cientifica y Técnica (DGICYT, Spain).