The occurrence of an extra-plastidial isoform of ADP-glucose (Glc) pyrophosphorylase (AGPase) among starch-storing organs was investigated in two ways. First, the possibility that an extra-plastidial isoform arose during the domestication of cereals was studied by comparing the intracellular distribution of enzyme activity and protein in developing endosperm of noncultivated Hordeum species with that previously reported for cultivated barley (Hordeum vulgare). As in cultivated barley, the AGPase of H. vulgare subsp. spontaneum and Hordeum murinum endosperm is accounted for by a major extra-plastidial and a minor plastidial isoform. Second, the ratio of ADP-Glc to UDP-Glc was used as an indication of the intracellular location of the AGPase activity in a wide range of starch-synthesizing organs. The ratio is expected to be high in organs in which UDP-Glc and ADP-Glc are synthesized primarily in the cytosol, because the reactions catalyzed by AGPase and UDP-Glc pyrophosphorylase will be coupled and close to equilibrium. This study revealed that ADP-Glc contents and the ratio of ADP-Glc to UDP-Glc were higher in developing graminaceous endosperms than in any other starch-storing organs. Taken as a whole the results indicate that an extra-plastidial AGPase is important in ADP-Glc synthesis in graminaceous endosperms, but not in other starch-storing organs.

Thermogenesis in Arum species is induced by salicylic acid (SA) and caused by activation of the alternative respiration pathway and the alternative oxidase (AOX), resulting in heat production. The enzymes phosphoenolpyruvate carboxylase (PEPCase) and NAD-dependent malic enzyme (NAD-ME) also show dramatic increases in activity during thermogenesis and are essential for heat generation. In this current study, we characterized the timing and localization of changes in levels of AOX, NAD-ME, and PEPCase polypeptide accumulation, and changes in Ppc and Me mRNA accumulation, in various Arum tissues during prethermogenic development and during thermogenesis. In addition, changes in ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) gene expression were analysed at the level of rbcL protein and mRNA accumulation. AOX, PEPCase, and NAD-ME all increased only in clubs during development, increasing 5–6-fold by the latest prethermogenic stage and remained at this level. The induction of thermogenesis did not cause any changes in levels of AOX, indicating that SA does not affect levels of the enzyme itself, but instead must act to stimulate activity. Reported increases in NAD-ME activity during club development correlated closely with mRNA and protein accumulation, whereas PEPCase activity appears to be determined by post-transcriptional and post-translational processes. Interestingly, Rubisco protein and mRNA were found in relatively abundant amounts in clubs during early developmental stages, and disappeared rapidly as the thermogenic enzymes began to increase in abundance. The induction of thermogenesis by a synthetic inducer of plant pathogen resistance, 2,6-dichloroisonicotinic acid, as well as the involvement of SA and AOX in both processes, reinforces evidence for a link between mechanisms controlling disease resistance in all plants and thermogenic induction in Arum.

The aim of this work was to determine the effects of hypoxia on the major fluxes of carbohydrate metabolism in climacteric fruit of banana (Musa cavendishii Lamb ex Paxton). Hands of bananas, untreated with ethylene, were allowed to ripen in air at 21°C in the dark. When the climacteric began, fruit were transferred to 15 or 10% oxygen and were analysed once the climacteric peak had been reached 8–12 h later. The rates of starch breakdown, sucrose, glucose and fructose accumulation, and CO2 production were determined, as were the contents of hexose monophosphates, adenylates and pyruvate. In addition, the detailed distribution of label was determined after supplying [U-14C]-, [1-14C]-, [3,4-14C]- and [6-14C]glucose, and [U-14C]glycerol to cores of tissue under hypoxia. The data were used to estimate the major fluxes of carbohydrate metabolism. There was a reduction in the rate of respiration. The ATP/ADP ratio was unaffected but there was a significant increase in the content of AMP. In 15% oxygen only minor changes in fluxes were observed. In 10% oxygen starch breakdown was reduced and starch synthesis was not detected. The rate of sucrose synthesis decreased, as did the rate of re-entry of hexose sugars into the hexose monophosphate pool. There was a large increase in both the glycolytic flux and in the flux from triose phosphates to hexose monophosphates. It is argued that the increase in these fluxes is due to activation of pyrophosphate: fructose-6-phosphate 1-phosphotransferase, and that this enzyme has an important role in hypoxia. The results are discussed in relation to our understanding of the control of carbohydrate metabolism in hypoxia.

The aim of this work was to discover whether the respiration of wheat (Triticum aestivum L. cv. Huntsman) leaves, transferred to darkness after 7 h photosynthesis, showed an initial period of wasteful respiration. For young and old leaves, CO2 production and O2 uptake after 7 h photosynthesis were up to 56% higher than at the end of an 8-h night. The maximum catalytic activities of citrate synthase (EC 4.1.3.7), aconitase (EC 4.2.1.3), fumarase (EC 4.2.1.2) and cytochrome-c oxidase (EC 1.9.3.1) at the end of the day did not differ from those at the end of the night. Changes in the contents of glucose 6-phosphate, fructose-1,6-bisphosphate, dihydroxyacetone phosphate, and α-ketoglutarate did not as a group parallel the changes in the rate of respiration. The detailed distribution of label from [U-14C] sucrose supplied to leaves in the dark was similar at the end of the day and the end of the night. No correlation was observed between the rates of leaf respiration and extension growth. It is argued that the higher rate of respiration at the beginning of the night cannot be attributed to wasteful respiration.

Leucoplasts and amyloplasts were isolated on Nycodenz gradients from lysates of protoplasts of suspension cultures of soybean (Glycine max). The labelling of CO2 and starch was determined after incubating intact and lysed preparations of plastids with C-14-labelled substrates. The substrates were said to have been metabolized by the plastids if the labelling of the product was greater in the intact than in the lysed preparations. Leucoplasts and amyloplasts metabolized both [C-14]glucose 1-phosphate and [C-14]glucose 6-phosphate to (CO2)-C-14. In both types of plastid [C-14]glucose 6-phosphate was the better precursor. Neither substrate was converted to starch by intact leucoplasts. Amyloplasts converted [C-14]glucose 1-phosphate, but not [C-14]glucose 6-phosphate, to starch in the presence of exogeneous ATP. It is suggested that in these plastids the reaction catalysed by phosphoglucomutase is not at equilibrium.

The aim of this work was to discover whether genetic manipulation of 6-phosphofructokinase [EC 2.7.1.11; PFK(ATP)] influenced the rate of respiration of tuber tissue of Solanum tuberosum L. Transgenic plants were produced that contained the coding sequence of the Escherichia coli pfkA gene linked to a patatin promoter. Expression of this chimaeric gene in tubers resulted in a 14to 21-fold increase in the maximum catalytic activity of PFK(ATP) without affecting the activities of the other glycolytic enzymes. Tubers, and ‘aged’ disks of tuber tissue, from transformed plants showed no more than a 30% fall in the content of hexose 6-monophosphates; the other intermediates of glycolysis increased threeto eightfold. Fructose-2,6-bisphosphate was barely detectable in aged disks of transformed tubers. The relative rates of 14CO2 production from [1-14C]-and [6-14C]-glucose supplied to disks of transformed and control tubers were similar. Oxygen uptake and CO2 production by aged disks of transformed tubers did not differ significantly from those from control tubers. The same was true of CO2 production, in air, and in nitrogen, for tuber tissue. It is concluded that PFK(ATP) does not dominate the control of respiration in potato tubers.

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.

The major fluxes of carbohydrate metabolism were estimated during starch breakdown by ripening bananas (Musa cavendishii Lamb ex Paxton). Hands of bananas, untreated with ethylene, were allowed to ripen in the dark at 21° C. Production of CO2 and the contents of starch, sucrose, glucose and fructose of intact fruit were determined for a period of 10 d that included the climacteric. The detailed distribution of label was determined after supplying the following to cores of pulp from climacteric fruit: [U-14C]-, [1-14C]-, [3,4-14C]-and [6-14C]glucose, [U-14C]glycerol, 14CO2. The data obtained were used to estimate the following fluxes, values given as μmol hexose · (g FW)−1 · h−1 in parenthesis: starch to hexose monophosphates (5.9) and vice versa (0.4); hexose monophosphates to sucrose (7.7); sucrose to hexose (4.7); hexose to hexose monophosphate (3.8); glycolysis (0.5–1.6); triose phosphate to hexose monophosphates (0.14); oxidative pentose-phosphate pathway (0.48); CO2 fixation in the dark (0.005). These estimates are related to our understanding of carbohydrate metabolism during ripening.

The aim of this work was to discover how leucoplasts from suspension cultures of soybean (Glycine max L.) oxidize hexose monophosphates. Leucoplasts were isolated from protoplast lysates on a continuous gradient of Nycodenz with a yield of 28% and an intactness of 80%. Incubation of the leucoplasts with 14C-labelled substrates led to 14CO2 production, that was dependent upon leucoplast intactness, from [U-14C]glucose 6-phosphate, [U-14C]glucose 1-phosphate, [U-14C] fructose 6-phosphate and [U-14C]glucose+ATP, but not from [U-14C]fructose-1,6-bisphosphate or [U-14C]triose phosphate. The yield from [U-14C]glucose 6-phosphate was at least four times greater than that from any of the other substrates. When [1-14C]-, [2-14C]-, [3,4-14C]-, and [6-14C]glucose 6-phosphate were supplied to leucoplasts significant 14CO2 production that was dependent upon leucoplast intactness was found only for [1-14C]glucose 6-phosphate. It is argued that soybean cell leucoplasts oxidize glucose 6-phosphate via the oxidative pentose phosphate pathway with very little recycling, and that in these plastids glycolysis to acetyl CoA is negligible.

Tissue from developing tubers, mature tubers, and mature tubers that had been stored at 4°C, was killed and extracted with trichloroacetic acid in diethylether. Inorganic pyrophosphate was detected in the aqueous phase of the extracts with pyrophosphate fructose-6-phosphate-1-phosphotransferase. No pyrophosphate could be detected in extracts that had been pretreated with pyrophosphatase. Pyrophosphate contents of about 3–12 nmol/g fresh weight were found. It is suggested that potato tubers contain sufficient pyrophosphate to allow the enzyme UDPglucose pyrophosphorylase to convert UDPglucose to glucose-1-phosphate during cucrose breakdown.

The aim of this work was to determine the maximum catalytic activity and intracellular location of NADP+-linked malic enzyme (EC 1.1.1.40) in C3 plants. Appreciable activities, ranging from 80 to 712 nmol · (gFW)−1 · min−1, were found in a wide range of tissues (roots and leaves of Pisum sativum L., cotyledons of Cucurbit a pepo Alef., developing seeds of Brassica napus L., mesocarp of Persea americana Gaertn., and suspension cultures of Glycine max L.). Overall, activity showed a rough positive correlation with biosynthesis. Differential and density-gradient fractionation of extracts of the cotyledons of germinating marrow (C. pepo) and lysates of protoplasts of suspension cultures of G. showed that the enzyme had the same distribution as the plastid marker enzymes. It is suggested that in C3 plants NADP+-linked malic enzyme is confined to the plastids and involved in biosynthesis.

The aim of this work was to purify and characterize sucrose-phosphate synthase from 38 hr germinated seeds of Pisum sativum . Chromatography on ω-aminohexyl Sepharose 4B and PD 10 Sephadex G-25M followed by fast protein liquid chromatography on a Mono Q anion exchange column and a Superose 6 gel filtration column gave a preparation of specific activity 4.22 μmol min −1 mg −1 protein. SDS PAGE showed four major and five minor bands. Native molecular mass was 456 000. Activity was optimum at pH 7.0 and 5 mM magnesium chloride. Hyperbolic kinetics were found for UDPglucose, apparent K m 2.4 mM and, in the presence of glucose 6-phosphate, for fructose 6-phosphate. No inhibition was detected with either sucrose or sucrose phosphate. Glucose 6-phosphate and fructose-1,6-bisphosphate stimulated activity, but Pi and UDP were inhibitory.

This work provides further evidence that plants contain appreciable amounts of inorganic pyrophosphate (PPi), and that breakdown of phosphoribosyl pyrophosphate (PPRibP) does not contribute significantly to the PPi detected in plant extracts. Inorganic pyrophosphate in extracts of the roots of Pisum sativum L., clubs of the spadices of Arum maculatum L., and the developing endosperm of Zea mays L. was assayed with pyrophosphate fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90), and with sulphate adenyltransferase (EC 2.7.7.4). The two different assays gave the same value for PPi content, and for recovery of added PPi. It was shown that PPRibP is converted to PPi during the extraction of PPi. However, the amounts of PPRibP in clubs of A. maculatum and the developing endosperm of Z. mays were negligible in comparison with the contents of PPi.

This work was done to determine whether the inorganic-pyrophosphate (PPi) content of plant tissues changes when the rate of glycolysis is altered. Treatment of excised clubs of the spadix of Arum maculatum L. and root apices of Pisum sativum L. with 2,4-dinitrophenol increased the rates of respiration but had no detectable effects on PPi contents. When the two tissues were subjected to up to 60 min anoxia, no changes in PPi were detected. Anoxia was shown to lead to a fall in ATP and concomitant rises in ADP and AMP in pea roots. It is argued (i) that variation in the rate of glycolysis was not accompanied by detectable changes in PPi content, (ii) that this observation does not favour the view that pyrophosphate fructose 6-phosphate 1-phosphotransferase mediates appreciable entry into glycolysis, and (iii) that PPi content can be maintained when respiratory-chain phosphorylation is inhibited.

The aim of this work was to develop a method for the isolation of amyloplasts from the endosperm of developing grains of Triticum aestivum . Protoplasts were prepared by digesting endosperm with cellulase and pectinase. Procedures were developed for the lysis of the protoplasts by DEAE-dextran, and for fractionation of the lysate on a continuous gradient of Nycodenz. The latter gave amyloplast preparations that contained 20% of the plastid marker enzymes in the lysate, were not contaminated by microbodies or the endomembrane system, and showed less than 3% contamination by cytosol and 5% by mitochrondria. When removed from the gradient 54% of the amyloplasts remained intact.

The aim of this work was to discover whether the Ra locus in Pisum sativum affected the maximum catalytic activities of: starch synthase, ADPglucos