The response of plant metabolism to genetic engineering of specific enzymes varies from the predictably obvious to the almost undetectable. This is probably due to the existence of alternative pathways, and to the sharing of control between many of the enzymes of a given pathway. These features of plant metabolism make it difficult to predict how successful attempts to manipulate it will be.

Experiments with transgenic plants are beginning to unravel how the products of photosynthesis get into the phloem for transport throughout the living tissues of the plant.

The aim of this work was to compare the carbohydrate metabolism of suspension cultures of soybean (Glycine max), intolerant of anoxia, with that of cultures of rice (Oryza sativa), tolerant of anoxia. Soybean cells in anoxia showed no increase in fresh weight, dry weight or extractable protein, and labelled few proteins when supplied with [35S]methionine. There were modest (50%) increases in the maximum catalytic activities of sucrose synthase, phosphofructokinase, pyruvate kinase and phosphoenolpyruvate carboxylase. There was a three-fold increase in alcohol dehydrogenase and no detectable change in lactate dehydrogenase and pyrophosphate:fructose 6-phosphate 1-phosphotransferase [PFK(PPi)]. The rates of respiration (O2 uptake and CO2 production in air) and fermentation (CO2 production in nitrogen), all declined with time in anoxia and the cells died after six days in anoxia. Rice cells in anoxia showed small increases in weight and protein content, and labelled many proteins with [35S]methionine. Increased maximum catalytic activities were found for sucrose synthase (× 2), PFK(PPi) (× 6), pyruvate kinase (× 2), alcohol dehydrogenase (× 5) and lactate dehydrogenase (× 2). When rice cells were grown in anoxia, respiration declined steadily. Fermentation increased after four days in anoxia and then declined steadily. However, both respiration and fermentation were still appreciable even after 52 days in anoxia.

The aim of this work was to discover whether the cells of the bundle sheath of the leaves of maize (Zea mays) contained pyrophosphate:fructose-6-phosphate 1-phos-photransferase (PFP) and fructose 2,6-bisphosphate (Fru-2,6-P2). Physiologically active preparations of bundle sheath cells from leaves of 4- to 6-week-old plants showed activities of PFP, 6-phosphofructo-2-kinase (6-PF-2-K), and fructose-2,6-bisphosphatase (Fru-2,6-Pase) of 38, 1.8, and 15 nmol min−1 mg−1 chlorophyll, respectively, and contained 75 pmol mg−1 chlorphyll Fru-2,6-P2. For the above enzymes, and marker enzymes for the bundle sheath and for mesophyll cells, the ratios of the activities in leaf extracts to those in bundle sheath extracts were determined. The ratios for PFP, 6-PF-2-K, Fru-2,6-Pase, and Fru-2,6-P2 were intermediate between those found for the mesophyll markers and bundle sheath markers. The distribution of PFP activity after nonaqueous fractionation of leaves differed from that of the bundle sheath and mesophyll marker enzymes. It is argued that maize bundle sheaths can contain significant activity of PFP and amounts of Fru-2 ,6-P2.

The aim of this work was to establish the pathways of carbohydrate oxidation in the bundle sheaths of maize (Zea mays L.). Preparations of bundle sheaths were made, characterized, and allowed to metabolize specifically labelled [14C]glucose, [14C]pyruvate, [14C]acetate and [U-14C]malate for up to 5 hours in the dark. The patterns of 14CO2 production and the distribution of label are consistent with the simultaneous operation of glycolysis, the oxidative pentose phosphate pathway, and the citric acid cycle. It is suggested that the path of carbon in bundle sheath respiration is similar to that in other plant cells.

This work was carried out to investigate the relative roles of phosphofructokinase and pyrophosphate-fructose-6-phosphate 1-phosphotransferase during the increased glycolysis at the climacteric in ripening bananas (Musa cavendishii Lamb ex Paxton). Fruit were ripened in the dark in a continuous stream of air in the absence of ethylene. CO2 production, the contents of glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, phosphoenolpyruvate and PP(i); and the maximum catalytic activities of pyrophosphate-fructose-6-phosphate 1-phosphotransferase, 6-phosphofructokinase, pyruvate kinase and phosphoenolpyruvate carboxylase were measured over a 12-day period that included the climacteric. Cytosolic fructose-1,6-bisphosphatase could not be detected in extracts of climacteric fruit. The peak of CO2 production was preceded by a threefold rise in phosphofructokinase, and accompanied by falls in fructose 6-phosphate and glucose 6-phosphate, and a rise in fructose 1,6-bisphosphate. No change in pyrophosphate-fructose-6-phosphate 1-phosphotransferase or pyrophosphate was found. It is argued that phosphofructokinase is primarily responsible for the increased entry of fructose 6-phosphate into glycolysis at the climacteric.

Measurements of total tissue activity, and of the proportion of that activity confined to mitochondria, gave estimates of the maximum catalytic activities of citrate synthase, aconitase, isocitrate dehydrogenase (NAD+), fumarase, and malate dehydrogenase (NAD+) involved in the Krebs cycle in the developing spadices of Arum maculatum. Isocitrate dehydrogenase had the lowest activity and malate dehydrogenase the highest. The activity of each enzyme rose markedly during spadix development: comparison of these activities with flux in vivo showed that the cycle is subject to coarse control during development. Preparations of mitochondria from Arum spadix and from florets of cauliflower (Brassica oleracea) were used to determine the mass-action ratios of citrate synthase, aconitase, and fumarase during the oxidation of pyruvate in the presence of malate. Collectively, the measurements of enzymes and substrates suggest that in the mitochondria the reactions catalysed by citrate synthase and isocitrate dehydrogenase (NAD+) are markedly displaced from equilibrium in vivo, whereas those catalysed by aconitase, fumarase and malate dehydrogenase (NAD+) are close to equilibrium.

Abstract My remit embraces the whole of intermediary metabolism: my intention is to concentrate on the central emerging hypothesis that the control of sucrose metabolism is the key to partitioning in higher plants (Farrar 1992). In this hypothesis sucrose is seen both as the major product of photosynthesis and as the main form in which the non-photosynthetic cells of the plant receive their carbon and energy. Photosynthetic cells are regarded as exerting control over the rest of the plant by regulating the latter’s supply of carbon and energy. The non-photosynthetic or ‘sink’ cells of the plant, in turn, are seen as influencing the photosynthetic cells by an ability of sucrose or its products to diminish the rate of photosynthesis. This inter-relationship could form the basis of the allometric growth of plants illustrated by the constant ratio of the relative growth rates of shoot and root. l shall concentrate on leaf metabolism, as partitioning in sinks has recently been discussed in detail (Pollock et al. 1992).

The aim of this work was to discover whether fructose-1,6-bisphosphatase (FBPase) is present in higher-plant cells that synthesize storage starch. The following were examined: suspension cultures of soybean (Glycine max), tubers of potato (Solanum tuberosum), florets of cauliflower (Brassica oleracea), developing endosperm of maize and of sweet corn (Zea mays), roots of pea (Pisum sativum), and the developing embryos of round and wrinkled varieties of pea. Unfractionated extracts of each tissue readily converted fructose 1,6-bisphosphate to fructose 6-phosphate in assays for both plastidic and cytosolic FBPase. These conversions were not inhibited by 20 microM-fructose 2,6-bisphosphate. Except in extracts of pea embryos and sweet-corn endosperm, treatment with affinity-purified antibodies to pyrophosphate: fructose-6-phosphate 1-phosphotransferase reduced the above fructose 6-phosphate production to the rate found with boiled extracts. The antibody-resistant activity from sweet corn was slight. In immunoblot analyses, antibody to plastidic FBPase did not react positively with any protein in extracts of soybean cells, potato tuber, cauliflower florets, maize endosperm and pea roots. Positive reactions were found for extracts of embryos of both round and wrinkled varieties of peas and endosperm of sweet corn. For pea embryos, but not for sweet-corn endosperm, the Mr of the recognized protein corresponded to that of plastidic FBPase. It is argued that soybean cells, potato tuber, cauliflower florets, maize (var. White Horse Tooth) endosperm and pea roots lack significant activity of plastidic FBPase, but that this enzyme is present in developing embryos of pea. The data for sweet corn (var. Golden Bantam) are not decisive. It is also argued that, where FBPase is absent, carbon for starch synthesis does not enter the amyloplast as triose phosphate.

This work was done to test claims (Sangwan and Singh, Physiol. Plant. 73: 21-26) that the developing endosperm of wheat (Triticum aestivum L.) contains a cytosolic and a plastidic fructose- 1,6-bisphosphatase (EC 3.1.3.11; FBPase). Repetition of the procedure of Sangwan and Singh with extracts of developing endosperm of Triticum aestivum cv. Mercia produced two peaks of apparent FBPase activity on elution from DEAE-cellulose. Both peaks showed high activity of pyrophosphate:fructose-6-phos-phate 1-phosphotransferase [EC 2.7.1.90; PFK(PP(i) )]. The apparent FBPase activity in both peaks was stimulated by 20 μM fructose-2,6-bisphosphate and inhibited by antibodies to PFK(PP(i) ). Antibody to plastidic FBPase did not react positively in an immunoblot analysis with any protein of M(r) comparable to that of known FBPase in either peak. It is argued that the ability of each peak to convert fructose-1,6-bisphosphate to fructose-6-phosphate was due to PFK(PP(i) ). and that there remains no substantiated evidence for the presence of a plastidic FBPase in the developing endosperm of wheat.

The aim of this work was to discover the role of pyrophosphate : fructose-6-phosphate 1-phosphotransferase [PFK(PP;)]. Substrates and enzymes of sucrose breakdown were measured during starch accumulation by the endosperm of wild type and the sh 1 shrunken mutant of Zea mays L. The results suggested that the mutant depended primarily on alkaline invertase to break down sucrose, and showed that the activities of PFK(PPi) and UDPglucose pyrophosphorylase and contents of pyrophosphate and fructose2, 6-bisphosphate (Fru-2,6-P2) were no lower in the mutant than in the wild type. Appreciable activities of PFK(PPi), which were not responsive to Fru-2,6-P2, were found in the following red algae, which lack sucrose: Audouninella purpurea, Polysiphonia sp., Mastocarpus stellatus, Rhodymenia pseudopalmata. No PFK(PP;) was found in the bacterium Paracoccus denitrificans. Addition of sucrose to starved suspension cultures of Glycine max L. led to increases in oxygen uptake and Fru-2,6-P2 content. Glucose produced the same effects. It is argued that, although one of the functions of PFK(PP;) is production of pyrophosphate for sucrose breakdown via sucrose synthase, the key role of the enzyme is the maintenance of the cytosolic concentration of pyrophosphate according to the supply and demand of the latter.

The maximum catalytic activities of acid invertase, alkaline invertase and sucrose synthase were shown to exceed estimates of the rate of sucrose break

The effects of lowering the temperature from 25 degrees C to 2-8 degrees C on carbohydrate metabolism by plant cells are considered. Particular emphasis is placed on the mechanism of cold-induced sweetening in tubers of potato (Solanum tuberosum). Temperatures between 0 and 10 degrees C were shown to cause a marked reduction in the rate of respiration of a wide range of plant tissues. At these temperatures the ability of suspension cultures of soybean (Glycine max), and callus cultures and tubers of potato to metabolize [14C]glucose was appreciably diminished. The detailed distribution of 14C showed that lowering the temperature decreased the proportion of the metabolized [14C]glucose that entered the respiratory pathways and increased the proportion converted to sucrose. Pulse and chase experiments, in which [14C]glucose was supplied to potato tubers at 2 and 25 degrees C, showed that lowering the temperature led to accumulation of label in hexose 6-phosphates, which were subsequently converted to sucrose. The patterns of 14CO2 production from specifically labelled [14C]glucose supplied to soybean suspension cultures and disks of potato tuber suggested that lowering the temperature reduced the activity of glycolysis more than that of the oxidative pentose phosphate pathway. It is argued that the above experiments demonstrate that lowering the temperature not only reduces the rate of carbohydrate metabolism but also alters the relative activities of the different pathways involved. A disproportionate reduction in glycolysis at the lower temperatures is suggested. Mature tubers of many varieties of potato accumulate sucrose and hexose when stored between 2 and 10 degrees C. Starch is the source of carbon for this synthesis of sugar. We could not detect cytosolic fructose-1,6-bisphosphatase in potato tubers and suggest that carbon for sugar synthesis in the cold leaves the amyloplast, not as triose phosphate, but probably as a six-carbon compound. Evidence is presented that phosphofructokinase (EC 2.7.1.11) plays a major role in regulating the entry of hexose 6-phosphates into glycolysis in potato tubers. Phosphofructokinase was purified from potato tubers and shown to consist of four forms. Three of these forms were shown to have higher Q10 values over the range 2-6 degrees C than over the range 12-16 degrees C and are regarded as being cold-labile. No such cold-lability was detected for the key enzymes involved in sucrose synthesis and the oxidative pentose phosphate pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Six-day-old seedlings of Pisum sativum were incubated for 5 hr with their roots in [14C]glucose, the pulse, and then transferred to glucose for

Developing embryos from growing pods of round (Birte) and wrinkled (Greenshaft) varieties of Pisum sativum were studied during the period of rapid growth. No significant activity of UDP-glucose phosphorylase or of pyrophosphohydrolases for ATP or UTP was detected in extracts of embryos. Estimates of the maximum catalytic activities of UDP-glucose pyrophosphorylase and pyrophosphate: fructose 6-phosphate 1-phosphotransferase [PFK(PPi)] showed them to change during development roughly in parallel with that of sucrose synthase, and to exceed the maximum estimates of the rate of sucrose breakdown. Measurements of the embryo content of UTP, UDP-glucose, inorganic pyrophosphate, hexose monophosphates and fructose-1,6-bisphosphate suggest that the reactions catalysed by sucrose synthase, UDP-glucose pyrophosphorylase and PFK(PPi) approach equilibrium in vivo. Fructose-2,6-bisphosphate content showed a positive correlation with the rate at which the embryos converted sucrose to starch. Incubation of embryos in [U-14C]sucrose led to substantial labelling of UDP-glucose. It is suggested that sucrose synthase makes a major contribution to sucrose breakdown by forming UDP-glucose that is converted to glucose 1-phosphate by UDP-glucose pyrophosphorylase using pyrophosphate generated by PFK(PPi).

This work is an investigation of the roles of sucrose synthase, acid invertase and alkaline invertase in sucrose metabolism in developing embryos of cv. Birte and cv. Greenshaft of Pisum sativum. The detailed distribution of label from [U-14C] sucrose showed that Birte embryos converted more sucrose to starch and protein than did Greenshaft embryos. From 0.06 to 0.53 M [14C] sucrose, uptake by embryos of both varieties exceeded its metabolism. No acid invertase was found in embryos of either variety. The maximum catalytic activity of sucrose synthase was roughly ten times that of alkaline invertase: both activities rose steadily during development. The rates of uptake and metabolism of 14C]sucrose were used to estimate the rates of sucrose breakdown in vivo. The results suggest that both sucrose synthase and alkaline invertase contribute to this breakdown, with the former making the greater contribution.

Excised young shoots of Typha angustifolia were incubated with [U-14C]sucrose in air, nitrogen and mixtures containing 14, 11, 8, 6, 4, 2 and 1% oxygen. Total 14C metabolized was not significantly affected by oxygen concentration. The percentage of metabolized 14C recovered in the fractions that contained the major macromolecules was not reduced until the oxygen concentration reached 1% and was appreciable even in anoxia. Pulse and chase experiments with [14C]valine confirmed appreciable protein synthesis in anoxia and indicated that 90%. of the anoxically synthesized protein was in the cell-wall and the membrane fractions of the shoot. This behaviour of Typha shoots, which had no living connexion with the atmosphere, is contrasted with that of a wetland plant with well developed aerenchyma.

The aim of this work was to discover the gross intracellular distribution of protein formed during the anoxic germination and growth of seedlings of Oryza sativa. Seedlings were grown anoxically and aseptically and were labelled with [14C]valine 96–108 h after imbibition. Most of the protein labelled was in the coleoptile but up to 20 % was in the seeds. Carefully prepared homogenates of the labelled coleoptiles were fractionated by differential and sucrose density centrifugation. Whilst much of the labelled protein did not sediment at 70,000 x g, an appreciable proportion did so. Some of the latter co-purified with cell wall, endomembrane fragments, and mitochondria.