Tom ap Rees

Pathways of carbohydrate fermentation in the roots of marsh plants

We did this work to discover the pathways of carbohydrate fermentation in unaerated roots of three species of flood-tolerant plants, Ranunculus sceleratus, Glyceria maxima, and Senecio aquaticus. The experiments were done with the apical 1–2 cm of the roots and the results for the three species were similar. The maximum catalytic activities of alcohol dehydrogenase, lactate dehydrogenase, phosphoenolpyruvate carboxylase, NADP-dependent malic enzyme, and phosphofructokinase were appreciable and roughly comparable. Reduced aeration of the roots led to 1.5 to 5-fold increases in the maximum catalytic activities of alcohol dehydrogenase, small increases in those of lactate dehydrogenase in two species, and no increase in those of phosphoenolpyruvate carboxylase and phosphofructokinase. Phosphoenolpyruvate carboxykinase could not be detected. Metabolism of [U-14C]sucrose under anaerobic conditions by excised roots, grown without aeration, led to appreciable labelling of ethanol and alanine, slight but significant labelling of lactate, and minimal labelling of malate and related organic acids. Incubation of similar excised roots under anaerobic conditions for 4 h caused marked accumulation of ethanol, smaller accumulation of lactate, and no detectable accumulation of malate. We conclude that in all three species fermentation of carbohydrate results in the accumulation of predominant amounts of ethanol, smaller amounts of lactate, no significant quantities of malate, and probably appreciable amounts of alanine. Crawfords metabolic theory of flooding tolerance is held to be incompatible with these results.

Capacities of pea chloroplasts to catalyse the oxidative pentose phosphate pathway and glycolysis

Abstract The aim of this work was to measure the capacities of pea (Pisum sativum) shoot chloroplasts to catalyse the oxidative pentose phosphate pathway and glycolysis. Of the total activities in the unfractionated homogenates, appreciable proportions of those of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and phosphofructokinase, and smaller but significant proportions of those of phosphopyruvate hydratase and pyruvate kinase were recovered in crude preparations of chloroplasts, and co-purified with intact chloroplasts on sucrose gradients. The activities in the chloroplasts showed considerable latency that was closely correlated with chloroplast integrity. Phosphoglyceromutase activity in the above preparations of chloroplasts did not exceed that expected from cytoplasmic contamination. The mass-action ratio for phosphoglyceromutase in illuminated isolated chloroplasts differed markedly from the enzymes equilibrium constant. Isolated chloroplasts converted 2-phosphoglycerate to pyruvate. The enzyme activities of the chloroplasts were compared with the rates of respiration and starch breakdown in pea leaves in the dark. It is concluded that in the dark chloroplasts could metabolize all the products of starch breakdown and catalyse much of the respiration of pea shoots via the oxidative pentose phosphate pathway and/or glycolysis as far as 3-phosphoglycerate. It is suggested that pea shoot chloroplasts lack phosphoglyceromutase but contain some phosphopyruvate hydratase and pyruvate kinase.

Sugar metabolism in developing tubers of Solanum tuberosum

Abstract Regulation of the sugar content of the developing tubers of three varieties (King Edward, Maris Bard, Pentland Javelin) of Solanum tuberosum was investigated. Sucrose, glucose, fructose, UDP-glucose and fructose-2,6-bispbosphate were measured during tuber development as were the maximum catalytic activities of acid invertase, alkaline invertase, sucrose synthase, α-glucan phosphorylase, hexokinase, phospbofructokinase and pyrophosphate: fructose 6-phosphate 1-phosphotransferase [PFK(PPi)]. Sucrose was the dominant sugar and there was less fructose than glucose; the amounts of all three per gram fresh weight fell during tuber development. The activity of hexokinase per gram fresh weight declined during development but those of the other enzymes listed did not alter significantly. No change in the amounts of fructose-2,6-bisphosphate or UDP-glucose per gram fresh weight were found. The above measurements suggest that much of the sucrose translocated to the developing tuber is metabolized via sucrose syntbase to UDP-glucose that is converted to glucose 1-phosphate by UDP-glucose pyrophosphorylase using pyrophosphate generated by PFK (PPi).

Genetic manipulation of 6-phosphofructokinase in potato tubers

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.

Effects of anaerobiosis on carbohydrate oxidation by roots of Pisum sativum

Abstract The aim of this work was to discover the effects of anaerobiosis on the breakdown of sugars by the apical 6 mm of the roots of 5-day-old seedlings of Pisum sativum . Estimates of the maximum catalytic activities of alcohol dehydrogenase, lactate dehydrogenase, phosphoenolpyruvate carboxylase and NADP-specific malic enzyme showed them to be comparable to that of phosphofructokinase. Metabolism of sucrose-[U- 14 C] by excised apices was restricted by anoxia mainly to conversion to ethanol, CO 2 alanine and glycolytic intermediates. Measurements of metabolites over a period of 240 min after transfer of excised apices to nitrogen showed a marked and continual accumulation of ethanol, a smaller continual accumulation of alanine, a small initial rise in lactate and no detectable accumulation of malate or pyruvate. The rates of CO 2 production, of accumulation of ethanol and alanine, and of the labelling of these compounds by sucrose-[ 14 C] declined markedly during the first 240 min of anaerobiosis. The conclusion is that under anaerobic conditions carbohydrate metabolism in the pea root apex is largely restricted to alcoholic fermentation, and, to a lesser degree, to alanine production.

Activities of enzymes of sugar metabolism in cold-stored tubers of Solanum tuberosum

Abstract Storage of tubers of Solanum tuberosum at 10° or 2° for 15 days did not alter significantly the maximum catalytic activities of sucrose phosphate synthetase, sucrose synthetase, glucose-6-phosphate dehydrogenase, aldolase, and glyceraldehydephosphate dehydrogenase. The temperature coefficients of phosphofructokinase, glyceraldehydephosphate dehydrogenase, and pyruvate kinase from the tubers were shown to be higher between 2° and 10° than between 10° and 25°. The rate of sugar accumulation at 2° exceeded the activity of sucrose synthetase but was less than that of sucrose phosphate synthetase. It is suggested that sucrose accumulation at 2° is catalysed by sucrose phosphate synthetase, is not due to changes in the maximum catalytic activities of any of the above enzymes, but may be due, in part, to the susceptibility of key glycolytic enzymes to cold.

Effects of anoxia on growth and carbohydrate metabolism in suspension cultures of soybean and rice

Abstract 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 [ 35 S]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(PP i )]. The rates of respiration ( O 2 uptake and CO 2 production in air) and fermentation (CO 2 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 [ 35 S]methionine. Increased maximum catalytic activities were found for sucrose synthase (× 2), PFK(PP i ) (× 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.

Pathways of carbohydrate oxidation in leaves of Pisum sativum and Triticum aestivum

Abstract The aim of this work was to establish the pathways of carbohydrate oxidation used in the dark by leaves of Pisum sativum and Triticum aestivum . Segments of young and mature leaves of pea released the carbons of glucose-[ 14 C] as 14 CO 2 in the order 3,4 > 1 > 2 > 6 whereas in segments of young and mature leaves of wheat the order was 3,4 > 1 > 6 > 2. The detailed labelling of the constituents of mature leaves of wheat by glucose-[1- 14 C], -[2- 14 C], -[3,4- 14 C], and -[6- 14 C] was determined and showed that the high yield of CO 2 from C-6 relative to that from C-2 was due to release of C-6 during pentan synthesis. Estimates were made of the maximum catalytic activities of phosphofructokinase and glucose-6-phosphate dehydrogenase in pea and wheat leaves of three ages. The results of all the above investigations strongly indicate that both pea and wheat leaves in the dark oxidize carbohydrate via glycolysis and the pentose phosphate pathway with the latter accounting for no more than a third of the total. No evidence was obtained of any major change in the relative activities of the two pathways during the development of either type of leaf.

Fluxes of carbohydrate metabolism in ripening bananas

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.

CARBOHYDRATE METABOLISM IN DEVELOPING POTATOES

In this essay we consider the extent to which we know the sequence and organization of the pathways responsible for the immediate metabolisto of the sugar that is delivered to the developing tubers of potato plants. The topic is important because such knowledge is essential ifwe are to make rational attempts to alter the composition and behavior of this crop. In order to take advantage of the ability of molecular biologists to transform potato plants we must be able to identify which genes to transform to produce a given effect; that is, we must discover how metabolism of the tuber is controlled. The initial requirement for the study of control is to establish the relevant pathways and their organization. The first point to grasp about the investigation of potato tuber metabolism is that it is exceedingly difficult. The problems of measuring enzymes and substrates, difficult in plants in general (2), ate even more so in potatoes. The causes of this interactability are not clearly established, but are almost certainly due to the release and formation of inhibitory phenolic substances during the preparation of extracts, and the persistence in such extracts of robust and active phosphatases. Whatever the causes, the effects can be spectacularly misleading. For example, attempts to compare the maximum c• activities of glucose-6-phosphate dehydrogenase and phosphofructokinase in mature tubers showed that only 10% of the dehydrogenase but as muchas 70% of the kinase were lost during extraction (38). Taken at their face value activities in such extracts would give a totally erroneous view of the tubers capacity to catalyze these two reactions. Similar pitfalls occur when measuring substrates. A method that gives accurate values for the amount of fructose 6-phosphate in tubers led to complete loss of fructose-l,6-bisphosphate (11). These, and other observations (2), make it mandatory to authenticate estimates of enzyme activity and substrate content with recovery experiments designed to measure the extent to which pure enzyme or substrate added to tuber tissue survives extraction and assay. The design of such experiments has been discussed in detail (2, 3). Data presented in this essay meet these requirements. The difficulties of working with potatoes mean that many of the key observations, on which our understanding of carbohydrate metabolism in non-