Regulation of fructose-1,6-biphosphatase activity in intact chloroplasts. Studies of the mechanism of inactivation.

1. The aim of this work was to investigate the mechanism of dark inactivation of fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) in isolated intact chloroplasts of Triticum aestivum. 2. Dark inactivation of the enzyme, which was rapid under aerobic conditions, was prevented under anaerobic conditions when chloroplasts were incubated in the absence of an electron acceptor. Electron acceptors such as oxaloacetate readily brought about inactivation under anaerobic conditions whether chloroplasts were illuminated or in the dark. Inactivation of the enzyme also occurred if illuminated or darkened anaerobic chloroplasts were exposed to oxygen. 3. Pyocyanine, which catalyses a cyclic electron flow around Photosystem I, also caused inactivation of the enzyme in illuminated, anaerobic chloroplasts. 4. It is proposed that the activity of fructose-1,6-bisphosphatase is regulated by the availability of electrons, and thus by electron acceptors, and that dark inactivation may occur by a direct reversal of the activation process.

Phosphoenolpyruvate carboxykinase and gluconeogenesis in cotyledons of Cucurbita pepo.

1. The aim of this work was to investigate the role of phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating) EC 4.1.1.49) in the conversion of fat to sugar by the cotyledons of seedlings of Cucurbita pepo. 2. The enzyme was partially purified from the cotyledons of 5-day-old seedlings. The Michaelis constants for oxaloacetate and ATP were 56 and 119 micron, respectively. The decarboxylation reaction was optimum at pH 7.4. A range of intermediary metabolites did not affect the activity of the enzyme, but 3-mercaptopicolinic acid at micron concentrations was an effective inhibitor. 3. Centrifugation of extracts of 5-day-old cotyledons sedimented appreciable proportions of the ribuloseibisphosphate carboxylase, isocitrate lyase and fumarate hydratase present but very little of the phosphoenolpyruvate carboxykinase. 4. Measurements of phosphoenolpyruvate carboxykinase of cotyledons during germination showed that the maximum catalytic activity exceeded, and changed coincidently with, the rate of gluconeogenesis. 5. 3-Mercaptopicolinic acid inhibited gluconeogenesis from [1-14C]- and [2-14C]acetate supplied to excised cotyledons. The detailed distribution of 14C indicated inhibition of the conversion of oxaloacetate to phosphoenolpyruvate. 6. It is concluded that in marrow cotyledons phosphoenolpyruvate carboxykinase is in the soluble phase of the cytoplasm and catalyses a component reaction of gluconeogenesis.

Identification of the regulatory steps in gluconeogenesis in cotyledons of Cucurbita pepo.

1. The aim of this work was to discover the steps at which the conversion of oxaloacetate to glucose 6-phosphate during gluconeogenesis is regulated in the cotyledons of 5-day-old seedlings of Cucurbita pepo. 2. We estimated the maximum catalytic activities of all the enzymes in the above sequence and also the amounts of their substrates present in vivo. The results show that the reactions catalysed by fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase are the only ones in the sequence that are substantially displaced from equilibrium in vivo. 3. We also determined the effects of 3-mercaptopicolinic acid, an inhibitor of gluconeogenesis, on the amounts of the gluconeogenic intermediates present in vivo. The results show that the enzyme system, fructose-1,6-bisphosphatase: phosphofructokinase, and the system phosphoenolpyruvate carboxykinase: phosphoenolpyruvate carboxylase make major contributions to the regulation of gluconeogenesis in the cotyledons. 4. Possible mechanisms for the above regulation are discussed.

A Spatial Analysis of Physiological Changes Associated with Infection of Cotyledons of Marrow Plants with Cucumber Mosaic Virus.

Changes in host primary metabolism associated with the compatible interaction between cucumber mosaic virus and cotyledons of the marrow plant (Cucurbita pepo L.) have been localized, first by measuring activities of key enzymes in infected and uninfected regions of the cotyledon, and second by histochemical techniques applied to tissue prints of the infected region. A series of progressive metabolic changes occurs within the expanding infected lesion. Virus replication and the synthesis of viral protein at the periphery creates a strong sink demand associated with increased activities of anaplerotic enzymes, increased photosynthesis, and starch accumulation. Inside the lesion, when the synthesis of virus has declined, photosynthesis is reduced, starch is mobilized, and the emphasis of metabolism is shifted toward glycolysis and mitochondrial respiration. These changes are associated spatially with the onset of chlorosis. A decrease in total protein synthesis in this inner zone could be instrumental in some or all of these changes, leading to symptoms of viral infection.

Leaf-Atmosphere NH3 Exchange in Barley Mutants with Reduced Activities of Glutamine Synthetase.

Mutants of barley (Hordeum vulgare L. cv Maris Mink) with 47 or 66% of the glutamine synthetase (GS) activity of the wild type were used for studies of NH3 exchange with the atmosphere. Under normal light and temperature conditions, tissue NH4+ concentrations were higher in the two mutants compared with wild-type plants, and this was accompanied by higher NH3 emission from the leaves. The emission of NH3 increased with increasing leaf temperatures in both wild-type and mutant plants, but the increase was much more pronounced in the mutants. Similar results were found when the light intensity (photosynthetic photon flux density) was increased. Compensation points for NH3 were estimated by exposing intact shoots to 10 nmol NH3 mol-1 air under conditions with increasing temperatures until the plants started to emit NH3. Referenced to 25[deg]C, the compensation points were 5.0 nmol mol-1 for wild-type plants, 8.3 nmol mol-1 for 47% GS mutants, and 11.8 nmol mol-1 for 66% GS mutants. Compensation points for NH3 in single, nonsenescent leaves were estimated on the basis of apoplastic pH and NH4+ concentrations. These values were 0.75, 3.46, and 7.72 nmol mol-1 for wild type, 47% GS mutants, and 66% GS mutants, respectively. The 66% GS mutant always showed higher tissue NH4+ concentrations, NH3 emission rates, and NH3 compensation points compared with the 47% GS mutant, indicating that NH4+ release was curtailed by some kind of compensatory mechanism in plants with only 47% GS activity.

Purification, and phosphorylation in vivo and in vitro, of phosphoenolpyruvate carboxykinase from cucumber cotyledons.

Phosphoenolpyruvate carboxykinase (PEPCK) with a subunit molecular mass of 74 kDa has been purified 450-fold to homogeneity from the cotyledons of cucumber (Cucumis sativus L.). This is the first purification of the native form of the enzyme from any plant tissue. Incubation of the purified enzyme with [gamma-32P]ATP and either phosphoenolpyruvate-carboxylase kinase or mammalian cAMP-dependent protein kinase led to labelling of the enzyme in a part of the molecule separate from the active site. This was reversed by incubation with protein phosphatase 2A. Cotyledons of cucumber seedlings were also supplied with 32Pi. Homogenates of such cotyledons contained a heavily labelled polypeptide which was confirmed as PEPCK by immunoprecipitation. Labelling of PEPCK by 32Pi in darkened cotyledons was reversed by illumination.

Regulation of photosynthetic CO2-pathway enzymes by light and other factors.

The regulatory properties of enzymes of the pathway of CO2 fixation are discussed in relation to changes in regulatory parameters with changing light, CO2 and temperature.

The intercellular compartmentation of metabolites in leaves of Zea mays L.

Sap extracted from attached leaves of two-to three-week-old maize plants witt the aid of a roller device was almost devoid of bundle-sheath contamination as judged by the distribution of mesophyll and bundle-sheath markers. The extraction could be done very rapidly (less than 1 s) and the extract immediately quenched in HClO4 or reserved for enzyme assay. Comparison of the contents of metabolites in intact leaves and in the leaf extract allowed estimation of the distribution of metabolites between the bundle-sheath and the mesophyll compartments. Substantial amounts of metabolites such as malate and amino acids were present in the non-photosynthetic cells of the midrib. In the illuminated leaf, triose phosphate was predominantly located outside the bundle-sheath while the major part of the 3-phosphoglycerate was in the bundle sheath. The results indicate the existence of concentration gradients of triose phosphate and 3-phosphoglycerate in the leaf which are capable of maintaining carbon flow between the mesophyll and bundle-sheath cells during photosynthesis. There was no evidence for the existence of a gradient of pyruvate between the bundle-sheath and the mesophyll cells.

Dark fixation of CO2 during gluconeogenesis by the cotyledons of Cucurbita pepo L.

We did this work to discover the pathway of CO2 fixation into sugars in the dark during gluconeogenesis by the cotyledons of 5-day-old seedlings of Cucurbita pepo L. We paid particular attention to the possibility of a contribution from ribulosebisphosphate carboxylase. The detailed distribution of (14)C after exposure of excised cotyledons to (14)CO2 in the dark was determined in a series of pulse and chase experiments. After 4s in (14)CO2, 89% of the (14)C fixed was in malate and aspartate. In longer exposures, and in chases in (12)CO2, label appeared in alanine, phosphoenolpyruvate, 3-phosphoglycerate and sugar phosphates, and accumulated in sugars. The transfer of label from C-4 acids to sugars was restricted by inhibition of phosphoenolpyruvate carboxykinase in vivo by 3-mercaptopicolinic acid. We conclude as follows. Initial fixation of CO2 in the dark is almost entirely into phosphoenolpyruvate, probably via phosphoenolpyruvate carboxylase (EC 4.1.1.31) which we showed to be present in appreciable amounts. Incorporation into sugars occurs chiefly, if not completely, as a result of randomization of the carboxyl groups of the C-4 acids and subsequent conversion of the oxaloacetate to sugars via the accepted sequence for gluconeogenesis. Ribulosebisphosphate carboxylase appears to make very little contribution to sugar synthesis from fat.

Isolation of Protoplasts and Chloroplasts from Flag Leaves of Triticum aestivum L.

Intact protoplasts and chloroplasts have been isolated from mature flag leaves of wheat (Triticum aestivum L.). Both showed high rates of photosynthesis, the best of which equaled those observed in the parent tissue (greater than 150 micromoles O(2) per milligram chlorophyll per hour). The presence of ethylenediaminetetraacetate and an alkaline medium (pH 8.4) were required in the isolation and assay for the achievement of maximum rates of photosynthesis by chloroplasts. Photosynthesis by isolated chloroplasts was inhibited at very low concentrations of external orthophosphate.

The relationship between contents of photosynthetic metabolites and the rate of photosynthetic carbon assimilation in leaves of Amaranthus edulis L.

The relationship between the gas-exchange characteristics of attached leaves of Amaranthus edulis L. and the contents of photosynthetic intermediates was examined in response to changing irradiance and intercellular partial pressure of CO2. After determination of the rate of CO2 assimilation at known intercellular CO2 pressure and irradiance, the leaf was freeze-clamped and the contents of ribulose-1,5-bisphosphate, glycerate-3-phosphate, fructose-1,6-bisphosphate, glucose-6-phosphate, fructose-6-phosphate, triose phosphates, phosphoenolpyruvate, pyruvate, oxaloacetate, aspartate, alanine, malate and glutamate were measured. A comparison between the sizes of metabolite pools and theoretical calculations of metabolite gradients required for transport between the mesophyll and the bundle-sheath cells showed that aspartate, alanine, glycerate-3-phosphate and triose phosphates were present in sufficient quantities to support transport by diffusion, whereas pyruvate and oxaloacetate were not likely to contribute appreciably to the flux of carbon between the two cell types. The amounts of ribulose-1,5-bisphosphate were high at low intercellular partial pressures of CO2, and fell rapidly as the CO2-assimilation rate increased with increasing intercellular partial pressures of CO2, indicating that bundle-sheath CO2 concentrations fell at low intercellular partial pressures of CO2. In contrast, the amount of phosphoenolpyruvate and of C4-cycle intermediates declined at low intercellular partial pressures of CO2. This behaviour is discussed in relation to the co-ordination of carbon assimilation between the Calvin and C4 cycles.

Limitation of photosynthesis by changes in temperature : Factors affecting the response of carbon-dioxide assimilation to temperature in barley leaves.

The aim of this work was to examine the effect of abrupt changes in temperature in the range 5 to 30°C upon the rate of photosynthetic carbon assimilation in leaves of barley (Hordeum vulgare L.). Measurement of the CO2-assimilation rate in relation to the intercellular partial pressure of CO2 at different temperatures and O2 concentrations and at saturating irradiance showed that as the temperature was decreased photosynthesis was saturated at progressively lower CO2 partial pressures and that the transition between the CO2-limited and ribulose-1,5-bisphosphate-regeneration-limited rate became more abrupt. Feeding of orthophosphate to leaves resulted in an increased rate of CO2 assimilation at lower temperatures at around ambient or higher CO2 partial pressures both in 20% O2 and in 2% O2 and it removed the abruptness in the transition between the CO2-limited and ribulose-1,5-bisphosphate-regeneration-limited rates. Phosphate feeding tended to inhibit carbon assimilation at higher temperatures. The response of carbon assimilation to temperature was altered by feeding orthophosphate, by changing the concentrations of CO2 or of O2 or by leaving plants in the dark at 4°C for several hours. Similarly, the response of carbon assimilation to phosphate feeding or to changes in 2% O2 was altered by leaving the plants in the dark at 4°C. The mechanism of limitation of photosynthesis by an abrupt lowering of temperature is discussed in the light of the results.

Some relationships between contents of photosynthetic intermediates and the rate of photosynthetic carbon assimilation in leaves of Zea mays L.

The relationship between the gas-exchange characteristics of attached leaves of Zea mays L. and the contents of photosynthetic intermediates was examined at different intercellular partial pressure of CO2 and at different irradiances at a constant intercellular partial pressure of CO2. (i) The behaviour of the pools of the C4-cycle intermediates, phosphoenolpyruvate and pyruvate, provides evidence for light regulation of their consumption. However, light regulation of phosphoenolpyruvate carboxylase does not influence the assimilation rate at limiting intercellular partial pressures of CO2. (ii) A close correlation between the pools of phosphoenolpyruvate and glycerate-3-phosphate exists under many different flux conditions, consistent with the notion that the pools of C4 and C3 cycles are connected via the interconversion of glycerate-3-phosphate and phosphoenolpyruvate. (iii) The ratio of triose-phosphate to glycerate-3-phosphate is used as an indicator of the availability of ATP and NADPH. Changes of this ratio with CO2 and with irradiance are compared with results obtained in C3 leaves and indicate that the mechanism of regulation of carbon assimilation by light in leaves of C4 plants may differ from that in C3 plants. (iv) The behaviour of the ribulose-1,5-bisphosphate pool with CO2 and irradiance is contrasted with the behaviour of these pools measured in leaves of C3 plants.

Factors influencing the capacity for photosynthetic carbon assimilation in barley leaves at low temperatures.

The aim of this work was to examine the effect upon photosynthetic capacity of short-term exposure (up to 10 h) to low temperatures (5° C) of darkened leaves of barley (Hordeum vulgare L.) plants. The carbohydrate content, metabolite status and the photosynthetic rate of leaves were measured at low temperature, high light and higher than ambient CO2. Under these conditions we could detect whether previous exposure of leaves to low temperature overcame the limitation by phosphate which occurs in leaves of plants not previously exposed to low temperatures. The rates of CO2 assimilation measured at 8° C differed by as much as twofold, depending upon the pretreatment. (i) Leaves from plants which had previously been darkened for 24 h had a low content of carbohydrate, had the lowest CO2-assimilation rates at low temperature, and photosynthesis was limited by carbohydrate, as shown by a large stimulation of photosynthesis by feeding glucose, (ii) Leaves from plants which had previously been illuminated for 24 h and which contained large carbohydrate reserves showed an accumulation of phosphorylated intermediates and higher CO2-assimilation rates at low temperature, but nevertheless remained limited by phosphate, (iii) Maximum rates of CO2 assimilation at low temperature were observed in leaves which had intermediate reserves of carbohydrate or in leaves which were rich in carbohydrate and which were also fed phosphate. It is suggested that carbohydrate reserves potentiate the system for the achievement of high rates of photosynthesis at low temperatures by accumulation of photosynthetic intermediates such as hexose phosphates, but that this potential cannot be realised if, at the same time, carbohydrate accumulation is itself leading to feedback inhibition of photosynthesis.

Regulation of phosphoenolpyruvate carboxylase activity in maize leaves.

The aim of this work was to investigate how light regulates the activity of phosphoenolpyruvate carboxylase in vivo in C(4) plants. The properties of phosphoenolpyruvate carboxylase were investigated in extracts which were rapidly prepared (in less than 30 seconds) from darkened and illuminated leaves of Zea mays. Illumination resulted in a significant decrease in the S(0.5)(phosphoenolpyruvate) but there was no change in V(max). The form of the enzyme from illuminated leaves was less sensitive to malate inhibition than was the form from darkened leaves. At low concentrations of phosphoenolpyruvate, the activity of the enzyme was strongly stimulated by glucose-6-phosphate, fructose-6-phosphate, triose-phosphate, alanine, serine, and glycine and was inhibited by organic acids. The enzyme was assayed in mixtures of metabolites at concentrations believed to be present in the mesophyll cytosol in the light and in the dark. It displayed low activity in a simulated ;dark' cytosol and high activity in a simulated ;light' cytosol, but activities were different for the enzyme from darkened compared to illuminated leaves.

Stimulation of photosynthesis by 2% oxygen at low temperatures is restored by phosphate.

The effect of phosphate feeding on the influence of low (2%) oxygen on photosynthetic carbon assimilation has been investigated in leaf discs of spinach (Spinacia oleracea L.) at 12°C. The following observations were made. First, after the transition from 20% O2 to 2% O2, the rate of CO2 uptake was inhibited at CO2 concentrations between about 250 and about 800 µl CO2·l(-1). Second, phosphate feeding stimulated the rate of CO2 uptake in 20% O2 at higher concentrations of CO2 (500-900 µl·l(-1)). Third, phosphate feeding stimulated the rate of CO2 uptake in 2% O2 at all but the highest (900 µl·l(-1)) and lowest 74 (µl·l(-1)) concentrations of CO2 employed. Phosphate thereby restored the stimulation of photosynthesis by 2% O2 and it did so over a wide range of lower temperatures. Fourth, oscillatory behaviour, however generated, was dampened by phosphate feeding, even at very low concentrations of CO2. Contents of leaf metabolites were measured during the transition to 2% O2 in control and phosphate-fed leaf discs. During this period the ratio glycerate-3-phosphate/triose phosphate rose steeply, but fell again only in the phosphate-treated leaf discs. These data, taken together with measured ATP/ADP ratios, showed that assimilatory power, the ratio [ATP]·[NAD(P)H]/[ADP]·[Pi]·[NAD(P)], decreased when leaves were exposed to 2% O2, but that this decrease was minimised by previous feeding of phosphate. The mechanism of phosphate limitation is discussed in the light of the results.

Influence of low temperature on respiration and contents of phosphorylated intermediates in darkened barley leaves.

The aim of this work was to examine the effect of exposure of leaves to low temperatures (5 degrees C) upon the contents of phosphorylated intermediates and respiration in darkened barley (Hordeum vulgare L.) plants which differed in their carbohydrate status. In leaves that had previously been illuminated for 24 hours, there was a large increase in amounts of phosphorylated metabolites at 5 degrees C during the first 3 hours of darkness, compared with control plants kept at 30 degrees C. Hexose phosphates accounted for about two-thirds of this increase, which reached a peak after about 3 hours. At higher temperatures, there was a peak in the amount of fructose 2,6-bisphosphate and the rate of respiration which accompanied the transient increase in phosphorylated intermediates. At 5 degrees C the increase in phosphorylated intermediates was not accompanied by appreciable changes in fructose 2,6-bisphosphate, and there was a rapid decline in the rate of respiration. Leaves that had previously been darkened for 24 hours and that were low in carbohydrate failed to accumulate phosphorylated intermediates when exposed to low temperatures. The results are discussed with respect to the acclimation of carbohydrate metabolism to low temperatures. The results suggest that respiratory carbohydrate metabolism is strictly controlled even when the carbohydrate supply and glycolytic intermediates are abundant. The possibility that accumulation of hexose phosphates may be involved in acclimation of metabolism to low temperature is discussed.

Phosphorylation of phosphoenolpyruvate carboxykinase in plants. Studies in plants with C4 photosynthesis and Crassulacean acid metabolism and in germinating seeds.

We have previously shown that phosphoenolpyruvate carboxykinase (PEPCK) is phosphorylated in vivo in the cotyledons of darkened cucumber seedlings and that phosphorylation is reversed by light [Walker and Leegood (1995) FEBS Lett. 362, 70-74]. In this study the molecular mass of PEPCK was estimated in a range of gluconeogenic seedlings and in leaves of C4 plants and plants with Crassulacean acid metabolism (CAM). Phosphorylation of PEPCK was studied in these plants by feeding tissues with [32P]Pi and assessing phosphorylation by SDS/PAGE and autoradiography of either total proteins or of immunoprecipitated protein. In gluconeogenic seedlings and most CAM plants PEPCK had a molecular mass of 74 kDa, whereas in C4 grasses the molecular mass of PEPCK was always smaller and varied from 67-71 kDa. In all gluconeogenic seedlings and leaves of CAM plants PEPCK was phosphorylated, but it was not phosphorylated in all species of C4 grasses studied. In CAM plants, phosphorylation of PEPCK occurred at night and dephosphorylation occurred during the day. In C4 grasses phosphorylation occurred when leaves were darkened and the enzyme was dephosphorylated following illumination, but it was only phosphorylated in those plants with larger (71 kDa) molecular mass forms of PEPCK.

Modulation of NADP-Malate Dehydrogenase Activity in Maize Mesophyll Chloroplasts.

When intact mesophyll chloroplasts of Zea mays var Kelvedon Glory were illuminated, activation of NADP-malate dehydrogenase occurred. Activity declined rapidly on darkening. Light activation of the enzyme was very much greater in the presence of pyruvate ( approximately 10- to 20-fold) than with the electron acceptors 3-phosphoglycerate or oxaloacetate present ( approximately 2-fold). Following preillumination in the presence of pyruvate, addition of 3-phosphoglycerate, oxaloacetate, or nitrite substantially diminished the activity of NADP-malate dehydrogenase. In these circumstances, with pyruvate and 3-phosphoglycerate present, activity could be restored by the addition of nigericin or dihydroxyacetone phosphate. Nigericin also restored activity with both oxaloacetate and pyruvate present. The effect of nitrite was more marked in the presence of low concentrations of DCMU.These observations are discussed in terms of the dependence of enzyme activity upon the redox state of ferredoxin and electron carriers; the redox state of the latter was estimated by analysis of the DCMU-induced relaxation kinetics of chlorophyll fluorescence quenching in the presence of different substrates.

Regulation of fructose-1,6-bisphosphatase activity in leaves.

Fructose-1,6-bisphosphatase (EC 3.1.3.11) activity increased markedly (greater than 10-fold) upon illumination of wheat leaves. Darkening caused a relatively slow but complete reversal of light activation. The effects of O2 and CO2 concentration and light intensity on fructose-bisphosphatase activation were measured. In ratelimiting light, 2% O2 stimulated enzyme activity, whereas varying the CO2 concentration had little effect. In saturating light, lowering the oxygen tension had no effect, but CO2 at near-saturating concentrations for photosynthesis inhibited enzyme activity. Dark inactivation of the enzyme was completely prevented by incubation of leaves in N2, but was facilitated by O2, indicating that O2 is the major oxidant in darkened leaves. It is argued that while fructose bisphosphatase is redox-regulated in leaves, modulation of enzyme activity by this mechanism is unlikely to contribute to the regulation of CO2 fixation in leaves.