Banana ripening: implications of changes in glycolytic intermediate concentrations, glycolytic and gluconeogenic carbon flux, and fructose 2,6-bisphosphate concentration

In ripening banana (Musa sp. [AAA group, Cavendish subgroup] cv Valery) fruit, the concentration of glycolytic intermediates increased in response to the rapid conversion of starch to sugars and CO₂. Glucose 6-phosphate (G-6-P), fructose 6-phosphate (Fru 6-P), and pyruvate (Pyr) levels changed in synchrony, increasing to a maximum one day past the peak in ethylene synthesis and declining rapidly thereafter. Fructose 1,6-bisphosphate (Fru 1,6-P₂) and phosphoenolpyruvate (PEP) levels underwent changes dissimilar to those of G 6-P, Fru 6-P, and Pyr, indicating that carbon was regulated at the PEP/Pyr and Fru 6-P/Fru 1,6-P₂ interconversion sites. During the climacteric respiratory rise, gluconeogenic carbon flux increased 50- to 100-fold while glycolytic carbon flux increased only 4- to 5-fold. After the climacteric peak in CO₂ production, gluconeogenic carbon flux dropped dramatically while glycolytic carbon flux remained elevated. The steady-state fructose 2,6-bisphosphate (Fru 2,6-P₂) concentration decreased to ½ that of preclimacteric fruit during the period coinciding with the rapid increase in gluconeogenesis. Fru 2,6-P₂ concentration increased thereafter as glycolytic carbon flux increased relative to gluconeogenic carbon flux. It appears likely that the initial increase in respiration in ripening banana fruit is due to the rapid influx of carbon into the cytosol as starch is degraded. As starch reserves are depleted and the levels of intermediates decline, the continued enhancement of respiration may, in part, be maintained by an increased steady-state Fru 2,6-P₂ concentration acting to promote glycolytic carbon flux at the step responsible for the interconversion of Fru 6-P and Fru 1,6-P₂.     

Regulation of sedoheptulose-1,7-bisphosphatase by sedoheptulose-7-phosphate and glycerate,and of fructose-1,6-bisphosphatase by glycerate in spinach chloroplasts

Using partially purified sedoheptulose-1,7-bisphosphatase from spinach (Spinacia oleracea L.) chloroplasts the effects of metabolites on the dithiothreitoland Mg²⁺-activated enzyme were investigated. A screening of most of the intermediates of the Calvin cycle and the photorespiratory pathway showed that physiological concentrations of sedoheptulose-7-phosphate and glycerate specifically inhibited the enzyme by decreasing its maximal velocity. An inhibition by ribulose-1,5-bisphosphate was also found. The inhibitory effect of sedoheptulose-7-phosphate on the enzyme is discussed in terms of allowing a control of sedoheptulose-1,7-bisphosphate hydrolysis by the demand of the product of this reaction. Subsequent studies with partially purified fructose-1,6-bisphosphatase from spinach chloroplasts showed that glycerate also inhibited this enzyme. With isolated chloroplasts, glycerate was found to inhibit CO₂ fixation by blocking the stromal fructose-1,6-bisphosphatase. It is therefore possible that the inhibition of the two phosphatases by glycerate is an important regulatory factor for adjusting the activity of the Calvin cycle to the ATP supply by the light reaction.Abbreviations DTT dithiothreitol - FBPase fructose-1,6-bisphosphatase - Fru-1,6-P₂ fructose-1,6-bisphosphate - Fru-6-P fructose-6-phosphate - 3-PGA 3-phosphoglycerate - Ru-1,5-P₂ ribulose-1,5-bisphosphate - Ru-5-P ribulose-5-phosphate - SBPase sedoheptulose-1,7-bisphosphatase - Sed-1,7-P₂ sedoheptulose-1,7-bisphosphate - Sed-7-P sedoheptulose-7-phosphate This work was supported by the Deutsche Forschungsgemein-schaft.     

Perturbation of photosynthesis in spinach leaf discs by low concentrations of methyl viologen

Spinach leaf discs were floated on methyl-viologen solutions (5–200 nmol·l^-1) and the effect on photosynthetic metabolism was then investigated under conditions of saturating CO₂. Methyl viologen led to increased non-photochemical quenching, and the ATP/ADP ratio increased from <2 to >10. Comparison of the apparent quantum yield and non-photochemical quenching indicated that these concentrations of methyl viologen were only catalysing a marginal electron flux, and that the decrease in quantum yield was mainly the result of pH-triggered energy dissipation. Similar changes were also obtained after supplying tentoxin to inhibit the chloroplast ATP synthase and increase the energisation of the thylakoids. The photosystem-II acceptor, Q_A, was monitored by photochemical fluorescence quenching, and became more reduced. In contrast, the activation of NADP-malate dehydrogenase decreased, showing that the acceptor side of photosystem I becomes more oxidised. Similar changes were observed after supplying tentoxin. It is concluded that increased thylakoid energisation can lead to a substantial restriction of linear electron transport. Analysis of metabolite levels showed that glycerate-3-phosphate reduction was imporved, but that there was a large accumulation of triose phosphates and fructose-1,6-bisphosphate. This is the consequence of an inhibition of the regeneration of ribulose-1,5-bisphosphate, caused by inactivation of the stromal fructose-1,6-bisphosphatase and, to a lesser extent, phosphoribulokinase. Methyl viologen also led to inactivation of sucrose-phosphate synthase, and abolished the response of fructose-2,6-bisphosphate to rising rates of photosynthesis. This provides evidence for a primary role of glycerate-3-phosphate in controlling the activity of fructose-6-phosphate, 2-kinase and, thence, the fructose-2,6-bisphosphate concentration as the rate of photosynthesis increases. It is concluded that the very moderate ATP/ADP ratios found in chloroplasts are the results of constraints on the operation of ATP synthase. They can be increased if the thylakoid energisation is increased. However, the increased energisation acts directly or indirectly to disrupt many other aspects of photosynthetic metabolism including linear electron transport, activation of the Calvin cycle, and the control of sucrose and starch synthesis.Abbreviations and symbols Frul,6P₂ (Fru1,6Pase) fructose-1,6-bisphosphate(ase) - Fru2,6P, (Fru2,6Pase) fructose-2,6-bisphosphate(-ase) - Fru6P fructose-6-phosphate - Glc6P glucose-6-phosphate - Pi inorganic phosphate - PSI and PSII photosystems I and II - qE high energy' quenching of chlorophyll fluorescence - PGA glycerate-3-phosphate - Q_A primary stable acceptor of PSII - Ru5P (Ru1,5P₂) ribulose-5-phosphate (-1,5-bisphosphate) - SPS sucrose-phosphate synthase - triose P dihydroxyacetone phosphate plus glyceraldehyde-3-phosphate - _s apparent quantum yield Dedicated to Professor E. Latzko on the occasion of his 65th birthday     

Feedback-limited photosynthesis and regulation of sucrose-starch accumulation during cold acclimation and low-temperature stress in a spring and winter wheat

Regulation of sucrose-starch accumulation and its effect on CO₂ gas exchange and electron transport were studied in low-temperature-stressed and cold-acclimated spring (Katepwa) and winter (Monopol) cultivars of wheat (Triticum aestivum L.). Low-temperature stress of either the spring or winter cultivar was associated with feedback-limited photosynthesis as indicated by a 50–60% reduction in CO₂ assimilation rates, twofold lower ATP/ADP ratio, and threefold lower electron transport rate than 20°C-grown control plants. However, no limitations were evident at the level of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) in low-temperature-stressed plants. Cold acclimation of the spring cultivar resulted in similar feedback-limited photosynthesis observed during low-temperature stress. In contrast, cold acclimation of the winter cultivar resulted in an adjustment of CO₂ assimilation rates to that of control plants. However, we show, for the first time, that this capacity to adjust CO₂ assimilation still appeared to be associated with limited triose phosphate utilisation, a twofold lower ATP/ADP ratio, a reduction in electron transport rates but no restriction at the level of Rubisco compared to controls grown at 20°C. Thus, contrary to previous suggestions, we conclude that cold-acclimated Monopol appears to exhibit feedback limitations at the level of electron transport characteristic of cold-stressed plants despite the maintenance of high rates of CO₂ assimilation. Furthermore, the differential capacity of the winter cultivar to adjust CO₂ assimilation rates was associated with higher levels of sucrose accumulation and a threefold higher sucrose-phosphate synthase activity despite an apparent limitation in triose phosphate utilisation.Abbreviations AGPase ADP-glucose pyrophosphorylase - FBPase fructose-1,6-bisphosphatase - Fru 6-P fructose 6-phosphate - Fru 1,6-BP fructose 1,6-bisphosphate - Glc 6-P glucose 6-phosphate - PGA 3-phosphoglyceric acid - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - RuBP ribulose 1,5-bisphosphate - SPS sucrose-phosphate synthase - Triose-P triose phosphate     

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.     

Decreased ribulose-1,5-bisphosphate carboxylase-oxygenase in transgenic tobacco transformed with “antisense” rbcS

Experiments were carried out to determine how decreased expression of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) affects photosynthetic metabolism in ambient growth conditions. In a series of tobacco (Nicotiana tabacum L.) plants containing progressively smaller amounts of Rubisco the rate of photosynthesis was measured under conditions similar to those in which the plants had been grown (310 mol photons · m^–2 · s^–1, 350 bar CO₂, 22° C). (i) There was only a marginal inhibition (6%) of photosynthesis when Rubisco was decreased to about 60% of the amount in the wildtype. The reduced amount of Rubisco was compensated for by an increase in Rubisco activation (rising from 60 to 100%), with minor contributions from an increase of its substrates (ribulose-1,5-bisphosphate and the internal CO₂ concentration) and a decrease of its product (glycerate-3-phosphate). (ii) The decreased amount of Rubisco was accompanied by an increased ATP/ADP ratio that may be causally linked to the increased activation of Rubisco. An increase of highenergy-state chlorophyll fluorescence shows that thylakoid membrane energisation and high-energy-state-dependent energy dissipation at photosystem two had also increased. (iii) A further decrease of Rubisco (in the range of 50–20% of the wildtype level) resulted in a strong and proportional inhibition of CO₂ assimilation. This was accompanied by a decrease of fructose-1,6-bisphosphatase activity, coupling-factor 1 (CF₁)-ATP-synthase protein, NADP-malate dehydrogenase protein, and chlorophyll. The chlorophyll a/b ratio did not change, and enolase and sucrose-phosphate synthase activity did not decrease. It is argued that other photosynthetic enzymes are also decreased once Rubisco decreases to the point at which it becomes strongly limiting for photosynthesis. (iv) It is proposed that the amount of Rubisco in the wildtype represents a balance between the demands of light, water and nitrogen utilisation. The wildtype overinvests about 15% more protein in Rubisco than is needed to avoid a strict Rubisco limitation of photosynthesis. However, this excess Rubisco allows the wildtype to operate with lower thylakoid energisation, and decreased high-energy-state-dependent energy dissipation, hence increasing light-use efficiency by about 6%. It also allows the wildtype to operate with a lower internal CO₂ concentration in the leaf and a lower stomatal conductance at a given rate of photosynthesis, so that instantaneous water-use efficiency is marginally (8%) increased.Abbreviations C_i CO₂ concentration in the air spaces within the leaf - CF₁ coupling factor 1 - Chl chlorophyll Fru1 - 6bisP fructose-1,6-bisphosphate - F_m fluorescence yield with a saturating pulse in dark-adapted material - F_o ground-level of fluorescence obtained using a weak non-actinic modulated beam in the dark - PGA glycerate-3-phosphate - rbcS gene for the nuclear-encoded small subunit of Rubisco - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - Ru1, 5bisP ribulose-1,5-bisphosphate     

Banana Ripening: Implications of Changes in Internal Ethylene and CO(2) Concentrations, Pulp Fructose 2,6-Bisphosphate Concentration, and Activity of Some Glycolytic Enzymes

In ripening banana (Musa acuminata L. [AAA group, Cavandish subgroup] cv. Valery) fruit, the steady state concentration of the glycolytic regulator fructose 2,6-bisphosphate (Fru 2,6-P₂) underwent a transient increase 2 to 3 hours before the respiratory rise, but coincident with the increase in ethylene synthesis. Fru 2,6-P₂ concentration subsequently decreased, but increased again approximately one day after initiation of the respiratory climacteric. This second rise in Fru 2,6-P₂ continued as ripening proceeded, reaching approximately five times preclimacteric concentration. Pyrophosphate-dependent phosphofructokinase glycolytic activity exhibited a transitory rise during the early stages of the respiratory climacteric, then declined slightly with further ripening. Cytosolic fructose 1,6-bisphosphatase activity did not change appreciably during ripening. The activity of ATP-dependent phosphofructokinase increased approximately 1.6-fold concurrent with the respiratory rise. A balance in the simultaneous glycolytic and gluconeogenic carbon flow in ripening banana fruit appears to be maintained through changes in substrate levels, relative activities of glycolytic enzymes and steady state levels of Fru 2,6-P₂.     

Acclimation of photosynthesis to low temperature in Spinacia oleracea L. II. Effects of nitrogen supply

The photosynthetic capacity of leaves of N-sufficent plantsof Spinacia oleracea L. increases following transfer a constanttemperature of 10C for 10 d compared to plants maintained at25C. The effects of nitrogen nutrition on this low temperatureacclimation have been investigated in respect of CO₂ assimilation,the activities and activation states of key enzymes and thepartitioning of recently fixed carbon. N-deficiency greatlyrestricted acclimation of photosynthetic CO₂ assimilation tolow temperature at both ambient and at saturating CO₂ concentrations,indicating a restriction on accilmatory changes in both ribulose1,5-bisphosphatecarboxylase-oxygenase (Rubisco) and the reactions of ribulose1,5-bisphosphateregeneration. Nitrogen limitation led to an increase in thepartitioning of recently-fixed carbon into starch. Total proteinincreased during acclimation in both N-sufficient and N-deficientleaves and was much less affected than were the activities ofenzymes. Increases in the activation state of Rubisco and thestromal fructose-1,6-bisphosphatase occurred in response tolow temperature, but increases in the activities of Rubisco,sucrose-phosphate synthase or the cytosolic fructose1,6-bisphosphatasecould not be sustained in N-deficient plants throughout theperiod of acclimation, although the activities of these enzymesdeclined less precipitately than in non-acclimated N-deficientplants. These data are all consistent with the view that increasesin the activities of key enzymes of carbon assimilation area pre-requisite for acclimation to low temperature and thatthese increases are restricted under N-limitation. Key words: Low temperature, nitrogen, photosynthesis, Rubisco, sucrose-phosphate synthase     

Coarse control of sucrose-phosphate synthase in leaves: Alterations of the kinetic properties in response to the rate of photosynthesis and the accumulation of sucrose

It has been investigated whether diurnal rhythms of sucrose-phosphate synthase (SPS) are involved in controlling the rate of photosynthetic sucrose synthesis. Extracts were prepared from spinach (Spinacia oleracea L.) and barley (Hordeum vulgare L.) leaves and assayed for enzyme activity. The activity of SPS increased in parallel with a rising rate of photosynthesis, and was increased by feeding mannose and decreased by supplying inorganic phosphate. In leaf material where sucrose had accumulated during the photoperiod or when sucrose was supplied exogenously, SPS activity decreased. During a diurnal rhythm, SPS activity increased after illumination, declined gradually during the light period, decreased further after darkening and then recovered gradually during the night. These changes did not involve an alteration of the maximal activity, but were caused by changes in the kinetic properties, revealed as a change in sensitivity to inhibition by inorganic phosphate. In experiments which modelled the response of SPS to changing metabolite concentrations, it was shown that these alterations of kinetic properties would strongly modify the activity of SPS in vivo. It is proposed that SPS can exist in kinetically distinct forms in vivo, and that the distribution between these forms can be rapidly altered. As the rate of photosynthesis increases there is an activation of SPS, which may be directly or indirectly linked to changes in the availability of P_i. This activation can be modified by factors related to the accumulation of sucrose. Under normal conditions there is a balance between these factors, and the leaf contains a mixture of the different forms of SPS.Abbreviations Chl chlorophyll - Frul,6bisP fructose-1,6-bisphosphate - Fru2,6bisP fructose-2,6-bisphosphate - Fru6P fructose-6-phosphate - Fru1,6bisPase fructose-1,6-bisphosphatase - Fru6P 2kinase fructose-6-phosphate, 2kinase - Fru2,6bisPase fructose-2,6-bisphosphatase - Glc6P glucose-6-phosphate - P_j inorganic phosphate - SPS sucrose-phosphate synthase - UDPGLc uridine 5-diphosphate glucose     

The Effect of Elevated Concentrations of Fructose 2,6-Bisphosphate on Carbon Metabolism during Deacidification in the Crassulacean Acid Metabolism Plant Kalanchöe daigremontiana

In C₃ plants, the metabolite fructose 2,6-bisphosphate (Fru 2,6-P₂) has an important role in the regulation of carbon partitioning during photosynthesis. To investigate the impact of Fru 2,6-P₂ on carbon metabolism during Crassulacean acid metabolism (CAM), we have developed an Agrobacterium tumefaciens-mediated transformation system in order to alter genetically the obligate CAM plant Kalanchöe daigremontiana. To our knowledge, this is the first report to use genetic manipulation of a CAM species to increase our understanding of this important form of plant metabolism. Transgenic plants were generated containing a modified rat liver 6-phosphofructo-2-kinase gene. In the plants analyzed the activity of 6-phosphofructo-2-kinase ranged from 175% to 198% of that observed in wild-type plants, resulting in Fru 2,6-P₂ concentrations that were 228% to 350% of wild-type plants after 2 h of illumination. A range of metabolic measurements were made on these transgenic plants to investigate the possible roles of Fru 2,6-P₂ during Suc, starch, and malic acid metabolism across the deacidification period of CAM. The results suggest that Fru 2,6-P₂ plays a major role in regulating partitioning between Suc and starch synthesis during photosynthesis. However, alterations in Fru 2,6-P₂ levels had little effect on malate mobilization during CAM fluxes.     

The Role of Ribulose-1,5-Bisphosphate Regeneration in the Induction Requirement of Photosynthetic CO(2) Exchange under Transient Light Conditions

The temporally variable light environment of natural plant canopies presents distinct limitations to carbon assimilation, partially as a result of the photosynthetic induction requirement that develops when leaves are shaded. This study was undertaken with soybean (Glycine max L.) leaves to further identify factors contributing to the activation state of the fast component of induction during low photosynthetic photon flux density (PPFD) periods. Determination of pool sizes of carbon reduction cycle intermediates at low light and upon return to saturating light indicated that different limitations to photosynthetic activity arise over the time course of a 10-minute low PPFD period. Photosynthetic activity upon reillumination was limited by the regeneration of ribulose 1,5-P₂. There was an increase in the levels of fructose 1,6-P₂, sedoheptulose 1,7-P₂, triose-P, ribose 5-P, and ribulose 5-P pools, indicating inactivation of stromal enzymes, most notably fructose 1,6-bisphosphatase, sedoheptulose 1,7-bisphosphatase, and ribulose 5-P kinase. The fast-induction component was the most important factor limiting assimilation during rapid, brief light transients, during which the decay of the slow component was minimal. This may be particularly significant for upper leaves in soybean canopies that generally experience very rapid light transients.     

In vitro activation of phosphoglucomutase by fructose 2,6-bisphosphate

The hexose bisphosphate activation of phosphoglucomutase was investigated with both plant (pea and mung bean) and animal (rabbit muscle) sources of the enzyme. Plant phosphoglucomutase was purified about 50-fold from seeds, and to a lesser extent, from seedlings of Pisum sativum L. cv Grenadier and seedlings of Phaseolus aureus. It was found that the plant enzyme was isolated in a mostly dephosphorylated form while commercial rabbit muscle phosphoglucomutase was predominantly in the phosphorylated form. Activation studies were done using the dephosphorylated enzymes. The range of activation constant (K_a) values were obtained for each bisphosphate were: for glucose 1-6-P₂, 0.5 to 1.8; fructose 2,6-P₂, 6 to 11.7; and fructose 1,6-P₂, 7 micromolar, respectively. Fructose 2,6-P₂ is known to occur in both plant and animal tissues at changing levels encompassing the K_a values found in this study; hence, these results implicate fructose 2,6-P₂ as a natural activator of phosphoglucomutase, particularly in plants. Also, glucose 1,6-P₂ has not been found in plants, and the method for measuring glucose 1,6-P₂ by monitoring the activation of phosphoglucomutase is not specific.     

Ribulose-1,5-bisphosphate carboxylase-oxygenase,other Calvin-cycle enzymes,and chlorophyll decrease when glucose is supplied to mature spinach leaves via the transpiration stream

The inhibition of photosynthesis after supplying glucose to detached leaves of spinach (Spinacia oleracea L.) was used as a model system to search for mechanisms which potentially contribute to the sink regulation of photosynthesis. Detached leaves were supplied with 50 mM glucose or water for 7 d through the transpiration stream, holding the leaves in low irradiance (16 mol photons · m^–2 · s^–1) and a cycle of 9 h light/15 h darkness to prevent any endogenous accumulation of carbohydrate. Leaves supplied with water only showed marginal changes of photosynthesis, respiration, enzyme levels or metabolites. When leaves were supplied with 50 mM glucose, photosynthesis was gradually inhibited over several days. The inhibition was most marked when photosynthesis was measured in saturating irradiance and ambient CO₂, less marked in saturating irradiance and saturating CO₂, and least marked in limiting irradiance. There was a gradual loss of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) protein, fructose-1,6-bisphosphatase, NADP-glyceraldehyde-3-phosphate dehydrogenase and chlorophyll. The inhibition of photosynthesis was accompanied by a large decrease of glycerate-3-phosphate, an increase of triose-phosphates and fructose-1,6-bisphospate, and a small decrease of ribulose-1,5-bisphosphate. The stromal NADPH/NADP ratio increased (as indicated by increased activation of NADP-malate dehydrogenase), and the ATP/ADP ratio increased. Chlorophyll-fluorescence analysis indicated that thylakoid energisation was increased, and that the acceptor side of photosystem II was more reduced. Similar results were obtained when glucose was supplied by floating leaf discs in low irradiance on glucose solution, and when detached spinach leaves were held in high light to produce an endogenous accumulation of carbohydrate. Feeding glucose also led to an increased rate of respiration. This was not accompanied by any changes of pyruvate kinase, phosphofructokinase, or pyrophosphate: fructose-6-phosphate phosphotransferase activity. There was a decrease of phosphoenolpyruvate, glycerate-3-phosphate and glycerate-2-phosphate, an increase of pyruvate and triose-phosphates, and an increased ATP/ADP ratio. These results show (i) that accumulation of carbohydrate can inhibit photosynthesis via a long-term mechanism involving a decrease of Rubisco and other Calvin-cycle enzymes and (ii) that respiration is stimulated due to an unknown mechanism, which increases the utilisation of phosphoenolpyruvate.Abbreviations and Symbols C_i CO₂ concentration in the air space within the leaf - F_m fluorescence yield with a saturating pulse in dark-adapted material - F_o ground level of fluorescence using a weak non-actinic modulated beam in the dark - Fru1,6bisP fructose-1,6-bisphosphate - Fru1,6Pase fructose-1,6-bisphosphatase - Fru2,6bisP fructose-2,6-bisphosphate - IRGA infrared gas analyser - NAD-MDH NAD-dependent malate dehydrogenase - NADP-MDH NADP-dependent malate dehydrogenase - NADP-GAPDH NADP-dependent glyceraldehyde-3-phosphate dehydrogenase - PEP phosphoenolpyruvate - PFK phospho-fructokinase - PFP pyrophospate: fructose-6-phosphate-phosphotransferase - 3-PGA glycerate-3-phospate - P_i inorganic phosphate - Ru1,5bisP ribulose 1,5-bisphosphate - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - triose-phosphates sum of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate This research was supported by the Deutsche Forschungsgemeinschaft (SFB 137).     

Conversion of l-Sorbosone to l-Ascorbic Acid by a NADP-Dependent Dehydrogenase in Bean and Spinach Leaf

An NADP-dependent dehydrogenase catalyzing the conversion of l-sorbosone to l-ascorbic acid has been isolated from Phaseolus vulgaris L. and Spinacia oleracea L. and partially purified. It is stable at −20°C for up to 8 months. Molecular masses, as determined by gel filtration, were 21 and 29 kilodaltons for bean and spinach enzymes, respectively. K_m for sorbosone were 12 ± 2 and 18 ± 2 millimolar and for NADP⁺, 0.14 ± 0.05 and 1.2 ± 0.5 millimolar, for bean and spinach, respectively. Lycorine, a purported inhibitor of l-ascorbic acid biosynthesis, had no effect on the reaction.     

Influences of leaf temperature on photosynthetic carbon metabolism in wheat

Net photosynthetic assimilation rate (A), extractable activities of three photosynthetic enzymes, and the concentrations of six metabolites were determined for wheat (Tricum aestivum L.) leaves as leaf temperature was varied under photorespiring (350 microliters per liter CO₂ and 21% O₂) and under nonphotorespiring conditions (800 microliters per liter CO₂ and 2% O₂). The extractable activity of ribulose-1,5-bisphosphate carboxylase (Rubisco) and fructose-1,6-bisphosphatase declined with increasing leaf temperature from 15 to 45°C. Leaf concentrations of ribulose-1,5-bisphosphate (RuBP) declined slightly between 15 and 25°C but increased to a level which is 4 to 5 times the binding site concentration of Rubisco at leaf temperatures of 35 and 45°C. Leaf concentrations of 3-phosphoglycerate, fructose-6-phosphate, and glucose-6-phosphate all declined with increasing leaf temperature. Outside of the limitations imposed by photorespiration, it is proposed that under high light and at suboptimal temperatures, A is limited by rate of utilization of triose phosphate; at optimal temperatures, by the availability of substrate (CO₂ and RuBP) under photorespiring conditions or utilization of triose phosphate under nonphotorespiring conditions; and at supraoptimal temperatures, by the activation state of Rubisco.     

Control of photosynthate partitioning in spinach leaves

Experiments were carried out to estimate the elasticity coefficients and thence the distribution of control of sucrose synthesis and photosynthate partitioning between cytosolic fructose-1,6-bisphosphatase and sucrose-phosphate synthase (SPS), by applying the dualmodulation method of Kacser and Burns (1979, Biochem. Soc. Trans. 7, 1149–1161). Leaf discs of spinach (Spinacia oleracea L.) were harvested at the beginning and end of the photoperiod and illuminated at five different irradiances to alter (i) the extent of feedback inhibition and (ii) the rate of photosynthesis. The rate of CO₂ fixation, sucrose synthesis and starch synthesis were measured and compared with the activation of SPS, and the levels of fructose-2,6-bisphosphate (Fru2,6bisP) and metabolites. Sucrose synthesis increased progressively with increasing irradiance, accompanied by relatively large changes of SPS activity and Fru2,6bisP, and relatively small changes of metabolites. At each irradiance, leaf discs harvested at the end of the photoperiod had (compared with leaf discs harvested at the beginning of the photoperiod) a decreased rate of sucrose synthesis, increased starch synthesis, decreased SPS activity, increased Fru2,6bisP, a relatively small (20%) increase of most metabolites, no change of the glycerate-3-phosphate: triose-phosphate ratio, a small increase of NADPmalate dehydrogenase activation, but no inhibition of photosynthesis. The changes of sucrose and starch synthesis were largest in low light, while the changes of SPS and Fru2,6bisP were as large, or even larger, in high light. It is discussed how these results provide evidence that the control of sucrose synthesis is shared between SPS and fructose-1,6-bisphosphatase, and provide information about the in-vivo response of these enzymes to changes in the levels of their substrates and effectors. At low fluxes, feedback regulation is very effective at altering partitioning. In high light, changes of SPS activation and Fru2,6bisP can be readily overriden by increasing levels of metabolites.     

Higher-plant chloroplast and cytosolic fructose-1,6-bisphophosphatase isoenzymes: origins via duplication rather than prokaryote-eukaryote divergence

Full-size cDNAs encoding the precursors of chloroplast fructose-1,6-bisphosphatase (FBP), sedoheptulose-1,7-bisphosphatase (SBP), and the small subunit of Rubisco (RbcS) from spinach were cloned. These cDNAs complete the set of homologous probes for all nuclear-encoded enzymes of the Calvin cycle from spinach (Spinacia oleracea L.). FBP enzymes not only of higher plants but also of non-photosynthetic eukaryotes are found to be unexpectedly similar to eubacterial homologues, suggesting a eubacterial origin of these eukaryotic nuclear genes. Chloroplast and cytosolic FBP isoenzymes of higher plants arose through a gene duplication event which occurred early in eukaryotic evolution. Both FBP and SBP of higher plant chloroplasts have acquired substrate specificity, i.e. have undergone functional specialization since their divergence from bifunctional FBP/SBP enzymes of free-living eubacteria.Abbreviations FBP fructose-1,6-bisphosphatase - SBP sedoheptulose-1,7-bisphosphatase - FBA fructose-1,6-bisphosphate aldolase     

Influence of Oxygen and Temperature on the Dark Inactivation of Pyruvate, Orthophosphate Dikinase and NADP-Malate Dehydrogenase in Maize

The influence of oxygen and temperature on the inactivation of pyruvate, Pi dikinase and NADP-malate dehydrogenase was studied in Zea mays. O₂ was required for inactivation of both pyruvate, Pi dikinase and NADP-malate dehydrogenase in the dark in vivo. The rate of inactivation under 2% O₂ was only slightly lower than that at 21% O₂. The in vitro inactivation of pyruvate, Pi dikinase, while dependent on adenine nucleotides (ADP + ATP), did not require O₂.

The postillumination inactivation of pyruvate, Pi dikinase in leaves was strongly dependent on temperature. As temperature was decreased in the dark, there was a lag period of increasing length (e.g. at 17°C there was a lag of about 25 minutes) before inactivation proceeded. Following the lag period, the rate of inactivation decreased with decreasing temperature. The half-time for dark inactivation was about 7 minutes at 32°C and 45 minutes at 17°C. The inactivation of pyruvate, Pi dikinase in vitro following extraction from illuminated leaves was also strongly dependent on temperature, but occurred without a lag period. In contrast, NADP-malate dehydrogenase was rapidly inactivated in leaves (half-time of approximately 3 minutes) during the postillumination period without a lag, and there was little effect of temperature between 10 and 32°C. The results are discussed in relation to known differences in the mechanism of activation/inactivation of the two enzymes.

     

Physiological relevance of fructose 2,6-bisphosphate in the regulation of spinach leaf pyrophosphate:fructose 6-phosphate 1-phosphotransferase

A major problem in defining the physiological role of pyrophosphate:fructose 6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90) is the 1,000-fold discrepancy between the apparent affinity of PFP for its activator, fructose 2,6-bisphosphate (Fru-2,6-P₂), determined under optimum conditions in vitro and the estimated concentration of this signal metabolite in vivo. The aim of this study was to investigate the combined influence of metabolic intermediates and inorganic phosphate (Pi) on the activation of PFP by Fru-2,6-P₂. The enzyme was purified to near-homogeneity from leaves of spinach (Spinacia oleracea L.). Under optimal in vitro assay conditions, the activation constant (K _a) of spinach leaf PFP for Fru-2,6-P₂ in the glycolytic direction was 15.8 nM. However, in the presence of physiological concentrations of fructose 6-phosphate, inorganic pyrophosphate (PPi), 3-phosphoglycerate (3PGA), phosphoenolpyruvate (PEP), ATP and Pi the K _a of spinach leaf PFP for Fru-2,6-P₂ was up to 2000-fold greater than that measured in the optimised assay and V _max decreased by up to 62%. Similar effects were observed with PFP purified from potato (Solanum tuberosum L.) tubers. Cytosolic metabolites and Pi also influenced the response of PFP to activation by its substrate fructose 1,6-bisphosphate (Fru-1,6-P₂). When assayed under optimum conditions in the gluconeogenic direction, the K _a of spinach leaf PFP for Fru-1,6-P₂ was approximately 50 μM. Physiological concentrations of PPi, 3PGA, PEP, ATP and Pi increased K _a up to 25-fold, and decreased V _max by over 65%. From these results it was concluded that physiological concentrations of metabolites and Pi increase the K _a of PFP for Fru-2,6-P₂ to values approaching the concentration of the activator in vivo. Hence, measured changes in cytosolic Fru-2,6-P₂ levels could appreciably alter the activation state of PFP in vivo. Moreover, the same levels of metabolites increase the K _a of PFP for Fru-1,6-P₂ to an extent that activation of PFP by this compound is unlikely to be physiologically relevant. Received: 21 July 2000 / Accepted: 15 September 2000