The photosynthetic induction response in wheat leaves: net CO2 uptake,enzyme activation,and leaf metabolites

The photosynthetic induction response was studied in whole leaves of wheat (Triticum aestivum L.) following 5-min, 30-min and 10-h dark periods. After the 5-min dark treatment there was a rapid burst in the rate of photosynthesis upon illumination (half of maximum after 30s), followed by a slight decrease after 1.5 more min and then a gradual rise to the maximum rate. During this initial burst in photosynthesis, there was a rapid rise in the level of 3-phosphoglycerate (PGA) and a high PGA/triose-phosphate (triose-P) ratio was obtained. In addition, after the 5-min dark treatment, ribulose-1,5-bisphosphate carboxylase (Rubisco, EC 4.1.1.39), ribulose-5-phosphate kinase (EC 2.7.1.19) and chloroplastic fructose-1,6-bisphosphatase (EC 3.1.3.11) maintained a relatively high state of activation, and maximum activation occurred within 1 min of illumination. The results indicate there is a high capacity for CO₂ fixation in the cycle upon illumination but attaining maximum rates requires an increase in the ribulose-1,5-bisphosphate (RuBP) pool (adjustment in triose-P utilization for carbohydrate synthesis versus RuBP synthesis). With both the 30-min and 10-h dark pretreatments there was only a slight rise in photosynthesis upon illumination, followed by a lag, then a gradual increase to steady-state (half-maximum rate after 6 min). In contrast to the 5-min dark treatment, the level of PGA was low and actually decreased initially, whereas the level of RuBP increased and was high during induction, indicating that Rubisco is limiting. This regulation via the carboxylase was not reflected in the initial extractable activity, which reached a maximum by 1 min after illumination. The light activation of chloroplastic fructose-1,6-bisphosphatase in leaves darkened for 30 min and 10 h prior to illumination was relatively slow (reaching a maximum after 8 min). However, this was not considered to limit carbon flux through the carbon-fixation cycle during induction since RuBP was not limiting. When photosynthesis approached the maximum steady-state rate, a high PGA/triose-P ratio and a high PGA/RuBP ratio were obtained. This may allow a high rate of photosynthesis by producing a favorable mass-action ratio for the reductive phase (the conversion of PGA to triose phosphate) while stimulating starch and sucrose synthesis.Abbreviations Chl chlorophyll - FBP fructose-1,6-bisphosphate - FBPase fructose-1,6-bisphosphatase - Fru6P fructose-6-phosphate - Glc6P glucose-6-phosphate - PGA 3-phosphoglycerate - Pi inoganic phosphate - Rubisco RuBP carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - Ru5P ribulose-5-phosphate - triose-P triose phosphates (dihydroxyacetone phosphate+glyceraldehyde-3-phosphate)     

Regulation of photosynthetic carbon metabolism during phosphate limitation of photosynthesis in isolated spinach chloroplasts

Intact chloroplasts isolated from spinach were illuminated in the absence of inorganic phosphate (Pi) or with optimum concentrations of Pi added to the reaction medium. In the absence of Pi photosynthesis declined after the first 1–2 min and was less than 10% of the maximum rate after 5 min. Export from the chloroplast was inhibited, with up to 60% of the ¹⁴C fixed being retained in the chloroplast, compared to less than 20% in the presence of Pi. Despite the decreased export, chloroplasts depleted of Pi had lower levels of triose phosphate while the percentage of total phosphate in 3-phosphoglycerate was increased. Chloroplast ATP declined during Pi depletion and reached dark levels after 3–4 min in the light without added Pi. At this point, stromal Pi concentration was 0.2 mM, which would be limiting to ATP synthesis. Addition of Pi resulted in a rapid burst of oxygen evolution which was not initially accompanied by net CO₂ fixation. There was a large decrease in 3-phosphoglycerate and hexose plus pentose monophosphates in the chloroplast stroma and a lesser decrease in fructose-1,6-bisphosphate. Stromal levels of triose phosphate, ribulose-1,5-bisphosphate and ATP increased after resupply of Pi. There was an increased export of ¹⁴-labelled compounds into the medium, mostly as triose phosphate. Light activation of both fructose-1,6-bisphosphatase and ribulose-1,5-bisphosphate carboxylase was decreased in the absence of Pi but increased following Pi addition.It is concluded that limitation of Pi supply to isolated chloroplasts reduced stromal Pi to the point where it limits ATP synthesis. The resulting decrease in ATP inhibits reduction of 3-phosphoglycerate to triose phosphate via mass action effects on 3-phosphoglycerate kinase. The lack of Pi in the medium also inhibits export of triose phosphate from the chloroplast via the phosphate transporter. Other sites of inhibition of photosynthesis during Pi limitation may be located in the regeneratige phase of the reductive pentose phosphate pathway.Abbreviations FBP Fructose-1,6-bisphosphate - FBPase Fructose-1,6-bisphosphatase - MP Hexose plus pentose monophosphates - PGA 3-phosphoglycerate - Pi inorganic orthophosphate - RuBP ribulose-1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate carboxylase - TP Triose Phosphate     

A comparative study of metabolite levels in plant leaf material in the dark

Metabolite levels have been compared in the dark and during photosynthesis in leaves and protoplasts from spinach, pea, wheat and barley. In protoplasts the subcellular distribution was also studied. The levels of triose phosphates and sugar bisphosphates were high in the light and low in the dark. The hexose phosphates and 3-phosphoglycerate levels in the dark were very variable depending on the plant material. In most conditions, hexose phosphates and triose phosphates were mainly in the extrachloroplast compartment, while 3-phosphoglycerate and the sugar bisphosphates were mainly in the chloroplast compartment. Leaves always had a very low triose phosphate: 3-phosphoglycerate ratio in the dark, but in protoplasts this ratio was higher. Detailed studies with spinach showed that metabolite levels were very dependent on the availability of carbohydrate in the leaf, particularly starch. Starch mobilisation is not controlled just by the availability of inorganic phosphate and accumulation of phosphorylated intermediates. Hydrolysis of starch may provide precursors for sucrose synthesis while phosphorolysis leads to provision of substrates for respiration. Starch breakdown generates high enough levels of hexose phosphate to support substantial rates of sucrose synthesis in the dark. Respiration is not greatly increased when metabolite levels are high during starch mobilisation. Higher levels of metabolites shorten the length of the induction phase of photosynthesis.Abbreviations Chl chlorophyll - DHAP dihydroxyacetone phosphate - Fru2,6bisP fructose-2,6-bisphosphate - NMR nuclear magnetic resonance - PGA 3-phosphoglyceric acid - Pi inorganic phosphate - RuBP ribulose-1,5-bisphosphate - UDPGlc uridine-5-diphosphate glucose     

Reversible inhibition of the calvin cycle and activation of oxidative pentose phosphate cycle in isolated intact chloroplasts by hydrogen peroxide

Hydrogen peroxide (6x10^-4 M) causes a 90% inhibition of CO₂-fixation in isolated intact chloroplasts. The inhibition is reversed by adding catalase (2500 U/ml) or DTT (10 mM). If hydrogen peroxide is added to a suspension of intact chloroplasts in the light, the incorporation of carbon into hexose- and heptulose bisphosphates and into pentose monophosphates is significantly increased, whereas; carbon incorporation into hexose monophosphates and ribulose 1,5-bisphosphate is decreased. At the same time formation of 6-phosphogluconate is dramatically stimulated, and the level of ATP is increased. All these changes induced by hydrogen peroxide are reversed by addition of catalase or DTT. Additionally, the conversion of [¹⁴C]glucose-6-phosphate into different metabolites by lysed chloroplasts in the dark has been studied. In presence of hydrogen peroxide, formation of ribulose-1,5-bisphosphate is inhibited, whereas formation of other bisphosphates,of triose phosphates, and pentose monophosphates is stimulated. Again, DTT has the opposite effect. The release of ¹⁴CO₂ from added [¹⁴C]glucose-6-phosphate by the soluble fraction of lysed chloroplasts via the reactions of oxidative pentose phosphate cycle is completely inhibited by DTT (0.5 mM) and re-activated by comparable concentrations of hydrogen peroxide. These results indicate that hydrogen peroxide interacts with reduced sulfhydryl groups which are involved in the light activation of enzymes of the Calvin cycle at the site of fructose- and sedoheptulose bisphophatase, of phosphoribulokinase, as well as in light-inactivation of oxidative pentose phosphate cycle at the site of glucose-6-phosphate dehydrogenase.Abbreviations ADPG ADP-glucose - DHAP dihydroxyacetone phosphate - DTT dithiothreitol - FBP fructose-1,6-bisphosphate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - HMP hexose monophosphates (fructose-6-phosphate, glucose-6-phosphate, glucose-1-phosphate) - 6-PGI 6-phosphogluconate - PMP pentose monophosphates (xylulose-5-phosphate, ribose-5-phosphate, ribulose-5-phosphate) - RuBP ribulose-1,5-bisphosphate - S7P sedoheptulose-7-phosphate - SBP sedoheptulose-1,7-bisphosphate Dedicated to Prof. Dr. W. Simonis on the occasion of his 70th birthday     

Do non-equilibrium reactions in the glucose-to-triose phosphate pathway exist in the liver?

The circadian changes in the contents of intermediates of the initial reactions of the glycolytic pathway in pigeon liver were studied. the concentrations of glucose, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate and triose phosphates were found to change synchronously, being maximal at the dark time and minimal during the light daytime. The glycogen content in the liver decreased steadily between 12.00 and 09.00. The diurnal variations in the concentrations of metabolite pairs (glucose and glucose-6-phosphate, glucose-6-phosphate and fructose-6-phosphate, fructose-6-phosphate and fructose-1.6-diphosphate, fructose-1.6-diphosphate and triose phosphates) appeared to correlate significantly. The results obtained suggest that in the liver at least there are no limiting i. e. physiologically non-equilibrium reactions in the carbohydrate metabolic pathway from glucose to triose phosphates.     

Leaf Phosphate Status, Photosynthesis and Carbon Partitioning in Sugar Beet: II. Diurnal Changes in Sugar Phosphates, Adenylates, and Nicotinamide Nucleotides

Sugar Beets (Beta vulgaris L. cv F58-554H1) were cultured hydroponically in growth chambers. Leaf orthophosphate (Pi) levels were varied nutritionally. The effect of decreased leaf phosphate (low-P) status was determined on the diurnal changes in the pool sizes of leaf ribulose 1,5-bisphosphate (RuBP), 3-phosphoglycerate (PGA), triose phosphate, fructose 1,6-bisphosphate, fructose-6-phosphate, glucose-6-phosphate, adenylates, nicotinamide nucleotides, and Pi. Except for triose phosphate, low-P treatment caused a marked reduction in the levels of leaf sugar phosphates (on a leaf area basis) throughout the diurnal cycle. Low-P treatment decreased the average leaf RuBP levels by 60 to 69% of control values during the light period. Low-P increased NADPH levels and NADPH/NADP⁺ ratio but decreased ATP; the ATP/ADP ratio was unaffected. Low P treatment caused a marked reduction in RuBP regeneration (RuBP levels were half the RuBP carboxylase binding site concentration) but did not depress PGA reduction to triose phosphate. These results indicate that photosynthesis in low-P leaves was limited by RuBP regeneration and that RuBP formation in low-P leaves was not limited by the supply of ATP and NADPH. We suggest that RuBP regeneration was limited by the supply of fixed carbon, an increased proportion of which was diverted to starch synthesis.     

Carbon metabolism of chloroplasts in the dark: Oxidative pentose phosphate cycle versus glycolytic pathway

The conversion of U-labelled [¹⁴C]glucose-6-phosphate into other products by a soluble fraction of lysed spinach chloroplasts has been studied. It was found that both an oxidative pentose phosphate cycle and a glycolytic reaction sequence occur in this fraction. The formation of bisphosphates and of triose phosphates was ATP-dependent and occurred mainly via a glycolytic reaction sequence including a phosphofructokinase step. The conversion, of glucose-6-phosphate via the oxidative pentose phosphate cycle stopped with the formation of pentose monophosphates. This was found not to be because of a lack in transaldolase (or transketolase) activity, but because of the high concentration ratios of hexose monophosphate/pentose monophosphate used in our experiments for simulating the conditions in whole chloroplasts in the dark. Some regulatory properties of both the oxidative pentose phosphate cycle and of the glycolytic pathway were studied.Abbreviations DHAP dihydroxyacetone phosphate - GAP 3-phosphoglyceraldehyde - PGA 3-phosphoglycerate - HMP hexose monophosphates - including F6P fructose-6-phosphate - G6P glucose-6-phosphate - GIP glucose-1-phosphate - 6-PGL phosphogluconate - PMP pentose monophosphates - including R5P ribose-5-phosphate - Ru5P ribulose-5-phosphate - X5P xylulose-5-phosphate - E4P erythrose-4-phosphate - S7P sedoheptulose-7-phosphate - FBP fructose-1,6-bisphosphate - SBP sedoheptulose-1,7-bisphosphate - RuBP ribulose-1,5-bisphosphate     

Effect of osmotic stress on photosynthesis studied with the isolated spinach chloroplast : site-specific inhibition of the photosynthetic carbon reduction cycle

The effects of reduced osmotic potential on the photosynthetic carbon reduction cycle were investigated by monitoring photosynthetic processes of spinach (Spinacia oleracea L. var. Long Standing Bloomsdale) chloroplasts exposed to increased assay medium sorbitol concentrations. CO₂ assimilation was found to be inhibited at 0.67 molar sorbitol by about 60% from control rates at 0.33 molar sorbitol. This level of stress inhibition was greater than that affecting the reductive phase of the cycle; glycerate 3-phosphate reduction was inhibited at 0.67 molar by 27 to 40%. Sorbitol (0.67 molar) inhibited the rate of O₂ evolution at saturating and limiting concentrations of NaHCO₃, and extended the lag phase of O₂ evolution. This indicated that factors which are rate-limiting to the photosynthetic process are adversely affected by reduced osmotic potential.

Analysis of photosynthetic products following CO₂ fixation in 0.33 molar sorbitol and 0.67 molar sorbitol indicated that reduced osmotic potential facilitated increases in the levels of fructose 1,6-bisphosphate and triose phosphates with reductions in glucose 6-phosphate and fructose 6-phosphate, implicating fructose 1,6-bisphosphatase as a site of osmotic stress. Osmotic inhibition of the reductive portion (glycerate 3-phosphate to triose phosphate) of the photosynthetic carbon reduction cycle was partially attributed to feedback inhibition by the product, triose phosphate, on glycerate 3-phosphate reduction. A saturating concentration of ribose 5-phosphate partially overcame osmotic inhibition of CO₂-supported O₂ evolution, indicating another but apparently less severe site of stress inhibition in the sequence of ribose 5-phosphate to glycerate 3-phosphate.

     

Short-term water stress leads to a stimulation of sucrose synthesis by activating sucrose-phosphate synthase

The aim of this work was to identify which aspects of photosynthetic metabolism respond most sensitively to leaf water deficit. Spinach (Spinacia oleracea L.) leaf discs were floated on sorbitol concentrations of increasing molarity and changes of the protoplast volume were estimated using [¹⁴C]sorbitol and ³H₂O penetration. Detached leaves were also wilted until 10% of their fresh weight was lost. Photosynthesis was studied at very high external CO₂ concentrations, to eliminate the effect of closing stomata. There was no large inhibition of CO₂ fixation after wilting leaves, or until the external water deficit was greater than-1.2 MPa. However, partitioning changed markedly at these moderate water deficits: more sucrose and less starch was made. When an inhibition of CO₂-saturated photosynthesis did appear at a water deficit of-2.0 MPa and above, measurements of chlorophyll-fluorescence quenching and metabolite levels showed the thylakoid reactions were not especially susceptible to short-term water stress. The inhibition was accompanied by a small increase of the triose phosphate: ribulose-1,5-bisphosphate ratio, showing regeneration of ribulose-1,5-bisphosphate was affected. However, there was also a general increase of the estimated concentrations of most metabolites, indicating that there is no specific site for the inhibition of photosynthesis. Increasing water deficit led to a large increase of fructose-2,6-bisphosphate. This is explained in terms of a simultaneous increase of fructose-6-phosphate and inorganic phosphate as the cell shrinks. The high fructose-2,6-bisphosphate led to the accumulation of triose phosphates, and the potential significance of this for protection against photoinhibition is discussed. There was an increase in the extractable activity of sucrose-phosphate synthase. This was only detected when the enzyme was assayed in conditions which distinguish between different kinetic forms which have previously been identified in spinach leaves. It is proposed that activation of sucrose-phosphate synthase is one of the first sites at which spinach leaves respond to a rising water deficit. This could be of importance for osmoregulation.Abbreviations Chl chlorophyll - Fru1,6bisP fructose-1,6-bisphosphate - Fru2,6bisP fructose-2,6-bisphosphate - Fru6P fructose-6-phosphate - Glc6P glucose-6-phosphate - PGA glycerate-3-phosphate - Pi inorgamic phosphate - Ru1,5bisP ribulose-1,5-bisphosphate - SPS sucrose-phosphate synthase - triose-P sum of glyceraldehyde-3-phosphate and dehydroxyacetone phosphate - UDPGlc uridine diphosphoglucose     

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     

Control of photosynthetic sucrose synthesis by fructose-2,6-bisphosphate

The metabolite levels in the mesophyll of leaves of Zea mays L. have been compared with the regulatory properties of the cytosolic fructose-1,6-bisphosphatase from the mesophyll to show how withdrawal of triose phosphate for sucrose synthesis is reconciled with generation of the high concentrations of triose phosphate which are needed to allow intercellular diffusion of carbon during photosynthesis. i) A new technique is presented for measuring the intercellular distribution of metabolites in maize. The bundle-sheath and mesophyll tissues are partially separated by differential homogenization and filtration through nylon nets under liquid nitrogen. ii) considerable gradients of 3-phosphoglycerate, triose phosphate, malate and phosphoenolpyruvate exist between the mesophyll and bundle sheath which would allow intercellular shuttles to be driven by diffusion. These gradients could result from the distribution of electron transport and the Calvin cycle in maize leaves. iii) consequently, the mesophyll contains high concentrations of triose phosphate and fructose-1,6-bisphosphate. iv) Most of the regulator metabolite fructose-2,6-bisphosphate, is present in the mesophyll. v) The cytosolic fructose-1,6-bisphosphatase has a lower substrate affinity than that found for the enzyme from C₃ species, especially in the presence of inhibitors like fructose-2,6-bisphosphate. vi) This lowered affinity for substrate makes it possible to reconcile use of triose phosphate for sucrose synthesis with the maintenance of the high concentration of triose phosphate in the mesophyll needed for operation of photosynthesis in this species.Abbreviations DHAP Dihydroxyacetonephosphate - Fru1,6-bisP fructose-1,6-bisphosphate - Fru2,6bisP fructose-2,6-bisphosphate - PEP(Case) phosphoenolpyruvate (carboxylase) - PGA 3-phosphoglycerate - Rubisco ribulose-1,5-bisphosphate carboxylase     

Effect of photosynthetic intermediates on the magnesium inhibition of oxygen evolution by barley chloroplasts

Millimolar concentrations of Mg²⁺ inhibited CO₂-dependent O₂ evolution by barley (Hordeum vulgare L.) chloroplasts and also prevented the activation of NADP-glyceraldehyde-3-phosphate dehydrogenase, ribulose-5-phosphate kinase, and fructose-1,6-diphosphatase by light in intact chloroplasts. When added in the dark, 3-phosphoglycerate prevented the inhibition of O₂ evolution by Mg²⁺ and reduced the Mg²⁺ inhibition of enzyme activation by light. Fructose 1,6-diphosphate and ribulose 5-phosphate also prevented the inhibition of O₂ evolution by Mg²⁺ whereas glucose 1-phosphate, glucose 6-phosphate, ribulose 1,5-diphosphate, and citrate had no effect. Phosphoenolpyruvate gave an intermediate response. Metabolites that prevented the Mg²⁺ inhibition of O₂ evolution shortened the lag phase of CO₂-dependent O₂ evolution in the absence of M²⁺. Loading chloroplasts in the dark with 3-phosphoglycerate reduced both the lag phase of O₂ evolution and the inhibition of O₂ evolution by Mg²⁺. The results suggested that Mg²⁺ inhibition was lessened either by external metabolites that compete with inorganic phosphate for transport into the chloroplast or by a high concentration of internal metabolites.     

Autocatalysis and light activation of enzymes in relation to photosynthetic induction in wheat chloroplasts

In order to study the relative contributions of the autocatalytic increase in the level of substrates and the light activation of enzymes to the control of the induction phase or “lag” in wheat chloroplasts, we measured the light-induced reductive activation of fructose 1,6-bisphosphatase, phosphoglycerate kinase, NADP⁺-dependent glyceraldehyde-phosphate dehydrogenase, ribulose 1,5-bisphosphate carboxylase, and phosphoribulokinase in isolated chloroplasts. Each was rapidly activated to levels more than adequate to support the maximum rate of photosynthesis. Induction in wheat chloroplasts is characterized by a period of about 1 min during which no O₂ is evolved. If small quantities of intermediates such as dihydroxyacetone phosphate (DHAP) or 3-phosphoglycerate (PGA) are added, maximum rates of photosynthesis are achieved within the first minute of illumination. The presence of PGA did not affect the activation of any of the above-mentioned enzymes. Each of the enzymes was therefore capable of sustaining maximum rates of photosynthesis in the presence of PGA, even though there was no O₂ evolution from those chloroplasts incubated with CO₂ alone as substrate. The inclusion of PGA did not give rise to abnormally high levels of DHAP, FBP, or fructose 6-phosphate in the stroma. We conclude that the levels of substrates or cofactors are the principal, if not the sole, determinants of the rate of photosynthetic carbon assimilation during induction in wheat chloroplasts.     

Evidence for extracellular enzymic activity of the isolated perfused rat heart

1. The dissimilation of a number of externally added hexose phosphates and 5′-nucleotides by the perfused rat heart is described, and non-specific esterase and 5′-nucleotidase activity associated with the superficial cell membrane or vascular system has been demonstrated. 2. The rate of production of ¹⁴CO₂ from [U-¹⁴C]glucose 6-phosphate suggests that oxidation occurred after hydrolysis to glucose. The incorporation of isotope from [U-¹⁴C]glucose 6-phosphate into glycogen was small, and similar to that obtained with [U-¹⁴C]glucose as substrate. 3. Glucose 6-phosphate was also partially isomerized to fructose 6-phosphate. Similarly, fructose 6-phosphate was converted mainly into glucose 6-phosphate, but also into glucose and inorganic phosphate. When fructose 1,6-diphosphate was added to the perfusate, a mixture of glucose 6-phosphate, fructose 6-phosphate and triose phosphates accumulated in the medium approximately in the equilibrium proportions of the phosphohexose-isomerase and triose phosphate-isomerase reactions, together with inorganic phosphate and some glucose. Glucose 1-phosphate was hydrolysed to glucose, but was not converted into glucose 6-phosphate. Leakage of enzymes out into the perfusion fluid did not occur. 4. This demonstration that phosphohexose isomerase, triose phosphate isomerase and aldolase may react with extracellular substrates at an appreciable rate suggests that these enzymes are attached to the cell membrane.     

Co-operative activation of chloroplast fructose-1,6-bisphosphatase by reductant,pH and substrate

Intact chloroplasts capable of high rates of photosynthesis fail to reduce CO₂ when illuminated in the absence of oxygen. While anaerobiosis limits proton gradient formation leading to ATP deficiency (Ziem-Hanck, U. and Heber, U. (1980) Biochim. Biophys. Acta 591, 266–274), light activation of fructose-1,6-bisphosphatase was also inhibited by anaerobiosis, whereas light activation of NADP-malate dehydrogenase was stimulated by anaerobiosis, indicating that reductant was still available for light activation. The chloroplast pool of NADP was largely reduced during illumination under anaerobiosis and electron transport to oxaloacetate was not inhibited by anaerobic conditions. Significant light activation of fructose-bisphosphatase was observed in anaerobic chloroplasts with 3-phosphoglycerate as substrate, but not with dihydroxyacetone phosphate (3-phosphoglycerate supports electron transport and hence proton gradient formation). In the absence of added substrates, illumination of anaerobic chloroplasts resulted in some light activation of fructose-bisphosphatase when the pH of the medium was increased. Under these conditions, light activation was stimulated by dihydroxyacetone phosphate. Dihydroxyacetone phosphate added together with oxaloacetate allowed light activation of fructose-bisphosphatase in anaerobic chloroplasts, while neither substrate added alone was effective. Formation of a transthylakoid proton gradient can therefore substitute for an alkaline suspension medium by causing an alkaline shift of the stromal pH on illumination. The data are interpreted as indicating that fructose-bisphosphatase, but not NADP-malate dehydrogenase, requires an alkaline pH and the presence of substrate for rapid reductive light activation and they bear on the interpretation of the lag observed in photosynthesis in chloroplasts and leaves on illumination after a prolonged dark period.     

Phosphate sequestration by glycerol and its effects on photosynthetic carbon assimilation by leaves

Glycerol induced a limitation on photosynthetic carbon assimilation by phosphate when supplied to leaves of barley (Hordeum vulgare L.) and spinach (Spinacia oleracea L.). This limitation by phosphate was evidenced by (i) reversibility of the inhibition of photosynthesis by glycerol by feeding orthophosphate (ii) a decrease in light-saturated rates of photosynthesis and saturation at a lower irradiance, (iii) the promotion of oscillations in photosynthetic CO₂ assimilation and in chlorophyll fluorescence, (iv) decreases in the pools of hexose monophosphates and triose phosphates and increases in the ratio of glycerate-3-phosphate to triose phosphate, (v) decreased photochemical quenching of chlorophyll fluorescence, and increased non-photochemical quenching, specifically of the component which relaxed rapidly, indicating that thylakoid energisation had increased. In barley there was a massive accumulation of glycerol-3-phosphate and an increase in the period of the oscillations, but in spinach the accumulation of glycerol-3-phosphate was comparatively slight. The mechanism(s) by which glycerol feeding affects photosynthetic carbon assimilation are discussed in the light of these results.Abbreviations Chl chlorophyll - C _i intercellular concentration of CO₂ - P phosphate - PGA glycerate-3-phosphate - Pi orthophosphate - triose-P sum of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate     

Photosynthesis and ribulose 1,5-bisphosphate levels in intact chloroplasts

The response of ribulose 1,5-bisphosphate levels and CO(2) fixation rates in isolated, intact spinach chloroplasts to pyrophosphate, triose phosphates, dl-glyceraldehyde, O(2), catalase, and irradiance during photosynthesis has been studied. Within 1 minute in the light, a rapid accumulation of ribulose bisphosphate was measured in most preparations of intact chloroplasts, and this subsequently dropped as CO(2) fixation increased. Pyrophosphate, triose phosphates, and catalase increased CO(2) fixation and also the levels of ribulose bisphosphate. CO(2) fixation was inhibited by dl-glyceraldehyde and O(2) with corresponding decreases in ribulose bisphosphate. When the rate of photosynthesis decreased at limiting irradiances (low light), the level of ribulose bisphosphate in the chloroplast did not always decrease, suggesting that ribulose bisphosphate was not limiting CO(2) fixation under these conditions. When triose phosphates (fructose bisphosphate plus aldolase) were added to suspensions of chloroplasts at low irradiances, ribulose bisphosphate increased while CO(2) fixation decreased. These observations provide considerable evidence that high ribulose bisphosphate levels clearly are not solely sufficient to permit rapid rates of CO(2) fixation, but that factors other than ribulose bisphosphate concentration are overriding the control of photosynthesis.Isolated chloroplasts are capable of using carbon reserves to produce considerable ribulose bisphosphate. Upon illumination in the absence of CO(2) and O(2), intact chloroplasts produced up to 13 millimolar ribulose bisphosphate.     

Measurement of Metabolites Associated with Nonaqueously Isolated Starch Granules from Immature Zea mays L. Endosperm

Starch granules with associated metabolites were isolated from immature Zea mays L. endosperm by a nonaqueous procedure using glycerol and 3-chloro-1,2-propanediol. The soluble extract of the granule preparation contained varying amounts of neutral sugars, inorganic phosphate, hexose and triose phosphates, organic acids, adenosine and uridine nucleotides, sugar nucleotides, and amino acids. Based on the metabolites present and on information about translocators in chloroplast membranes, which function in transferring metabolites from the chloroplast stroma into the cytoplasm, it is suggested that sucrose is degraded in the cytoplasm, via glycolysis, to triose phosphates which cross the amyloplast membrane by means of a phosphate translocator. It is further postulated that hexose phosphates and sugars are produced from the triose phosphates in the amyloplast stroma by gluconeogenesis with starch being formed from glucose 1-phosphate via pyrophosphorylase and starch synthase enzymes. The glucose 1-phosphate to inorganic phosphate ratio in the granule preparation was such that starch synthesis by phosphorylase is highly unlikely in maize endosperm.     

The ATP/2e ratio during photosynthesis in intact spinach chloroplasts.

Addition of ribose-5-phosphate to intact spinach chloroplasts in the absence of added P_i resulted in a conversion of part of the Benson-Calvin cycle into a linear sequence so that triose phosphate accumulated during CO₂ fixation stoichiometrically with the O₂ evolved (triose phosphate / O₂ ratio was 2.0). The fortunate consequence of this effect is that the $\text{ATP}\text{2e}$ ratio may be calculated from the 3-phosphoglycerate and triose phosphate accumulated and the O₂ evolved. In this way the $\text{ATP}\text{2e}$ ratio was shown to be 2.0, with cyclic or pseudocyclic phosphorylation contributing less than 9% to the total phosphorylation.     

The redox state of the NADP system in illuminated chloroplasts

Pyridine nucleotide levels were measured in intact spinach chloroplasts. The NADPH/NADP ratio was close to unity in darkened chloroplasts. On illumination, chloroplast NADP levels decreased rapidly. The decrease was more prominent at low than at high light intensities. In the presence of bicarbonate, NADP subsequently increased to reach a steady-state level. The kinetics of the increase were related in general, but not in detail, to the lag phase of photosynthesis. In the steady state, chloroplast NADP was sometimes, particularly during photosynthesis at high light intensities, less reduced in the light than in the dark. In the dark-light transition, phosphoglycerate reduction is driven by increases in the ratios NADPH/NADP and ATP/ADP. When photosynthesis accelerates after the initial lag phase, the NADPH/NADP ratio decreases and a high ratio of phosphoglycerate to triose phosphate becomes an important factor in driving carbon reduction. Under photosynthetic flux conditions, the redox state of the chloroplast NADP system appeared to be governed largely by the chloroplast ratio of phosphoglycerate to dihydroxyacetone phosphate and by the phosphorylation potential [ATP]/[ADP] [P_i]. The inhibitor of cyclic electron transport, antimycin A, increased reduction of the chloroplast NADP system. Even when reduction was almost complete in the presence of 5 μM antimycin A, photosynthesis was still significant at low light intensities. Electrons appeared to be effectively distributed between the cyclic electron-transport pathway and the noncyclic route to NADP at NADPH/NADP ratios as low as about 1. When bicarbonate was absent, the NADP system remained largely reduced in the light. The energy-transfer inhibitor, Dio-9, and uncouplers and agents which interfered with pH regulation of the Calvin cycle increased reduction of the NADP system while decreasing photosynthesis.