Simulation of in situ freezing damage of the photosynthetic apparatus by freezing in vitro of thylakoids suspended in complex media

Chloroplast thylakoid membranes were isolated from leaves of unhardened and cold-acclimated spinach (Spinacia oleracea L.). For freezethaw treatment, the membranes were suspended in complex media composed to simulate the solute concentrations in the chloroplast stroma in the unhardened and hardened states of the leaves. In particular, high concentrations of amino acids were applied for simulating the hardened state. After frost treatment, photosynthetic activities and chlorophyll fluorescence parameters of the thylakoids were tested to determine the degree of freezing damage. The results revealed a pattern of freezing injury similar to that observed upon frost treatment of thylakoids in situ. A major manifestation of damage was the inhibition of photosynthetic electron transport. Uncoupling of photophosphorylation, which is the dominating effect of freezing of thylakoids suspended in binary solutions (e.g., containing one sugar and one inorganic salt), was also visible but less pronounced in the complex media. Thylakoids obtained from cold-acclimated leaves did not exhibit an increased frost tolerance in vitro, as compared with thylakoids from unhardened plants. The results, furthermore, indicated a strong protective effect of free amino acids at the concentrations and composition found in chloroplasts of hardened leaves. The presence of inorganic salts in the complex media slightly stabilized rather than damaged the membranes during freezing. It is concluded that inactivation of thylakoids in situ may be understood as the destabilizing action of the combined solutes surrounding the thylakoids, occurring when solute concentration is raised due to freezing of water.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Hepes 4-(2-hydroxyethyl)-1-piper-azineethanesulfonic acid - PSI photosystem I - PSII photosystem II     

Salt treatment induces frost hardiness in leaves and isolated thylakoids from spinach

Frost hardiness of spinach (Spinacia oleracea L.) leaves was increased by high concentrations of NaCl in the hydroponic culture medium. Freezing damage was determined by measurement of slow chlorophyll fluorescence quenching after freezing of leaves. Both the osmolality of the leaf sap and forst hardiness of the leaves were linearly correlated with the salt concentration in the hydroponic culture medium. Freezing damage occurred, irrespective of the extent of frost hardening, when dehydration of cells during extracellular ice formation decreased cellular volume to approximately 14% of the volume of unfrozen cells. The resistance of isolated, washed thylakoids against mechanical and chemical damage by freezing was investigated. Chemical damage by freezing caused by salt accumulation was measured as release of chloroplast coupling factor (CF1; EC 3.6.1.3), and mechanical damage was measured as release of the lumenal protein plastocyanin from the membranes during an in-vitro freeze-thaw cycle. Isolated thylakoids from salt-treated frost-hardy spinach and those from plants hardened under natural conditions did not exhibit improved tolerance against chemical freezing stress exerted by high salt concentrations. They were, however, more hardy than thylakoids from unhardened control leaves against mechanical damage by freezing.Abbreviation CF1 peripheral part of chloroplast coupling factor ATPase     

Hydration-state-responsive proteins link cold and drought stress in spinach

Spinach (Spinacia oleracea L.) seedlings exposed to low nonfreezing temperatures (0–10° C) that promote cold acclimation, synthesize a variety cold-acclimation proteins and at the same time acquire a greater ability to withstand cellular dehydration imposed by the freezing of tissue water. Two of these proteins (160 and 85 kDa) become more abundant over time at low temperature. In addition, a small decline in tissue water status from a maximally hydrated state also appears to be associated with an initiation of the accumulation of these proteins at a noninductive temperature. Imposing a severe water stress on young seedlings grown at 25° C by withholding water leads to substantial accumulation of the 160- and 85-kDa proteins, and maximal induction of freezing tolerance. This evidence implies that responses to cold acclimation and water stress involve common mechanisms, and further establishes the linkage of these two proteins with stresses having an osmotic component.Abbreviations ABA abscisic acid - CAP cold-acclimation protein - kDa kilodaltons We thank T. Sinclair and K. Cline for critical reading and discussions, N. Denslow for assistance with protein sequencing methods, and L. Greene, S. Henry for preparing the monoclonal antibodies. The work was made possible by support from the USDA Competitive Grants Program No. 90-37280-5527, the Institute for Food and Agricultural Sciences, and through access to the protein sequencing and hybridoma facilities of the Interdisciplinary Center for Biotechnology Research at the University of Florida. Florida Agricultural Experiment Station Journal Series R-02399.     

Freezing injury in cold-acclimated and unhardened spinach leaves

Spinach plants (Spinacia oleracea L.) were frost-hardened by cold-acclimation to 1° C or kept in an unhardy state at 20°/14° C in phytotrons. Detached leaves were exposed to temperatures below 0°C. Rates of photosynthetic CO₂ uptake by the leaves, recorded after frost treatment, served as a measure of freezing injury. Thylakoid membranes were isolated from frost-injured leaves and their photosynthetic activities tested. Ice formation occurred at about-4° to-5° C, both in unhardened and cold-acclimated leaves. After thawing, unhardened leaves appeared severely damaged when they had been exposed to-5° to-8° C. Acclimated leaves were damaged by freezing at temperatures between-10° to-14° C. The pattern of freezing damage was complex and appeared to be identical in hardened and unhardened leaves: 1. Inactivation of photosynthesis and respiration of the leaves occurred almost simultaneously. 2. When the leaves were partly damaged, the rates of photosynthetic electron transport and noncyclic photophosphorylation and the extent of light-induced H⁺ uptake by the isolated thylakoids were lowered at about the same degree. The dark decay of the proton gradient was, however, not stimulated, indicating that the permeability of the membrane to-ward protons and metal cations had not increased. 3. As shown by partial reactions of the electron transport system, freezing of leaves predominantly inhibited the oxygen evolution, but photosystem II and photosystem I-dependent electron transport were also impaired. 4. Damage of the chloroplast envelope was indicated by a decline in the percentage of intact chloroplasts found in preparations from injured leaves. The results are discussed in relation to earlier studies on freezing damage of thylakoid membranes occurring in vitro.Abbreviations Chl chlorophyll - DCPIP 2,6-dichlorophenol indophenol - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - MES 2(N-morpholino) ethane sulfonic acid     

Metabolite levels in the stroma of spinach chloroplasts exposed to osmotic stress: Effects of the pH of the medium and exogenous dihydroxyacetone phosphate

The levels of stromal photosynthetic intermediates were measured in isolated intact spinach (Spinacia oleracea L.) chloroplasts exposed to reduced osmotic potentials. Stressed chloroplasts showed slower rates of metabolite accumulation upon illumination than controls. Relative to other metabolites sedoheptulose-1,7-bisphosphate (SBP) and fructose-1,6-bisphosphate (FBP) accumulated in the stroma in the stressed treatments. Under these conditions 3-phosphoglycerate (3-PGA) efflux to the medium was restricted. Chloroplasts previously incubated with [³²P]KH₂PO₄ and [³²P]dihydroxyacetone phosphate ([³²P]DAP) in the dark were characterized by very high FBP and SBP levels prior to illumination. Metabolism of these pools upon illumination increased with increasing pH of the medium but was consistently inhibited in osmotically stressed chloroplasts. The responses of stromal FBP and SBP pools under hypertonic conditions are discussed in terms of both inhibited light activation of fructose-1,6-bisphosphatase (EC 3.1.3.11) and sedoheptulose-1,7-bisphosphatase (EC 3.1.3.37), and likely increases in stromal ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) active-site concentrations.Abbreviations and symbols DAP dihydroxyacetone phosphate - FBP fructose-1,6-bisphosphate - PGA 3-phosphoglycerate - RuBP ribulose-1,5-bisphosphate - SBP sedoheptulose-1,7-bisphosphate - _s osmotic potential     

Proteins from frost-hardy leaves protect thylakoids against mechanical freeze-thaw damage in vitro

We have isolated protein fractions from cold-acclimated, frost-hardy cabbage (Brassica oleracea L.) and spinach (Spinacia oleracea L.) leaves which protect isolated thylakoids from non-hardy spinach against mechanical membrane rupture during an in-vitro freeze-thaw cycle. No protective activity was found in similar preparations from non-hardy leaves. The proteins protected the membranes from damage by reducing their solute permeability during freezing and by increasing their expandability during thawing. The proteins act by increasing the resistance of the membranes against the osmotic stress to which they are exposed during a freeze-thaw cycle. In the absence of cryoprotectants this stress results in membrane rupture.This investigation was supported by the Deutsche Forschungsge-meinschaft.     

High concentrations of the compatible solute glycinebetaine destabilize model membranes under stress conditions

Compatible solutes are accumulated by diverse organisms in response to environmental stresses such as drought, salt, or cold. Glycinebetaine (Bet) is such a solute that is accumulated by many plants and microorganisms to high concentrations under stress conditions. It is an osmoprotectant in bacteria and stabilizes both soluble and peripherally membrane-bound proteins in vitro. Here, the effects of Bet on the stability of model lipid membranes are compared to the effects of two other compatible solutes, sucrose and trehalose. Both in the presence of 1M NaCl and during freezing to -20 degrees C, Bet is highly destabilizing to liposomes containing nonbilayer lipids, while the disaccharides are either protective or, in some cases, much less destabilizing. The destabilizing effect of Bet is more pronounced in membranes containing the nonbilayer galactolipid monogalactosyldiacylglycerol from plant chloroplasts than in membranes containing the nonbilayer phospholipid phosphatidylethanolamine. The most dramatic differences between the sugars and Bet were observed in liposomes made from a combination of lipids resembling plant chloroplast thylakoid membranes. Measurements with the dye merocyanine 540 indicate that the water-membrane interface was affected in opposite directions by the presence of high concentrations of sucrose or Bet. The dynamics of the lipids, however, were not differentially affected by the solutes, making direct solute-lipid interactions an unlikely explanation for the different effects on stability. The data offer an explanation, why Bet at high concentrations achieved during exogenous feeding of leaf tissues can be detrimental to cellular stability and survival under stress, while bacterial membranes that contain phosphatidylethanolamine instead of monogalactosyldiacylglycerol, or cyanobacteria that contain highly saturated monogalactosyldiacylglycerol are less susceptible.     

Genetic engineering of the biosynthesis of glycinebetaine enhances thermotolerance of photosystem II in tobacco plants

Genetically engineered tobacco (Nicotiana tabacum L.) with the ability to accumulate glycinebetaine was established. The wild type and transgenic plants were exposed to heat treatment (25–50°C) for 4 h in the dark and under growth light intensity (300 μmol m⁻² s⁻¹). The analyses of oxygen-evolving activity and chlorophyll fluorescence demonstrated that photosystem II (PSII) in transgenic plants showed higher thermotolerance than in wild type plants in particular when heat stress was performed in the light, suggesting that the accumulation of glycinebetaine leads to increased tolerance to heat-enhanced photoinhibition. This increased tolerance was associated with an improvement on thermostability of the oxygen-evolving complex and the reaction center of PSII. The enhanced tolerance was caused by acceleration of the repair of PSII from heat-enhanced photoinhibition. Under heat stress, there was a significant accumulation of H₂O₂, O₂⁻ and catalytic Fe in wild type plants but this accumulation was much less in transgenic plants. Heat stress significantly decreased the activities of catalase, ascorbate peroxidase, glutathione reductase, dehydroascorbate reductase, and monodehydroascorbate reductase in wild type plants whereas the activities of these enzymes either decreased much less or maintained or even increased in transgenic plants. In addition, heat stress increased the activity of superoxide dismutase in wild type plants but this increase was much greater in transgenic plants. Furthermore, transgenic plants also showed higher content of ascorbate and reduced glutathione than that of wild type plants under heat stress. The results suggest that the increased thermotolerance induced by accumulation of glycinebetaine in vivo was associated with the enhancement of the repair of PSII from heat-enhanced photo inhibition, which might be due to less accumulation of reactive oxygen species in transgenic plants.     

Freezing of isolated thylakoid membranes in complex media. V. Inactivation and protection of electron transport reactions

When chloroplast thylakoid membranes isolated from spinach leaves (Spinacia oleracea L. cv. Monatol) were frozen in media containing the predominant inorganic electrolytes of the chloroplast stroma, linear photosynthetic electron transport became progressively inhibited. After onset of freezing, both PSII- and PSI-mediated electron flow were inactivated almost to the same extent. Prolonged storage of the membranes in the frozen state increased damage to PSII relative to PSI activity. Under these conditions, a preferential injury of the water oxidation system was not observed. In thylakoids stored at 0 °C, PSI activity remained fairly unimpaired but inactivation of PSII occurred with strongest inhibition at the oxidizing side.The addition of low-molecular-weight cryoprotectants such as glycerol, sugars, certain amino acids and carbonic acids to thylakoid suspensions prior to freezing provided almost complete preservation of PSI activity and considerable but incomplete stabilization of PSII.Abbreviations BQ 1,4-benzoquinone - Chl chlorophyll - DAD 1,4-diamino-2,3,5,6-tetramethylbenzene - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCPIP 2,6-dichlorophenolindophenol - DMBQ 2,5-dimethyl-p-benzoquinone - DPC 1,5-diphenylcarbazide - Hepes 4-(2-hydroxyethyl)-1-piperazineeth-anesulfonic acid - MV methylviologen - PD 1,4-diaminobenzene - SOD superoxide dismutase (EC 1.15.1.1) - TMHQ tetramethyl-p-hydroquinone - TMPD N,N,N,N-tetramethyl-1,4-diaminobenzene - Tris 2-amino-2-(hydroxymethyl)-1,3-propandiol Dedicated to Professor Dr. Wilhelm Simonis, Würzburg, on the occasion of his 80th birthday     

Intracellular localization of serine acetyltransferase in spinach leaves

Intact chloroplasts isolated from spinach leaves by a combination of differential and Percoll density gradient centrifugation and free of mitochondrial and peroxisomal contamination contained about 35% of the total leaf serine acetyltransferase (EC 2.3.1.30) activity. No appreciable activity of the enzyme could be detected in the gradient fractions containing broken chloroplasts, mitochondria, and peroxisomes. L-cysteine added to the incubation mixture at 1 mM almost completely inhibited serine acetyltransferase activity, both of leaf and chloroplast extracts. D-cysteine was much less inhibitory. L-cystine up to 5 mM and O-acetyl-L-serine up to 10 mM had no effect on the enzyme activity. When measured at pH 8.4, the enzyme extracted from the leaves had a K _m for L-serine of 2.4, the enzyme from the chloroplasts a K _m of 2.8 mM.Abbreviations NAS N-acetyl-L-serine - NADP-GPD NADP-dependent glyceraldehyde-3-phosphate dehydrogenase - OAS O-acetyl-L-serine - OASSase O-acetyl-L-serine sulfhydrylase - 3-PGA D-3-phosphoglycerate - SATase serine acetyltransferase     

Effect of growth regulators and role of roots in sex expression in spinach

When 7-d-old plantlets of spinach (Spinacia oleracea L.) were immersed with their roots for 24 h in 25 mg/l gibberellic acid (GA₃), or 15 mg/l 6-benzylaminopurine (6-BAP), or 15 mg/l indole-3-acetic acid (IAA), or 10 mg/l abscisic acid (ABA) and subsequently grown on long (18-h) days, the ratio of plants with male and female flowers, which in the controls was almost 1:1 (48 and 52%, respectively), was greatly altered. The treatments with 6-BAP, IAA and ABA raised the percentage of female plants to 88, 76 and 71%, respectively; the GA₃ treatment increased the percent of male plants to 79%. When young, vegetative spinach plants (3 visible leaves) grown in 18-h days were cut a the root neck, and the shoots grown with their bases in nutrient solution, with adventitious roots either being allowed to develop or being systematically removed, 85% of the plants without roots became males, 85% of those with roots became females. But if the cut shoots were first, for 28 h, placed in a 15-mg/l 6-BAP solution and then grown in the absence of roots, the percent of female plants was restored to 84. These results fully agree with those obtained previously with hemp, namely, that plant growth regulators exert a regulating effect on the sex expression of dioecious plants when applied through the roots in early stages of development; that the root system plays an important role in determining the sex of these plants, that this role of the roots is associated with the synthesis of cytokinins in them. Dioecious short- and long-day plants do not differ in these respects.     

Effects of freezing on isolated plant mitochondria

Mitochondria isolated from spinach leaves (Spinacia oleracea L.) and potato tubers (Solanum tuberosum L.) were partly injured when subjected to freezing for 2 to 4 h at-25°C in salt solutions in the absence of cryoprotectants. Damage was manifested by the inactivation of respiratory properties and increase in the permeability of the mitochondrial membranes. Decrease in respiratory control indicated that the control mechanism of the electron transport chain was influenced by freezing. Oxidative phosphorylation was only slightly more affected than electron transport. The inactivation of the membrane systems was caused by an increase in the concentration of membrane-toxic solutes. This was confirmed by treatment of the organelles at 0°C in solutions of high salt concentrations. When sugar was present in the course of freezing, mitochondria were partly or completely protected. On a molar basis, sucrose was more effective in membrane protection than glucose. Under certain conditions amino acids, e.g., proline and hydroxyproline, also stabilized isolated mitochondria during freezing.Abbreviations BSA bovine albumin - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - MOPS 2-N-morpholinopropane sulfonic acid - PVP polyvinyl pyrrolidone - RC respiratory control - Tris tris (hydroxymethyl) aminomethane     

Photosynthesis under osmotic stress

The reversibility of the inhibition of photosynthetic reactions by water stress was examined with four systems of increasing complexity—stromal enzymes, intact chloroplasts, mesophyll protoplasts, and leaf slices. The inhibition of soluble chloroplast enzymes by high solute concentrations was instantly relieved when solutes were properly diluted. In contrast, photosynthesis was not restored but actually more inhibited when isolated chloroplasts exposed to hypertonic stress were transferred to conditions optimal for photosynthesis of unstressed chloroplasts. Upon transfer, chloroplast volumes increased beyond the volumes of unstressed chloroplasts, and partial envelope rupture occurred. In protoplasts and leaf slices, considerable and rapid, but incomplete restoration of photosynthesis was observed during transfer from hypertonic to isotonic conditions. Chloroplast envelopes did not rupture in situ during water uptake. It is concluded that inhibition of photosynthesis by severe water stress is at the biochemical level brought about in part by reversible inhibition of chloroplast enzymes and in part by membrane damage which requires repair mechanisms for reversibility. Both soluble enzymes and membranes appear to be affected by the increased concentration of internal solutes, which is caused by dehydration.     

Guanosine triphosphate-binding proteins on the plasmalemma of spinach leaf cells

The molecular mechanism of light perception through phytochrome is not well understood. This red-light photosensor has been implicated in various physiological processes, including the photoinduction of flowering. A few recent studies have shown that phytochrome initiates signal transduction chains via guanosine triphosphate (GTP)-binding proteins (G-proteins). We show here by different approaches that G-proteins exist in spinach (Spinacia oleracea L. cv. Nobel). Binding of GTP on the plasmalemma has been partially characterized and its possible regulation by red light examined by in-vitro assays. These experiments indicate a clear regulation of GTP binding by red light and also by Mastoparan. At least three G-proteins or protein subunits were found to be associated with the plasmalemma of leaf cells. The use of an antibody raised against an animal Gβ subunit confirmed the presence of heterotrimeric G-proteins. Separation of a crude membrane extract by free-flow electrophoresis also showed that some G-proteins could exist on the tonoplast.     

The reactivation of nitrate reductase from spinach (Spinacea oleracea L.) inactivated by NADH and cyanide: effects of peroxidase and associated systems

Nitrate reductase of spinach (Spinacea oleracea L.) leaves which had been inactivated in vitro by treatment with NADH and cyanide, was reactivated by incubation with oxidant systems and measured as FMNH₂-dependent activity. Ferricyanide, a purely chemical oxidant, produced rapid maximal reactivation (100%) which was 90% complete in less than 3 min. Reactivation occurred slowly and less completely (30–75% in 30 or 60 min) when the enzyme was incubated with pure horseradish peroxidase alone, depending on using one or 20 units and time. Addition of glucose and glucose oxidase to generate hydrogen peroxide increased reactivation slightly (10–15%) with 20 units of peroxidase but more (30–50%) with one unit and to 75–90% of ferricyanide values. Adding catalase decreased reactivation by more than half either with or without glucose oxidase. Glucose and glucose oxidase alone did not cause reactivation. Addition of superoxide dismutase increased reactivation from 50–75% of ferricyanide values with one unit of peroxidase alone but had no effect on greater reactivation obtained in the presence of glucose oxidase. The addition of p-cresol and manganese together increased reactivation with one unit of peroxidase and in the presence of glucose oxidase by about double, but omission of manganese had no effect. However, as shown previously, although trivalent manganese was formed, the residual presence of manganous ions inhibited reactivation. Nevertheless, peroxidase systems either alone or with additionally generated hydrogen peroxide can induce substantial reactivation of nitrate reductase in physiologically relevant conditions.Abbreviations EDTA ethylenediaminetetraacetic acid - FMN flavine mononucleotide     

Water stress provokes a generalized increase in phosphatidylcholine turnover in barley leaves

Water and salt stress promote betaine accumulation in leaves of barley (Hordeum vulgare L.) by accelerating the de-novo synthesis of betaine, via choline. Previous radiotracer kinetic studies have implicated stress-enhanced turnover of the choline moiety of phosphatidylcholine (PC) as a major source of choline for betaine synthesis. Two approaches have therefore been followed to show whether stress-induced PC turnover is a cellor organelle-specific phenomenon, or a generalized one. In the first approach, [³H]ethanolamine of high specific activity was supplied to second leaves of unstressed and water-stressed barley plants; after 1 h, paired sections of tissue were excised from each leaf, one for extraction and analysis of [³H]metabolites and the other for autoradiography. The³H-activity remaining in the leaf tissue after washing out the water-soluble³H-metabolites during preparation for autoradiography was taken to be mainly in phospholipids. In unstressed leaves, [³H]phosphatidylethanolamine (PE) was the major labeled phospholipid, whereas there were approximately equal amounts of [³H]PE and [³H]PC in stressed leaves. At the light-microscope level, silver grains were associated with all living cells in both unstressed and stressed leaves; grains were concentrated in the cytoplasmic regions of highly vacuolate mesophyll cells, and were distributed throughout densely cytoplasmic vascular parenchyma. At the electron-microscope level, silver grains were not confined to any particular types of membranes in unstressed or stressed leaves. In the second approach, second leaves of stressed plants received a 1-h pulse of [¹⁴C]ethanolamine, and were then homogenized. The brei was subjected to sucrose density gradient centrifugation. The specific radioactivity of [¹⁴C]PC was quite similar in the gradient fractions, whether they contained microsomes or mitochondrial plus chloroplast membranes. We infer that stress does not enhance the turnover of any structurally discrete class of PC, but rather stimulates PC turnover in several or all classes of membranes in most cells of the leaf.Abbreviations and symbols PE phosphatidylethanolamine - PC phosphatidylcholine - PMME phosphatidylmonomethylethanolamine - PDME phosphatidyldimethylethanolamine - TLC thin-layer chromatography - _leaf leaf water potential     

The relationship between the activation state of sucrose-phosphate synthase and the rate of CO2 assimilation in spinach leaves

The relationship between the gas-exchange characteristics of spinach (Spinacia oleracea L.) leaves and the activation state of sucrose-phosphate synthase was examined at different intercellular partial pressures of CO₂ at two different photon flux densities. There was a strong positive correlation between the activation state of sucrose-phosphate synthase and the assimilation rate. The relationship was the same at both photon flux densities, indicating that the activation state of the enzyme is determined by a product of carbon assimilation, rather than directly by light.Abbreviations A assimilation rate for CO₂ - p _i intercellular CO₂pressure - PFD photon flux density - SPS sucrose-phosphate-synthase - Glc6P glucose-6-phosphate - Fru6P fructose-6-phosphate A.B. was the recipient of a visiting fellowship from the National Research Council of the Italy. This work was also supported by the Science and Engineering Research Council and the Agricultural and Food Research Council, UK.     

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     

Enzyme activities in concentrated solutions of glycinebetaine and other solutes

The activities of a number of enzymes in concentrated solutions of glycinebetaine and other solutes have been studied. Glycinebetaine, in contrast to electrolytes such as NaCl, was found to be noninhibitory up to 500 mM. This is compatible with the postulated role of glycinebetaine in cytoplasmic osmoregulation. Partial protection against NaCl inhibition was afforded by glycinebetaine in some cases. More detailed studies on glycinebetaine —NaCl-enzyme interactions were carried out using malate dehydrogenase (decarboxylating) from Hordeum vulgare.Abbreviations TES N-tris[hydroxymethyl]methyl-2-aminoethane sulphonic acid - MES 2[N-Morpholino]ethane sulphonic acid