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     

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.     

The role of monovalent cations for photosynthesis of isolated intact chloroplasts

The role of monovalent cations in the photosynthesis of isolated intact spinach chloroplasts was investigated. When intact chloroplasts were assayed in a medium containing only low concentrations of mono- and divalent cations (about 3 mval l^-1), CO₂-fixation was strongly inhibited although the intactness of chloroplasts remained unchanged. Addition of K⁺, Rb⁺, or Na⁺ (50–100 mM) fully restored photosynthesis. Both the degree of inhibition and restoration varied with the plant material and the storage time of the chloroplasts in low-salt medium. In most experiments the various monovalent cations showed a different effectiveness in restoring photosynthesis of low-salt chloroplasts (K⁺>Rb⁺>Na⁺). Of the divalent cations tested, Mg²⁺ also restored photosynthesis, but to a lesser extent than the monovalent cations.In contrast to CO₂-fixation, reduction of 3-phosphoglycerate was not ihibited under low-salt conditions. In the dark, CO₂-fixation of lysed chloroplasts supplied with ATP, NADPH, and 3-phosphoglycerate strictly required the presence of Mg²⁺ but was independent of monovalent cations. This finding excludes a direct inactivation of Calvin cycle enzymes as a possible basis for the inhibition of photosynthesis under low-salt conditions.Light-induced alkalization of the stroma and an increase in the concentration of freely exchangeable Mg²⁺ in the stroma, which can be observed in normal chloroplasts, did not occur under low-salt conditions but were strongly enhanced after addition of monovalent cations (50–100 mM) or Mg²⁺ (20–50 mM).The relevance of a light-triggered K⁺/H⁺ exchange at the chloroplast envelope is discussed with regard to the light-induced increase in the pH and the Mg²⁺ concentration in the stroma, which are thought to be obligatory for light activation of Calvincycle enzymes.     

Photosynthesis of isolated chloroplasts and protoplasts under osmotic stress

1. Isolated intact spinach chloroplasts respond to changes of the sorbitol concentration of the suspending medium as near-perfect osmometers within a large range of osmotic potentials. Under isotonic conditions (=9–10 bar), their average osmotic volume is 24 m³ and the total volume 36 m³. The osmotic volume can be increased to 63 m³ by lowering the sorbitol concentration until a critical osmotic potential of =4 bar is reached. Below that value chloroplasts rupture. Between 10 bar and 4 bar, volume changes are reversible. 2. Increasing the chloroplast volume above 24 m³ causes inhibition of photosynthesis, with 50% inhibition occurring at an osmotic potential of =5–6 bar. This corresponds to an osmotic volume of 45–55 m³. Depending on the duration of hypotonic treatment, inhibition of photosynthesis is more or less reversible. 3. Between 4 and 10 bar, the chloroplast envelope exhibits a very low permeability for ferricyanide, many metabolites, and soluble stroma proteins. 4. Electron transport is not inhibited by swelling of chloroplasts. Also, the ATP/ADP-ratio remains unchanged. 5. The solute concentration in the chloroplasts appears to be optimal for photosynthesis at 10 bar. Increasing the chloroplast volume causes inhibition of photosynthesis by dilution effects.     

Calcium binding by spinach stromal proteins

Calcium binding to spinach (Spinacia oleracea L.) stromal proteins was examined by dual-wavelength spectrophotometry using the metallochromic indicator tetramethylmurexide. The data are consistent with the existence of at least two, probably independent, classes of binding sites. The total number of binding sites varied between 90–155 nmol·mg^–1 protein with average binding constants of 1.1–2.7·mM^–1. Both Mg²⁺ and La³⁺ inhibited calcium binding competitively, with average inhibitor constants of 0.26·mM^–1 and 39.4·mM^–1, respectively; an increase in the potassium concentration up to 50 mM had no effect. In a typical experiment a decrease in pH (7.8 to 7.1) resulted in a decrease in the total number of calcium binding sites from 90 to 59 nmol·mg^–1 protein, but in an increase of the average affinity from 2.7 to 4.5·mM^–1. Calculations, using these data and those of Gross and Hess (1974, Biochim. Biophys. Acta 339, 334–346) for binding site I of washed thylakoid membranes, showed that the free-Ca²⁺ concentration in the stroma under dark conditions, pH 7.1, is higher than under light conditions, pH 7.8. The physiological relevance of the observed calcium binding by stromal proteins is discussed.Abbreviations Ca _b ²⁺ bound calcium - Ca _f ²⁺ free calcium     

Photosynthetic oxygen evolution and chlorophyll fluorescence in intact isolated chloroplasts on a solid support: the influence of orthophosphate

We devised recently a method to trap intact isolated chloroplasts on a solid support consisting of membrane filters made of cellulose nitrate (Cerovi et al., 1987, Plant Physiol. 84, 1249–1251). The addition of alkaline phosphatase to the reaction medium enabled continuous photosynthesis by spinach (Spinacia oleracea L.) chloroplasts to be sustained by hydrolysis of newly produced and exported triose phosphates and recycling of orthophosphate. In this system, simultaneous measurements of chlorophyll fluorescence and oxygen evolution were performed and their dependence on orthophosphate concentration was investigated. Optimal photosynthesis was obtained at a much higher initial orthophosphate concentration (2–4 mM) compared to intact chloroplasts in suspension. Secondary kinetics of chlorophyll fluorescence yield were observed and were shown to depend on the initial orthophosphate concentration.Abbreviations Chl chlorophyll - CSS intact isolated chloroplasts on solid support - ICS intact isolated chloroplasts in suspension - P_i orthophosphate - v rate of O₂ evolution - PPFD photosynthetic photon flux density The authors wish to thank Dr. Marijana Plesniar, from the University of Novi Sad, for stimulating discussions. This work was supported by the Fond for Science of the Republic of Serbia. Z.G.C.'s visit to the Robert Hill Laboratory was supported by the British Council and the University of Sheffield.     

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     

The relationship between phosphate status and photosynthesis in leaves

Spinach (Spinacia oleracea L.) and barley (Hordeum vulgare L.) were grown in hydroponic culture with varying levels of orthophosphate (Pi). When leaves were fed with 20 mmol·l^–1 Pi at low CO₂ concentrations, a temporary increase of CO₂ uptake was observed in Pi-deficient leaves but not in those from plants grown at 1 mmol·l^–1 Pi. At high concentrations of CO₂ (at 21% or 2% O₂) the Pi-induced stimulation of CO₂ uptake was pronounced in the Pi-deficient leaves. The contents of phosphorylated metabolites in the leaves decreased as a result of Pi deficiency but were restored by Pi feeding. These results demonstrate that there is an appreciable capacity for rapid Pi uptake by leaf mesophyll cells and show that the effects of long-term phosphate deficiency on photosynthesis may be reversed (at least temporarily) within minutes by feeding with Pi.Abbreviation Pi orthophosphate     

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

The effect of phosphate feeding on the influence of low (2%) oxygen on photosynthetic carbon assimilation has been investigated in leaf discs of spinach (Spinacia oleracea L.) at 12°C. The following observations were made. First, after the transition from 20% O₂ to 2% O₂, the rate of CO₂ uptake was inhibited at CO₂ concentrations between about 250 and about 800 l CO₂·l^-1. Second, phosphate feeding stimulated the rate of CO₂ uptake in 20% O₂ at higher concentrations of CO₂ (500–900 l·l^-1). Third, phosphate feeding stimulated the rate of CO₂ uptake in 2% O₂ at all but the highest (900 l·l^-1) and lowest 74 (l·l^-1) concentrations of CO₂ employed. Phosphate thereby restored the stimulation of photosynthesis by 2% O₂ and it did so over a wide range of lower temperatures. Fourth, oscillatory behaviour, however generated, was dampened by phosphate feeding, even at very low concentrations of CO₂. Contents of leaf metabolites were measured during the transition to 2% O₂ in control and phosphate-fed leaf discs. During this period the ratio glycerate-3-phosphate/triose phosphate rose steeply, but fell again only in the phosphate-treated leaf discs. These data, taken together with measured ATP/ADP ratios, showed that assimilatory power, the ratio [ATP]·[NAD(P)H]/[ADP]·[Pi]·[NAD(P)], decreased when leaves were exposed to 2% O₂, but that this decrease was minimised by previous feeding of phosphate. The mechanism of phosphate limitation is discussed in the light of the results.Abbreviations C_i intercellular concentration of CO₂ - RuBP ribulose-1,5-bisphosphate     

Multiplicity of soluble glucan-synthase activity in spinach leaves: Enzyme pattern and intracellular location

Buffer-extractable proteins from leaves of Spinacia oleracea L. were separated by non-denaturing polyacrylamide gel electrophoresis. Gels were stained for adenosine diphosphoglucose (ADPglucose)-dependent glucan-synthase (GS) activity (EC 2.4.1.21). Three major forms of activity were observed. No staining was detectable when ADPglucose was replaced by an equimolar concentration of either uridine, guanosine or cytosine diphosphoglucose. Two of the three GS forms exhibited both primed and citrate-stimulated unprimed activity whereas one enzyme form was strictly dependent upon the presence of an exogenous glucan. For intracellular localization, mesophyll protoplasts and intact chloroplasts were isolated and their enzyme pattern was compared with that of the leaf extract. Intactness and purity of the chloroplast preparations were ascertained by polarographic measurement of the ferricyanide- or CO₂-dependent oxygen evolution, by determination of marker-enzyme activities, and by electrophoretic evaluation of the content of chloroplast- and cytosol-specific glucanphosphorylase forms (EC 2.4.1.1). The three GS forms were present in mesophyll protoplasts. Intact chloroplasts possessed both primer-independent enzyme forms but lacked the primer-dependent one. The latter form was enriched in supernatant fractions of leaf homogenates when the intact chloroplasts had been pelleted by centrifugation. Thus, in spinach-leaf mesophyll cells soluble ADPglucose-dependent GS is located both inside and outside the chloroplast.Abbreviations GS glucan synthase - PAGE polyacrylamide gel electrophoresis This work has been made possible by grants from the Deutsche Forschungsgemeinschaft and from the Minister für Wissenschaft und Forschung des Landes Nordrhein-Westfalen. The authors gratefully acknowledge the generous permission to use the laser densitometer of Professor Dr. W. Barz (Biochemie der Pflanzen, Universität Münster, FRG). They are indebted to Dr. H.-J. Witt (Pflanzenphysiologie, Universität Kassel, FRG) for helpful discussions and to Mr. W. Lamkemeyer for skilfull technical assistance.     

Non-photochemical quenching of chlorophyll a fluorescence in isolated chloroplasts under conditions of stressed photosynthesis

Non-photochemical quenching of chlorophyll a fluorescence after short-time light, heat and osmotic stress was investigated with intact chloroplasts from Spinacia oleracea L. The proportions of non-photochemical fluorescence quenching (q _N ) which are related (q _E ) and unrelated (q _I ) to the transthylakoid proton gradient (pH) were determined. Light stress resulted in an increasing contribution of q _Ito total q _N.The linear dependence of q. _Eand pH, as seen in controls, was maintained. The mechanisms underlying this type of quenching are obviously unaffected by photoin-hibition. In constrast, q _Ewas severely affected by heat and osmotic stress. In low light, the response of q _Eto changes in pH was enhanced, whereas it was reduced in high light. The data are discussed with reference to the hypothesis that q _Eis related to thermal dissipation of excitation energy from photosystem II. It is shown that q _Eis not only controlled by pH, but also by external factors.Abbreviations and symbols 9-AA 9-aminoacridine - F _o basic chlorophyll fluorescence - F _o variable chlorophyll fluorescence - L ₂ saturating light pulse - PS photosystem - q _E pH-dependent, non-photochemical quenching of fluorescence - q _I pH-independent, non-photochemical quenching - q _N entire non-photochemical quenching - q _Q photochemical quenching     

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     

Fast photoacoustic transients from dark-adapted intact leaves: oxygen evolution and uptake pulses during photosynthetic induction — a phenomenology record

Using a photoacoustic technique it has been possible to observe fast oxygen evolution and uptake transients at a high time resolution (approx. 0.2 s), when a dark-adapted leaf is reilluminated. There is initially a rapid pulse of oxygen evolution, correlated with the initial fluorescence rise (total duration under the experimental conditions used about 1–2 s), corresponding presumably to the photoreduction of the plastoquinone pool. This phenomenon may be utilized to calibrate the oxygen-evolution photoacoustic signal. The first pulse is followed by a series of slower bursts of oxygen uptake and evolution, reflecting various pools which are expressed following sequential activation of various parts of the photosynthetic apparatus, until achievement of a steady state.Abbreviations and symbols RuBP ribulose-1,5-bisphosphate - PSI, PSII photosystems I, II - F_o, F_m, F(t) initial, maximum and instantaneous chlorophyll fluorescence emission     

Inhibition of photosynthetic reactions by light

Illumination of isolated intact chloroplasts of Spinacia oleracea L. for 10 min with 850 W m^-2 red light in the absence of substrate levels of bicarbonate caused severe inhibition of subsequently measured photosynthetic activities. The capacity of CO₂-dependent O₂ evolution and of non-cyclic electron transport were impaired to similar degrees. This photoinactivation was prevented by addition of bicarbonate which allowed normal carbon metabolism to proceed during preillumination. Photoinhibition of electron transport was observed likewise upon illumination of intact or broken chloroplasts when efficient electron acceptors were absent. Addition of uncouplers did not influence the extent of inhibition. Studies of partial electron-transport reactions indicated that the activity of both photosystems was affected by light. In addition, the water-oxidation system or its connection to photosystem II seemed to be impaired. Preillumination did not cause uncoupling of photophosphorylation. Chlorophyll-fluorescence data obtained at room temperature and at 77 K are consistent with the view that photosystem-II reaction centers were altered. Addition of superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6) or 1,4-diazabicyclo(2,2,2)octane to isolated thylakoids prior to preillumination substantially diminished photoinhibition. This result shows that reactive oxygen species were involved in the damage. It is concluded that bright light, which normally does not damage the photosynthetic apparatus, may exert the described destructive effects under conditions that restrict metabolic turnover of photosynthetic energy.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PSI photosystem I - PSII photosystem II     

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     

Synthesis of acyl-CoAs by isolated spinach chloroplasts in relation to added CoA and ATP

The kinetics of incorporation of [2-¹⁴C] acetate into lipids and acyl-CoAs in relation to added CoA and ATP by isolated spinach chloroplasts have been examined. The effect of the concentration of these cofactors on lipid and acyl-CoA synthesis was also studied. In the absence of cofactors, or when only one was present, the incorporation was very low and went mainly into lipids. When both cofactors were present a strong stimulation of both activities occurred. After 25 min, acyl-CoAs were more strongly labeled than lipids and both activities continued linearly for at least 60 min.Abbreviations ACP acyl carrier protein - FFA free fatty acids     

Phosphorylation of chloroplast membrane proteins partially protects against photoinhibition

Thylakoids isolated from peas (Pisum sativum cv. Kelvedon Wonder) and phosphorylated by incubation with ATP have been compared with non-phosphorylated thylakoids in their sensitivity to photoinhibition by exposure to illumination in vitro. Assays of the kinetics of fluorescence induction at 20° C and the fluorescence emission spectra at-196° C indicate a proportionally larger decrease in fluorescence as a result of photoinhibitory treatment of non-phosphorylated compared with phosphorylated thylakoids. It is concluded that protein phosphorylation can afford partial protection to thylakoids exposed to photoinhibitory conditions.Abbreviations and symbols DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F ₀ Level of chlorophyll fluorescence when photosystem 2 traps are open - F _m Level of chlorphyll fluorescence when photosystem 2 traps are closed - P Maximum level of fluorescence reached in the absence of DCMU - PSI (II) photosystem I(II)     

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     

Protective systems against active oxygen species in spinach: response to cold acclimation in excess light

Spinach (Spinacia oleracea L.) plants were acclimated to 1° C or maintained at 18° C under the same light regime (260–300 mol photons·m^–2·s^–1). The cold acclimation led to several metabolic and biochemical changes that apparently include improved protection of the photosynthetic apparatus against active oxygen species. In particular, cold-acclimated leaves exhibited a considerably higher ascorbate content and significantly increased activities of superoxide dismutase, ascorbate peroxidase, and monodehydroascorbate reductase in the chloroplasts. The level of dehydroascorbate reductase did not alter. Catalase activity decreased. The photosynthetic pigment composition of cold-acclimated spinach was characterized by increased levels of the xanthophylls lutein + zeaxanthin and violaxanthin. The observed changes are discussed in terms of their possible relevance for plant resistance to photoinhibition at chilling temperatures.Abbreviations DHA dehydroascorbate - GSH reduced glutathione - MDA monodehydroascorbate - SOD superoxide dismutase The authors thank the Deutsche Forschungsgemeinschaft for financial support of this study.