Effects of Crosslinking Agent-DFDNB on Energy-transducing Reactions of Chloroplast Thylakoid Membrane Proteins

The effects of crosslinking agent-DFDNB (difluoro dinitro benzene) on functions of chloroplast thylakoid membrane proteins were investigated. DFDNB inhibited activities of PSP and membrane-bound ATPase in chloroplasts. It decreased proton uptake of light-inducted chloroplast thylakoids and the relative value of fluorescence quenching of 9-aminoacridine, and inhibited the rate of fast electrogenic phase of absorption change at 515 nm in chloroplasts. In addition, the isolated CF1-ATPase was crosslinked with DFDNB. The pattern of polymers of crosslinked CF1-ATPase was observed on SDS-PAGE.     

ATP-induced quenching of chlorophyll fluorescence in chloroplasts of higher plants. Dependence on structural properties of the membranes

The ATP-induced quenching of chlorophyll fluorescence in chloroplasts of higher plants is shown to be inhibited when the mobility of the protein complexes into the thylakoid membranes is reduced. Its occurrence also requires the presence of LHC complexes and the ability of the membranes to unstack.These observations, in addition to a slight increase of charge density of the surface-as indicated by 9-aminoacridine fluorescence and high salt-induced chlorophyll fluorescence studies-and partial unstacking of the membranes-as monitored by digitonin method and 540 nm light scattering changes-after phosphorylation, suggest that the ATP-induced quenching of chlorophyll fluorescence could reflect some lateral redistribution of membrane proteins in the lipid matrix of the thylakoids.Abbreviations ATP adenosine triphosphate - 9-AA 9-aminoacridine - Chl chlorophyll - EDTA ethylenediaminetetraacetate - GDA glutaraldehyde - Hepes N-2-hydroxyethylpiperazine-N-2-ethane-sulphonic acid - LHC light-harvesting chlorophyll a/b complex PS photosystem     

Effect of redox and chelating agents on the properties of chloroplast ATPase

The effect of redox and chelating reagents on the ATPase and ATP-synthetase activity in chloroplast membranes as well as the ATPase activity of isolated CF1-coupling factor from chloroplasts has been studied. The Mg2+-ATPase in thylakoid membranes and isolated Ca2+-ATPase is stimulated by dithionite. In the presence of reduced glutathione the effect of dithionite is similar to those of prolonged illumination or heating. Dichlorophenolindophenol partially inhibits this activity as well as citrate, tenoyltrifluoroacetone and the excess fo ATP. Photophosphorylation in chloroplast lamellae is inhibited with dithionite. It is suggested that the membrane bound ATPase from chloroplasts may be in two structural states which differ in their enzymic activity and in the coupling to electron transfer in membrane. The transitions between these states can be induced by redox reagents.     

Flow cytometry of spinach chloroplasts : determination of intactness and lectin-binding properties of the envelope and the thylakoid membranes

Intact spinach (Spinacia oleracea) chloroplasts, thylakoid membranes, and inside-out or right-side-out thylakoid vesicles have been characterized by flow cytometry with respect to forward angle light scatter, right angle light scatter, and chlorophyll fluorescence. Analysis of intact chloroplasts with respect to forward light scatter and the chlorophyll fluorescence parameter revealed the presence of truly “intact” and “disrupted” chloroplasts. The forward light scatter parameter, normally considered to reflect object size, was instead found to reflect the particle density. One essential advantage of flow cytometry is that additional parameters such as Ricinus communis agglutinin (linked to fluorescein isothiocyanate) fluorescence can be determined through logical conditions placed on bit-maps, amounting to an analytical purification procedure. In the present case, chloroplast subpopulations with fully preserved envelopes, thylakoid membrane, and inside-out or right-side-out thylakoid membranes vesicles can be distinguished. Flow cytometry is also a useful tool to address the question of availability of glycosyl moities on the membrane surfaces if one keeps in mind that organelle-to-organelle interactions could be partially mediated through a recognition process. A high specific binding of R. communis agglutinin and peanut lectin to the chloroplast envelope was detected. This showed that galactose residues were exposed and accessible to specific lectins on the chloroplast surface. No exposed glucose, fucose, or mannose residues could be detected by the appropriate lectins. Ricin binding to the intact chloroplasts caused a strong aggregation. Disruption of these aggregates by resuspension or during passage in the flow cytometer induced partial breakage of the chloroplasts. Only minor binding of R. communis agglutinin and peanut lectin to the purified thylakoid membranes was detected; the binding was found to be low for both inside-out and right-side-out vesicles of the thylakoid membranes.     

The effects of reduced amounts of lipid unsaturation on chloroplast ultrastructure and photosynthesis in a mutant of Arabidopsis

A mutant of Arabidopsis thaliana with reduced content of C_18:3 and C_16:3 fatty acids in membrane lipids exhibited a 45% reduction in the cross-sectional area of chloroplasts and had a decrease of similar magnitude in the amount of chloroplast lamellar membranes. The reduction in chloroplast size was partially compensated by a 45% increase in the number of chloroplasts per cell in the mutant. When expressed on a chlorophyll basis the rates of CO₂-fixation and photosynthetic electron transport were not affected by these changes. Fluorescence polarization measurements indicated that the fluidity of the thylakoid membranes was not significantly altered by the mutation. Similarly, on the basis of temperature-induced fluorescence yield enhancement measurements, there was no significant effect on the thermal stability of chlorophyll-protein complexes in the mutant. These observations suggest that the high content of trienoic fatty acids in chloroplast lipids may be an important factor regulating organelle biogenesis but is not required to support normal levels of the photosynthetic activities associated with the thylakoid membranes.     

High-temperature damage and acclimation of the photosynthetic apparatus

The influence of mono- (K⁺) and divalent (Mg²⁺) cations and protons (pH) on the temperature sensitivity of thylakoid membranes was investigated in three groups of young bean plants (control, heat-acclimated and non-acclimated). Thylakoid-membrane function was monitored by second and millisecond delayed fluorescence and 9-aminoacridine fluorescence quenching. It was established that metal ions at investigated concentrations decreased the thermostability of the photosynthetic parameters — an increase of MgSO₄ concentration from 0.1 to 20 mM decreased the temperature of their half-inactivation (T50) by 13°C. At the same time the pH dependence of the thermal stability of these parameters showed a maximum at pH 5.5–6.5. The half-inactivation temperatures of those photosynthetic parameters connected with the ability of the thylakoid membrane to form light-induced proton gradients increased by 6–7°C in the heat-acclimated plants compared with the control. It was assumed that the temperature inactivation of photosynthetic electron transfer and the energization of the thylakoid membrane was determined both by the thermoinduced dissociation of the light-harvesting chlorophyll a/b protein complex from PSII, leading to destruction of the excitation energy transfer to the reaction centres, and by the thermal denaturation of the membrane-protein components. The rate of these processes was probably controlled by the size of the negative surface charge and the viscosity of the thylakoid membrane.Abbreviations 9-AA 9-aminoacridine - DF delayed fluorescence - LHCP light-harvesting chlorophyll a/b protein complex - PSI (II) photosystem I (II) - T50 temperature of 50% inhibition of photosynthetic parameter - Tricine N-[2-hydroxy-1, 1-bis(hydroxymethyl)ethyl] glycine     

ΔpH-dependent chlorophyll fluorescence quenching indicating a mechanism of protection against photoinhibition of chloroplasts

Intact isolated spinach chloroplasts were subjected to photoinhibitory conditions (high light and lack of CO₂). Photoinhibition of the electron transport system was considerably diminished when the chloroplasts were in a low-fluorescent state related to a high proton gradient across the thylakoid membranes, as compared to a high-fluorescent state in which ΔpH-dependent fluorescence quenching was abolished by addition of uncouplers. The hypothesis is discussed that in chloroplasts exposed to excess light, photoinhibition is partly prevented by increased thermal dissipation of excitation energy, as expressed by ΔpH-dependent (‘energy-dependent’) chlorophyll a fluorescence quenching.     

Clotrimazole对叶绿体ATPase功能的影响及其作用部位

clotrimazole能抑制 DTT+光激活的类囊体膜上Mg~(2+)—ATPase的活力。这种抑制属于可逆非竞争性抑制。进一步的实验还表明clotrimazole可以消除 9—AA光下荧光粹灭指示的正常类囊体及DCCD重组残缺膜的跨膜质子梯度。卵磷脂可以减缓 clotrimazole对9—AA荧光粹灭的抑制作用。clotrimazole还能抑制DTT加热激活的游离CF_1 Ca~(2+)—ATPase的活力。根据以上结果我们推测 clotrimazole在类囊体上可能有两个作用部位,一个在类囊体膜脂;另一个在CF_1。     

ATP-induced quenching of chlorophyll fluorescence in chloroplasts of higher plants. Dependence on structural properties of the membranes

The ATP-induced quenching of chlorophyll fluorescence in chloroplasts of higher plants is shown to be inhibited when the mobility of the protein complexes into the thylakoid membranes is reduced. Its occurrence also requires the presence of LHC complexes and the ability of the membranes to unstack. These observations, in addition to a slight increase of charge density of the surface—as indicated by 9-aminoacridine fluorescence and high salt-induced chlorophyll fluorescence studies—and partial unstacking of the membranes—as monitored by digitonin method and 540 nm light scattering changes—after phosphorylation, suggest that the ATP-induced quenching of chlorophyll fluorescence could reflect some lateral redistribution of membrane proteins in the lipid matrix of the thylakoids.     

Chloroplast biogenesis. Regulation of lipid transport to the thylakoid in chloroplasts isolated from expanding and fully expanded leaves of pea

To study the regulation of lipid transport from the chloroplast envelope to the thylakoid, intact chloroplasts, isolated from fully expanded or still-expanding pea (Pisum sativum) leaves, were incubated with radiolabeled lipid precursors and thylakoid membranes subsequently were isolated. Incubation with UDP[(3)H]Gal labeled monogalactosyldiacylglycerol in both envelope membranes and digalactosyldiacylglycerol in the outer chloroplast envelope. Galactolipid synthesis increased with incubation temperature. Transport to the thylakoid was slow below 12 degrees C, and exhibited a temperature dependency closely resembling that for the previously reported appearance and disappearance of vesicles in the stroma (D.J. Morré, G. Selldén, C. Sundqvist, A.S. Sandelius [1991] Plant Physiol 97: 1558-1564). In mature chloroplasts, monogalactosyldiacylglycerol transport to the thylakoid was up to three times higher than digalactosyldiacylglycerol transport, whereas the difference was markedly lower in developing chloroplasts. Incubation of chloroplasts with [(14)C]acyl-coenzyme A labeled phosphatidylcholine (PC) and free fatty acids in the inner envelope membrane and phosphatidylglycerol at the chloroplast surface. PC and phosphatidylglycerol were preferentially transported to the thylakoid. Analysis of lipid composition revealed that the thylakoid contained approximately 20% of the chloroplast PC. Our results demonstrate that lipids synthesized at the chloroplast surface as well as in the inner envelope membrane are transported to the thylakoid and that lipid sorting is involved in the process. Furthermore, the results also indicate that more than one pathway exists for galactolipid transfer from the chloroplast envelope to the thylakoid.     

Effect of magnesium and calcium ions on photoinduced lipid peroxidation and thylakoid breakdown of cell-free chloroplasts

Aging of cell-free chloroplasts at pH 7.0 and 9.0 causes a decline in the level of photosynthetic pigments, quenching of chlorophyll a fluorescence and enhancement in fluorescence polarization. These changes are correlated with photoinduced enhancement of thylakoid lipid peroxidation. The alkaline earth metal cations, namely magnesium and calcium, show opposite actions on lipid peroxidation and modulate thylakoid disorganisation differently. Magnesium ion may stabilise thylakoid membrane by retarding lipid peroxidation. It lowers aging-induced quenching of fluorescence intensity and enhancement of fluorescence polarization. Calcium ion, on the other hand, stimulates disorganisation of thylakoid membranes. It enhances membrane lipid peroxidation, quenching of chlorophyll a fluorescence intensity and fluorescence polarization.     

The effects of external electric field on the electron transport system of spinach chloroplasts

Since the thylakoid membranes of an active chloroplast are constantly exposed to the electric fields generated by the electron transport system inside the membranes, we have studied the effects of pretreating chloroplasts of spinach ( Spinacia oleracea L.) leaves with an external AC (alternating current) electric field on their electron transport system. It was found that a few minutes electric field pretreatment (333 V cm^-1 across chloroplast samples), especially at low frequency, irreversibly inhibited the activity of photosystem II (PSII), but under certain conditions, stimulated that of photosystem I (PSI). From the measurements of fluorescence from PSII, we ascribe the inhibition to a lesion close to its reaction center P680, leading to increased dissipation of excitation energy to heat. The effect on PSI was investigated by the reduction of its reaction center, P700 by various artificial donors. We suggest that the stimulative effect can be attributed to a positive shift of the surface charge density of thylakoid membranes that brings about an increase in the accessibility of exogenous electronegative donors.     

Photosynthetic control, “energy-dependent” quenching of chlorophyll fluorescence and photophosphorylation under influence of tertiary amines

The effects of the tertiary amines tetracaine, brucine and dibucaine on photophosphorylation and control of photosynthetic electron transport in isolated chloroplasts of Spinacia oleracea were investigated. Tertiary amines inhibited photophosphorylation while the related electron transport decreased to the rates, observed under non-phosphorylating conditions. Light induced quenching of 9-aminoacridine fluorescence and uptake of ¹⁴C-labelled methylamine in the thylakoid lumen declined in parallel with photophosphorylation, indicating a decline of the transthylakoid proton gradient. In the presence of ionophoric uncouplers such as nigericin, no effect of tertiary amines on electron transport was seen in a range of concentration where photophosphorylation was inhibited. Under the influence of the tertiary amines tested, pH-dependent feed-back control of photosystem II, as indicated by energy-dependent quenching of chlorophyll fluorescence, was unaffected or even increased in a range of concentration where 9-aminoacridine fluorescence quenching and photophosphorylation were inhibited. The data are discussed with respect to a possible involvement of localized proton flow pathways in energy coupling and feed-back control of electron transport.Abbreviations 9-AA 9-aminoacridine - J _e flux of photosynthetic electron transport - PC photosynthetic control - pH₁ H⁺ concentration in the thylakoid lumen - pmf proton motive force - _P potential quantum yield of photochemistry of photosystem II (with open reaction centers) - Q _A primary quinone-type electron acceptor of photosystem II - q _Q photochemical quenching of chlorophyll fluorescence - q _E energy-dependent quenching of chlorophyll fluorescence - q _AA light-induced quenching of 9-amino-acridine fluorescence     

Paraquat tolerant and susceptible perennial ryegrasses: effects of paraquat treatment on carbon dioxide uptake and ultrastructure of photosynthetic cells

Abstract. Paraquat treatment of susceptible Lolium perenne seedlings (cultivar Kent Indigenous) rapidly inhibited CO₂ uptake and after 1 h chloroplasts exhibited abnormal fusions of the thylakoid membranes. Further ultrastructural changes occurred within the chloroplasts until 8 h after treatment, when cytoplasmic damage also became evident. The localization of primary damage within the chloroplast differs from previous reports of paraquat toxicity in other species. Paraquat treatment of tolerant L. perenne seedlings (line PRP IX) resulted in little change in CO₂ uptake and ultrastructural effects were generally confined to the gradual development of deposits in the chloroplast stroma. These observations are discussed in relation to the mechanism of action of the herbicide and the proposed mechanism of paraquat tolerance in L. perenne.     

The effects of tetraphenylboron on energy-linked reactions in spinach chloroplasts

1. The inhibitory action of tetraphenylboron, a lipid-soluble anion, on the proton uptake, the photophosphorylation and the light-induced quenching of the fluorescence of 9-aminoacridine by spinach chloroplasts was studied.2. The inhibition of the three processes by tetraphenylboron was transient; the proton uptake was affected to a much smaller extent than either the photophosphorylation or the fluorescence quenching.3. The inhibitory effects of tetraphenylboron on the proton uptake and the fluorescence quenching of 9-aminoacridine were qualitatively the same in CF₁-depleted chloroplasts, that were recoupled with N,N′-dicyclohexylcarbodiimide (DCCD).4. The reversal of the fluorescence quenching of 9-aminoacridine upon addition of tetraphenylboron in the light was found to be very fast, being completed within the response time of the apparatus.5. The presence of tetraalkylammonium salts in the incubation medium prevented the inhibitory effect of tetraphenylboron.6. Tetraphenylboron disappeared from the chloroplast suspension in a light-dependent irreversible way; in the dark no ‘ptake’ of tetraphenylboron could be detected.7. The effects of tetraphenylboron may be explained by the presence of groups with a high affinity for tetraphenylboron in the membrane; these groups become protonated upon illumination of the chloroplasts.     

The role of different thylakoid glycolipids in the function of reconstituted chloroplast ATP synthase

ATPase activity of CF₀CF₁ from spinach chloroplasts is specifically stimulated by chloroplast lipids (Pick, U., Gounaris, K., Admon, A. and Barber, J. (1984) Biochim. Biophys. Acta 765, 12–20). The association of CF₀-CF₁ with isolated lipids and their mixtures has been examined by analyzing the stimulation of ATPase and ATP-P_i exchange activities, by binding studies and by measurement of proton conductance of reconstituted proteoliposomes. Monogalactosyldiacylglycerol is the only chloroplast lipid which by itself activates ATP hydrolysis. A mild saturation of the fatty acids of the lipid partially inhibits the activation. CF₀-CF₁ has a higher binding capacity for monogalactosyldiacylglycerol (1.5 mg/mg protein) than for other thylakoid glycolipids. However, ATPase activation is not correlated with the amount of bound lipid but rather with its type. For the same amount of bound lipid, monogalactosyldiacylglycerol best activates ATP hydrolysis, while the acidic lipids phosphatidylglycerol and sulphoquinovosyldiacylglycerol inhibit ATPase activity. Optimal activation of ATP-P_i exchange requires, in addition to monogalactosyldiacylglycerol, digalactosyldiacylglycerol and sulphoquinovosyldiacylglycerol at a ratio of 6:3:1, respectively. Correlations between proton conductance, ATP-P_i exchange and uncoupler stimulation of ATPase activity indicate that sulphoquinovosyldiacylglycerol reduces the permeability of the proteoliposomes to protons. The results suggest that: (a) association of CF₀-CF₁ with polyunsaturated monogalactosyldiacylglycerol greatly stimulates ATPase activity; (b) reconstitution of coupled CF₀-CF₁ proteoliposomes requires a careful balance of the natural glycolipids of thylakoid membranes in similar proportions to their occurrence in chloroplasts, and (c) sulphoquinovosyldiacylglycerol may control the permeability of chloroplast membranes to protons.     

低温弱光下类囊体腔内质子由CF_0渗漏引起黄瓜类囊体耦联度降低

毫秒延迟发光测定结果表明低温弱光处理黄瓜叶片导致类囊体原位 (in situ)耦联度显著降低。DCCD可以恢复低温弱光处理的黄瓜叶片的毫秒延迟发光的慢相强度和反映类囊体膜质子吸收的 9- AA(9- Aminoacridine)荧光猝灭能力 ,说明类囊体耦联度降低的原因是质子由 CF0 大量快速渗漏。进一步研究结果表明 ,活性氧和 CF1的脱落不是低温弱光引起黄瓜类囊体耦联度降低的根本原因。     

Study of membrane-bound ribosomes during pea chloroplasts formation]

Membrane-bound ribosomes of chloroplasts, isolated from pea seedlings during grana formation, can be partially liberated by 0.5 M KCl and 0.001 M puromycin. In case of mature chloroplasts, after the completion of grana formation process these agents are inefficient, and liberation of ribosomes and polyribosomes may be achieved only after solubilization of thylakoid membranes by 1% Triton X-100. Electron microscopic study of the heavy membrane fraction of young chloroplasts reveals electron-transparent membranes, containing rings and discs of thylakoids with a diameter of about 2 mum. These rings are liberated together with ribosomes under the action of 0.5 M KCl; Triton X-100 liberates equally-sized annular polyribosomes. The rings detected in chloroplast membranes at early stages of development are regarded as structures, precursor grana thylakoids, and the annular polyribosomes included into them as immediate participants of thylakoid morphogenesis.     

Response of senescing rice leaves to flooding stress

Flooding stress (FS) induced changes in pigment and protein contents and in photochemical efficiency of thylakoid membranes of chloroplasts were investigated during senescence of primary leaves of rice seedlings. Leaf senescence was accompanied by loss in 2,6-dichlorophenolindophenol (DCPIP) photoreduction, rate of oxygen evolution, quantum yield of photosystem 2 with an increase in MDA accumulation, and non-photochemical quenching (NPQ) of chlorophyll fluorescence. These changes were further aggravated when the leaves during this period experienced FS. The increase in NPQ value under stress may indicate photosynthetic adaptation to FS.     

The response of ultrastructure and function of chloroplasts from cycads to doubled CO2 concentration

This study examines the effects of doubled CO₂ concentration on the ultrastructure and function of chloroplasts from cycads and, for control from two other herbaceous angiosperms. Under a doubled CO₂ concentration condition, the chloroplast ultrastructure of the two cycads (Cycas multipinnata with a shade-type chloroplast andC. panzhihuaensis with a sun-type chloroplast) changed little: The conformation of the thylakoid membrane system kept well, and almost no starch grains accumulated. In contrast, under the same conditions the chloroplast ultrastructure of soybean and foxtail millet changed considerably, with starch grains accumulating in their chloroplasts and some of thylakoids (especially stroma thylakoid) membranes being destroyed to some degree by the more numerous and larger starch grains that accumulated in the chloroplasts. Interestingly, the changes in the ultrastructure of the chloroplasts from the two cycads was correlated with the 77K fluorescence emission spectra of their chlorophyll; i.e., the F685/F734 (PS II / PS I) ratio within the chloroplasts, which were minimal. The absorption spectrum showed decreases in the red and blue peaks. These changes in the absorption spectrum may be related to changes in the structural arrangement of the thylakoid membranes. Preliminarily, this experimental result shows that the cycads may adapt themselves to environmental changes under doubled CO₂ concentration in the coming centuries. However, more studies on this aspect are necessary.