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1.
《BBA》1986,851(2):322-326
We have used trivalent lanthanide metal cations in the buffering media of pea chloroplasts to probe the stacking arrangement of thylakoid membranes and the spatial distribution of chlorophyll-protein complexes of Photosystems I and II. Measurements of steady-state chlorophyll fluorescence emission spectra of pea chloroplasts at room temperature demonstrate that, within this tripositive valency group, the extent of membrane appression is a function of hydrated metal ionic radius. These results are in agreement with a recent investigation using monovalent and divalent metal cations (Karukstis, K.K. and Sauer, K. (1985) Biochim. Biophys. Acta 806, 374–389). In addition, the lanthanide cation concentration effective in producing the maximum chlorophyll fluorescence intensity upon grana formation is dependent on hydrated ionic size. The current investigation supports the proposed hypothesis that cation screening ability defines the extent of intermembrane separation as well as the extent of lateral distribution of chlorophyll-protein complexes. 相似文献
2.
W.S. Chow S.W. Thorne J.T. Duniec M.J. Sculley N.K. Boardman 《Archives of biochemistry and biophysics》1980,201(1):347-355
The differential action of digitonin on stacked and unstacked chloroplast thylakoids was used to investigate the molecular interactions between thylakoid membranes. The yield of the heavy fraction which is obtained from chloroplasts after digitonin incubation and differential centrifugation was taken as a measure of the degree or tightness of membrane appression. The effects of various mono-, di-, and trivalent cations on the yield of the heavy fraction were studied, and the results interpreted in terms either of electrostatic screening or ion binding to the thylakoid membrane surface: Although there was some degree of cation specificity in the degree of thylakoid appression indicative of cation binding, the nonspecific screening effect was much more important in determining the overall balance of forces. It is postulated that stacking occurs in regions of low net surface charge density, with a possible segregation of excess negative charges into nonstacked regions. 相似文献
3.
Factors that may influence the extent of thylakoid membrane appression have been examined using lettuce (Lactuca sativa cv. Celtuce) grown under different irradiances. Electron microscopy and salt-induced chlorophyll fluorescence suggest that the percentage of membrane appression is increased in plants grown in low light (20 Wm–2) compared with those grown in high light (150 Wm–2). In high light plants surface charge, as measured by 9-aminoacridine, was found to be twice that measured in low light plants. There was a similar difference in ATPase activity of CF1 and in light saturated photophosphorylation. The chlorophyll content of LHC-2 as a proportion of the total chlorophyll was greatest in thylakoids of low light plants. Measurement of non-cyclic photophosphorylation rates suggested that membrane appression has a stimulatory role in the photophosphorylation process. The importance of these inter-related factors for the mechanism of thylakoid appression is discussed.Abbreviations PS
photosystem
- chl
chlorophyll
- LHC-2
light harvesting chlorophyll-protein complex serving PS 2
- CF1
coupling factor 1
- NADP
nicotinamide-adenine dinucleotide phosphate 相似文献
4.
Analyses of chlorophyll fluorescence induction kinetics from DCMU-poisoned thylakoids were used to examine the contribution of the light-harvesting chlorophyll a/b protein complex (LHCP) to Photosystem II (PS II) heterogeneity. Thylakoids excited with 450 nm radiation exhibited fluorescence induction kinetics characteristic of major contributions from both PS II and PS IIβ centres. On excitation at 550 nm the major contribution was from PS IIβ centres, that from PS II centres was only minimal. Mg2+ depletion had negligible effect on the induction kinetics of thylakoids excited with 550 nm radiation, however, as expected, with 450 nm excitation a loss of the PS II component was observed. Thylakoids from a chlorophyll-b-less barley mutant exhibited similar induction kinetics with 450 and 550 nm excitation, which were characteristic of PS IIβ centres being the major contributors; the PS II contribution was minimal. The fluorescence induction kinetics of wheat thylakoids at two different developmental stages, which exhibited different amounts of thylakoid appression but similar chlorophyll a/b ratios and thus similar PS II:LHCP ratios, showed no appreciable differences in the relative contributions of PS II and PS IIβ centres. Mg2+ depletion had similar effects on the two thylakoid preparations. These data lead to the conclusion that it is the PS II:LHCP ratio, and probably not thylakoid appression, that is the major determinant of the relative contributions of PS II and PS IIβ to the fluorescence induction kinetics. PS II characteristics are produced by LHCP association with PS II, whereas PS IIβ characteristic can be generated by either disconnecting LHCP from PS II or by preferentially exciting PS II relative to LHCP. 相似文献
5.
The time-resolved chlorophyll fluorescence emission of higher plant chloroplasts monitors the primary processes of photosynthesis and reflects photosynthetic membrane organization. In the present study we compare measurements of the chlorophyll fluorescence decay kinetics of the chlorophyll-b-less chlorina-f2 barley mutant and wild-type barley to investigate the effect of alterations in thylakoid membrane composition on chlorophyll fluorescence. Our analysis characterizes the fluorescence decay of chlorina-f2 barley chloroplasts by three exponential components with lifetimes of approx. 100 ps, 400 ps and 2 ns. The majority of the chlorophyll fluorescence originates in the two faster decay components. Although photo-induced and cation-induced effects on fluorescence yields are evident, the fluorescence lifetimes are independent of the state of the Photosystem-II reaction centers and the degree of grana stacking. Wild-type barley chloroplasts also exhibit three kinetic fluorescence components, but they are distinguished from those of the chlorina-f2 chloroplasts by a slow decay component which displays cation- and photo-induced yield and lifetime changes. A comparison is presented of the kinetic analysis of the chlorina-f2 barley fluorescence to the decay kinetics previously measured for intermittent-light-grown peas (Karukstis, K. and Sauer, K. (1983) Biochim. Biophys. Acta 725, 384–393). We propose that similarities in the fluorescence decay kinetics of both species are a consequence of analogous rearrangements of the thylakoid membrane organization due to the deficiencies present in the light-harvesting chlorophyll complex. 相似文献
6.
Fluorescence changes in isolated broken chloroplasts and the involvement of the electrical double layer 总被引:1,自引:0,他引:1
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We studied the effects of a variety of cations on chlorophyll fluorescence yield of broken chloroplasts prepared under carefully controlled ionic conditions. In the absence of light-induced electron transport and associated proton pumping, two types of cation-induced chlorophyll fluorescence changes could be distinguished in broken chloroplasts. These are termed "reversible" and "irreversible" fluorescence yield changes. Reversible fluorescence yield changes are characterized by antagonistic effects of monovalent and divalent cations and are prevented by the presence of 5 mM Mg2+ in the suspending media. Reversible-type fluorescence yield changes show little or no dependence on the structure, lipid solubility, or coordination number of the cation, but depend strictly on the net positive charge carried by the ion. It is proposed that these fluorescence changes are brought about through the interaction of monovalent or divalent cations with an electrical double layer at the interface of the outer surface of the thylakoid membrane and the surrounding aqueous solution. The results are interpreted in terms of the Gouy-Chapman theory of the diffuse double layer, indicating that the thylakoid outer surface bears an excess fixed negative charge density of about 2.5 muC/cm2, or approximately 1 negative charge per 640 A2 of membrane surface. Chlorophyll fluorescence quenching in isolated broken chloroplasts suspended in media containing 5 mM MgCl2 is also observed on addition of certain polyvalent cations to the medium. This type of cation-induced fluorescence change appears to be largely irreversible and may occur through specific binding of the cation to the thylakoid as a result of the high electrostatic attraction exerted by the negatively charged membrane surface. 相似文献
7.
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. 相似文献
8.
Experiments are presented to show that the phosphorylation of the light-harvesting chlorophyll complex (LHC) induces structural reorganisation within the thylakoid membrane in response to the introduction of additional negative surface charges. The effect of cations of different valency on chlorophyll fluorescence measurements indicates that LHC-phosphorylation-induced reorganisation involves a change in the electrostatic screening capability of the added cation. At intermediate levels of cations (e.g., 1 or 2 mM Mg2+), which substantially stack non-phosphorylated membranes, it was found that membrane phosphorylation caused considerable unstacking as monitored by light scattering and electron microscopy. Concomitant with this was a large decrease in chlorophyll fluorescence indicative of randomisation of chlorophyll protein complexes which would result in an increase in energy transfer between the photosystems as well as an absorption cross-section change. At higher concentrations (e.g., above 5 mM Mg2+) a persistent ATP-induced decrease in chlorophyll fluorescence has been attributed to the displacement of charged phosphorylated LHC from the appressed granal to the non-appressed stromal lamellae, thus decreasing the absorption cross-section of Photosystem II. Under these circumstances only a small degree of unstacking was detected by light scattering and measurements of the percentage of thylakoid length which is stacked to form grana. However, when considered on a surface area basis, the structural changes observed can qualitatively account for the magnitude of the chlorophyll fluorescence quenching due to the lateral diffusion of LHC. 相似文献
9.
Using measurements of the kinetics of chlorophyll a fluorescence emission, we have investigated the development of the photosynthetic membrane during etioplast-to-chloroplast differentiation. The chlorophyll fluorescence decay kinetics of pea chloroplasts from plants grown under intermittent (2 min light-118 min dark) and continuous light regimes were monitored with a single-photon timing system with picosecond resolution. We have associated the changes in the fluorescence yields and decay kinetics with known structural and organizational developmental phenomena in the chloroplast. This correlation provides a more detailed assignment of the origins of the fluorescence decay components than has been previously obtained by studying only mature chloroplasts. In particular, our analysis of the variable kinetics and multiexponential character of the fluorescence emission during thylakoid development focuses on the organization of photosynthetic units and the degree of communication between reaction centers in the same photosystem. Our results further demonstrate that the age of etiolated tissue is critical to plastid development. 相似文献
10.
Analyses of chlorophyll fluorescence induction kinetics from DCMU-poisoned thylakoids were used to examine the contribution of the light-harvesting chlorophyll
protein complex (LHCP) to Photosystem II (PS II) heterogeneity. Thylakoids excited with 450 nm radiation exhibited fluorescence induction kinetics characteristic of major contributions from both PS IIα and PS IIβ centres. On excitation at 550 nm the major contribution was from PS IIβ centres, that from PS IIα centres was only minimal. Mg2+ depletion had negligible effect on the induction kinetics of thylakoids excited with 550 nm radiation, however, as expected, with 450 nm excitation a loss of the PS IIα component was observed. Thylakoids from a chlorophyll-b-less barley mutant exhibited similar induction kinetics with 450 and 550 nm excitation, which were characteristic of PS IIβ centres being the major contributors; the PS IIα contribution was minimal. The fluorescence induction kinetics of wheat thylakoids at two different developmental stages, which exhibited different amounts of thylakoid appression but similar chlorophyll
ratios and thus similar PS II:LHCP ratios, showed no appreciable differences in the relative contributions of PS IIα and PS IIβ centres. Mg2+ depletion had similar effects on the two thylakoid preparations. These data lead to the conclusion that it is the PS II:LHCP ratio, and probably not thylakoid appression, that is the major determinant of the relative contributions of PS IIα and PS IIβ to the fluorescence induction kinetics. PS IIα characteristics are produced by LHCP association with PS II, whereas PS IIβ characteristic can be generated by either disconnecting LHCP from PS II or by preferentially exciting PS II relative to LHCP. 相似文献
11.
The increase of chlorophyll fluorescence yield in chloroplasts in a 12.5 Hz train of saturating single turnover flashes and the kinetics of fluorescence yield decay after the last flash have been analyzed. The approximate twofold increase in Fm relative to Fo, reached after 30-40 flashes, is associated with a proportional change in the slow (1-20 s) component of the multiphasic decay. This component reflects the accumulation of a sizeable fraction of QB-nonreducing centers. It is hypothesized that the generation of these centers occurs in association with proton transport across the thylakoid membrane. The data are quantitatively consistent with a model in which the fluorescence quenching of QB-nonreducing centers is reversibly released after second excitation and electron trapping on the acceptor side of Photosystem II. 相似文献
12.
13.
Cation-dependent changes in the thylakoid membrane appression of the diatom Thalassiosira pseudonana
Diatoms show a special organisation of their plastid membranes, such that their thylakoids span the entire plastid in bands of three. While in higher plants the interaction of the light harvesting complex II and photosystem II with divalent cations (especially Mg2+) was found to take part in the interplay of electrostatic attraction and repulsion in grana membrane appression, for diatoms the key players in maintaining proper membrane distances were not identified so far. In this work, we investigated the changes in the thylakoid architecture of Thalassiosira pseudonana in reaction to different salts by using circular dichroism and fluorescence spectroscopy in combination with other techniques. We show that divalent cations have an important influence on optimal pigment organisation and thus also on maintaining membrane appression. Thereby, monovalent cations are far less effective. The concentration needed is in a physiological range and fits well with the values obtained for higher plant grana stacking, despite the fact that strict protein segregation as seen in higher plant grana is missing. 相似文献
14.
Studies on Cation-induced Thylakoid Membrane Stacking, Fluorescence Yield, and Photochemical Efficiency 总被引:2,自引:1,他引:1
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Trypsin digestion of photosynthetic membranes isolated from spinach (Spinacia oleracea L.) leaves eliminates the cation stimulation of chlorophyll fluorescence. High concentrations of cations protect the fluorescence yield against trypsin digestion, and the cation specificity for this protection closely resembles that required for the stimulation of fluorescence by cations. Trypsin digestion reverses cation-induced thylakoid stacking, and the time course of this effect seems to parallel that of the reversal of cation fluorescence. High concentrations of cations protect thylakoid stacking and cation-stimulated fluorescence alike. The cation stimulation of photosytem II photochemistry remains intact after trypsinization has reversed both cation-induced thylakoid stacking and fluorescence yield. It is concluded that cation-stimulated fluorescence yield, and not the cation stimulation of photosystem II photochemistry, is associated with thylakoid membrane stacking. 相似文献
15.
Investigations of the role of the main light-harvesting chlorophyll- protein complex in thylakoid membranes. Reconstitution of depleted membranes from intermittent-light-grown plants with the isolated complex 总被引:1,自引:0,他引:1
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《The Journal of cell biology》1984,98(1):163-172
The functions of the light-harvesting complex of photosystem II (LHC- II) have been studied using thylakoids from intermittent-light-grown (IML) plants, which are deficient in this complex. These chloroplasts have no grana stacks and only limited lamellar appression in situ. In vitro the thylakoids showed limited but significant Mg2+-induced membrane appression and a clear segregation of membrane particles into such regions. This observation, together with the immunological detection of small quantities of LHC-II apoproteins, suggests that the molecular mechanism of appression may be similar to the more extensive thylakoid stacking seen in normal chloroplasts and involve LHC-II polypeptides directly. To study LHC-II function directly, a sonication- freeze-thaw procedure was developed for controlled insertion of purified LHC-II into IML membranes. Incorporation was demonstrated by density gradient centrifugation, antibody agglutination tests, and freeze-fracture electron microscopy. The reconstituted membranes, unlike the parent IML membranes, exhibited both extensive membrane appression and increased room temperature fluorescence in the presence of cations, and a decreased photosystem I activity at low light intensity. These membranes thus mimic normal chloroplasts in this regard, suggesting that the incorporated LHC-II interacts with photosystem II centers in IML membranes and exerts a direct role in the regulation of excitation energy distribution between the two photosystems. 相似文献
16.
We have investigated the influence of chloroplast organization on the nature of chemical reductive titrations of Photosystem II fluorescence decay kinetics in spinach chloroplasts. Structural changes of the chloroplast membrane system were induced by varying the ionic environment of the thylakoids. A single-photon timing system with picosecond resolution monitored the kinetics of the chlorophyll a fluorescence emission. At all ionic concentrations studied, we have observed biphasic potentiometric titration curves of fluorescence yield; these have been interpreted to be suggestive of electron acceptor Q heterogeneity (Karukstis, K.K. and Sauer, K. (1983) Biochim. Biophys. Acta 722, 364–371; Cramer, W.A. and Butler, W.L. (1969) Biochim. Biophys. Acta 172, 503–510). A direct relation is observed between the Em value of the low-potential component of Q and the Mg2+ concentration of the chloroplast suspending medium. We have attributed these midpoint potential variations to the thylakoid structural rearrangements involved in cation-regulated grana stacking. Ionic effects on the fluorescence decay kinetics at the redox transitions are discussed in terms of the heterogeneity of Photosystem II units (α- and β-centers) and the mechanism of deexcitation at a closed reaction center (fluorescence or nonradiative decay). 相似文献
17.
The amphipathic fluorescence probe, 2-p-toluidinonaphthalene-6-sulphonate has been used to investigate the surface electrical properties of chloroplast thylakoid membranes. The fluorescence yield of 2-p-toluidinonaphthalene-6-sulphonate in aqueous solution increases on addition of hypotonically shocked chloroplast, and the emission maximum shifts towards the blue to 440 nm, although the emission spectrum is somewhat distorted by chloroplast pigment absorption. The intensity of 2-p-toluidinonaphthalene-6-sulphonate fluorescence is further increased on adding salts to the membrane suspension, and changes of greater than 100% are routinely observed. Similar observations have also been made with soya bean phospholipid (azolectin) liposomes. The magnitude of the fluorescence increase is dependent on membrane concentration, being more pronounced at high surface area/suspending volume ratios. The effect of salt addition appears to be that of shielding the fixed negative charges on the membrane surface, thus increasing the fraction of 2-p-toluidinonaphthalene-6-sulphonate molecules at the surface, where the 2-p-toluidinonaphthalene-6-sulphonate has a higher fluorescence yield than in free aqueous solution. This concept is supported by the fact that the effectiveness of salts in increasing 2-p-toluidinonaphthalene-6-sulphonate fluorescence is as predicted by classical electrical double layer theory: governed mainly by the charge carried by the cation with an order of effectiveness C3+ greater than C2+ greater than C+, and not by the chemical nature of the cation or by the nature of its co-ion. It has been argued that the chlorophyll fluorescence yield, controlled by the cation composition of the suspending medium follows the total diffusible positive charge density at the thylakoid membrane surface (Barber, J., Mills, J. and Love, A. (1977) Febs. Lett. 74, 174--181). Although the cation induced 2-p-toluidinonaphthalene-6-sulphonate and chlorophyll fluorescence yield changes show similar characteristics, there are also distinct differences between the two phenomena particularly when cations are added to chloroplasts initially suspended in a virtually cation-free medium. Therefore it is concluded that although both 2-p-toluidinonaphthalene-6-sulphonate and chlorophyll fluorescence yields are governed by the electrical properties of the thylakoid membrane surface, the mechanism controlling their cation sensitivity is not the same. 相似文献
18.
Eight chlorophyll b deficient nuclear mutants of pea (Pisum sativum L.) have been characterized by low temperature fluorescence emission spectra of their leaves and by the ultrastructure, photochemical activities and polypeptide compositions of the thylakoid membranes. The room temperature fluorescence induction kinetics of leaves and isolated thylakoids have also been recorded. In addition, the effects of Mg2+ on the fluorescence kinetics of the membranes have been investigated. The mutants are all deficient in the major polypeptide of the light-harvesting chlorophyll a/b protein of photosystem II. The low temperature fluorescence emission spectra of aurea-5106, xantha-5371 and –5820 show little or no fluorescence around 730 nm (photosystem I fluorescence), but possess maxima at 685 and 695 nm (photosystem II fluorescence). These three mutants have low photosystem II activities, but significant photosystem I activities. The long-wavelength fluorescence maximum is reduced for three other mutants. The Mg2+ effect on the variable component of the room temperature fluorescence (685 nm) induction kinetics is reduced in all mutants, and completely absent in aurea-5106 and xantha-5820. The thylakoid membranes of these 2 mutants are appressed pairwise in 2-disc grana of large diameter. Chlorotica-1-206A and–130A have significant long-wavelength maxima in the fluorescence spectra and show the largest Mg2+ enhancement of the variable part of the fluorescence kinetics. These two mutants have rather normally structured chloroplast membranes, though the stroma regions are reduced. The four remaining mutants are in several respects of an intermediate type.Abbreviations Chl
chlorophyll
- CPI
Chi-protein complex I, Fo, Fv
- Fm
parameters of room temperature chlorophyll fluorescence induction kinetics
- F685, F695 and F-1
components of low temperature chlorophyll emission with maximum at 685, 695 and ca 735 nm, respectively
- PSI
photosystem I
- PSII
photosystem II
- LHCI and LHCII
light-harvesting chlorophyll a/b complexes associated with PSI and PSII, respectively
- SDS
sodium dodecyl sulfate 相似文献
19.
Heat-induced changes of chlorophyll fluorescence in isolated chloroplasts and related heat-damage at the pigment level 总被引:14,自引:0,他引:14
The heat-induced changes of chlorophyll fluorescence excitation and emission properties were studied in isolated chloroplasts of Larrea divaricata Cav. An analysis of the temperature dependency of fluorescence, under Fo and Fmax conditions, of temperature-jump fluorescence induction kinetics, and of 77 degrees K emission spectra of preheated chloroplasts revealed two major components in the heat-induced fluorescence changes: (1) a fluorescence rise, reflecting the block of Photosystem II reaction centers; and (2) a fluorescence decrease, caused by the functional separation of light-harvesting pigment protein complex from the rest of the pigment system. Preferential excitation of chlorophyll a around 420 nm, produced a predominant fluorescence rise. Preferential excitation of chlorophyll b, at 480 nm, gives a predominant fluorescence decrease. It is proposed that the overlapping of the fluorescence decrease on the somewhat faster fluorescence rise, results in the biphasic fluorescence rise kinetics observed in isolated chloroplasts. Both the rise component and the decay component are affected by the thermal stability of the chloroplasts, acquired during growth of the plants in different thermal environments. Mg2+ enhances the stability against heat-damage expressed in the decrease component, but has no effect on the rise component. Heat pretreatment leads to a decrease of the variable fluorescence in the light-induced 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) rise curve, but no change in half-rise time is observed. It is concluded that the block of Photosystem II reaction centers precedes the loss of the light-harvesting pigment protein complex. However, the approximately antiparallel heat-induced Fmax decrease and Fo increase suggest a common cause for the two events. A heat-induced perturbation of the thylakoid membrane is discussed. 相似文献
20.
The increase of chlorophyll fluorescence yield in chloroplasts in a 12.5 Hz train of saturating single turnover flashes and the kinetics of fluorescence yield decay after the last flash have been analyzed. The approximate twofold increase in Fm relative to Fo, reached after 30-40 flashes, is associated with a proportional change in the slow (1-20 s) component of the multiphasic decay. This component reflects the accumulation of a sizeable fraction of QB-nonreducing centers. It is hypothesized that the generation of these centers occurs in association with proton transport across the thylakoid membrane. The data are quantitatively consistent with a model in which the fluorescence quenching of QB-nonreducing centers is reversibly released after second excitation and electron trapping on the acceptor side of Photosystem II. 相似文献