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1.
Experiments are presented to show that the phosphorylation of the light-harvesting chlorophyll ab-protein 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   

2.
The effect of Mg2+ concentration and phosphorylation of light-harvesting chlorophyll ab-protein on various chlorophyll fluorescence induction parameters of isolated pea thylakoids has been studied. (1) Lowering the Mg2+ concentration from 3 to 0.4 mM decreases only the variable fluorescence (Fv) and the area above the induction curve while at the same time increasing the slow exponential component of the rise (βmax). (2) A further decrease in Mg2+ concentration from 0.4 to 0 mM decreases the initial (F0) fluorescence level such that the ratio FvFm increases slightly as does the area above the induction curve and βmax. (3) Thylakoid membranes, phosphorylated at 5 mM Mg2+, show an equal decrease in Fv and F0, no change in the area above the induction curve and an increase in βmax. At 2 mM Mg2+, however, phosphorylation induced a more extensive quenching of Fv so that the FvFm ratio was lowered and the area above the induction curve decreased while βmax increased. (4) When phosphorylated membranes were subsequently suspended in an Mg2+-free medium the effect on F0 due to phosphorylation was found to be additive to that due to the absence of Mg2+. The effect of membrane phosphorylation on fluorescence is discussed in relation to the control of excitation energy distribution and shows that different mechanisms operate depending on the background Mg2+ levels. At high Mg2+ the phosphorylation seems to affect the absorption cross-section of Photosystem II while at lower Mg2+ levels there is an additional effect of increased spillover from Photosystem II to I.  相似文献   

3.
Single-photon timing with picosecond resolution is used to investigate the effect of Mg2+ on the room-temperature fluorescence decay kinetics in broken spinach chloroplasts. In agreement with an earlier paper (Haehnel, W., Nairn, J.A., Reisberg, P. and Sauer, K. (1982) Biochim. Biophys. Acta 680, 161–173), we find three components in the fluorescence decay both in the presence and in the absence of Mg2+. The behavior of these components is examined as a function of Mg2+ concentration at both the F0 and the Fmax fluorescence levels, and as a function of the excitation intensity for thylakoids from spinach chloroplasts isolated in the absence of added Mg2+. Analysis of the results indicates that the subsequent addition of Mg2+ has effects which occur at different levels of added cation. At low levels of Mg2+ (less than 0.75 mM), there appears to be a decrease in communication between Photosystem (PS) II and PS I, which amounts to a decrease in the spillover rate between PS II and PS I. At higher levels of Mg2+ (about 2 mM), there appears to be an increase in communication between PS II units and an increase in the effective absorption cross-section of PS II, probably both of these involving the chlorophyll ab light-harvesting antenna.  相似文献   

4.
Kenneth Leto  Charles Arntzen 《BBA》1981,637(1):107-117
Despite the total loss of Photosystem II activity, thylakoids isolated from the green nuclear maize mutant hcf1-3 contain normal amounts of the light-harvesting chlorophyll ab pigment-protein complex (LHC). We interpret the spectroscopic and ultrastructural characteristics of these thylakoids to indicate that the LHC present in these membranes is not associated with Photosystem II reaction centers and thus exists in a ‘free’ state within the thylakoid membrane. In contrast, the LHC found in wild-type maize thylakoids shows the usual functional association with Photosystem II reaction centers. Several lines of evidence suggest that the free LHC found in thylakoids isolated from hcf1-3 is able to mediate cation-dependent changes in both thylakoid appression and energy distribution between the photosystems: (1) Thylakoids isolated from hcf1-3 and wild-type seedlings exhibit a similar Mg2+-dependent increase in the short/long wavelength fluorescence emission peak ratio at 77 K. This Mg2+ effect is lost following incubation of thylakoids isolated from either source with low concentrations of trypsin. Such treatment results in the partial proteolysis of the LHC in both membrane types. (2) Thylakoids isolated from both hcf1-3 and wild-type seedlings show a similar Mg2+ dependence for the enhancement of the maximal yield of room temperature fluorescence and light scattering; both Mg2+ effects are abolished by brief incubation of the thylakoids with low concentrations of trypsin (3) Mg2+ acts to reduce the relative quantum efficiency of Photosystem I-dependent electron transport at limiting 650 nm light in thylakoids isolated from hcf1-3. (4) The pattern of digitonin fractionation of thylakoid membranes, which is dependent upon structural membrane interactions and upon LHC in the thylakoids, is similar in thylakoids isolated from both hcf1-3 and wild-type seedlings. We conclude that the surface-exposed segment of the LHC, but not the LHC-Photosystem II core association, is necessary for the cation-dependent changes in both thylakoid appression and energy distribution between the two photosystems, and that the LHC itself is able to transfer excitation energy directly to Photosystem I in a Mg2+-dependent fashion in the absence of Photosystem II reaction centers. The latter phenomenon is equivalent to a cation-induced change in the absorptive cross-section of Photosystem I.  相似文献   

5.
A highly purified light-harvesting pigment-protein complex (LHC) was obtained by fractionation of cation-depleted chloroplast membranes using the nonionic detergent, Triton X-100. The isolated LHC had a chlorophyll ab ratio of 1.2 and exhibited no photochemical activity. SDS-polyacrylamide gel electrophoresis of the LHC revealed three polypeptides in the molecular weight classes of 23, 25, and 30 × 103. Antibodies were prepared against the LHC and their specificity was established. The effect of the α-LHC (antibodies to LHC) on salt-mediated changes in PS I and PS II photochemistry, Chl α fluorescence inductions, and 77 °K fluorescence emission spectra was investigated. The results show that: (i) The Mg2+-induced 20% decrease in photosystem I (PS I) quantum yield observed in control chloroplasts was blocked by the presence of the α-LHC antibody, (ii) The Mg2+-induced 70% increase in photosystem II (PS II) quantum yield of control chloroplasts was reduced 35% for plastids in the presence of α-LHC antibody, (iii) The Mg2+-induced increase in room-temperature variable fluorescence was reduced 60% by α-LHC antibody, (iv) The Mg2+-induced increase in the F685F730 emission peak ratio at 77 °K was inhibited 50% in the presence of α-LHC antibody. These results provide direct evidence for the involvement of the light-harvesting complex in cation regulation of energy redistribution between the photosystems. The fact that the α-LHC antibody does not fully block Mg2+-induced PS II increases or chlorophyll fluorescence increases supports the concept that Mg2+ has two mechanisms of action: one effect on energy distribution and a second direct effect on photosystem II centers.  相似文献   

6.
The extraction of chlorophyll-protein (CP) complexes from thylakoids by the detergent octyl glucoside is strongly affected by pretreatment of the thylakoids with trypsin or cations. In these experiments, washed thylakoids were incubated in the presence of 0.5 μm to 5 mm Mg2+, pelleted, and extracted with octyl glucoside (30 mm). Increasing amounts of Mg2+ depressed extractability of all CP complexes, but especially the chlorophyll a + b-containing light-harvesting complex (LHC). This cation effect is observed with other cations which promote thylakoid stacking (5 mm Mn2+ or Ca2+, 50 mm Na+). However, the effect is not merely due to stacking, since low concentrations of Mg2+ (0.5 μmto 0.5 mm) have a marked effect on extractability but have no effect on light scattering (OD 550 nm), an indicator of stacking. Furthermore, trypsin treatment of thylakoids stacked with 5 mm Mg2+ caused a significant reversal of stacking, but had little effect on extractability. Trypsin treatment of unstacked membranes resulted in increased extractability of all CP complexes, but especially of the LHC. Cation-treated membranes are also significantly different from those “stacked” at pH 4.5. While the latter do show decreased extractability, there is no change in the chlorophyll ab ratio of the extract, and the membranes cannot be “unstacked” with trypsin. We conclude that octyl glucoside extractability reflects the lateral interaction of CP complexes with each other and with other components in the same plane of the membrane. It is clear that divalent cations have several effects on thylakoid membranes, not all of which are due to their ability to promote stacking.  相似文献   

7.
The addition of ATP to thylakoids isolated from Chlorella vulgaris is shown to lead to a quenching of fluorescence originating from Photosystem II and phosphorylation of chlorophyll achlorophyll b light-harvesting protein (LHCP) directly analogous to that reported for higher-plant chloroplasts. The time courses of these two processes are shown to be identical. Parallel measurements of ATP-induced changes in the fluorescence properties of isolated algal thylakoids and light-driven (State 1 / State 2 changes) in whole cells strongly support the idea that LHCP phosphorylation plays an important role in State 2 adaptation under in vivo conditions.  相似文献   

8.
In strong illumination, 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU)-poisoned chloroplasts exhibit a high yield of chlorophyll fluorescence while P-700 turnover, proton uptake, and phosphorylation are inhibited and a pH gradient is undetectable. When 10 μM N-methylphenazonium methosulfate (PMS) is included, the fluorescence yield in light is substantially reduced, and when 100 μM ascorbate is also included, the yield is diminished approximately to the level in darkness. Only very slight increases in P-700 turnover and proton uptake (but no detectable pH gradient) accompany the fluorescence yield decline.When 10 μM PMS and 15 mM ascorbate are added to poisoned chloroplasts (the oxygen concentration being greatly reduced), P-700 turnover, proton uptake, the pH gradient and phosphorylation all reach high levels. In this case, the yield of chlorophyll fluorescence is low and is the same in both light and dark. Further addition of an uncoupler eliminates proton uptake, the pH gradient and phosphorylation but does not significantly elevate the fluorescence yield. From these observations we suggest that, in DCMU-poisoned chloroplasts, the fluorescence quenching with PMS occurs by a mechanism unrelated to the generation of a phosphorylation potential.With chloroplasts unpoisoned by DCMU, PMS quenches fluorescence and considerably stimulates proton uptake, the pH gradient and phosphorylation. However, in this case, PMS serves to restore net electron transport.  相似文献   

9.
Peter Horton  Michael T. Black 《BBA》1983,722(1):214-218
Fluorescence induction curves in chloroplasts phosphorylated by the thylakoid protein kinase activated at low light intensity and high chlorophyll concentration have been measured. At 5 mM Mg2+, phosphorylation did not preferentially quench variable fluorescence. At 1 mM, preferential quenching of variable fluorescence was observed, indicating a second effect of phosphorylation at low Mg2+ (Horton, P. and Black, M.T. (1982) Biochim. Biophys. Acta 680, 22–27). Comparison of the extent of fluorescence decrease and the resulting ratio of variable to maximum fluorescence after phosphorylation and after lowering Mg2+ concentration demonstrated a difference between these two mechanisms of lowering of fluorescence. The significance of these results in terms of how phosphorylation may alter membrane organization is discussed.  相似文献   

10.
11.
《BBA》1987,893(3):480-489
Room temperature chlorophyll fluorescence lifetime measurements using single photon counting and low-intensity laser excitation have been carried out on photosynthetic systems which have undergone protein reorganisation by an in vivo state 1-state 2 transition, protein phosphorylation and the absence of Mg2+. Analysis of the global changes in average lifetime and total fluorescence yield suggest that each treatment brings about a decrease in Photosystem (PS) II absorption cross-section but that this mechanism of energy redistribution accounts for different proportions of the total fluorescence quenching in the various cases. Further analysis of the overall fluorescence decay into individual kinetic components was carried out using a four-exponential model. The state transition did not alter the lifetimes of the four components but decreased the fluorescence yield of the long-lived decay, at both F0 and FM, by 24% and increased the yield of the rapid components. Such changes infer that there is a decrease in PS II absorption cross-section and an increase in PS I excitation on going from state 1 to state 2. Furthermore, these alterations show that the 500 ps component (at F0) gives rise to the 2 ns decay (at FM). After in vitro protein phosphorylation at 5 mM Mg2+, the changes are very similar to those brought abought by a state transition, except that both long-lived kinetic components exhibit a decrease in yield. When protein phosphorylation was carried out at 2 mM Mg2+ a slight decrease in the lifetimes of the two slow components was observed, with a further decrease in the yield of the 2.3 ns decay and a larger increase in the yields of the two rapid decays. Although the fluorescence quenching brought about by the absence of Mg2+ (57%) was the largest of all the treatments, only a small part could be explained by a decrease in PS II absorption cross-section (17%). The absence of Mg2+ led to a decrease in the lifetimes and yields of the two long-lived decays. A careful comparison of the characteristics of the slowest component in the presence and absence of 5 mM Mg2+ on closing the PS II traps suggest that this decay has different origins in the two cases.  相似文献   

12.
S. Köster  U. Heber 《BBA》1982,680(1):88-94
Upon illumination of suspensions of intact chloroplasts, fluorescence of 9-aminoacridine was quenched, light scattering was increased, chlorophyll fluorescence was decreased after an initial increase, and chloroplast ATPADP ratios were increased. The response of 9-aminoacridine fluorescence quenching and light scattering to light intensity, anaerobiosis and inhibition of electron transport by DCMU was similar to that shown by chloroplast ATPADP ratios. It is discussed under what conditions 9-aminoacridine fluorescence quenching or light scattering can be used to monitor changes in the phosphorylation state of the chloroplast adenylate system.  相似文献   

13.
1. Phosphorylation of chloroplast membranes by illumination in the presence of ATP results in a 15–20% increase in the rate of Photosystem I electron transfer at low light intensity. 2. Phosphorylated membranes when depleted of Mg2+ and resuspended in a low salt medium still show a 17% lower yield of Photosystem II fluorescence than do unphosphorylated membranes. A 31% difference is seen after restoration of the maximal yield by addition of Mg2+. 3. The concentration of Mg2+ required to induce a half-maximal increase in fluorescence is 0.9 mM for control and 1.8 mM for phosphorylated chloroplasts. Phosphorylation at 1 mM Mg2+ can therefore cause more than double the amount of decrease in fluorescence yield from Photosystem II compared to phosphorylation at 5 mM. 4. The above results are discussed in terms of the mechanism of the ATP-induced fluorescence changes and a suggestion is made that the apparent interaction between phosphorylation and Mg2+ concentration may be a physiologically important phenomenon.  相似文献   

14.
The yield of P-700 photooxidation has been studied in isolated chloroplast membranes by measuring the extent of the flash-induced absorption increase at 820 nm (ΔA820) in the microsecond time range. The extent of ΔA820 induced by non-saturating laser flashes was increased by the following treatments. (1) Suspension of chloroplast membranes in Mg2+ free medium (plus 15 mM K+) which leads to unstacking of grana (as detected by a decrease in chlorophyll fluorescence). (2) Reduction of Q, the primary acceptor of Photosystem II, in the presence of 20 μM 3-(3,4 dichlorophenyl)-1,1-dimethylurea by a saturating xenon flash, fired 300 ms before the laser flash. (3) Phosphorylation of light harvesting chlorophyll ab-protein complex, which occurs in the presence of ATP after activation of protein kinase in the dark with NADPH and ferredoxin. We conclude that the Mg2+ concentration, the redox state of Q and the protein-phosphorylation all can control the photochemical efficiency of P-700 photooxidation in isolated chloroplasts, and we discuss these results in relation to control of excitation energy distribution between the two photosystems. We also discuss the significance of these results in relation to the regulation of photosynthetic electron transport in vivo.  相似文献   

15.
W.S. Chow  J. Barber 《BBA》1980,593(1):149-157
Salt-induced changes in thylakoid stacking and chlorophyll fluorescence do not occur with granal membranes obtained by treatment of stacked thylakoids with digitonin. In contrast to normal untreated thylakoids, digitonin prepared granal membranes remain stacked under all ionic conditions and exhibit a constant high level of chlorophyll fluorescence. However, unstacking of these granal membranes is possible if they are pretreated with either acetic anhydride or linolenic acid.Trypsin treatment of the thylakoids inhibits the salt induced chlorophyll fluorescence and stacking changes but stacking of these treated membranes does occur when the pH is lowered, with the optimum being at about pH 4.5. This type of stacking is due to charge neutralization and does not require the presence of the 2000 dalton fragment of the polypeptide associated with the chlorophyll achlorophyll b light harvesting complex and known to be lost during treatment with trypsin (Mullet, J.E. and Arntzen, C.J. (1980) Biochim. Biophys. Acta 589, 100–117).Using the method of 9-aminoacridine fluorescence quenching it is argued that the surface charge density, on a chlorophyll basis, of unstacked thylakoid membranes is intermediate between digitonin derived granal and stromal membranes, with granal having the lowest value.The results are discussed in terms of the importance of surface negative charges in controlling salt induced chlorophyll fluorescence and thylakoid stacking changes. In particular, emphasis is placed on a model involving lateral diffusion of different types of chlorophyll protein complex within the thylakoid lipid matrix.  相似文献   

16.
Jajoo  Anjana  Bharti  Sudhakar 《Photosynthetica》2000,37(4):529-535
Cations such as Mg2+ regulate spillover of absorbed excitation energy mainly in favour of photosystem (PS) 2. Effect of low concentration (<10 mM) of the monovalent cation Na+ on chlorophyll (Chl) a fluorescence was completely overridden by divalent cation Mg2+ (5 mM). Based on Chl a fluorescence yield and 77 K emission measurements, we revealed the role and effectiveness of anions (Cl-, SO4 2-, PO4 3-) in lowering the Mg2+-induced PS2 fluorescence. The higher the valency of the anion, the lesser was the expression of Mg2+ effect. Anions may thus overcome Mg2+ effects up to certain extent in a valency dependent manner, thereby diverting more energy to PS1 even in the presence of MgCl2. They may do so by reversing Mg2+-induced changes.  相似文献   

17.
18.
The effects of protein phosphorylation and cation depletion on the electron transport rate and fluorescence emission characteristics of photosystem I at two stages of chloroplast development in light-grown wheat leaves are examined. The light-harvesting chlorophyll a/b protein complex associated with photosystem I (LHC I) was absent from the thylakoids at the early stage of development, but that associated with photosystem II (LHC II) was present. Protein phosphorylation produced an increase in the light-limited rate of photosystem I electron transport at the early stage of development when chlorophyll b was preferentially excited, indicating that LHC I is not required for transfer of excitation energy from phosphorylated LHC II to the core complex of photosystem I. However, no enhancement of photosystem I fluorescence at 77 K was observed at this stage of development, demonstrating that a strict relationship between excitation energy density in photosystem I pigment matrices and the long-wavelength fluorescence emission from photosystem I at 77 K does not exist. Depletion of Mg2+ from the thylakoids produced a stimulation of photosystem I electron transport at both stages of development, but a large enhancement of the photosystem I fluorescence emission was observed only in the thylakoids containing LHC I. It is suggested that the enhancement of PS I electron transport by Mg2+-depletion and phosphorylation of LHC II is associated with an enhancement of fluorescence at 77 K from LHC I and not from the core complex of PS I.  相似文献   

19.
J. Barber  G.F.W. Searle  C.J. Tredwell 《BBA》1978,501(2):174-182
The MgCl2-induced chlorophyll fluorescence yield changes in broken chloroplasts, suspended in a cation-free medium, treated with 3,-(3′,4′-dichlorophenyl)-1,1-dimethylurea and pre-illuminated, has been investigated on a picosecond time scale. Chloroplasts in the low fluorescing state showed a fluorescence decay law of the form exp ?At12, where A was found to be 0.052 ps?12, and may be attributed to the rate of spillover from Photosystem II to Photosystem I. Addition of 10 mM MgCl2 produced a 50% increase in the steady-state fluorescence quantum yield and caused a marked decrease in the decay rate. The fluorescence decay law was found to be predominantly exponential with a 1/e lifetime of 1.6 ns. These results support the hypothesis that cation-induced changes in the fluorescence yield of chlorophyll are related to the variations in the rate of energy transfer from Photosystem II to Photosystem I, rather than to changes in the partitioning of absorbed quanta between the two systems.  相似文献   

20.
Low concentrations of Mg2+ (concn < 10 mm) generate structural changes in delipidated spinach chloroplast lamellae, that appear as changes in the fluorescence yield of native tryptophyl residues and of the externally added polarity probe magnesium 1-anilinonaphthalene-8-sulfonate.The delipidated lamellae, consisting essentially of structural protein monomers and aggregates, bind magnesium 1-anilinonaphthalene-8-sulfonate to the extent of 126 ± 13 nmol/mg protein, and with a dissociation constant KD = 167 μM. Bound ANS fluoresces at 458 nm with a quantum yield Φ = 0.121. Tryptophyls sensitize the fluorescence of bound ANS with a maximal efficiency Tmax = 0.85. Assuming completely random orientation of the interacting chromophores, an interchromophore separation R = 17.3 A? is calculated. Only two-thirds of the membrane tryptophyls have ANS-binding sites in their vicinity.Mg2+ binds to the delipidated membranes with a dissociation constant KD = 2 mM. The binding is attended by enhancement of magnesium 1-anilinonaphthalene-8-sulfonate fluorescence, and deenhancement of tryptophyl fluorescence, while the efficiency of interchromophore excitation transfer increases only slightly. These effects suggest that Mg2+ generates a structural change which lowers the polarity of the membrane region where tryptophyl and magnesium 1-anilinonaphthalene-8-sulfonate are situated, but which has a minor effect only on the interchromophore separation.  相似文献   

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