Search

α-Tocopherol is a lipophilic antioxidant that is an efficient scavenger of singlet oxygen. We investigated the role of α-tocopherol in the protection of photosystem II (PSII) from photoinhibition using a mutant of the cyanobacterium Synechocystis sp. PCC 6803 that is deficient in the biosynthesis of α-tocopherol. The activity of PSII in mutant cells was more sensitive to inactivation by strong light than that in wild-type cells, indicating that lack of α-tocopherol enhances the extent of photoinhibition. However, the rate of photodamage to PSII, as measured in the presence of chloramphenicol, which blocks the repair of PSII, did not differ between the two lines of cells. By contrast, the repair of PSII from photodamage was suppressed in mutant cells. Addition of α-tocopherol to cultures of mutant cells returned the extent of photoinhibition to that in wild-type cells, without any effect on photodamage. The synthesis de novo of various proteins, including the D1 protein that plays a central role in the repair of PSII, was suppressed in mutant cells under strong light. These observations suggest that α-tocopherol promotes the repair of photodamaged PSII by protecting the synthesis de novo of the proteins that are required for recovery from inhibition by singlet oxygen.

Inhibition of the activity of photosystem II (PSII) under strong light is referred to as photoinhibition. This phenomenon is due to an imbalance between the rate of photodamage to PSII and the rate of the repair of damaged PSII. In the “classical” scheme for the mechanism of photoinhibition, strong light induces the production of reactive oxygen species (ROS), which directly inactivate the photochemical reaction center of PSII. By contrast, in a new scheme, we propose that photodamage is initiated by the direct effect of light on the oxygen-evolving complex and that ROS inhibit the repair of photodamaged PSII by suppressing primarily the synthesis of proteins de novo. The activity of PSII is restricted by a variety of environmental stresses. The effects of environmental stress on damage to and repair of PSII can be examined separately and it appears that environmental stresses, with the exception of strong light, act primarily by inhibiting the repair of PSII. Studies have demonstrated that repair-inhibitory stresses include CO2 limitation, moderate heat, high concentrations of NaCl, and low temperature, each of which suppresses the synthesis of proteins de novo, which is required for the repair of PSII. We postulate that most types of environmental stress inhibit the fixation of CO2 with the resultant generation of ROS, which, in turn, inhibit protein synthesis.

Irreversible photoinhibition of photosystem II (PSII) occurred when Synechocystis sp. PCC 6803 cells were exposed to very strong light for a prolonged period. When wild-type cells were illuminated at 20 degrees C for 2 h with light at an intensity of 2,500 micromol photons m(-2) s(-1), the oxygen-evolving activity of PSII was almost entirely and irreversibly lost, whereas the photochemical reaction center in PSII was inactivated only reversibly. The extent of irreversible photoinhibition was enhanced at lower temperatures and by the genetically engineered rigidification of membrane lipids. Western and Northern blotting demonstrated that, after cells had undergone irreversible photoinhibition, the precursor to D1 protein in PSII was synthesized but not processed properly. These observations may suggest that exposure of Synechocystis cells to strong light results in the irreversible photoinhibition of the oxygen-evolving activity of PSII via impairment of the processing of pre-D1 and that this effect of strong light is enhanced by the rigidification of membrane lipids.

The Photosystem II complex (PSII) is susceptible to inactivation by strong light, and the inactivation caused by strong light is referred to as photoinactivation or photoinhibition. In photosynthetic organisms, photoinactivated PSII is rapidly repaired and the extent of photoinactivation reflects the balance between the light-induced damage (photodamage) to PSII and the repair of PSII. In this study, we examined these two processes separately and quantitatively under stress conditions in the cyanobacterium Synechocystis sp. PCC 6803. The rate of photodamage was proportional to light intensity over a range of light intensities from 0 to 2000 microE m(-2) s(-1), and this relationship was not affected by environmental factors, such as salt stress, oxidative stress due to H2O2, and low temperature. The rate of repair also depended on light intensity. It was high under weak light and reached a maximum of 0.1 min(-1) at 300 microE m(-2) s(-1). By contrast to the rate of photodamage, the rate of repair was significantly reduced by the above-mentioned environmental factors. Pulse-labeling experiments with radiolabeled methionine revealed that these environmental factors inhibited the synthesis de novo of proteins. Such proteins included the D1 protein which plays an important role in the photodamage-repair cycle. These observations suggest that the repair of PSII under environmental stress might be the critical step that determines the outcome of the photodamage-repair cycle.

The Fad12 mutant of Synechocystis sp. PCC 6803 has a defect in the desA gene for delta 12 acyl-lipid desaturase. We identified a change in the nucleotide sequence of the structural gene for the desaturase, in which a leucine codon has been converted to a stop codon. Western blot analysis revealed that the delta 12 acyl-lipid desaturase was localized in both plasma membranes and thylakoid membranes of wild-type cells but was absent from both types of membrane in Fad12 cells. These findings suggest that the desaturation of fatty acids takes place in both types of membrane in Synechocystis sp. PCC 6803. The mutation in the delta 12 desaturase did not affect the lipid composition of thylakoid and plasma membranes, but it changed the fatty acid composition of lipids in similar ways in both types of membrane.

The major phospholipid, phosphatidylethanolamine (PE), of Vibrio sp. strain ABE-1 contains a unique trans-unsaturated fatty acid, 9-trans-hexadecenoic acid (16:1(9t], at the sn-2 position of the glycerol moiety. The major molecular species of PE that contain 16:1(9t) at the sn-2 position have either 9-cis-hexadecenoic acid (16:1(9c] or hexadecanoic acid (16:0) at the sn-1 position. The transition temperatures of the liquid-crystal to the gel phase of 16:1(9c)/16:1(9t)-PE and 16:0/16:1(9t)-PE were -3 degrees C and 38 degrees C, respectively, temperatures that were 31 degrees C and 18 degrees C higher than the corresponding temperatures for 16:1(9c)/16:1(9c)-PE and 16:0/16:1(9c)-PE. The proportion of 16:1(9c)/16:1(9t)-PE and 16:0/16:1(9t)-PE increased significantly in cells grown at 20 degrees C over that in cells grown at 5 degrees C. When cells grown at 5 degrees C were incubated at 20 degrees C in the presence of cerulenin, an inhibitor of the synthesis de novo of fatty acids, the level of 16:1(9t) increased almost in parallel with a concomitant decrease in the level of 16:1(9c) at the sn-2 position. These results suggest that 16:1(9c) is converted to 16:1(9t) by the cis/trans isomerization of the double bond in the fatty acid. This conversion is discussed as a possible strategy for adaptation by bacteria to changes in temperature.

The phase transition in the lipid phase of aqueous dispersions of diacylglyceryltrimethylhomoserine (DGTS) was measured by fluorescence depolarization of parinaric acid and differential scanning calorimetry. In both techniques, the phase transition temperatures (Tm) of 1-palmitoyl-2-stearoyl DGTS and of 1,2-distearoyl DGTS were 53 and 59°C, respectively. Each of these Tm values was significantly higher than the Tm value of phosphatidylcholine with an identical combination of fatty acids. This suggests that the intermolecular interactions of DGTS molecules are slightly different from those of phosphatidylcholine molecules.

1. 1. The structure of intracytoplasmic membranes of a photosynthetic bacterium Chromatium vinosum and a nitrogen-fixing bacterium Azotobacter vinelandii was studied by chemical modification of amino groups of phospatidylethanolamine and proteins with trinitrobenzensulfonate. 2. 2. Almost all the constituents of intracytoplasmic membranes of C. vinosum were solubilized in a mixture of chloroform, methanol and trichloroacetic acid. One-third of proteins in the intracytoplasmic membranes of C. vinosum was found solubilized in a mixture of chloroform and methanol. By using a column chromatography with Sephadex LH-20 in organic solvents, the unmodified as well as the trinitrophenylated proteins and also the trinitrophenylated phosphatidylethanolamine were separated from the other colored substances. 3. 3. In the chemical modification of the intracytoplasmic membrane preparations, 30% of phosphatidylethanolamine and 15% of protein amino groups in C. vinosum and 45% of phosphatidylethanolamine and 20% of protein amino groups in A. vinelandii were estimated to be exposed to the aqueous phase. In the single-layered liposomes composed of phosphatidylethanolamine and phospatidylglycerol with a ratio of 2:1, 40% of phosphatidylethanolamine were estimated to be exposed to the aqueous phase.

The temperatures of the lipid phase transition at which the solid phase disappears were determined by using the X-ray diffraction method in thylakoid membranes of the blue-green alga, Anacystis nidulans. The temperatures were determined as 26 and 16 degrees C for cells grown at 38 and 28 degrees C, respectively.

Absorption and fluorescence spectra in the red region of water-soluble chlorophyll proteins, Lepidium CP661, CP663 and Brassica CP673, pigment System II particles of spinach chloroplasts and chlorophyll a in diethylether solution at 25 degrees C were analyzed by the curve-fitting method (French, C.S., Brown, J.S. and Lawrence, M.C. (1972) Plant Physiol 49, 421--429). It was found that each of the chlorophyll forms of the chlorophyll proteins and the pigment System II particles had a corresponding fluorescence band with the Stokes shift ranging from 0.6 to 4.0 nm. The absorption spectrum of chlorophyll a in diethylether solution was analyzed to one major band with a peak at 660.5 nm and some minor bands, while the fluorescence spectrum was analyzed to one major band with a peak at 664.9 nm and some minor bands. A mirror image was clearly demonstrated between the resolved spectra of absorption and fluorescence. The absorption spectrum of Lepidium CP661 was composed of a chlorophyll b form with a peak at 652.8 nm and two chlorophyll a forms with peaks at 662.6 and 671.9 nm. The fluorescence spectrum was analyzed to five component bands. Three of them with peaks at 654.8, 664.6 and 674.6 nm were attributed to emissions of the three chlorophyll forms with the Stokes shift of 2.0--2.7 nm. The absorption spectrum of Brassica CP673 had a chlorophyll b form with a peak at 653.7 nm and four chlorophyll a forms with peaks at 662.7, 671.3, 676.9 and 684.2 nm. The fluorescence spectrum was resolved into seven component bands. Four of them with peaks at 666.7, 673.1, 677.5 and 686.2 nm corresponded to the four chlorophyll a forms with the Stokes shift of 0.6--4.0 nm. The absorption spectrum of the pigment System II particles had a chlorophyll b form with a peak at 652.4 nm and three chlorophyll a forms with peaks at 662.9, 672.1 and 681.6 nm. The fluorescence spectrum was analyzed to four major component bands with peaks at 674.1, 682.8, 692.0 and 706.7 nm and some minor bands. The former two bands corresponded to the chlorophyll a forms with peaks at 672.1 and 681.6 nm with the Stokes shift of 2.0 and 1.2 nm, respectively. Absorption spectra at 25 degrees C and at --196 degrees C of the water-soluble chlorophyll proteins were compared by the curve-fitting methods. The component bands at --196 degrees C were blue-shifted by 0.8--4.1 nm and narrower in half widths as compared to those at 25 degrees C.

The photosynthetic electron transport and phosphorylation reactions were measured in the room temperature region in the thylakoid membranes prepared from the blue-green alga, Anacystis nidulans . The Arrhenius plot of the Hill reaction with 2,6-dichlorophenolindophenol showed a distinct break of straight lines at 21°C in the membranes from cells grown at 38°C, and at 12°C in those from cells grown at 28°C. The Arrhenius plot of the Hill reaction with ferricyanide showed a break at 13°C in the membranes from cells grown at 38°C, and at 7°C in those from cells grown at 28°C. On the other hand, the Arrhenius plot of the System I reaction with methylviologen as an electron acceptor and 2,6-dichlorophenolindophenol and ascorbate as an electron donor system was composed of a straight line in the membranes from cells grown at 28°C as well as at 38°C. The Arrhenius plot of the System II reaction measured by the ferricyanide reduction mediated by silicotungstate in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea also showed a break at 11°C in the membranes from cells grown at 38°C. The Arrhenius plot of the phosphorylation mediated by N -methylphenazonium methylsulfate showed a break at 21°C in the membranes from cells grown at 38°C and at 12°C in those from cells grown at 28°C. The Arrhenius plot of the phosphorylation mediated by the System I reaction showed a break at 24°C in the membranes from cells grown at 38°C. The characteristic features in the Arrhenius plots of the photosynthetic electron transport and phosphorylation reactions are discussed in terms of the transition of physical phase of the thylakoid membrane lipids.

1. 1. Kinetics of the light-induced spectral shift of carotenoids were studied by changing temperature of cells of the thermophilic alga Cyanidium caldarium . Below the temperature of phase transition of thylakoid membrane lipids at 9 °C, the dark recovery of the carotenoid shift after several seconds of illumination was composed of a fast and a slow decay component. Above the phase transition temperature only the slow decay component was seen. 2. 2. In chloroplasts of a desert plant, Tidestromia oblongifolia , the fluorescence versus temperature curve indicated that the phase transition of the thylakoid membrane lipids occurred at about 5 °C. Below the phase transition temperature the kinetics of dark decay of the spectral shift of carotenoids at 520 nm in leaves was composed of a fast and a slow component. Above the phase transition temperature only the slow decay component was present. 3. 3. The light-induced spectral shift of carotenoids was measured at 520 nm in leaves of the chilling-sensitive plants, tomato and bean. The Arrhenius plots of dark recovery of the light-induced changes showed characteristic breaks at 10 and 12 °C in tomato leaves grown at 15 and 25 °C, respectively, and at 15 °C in a bean leaf. In leaves of the chilling-resistant plants lettuce and spinach, on the other hand, the dark recovery of the carotenoid shift showed no breaks in the Arrhenius plots. 4. 4. If the light-induced spectral shift of carotenoids in the photosynthetic membrane is caused by a production of a transmembrane electric field, then the dark recovery of this shift is an indicator of the disappearance of this accumulated electric charge. It is concluded that the diffusion of ions through thylakoid membranes is greatly affected by the physical phase of membrane lipids and that the membrane becomes leaky to the ions when the lipids are in the phase separation state.

Chymotrypsin and Staphylococcus aureus (strain V8) proteinase eliminated sixteen and eighteen amino acid residues, respectively, from the amino-terminal side of the extrinsic 33 kDa protein of the oxygen-evolving Photosystem II (PS II) complex of spinach. The carboxy-terminus of the resultant large fragments was found to be identical with that of the intact protein. Neither fragment could rebind to PS II membranes depleted of all the extrinsic proteins. Circular dichroism spectroscopy did not reveal any major conformational change within the two fragments. These results suggest that the amino-terminal region of the 33 kDa protein contains a domain essential for binding to the PS II complex.

The effects of divalent metal ions on the yields of chlorophyll a fluorescence were investigated in isolated spinach chloroplasts at room and liquid nitrogen temperatures. Mg 2+ , Ca 2+ , Sr 2+ , Ba 2+ and Mn 2+ increased the yields of fluorescence emission at 684 and 695 nm from pigment system II and decreased that at 735 nm from pigment system I. Al 3+ showed similar but less significant effects on the fluorescence yields. Zn 2+ and Cd 2+ showed no significant effect on the fluorescence yields at concentrations lower than 5 mM. In accordance with the results of our previous study concerning the effects of Mg 2+ on the excitation transfer in the chloroplasts, it was concluded that ions of alkaline earths and manganese suppress the excitation transfer from bulk chlorophyll a of pigment system II to that of pigment system I.

1. Delayed light of chlorophyll emitted at 0.1–3.9 ms after cessation of repetitive flash light was studied at temperatures between +40 and −196 °C in isolated spinach chloroplasts. 2. Induction kinetics of delayed light varied depending on temperature. It was found to be composed of two phases; one was an initial rapid rise followed by a rather fast decline to a low steady state level (fast phase), and the other was a slow increase after the initial rapid rise to the maximum followed by an insignificant slow decrease to a high steady state level (slow phase). The fast phase existed between −175 and 40 °C with the maximum at −40 °C, while the slow phase, between 0 and 40 °C with the maximum at 25 °C. 3. The intensity of delayed light at −175 °C was found to be less than one fiftieth that at 0 °C, and no delayed light emission was observed at −196 °C within experimental accuracy. This is in contrast to the results reported by Tollin, G., Fujimori, E. and Calvin, M. ((1958) Proc. Natl. Acad. Sci. U.S. 44, 1035–1047) in which the intensity of delayed light measured at −170 °C was about a half that at 0 °C. 4. The induction of delayed light measured at −96 °C was found to be significantly suppressed by the preillumination at −196 °C. This finding suggests that the primary photochemical event still survives at −196 °C without emission of delayed light. 5. Decay kinetics of delayed light after the flash excitation revealed the presence of at least two decay components. A slow decay component with a half decay time of several tens of milliseconds was observed at temperatures higher than 0 °C. A fast decay component with a half decay time of about 0.2 ms was observed at temperatures between −120 and 25 °C. The decay rate of this component was slightly retarded on cooling. 6. The System II particles derived from spinach chloroplasts with digitonin treatment showed a temperature dependence of delayed light similar to that of the chloroplasts. System I particles, on the other hand, scarcely emitted the delayed light at any temperature between 40 and −196 °C.

The time course of changes in delayed light emission measured at 1.2 msec (average) after each excitation flash during intermittent illumination of spinach chloroplasts showed a complicated time-dependent change. In this time course, the existence of at least two components was found. One which developed slowly during illumination was selectively and thoroughly eliminated by addition of the uncouplers methylamine or a low concentration of carbonylcyanide 3-chlorophenylhydrazone (CCCP), or by uncoupling treatments of chloroplasts such as EDTA washing, sonic oscillation of chloroplasts and addition of a low concentration of Triton X-100. This component was found to be completely suppressed by addition of inhibitors of electron transport, 3-(4′-chlorophenyl)-1,1-dimethylurea (CMU), 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU) and o -phenanthroline. A suspending medium of pH 6.0 was optimal for the development of the component. The other component which developed rapidly at the onset of illumination was suppressed by the inhibitors CMU, DCMU, o -phenanthroline and high concentrations of CCCP. The highest yield of this component was observed at pH 4. The mechanism of the delayed light emission was discussed on the basis of the assumption that it is ultimately excited by a reverse reaction of the electron flow through Photoreaction II. It was concluded that the former, slowly developing component of delayed light emission is related to the amounts of accumulated highenergy intermediate or state of photophosphorylation, and the latter, fast-developing component to the amounts of reduced and oxidized products formed during illumination by Photoreaction II.

Light-induced changes of absorbance were measured in Fraction 1 particles prepared from spinach chloroplasts according to a method developed by Michel and Michel-Wolwertz 6 that employs breakage of chloroplasts in the French pressure cell and centrifugation of the resulting fragments on a sucrose density gradient. Absorbance changes were measured in the presence of ascorbate and 2,3,5,6-tetramethyl-1,4-phenylenediamine as an electron donating system. In addition to the oxidation of P700 and cytochrome f, that was investigated in our previous study11, three other light-induced absorbance changes were observed; reduction of cytochrome b with a peak at 562 nm, the so-called 515-nm change showing an absorbance increase at 515 nm and a decrease at 480 nm, and a broad band of unknown absorbance increase covering wave-lengths from 490 to 570 nm. Uncouplers such as carbonylcyanide-m-chlorophenylhydrazone and gramicidin D inhibited the 515-nm change under continuous light and accelerated the dark decay of flash-light-induced change. The other broad absorbance change was insensitive to these inhibitors.

1. 1. Effects of metal ions on the yields of chlorophyll a fluorescence were investigated in spinach chloroplasts. At room and liquid-nitrogen temperatures, addition of Mg2+ increased the yields of two emissions of chlorophyll a, having peaks at 684 and 695 nm which were emitted from pigment system II and decreased the yield of another emission of chlorophyll a at about 735 nm which was emitted from pigment system I. Mg2+ also changed the pattern of the fluorescence induction at room temperature to increase the variable component of fluorescence. 2. 2. Effects of Mg2+ on the quantum yields of the two photoreactions were investigated in a region of weak light intensities. Mg2+ accelerated the rate of photoreaction II measured by the Hill reaction with 2,6-dichlorophenolindophenol and depressed the rate of photoreaction I measured by the NADP+ reduction in the presence of reduced 2,6-dichlorophenolindophenol and 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea. 3. 3. These facts indicate that Mg2+ controls the distribution of excitation energy between the two pigment systems and that the control consists mainly in change in rate of excitation transfer from the bulk chlorophyll a molecules in pigment system II to those in pigment system I; namely, Mg2+ suppresses the spillover of excitation energy from pigment system II to pigment system I.

1. 1. The induction of chlorophyll a fluorescence was measured at 690 nm in isolated spinach chloroplasts at liquid N2 temperature. The effects of preillumination of the chloroplasts with varied durations of flashes were investigated. When the degree of fluorescence induction was represented by the extent of the variable component of fluorescence, a 10-μs flash at a saturating intensity eliminated 13% of the induction, and a 20-ms flash eliminated 50%. 2. 2. An oxidant, ferricyanide, markedly decreased the fluorescence yield at the steady-state level but little at the initial rise level. 3. 3. The dark recovery of the fluorescence induction at liquid N2 temperature was found to be slow and only partial. The maximum recovery was reached after 20 min of dark time. 4. 4. The following scheme is presented for the reaction at the reaction center of System 2 at liquid N2 temperature. X and X− are the primary electron acceptors in the oxidized and reduced forms, respectively. Y and Y+ are the primary electron donors in the reduced and oxidized forms, respectively. The (X Y) state is converted to (X− Y+) by consuming the excitation energy. The (X− Y+) state formed is degradated either to (X Y) with loss of energy or to (X− Y) with reduction of Y+ by a secondary electron donor. It is suggested that all the reaction centers are in the (X− Y) state at the maximum fluorescence yield. Kinetic analysis indicates that the rate constants of Reaction 1 and 2 at liquid N2 temperature are 130 s−1 and 20 s−1, respectively, and the life time of the (X− Y+) state is 4.5 ms.