Characterization of a non-detergent PS II-cytochrome b/f preparation (BS)

A non-detergent photosystem II preparation, named BS, has been characterized by countercurrent distribution, light saturation curves, absorption spectra and fluorescence at room and at low temperature (–196°C). The BS fraction is prepared by a sonication-phase partitioning procedure (Svensson P and Albertsson P-Å, Photosynth Res 20: 249–259, 1989) which removes the stroma lamellae and the margins from the grana and leaves the appressed partition region intact in the form of vesicles. These are closed structures of inside-out conformation. They have a chlorophyll a/b ratio of 1.8–2.0, have a high oxygen evolving capacity (295 mol O₂ per mg chl h), are depleted in P700 and enriched in the cytochrome b/f complex. They have about 2 Photosystem II reaction centers per 1 cytochrome b/f complex.The plastoquinone pool available for PS II in the BS vesicles is 6–7 quinones per reaction center, about the same as for the whole thylakoid. It is concluded, therefore, that the plastoquinone of the stroma lamellae is not available to the PS II in the grana and that plastoquinone does not act as a long range electron transport shuttler between the grana and stroma lamellae.Compared with Photosystem II particles prepared by detergent (Triton X-100) treatment, the BS vesicles retain more cytochrome b/f complex and are more homogenous in their surface properties, as revealed by countercurrent distribution, and they have a more efficient energy transfer from the antenna pigments to the reaction center.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F_v variable fluorescence - LHC light-harvesting complex - PpBQ phenyl-p-benzoquinone - PQ plastoquinone pool - P700 reaction center of PS I - PS I, PS II Photosystem I, II - Q_A first bound plastoquinone accepter - RC reaction centre     

Quantitative separation of spinach thylakoids into Photosystem II-enriched inside-out vesicles and Photosystem I-enriched right-side-out vesicles

Yeda press disruption of thylakoids in the presence of magnesium followed by aqueous polymer two-phase partitioning fractionated the total thylakoid membrane material into two distinctly different fractions. One fraction comprised approx. 60% of the material on a chlorophyll basis and contained inside-out vesicles while the other fraction (40%) contained right-side-out vesicles. The sidedness of the vesicles was determined from the direction of their light-induced proton translocation. The inside-out vesicles showed a pronounced Photosystem (PS) II enrichment as judged by their high PS II and low PS I activities. Moreover, they showed a high ratio between the PS II reaction centre chlorophyll-protein complex and the PS I reaction centre chlorophyll-protein complex (CP I). The chlorophyll $\text{a}\text{b}$ ratio was as low as 2.3 compared to 3.2 for the starting material. In contrast, the right-side-out vesicles showed a pronounced PS I enrichment. Their chlorophyll $\text{a}\text{b}$ ratio was 4.3–4.9. The tight stacking induced by Mg²⁺ allows a quantitative formation of inside-out vesicles from the appressed thylakoid regions while mainly non-appressed thylakoids turn right-side-out. The possibility of fractionating all of the thylakoid material into two sub-populations with markedly different composition with respect to PS I and PS II argues against a close physical association between the two photosystems and in favour of their spatial separation in the plane of the membrane. This fractionation procedure, which can be completed within 1 h and gives high yields of both PS II inside-out thylakoids and PS I right-side-out thylakoids, should be very useful for facilitating and improving studies on both the transverse and lateral organization of the thylakoid membrane.     

Rapid and simple isolation of pure photosystem II core and reaction center particles from spinach

Pure and active oxygen-evolving PS II core particles containing 35 Chl per reaction center were isolated with 75% yield from spinach PS II membrane fragments by incubation with n-dodecyl--D-maltoside and a rapid one step anion-exchange separation. By Triton X-100 treatment on the column these particles could be converted with 55% yield to pure and active PS II reaction center particles, which contained 6 Chl per reaction center.Abbreviations Bis-Tris bis[2-hydroxyethyl]imino-tris[hydroxymethyl]methane - Chl chlorophyll - CP29 Chl a/b protein of 29 kDa - Cyt b ₅₅₉ cytochrome b ₅₅₉ - DCBQ 2,5-dichloro-p-benzo-quinone - LHC II light-harvesting complex II, predominant Chl a/b protein - MES 2-[N-Morpholino]ethanesulfonic acid - Pheo pheophytin - PS H photosystem II - Q_A bound plastoquinone, serving as the secondary electron acceptor in PS II (after Pheo) - SDS sodiumdodecylsulfate     

A single step separation of PS 1, PS 2 and chlorophyll-antenna particles from spinach chloroplasts

After solubilization of photosynthetic membranes by digitonin, three main protein pigment complexes were isolated by electrophoresis with deoxycholate as detergent.The band with the slowest mobility, fraction 1, had PS 1 activity and was devoid of PS 2 activity. This fraction was four times enriched in P700 when compared with chloroplasts. Fraction 1 had little chl b, a long wavelength absorption maximum in the red, a maximum of low temperature emission fluorescence at 730nm, and a circular dichroism spectrum characteristic of PS 1 enriched fraction.Fraction 2 exhibited a PS 2 activity and no PS 1 activity. It was enriched five times in PS 2 reaction centre and had little chl b and carotenoids. The absorption maximum was at 674 nm and the low temperature fluorescence emission maximum was at 700 nm. Fraction 2 might be useful PS 2 enriched particle because of the great stability of this fraction with regard to photochemical activity and also rapidity and simplicity of its preparation.Fraction 3, which had the fastest migration, was devoid of photochemical activities; It was rich in chl b and had the fluorescence and the circular dichroism spectrum characteristic of an antenna complex.Abbreviations PS 1 (2) photosystem 1 (2) - chl chlorophyll - car carotenoid - Q primary plastoquinone electron acceptor - P700 primary electron donor of PS 1 - P680 primary electron donor of PS 2 - K₃Fe(CN)₆ potassium ferricyanide - DCMU dichlorophenyldimethylurea - DCPIP dichlorophenolindophenol - DPC diphenyl-carbazide     

Development of Photosystem II in dark grown Chlamydomonas reinhardtii. A light-dependent conversion of PS IIβ, QB-nonreducing centers to the PS IIα, QB-reducing form

The green alga Chlamydomonas reinhardtii is a facultative heterotroph and, when cultured in the presence of acetate, will synthesize chlorophyll (Chl) and photosystem (PS) components in the dark. Analysis of the thylakoid membrane composition and function in dark grown C. reinhardtii revealed that photochemically competent PS II complexes were synthesized and assembled in the thylakoid membrane. These PS II centers were impaired in the electron-transport reaction from the primary-quinone electron acceptor, Q_A, to the secondary-quinone electron acceptor, Q_B (Q_B-nonreducing centers). Both complements of the PS II Chl a–b light harvesting antenna (LHC II-inner and LHC II-peripheral) were synthesized and assembled in the thylakoid membrane of dark grown C. reinhardtii cells. However, the LHC II-peripheral was energetically uncoupled from the PS II reaction center. Thus, PS II units in dark grown cells had a -type Chl antenna size with only 130 Chl (a and b) molecules (by definition, PS II units lack LHC II-peripheral). Illumination of dark grown C. reinhardtii caused pronounced changes in the organization and function of PS II. With a half-time of about 30 min, PS II centers were converted froma Q_B-nonreducing form in the dark, to a Q_B-reducing form in the light. Concomitant with this change, PS II units were energetically coupled with the LHC II-peripheral complement in the thylakoid membrane and were converted to a PS II form. The functional antenna of the latter contained more than 250 Chl(a+b) molecules. The results are discussed in terms of a light-dependent activation of the Q_A-Q_B electron-transfer reaction which is followed by association of the PS II unit with a LHC II-peripheral antenna and by inclusion of the mature form of PS II (PS II) in the membrane of the grana partition region.Abbreviations Chl chlorophyll - PS photosystem - Q_A primary quinone electron acceptor of PS II - Q_B secondary quinone electron acceptor of PS II - LHC light harvesting complex - F₀ non-variable fluorescence yield - F_plf intermediate fluorescence yield plateau leyel - F_max maximum fluorescence yield - F_i initial fluorescence yield increase from F₀ to F_pl (F_pl–F₀) - F_v total variable fluorescence yield (F_m–F0) - DCMU dichlorophenyl-dimethylurea     

Decrease of polypeptides in the PS I antenna complex with increasing growth irradiance in the red alga Porphyridium cruentum

Thylakoids isolated from cells of the red alga Porphyridium cruentum exhibit an increased PS I activity on a chlorophyll basis with increasing growth irradiance, even though the stoichiometry of Photosystems I and II in such cells shows little change (Cunningham et al. (1989) Plant Physiol 91: 1179–1187). PS I activity was 26% greater in thylakoids of cells acclimated at 280 mol photons · m^–2 · s^–1 (VHL) than in cells acclimated at 10 mol photons · m^–2 · s^–1 (LL), indicating a change in the light absorbance capacity of PS I. Upon isolating PS I holocomplexes from VHL cells it was found that they contained 132±9 Chl/P700 while those obtained from LL cells had 165±4 Chl/P700. Examination of the polypeptide composition of PS I holocomplexes on SDS-PAGE showed a notable decrease of three polypeptides (19.5, 21.0 and 22 kDa) in VHL-complexes relative to LL-complexes. These polypeptides belong to a novel LHC I complex, recently discovered in red algae (Wolfe et al. (1994a) Nature 367: 566–568), that lacks Chl b and includes at least six different polypeptides. We suggest that the decrease in PS I Chl antenna size observed with increasing irradiance is attributable to changes occurring in the LHC I-antenna complex. Evidence for a Chl-binding antenna complex associated with PS II core complexes is lacking at this point. LHC II-type polypeptides were not observed in functionally active PS II preparations (Wolfe et al. (1994b) Biochimica Biophysica Acta 1188: 357–366), nor did we detect polypeptides that showed immunocross-reactivity with LHC II specific antisera (made to Chlamydomonas and Euglena LHC II).Abbreviations Bis-Tris bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane - DCPIP 2,6-dichlorophenol indophenol - -dm dodecyl--d-maltoside - HL high light of 150 mol photons · m^–2 · s^–1 - LGB lower green band - LHC I light-harvesting complex of PS I - LHC II light-harvesting complex of PS II - LL low light of 10 mol photons · m^–2 · s^–1 - ML medium light of 50 mol photons · m^–2 · s^–1 - MES 2-(N-morpholino) ethanesulfonic acid - P700 reaction center of PS I - PFD photon flux density - Trizma tris(hydroxymethyl)aminomethane - UGB upper green band - VHL very high light of 280 mol photons · m^–2 · s^–1     

Calculation of absolute photosystem I absorption cross-sections from P700 photo-oxidation kinetics

A procedure is described which permits determination of the absolute absorption cross-section of a photosynthetic unit from the kinetics of reaction center photo-oxidation under weak, continuous actinic illumination. The method was first tested on a simple model compound of known absorption cross-section. We then applied the technique to absorption cross-section and functional antenna size measurements in photosystem I (PS I). A kinetic model is presented that can be used to fit P700 photo-oxidation measurements and extract the effective photochemical rate constant. The procedure is shown to properly correct for sample scattering and for the presence of heterogeneous absorbers (pigments not functionally coupled to P700). The relevance of these corrections to comparisons of antenna size using techniques that measure relative absorption cross-sections is discussed. Measurements on pea thylakoids in the presence and absence of 5 mM MgCl₂ show a 45% increase in PS I absorption cross-section in unstacked thylakoids. Analysis of detergent-isolated native PS I preparations (200 chlorophyll a+b/P700) clearly indicate that the preparation contains a broad distribution of antenna sizes. Finally, we confirm that Chlamydomonas reinhardtii strain LM3-A4d contains a PS I core antenna complex which binds only 60 chlorophyll a/P700, about half the functional size of the wild type complex. Limitations associated with calculation of functional antenna size from cross-section measurements are also discussed.Abbreviations PS photosystem - PS I-200 detergent-isolated photosystem I preparation containing about 200 Chl a+b/P700 - A _xxx absorbance at xxx nm - absolute absorption cross-section - I _a rate of light absorption - In _o incident actinic light intensity - _p quantum yield of photochemistry - k _eff effective rate constant for P700 photo-oxidation measured under conditions of limiting actinic intensity - k _r rate constant for P700⁺ reduction     

Normal-phase HPLC quantitation of chlorophyll a′ and phylloquinone in Photosystem I particles

Fractionated Photosystem (PS) I particles consisting of six, five or two core proteins were analyzed by HPLC for chlorophyll (Chl) a and phylloquinone (PhQ). Each particle had a Chl a/P700 molar ratio of 50–55 and contained ca. 2 molecules of Chl a per P700. Deliberate control of eluent composition led to isolated elution of PhQ and -carotene in the normal-phase chromatogram. Based on these a simple HPLC procedure has been established to determine the PhQ/P700 molar ratio, which was ca. 2 for the larger two PS I particles and ca. 1 for the smallest particle, in line with previous reports.     

Photoinhibition of Photosystem I electron transfer activity in isolated Photosystem I preparations with different chlorophyll contents

Photoinhibition of the light-induced Photosystem I (PS I) electron transfer activity from the reduced dichlorophenol indophenol to methyl viologen was studied. PS I preparations with Chl/P700 ratios of about 180 (PS I-180), 100 (PS I-100) and 40 (PS I(HA)-40) were isolated from spinach thylakoid membranes by the treatments with Triton X-100, followed by sucrose density gradient centrifugation and hydroxylapatite column chromatography. White light irradiation (1.1 × 10⁴E m^–2 s^–1) of PS I-180 for 2 hours bleached 50% of the chlorophyll and caused a 58% decrease in the electron transfer activity with virtually no loss of the primary donor, P700. The flash-induced absorbance change showed the decay phase with a half time of about 10 s that was attributed to the P700 triplet, suggesting that the photoinhibitory light treatment caused the destruction of the PS I acceptor(s), F_x and possibly A₁. PS I-100 was similarly photobleached by the irradiation and the electron transfer activity decreased. There was, however, no apparent photoinhibition of the electron transport activity in PS I(HA)-40. Photoinhibition similar to that seen in PS I-180 also occurred in membrane fragments that were isolated without any detergent from a PS II-deficient mutant strain of the cyanobacterium Synechocystis sp. PCC 6803. PS I-180 was not photoinhibited under anaerobic conditions. The production of superoxide and fatty acid hydroperoxide during white light irradiation was significantly greater in PS I-180 than in PS I(HA)-40. The mechanism of photoinhibition in PS I preparations is discussed in relation to the formation of toxic oxygen molecules.Abbreviations A₀,A₁ primary and secondary electron acceptors of PS I - CD circular dichroism - DCPIP 2,6-dichlorophenol indophenol - F_A, F_B, F_X iron-sulfur centers A, B, X - HA hydroxylapatite - LHCI lightharvesting complex of PS I - MDA malondialdehyde - MV methyl viologen - Na-Asc sodium L-ascorbate - P700 primary electron donor of PS I - PFD photon flux density - PS I-A and PS I-B psaA and psaB gene products - TBA thiobarbituric acid     

Photosystem 2-activity and thylakoid membrane polypeptides of <Emphasis Type="Italic">in vitro</Emphasis> cultured chrysanthemum as affected by NaCl

Long-term (30 d) effects of 100, 200, 300, and 400 mM NaCl on photosystem 2 (PS 2)-mediated electron transport activity and content of D1 protein in the thylakoid membranes of chrysanthemum (Dendranthema grandiflorum) cultured in vitro at low irradiance 20 μmol(photon) m⁻² s⁻¹ were investigated. 100 mM NaCl increased contents of chlorophylls (Chl) a and b, carotenoids (Car; xanthophylls + carotenes), and the ratio of Chl a/b, and Car/Chl a+b. However, further increase in NaCl concentration led to the significant reduction in the contents of Chl a, and Chl b, and increase in the ratio of Chl a/b and Car/Chl a+b. NaCl treatment decreased the PS 2-mediated electron transport activity and contents of various thylakoid membrane polypeptides including D1 protein.     

Photosystem II chlorophyll a fluorescence lifetimes and intensity are independent of the antenna size differences between barley wild-type and chlorina mutants: Photochemical quenching and xanthophyll cycle-dependent nonphotochemical quenching of fluorescence

Photosystem II (PS II) chlorophyll (Chl) a fluorescence lifetimes were measured in thylakoids and leaves of barley wild-type and chlorina f104 and f2 mutants to determine the effects of the PS II Chl a+b antenna size on the deexcitation of absorbed light energy. These barley chlorina mutants have drastically reduced levels of PS II light-harvesting Chls and pigment-proteins when compared to wild-type plants. However, the mutant and wild-type PS II Chl a fluorescence lifetimes and intensity parameters were remarkably similar and thus independent of the PS II light-harvesting antenna size for both maximal (at minimum Chl fluorescence level, F_o) and minimal rates of PS II photochemistry (at maximum Chl fluorescence level, F_m). Further, the fluorescence lifetimes and intensity parameters, as affected by the trans-thylakoid membrane pH gradient (pH) and the carotenoid pigments of the xanthophyll cycle, were also similar and independent of the antenna size differences. In the presence of a pH, the xanthophyll cycle-dependent processes increased the fractional intensity of a Chl a fluorescence lifetime distribution centered around 0.4–0.5 ns, at the expense of a 1.6 ns lifetime distribution (see Gilmore et al. (1995) Proc Natl Acad Sci USA 92: 2273–2277). When the zeaxanthin and antheraxanthin concentrations were measured relative to the number of PS II reaction center units, the ratios of fluorescence quenching to [xanthophyll] were similar between the wild-type and chlorina f104. However, the chlorina f104, compared to the wild-type, required around 2.5 times higher concentrations of these xanthophylls relative to Chl a+b to obtain the same levels of xanthophyll cycle-dependent fluorescence quenching. We thus suggest that, at a constant pH, the fraction of the short lifetime distribution is determined by the concentration and thus binding frequency of the xanthophylls in the PS II inner antenna. The pH also affected both the widths and centers of the lifetime distributions independent of the xanthophyll cycle. We suggest that the combined effects of the xanthophyll cycle and pH cause major conformational changes in the pigment-protein complexes of the PS II inner or core antennae that switch a normal PS II unit to an increased rate constant of heat dissipation. We discuss a model of the PS II photochemical apparatus where PS II photochemistry and xanthophyll cycle-dependent energy dissipation are independent of the Peripheral antenna size.Abbreviations Ax antheraxanthin - BSA bovine serum albumin - c_x lifetime center of fluorescence decay component x - CP chlorophyll binding protein of PS II inner antenna - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DTT dithiothreitol - f_x fractional intensity of fluorescence lifetime component x - F_m, F_m maximal PS II Chl a fluorescence intensity with all Q_A reduced in the absence, presence of thylakoid membrane energization - F_o minimal PS II Chl a fluorescence intensity with all Q_A oxidized - F_v=F_m–F_o variable level of PS II Chl a fluorescence - HPLC high performance liquid chromatography - k_A rate constant of all combined energy dissipation pathways in PS II except photochemistry and fluorescence - k_F rate constant of PS II Chl a fluorescence - LHCIIb main light harvesting pigment-protein complex (of PS II) - N_pig mols Chl a+b per PS II - NPQ=(F_m/F_m–1) nonphotochemical quenching of PS II Chl a fluorescence - PAM pulse-amplitude modulation fluorometer - PFD photon-flux density, mols photons m^–2 s^–1 - PS II Photosystem II - P680 special-pair Chls of PS II reaction center - Q_A primary quinone electron acceptor of PS II - V_x violaxanthin - w_x width at half maximum of Lorentzian fluorescence lifetime distribution x - Zx zeaxanthin - pH trans-thylakoid proton gradient - % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXafv3ySLgzGmvETj2BSbqef0uAJj3BZ9Mz0bYu% H52CGmvzYLMzaerbd9wDYLwzYbItLDharqqr1ngBPrgifHhDYfgasa% acOqpw0xe9v8qqaqFD0xXdHaVhbbf9v8qqaqFr0xc9pk0xbba9q8Wq% Ffea0-yr0RYxir-Jbba9q8aq0-yq-He9q8qqQ8frFve9Fve9Ff0dme% GabaqaaiGacaGaamqadaabaeaafiaakeaacqGH8aapcqaHepaDcqGH% +aGpdaWgaaWcbaGaamOraiaad2gaaeqaaaaa!4989!\[< \tau > _{Fm}\],% MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXafv3ySLgzGmvETj2BSbqef0uAJj3BZ9Mz0bYu% H52CGmvzYLMzaerbd9wDYLwzYbItLDharqqr1ngBPrgifHhDYfgasa% acOqpw0xe9v8qqaqFD0xXdHaVhbbf9v8qqaqFr0xc9pk0xbba9q8Wq% Ffea0-yr0RYxir-Jbba9q8aq0-yq-He9q8qqQ8frFve9Fve9Ff0dme% GabaqaaiGacaGaamqadaabaeaafiaakeaacqGH8aapcqaHepaDcqGH% +aGpdaWgaaWcbaGaamOraiaad+gaaeqaaOGaeyypa0Zaaabqaeaaca% WGMbWaaSbaaSqaaiaadIhaaeqaaOGaam4yamaaBaaaleaacaWG4baa% beaaaeqabeqdcqGHris5aaaa!50D3!\[< \tau > _{Fo} = \sum {f_x c_x }\] average lifetime of Chl a fluorescence calculated from a multi-exponential model under F_m, F_o conditions     

The effect of dynamic light regimes on Chlorella

The patterns of diurnal variations in pigmentation and optical cross-section were compared for two cyclostat cultures of Chlorella pyrenoidosa, where the dynamics of the photoperiod differed. Populations were light-limited, nutrient rich and growing on an 8:16 light-dark (LD) cycle. One light regime was an 8 h sine function of the light period (sinusoidal culture), while the second had an 1 h sine function super-imposed on the 8 hour sine function (oscillating sinusoidal culture). Hourly samples were taken throughout a 12 h period including the light period. Determinations were made of chlorophyll (Chl) a and b abundance, in vivo absorption spectra, cell number and volume and used to derive both cell-specific (cell) and optical chlorophyll specific (chl) cross sections, as well as the absorption efficiency, Q, of the cells. The results indicate that C. pyrenoidosa is capable of adapting to dynamics in light intensity within an 8 h photoperiod. The sinusoidal culture showed a constant decrease in the Chl a/b ratio of 28% while the total Chl content per cell increased slightly and chl and Q remained constant, suggesting coordinated changes in reaction centers and light harvesting complexes. Over the oscillating photoperiod, however, the second culture displayed a diurnal variation in Chl a/b ratio, a 20% increase in chl and an apparent oscillation in Q. These observations suggest that an oscillating photoperiod promoted the capability of Chl molecules to collect light and that the fractional area of all Chl molecules exposed to the photon flux is inversely related to the photon flux.     

Analysis of xanthophyll cycle carotenoids and chlorophyll fluorescence in light intensity-dependent chlorophyll-deficient mutants of wheat and barley

Three light intensity-dependent Chl b-deficient mutants, two in wheat and one in barley, were analyzed for their xanthophyll cycle carotenoids and Chl fluorescence characteristics under two different growth PFDs (30 versus 600 mol photons·m^–2 s^–1 incident light). Mutants grown under low light possessed lower levels of total Chls and carotenoids per unit leaf area compared to wild type plants, but the relative proportions of the two did not vary markedly between strains. In contrast, mutants grown under high light had much lower levels of Chl, leading to markedly greater carotenoid to Chl ratios in the mutants when compared to wild type. Under low light conditions the carotenoids of the xanthophyll cycle comprised approximately 15% of the total carotenoids in all strains; under high light the xanthophyll cycle pool increased to over 30% of the total carotenoids in wild type plants and to over 50% of the total carotenoids in the three mutant strains. Whereas the xanthophyll cycle remained fairly epoxidized in all plants grown under low light, plants grown under high light exhibited a considerable degree of conversion of the xanthophyll cycle into antheraxanthin and zeaxanthin during the diurnal cycle, with almost complete conversion (over 90%) occurring only in the mutants. 50 to 95% of the xanthophyll cycle was retained as antheraxanthin and zeaxanthin overnight in these mutants which also exhibited sustained depressions in PS II photochemical efficiency (F_v/F_m), which may have resulted from a sustained high level of photoprotective energy dissipation activity. The relatively larger xanthophyll cycle pool in the Chl b-deficient mutant could result in part from the reported concentration of the xanthophyll cycle in the inner antenna complexes, given that the Chl b-deficient mutants are deficient in the peripheral LHC-II complexes.Abbreviations A antheraxanthin - Chl chlorophyll - F_o and F_m minimal yield (at open PS II reaction centers) and maximal yield (at closed centers) of chlorophyll fluorescence in darkness - F level of fluorescence during illumination with photosynthetically active radiation - F_m maximal yield (at closed centers) of chlorophyll fluorescence during illumination with photosynthetically active radiation - (F_m–F)/F_m actual efficiency of PS II during illumination with photosynthetically active radiation - F_v/F_m+(F_m–F_o)/F_m intrinsic efficiency of PS II in darkness - LHC_II light-harvesting chlorophyll-protein complex of Photosystem II - PFD photon flux density (between 400 and 700 nm) - PS I Photosystem I - PS II Photosystem II - V violaxanthin - Z zeaxanthin     

Adenocarcinoma of the human exocrine pancreas: presence of secretin and caerulein receptors

We have measured the cytochrome compositions of subfractions derived from appressed and non-appressed thylakoids by centrifugation and aqueous two-phase partition. Cytochrome $\text{b}$-559 (HP) was not detectable in the fraction derived from non-appressed thylakoids. Cytochromes $\text{f}\text{,}\text{b}$-563 and $\text{b}$-559 (LP) were all evenly distributed throughout the thylakoid membrane. This distribution points to plastocyanin as a possible lateral shuttle of reducing equivalents between spatially separated photosystems.Cytochrome $\text{f}$ was accessible to externally added plastocyanin in the inside-out vesicles but not in vesicles of normal sidedness. This strongly supports a location at the inner side of the thylakoid membrane. Cytochrome $\text{b}$-563 was slowly reduced by dithionite in vesicles with both normal and inside-out orientation suggesting a location within the membrane interior.     

Plastocyanin stimulation of whole chain and photosystem I electron transport in inside-out thylakoid vesicles

Inside-out and right-side-out thylakoid vesicles were isolated from spinach chloroplasts by aqueous-polymer two-phase (dextran/polyethylene glycol) partitioning. Externally added plastocyanin stimulated the whole-chain and PSI electron transport rates in the inside-out thylakoid vesicles by about 500 and 350%, respectively, compared to about 50% stimulation for both assays in the fraction enriched in right-side-out vesicles. No apparent stimulation by plastocyanin was observed in unbroken Class II thylakoids. The electron transport between PSII and PSI in inside-out thylakoid vesicles appears to be interrupted due to plastocyanin release from the thylakoids by the Yeda press treatment, but it was restored by externally added plastocyanin. The P700 content of the inside-out membrane preparations, measured by chemical and photochemical methods, was 1 P700 per 1100 to 1500 chlorophylls while it was about 1 P700 per 500 chlorophylls for the right-side-out vesicles. The data presented support the concept of lateral heterogeneity of PS I and II in thylakoid membranes, but does not support a virtual or total absence of PSI in the appressed grana partitions. Further, the heterogeneity does not appear to be as extreme as suggested earlier. Although PSI is somewhat depleted in the appressed grana membrane region, there is adequate photochemically active P700, when sufficient plastocyanin is available, to effectively couple PSI electron transfer with the preponderant PSII in linear electron transport.     

Adaptation of the thylakoid membranes of pea chloroplasts to light intensities. II. Regulation of electron transport capacities,electron carriers,coupling factor (CF1) activity and rates of photosynthesis

The electron transport rates of photosystems II and I, amounts of electron carriers, coupling factor activity and photosynthetic rates were investigated in thylakoids isolated from pea plants grown under a wide range of light intensities (16 h light-8 h dark). The electron transport rates of PS II and PS I, as partial reactions or in whole chain, and coupling factor activity on a unit chlorophyll basis, all increased as the light intensity available for growth was altered from a very low intensity of 10 E m^-2s^-1 to a high intensity of 840 E m^-2s^-1. Similarly, there were increases in the amounts of atrazine binding sites, plastoquinine, cytochrome f and P700 per unit chlorophyll; significantly, the amounts of reaction centres of PS II and PS I were not equal at any light intensity. The rate of change of all parameters with respect to light intensity could be represented by two straight lines of different slopes which met at a transition point corresponding to approximately 200 E m^-2s^-1 during growth. These photoadaptations were similar to those observed for both the relative distribution of chlorophyll in chlorophyll-protein complexes and the chl a/chl b ratios [Leong and Anderson, 1984, Photosynthesis Research 5:117–128]. Since these thylakoid components and functions were affected in the same direction by light intensity during growth and all show linear relationships with chl a/chl b ratios, it indicates that they are closely regulated and markedly well co-ordinated. Plants compensate for the limited amount of low light intensities by drastically increasing the light-harvesting antenna unit size of photosystem II and to a lesser extent that of photosystem I. Changes in the composition of the thylakoid membranes exert a regulatory effect on the overall photosynthetic rate up to approximately 450 E m^-2s^-1.Abbreviations chl chlorophyll - cyt cytochrome - PQ plastoquinone - PS photosystem     

Counter-current distribution of sonicated inside-out thylakoid vesicles

Summary Inside-out thylakoid vesicles were isolated from spinach chloroplasts, and fragmented by sonication. Different fragments were separated by counter-current distribution and analyzed for chlorophyll and P700. The inside-out vesicles had a chlorophyll a/b ratio of 2.2–2.4 (original chloroplasts 2.8–3.0). After further fragmentation of the inside-out vesicles by sonication and separation by countercurrent distribution three populations of vesicles were obtained having chlorophyll a/b ratios of 1.7, 1.9 and 2.5 respectively. The P-700 was depleted in fractions with lower chlorophyll a/b ratio and was nearly absent in the fraction having a chlorophyll a/b ratio of 1.7 (chlorophyll/P700 > 4500 mol/mol). That PSII membrane vesicles, with such a low chlorophyll a/b ratio and lacking PSI, can be prepared by a non-detergent method provides strong support for the notion that PSI and PSII are segregated along the thylakoid membrane.A plot of P700 per chlorophyll against chlorophyll b/(a+b) fits a straight line connecting the pure PSI membrane (chlorophyll a/b = 6; P700/chlorophyll = 5.6 mmol/mol) with the pure PSII membrane (chlorophyll a/b = 1.7; P700 = 0). These two membranes can be considered as separate phases of a two-dimensional phase system. Models for the thylakoid membrane are discussed.Abbreviations PSI Photosystem I - PSII Photosystem II - PEG Polyethylene Glycol - P700 Reaction Center of PSI     

Conversion of everted thylakoids into vesicles of normal sidedness exposing the outer grana partition membrane surface

Inside-out spinach thylakoid vesicles can be isolated by aqueous polymer two-phase partition following mechanical disruption of spinach chloroplast lamellae (Andersson, B and Åkerlund, H.-E. (1978) Biochim. Biophys. Acta 503, 462–472) and a mechanism for their formation has been experimentally supported (Andersson B., Sundby, C. and Albertsson, P.-Å. (1980) Biochim. Biophys. Acta 599, 391–402). Upon disruption, inside-out vesicles may form under stacking conditions, e.g., in 5 mM MgCl₂ or 150 mM NaCl, while disruption under destacking conditions, i.e., low concentrations of monovalent cations, gives only right-side-out vesicles. This study deals with the sidedness stability of the isolated inside-out thylakoid vesicles when stored or disrupted by sonication in various ionic environments. The sidedness of thylakoid vesicles was determined by their partition behaviour in an aqueous polymer phase system, direction of proton translocation and aggregation response (stacking) upon addition of MgCl₂. The results show that no spontaneous change from everted to normal sidedness occurs upon storage of the inside-out thylakoids. In contrast, sonication of these vesicles under destacking conditions (5 mM NaCl) results in a nearly complete transformation to right-side-out orientation. Also, in the presence of 5 mM MgCl₂ or 150 mM NaCl, sonication induced a change in sidedness of the inside-out vesicles but to a lesser extent. The stabilizing effect on the everted sidedness by cations was shown to be a result of preventing vesicle fragmentation by maintaining internal thylakoid appresions rather than by influencing the membrane curvature during resealing. Once released from an appressed state by overcoming the stacking forces, an opened thylakoid membrane shows an absolute preference for turning right-side-out in all media tested. These results strongly support the proposed formation mechanism, in which pairs of neighbouring grana membranes after disruption reseal with each other promoted by their close proximity. Since the inside-out vesicles derive from the grana appressions, their transformation back to normal sidedness exposes the outer membrane surface of appressed thylakoids. This region of the thylakoid membrane is normally hidden in the grana appressions and removal of grana leads concomitantly to lateral intermixing with non-appressed thylakoid components. Thus the current isolation of right-sided vesicles derived from the grana appressions should be a new tool for studies on the molecular organization of the thylakoid membrane.     

Linear Dichroism,Fluorescence Polarization,and Path of the Thermal Deactivation of Excited Cyanobacterial (Synechococcus Elongatus) Photosystem 1 Immobilized and Oriented in Polymer Films

Pigment-protein complexes enriched in photosystem 1 (PS1) and, for comparison, enriched in photosystem 2 (PS2) were isolated from the cyanobacterium Synechococcus elongatus Nag. f. thermalis Geitl. They were immobilized and oriented in the polyvinyl alcohol (PVA) films, and studied by linear dichroism (LD), fluorescence polarization (FP), photoacoustic spectroscopy (PAS), and polarized photoacoustic spectroscopy (PAS and PAS). The LD signal of -carotene in the region with maximum at 500 nm was positive in the PS1 complex. The maximum value of fluorescence polarization (FP) in the measured photosynthetic pigment region was 1.25 and was similar to higher plant values. Carotenoids exhibited different efficiencies of thermal deactivation (max. at 500 nm) in PS1 and PS2. The thermal deactivation efficiency of carotenoids in comparison with that of chlorophyll (Chl) a at its red absorbance maximum was much higher in PS1 than in PS2 complexes. Cyanobacterial complexes did not contain Chl b, interpretation of the LD, PAS, and FP results is thus easier and can be compared with PS1 and PS2 values of higher plants, especially with Chl b-less mutant values.     

Acclimation of barley to changes in light intensity: chlorophyll organization

Barley seedlings (Hordeum vulgare L. cv. Boone) were grown at 20°C with a 16h/8h light/dark cycle of either high (H) intensity (550 mole m^-2 s^-1) or low (L) intensity (55 mole m^-2 s^-1) white light. Plants were transferred from high to low (H L) or low to high (L H) light intensity at various times from 4 to 8 d after leaf emergence from the soil. Primary leaves were harvested at the beginning of the photoperiod and a 3 cm apical segment removed for analysis. H control plants had greater chlorophyll (Chl) per leaf area and higher Chl a/b ratios than L controls. Analysis of Chl-protein complexes revealed that H and L plants had the same percentage of total Chl (62–65%) associated with Photosystem II (PS II), but that the organization of Chl within PS II was different. H plants contained lower levels of light-harvesting complex (LHC-II) and higher levels of the PS II complex CPa compared with L plants. Leaf Chl content and Chl organization within PS II were sensitive to changes in light intensity. In H L plants, leaf Chl content decreased, Chl a/b ratio decreased, and a redistribution of Chl from CPa to LHC-II occurred during acclimation to low light. Acclimation of L H plants to high light involved an increase in leaf Chl content, an increase in Chl a/b ratio, and a decrease in LHC-II. In contrast, the level of photosystem I related Chl-protein complexes (CP1 + CP1a) was similar in all light treatments. The light acclimation process occurred slowly over a period of 6 to 8 d in H L and L H plants.Abbreviations DMF dimethylformamide - H control plants grown under high light intensity - H L plants transferred from high to low light intensity - L control plants grown under low light intensity - L H plants transferred from low to high light intensity Cooperative investigations of the United States Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh, NC. Paper No. 11989 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7643, USA.