On the molecular mechanism of light-induced D1 protein degradation in photosystem II core particles.

The mechanism of D1 protein degradation was investigated during photoinhibitory illumination of isolated photosystem II core preparations. The studies revealed that a proteolytic activity resides within the photosystem II core complex. A relationship between the inhibition of D1 protein degradation and the binding of the highly specific serine protease inhibitor diisopropyl fluorophosphate to isolated complexes of photosystem II was observed, evidence that this protease is of the serine type. Using radiolabeled inhibitor, it was shown that the binding site, representing the active serine of the catalytic site, is located on a 43-kDa polypeptide, probably the chlorophyll a protein CP43. The protease is apparently active in darkness, with the initiation of breakdown being dependent on high light-induced substrate activation. The proteolysis, which has an optimum at pH 7.5, gives rise to primary degradation fragments of 23 and 16 kDa. In addition, D1 protein fragments of 14, 13, and 10 kDa were identified. Experiments with phosphate-labeled D1 protein and sequence-specific antisera showed that the 23- and 16-kDa fragments originate from the N- and C-termini, respectively, suggesting a primary cleavage of the D1 protein at the outer thylakoid surface in the region between transmembrane helices D and E.     

Regulation of Photosystem II core protein phosphorylation at the substrate level: Light induces exposure of the CP43 chlorophyll a protein complex to thylakoid protein kinase(s)

Light induces conformational changes in the CP43 chl-a-protein antenna complex in isolated PS II core-complexes exposing phosphorylation site(s) to PS II core-associated protein kinase(s), to added solubilized thylakoid protein kinase(s), as well as to tryptic cleavage. The substrate-activation effect is demonstrated by exposure of the PS II cores to light during the kinase assay as well as by preillumination of the PS II cores in which the endogenous kinase(s) has been inactivated by treatment with N-ethylmaleimid. In the latter case, phosphorylation was performed in darkness following addition of the solubilized protein kinase(s). The solubilized protein kinase(s) does not require light activation. The apparent molecular masses of the main protein kinase(s) associated with the PS II cores (about 31–35 kDa and 45 kDa) differ from that of the major protein kinase present in solubilized preparations obtained from spinach thylakoids (64 kDa). The light-induced exposure of CP43 increases with the light intensity in the range of 20–100 μmol photons m⁻² s⁻¹ as demonstrated by preillumination of N-ethylmaleimid treated cores followed by addition of the solubilized protein kinase(s) and performing the phosphorylation assay in darkness. This revised version was published online in June 2006 with corrections to the Cover Date.     

Isolation and characterization of photosystem II from the filamentous sporophyte of Porphyra yezoensis

Thylakoid membranes were isolated and purified from diploid filamentous sporophytes of Porphyra yezoensis Ueda using sucrose density gradient ultracentrifugation (SDGUC). After thylakoid membranes were solubilized with SDS, the phtosystem II (PSII) particles with high 2, 6-dichloroindophenol (DCIP) photoreduction activity were isolated by SDGUC. The absorption and fluorescence spectra, DCIP photoreduction activity and oxygen evolution activity of the thylakoid membranes and PSII particles were determined. The polypeptide composition of purified PSII particles was distinguished by SDS-PAGE. Results showed that PSII particles of sporophytes differed from the gametophytes in spectral properties and polypeptide composition. Apart from 55 kDa D1-D2 heterodimer, CP47, CP43, 33 kDa protein, D1, D2, cyt b559 and 12 kDa protein were identified from PSII particles from sporophytes; a new 102 kDa protein was also detected. However, cyt c-550, 20 kDa, 14 kDa and 16 kDa proteins found in PSII particles from gametophytes were not detected in the sporophytes.     

Autotrophic cells of the <Emphasis Type="Italic">Synechocystis psbH</Emphasis> deletion mutant are deficient in synthesis of CP47 and accumulate inactive PS II core complexes

Cells of the psbH deletion mutant IC7 of the cyanobacterium Synechocystis PCC 6803 grown in the absence of glucose contain strongly reduced levels of chlorophyll when compared with cells grown in the presence of glucose, or compared with wild-type (WT) cells. Low-temperature fluorescence emission spectra revealed decreased content of both active PS II (Photosystem II) and PS I (Photosystem I) complexes. Analysis of thylakoid membrane complexes of IC7 by native electrophoresis showed a similar set of chlorophyll–proteins, namely a PS II core complex and trimeric and monomeric PS II complexes, as in WT. However, in contrast to WT, the ³⁵S-methionine protein labeling pattern of the mutant exhibited no preferential labeling of the D1 protein in the PS II core complexes, and the labeled D1 and D2 proteins accumulated predominantly in the PS II reaction center lacking CP47. The results show that in autotrophically grown cells of the psbH deletion mutant, selective D1 turnover is inhibited and synthesis of CP47 becomes a limiting step in the PS II assembly.     

The distribution of Photosystem I and Photosystem II polypeptides between the cytoplasmic and thylakoid membranes of cyanobacteria

Abstract In a previous study we found that the 33 kDa extrinsic polypeptide of Photosystem II is present in both the cytoplasmic and thylakoid membranes of cyanobacteria, but forms part of a functional complex only in the latter [Smith et al. (1987) Mol. Microbiol. 6, 1821–1827]. In order to determine if this phenomenon is restricted to the 33 kDa polypeptide we have extended this study in Anacystis nidulans to include a number of other polypeptides of Photosystem I and Photosystem II. We have found that D1 and possibly PsaC are present in both membranes, CP43 and CP47 are confined to the thylakoid membranes, and the distribution of PsaD and PsaE is dependent upon the growth stage of the cyanobacteria.     

Regeneration of the high-affinity manganese-binding site in the reaction center of an oxygen-evolution deficient mutant of Scenedesmus by protease action

The O₂-evolution deficient mutant (LF-1) of Scenedesmus obliquus inserts an unprocessed D1 protein into the thylakoid membrane and binds less than half the wild type (WT) level of Mn. LF-1 photosystem II (PS II) membrane fragments lack that part of the high-affinity Mn²⁺-binding site found in WT membranes which may be associated with histidine residues on the D1 protein (Seibert et al. 1989 Biochim Biophys Acta 974: 185–191). Hsu et al. (1987 Biochim Biophys Acta 890: 89–96) purport that the high-affinity site (characterized by competitive inhibition of DPC-supported DCIP photoreduction by M concentrations of Mn²⁺) in Mn-extracted PS II membranes is also the binding site for Mn functional in O₂ evolution. Proteases (papain, subtilisin, and carboxypeptidase A) can be used to regenerate the high-affinity Mn²⁺-binding site in LF-1 PS II membranes but not in thylakoids. Experiments with the histidine modifier, DEPC, suggest that the regenerated high-affinity Mn²⁺-binding sites produced by either subtilisin or carboxypeptidase A treatments were the same sites observed in WT membranes. However, none of the protease treatments produced LF-1 PS II membranes that could be photoactivated. Reassessment of the processing studies of Taylor et al. (1988 FEBS Lett 237: 229–233) lead us to believe that their procedure also does not result in substantial photoactivation of LF-1 PS II membranes. We conclude that (1) the unprocessed carboxyl end of the D1 protein in LF-1 is located on the lumenal side of the PS II membrane, (2) the unprocessed fragment physically obstructs or perturbs that part of the high-affinity Mn²⁺-binding site undetectable in LF-1, and (3) the D1 protein must be processed at the time of insertion into the membrane for normal O₂-evolution function to result.Abbreviations Chl chlorophyll - DCBQ 2,6-dichloro-1,4-benzoquinone - DCIP 2,6-dichlorophenol indophenol - DEPC diethylpryocarbonate - DPC 1,5-diphenylcarbazide - HEPES 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid - LDS-PAGE lithium dodecylsulfate polyacrylamide gel electrophoresis - LF-1 a low-fluorescent mutant of Scenedesmus obliquus - MES 4-morpholineethanesulfonic acid - PS II photosystem II - PMSF phenylmethylsulfonyl fluoride - RC photosystem II reaction center - Tris tris(hydroxymethyl)aminomethane - WT wild type Operated by the Midwest Research Institute for the U.S. Department of Energy under contract DE-AC-02-83CH10093.     

Effect of Irradiance on the Thermal Stability of Thylakoid Membrane Isolated from Acclimated Wheat Leaves

Thermal stability of thylakoid membranes isolated from acclimated and non-acclimated wheat (Triticum aestivum L. cv. HD 2329) leaves under irradiation was studied. Damage to the photosynthetic electron transport activity was more pronounced in thylakoid membranes isolated from non-acclimated leaves as compared to thylakoid membrane isolated from acclimated wheat leaves at 35 °C. The loss of D1 protein was faster in non-acclimated thylakoid membrane as compared to acclimated thylakoid membranes at 35 °C. However, the effect of elevated temperature on the 33 kDa protein associated with oxygen evolving complex in these two types of thylakoid membranes was minimal. Trypsin digestion of the 33 kDa protein in the thylakoid membranes isolated from control and acclimated seedlings suggested that re-organisation of 33 kDa protein occurs before its release during high temperature treatment.     

Photosystem II solubilizes as a monomer by mild detergent treatment of unstacked thylakoid membranes*

We studied the aggregation state of Photosystem II in stacked and unstacked thylakoid membranes from spinach after a quick and mild solubilization with the non-ionic detergent n-dodecyl-α,D-maltoside, followed by analysis by diode-array-assisted gel filtration chromatography and electron microscopy. The results suggest that Photosystem II (PS II) isolates either as a paired, appressed membrane fragment or as a dimeric PS II-LHC II supercomplex upon mild solubilization of stacked thylakoid membranes or PS II grana membranes, but predominantly as a core monomer upon mild solubilization of unstacked thylakoid membranes. Analysis of paired grana membrane fragments reveals that the number of PS II dimers is strongly reduced in single membranes at the margins of the grana membrane fragments. We suggest that unstacking of thylakoid membranes results in a spontaneous disintegration of the PS II-LHC II supercomplexes into separated PS II core monomers and peripheral light-harvesting complexes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Long-lived primary radical pair state detected by time-resolved fluorescence and absorption spectroscopy in an isolated Photosystem two core

A Photosystem two (PS II) core preparation containing the chlorophyll a binding proteins CP 47, CP 43, D1 and D2, and the non-chlorophyll binding cytochrome-b559 and 33 kDA polypeptides, has been isolated from PS II-enriched membranes of peas using the non-ionic detergent heptylthioglucopyranoside and elevated ionic strengths. The primary radical pair state, P680⁺Pheo^-, was studied by time-resolved absorption and fluorescence spectroscopy, under conditions where quinone reduction and water-splitting activities were inhibited. Charge recombination of the primary radical pair in PS II cores was found to have lifetimes of 17.5 ns measured by fluorescence and 21 ns measured by transient decay kinetics under anaerobic conditions. Transient absorption spectroscopy demonstrated that the activity of the particles, based on primary radical pair formation, was in excess of 70% (depending on the choice of kinetic model), while time-resolved fluorescence spectroscopy indicated that the particles were 91% active. These estimates of activity were further supported by steady-state measurements which quantified the amount of photoreducible pheophytin. It is concluded that the PS II core preparation we have isolated is ideal for studying primary radical pair formation and recombination as demonstrated by the correlation of our absorption and fluorescence transient data, which is the first of its kind to be reported in the literature for isolated PS II core complexes from higher plants.Abbreviations CP 43 and CP 47 chlorophyll binding proteins of PS II having apparent molecular weights on SDS-PAGE of 43 kDa and 47 kDa, respectively - D1 and D2 polypeptides PS II reaction centre polypeptides encoded by the psbA and psbD genes, respectively - HPLC high performance liquid chromatography - PS II Photosystem two - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - P680 primary electron donor of PS II - Pheo phenophytin a - SPC single photon counting - PBQ phenyl-p-benzoquinone - DPC 1,5-diphenylcarbazide AFRC Photosynthesis Research Group, Department of Biochemistry     

Photosynthetic water oxidation: The protein framework

Approximately 20 protein subunits are associated with the PS II complex, not counting subunits of peripheral light-harvesting antenna complexes. However, it is not yet established which proteins specifically are involved in the water-oxidation process. Much evidence supports the concept that the D1/D2 reaction center heterodimer not only plays a central role in the primary photochemistry of Photosystem II, but also is involved in electron donation to P680 and in ligation of the manganese cluster. This evidence includes (a) the primary donor to P680 has been shown to be a redox-active tyrosyl residue (Tyr161) in the D1 protein, and (b) site-directed mutagenesis and computer-assisted modeling of the reaction center heterodimer have suggested several sites with a possible function in manganese ligation. These include Asp170, Gln165 and Gln189 of the D1 protein and Glu69 of the D2 protein as well as the C-terminal portion of the mature D1 protein. Also, hydrophilic loops of the chlorophyll-binding protein CP43 that are exposed at the inner thylakoid surface could be essential for the water-splitting process.In photosynthetic eukaryotes, three lumenal extrinsic proteins, PS II-O (33 kDa), PS II-P (23 kDa) and PS II-Q (16 kDa), influence the properties of the manganese cluster without being involved in the actual catalysis of water oxidation. The extrinsic proteins together may have multiple binding sites to the integral portion of PS II, which could be provided by the D1/D2 heterodimer and CP47. A major role for the PS II-O protein is to stabilize the manganese cluster. Most experimental evidence favors a connection of the PS II-P protein with binding of the Cl^- and Ca²⁺ ions required for the water oxidation, while the PS II-Q protein seems to be associated only with the Cl^- requirement. The two latter proteins are not present in PS II of prokaryotic organisms, where their functions may be replaced by a 10–12 kDa subunit and a newly discovered low-potential cytochrome c-550.Abbreviations PS II Photosystem II - PCC Pasteur Culture Collection     

Reversible phosphorylation and turnover of the D1 protein under various redox states of Photosystem II induced by low temperature photoinhibition

Reversible phosphorylation and turnover of the D1 protein in vivo were studied under low-temperature photoinhibition of pumpkin leaves and under subsequent recovery at low light at 4 °C or 23 °C. The inactivation of PS II and photodamage to D1 were not enhanced during low-temperature photoinhibition when compared to that at room temperature. The PS II repair cycle, however, was completely blocked at 4 °C at the level of D1 degradation. Both the recovery of the photochemical activity of PS II and the degradation of the damaged D1 protein at low light at 23 °C were delayed about 1 hour after low-temperature photoinhibition, suggesting that in addition to the decrease in catalytic turnover of the enzyme, the protease was specifically inactivated in vivo at low temperature. The effect of low temperature on the other regulatory enzymes of PS II repair, protein kinase and phosphatase [Rintamäki et al. (1996) J Biol Chem 271: 14870-14875] was variable. The D1 protein kinase was operational at low temperature while dephosphorylation of the D1 protein seemed to be completely inhibited during low temperature treatment. Under subsequent recovery conditions at low light and 23 °C, the high phosphorylation level of D1 was sustained in leaf discs photoinhibited at low temperature, despite the recovery of the phosphatase activity. This high phosphorylation level of D1 was due to the persistently active kinase. The D1 kinase, previously shown to get activated by reduction of plastoquinone, was, however, found to be maximally active already at relatively low redox state of the plastoquinone pool. We suggest that phosphorylation of PS II centers increases the stability of PS II complexes and concomitantly improves their survival under stress conditions.     

Implication of D1 degradation in phosphorylation-induced state transitions

State transitions and lateral migration of phosphorylated mobile-LHC II upon thylakoid unstacking have been reported as being interdependent. However, now the thyakoid unstacking event can be separated from the thyakoid phosphorylation and the associated F730/F685 enhancement by using the serine-type-protease inhibitor benzamidine. Thus, lateral migration appears not be necessary, and it can be shown that LHC II-rich fragments, originating in peripheral granal membranes, can be released by digitonin although in reduced amounts. On the other hand, phosphorylation of thylakoid proteins greatly stimulates the light-induced D1 degradation, which is observed in chloroplasts phosphorylated even at very low light (15 µmol m^–2s^–1). Thylakoid pretreatment with FSBA (the PS II protein-kinase inhibitor) blocks the light-induced and ATP-stimulated D1 degradation, and the F730/F685 ratio increase; this suggests that the dissociation of the PS II unit, resulting from the introduction of repulsive negative charges ( ATP groups) into LHC II and PS II core proteins, leads to D1 degradation. In chloroplast samples transferred to darkness following short-time phosphorylation, the D1 level is recovered. The results suggest that disassembly of PS II and D1 degradation occur parallel to State transitions. The removal of outer phospho-LHC II from PS II and its association with PS I at the periphery of grana may allow D1 degradation and increased light utilization by PS I, while net de novo synthesis of D1, stimulated by ATP, may lead to the assembly of new PS II units which could bind dephosphorylated LHC II in the dark, resulting in increased light utilization by PS II.     

Quality control of Photosystem II under light stress – turnover of aggregates of the D1 protein in vivo

When photodamaged under excessive light, the D1 protein is digested and removed from Photosystem (PS) II to facilitate turnover of the protein. In vitro studies have shown that part of the photodamaged D1 protein forms aggregates with surrounding polypeptides before being digested by a protease(s) in the stroma [Yamamoto Y (2001) Plant Cell Physiol 42: 121–128]. The aim of this study was to examine whether light-induced aggregation of the D1 protein also occurs in vivo. The following results were obtained: (1) PS II activity in spinach leaves was significantly inhibited by weak illumination (light intensity, 20–100 μE m⁻² s⁻¹), as monitored by chlorophyll fluorescence Fv/Fm, when the leaves were kept at higher temperatures (35–40 °C); (2) aggregation of the D1 protein, as well as cleavage of the protein, was detected in thylakoids isolated from spinach leaves that had been subjected to heat/light stress; (3) aggregates of the D1 protein disappeared after incubation of the leaves at 25 °C in the dark or under illumination with weak light. Since it is dependent on the presence of oxygen, aggregation of the D1 protein is probably induced by reactive oxygen species produced in thylakoids upon illumination at elevated temperatures. Consistent with this notion, singlet oxygen production in thylakoid samples under illumination was shown to be stimulated significantly at higher temperatures.     

Thylakoid protein phosphorylation is suppressed by 'free radical scavengers'. Correlation between PS II core protein degradation and thylakoid protein phosphorylation

Recent work has shown that the light-induced PS II core protein degradation, as monitored by immunostain reduction on Western blots, was stimulated even at low light during phosphorylation of thylakoid proteins in the presence of NaF, and that the thylakoid kinase inhibitor FSBA blocked completely the light- and ATP-stimulated degradation [Georgakopoulos and Argyroudi-Akoyunoglou (1997) Photosynth Res 53: 185–195]. To assess whether D1, D2 or both proteins are degraded, antibodies raised against D1/D2, or the D-E loop of D1 were used. Greatest immunostain reduction was observed with antibodies raised against D1/D2, immunostaining a 34 kDa protein on blots of 15% polyacrylamide-6 M urea gels, suggesting that the phosphorylation-induced degradation may be mainly directed against D2. To see how protein phosphorylation might be implicated in PS II core protein degradation we further tested the effect of free radical scavengers, on thylakoid protein phosphorylation. Active oxygen scavengers like n-propyl gallate, histidine, and imidazole, shown earlier to inhibit high light-induced D1 degradation, also suppressed the phosphorylation of thylakoid proteins; on the other hand, NaN3 and D-mannitol, known to stimulate light- induced D1 degradation did not suppress protein phosphorylation, whereas superoxide dismutase and catalase, known also to inhibit high light-induced D1 degradation, did not affect thylakoid protein phosphorylation. In addition, the ATP-induced degradation was also observed in the dark under conditions of kinase activation, and in the light under anaerobic conditions, that block light-induced degradation, whereas it was reduced in the absence of NaF, the phosphatase inhibitor. The results point to the involvement of a proteolytic system in PS II core protein degradation, which is active in its phosphorylated state.     

Carotenoids in photosynthesis: Protection of D1 degradation in the light

Photosynthesis has been determined with mutants of Anacystis which form different amounts of carotenoids. With these cultures a highly significant correlation between photosynthetic oxygen evolution and the amounts of synthesized carotenoids was observed. In addition, the influence of carotenoids on light-dependent degradation of thylakoid proteins was investigated with Scenedesmus cultures grown in darkness in the presence of norflurazon, an inhibitor of carotenoid biosynthesis. Pre-illumination of cells resulted in decrease of photosynthetic activity accompanied by loss of the D1 protein. This effect is dependent on the length of illumination, and the light intensity, and increased when carotenoid content was lowered during previous growth of the norflurazon-treated cultures.Abbreviations BSA bovine serum albumin - D1 32 kDa Q_B-binding protein - EDTA ethylenediaminetetraacetic acid - LHCII light-harvesting complex II - PMSF phenylmethylsulfonyl fluoride - PS photosystem - tricine N-[tris(hydroxymethyl)methyl] glycine     

A Fourier transform infrared spectroscopic study of the effects of hydrogen peroxide and high light on protein conformations of Photosystem II

Degradation of the reaction center-binding protein D1 of Photosystem II (PS II) during photoinhibition is dependent on the action of active oxygen species and/or D1-specific proteases. Protein conformational changes may be involved in the process of D1 degradation. In the present study, we determined the effect of H₂O₂ on spinach PS II-enriched membranes and core complexes with respect to electron transport, Mn content and protein secondary structural changes as measured by Fourier transform infrared (FTIR) spectroscopy. H₂O₂ is effective in removing catalytic Mn in PS II, especially in PS II core complexes depleted of OEC18 and OEC24, impairing the donor-side. By quantitative analysis of the amide I band (1600 – 1700 cm^-1) with both aqueous and dehydrated PS II samples, we found that no significant secondary structural changes are associated with H₂O₂ treatment in the dark, even though there is some cleavage of the D1 protein by H₂O₂ treatment as determined by Western analysis with specific antibodies. In contrast, a large decrease in the -helices in the PS II core occurs, with or without H₂O₂ treatment, after 20 min strong illumination and there is more extensive degradation of the D1 protein. Our results suggest that high light enhances the cleavage of the D1 protein which is reflected in the large protein secondary structural changes in PS II detected by FTIR measurements.     

D1 protein of photosystem II: The light sensor in chloroplasts

Light, controls the “blueprint” for chloroplast development, but at high intensities is toxic to the chloroplast. Excessive light intensities inhibit primarily photosystem II electron transport. This results in generation of toxic singlet oxygen due to impairment of electron transport on the acceptor side between pheophytin and Q_B -the secondary electron acceptor. High light stress also impairs electron transport on the donor side of photosystem II generating highly oxidizing species Z⁺ and P680⁺. A conformationsl change in the photosystem II reaction centre protein Dl affecting its Q_B-binding site is involved in turning the damaged protein into a substrate for proteolysis. The evidence indicates that the degradation of D1 is an enzymatic process and the protease that degrades D1 protein has been shown to be a serine protease Although there is evidence to indicate that the chlorophyll a-protein complex CP43 acts as a serine-type protease degrading Dl, the observed degradation of Dl protein in photosystem II reaction centre particlesin vitro argues against the involvement of CP43 in Dl degradation. Besides the degradation during high light stress of Dl, and to a lesser extent D2-the other reaction centre protein, CP43 and CP29 have also been shown to undergo degradation. In an oxygenic environment, Dl is cleaved from its N-and C-termini and the disassembly of the photosystem II complex involves simultaneous release of manganese and three extrinsic proteins involved in oxygen evolution. It is known that protein with PEST sequences are subject to degradation; D1 protein contains a PEST sequence adjacent to the site of cleavage on the outer side of thylakoid membrane between helices IV and V. The molecular processes of “triggering” of Dl for proteolytic degradation are not clearly understood. The changes in structural organization of photosystem II due to generation of oxy-radicals and other highly oxidizing species have also not been resolved. Whether CP43 or a component of the photosystem II reaction centre itself (Dl. D2 or cy1 b559 subunits), which may be responsible for degradation of Dl, is also subject to light modification to become an active protease, is also not known. The identity of proteases degrading Dl, LHCII and CP43 and C29 remains to be established     

Site-directed mutagenesis of the psbC gene of photosystem II: isolation and functional characterization of CP43-less photosystem II core complexes

Two mutants of Synechocystis PCC 6803 lacking the psbC gene product CP43 were constructed by site-directed mutagenesis. Analysis of cells and thylakoid membranes of these mutants indicates that PS II reaction centers accumulate to a concentration of about 10% of that of WT cells. PS II core complexes isolated from mutants lacking the CP43 subunit show light-driven electron transfer from the secondary electron donor Z to the primary quinone electron acceptor QA with a quantum yield similar to that of wild type, indicating that CP43 is not required for binding or function of QA. The use of mutants for the removal of CP43 thus avoids the loss of QA function associated with biochemical extraction of CP43 from intact core complexes. Both absorbance and fluorescence emission maxima of the mutant complexes show a blue shift in comparison to the WT PS II core complex, indicating that the absorbance spectrum of CP43 is red-shifted relative to that of the remainder of the core complex. The antenna size of these CP43-less complexes is about 70% of that of WT, indicating that approximately 15 chlorophyll molecules are bound by CP43. The molecular mass of the PS II complex, including the detergent shell, shifts from 310 +/- 15 kDa in WT to 285 +/- 15 kDa in the CP43-less mutants.     

Chlorophyll fluorescence changes at high temperatures induced by linear heating of greening barley leaves

A relative decrease of the high temperature part (above 60°C) of the chlorophyll fluorescence temperature curve during 3 h to 10 h greening period of barley (Hordeum vulgare L.) leaves was found to be concomitant to a decrease of Chl alb ratio and to a gradual increase of LHCP/core ratio found by electrophoresis and the ratio of granal to total length of thylakoid membranes. It is suggested that the high temperature part of the fluorescence temperature curve depends inversely on the relative amount of LHC II in thylakoid membranes.Abbreviations Chl a(b) chlorophyll a(b) - CPa chlorophyll a protein complex of PS II - CP1 P700 chlorophyll a protein complex of PS I - FP free pigments - FTC fluorescence temperature curve - F(T30) fluorescence intensity at 30°C - LHC II light harvesting complex II - LHCP light harvesting chlorophyll protein - LHCP3 (LHCPm) monomeric form of LHC II - LHCPo oligomeric form of LHC II complex - M1 first maximum of FTC - M2 second maximum (region) of FTC - PAA polyacrylamide - PAR photosynthetically active radiation - PS I(II) Photosystem I(II) - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Isolation of PS II reaction centre and its relationship to the minor chlorophyll-protein complexes

Evidence is presented for the identification of the chlorophyll- protein complex CPa-1 (CP 47) as the reaction centre of photosystem II (PS II). We have developed a simple, rapid method using octyl glucoside solubilization to obtain preparations from spinach and barley that are highly enriched in PS II reaction centre activity (measured as the light-driven reduction of diphenylcarbazide by 2,6-dichlorophenolindophenol). These preparations contain only the two minor chlorophyll-protein complexes CPa-1 and CPa-2. During centrifugation on a sucrose density gradient, there is a partial separation of the two CPa complexes from each other, and a complete separation from other chlorophyll-protein complexes. The PS II activity comigrates with CPa-1 but not CPa-2, strongly suggesting that the former is the reaction centre complex of PS II. Reaction centre preparations are sensitive to the herbicide 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), but only at much higher concentrations than those required to inhibit intact thylakoid membranes. A model of PS II incorporating our current knowledge of the chlorophyll-protein complexes is presented. It is proposed that CPa-2 and the chlorophyll a + b complex CP 29 may function as internal antenna complexes surrounding the reaction centre, with the addition of variable amounts of the major chlorophyll a + b light-harvesting complex.