Protein synthesis and phosphorylation of light-harvesting apoproteins in normal and chlorophyll b-deficient Chlamydomonas reinhardii

Phosphorylation of polypeptides in whole cells and in chloroplasts of different strains of Chlamydomonas reinhardii was studied. Phosphorylation in vivo was strongly reduced when cytoptasmic protein synthesis was inhibited either by anisomycin or by cycloheximide. In isolated chloroplasts these two inhibitors had no effect on labelling. The incorporation of [³²P]-phosphate into one of the apoproteins of the light-harvesting chlorophyll a/b -protein complex (LHC 2) was also studied in relation to its synthesis. In vivo, in a chlorophyll b -deficient mutant and in its parent strain we found a pronounced relationship between synthesis and phosphorylation of this LHC 2-apoprotein. Our results suggest that LHC 2-apoproteins, newly synthesized in the cytoplasm, are preferentially phosphorylated after synthesis. Together with the observation that phosphorylation still occurs in isolated chloroplasts we conclude that in vivo at least two levels of phosphorylation of the LHC 2-apoproteins have to be clearly differentiated. One level involves the phosphorylation of existing and the other of newly synthesized polypeptides. The biological significance of phosphorylation of the LHC 2-apoproteins in vivo and probably also of other thylakoid polypeptides is complex and not restricted to regulation of energy distribution between photosystems 1 and 2.     

Functional and mutational analysis of the light-harvesting chlorophyll a/b protein of thylakoid membranes

The precursor for a Lemna light-harvesting chlorophyll a/b protein (pLHCP) has been synthesized in vitro from a single member of the nuclear LHCP multigene family. We report the sequence of this gene. When incubated with Lemna chloroplasts, the pLHCP is imported and processed into several polypeptides, and the mature form is assembled into the light-harvesting complex of photosystem II (LHC II). The accumulation of the processed LHCP is enhanced by the addition to the chloroplasts of a precursor and a co-factor for chlorophyll biosynthesis. Using a model for the arrangement of the mature polypeptide in the thylakoid membrane as a guide, we have created mutations that lie within the mature coding region. We have studied the processing, the integration into thylakoid membranes, and the assembly into light-harvesting complexes of six of these deletions. Four different mutant LHCPs are found as processed proteins in the thylakoid membrane, but only one appears to have an orientation in the membrane that is similar to that of the wild type. No mutant LHCP appears in LHC II. The other two mutant LHCPs cannot be detected within the chloroplasts. We conclude that stable complex formation is not required for the processing and insertion of altered LHCPs into the thylakoid membrane. We discuss the results in light of our model.     

In vitro synthesis and assembly of photosystem II proteins of spinach chloroplasts

The synthesis and assembly of photosystem II (PS II) proteins of spinach chloroplasts were investigated in three different in vitro systems, i.e., protein synthesis in isolated chloroplasts (in organello translation), read-out translation of thylakoid-bound ribosomes, and transport of translation products from spinach leaf polyadenylated RNA into isolated chloroplasts. Polyacrylamide gel electrophoresis of labeled thylakoid polypeptides in the presence of sodium dodecyl sulfate revealed that the first two systems were capable of synthesizing the reaction center proteins of PS II (47 and 43 kDa), the herbicide-binding protein, and cytochrome b559. The reaction center proteins synthesized in organello were shown to bind chlorophyll and to assemble properly into the PS II core complex. One of the reaction center proteins translated by the thylakoid-bound ribosomes (47 kDa) was also found to be integrated in situ into the complex but was lacking bound chlorophyll. Incorporation of radioactivity into the three extrinsic proteins of the oxygen-evolution system (33, 24, and 18 kDa) was detected only when intact chloroplasts were incubated with the translation products from polyadenylated RNA, showing that these proteins are coded for by nuclear DNA. The occurrence of a precursor polypeptide 6 kDa larger than the 33-kDa protein was immunochemically detected in the translation products.     

The site of inhibition of the chloroplast electron-transport system by 2,3-dithiopropan-1-ol (BAL)

BAL (2,3-dithiopropan-1-ol) treatment of chloroplasts has previously been reported to induce a block in electron transport from water to NADP+ at a site preceding plastocyanin [Belkin et al. (1980) Biochim. Biophys. Acta 766, 563-569]. In the present work the block was further characterized. The following properties of BAL treatment are described. Inhibition of electron transport from water to lipophilic acceptors but not to silicomolybdate. Inhibition of the slow, sigmoidal phase of chlorophyll a fluorescence induction. Inability of N,N,N',N',-tetramethyl-p-phenylenediamine to bypass the inhibition of NADP+ photoreduction with water as the electron donor. Inhibition of electron transport from externally added quinols to NADP+. Inhibition of cytochrome f reduction by photosystem II, but not its oxidation by photosystem I. Inhibition of cytochrome b6 turnover and cytochrome f rereduction after single-turnover flash illumination under cyclic electron-flow conditions. The BAL-induced block is therefore located between the secondary quinone acceptor (QB) and the cytochrome b6f complex. It was further found that (a) the isolated cytochrome complex is not inhibited after BAL treatment; (b) BAL-reacted plastoquinone-1 inhibits electron transport in chloroplasts; (c) BAL does not inhibit electron transport in chromatophores of Rhodospirilum rubrum or Rhodopseudomonas capsulata. It is suggested that the inhibition of electron transport in chloroplasts results from specific reaction of BAL with the endogenous plastoquinone.     

Changes in the carotenoid composition of chloroplast membranes from Chlamydomonas reinhardtii double mutants with alterations of various sites in photosystem II

The variable fluorescence and polypeptide and carotenoid compositions of the chlorophyll b-deficient mutant C-48 of the unicellular green alga Chlamydomonas reinhardtii and its double mutants without chlorophyll b and with inactive photosystem II were compared with those of the wild-type algal cells. Studying variable fluorescence demonstrated the alterations at the donor side (AC-121), the acceptor side (AC-234) or immediately in the photosystem II reaction centre (AC-184, AC-864). Gel electrophoresis showed that the absence of chlorophyll b in all mutants was due to the lack of 26, 28 and 31 kDa polypeptides in the light-harvesting chlorophyll a/b-protein complex II (LHC II). As a result of the second mutation, the chlorophyll a-protein complex of photosystem II did not form in chloroplast membranes. The disassembly of this complex in the mutants AC-121, AC-234 and AC-864 was related to the deficiency of both polypeptides of the reaction centre (30 and 32 kDa) and polypeptides of the water-oxidizing system (18, 23 and 34 kDa). Besides the loss of these polypeptides, the contents of polypeptides with molecular masses of 47 and 51 kDa decreased in the double mutant AC-184. Substantial changes were revealed in the carotenoid composition of the double mutants. We observed the considerable accumulation of carotenes that accompanied alterations in the donor (mutant AC-121) or acceptor (mutant AC-234) sides of PS II. In the first case, beta-carotene predominantly accumulated (87%); in the second case, it was alpha-carotene (52%). Alterations in the PS II reaction centre (mutants AC-184, AC-864) caused accumulation of xanthophylls, mainly lutein (38-41%). We suppose that alterations in different parts of the PS II chloroplast membrane lead to substantial changes in the carotenoid composition.     

Cytochrome b6f complex is required for phosphorylation of light-harvesting chlorophyll a/b complex II in chloroplast photosynthetic membranes

The light-harvesting chlorophyll a/b complex (LHC II) and four photosystem II (PS II) core proteins (8.3, 32, 34 and 44 kDa) become phosphorylated in response to reduction of the intersystem electron transport chain of green plant chloroplasts. Previous studies indicated that reduction of the plastoquinone (PQ) pool is the key event in kinase activation. However, we show here that, unlike PS II proteins, LHC II is phosphorylated only when the cytochrome b6f complex is active. Two lines of evidence support this conclusion. (1) 2,5-Dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and the 2,4-dinitrophenyl ether of iodonitrothymol (DNP-INT), which are known to block electron flow into the cytochrome complex, selectively inhibit LHC II phosphorylation in spinach thylakoids. (2) The hcf6 mutant of maize, which contains PQ but lacks the cytochrome b6f complex, phosphorylates the four PS II proteins but fails to phosphorylate LHC II in vivo or in vitro.     

Evidence for the role of surface-exposed segments of the light-harvesting complex in cation-mediated control of chloroplast structure and function.

Chloroplast membranes contain a light-harvesting pigment-protein complex (LHC) which binds chlorophylls a and b. A mild trypsin digestion of intact thylakoid membranes has been utilized to specifically alter the apparent molecular weights of polypeptides of this complex. The modified membrane preparations were analyzed for altered functional and structural properties. Cation-induced changes in room temperature fluorescence intensity and low temperature chlorophyll fluorescence emission spectra, and cation regulation of the quantum yield of photosystem I and II partial reactions at limiting light were lost following the trypsin-induced alteration of the LHC. Electron microscopy revealed that cations can neither maintain nor promote grana stacking in membranes which have been subjected to mild trypsin treatment. Freeze-fracture analysis of these membranes showed no significant differences in particle density or average particle size of membrane subunits on the EF fracture face; structural features of the modified lamellae were comparable to membranes which had been unstacked in a “low salt” buffer. Digitonin digestion of trypsin-treated membranes in the presence of cations followed by differential centrifugation resulted in a subchloroplast fractionation pattern similar to that observed when control chloroplasts were detergent treated in cation-free medium. We conclude that: (a) the initial action of trypsin at the thylakoid membrane surface of pea chloroplasts was the specific alteration of the LHC polypeptides, (b) the segment of the LHC polypeptides which was altered by trypsin is necessary for cation-mediated grana stacking and cation regulation of membrane subunit distribution, and (c) cation regulation of excitation energy distribution between photosystem I and II involves the participation of polypeptide segments of the LHC which are exposed at the membrane surface.     

Functional assembly in vitro of phycobilisomes with isolated photosystem II particles of eukaryotic chloroplasts

Phycobiliproteins obtained by dissociation of phycobilisomes were reassociated in vitro with intact thylakoids or isolated photosystems I and II preparations obtained from cyanophytes (prokaryotes) or green algae (eukaryotes) to form bound phycobilisome complexes. Energy transfer from Fremyella diplosiphon phycobiliproteins to chlorophyll a of reaction centers I and II was measured in: complexes containing intact thylakoids of the cyanophytes F. diplosiphon or Anacystis nidulans and the eukaryotic algae Euglena gracilis and mutants of Chlamydomonas reinhardtii; complexes containing isolated photosystem II particles of A. nidulans or C. reinhardtii; and complexes containing reaction center I of F. diplosiphon or C. reinhardtii. Energy transfer from phycoerythrin to chlorophyll a of photosystem II could be demonstrated in complexes containing phycobilisomes bound to cyanophyte thylakoids or isolated photosystem II particles of A. nidulans or C. reinhardtii. Bound phycobilisomes did not transfer energy to photosystem II within green algae thylakoids containing altered forms of light-harvesting chlorophyll a/b-protein complex (LHC) II antenna, reduced amounts of LHC II, or chlorophyll b, or chlorophyll b-less mutants, nor to chlorophyll a of photosystem I of intact thylakoids or isolated reaction centers. We conclude that phycobilisomes can form a specific and functional association with photosystem II particles of both cyanophytes and eukaryotic thylakoids. This interaction appears to be hindered by the presence of LHC II antenna in the eukaryotic thylakoids.     

Polypeptides belonging to each of the three major chlorophyll a + b protein complexes are present in a chlorophyll-b-less barley mutant

Polypeptides of the three major chlorophyll a + b protein complexes were detected in a chlorophyll-b-less barley mutant (chlorina f2) using immunological techniques. Antibodies to CP Ia, a photosystem I complex containing both the reaction center (CP I) and the chlorophyll a + b antenna (LHCI), detected substantial amounts of LHCI polypeptides in mutant thylakoids. Some polypeptides of the two photosystem-II-associated chlorophyll a + b complexes, CP 29 and LHCII, were also detected using antibodies raised against these complexes. The CP 29 apoprotein and the minor 25-kDa polypeptide of LHCII were present in amounts that could be seen by Coomassie blue staining. In contrast, the two major polypeptides of LHCII were greatly diminished in amount, and one of them may be completely absent. These data suggest that the absence of chlorophyll b may have differing effects on the synthesis, processing or turnover of the various chlorophyll a + b binding polypeptides. They also show that these polypeptides can be inserted into thylakoids in the absence of Chl b, and that significant amounts of some of them are accumulated in the mutant thylakoids.     

Conversion of Chlorophyll b to Chlorophyll a and the Assembly of Chlorophyll with Apoproteins by Isolated Chloroplasts

The photosynthetic apparatus is reorganized during acclimation to various light environments. During adaptation of plants grown under a low-light to high-light environment, the light-harvesting chlorophyll a/b-protein complexes decompose concomitantly with an increase in the core complex of photosystem II. To study the mechanisms for reorganization of photosystems, the assembly of chlorophyll with apoproteins was investigated using isolated chloroplasts. When [14C]chlorophyllide b was incubated with chloroplasts in the presence of phytyl pyrophosphate, it was esterified and some of the [14C]chlorophyll b was converted to [14C]chlorophyll a via 7-hydroxymethyl chlorophyll. [14C]Chlorophyll a and b were incorporated into chlorophyll-protein complexes. Light-harvesting chlorophyll a/b-protein complexes of PSII had a lower [14C]chlorophyll a to [14C]chlorophyll b ratio than P700-chlorophyll a-protein complexes, indicating the specific binding of chlorophyll to apoproteins in our systems. 7-Hydroxymethyl chlorophyll, an intermediate molecule from chlorophyll b to chlorophyll a, did not become assembled with any apoproteins. These results indicate that chlorophyll b is released from light-harvesting chlorophyll a/b-protein complexes of photosystem II and converted to chlorophyll a via 7-hydroxymethyl chlorophyll in the lipid bilayer and is then used for the formation of core complexes of photosystems. These mechanisms provide the fast, fine regulation of the photosynthetic apparatus during construction of photosystems.     

Studies of chloroplast thylakoid proteins using monoclonal antibodies

Several monoclonal antibodies have been produced against partially purified photosystem I reaction center complexes isolated from spinach chloroplasts. One of the clones was shown to be highly specific for the 28,000 and 27,000 dalton subunits of purified light harvesting chlorophyll a/b binding complex. Studies with thylakoids suggest at least a portion of the light harvesting chlorophyll a/b binding protein molecules are exposed on a normally inaccessible surface of the membrane.     

Antibodies to the photosystem I chlorophyll a + b antenna cross-react with polypeptides of CP29 and LHCII

A chlorophyll (a + b)--protein complex associated with photosystem I (PSI) was isolated from a larger PSI complex (CPIa) produced by electrophoresis of barley thylakoids solubilized with 300 mM octyl glucoside. It had an apparent Mr of 35,000-43,000 on 7.5% and 10% acrylamide gels respectively, and a chlorophyll a/b ratio of 2.5 +/- 1.5. Denaturation released four polypeptides migrating between 21-24 kDa. They were well separated from the polypeptides of the two photosystem II chlorophyll a + b antenna complexes: LHCII (25-27 kDa) and CP29 (28-29 kDa). In order to study the PSI antenna complex, antibodies were raised against highly purified CPIa. The antigen appeared to be pure when electrophoresed, blotted and reacted with its antiserum, i.e. anti-CPIa detected only the 64-66-kDa CPI apoprotein and the four 21-24 kDa antenna polypeptides. However, when blotted against the whole spectrum of thylakoid proteins, it cross-reacted with both LHCII and CP29 apoproteins. Removal of anti-CPI activity from the anti-CPIa did not affect these cross-reactions, showing that they were not due to antibodies directed against CPI. To show that the same antibody population was reacting with both the photosystem I and photosystem II antenna polypeptides, anti-CPIa was adsorbed onto highly purified CPIa on nitrocellulose. The bound antibody was eluted and used again in a Western blot against whole thylakoid proteins. This selected antibody population showed the same relative strength of reaction with photosystem I and photosystem II antenna polypeptides as the original antibody population had. Similar observations have been made with antibodies to the two photosystem II antenna complexes. We therefore conclude that there are antigenic determinants in common among the chlorophyll a + b binding polypeptides, and predict that there could be amino acid sequence similarities.     

Investigation of the spatial relationships between photosystem 2 polypeptides by reversible crosslinking and diagonal electrophoresis

Nearest neighbour relationships within the LHC2-PS2 complex were investigated by using the reversible crosslinking agent dithiobis(succinimidyl propionate) (DSP). This was accomplished by treating PS2-enriched membranes, prepared from chloroplasts of Pisum sativum, with the crosslinker followed by diagonal electrophoresis of the solubilised polypeptides.Analysis of the off-diagonal spot patterns produced by crosslinker cleavage and second dimension electrophoresis was made on the basis of: staining with Coomassie blue or silver, labelling with [³⁵S]-methionine, and sensitivity to 1 M NaCl washing. It was concluded that LHC2 polypeptides crosslinked with several components of the PS2 complex and that the extrinsic polypeptides associated with water oxidation, having approximate molecular weights of 16 and 23 kDa, crosslink to form homodimers. The latter finding suggests that there may be more than one copy of each of these polypeptides per PS2 complex.Abbreviations DMSO dimethylsulphoxide - DSP dithiobis(succinimidylpropionate) - DCPIP dichlorophenolindophenol - SDS sodium dodecylsulphate - PS2 photosystem 2 - LHC2 light harvesting chlorophyll a/b complex associated with photosystem 2 - MES 2[N-morpholino] ethanesulphonic acid     

The atypical chlorophyll a/b/c light-harvesting complex of Mantoniella squamata: molecular cloning and sequence analysis

cDNA species encoding precursor polypeptides of the chlorophyll a/b/c light-harvesting complex (LHC) of Mantoniella squamata were cloned and sequenced. The precursor polypeptides have molecular weights of 24.2 kDa and are related to the major chlorophyll a/b polypeptides of higher plants. Southern analysis showed that their genes belong to the nuclear encoded Lhc multigene family; the investigated genes most probably do not contain introns. The chlorophyll a/b/c polypeptides contain two highly conserved regions common to all LHC polypeptides and three hydrophobic α-helices, which span the thylakoid membrane. The first membrane-spanning helix, however, is not detected by predictive methods: its atypical hydrophilic domains may bind the chlorophyll c molecules within the hydrophobic membrane environment. Homology to LHC 11 of higher plants and green algae is specifically evident in the C-terminal region comprising helix III and the preceding stroma-exposed domain. The N-terminal region of 29 amino acids resembles the structure of a transit sequence, which shows only minor similarities to those of LHC II sequences. Strikingly, the mature light-harvesting polypeptides of M. squamata lack an N-terminal domain of 30 amino acids, which, in higher plants, contains the phosphorylation site of LHC 11 and simultaneously mediates membrane stacking. Therefore, the chlorophyll a/b/c polypeptides of M. squamata do not exhibit any light-dependent preference for photosystem I or 11. The lack of this domain also indicates that the attractive forces between stacked thylakoids are weak.     

Assembly of the D1 precursor in monomeric photosystem II reaction center precomplexes precedes chlorophyll a-triggered accumulation of reaction center II in barley etioplasts

Assembly of plastid-encoded chlorophyll binding proteins of photosystem II (PSII) was studied in etiolated barley seedlings and isolated etioplasts and either the absence or presence of de novo chlorophyll synthesis. De novo assembly of reaction center complexes in etioplasts was characterized by immunological analysis of protein complexes solubilized from inner etioplast membranes and separated in sucrose density gradients. Previously characterized membrane protein complexes from chloroplasts were utilized as molecular mass standards for sucrose density gradient separation analysis. In etiolated seedlings, induction of chlorophyll a synthesis resulted in the accumulation of D1 in a dimeric PSII reaction center (RCII) complex. In isolated etioplasts, de novo chlorophyll a synthesis directed accumulation of D1 precursor in a monomeric RCII precomplex that also included D2 and cytochrome b(559). Chlorophyll a synthesis that was chemically prolonged in darkness neither increased the yield of RCII monomers nor directed assembly of RCII dimers in etioplasts. We therefore conclude that in etioplasts, assembly of the D1 precursor in monomeric RCII precomplexes precedes chlorophyll a-triggered accumulation of reaction center monomers.     

Seasonal Effects on Chlorophyll-Protein Complexes Isolated from P-inus silvestris

The seasonal changes in the relative distribution of P700 chlorophyll-protein complex a₁ and light harvesting chlorophyll-protein complex a/b were studied in a natural stand of Pinus silvestris. Similar measurements were made after artificial photobleaching of chlorophyll in pine seedlings or in isolated pine chloroplasts. The chlorophyll-protein complexes were solubilized by sodium dodecyl sulphate and separated by polyacrylamide gel electrophoresis. When autumn and winter destruction of chlorophyll occurs, the chlorophyll a antenna associated with P700 in photosystem 1 (P700-CPa₁) is relatively more affected than the light harvesting complex, which lacks a reaction centre. These results are further supported by low-temperature fluorescence emission properties of isolated chloroplasts presented in this work and elsewhere. The destruction of chlorophyll in stressing autumn and winter climates is most probably caused by photosensitized oxidation of chlorophyll.     

Analysis of the pigment stoichiometry of pigment-protein complexes from barley (Hordeum vulgare). The xanthophyll cycle intermediates occur mainly in the light-harvesting complexes of photosystem I and photosystem II.

The carotenoid zeaxanthin has been implicated in a nonradiative dissipation of excess excitation energy. To determine its site of action, we have examined the location of zeaxanthin within the thylakoid membrane components. Five pigment-protein complexes were isolated with little loss of pigments: photosystem I (PSI); core complex (CC) I, the core of PSI; CC II, the core of photosystem II (PSII); light-harvesting complex (LHC) IIb, a trimer of the major light-harvesting protein of PSII; and LHC IIa, c, and d, a complex of the monomeric minor light-harvesting proteins of PSII. Zeaxanthin was found predominantly in the LHC complexes. Lesser amounts were present in the CCs possibly because these contained some extraneous LHC polypeptides. The LHC IIb trimer and the monomeric LHC II a, c, and d pigment-proteins from dark-adapted plants each contained, in addition to lutein and neoxanthin, one violaxanthin molecule but little antheraxanthin and no zeaxanthin. Following illumination, each complex had a reduced violaxanthin content, but now more antheraxanthin and zeaxanthin were present. PSI had little or no neoxanthin. The pigment content of LHC I was deduced by subtracting the pigment content of CC I from that of PSI. Our best estimate for the carotenoid content of a LHC IIb trimer from dark-adapted plants is one violaxanthin, two neoxanthins, six luteins, and 0.03 mol of antheraxanthin per mol trimer. The xanthophyll cycle occurs mainly or exclusively within the light-harvesting antennae of both photosystems.     

Stoichiometries of electron transport complexes in spinach chloroplasts

The stoichiometric relationship among photosystem II complexes, photosystem I complexes, cytochrome b/f complexes, high-potential cytochrome b-559, and chlorophyll in spinach chloroplasts has been determined. Two features of this data stand out, in contrast to currently proposed stoichiometries in which the ratio of photosystem II to photosystem I is reported to be 2:1 and the chlorophyll to reaction center ratio to be as low as 260:1. Using a variety of techniques it was found that the stoichiometry of photosystem II:photosystem I:cytochrome b/f complex was 1:1:1, within 10%, and that the ratio of total chlorophyll to these components was 600:1, also within 10%. A ratio of two high-potential cytochrome b-559 molecules per 640 chlorophyll, or two molecules per photosystem II reaction center, was found. These ratios were remarkably constant regardless of the time of year or the source of the spinach. The concentration of photosystem II complexes was determined using a pH electrode to measure the flash-induced proton release resulting from water oxidation. The photosystem I reaction center concentration was measured by two different techniques that compared favorably. In the first method a pH electrode was used to measure the amount of flash-induced proton consumption associated with the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive oxidation of N,N,N',N'- tetramethylphenylenediamine , resulting in the production of hydrogen peroxide. In the second method the amount of P700 oxidized by far-red light was determined using dual-wavelength spectroscopy. The concentration of the cytochrome b/f complex was determined assuming 1 mol of cytochrome f per complex. The concentration of cytochrome f was measured spectroscopically by its light-induced turnover and by chemical difference spectra. The concentration of high-potential cytochrome b-559 was determined by chemical difference spectra. In addition to these studies, the light-induced absorbance change exhibiting a peak at 323 nm that has been attributed to the reduction of the primary quinone acceptor of photosystem II has been investigated. This measurement frequently has been used to quantitate the photosystem II to chlorophyll ratio. However, in view of these results it is argued that this technique significantly overestimates the photosystem II concentration.     

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.     

Spectral anisotropy of chloroplasts, subchloroplast particles and pigment-protein complexes

Anisotropic properties of pea chloroplasts, subchloroplast fragments (photosystem 1 particles and pigment-protein complexes) and the blue-green algae oriented in polyacrylamide gel were investigated. It was shown that linear dichroism spectra of chloroplasts are the superposition of the corresponding spectra for the main light harvesting complex (HMLC) and P 700 chlorophyll a--protein complex (CP 1). Anisotropic properties of the photosystem 1 particles and blue-green algae are mainly caused by CP 1 anisotropy. Qy-transition moments tend to perpendicular orientation to the membrane plane for the Chl. b 649, Chl. a 660 and parallel orientation--for Chl. b 654, Chl. a 682. The degree of Qy-transition moments parallel orientation is higher for the longwave forms (Chl. a 690, Chl. a 702, Chl. a 712), than for the shortwave ones and coincides with this degree for the reaction centre pigment P 700 transition moment. It is suggested that the specific orientation of the pigment-protein complexes in the chloroplast membrane is important for the regulation of the spillover between two photosystems.