Ultrastructure and freeze-fracture studies of the thylakoids ofMantoniella squamata (Prasinophyceae)

Summary The ultrastructure and the supramolecular organization of the thylakoids of the small green flagellate,Mantoniella squamata, were examined in thin sections and freeze-fracture preparations. The whole chloroplast is tightly packed with thylakoids, which show a pattern of meandering, branching and/or anastomosing membranes. In freeze-fracture preparations only two fracture-faces can be distinguished: the PF- and the EF-face. The PF-face has a much higher particle density than the EF-face (PF: 4086 particles/m²; EF: 865 particles/m²). The EF-face is not as uniform as the PF-face. The areas which are packed with particles probably correspond to closely appressed thylakoid regions or adhesive patches, noticed in thin sections in some areas. The mean particle size on both faces is also different (EF: 10.5 nm; PF: 8.6 nm), but no information about the classification of the particles to special protein complexes is available at this time.Abbreviations chl chlorophyll - EF exoplasmic fracture face - ER endoplasmic reticulum - LHC light-harvesting chlorophyll-protein complex - PF protoplasmic fracture face - PS I photosystem I - PS II photosystem II     

Ultrastructure of cell wall and photosynthetic apparatus of the phycobilisome-less Synechocystis sp. strain BO 8402 and phycobilisome-containing derivative strain BO 9201

The ultrastructures of two closely related strains of a novel diazotrophic cyanobacterium, Synechocystis sp. BO 8402 and BO 9201, were examined using ultrathin sections and freeze-fracture electron microscopy. Cells of both strains were surrounded by an unusual thick peptidoglycan layer. Substructures in the layer indicated the presence of microplasmodesmata aligned perpendicular to the free cell surface and in the septum of dividing cells. Synechocystis sp. strain BO 8402 contained lobed, electronopaque, highly fluorescent inclusion bodies consisting of phycocyanin-linker complexes. The thylakoids lacked phycobilisomes and accommodated, in addition to randomly distributed exoplasmic freeze-fracture particles, patches of two-dimensionally ordered arrays of dimeric photosystem II particles in the exoplasmic fracture face. Determination of photosystem I and photosystem II suggested an increase of photosystem II in strain BO 8402. Strain BO 9201 performed phycobilisome-supported photosynthesis and showed rows of dimeric photosystem II particles in the exoplasmic fracture face. Corresponding particle-free grooves in the protoplasmic fracture face were lined by a class of large particles tentatively assigned as trimers of photosystem I. The different lateral organization of protein complexes in the thylakoid membranes and the fine structure of the cell wall are discussed with respect to absorption cross-section of photosynthesis and nitrogen fixation.Abbreviations EF Exoplasmic freeze-fracture face - P 700 Reaction centre chlorophyll of photosystem I - PF Protoplasmic freeze-fracture face - PS I Photosystem I - PS II Photosystem II     

The action of lipase on chloroplast membranes: II. Polypeptide patterns of bean galactolipase- and phospholipase A2-treated thylakoid membranes

Thylakoid membranes obtained from bean chloroplasts treated with bean galactolipase or phospholipase A₂ (from Crotalus terr. terr.) showed marked changes in their polypeptide patterns when separated on SDS-PAGE. The obtained results have been discussed with regard to the relationship between chloroplast lipids and polypeptides originating from chlorophyll-protein complexes of bean thylakoids. A coexistence between galactolipids and the peripheral antennae in PS I complex and LHCP³ as well as a conspicuous role of phospholipids in PSI and PSII centre chlorophyll-protein complexes has to be underlined.Abbreviations CP1 chlorophyll a-protein complex of PSI - CPa chlorophyll a-protein complex of PSII - D₁₀ digitonin subchloroplast particles enriched in PSII - D₁₄₄ digitonin subchloroplast particles enriched in PSI - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - LHCP^1–3 light harvesting chlorophyll a/b protein complexes - PAGE polyacrylamide gel electrophoresis - PSI photosystem I - PSII photosystem II - SDS sodium dodecyl sulphate - TCA trichloroacetic acid - Tricine N-Tris-(hydroxymethyl)-methylglycine - Tris Tris-(hydroxymethyl)-aminomethan     

The action of lipase on chloroplast membranes: II. Polypeptide patterns of bean galactolipase- and phospholipase A2-treated thylakoid membranes

Thylakoid membranes obtained from bean chloroplasts treated with bean galactolipase or phospholipase A₂ (from Crotalus terr. terr.) showed marked changes in their polypeptide patterns when separated on SDS-PAGE. The obtained results have been discussed with regard to the relationship between chloroplast lipids and polypeptides originating from chlorophyll-protein complexes of bean thylakoids. A coexistence between galactolipids and the peripheral antennae in PS I complex and LHCP³ as well as a conspicuous role of phospholipids in PSI and PSII centre chlorophyll-protein complexes has to be underlined.Abbreviations CP1 chlorophyll a-protein complex of PSI - CPa chlorophyll a-protein complex of PSII - D₁₀ digitonin subchloroplast particles enriched in PSII - D₁₄₄ digitonin subchloroplast particles enriched in PSI - DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethylurea - LHCP^1-3 light harvesting chlorophyll a/b protein complexes - PAGE polyacrylamide gel electrophoresis - PSI photosystem I - PSII photosystem II - SDS sodium dodecyl sulphate - TCA trichloroacetic acid - Tricine N-Tris-(hydroxymethyl)-methylglycine - Tris Tris-(hydroxymethyl)-aminomethan     

Structural and functional self-organization of Photosystem II in grana thylakoids

The biogenesis of the well-ordered macromolecular protein arrangement of photosystem (PS)II and light harvesting complex (LHC)II in grana thylakoid membranes is poorly understood and elusive. In this study we examine the capability of self organization of this arrangement by comparing the PSII distribution and antenna organization in isolated untreated stacked thylakoids with restacked membranes after unstacking. The PS II distribution was deduced from freeze-fracture electron microscopy. Furthermore, changes in the antenna organization and in the oligomerization state of photosystem II were monitored by chlorophyll a fluorescence parameters and size analysis of exoplasmatic fracture face particles. Low-salt induced unstacking leads to a randomization and intermixing of the protein complexes. In contrast, macromolecular PSII arrangement as well as antenna organization in thylakoids after restacking by restoring the original solvent composition is virtually identical to stacked control membranes. This indicates that the supramolecular protein arrangement in grana thylakoids is a self-organized process.     

Low-light-induced formation of semicrystalline photosystem II arrays in higher plant chloroplasts

Remodeling of photosynthetic machinery induced by growing spinach plants under low light intensities reveals an up-regulation of light-harvesting complexes and down-regulation of photosystem II and cytochrome b6f complexes in intact thylakoids and isolated grana membranes. The antenna size of PSII increased by 40-60% as estimated by fluorescence induction and LHCII/PSII stoichiometry. These low-light-induced changes in the protein composition were accompanied by the formation of ordered particle arrays in the exoplasmic fracture face in grana thylakoids detected by freeze-fracture electron microscopy. Most likely these highly ordered arrays consist of PSII complexes. A statistical analysis of the particles in these structures shows that the distance of neighboring complexes in the same row is 18.0 nm, the separation between two rows is 23.7 nm, and the angle between the particle axis and the row is 26 degrees . On the basis of structural information on the photosystem II supercomplex, a model on the supramolecular arrangement was generated predicting that two neighboring complexes share a trimeric light-harvesting complex. It was suggested that the supramolecular reorganization in ordered arrays in low-light grana thylakoids is a strategy to overcome potential diffusion problems in this crowded membrane. Furthermore, the occurrence of a hexagonal phase of the lipid monogalactosyldiacylglycerol in grana membranes of low-light-adapted plants could trigger the rearrangement by changing the lateral membrane pressure.     

Structural and functional self-organization of Photosystem II in grana thylakoids

The biogenesis of the well-ordered macromolecular protein arrangement of photosystem (PS)II and light harvesting complex (LHC)II in grana thylakoid membranes is poorly understood and elusive. In this study we examine the capability of self organization of this arrangement by comparing the PSII distribution and antenna organization in isolated untreated stacked thylakoids with restacked membranes after unstacking. The PS II distribution was deduced from freeze-fracture electron microscopy. Furthermore, changes in the antenna organization and in the oligomerization state of photosystem II were monitored by chlorophyll a fluorescence parameters and size analysis of exoplasmatic fracture face particles. Low-salt induced unstacking leads to a randomization and intermixing of the protein complexes. In contrast, macromolecular PSII arrangement as well as antenna organization in thylakoids after restacking by restoring the original solvent composition is virtually identical to stacked control membranes. This indicates that the supramolecular protein arrangement in grana thylakoids is a self-organized process.     

Phycoerythrin is absent from the pyrenoid of Porphyridium cruentum: photosynthetic implications

The thylakoid lamellae which traverse the pyrenoid of the unicellular red alga Porphyridium cruentum (Agardh) Nägeli appear to lack phycobilisomes. We have confirmed by immuno-electron microscopy that phycoerythrin (PE), an important structural component of the phycobilisomes of red algae, is absent from the pyrenoid. To characterize pyrenoid thylakoids further, electron-microscopic cytochemical methods were employed to detect photosystem activity. Photosystem (PS) I activity was demonstrated in both stromal and pyrenoid thylakoids by the photooxidation of 3,3-diaminobenzidine. In contrast, the localization of photoreduced distyryl nitroblue tetrazolium demonstrated that PSII activity was restricted to stromal thylakoids. The observed partitioning of PE and PSII activity within the plastid may be related to another observation, that being the localization of nearly all ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) within the pyrenoid of this alga. It is possible that the pyrenoid of P. cruentum functions as a specific metabolic compartment where CO₂ fixation is enhanced by the absence of photosynthetic O₂ evolution.Abbreviations DAB 3,3-diaminobenzidine-4HCl - DS-NBT distyryl nitroblue tetrazolium chloride - EF exoplasmic face - LSU large subunit of RuBisCO - PE phycoerythrin - PS photosystem - RuBisCO ribulose-1,5-bisphosphate carboxylase/oxygenase We thank Drs. Jacqueline Fleck (CNRS, Strasbourg) and Robert MacColl (New York State Department of Health, Albany) for providing us with the antibodies used in this study. We also thank Dr. C.E. Smith for use of the Zeiss MOP-3 digital analyser and Dr. Geneviève Bricheux for kindly providing Lowicryl-embedded samples of P. cruentum. Aatrex^® was kindly donated by Ciba-Geigy. This research was supported by the Natural Sciences and Engineering Research Council of Canada (grant No. A-2921).     

Excitation-energy transfer in heterocysts isolated from the cyanobacterium Anabaena sp. PCC 7120 as studied by time-resolved fluorescence spectroscopy

Heterocysts are formed in filamentous heterocystous cyanobacteria under nitrogen-starvation conditions, and possess a very low amount of photosystem II (PSII) complexes than vegetative cells. Molecular, morphological, and biochemical characterizations of heterocysts have been investigated; however, excitation-energy dynamics in heterocysts are still unknown. In this study, we examined excitation-energy-relaxation processes of pigment-protein complexes in heterocysts isolated from the cyanobacterium Anabaena sp. PCC 7120. Thylakoid membranes from the heterocysts showed no oxygen-evolving activity under our experimental conditions and no thermoluminescence-glow curve originating from charge recombination of S₂Q_A^?. Two dimensional blue-native/SDS-PAGE analysis exhibits tetrameric, dimeric, and monomeric photosystem I (PSI) complexes but almost no dimeric and monomeric PSII complexes in the heterocyst thylakoids. The steady-state fluorescence spectrum of the heterocyst thylakoids at 77 K displays both characteristic PSI fluorescence and unusual PSII fluorescence different from the fluorescence of PSII dimer and monomer complexes. Time-resolved fluorescence spectra at 77 K, followed by fluorescence decay-associated spectra, showed different PSII and PSI fluorescence bands between heterocysts and vegetative thylakoids. Based on these findings, we discuss excitation-energy-transfer mechanisms in the heterocysts.     

Ultrastructural organization of chloroplast membranes in mutants of Pisum sativum L. with impaired activity in the photosystems

The ultrastructural organization and the photosynthesis reactions of chloroplast membranes were studied in three lethal mutants of Pisum sativum, Chl-1, Chl-19 and Chl-5, all lacking the capacity to evolve oxygen. The rates of 2,6-dichloroindophenol reduction, delayed fluorescence and electron-spin-resonance signal 1 indicate that Chl-1 and Chl-19 have an impaired activity in photosystem II (PS II), while in Chl-5 the electron transport is blocked between PS I and the reactions of CO₂ fixation. Ultrathin sectioning demonstrates the presence of giant grana in the chloroplasts of Chl-1 and Chl-19, while the chloroplast structure of the Chl-5 is very similar to that of the wild-type. The grana of the Chl-19 mutant contain large multilamellar regions of tightly packed membranes. When the chloroplast membranes were studied by freeze-fracture, the exoplasmic and protoplasmic fracture faces (EF and PF, respectively) in both stacked and unstacked membranes were found to show large differences in particle concentrations and relative population area (per m²), and also in particle size distribution, between all mutant chloroplast membranes and the wild-type. A close correlation between increasing k_mt (ratio of particle concentrations on PF/EF) and PS II activity was observed. The differences in particle concentrations on both fracture faces in different regions of the intact chloroplast membranes of the wild-type are the consequence of a rearrangement of existing membrane components by lateral particle movements since quantitative measurements demonstrate almost complete conservation of intramembrane particles in number and size during the stacking of stroma thylakoid membranes. The results indicating particle movements strongly support the concept that the chloroplast membranes have a highly dynamic structure.Abbreviations DPIP 2,6-dichloroindophenol - EF and PF exoplasmic and protoplasmic fracture faces, respectively - PS I and PS II photosystems I and II, respectively     

Membranes and organelles of dehydratedSelaginella andTortula retain their normal configuration and structural integrity

Summary Dry (7–10% water content) leaves of the spikemossSelaginella lepidophylla (resurrection plant) and of the desiccationtolerant moss,Tortula ruralis were examined by freeze fracture electron microscopy. As has been described for dry seeds, the cells of these dehydrated leaves were shrunken, with highly convoluted walls and membranes. The membranes of all samples had a lipid bilayer organization with dispersed intramembranous particles (IMPs). Lipid droplets were very closely associated with the plasmamembrane. Chloroplasts were surrounded by a double membrane envelope and contained well-organized grana. Mitochondria were irregular in outline, and endoplasmic reticulum and cytoplasmic vesicles were present.Abbreviations ABA abscisic acid - EF exoplasmic fracture - FTIR Fourier transform infrared analysis - H_II hexagonal II - IMPs intramembranous particles - MGDG monogalactosyl diacylglycerol - NMR nuclear magnetic resonance - PE phosphatidylethanolamine - PF protoplasmic fracture - PS I photosystem I - PS II photosystem II     

Development of chloroplast membranes during light induced greening ofChlorella fusca g-2

Summary The development of thylakoid protein complexes during light induced greening of a mutant ofChlorella fusca was studied. Separation of chlorophyll-protein complexes and thylakoid polypeptides by LDS-polyacrylamide gel electrophoresis show that cells grown in the dark contain proteins belonging to coupling factor and cytochrome f/b₆ complex. Parts of both reaction centers are present also. The antennae complexes are specifically lost in yellow cells. The changes in polypeptide pattern at different stages of development in the light are related to ultrastructural changes. The beginning of membrane appression can be correlated with the appearance of the light-harvesting complex II. While the average diameter of EF-particles increases throughout the greening process, their densitiesapart from the rearrangement due to membrane stacking-remain fairly constant. The kinetics of EF_u-particle enlargement are different from those of EF_s-particles.PF-faces in thylakoids grown in the dark contain particles of uniform diameter but some of them protrude more from the fracture plane than do their neighbors. During the first hours of greening, their density increases and two classes develop. From the beginning of membrane stacking, the composition of PF_u-faces remains constant and PF_s-particles increase in number for some time.Results are discussed on the basis of present knowledge of structurefunction relations in thylakoids.Abbreviations CF _o intrinsic membrane complex of the coupling factor - EF, EF _s ,EF _u exoplasmic fracture face, stacked and unstacked region, respectively - LDS lithium dodecyl sulfate - LHCII light-harvesting complex of Photosystem II - PF, PF _s ,PF _u protoplasmic fracture face, stacked and unstacked region, respectively - PS I andPS II Photosystem I and Photosystem II     

Further identification of the exoplasmic face particles on the freeze-fractured thylakoid membranes: a study using double and triple mutants from Chlamydomonas reinhardtii lacking various photosystem II subunits and the cytochrome b6/f complex.

About 20% of the exoplasmic face (EF) particles present in the freeze-fractured thylakoid membranes of the wild type strain of Chlamydomonas reinhardtii remain in mutants lacking photosystem II (PSII) because of the absence of either one of the two PSII subcomplexes CP43 or D1/D2/CP47. We show that about half of these residual EF particles can be accounted for by PSII subcomplexes still present in such mutants, and by cytochrome (cyt) b6/f complexes. Analysis of double mutants lacking both types of protein complexes points to an association of cyt b6/f complexes with PSII subcomplexes in some of these EF particles and to a requirement in cyt b6/f complexes for the translocation of each of the two PSII subcomplexes (the CP43 subunit and the D1/D2/CP47 subcomplex) from the unstacked to the stacked regions of the thylakoid membranes.     

Ultrastructure of Acaryochloris marina, an oxyphotobacterium containing mainly chlorophyll d

We present a detailed investigation of the ultrastructure of the chlorophyll a/d-containing unicellular oxyphotobacterium Acaryochloris marina, combining light and transmission electron microscopy and showing freeze fractures of this organism for the first time. The cells were 1.8-2.1 microm x 1.5-1.7 microm in size. The cell envelope consisted of a peptidoglycan layer of approximately 10 nm thickness combined with an outer membrane. Cell division was intermediate between the constrictive and the septum type. The nucleoplasm, which contained several carboxysomes, was surrounded by 7-11 concentrically arranged thylakoids, which were predominantly stacked, with the exception of distinct areas where phycobiliproteins were located. The thylakoids were perforated by channel-like structures connecting the central and peripheral portions of the cytoplasm and not yet observed in other organisms. In freeze fractures, the protoplasmic fracture faces of thylakoid membranes were densely covered with particles of inhomogenous size. The particle size histogram peaked at 10-11, 13 and 18 nm. The 18-nm particles are assumed to represent photosystem I trimers. The particles on exoplasmic fracture faces, proposed to represent photosystem II complexes, were significantly larger than the corresponding particles of cyanobacteria and clustered to form large aggregates. This kind of arrangement is unique among photosynthetic organisms.     

Structure of the Thylakoids and Envelope Membranes of the Cyanelles of Cyanophora paradoxa

The cyanelles of Cyanophora paradoxa Korsch. are photosynthetically active obligate endosymbionts in which phycobiliproteins serve as the major accessory pigments. Freeze-fracture electron micrographs of thylakoids in isolated cyanelles reveal long parallel rows of particles covering most of the E-face, while a more random particle arrangement is evident in some areas. The center-to-center spacing of particles within these rows is about 10 nanometers. Their mean diameter was measured at 9.4 nanometers. The particles on the P-face have a mean diameter of 7.2 nanometers. Thylakoids that retained nearly the full complement of phycobiliproteins (determined spectrophotometrically and by gel electrophoresis) were isolated from the cyanelles. In thin sections of these preparations, rows of disc-shaped phycobilisomes are evident on the surface of the thylakoids. The spacing of the rows of phycobilisomes corresponds to that of the rows of E-face particles (approximately 45 nanometers, center to center). The periodicity of the disc-shaped phycobilisomes within a row is 10 nanometers suggesting a one-to-one association between phycobilisomes and E-face particles.

In addition, visualization of the protoplasmic surface (PS) of isolated thylakoids by freeze-etch electron microscopy shows that rows of disc-shaped phycobilisomes are aligned directly above rows of particles exhibiting two subunits, presumably the P-surface projections of the 10-nanometer intramembrane particles. These observations, together with earlier studies indicating that the 10-nanometer E-face particles probably represent photosystem II (PSII) complexes, suggest that phycobilisomes are positioned on the thylakoid surface in direct contact with PSII centers within the thylakoid membrane.

The inner envelope membrane of the cyanelles, observed in freeze-fracture replicas, resembles cyanobacterial plasma membranes and is dissimilar to the chloroplast envelope membranes of red or green algae. The envelope of isolated cyanelles exhibits two additional layers: (a) a 5- to 7-nanometer-thick layer that lies adjacent to the inner membrane and which seems to correspond to the peptidoglycan layer of cyanobacteria; and (b) a layer external to the purported peptidoglycan layer that exhibits fracture faces similar to those of the lipopolysaccharide layer of gram negative bacteria. Our findings indicate that the supramolecular architecture of cyanelles differs only slightly from free-living cyanobacteria to which they are presumably related.

     

Developmental Loss of Photosystem II Activity and Structure in a Chloroplast-Encoded Tobacco Mutant, Lutescens-1

Lutescens-1, a tobacco mutant with a maternally inherited dysfunction, displayed an unusual developmental phenotype. In vivo measurement of chlorophyll fluorescence revealed deterioration in photosystem II (PSII) function as leaves expanded. Analysis of thylakoid membrane proteins by polyacrylamide gel electrophoresis indicated the physical loss of nuclear- and chloroplast-encoded polypeptides comprising the PSII core complex concomitant with loss of activity. Freeze fracture electron micrographs of mutant thylakoids showed a reduced density, compared to wild type, of the EF_s particles which have been shown previously to be the structural entity containing PSII core complexes and associated pigment-proteins. The selective loss of PSII cores from thylakoids resulted in a higher ratio of antenna chlorophyll to reaction centers and an altered 77 K chlorophyll fluorescence emission spectra; these data are interpreted to indicate functional isolation of light-harvesting chlorophyll a/b complexes in the absence of PSII centers. Examination of PSII reaction centers (which were present at lower levels in mutant membranes) by monitoring the light-dependent phosphorylation of PSII polypeptides and flash-induced O₂ evolution patterns demonstrated that the PSII cores which were assembled in mutant thylakoids were functionally identical to those of wild type. We conclude that the lutescens-1 mutation affected the correct stoichiometry of PSII centers, in relation to other membrane constituents, by disrupting the proper assembly and maintenance of PSII complexes in lutescens-1 thylakoid membranes.     

State 1-state 2 adaptation in the cyanobacteria Synechocystis PCC 6714 wild type and Synechocystis PCC 6803 wild type and phycocyanin-less mutant

The mechanism of excitation energy distribution between the two photosystems (state transitions) is studied in Synechocystis 6714 wild type and in wild type and a mutant lacking phycocyanin of Synechocystis 6803. (i) Measurements of fluorescence transients and spectra demonstrate that state transitions in these cyanobacteria are controlled by changes in the efficiency of energy transfer from PS II to PS I (spillover) rather than by changes in association of the phycobilisomes to PS II (mobile antenna model). (ii) Ultrastructural study (freeze-fracture) shows that in the mutant the alignment of the PS II associated EF particles is prevalent in state 1 while the conversion to state 2 results in randomization of the EF particle distribution, as already observed in the wild type (Olive et al. 1986). In the mutant, the distance between the EF particle rows is smaller than in the wild type, probably because of the reduced size of the phycobilisomes. Since a parallel increase of spillover is not observed we suggest that the probability of excitation transfer between PS II units and between PS II and PS I depends on the mutual orientation of the photosystems rather than on their distance. (iii) Measurements of the redox state of the plastoquinone pool in state 1 obtained by PS I illumination and in state 2 obtained by various treatments (darkness, anaerobiosis and starvation) show that the plastoquinone pool is oxidized in state 1 and reduced in state 2 except in starved cells where it is still oxidized. In the latter case, no important decrease of ATP was observed. Thus, we propose that in Synechocystis the primary control of the state transitions is the redox state of a component of the cytochrome b ₆/f complex rather than that of the plastoquinone pool.Abbreviations DCCD dicyclohexylcarbodiimide - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - EF exoplasmic face - PQ plasto-quinone - PS photosystem - PBS phycobilisome     

Deletion mutations in a long hydrophilic loop in the photosystem II chlorophyll-binding protein CP43 in the cyanobacterium Synechocystis sp. PCC 6803

In order to investigate the role and function of the hydrophilic region between transmembrane regions V and CI in the photosystem II core antenna protein CP43, we introduced eight different deletions in psbC of Synechocystis sp; PCC 6803 resulting in a loss of 7–11 codons in evolutionary conserved domains in this region. All deletions resulted in an obligate photoheterotrophic phenotype (requirement of glucose for cell growth) and the absence of any detectable oxygen evolution activity. The various deletion mutations showed a different impact on the amount of CP43 in the thylakoid, ranging from wild-type levels of (a now slightly smaller) CP43 to no detectable CP43 at all. All deletions led to a decrease in the amount of the D1 and D2 proteins in the thylakoids with a larger effect on D2 than on D1. CP47, the other major chlorophyll-binding protein, was present in reduced but significant amounts in the thylakoid. Herbicide binding (diuron) was lost in all but one mutant indicating the PSII components are not assembled into functionally intact complexes. Fluorescence-emission spectra confirmed this notion. This indicates that the large hydrophilic loop of CP43 plays an important role in photosystem II, and even though a shortened CP43 is present in thylakoids of most mutants, functional characteristics resemble that of a mutant with interrupted psbC.Abbreviations CP chlorophyll-binding protein - DCPIP 2,6-dichlorophenolindophenol - DPC diphenylcarbazide - ferricyanide K₃Fe(CN)₆ - HEPES N-(2-hydroxyelthyl)piperazine-N-(2-hydroxypropane sulfonic acid) - MES 2-(N-morpholino)-ethanesulfonic acid - PCC Pasteur Culture Collection - PCR polymerase chain reaction - PS photosystem - Q_A first quinone acceptor in PSII - Q_B second quinone acceptor in PSII - Z redox-active tyrosine (Y161) in D1 serving as electron carrier between the Mn cluster and P680     

Quantitative correlation of peripheral and intrinsic core polypeptides of photosystem II with photosynthetic electron-transport activity ofAcetabularia mediterranea in red and blue light

The high photosynthetic activity (O₂ production and CO₂ consumption) ofAcetabularia mediterranea Lamour. (=A. acetabulum (L.) Silva) characteristic of cells cultured in white light decreases slowly when cells are kept in continuous red light, and is less than 20% of the original activity after three weeks. Subsequent blue irradiation restores the original activity completely within 3–5 d. The polypeptide composition of the thylakoids from cells grown in either red or blue light and after transfer from red to blue light was analyzed mainly with regards to photosystem II (PSII). The P700-containing reaction-centre complex of photosystem I, CPI, showed only minor quantitative alterations as a consequence of the growth-light quality, which correlated well with the activity of photosystem I under these conditions. In PSII, no drastic changes occurred in the quantity of the reaction-centre components D1 (herbicide-binding polypeptide) and D2, as determined by immunoblots. Likewise, the proteins associated with the water-splitting apparatus did not change detectably in thylakoids from red- or blue-light-treated cells (the 16-kDa component could not be found inAcetabularia thylakoids). The level of the major light-harvesting complex was completely unaffected by the light quality. In contrast, the quantities of the chlorophyll a-protein complexes of the core antenna, CP43 and CP47 (and probably CP29), changed, with kinetics similar to those of total photosynthetic activity. We postulate that the function of the PSII antenna became increasingly impaired in the absence of blue light (i.e. in red light), while blue light had a restoring effect. The peripheral antenna, comprising the light-harvesting complexes, is probably functionally connected with the reaction-centre chlorophylls via the core antenna chlorophyll-protein complexes (CP43, CP47 and probably CP29). A deficiency of these complexes would lead to uncoupling of antenna and reaction centre in the majority of PSII complexes after long periods of red-light treatment.     

The photosynthetic apparatus of C3 and CAM-induced Mesembryanthemum crystallinum L.

Accompanying the CAM induction of Mesembryanthemum crystallinum L. grown in high salinity there are changes in the enzymes of carbon metabolism. However, there are no changes in the electron transport activities, Chla/b ratios or in the distribution of chlorophyll amongst the various pigment-protein complexes of isolated thylakoids. Hence with CAM induction there are no changes in the photochemical apparatus of M. crystallinum thylakoids. Despite comparable amounts of chlorophylla/b-proteins of photosystem II to those found in typical C₃ sun plants, both the C₃ and CAM M. crystallinum chloroplasts have relatively more photosystem II, and, concommitantly, less photosystem I complex. This is consistent with greater fluorescence emission at 685 and 695 nm, and lower emission at 735 nm (measured at 77 K) than typically found for C₃ plants, whether sun or shade species. Photoinhibition of isolated C₃ and CAM thylakoids by white light led to comparable decreases in electron transport capacities and fluorescence emission at 77 K with photosystem II being more affected than PSI. We suggest however, that the presence of more core PSII complexes relative to PSI complexes in this CAM-inducible plant, may provide an additional strategy to mitigate photoinhibition in the short-term.