The Supramolecular Architecture in Chloroplast Thylakoid Membranes of Warigal Wheat

Thylakoid membranes of chloroplast from first leaf and flag leaf of wheat Warigal were examined by freeze-fracture and rotary shadowing etectron microscopy. The shape, size, density and size distribution of freeze-fracture partieles of their four faces were measured and plotted as three-dimensional histograms by a Hewlett-packard 9874 A digitizer with a HP 9845 B Computer and HP 9872 C plotter. When comparisons were made among different fracture faces and between the corresponding faces of the first leaf and the flag leaf, we found that the supramolecular architecture on the four fracture faces of the flag leaf differs from that on the corresponding faces of the chloroplast thytakoid membranes of the first leaf. The most significant difference was that the EFs particles contain the photosystem Ⅱ reaction centres associated with LHCP and the PFs particles were mostly light-harvesting complex. There was a 15% increase in EFs particle density, a 22% increase in PFs particle density and a 28% increase in EFu particle density. The large PFu particles contained the photosystem Ⅰ reaction centre and the flag lcaves contained 5% more than the first leaves. In addition, the stacking of thylakoid membranes in the flag leaf was 5% more than those in the first leaf.Thus, it provides theoretical basis for the fact that the flag leaf has higher photosynthetic rate.     

Effects of Photoperiod on Supramolecular Architecture of Thylakoid Membranes from a Rice Mutant (Oryza sativa Nongken 58S)

Plants of a rice mutant (Hubei photoperiod-sensitive genic male-sterile rice, Oryza sativa L. Nongken 58S) and its wild type cv. Nongken 58 were cultured in natural summer conditions in Beijing. After induction of proper photoperiods small panicle at the stem tip emerged and developed to the stage of secondary rachis-branch and spikelet primordium formation. Subsequently, part of the rice plants received long day (LD), i.e. 10 h of day-light treatment followed by 5 h of white fluorescent illumination with 1～2 Wm-2) . The others were exposed to daylight for 10 h alternating with a 14 h of dark period as short day (SD) treatment. After 10 days of the photoperiodic treatments, the chloroplast ultrastructure of the first leave below the flag leaf was examined by freeze-fracture rotary and unidirectionally shadowed electron microscopy. At anthesis stage, Nongken 58S plants with LD treatment showed complete pollen sterility, while the same plants with SD treatment exhibited normal fertility. And fertility of Nongken 58 was not affected by photoperiod treatments. The results from electron microscopic observation showed no significant effects of either SD or LD treatment on the freeze-fractured uhrastructure of thylakoid membranes in Nongken 58. No significant difference in particle density and size distribution was found on stacked and unstacked thylakoid membrane regions of the Nongken 58S-SD and those of Nongken 58 rice. However, the particle density of the endoplasmic fracture face in the staked region (EFs) and protoplasmic fracture face in the staked region (PFs) faces detected from the leaf thylakoid membranes of Nongken 58S-SD rice was significantly higher than that of the corresponding faces from Nongken 58S-LD. In some cases much more particles on EFs faces of thylakoid membranes isolated from Nongken 58S-SD rice appeared as paracrystalline particle array, indicating increases in the number of PS Ⅱ reaction centres, LHC I and Cyt b6/f per unit area of thylakoid membrane. The particle density of the endoplasmic fracture face in the unstaked region (EFu) and protoplasmic fracture face in the unstaked region (PFu) faces from unstacked thylakoid membranes of Nongken 58S-LD was less than that of the corresponding faces from Nongken 58S-SD. And the particle density of PFu faces from margin and end of the membranes of the grana thylakoids of LD-treated Nongken 58S leaves was also less than that of unstacked thylakoid membranes from SDtreated rice. In severe cases, most of the particles on endoplasmic fracture face in the unstaked region (EFu) and protoplasmic fracture face in the unstaked region (PFu) faces were even missing, indicating a decrease in the numbers of photosystem Ⅰ , LHCⅠ , Cyt b6/f and ATPase per unit area of' thylakoid membrane. The above results could further provide an augmentation for explaning the photoperiod-sensitive genic male-sterility.     

The light-harvesting chlorpohyll-protein complex of photosystem II. Its location in the photosynthetic membrane

We have investigated the structure of the photosynthetic membrane in a mutant of barley known to lack a chlorophyll-binding protein. This protein is thought to channel excitation energy to photosystem II, and is known as the "light-harvesting chlorophyll-protein complex." Extensive stacking of thylakoids into grana occurs in both mutant and wild-type chloroplasts. Examination of membrane internal structure by freeze-fracturing indicates that only slight differences exist between the fracture faces of mutant and wild-type membranes. These differences are slight reductions in the size of particles visible on the EFs fracture face, and in the number of particles seen on the PFs fracture face. No differences can be detected between mutant and wild-type on the etched out surface of the membrane. In contrast, tetrameric particles visible on the etched inner surface of wild-type thylakoids are extremely difficult to recognize on similar surfaces of the mutant. These particles can be recognized on inner surfaces of the mutant membranes when they are organized into regular lattices, but these lattices show a much closer particle-to-particle spacing than similar lattices in wild-type membranes. Although several interpretations of these data are possible, these observations are consistent with the proposal that the light-harvesting chlorophyll-protein complex of photosystem II is bound to the tetramer (which is visible on the EFs face as a single particle) near the inner surface of the membrane. The large tetramer, which other studies have shown to span the thylakoid membrane, may represent an assembly of protein, lipid, and pigment comprising all the elements of the photosystem II reaction. A scheme is presented which illustrates one possibility for the light reaction across the photosynthetic membrane.     

FREEZE FRACTURE STUDY OF THYLAKOIDS OF FUCUS SERRATUS1

Freeze-fractured thylakoids of Fucus serratus L. exhibit three types of faces with a particle density analogous to that of EFs, EFu and PF faces of green plants. However the particle size distribution is unimodal in the three types with a mean of about 8 nm. No obvious distinction between PFs and PFu faces could be detected. The absence on EFs faces of the distinct class of large particles (>13 nm) existing in green plant thylakoids implies a unique organization of pigment proteins, especially of the light-harvesting complexes.     

Hierarchical Response of Light Harvesting Chlorophyll-Proteins in a Light-Sensitive Chlorophyll b-Deficient Mutant of Maize

The light-sensitive chlorophyll b (Chl b)-deficient oil yellow-yellow green (OY-YG) mutant of maize (Zea mays) grown under conditions of high light exhibits differential reductions in the accumulation of the three major Chl b-containing antenna complexes and characteristic changes in thylakoid architecture. When observed by freeze-fracture electron microscopy, the most notable changes in the OY-YG thylakoid structure are: (a) a major reduction in the number of 8 nanometer particles of the protoplasmic fracture face of stacked membrane regions (PFs) paralleled by a 60% reduction in the chlorophyll-proteins (CP) associated with the peripheral light harvesting complex (LHCII) for photosystem II (PSII) and which give rise to the LHCII oligomer/monomer (CPII^*/CPII) bands on mildly dissociated green gels; (b) a sizable decrease in the proportion of 11 to 13 nanometer particles of the protoplasmic fracture face of unstacked membrane regions (PFu) that parallels the loss of light harvesting complex I (LHCI) antennae from photosystem I (PSI) centers and a 40% reduction of the band containing CP1 and LHCI (CPI^*) on mildly dissociating green gels; (c) an unchanged or slightly increased average size of particles of the exoplasmic fracture face of stacked (or appressed) membrane regions (EFs) along with a relative increase in CP29, the postulated bound LHC of PSII, and of CP47 and CP43, PSII core antenna complexes. This latter result sets the OY-YG mutant apart from all other Chl b-deficient mutants studied to date, all of which possess EFs particles that are substantially reduced in size. Based on these findings, we postulate that the bound LHCII associated with EFs particles consists mostly of CP29 chlorophyll proteins and very little, if any, CPII^*/CPII chlorophyll proteins. Indeed, the CPII^*/CPII chlorophyll proteins may be exclusively associated with the `peripheral' LHCII units that give rise to 8 nanometer PF particles. The differential effect of the Chl b deficiency on the accumulation of the three main antenna complexes (CPII^*/CPII>CPI^*>CP29) suggests, furthermore, that there is a hierarchy among Chl b-binding proteins, and that this hierarchy might be an integral part of long-term photoregulation mediating Chl b partitioning in the chloroplast.     

Changes of Freeze-Fracture Ultrastructure and Light Harvesting Chlorophyll Protein Complex in Thylakoid Membranes During Ontogeny of Maize

Freeze-fracture electron microscopy enables us to observe and count the freeze-fracture particles which correspond to the different functional components of thylakoid membranes. The present paper reports the observation on freeze-fracture ultrastructure of thylakoid membranes and the analysis of proteins by the SDS-polyacrylamide gel electrophoresis within the membranes from differenly located leaves of maize. In the past, we found that the leaies subtending the ear of maize had a much higher chlorophyll content, a lower chlorophyll a/b ratio and more staking thylakoid membranes and provided the photosynthetic energy used to fill the maize seeds more than that of other leaves. Recently, we have further found that the particle densities of all four faces of thylakoid membranes from the ear leaf were the highest, than those, successively, from the terminal leaf, and the fifth leaf (from the base of the plant). The particle densities on all four fracture faces of thylakoid membranes isolated from the ear leaves of maize were significantly higher than those from the terminal leaves with the increases of 19% in EFs, 28% in PFs and 20% in PFu. Increases in particle densities on the PFs, EFs and PFu faces result in increased densities of LHCP II, PSⅡ and PSI reactions centres, respectively. It is significant that this supramolecular architecture of the ear leaves is consistent with our analytical results of the SDS-polyacrylamide gel electrophoresis within the membranes (a detailed report in another paper). The contents of major polypeptides of 21 kD (LHCP Ⅰ) and 25 kD (LHCP Ⅱ) in thylakoid membranes from the ear leaves were more than those from the terminal leaves. The characteristics of both supramolecular architecture and polypeptide components are in favour of absorbing, transferring, distributing and conversing light energy in the course of photosynthesis of the ear leaves in maize.     

Spatial relationship of photosystem I, photosystem II, and the light- harvesting complex in chloroplast membranes

We have previously demonstrated (Armond, P. A., C. J. Arntzen, J.-M. Briantais, and C. Vernotte. 1976. Arch. Biochem. Biophys. 175:54-63; and Davis, D. J., P. A. Armond, E. L. Gross, and C. J. Arntzen. 1976. Arch. Biochem. Biophys. 175:64-70) that pea seedlings which were exposed to intermittent illumination contained incompletely developed chloroplasts. These plastids were photosynthetically competent, but did not contain grana. We now demonstrate that the incompletely developed plastids have a smaller photosynthetic unit size; this is primarily due to the absence of a major light-harvesting pigment-protein complex which is present in the mature membranes. Upon exposure of intermittent- light seedlings to continuous white light for periods up to 48 h, a ligh-harvesting chlorophyll-protein complex was inserted into the chloroplast membrane with a concomitant appearance of grana stacks and an increase in photosynthetic unit size. Plastid membranes from plants grown under intermediate light were examined by freeze-fracture electron microscopy. The membrane particles on both the outer (PF) and inner (EF) leaflets of the thylakoid membrane were found to be randomly distributed. The particle density of the PF fracture face was approx. four times that of the EF fracture face. While only small changes in particle density were observed during the greening process under continuous light, major changes in particle size were noted, particularly in the EF particles of stacked regions (EFs) of the chloroplast membrane. Both the changes in particle size and an observed aggregation of the EF particles into the newly stacked regions of the membrane were correlated with the insertion of light-harvesting pigment- protein into the membrane. Evidence is presented for identification of the EF particles as the morphological equivalent of a "complete" photosystem II complex, consisting of a phosochemically active "core" complex surrounded by discrete aggregates of the light-harvesting pigment protein. A model demonstrating the spatial relationships of photosystem I, photosystem II, and the light-harvesting complex in the chloroplast membrane is presented.     

Freeze-etched membrane faces and photosynthetic activity in normal and mutantTradescantia chloroplasts

Summary Membrane structure and photosynthetic activity was investigated in normal and mutant plastids ofTradescantia albiflora cv.aureo-vittata. In the stacked membrane regions (the macrograna) of mutant plastids, the B fracture faces lack both 170 Å particles and photosystem II (PS II) activity. The C face has the normal 110 Å particles, and photosystem I (PS I) activity is also similar to that in normal chloroplasts. In dilated macrograna the particle size on the C face significantly decreases, and as progressive plastid destruction occurs so PS I activity also disappears. It has been concluded that the integrity of B face particles is related to PS II activity, rather than for membrane stacking. A similar correlation seems to be valid for C face particles and PS I activity.     

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     

A COMPARISON OF CHLOROPLAST MEMBRANE SURFACES VISUALIZED BY FREEZE-ETCH AND NEGATIVE STAINING TECHNIQUES; AND ULTRASTRUCTURAL CHARACTERIZATION OF MEMBRANE FRACTIONS OBTAINED FROM DIGITONIN-TREATED SPINACH CHLOROPLASTS

Spinach chloroplast lamellae were washed free of negatively staining surface particles (carboxydismutase and coupling factor protein) and the resulting smooth-surfaced lamellae still showed the usual large (175 A) and small (110 A) particles seen by freeze-etching. Therefore, the freeze-fracture plane probably occurs along an internal surface of the chloroplast membrane. Fractions obtained by differential centrifugation of digitonin-treated chloroplast membranes were studied by negative staining, thin sectioning, and freeze-etching techniques for electron microscopy. The material sedimenting between 1,000 g and 10,000 g, enriched in photosystem II activity, was shown to consist of membrane fragments. These freeze-etched membrane fragments were found to have large particles on most of the exposed fracture faces. The large particles had the same size and distribution pattern as the 175 A particles seen in intact chloroplast membranes. The material sedimenting between 50,000 g and 144,000 g, which had only photosystem I activity, was found to consist of particles in various degrees of aggregation. Freeze-etching of this fraction revealed only small particles corresponding to the 110 A particles seen in intact chloroplasts. A model is presented suggesting that chloroplast lamellar membranes have a binary structure, which digitonin splits into two components. The two membrane fragments have different structures, revealed by freeze-etching, and different photochemical and biochemical functions.     

Alteration of thylakoid membrane structure in Brassica rapa ssp. oleifera during ageing in high and low light

Abstract Alterations in the composition and structure of thylakoids were studied in Brassica rapa ssp. oleifera grown under high and low irradiance (800 μmol m^?2 s^?1 and 80 μmol m^?2 s^?1). During ageing, both high and low light induced a decrease in total protein particle density and in the relative amount of 80–90 Å cytochrome b6/f and 90–100 Å ATP-synthetase. The density of PSII complexes in stacked (EFs) and unstacked (EFu) thylakoids also decreased. In high light, a shift was noted towards smaller PSII complexes in the EFs face with decreasing attached antenna complex CP29, but the relative amount of the antenna chlorophyll a-protein complexes of photosystem II (CPa) remained stable. In contrast, the proportion of peripheral LHCH on the PFs face and the density of PFs particles increased together with an increase in grana size. In low light, a shift occurred towards larger PSII complexes on the EFs face, along with a decrease in the proportion of CPa complexes and the PFs particle density (peripheral LHCH), though a marked increase was observed in the proportion of chlorophyll a/b-protein complexes in SDS-PAGE. The amount of photosystem I in green gel remained fairly stable, although the density of PFu particles (including PSI) increased in low and slightly diminished in high light. The results indicate that the organization of thylakoid components depends strongly on the light conditions and stage of development.     

Freeze-fracture studies on barley plastid membranes. VII. Structural changes associated with phosphorylation of the light-harvesting complex

Chloroplast thylakoid membranes were isolated from barley at room temperature under redox conditions which ensured that the light-harvesting complex was either non-phosphorylated or phosphorylated. The ultrastructural appearance of these membranes was characterised by rotary shadowed, freeze-fracture electron microscopy. Upon phosphorylation, there was a slight (5%) decrease in the extent of thylakoid stacking, as evidenced by an increase in EFu face particle density. It was concluded from detailed measurements of particle density and size distribution that phosphorylation of the light-harvesting complex results in the movement of some of the Photosystem II EFs particles and some of the PFs particles containing the light-harvesting complex from grana to stroma membranes. There was also a slight increase in PFs particle size and the appearance of a population of large particles on this face, which may be due to conformational changes in the light-harvesting complex or to the movement of some Photosystem I particles from stroma to grana membranes.     

Studies on the cytochrome b6/f complex. II. Localization of the complex in the thylakoid membranes from spinach and Chlamydomonas reinhardtii by immunocytochemistry and freeze-fracture analysis of b6/f mutants

The distribution of the b₆/f complex among stacked and unstacked thylakoid membranes was studied by immunocytochemistry and freeze-fracture analysis of mutants of Chlamydomonas reinhardtii lacking the complex. Immunogold labeling demonstrates the presence of b₆/f complex in both regions of the thylakoid membrane in spinach and in C. reinhardtii. Numerous modifications were observed in the ultrastructure of the thylakoid membranes of mutants from C. reinhardtii lacking the complex. These modifications are consistent with the presence of b₆/f complexes in different states of association in the stacked and unstacked regions of the thylakoid membrane. In particular we present evidence for an association of some b₆/f complexes with the reaction centers of Photosystem I and II in large PFu and EFs particles, respectively.     

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.     

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     

The effects of galactolipid depletion on the structure of a photosynthetic membrane

The galactolipids monogalactosyldiglyceride and digalactosyldiglyceride together comprise more than 77% of the photosynthetic membrane lipids of higher plant chloroplasts. We have isolated a lipase from the chloroplasts of runner beans (Phaseolus vulgaris) which is highly specific for these galactolipids. This galactolipase promotes the hydrolysis of monogalactosyldiglyceride and digalactosyldiglyceride, in the process liberating two free fatty acids into the membrane bilayer, leaving the residual galactosyl glyceride group to diffuse into the aqueous bulk phase. Isolated spinach photosynthetic membranes were treated with this enzyme preparation and changes in membrane composition were studied with thin layer chromatography (for lipids), gel electrophoresis (proteins), and freeze-etching (membrane structure). After 30 min of lipolysis, nearly 100% of the galactolipids had been converted into membrane-associated fatty acids and water-soluble galactosyl glycerides. SDS PAGE showed that two proteins, one of which is possibly associated with the reaction center of photosystem II, were removed by the treatment. Despite the minor nature of changes in membrane protein composition, freeze-fracture and freeze-etch studies showed that striking changes in membrane structure had taken place. The large freeze-fracture particle on the E fracture face had disappeared in stacked regions of the membrane system. In addition, a tetrameric particle visible at the inner surface of the membrane had apparently dissociated into individual monomeric particles. The fact that these two structures are so dramatically affected by the loss of galactolipids strongly suggests that these lipids play a crucial role in maintaining their structure. Both structures are believed to be different views of the same transmembrane unit: a membrane-spanning complex associated with photosystem II. Our results are consistent with two possible interpretations: the intramembrane particles may be lipidic in nature, and hence lipolysis causes their disappearance; or galactolipids are necessary for the organization of a complex photosystem II-associated structure which is composed of a number of different molecular species.     

Structural analysis of photosystem II in far-red-light-adapted thylakoid membranes. New crystal forms provide evidence for a dynamic reorganization of light-harvesting antennae subunits.

We studied two-dimensional crystals of the major pigment-protein complex, photosystem II, in far-red-light-adapted thylakoid membranes of the viridis-zb63 mutant of barley. Significantly larger grana membranes were produced with an increased synthesis of the entire photosystem II complex. These red-light-adapted membranes also contained two-dimensional crystals with a high frequency. Three different crystal forms of photosystem II were observed, providing the following data which further our understanding of the architecture of the native complex. (a) The oligomeric form of photosystem II in the membrane was monomeric in all crystal forms, but with a clear non-crystallographic pseudo-twofold symmetry. This was more apparent on the lumenal face of the complex. (b) The variability of unit cell contacts in different crystal forms implied that the peripheral light-harvesting antenna complex and the core of the complex were loosely connected. These peripheral subunits were predicted to rearrange so that they can either encircle the core complex or associate in parallel channels separated by lines of core complexes. (c) Grana membranes were found to retain a double-layered inside-out character, with a stromal face-to-stromal face packing. However, the presence of a crystal in one membrane did not necessarily impose crystallinity on its pair.     

A freeze-fracture and concanavalin A-binding study of the membrane of cleaving Xenopus embryos.

The freeze-fracture appearance and concanavalin A-binding capacity of the plasma membrane of cells of the cleaving Xenopus embryo have been examined up to the 16-cell stage. It was found that membrane on the outer surface of the embryo, which faces the vitelline membrane and is remote from cleavage furrows, and membrane in the shallow regions of the furrow possessed a high population of intramembranous particles on the PF-face (1171 per mum2). The EF-face of these membranes showed a lower particle population (245 per mum2). By contrast, membrane deep in the furrow and bounding the blastocoel did not display a face with high particle numbers. Both faces of this membrane, which is newly exposed as the furrow grows, were relatively poorly supplied with particles (93 per mum2). Therefore it appears that, in this tissue, newly added membrane possesses fewer intramembranous particles than the pre-existing membrane. Concanavalin A, as detected cytochemically using peroxidase and haemocyanin techniques, bound extensively to both particle-rich and particle-poor membrane. Thus there was no correlation between intramembranous particle frequency and degree of concanavalin A binding.     

Biogenesis of chloroplast membranes: XIV. Inhomogeneity of membrane protein distribution in photosystem particles obtained from Chlamydomonas reinhardi. Y-l

Treatment of chloroplast membranes of Chlamydomonas reinhardi with Triton-× 100 yielded membrane particles which were resolved into three bands on discontinuous sucrose gradients. One of these was enriched in the chlorophyll absorption and fluorescence properties and photosynthetic activities consistent with photosystem I enrichment, while another had the chlorophyll absorption and fluorescence properties expected to photosystem II enriched particles. The third type of particle was enriched in chlorophyll species which are probably the bulk chlorophylls of photosystem I. Analysis of the proteins of these fractions by polyacrylamide electrophoresis indicated substantial differences, the most striking being that the photosystem II particle type was greatly enriched in the major species of chloroplast membrane protein. Previous work has shown this to be an important protein controlling membrane assembly. This protein was depleted in the photosystem I particle type. We interpret this data to indicate a lack of homogeneity in the distribution of membrane proteins in the chloroplast membranes of Chlamydomonas, at the level of the two photosystems.