ATP Synthase of Chloroplast Thylakoid Membranes: A More in Depth Characterization of Its ATPase Activity

In contrast to everted mitochondrial inner membrane vesicles and eubacterial plasma membrane vesicles, the ATPase activity of chloroplast ATP synthase in thylakoid membranes is extremely low. Several treatments of thylakoids that unmask ATPase activity are known. Illumination of thylakoids that contain reduced ATP synthase (reduced thylakoids) promotes the hydrolysis of ATP in the dark. Incubation of thylakoids with trypsin can also elicit higher rates of ATPase activity. In this paper the properties of the ATPase activity of the ATP synthase in thylakoids treated with trypsin are compared with those of the ATPase activity in reduced thylakoids. The trypsin-treated membranes have significant ATPase activity in the presence of Ca²⁺, whereas the Ca²⁺-ATPase activity of reduced thylakoids is very low. The Mg²⁺-ATPase activity of the trypsinized thylakoids was only partially inhibited by the uncouplers, at concentrations that fully inhibit the ATPase activity of reduced membranes. Incubation of reduced thylakoids with ADP in Tris buffer prior to assay abolishes Mg²⁺-ATPase activity. The Mg²⁺-ATPase activity of trypsin-treated thylakoids was unaffected by incubation with ADP. Trypsin-treated membranes can make ATP at rates that are 75–80% of those of untreated thylakoids. The Mg²⁺-ATPase activity of trypsin-treated thylakoids is coupled to inward proton translocation and 10 mM sulfite stimulates both proton uptake and ATP hydrolysis. It is concluded that cleavage of the γ subunit of the ATP synthase by trypsin prevents inhibition of ATPase activity by the ε subunit, but only partially overcomes inhibition by Mg²⁺ and ADP during assay.     

Immunogold localization of photosystem I and photosystem II light-harvesting complexes in cryptomonad thylakoids

Summary— The molecular organization of the thylakoids of Cryptomonas rufescens was studied by immunoelectron microscopy employing antibodies against photosystem (PS)-I and two PS-II antenna proteins. The PS-I complex and the 19-kDa chlorophyll a/c light-harvesting (LH) protein are both localized along the length of the thylakoid membranes. The external membranes of the paired thylakoids are enriched in PS-I whereas the chlorophyll a/c LH protein is more concentrated in the internal or appressed membranes. However, unlike the situation in higher plants and Chlamydomonas, there is not a marked asymmetry in the concentration of PS-I and chorophyll a/c LH protein in the two types of membranes. Double labelling studies of sections and isolated PE-PS-II particles with anti-phycoerythrin and anti-LH confirmed that phycoerythrin is localized in the thylakoid lumen and that this pigment exists in two forms, a fraction closely associated with the thylakoid membranes and another fraction free in the lumen. These results confirm the uniqueness of cryptomonad thylakoids.     

IMMUNOLOGICAL AND ULTRASTRUCTURAL CHARACTERIZATION OF THE PHOTOSYNTHETIC COMPLEXES OF THE PROCHLOROPHYTE PROCHLOROCOCCUS (OXYCHLOROBACTERIA)

Ultrastructural features and immunological properties of some thylakoid proteins were examined in two strains of the prochlorophyte Prochlorococcus and compared to those of other photosynthetic prokaryotes and eukaryotes. Both strains exhibited two or three rows of tightly appressed thylakoidal membranes, located at the cell periphery. However, thylakoids were concentrically arranged in the strain from the Sargasso Sea (SARG) and horseshoe-shaped in the Mediterranean isolate (CCMP 1378). Although lacking phycobilisomes, both cell types shared with cyanobacteria the presence of carboxysome-like structures and glycogen granules as storage compounds. The main thylakoid polypeptides separated by sodium dodecyl sulfate—polyacrylamide gel electrophoresis were characterized by Western blotting using several antibodies. The 30-kDa polypeptide of the light-harvesting complex (LHC) of Prochlorococcus showed a weak positive immunological cross-reaction with an antibody raised against the 32-kDa apoprotein of the LHC of the prochlorophyte Prochlorothrix hollandica. In contrast, it showed no immunological relationships with the chlorophyll a/b (Chl a/b) LHCs of green algae and higher plants. Protein membranes from Prochlorococcus strongly cross-reacted with antibodies raised against reaction center polypeptides of photosystems II and I (PSs II and I) of other photosynthetic organisms, confirming the high degree of conservation of these basic compounds of the photosynthetic machinery during evolution. Immunolocalization of thylakoid proteins showed that the LHC proteins, the major PS II reaction center proteins (CP 43 and D2), and the PS I reaction center proteins were equally distributed within the thylakoid membranes in contrast to the segregation observed in higher plants and green alga thylakoids. We also identified ribulose-1, 5-bisphosphate carboxylase/oxygenase in the carboxysomes. These results suggest that Prochlorococcus is more closely related to cyanobacteria than to green plastids even though it contains Chl b.     

COMPARATIVE ULTRASTRUCTURE OF CULTURED SPECIES OF TREBOUXIA1,2

Five species of cultured Trebouxia—T. anticipata, T. decolorans, T. erici, T. gelatinosa, and T. impressa—were examined with the electron microscope. A comparative examination of their pyrenoids revealed pyrenoglobuli associated with single pyrenoid thylakoids. The pyrenoids of T. decolorans, T. erici, and T. gelatinosa possess single thylakoids that cross or deeply penetrate the pyrenoid matrix and are often disposed in parallel arrays. T. anticipata possesses both single and double pyrenoid thylakoids within the matrix. T. impressa possesses vesiculate invaginations of thylakoid membranes into the pyrenoid matrix. The phycobiont. T. erici was examined in detail at the light and electron microscopic levels for pyrenoid alterations associated, with varied environmental regimes and with cell division. A greater amount of starch is present in cells grown in organic culture at 215 lux light intensity than in cells of similar size grown at 1075 or 3600 lux. Pyrenoglobuli are present throughout the life cycle and occur both in aplanospores and in zoospores.     

ULTRASTRUCTURE OF THE RED TIDE DINOFLAGELLATE GYMNODINIUM BREVE. I. GENERAL DESCRIPTION1,2,3

Gymnodimium breve Davis, an unarmored marine dinoflagellate has a cell covering (theca) composed of four membranes. The inner two membranes represent a vesicular layer and in tangential section, the theca appears composed of polygonal areas. Unusual threat ridges are located in the cingular region between the epi- and hypocone. This osmotically sensitive species is extremely vesiculate with dispersed areas of cytoplasm containing typical eukaryotic organelles as well as other organelles found only in dinoflagellates. The non-vesiculated cytoplasm is continuous in serial sections. The chloroplasts can contain either quasi-radial or parallel lamellae typically consisting of three thylakoids each. The pyrenoid is multiple-stalked and lacks a starch cap. The dinophycean pusule is simple and similar to those found in several unarmored marine species. The nucleus is typically dinophycean but the chromosomes appear to lack nonfibrillar material.     

Immunocytochemical localization of photosystem I and the fucoxanthin-chlorophylla/c light-harvesting complex in the diatomPhaeodactylum tricornutum

Summary iserum against two polypeptides of the major fucoxanthin-chlorophylla/c light-harvesting complex of the diatomPhaeodactylum tricornutum and heterologous antiserum against purified photosystem I particles of maize were used to localize these two complexes on the thylakoid membranes ofP. tricornutum. As in many chromophyte algae, the thylakoids are loosely appressed and organized into extended bands of three, giving a ratio of 21 for appressed versus non-appressed membranes. Immunoelectron microscopy demonstrated that the fucoxanthin-chlorophylla/c light-harvesting complex, which is believed to be associated with photosystem II, was equally distributed on the appressed and non-appressed thylakoid membranes. Photosystem I was also found on both types of membranes, but was slightly more concentrated on the two outer non-appressed membranes of each band. Similarly, photosystem I activity, as measured by the photooxidation of 3,3-diaminobenzidine, was higher in the outer thylakoids than in the central thylakoid of each band. We conclude that the thylakoids of diatoms differ from those of green algae and higher plants in their macromolecular organization as well as in their morphological arrangement.Abbreviations BSA bovine serum albumin - DAB 3,3-diaminobenzidine - FCPC fucoxanthin-chlorophylla/c light-harvesting complex - LHC light-harvesting complex - PBS phosphate-buffered saline - PS photosystem     

Immunogold localization of light-harvesting and photosystem I complexes in the thylakoids ofFucus serratus (Phaeophyceae)

Summary The repartition of light-harvesting complex (LHC) and photosystem I (PS I) complex has been examined in isolated plastids ofFucus serratus by immunocytochemical labelling. LHC is distributed equally all along the length of thylakoid membranes, without any special repartition in the appressed membranes of the three associated thylakoids ofFucus. PS I is present on all the thylakoid membranes, but the external membranes of the three associated thylakoids are largely enriched relatively to the inner ones. This specific repartition of PSI on non-appressed membranes can be compared to the localization of PSI on stroma thylakoid membranes of higher plants and green algae. Consequently, although they share some common features with those of higher plants and green algae, the appressions of thylakoids in brown algae has neither the same structure nor the same functional role as typical grana stacked membranes in the repartition of the harvested energy.Abbreviations BSA bovine serum albumin - GAR goat anti-rabbit immunoglobulin G - LHC light-harvesting complex - PBS phosphatebuffered saline - PS I photosystem I - PS II photosystem II     

The effects of cations and trypsin on extraction of chlorophyll-protein complexes by octyl glucoside

The extraction of chlorophyll-protein (CP) complexes from thylakoids by the detergent octyl glucoside is strongly affected by pretreatment of the thylakoids with trypsin or cations. In these experiments, washed thylakoids were incubated in the presence of 0.5 μm to 5 mm Mg²⁺, pelleted, and extracted with octyl glucoside (30 mm). Increasing amounts of Mg²⁺ depressed extractability of all CP complexes, but especially the chlorophyll a + b-containing light-harvesting complex (LHC). This cation effect is observed with other cations which promote thylakoid stacking (5 mm Mn²⁺ or Ca²⁺, 50 mm Na⁺). However, the effect is not merely due to stacking, since low concentrations of Mg²⁺ (0.5 μmto 0.5 mm) have a marked effect on extractability but have no effect on light scattering (OD 550 nm), an indicator of stacking. Furthermore, trypsin treatment of thylakoids stacked with 5 mm Mg²⁺ caused a significant reversal of stacking, but had little effect on extractability. Trypsin treatment of unstacked membranes resulted in increased extractability of all CP complexes, but especially of the LHC. Cation-treated membranes are also significantly different from those “stacked” at pH 4.5. While the latter do show decreased extractability, there is no change in the chlorophyll $\text{a}\text{b}$ ratio of the extract, and the membranes cannot be “unstacked” with trypsin. We conclude that octyl glucoside extractability reflects the lateral interaction of CP complexes with each other and with other components in the same plane of the membrane. It is clear that divalent cations have several effects on thylakoid membranes, not all of which are due to their ability to promote stacking.     

Observations on the ultrastructure of representatives of the genera Hemiselmis and Chroomonas (Cryptophyceae)

Hemiselmis rufescens Parke and three species of the genus Chroomonas have been examined by electron microscopy. They demonstrate certain characteristic features of the Cryptophyceae, such as the presence of trichocysts and the arrangement of thylakoids in pairs in the plastid. The prominent pyrenoid of the genus Chroomonas is penetrated longitudinally by a tongue of cytoplasmic matrix which originates from between the two pairs of plastid membranes. In the genus Hemiselmis, however, the pyrenoid is traversed by a pair of thylakoids. The nucleus, Golgi apparatus and Corps de Maupas also occupy characteristic positions. These features indicate a close relationship between these two genera and the other major genus Cryptomonas, and support the suggestion that the Cryptophyceae is a discrete taxonomic group.     

Desiccation‐induced alterations in surface topography of thylakoids from resurrection plant Haberlea rhodopensis studied by atomic force microscopy,electrokinetic and optical measurements

With their ability to survive complete desiccation, resurrection plants are a suitable model system for studying the mechanisms of drought tolerance. In the present study, we investigated desiccation‐induced alterations in surface topography of thylakoids isolated from well‐hydrated, moderately dehydrated, severely desiccated and rehydrated Haberlea rhodopensis plants by means of atomic force microscopy (AFM), electrokinetic and optical measurements. According to our knowledge, so far, there were no reports on the characterization of surface topography and polydispersity of thylakoid membranes from resurrection plants using AFM and dynamic light scattering. To study the physicochemical properties of thylakoids from well‐hydrated H. rhodopensis plants, we used spinach thylakoids for comparison as a classical model from higher plants. The thylakoids from well‐hydrated H. rhodopensis had a grainy surface, significantly different from the well‐structured spinach thylakoids with distinct grana and lamella, they had twice smaller cross‐sectional area and were 1.5 times less voluminous than that of spinach. Significant differences in their physicochemical properties were observed. The dehydration and subsequent rehydration of plants affected the size, shape, morphology, roughness and therefore the structure of the studied thylakoids. Drought resulted in significant enhancement of negative charges on the outer surface of thylakoid membranes which correlated with the increased roughness of thylakoid surface. This enhancement in surface charge density could be due to the partial unstacking of thylakoids exposing more negatively charged groups from protein complexes on the membrane surface that prevent from possible aggregation upon drought stress.     

ULTRASTRUCTURAL CHANGES IN SUNFLOWER CHLOROPLASTS FOLLOWING INOCULATION WITH PSEUDOMONAS SYRINGAE PV. TAGETIS

Severe chlorosis and ultrastructural modifications of chloroplasts occur in sunflower in response to infection by Pseudomonas syringae pv. tagetis. Chlorosis became apparent within 2 days after the cotyledons of 10-day-old sunflower seedlings were inoculated with the bacteria. The first symptoms generally appeared in the center of leaves at the second node above the cotyledons. Leaves above the second node lost essentially all of their pigmentation but remained turgid and continued to expand. Grana thylakoids became dilated and separated from the granal stacks. These thylakoid membranes did not chemically breakdown as in the case in chromoplast formation or normal chloroplast senescence. Both grana and stroma thylakoid membranes coalesced to form a large membrane sheet within the plastid. The ultrastructural changes are unlike those reported to be caused by other chlorosis-inducing bacteria or chlorosis associated with normal senescence.     

Insights into the complex 3-D architecture of thylakoid membranes in the unicellular cyanobacterium Cyanothece sp. ATCC 51142

In cyanobacteria and chloroplasts, thylakoids are the complex internal membrane system where the light reactions of oxygenic photosynthesis occur. In plant chloroplasts, thylakoids are differentiated into a highly interconnected system of stacked grana and unstacked stroma membranes. In contrast, in cyanobacteria, the evolutionary progenitors of chloroplasts, thylakoids do not routinely form stacked and unstacked regions, and the architecture of the thylakoid membrane systems is only now being described in detail in these organisms. We used electron tomography to examine the thylakoid membrane systems in one cyanobacterium, Cyanothece sp. ATCC 51142. Our data showed that thylakoids form a complicated branched network with a rudimentary quasi-helical architecture in this organism. A well accepted helical model of grana-stroma architecture of plant thylakoids describes an organization in which stroma thylakoids wind around stacked granum in right-handed spirals. Here we present data showing that the simplified helical architecture in Cyanothece 51142 is lefthanded in nature. We propose a model comparing the thylakoid membranes in plants and this cyanobacterium in which the system in Cyanothece 51142 is composed of non-stacked membranes linked by fret-like connections to other membrane components of the system in a limited left-handed arrangement.Key words: cyanobacteria, Cyanothece 51142, thylakoid membrane, electron tomography, chloroplast     

Intracellular localization of glutamine synthetase in Chlorogloeopsis fritschii

After differential centrifugation of cell-free extracts of Chlorogloeopsis fritschii, 71% of the original glutamine synthetase (GS) activity was associated with the thylakoids, while little activity was detected in the cytoplasmic membranes. Monospecific antiserum to a purified GS inhibited 88% of the enzyme activity in solubilized thylakoid membranes. An antiserum raised against thylakoids gave 81% inhibition. However, using intact thylakoid membranes, only 7% inhibition was obtained with the GS antiserum, indicating that GS is located inside the thylakoid membranes.The author is with the Department of Biological Sciences, University of Science and Technology, Irbid, Jordan     

LIPID MODIFICATIONS RELATED TO ENCYSTMENT AND EXCYSTMENT OF CRYPTOMONAS RUFESCENS SKUJA (CRYPTOPHYCEAE)1

The lipids of Cryptomonas rufescens (Skuja) cells have been analyzed. Quantitative changes of polar and neutral lipids were observed during cell encystment, induced by cultures in a nitrogen-deficient medium. During encystment, thylakoids disappeared while unsaturated galactolipids, characteristics of chloroplast membranes, decreased and neutral lipids accumulated in the cytoplasm. When excystment was induced, the reversal of the phenomenon was observed while thylakoids containing galactolipids were formed.     

Are the photosynthetic membranes of cryptophyte algae inside out?

Summary Cryptomonads are unicellular algae with a unique photosynthetic apparatus, both in structure and pigment composition. A cryptomonad,Rhodomonas lens (R. lens), was studied by conventional electron microscopy, freeze-fracture, and freeze-etch in order to determine whether the thylakoids of this alga are everted with respect to those of other plants, as has been postulated (Gantt et al. 1971,Gantt 1979, 1980) as a means to compensate for the location of the cryptomonad light-harvesting apparatus on the opposite side of the thylakoid membrane from that of related algae. We have characterized the thylakoids of this alga and conclude that they are not everted, but are oriented in the same manner as those of other algae and green plants. Implications for energy transfer are discussed.     

Characteristics of Photosystem I Complexes in the End Membranes of Pea Granal Thylakoids

Two fractions of the light fragments enriched in the photosystem I (PSI) complexes were obtained from pea (Pisum sativum L.) thylakoids by digitonin treatment and subsequent differential centrifugation. The ratio of chlorophyll a to chlorophyll b, chlorophyll/P700 spectra of low-temperature fluorescence, and excitation spectra of long-wave fluorescence were measured. These characteristics were shown to be different due to variation in the size and composition of the light-harvesting antenna of PSI complexes present in the particles obtained. The larger antenna size of one of the fractions was related to the incorporation of the pool of light-harvesting complex II (LHCII). A comparison with the data available allowed us to identify these particles as fragments of intergranal thylakoids and end membranes of granal thylakoids. The suggestion that an increase in the PSI light-harvesting antenna in intergranal thylakoids is related to the attachment of phosphorylated LHCII is discussed.     

Growth and development of winter rye at cold-hardening temperatures results in thylakoid membranes with increased sensitivity to low concentrations of osmoticum

Chloroplasts developed at cold-hardening (5°C) and non-hardening temperatures (20°C) were compared with respect to the stability of photosynthetic electron transport activities, the capacity to produce and maintain a H⁺ gradient and the capacity fat photophosphorylation as a function of resuspension in the presence or absence of osmoticum. The results for electron transport indicate that whole chain, photosystem I and pfaotosystem II activities in non-hardened chloroplast thyalkoids were unaffected by resuspension in the presence of high or low osmoticum. In contrast, the same electron transport activities in cold-hardened chloroplast thylakoids exhibited a 3- to 4-fold decrease in activity when resuspended in the presence of low osmoticum. Impairment of electron transport through photosystem II of cold-hardened thylakoids resuspended in the presence of low osmoticum was supported by room temperature fluorescence induction kinetics. Since the presence of Mn²⁺ partially overcame this inhibition, it is concluded that this osmotically-induced inhibition of PSII activity in cold-hardened chloroplast thylakoids may, in part, be due to damage to the H₂O-splitting side of photosystem II. Both the initial rate and the maximum capacity for cyclic photophosphorylation were significantly inhibited in cold-hardened as compared to non-hardened thylakoids upon resuspension in the presence of low concentrations of osmoticum. This was correlated with an inability of the cold-hardened chloroplast thylakoids to maintain a significant transrnembrane H⁺ gradient. The results indicate that cold-hardened thylakoid membranes required an osmotic concentration (0.8 M) twice as high as non-hardened thylakoids (0.4 M) to produce the same initial rate of H⁺ uptake. In addition, the capacity to produce a proton gradient in cold-hardened thylakoids was less stable than that in non-hardened thylakoids regardless of the osmotic concentration tested. It is concluded that development of rye thylakoid membranes at low temperature results in a differential sensitivity to low osmoticum and thus extreme caution should be exercised when comparing the structure and function of isolated thylakoids developed under contrasting thermal regimes.     

SPECIES-SPECIFIC DIFFERENCES OF PYRENOIDS IN CHLORELLA (CHLOROPHYTA)1

The structure of the pyrenoid supports the separation of Chlorella species into two groups based on cell wall chemistry and suggests evolutionary relationships. Chlorella species with a glucan-type wall exhibit quite diverse pyrenoid structures, which may indicate that these species are not closely related. Those species with glucosamine cell walls (C. kessleri, C. sorokiniana, C. vulgaris) are virtually identical in pyrenoid morphology, indicating a closer evolutionary relationship. In the species with glucosamine walls, the thylakoid that penetrates into the pyrenoid matrix, is unijormly double-layered. Pyrenoids in the species with glucan walls show various features: 1) a pyrenoid matrix only, 2) a pyrenoid traversed by a few discs of double thylakoids with many adhering pyrenoglobuli, 3) a pyrenoid penetrated with tubelike structures or 4) a pyrenoid penetrated with many single undulating thylakoids. The pyrenoid structure of the symbiotic Chlorella in Paramecium bursaria resembles those of free-living Chlorella with glucosamine walls.     

The fluidity of chloroplast thylakoid membranes and their constituent lipids: A comparative study by ESR

Comparative measurements were made of the fluidity of chloroplast thylakoids, total membrane lipids and polar lipids utilizing the order parameter and motion of spin labels.No significant differences were found in the fluidity of membranes or total membrane lipids from a wild type and a mutant barley (Hordeum vulgare chlorina f₂ mutant) which lacks chlorophyll $\text{b}$ and a 25 000 dalton thylakoid polypeptide. Redistribution of intrinsic, exoplasmic face (EF) membrane particles by unstacking thylakoid membranes in low salt medium also had no effect on membrane fluidity. However, heating of isolated thylakoids decreased membrane fluidity.The fluidity of vesicles composed of membrane lipids is much greater than that of the corresponding membranes. Fluidity of the membranes, however, increased during greening indicating that the rigidity of the membranes, compared with that of total membrane lipids, is not caused by chlorophyll or its associated peptides. It is concluded that the restriction of motion in the acyl chains in the thylakoids is not caused by chlorophyll or the major intrinsic polypeptide but by some other protein components.     

A chlorophyll b-less mutant of Chlamydomonas reinhardii lacking in the light-harvesting chlorophyll complex but not in its apoproteins

The mutant pg 113, derived from Chlamydomonas reinhardii, arg₂⁻ mt⁺ (parent strain), completely lacks chlorophyll (Chl) b but is still able to grow under autotrophic conditions. The light-harvesting Chl complex (LHCP) is absent. This is shown (a) by the lack of the corresponding signal in the CD spectrum of thylakoids and (b) by the absence of the band of the LHCP after electrophoresis of partially solubilized thylakoid membranes on lithium dodecyl sulfate polyacrylamide gels. All the other chlorophyll-protein complexes are present. In spite of the absence of the LHCP, all the polypeptide components of this complex are present in the mutant in the same ratios as in the parent strain, although in slightly reduced amounts. The LHC apoproteins are synthesized, processed and transported into the thylakoid membrane of the mutant. Moreover, the phosphorylation of thylakoid membrane polypeptides, which is related to the regulation of the energy distribution between Photosystem I and II, is the same in the mutant and in the parent strain, indicating that phosphorylation is not dependent on the presence of Chl b. Electron micrographs of thin sections of whole cells show that there are stacked regions of thylakoids in both the mutant and the parent strain chloroplasts. However, in the mutant, stacks are located near the chloroplast envelope, while long stretches or sometimes circles of unstacked membranes are found in the interior, mostly around the pyrenoid.