Three-dimensional ultrastructure of a unicellular cyanobacterium

The first complete three-dimensional ultrastructural reconstruction of a cyanobacterium was accomplished with high-voltage electron microscopy and computer-aided assembly of serial sections. The precise arrangement of subcellular features within the cell body was very consistent from one cell to another. Specialized inclusion bodies always occupied specific intracellular locations. The photosynthetic thylakoid membranes entirely surrounded the central portion of the cytoplasm, thereby compartmentalizing it from the rest of the cell. The thylakoid membranes formed an interconnecting network of concentric shells, merging only at the inner surface of the cytoplasmic membrane. The thylakoids were in contact with the cytoplasmic membrane at several locations, apparently to maintain the overall configuration of the thylakoid system. These results clarified several unresolved issues regarding structure-function relationships in cyanobacteria.     

Unique thylakoid membrane architecture of a unicellular N2-fixing cyanobacterium revealed by electron tomography

Cyanobacteria, descendants of the endosymbiont that gave rise to modern-day chloroplasts, are vital contributors to global biological energy conversion processes. A thorough understanding of the physiology of cyanobacteria requires detailed knowledge of these organisms at the level of cellular architecture and organization. In these prokaryotes, the large membrane protein complexes of the photosynthetic and respiratory electron transport chains function in the intracellular thylakoid membranes. Like plants, the architecture of the thylakoid membranes in cyanobacteria has direct impact on cellular bioenergetics, protein transport, and molecular trafficking. However, whole-cell thylakoid organization in cyanobacteria is not well understood. Here we present, by using electron tomography, an in-depth analysis of the architecture of the thylakoid membranes in a unicellular cyanobacterium, Cyanothece sp. ATCC 51142. Based on the results of three-dimensional tomographic reconstructions of near-entire cells, we determined that the thylakoids in Cyanothece 51142 form a dense and complex network that extends throughout the entire cell. This thylakoid membrane network is formed from the branching and splitting of membranes and encloses a single lumenal space. The entire thylakoid network spirals as a peripheral ring of membranes around the cell, an organization that has not previously been described in a cyanobacterium. Within the thylakoid membrane network are areas of quasi-helical arrangement with similarities to the thylakoid membrane system in chloroplasts. This cyanobacterial thylakoid arrangement is an efficient means of packing a large volume of membranes in the cell while optimizing intracellular transport and trafficking.     

Isolation and Characterization of the Thylakoid Membranes from the NaCl-Resistant (NaClr) Mutant Strain of the Cyanobacterium Anabaena variabilis

NaCl-induced changes in the thylakoid membrane of wild-type Anabaena variabilis and its NaCl^r mutant strain have been studied. Biochemical characterization of the thylakoid membrane was done by taking its absorption and fluorescence spectra at different wavelength. The thylakoid membranes of both strains were isolated by mechanical disruption of the freeze-dried and lysozyme-treated cells, followed by differential and density gradient centrifugation. The light absorption spectra of the thylakoid membrane showed three and two peaks in NaCl^r mutant strain and its wild-type counterpart respectively at wavelengths of 400–850 nm. These peaks revealed that the thylakoid membrane contains a large amount of carotenoid and chlorophyll a. Fluorescence emission spectra of thylakoid membrane of NaCl^r mutant and its wild-type strain at excitation wavelength of 335 nm showed two different peaks, one at 340 nm and the other at 663 nm respectively. The light absorption and fluorescence spectra of the thylakoid membrane also revealed that the membrane contained carotenoid pigment, chlorophyll (Chl) a, and a pigment with an emission peak at 335 nm. The HPLC analysis of the pigments of the thylakoid membrane indicates that the NaCl^r mutant strain under NaCl stress contained an additional peak for the carotenoid pigment, which was lacking in its wild-type counterpart. The major peak in thylakoid membrane was that of echinenone and β-carotene. Whereas the polypeptide composition of thylakoid membrane differed in the wild-type and its NaCl^r mutant strain, no difference in the cell wall protein pattern was observed in both strains. The thylakoid membrane of NaCl^r mutant strain contained two additional protein bands that were absent in its wild-type counterpart. The thylakoid membrane of the wild-type and its NaCl^r mutant strain also showed morphological variations under NaCl stress. Received: 14 April 2000 / Accepted: 23 May 2000     

Fine structural observations of the cotyledons in germinating seeds ofPelargonium XHortorum Bailey: With normal and mutant plastids

Summary The fine structural changes in cotyledon cells of germinatingPelargonium seeds are studied on the first to fifth day of seedling emergence. Initially there is a rapid change in the cell fine structure, marked most conspicuously by the progressive liberation of the lipid and protein food reserves and the formation of an extensive thylakoid system within the plastids, but gradually the cells start to senesce. The subcellular changes in mutant cells are similar except that the plastids lack the usual thylakoid system and develop only deranged prolamellar bodies. They may store starch and they possess plastoglobuli, but seem not to contain plastid ribosomes. In rare mixed cells normal and mutant plastids remain quite distinct.     

Three-dimensional organization of higher-plant chloroplast thylakoid membranes revealed by electron tomography

The light-harvesting and energy-transducing functions of the chloroplast are performed within an intricate lamellar system of membranes, called thylakoid membranes, which are differentiated into granum and stroma lamellar domains. Using dual-axis electron microscope tomography, we determined the three-dimensional organization of the chloroplast thylakoid membranes within cryo-immobilized, freeze-substituted lettuce (Lactuca sativa) leaves. We found that the grana are built of repeating units that consist of paired layers formed by bifurcations of stroma lamellar sheets, which fuse within the granum body. These units are rotated relative to each other around the axis of the granum cylinder. One of the layers that makes up the pair bends upwards at its edge and fuses with the layer above it, whereas the other layer bends in the opposite direction and merges with the layer below. As a result, each unit in the granum is directly connected to its neighbors as well as to the surrounding stroma lamellae. This highly connected morphology has important consequences for the formation and function of the thylakoid membranes as well as for their stacking/unstacking response to variations in light conditions.     

Stimulation of photosynthesis and growth of photoautotrophically cultured plant cells by choline and its analogs

The effects of choline and its analogs, allylcholine and benzylcholine, on the photosynthesis and on the cell growth were examined using photoautotrophically, photomixotrophically and heterotrophically cultured cells. The addition of choline and its analogs stimulated the cellular photosynthetic activity and enhanced the dry weight increase in both photoautotrophic and photomixotrophic cells. However, the growth of heterotrophic cells did not increase by the addition of choline and choline analogs. The photosynthetic electron transport activity in thylakoid membrane was enhanced when cells were treated with choline and choline analogs, suggesting that thylakoid membranes are the initial site of the stimulation of cellular photosynthesis. The stimulatory effect of choline and choline analogs was sustained even after 3 week-culture. Among the choline analogs tested, benzylcholine showed the most quick effect and was effective at a lower concentration (1 mg/l) than choline (10 mg/l).Abbreviations GA₃ gibberellin A₃     

Separation and Characterization of Thylakoid and Cell Envelope of the Blue-green Alga (Cyanobacterium) Anacystis nidulans

The thylakoid and the cell envelope of the blue-green alga Anacystisnidulans were separated by mechanical disruption of lysozyme-treatedcells followed by differential and density gradient centrifugation.The prepared envelope was composed of an outer membrane, a peptidoglycanlayer and possibly a part of the cytoplasmic membrane. The preparedthylakoid retained the size and intricate structure typicalof the thylakoid membrane of this alga. Light absorption andfluorescence spectra revealed that the envelope contained carotenoids,a pigment with an absorption maximum at 748 nm (P750), and asmall amount of pheophytin-like pigment with an absorption maximumat 673 nm. The thylakoid contained chlorophyll a and carotenoidsbut no P750. The thylakoid contained five kinds of carotenoids,the major ones being rß-carotene and zeaxanthin, whereasthe cell envelope contained two kinds of carotenoids, zeaxanthinand nostoxanthin. Four kinds of lipids, abundant in the blue-greenalgae, were present in both the thylakoid and the cell envelope.However, the content of sulfolipid was very low in the cellenvelope. The polypeptide compositions differed between thethylakoid and the cell envelope. Similarities between blue-greenalgal cells and eukaryotic chloroplasts are discussed with respectto the spectrophotometric and biochemical characteristics ofthe thylakoid and the envelope. (Received March 7, 1981; Accepted May 22, 1981)     

The three-dimensional structure of the cyanobacterium Synechocystis sp. PCC 6803

To advance our knowledge of the model cyanobacterium Synechocystis sp. PCC 6803 we investigated the three-dimensional organization of the cytoplasm using standard transmission electron microscopy and electron tomography. Electron tomography allows a resolution of ~5 nm in all three dimensions, superior to the resolution of most traditional electron microscopy, which is often limited in part by the thickness of the section (70 nm). The thylakoid membrane pairs formed layered sheets that followed the periphery of the cell and converged at various sites near the cytoplasmic membrane. At some of these sites, the margins of thylakoid membranes associated closely along the external surface of rod-like structures termed thylakoid centers, which sometimes traversed nearly the entire periphery of the cell. The thylakoid membranes surrounded the central cytoplasm that contained inclusions such as ribosomes and carboxysomes. Lipid bodies were dispersed throughout the peripheral cytoplasm and often juxtaposed with cytoplasmic and thylakoid membranes suggesting involvement in thylakoid maintenance or biogenesis. Ribosomes were numerous and mainly located throughout the central cytoplasm with some associated with thylakoid and cytoplasmic membranes. Some ribosomes were attached along internal unit-membrane-like sheets located in the central cytoplasm and appeared to be continuous with existing thylakoid membranes. These results present a detailed analysis of the structure of Synechocystis sp. PCC 6803 using high-resolution bioimaging techniques and will allow future evaluation and comparison with gene-deletion mutants.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Fluorescence staining of live cyanobacterial cells suggest non-stringent chromosome segregation and absence of a connection between cytoplasmic and thylakoid membranes

Background  

In spite of their abundance and importance, little is known about cyanobacterial cell biology and their cell cycle. During each cell cycle, chromosomes must be separated into future daughter cells, i.e. into both cell halves, which in many bacteria is achieved by an active machinery that operates during DNA replication. Many cyanobacteria contain multiple identical copies of the chromosome, but it is unknown how chromosomes are segregated into future daughter cells, and if an active or passive mechanism is operative. In addition to an outer and an inner cell membrane, cyanobacteria contain internal thylakoid membranes that carry the active photosynthetic machinery. It is unclear whether thylakoid membranes are invaginations of the inner cell membrane, or an independent membrane system.     

Dynamical localization of a thylakoid membrane binding protein is required for acquisition of photosynthetic competency

Vipp1 is highly conserved and essential for photosynthesis, but its function is unclear as it does not participate directly in light‐dependent reactions. We analyzed Vipp1 localization in live cyanobacterial cells and show that Vipp1 is highly dynamic, continuously exchanging between a diffuse fraction that is uniformly distributed throughout the cell and a punctate fraction that is concentrated at high curvature regions of the thylakoid located at the cell periphery. Experimentally perturbing the spatial distribution of Vipp1 by relocalizing it to the nucleoid causes a severe growth defect during the transition from non‐photosynthetic (dark) to photosynthetic (light) growth. However, the same perturbation of Vipp1 in dark alone or light alone growth conditions causes no growth or thylakoid morphology defects. We propose that the punctuated dynamics of Vipp1 at the cell periphery in regions of high thylakoid curvature enable acquisition of photosynthetic competency, perhaps by facilitating biogenesis of photosynthetic complexes involved in light‐dependent reactions of photosynthesis.     

Accumulation of plant galactolipid affects cell morphology of Escherichia coli

Monogalactosyldiacylglycerol (MGDG) is a major constituent of thylakoid membrane in chloroplasts. Therefore, it is considered to have an important role in the maintenance of the complicated structure of the thylakoid membrane. We have succeeded in cloning the enzyme for MGDG synthesis and overexpressed it in Escherichia coli. In this study we analyzed the morphology of the E. coli harboring the gene. The fatty acid composition of its membrane lipids did not differ between the wild type and transformant, except for the appearance of MGDG. However, transformant cells appeared to be elongated. DAPI staining revealed the entire intracellular region of filamentous cells to be stained; therefore, the elongation of the cells is probably due to a defect in cell division. Atomic force microscopy revealed that the transformant had a smooth but scratched surface. It was concluded that the excessive accumulation of a non-bilayer lipid, MGDG, interfered with the translocation of proteins across the plasma membrane, including those for cell division.     

In vitro generation of three-dimensional renal structures

End-stage renal disease is currently being treated effectively by transplantation. However, increasing demand and donor shortage make this treatment challenging. Recent advances in cell-based therapies have provided potential opportunities to alleviate the current challenges of donor shortage. In this study we developed a system to generate renal structures in vitro using primary kidney cells. This system involves the cultivation of expanded primary renal cells in a three-dimensional collagen-based culture system. After one week of growth, individual renal cells began to form renal structures resembling tubules and glomeruli. Histologically, these structures show phenotypic resemblance to native kidney structures. The reconstituted tubules stained positively for Tamm-Horsfall protein, which is expressed in the thick ascending limb of Henle's Loop and distal convoluted tubules. These results show that renal structures can be reconstituted in a three-dimensional culture system, which may eventually be used for renal cell therapy applications.     

Biogenic isoprene emission as expression of dissipativity,a fundamental cell property

Energy dynamics of isoprene biosynthesis and the mechanism of isoprene emission are discussed in view of their fundamental role in dissipativity of living cells. The significance of basic principles of colloidal chemistry for biological energy conversion is emphasized. The idea is put forward of the existence in living cells of the universal energy-dynamic structural unit, termed “biological micelle,” that accounts for the transport and distribution of protons over the cell volume. This unit is responsible for the creation and maintenance of physiological pH at any metabolically active site within the cell. Particular attention is paid to the involvement of F-type ATPase in the active proton transport from the thylakoid interior to the F1 domain of ATP-synthase and to recycling of protons from the outer cell surface to the thylakoid lumen due to H+-pumping activity of the thylakoid ATPase. The mechanism responsible for the outflow of entropy deS through the production of isoprene by protonation of dimethylallyl pyrophosphate (DMAPP) has been found. The stable steady-state condition of any thermodynamic system, including the living system, is correlated with the maximum entropy production. The rate of isoprene emission increases with temperature, which compensates for the decrease in outflow of thermal entropy deS. When the ambient temperature is increased, the sum of deS removed as heat and deS removed with isoprene emission remains constant. Thus, photobiosynthesis of isoprene is a special case of the entropy deS dissipation that provides a stable stationary state to the cell.     

鱼腥藻类囊体膜及其性质的研究

研究了鱼腥藻(Anabaena azollae Strasb.)的光合膜及其性质,结果如下:(1)以培养液为反应介质,测出鱼腥藻细胞的光合放氧,这种放氧受电子传递抑制剂DCMU的抑制。1mmol/L的NH_4Cl既延迟光合放氧的诱导期又抑制光合放氧速率。(2)在700～300nm波长范围,鱼腥藻出现4个吸收高峰,分别位于680、625、480和440nm处。其中625nm的吸收峰应属蓝绿藻的特有吸收峰。(3)通过高压氮气破碎法,并结合超声波处理,可以破碎鱼腥藻细胞壁,并从中分离出鱼腥藻类囊体膜制剂。测定表明,这种膜制剂具有进行光合磷酸化的能力,同时亦有膜上ATP酶水解ATP的能力。(4)利用此膜制剂,分离并部分纯化出ATP酶,在SDS-PAGE图谱上,此酶的两种小亚基(δ和ε亚基)的分子量分别低于菠菜叶绿体ATP酶的δ和ε亚基,但两种酶的α和β两种大亚基的分子量相近。以上结果提示,鱼腥藻具有进行光合作用的内在机构,这种机构在组分及其性质上与其它种类光合膜的异同是值得深入研究的课题。     

Assembly of Two Subunits of the Cyanobacterial Photosystem I on the n-Side of Thylakoid Membranes

Photosystem I contains several peripheral membrane proteins that are located on either positive (luminal) or negative (stromal or cytoplasmic) sides of thylakoid membranes of chloroplasts or cyanobacteria. Incorporation of two peripheral subunits into photosystem I of the cyanobacterium Synechocystis species PCC 6803 was studied using a reconstitution system in which radiolabeled subunits II (PsaD) and IV (PsaE) were synthesized in vitro and incubated with the isolated thylakoid membranes. After such incubation, the subunits were found in the membranes and were resistant to digestion with proteases and removal by 2 molar NaBr. All of the radioactive proteins incorporated in the membrane were found in the photosystem I complex. The subunit II was assembled specifically into cyanobacterial thylakoid membranes and not into Escherichia coli cell membranes or thylakoid membranes isolated from spinach. The assembly process did not require ATP or proton motive force, and it was not stimulated by ATP. The assembly of subunits II and IV into thylakoid membranes isolated from the strain AEK2, which lacks the gene psaE, was increased two- to threefold. The incorporation of subunit II was 15 to 17 times higher in the thylakoids obtained from the strain ADK3 in which the gene psaD has been inactivated. However, assembly of subunit IV in the same thylakoids was reduced by 65%, demonstrating that the presence of subunit II is required for the stable assembly of subunit IV. Large deletions in subunit II prevented its incorporation into thylakoids and assembly into photosystem I, suggesting that the overall conformation of the protein rather than a specific targeting sequence is required for its assembly into photosystem I.     

Biogenesis of thylakoid networks in angiosperms: knowns and unknowns

Aerobic life on Earth depends on oxygenic photosynthesis. This fundamentally important process is carried out within an elaborate membranous system, called the thylakoid network. In angiosperms, thylakoid networks are constructed almost from scratch by an intricate, light-dependent process in which lipids, proteins, and small organic molecules are assembled into morphologically and functionally differentiated, three-dimensional lamellar structures. In this review, we summarize the major events that occur during this complex, largely elusive process, concentrating on those that are directly involved in network formation and potentiation and highlighting gaps in our knowledge, which, as hinted by the title, are substantial.     

Localization of phycoerythrin at the lumenal surface of the thylakoid membrane in Rhodomonas lens

The thylakoids of cryptomonads are unique in that their lumens are filled with an electron-dense substance postulated to be phycobiliprotein. In this study, we used an antiserum against phycoerythrin (PE) 545 of Rhodomonas lens (gift of R. MacColl, New York State Department of Health, Albany, NY) and protein A-gold immunoelectron microscopy to localize this light-harvesting protein in cryptomonad cells. In sections of whole cells of R. lens labeled with anti-PE 545, the gold particles were not uniformly distributed over the dense thylakoid lumens as expected, but instead were preferentially localized either over or adjacent to the thylakoid membranes. A similar pattern of labeling was observed in cell sections labeled with two different antisera against PE 566 from Cryptomonas ovata. To determine whether PE is localized on the outer or inner side of the membrane, chloroplast fragments were isolated from cells fixed in dilute glutaraldehyde and labeled in vitro with anti-PE 545 followed by protein A-small gold. These thylakoid preparations were then fixed in glutaraldehyde followed by osmium tetroxide, embedded in Spurr, and sections were labeled with anti-PE 545 followed by protein A-large gold. Small gold particles were found only at the broken edges of the thylakoids, associated with the dense material on the lumenal surface of the membrane, whereas large gold particles were distributed along the entire length of the thylakoid membrane. We conclude that PE is located inside the thylakoids of R. lens in close association with the lumenal surface of the thylakoid membrane.     

Selection of an atrazine-resistant tobacco cell line having a mutant psbA gene

Summary A mutant cell line that shows high resistance to the photosynthesis-inhibiting herbicide atrazine was selected from cultured photomixotrophic Nicotiana tabacum cv. Samsun NN cells by repeated exposure to toxic levels of the herbicide. This resistance was confirmed by measurements of Hill reaction activity in isolated thylakoid membranes. Nucleotide sequencing revealed that the resistant cell line had a point mutation in its chloroplast psbA gene. The 264th codon, AGT (serine) was changed to ACT (threonine) in this mutant. This new type of mutation also conferred moderate cross-resistance to diuron and subsequently was stable in the absence of continued selection pressure.     

Hg2+对海马齿叶肉细胞超微结构的影响

以红树林植物海马齿为材料,将生长一致的海马齿水培苗放到含有不同浓度Hg2+的营养液中进行Hg2+胁迫,用透射电镜观察海马齿叶肉细胞超微结构对不同浓度Hg2+胁迫的响应,以明确重金属汞对海马齿叶肉细胞超微结构的影响,探讨海马齿耐汞机制。结果表明:重金属汞能造成海马齿叶肉细胞不同程度的伤害,主要表现为对叶肉细胞中的叶绿体、线粒体、细胞核以及膜系统的伤害。随着Hg2+浓度不断升高,其叶绿体数目不断减少,形状由船型变成长形以及出现一些巨型叶绿体,类囊体系统受到伤害、基粒片层变得模糊不清。线粒体数目由于Hg2+浓度的不同而不同,形状由棒状变成圆形及椭圆形,线粒体双层膜结构与嵴变得模糊不清。细胞核也受到不同程度的伤害,核仁由一个变成多个,最后消失;同时细胞膜也受到伤害,主要表现为,不断的向胞内形成膜突起再形成空泡。最后在高浓度Hg2+胁迫下,随着叶肉细胞内细胞器的不断减少,最终造成细胞解体死亡。     

淹水对玉米叶片细胞超微结构的影响

对淹水过程中玉米（Ｚｅａ ｍａｙｓ Ｌ．）叶片细胞超微结构的变化进行连续观察。淹水２ｈ后,液泡膜发生明显内陷。淹水６ｈ后,液泡膜内陷加剧,呈极度松弛状态;叶发体被膜局部向外突出一个由单层膜包裹的泡状结构。淹水１２ｈ后,液泡膜局部破裂;叶绿体被膜破坏加剧,成为一松弛的单膜结构,同时,基质类囊体出现空泡化。淹水１８ｈ后,叶绿体的破坏进一步加剧：被膜完全消失,基质类囊体开始消化;同时,线粒体膜和核膜也开