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Biogenesis of the complex 3D architecture of plant thylakoids remains an unsolved problem. Here, we analyzed this process in chloroplasts of germinating Arabidopsis thaliana cotyledons using 3D electron microscopy and gene expression... more
Biogenesis of the complex 3D architecture of plant thylakoids remains an unsolved problem. Here, we analyzed this process in chloroplasts of germinating Arabidopsis thaliana cotyledons using 3D electron microscopy and gene expression analyses of chloroplast proteins. Our study identified a linear developmental sequence with five assembly stages: tubulo-vesicular pro-thylakoids (24 HAI; hours after imbibition); sheet-like pre-granal thylakoids that develop from the pro-thylakoids (36 HAI); proliferation of pro-grana stacks with wide tubular connections to the originating pre-grana thylakoids (60 HAI); structural differentiation of pro-grana stacks and expanded stroma thylakoids (84 HAI); conversion of the pro-grana stacks into mature grana stacks (120 HAI). Development of the planar pre-granal thylakoids and the pro-grana membrane stacks coincides with the appearance of thylakoid-bound polysomes and photosystem II (PSII) complex subunits at 36 HAI. ATP synthase, cytochrome b6f and li...
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The preprophase band (PPB) is a cytokinetic apparatus that determines the site of cell division in plants. It originates as a broad band of microtubules (MTs) in G2 and narrows to demarcate the future division site during late prophase.... more
The preprophase band (PPB) is a cytokinetic apparatus that determines the site of cell division in plants. It originates as a broad band of microtubules (MTs) in G2 and narrows to demarcate the future division site during late prophase. Studies with fluorescent probes have shown that PPBs contain F-actin during early stages of their development but become actin depleted in late prophase. Although this suggests that actins contribute to the early stages of PPB formation, how actins contribute to PPB-MT organization remains unsolved. To address this question, we used electron tomography to investigate the spatial relationship between microfilaments (MFs) and MTs at different stages of PPB assembly in onion cotyledon epidermal cells. We demonstrate that the PPB actins observed by fluorescence microscopy correspond to short, single MFs. A majority of the MFs are bound to MTs, with a subset forming MT-MF-MT bridging structures. During the later stages of PPB assembly, the MF-mediated lin...
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The complexity of the thylakoid membrane frequently necessitates its separation into component parts before meaningful analysis may be carried out. Thylakoid fractionation, typically via disruption of the bilayer with detergents, and... more
The complexity of the thylakoid membrane frequently necessitates its separation into component parts before meaningful analysis may be carried out. Thylakoid fractionation, typically via disruption of the bilayer with detergents, and subsequent manipulations required for various analytical techniques, always raises the question of the extent to which the solubilized experimental material is representative of the in vivo state. We report here on the application of a new native green gel system to five higher plant species. This system resolves as many as eighteen chlorophyll-protein complexes with very little release of free pigment and with a degree of preservation of subunit-subunit interactions that has not been obtainable with previous systems, suggesting that this system yields a more accurate picture of the native condition of the membrane. The system was originallydeveloped for use with Chlamydomonas thylakoids, from which up to twenty chlorophyll-protein complexes may be resolved (Allen and Staehelin, these Proceedings).
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Research Interests: Plant Biology, Biology, Cell Biology, Medicine, Endocytosis, and 6 morePlant, Onion, Cytokinesis, Onions, Clathrin, and Plant Epidermis
We have investigated the process of somatic-type cytokinesis in Arabidopsis (Arabidopsis thaliana) meristem cells with a three-dimensional resolution of ∼7 nm by electron tomography of high-pressure frozen/freeze-substituted samples. Our... more
We have investigated the process of somatic-type cytokinesis in Arabidopsis (Arabidopsis thaliana) meristem cells with a three-dimensional resolution of ∼7 nm by electron tomography of high-pressure frozen/freeze-substituted samples. Our data demonstrate that this process can be divided into four phases: phragmoplast initials, solid phragmoplast, transitional phragmoplast, and ring-shaped phragmoplast. Phragmoplast initials arise from clusters of polar microtubules (MTs) during late anaphase. At their equatorial planes, cell plate assembly sites are formed, consisting of a filamentous ribosome-excluding cell plate assembly matrix (CPAM) and Golgi-derived vesicles. The CPAM, which is found only around growing cell plate regions, is suggested to be responsible for regulating cell plate growth. Virtually all phragmoplast MTs terminate inside the CPAM. This association directs vesicles to the CPAM and thereby to the growing cell plate. Cell plate formation within the CPAM appears to be ...
Research Interests: Genetics, High Pressure, Plant Biology, Biology, Scanning Electron Microscopy, and 15 moreCell Biology, Polysaccharides, Cell Division, Microtubules, Arabidopsis, Pressure, Cytokinesis, Vesicle, Plant cell, Cell Wall, X ray Computed Tomography, Freezing, Meristem, Biochemistry and cell biology, and Somatic Cells
Chlorophyll a/b light-harvesting complexes (chl a/b LHC) and photosystem II (PSII) cores were isolated from an octyl glucoside-containing sucrose gradient after solubilization of barley thylakoid membranes with Triton X-100 and octyl... more
Chlorophyll a/b light-harvesting complexes (chl a/b LHC) and photosystem II (PSII) cores were isolated from an octyl glucoside-containing sucrose gradient after solubilization of barley thylakoid membranes with Triton X-100 and octyl glucoside. No cation precipitation step was necessary to collect the chl a/b LHC. PAGE under mildly denaturing and fully denaturing conditions showed that the chl a/b LHC fraction contained chlorophyll-protein complexes CP27, CP29, and CP64. The PSII core material contained CP43 and CP47, and little contamination by other nonpigmented polypeptides. Freeze-fracture electron microscopy of the chl a/b LHC after reconstitution into digalactosyldiglyceride (DG) or phosphatidylcholine (PC) vesicles showed that the protein particles (approximately 7.5 +/- 1.6 nm) were approximately 99 and 90% randomly dispersed, respectively, in the liposomes. Addition of Mg++ produced particle aggregation and membrane adhesion in chl a/b LHC-DG liposomes in a manner analogous...
Research Interests: Photosynthesis, Biology, Phospholipids, Cell Biology, Medicine, and 12 moreBiological Sciences, Chlorophyll, Liposomes, Protein Complex Detection, Glycolipids, The cell, Photosystem II, Chlorophyll a, Chloroplasts, Freeze-fracturing, Medical and Health Sciences, and Pharmacology and pharmaceutical sciences
Freeze-fracture replicas of the photosynthetic prokaryote Prochloron sp., collected at Coconut Island, Hawaii, show that the thylakoids are differentiated into stacked and unstacked regions much like the thylakoids of green algae and... more
Freeze-fracture replicas of the photosynthetic prokaryote Prochloron sp., collected at Coconut Island, Hawaii, show that the thylakoids are differentiated into stacked and unstacked regions much like the thylakoids of green algae and higher plants. On the exoplasmic (E) fracture face, the particle density is greater in stacked regions (≈3100 particles/μm 2 ) than in unstacked regions (≈925 particles/μm 2 ). On the complementary protoplasmic (P) fracture face, the particle density is lower in stacked regions (≈2265 particles/μm 2 than in unstacked regions (≈4980 particles/μm 2 ). The histogram of the diameters of E face particles in unstacked regions shows a single broad peak centered at 80 Å. In stacked regions, four distinct peaks, at 75, 105, 130, and 160 Å, are observed. These size classes are virtually identical to those found on E faces of thylakoids of the green alga Chlamydomonas and of greening pea chloroplasts. In the latter systems, the different size classes of E face par...
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Freeze-fracture electron microscopy of propane-jet-frozen samples has been employed to investigate vesicle-mediated secretion and membrane recycling events in carrot (Daucus carota L.) and sycamore maple (Acer pseudoplatanus L.)... more
Freeze-fracture electron microscopy of propane-jet-frozen samples has been employed to investigate vesicle-mediated secretion and membrane recycling events in carrot (Daucus carota L.) and sycamore maple (Acer pseudoplatanus L.) suspension-culture cells. Stabilization of the cells by means of ultrarapid freezing has enabled us to preserve the cells in a turgid state and to visualize new intermediate membrane configurations related to these events. Indeed, many of the observed membrane configurations, such as flattened membrane vesicles with slit-shaped membrane fusion sites and horseshoe-shaped membrane infoldings, appear to result from the action of turgor forces on the plasma membrane. Individual cells exhibited great variations in numbers and types of membrane configurations postulated to be related to secretion and membrane-recycling events. In the majority of cells, the different membrane profiles displayed a patchy distribution, and within each patch the membrane configurations tended to be of the same stage. This result indicates that secretory events are triggered in domains measuring from 0.1 to about 10 μm in diameter. Based on an extensive analysis of the different membrane configurations seen in our samples, we have formulated the following model of vesicle-mediated secretion in plant cells: Fusion of a secretory vesicle with the plasma membrane leads to the formation of a single, narrow-necked pore that increases in diameter up to about 60 nm. During discharge, the vesicle is flattened, forming a disc-shaped structure perpendicular to the plane of the plasma membrane. As the vesicle is flattened, the pore is converted to a slit, the maximum length of which coincides with the diameter of the flattened vesicle. The flattened vesicle then tips over and concomitantly the plasma-membrane slit becomes curved into a horseshoe-shaped configuration as it extends along the outer margins of the tipped-over vesicle. Some coated pits are present interspersed between the above-mentioned structures, but their numbers appear insufficient to account for an exclusively endocytotic mechanism of membrane recycling. Instead, our micrographs are more consistent with a mixed mode of recycling of membrane components to the cortical endoplamic reticulum and to Golgi cisternae that involves both internalization of membrane by endocytosis and of individual lippid molecules by unknown mechanisms (lipid exchange proteins?). To this end, overall flattening out of the horseshoe-shaped membrane infoldings is accompanied by a retraction and reduction in size of their central, tongue-like structure.
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We have investigated the three-dimensional (3D) architecture of the thylakoid membranes of Arabidopsis (Arabidopsis thaliana), tobacco (Nicotiana tabacum), and spinach (Spinacia oleracea) with a resolution of approximately 7 nm by... more
We have investigated the three-dimensional (3D) architecture of the thylakoid membranes of Arabidopsis (Arabidopsis thaliana), tobacco (Nicotiana tabacum), and spinach (Spinacia oleracea) with a resolution of approximately 7 nm by electron tomography of high-pressure-frozen/freeze-substituted intact chloroplasts. Higher-plant thylakoids are differentiated into two interconnected and functionally distinct domains, the photosystem II/light-harvesting complex II-enriched stacked grana thylakoids and the photosystem I/ATP synthase-enriched, nonstacked stroma thylakoids. The grana thylakoids are organized in the form of cylindrical stacks and are connected to the stroma thylakoids via tubular junctions. Our data confirm that the stroma thylakoids are wound around the grana stacks in the form of multiple, right-handed helices at an angle of 20° to 25° as postulated by a helical thylakoid model. The junctional connections between the grana and stroma thylakoids all have a slit-like archite...
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Three light intensity-dependent Chl b-deficient mutants, two in wheat and one in barley, were analyzed for their xanthophyll cycle carotenoids and Chl fluorescence characteristics under two different growth PFDs (30 versus 600 μmol... more
Three light intensity-dependent Chl b-deficient mutants, two in wheat and one in barley, were analyzed for their xanthophyll cycle carotenoids and Chl fluorescence characteristics under two different growth PFDs (30 versus 600 μmol photons·m(-2) s(-1) incident light). Mutants grown under low light possessed lower levels of total Chls and carotenoids per unit leaf area compared to wild type plants, but the relative proportions of the two did not vary markedly between strains. In contrast, mutants grown under high light had much lower levels of Chl, leading to markedly greater carotenoid to Chl ratios in the mutants when compared to wild type. Under low light conditions the carotenoids of the xanthophyll cycle comprised approximately 15% of the total carotenoids in all strains; under high light the xanthophyll cycle pool increased to over 30% of the total carotenoids in wild type plants and to over 50% of the total carotenoids in the three mutant strains. Whereas the xanthophyll cycle remained fairly epoxidized in all plants grown under low light, plants grown under high light exhibited a considerable degree of conversion of the xanthophyll cycle into antheraxanthin and zeaxanthin during the diurnal cycle, with almost complete conversion (over 90%) occurring only in the mutants. 50 to 95% of the xanthophyll cycle was retained as antheraxanthin and zeaxanthin overnight in these mutants which also exhibited sustained depressions in PS II photochemical efficiency (Fv/Fm), which may have resulted from a sustained high level of photoprotective energy dissipation activity. The relatively larger xanthophyll cycle pool in the Chl b-deficient mutant could result in part from the reported concentration of the xanthophyll cycle in the inner antenna complexes, given that the Chl b-deficient mutants are deficient in the peripheral LHC-II complexes.
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We have expressed a fusion protein formed between the avian infectious bronchitis virus M protein and the bacterial enzyme beta-glucuronidase in transgenic tobacco cells. Electron microscope images of such cells demonstrate that... more
We have expressed a fusion protein formed between the avian infectious bronchitis virus M protein and the bacterial enzyme beta-glucuronidase in transgenic tobacco cells. Electron microscope images of such cells demonstrate that overexpression of this fusion protein gives rise to a type of endoplasmic reticulum membrane domain in which adjacent membranes become zippered together apparently as a consequence of the oligomerizing action of beta-glucuronidase. These zippered (Z-) membranes lack markers of the endoplasmic reticulum (NADH cytochrome c reductase and ribosomes) and accumulate in the cells in the form of multilayered scroll-like structures (up to 2 micrometers in diameter; 20-50 per cell) without affecting plant growth. The discovery of Z-membranes has broad implications for biology and biotechnology in that they provide a means for accumulating large quantities of recombinant membrane proteins within discrete domains of native membranes.
Research Interests: Biology, Tobacco, Scanning Electron Microscopy, Membrane Proteins, Transmission Electron Microscopy, and 15 moreMedicine, Multidisciplinary, Organelles, Cytochrome C, Endoplasmic Reticulum, Plant growth, Glucuronidase, Enzyme, Base Sequence, Membrane Protein, Cell free System, Recombinant Proteins, Fusion Protein, Electron Microscope, and Molecular Sequence Data
... Plastoglobuli contain large amounts of carotenoids and plastoquinone and usually accumulate when development is arrested and during leaf senescence (Lichtenthaler, 1968; Dahlin andRyberg, 1986). II. ... Page 4. 14 L. Andrew Staehelin... more
... Plastoglobuli contain large amounts of carotenoids and plastoquinone and usually accumulate when development is arrested and during leaf senescence (Lichtenthaler, 1968; Dahlin andRyberg, 1986). II. ... Page 4. 14 L. Andrew Staehelin and Georg WM van der Staay ...
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A chlorophyll-protein complex of chloroplast membranes, which simultaneously serves as light-harvesting antenna and membrane adhesion factor, undergoes reversible, lateral diffusion between appressed and nonappressed membrane regions... more
A chlorophyll-protein complex of chloroplast membranes, which simultaneously serves as light-harvesting antenna and membrane adhesion factor, undergoes reversible, lateral diffusion between appressed and nonappressed membrane regions under the control of a protein kinase. The phosphorylation-dependent migration process regulates the amount of light energy that is delivered to the reaction centers of photosystems I and II (PS I and PS II), and thereby regulates their rate of turnover. This regulatory mechanism provides a rationale for the finding that the two photosystems are physically separated in chloroplast membranes (PS II in appressed, grana membranes, and PS I in nonappressed, stroma membranes). The feedback system involves the following steps: a membrane-bound kinase senses the rate of PS II vs. PS I turnover via the oxidation-reduction state of the plastoquinone pool, which shuttles electrons from PS II via cytochrome f to PS I. If activated, the kinase adds negative charge (phosphate) to a grana-localized pigment-protein complex. The change in its surface charge at a site critical for promoting membrane adhesion results in increased electrostatic repulsion between the membranes, unstacking, the lateral movement of the complex to adjacent stroma membranes, which differ in their functional composition. The general significance of this type of membrane regulatory mechanism is discussed.