Changes in Molecular Organization of Thylakoid Membranes during the Cell Cycle of Scenedesmus obliquus

Cultures of Scenedesmus obliquus, synchronized by a light-darkregime of 14 : 10 hours, show distinct changes in their photosyntheticactivity with a maximum at the 8th h and a minimum at the 16thh after onset of illumination. The activity differences occurunder limiting light-intensities as well as under saturatinglight-intensities, indicating at least two different factorsregulating photosynthesis during the cell cycle. Based on differences in the ratio of oligomeric to monomericpigment-protein complexes between 8 and 16 h old cells, andMg²⁺-induced effects on the fluorescence properties of chloroplastparticles deriving from 8 and 16 h old cells, changes in themolecular organization of the photosynthetic apparatus are documented.These changes might regulate energy distribution within thephotosynthetic apparatus during ontogenetic development andare supposed to be responsible for the differences in activityunder limiting light-intensities. By analysis of pure PS II-particles from cells with maximaland minimal photosynthetic activity it could be demonstratedthat changes occurring in PS II proper account for the differencesin photosynthetic capacity measured under saturing light. Amodel demonstrating the changes in the molecular organizationof thylakoid membranes occurring during ontogenetic developmentof Scenedesmus obliquus is presented. ¹ This paper is dedicated to the memory of Hiroshi Tamiya, apioneer in the field of plant physiology and a fatherly friend. (Received December 27, 1985; Accepted June 18, 1986)     

Phase Transition Temperatures Determined for Chloroplast Thylakoid Membranes and for Liposomes Prepared from Charged Lipids Extracted from Thylakoid Membranes of Higher Plants

Lipid phase separation temperatures of intact thylakoid membranesfrom a number of chilling sensitive plants were measured usingchlorophyll a as the intrinsic fluorescent probe. The phospho-and sulfolipids were extracted from the thylakoid lamellae ofthese plants and purified by silicic acid column and thin layerchromatographies. These separated lipids were eluted and recombinedto give a total charged anionic thylakoid lipid fraction thatwas used to prepare liposomes containing purified chlorophylla as the fluorescent probe. The phase separation temperaturesof these liposomes were compared to phase separation temperaturesin intact thylakoid membranes isolated from the same plants. The chilling-sensitive plants—corn, pepper, tomato andwater hyacinth — showed phase separation temperaturesranging from 9 to 19°C for both the liposomes and the thylakoidmembranes. In addition, low temperature phase separations wereseen from –21 to –27°C. Mimulus, which is notas chilling sensitive as the former plants, had a phase separationtemperature near 0 to 2.5°C and at –27°C. In general,there was a good agreement between the phase separation temperaturesof intact thylakoids and the purified anionic lipid fractionextracted from these thylakoids. Similar results were obtained using either trans-parinaric acidor chlorophyll a as the fluorescent probe in liposomes madefrom anionic thylakoid lipids or in liposomes prepared frompure dimyristoyl phosphatidyl choline, distearoyl phosphatidylcholine, or mixtures of equal amounts of these phospholipids. ¹ CIW-DPB Publication # 728. ³ Present address: Laboratory of Experimental Physics, Departmentof Biophysics, State University of Utrecht, Princetonplein 5,Utrecht, The Netherlands. (Received January 18, 1981; Accepted July 2, 1981)     

Light Activation of Rubisco by Rubisco Activase and Thylakoid Membranes

A reconstituted system comprising ribulose bisphosphate carboxylase/oxygenase(rubisco), rubisco activase, washed thylakoid membranes, andATP was used to demonstrate a light-dependent stimulation ofrubisco activation. ATP, ribulose bisphosphate, H⁺, and Mg²⁺concentrations are normally light-dependent variables in thechloroplast but were maintained at pre-determined levels. Resultsindicated that rubisco activase and washed thylakoid membranesare sufficient to catalyze light stimulation of rubisco activationwith the reconstituted system, and that rubisco activase isrequired for this light stimulation. The washed thylakoid membranesdid not exhibit rubisco activase activity, nor was rubisco activaseprotein detected immunologically. Light-dependent activationof rubisco in the reconstituted system was similar in whole-chainand PS I electron transport reactions, and saturated at approximately100 µmol photons m^–2 s^–1. ¹ Present address: Department of Biological Sciences, LouisianaTech University, Ruston, LA 71272, U.S.A.     

Effects of Nano-anatase on Spectral Characteristics and Distribution of LHCII on the Thylakoid Membranes of Spinach

In the article, we report that effects of nano-anatase on the spectral characteristics and content of light-harvesting complex II (LHCII) on the thylakoid membranes of spinach were investigated. The results showed that nano-anatase treatment could increase LHCII content on the thylakoid membranes of spinach and the trimer of LHCII; nano-anatase could enter the spinach chloroplasts and bind to PSII. Meanwhile, spectroscopy assays indicated that the absorption intensity of LHCII from nano-anatase-treated spinach was obviously increased in the red and the blue region, fluorescence quantum yield near 685 nm of LHCII was enhanced, the fluorescence excitation intensity near 440 and 480 nm of LHCII significantly rose and F ₄₈₀/F ₄₄₀ ratio was reduced. Oxygen evolution rate of PSII was greatly improved. Together, nano-anatase promoted energy transferring from chlorophyll (chl) b and carotenoid to chl a, and nano-anatase TiO₂ was photosensitized by chl of LHCII, which led to enhance the efficiency of absorbing, transferring, and converting light energy.     

A Parallel Study of Pigment Bleaching and Cytochrome Breakdown during Aging of Thylakoid Membranes

Aging of freshly isolated thylakoid membranes from spinach leaves(Spinacia oleracea L.) leads to dramatic alterations in boththe cytochrome (b_{559 (HP)} and f) composition and pigment (chlorophyllsa and b and ß-carotene) content. These changes occurat a faster rate under anaerobic conditions or after heatingthylakoid membranes, and in light as well as in darkness. Inaddition, when thylakoid membranes are heated at 78°C for8 min, or incubated in the presence of an emulsion of linoleicacid, a huge decrease in both cytochrome (particularly cyt.b_{559 (HP)}) and pigment contents occur. Whatever the experimentalconditions, cytochrome b_{559 (HP)} destruction occurs as soonthe aging process starts. Conversely, pigment bleaching is detectableafter an initial lag phase of about 60–70 min. Then, thetwo processes (cytochrome breakdown and bleaching of pigments)appear to take place in parallel. The addition of salicylhydroxamicacid or 8-hydroxy-quinoline, two radical scavenger components,to the aging medium strongly reduces the rate and extent ofcytochrome breakdown and pigment bleaching. On the basis of these results, a tentative scheme accountingfor the bleaching of pigments and the breakdown of cytochromesduring aging in vitro of thylakoid membranes is proposed. Itis suggested that these changes are mediated via a non-enzymaticmechanism in which free radicals could be implicated. The possiblerole of free radicals inducing ultrastructural changes at thelevel of chloroplast membranes in senescent leaves is also considered. (Received October 11, 1985; Accepted January 24, 1986)     

Spectral Fingerprinting of Individual Cells Visualized by Cavity-Reflection-Enhanced Light-Absorption Microscopy

The absorption spectrum of light is known to be a “molecular fingerprint” that enables analysis of the molecular type and its amount. It would be useful to measure the absorption spectrum in single cell in order to investigate the cellular status. However, cells are too thin for their absorption spectrum to be measured. In this study, we developed an optical-cavity-enhanced absorption spectroscopic microscopy method for two-dimensional absorption imaging. The light absorption is enhanced by an optical cavity system, which allows the detection of the absorption spectrum with samples having an optical path length as small as 10 μm, at a subcellular spatial resolution. Principal component analysis of various types of cultured mammalian cells indicates absorption-based cellular diversity. Interestingly, this diversity is observed among not only different species but also identical cell types. Furthermore, this microscopy technique allows us to observe frozen sections of tissue samples without any staining and is capable of label-free biopsy. Thus, our microscopy method opens the door for imaging the absorption spectra of biological samples and thereby detecting the individuality of cells.     

Selective Photoinhibition of Photosystem I in Isolated Thylakoid Membranes from Cucumber and Spinach

The site of photoinhibition at low temperatures in leaves ofa chilling-sensitive plant, cucumber, is photosystem I [Terashimaet al. (1994) Planta 193: 300]. As described herein, selectivephotoinhibition of PSI can also be induced in isolated thylakoidmembranes in vitro. Inhibition was observed both at chillingtemperatures and at 25°C, and not only in the thylakoidmembranes isolated from cucumber, but also in those isolatedfrom a chilling-tolerant plant, spinach. Comparison of theseobservations in vitro to the earlier results in vivo indicatesthat (1) photoinhibition of PSI is a universal phenomenon; (2)a mechanism exists to protect PSI in vivo; and (3) the protectivemechanism is chilling-sensitive in cucumber. The chilling-sensitivecomponent seems to be lost during the isolation of thylakoidmembranes. Very weak light (10–20µmol m^-2 s^-1) wassufficient to cause the inhibition of PSI. About 80% of theoxygen-evolving activity by PSII was maintained even after theactivity of PSI had decreased by more than 70%. This is thefirst report of the selective photoinhibition of PSI in vitro. (Received March 1, 1995; Accepted April 26, 1995)     

Transformations of the Cell Center during Cell Differentiation

A question was posed as to how the multicomponent and polyfunctional organelle dynamically changes during metazoan ontogenesis. The centrosome structure is gradually formed and its functions are switched on during early embryogenesis, one of which is the cell center formation. During cell differentiation, the condition of the cell center and surrounding structures may be different: first, the cell center is quite distinct; second, the cell center is absent due to redistribution of the microtubule organizing centers; third, the cell center disappears due to reversible or irreversible inactivation of the centrosome and other centers of microtubule organization. The assembly of the Golgi complex does not depend directly to the cell center presence. In some cell types, the Golgi complex is topologically associated with the cell center, while in others it exists as individual dictyosomes despite the cell center presence. In some other cell types, the common Golgi complex is assembled without the cell center, but in the presence of microtubules that are formed by noncentrosome centers of microtubule organization. In still others, degradation of both the cell center and the common Golgi complex takes place in the case of centrosome inactivation.     

Composition and Function of Thylakoid Membranes from Grana-rich and Grana-deficient Chloroplast Mutants of Barley

Chlorophyll-deficient barley (Hordeum vulgare) mutants were studied that had chlorophyll a/b ratios either higher or lower than the wild type. Mutants with high ratios (>5.2) had a reduced proportion of their photosynthetic lamellae appressed into grana (“grana-deficient” mutants) compared with wild type (chlorophyll a/b = 3.2), while the majority of lamellae in the chloroplasts with low chlorophyll a/b ratios (2.0-2.4) were organized into grana (“grana-rich” mutants).     

Induction of the H+ Release from Thylakoid Membranes by Illumination in the Presence of Protonophores at High Concentrations

The generally observed light-induced uptake of protons intothe thylakoid lumen is diminished by adding protonophores. Insteadof the H⁺ uptake, the release of protons was observed duringillumination in the presence of various protonophores at highconcentrations, namely, 1 µM nigericin, 10 µM carbonylcyanidem-chlorophenylhydrazone or 30 µM gramicidin. An uncoupler,NH₄C1 (4 mM), and a detergent, Triton X-100 (0.02%), also inducedthe H⁺ release but a K⁺ ionophore, valinomycin, did not. Theamount of H⁺ released reached about 100 nmol H⁺ (mg Chl)^–1at pH 7.5 under continuous illumination. The rate of the H⁺release was similar to that of the conventional H⁺ uptake butits dark relaxation was much slower than that of the H⁺ uptake.We compared the H⁺ release in protonophore-added thylakoidswith the previously reported H⁺ release in coupling factor 1(CF₁-depleted thylakoids. The H⁺ release in thylakoids withnigericin showed similar characteristics to that in CF₁-depletedthylakoids in terms of their responses to pH, phenazine methosulfateand light intensity. Both types of H⁺ release were relativelyinsensitive to DCMU and were stimulated somewhat by DCMU atlow concentrations (around 200 nM). Nigericin did not inhibitthe superoxide dismutase activity of the membranes. These resultsindicate that the H⁺ release in protonophore-added thylakoidsand that in CF₁ depleted thylakoids involve the same mechanismand that water-derived protons from PS II that result from animpairment of the activity of superoxide dismutase, as previouslyproposed, are not involved. Judging from the rate of electronflow and the lumenal acidification under the illumination, weconclude that the H⁺ release is a light-dependent scalar processwhich can be observed in thylakoid membranes with high H⁺ permeability.The H⁺ release of this type was not observed in mitochondriafrom rat liver or in chromatophores from Rhodobacter sphaeroides. (Received November 29, 1990; Accepted June 27, 1991)     

Stimulation of Antioxidant Enzymes and Lipid Peroxidation by UV-B Irradiation in Thylakoid Membranes of Wheat

In wheat seedlings (Triticum aestivum L. cv. 2329) oxidative stress caused by UV-B radiation led to lipid peroxidation of thylakoid membrane; it was expressed in term of malondialdehyde (MDA) formation. The peroxidation of lipids of thylakoid membrane in isolated chloroplasts was prevented when flavonoids quercetin and rutin were supplied into the incubation medium. The activities of superoxide dismutase, ascorbate peroxidase, and catalase increased during the first hours of UV-B exposure. A comparative study of UV-B and temperature effects showed different profiles of the antioxidant enzymes and MDA, suggesting that these two stresses have distinct sites of action. In addition to quantitative increase in flavonoids, qualitative change in flavonoid composition was also marked during UV-B stress, and a new peak at 330 nm was found as compared to control. This revised version was published online in July 2006 with corrections to the Cover Date.     

The Behaviour of Lysosome-like Particles during Cell Differentiation

The presence of lysosome-like particles has been demonstratedhistochemically in the various tissues of the root tips of Viciafaba. The behaviour of the enzyme acid phosphatase has beeninvestigated in cells from the root cap and developing protoxylemelements. The evidence presented may be interpreted as supportingthe concept that self digestion of the cell contents occursby the release of hydrolytic enzymes from the lysosomes in thecells studied.     

Penetration of Host Cell Membranes by Adenovirus 2

Highly purified human adenovirus type 2 directly penetrated the plasma membranes of KB cells. The process of membrane penetration resulted in the appearance of large numbers of adenovirions free in the cytoplasm of the infected cells. The virions underwent a morphological change as they penetrated the cell surface. Penetration of the plasma membranes and the accompanying alteration in virion morphology was dependent on a function associated with the intact cells, because neither event occurred when purified virions were added to isolated cell membranes. Inactivation of the adenovirions with heat or antibodies before inoculation of the cells reduced the infectivity of the virus population and prevented the appearance of virions free in the cytoplasm. The inactivation of the virions did not significantly reduce the number of virus particles which were found in cell vacuoles and pinocytotic vesicles.     

Probing Orientational Behavior of MHC Class I Protein and Lipid Probes in Cell Membranes by Fluorescence Polarization-Resolved Imaging

Steady-state polarization-resolved fluorescence imaging is used to analyze the molecular orientational order behavior of rigidly labeled major histocompatibility complex class I (MHC I) proteins and lipid probes in cell membranes of living cells. These fluorescent probes report the orientational properties of proteins and their surrounding lipid environment. We present a statistical study of the molecular orientational order, modeled as the width of the angular distribution of the molecules, for the proteins in the cell endomembrane and plasma membrane, as well as for the lipid probes in the plasma membrane. We apply this methodology on cells after treatments affecting the actin and microtubule networks. We find in particular opposite orientational order changes of proteins and lipid probes in the plasma membrane as a response to the cytoskeleton disruption. This suggests that MHC I orientational order is governed by its interaction with the cytoskeleton, whereas the plasma membrane lipid order is governed by the local cell membrane morphology.     

Formation of Postsynaptic-Like Membranes during Differentiation of Embryonic Stem Cellsin Vitro

To analyze the formation of neuromuscular junctions, mouse pluripotent embryonic stem (ES) cells were differentiated via embryoid bodies into skeletal muscle and neuronal cells. The developmentally controlled expression of skeletal muscle-specific genes coding for myf5, myogenin, myoD and myf6, α₁subunit of the L-type calcium channel, cell adhesion molecule M-cadherin, and neuron-specific genes encoding the 68-, 160-, and 200-kDa neurofilament proteins, synaptic vesicle protein synaptophysin, brain-specific proteoglycan neurocan, and microtubule-associated protein tau was demonstrated by RT-PCR analysis. In addition, genes specifically expressed at neuromuscular junctions, the γ- and ?-subunits of the nicotinic acetylcholine receptor (AChR) and the extracellular matrix protein S-laminin, were found. At the terminal differentiation stage characterized by the formation of multinucleated spontaneously contracting myotubes, the myogenic regulatory gene myf6 and the AChR ?-subunit gene, both specifically expressed in mature adult skeletal muscle, were found to be coexpressed. Only the terminally differentiated myotubes showed a clustering of nicotinic acetylcholine receptors (AChR) and a colocalization with agrin and synaptophysin. The formation of AChRs was also demonstrated on a functional level by using the patch clamp technique. Taken together, our results showed that during ES cell differentiationin vitroneuron- and muscle-specific genes are expressed in a developmentally controlled manner, resulting in the formation of postsynaptic-like membranes. Thus, the embryonic stem cell differentiation model will be helpful for studying cellular interactions at neuromuscular junctions by “loss of function” analysisin vitro.     

Alteration of the Cell Surface during the Vegetative Cell Cycle of the Unicellular Green Alga Chlamydomonas reinhardtii

Alterations of the cell surface during the vegetative cell cycleof the unicellular green alga Chlamydomonas reinhardtii wereinvestigated using polyclonal antibodies against the purifiedand subsequently deglycosylated insoluble cell wall componentand against a 100 kDa polypeptide of the deglycosylated, chaotrope-solublewall fraction, respectively. Both antibodies recognized epitopeswithin the non-glycosylated domains of a ‘150 kDa’chaotrope-soluble glycoprotein (=GP3B) localized in the outerlayers of the C. reinhardtii cell wall. Immunofluorescence studiesindicated that both antibodies reacted with the surface of ‘late’sporangia (harvested 1 h before liberation of the zoospores),but not with the cell surfaces of released zoospores, growingcells and young sporangia, respectively. After pretreatmentwith aqueous LiCl, however, the cell surfaces of zoospores,growing cells and young sporangia became accessible to theseparticular antibodies. Highly purified preparations of the insolublewall fraction revealed strong immunofluorescence with both antibodiesbut not with the corresponding preimmune sera. Based on thesedata, we concluded that the antigenic sites of the insolubleglycoprotein framework of the C. reinhardtii wall are maskedby LiCl-soluble glycoproteins in single cell stages and youngsporangia, but not or to a lesser extent in the case of themother walls of ‘late’ sporangia. The conclusionwas supported by findings that (I) the multilayered structureof the mother-cell wall was disturbed in ‘late’,but not in young sporangia and that (II) the amounts of chaotropesolublecell wall glycoproteins present in the LiCl-extracts from intactsporangia decreased during ripening of the sporangia. (Received January 10, 1996; Accepted May 27, 1996)