CHLOROPLAST DEVELOPMENT IN THE GERMINATING SAFFLOWER (CARTHAMUS TINCTORIUS) COTYLEDON

Proplastids in the mesophyll cells of the cotyledons of mature seeds of safflower are irregular in shape and compressed in narrow corners between the large inclusion bodies, oil vacuoles and protein bodies. The proplastids contain a few irregular internal membranes. During dark germination, sheets or sac-like membranes are produced by the activity of the inner component of the proplastid envelope. These continuous membranes become reticulate and aggregate to the center of the proplastid to form after seven days' germination a quasicrystalline prolamellar body. The membranes are at first irregularly arranged and are of two sorts: those found in the interior of the developing prolamellar body, composed of laterally connected spherical profiles, and those on the periphery of the prolamellar body, which are continuous smooth sheets. The prolamellar body in these dark-germinated proplastids reverts after 3 hr of illumination to the irregularly arranged membranous structure of the 5-day dark germination stage. After 6 hr of illumination membranes grow from the prolamellar body forming concentric loops which, in cross section, appear as concentric circles. These membranes must be nested semi-spheroids. Small grana appear immediately on these looped membranes close to the prolamellar body. With further illumination additional grana develop along the looped membranes in close proximity to the slowly disappearing prolamellar body. Grana increase in size and number along the looped intergranal membranes. The prolamellar body disappears after 15 hr of illumination. The interconnecting fret membranes, sparse at the 15-hr stage, increase and after 24-hr illumination result in the typical grana fretwork system of the mature chloroplast. Membranes are continuously being produced by the invagination of the inner member of the plastid envelope.     

Chloroplast fine structure in the japonica-2 maize mutant exposed to continuous illumination. 1. The green tissues.

Exposure to continuous illumination causes the appearance of numerous plastoglobuli in the stroma of both the mesophyll and bundle sheath chloroplasts of the green tissues of the leaves of the japonica-2 mutant of maize. In the pale green tissues the thylakoids have markedly swollen membranes. Another feature of the plastids exposed to continuous illumination is the heavy accumulation of starch. The japonica-2 chloroplasts show a different sensitivity to light, the chloroplasts of the pale green tissues being affected more markedly than the ones of the dark green tissues, and the bundle sheath chloroplasts more than those of the mesophyll. The effects of continuous illumination may be interpreted as an acceleration of chloroplast ontogenesis.     

THE MOLECULAR ORGANIZATION OF CHLOROPLAST MEMBRANES

The presence of subunits in chloroplast membranes is suggested by polarization, fluorescence, and X-ray studies. Subunits (quantasomes) may be observed in the electron microscope on dried shadowed membranes and in replicas of membranes produced by the freeze-etching technique. Regular subunits are also observed with the electron microscope in thin sections of chloroplast membranes. Chemical considerations suggest that many membranes are composed of lipoprotein subunits. Thin sections reveal two types of chloroplast membranes, the fret membranes composed of one layer of subunits, and the partitions composed of two layers of subunits. Chloroplast membranes consist of about 45% protein and 55% lipid. Some 80% of the lipids are the highly surfactant glycolipids. In this paper the subunits are visualized as assymetric lipoproteins, probably having a protein core surrounded by components determined by the nature and environment of the membrane. Since the stroma, fret channels, and loculi contain aqueous materials, it is further postulated that the membranes bordering these spaces bind the highly surfactant glycolipids. The region between the two rows of subunits in the partition appears to be highly hydrophobic, rich in chlorophyll, and low in glycolipids. Some chlorophyll also may occur within the subunits both in the partitions and in the fret membranes. Since four subunits appear to comprise a quantasome, at least two types of forces, inter- and intra-quantasome forces, bind the subunits together in sheets. Chloroplast membranes thus differ from a “unit membrane” in two important respects: (1) they must be an aggregate of globular subunits, and (2) the lipoprotein subunits consist of a protein matrix which binds the chlorophylls and lipids by hydrophobic association with their hydrocarbon moieties.     

Continuity of the Chloroplast Membrane Systems in Zea mays L

Ultrastructural studies of the chloroplasts of the normal, yellow-green, and pale green phenotypes of Zea mays L. indicate that the internal membrane system is continuous with the plastid envelop. The intramembraneous spaces, loculi, and fret channels are also continuous with inner component of the plastid envelop. High energy compounds or other photosynthates, formed in the grana or frets are thus separated from both stroma and cytoplasm by a single membrane, either the fret membrane or the outer component of the plastid envelop. Since this type of plastid ultrastructure is apparently found only in plants exhibiting the Hatch and Slack pathways of photosynthesis there may be a relation between plastid ultrastructure and the pathways of photosynthetic carbon fixation.     

Ultrastructural studies of isolatedZea mays L. Chloroplasts

Summary The ultrastructure of isolated mesophyll chloroplasts ofZea mays L. was studied using the shadow casting technique. Grana and interconnecting fret membranes were observed in the same basic arrangement as they appear in thin sections. Quantasome units, as previously described by other authors for other species, were detected. Portions of the peripheral reticulum and envelope were also observed. Swollen membranes, comparable to those observed when isolated plastids are placed in water, were frequent in the preparations. It can be assumed, then, that in any preparation of this nature the hydrophillic spaces will absorb a large amount of water, swelling the intramembranous spaces and giving the membranes the appearance of large vesicles which will appear as flattened sacs when dried on the electron microscope grids. As there is no indication of any quantasome pattern in these membranes it is assumed that the particle arrangement of these membranes was altered during the procedure. It was not possible to determine whether the membranes observed in the swollen vesicles belonged to the grana or frets. A change in the morphology of the membranes during the processing may be observed with the light microscope. Recognition of this change is frequently difficult in electron micrographs, consequently, membranes with similar appearance under the electron microscope may actually have arisen from different portions of the plastid membrane systems.     

The mitotic apparatus. Identification of the major soluble component of the glycol-isolated mitotic apparatus

Particles having ribosome-like characteristics are described in proplastids of dark-grown wheat seedlings as the membranes of the prolamellar body become transformed, under the influence of light, into grana and fret membranes. Three arrangements of particles were noted: (1) a random distribution of discrete particles; (2) particles occurring in helices or parallel rows; and (3) particles arranged in rough squares with six to eight particles per side. It is possible that the third type of particle is a cross-section of long parallel rods. A particle ranges in size from 170 to 220 A, those of group three being somewhat smaller. The particulates vary from diamond shaped with smooth surfaces to circular with irregular surfaces. These particles have the characteristics of ribosomes as visualized by the electron microscope: they are preserved by glutaraldehyde and osmium tetroxide, they stain intensely with uranyl acetate, and are digested by RNase. Their properties do not coincide with those of viruses, smog-induced particles, stromacenter particles, or phytoferritin. They are frequently adjacent to membranes but never attached to membranes. The involvement of ribosomes in membrane development is discussed.     

Light-dependent degradation of the D1 protein in photosystem II is accelerated after inhibition of the water splitting reaction

Strong illumination of oxygen-evolving organisms inhibits the electron transport through photosystem II (photoinhibition). In addition the illumination leads to a rapid turnover of the D1 protein in the reaction center of photosystem II. In this study the light-dependent degradation of the D1 reaction center protein and the light-dependent inhibition of electron-transport reactions have been studied in thylakoid membranes in which the oxygen evolution has been reversibly inhibited by Cl- depletion. The results show that Cl(-)-depleted thylakoid membranes are very vulnerable to damage induced by illumination. Both the D1 protein and the inhibition of the oxygen evolution are 15-20 times more sensitive to illumination than in control thylakoid membranes. The presence, during the illumination, of the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) prevented both the light-dependent degradation of the D1 protein and the inhibition of the electron transport. The protection exerted by DCMU is seen only in Cl(-)-depleted thylakoid membranes. These observations lead to the proposal that continuous illumination of Cl(-)-depleted thylakoid membranes generates anomalously long-lived, highly oxidizing radicals on the oxidizing side of photosystem II, which are responsible for the light-induced protein damage and inhibition. The presence of DCMU during the illumination prevents the formation of these radicals, which explains the protective effects of the herbicide. It is also observed that in Cl(-)-depleted thylakoid membranes, oxygen evolution (measured after the readdition of Cl-) is inhibited before electron transfer from diphenylcarbazide to dichlorophenolindophenol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid-protein interactions in stacked and destacked thylakoid membranes and the influence of phosphorylation and illumination. Spin label ESR studies

The effects of membrane destacking, protein phosphorylation, and continuous illumination have been studied in pea thylakoid membranes using ESR spectroscopy of an incorporated spin-labelled phosphatidylglycerol. This spin-labelled analogue of an endogenous thylakoid lipid has previously been shown to exhibit a selectivity of interaction with thylakoid proteins. Neither destacking, phosphorylation nor illumination was found to change the ESR spectra appreciably, suggesting that for phosphatidylglycerol at least, neither the number of protein-associated membrane lipids nor their pattern of selectivity was altered. The redistribution of the thylakoid protein complexes in the membrane, under these various conditions, therefore takes place with conservation of the properties of the lipid/protein interface.     

Active and resting states of the O2-evolving complex of photosystem II

During dark adaptation, a change in the O2-evolving complex (OEC) of spinach photosystem II (PSII) occurs that affects both the structure of the Mn site and the chemical properties of the OEC, as determined from low-temperature electron paramagnetic resonance (EPR) spectroscopy and O2 measurements. The S2-state multiline EPR signal, arising from a Mn-containing species in the OEC, exhibits different properties in long-term (4 h at 0 degrees C) and short-term (6 min at 0 degree C) dark-adapted PSII membranes or thylakoids. The optimal temperature for producing this EPR signal in long-term dark-adapted samples is 200 K compared to 170 K for short-term dark-adapted samples. However, in short-term dark-adapted samples, illumination at 170 K produces an EPR signal with a different hyperfine structure and a wider field range than does illumination at 160 K or below. In contrast, the line shape of the S2-state EPR signal produced in long-term dark-adapted samples is independent of the illumination temperature. The EPR-detected change in the Mn site of the OEC that occurs during dark adaptation is correlated with a change in O2 consumption activity of PSII or thylakoid membranes. PSII membranes and thylakoid membranes slowly consume O2 following illumination, but only when a functional OEC and excess reductant are present. We assign this slow consumption of O2 to a catalytic reduction of O2 by the OEC in the dark. The rate of O2 consumption decreases during dark adaptation; long-term dark-adapted PSII or thylakoid membranes do not consume O2 despite the presence of excess reductant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Light-induced generation of membrane potential in intact chloroplasts suspended in H2O or D2O media

The membrane potential changes induced by short flashes and continuous light were investigated in isolated chloroplasts of Peperomia metallica suspended in H2O- or D2O media. The potential generated in H2O-suspended chloroplasts by a single turnover flash is approximately two times lower than the maximal level of potential induced by continuous light. The photoelectric response of D2O-suspended chloroplasts differs from that of H2O-suspended chloroplasts by an increased amplitude and a prolonged phase of the potential rise. Te dark decay of the potential proceeds 2-3 times slower in the D2O-suspended chloroplasts as compared to the H2O-suspended chloroplasts. The magnitude of the flash-induced potential is somewhat lower for the chloroplasts in D2O than for the chloroplasts in the H2O medium. The results obtained suggest that the substitution of H2O for D2O results in a decrease of the ionic conductance and an increase of stability of thylakoid membranes. It was shown that the rise of electrical potential under continuous illumination proceeds in two stages. The difference kinetics of membrane potential changes are observed under conditions of separate activity of two systems of photosynthesis.     

Structural and functional modifications of the manganese cluster in Ca(2+)-depleted S1 and S2 states: electron paramagnetic resonance and X-ray absorption spectroscopy studies.

The effect of extraction of weakly bound Ca2+ by low-pH treatment on the O2-evolving apparatus was studied by use of low-temperature electron paramagnetic resonance (EPR) and X-ray absorption spectroscopy. In low-pH-treated PSII membranes, an S2 EPR multiline signal with modified line shape was induced by illumination at 0 degrees C, but its signal amplitude decreased upon lowering the excitation temperature with concomitant oxidation of cytochrome (cyt) b-559 in place of Mn. The half-inhibition temperature for formation of the modified multiline signal was found at -33 degrees C, which was much higher than that for formation of the normal S2 state in untreated control membranes. Signal IIf was normally induced down to -30 degrees C, but its dependence on excitation temperature was different from that for modified S2. This was interpreted as indicating that the low-temperature blockage of modified S2 formation is due to the incapability of electron abstraction from the Mn cluster. The Mn K-edge of X-ray absorption near-edge structure (XANES) spectrum shifted to lower energy by 0.8 eV after low-pH treatment, but the shift was reversed by addition of Ca2+. Upon illumination at 0 degrees C of treated membranes, the K-edge energy was up-shifted by 0.8 eV, but was not upon illumination at 210 K. These results were interpreted as indicating that extraction of weakly bound Ca2+ by low-pH treatment gives rise to structural and functional modulations of the Mn cluster.     

A link between rhodopsin and disc membrane cyclic nucleotide phosphodiesterase. Action spectrum and sensitivity to illumination.

Frog (Rana pipiens) rod outer segment disc membranes contain guanosine 3',5'-cyclic monophosphate phosphodiesterase (EC 3.1.4.1.c) which, in the presence of ATP, is stimulated 5- to 20-fold by illumination. The effectiveness of monochromatic light of different wavelengths in activating phosphodiesterase was examined. The action spectrum has a maximum of 500 nm, and the entire spectrum from 350 to 800 nm closely matches the absorption spectrum of rhodopsin, which is apparently the pigment which mediates the effects of light on phosphodiesterase activity. trans-Retinal alone does not mimic light. Half-maximal activation of the phosphodiesterase occurs with a light exposure which bleaches 1/2000 of the rhodopsins. Half-maximal activation can also be achieved by mixing 1 part of illuminated disc membranes in which the rhodopsin is bleached with 99 parts of unilluminated membranes. Regeneration of bleached rhodopsin by addition of 11-cis-retinal is illuminated disc membranes reverses the ability of these membranes to activate phosphodiesterase in unilluminated membranes. If the rhodopsin regenerated by 11-cis-retinal is illuminated again, it regains the ability to activate phosphodiesterase. These studies show that the levels of cyclic nucleotides in vetebrate rod outer segments are regulated by minute amounts of light and clearly indicate that rhodopsin is the photopigment whose state of illumination is closely linked to the enzymatic activity of disc membrane phosphodiesterase.     

Biochemical aspects of the visual process. XXIII. Sulfhydryl groups and rhodopsin photolysis

1. The number of exposed sulfhydryl groups in cattle rod photoreceptor membranes has been determined in suspension and after solubilization in various detergents both before and after illumination.2. In suspensions, two sulfhydryl groups are modified per mole of rhodopsin, both by Ellman's reagent 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide, while no extra SH groups are uncovered upon illumination. Neither reagent affects the spectral integrity of rhodopsin at 500 nm and the recombination capacity is retained upon modification of both rhodopsin and opsin.3. However, in detergents (digitonin, Triton X-100 and cetyltrimethylammonium bromide (CTAB)) 2–3 additional sulfhydryl groups appear upon illumination, in agreement with earlier reports.4. A total number of six sulfhydryl groups and two disulfide bridges are found in rod photoreceptor membranes, expressed per mole of rhodopsin.5. DTNB reacts somewhat faster with membrane suspensions after than before illumination. The less reactive sulfhydryl modifying agents O-methylisourea and methyl-p-nitrobenzene sulfonate show a similar behavior.6. It is concluded that illumination of rhodopsin in vivo will not uncover additional SH groups, although the reactivity of one exposed SH group may increase somewhat. These findings also exclude a role of SH groups in the covalent binding of the chromophore.     

Reconstitution of chlorophyllide formation by isolated etioplast membranes.

1. The reconstitution of chlorophyllide biosynthesis by barley etioplast membranes is described. 2. The process is dependent on the additon of NADPH and protochlorophyllide and on illumination, which can be either continuous or intermittent. 3. The reconstituted process involves spectroscopically similar intermediates to the native reaction in whole leaves. 4. Steps in the process are an initial enzymic formation in the dark of a photoactive complex, P638/652 (probably a ternary protochlorophyllide-NADPH-enzyme complex), followed by a very rapid light-dependent hydrogen transfer from the NADPH to the protochlorophyllide giving chlorophyllide giving chlorophyllide, finally releasing the enzyme for repeating the process. 5. A continuous assay for the system regenerating complex P638/652 was devised on the basis of monitoring chlorophyllide formation. 6. The pH optimum of the reaction is at 6.9 and Km values for protochlorophyllide and NADPH are 0.46 and 35 micron respectively. 7. The reaction is associated specifically with the etioplast membrane fraction. 8. Activities of the system assayed in vitro are more than adequate to account for rates of chlorophyll formation in vivo.     

光照,湿度和培养基对苎麻疫霉卵孢子产生量的影响

采用３因素随机区组设计研究了光照、湿度和培养基对苎麻疫霉（Ｐｈｙｔｏｐｈｔｈｏｒａｂｏｅｈｍｅｒｉａｅ）卵孢子产生量的影响．结果表明,各因子对卵孢子产生量的影响效应大小次序为培养基＞光照＞湿度,其中培养基和光照两因子影响均在０．０１水平上显著．在供试的４种常用培养基上,卵孢子产生量的大小次序为：ＳＬＡ培养基（ＳＬＡ）＞利马豆培养基（ＬＢＡ）＞Ｖ６汁培养基（Ｖ６Ａ）＞澄清的Ｖ６汁培养基（Ｖ６Ｂ）．在设置的３种光照条件中,卵孢子产生量以连续黑暗处理最高,连续光照最低,光照与黑暗交替处理居中．３个试验因子间的所有互作对卵孢子产生量均有极显著的影响．在不同光照、湿度、培养基组合中,卵孢子产生量以低湿＋连续黑暗＋ＳＬＡ组合最高,低湿＋连续光照＋Ｖ６Ｂ组合最低．     

Membrane-potential- and surface-potential-induced absorbance changes of merocyanine dyes added to chromatophores from Rhodopseudomonas sphaeroides

(1) Three analogs of merocyanine dyes added to suspensions of chromatophore vesicles showed absorbance changes responding to the change in surface potential induced by salt addition and to the change in membrane potential induced by illumination. (2) The extent of the light-induced absorbance changes of the dyes was linearly related, in the presence and absence of uncouplers, to that of carotenoid spectral shift which is an intrinsic probe of the intramembrane electric field. (3) Comparison of the merocyanine absorbance changes induced by salt addition with those induced by illumination indicated that the surface potential change in the outer surface of chromatophore membranes during illumination was very small. (4) Judging from the spectra of these absorbance and from the low permeabilities of the dyes to membrane, the absorbance change are attributed to change in distribution of the dyes between the medium and the outer surface region in chromatophore membranes. The extent of the light-induced absorbance changes of merocyanine dyes depended on the salt concentration of the medium. The types of dependence were different among three merocyanine analogs. This is explained by the mechanism mentioned above assuming appropriate parameters. It is suggested that, under continuous illumination, an equilibrium of the electrochemical potential of H⁺ is reached between the bulk aqueous phase and the outer surface region in the membrane where the merocyanine dyes are distributed.     

E.p.r. studies of photolysis of nitrosyl haemoglobin at low temperatures

Photolysis of HbNO has been studied from 6.2 K to 15.5 K by electron spin resonance during and after continuous illumination. Non-exponential kinetics of both dissociation and reassociation of NO was observed. The prolonged illumination separates the fast and slow ligands. This picture is consistent with NO tunnelling from two sites at different distances from the bound position. This result is obtained using a model of a sum of two exponentials or of conformational substates.     

Platymonas subcordiformis Channelrhodopsin-2 (PsChR2) Function: II. RELATIONSHIP OF THE PHOTOCHEMICAL REACTION CYCLE TO CHANNEL CURRENTS*

Channelrhodopsins, such as the algal phototaxis receptor Platymonas subcordiformis channelrhodopsin-2 (PsChR2), are light-gated cation channels used as optogenetic tools for photocontrol of membrane potential in living cells. Channelrhodopsin (ChR)-mediated photocurrent responses are complex and poorly understood, exhibiting alterations in peak current amplitude, extents and kinetics of inactivation, and kinetics of the recovery of the prestimulus dark current that are sensitive to duration and frequency of photostimuli. From the analysis of time-resolved optical absorption data, presented in the accompanying article, we derived a two-cycle model that describes the photocycles of PsChR2. Here, we applied the model to evaluate the transient currents produced by PsChR2 expressed in HEK293 cells under both fast laser excitation and step-like continuous illumination. Interpretation of the photocurrents in terms of the photocycle kinetics indicates that the O states in both cycles are responsible for the channel current and fit the current transients under the different illumination regimes. The peak and plateau currents in response to a single light step, a train of light pulses, and a light step superimposed on a continuous light background observed for ChR2 proteins are explained in terms of contributions from the two parallel photocycles. The analysis shows that the peak current desensitization and recovery phenomena are inherent properties of the photocycles. The light dependence of desensitization is reproduced and explained by the time evolution of the concentration transients in response to step-like illumination. Our data show that photocycle kinetic parameters are sufficient to explain the complex dependence of photocurrent responses to photostimuli.     

Pinealectomy and sexual rhythm in female rats.

The role of the pineal gland in inducing and maintaining the persistent estrus of rats exposed to continuous illumination was examined. Under the cyclic illumination, the pinealectomy or sham-operation revealed no effect on the estrous cycle, although weights of the ovaries, adrenals and hypophysis were slightly but definitely greater in the pinealectomized animals. In rats exposed to the continous illumination immediately after pinealectomy or sham-operation, both groups exhibited the persistent estrous states soon after the change of lighting condition. In these rats, neither the degree of persistent estrus nor the organ weights a autopsy showed any significant difference between the groups. Moreover, the pinealectomy could not alter the incidence of estrous in persistent estrous rats which had been established already by the continuous illumination.     

Energization and ultrastructural pattern of thylakoids formed under periodic illumination followed by continuous light

Bean leaves grown under periodic illumination (56 cycles of 2 min light and 98 min darkness) were subsequently exposed to continuous illumination, and in connection with granum formation and accumulation of the light-harvesting pigment-protein complex thermoluminescence and light-induced shrinkage of thylakoid membranes were studied. Juvenile chloroplasts with large double sheets of thylakoids obtained under periodic light exhibited low temperature spectra of polarized fluorescence yielding fluorescence polarization (FP) values < 1 at 695 nm, characteristic for pheophytin emission. In the course of maturation under continuous light when normal grana appeared and the chlorophyll a/b light-harvesting photosystem II complex was incorporated into the membrane, at 695 nm the relative intensity of fluorescence dropped and FP changed to a value of > 1, suggesting an overlap between the emission of pheophytin and that of the chlorophyll a/b light-harvesting photosystem II complex. Thermoluminescence glow curves recorded with juvenile thylakoids displayed a relatively high proportion of emission at low temperatures (around -10°C) while with mature chloroplasts, more thermoluminescence originated from energetically deeper traps (discharged around 28°C). This means that during thylakoid development the capacity of the membrane to stabilize the separated charges increases, which might be favourable for the ultimate conservation of energy. The more extensive energization of mature thylakoids was also indicated by a light-induced decrease in the thickness of the membranes upon illumination; a change which could not be detected in juvenile thylakoids.Abbreviations EDTA ethylenediamine tetraacetic acid - Hepes 4-(2-hydroxy ethyl)-1-piperazine ethane sulfonic acid Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.