Characterization of the photosynthetic electron transport chain in normal and photobleachedAnabaena cylindrica by flash spectroscopy

Electron transport of normal and photobleachedAnabaena cylindrica was studied using spectral and kinetic analyses of absorbance transients induced by single turnover flashes. Between 500 and 600 nm two positive bands (540 and 566 nm) and two negative bands (515 and 554 nm) were found. Absorbance changes at 515 and 540 nm were partly characterized. None of these absorbance changes represent an electrochromic shift. Absorbance changes at 554 and 566 nm correspond to the oxidation of cytochromef and the reduction of cytochromeb ₅₆₃, respectively. We found a very slight 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) sensitivity of cytochromef in normal cells, while DCMU was completely ineffective for cytochromef reduction in photobleached cells. The absorbance change of cytochromeb ₅₆₃ increased, while the absorbance change of cytochromef was smaller than in normal cells. The increased O₂ evolution in photobleached cells and the negligible electron transport via cytochromef suggest the participation of other electron acceptor(s) in the electron-transport chain of photobleachedAnabaena cylindrica.     

Growth,fluorescence, photosynthetic O<Subscript>2</Subscript> production and pigment content of salt adapted cultures of <Emphasis Type="Italic">Arthrospira</Emphasis> (<Emphasis Type="Italic">Spirulina</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis>

The effect of salt concentration (NaCl) on growth, fluorescence, photosynthetic activities and pigment content of the cyanobacterium Arthrospira platensis has been investigated over 15 days. It has been observed that high NaCl concentration induces an increase of the growth, photosynthetic efficiency (α), phycobilin/chlorophyll ratio and a slight decrease of dark respiration and compensation points. Moreover, high NaCl concentration enhances photosystem II (PSII) activity compared to photosystem I (PSI). Results show that the phycobilin-PSII energy transfer compared to the chlorophyll-PSII (F_695,600/F_695,440) increases. However, data obtained about the maximal efficiency of PSII photochemistry are controversial. Indeed, the Fv/Fm ratio decreases in salt adapted cultures, while at the same time the trapping flux per PSII reaction center (TR₀/RC) and the probability of electron transport beyond Q_A (₀) remain unchanged at the level of the donor and the acceptor sites of PSII. This effect can be attributed to the interference of phycobilin fluorescence with Chl a when performing polyphasic transient measurements.     

DNA replication in soybean protoplasts and suspension-cultured cells: Comparison of exponential and fluorodeoxyuridine synchronized cultures

Cell-suspension cultures of soybean (Glycine max (L.) Merr., line SB-1) have been used to study DNA replication. Cells or protoplasts incorporate either radioactive thymidine or 5-bromodeoxyuridine (BUdR) into DNA. The DNA has been extracted as large molecules which can be visualized by autoradiography. Nuclei were isolated and lysed on slides thus avoiding degradation of DNA by a cytoplasmic endonuclease. The autoradiograms demonstrated that DNA synthesis occurs at several sites tandemly arranged on single DNA molecules separated by center to center distances ranging from 10 to 30 m. Velocity sedimentations through alkaline gradients confirm the lengths of the replicated regions seen in autoradiograms. By using velocity sedimentation it also has been possible to demonstrate that replication proceeds by the synthesis of very small (4–6S) DNA intermediates which join to form the larger, replicon-size pieces seen in autoradiograms. Both small (4–6S) and large (20–30S) intermediates are observed in synchronized and exponential cultures. However, after synchronization with fluorodeoxyuridine (FUdR) the rate of DNA synthesis is reduced. Since the size of intermediates is not reduced by FUdR treatment, it is concluded that the slower rate of replication results from a reduction in the number of tandem replication units but not in the rate at which they are elongated. After FUdR treatment, the density analogue of thymidine, BUdR, can be substituted for almost all of the thymidine residue in DNA, resulting in a buoyant density increase (in CsCl) from 1.694 to 1.747 g/cm³. Using this density analogue it is possible to estimate the amount of template DNA attached to new replication sites. When this is done, it can be shown that synchronized cells initiate replication at about 5,000 different sites at the beginning of S. (Each such site will replicate to an average length of 20 m.) Use of BUdR also substantiates that at early stages of replication, very small replicated regions (<8S) exist which are separated by unreplicated segments of DNA which replicate at a later time. Most of these conclusions agree with the pattern of DNA replication established for animal cells. However, a major difference appears to be that after prolonged inhibition of soybean cell replication with FUdR, very small, as well as replicon-size intermediates accumulate when replication is restored. This indicates that regulation of replication in these cells may be different from animal cells.Abbreviations BUdR 5-Bromodeoxyuridine - FUdR 5-Fluorodeoxyuridine     

NADH as electron donor for the photosynthetic membrane of Chlamydomonas reinhardii

A chloroplast fraction from Chlamydomonas reinhardii cells can oxidize NADH in the light, unlike chloroplasts of higher plants. The Chlamydomonas preparation catalyzes electron flow from NADH to methylviologen or ferredoxin to evolve hydrogen (in the presence of a hydrogenase) or take up oxygen. The NADH photooxidation is sensitive to rotenone, dibromothymoquinone and dicyclohexylcarbodiimide. This suggests that a rotenone sensitive NADH dehydrogenase is coupled on the plastoquinone reduction site of the potosynthetic electron flow system. On sonication of the particles NADH photooxidation is lost but may be restored by a protein fraction from an acetone extract plus plastocyanin.Abbreviations DAD diaminodurene - DCCD dicyclohexylcarbodiimide - DCMU (3,3-dichlorphenyl)-N·N dimethyl urea - DBMIB dibromothymoquinone - DNP-INT dinitro-phenylether of 2-iodo-4-nitrothymol - MV methylviologen - chl chlorophyll Dedicated to Professor Dr. O. Kandler on the occasion of his 60th birthday     

Investigations on the cell cycle of haploid and diploid tissue cultures of Datura innoxia Mill. and its synchronization

Using a ¹⁴C/³H double-labelling technique, the influence of kinetic on the length of the cell cycle of meristematic cells in haploid and diploid callus cultures of Datura innoxia was determined. The total length of the cell cycle of haploid cells as compared to that of diploid cells was reduced by 2.3 h (-kinetin) or 1.4 h (+kinetin). Furthermore, the addition of kinetin to the nutrient solution also reduces cell cycle duration at both ploidy levels. For synchronization of the cell cycle, a fluorodesoxyuridine/thymidine system was successfully employed. Apparently, the reduction of total cell cycle duration of cycling cells due to treatment with kinetin occurred at the expense of the G₁phase. Nevertheless, kinetin seems to exert an influence on the transition of cells from the G₂ into the M phase as well.Abbreviations FUdR fluorodeoxyuridine - HU hydroxyurea - IAA nidole acetic acid     

One-carbon metabolism in synchronized cultures of Euglena gracilis

The levels of folate derivatives in division synchronized cultures of Euglena gracilis Klebs (strain Z) increased rapidly on a per cell basis durin     

DNA synthesis by Escherichia coli B/r/l synchronized and grown under conditions of slow growth

Escherichia coli B/r/l was synchronized by a novel method and its growth was followed in a minimal salts medium containing glucose, acetate, aspartate or succinate as the sole carbon source. Thymine incorporation experiments showed agreement with the Cooper-Helmstetter model for DNA synthesis, during the division cycle, both in glucose grown culture with a doubling time 57.5 min and in acetate, aspartate and succinate where the doubling time was extended up to 90 min. The ratio C/C+D was identical or close to that predicted by the model. Prolonged growth of the synchronized cultures prior to each experiment was practised in order to ensure their physiological state without causing any considerable deterioration of synchrony.     

An improved method of measuring photosynthetic electron transport in Euglena under in vivo conditions

A method is described to measure photochemical activity in intact cells of Euglena under in vivo conditions. The method employs a cell wall digesting enzyme (cellulysin) to induce enough permeability in the cell walls and membranes in order to allow dyes, commonly used to investigate light-dependent electron transport reactions to enter, but without inducing a concomittant efflux of metabolites. Between 1 and 2 h of incubation in 5% (w/v) cellulysin provided conditions which allowed measurement of photosystem I-, II- and I+II-dependent electron transport with rates up to 600% higher than in control cells; whereas other cell wall degrading enzymes (cellulase and pectinase) still did not increase the entry of the dyes. Cellulysin up to 2 h of incubation had little or no effect on whole cell respiration, photosynthetic O₂ evolution, or the export of potassium and (¹⁴C) labeled compounds out of cells; therefore cellulysin obviously did not change the normal habit or physiology of Euglena. Cellulysin (4 h digestion), cellulase and pectinase (2–4 h of incubation) on the other hand led to a lowering of respiration and light-dependent O₂ evolution, and increased the efflux of K⁺, but apparently decreased that of (¹⁴C)labeled fixation products.Abbreviations DBMIB dibromothymoquinone - DCPIP 2,6-dichlorophenol-indophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMMIB 2,3-dimethyl-5,6-methylenedioxy-p-benzoquinone - MV methylviologen - PSI photosystem I - PS II photosystem II     

Cell cycle analysis of tumour-derived cultures ofCrepis capillaris: A kinetic analogy with proliferation of animal tumour cells

Summary Analysis of the cell cycle by three methods has revealed unusual kinetics of proliferation in tumour derived suspensions ofCrepis capillaris. The different methods of analysis yield different estimates of cycle phase durations, and such discrepancies have been explained in terms of low growth fractions with rapid total cycle traverse. Specifically, confidence in the estimation of G₂ duration by the fraction of labelled mitosis analysis, and comparison with shorter G₂ estimates obtained by the two other methods, suggests that cells drop out in G₁. However, cells which do not drop out of the proliferative compartment traverse G₁ extremely rapidly. Extremely short cell cycle durations in which the G₁ phase is virtually non-existent are uncharacteristic of plant cell suspension cultures, in which the G₁ phase has previously been shown to be extended as compared with meristematic root tip cells. A model has been proposed in which a central core of rapidly dividing cells continuously loses cells into a subpopulation of resting or G₀ cells with the G₁ DNA content. Similarities between plant and animal tumours with respect to cell growth and division are discussed.     

Ribulose-1,5-bisphosphate regeneration limitation in rice leaf photosynthetic acclimation to elevated CO2

Our previous study has demonstrated that both RuBP carboxylation limitation and RuBP regeneration limitation exist simultaneously in rice grown under free-air CO₂ enrichment (FACE, about 200 μmol mol⁻¹ above the ambient air CO₂ concentration) conditions [G.-Y. Chen, Z.-H. Yong, Y. Liao, D.-Y. Zhang, Y. Chen, H.-B. Zhang, J. Chen, J.-G. Zhu, D.-Q. Xu, Photosynthetic acclimation in rice leaves to free-air CO₂ enrichment related to both ribulose-1,5-bisphosphate carboxylase limitation and ribulose-1,5-bisphosphate regeneration limitation. Plant Cell Physiol. 46 (2005) 1036–1045]. To explore the mechanism for forming of RuBP regeneration limitation, we conducted the gas exchange measurements and some biochemical analyses in FACE-treated and ambient rice plants. Net CO₂ assimilation rate (A_net) in FACE leaves was remarkably lower than that in ambient leaves when measured at the same CO₂ concentration, indicating that photosynthetic acclimation to elevated CO₂ occurred. In the meantime the maximum electron transport rate (ETR) (J_max), maximum carboxylation rate (V_cmax) in vivo, and RuBP contents decreased significantly in FACE leaves. The whole chain electron transport rate and photophosphorylation rate reduced significantly while ETR of photosystem II (PSII) did not significantly decrease and ETR of photosystem I (PSI) was significantly increased in the chloroplasts from FACE leaves. Further, the amount of cytochrome (Cyt) f protein, a key component localized between PSII and PSI, was remarkably declined in FACE leaves. It appears that during photosynthetic acclimation the decline in the Cyt f amount is an important cause for the decreased RuBP regeneration capacity by decreasing the whole chain electron transport in FACE leaves.     

Reversible photoinhibition of unhardened and cold-acclimated spinach leaves at chilling temperatures

The photoinhibition of photosynthesis at chilling temperatures was investigated in cold-acclimated and unhardened (acclimated to +18° C) spinach (Spinacia oleracea L.) leaves. In unhardened leaves, reversible photoinhibition caused by exposure to moderate light at +4° C was based on reduced activity of photosystem (PS) II. This is shown by determination of quantum yield and capacity of electron transport in thylakoids isolated subsequent to photoinhibition and recovery treatments. The activity of PSII declined to approximately the same extent as the quantum yield of photosynthesis of photoinhibited leaves whereas PSI activity was only marginally affected. Leaves from plants acclimated to cold either in the field or in a growth chamber (+1° C), were considerably less susceptible to the light treatment. Only relatively high light levels led to photoinhibition, characterized by quenching of variable chlorophyll a fluorescence (F_V) and slight inhibition of PSII-driven electron transport. Fluorescence data obtained at 77 K indicated that the photoinhibition of cold-acclimated leaves (like that of the unhardened ones) was related to increased thermal energy dissipation. But in contrast to the unhardened leaves, 77 K fluorescence of cold-acclimated leaves did not reveal a relative increase of PSI excitation. High-light-treated, cold-acclimated leaves showed increased rates of dark respiration and a higher light compensation point. The photoinhibitory fluorescence quenching was fully reversible in low light levels both at +18° C and +4° C; the recovery was much faster than in unhardened leaves. Reversible photoinhibition is discussed as a protective mechanism against excess light based on transformation of PSII reaction centers to fluorescence quenchers.Abbreviations F_O initial fluorescence - F_M maximal fluorescence - F_V devariable fluorescence (f_m-f_o) - PFD photon flux density - PS photosystem - SD standard deviation The authors thank the Deutsche Forschungsgemeinschaft and the Academy of Finland for financial support.     

The cell cycle of Bacillus subtilis as studied by electron microscopy

Bacillus subtilis strain Marburg was grown exponentially with a doubling time of 65 min. To follow the time course of various cell cycle events, cells were collected by agar filtration and were then classified according to length. The DNA replication cycle was determined by a quantitative analysis of radioautograms of tritiated thymidine pulse labeled cells. The DNA replication period was found to be 45 min. This period is preceded and followed by periods without DNA synthesis of about 10 min.The morphology and segregation of nucleoplasmic bodies was studied in thin sections. B. subtilis contains two sets of genomes. DNA replication and DNA segregation seem to go hand in hand and DNA segregation is completed shortly after termination of DNA replication.Cell division and cell separation were investigated in whole mount preparations (agar filtration) and in thin sections. Cell division starts about 20 min after cell birth; cell separation starts at about 45 min and before completion of the septum.     

The origin and organization of cortical microtubules during the transition between M and G1 phases of the cell cycle as observed in highly synchronized cells of tobacco BY-2

The organization of microtubules (MTs) during the transition from the M phase to the G₁ phase of the cell cycle was followed in highly synchronized suspension-cultured cells ofNicotiana tabacum L. (tobacco BY-2) by sequential treatment of cells with aphidicolin and propyzamide. Short MTs were first formed in the perinuclear regions at the expense of phragmoplasts, but when these short MTs elongated to reach the cell cortex, they grew parallel to the long axis and towards the distal end of the cells. As soon as, or shortly before the tips of elongated MTs reached the distal end, transverse cortical MTs were formed in the region proximal to the division plane. Thereafter, almost all cells retained cortical MTs which were transversely orientated to the long axis of cells and could be observed in the G₁ phase. Thus, in the organization of cortical MTs, there are two steps that have been overlooked thus far. This novel observation provides a new scheme for the organization of cortical MTs, which could unify two contrasting hypotheses, i.e. organization in the perinuclear regions versus that in the cell cortex. These observations are discussed in relation to the microtubule-organizing center of plant cells.     

Observations of mitochondria and mitochondrial nuclei by double staining with rhodamine-123 and DAPI in synchronous cultures ofCatharanthus roseus

Mitochondria in cells ofCatharanthus roseus (L.) G. Don in synchronous cell division cultures were observed by double staining using fluorescence microscopy. The cells were stained with 4′-6-diamidino-2-phenylindole (DAPI) first and subsequently stained with rhodamine 123 (r-123). Immediately after staining with r-123, yellowishgreen, elongated and moving mitochondria were observed upon excitation at 485 nm. When the excitation filters were replaced by a UV filter (360 nm), 1 to 7 mitochondrial nucleoids were visible in each mitochondrion in the same field. Changes in the lengths of mitochondria during the cell cycle obtained from the observations under fluorescence microscopy by this staining method suggest the occurrence of multiplication of mitochondria concurrent with the cell cycle ofC. roseus.     

Characterization of Rhodopseudomonas capsulata

Thirty-three strains of Rhodopseudomonas capsulata have been studied in order to develop a more comprehensive characterization of the species. On the basis of morphological, nutritional, physiological and other properties, the characteristics of an ideal biotype have been defined, which can be used to distinguish Rps. capsulata from similar purple bacteria. In this connection, two properties of Rps. capsulata are of particular note: a) sensitivity to penicillin G is 10³–10⁵ times greater than that shown by closely related species, and b) all strains examined are susceptible to lysis by one or more strains of host species-specific virulent bacteriophages. It appears that members of the species Rps. capsulata form a stringent taxonomic grouping.     

Respiratory control over photosynthetic electron transport in chloroplasts of higher-plant cells: evidence for chlororespiration

Flash-induced primary charge separation, detected as electrochromic absorbance change, the operation of the cytochrome b/f complex and the redox state of the plastoquinone pool were measured in leaves, protoplasts and open-cell preparations of tobacco (Nicotiana tabacum L.), and in isolated intact chloroplasts of peas (Pisum sativum L.). Addition of 0.5–5 mM KCN to these samples resulted in a large increase in the slow electrochromic rise originating from the electrogenic activity of the cytochrome b/f complex. The enhancement was also demonstrated by monitoring the absorbance transients of cytochrome f and b ₆ between 540 and 572 nm. In isolated, intact chloroplasts with an inhibited photosystem (PS) II, low concentrations of dithionite or ascorbate rendered turnover of only 60% of the PSI reaction centers, KCN being required to reactivate the remainder. Silent PSI reaction centers which could be reactivated by KCN were shown to occur in protoplasts both in the absence and presence of a PSII inhibitor. Contrasting spectroscopic data obtained for chloroplasts before and after isolation indicated the existence of a continuous supply of reducing equivalents from the cytosol.Our data indicate that: (i) A respiratory electron-transport pathway involving a cyanide-sensitive component is located in chloroplasts and competes with photosynthetic electron transport for reducing equivalents from the plastoquinone pool. This chlororespiratory pathway appears to be similar to that found in photosynthetic prokaryotes and green algae. (ii) There is an influx of reducing equivalents from the cytosol to the plastoquinone pool. These may be indicative of a complex respiratory control of photosynthetic electron transport in higher-plant cells.Abbreviations and symbols A₅₁₅ flash-induced electrochromic absorbance change at 515 nm - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PS photosystem - SHAM salicylhydroxamic acid     

A Brownian dynamics computational study of the interaction of spinach plastocyanin with turnip cytochrome f: the importance of plastocyanin conformational changes

Brownian Dynamics (BD) computer simulations were used to study electrostatic interactions between turnip cytochrome f (cyt f) and spinach plastocyanin (PC). Three different spinach PC structures were studied: The X-ray crystal structure of Xue and coworkers [(1998) Protein Sci 7:2099–2105] and the NMR structure of Musiani et al. [(2005) J Biol Chem 280:18833–18841] and Ubbink and co-workers [(1998) Structure 6:323–335]. Significant differences exist in the backbone conformation between the PC taken from Ubbink and coworkers and the other two PC structures particularly the regions surrounding G10, E59–E60, and D51. Complexes formed in BD simulations using the PC of Ubbink and colleagues had a smaller Cu–Fe distance than the other two. These results suggest that different PC conformations may exist in solution with different capabilities of forming electron-transfer-active docks. All three types of complexes show electrostatic contacts between D42, E43, and D44 on PC and K187 on cyt f as well as between E59 on PC and K58 on cyt f. However, the PC of Ubbink and coworkers reveals additional contacts between D51 and cyt f as a result of the difference in backbone configuration. A second minor complex component was observed for the PC of Ubbink and co-workers and Xue and co-workers which had contacts between K187 on cyt f and E59 and E60 on PC rather than between K187 on cyt f and D42-D44 on PC as observed for the major components. This second type of complex may represent an earlier complex which rearranges to form a final complex capable of electron transfer. Professor Elizabeth L. Gross, Professor Emeritus of Biochemistry, The Ohio State University, passed away on June 27, 2007.     

The integration of a major hepatitis B virus gene into cell-cycle synchronized carrot cell suspension cultures and its expression in regenerated carrot plants

The percentage of cells successfully transformed by Agrobacteria is usually very low (not more than 10% and usually much less; Thomas et al., 1989). In this report we show that in carrot (Daucus carota L., ssp. sativus) cell suspensions transformation efficiency was strongly improved by using cell cycle synchronized cells. Fluorodesoxyuridine (FDU) was added for 24 h to inhibit thymidine synthesis. This blocked the cell cycle at the transition from G1- to S-phase. Then the block was released by applying thymidine. A high rate of transformation was obtained when Agrobacterium tumefaciens was added concurrently with thymidine. As examples of efficient and long term foreign gene expression in transgenic cells, the reporter enzyme -glucuronidase (GUS) as a model as well as the major hepatitis B virus surface protein were used. Both genes were linked to the MAS promoter. In carrot cell suspensions containing the viral gene, the corresponding viral protein was produced. In roots of mature transgenic carrot plants generated through somatic embryogenesis and raised in soil as well as in callus cultures derived thereof, the viral protein was also produced.     

A simple procedure for the isolation of pure nuclei from carrot embryos in synchronized cultures

A simple method is presented for the isolation of nuclei from somatic embryos of carrot (Daucus carota L.), which is applicable to small amounts of material in synchronized culture. The method employs buffers containing a high concentration of glycerol to stabilize the structure of the nuclei. Purification was carried out by centrifugation using preformed Percoll gradients. Treatment with cell wall-degrading enzymes prior to homogenization improved the efficiency of isolation and permitted a reproducible yield of nuclei. The pure preparations were obtained with an efficiency of approximately 60%. The isolated nuclei retained their morphological characteristics as demonstrated by phase — contrast and electron microscopy. Nuclear proteins displayed the expected species of histones by two-dimensional gel electrophoresis. The isolated nuclei showed high RNA polymerase activity.