Redox-state of intactNitella cells: dependency on intracellular pH and photosynthesis

Summary The present paper describes the construction and properties of a Pt/Ir-semi-microelectrode and its application as a redoxsensitive electrode in intact cells of the giant algaNitella. For compartmental analysis of the stationary redox-state voltage (E_RED), a value reflecting the interaction of the dominant redox couples with a Pt/Ir-electrode, the redox-sensitive electrode was inserted into the vacuole of leaf cells or cytoplasm enriched fragments (CEF) fromNitella internodal cells. After correction for the membrane voltage, measured with a second, conventional voltage electrode, E_RED values of+237±93mVand+419±51 mV with respect to a normal H⁺-electrode were obtained for cytoplasm and vacuole, respectively. The redox-state of the cell culture medium was+604 mV. The steady state E_RED in the cytoplasm can be perturbed by experimental treatments: indirect acidification of the cytoplasm by an external pH jump from 7.5 to 5.8 and direct acidification, by acid loading with 5 mM butyrate, both resulted in a positive shift of E_RED, i.e., to an increase in cytoplasmic oxidation. At the same time the membrane depolarized electrically following the external pH jump, but hyperpolarized in response to acid loading. The data demonstrate the direct dependence of cytoplasmic redox state on intracellular pH, probably due to enhanced oxidation of protonated redox couples favoured by mass action. The electrical membrane voltage changes were not correlated with the shift in cytoplasmic E_RED. This demonstrated that redox energy does not determine the electrical membrane voltage. Cytoplasmic E_RED was also affected by photosynthesis. When CEFs were transferred from light to dark, or exposed to 10M 3-(3,4-dichlorophenyl)-1,l-dimethylurea (DCMU), E_RED shifted negatively (more reduced) by 6.4±4.5mV or 4.2±2mV, respectively. These data compare favourably with biochemical estimates of cytoplasmic pyridin nucleotides which also show an increase in cytoplasmic reduction in the dark. Therefore, it is unlikely that diffusable reducing equivalents are supplied to the cytoplasm from photosynthetically-active chloroplasts to act as secondary messengers.Abbreviations E_M transmembrane voltage - E_RED redox-state voltage - E₀ midpoint-redox-voltage - APW artificial pond water - CEF cytoplasm enriched fragment     

Cytological characterization of isolated sperm cells of perennial ryegrass (Lolium perenne L.)

Summary The cytoplasmic content and the distribution of intramembrane particles (IMPs) of the plasma membrane of isolated sperm cells of perennial ryegrass (Lolium perenne L.) have been characterized using flow cytometry, transmission electron microscopy, confocal scanning laser microscopy and freeze-fracture studies. The isolated haploid sperm cells contain a variety of cell organelles with the exception of microtubules. Proplastids and plastids with starch were observed, although only rarely. Vacuoles containing remnants of organelles and stacked lamellae of endoplasmic reticulum with cytoplasmic inclusions were observed frequently, indicating that autophagy takes place. The number of mitochondria varies from 11 to 26 with an average of 17. Generally, the nucleus has a lobed shape and displays various interphasic stages of chromatin condensation. The analysis of the number of mitochondria and the nuclear state did not show evidence of sperm cell dimorphism. The cytological variability observed, could be explained by differences in developmental stages already present in vivo at the moment of isolation. No correlation between the number of mitochondria and the nuclear cross-sectioned area and/or the condensation state of the chromatin could be found. The density of intramembrane particles of the plasma membrane on the exoplasmic fracture face is more than twice that on the protoplasmic fracture face. That is the opposite of what was found for sporophytic cells of perennial ryegrass. These results are discussed in relation to the potential use of these cells for biotechnology and developmental studies.     

Competition between electron acceptors in photosynthesis: Regulation of the malate valve during CO2 fixation and nitrite reduction

For maximal rates of CO₂ assimilation in isolated intact spinach chloroplasts the generation of the adequate NADPH/ATP ratio is achieved either by cyclic electron flow around photosystem I or by linear electron transport to oxaloacetate, nitrite or oxygen (Mehler-reaction). The interrelationships between these poising mechanisms turn out to be strictly hierarchical. In the presence of antimycin A, an inhibitor of ferredoxin-dependent cyclic electron transport, the reduction of both, oxaloacetate and nitrite, but not that of oxygen restores CO₂ fixation. When oxaloacetate and nitrite are added at low concentrations simultaneously during steady-state CO₂ fixation, the reduction of nitrite is clearly preferred over the reduction of oxaloacetate, but CO₂ fixation is not influenced. Nitrite reduction is not decreased upon addition of oxaloacetate, but vice versa. This is due to the regulation of NADP-malate dehydrogenase activation by electron pressure via the ferredoxin/thioredoxin system on the one hand, and by the NADPH/(NADP+NADPH) ratio (anabolic reduction charge, ARC) on the other hand. Thus the closing of the malate valve prevents drainage of reducing equivalents from the chloroplast (1) when a low ARC indicates a high demand for NADPH in the stroma and (2) when nitrite reduction reduces the electron pressure at ferredoxin. The malate valve is opened when cyclic electron transport is inhibited by antimycin A. Under these conditions the rate of malate formation is higher than in the absence of the inhibitor even in the presence of oxaloacetate, thus indicating that the regulation of the malate valve functions at various redox states of the acceptor side of Photosystem I.Abbreviations ARC anabolic reduction charge (NADPH/(NADP+NADPH)) - Chl chlorophyll - DTT dithiothreitol; Fd-ferredoxin - NADP-MDH NADP-malate dehydrogenase - OAA oxaloacetate - PS photosystem - qN non-photochemical quenching - qP photochemical quenching - _E quantum efficiency of PS II Dedicated to Prof. Dr. Hans Walter Heldt on the occasion of his 60th birthday.     

Streptococcal erythrogenic toxin type C is not a phosphorylated protein. Description of two different purification procedures and investigation of its phosphorylation state

Abstract Erythrogenic toxin type C (ETC) from different streptococcal group A strains was successively purified by absorption on phenylsepharose, acidic dialysis of the eluate at 40% saturated ammonium sulphate solution, CM-Sepharose chromatography, finally by immunoaffinity chromatography on monoclonal antibodies. Second, after growing of bacteria in the presence of [³²P]orthophosphate to phosphorylate ETC, the ETC was purified with phenylsepharose following immunoaffinity chromatography. The occurrence of phosphoamino acids in the purified ETC was investigated by an immunoassay. No phosphoamino acids could be detected in the ETC molecule. Also after radiolabelling with ³²P it was not possible to demonstrate a radioactive signal. The treatment with alkaline phosphatase has no influence on the mitogenicity or position of ETC in isoelectric focusing. The results obtained led to the conclusion that in contrast to the literature, ETC is not a phosphorylated protein.     

Thermodynamics of staphylococcal nuclease denaturation. II. The A-state.

Staphylococcal nuclease, at low pH and in the presence of high salt concentrations, has previously been proposed to exist in a partially folded or molten globule form called the "A-state" (Fink et al., 1993, Protein Sci 2:1155-1160). We have found that the A-state of nuclease at pH 2.1 in the presence of moderate to high salt concentrations and at low temperature exists in a substantially folded form structurally more similar to a native state. The A-state has the far-UV circular dichroism spectra characteristic of the native protein, which indicates that it has a large degree of secondary structure. Upon heating, the A-state denatures with a sigmoidal change in far-UV ellipticity and an observable peak in a differential scanning calorimeter trace, indicating that it is thermodynamically distinct from the denatured state. Three different mutations in a residue normally buried in the protein's core stabilize or destabilize the A-state in the same way as they affect the denaturation of the native state. The A-state must, therefore, contain at least some tertiary packing of side chains. Unlike the native state, which shows cold denaturation at low temperatures, the A-state is most stable at temperatures below 0 degrees C.     

Monte carlo simulations of protein folding. II. Application to protein A,ROP, and crambin

The hierarchy of lattice Monte Carlo models described in the accompanying paper (Kolinski, A., Skolnick, J. Monte Carlo simulations of protein folding. I. Lattice model and interaction scheme. Proteins 18:338–352, 1994) is applied to the simulation of protein folding and the prediction of 3-dimensional structure. Using sequence information alone, three proteins have been successfully folded: the B domain of staphylococcal protein A, a 120 residue, monomeric version of ROP dimer, and crambin. Starting from a random expanded conformation, the model proteins fold along relatively well-defined folding pathways. These involve a collection of early intermediates, which are followed by the final (and rate-determining) transition from compact intermediates closely resembling the molten globule state to the native-like state. The predicted structures are rather unique, with native-like packing of the side chains. The accuracy of the predicted native conformations is better than those obtained in previous folding simulations. The best (but by no means atypical) folds of protein A have a coordinate rms of 2.25 Å from the native Cα trace, and the best coordinate rms from crambin is 3.18 Å. For ROP monomer, the lowest coordinate rms from equivalent Cαs of ROP dimer is 3.65 Å. Thus, for two simple helical proteins and a small α/β protein, the ability to predict protein structure from sequence has been demonstrated. © 1994 John Wiley & Sons, Inc.     

Photosynthetic gas exchange response of poplars to steady-state and dynamic light environments

The steady-state and dynamic photosynthetic response of two poplar species (Populus tremuloides and P. fremontii) to variations in photon flux density (PFD) were observed with a field portable gas exchange system. These poplars were shown to be very shade intolerant with high light saturation (800 to 1300 mol photons m^–2 s^–1) and light compensation (70 to 100 mol m^–2 s^–1) points. Understory poplar leaves showed no physiological acclimation to understory light environments. These plants become photosynthetically induced quickly (10 min). Activation of Rubisco was the primary limitation for induction, with stomatal opening playing only a minor role. Leaves maintained high stomatal conductances and stomata were unresponsive to variations in PFD. Leaves were very efficient at utilizing rapidly fluctuating light environments similar to those naturally occurring in canopies. Post-illumination CO₂ fixation contributed proportionally more to the carbon gain of leaves during short frequent lightflecks than longer less frequent ones. The benefits of a more dynamic understory light environment for the carbon economy of these species are discussed.     

An ecosystems approach to base-rich freshwater wetlands,with special reference to fenlands

A survey of base-rich wetlands in The Netherlands is presented. The main area of their occurrence is the low-lying Holocene part of the country, until some thousand years ago a large and coherent wetland landscape: the Holland wetland. The development of various parts of the Holland wetland into marshes, fens and bogs can be understood from hydrological relations in mire basins, as recognized in the distinction of primary, secondary and tertiary mire basin stages. Presently, the remnants of the Holland wetland are separate base-rich wetlands. The succession of their vegetation reflects various abiotic conditions and human influences. Three main developmental periods are distinguished as regards these factors. The first, geological period of mire development is seen as a post-glacial relaxation, with the inertia due to the considerable mass of wetland as a stabilizing factor. Biological “grazing” influences, as an aspect of utilization by humans, converted base-rich wetlands to whole new types in the second, historical period. Presently, mass and harvesting have decreased in importance, and actual successions in terrestrializing turbaries seem to reflect rapidly changing environmental conditions. Human control could well become the most important factor in the future development of wetland nature. The present value of open fen vegetation strongly depends on the continuation of the historical harvesting. The development of wooded fen may help to increase the mass of wetland in the future. Best results in terms of biodiversity are expected when their base state is maintained through water management. The vegetation and hydrology of floating fens in terrestrializing turbaries is treated in some more detail. Various lines and phases in the succession are distinguished. Open fen vegetation at base-rich, yet nutrient-poor sites is very rich in species threatened elsewhere. The fast acidification of certain such fens is attributed to hydrological and management factors. This acidification is illustrated in the profile of a floating raft sample. At the scale of these small fens, the elemental structure comprising base-rich fen, transitional fen and bog vegetation, is not as stable as it was in the large Holland wetland. A critical role seems to be played by the supply of bases with the water influx. The changing base state is supposed to change the nutrient cycling to such an extent that it would be correct to call this trophic excitation of the ecosystem, rather than just eutrophication. Eutrophication indicates a quantitative reaction to an increased nutrient supply, the internal system being unaltered. The drainage of fens, resulting in an increased productivity of the vegetation, provides another example of excitation, to the effect that the functional system is dramatically changed internally.     

Environmental and physiological factors influencing phototactic flight of Bemisia tabaci

Abstract. Experiments were conducted in a glasshouse and in the laboratory to determine the environmental and physiological parameters that affect flight behaviour of Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae). The number of whiteflies taking off and exhibiting a positive response to sky light in the glasshouse was greatest from 08.30 to 10.00 hours. During peak flight activity less than 5% of the population engaged in phototactic orientation. Temperature was the best single predictor for the phototactic response, accounting for 75% of the variability in whitefly ascent. Sex ratios were determined for individuals remaining on poinsettia, Euphorbia pulcherrima (Willd.), and for individuals that exhibited phototactic orientation; both groups deviated from an expected 1:1 ratio. Males were more prevalent on the plants (1:0.76), whereas females were much more prevalent (1:3.02) among the whiteflies responding to sky light. A higher percentage of the females displaying a phototactic response contained eggs when compared to females remaining on poinsettia (87 v 65%). The two groups of females did not differ significantly in their weights, but males that remained on the plants were heavier than males that responded to sky light. Mark–recapture studies and experiments where phototactic individuals were removed from the population established that the response was short-lived. Whiteflies exhibiting a phototactic response in the glasshouse were more likely to exhibit long-duration, phototactic flights in a vertical flight chamber when compared to individuals that remained on poinsettia (80.7 ± 6.7 v 36.0 ± 5.8% phototactic response; 7.0 ±3.2 v 0.7±0.2min flights). There was also less deviation in flight across the horizontal plane among the individuals that exhibited a positive response to sky light in the glasshouse in comparison to individuals that remained on their host. However, initial rates of climb were not significantly different between the two groups of whiteflies.     

Maximal ecological diversity exceeds evolutionary diversity in model ecosystems

Understanding community saturation is fundamental to ecological theory. While investigations of the diversity of evolutionary stable states (ESSs) are widespread, the diversity of communities that have yet to reach an evolutionary endpoint is poorly understood. We use Lotka–Volterra dynamics and trait-based competition to compare the diversity of randomly assembled communities to the diversity of the ESS. We show that, with a large enough founding diversity (whether assembled at once or through sequential invasions), the number of long-time surviving species exceeds that of the ESS. However, the excessive founding diversity required to assemble a saturated community increases rapidly with the dimension of phenotype space. Additionally, traits present in communities resulting from random assembly are more clustered in phenotype space compared to random, although still markedly less ordered than the ESS. By combining theories of random assembly and ESSs we bring a new viewpoint to both the saturation and random assembly literature.