Effects of ozone on cell organelles of alfalfa (Medicago sativa L.) seedlings

Seedlings of alfalfa (Medicago sativa L.) were exposed to different concentrations of atmospheric ozone (20–30 (control), 40–60, 65–80, and 85–120 ppb) in four distinct areas in the Riyadh region, so as to decide how ozone affected some of the seedling cellular organelles. Results acquired utilizing transmission electron microscopy demonstrated certifiable impacts to exist on the cell organelles in the tissues of both the leaf mesophyll and stem cortex; contrasted with control plants, the chloroplasts seemed enlarged, irregular, different sizes, decomposed, and possibly dissolved, while the plastoglobules seemed deformed, more widely spaced, and enlarged, also the vacuoles contained no clear non-living components. Moreover, some parts of the cytoplasmic membranes were ruptured, with only a few vesicles created at all concentrations, particularly in plants exposed to concentrations of 65–80 and 85–120 ppb, while no effects were noted in these organelles in control plants or plants exposed to 40–60 ppb. High concentrations (85–120 ppb) led to enlarged, irregularly shaped nuclei and chromatin intensification; however, no clear effects of ozone were noted on the shapes of chloroplast starch grains or the mitochondria in leaf mesophyll and cortex cells in the stem. The high ozone concentrations can cause negative effects on the growth of alfalfa seedlings, leading to imbalances in their vital functions and acceleration of aging, thus potentially decreasing the total plant yield. The discoveries hence propose that alfalfa plants should not be planted near polluted areas, and that they can be utilized as bioindicators of air pollution by ozone.     

Ultrastructural study of two rumen fungi:Piromonas communis andSphaeromonas communis

Two species of strictly anaerobic rumen fungi,Sphaeromonas communis andPiromonas communis, were examined by light and electron microscopy (scanning and transmission). Although morphologically different (vegetative body, number of flagella per zoospore), the ultrastructure of these two microorganisms was rather similar to that ofNeocallimastix frontalis andN. patriciarum. Two types of organelles were regularly found, i.e., isolated or associated ribosomes in the form of aggregates and hydrogenosome-like organelles with an amorphous content that may be involved in energy generation for these mitochondria-free strictly anaerobic fungi. UnlikeN. frontalis, the distribution of organelles was homogenous.     

Male red deer (<Emphasis Type="Italic">Cervus elaphus</Emphasis>) dispersal during the breeding season

Breeding dispersal can be of significant ecological and evolutionary importance. Yet, it is seldom considered in mammals. I present data on male red deer (Cervus elaphus) movements between sub-populations in southern Sweden during the rut. I investigated whether these movements could be breeding dispersal driven by mate competition. During the ruts of 1998–2009, I recorded 91 movements of males. The longest movement distance was 18.5 km. Dispersal was not restricted to yearlings or sub-adults, but also observed among adult stags. Of 91 movements observed, 7 were made by yearlings, 46 by sub-adults and 38 by adults. There was a significant move among yearlings and sub-adults towards areas with a higher ratio of females/adult males and towards areas with more females. The movements between rutting areas thereby seemed driven by sexual competition.     

Biochemical and Ultrastructural Changes in Tetrahymena pyriformis During Starvation*

SYNOPSIS. Certain of the ultrastructural and biochemical changes occurring during the first 25 hr of starvation in Tetrahymena pyriformis were studied. Ultrastructurally, numerous profiles of degenerating mitochondria were seen in the early stages of starvation. The presence of oxidizable substrate such as glucose and acetate did not prevent this degeneration. Numerous large nucleoli were formed, many of which seemed to be passing into the cytoplasm as forming autophagic vacuoles. There was a transient increase in Oil Red O-positive bodies, presumably lipid (triglycerides). The extent and duration of this increase were pronounced in the presence of acetate. The lipid droplets appeared to arise within the cisternae of the endoplasmic reticulum. Lipid reserves were apparently utilized prior to carbohydrates, as the disappearance of lipid droplets preceded glycogen utilization, both in the presence of acetate and in the absence of exogenous substrate. A considerable loss of cellular protein also occurred. In cells from inorganic medium supplemented with glucose, glycogen occupied much of the cell, leaving only islands of cell organelles. Acid phosphatase was localized, ultrastructurally, mainly in autophagic vacuoles which contained mitochondria and other cell organelles, and in association with small, double-membraned structures which seemed to be sequestering small areas of cytoplasm. Such sequestered areas also appeared within larger autophagic vacuoles. Residual bodies containing concentric whorls of myelin-like membranes surrounding a more solid core accumulated during starvation. Acid phosphatase activity decreased in amount but not in specific activity. The specific activity of cathespin doubled or tripled, but there was little change in total enzyme.     

THE FINE STRUCTURE OF OOGENESIS IN MARCHANTIA POLYMORPHA

Archegonium development, beginning with the archegonial initial and culminating in the mature egg, was studied with the electron microscope. The ultrastructural features of the beginning stages in development of the archegonium are relatively similar to one another. Plasmodesmata occur between all adjacent cells at this time. After the secondary central cell is formed these protoplasmic connections are lost, and both axial and parietal cell lineages begin to show signs of ultrastructural differentiation. The mature egg is characterized by cytoplasm rich in ribosomes and larger organelles. Mitochondria and simplified plastids commonly display a juxtaposed association. As far as could be ascertained the numerous plastids and mitochondria in the egg of Marchantia arise through division of preexisting organelles and are not formed anew from evaginations of the nucleus. Blebbing of the nucleus produces polymorphic organelles which appear to be pinched off into the cytoplasm. The mature egg also contains vacuoles and lipid bodies toward its periphery, while dictyosomes and extensive endoplasmic reticulum occur throughout. The space between the wall cells and the mature egg appears to contain an amorphous substance. No extra membrane was observed around the mature egg.     

Changes in the lysosome structure during the formation of zoospores inTrebouxia potteri

Summary Changes in the lysosome structures were examined by electron microscopy during the formation of zoospores inTrebouxia potteri. Lysosomes in vegetative cells were homogeneously filled with electron-dense material. At the beginning of zoospore formation, lysosomes invaginated or evaginated to take up mitochondria, ER, or cytoplasmic ground plasma. The ingested organelles became disorganized within the lysosomes. During this disruption of these organelles, the lysosomal contents became heterogeneous, suggesting a decrease in the amount of enzymes within the lysosomes. Golgi bodies and ER seemed to be involved with the disruption of the organelles, probably supplying some substances necessary for the functioning of the lysosomes. Amount of electron-dense materials decreased and, finally, only one to three small spherical aggregates remained in the lysosomes. Then the lysosomes appeared to shrink via loss of watery substances or cutting off of electron-transparent regions. After these changes in lysosome structure, nuclei started to divide successively for formation of the zoospores. The possibility is proposed that the drastic cytoplasmic changes operated by lysosomes trigger the following morphogenetic events in the formation of zoospores.Abbreviations ER endoplasmic reticulum - TGN trans Golgi network     

Cellular Damage to the Callus Cells of Potato Subjected to Freezing

Callus cells of potato (Solanum tuberosum L.) cv. Désirée were exposed to various subzero temperatures and examined for the freezing damage. In the cells subjected to –3 °C, plasma membranes appeared to be intact, while tonoplast seemed to be damaged and organelles to be swollen. After freezing at –6 °C, the damage became severe and plasma membranes were ruptured. After exposure to –10 °C, the damage was so severe that the cell organelles could not be recognised and cytoplasm became fragmented.     

Microbodies und Diaminobenzidin-Reaktion in den Acetat-Flagellaten Polytomella caeca und Chlorogonium elongatum

Summary In two forms of acetate flagellates, the colourless Volvocale Polytomella caeca and the green Volvocale Chlorogonium elongatum, cell organelles can be demonstrated which are ultrastructurally similar to microbodies of higher organisms. The organelles do not have a close association with the endoplasmic reticulum and are located in the peripheral cytoplasm between the elongated mitochondria. In Polytomella they exhibit more or less spherical profiles in section and have a maximum diameter of approximately 0.2–0.25 . In Chlorogonium the organelles occasionally have an elongated shape and are larger than in Polytomella. Employing the electron microscopic cytochemical reagent diaminobenzidine (DAB)/H₂O₂ to localize the microbodial marker enzyme catalase in these organelles, it was found that no accumulation of the electron-opaque product occurs in the microbodies either at alkaline or neutral pH or at room temperature or 37° C. Only the cristae of mitochondria are stained with the DAB reaction caused by cytochrome oxidase and possibly by a cytochrome peroxidase.Organelles of Polytomella caeca containing catalase or cytochrome oxidase can be separated by rate centrifugation of a crude particulate fraction on a sucrose gradient (Gerhardt, 1971). The particles isolated from the peak of catalase activity show the same fine structural characteristics as the microbodies in situ do. But again, there is no detectable staining of these organelles by the DAB/H₂O₂ reaction.The identity of the microbody-like particles in Polytomella caeca and Chlorogonium elongatum with microbodies in general is deduced despite the negative results in cytochemical localization of catalase in these organelles.     

bcd: A mutation affecting the width of organelle domains in the cortex of Tetrahymena thermophila

Summary A single-gene recessive mutation, bcd (broadened cortical domains), of Tetrahymena thermophila is characterized by a variable broadening of the spatial domains within which cortical organelles, including both the contractile vacuole pores (CVP) and oral apparatus (OA), are formed. The phenotype is not temperature-sensitive. During the development of the organelles of the mutant prior to cell division, extra CVPs and extra oral primordia (OP) appear near ciliary rows adjacent to the rows at which these structures normally form. In the later stages of development, some, but not all, of these extra structures are resorbed, or in the case of the oral domain, multiple adjacent OPs may be completely or partially integrated into a single enlarged OA. When multiple OAs persist, one or more of these may display a reversed orientation reminiscent of those encountered in janus mutants. However, unlike janus, bcd cells do not express any sign of a mirror-image global organization.Our results can best be accounted for by postulating that the bcd mutation affects some common determinant of the widths of both CVP and OA domains. Studies are in progress which explore the relationship between this width-determining mechanism(s) and the mechanism(s) determining the location of cortical organelles around the cell circumference.     

Alteration of epithelial cell lysosomal integrity induced by bacterial cholesterol‐dependent cytolysins

Bacterial pathogens can interfere during infection with host cell organelles, such as mitochondria, the endoplasmic reticulum‐Golgi system or nuclei. As important cellular functions are often compartmentalized in these organelles, their targeting allows pathogens to manipulate key host functions during infection. Here, we identify lysosomes as a new class of organelles targeted by the pathogenic bacterium Listeria monocytogenes. We demonstrate that extracellular Listeria, via secretion of the pore‐forming toxin listeriolysin O, alters lysosomal integrity in epithelial cells but not in macrophages. Listeriolysin O induces lysosomal membrane permeabilization and release of lysosomal content, such as cathepsins proteases, which remain transiently active in the host cytosol. We furthermore show that other bacterial pore‐forming toxins, such as perfringolysin O and pneumolysin, also induce lysosomes alteration. Together, our data unveil a novel activity of bacterial cholesterol‐dependent cytolysins.     

Differential effects of polymers PVP90 and Ficoll400 on storage stability and viability of Lactobacillus coryniformis Si3 freeze‐dried in sucrose

Aims: To investigate the effect of freeze‐dried Lactobacillus coryniformis Si3 on storage stability by adding polymers to sucrose‐based formulations and to examine the relationship between amorphous matrix stability and cell viability. Methods and Results: The resistance to moisture‐induced sucrose crystallization and effects on the glass transition temperature (T_g) by the addition of polymers to the formulation were determined by different calorimetric techniques. Both polymers increased the amorphous matrix stability compared to the control, and poly(vinyl)pyrrolidone K90 was more effective in increasing amorphous stability than Ficoll 400. The viability of Lact. coryniformis Si3 after storage was investigated by plate counts following exposure to different moisture levels and temperatures for up to 3 months. The polymers enhanced the cellular viability to different degrees, dependent upon polymer and storage condition. Conclusions: Polymers can be used to enhance the stability of freeze‐dried Lact. coryniformis Si3 products, but cell viability and matrix stability do not always correlate. The general rule of thumb to keep a highly amorphous product 50° below its T_g for overall stability seemed to apply for this type of bacterial products. We showed that by combining thermal analysis with plate counts, it was possible to determine storage conditions where cell viability and matrix stability were kept high. Significance and Impact of the Study: The results will aid in the rational formulation design and proper determination of storage conditions for freeze‐dried and highly amorphous lactic acid bacteria formulations. We propose a hypothesis of reason for different stabilizing effects on the cells by the different polymers based on our findings and previous findings.     

X-ray diffraction studies on melanins in lyophylized melanosomes

A series of experiments was performed on lyophylized melanosomes in order to analyze the melanin in the natural state as polymerized into these organelles and to verify in such biological amorphous material the possibility of obtaining intensity scattering curves from which Bragg distances can be calculated. The results confirm the feasibility of the method and show that melanins in melanosomes maintain many structural features of the purified form and that the biochemical composition of the organelles can be responsible for the observed differences in the diffractograms. The presence in melanosomes of supramolecular paracrystalline aggregates was also clearly demonstrated.     

Predation on northern shrimp (Pandalus borealis) by three gadoid species

Northern shrimp (Pandalus borealis) is targeted by commercial fisheries, but is also an important prey in the marine food web. In this study, stomach content data were used to study predation on shrimp by three gadoid species, cod (Gadus morhua), haddock (Melanogrammus aeglefinus) and whiting (Merlangius merlangus), in six inshore areas around Iceland. The results showed that shrimp was more important in the diet of cod compared with the other two predators. However, the overall predation pressure was similar for cod and haddock due to the high abundance of haddock. Therefore, even though shrimp is not the primary source of food for haddock, this species can have a substantial effect on shrimp stocks when haddock abundance is high. In addition, while cod and haddock did not select for any particular size of shrimp, whiting seemed to preferentially target juvenile shrimp. The results suggest that due to the overall effects of these three predators on shrimp stocks, gadoids need to be included in the management of shrimp stocks as predation is one of the major drivers in the development of this important prey stock.     

ULTRASTRUCTURE OF THE PSEUDOCAPILLITIUM AND SPORES OF THE MYXOMYCETE LYCOGALA EPIDENDRUM. LICEALES

The pseudocapillitium and spores of L. epidendrum were studied by transmission (TEM) and scanning electron microscopy (SEM). The SEM reveals that the pseudocapillitial surface is covered by bands of “wartlike” processes that alternate with non-ornamented regions. Otherwise, the pseudocapillitium is a hollow structure composed of three regions. The outer region is thin, electron dense and continuous with many irregular processes. Internal to this area is an amorphous region containing scattered electron dense material. The innermost region of the pseudocapillitium is thin, inconspicuous and usually electron dense. L. epidendrum possesses spores that are covered by a surface reticulum consisting of polygonal areas which are continuous with the outermost spore layer. The outer spore layer is thin and electron dense. The inner spore layer is an electron transparent region that contains granular or fibrillar components. Sections of spores showed a dense cytoplasm possessing most of the usual organelles along with microtubules and microbodies.     

Tobacco pollen tubes – a fast and easy tool for studying lipid droplet association of plant proteins

In recent years, lipid droplets have emerged as dynamic organelles rather than inactive storage sites for triacylglycerol. The number of proteins known to be associated with lipid droplets has increased, but remains small in comparison with those found with other organelles. Also the mechanisms of how lipid droplets are recognized and bound by proteins need deeper investigation. Here, we present a fast, simple and inexpensive approach to assay proteins for their association with lipid droplets in vivo that can help to screen protein candidates or mutated variants of proteins for their association in an efficient manner. For this, a system to transiently transform Nicotiana tabacum pollen grains was used because these naturally contain lipid droplets. We designed vectors for fast cloning of genes as fusions with either mVenus or mCherry. This allowed us to assay colocalization with lipid droplets stained with Nile Red and Bodipy 505/515, respectively. We successfully tested our system not only for proteins from Arabidopsis thaliana, but also for proteins from the moss Physcomitrella patens and the alga Chlamydomonas reinhardtii. The small size of the vector used allows easy exchange of codons by site‐directed mutagenesis. We used this to show that two proline residues in the proline knot of a caleosin are not essential for the binding of lipid droplets. We also demonstrated that peroxisomes are not associated with the lipid droplets in tobacco pollen tubes, which reduces the risk of false interpretation of microscopic data in our system.     

The <Emphasis Type="Italic">Arabidopsis</Emphasis> calmodulin-like proteins AtCML30 and AtCML3 are targeted to mitochondria and peroxisomes,respectively

Calmodulin (CaM) is a ubiquitous sensor/transducer of calcium signals in eukaryotic organisms. While CaM mediated calcium regulation of cytosolic processes is well established, there is growing evidence for the inclusion of organelles such as chloroplasts, mitochondria and peroxisomes into the calcium/calmodulin regulation network. A number of CaM-binding proteins have been identified in these organelles and processes such as protein import into chloroplasts and mitochondria have been shown to be governed by CaM regulation. What have been missing to date are the mediators of this regulation since no CaM or calmodulin-like protein (CML) has been identified in any of these organelles. Here we show that two Arabidopsis CMLs, AtCML3 and AtCML30, are localized in peroxisomes and mitochondria, respectively. AtCML3 is targeted via an unusual C-terminal PTS1-like tripeptide while AtCML30 utilizes an N-terminal, non-cleavable transit peptide. Both proteins possess the typical structure of CaMs, with two pairs of EF-hand motifs separated by a short linker domain. They furthermore display common characteristics, such as calcium-dependent alteration of gel mobility and calcium-dependent exposure of a hydrophobic surface. This indicates that they can function in a similar manner as canonical CaMs. The presence of close homologues to AtCML3 and AtCML30 in other plants further indicates that organellar targeting of these CMLs is not a specific feature of Arabidopsis. The identification of peroxisomal and mitochondrial CMLs is an important step in the understanding how these organelles are integrated into the cellular calcium/calmodulin signaling pathways.     

Physical and chemical characteristics of shallow embayments on the southern coast of Finland

Regular water-quality monitoring is yielding a great number of hydrographical and chemical data regarding the brackish Baltic Sea. However, present monitoring programmes often do not include shallow and sheltered near-shore areas, which are adjacent to human population and used for recreation purposes. We collected new near-shore water quality data from the southern coast of Finland that will allow the definition of the present state of these systems and may serve as reference data for possible future monitoring programmes. The data set comprises 45 small and shallow embayments with a mean surface area of 211 ha and a mean depth of 4.3 m. According to redundancy analysis (RDA), catchment land use explained 20.3% of the variance in the measured water chemistry. Agriculture had the strongest influence on the water quality of the study sites, capturing 13.1% of the variance. Nitrogen seemed to be strongly related to agriculture, whereas phosphorus was correlated with urban areas and industry. Higher than average nutrient concentrations regularly occurred at the mouths of large rivers, or close to human settlement, emphasising the significance of local sources to the nutrient concentrations of coastal waters. No increasing trend in nutrient or chlorophyll-a concentrations was observed from west to east in these shallow and sheltered nearshore areas. This contrasts with earlier results from pelagic samplings that report a marked influence of the River Neva and St. Petersburg on the nutrient status of the Gulf of Finland. Because these shallow embayments are strongly influenced by the local nutrient load, continued measures must be undertaken to reduce the nutrient load from land to prevent the continued deterioration and eutrophication of the nearshore, coastal waters.     

Genetic discontinuities and disequilibria in recently established populations of the plant pathogenic fungus Mycosphaerella fijiensis

Dispersal processes of fungal plant pathogens can be inferred from analysis of spatial genetic structures resulting from recent range expansion. The relative importance of long‐distance dispersal (LDD) events vs. gradual dispersal in shaping population structures depends on the geographical scale considered. The fungus Mycosphaerella fijiensis, pathogenic on banana, is an example of a recent worldwide epidemic. Founder effects in this species were detected at both global and continental scale, suggesting stochastic spread of the disease through LDD events. In this study, we analysed the structure of M. fijiensis populations in two recently (∼1979–1980) colonized areas in Costa Rica and Cameroon. Isolates collected in 10–15 sites distributed along a ∼250‐ to 300‐ km‐long transect in each country were analysed using 19 microsatellite markers. We detected low‐to‐moderate genetic differentiation among populations in both countries and isolation by distance in Cameroon. Combined with historical data, these observations suggest continuous range expansion at the scale of banana‐production area through gradual dispersal of spores. However, both countries displayed specific additional signatures of colonization: a sharp discontinuity in gene frequencies was observed along the Cameroon transect, while the Costa Rican populations seemed not yet to have reached genetic equilibrium. These differences in the genetic characteristics of M. fijiensis populations in two recently colonized areas are discussed in the light of historical data on disease spread and ecological data on landscape features.     

Chilling stability of microtubules in root-tip cells of cucumber

The chilling stability of microtubules was investigated in root-tip cells of cucumber (Cucumis sativus L.) using immunofluorescence labeling and a confocal laser scanning microscope. After low temperature (4°C) treatment for 5 h, we found not only chilling-stable cortical microtubules under the plasma membrane of the root-tip cells, but also chilling-stable punctate microtubules in the cytoplasm, which might be the microtubules associated with organelles. 6-Dimethylaminopurine (6-DMAP), an inhibitor of protein kinase, reduced the chilling stability of the cortical microtubules in the cells. When the root-tip cells were restored to normal growth temperature (28°C), the recovery of the microtubules seemed to be closely associated with the microtubules still remaining in these cells after chilling treatment.Abbreviations 6-DMAP 6-Dimethylaminopurine - DMSO Dimethyl sulfoxide - FITC Fluorescein isothiocyanate - MTOCs Microtubule-organizing centers - PPB Pre-prophase bandCommunicated by H. WangJ.-L. Zhao and X.-J. Li are considered joint first authors     

A LIGHT- AND ELECTRON-MICROSCOPIC STUDY OF PRIMARY HETEROGENEITY IN THE EGGS OF TWO BROWN ALGAE

The mature eggs of Zonaria farlowii and Dictyota binghamiae exhibit a distinctively symmetrical arrangement of organelles. In mature oogonia plastids occur in two groups in the central region of the cell and vacuoles of several types are arranged in peripheral layers. Before the mature egg is released, an amorphous “mucilage layer” developes inside the wall of the oogonium. After fertilization the plastids become uniformly distributed. When the eggs of Zonaria develop parthenogenetically the primary heterogeneity is retained. The symmetrical arrangement of membranes seen in the oocytes of these plants may represent an organizing system that influences cytodifferentiation and perhaps subsequent embryology.