Formation and Growth of Bryopsis hypnoides Lamouroux Regenerated from Its Protoplasts

Tissue culture, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and spectra analysis were used for studying the aggregation mechanism of protoplasts from Bryopsis hypnoides Lamouroux and the discrepancy between the protoplast-regenerated plants and the wild type. The aggregation of protoplasts from B. hypnoides was observed in natural seawater and artificial seawater with different pH values, and the location and mechanism of the materials causing the aggregation were also studied. Results showed that the protoplasts could aggregate into some viable spheres in natural seawater and subsequently grow into mature individuals. Aggregation of the protoplasts depended exclusively upon the pH value (6-11), and the protoplasts aggregated best at pH 8-9. Some of the extruded protoplasts were separated into two parts by centrifugation: the pellet (PO) and the supernatant (PL). The PO could aggregate in artificial seawater (pH8.3) but not in PL. No aggregation was found in PO cultured in natural seawater containing nigericin, which can dissipate the proton gradients across the membrane. These experiments suggest that the aggregation of protoplasts is proton-gradient dependent and the materials causing the aggregation were not in the vacuolar sap, but located on the surface or inside the organelles. Furthermore, the transfer of the materials across the membrane was similar to △pH-based translocafion (△pH/TAT) pathway that occurs in the chloroplasts of higher plants and bacteria. Obvious discrepancies in both the total soluble proteins and the ratio of chlorophyll a to chlorophyll b between the regenerated B. hypnoides and the wild type were found, which may be related to the exchange of genetic material during aggregation of the organelles. In the process of development, diatom Amphora coffeaeformis Agardh attached to the protoplast aggregations, retarding their further development, and once they were removed, the aggregations immediately germinated, which showed that diatoms can affect the development of other algae.     

Culturing the Segments of Bryopsis hypnoides Lamouroux Thalli Regenerated from Protoplast Aggregations

Bryopsis hypnoides Lamouroux was regenerated In vitro from the protoplasm squeezed out from wild algae and the regenerated Individuals had an advantage over Indlvlduala from the wild In terms of growth. Culturing of segments of thalll also showed that segments from the regenerated algae grew better than those from individuals from the wild. The segment that corresponds to a part of a cell, at least a part of the protoplasm, can develop Into a mature Individual, Including rhlzold and thallus, suggesting that a multlnucleate alga, such as B. hypnoides, Is different from higher plants, whose totlpotancy Is based on an Intact cell （or protoplasm）. Further cultivation of mature Individuals from segments Indicated that the organelles In the thallus had two ways In which they could survive when the alga was on the decline： （Ⅰ） the organelles were transferred Into a ball with a gelatinous envelope through a formed pipe and the ball seemed to be capable of being propagated; and （Ⅱ） the organelles were aggregated In the thallus and then moved to the outside. An Interesting result Is that one organelle aggregation located outside the thallus germinated and developed Into a mature alga, although most organelle aggregations gradually lost vitality and died. The results of the present study reveal that the aggregation of organelles can regenerate a stronger organism than Individuals from the wild owing to the complete exchange of genetic material and may possibly enable organelles to survive in unfavorable surroundings.     

Assembly of the subcellular parts of <Emphasis Type="Italic">Bryopsis hypnoides</Emphasis> Lamouroux into new protoplasts

The chloroplasts, mitochondria, and protoplasm devoid of mature chloroplasts (PMC) of Bryopsis hypnoides Lamouroux were isolated by low-speed and sucrose density centrifugation. The PMC aggregated in artificial seawater, and then protoplasts without mature chloroplasts (PtMCs) were formed. Transmission electron microscopy and cytochemical studies indicated that there were mitochondria, nuclei, vesicles, and other small cell organelles in the PtMCs. Scanning electron microscopy showed that there were holes on the surface of 1-h PtMCs and then fewer holes on the surface of 24-h PtMCs, suggesting that a healing process occurred. The plasma membrane was formed over the surface of the PtMCs. However, the cell wall was not regenerated, and the newly formed PtMCs were ruptured and died in 3 days. Light intensity during alga maintenance before use influenced significantly (one-way ANOVA, P < 0.0001) on the number of PtMCs formed; the highest number of PtMCs was formed at 20μmol/(m² s). When isolated chloroplasts were transferred into seawater, there were only two or three chloroplasts aggregated together. However, isolated mitochondria and the mixed six layers of cell organelles (separated by sucrose density centrifugation) could not aggregate in the artificial seawater. This indicates that the conjunction of cell organelles is important for their aggregation. Published in Russian in Fiziologiya Rastenii, 2009, Vol. 56, No. 1, pp. 123–130. The text was submitted by the autors in English.     

Assembly of the protoplasm of Codium fragile (Bryopsidales, Chlorophyta) into new protoplasts

The cell organelles of the coenocytic alga Codium fragile (Sun) Hariot aggregated rapidly and protoplasts were formed when its protoplasm was extruded out in seawater. Continuous observation showed that there were long and gelatinous threads connecting the cell organelles. The threads contracted, and thus the cell organelles aggregated into protoplasmic masses. The enzyme digestion experiments and Coomassie Brilliant Blue and Anthrone stainings showed that the long and gelatinous threads involved in the formation of the protoplasts might Include protein and saccharides as structure components. Nile Red staining Indicated that the protoplast primary envelope was non-lipid at first, and then lipid materials Integrated Into its surface gradually. The fluorescent brightener staining Indicated that the cell wall did not regenerate in the newly formed protoplasts and they all disintegrated within 72 h after formation. Transmission electron microscopy of the cell wall of wild C. fragile showed electron-dense material embedded in the whole cell wall at regular intervals. The experiments indicated that C. fragile would be a suitable model alga for studying the formation of protoplasts.     

Cellular Fractionation of Chlamydomonas reinhardii with Emphasis on the Isolation of the Chloroplast

A method for cellular fractionation of Chlamydomonas reinhardii, SAG 11-32/b, and isolation of intact chloroplasts from synchronized cells of the alga is described. The procedure for cell fractionation comprises essentially four steps: (1) protoplast production with autolysine; (2) lysis of the protoplasts with digitonin; (3) aggregation of broken protoplasts; and (4) separation of organelles by differential centrifugations.

Replacing the differential centrifugations (step 4) by Percoll cushion centrifugations yields intact chloroplasts. Starting with 100 milliliters of an algal culture containing 3000 micrograms chlorophyll, intact chloroplasts with 100 to 200 micrograms of chlorophyll can be isolated. Envelope integrity is about 90% (ferricyanide assay). Examination of the chloroplasts by electron microscopy and marker enzyme activities indicated some mitochondrial and cytoplasmic contamination.

     

Age-specific aggregation of wild juvenile Atlantic salmon Salmo salar at cool water sources during high temperature events

This study quantified the use of cool water sources by wild 0+, 1+ and 2+ year Atlantic salmon Salmo salar during high water temperatures ( i.e. >23° C) in summer 1995 and 2004. During these events, 0+ year Atlantic salmon did not aggregate or increase in abundance in cool water sites. Interestingly, 1+ and 2+ year Atlantic salmon numbers increased in cool water sites. In addition, these older juveniles formed numerous, discrete aggregations along the plume created by a tributary with aggregation locations being similar between years. Aged 2+ year fish aggregations were at the coolest sources whereas 1+ year aggregations were in locations cooler than the main river. Fish in aggregations on average used deeper sections (average depth: 380 mm) compared with the coolest available habitat in the thermal plume (average depth: 230 mm). Hence, during high temperature events, older juvenile Atlantic salmon moved to cooler water sites and then aggregated in deeper microhabitats.     

Genetic structure of an Apis dorsata population: the significance of migration and colony aggregation

Eight microsatellite loci were used to investigate the genetic structure of the giant honeybee (Apis dorsata) population in northeast India. This species migrates seasonally between summer and winter nesting sites, and queens appear to return to their previously occupied site. Furthermore, there is a strong tendency for colonies of this species to aggregate at perennially utilized nesting sites that may be shared by more than 150 colonies. These behavioral features suggest that colonies within aggregations should be more related than random colonies, but that the long-distance migration could act to minimize genetic differentiation both between geographical areas and within aggregations. Our genetic study supports these conjectures arising from natural history. A. dorsata aggregations are comprised of colonies that share more alleles than expected by chance. Although queens heading neighboring colonies are not close relatives, fixation indices show significant genetic differentiation among aggregation sites. However, there appears to be sufficient gene flow among aggregations to prevent high degrees of relatedness developing between colonies within aggregations. The results also suggest that there is significant population structuring between geographical regions, although the level of structuring caused by aggregation exceeds the differentiation attributable to geographic region.     

Seawater-resistant, non-spherical protoplasts from seagrass leaves

Two distinct types occurred among enzymatically isolated protoplasts from leaves of eelgrasses ( Zostera marina L., Z. japonica Ascherson and Phyllospadix iwatensis Makino). Spherical protoplasts with a smooth cell membrane were obtained only from young leaf tissues at the basal portions of blades protected from seawater by tightly enclosing sheaths. Non-spherical protoplasts had a highly invaginated cell membrane and were obtained from mature leaf blades, where the cells also in situ have this type of membrane. The protoplasts from mature leaves were rather rigid in shape and resistant to wide ranges of osmotic potential and salinity without change in their non-spherical shape, while the spherical protoplasts were rapidly destroyed in seawater. Detergents lysed the spherical protoplasts but not the non-spherical ones, suggesting that the highly invaginated enclosing structures of the non-spherical protoplasts contained detergent-resistant materials. Thus, the seagrass leaf cells develop seawater resistance, and this change alters the nature of the enclosing structures during the growth of the leaf blades. The non-membranous enclosing structures and their characteristic materials in the mature leaf cells remain to be defined.     

Photosynthetic performance of regenerated protoplasts from disintegrated cells of <Emphasis Type="Italic">Bryopsis hypnoides</Emphasis> (Chlorophyta)

The photosynthetic performances of regenerated protoplasts of Bryopsis hypnoides, which were incubated in seawater for 1, 6, 12, and 24 h, were studied using chlorophyll (Chl) fluorescence and oxygen measurements. Results showed that for the regenerated protoplasts, the pigment content, the ratios of photosynthetic rate to respiration rate, the maximal photosystem II (PSII) quantum yield (F_v/F_m), and the effective PSII quantum yield (Φ_PSII) decreased gradually along with the regeneration progress, indicated that during 24 h of regeneration there was a remarkable reduction in PSII activity of those newly formed protoplasts. We assumed that during the cultivation progress the regenerated protoplasts had different photosynthetic vigor, with only some of them able to germinate and develop into mature thalli. The above results only reflected the photosynthetic features of the regenerated protoplasts at each time point as a whole, rather than the actual photosynthetic activity of individual aggregations. Further investigation suggested a relationship between the size of regenerated protoplasts and their viability. The results showed that the middle-sized group (diameter 20–60 μm) retained the largest number of protoplasts for 24 h of growth. The changes in F_v/F_m and Φ_PSII of the four groups of differently sized protoplasts (i.e. < 20, 20–60, 60–100, and > 100 μm) revealed that the protoplasts 20–60 μm in diameter had the highest potential activity of the photosynthetic light energy absorption and conversion for several hours.     

Characterization of large basking shark Cetorhinus maximus aggregations in the western North Atlantic Ocean

Cetorhinus maximus aggregations recorded during extensive aerial survey efforts off the north‐eastern United States between 1980 and 2013 included aggregations centring on sightings with group sizes of at least 30 individuals. These aggregations occurred in summer and autumn months and included aggregation sizes of up to 1398 individuals, the largest aggregation ever reported for this species. The aggregations were associated with sea surface temperatures of 13–24° C and chlorophyll‐a concentrations of 0·4–2·6 mg m^?3 and during one aggregation, a high abundance of zooplankton prey was present. Photogrammetric tools allowed for the estimation of total body lengths ranging between 4 and 8 m. Characterization of these events provides new insight into the potential biological function of large aggregations in this species.     

A BROAD SPECTRUM ARTIFICIAL SEA WATER MEDIUM FOR COASTAL AND OPEN OCEAN PHYTOPLANKTON1

A new artificial seawater medium has been tested with 83 strains of coastal and open ocean phytoplankton from 11 different algal classes. The cultures were carried through four transfers, representing a period of eight weeks for most species. Only three species could not be maintained in the enriched artificial seawater, and 16 species, mainly from the Prymnesiophyceae and Dinophyceae, had reduced final cell yields compared to those grown in enriched natural seawater. Since 77% of the species tested grew equally well in enriched artificial or natural seawater and more than 95% could be maintained in the artificial medium, this recipe is useful over a broad spectrum of species. The artificial seawater base was enriched with a modified ES enrichment solution; the primary modifications were the omission of Tris and the addition of Si. Enriched medium was autoclaved without precipitation by lowering the pH before autoclaving. This was accomplished by adding equimolar amounts of Na-HCO₃ and HCl which produced NaCl and CO₂ during the heating process. When no pH buffer was used, precipitation could only be avoided by autoclaving the artificial seawater base as two separate salt solutions (with Ca and Sr separated from CO₃^?2 and SO₄^?2), cooling, mixing and aseptically adding the sterilized enrichment solution.     

MOLECULAR CHARACTERIZATION OF THE LECTIN,BRYOHEALIN, INVOLVED IN PROTOPLAST REGENERATION OF THE MARINE ALGA BRYOPSIS PLUMOSA (CHLOROPHYTA)1

When a coenocytic cell of the green alga Bryopsis plumosa (Hudson) C. Agardh was cut open and the cell contents expelled, the cell organelles agglutinated rapidly in seawater to form protoplasts. This process was mediated by a lectin, Bryohealin. The full sequence of the cDNA encoding Bryohealin was obtained, which consisted of 1,101 base pairs (bp), with 24 bp of 5′ untranslated region (UTR) and 201 bp of 3′ UTR. It had an open reading frame (ORF) of 771 bp encoding 257 amino acid residues. A signal peptide consisted of 22 amino acids presented before the start codon of Bryohealin, indicating that this lectin was a vacuolar (storage) protein. The C‐terminal sequence of Bryohealin was composed of antibiotic domains, suggesting that this lectin could perform two functions: (i) aggregation of cell organelles in seawater and (ii) protection from bacterial contamination for successful protoplast regeneration. The BLAST search result showed that Bryohealin had little sequence homology with any known plant lectins, but rather resembled animal lectins with fucolectin domains. The expression of recombinant Bryohealin (rBryohealin) was obtained in the Escherichia coli system.     

In vitro reconstitution of fish melanophore pigment aggregation

Movement and positioning of melanophore pigment organelles depend on microtubule- and actin-dependent motors, but the regulation of these forces are poorly understood. Here, we describe a cell free and fixed time motility assay for the study of the regulation of microtubule-dependent pigment organelle positioning in vitro. The assay involves introduction of microtubule-asters assembled in clam oocyte lysates into lysates prepared from Fundulus heteroclitus melanophores with either aggregated or dispersed pigment. When asters were introduced in lysates prepared from melanophores with dispersed pigment, pigment organelles bound astral microtubules and were evenly distributed throughout the aster. In contrast, when asters were introduced into lysates prepared from melanophores with aggregated pigment, pigment organelles accumulated around the centrosome, mimicking a pigment aggregate. The addition of anti-dynein intermediate chain antibody (m74-1), previously shown to interfere with binding of dynactin to dynein and thereby causing detachment of dynein from organelles, blocked the ATP-dependent aggregation of pigment in vitro and induced a depletion of pigment from the centrosomal area. The results show that dynein is essential for pigment aggregation and involved in maintenance of evenly dispersed pigment in vitro, analogous to cellular evidence, and suggest a possible role for dynactin in these processes as well.     

亚热带退化森林不同恢复方式对土壤团聚体胶结物质及稳定性的影响

退化森林的恢复一直是林业和生态学研究的热点。良好的森林土壤结构有助于林木生长发育,作为土壤最基本的结构单元,土壤团聚体组成和稳定性是衡量土壤肥力和质量的重要指标,其团聚结构的形成依靠土壤胶结物质。然而,土壤胶结物质与团聚体稳定性之间的关系尚不确定。为探明亚热带退化森林的不同恢复方式对团聚体稳定性的影响及其潜在机制,以自然恢复的次生林为对照(CK),选取了亚热带三种常见的人工林：一代杉木林(P1)、二代杉木林(P2)和黄山松林(P3),测定土壤团聚体稳定性及其胶结物质的含量,并分析了各胶结物质对团聚体稳定性的影响。研究发现：不同森林恢复方式显著影响了土壤pH值、碳氮比、速效磷含量、团聚体的组成和稳定性。所有森林类型中,三种人工林的团聚体稳定性显著高于CK,P2的大团聚体所占比例最大,团聚体稳定性最高。恢复方式显著影响了土壤游离氧化铁含量、菌根密度和易提取球囊霉素相关土壤蛋白(EEG)含量。团聚体稳定性与游离氧化铁含量、菌根密度呈正相关,但与EEG含量呈负相关。研究结果表明,亚热带退化森林的人工恢复比自然恢复更有助于增强土壤团聚体稳定性,土壤游离氧化铁、菌根密度和EEG是显著影响团聚体稳定...     

The aggregating behaviour of <Emphasis Type="Italic">Argiope radon</Emphasis>, with special reference to web decorations

The existence of aggregations in taxa that are normally solitary poses questions regarding the costs and benefits of group living. Most orb-web spiders are solitary and are aggressive to conspecifics, but a few species aggregate in large numbers. These spiders benefit by enhancing the prey interception potential, but also suffer costs of increased predation and parasitism. In this study, we report on the natural history characteristics of the orb-web spider, Argiope radon, which not only lives in aggregations but also builds silk decorations. Our results show that A. radon aggregates facultatively and that the main benefit of aggregation that we could identify is enhanced mating potential. We also show that decorations built by A. radon are highly visible to both model prey and predator, and suggest that solitary individuals with longer and more frequent decorations may offset the foraging advantage of being in aggregations.     

Domestication and fitness in the wild: A multivariate view

Domesticated species continually escaping and interbreeding with wild relatives impose a migration load on wild populations. As domesticated stocks become increasingly different as a result of artificial and natural selection in captivity, fitness of escapees in the wild is expected to decline, reducing the effective rate of migration into wild populations. Recent theory suggest that this may alleviate and eventually eliminate the resulting migration load. I develop a multivariate model of trait and wild fitness evolution resulting from the joint effects of artificial and natural selection in the captive environment. Initially, the evolutionary trajectory is dominated by the effects of artificial selection causing a fast initial decline in fitness of escapees in the wild. In later phases, through the counteracting effects of correlational multivariate natural selection in captivity, the mean phenotype is pushed in directions of weak stabilizing selection, allowing a sustained response in the trait subject to artificial selection. Provided that there is some alignment between the adaptive landscapes in the wild and in captivity, these phases are associated with slower rates of decline in wild fitness of the domesticated stock, suggesting that detrimental effects on wild populations are likely to remain a concern in the foreseeable future.     

Scanning electron microscopy of barley protoplasts

Summary Scanning electron micrographs of barley protoplasts were compared using various preparatory techniques. Numerous features were observed which turned out to be artifactual characteristics of the processing procedure used in collecting and dehydrating the samples. The most successful technique gave protoplasts which presumably maintained their natural structural integrity, as judged by retention of sphericity and absence of holes in the plasma membrane. The relative numbers of fragmented protoplasts and cellular organelles was also greatly reduced.     

When size does matter: organelle size influences the properties of transport mediated by molecular motors

Background

Organelle transport is driven by the action of molecular motors. In this work, we studied the dynamics of organelles of different sizes with the aim of understanding the complex relation between organelle motion and microenvironment.

Methods

We used single particle tracking to obtain trajectories of melanosomes (pigmented organelles in Xenopus laevis melanophores). In response to certain hormones, melanosomes disperse in the cytoplasm or aggregate in the perinuclear region by the combined action of microtubule and actin motors.

Results and conclusions

Melanosome trajectories followed an anomalous diffusion model in which the anomalous diffusion exponent (α) provided information regarding the trajectories' topography and thus of the processes causing it. During aggregation, the directionality of big organelles was higher than that of small organelles and did not depend on the presence of either actin or intermediate filaments (IF). Depolymerization of IF significantly reduced α values of small organelles during aggregation but slightly affect their directionality during dispersion.

General significance

Our results could be interpreted considering that the number of copies of active motors increases with organelle size. Transport of big organelles was not influenced by actin or IF during aggregation showing that these organelles are moved processively by the collective action of dynein motors. Also, we found that intermediate filaments enhance the directionality of small organelles suggesting that this network keeps organelles close to the tracks allowing their efficient reattachment. The higher directionality of small organelles during dispersion could be explained considering the better performance of kinesin-2 vs. dynein at the single molecule level.     

Chlorophyllase is a rate-limiting enzyme in chlorophyll catabolism and is posttranslationally regulated

Chlorophyll is a central player in harvesting light energy for photosynthesis, yet the rate-limiting steps of chlorophyll catabolism and the regulation of the catabolic enzymes remain unresolved. To study the role and regulation of chlorophyllase (Chlase), the first enzyme of the chlorophyll catabolic pathway, we expressed precursor and mature versions of citrus (Citrus sinensis) Chlase in two heterologous plant systems: (1) squash (Cucurbita pepo) plants using a viral vector expression system; and (2) transiently transformed tobacco (Nicotiana tabacum) protoplasts. Expression of full-length citrus Chlase resulted in limited chlorophyll breakdown in protoplasts and no visible leaf phenotype in whole plants, whereas expression of a Chlase version lacking the N-terminal 21 amino acids (ChlaseDeltaN), which corresponds to the mature protein, led to extensive chlorophyll breakdown in both tobacco protoplasts and squash leaves. ChlaseDeltaN-expressing squash leaves displayed a dramatic chlorotic phenotype in plants grown under low-intensity light, whereas under natural light a lesion-mimic phenotype occurred, which was demonstrated to follow the accumulation of chlorophyllide, a photodynamic chlorophyll breakdown product. Full-length and mature citrus Chlase versions were localized to the chloroplast membrane fraction in expressing tobacco protoplasts, where processing of the N-terminal 21 amino acids appears to occur. Results obtained in both plant systems suggest that Chlase functions as a rate-limiting enzyme in chlorophyll catabolism controlled via posttranslational regulation.     

Early stages of salmon calcitonin aggregation: effect induced by ageing and oxidation processes in water and in the presence of model membranes

The natural ageing- and hydrogen peroxide-induced aggregation of salmon calcitonin were studied in water and in the presence of dipalmitoylphosphatidylcholine (DPPC) liposomes. The early stages of the aggregation process at low protein concentration were investigated by means of Circular Dichroism spectroscopy (CD) and conventional and immunogold labelling Transmission Electron Microscopy (TEM). In buffered water solution, salmon calcitonin showed a two-stage conformational variation related to fibril formation and phase-separation of larger aggregates. A first stage, characterised by small conformational changes but a decrease in dichroic band intensity, was followed by a second stage, 6 days after, leading to higher conformational variations and aggregations. Salmon calcitonin showed a distinct modification in the secondary structure and aggregate morphology in the presence of hydrogen peroxide with respect to natural ageing, indicating that the two aggregation processes (natural and chemical-induced) followed a distinct mechanism. The oxidised forms of the peptide were separated by liquid chromatography. The same study was performed in the presence of DPPC liposomes. The results obtained by conventional and immunogold labelling TEM evidenced that salmon calcitonin in buffered water solution essentially does not enter the liposomes but forms around them a fibril network characterised by the same conformational changes after 6 days. The oxidised sample in the presence of liposomes showed a "fibrils hank", separated from liposomes. The presence of liposomes did not affect either the aggregation or the conformational modifications yet observed by TEM and CD in water solution.