Detection of intracellular macromolecule behavior under strong magnetic fields by linearly polarized light

Strong static magnetic fields on the order of 10 T have a diamagnetic force on cell components and generate a clear alignment of a smooth muscle cell assembly, parallel to the direction of the magnetic fields within an exposure period of 3 days. This work shows the effects of diamagnetic torque forces on cell component motion. Linearly polarized light was utilized to detect the displacement of intracellular macromolecules. The polarized light passed through a mass of cells in a magnetic field, and transmission of the light increased and reached a plateau 2 h after the beginning of magnetic field exposure at 14 T. However, no distinct change was observed in transmission of the light under zero magnetic field exposure. The change in polarized light intensity through the lamellar cell assembly under magnetic fields corresponds to behavioral changes in cell components. It was speculated that intracellular macromolecules rotated and showed a displacement due to diamagnetic torque forces during 2-3 h of magnetic field exposure at 14 T.     

Electron microscopic observations of cell regeneration from cultured protoplasts ofAmmi visnaga

Summary Protoplasts ofAmmi visnaga initiated cell wall formation within 2 days in culture; after 13 days the new cells were enclosed by a cell wall similar to the walls on the original cultured cells. Budding occurred in protoplasts with little or no detectable cell wall. No evidence was obtained for direct participation of any organelle in cell wall formation. The cytoplasm of regenerating cells contained numerous organelles and appeared typical of actively growing plant cells; they were easily distinguished from degenerate cells and protoplasts. While coated vesicles were common, spiny vesicles occurred in only a few cells. Sustained cell division yielded multicellular aggregates. Multinucleate protoplasts, formed by spontaneous fusion, did not divide; some of them contained annulate lamellae with few pore complexes.Supported by the National Research Council of Canada, Grant A6304.     

Presence of a plant cell wall is not required for transformation of Nicotiana by Agrobacterium tumefaciens

Agrobacterium has been used to transform zero to six-day-old cell wall nonregenerating (CWNR) and cell wall regenerating (CWR) leaf protoplasts of tobacco. Transformed cells were selected by phoytohormone autotrophic growth and were verified by detection of the presence of lysopine dehydrogenase. Transformation frequencies in CWNR protoplasts were at least as high as those in CWR protoplasts, indicating that a plant cell wall is not required for the process of crown gall tumorigenesis. Transformation frequencies were highest in two-day-old protoplasts. This age coincides with the onset of DNA synthesis and the first mitosis within the cell populations. We suggest that the initiation of cell cycle activity may be important for the transformation process.     

Isolation and characterization of four cell wall purple acid phosphatase genes from tobacco cells

Four full-length cDNAs were isolated from a cDNA library prepared from tobacco cultured cells and designated NtPAP4, NtPAP12, NtPAP19 and NtPAP21, which could correspond to purple acid phosphatase (PAP). Levels of both NtPAP12 and NtPAP21 mRNA in the protoplasts immediately increased after the protoplasts were transferred to a medium for cell wall regeneration, and the accumulation of the mRNA was correlated with cell wall regeneration for 3 h. It is likely that the NtPAP12 and NtPAP21 gene products are wall-bound PAPs at the early stage of regenerating walls in tobacco protoplasts.     

Ultrastructural and biochemical aspects of cell wall reconstitution in recalcitrant (grapevine) and regenerating (tobacco) leaf protoplasts

Summary To identify possible reasons that may contribute to recalcitrance in plant protoplasts, the time course of new cell wall deposition was studied by scanning electron microscopy in protoplasts of a recalcitrant species, the grapevine. Results showed that microfibrils were developed after 2 days of culture, that complete cell wall formation occurred on Day 6 to 7 of protoplast culture, and its ultrastructural appearance was identical to that of grapevine leaf-derived callus cells. In addition, a comparative study was undertaken on [U-¹⁴C]glucose uptake and incorporation in ethanol-soluble, cellulosic, and noncellulosic polysaccharide fractions in protoplasts of grapevine and of a readily regenerating species, tobacco, during culture. There was a significantly higher [U-¹⁴C]glucose uptake by tobacco than by grapevine protoplasts. The label distribution in the ethanol-soluble, cellulosic, and noncellulosic fractions of newly synthesized cell walls differed quantitatively between the two species. In particular, the labeled glucose incorporated in the noncellulosic cell wall fraction was threefold greater in tobacco than in grapevine protoplasts. Differences were also revealed in the monosaccharide composition of this fraction between the two species. Addition of dimethyl sulfoxide to the culture medium resulted in a dramatic increase in [U-¹⁴C]glucose uptake by grapevine protoplasts, whereas it exhibited a limited effect in tobacco protoplasts. It showed no effect on the ultrastructural characteristics of new cell wall nor on the incorporation rate of labeled glucose in the cellulosic and noncellulosic cell wall fractions.     

Use of RAPD-PCR to isolate a species specific DNA probe for Phytophthora cinnamomi

Abstract Aculeacin A and papulacandin B block cell wall regeneration in Candida albicans protoplasts at an intermediate step in which the protoplasts have not yet synthesized the rigid structure of the cell wall and are therefore still osmotically sensitive. In the presence of the antibiotics, total synthesis of glucan is not significantly lowered with respect to control cells, although most of it appears either in the culture medium or in the regenerating wall as alkali-soluble glucan. Thus, it is proposed that echinocandins (such as aculeacin A) and papulacandins may not inhibit glucan synthesis per se but instead inhibit its incorporation into the supramolecular organization of the cell wall.     

Molecular weight distribution of cellulose in primary cell walls

The distribution pattern of the degree of polymerization (DP) of cellulose present in the cell walls of mesophyll- and suspension-cultured cells of tobacco was compared to that of newly synthesized ¹⁴C-labeled cellulose from regenerating tobacco protoplasts and suspension-cultured cells. The cellulose was nitrated, and, after fractionation according to differences in solubility in acetone/water, the DP pattern of labeled or unlabeled cellulose nitrate was determined by viscosity measurements. A low (DP<500) and high DP-fraction (DP>2500) of cellulose were predominant in the cell walls of protoplasts, suspension — cultured cells, and mesophyll cells. The average DP of the high molecular weight fraction of cellulose in the cell walls of mesophyll was higher (DP4,000) than in protoplasts or suspension — cultured cells (DP 2,500-3,000). In all cell walls tested, minor amounts of cellulose molecules with a broad spectrum of a medium DP were present. Pulse — chase experiments with either protoplasts or suspension —cultured cells showed that a large proportion of the low and medium DP-cellulose are a separate class of structural components of the cellulose network. The results are discussed in relation to the organization of cellulose in the primary cell wall.Abbreviations DP degree of polymerisation - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid     

Effect of magnetic field exposure on calcium channel currents using patch clamp technique

Calcium influxes through the membrane of PC-12D cells were measured under exposure to DC biased AC magnetic fields in resonant conditions of the ion cyclotron and the ion parametric resonance hypotheses and compared with influxes in cells without exposure to the magnetic field. After cancellation of the geomagnetic field, the cells were exposed to the horizontal fields generated by a current sheet, a planar sheet of conductor which generated a satisfactorily homogeneous horizontal magnetic field on the stage of a microscope without hindering treatment of a cell under observation. At or near any resonant conditions, no change in calcium influx could be detected under standard patch clamp conditions.     

Electron microscopy study of cell structures and their changes during growth and regeneration ofSchizosaccharomyces pombe protoplasts

The submicroscopic structure of growing and regeneratingSchizosaccharomyces pombe protoplasts cultivated in solid and liquid medium was studied by means of ultrathin sectioning. The protoplasts regenerate within 24 hours. Shortly before growth commences, rudiments of the new cell wall can be identified on the protoplast surface. Simultaneously, a large number of dictyosomes appears in the cytoplasm and decreases as synthesis of the new wall progresses. An increase occurs in the number of endoplasmic reticulum membranes some of which are arranged parallel with the cytoplasm membrane of the protoplast. Throughout the whole time of regeneration the protoplasts contain only one nucleus. The nucleo-cytoplasm ratio of growing and regenerating protoplasts is lower than in intact cells. The number of mitochondria falls at the outset of regeneration and does not rise again until towards the end.     

Differences in Protein Synthesis and Peroxidase isoenzymes between recalcitrant and regenerating ProtoPlasts

The reasons for the inability of recalcitrant mesophyll protoplasts to divide and re-enter the cell cycle are unknown. Changes in protein profile, indole-3-acetic acid (IAA)-oxidase and peroxidase activities, and isoenzymes were compared in protoplasts of recalcitrant grapcvine ( Vitis vinifera ) L. cv. Sultanina) and regenerating tobacco ( Nicotiana tabacum ) L. cv. Xanthi). Using [³⁵S]-methionine. SDS-PAGE and two-dimensional separation of proteins, differences in protein profile during protoplast culture were assessed. The changes in the de novo synthesized proteins were both qualitative and quantitative between the two species. The number of proteins which changed was double in tobacco compared to grapevine protoplasts. Peroxidase and IAA-oxidase activities increased significantly in tobacco protoplasts during culture whereas in grapevine they remained low. In tobacco protoplasts. 3 and 7 basic and acidic peroxidases, respectively, were induced during protoplast culture. which were not detected in the intact leaf, whereas in grapevine no new peroxidases were induced during protoplast culture.     

Influence of growth conditions on the composition of cell wall polysaccharides from cultured tobacco cells

The sugar composition of cell wall polysaccharides of two tobacco varieties obtained from mesophyll, regenerating protoplasts and cells grown under various conditions were compared. Regenerating protoplasts developed an unusual cell wall with a low cellulose and a high non-cellulosic glucan content. In the presence of different phytohormones compact and friable calli were obtained with cell walls containing low and high arabinose/xylose ratios. The cell walls of compact calli were comparable to those of genuine mesophyll cells. The sugar constituents of cell walls obtained from cells grown in liquid media were different from those of solid calli. The cell wall composition of suspension cultured cells was hardly affected by various combinations of phytohormones, but was altered by high osmolarity of the medium.     

Changes in the plasma membrane of regenerating protoplasts of Candida albicans as revealed by freeze-fracture electron microscopy

Modifications occurring in the plasma membrane and their relationship to newly synthesized microfibrils were examined in regenerating protoplasts of Candida albicans by freeze-fracture electron microscopy. Freshly prepared protoplasts showed no residual wall material, and long invaginations covered the surface of the plasma membrane. Analysis of the external face (E-face) of the plasma membrane showed a significant decrease in the number of intramembranous particles (IMP) in comparison with the original cells. After 40 min incubation in regeneration medium, newly synthesized microfibrils which seemed to originate from protrusions in the plasma membrane were observed. The plasma membrane showed important modifications with respect to IMP. After 3 h 45 min, the cells were covered by an abnormal wall which showed isolated fibrils partially embedded in the matrix material. The plasma membrane of these partially regenerated protoplasts was similar to that of original cells. After 8 h, regeneration of the protoplasts seemed to be complete as no differences from the original cells were detected in the plasma membrane or the wall. Calcofluor white altered the deposition of wall polymers during regeneration, but did not modify the plasma membrane of the protoplasts.     

Autolytic Formation of Protoplasts (Autoplasts) of Streptococcus faecalis 9790: Release of Cell Wall, Autolysin, and Formation of Stable Autoplasts

A system for the formation of apparently wall-free protoplasts from exponential-phase cells of Streptococcus faecalis ATCC 9790 in the absence of added lytic enzymes was developed. Exponential-phase cells suspended in 0.04 M ammonium acetate, pH 6.7, 1 mM magnesium acetate, and 0.5 M sucrose become osmotically fragile within 1 to 1.5 h due to the action of the native, autolytic enzyme on the cell wall peptidoglycan. However, maximal cell wall loss occurred much more slowly, being complete only after 3 to 6 h. Under these conditions, the autolytically formed protoplasts (autoplasts) remained intact for prolonged periods (up to 24 h) with less than 5% of their deoxyribonucleic acid, ribonucleic acid, and protein lost during the first 6 h. During dissolution of the cell wall, release of autolytic enzyme to the supernatant fluid began after 60% of the wall was lost. The addition of trypsin to the incubation mixture increased the rate of attainment of osmotic fragility and cell wall loss two- to threefold, apparently due to the activation of the latent form of the autolysin. Electron microscopy was used to confirm cell wall loss and the presence of intact protoplasts at the end of the incubation periods.     

Regeneration of plantlets fromEnteromorpha (Ulvales,Chlorophyta) protoplasts in axenic culture

High yields of protoplasts have been obtained from vegetative thalli of three species ofEnteromorpha by enzymatic degradation of the cell wall. Several commercial and crude enzymes prepared from the digestive system and hepatopancrease of abalone and top-shell were tested at different concentrations and combinations to evaluate the yield. Commercial enzymes in combination with either abalone or top-shell crude enzymes, consistently produced a high yield of protoplasts from all three species. High regeneration rate (85–95%) occurred in the protoplasts cultured at a density greater than 1.72 × 10³ cells cm⁻² at 20 and 25°C. Light intensities tested in the present study did not affect protoplast wall formation and regeneration. Protoplasts, after regenerating the cell wall, followed different types of developmental patterns under identical culture conditions. In one type some cells underwent repeated cell divisions and formed a round and oval shaped hollow thallus with a single layer of cells. In the second type many cells underwent one or two cell divisions (occasionally no division) and soon matured and discharged many motile spores, which on germination grew into normal plantlets. In the third type some cells divided irregularly to form a mass of callus-like cells (exceptE. prolifera). Culture medium supplemented with either mannitol, sorbitol, dextrose, saccharose or NaCl at higher concentrations (> 0.4 M) inhibited cell division and further differentiation in all species. author for correspondence     

Protoplast isolation and culture from carob (Ceratonia siliqua) hypocotyls: ability of regenerated protoplasts to produce mannose-containing polysaccharides

The aim of this study was to isolate protoplasts from carob (Ceratonia siliqua L.) embryonic tissues with the ability to regenerate cell walls, divide and synthesize galactomannan, a valuable polysaccharide for industry. Protoplasts isolated from carob hypocotyl hooks regenerated cell walls within 24 h. The first divisions of the regenerated cells were observed after 2 days of culture. The highest percentage that successfully divided was achieved when the seedlings were grown under diffuse light, the hypocotyl hooks were plasmolysed for 1 h before incubation in the protoplast isolation solution and the protoplasts were cultured under diffuse light. After 9 days of culture, cell clusters, consisting of eight cells, had been produced, which underwent further mitotic divisions and which were expected to lead to callus formation. Polysaccharide and oligosaccharide synthesis during protoplast regeneration was studied by radiolabelling with exogenous d ‐[U‐¹⁴C]glucose, d ‐[U‐¹⁴C]mannose or d ‐[2‐³H]mannose, which gave rise to uniform, moderately specific and highly specific labelling, respectively. As revealed by the radioactivity distribution in cell wall monosaccharides, the regenerants deposited new wall polymers that differed markedly from those being synthesized by the hypocotyls from which the protoplasts had been isolated. The regenerants deposited large amounts of callose and smaller amounts of galactose‐, arabinose‐ and mannose‐containing polymers. The latter included glucuronomannan, as demonstrated by a new method involving partial acid hydrolysis followed by β‐glucuronidase (EC 3.2.1.31) digestion. The regenerating protoplasts also released soluble extracellular carbohydrates: polysaccharides which appeared to be mainly acidic arabinogalactans, and oligosaccharides which were mainly neutral and contained glucose, galactose and mannose. We conclude that regenerating carob protoplasts are a useful system for studying carbohydrate secretion, including mannose‐rich poly‐ and oligosaccharides.     

The effect of 8 days of microgravity on regeneration of intact plants from protoplasts

The growth and development of protoplasts of rapeseed (Brassica napus L. cv Line) and carrot (Daucus carota L. cv. Navona) were studied onboard the Space Shuttle‘Discovery’during an 8-day International Microgravity Laboratory [IML-l) mission in January 1992. The Flight experiments were carried out in‘Biorack'. a fully controlled cell biological experimental facility. under microgravity conditions and in a l-g centrifuge. Parallel experiments were performed in a‘Biorack’module on the ground. After retrieval, some samples were subcultured on appropriate media and analysed for callus growth and regeneration to intact plants. The remainder were used for biochemical analysis. Samples fixed on board the Space Shuttle were kept in l% glutaraldehyde fixative at 4°C for 3–7 days for microscopy analysis after retrieval. Protoplasts exposed to microgravity conditions showed a delay in cell wall synthesis. Cells were swollen in appearance and formed cell aggregates with only few cells. Callus were obtained from protoplasts cultured under microgravity (Fogl). on the l-g centrifuge on board the shuttle (Flg), under normal l-g conditions on the ground (G1g) and on a centrifuge on the ground giving 1.4 g (Gl.4g). Regeneration of intact rapeseed plants was obtained from Flg. Glg and G1.4g. However, no plants were regenerated from protoplasts exposed to microgravity (Fog). Biochemical analysis indicated that the microgravity samples (Fog displayed a reduced packed cell volume, an increased concentration of soluble proteins per cell, and a reduced specific activity of peroxidase in the cytoplasm. Morphometric analysis of fixed samples demonstrated that 3-day old protoplasts under microgravity conditions were significantly larger than protoplasts kept on the l-g centrifuge in space. UItrastructural analysis by transmission electron microscopy showed that protoplasts exposed to microgravity conditions for 3 days had larger vacuoles and a slightly reduced starch content compared to Flg cells. Cell aggregates formed under microgravity conditions (Fog) had an average of 2–I cells per aggregate while aggregates formed under Flg had 8–12 cells.     

Vectorial and nonvectorial transphosphorylation catalyzed by enzymes II of the bacterial phosphotransferase system

The plasmolytic response of Bacillus licheniformis 749/C cells to the increasing osmolarity of the surrounding medium was quantitated with stereological techniques. Plasmolysis was defined as the area (in square micrometers) of the inside surface of the bacterial wall not in association with bacterial membrane per unit volume (in cubic micrometers) of bacteria. This plasmolyzed surface area was zero when the cells were suspended in a concentration of sucrose solution lower than 0.5 M, but increased linearly when the sucrose molarity rose above 0.5 M, reaching a plateau value of 3.61 micrometers2/micrometers3 in 2 M sucrose. In contrast, when the bacterial cells were treated with lysozyme plasmolysis increased abruptly from 0.06 micrometers2/micrometers3 in 0.75 M sucrose to 4.09 micrometers2/micrometers3 in 1 M sucrose. When the time of exposure was prolonged, the degree of plasmolysis increased gradually for the duration of the experiment (30 min) after exposure to 1 M sucrose without lysozyme, whereas with lysozyme plasmolysis reached a maximum (4.09 micrograms2/micrometers3) in 2 to 5 min. The examination of ultrastructure showed that the protoplast bodies of lysozyme-treated cells in 1 M sucrose and untreated cells in 2 M sucrose are maximally retracted from the intact wall of the bacteria; hardly any retraction of protoplasts could be seen for untreated cells in 1 M sucrose. The data suggest that the B. licheniformis cells are isoosmotic to 800 to 1,100 mosM solutions, but are able to withstand much greater osmotic pressure with no signs of plasmolysis because the cell wall and the plasma membrane are held in close association, perhaps by a covalent bond. It is likely that lysozyme weakens this bond by degradation of the peptidoglycan layer. Cellular autolysis also weakens this wall-membrane association.     

Heavy metal uptake by intact cells and protoplasts of Aureobasidium pullulans

Protoplasts prepared from yeast-like cells, hyphae and chlamydospores of Aureobasidium pullulans can take up heavy metals such as Zn²⁺, Co²⁺, Cd²⁺ and Cu²⁺. In relation to intact cells, the sensitivity of protoplasts to Cu²⁺ and Cd²⁺ was increased although chlamydospore protoplasts were more tolerant than yeast-like cell protoplasts. Surface binding of metals was reduced in protoplasts as compared with intact cells and this reduction was particularly evident for chlamydospore protoplasts. At the highest concentrations used, uptake of Zn²⁺, Co²⁺ and Cd²⁺ by yeast-like cell protoplasts was greater than that observed in intact cells which may have been due to toxicity, especially for Cd²⁺, resulting in increased membrane permeability, though for Zn²⁺ and Co²⁺ some barrier effect of the cell wall could not be completely discounted. Chlamydospore protoplasts were capable of intracellular metal uptake, unlike intact chlamydospores, and for Zn²⁺, uptake appeared to be via a different system less specific than that of the other cell types. For chlamydospores, the use of protoplasts confirmed the importance of the cell wall in preventing entry of metal ions into the cell.     

Tobacco zygotic embryogenesis in vitro: the original cell wall of the zygote is essential for maintenance of cell polarity, the apical-basal axis and typical suspensor formation

We have developed a reliable in vitro zygotic embryogenesis system in tobacco. A single zygote of a dicotyledonous plant was able to develop into a fertile plant via direct embryogenesis with the aid of a co-culture system in which fertilized ovules were employed as feeders. The results confirmed that a tobacco zygote could divide in vitro following the basic embryogenic pattern of the Solanad type. The zygote cell wall and directional expansion are two critical points in maintaining apical-basal polarity and determining the developmental fate of the zygote. Only those isolated zygotes with an almost intact original cell wall could continue limited directional expansion in vitro, and only these directionally expanded zygotes could divide into typical apical and basal cells and finally develop into a typical embryo with a suspensor. In contrast, isolated zygote protoplasts deprived of cell walls could enlarge but could not directionally elongate, as in vivo zygotes do before cell division, even when the cell wall was regenerated during in vitro culture. The zygote protoplasts could also undergo asymmetrical division to form one smaller and one larger daughter cell, which could develop into an embryonic callus or a globular embryo without a suspensor. Even cell walls that hung loosely around the protoplasts appeared to function, and were closely correlated with the orientation of the first zygotic division and the apical-basal axis, further indicating the essential role of the original zygotic cell wall in maintaining apical-basal polarity and cell-division orientation, as well as subsequent cell differentiation during early embryo development in vitro.     

抗坏血酸对花生原生质体分离过程中膜损伤的保护作用

酶解处理使花生叶肉细胞原生质体膜脂质过氧化产物丙二醛(MDA)积累,添加抗坏血酸(ASA)能降低MDA的积累。未添加ASA时,酶解初期,过氧化物酶(POD)、过氧化氢酶(CAT)活性上升,说明原生质体存在各种抵御不良变化的机制,但酶解时间加长,POD活性下降。添加ASA能使超氧化物歧化酶(SOD)活性明显上升。试验结果说明:在原生质体分离过程中会导致膜损伤,细胞自身存在一个响应的防御机制,添加ASA能降低酶解过程对原生质膜的损伤。