Plasmolysis as a Tool to Reveal Lead Localization in the Apoplast of Root Cells

In order to analyze the distribution of lead between cell walls and plasmalemma, two-day-old maize seedlings (Zea mays L.) were incubated for 24 h on a solution of lead nitrate at a concentration causing 50% inhibition of root growth (10^–5 M). Using the histochemical technique (precipitation of lead dithizonate), the distribution of lead in plasmolyzed and nonplasmolyzed cells of the root cortex was compared. This allowed us to separate the lead bound by cell walls from the lead located on the protoplast surface and in the periplasmic space. The plasmolysis was conducted prior to histochemical reaction by the incubation of seedling roots in 0.6 M sucrose solution for 30 min. The lead precipitates were located in cell walls and on the surface of protoplast. A small amount of lead was found in periplasmic space of some cells in root cortex. It is suggested that the lead is bound not only to the cell wall matrix but also to the plasmalemma.     

The use of TTC reduction assay for assessment of Gentiana spp. cell suspension viability after cryopreservation

This study was aimed at improving the 2,3,5-triphenyl-tetrazoliumchloride (TTC) reduction test for initial assessment of cell survival after cryopreservation. Experiments were carried out on three embryogenic cell suspensions of different ages: 9-year-old Gentiana tibetica (King ex Hook. F.), 2-year-old G. kurroo (Royle), and 1-year-old G. cruciata (L.). The suspensions were maintained in MS medium supplemented with 1.0 mg 1⁻¹ 3,6-dichloro-o-anisic acid, 0.1 mg 1⁻¹ naphthaleneacetic acid, 2.0 mg l⁻¹ 6-benzylaminopurine, 80.0 mg 1⁻¹ adenine sulphate and 0.09 M sucrose. Four weeks before freezing, part of the tissue was subcultured to the same medium with sucrose concentrations elevated from 0.09 M (3%sMS) to 0.175 M (6%sMS) or 0.26 M (9%sMS). In freezing treatments without cryoprotection, tissue was plunged directly into liquid nitrogen (LN) or cooled gradually. In freezing treatments with cryoprotection, the cells were pretreated with 1 M sucrose, or with 0.4 M sorbitol + 0.25 M proline or + 0.08 M DMSO, or with vitrification solution (PVS2). Encapsulation was another variant. TTC reduction activity was spectrophotometrically assessed immediately, 1, 3, 5, 24 and 48 h after thawing. Cells without cryoprotection were lethally damaged, but TTC reduction activity in those cells ranged from 6.5% (tissue from 3%sMS) to 73 % (tissue from 9%sMS) directly after thawing. Formazan production was reduced to zero after 24 h. The TTC test showed 50% formazan content immediately after thawing of DMSO-protected G. tibetica tissue, but only 22.47% after 24 h and 2.9% after 48 h. Ultrastructural analysis of those cells showed lethal damage in many of them. For the PVS2 treatment, the formazan content was similar in samples analyzed directly after thawing and 24 h later. Cells treated with PVS2 did not show structural disturbances. Encapsulated cell aggregates of G. cruciata treated with concentrations of sucrose increasing up to 1 M produced 2.6 times more formazan. When applied at least 48 h after thawing, the TTC test can reflect cell viability and can be used to compare the effectiveness of cryoprotectant performance and freezing protocols, but it must be carefully evaluated, with appropriate controls.     

The Azolla-Anabaena azollae relationship

The heterocystous blue-green alga, Anabaena azollae, was isolated from the leaf cavities of the water fern, Azolla caroliniana, where it occurs as an endophyte. The isolated alga was capable of light dependent CO₂ fixation and acetylene reduction. Aerobic dark acetylene reduction occurred and was dependent upon endogenous substrates. Vegetative cells of the alga reduced nitro-blue tetrazolium chloride (NBT) to blue formazan. Heterocysts did not. Heterocysts reduced triphenyl tetrazolium chloride (TTC) to red formazan faster than vegetative cells. Reduction of TTC by both heterocysts and vegetative cells was much more rapid than has been reported for free-living heterocystous blue-green algae. Both NBT and TTC inhibited acetylene reduction and CO₂ fixation. The inhibition by TTC was more closely correlated to the time of exposure of the cells to the reagent and to the amount of deposition per cell than to the number of cells containing red formazan. No differential inhibition of acetylene reduction versus CO₂ fixation was observed. Autoradiography showed that CO₂ fixation occurred only in vegetative cells. Heterocysts caused a darkening of nuclear emulsions (chemography). This observation has been employed by others as an index of reducing activity in these cells. DCMU inhibited the acetylene reducing capacity of alga isolated from dark pretreated fronds more rapidly and to a greater extent than that in alga isolated from light pretreated fronds. Ammonia in excess of 5 mM was required before any inhibition of acetylene reduction was observed under either aerobic or anaerobic conditions in the light.     

Ultrastructural aspects of leaves inFestuca ovina andPoa sphondylodes (C-3 Poaceae)

Structural aspects of the leaves of two common festucoids,Festuca ovina andPoa sphondylodes, have been examined employing the electron microscopy. The nature of vascular bundles and of sheaths that surround vascular tissues was discussed in the study. The festucoids exhibited a non-Kranz C-3 anatomy with more than four mesophyll cells separating the bundle sheaths of a leaf blade. Vascular tissues in theseFestuca andPoa leaves were surrounded by a double sheath: an inner distinct mestome sheath (MST) and an outer indistinctive layer of parenchymatous bundle sheath (PBS) cells. The PBS cells were much larger than the MST and had thin walls. The MST cells were relatively small and rectangular inP. sphondylodes and more or less hexangular in transverse sections ofF. ovina. InP. sphondylodes, MST had conspicuously thickened inner tangential walls with asymmetrically uninterrupted suberized lamellae in radial and tangential walls. In most differentiated MST cells, all walls were highly suberized. During suberin deposition, MST cells were quite vacuolated and most of the cytoplasm was present as a thin peripheral layer. However, MST walls inF. ovina revealed very thin suberized lamellae with translucent striations. No chloroplasts were detected inP. sphondylodes, whereas the MST inF. ovina contained small chloroplasts. Plasmodesmata were well developed in the primary pit fields of walls between MST and vascular cells, and between adjacent MST cells. Plasmodesmata were less frequent in the walls between the inner and outer sheath cells. Suberized lamellae were totally absent from the PBS cell walls in all veins. External to the PBS, the mesophyll comprised thin walled cells with abundant intercellular spaces. Peripherally arranged chloroplasts in the mesophyll were numerous and often larger than those of PBS and MST cells. Characteristics associated with C-3 and other ultrastructural features were also discussed in the study.     

2, 3, 5-triphenyl tetrazolium chloride (TTC) reduction as exponential growth phase marker for mammalian cells in culture and for myeloma hybridization experiments

Triphenyl tetrazolium chloride in vitro reduction by cells produces a red formazan pellet which can be extracted and measured. We have shown that such reduction is associated with animal cell growth, and particularly with the specific growth rate, so the measurement of Triphenyl tetrazolium chloride reduction is proposed as a physiological marker of the exponential growth of cultured cells. Further application of this technique is shown using this Redox reaction for estimating plasmacytoma fusion potential for hybridoma cell line production.Abbreviations TTC 2,3,5-Triphenyl Tetrazolium Chloride     

Responses of Chrysanthemum Cells to Mechanical Stimulation Require Intact Microtubules and Plasma Membrane–Cell Wall Adhesion

Plant cells are highly susceptible and receptive to physical factors, both in nature and under experimental conditions. Exposure to mechanical forces dramatically results in morphological and microstructural alterations in their growth. In the present study, cells from chrysanthemum (Dendranthema morifolium) were subjected to constant pressure from an agarose matrix, which surrounded and immobilized the cells to form a cell-gel block. Cells in the mechanically loaded blocks elongated and divided, with an axis preferentially perpendicular to the direction of principal stress vectors. After a sucrose-induced plasmolysis, application of peptides containing an RGD motif, which interferes with plasma membrane-cell wall adhesion, reduced the oriented growth under stress conditions. Moreover, colchicines, but not cytochalasin B, abolished the effects of mechanical stress on cell morphology. Cellulose staining revealed that mechanical force reinforces the architecture of cell walls and application of mechanical force, and RGD peptides caused aggregative staining on the surface of plasmolyzed protoplasts. These results provide evidence that the oriented cell growth in response to compressive stress requires the maintenance of plasmalemma-cell wall adhesion and intact microtubules. Stress-triggered wall development in individual plant cells was also demonstrated.     

Postpollination-prezygotic ovular secretions into the micropylar canal inPseudotsuga menziesii (Pinaceae)

Ovular morphology was examined ultrastructurally inPseudotsuga menziesii to determine the effects of the ovule on pollen development. Vesicles containing lipid-like substances traverse cell walls of the inner epidemis of the integument and release their contents at the integument surface to form the integumentary membrane. A major aqueous secretion from the integument into the micropylar canal is proposed to occur by the movement of the integumentary membrane and its invaginations towards the center of the micropylar canal. The cellular degeneration of the nucellar apex results from the breakdown of vacuoles. After this degeneration, electron-dense substances move from the prothallial cells of the female gametophyte towards the nucellus, and many morphological changes in the nucellus, prothallial cells, and micropylar canal take place simultaneously. We interpret these changes to result from another major secretion from the prothallial cells. Egg cytoplasm appears to disorganize for a short time. Simultaneously, substantial amounts of electron dense-substances in the prothallial cells and lipid-like substances in surface cell walls of the female gametophyte move towards the nucellus as components of the third major secretion.     

Freezing behavior of xylem ray parenchyma cells in softwood species with differences in the organization of cell walls

Summary By cryo-scanning electron microscopy we examined the effects of the organization of the cell walls of xylem ray parenchyma cells on freezing behavior, namely, the capacity for supercooling and extracellular freezing, in various softwood species. Distinct differences in organization of the cell wall were associated with differences in freezing behavior. Xylem ray parenchyma cells with thin, unlignified primary walls in the entire region (all cells inSciadopitys verticillata and immature cells inPinus densiflora) or in most of the region (mature cells inP. densiflora and all cells inP. pariflora var.pentaphylla) responded to freezing conditions by extracellular freezing, whereas xylem ray parenchyma cells with thick, lignified primary walls (all cells inCrytomeria japonica) or secondary walls (all cells inLarix leptolepis) in most regions responded to freezing by supercooling. The freezing behavior of xylem ray parenchyma cells inL. leptolepis changed seasonally from supercooling in summer to extracellular freezing in winter, even though no detectable changes in the organization of cell walls were apparent. These results in the examined softwood species indicate that freezing behavior of xylem ray parenchyma cells changes in parallel not only with clear differences in the organization of cell walls but also with subtle sub-electron-microscopic differences, probably, in the structure of the cell wall.     

Organization of the actin cytoskeleton during pollen development inGasteria verrucosa (Mill.) H. Duval visualized with rhodamine-phalloidin

The three-dimensional organization of the microfilamental cytoskeleton of developingGasteria pollen was investigated by light microscopy using whole cells and fluorescently labelled phalloidin. Cells were not fixed chemically but their walls were permeabilized with dimethylsulphoxide and Nonidet P-40 at premicrospore stages or with dimethylsulphoxide, Nonidet P-40 and 4-methylmorpholinoxide-monohydrate at free-microspore and pollen stages to dissolve the intine.Four strikingly different microfilamentous configurations were distinguished. (i) Actin filaments were observed in the central cytoplasm throughout the successive stages of pollen development. The network was commonly composed of thin bundles ramifying throughout the cytoplasm at interphase stages but as thick bundles encaging the nucleus prior to the first and second meiotic division. (ii) In released microspores and pollen, F-actin filaments formed remarkably parallel arrays in the peripheral cytoplasm. (iii) In the first and second meiotic spindles there was an apparent localization of massive arrays of phalloidin-reactive material. Fluorescently labelled F-actin was present in kinetochore fibers and pole-to-pole fibers during metaphase and anaphase. (iv) At telophase, microfilaments radiated from the nuclear envelopes and after karyokinesis in the second meiotic division, F-actin was observed in phragmoplasts.We did not observe rhodamine-phalloidin-labelled filaments in the cytoplasm after cytochalasin-B treatment whereas F-actin persisted in the spindle. Incubation at 4° C did not influence the existence of cytoplasmic microfilaments whereas spindle filaments disappeared. This points to a close interdependence of spindle microfilaments and spindle tubules.Based on present data and earlier observations on the configuration of microtubules during pollen development in the same species (Van Lammeren et al., 1985, Planta165, 1-11) there appear to be apparent codistributions of F-actin and microtubules during various stages of male meiosis inGasteria verrucosa.Abbreviation DMSO dimethylsulfoxide     

Permeability of Bacteria to Inorganic Cations. A Comparison between a Psychrophilic Achromobacter Strain and Escherichia coli

The permeability of a psychrophilic Acbromobacter strain to the chlorides of Na, K, Mg and Ca was investigated with light-scattering technique. Comparisons, were made with cells of Escherichia coli B. Cells of both strains suspended in “water were plasmolyzed by 0.1 or 0.2 M solutions of MgCl₂ or CaCl₂ without subsequent deplasmolysis. NaCl or KCl also plasmolyzed the cells, but deplasmolysis followed.” When suspended in growth medium E. coli became completely de-plasmolyzed., whereas the psychrophile still excluded MgCl₂ and CaCl₂ to a great extent. The plasmolysis and deplasmolysis were reversible. Electron micrographs of the psychrophile exposed to CaCl₂ confirmed the presence of plasrnolysis.     

Formation of sperm cells inGalium mollugo (Rubiaceae),Trichodiadema setuliferum (Aizoaceae), andAvena sativa (Poaceae)

The formation of sperm cells has been examined ultrastructurally in the tricellular pollen grains ofGalium mollugo L. (Rubiaceae).Trichodiadema setuliferum Schwantes (Aizoaceae), andAvena sativa L. (Poaceae). After detachement from the intine the generative cell of all three species lies free within the vegetative cytoplasm. The two sperm cells are built inTrichodiadema andAvena by a single separating wall, while inGalium mollugo two independent walls are formed. However, both mechanisms separate the two male gametes completely.     

High resolution shadowing of Mycobacterium leprae

Metal shadow casting techniques for transmission electron microscopic examination was used to determine the morphological characteristics of Mycobacterium leprae in untreated and treated patients. This technique is used to visualize bacterial surface structures by thermal evaporation of platinum alloys under moderate vacuum. This method gives a high contrast image at relatively low resolution and is useful for correlating micro-morphology quantitatively to early therapeutic effects of anti-leprosy drugs. Using these techniques in untreated cases, the surface structures of M. leprae were uniformly filled with relatively homogenous protoplasm surrounded by a cell wall. Most of the bacilli had thick cell walls with prominent banded and fibrous structures on the surface of the cell body. The cell wall was not detached in any of the solid bacilli in untreated cases. The bacilli varied in size and some of them were swollen in their mid-portion. Some bacilli were very short and completely filled with cytoplasm; therefore, these short bacilli were counted as solid bacilli in electron microscopic morphological index (EM-MI) determination. During treatment, mainly the cytoplasms of the bacilli were affected, and degeneration was observed. Ultrastructurally, the cytoplasm was shrunken and detached from the cell wall indicating mild degeneration. After moderate degeneration, the cytoplasm appeared fragmented. In advanced degeneration, all structures except the cell walls collapsed completely and no fibrous or band structures were visible on the surfaces of the cell walls. Therefore, these bacilli were counted as non-solid bacilli for EM-MI determination. This study shows that transmission electron shadowing gives more accurate counts than standard light microscopy of intact M. leprae bacilli in patient specimens.     

四唑(Tetrazolium)对固氮鱼腥藻固氮和氢代谢的影响

固氮鱼腥藻(Anabaena azotica Ley)细胞能还原无色的TTC和NBT分别成为红色或蓝色的甲(月朁)(formazan)沉淀。异形胞还原TTC的速率高于营养细胞。前异形胞及异形胞附近的营养细胞对NBT的还原作用最强。而异形胞对NBT不起还原作用。无论在异形胞形成红色甲(月朁)或在营养细胞形成蓝色甲(月朁)后都抑制固氮酶活性。NBT甲(月朁)对固氮酶活性的抑制作用大于TTC甲(月朁),因为NBT氧化还原电位低于TTC。 TTC和NBT两者都明显地抑制固氮鱼腥藻完整细胞的放氢。因鱼腥藻的放氢是由固氮酶催化的结果。四唑抑制放氢推想是由于它截取了固氮酶催化系统中的电子的缘故。固氮微生物(包括蓝色细菌和根瘤菌)对四唑还原与吸氢酶之间有无相关是一个争论的问题。一些学者认为分离豆科植物体的一些根瘤菌株培养于含有TTC的琼脂培养基,如还原,便可证明这些根瘤菌株能氧化氢;换言之,应用TTC的还原可作为一些根瘤菌的菌落具有吸氢酶的验证。相反,我们发现固氮鱼腥藻还原TTC和NBT之后,都没有影响吸氢的能力。因此,我们推想固氮鱼腥藻对四唑之还原与吸氢酶是没有直接的关系。     

Some fine structural observations on the rhizome ofDendroligotrichum (Bryophyta)

Summary The underground portion (rhizome) of the gametophytic axis ofD. dendroides bears anatomical resemblances to a triarch dicotyledonous root. The similarities include: 1. an epidermis producing epidermal appendages; 2. a cortex with endodermoid layer; and 3. a tri-radiate arrangement of the food and water-conducting tissue. Histochemical observations reveal that the entire radial and transverse walls of the endodermoid cells are encrusted with amorphous deposits, probably of polyphenolic nature. Casparian bands are not present as reported by earlier workers. The radial walls exhibit a fine structure of alternating electron-dense and electron-opaque lamellae. In plasmolyzed cells the plasmalemma does not adhere to the radial wall. Plasmodesmatal connections were observed in the radial and outer tangential walls of the endodermoid cells, but not in the inner tangential walls. These features of the endodermoid layer ofD. dendroides are discussed in relationship to the structure and function of the endodermis of vascular plants.     

Osmotic induced stimulation of the reduction of the viability dye 2,3,5-triphenyltetrazolium chloride by maize roots and callus cultures

Live cells can reduce colorless 2,3,5-triphenyltetrazolium chloride (TTC) to a red insoluble compound, formazan. Maize (Zea mays) callus, when osmotically stressed by 0.53 mol/L mannitol, produced 7-times or more formazan than untreated control callus. This result was seen with all osmotica tested and could not be attributed to differences in TTC uptake rate or accumulation, increased respiration rate as measured by O2 uptake, or to de novo protein synthesis. Increased formazan production could be detected after 2.5 h of exposure to osmotic stress and leveled off after 48 h of exposure. The increased formazan production was only detected when callus was moved from high osmotic medium to low osmotic, TTC-containing medium. Abscisic acid increased TTC reduction only when added in combination with 0.53 mol/L mannitol. Incubation of maize seedling roots with 0.53 mol/L mannitol also increased formazan production as seen visually. Further studies are needed to determine the cause of the increased formazan production. These results show that TTC viability measurements must be carefully evaluated with appropriate controls to confirm their validity.     

Cellular changes during heat shock induction and embryo development of cultured microspores ofBrassica napus cv. Topas

Summary Brassica napus cv. Topas microspores, isolated and cultured near the time of the first pollen mitosis and subjected to a heat treatment of 24 h, can be induced to develop into haploid embryos. This is a study of microspore structure during induction and embryo determination. Early during the 32.5 °C incubation period the nucleus moved away from the edge of the cell, and granules, 30 to 60 nm in diameter, appeared in the mitochondria and as a cluster in the cytoplasm. Cells divided symmetrically and at the end of the heat treatment, acquired the features of induced bicellular structures described previously. The features persisted as the cells divided randomly within the exine for 4–7 days following heat induction. Multicellular structures released from the exine underwent periclinal divisions resulting in protoderm differentiation of the globular embryo, thus determining embryo development. The cytoplasm of early heart-stage embryos contains abundant polyribosomes. Non-embryogenic development was indicated by large accumulations of starch and/or lipid and thickened cell walls or an unorganized pattern of cell division following release of the multicellular structures from the exine. Embryogenesis is discussed in terms of induction, embryo determination and development.     

Scanning electron microscopy of cells and protoplasts ofSaccharomyces rouxii

Cells ofSaccharomyces rouxii from a normal broth culture were subjected to a high osmotic pressure (2 M KCl), fixed in 3% glutaraldehyde fortified with 2 M KCl, and then processed routinely for examination in a scanning electron microscope. Micrographs revealed birth and bud scars typical for the genus and an apparently undamaged surface topography. Protoplasts were prepared from the same material by digestion of cell walls with snail gut enzymes in the presence of 2 M KCl. Naked protoplasts were obtained and these exhibited surface invaginations. In addition, spheroidal protrusions were noted and these structures were equated with the periplasmic bodies previously described by transmission electron microscopy. The propensity for periplasmic body formation inSaccharomyces rouxii is contrasted with otherSaccharomyces species and the circumstantial evidence that relates periplasmic bodies to cryptic β-fructofuranosidase inS. rouxii is briefly discussed.     

Electron microscopic observation of cell wall structure during appressorium formation in Colletotrichum lagenarium

Summary The formation of cell walls during the appressorium formation inColletotrichum lagenarium was observed by electron microscope on the materials prepared by replicas and sectioning. The outer layer of conidia cell walls ruptured at the time of germination and the inner layer bulged out to form a germ tube. The germ tubes and primordia of appressoria had smooth surface and were consisted of one-layered cell wall. However, as the appressorium matured, the electron dense materials appeared on the outer surface of the cell wall which grew into granules. These granules are believed to form the outer layer of appressoria. The under side of the appressorium in contact with the glass surface showed a round pore.Contribution No. 191.     

Microfibril orientation in plant cell walls

Summary The distribution of particles on the surface of the plasmalemma in the collenchyma of Apium graveolens was studied by the freeze-etching technique. The aim was to determine whether the distribution of particles was related to the known longitudinal or transverse orientation of cellulose microfibrils in different layers of the walls of these cells. Preliminary statistical studies have shown no obvious correlation between particle distribution and microfibril orientation although the distribution appeared uniform rather than random. Qualitatively, the particle distribution on the plasmalemma of differentiating xylem fibres of Eucalyptus maculata and of the cortical parenchyma of Avena sativa coleoptiles appeared to be similar to that observed on the plasmalemma of Apium. No correlation between the particle distribution and the microfibril orientation known to exist in the walls of these cells could be discerned.The orientation of microtubules in the cytoplasm of collenchyma cells of Apium graveolens was parallel to the microfibril orientation in many instances, but exceptions were noted. A possible interpretation for this variation is discussed. It is concluded that the microtubules are the structures which are most likely to be involved in determining microfibril orientation in the cell wall.     

Ooplasmic segregation and axis formation in the polychaeteNereis virens embryo

Ooplasmic segregation is of great importance in the development of Annelida. The mechanisms of this process are very diverse in different groups of polychaetes, oligochaetes, and leeches (Fernandezet al., 1998). Ooplasmic segregation inNereis virens is connected with the first meiotic spindle formation and animal-vegetative axis appearance. Spherical polyaxial symmetry of the oocyte transforms into radial stratified symmetry in the course of ooplasmic segregation. There are two main steps of ooplasmic segregation inNereis virens. The first step begins after the cortical reaction when the central clear cytoplasm reaches the surface of the oocyte. The movement of the cytoplasm is sensitive to nocodazole, colchicine, and cytochalasin B and appears to be mediated by microtubules and, partly, by microfilaments. The second step is not sensitive to the microtubule inhibitors and is mediated mainly by actin filaments. Ooplasmic segregation inNereis virens may be considered as a primitive form of ooplasmic segregation in Annelida.