UTILIZATION OF SEM AND FREEZE-ETCH TECHNIQUES IN THE STUDY OF HYPNOSPORES1

Hypnospores, a type of sculptured-walled resting spore formed by some zoosporic chlorophycean and chrysophycean algae, have been viewed at the ultrastructural level using thin section, SEM, and freeze-etch techniques. The “spiny” wall of the hypnospore, as seen in the light microscope, appears as a raised, scalloped surface at the ultrastructural level. The PAS-positive wall is composed of 2 distinct layers. Prior to the formation of the wall, vacuoles form in the cytoplasm, lipid deposits are detectable, and typical cytoplasmic organelles apparently degenerate. The entire process is completed in less than 24 hr.     

Low temperature-induced modifications in cell ultrastructure and localization of phenolics in winter oilseed rape (Brassica napus L. var. oleifera L.) leaves

Acclimation of winter oilseed plants in the cold (i.e. at temperatures >0 degrees C) followed by short exposure to sub-lethal freezing temperatures resulted in pronounced ultrastructural changes of leaf epidermal and mesophyll cells. The following major changes were observed upon acclimation at 2 degrees C: increased thickness of cell walls; numerous invaginations of plasma membranes; the appearance of many large vesicles localized in the cytoplasm in close proximity to the central vacuole; the occurrence of abundant populations of microvesicles associated with the endoplasmic reticulum (ER) cisternae or located in the vicinity of dictyosomes; and the occurrence of paramural bodies and myelin-like structures. In addition, large phenolic deposits were observed in the vicinity of the plasma membrane and membrane-bound organelles such as chloroplasts, large vesicles or cytoplasm/tonoplast interfaces. Transient freezing (-5 degrees C for 18 h) of the cold-acclimated leaves led to reversible disorganization of the cytoplasm and to pronounced structural changes of the cellular organelles. Chloroplasts were swollen, with the stroma occupying one half of their volume and the thylakoid system being displaced to the other half. Large phenolic aggregates disappeared but distinct layers of phenolic deposits were associated with mitochondrial membranes and with chloroplast envelopes. In frost-thawed cells recovered at 2 degrees C for 24 h, dictyosomes and dictyosome- or ER-derived small vesicles reappeared in the ribosome-rich cytoplasm. Aberrations in the structure of chloroplasts and mitochondria were less pronounced. Few phenolic deposits were seen as small grains associated with chloroplast envelopes and vesicle membranes. These observations demonstrate that plants undergo different changes in cell ultrastructure depending on whether they are subjected to chilling or freezing temperatures. Results are discussed in relation to membrane recycling and the possible role of phenolics during the first and second stages of plant acclimation at low temperature.     

Ultrastructure of endothelium in ovules of Penstemon gentianoides Poir. (Scrophulariaceae) at mature embryo sac phase

In this study ultrastructural differences between endothelial cells of different location in Penstemon gentianoides have been examined with electron microscope at mature embryo sac phase. Embryo sac is of the Polygonum type and surrounded by endothelium except the micropylar region. The cuticle is located primarily around the chalazal three-fourths of the embryo sac. Endothelium cells around the chalaza and toward the micropylar region are rich in cytoplasmic organelles. The cytoplasm of endothelial cells near the central cell has large vacuoles and few organelles. There are also plasmodesmas on the anticlinal walls of endothelial cells. The endothelium and the micropylar integumentary cells play a role in transport of metabolites into the embryo sac.     

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.     

Chloroplast response to low leaf water potentials: I. Role of turgor

The effect of decreases in turgor on chloroplast activity was studied by measuring the photochemical activity of intact sunflower (Helianthus annuus L. cv. Russian Mammoth) leaves having low water potentials. Leaf turgor, calculated from leaf water potential and osmotic potential, was found to be affected by the dilution of cell contents by water in the cell walls, when osmotic potentials were measured with a thermocouple psychrometer. After the correction of measurements of leaf osmotic potential, both the thermocouple psychrometer and a pressure chamber indicated that turgor became zero in sunflower leaves at leaf water potentials of −10 bars. Since most of the loss in photochemical activity occurred at water potentials below −10 bars, it was concluded that turgor had little effect on the photochemical activity of the leaves.     

QUANTITATIVE ULTRASTRUCTURE AND PROTEIN COMPOSITION OF DATE PALM (PHOENIX DACTYLIFERA) SEEDS: A COMPARATIVE STUDY OF ENDOSPERM VS. EMBRYO

Comparative studies on the ultrastructure and protein composition of the embryo and endosperm of date palm (Phoenix dactylifera L.) were conducted. Cells of the embryo cotyledon and endosperm function in reserve storage and contained cell walls, nuclei, and cytoplasm rich in lipid and protein bodies. Morphometric analysis from light and electron micrographs showed that the cell walls of the endosperm occupied 65% of the total cell volume, but only 6% in the embryo. The protein bodies of the endosperm accounted for 11%, whereas those of the embryo occupied more than half of the total cell volume. The volume of organelles and organelle-free cytoplasm in the endosperm was negligible, suggesting that most of the extractable endosperm proteins are localized in the protein bodies. Extractable proteins in the embryo may come from cytoplasm, protein bodies, and other organelles. The endosperm contains relatively lower amounts of proteins than does the embryo. Proteins extracted from both tissues were compared using SDS-polyacrylamide gel electrophoresis, tube gel isoelectric focusing, and two-dimensional electrophoresis. Proteins of both the tissues were heterogeneous in molecular mass and charge. The majority of the proteins were similar in molecular mass and charge in the two tissues, suggesting that most of the storage proteins are probably the same. However, there were also several embryo- and endosperm-specific proteins apparent in both the first- and second-dimension gels. The endosperm-specific proteins may play an important role in germination and seedling development.     

ULTRASTRUCTURAL ALTERATIONS IN OAK LEAVES PARASITIZED BY TAPHRINA CAERULESCENS

Leaves of Quercus velutina Lam. parasitized by Taphrina caerulescens (Desm.) Tulanse were examined with the electron microscope to ascertain cellular aberrations that were induced by the fungus. Major changes were most apparent in the lower epidermis, where cellular divisions and enlargement were common, the cell walls becoming irregularly thickened. Many of the ultrastructural features of the diseased cells resembled those of meristematic cells; the absence of a large central vacuole, a disproportionately large nucleus, and an abundance of cytoplasmic organelles. Chloroplasts with large amounts of starch were also routinely observed.     

Ultrastructural studies of the effects produced by some amino acid metal systems on escherichia coli B

Escherichia coli cells were grown in the presence of L-serine and gallium(III) nitrate a different molar ratios. Under these conditions ultrastructural changes were observed in the cells when examined under the electron microscope. Although some changes were seen inside the cell the major modifications were observed at the cell surface. These changes appeared to involve both the cell and the peptidoglycan layer. Autoradiography at the electron microscope level undertaken with similar mixtures and containing L-(3 - 3H) serine showed silver grains at or near the cell surface. In some cases, surface modifications were so pronounced that they resulted in the E. coli appearing as sheets of cells.No cell surface changes were detected when mixtures of L-serine and potassium tetrachloropalladate(II) were used as modifying agents. With the palladium(II) mixtures all changes observed were intracellular. These modifications included the appearance of membrane-bound vehicles, clumping of the cytoplasm and changes in the nucleoplasm. Autoradiography carried out in the presence of L-(3 - 3H) serine showed a significant proportion of silver grains over the nuclear region. A pure palladium(II) complex of L-serine was examined as a modifying agent in the concentration range 1–9 μ/cm³ resulting in very pronounced modification of the cells when exposed to higher concentrations.     

Changes in the arrangement of cellulose microfibrils associated with the cessation of cell expansion in tracheids

 The relationship between the cessation of cell expansion and formation of the secondary wall was investigated in the early-wood tracheids of Abies sachalinensis Masters by image analysis and field emission scanning electron microscopy. The area of the lumen and the length of the perimeter of the lumen of differentiating tracheids increased from the cambium towards the xylem. These increases had just ceased in the case of tracheids closest to the cambium in which birefringence was first detected by observations with a polarizing light microscope. Cellulose microfibrils (MFs) deposited on the innermost surfaces of radial walls were not well ordered during the expansion of cells, but well ordered MFs were deposited at the subsequent stage of cell wall formation. The first well ordered MFs were oriented in an S-helix. The well ordered MFs had already been deposited at the tracheids where birefringence was first detected under the polarizing light microscope. These results indicate that the deposition of the well ordered MFs, namely, the formation of the secondary wall, begins before the cessation of cell expansion of tracheids. Therefore, it seems that the expansion of tracheids is restricted by the deposition of the secondary wall because the cell walls become rigid simultaneously with the development of the secondary wall and, therefore, the yield point of cell walls exceeds the turgor pressure of the cell. Received: 3 July 1996 / Accepted: 24 September 1996     

The structure and composition of aleurone grains in the barley aleurone layer

Summary Cytochemical methods have been used in conjunction with light and electron microscopy to determine the nature of the inclusions in aleurone grains of barley aleurone layers. Two kinds of inclusions were found: (1) Globoids within globoid cavities which were not enclosed by a membrane: the globoids stained red with toluidin blue due to the presence of phytin, and with lipid stains; (2) Protein-carbohydrate bodies which stained green with toluidin blue. The characteristics of globoids and protein-carbohydrate bodies as seen in the electron microscope are described in detail using both glutaraldehyde- and permanganatefixed tissues. The protein-carbohydrate body was identified by silver-hexaminestaining; this was not caused by carbohydrate but by some component which stained green in toluidin blue and which also occurred in cell walls in a thin band adjacent to the cytoplasm. The characteristics of both bodies are discussed in relation to apparent confusion in their identities in previous electron-microscope studies.     

Ultrastructural characteristics of the cortex and of the membranelles of the ciliate Espejoia mucicola (Oligohymenophora, Hymenostomata, Tetrahymenina)]

It was shown in a detailed study of the cortex and buccal organelles of Espejoia that, at the ultrastructural level, the general plan of organization of this ciliate conforms to that of Tetrahymenina. Specific variations seen in the cortex and membranelles bring out the very pronounced affinities of Espejoia to the genus Glaucoma and other related ciliates. In view of the foregoing and on the basis of reccent findings on morphogenesis, we confirm the taxonomic position of Espejoia mucicola among Tetrahymenina in the family Glaucomidae.     

Subcellular localization of chromium and nickel in root cells of Allium cepa by EELS and ESI

The ultrastructural investigation of the root cells of Allium cepa L. exposed to two different concentrations of chromium + nickel (Cr+Ni) (10 micromol/L and 100 micromol/L) revealed that toxic symptoms were induced by increasing heavy metal concentration and treatment time. Several significant ultrastructural changes were caused by 100 micromol/L Cr+Ni - deposition of electron dense material in cell walls; larger vacuolar precipitates surrounded by membranes inside vacuoles; increment of disintegrated organelles and high vacuolization in cytoplasm. The localization of the precipitates in which the metal ions were detected by electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) was investigated. Chromium and nickel were localized in the electron dense precipitates of the root cells exposed to only 100 micromol/L Cr+Ni. None were found in the root cells exposed to 10 micromol/L Cr+Ni. Higher amounts of Cr+Ni were mainly accumulated in the cell walls and vacuoles of the fourth or fifth cortical layer.     

Mise en evidence d'une activité catalasique dans les peroxysomes de Micrasterias fimbriata (Ralfs)

Summary By ultrastructural methods, cytology, and cytochemistry it is shown that peroxysomes are present during all stages of the life cycle of the green unicellular alga Micrasterias fimbriata, cultivated on mineral medium. These organelles, surrounded by a single membrane, are in connection with endoplasmic reticulum. In full-grown cells, they are preferentially situated near chloroplasts and cell walls. The number of peroxisomes increase before cellular division and the organelles flow into the young bulge in front of the chloroplast.Application of a modified Graham and Karnosky's medium using DAB at pH 9 shows that an important activity of catalase is present not only at the level of peroxisomes but also at the level of the cell walls and certain Golgi vesicles.The topographic relations of peroxisomes with different cellular organelles and their possible functions in cell wall or mucus synthesis are discussed.     

Changes in calcium signalling, gravitropism, and statocyte ultrastructure in pea roots induced by calcium channel blockers

The effect of Ca2+ channel blockers (D600 and nicardipine) were investigated in our experiments on 5-day seedlings of pea. Nicardipine had more inhibiting effect on root elongation than D600. The Ca2+ channel blockers (CCB) depressed gravitropic response in roots. In root statocytes, the destruction of the polar arrangement of cell organelles and other changes were induced by 10(-5) M D600 or nicardipine treatment for 12 h. At ultrastructural level, there were observed a lack of polarity, pronounced vacuolization, and changes in dictyosome structure in treated statocytes. Cytochemical study indicated that Ca2+ ions were concentrated in the intracellular organelles and cell walls in statocytes treated with CCB similar to untreated control. The data suggest that the effects of the CCB that demonstrated the correlation between the loss of polarity in statocytes and altered root gravitropism may be functionally related to systems that regulate Ca2+ homeostasis, particularly Ca2+ channels.     

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.     

Potassium in the Apoplast of the Root Sink Region

Using carboxyfluorescein, a fluorochrome transported along the phloem, we demonstrated that symplasmic phloem unloading in the watermelon root occurred in the basal zone of the meristem adjusting to the elongation zone. In the similar zones of maize and pumpkin roots, a high level of potassium was detected by X-ray microanalysis in the cell walls and intercellular spaces. Potassium concentration in these compartments comprised two-thirds of that in the cytoplasm. Such proportion between potassium concentrations in the cytoplasm and apoplast was characteristic of both the cortex and stele. Since potassium is a dominant osmotically active component in root tissues, such a proportion between its intracellular and apoplastic concentrations provides for a low turgor pressure in the cells of the sink region, in the phloem in particular. This might increase a turgor pressure gradient along the translocation route between source and sink tissues, which is a driving force for phloem assimilate transport.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 591–599.Original Russian Text Copyright © 2005 by Krasavina, Burmistrova, Feshchenko, Nosov.     

An electron microscopic study of the mature megagametophyte in Zea mays

With light microscopy maize megagametophytes stained with Alcian blue-periodic acid-Schiff (AB-PAS) reveal acid or neutral polysaccharides in various cell walls. Comparative fine structural studies were made of permanganate- or OsO₄-fixed material. Organelle distribution is random in the vacuolate and multinucleate antipodal cells; organelles are abundant; starch is scarce. Antipodal cell walls have large openings forming several syncytia. Some walls are papillate. In the central cell (primary endosperm cell) a thin peripheral layer of cytoplasm surrounds the large vacuole; organelle number is moderate; starch is abundant. The central cell wall is also papillate adjacent to the antipodals and around the egg apparatus. In the synergids organelle distribution is non-random; nuclei and numerous organelles occupy the micropylar cytoplasm of each synergid; vacuoles dominate the chalazal cytoplasm of these cells. The filiform apparatus stains with AB-PAS and is composed of both lightly and darkly stained amorphous material. In the egg, organelle distribution is perinuclear with vacuoles proximal to the micropyle; mitochondria are large, abundant and polymorphic; starch is abundant. Nucleolar diameter is five times greater in the central cell and egg than in the antipodal cells and ten times greater than in the synergids. Plasmodesmata occur in all cell walls within the gametophyte, but none appear in the gametophyte wall itself. It is suggested that the antipodals and synergids might be secretory, the latter probably being involved in pollen tube attraction, and that stored metabolites in the central cell and egg cytoplasm support rapid increase in metabolism following fertilization.     

伊曲康唑对皮肤癣菌形态学影响研究

目的观察皮肤癣菌在伊曲康唑作用下的形态学变化。方法应用美国CLSI制订的标准M38-A方案进行伊曲康唑对皮肤癣菌的体外药敏试验,测定最小抑菌浓度（MIC）,将伊曲康唑作用前后的皮肤癣菌分别制成标本,在光学显微镜、扫描电子显微镜和透射电子显微镜下观察形态学变化。结果伊曲康唑作用于皮肤癣菌后,在光学显微镜下菌丝变得弯曲、短粗,顶端和局部出现膨大;扫描电子显微镜下菌丝变得弯曲、短粗、干瘪,顶端和局部出现膨大,有不规则分支,表面粗糙,有大小不等的凹陷;透射电子显微镜下菌丝变得皱缩,有凹陷,双层细胞壁结构消失或不完整,细胞膜不连续,皱缩细胞膜和细胞壁之间及胞浆内出现许多小的高电子密度颗粒,细胞器也变得不清晰。结论伊曲康唑使皮肤癣菌的形态发生明显变化。     

Fracture of plant tissues and walls as visualized by environmental scanning electron microscopy

The environmental scanning electron microscope (ESEM) provides a highly relevant and controllable environment in which to study hydrated systems without the artefacts of other highly prepared specimens. The instrument facilitates control of turgor through hydration using different chamber vapour pressures. Deformation of a simple plant tissue-upper epidermal layers in Allium cepa (onion)-was observed at the scale of the two principal failure mechanisms: cell breakage; and cell separation induced by treatment with a chelating agent. Cell rupture and release of contents occurred at cellular junctions ahead of an imposed growing notch, indicating that disruption of cells occurred remotely from the creation of a new surface. Cells that separated usually maintained their turgor and the separation process took place through progressive failure of middle lamellar material seen as strands between separating cells. These mechanisms were compared with the rupture of excised Chara corallina walls that occurred by formation and breakage of strands between separating wall layers. This study provides in situ visual characterization of wall rupture and cell separation at the microscopic level in hydrated plant material.     

The injury response of the neurones of Periplaneta Americana

Bilateral pairs of specific neurones in the metathoracic ganglion of the cockroach were used to study the ultrastructural changes which follow axon injury. The appearance of these matched pairs of cells was compared three days after the axons on one side of the ganglion only had been severed. The control or uninjured cell body appeared normal, but changes were observed in the neurones whose axon had been cut. These changes included a general increase in the number of ribosomes throughout the cytoplasm, dilatation of the cisternae of the endoplasmic reticulum and a reduction in size of the Golgi bodies. No ultrastructural features were seen which could correspond to the perinuclear ring of RNA observed histochemically with the light microscope. The possible nature of this RNA is discussed.