Immunogold labelling of actin on sections of freeze-substituted plant cells

Summary We have successfully combined the superior ultrastructural preservation capabilities of rapid freeze fixation and freeze substitution (RF-FS) with immunogold antibody localization techniques to label microfilament (MF) bundles with monoclonal antibody to actin in two different plant tissues:Nicotiana pollen tubes andDrosera tentacles. We have thus verified that the extensive MF bundles seen in these cells after RF-FS are composed of actin, a protein that is difficult to preserve by conventional fixation methods for electron microscopy.     

Microfilaments in the preprophase band of freeze substituted tobacco root cells

Summary The organization and distribution of microfilaments (MFs) in the preprophase bands (PPBs) of tobacco (Nicotiana tabacum L. var. Maryland Mammoth) root tip cells were studied with high pressure freezing and freeze-substitution methods. MFs were present predominantly as single filaments interspersed among microtubules (MTs) throughout the PPB. Some MFs appeared to be associated with MTs, whereas others were not. This is the first time that MFs have been demonstrated in the PPB at the electron microscope level.     

High pressure freezing and freeze substitution improve immunolabeling of S-locus specific glycoproteins in the stigma papillae ofBrassica

Summary High pressure freezing and freeze substitution methods significantly improve the antigenic preservation of S-locus specific glycoproteins (SLSG). The SLSG, which are implicated in the incompatibility response, are localized over the cell wall and cytoplasm. Labeling in the cytoplasm is mainly associated with dictyosomes and rough endoplasmic reticulum. Quantitative analysis show that in cryofixed papillae the labeling was enhanced by approximately 45% over the cell wall and approximately 90% over the dictyosomes compared to chemically fixed papillae.     

Sporangial structure inPhytophthora is disrupted after high pressure freezing

Summary The effects of high pressure on the ultrastructure of sporangia ofPhytophthora cinnamomi andP. palmivora have been examined by comparing sporangia frozen in a Balzers hyperbaric freezer or pressurized in a French pressure cell with sporangia plunge frozen at ambient pressure. Both freeze fixation methods provided excellent preservation of most cell structures, but one organelle type seen in plunge frozen material, the large peripheral vesicle (LPV), was not observed in high pressure frozen sporangia. Instead, these sporangia contained large irregularly shaped structures which exhibit the patterns of spatial distribution and, forP. cinnamomi, the monoclonal antibody binding characteristic of LPVs. These findings suggest that some factor of the hyperbaric freezing process causes LPVs to be degraded. Sporangia ofP. cinnamomi that had been pressurized in a French pressure cell also exhibited large structures with the spatial distribution and monoclonal antibody binding characteristic of LPVs. The apparent expansion of LPVs that follows from both pressurizing treatments causes considerable passive disruption of sporangial structure. This is the first report of a major disturbance of cell structure from use of the Balzers hyperbaric freezer, and reflects the lability, noted in previous work, of LPVs inPhytophthora.     

Endoplasmic reticulum in developing seeds of Vicia faba

The changes in endoplasmic reticulum (ER) morphology during seed development have been followed using a thick section electron microscope technique. The tissues were stained with a zinc iodineosmium tetroxide complex which preferentially accumulated in the lumen between double membranes. Sections up to 2 m in thickness were examined in a high voltage electron microscope (HVEM) with tilt facility to produce stereo pairs. The micrographs from HVEM showed an increase in the extent of interconnecting tubular and cisternal ER during the protein deposition phase of seed maturation with subsequent degeneration of the cisternae to a reticular form during the final seed maturation phase. No evidence of cisternal ER vesicles was found, instead our work suggests that such structures are artefacts of thin sectioning with the so-called vesicles representing the interconnection of cisternal and tubular ER. The results are discussed with reference to the transport of storage protein from its site of synthesis, the rough cisternal ER, to that of accumulation, the vacuolar protein bodies.Abbreviations ER endoplasmic reticulum - HVEM high voltage electron microscopy     

The hyphae ofUromyces appendiculatus within the leaf tissue after high pressure freezing and freeze substitution

Summary The fine structure of the intercellular dikaryotic hyphae of the biotrophic fungusUromyces appendiculatus was studied. High pressure freezing and freeze substitution were used to achieve a closer approximation of the native state than with conventional fixation and dehydration techniques. In addition to organelles previously described in rust fungi, heavily decorated multivesicular bodies (star bodies) were found close to the nuclei. Two types of tubular-vesicular complexes were distributed randomly within the cytoplasm of the hyphae. Furthermore, a more or less pronounced brush-like fibrillar layer on the hyphal walls was detected. The possibility that the latter two structures are correlated with the biotrophic phase of this fungus is discussed.Abbreviations TVC tubular-vesicular complex - MVB multivesicular body - M mitochondrion - N nucleus - NP nuclear pore - S septum - MT microtubule     

Microfilament organization and distribution in freeze substituted tobacco plant tissues

Summary The organization and distribution of microfilaments in freze substituted leaf tissues and root tips of tobacco plants (Nicotiana tabacum L. var. Maryland Mammoth) were investigated in detail. Three categories of microfilaments were recognized in interphase cells of all tissues including those in the root cap: (1) microfilament bundles; (2) single microfilaments; (3) cortical microtubuleassociated microfilaments. While the microfilament bundles appeared to be distributed throughout the cytoplasm, the single microfilaments were mainly confined to the cell periphery. All three categories of microfilaments were associated with various organelles. Our study indicates that the three categories of microfilaments are normal cytoskeletal components in higher plant cells. The implications of these findings are discussed.Abbreviations MFB microfilament bundle - SMF single microfilament - MAMF microtubule-associated microfilament - AAP actin-associated protein - MAP microtubule-associated protein - MES 2(N-morpholino)ethanesulfonic acid     

Cytoplasmic streaming inChara rhizoids

Summary In-vivo videomicroscopy ofChara rhizoids under 10^–4g demonstrated that gravity affected the velocities of cytoplasmic streaming. Both, the acropetal and basipetal streaming velocities increased on the change to microgravity. The endogenous difference in the velocities of the oppositely directed cytoplasmic streams was maintained under microgravity, yet the difference was diminished as the basipetal streaming velocity increased more than the acropetal streaming velocity. Direction and structure of microfilaments labeled by rhodamine-phalloidin had not changed after 6 min of microgravity.Abbreviations g gravitational acceleration - Nizemi slow rotating centrifuge microscope - Texus technological experiments under reduced gravity     

Active sliding movement of latex beads coated with skeletal muscle myosin onChara actin bundles

Summary Latex beads coated with rabbit skeletal muscle myosin were introduced by intracellular perfusion intoChara cells from which the tonoplasts had been removed. Mg · ATP dependent movement of the beads along files ofChara chloroplast layers was observed. The movement was in opposite directions on the two sides of the indifferent line, indicating that the movement was dependent on the polarity of the actin bundles. This suggests that the unknown factor responsible for generating the motive force for cytoplasmic streaming inChara endoplasm is myosin. The advantages of the present experimental system for studying the sliding mechanism of actomyosin are discussed.Abbreviations APW artificial pond water - ATP adenosine 5-triphosphoric acid - DTT dithiothreitol - EDTA ethylenediamine tetraacetic acid - EGTA ethyleneglycol-bis(-aminoethyl ether)N, N, N, N-tetraacetic acid - HMM heavy meromyosin - LMM light meromyosin - NEM N-ethylmaleimide - PIPES piperazine-N, N- bis(2-ethanesulfonic acid)     

Specialized formations of endoplasmic reticulum in parenchyma cells ofMimosa pudica L.

Summary Parenchyma cells ofMimosa pudica display close associations between two or more cisternae of the endoplasmic reticulum. These associations form simplified types of lamellar bodies in which inner paired lamellae have lost their ribonucleoprotein granules and are separated by a dense layer.     

Structural interactions of actin filaments and endoplasmic reticulum in honeybee photoreceptor cells

Summary Fluorescent phallotoxins and heavy meromyosin were used to reveal the organization of the actin cytoskeleton in honeybee photoreceptor cells, and the relationship of actin filaments to the submicrovillar, palisade-like cisternae of the endoplasmic reticulum (ER). Bundles of unipolar actin filaments (pointed end towards the cell center) protrude from the microvillar bases and extend through cytoplasmic bridges that traverse the submicrovillar ER. Within the cytoplasmic bridges, the filaments are regularly spaced and tightly apposed to the ER membrane. In addition, actin filaments are deployed close to the microvillar bases to form a loose web. Actin filaments are scarce in cell areas remote from the rhabdom; these areas contain microtubule-associated ER domains. The results suggest that the actin system of the submicrovillar cytoplasm shapes the submicrovillar ER cisternae, and that the distinct ER domains interact with different cytoskeletal elements.     

Membrane associated qualitative differences in cell ultrastructure of chemically and high pressure cryofixed plant cells

Membrane contrast can sometimes be poor in biological samples after high pressure freezing (HPF) and freeze substitution (FS). The addition of water to the FS-medium has been shown to improve membrane contrast in animal tissue and yeast. In the present study we tested the effects of 1% and 5% water added to the FS-medium (2% osmium with 0.2% uranyl acetate in anhydrous acetone) on the quality and visibility of membranes in high pressure frozen leaf samples of Cucurbita pepo L. plants and compared them to chemically fixed cells (3% glutaraldehyde post-fixed with 1% osmium tetroxide). The addition of water to the FS-medium drastically decreased the amounts of well preserved cells and did not significantly improve the quality nor visibility of membranes. In samples that were freeze substituted in FS-media containing 1% and 5% water the width of thylakoid membranes was found to be significantly increased of about 20% and the perinuclear space was up to 76% wider in comparison to what was found in samples which were freeze substituted without water. No differences were found in the thickness of membranes between chemically and cryofixed cells that were freeze substituted in the FS-medium without water. Nevertheless, in chemically fixed cells the intrathylakoidal space was about 120% wider than in cryofixed cells that were freeze substituted with or without water. The present results demonstrate that the addition of water to the FS-medium does not improve membrane contrast but changes the width of thylakoid membranes and the perinuclear space in the present plant material. The addition of water to the FS-medium is therefore not as essential for improved membrane contrast in the investigated plant samples as it was observed in cells of animal tissues and yeast cells.     

Gravitropic bending of cress roots without contact between amyloplasts and complexes of endoplasmic reticulum

The polar arrangement of cell organelles in Lepidium root statocytes is persistently converted to a physical stratification during lateral centrifugation (the centrifugal force acts perpendicular to the root long axis) or by apically directed centrifugation combined with cytochalasin-treatment. Lateral centrifugation (10 min, 60 min at 10\g or 50\g) causes displacement of amylplasts to the centrifugal anticlinal cell wall and shifting of the endoplasmic reticulum (ER) complex to the centripetal distal cell edge. After 60 min of lateral centrifugation at 10\g or 50\g all roots show a clear gravitropic curvature. The average angle of curvature is about 40° and corresponds to that of roots stimulated gravitropically in the horizontal position at 1\g in spite of the fact that the gravistimulus is 10-or 50-fold higher. Apically directed centrifugation combined with cytochalasin B (25 g\ml^-1) or cytochalasin D (2.5 g\ml^-1) incubation yields statocytes with the amyloplasts sedimented close to the centrifugal periclinal cell wall and ER cisternae accumulated at the proximal cell pole. Gravitropic stimulation for 30 min in the horizontal position at 1\g and additional 3 h rotation on a clinostat result in gravicurvature of cytochalasin B-treated centrifuged (1 h at 50\g) roots, but because of retarded root growth the angle of curvature is lower than in control roots. Cytochalasin D-treatment during centrifugation (20 min at 50\g) does not affect either root growth or gravicurvature during 3 h horizontal exposure to 1\g relative to untreated roots. As lateral centrifugation enables only short-term contact between the amyloplasts and the distal ER complex at the onset of centrifugation and apically directed centrifugation combined with cytochalasin-treatment even exclude any contact the integrity of the distal cell pole need not necessarily be a prerequisite for graviperception in Lepidium root statocytes.Abbreviations CB cytochalasin B - CD cytochalasin D - ER endoplasmic reticulum - g gravitational acceleration     

Novel ultrastructural observations of pea (Pisum sativum) root nodule cells by high-pressure freezing and propane-jet freezing techniques

Summary Pea (Pisum sativum) root nodule cells infected by the diazotrophRhizobium leguminosarum have been well characterized by chemical fixation techniques. Propane-jet freezing and high pressure freezing were used in this study to compare rapidly frozen and chemically fixed pea root nodule cells. Cells that had been incubated in 2-(N-morpholino)ethanesulfonic acid buffer and frozen with the propane-jet freezer were better preserved than cells that had been chemically fixed or frozen with the high-pressure freezer. Rapidly frozen infected nodule cells showed that the rough endoplasmic reticulum had a high frequency of associations with the peribacteroid membrane and the infection thread. The peribacteroid space also varied in size depending on the method of preservation; however, it was most reduced in size and devoid of inclusions in the propane-jet frozen tissue. The biological significance of these observations is discussed.Abbreviations HPF high-pressure freezing - MES 2-(N-morpholino)ethanesulfonic acid - PBM peribacteroid membrane - PBS peribacteroid space - PJF propane-jet freezing - RER rough endoplasmic reticulum     

Localization of gibberellic acid-induced acid phosphatase activity in the endoplasmic reticulum of barley aleurone cells with the electron microscope

A metal-salt precipitation method with p-nitrophenyl phosphate as substrate has been used to localize in the electron microscope acid phosphatase activity in isolated aleurone layers of barley (Hordeum vulgare L.), treated for 16 h in the presence or absence of gibberellic acid (GA₃). The paper confirms results obtained earlier with an azo-dye precipitation method of enzyme localization. In addition the results show for the first time that in GA₃-treated tissue enzyme activity is associated with the endoplasmic reticulum (ER), there being reaction product deposited in the ER cisternae. It is suggested that this activity represents new enzyme synthesized on ER in response to GA₃ and probably destined for secretion.Abbreviation ER endoplasmic reticulum     

Effect of supraoptimal temperatures on the function of the subcortical fibrils and an endoplasmic factor inNitella internodes

Summary The effect of supraoptimal temperatures onNitella cells was studied with special reference to the function of subcortical fibrils and an endoplasmic factor. Local heat-treatment (50 °C for 1 minute) of an internodal cell ofNitella disclosed that 1. the subcortical fibrils in the treated area remain normal, not affected by the treatment, 2. the subcortical fibrils alone produce no cytoplasmic streaming, 3. the endoplasm contains an extremely heat-labile factor which is indispensable for streaming, and 4. the stagnant endoplasm in the heat-treated area is neither coagulated nor gelated by heat.Preliminary reports appeared in Proc. 37th Annu. Meet. Bot. Soc. Jpn. P. 160 (1972, in Japanese) and in Abst. Annu. Meet. Jpn. Soc. Cell Biol. p. 57 (1975, in Japanese).     

Endoplasmic reticulum and crystalline fibrils in the root protophloem of Nymphoides peltata

Endoplasmic reticulum in the root protophloem of Nymphoides peltata (S.G. Gmel.) O. Kuntze changes form as sieve elements differentiate. In immature sieve elements the individual endoplasmic reticulum (ER) cisternae form large irregular aggregates in the cytoplasm. In older immature sieve elements the ER aggregates are more ordered and membranes in them are convoluted. Although convoluted ER predominates in immature sieve elements the ER of the mature sieve elements consists mainly of flattened stacks of ER cisternae. Some of these stacks of ER may be derived from the existing convoluted ER. Crystalline fibrils first appear in the cytoplasm of the sieve element when the ER starts to aggregate. The crystalline fibrils move to the parietal layer of the sieve element along with the aggregates of ER. A possible ontogenetic relationship between ER and crystalline fibrils is discussed.Abbreviation ER endoplasmic reticulum     

Improved ultrastructural preservation ofPetunia andBrassica ovules and embryo sacs by high pressure freezing and freeze substitution

Summary In order to improve the ultrastructural preservation of the female gametophyte ofPetunia x hybrida andBrassica napus we tested several cryofixation techniques and compared the results with those of conventional chemical fixation methods. Ovules fixed with glutaraldehyde and osmium tetroxide in the presence or absence of potassium ferrocyanide showed poor cell morphological and ultrastructural preservation. In ovules cryo-fixed by plunging into liquid propane, the cell morphology was well preserved. However, at the ultrastructural level structure-distorting ice crystals were detected in all tissues. Due to the large size of the ovules, cryofixation by plunging in liquid propane is not adequate for ultrastructural studies. In contrast,P. x hybrida andB. napus ovules cryo-fixed by high pressure freezing showed improved cell morphological as well as ultrastructural preservation of the embryo sac and the surrounding integumentary tissues. The contrast of the cellular membranes after freeze substitution with 2% osmium tetroxide and 0.1% uranyl acetate in dry acetone was high. At the ultrastructural level, the most prominent improvements were: straight plasma membranes which were appressed to the cell walls; turgid appearing organelles with smooth surface contours; minimal extraction of cytoplasmic and extracellular substances. In contrast to the chemically fixed ovules, in high pressure frozen ovules numerous microtubules and multivesicular bodies could be distinguished.