Uptake of Proteins by Plant Roots

The patterns of uptake of fluorescein-labelled lysozyme (Fl-lysozyme) by barley, maize, onion, tomato and vetch are similar as revealed by fluorescence microscopy. Penetration of the root cap and through the epidermis into the cortex increases with time of exposure and decreases with higher salt concentrations. In fact, one molar ethylammonium chloride can remove most of the absorbed protein from treated roots and the space observed to be stained by Fl-lysozyme in this manner can be visualized as “free space”. Results with sterile and non-sterile barley roots were indistinguishable. At low ionic strength, Fl-lysozyme can penetrate cells and complex with nucleoli. Such cell protoplasts appear “coagulated”. Uptake results with fluorescein per se were unlike those with protein. The uptake of a much larger molecule, ferritin, is confined to the epidermis and root cell walls. Localized, absorbed protein and root growth inhibition by basic proteins have yet to be related.     

The Effect of Histones and Other Basic Macromolecules on Cell Permeability and Elongation of Barley Roots

Low exogenous concentrations of calf-thymus histone, poly-l -lysine and lysozyme inhibit root elongation. These basic macromolecules (polycations) also affect cell permeability resulting in a leakage of ultraviolet light (UV)-absorbing materials and chloride ion from cells. Adenosine 5′-monophosphate was identified as one of the UV-absorbing compounds in the root exudate, by thin layer chromatography. These deleterious effects of polycations on root growth and permeability are reduced in the presence of calcium and other divalent cations. Calcium ion-histone interaction appears to exhibit competitive kinetics and suggests that both calcium and histone compete for attachment to negative sites on cell membrances.     

Antimicrobial Actions of Hexachlorophene: Lysis and Fixation of Bacterial Protoplasts

Hexachlorophene was found to be both a lytic and a fixative agent for protoplasts isolated from Bacillus megaterium. Concentrations of 50 to 100 mug of drug per mg of original cell dry weight were required to lyse 4.4 x 10(9) protoplasts (2 mg of original cell dry weight). At higher drug concentrations, protoplasts became fixed against osmotic stress and reduced in sensitivity to disruption by n-butanol. Lower drug concentrations caused proportionate lysis in the protoplast population. Intact cells lost the ability to become plasmolyzed at these same hexachlorophene concentrations. Nonplasmolyzed, drug-treated cells were resistant to the action of lysozyme, whereas plasmolyzed, drug-treated cells were sensitive. But the sensitivity of isolated cell walls to lysozyme digestion was not markedly altered by hexachlorophene treatment. These effects appeared to be secondary in the killing of cells by hexachlorophene because they occurred at concentrations higher than the minimum lethal concentration.     

UPTAKE BY AND FATE OF LYSOZYME IN ROOTS OF IASIONE MONTANA (CAMPANULACEAE)

A basic protein, lysozyme, labeled with fluorescein isothiocyanate, is readily taken up by roots of lasione montana. Most of the protein taken up is tightly bound to cell walls of the roots. Fluorescent protein is diluted in the growing region of a root as cells elongate and divide. Fluorescence remains in mature nongrowing regions and root cap cells for one to two weeks. Redistribution and translocation of the protein within the root is minimal or nil. A layer of chloroform-soluble material that prevents lysozyme from interacting with stem cell walls exposed to fluorescent lysozyme was found on stems of germinating Iasione montana.     

Structure and function of the microtubular cytoskeleton during endosperm development in wheat: an immunofluorescence study

Summary The three-dimensional structure of the microtubular cytoskeleton of developing wheat endosperm was investigated immunocytochemically. Semi-thin sections were prepared from polyethylene glycol embedded ovaries. At the free-nuclear stage the endosperm cytoplasm with regularly distributed nuclei surrounded a large central vacuole and exhibited an extensive network of fluorescent labelled microtubular assemblies radiating from each nucleus. As was found in other coenocytes, this particular and nuclear-dependent cytoskeletal configuration functions in the arrangement of nuclei and in the stabilization of the nuclear positions. At the beginning of cellularization of the endosperm the formation of vacuoles altered the radiating networks. It is likely that the radiating microtubular arrays function in the formation of phragmoplasts, independent of nuclear divisions. The formation of anticlinal cell walls, giving rise to openended cell cylinders, coincides with the occurrence of phragmoplast microtubular arrays which were demonstrated during the period of cell wall elongation. The microtubular system radiating from the nuclei in these cell cylinders anchored the nuclei in stage- and locus-specific positions. During the development of aleurone and inner endosperm cells, cell morphogenesis was related to earlier demonstrated types of microtubular configurations in the cortical cytoplasm. This suggests that a general mechanism is involved.Abbreviations A alveolus - AL aleurone layer - CE central endosperm - CV central vacuole - DAP days after pollination - END endosperm - FITC fluorescein isothiocyanate - GAR-FITC goat anti-rabbit antibodies conjugated with FITC - I integument - IE PC inner epidermis pericarp - II inner integument - N nucleus - NC nucellus cells - NE nucellar epidermis - NUC nucellus - OI outer integument - PBS phosphate buffered saline - PC pericarp - PEG polyethylene glycol - V vacuole     

A new fluorescent test for cell vitality using calcofluor white M2R

The fluorescent fabric-brightener dye, Calcofluor white M2R (CFW), can be used to distinguish between living and dead cells from a variety of animal and plant sources. CFW does not stain living mouse fibroblasts or trout red blood cells and stains only the cell walls in living cells from the epidermis of onion bulb scale, staminal hairs of Tradescantia, and longitudinal sections of broad bean stems and roots. Heat-killed plant or animal cells are recognized by their lightly stained cytoplasm and brightly stained nuclei. The optimum staining concentrations were very low (0.01% to 0.03%) and nontoxic. Using onion scale epidermis in which some cells had been killed by heating as a test system, and the plasmolysis-deplasmolysis rection as the ultimate test for cell vitality, results from CFW staining correctly predicted cell vitality for about 98% of the cells tested. This success rate was comparable to those for Evans blue, uranin or neutral red in this test system.     

A New Fluorescent Test for Cell Vitality Using Calcofluor White M2R

The fluorescent fabric-brightener dye, Calcofluor white M2R (CFW), can be used to distinguish between living and dead cells from a variety of animal and plant sources. CFW does not stain living mouse fibroblasts or trout red blood cells and stains only the cell walls in living cells from the epidermis of onion bulb scale, staminal hairs of Tradescantia, and longitudinal sections of broad bean stems and roots. Heat-killed plant or animal cells are recognized by their lightly stained cytoplasm and brightly stained nuclei. The optimum staining concentrations were very low (0.01% to 0.03%) and nontoxic. Using onion scale epidermis in which some cells had been killed by heating as a test system, and the plasmolysisdeplasmolysis rection as the ultimate test for cell vitality, results from CFW staining correctly predicted cell vitality for about 98% of the cells tested. This success rate was comparable to those for Evans blue, uranin or neutral red in this test system.     

Vital and Calcofluor staining ofCosmarium and its protoplasts

Summary Intact cells, protoplasts, primary and secondary walls ofCosmarium species were stained with 13 vital stains and with Calcofluor. The intracellular contents of viable cells and protoplasts were not stained with any of the test dyes although the primary and secondary walls often were stained. Protoplasts disintegrated after short periods in acid stains but did survive for up to 24 hours in several dilute basic dyes. Once the protoplast membrane was damaged, the nuclei and cytoplasm were brightly stained with most dyes, whereas the vacuoles remained unstained. Calcofluor was particularly useful in determining the porous structure and pattern of the primary walls.     

Maize calreticulin localizes preferentially to plasmodesmata in root apex

Using a polyclonal antibody raised against calreticulin purified and sequenced from maize, we performed an immunocytological study to characterize putative domain-specific subcellular distributions of endoplasmic reticulum (ER)-resident calreticulin in meristematic cells of maize root tip. At the light microscopy level, calreticulin was immunolocalized preferentially at cellular peripheries, in addition to nuclear envelopes and cytoplasmic structures. Punctate labelling at the longitudinal walls and continuous labelling at the transverse walls was characteristic. Immunogold electron microscopy revealed plasmodesmata as the most prominently labelled cell periphery structure. In order to further probe the ER-domain-specific distribution of maize calreticulin at plasmodesmata, root apices were exposed to mannitol-induced osmotic stress. Plasmolysis was associated with prominent accumulations of calreticulin at callose-enriched plasmodesmata and pit fields while the contracting protoplasts were depleted of calreticulin. In contrast, other ER-resident proteins recognized by HDEL peptide and BiP antibodies localized exclusively to contracted protoplasts. This finding reveals that, in plasmolysed cells, calreticulin enriched ER domains at plasmodesmata and pit fields are depleted of other ER-resident proteins containing the HDEL retention peptide.     

Compartmentation and transport of zinc in barley primary leaves as basic mechanisms involved in zinc tolerance

The heavy metal zinc was administered to barley seedlings by increasing its concentration in the hydroponic medium. The most dramatic effect was a severe inhibition of root elongation with little effect on root biomass production. The growth of primary leaves was little affected although the zinc content of the primary leaves increased several-fold. A detailed compartment analysis was performed for 10-d-old barley primary leaves. Under low zinc nutrition (2mmol m ⁻³), highest zinc contents were observed in the cytoplasm of mesophyll protoplasts. At inhibitory zinc concentrations in the hydroponic medium (400 μmol m ⁻³), zinc levels dramatically and preferentially increased in the apoplastic space. Elevated zinc levels were also observed in the epidermal cells, and to a lesser extent, in mesophyll vacuoles. The cytoplasmic content of mesophyll protoplasts was unchanged, indicating perfect zinc homeostasis within the leaf. In order to understand the transport mechanisms underlying the steady-state distribution profile, we used ⁶⁵Zn to conduct uptake experiments with leaves whose lower epidermis had been stripped. The leaves were placed on zinc solutions of varying concentrations containing ⁶⁵Zn for 5 min to 6 h. After the incubation, the leaves were fractionated into mesophyll and epidermis protoplasts and residue, the latter mainly representing cell wall. Adsorption of Zn to the extracellular matrix was 100 times faster than Zn uptake into the cells. By far the largest portion taken up into the mesophyll protoplasts rapidly appeared in the vacuolar compartment. These results demonstrate the importance of compartmentation and transport as homeostatic mechanisms within the leaves to handle high, possibly toxic, zinc levels in the shoot.     

The use of radioautography for investigating wall secretion in plant cells

Summary The paper summarises results of simple radioautographic experiments using tritiated glucoses to investigate wall secretion in plant cells. In outer root cap cells, labelled material was first concentrated in the Golgi bodies; it later appeared in vesicles, and was incorporated into the wall immediately under the plasmalemma. It finally collected mainly in the slime layer surrounding the root tip. Biochemical analyses have indicated that this material was pectic in nature. In inner root cap and epidermal cells, labelled material incorporated into the walls and also the cell plates of dividing cells was also apparently mainly derived from Golgi bodies. In meristematic (less differentiated) cells, however, the endoplasmic reticulum was more frequently labelled than the Golgi bodies near walls that had incorporated derivatives of labelled glucose. Considerable incorporation of labelled derivatives into the wall thickenings in coleoptile xylem cells was often detected; nearby elements of the endoplasmic reticulum were again frequently labelled in these cells and less often, Golgi bodies and the cytoplasm in the region occupied by microtubules contained radioactivity. Labelling of starch grains in the plastids was generally observed, but not in cells secreting large amounts of wall materials (outer root cap and older xylem cells); however, addition of larger amounts of exogenous glucose to outer root cap cells, following their incubation in tritiated glucose, promoted such incorporation. The paper finally sets forth some considerations on experimental techniques for radioautography that might be of more general application.     

Reassembly of anAcetabularia mediterranea cell from the nucleus,cytoplasm, and cell wall

Summary A. mediterranea cells capable of full morphogenesis were reassembled from nuclei, cytoplasm, and cell wall fraction.Reassembly was performed stepwise with the recombination of cytoplasm and cell walls and finally a nucleus was implanted.Reassembly of anucleate cells was carried out by means of retransplantation of their own cytoplasm or transplantation of cytoplasm from another cell. Combinations between cytoplasm and cell walls of dark or light maintained cells were prepared. The nuclei were always transferred from light maintained cells.     

Immunocytochemical localisation of auxin-binding proteins in coleoptiles and embryos ofZea mays L.

Summary The auxin-binding protein ABP-1 was localised immunocytochemically in coleoptiles and immature embryos ofZea mays. Two primary polyclonal antibodies raised against ABP-1 and secondary antibodies were either labelled with FITC or 10 nm gold particles for light microscopy, and with 10 nm gold particles for transmission electron microscopy. Light microscopy revealed that ABP-1 was localised in the epidermal cells of etiolated maize coleoptiles, in subepidermal parenchymatic mesophyll cells of the coleoptile and in the companion cells of the vascular bundles. Most labelling was found in the cytoplasm, less in nuclei and vacuoles and cell walls appeared negative. The region of the plasma membrane exhibited prominent labelling. Embryos showed low labelling throughout their tissues just after excision, but after culture for 7 days intensive labelling was found in the epidermis of the scutellum. Quantitative electron microscopy confirmed that ABP-1 was present in the cytoplasm of epidermal, mesophyll, and companion cells of coleoptiles. Gold particles were neither found in cell walls nor in the cuticle. Areas with ER and dictyosomes within epidermal and mesophyll cells of coleoptiles had a denser labelling with gold particles than elsewhere. Labelling at the plasma membrane, being the site where the auxin binds to the ABP, was observed at low levels in all cells examined, which is due to the method applied. Epidermal cells of embryos cultured for 5 days exhibited high levels of gold particles in ER and nuclei, and lower levels in the cytoplasm. The distribution is only partly in accordance with the model in which ABP is thought to cycle through the plant cell from the ER via the Golgi system towards the plasma membrane.Abbreviations ABP-1 auxin-binding protein 1 - BSA bovine serum albumin - 2,4-D 2,4-dichlorophenoxyacetic acid - EM electron microscopy - LM light microscopy - LR Write London resin white - PBS phosphate-buffered saline - PEG polyethylene glycol - TEM transmission electron microscopy     

Cellulose and pectin localization in roots of mycorrhizalAllium porrum: labelling continuity between host cell wall and interfacial material

Two different types of contacts (or interfaces) exist between the plant host and the fungus during the vesicular-arbuscular mycorrhizal symbiosis, depending on whether the fungus is intercellular or intracellular. In the first case, the walls of the partners are in contact, while in the second case the fungal wall is separated from the host cytoplasm by the invaginated host plasmamembrane and by an interfacial material. In order to verify the origin of the interfacial material, affinity techniques which allow identification in situ of cell-wall components, were used. Cellobiohydrolase (CBH I) that binds to cellulose and a monoclonal antibody (JIM 5) that reacts with pectic components were tested on roots ofAllium porrum L. (leek) colonized byGlomus versiforme (Karst.) Berch. Both probes gave a labelling specific for the host cell wall, but each probe labelled over specific and distinct areas. The CBH I-colloidal gold complex heavily labelled the thick epidermal cell walls, whereas JIM 5 only labelled this area weakly. Labelling of the hypodermis was mostly on intercellular material after treatment with JIM 5 and only on the wall when CBH I was used. Suberin bands found on the radial walls were never labelled. Cortical cells were mostly labelled on the middle lamella with JIM 5 and on the wall with CBH I. Gold granules from the two probes were found in interfacial material both near the point where the fungus enters the cell and around the thin hyphae penetrating deep into the cell. The ultrastructural observations demonstrate that cellulose and pectic components have different but complementary distributions in the walls of root cells involved in the mycorrhizal symbiosis. These components show a similar distribution in the interfacial material laid down around the vesicular-arbuscular mycorrhizal fungus indicating that the interfacial material is of host origin.     

Mating reaction in yeast protoplasts

Protoplasts prepared from complementary haploid strains ofSaccharomyces cerevisiae were studied with regard to their ability of conjugating. Neither fresh protoplasts nor the growing protoplasts possessing fibrillar walls exhibited sex specific agglutination or fusion. However, they were capable of inducing sexual activation in normal cells of opposite mating type. After completing the regeneration of cell walls the protoplasts could conjugate either with each other or with cells of opposite sex. The frequency of conjugations was low, about 1%, and was largely dependent on the degree of completition of the wall during regeneration. From the results the following conclusions may be drawn: 1. The initiation of mating is dependent on the integrity of the cell wall. 2. The sex specific morphogenetic changes do not occur in wall-less protoplasts but may happen after the protoplasts have regenerated their cell walls. 3. The lysis of cell walls does not occur until the walls come into close contact. 4. The fusion of plasma membranes in sex-activated protoplasts cannot be induced by artefucial agglutination.     

Anatomy and ultrastructure of epidermal cells in the haustorium of a parasitic flowering plant,Cuscuta japonica, during attachment to the host

Changes of epidermal cells in the haustorium of the parasiticCuscuta japonica during its attachment to the host plantimpatiens balsamina were studied with light and electron microscopy. In the transverse sections of dodder stems not in contact with the host, epidermal cells had rounded outlines. However, when haustorial initials developed in the cortex of the parasite stem at the contact site, the epidermal cells had more dense cytoplasm and conspicuous nuclei than before, and their outline was flat in the longitudinal section. As meristem cells developed from those initials, the epidermal cells became more elongated. When the haustorium was fully matured, the apical tips of the elongated epidermal cells at the contact site branched like toes, producing numerous projections via cell wall invaginations. This event caused spaces to form between the projections; coincidently, the surface area of the apical ends of the epidermal cells increased. The dense cytoplasm at those projections contained prominent nuclei and abundant other organelles, suggesting a active metabolism. Osmiophilic particles, releasing into the cell walls from the cytoplasm, were though to be associated with the loosening and elongating of the epidermal cell walls. Dense and homogeneous materials were secreted within the spaces between the projections. These materials could play an important role in cementing the haustorium onto the surface of the host organ.     

Epidermal Growth Factor and EGF Receptors are mainly expressed in the wound epidermis and proliferating ependyma of the regenerating tail of lizards

The presence of EGF and its receptor during tail regeneration in lizard has been assessed by immunoblotting and immunofluorescence to test whether this growth factor may be involved in the process. Immunolabelled bands at 8 and 42–46 kDa for EGF are detected in the regenerating tail. A main band at 45–50 kDa and other weaker bands at lower or higher molecular weight for the EGF receptor are also present. The results indicate that degraded forms of the protein are present although the specific nature of the different bands could not be determined. Immunofluorescence indicates that EGF-labelled cells and EGF receptor are especially seen in the wound epidermis and in the cytoplasm of ependymal cells. Numerous basal keratinocytes of the wound epidermis and apical epidermal peg contain labelled nuclei for EGFR, suggesting that activated receptor stimulates intense cell proliferation of the wound epidermis. Blastema and labelled myoblasts are occasionally detected in early differentiating muscles, but almost no labelled chondroblasts are present in the differentiating cartilaginous tube. The study indicates that EGF and its receptor are mainly present in epithelial cells in a form that allows them to regulate proliferation during tail regeneration.     

A histological and histochemical study of the cotyledons ofPhaseolus vulgaris L. during germination

Summary The cotyledon ofPhaseolus vulgaris L. comprises four tissues: epidermis, abaxial hypodermis, storage parenchyma, and procambium. A complex intercellular space system is present throughout the storage tissue and comprises about 16% of the cotyledon volume. All the cells contain protein bodies, and the hypodermis and storage parenchyma also contain starch grains. The epidermal cells are at the 2 C level of DNA, those of the hypodermis at the 4 C level, and the storage cells vary from 8 C to 32 C. During germination stomata differentiate in the epidermis. Reserve mobilization begins in the cells furthest from the epidermis and from the vascular tissue. Protein is removed from these cells with little or no coalescence of protein bodies. The DNA content of the nuclei decreases. The cell walls swell and then decrease in thickness as material is removed. Finally the nuclei and cytoplasm disappear and the cells collapse. In the cells near vascular bundles the protein bodies coalesce before losing their protein. The DNA content of the nuclei declines but nuclei and cytoplasm are still present at abscission. These cells do not collapse. Cytoplasmic RNA content is highest near the abaxial surface. Most of the RNA is removed during the first three days of germination.     

Involvement of JIM13- and JIM8-Responsive Carbohydrate Epitopes in Early Stages of Cell Wall Formation

Beta vulgaris L.). The spatial and temporal expression of both antigens was studied in suspension cells used as the source-tissue for protoplast isolation, in suspension- and mesophyll-derived protoplasts, and in cells which developed from both types of protoplast. Immunofluorescence and immunocytochemical-electron microscopic methods revealed that labeling was present in the cell walls of most suspension cells and also in the incipients of cell walls synthesized around the protoplasts. This signal became much more intense as rebuilding of the cell wall progressed during culture. Relatively weaker labeling was observed in the cytoplasm, where it was frequently associated with the vacuolar compartment. Signal intensity varied between individual cells of the same population and in successive stages of development, but was always stronger with JIM13 than with JIM8. The role of JIM13-responsive epitope in the development of suspension-derived protoplasts was further studied by its ability to bind antibody added to cultures of different ages. Both JIM8- and JIM13-responsive epitopes were widespread in sugar beet cells of different origin and stage of cell wall synthesis. These epitopes may play an important role in cell wall formation and growth under in vitro conditions. Received 17 August 1998/ Accepted in revised form 13 January 1999