Plasmolysed Protoplast Research Articles

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.

The root endophytic fungus Piriformospora indica requires host cell death for proliferation during mutualistic symbiosis with barley

Fungi of the recently defined order Sebacinales (Basidiomycota) are involved in a wide spectrum of mutualistic symbioses (including mycorrhizae) with various plants, thereby exhibiting a unique potential for biocontrol strategies. The axenically cultivable root endophyte Piriformospora indica is a model organism of this fungal order. It is able to increase biomass and grain yield of crop plants. In barley, the endophyte induces local and systemic resistance to fungal diseases and to abiotic stress. To elucidate the lifestyle of P. indica, we analyzed its symbiotic interaction and endophytic development in barley roots. We found that fungal colonization increases with root tissue maturation. The root tip meristem showed no colonization, and the elongation zone showed mainly intercellular colonization. In contrast, the differentiation zone was heavily infested by inter- and intracellular hyphae and intracellular chlamydospores. The majority of hyphae were present in dead rhizodermal and cortical cells that became completely filled with chlamydospores. In some cases, hyphae penetrated cells and built a meshwork around plasmolyzed protoplasts, suggesting that the fungus either actively kills cells or senses cells undergoing endogenous programmed cell death. Seven days after inoculation, expression of barley BAX inhibitor-1 (HvBI-1), a gene capable of inhibiting plant cell death, was attenuated. Consistently, fungal proliferation was strongly inhibited in transgenic barley overexpressing GFP-tagged HvBI-1, which shows that P. indica requires host cell death for proliferation in differentiated barley roots. We suggest that the endophyte interferes with the host cell death program to form a mutualistic interaction with plants.

Macrotubule‐dependent protoplast volume regulation in plasmolysed root‐tip cells ofTriticum turgidum: involvement of phospholipase D

The probable involvement of phospholipase D (PLD)/phosphatidic acid (PA) signalling in the hyperosmotic stress response of Triticum turgidum root cells was investigated by examining the effects of butanol-1, butanol-2, phosphatidylbutanol (PtdBut), N-acylethanolamine (NAE) and PA on the hyperosmotic response, the organization of the tubulin cytoskeleton and the accumulation of a phosphorylated p38-like mitogen-activated protein (MAP) kinase (phospho-p46) in plasmolysed root cells. The effects of all the treatments were assessed by differential interference contrast (DIC) microscopy of living cells, tubulin immunofluorescence, conventional transmission electron microscopy (TEM), tubulin immunogold localization, protoplast volume measurements and western blot analysis. Butanol-1 and NAE compromised the viability of plasmolysed cells, induced a marked reduction in the plasmolysed protoplast volume, and inhibited hyperosmotically induced tubulin macrotubule formation and the accumulation of phospho-p46. Exogenous PA reinforced the hyperosmotic response of T. turgidum root cells and positively affected tubulin macrotubule formation. Additionally, PA reduced the effects of butanol-1 in plasmolysed cells. Taken together, the data suggest that PLD-mediated PA synthesis occurs upstream of the accumulation of phospho-p46 to regulate hyperosmotically induced macrotubule formation in plasmolysed T. turgidum root cells.

Membrane?wall attachments in plasmolysed plant cells

Field emission scanning electron microscopy of plasmolysed Tradescantia virginiana leaf epidermal cells gave novel insights into the three-dimensional architecture of Hechtian strands, Hechtian reticulum, and the inner surface of the cell wall without the need for extraction. At high magnification, we observed fibres that pin the plasma membrane to the cell wall after plasmolysis. Treatment with cellulase caused these connecting fibres to be lost and the pinned out plasma membrane of the Hechtian reticulum to disintegrate into vesicles with diameters of 100-250 nm. This suggests that the fibres may be cellulose. After 4 h of plasmolysis, a fibrous meshwork that labelled with anti-callose antibodies was observed within the space between the plasmolysed protoplast and the cell wall by field emission scanning electron microscopy. Interestingly, macerase-pectinase treatment resulted in the loss of this meshwork, suggesting that it was stabilised by pectins. We suggest that cellulose microfibrils extending from strands of the Hechtian reticulum and entwining into the cell wall matrix act as anchors for the plasma membrane as it moves away from the wall during plasmolysis.

Hyperosmotic stress induces formation of tubulin macrotubules in root-tip cells of Triticum turgidum: their probable involvement in protoplast volume control.

Treatment of root-tip cells of Triticum turgidum with 1 M mannitol solution for 30 min induces microtubule (Mt) disintegration in the plasmolyzed protoplasts. Interphase plasmolyzed cells possess many cortical, perinuclear and endoplasmic macrotubules, 35 nm in mean diameter, forming prominent arrays. In dividing cells macrotubules assemble into aberrant mitotic and cytokinetic apparatuses resulting in the disturbance of cell division. Putative tubulin paracrystals were occasionally observed in plasmolyzed cells. The quantity of polymeric tubulin in plasmolyzed cells exceeds that in control cells. Root-tip cells exposed for 2-8 h to plasmolyticum recover partially, although the volume of the plasmolyzed protoplast does not change detectably. Among other events, the macrotubules are replaced by Mts, chromatin assumes its typical appearance and the cells undergo typical cell divisions. Additionally, polysaccharidic material is found in the periplasmic space. Oryzalin and colchicine treatment induced macrotubule disintegration and a significant reduction of protoplast volume in every plasmolyzed cell type examined, whereas cytochalasin B had only minor effects restricted to differentiated cells. These results suggest that Mt destruction by hyperosmotic stress, and their replacement by tubulin macrotubules and putative tubulin paracrystals is a common feature among angiosperms and that macrotubules are involved in the mechanism of protoplast volume regulation.

A plasmolytic cycle: The fate of cytoskeletal elements

In most plant cells, transfer to hypertonic solutions causes osmotic loss of water from the vacuole and detachment of the living protoplast from the cell wall (plasmolysis). This process is reversible and after removal of the plasmolytic solution, protoplasts can re-expand to their original size (deplasmolysis). We have investigated this phenomenon with special reference to cytoskeletal elements in onion inner epidermal cells. The main processes of plasmolysis seem to be membrane dependent because destabilization of cytoskeletal elements had only minor effects on plasmolysis speed and form. In most cells, the array of cortical microtubules is similar to that found in nonplasmolyzed states except that longitudinal patterns seen in some control cells were never observed in plasmolyzed protoplasts of onion inner epidermis. As soon as deplasmolysis starts, cortical microtubules become disrupted and only slowly regenerate to form an oblique array, similar to most nontreated cells. Actin microfilaments responded rapidly to the plasmolysis-induced deformation of the protoplast and adapted to its new form without marked changes in organization and structure. Both actin microfilaments and microtubules can be present in Hechtian strands, which, in plasmolyzed cells, connect the cell wall to the protoplast. Anticytoskeletal drugs did not affect the formation of Hechtian strands.

Phyllosphere fungi which capture wind-borne pollen grains. II. Hexacladium corynephorum gen. et sp.nov.

Hexacladium corynephorum gen. et sp.nov. is described. This fungus is parasitic on wind-borne pollen grains which it captures by means of short clavate hyphae with sticky tips. The six-branched conidia have similarly clavate ends, also capable of capturing pollen. A hollow sphere of mycelium forms between the plasmolysed protoplast and wall of the pollen grain. The function seems to be haustorial and it has not been observed to survive the dry season. Microsclerotia formed by the hyphae after conidiation are likely to be the main perennating structures.

Effects of Octylguanidine on Cell Permeability and Other Protoplasmic Properties of Allium cepa Epidermal Cells

Effects of octylguanidine (OG) were studied on the permeability of cells of the adaxial epidermis of Allium cepa bulb scales to water and methyl urea and on the protoplast surface. Interference of OG with the Ca(2+) and Al(3+) action on the cell surface was also investigated.Permeability of the cell membrane for water and methyl urea increased nearly three times in presence of OG. The effect of OG on cell permeability depended on its direct contact with the protoplast surface.The effect of OG on the interaction between the protoplast surface and the cell wall (wall attachment) was marked and rapid; OG (225 micromolar) decreased the time for protoplast detachment in hypertonic solutions from 420 to 120 seconds. The plasmolyzed protoplasts were immediately rounded off while the controls without OG remained heavily concave.A considerable increase in protoplast detachment time and a decrease in rounding percentage were found when cells were plasmolyzed after pretreatment with AlCl(3) (0.05 molar for 2 minutes). This effect was partially reversed by KCl which was further enhanced by addition of OG.Penetration of OG into the mesoplasm was manifested only after 10 to 15 minutes. Vacuolization and swelling of the protoplasm, fragmentation of the protoplast, and aggregation of the spherosomes, however, were observed only 30 minutes after transfer. No evidence for penetration of OG into the vacuole was found.The results support earlier suggestions that OG acts primarily on the protoplast surface by interacting with membrane proteins as well as with phospholipids. In several aspects, OG acts on the cell surface similarly to a surfactant.

THE FINE STRUCTURE OF THE PROTOPLAST IN PRIMARY TRACHEARY ELEMENTS OF THE CUCUMBER AFTER PLASMOLYSIS

SUMMARY A striped pattern consisting of lighter and darker bands can be seen under the light microscope on the surface of the cell in tracheary elements in the hypocotyl of the cucumber. After plasmolysis, a striped pattern can also be observed both on the surface of the protoplast and on the cell wall. The nature of the striped pattern on the plasmolysed protoplast was investigated by electron microscopical examination. In the non-plasmolysed cell, high densities of cisternae of the endoplasmic reticulum and of Golgi vesicles are found in the areas between the developing cell wall thickenings. At the same time, bands of microtubules are present along the plasmalemma around the thickenings. Comparing these results with the electron microscope picture of the plasmolysed cell, the following observations can be made: 1. the regular distribution of the endoplasmic reticulum and the Golgi vesicles has disappeared; all cell organelles are disorganized. 2. The bands of microtubules are still present at their original sites. 3. The surface of the plasmolysed protoplast does not become smooth after detachment from the cell wall. It shows low, somewhat irregular ridges. These ridges are the areas which were originally located between the thickenings. Several hypotheses which might explain the pattern observed under the light microscope are discussed.

APHANOMYCES PHYCOPHILUS IN CULTURE

APHANOMYCES PHYCOPHILUS De Bary, one of the rarer Saprolegniales, was first described by DeBary in 1860 from a collection made at Frankfort am Main. Since then it has been reported from Michigan by Kauffman (1915), from Indiana by Weatherwax (1914), from New York by Sparrow (1933), and from North Carolina by Couch (1926). Previous attempts to obtain saprophytic growth of this parasite have been unsuccessful. DeBary says that it is parasitic on Spirogyra and Zygnema and is not to be cultured on decaying Phanerogam remains and dead animals. The purpose of this paper is to describe experiments in which it has been possible to induce growth of Aphanomyces phycophilus on artificial culture media. GROWTH ON SPIROGYRA.-The hyphae, 8-15 ,. thick, grow lengthwise through the Spirogyra filament by penetration of the cross walls. The tip of the growing hypha upon coming in contact with the cross wall of the host becomes somewhat flattened and presses against it as the wall becomes thinner. When a hole has been dissolved in the wall, the hypha pushes through it rapidly into the adjoining cell, which becomes plasmolysed. One hypha was seen to grow through one cell, a distance of 150 , in 20 minutes. After traversing the length of the cell along the surface of the plasmolysed protoplast, the hypha sends down lateral branches among the chloroplasts. The main hypha may continue on into the next cell, or a lateral branch may penetrate the side wall and grow out some distance into the water and parasitize another Spirogyra filament. Oogonia, 40-50 , in diameter, are formed on the ends of short lateral branches and seem to be intramatrical as often as extramatrical: The oogonial wall is drawn out into a number of spines, 7-9 # in length, and is yellowbrown in color. The single brownish oospore, 24-31 , in diameter, is finely granular, with one or two rows of oil globules around the periphery. The curvedclavate antheridia develop on the ends of nearby lateral branches and often wind around the hyphae bearing oogonia. There are one to three antheridia on each oogonium. No spores were observed. ISOLATION AND CULTURE ON AGAR.-A fungus was found growing parasitically on Spirogyra in a collection made by Mrs. H. B. Gotass on April 16, 1938, from a small stream near Chapel Hill. The size of the hyphae, the mode of growth, the structure of the oogonia, and the antheridia all agreed with DeBary's description and figures of Aphanomyces phycophilus. It differed from Wille's (1899) description of A. nor-