The influence of culture conditions on mycorrhiza formation between the ectomycorrhizal fungus Pisolithus sp. and Afzelia africana Sm. seedlings

Interactions between an isolate of the ectomycorrhizal fungus Pisolithus sp. and Afzelia africana Sm. seedlings were studied at the structural and ultrastructural levels. Several different conditions were tested with or without sugar and in a sterile or nonsterile medium. In the growth cabinet, the A. africana/Pisolithus sp. interactions did not produce ectomycorrhizas. A fungal sheath was formed but no Hartig net, and an unusual host epidermal cell wall was observed. Hyphae of Pisolithus sp. induced modifications of epidermal cells of 15-day-old A. africana seedlings indicative of non-mycorrhizal interactions, such as wall thickening, wall ingrowth, papillae formation, degraded host wall material and the presence of intracellular hyphae. Wall ingrowth consisted of depositions of host cell wall materials giving a positive reaction for polysaccharides; however, wall thickenings and papillae showed no homogeneous reactions for polysaccharides. In glasshouse conditions, inocula of Pisolithus sp. in the form of spores or mycelia entrapped in peat-vermiculite added to sterilized soil produced typical ectomycorrhizae only with 6-month-old A. africana seedlings. Under these conditions, no conspicuous cell wall reactions occurred on A. africana roots. The results demonstrate that the establishment of an association between an ectomycorrhizal fungus and a potential host plant is strongly influenced by seedling age and/or environmental conditions. Therefore, in vitro synthesis is not a conclusive demonstration of a symbiotic relationship.     

Cytology of induced systemic resistance of tomato to Phytophthora infestans

The infection of tomato leaves by Phytophthora infestans was followed using cytological methods. Fungal ingress and plant reactions in untreated and induced resistant plants were studied. Systemic disease resistance was induced by a local pre-infection with the same fungus. Induction retarded fungal progress at the leaf surface, epidermis and in the mesophyll. The reduced numbers of germinated cysts indicate the presence of fungitoxic substances on the leaf surface of induced plants. Frequency of fungal penetration through the outer epidermal cell wall was reduced, but only in plants exhibiting a high level of induced resistance. Autofluor-escent material, indicating the presence of lignin-like substances, accumulated rapidly beneath some of the appressoria, but this plant response was similar in induced and non-induced plants. Staining with aniline blue indicated that callose deposition was not involved in induced resistance. Thus, none of the cytologically investigated plant reactions correlated with the reduced penetration frequency observed. In the mesophyll, however, the cytological picture corresponding to a hypersensitive reaction occurred more often in induced plants. It is concluded that reduction of disease severity by induction is the result of the combined action of several successive defence reactions.Dedicated to the memory of Professor H. Grisebach     

Role of cell-wall biogenesis in the initiation of auxin-mediated growth in coleoptiles of Zea mays L.

The involvement of cell-wall polymer synthesis in auxin-mediated elongation of coleoptile segments from Zea mays L. was investigated with particular regard to the growth-limiting outer epidermis. There was no effect of indole acetic acid (IAA) on the incorporation of labeled glucose into the major polysaccharide wall fractions (cellulose, hemicellulose) within the first 2 h of IAA-induced growth. 2,6-Dichlorobenzonitrile inhibited cellulose synthesis strongly but had no effect on IAA-induced segment elongation even after a pretreatment period of 24 h, indicating that the growth response is independent of the apposition of new cellulose microfibrils at the epidermal cell wall. The incorporation of labeled leucine into total and cell-wall protein of the epidermis was promoted by IAA during the first 30 min of IAA-induced growth. Inhibition of IAA-induced growth by protein and RNA-synthesis inhibitors (cycloheximide, cordycepin) was accompanied by an inhibition of leucine incorporation into the epidermal cell wall during the first 30 min of induced growth but had no effect on the concomitant incorporation of monosaccharide precursors into the cellulose or hemicellulose fractions of this wall. It is concluded that at least one of the epidermal cell-wall proteins fulfills the criteria for a growth-limiting protein induced by IAA at the onset of the growth response. In contrast, the synthesis of the polysaccharide wall fractions cellulose and hemicellulose, as well as their transport and integration into the growing epidermal wall, appears to be independent of growth-limiting protein and these processes are therefore no part of the mechanism of growth control by IAA.Abbreviations CHI cycloheximide - COR cordycepin - DCB 2,6-dichlorobenzonitrile - GLP growth-limiting protein(s) - IAA indole-3-acetic acid     

ECTODESMATA AND FOLIAR ABSORPTION

Franke , Wolfgang . (U. Bonn, Germany.) Ectodesmata and foliar absorption. Amer. Jour. Bot. 48(8): 683–691. Illus. 1961.—Plasmodesmata, called ectodesmata, in the outer walls of epidermal cells, have been investigated. Their occurrence and distribution in the epidermis of leaves of Plantago major and Helxine soleirolii have been examined in connection with foliar absorption. Leaf structures such as guard-cells, conical hairs, anticlinal walls and the epidermal cells adjacent to the leaf veins have been shown consistently to contain large numbers of ectodesmata, while in neighboring cells ectodesmata may be low in number or lacking. The same areas also are known to be especially pervious to water and dissolved dyes applied to the surface of the leaf. From special investigations, it appears that certain solutions that form visible crystals and precipitates in the outer wall enter the epidermal cell wall in localized pathways. The localization of these bodies coincides with that of ectodesmata. Therefore, it is concluded that the ectodesmata may be the pathways for transport of substances from the outside to the interior of tissues and vice versa. Nutrients applied to the surface of leaves are thought to enter by the same pathways, i.e., the ectodesmata, as those in which the penetration can be visibly detected. Some phenomena of foliar absorption which confirm this theory are explained in connection with the presence of ectodesmata.     

Fused organs in the adherent1 mutation in maize show altered epidermal walls with no perturbations in tissue identities

In the absence of wounding, the epidermis is only rarely involved in cell or organ fusion events; in fact, intact epidermal layers prevent graft unions. In Zea mays L. the mutation adherent1 (ad1) shows abnormal fusions between cells and organs. Fusions involve epidermal cells of vegetative and floral organs and occur early in the ontogeny of organs. Even so, epidermal cell types differentiate normally in the fused regions and internal tissue identities are maintained. In contrast, the extracellular matrix (cell wall and cuticle) of the epidermal cells is perturbed. Epidermal cell walls in adherent leaves are thicker than normal. Epicuticular wax particles appear reduced in size and number and altered in shape in mutant leaves. In addition, the outer epidermal cell walls of adherent leaves fluoresce when stained with aniline blue, a reagent that binds to callose. Immunolocalizations to specific cell wall epitopes suggest that pectins but not arabinogalactans may have a role in the fusion events. Taken together, these results suggest that the ad1 mutation results in cell-wall and epicuticular-wax defects similar to responses seen in wounding, pollination by incompatible pollen, or pathogen attack. Since cell wall components and epicuticular waxes are extracellular secreted products, the ad1 mutation may disrupt normal functioning and/or composition of the secretory pathway and its cargo. Received: 30 January 1998 / Accepted: 5 March 1998     

THE ULTRASTRUCTURE OF THE SALT GLANDS OF CYNODON AND DISTICHLIS (POACEAE)

The epidermal salt glands of the grasses Cynodon and Distichlis consist of a small outer cap cell and a large, flask-shaped basal cell. The wall of the basal cell is contiguous with those of the adjacent epidermal cells and underlying mesophyll cells. The basal cell is connected symplastically with all adjoining cells via plasmodesmata. The outer, protruding portion of the glands is covered by a cuticle continuous with that of the adjoining epidermal cells. However, the lateral cell walls of the glands are not incrusted by this cuticle. The cap cell wall has a loose, mottled appearance quite different from the compact striated appearance of the basal cell wall. The cap cell is characterized by dense cytoplasm containing many organelles and a varying number of small vacuoles. The basal cell cytoplasm is distinguished by the presence of an intricate system of paired membranes that are closely associated with mitochondria and microtubules. These membranes are infoldings of the plasmalemma that originate adjacent to the wall separating the cap and basal cells. The space enclosed by the paired membranes, therefore, is an extracellular channel that is open only in the direction of secretory flow. The consistent orientation of this system of paired membranes suggests that it represents a structural specialization which is directly and functionally involved in the secretory process. The close association of mitochondria and microtubules with the paired membranes implies that these structures are also functionally related to the secretory process. Finally, the results of this study indicate that these glands are ultrastructurally similar to those of Spartina and that the glands of these three grasses are structurally distinct from those of dicotyledonous plants.     

Ultrastructure of the body wall of three species of Grania (Annelida: Clitellata: Enchytraeidae)

De Wit P., Erséus C. and Gustavsson L.M. 2011. Ultrastructure of the body wall of three species of Grania (Annelida: Clitellata: Enchytraeidae). —Acta Zoologica (Stockholm) 92 : 1–11. The body wall of three species of Grania, including the cuticle, epidermis and the musculature, are studied using TEM. The cuticle is similar to previously studied enchytraeids, with an orthogonal grid pattern of collagen fibers. This pattern is also seen in Crassiclitellata, which has been suggested as the sister taxon of Enchytraeidae. Variation of epicuticular and fiber zone patterns seen in Naididae (formerly Tubificidae and Naididae) seem to be lacking in Enchytraeidae. The fiber thickness, however, varies between Grania species and may be a phylogenetically informative character. The epidermis consists of supporting cells, secretory cells and sensory cells. Basal cells, typical for Crassiclitellata, were not observed. The clitellum of Grania seems to consist of two types of gland cells, which develop from regular epidermal tissue. It is possible that more cell types exist in different regions of the clitellum, however. The body wall musculature is arranged somewhat differently from that of closely related taxa; this refers to the reduction of circular and outer, triangular longitudinal muscle fibers, while the inner, ribbon‐shaped longitudinal muscle fibers are well‐developed. A search was conducted for the cause of the peculiar green coloration of Grania galbina De Wit and Erséus 2007, and it was concluded that neither cyanobacteria nor epidermal pigment granules were present in the fixed material.     

On the mechanism of callose synthesis induction by metal ions in onion epidermal cells

Summary.  Metal ions induce the synthesis of callose in Allium cepa epidermal cells. Callose is deposited as single knoblike local accumulations, aggregates of knobs, or furrowed clusters tightly attached to the cell wall. The most effective metal is copper, it induces callose formation at micromolar concentrations. Agents acting on inositolphosphate metabolism, phospholipase inhibitors, calcium channel inhibitors, modulators of cytoplasmic calcium, or receptor antagonists influence callose synthesis. It is concluded that metal ions, especially Cu²⁺, initiate a signal transduction chain by activation of phospholipases and generation of inositol 1,4,5-trisphosphate, and that callose synthesis is a cellular defence reaction caused by the disturbance of intracellular calcium homeostasis. Received October 10, 2001; accepted September 16, 2002; published online March 11, 2003     

Dynamic microtubules under the radial and outer tangential walls of microinjected pea epidermal cells observed by computer reconstruction

By microinjecting rhodamine-conjugated pig brain tubulin into living pea stem epidermal cells it has been possible to follow cortical microtubules beneath the outer tangential wall (OTW) as they re-orientate from a transverse to a longitudinal alignment. Earlier immunofluorescence studies on fixed material have shown that parallel cortical microtubules circumnavigate the cell forming apparently continuous arrays which are transverse, oblique or longitudinal to the cell's long axis. If the array re-orientates as a whole then microtubules along the radial walls would be expected to share the alignment of those on the tangential walls. There are, however, reports that microtubules beneath the outer tangential wall have a different orientation from microtubules at the radial cell walls, raising important questions about the construction and behaviour of the array. Using computer-rotated stacks of optical sections collected by confocal scanning laser microscopy it has been possible to display the microtubules along radial as well as tangential walls of the same microinjected cells. These observations demonstrate for living epidermal cells that when microtubules are aligned longitudinally at the outer epidermal wall they remain oblique or transverse at the radial walls. The array may not therefore re-orientate as a whole but seems to undergo re-organization on only one cell face. However, despite the differing angles between the OTW and radial walls microtubules still form patterns which at the level of the confocal microscope are continuous from one cell face to another, around the cell.
It is concluded that some organizing principle attempts to establish overall organization at the cellular level but that this can be perturbed by local re-organization of dynamic microtubules in subcellular domains. This study emphasizes the importance of the outer epidermal wall and its associated cytoskeleton in initiating changes in the direction of cell expansion.     

Penetration of UV-B radiation in foliage: evidence that the epidermis behaves as a non-uniform filter

In some plants, particularly herbaceous species, a considerable proportion of incident ultraviolet-B radiation (UV-B, 280-320 nm) penetrates into the leaf mesophyll where it is potentially damaging to nucleic acids and the photosyn-thetic machinery. We used optical techniques to look at the spatial variation in UV-B penetration through the epidermis of foliage of two herbaceous species (Chenopodium album and Smilacina stellata)and a conifer (Picea pun-gens). Measurements of UV-B penetration in intact foliage with a fibre-optic microprobe revealed that 300 nm radiation reached 161±36μm (mean±SD) into leaves of C. album, 154±40μm in S. stellata and 17±2μm in P. pungens, with epidermal transmittance being 39±14%, 55±19% and 0%, respectively. A thin polymer film was developed which fluoresced blue when irradiated by UV-B. Fresh epidermal leaf peels were placed over the film and irradiated with UV-B, and microscopic examination of the film from below allowed us to determine the spatial pattern of UV-B penetration through the epidermis. In herbaceous species, film fluorescence below cell walls, but not epidermal and guard cell protoplasts indicated that UV-B transmittance was much greater through anticlinal cell wall regions than protoplasts. Ultraviolet-B transmittance through large areas of epidermal cells could be induced by plasmolysis. Epidermal transmittance was also relatively high through stomal pores (and what appear to be nuclei in Smilacina), but relatively low through stomatal guard cells. Results from the fluorescing film technique were substantiated by direct measurements of UV-B transmittance through epidermal peels with a fibre-optic microprobe run paradermally along the bottom or inner side of irradiated peels. In Smilacina, we estimate that UV-B epidermal transmittance was up to 90% through anticlinal cell wall regions, but <10% through protoplast areas. In contrast to herbaceous species, we did not detect any UV-B transmittance through the epidermis of P. pungens with either the fluorescing film or the fibre-optic microprobe technique. The epidermis appears to be a much more spatially uniform UV-B filter in conifers than in these herbaceous species.     

Development of Epidermis on Banana Fruits

Fruit epidermis ofMusa (AAB) cv. Poovan (S) remains single-layered throughout its development. There is no change in stomatal number but its frequency and index decrease due to slight increase in epidermal cell number and size. The external wall of the epidermal cells shows stratification of wall layers that is characteristic of normal epidermal cell with cuticle and epicuticular wax deposits. Surface wax deposits show qualitative and quantitative variations during fruit development and ripening.     

Chalara elegal-Infected Tobacco Roots: Ultrastructural Observations on in vitro Host-Fungus Relationships

Evidence, based on ultrastructural observations of stages involved in root infection oi Nicotiana tabacum cv. Xanthi n.c. in vitro by the black root rot fungus Chalara elegans, indicates that host cells from various layers react differently when challenged by the pathogenic fungus. All the host responses observed were associated with host cell wall modifications. Host reaction to fungal invasion occurring in the epidermal cells was limited to a disorganization of the cytoplasm. In the hypodermal cell layer, fibrillar cell wall outgrowths and wall thickenings were the earliest and the most obvious host reactions. In parenchymal cells, the host reacted by depositing papilla-like wall appositions directly adjacent to the infecting hyphae; with secondary infection of these cells, a densely staining material was laid down, mainly around the distal region of the infecting hyphae. In all these tissues, infection also led to disorganization of the host cytoplasm. Colonization of the endodermis did not lead to any rapid lethal modifications in either the host or the fungus, and a biotrophic-like state seemed to occur at this stage of the infection. No hyphal infection occurred in the central cylinder.     

Flavonol rhamnosylation indirectly modifies the cell wall defects of RHAMNOSE BIOSYNTHESIS1 mutants by altering rhamnose flux

Rhamnose is required in Arabidopsis thaliana for synthesizing pectic polysaccharides and glycosylating flavonols. RHAMNOSE BIOSYNTHESIS1 (RHM1) encodes a UDP‐l ‐rhamnose synthase, and rhm1 mutants exhibit many developmental defects, including short root hairs, hyponastic cotyledons, and left‐handed helically twisted petals and roots. It has been proposed that the hyponastic cotyledons observed in rhm1 mutants are a consequence of abnormal flavonol glycosylation, while the root hair defect is flavonol‐independent. We have recently shown that the helical twisting of rhm1 petals results from decreased levels of rhamnose‐containing cell wall polymers. In this study, we found that flavonols indirectly modify the rhm1 helical petal phenotype by altering rhamnose flux to the cell wall. Given this finding, we further investigated the relationship between flavonols and the cell wall in rhm1 cotyledons. We show that decreased flavonol rhamnosylation is not responsible for the cotyledon phenotype of rhm1 mutants. Instead, blocking flavonol synthesis or rhamnosylation can suppress rhm1 defects by diverting UDP‐l ‐rhamnose to the synthesis of cell wall polysaccharides. Therefore, rhamnose is required in the cell wall for normal expansion of cotyledon epidermal cells. Our findings suggest a broad role for rhamnose‐containing cell wall polysaccharides in the morphogenesis of epidermal cells.     

Peroxidase isozyme patterns in the skin of maturing tomato fruit

The cessation of tomato fruit growth is thought to be induced by an increase in the activity of enzymes which rigidify cell walls in the fruit skin. Peroxidase could catalyse such wall‐stiffening reactions, and marked rises in peroxidase activity were recently reported in skin cell walls towards fruit maturity. Peroxidase isoforms in the fruit are here analysed using native gel electrophoresis. New isoforms of apparent M_r 44, 48 and 53 kDa are shown to appear in cell walls of the fruit skin at around the time of cessation of growth. It is inferred that these isozymes are present in the cell wall in vivo. Fruit from a range of non‐ripening mutants were also examined. Some of these do not soften or ripen for many weeks after achieving their final size. The new isozymes were found in skin cell walls of mature fruit in each of these mutants, as in the wild‐type and commercial varieties. It is concluded that the late‐appearing isozymes are not associated with fruit ripening or softening, and are probably not ethylene‐induced. They may act to control fruit growth by cross‐linking wall polymers within the fruit skin, thus mechanically stiffening the walls and terminating growth.     

INCOMPLETE CYTOKINESIS IN FUNARIA STOMATA

The development of the one-celled condition in Funaria (Musci) stomata was investigated using light and electron microscopy. The guard cell parent cell is unusual in that it undergoes karyokinesis but incomplete cytokinesis. The septal wall, and the cell plate from which it forms, have incurved edges in contact with the polar cytoplasm. No evidence was found to support Haberlandt's claim that the stomate is initially two celled but undergoes wall resorption. Preprophase microtubule bands appear to be present in nonstomatal epidermal cells with normal cytokinesis, but the possibility is raised that they are absent in guard cell parent cells.     

Enzymatic processes involved in the incorporation of hydroxycinnamates into grass cell walls

Many plant species have one or more types of acylation of cell wall polymers. Grasses (Poaceae family) are unique with abundant acylation of specific cell wall polymers by hydroxycinnamates. The most common hydroxycinnamates found in a wide range of grasses are ferulates (trans-4-hydroxy-3-methoxycinnamate) and p-coumarates (trans-4-hydroxycinnamate). These two hydroxycinnamates are synthesized by the phenylpropanoid pathway. Though structurally related, they seem to have different functional roles within the cell wall. Ferulates have been shown to have a critical role in cross-linking cell wall components; forming links between structural polysaccharides and links between structural polysaccharides and lignin. They are incorporated into the cell wall by distinctly different mechanisms. Ferulic acid is incorporated into cell walls as ester linked substituents on arabinoxylans. The exact role p-coumarates play in plant cell walls is unknown, but it has been shown that p-coumaric acid is ester-linked to monolignols and shuttled out to the wall to become incorporated into newly forming lignin polymers. Both processes require the activity of specific hydroxycinnamoyl transferases utilizing CoA derivatives to drive the transferase reactions.     

Attachment of the parasitic weed dodder to the host

Summary.  The parasitic weed dodder (Cuscuta pentagona L.) invades a number of potential host species, but the mechanisms responsible for ensuring tight adhesion to the wide variety of host surfaces have yet to be identified. In this study, a battery of microscopy protocols is used to examine the host–parasite interface in an effort to deduce these mechanisms. As the dodder shoot approaches the host tissue, epidermal cells in the parasite shoot elongate and differentiate into secretory type trichomes. The trichome cell walls are malleable, allowing them to elongate towards the host and bend their walls to conform to the shape of the host cell surface. The presence of osmiophilic particles (probable cell-wall-loosening complexes) at far greater numbers than found in other species presages the expansion and malleable nature of the epidermal cells. In addition to the changes in cell shape, the dodder trichome cells secrete an electron-opaque cementing substance that covers the host–parasite interface. When probed with antibodies that recognize cell wall components, the cement reacted only with antibodies that recognize chiefly de-esterified pectins but not other common wall constituents. These data indicate that dodder utilizes both a cementing layer of pectin and a radically modified epidermal cell wall to secure the parasite to the perspective host. Received January 29, 2001 Accepted November 28, 2001     

Cell wall structure of the agarophytes Gracilaria tikvahiae and G. cornea (Rhodophyta) and penetration by the epiphyte Ulva lactuca (Chlorophyta)

Use of light, transmission, and scanning electronmicroscopes revealed that the epidermal cell wall ofthe red algal agarophytes Gracilaria tikvahiaeMcLachlan and G. cornea J. Agardh consists of adecklamelle and outer and inner wall layers. The twospecies differed, with G. cornea having asignificantly thicker outer wall and a more diffusedecklamelle. After induction, the zooids of Ulvalactuca would attach to glass slides and the twospecies of Gracilaria via an adhesion pad. Within a few days, 3–5 celled germlings penetrated thedecklamelle and outer wall layer of both basiphytes. By the time the epiphyte germlings reached the 15celled stage, they had penetrated the inner walllayer. The differences in epidermal cell wallconstruction between the two basiphytes may play arole in the ability of zooids of U. lactuca toattach in nature where epiphytization of G.cornea is infrequent.     

Ontogeny of external glands in the bladderwortUtricularia monanthos

Summary The development of external glands on traps and stolons ofU. monanthos has been studied using transmission electron microscopy. During early differentiation of the epidermis some cells remain narrow and develop a protuberance which subsequently divides into a terminal and a pedestal cell, with the remainder of the original cell forming the basal epidermal cell of the gland. The lateral wall of the pedestal cell soon becomes densely impregnated throughout its thickness, and this is followed by the formation of discontinuous cuticular deposits within the primary wall of the terminal cell. The outer wall of the terminal cell then usually undergoes extensive secondary wall thickening beginning with the formation of ingrowths which for a period characterize the cell as a transfer cell. Later, at the stage when traps begin capturing prey, these ingrowths are overlain by further layers of secondary wall material. Concomitantly, in the pedestal cell, wall ingrowths become fully differentiated on the outer transverse wall and persist throughout the remaining life of the gland.The function of external glands during early ontogeny is discussed. At the stage when the terminal cell is differentiated as a transfer cell it is suggested that the gland is mainly responsible for absorbing solutes from the external medium. Once traps commence capturing prey the gland may become modified for a rôle in water secretion, facilitated by the differentiation of the pedestal cell as a transfer cell, and by the formation of a thick outer wall in the terminal cell.     

Control of Auxin-induced Stem Elongathn by the Epidermis

Segments of the 4th and 5th internodes of light-grown pea seedlings were used for the study of control of stem elongation. With 5th internodes, at low turgor as well as at water saturation auxin primarily appeared to cause a change in cell wall properties of the epidermis but it showed little effect on expansion af the inner tissue. This was confirmed by comparison of expansion between peeled and unpeeled segments, split tests and by measurements of stress-relaxation properties of the epidermal cell wall. Segments with the central part re-moved elongated well in response to auxin, but the isolated epidermis showed neither auxin-induced elongation nor cell wall loosening. A fungal β-1,3-glucanase appeared, at least partly, to have a similar effect as that of auxin on elongation, by changing cell wall properties of the epidermal cell wall. Peeled segments of 4th internodes expanded very little and auxin had little effect on their epidermal cell wall properties.