Characterization of the epidermis from barley primary leaves

A method is described for isolating epidermal protoplasts from the primary leaves of barley (Hordeum vulgare L.). Epidermal protoplasts are lighter than mesophyll protoplasts because of their smaller ratio of cytoplasm to vacuole, and can be separated from the latter by density-gradient centrifugation after complete digestion of the leaves. We have started a basic characterization of the epidermal protoplast fraction in comparison with mesophyll protoplasts. Epidermal protoplasts had a mean diameter of 63.5 m, whereas that of mesophyll protoplasts was 35.7 m. Their respiratory oxygen consumption was not influenced by light. They contained acid hydrolases and cytoplasmic enzymes in relative activities different from those of mesophyll protoplasts. Their polypeptide pattern as judged from two-dimensional separations was, in principle, similar to that of mesophyll cells after elimination of the plastids from the latter by the preparation of vacuoplasts. However, in addition, a considerable number of epidermis-specific polypeptides were observed. Isolated epidermal protoplasts were viable and efficiently incorporated [³⁵S]methionine into newly synthesized proteins. The results show that epidermal protoplasts are suitable for the investigation of the physiological and molecular properties of epidermal cells in leaves.Abbreviation SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We are grateful to Professor U. Heber (Lehrstuhl Botanik 1, Würzburg) for his continuous support. This work was supported by the DFG and the University of Würzburg within the Sonderforschungsbereich 176.     

Covisualization in living onion cells of putative integrin, putative spectrin, actin, putative intermediate filaments, and other proteins at the cell membrane and in an endomembrane sheath

Summary Covisualizations with wide-field computational opticalsectioning microscopy of living epidermal cells of the onion bulb scale have evidenced two major new cellular features. First, a sheath of cytoskeletal elements clads the endomembrane system. Similar elements clad the inner faces of punctate plasmalemmal sites interpreted as plasmalemmal control centers. One component of the endomembrane sheath and plasmalemmal control center cladding is antigenicity-recognized by two injected antibodies against animal spectrin. Immunoblots of separated epidermal protein also showed bands recognized by these antibodies. Injected phalloidin identified F-actin with the same cellular distribution pattern, as did antibodies against intermediate-filament protein and other cytoskeletal elements known from animal cells. Injection of general protein stains demonstrated the abundance of endomembrane sheath protein. Second, the endomembrane system, like the plasmalemmal puncta, contains antigen recognized by an anti-₁ integrin injected into the cytoplasm. Previously, immunoblots of separated epidermal protein were shown to have a major band recognized both by this antibody prepared against a peptide representing the cytosolic region of ₁ integrin and an antibody against the matrix region of ₁ integrin. The latter antibody also identified puncta at the external face of protoplasts. It is proposed that integrin and associated transmembrane proteins secure the endomembrane sheath and transmit signals between it and the lumen or matrix of the endoplasmic reticulum and organellar matrices. This function is comparable to that proposed for such transmembrane linkers in the plasmalemmal control centers, which also appear to bind cytoskeleton and a host of related molecules and transmit signals between them and the wall matrix. It is at the plasmalemmal control centers that the endoplasmic reticulum, a major component of the endomembrane system, attaches to the plasma membrane.Abbreviations DiOC₆ 3,3-dihexyloxacarbocyanine iodide - GF Gaussian filtering - ML maximum likelihood (algorithm or method) - PBS phosphate-buffered saline     

Intracellular and intramitochondrial binding of lanthanum in dark degenerating midgut cells of a collembolan (Insect)

Summary Glycogen synthetase (2.4.1.11) forms I (independent or active) and D (dependent or passive) as well as the enzymes active in the transformation of the pathways, protein kinase and phosphatase transferase, were studied in the sensory cells and glycogen rich epidermal cells of the weakly electric fish Gnathonemus petersii (Mormyridae). For light microscopy an indirect cytochemical method which differentiated between glycogen originally present and that produced during incubation in the presence of UDPG was used. This differentiation was obtained by iodine, PAS and and amylases.Glycogen synthetase is present in the sensory cells in the I and D forms. The epidermal cells only contain the D form. Protein kinase (active ID) has only been found in the sensory cells but phosphatase transferase (active DI) has been found in both the epidermal cells and the sensory cells, but only within certain organs.Electron microscopy studies of glycogen synthetase I and D and protein kinase were restricted to the sensory cells only. As with the light microscope it was possible to differentiate between native glycogen and newly formed glycogen. This was done using ultrathin sections and staining with uranyl acetate, lead citrate or by the PATAg reaction.It was possible from these observations to locate precisely the positions of these enzymes. In fact, glycogen synthetase I and D are found both in the sensory cytoplasm and in the sensory cavity with the polysaccharide filaments. Protein kinase is also abundant in the sensory cytoplasm especially in the periphery of the cell near the microvillary border.     

The effect of humidity and light on cellular water relations and diffusion conductance of leaves ofTradescantia virginiana L.

Turgor (_p) and osmotic potential (_s) in epidermal and mesophyll cells, in-situ xylem water potential (-xyl) and gas exchange were measured during changes of air humidity and light in leaves ofTradescantia virginiana L., Turgor of single cells was determined using the pressure probe. Sap of individual cells was collected with the probe for measuring the freezing-point depression in a nanoliter osmometer. Turgor pressure was by 0.2 to 0.4 MPa larger in mesophyll cells than in epidermal cells. A water-potential gradient, which was dependent on the rate of transpiration, was found between epidermis and mesophyll and between tip and base of the test leaf. Step changes of humidity or light resulted in changes of epidermal and mesophyll turgor (_p-epi, _p-mes) and could be correlated with the transpiration rate. Osmotic potential was not affected by a step change of humidity or light. For the humidity-step experiments, stomatal conductance (g) increased with increasing epidermal turgor.g/_p-epi appeared to be constant over a wide range of epidermal turgor pressures. In light-step experiments this type of response was not found and stomatal conductance could increase while epidermal turgor decreased.Symbols E transpiration - g leaf conductance - w leaf/air vapour concentration difference - -epi water potential of epidermal cells - -mes water potential of mesophyll cells - -xyl water potential of xylem - _p-epi turgor pressure of epidermal cells - _p-mes turgor pressure of mesophyll cells - _s-epi osmotic potential of epidermal cells - _s-mes osmotic potential of mesophyll cells     

Scanning electron microscopical observations on epidermal wound healing in the PlanarianDugesia tigrina

Summary Epidermal wound healing in regeneratingDugesia tigrina (Planaria) has been studied using scanning electron microscopy (SEM). The normal epidermal surface and its differentiations have been descrebed. Observations on living material reveal the highly dynamic state of the wound in invididual animals and its more or less continously changing size due to the state of activity of the animals. These observations show good agreement with the SEM studies, which allow a clear delineation of cellular details of the wound, the wound margins and the apposing epidermal regions. These details are described. The over-all picture of planarian wound healing that emerges is briefly as follows: Epithelization is characterized by absence of proliferation from the old intact epidermis. Variable contraction of smooth muscle cells reduces the wound size to a certain extent. Simultaneously with this and also during a longer period epidermal cells adjacent to the wound are extending and some become highly attenuated. These two processes together are only to a certain degree effective in wound closure because of a definite epidermal cell deficit which is reflected in the emergence of an epidermal wound edge reflecting the maximal contribution of these two processes to an attempt to close the wound. Complete epithelization is effected by the operation of a third mechanism: Recruitment of cell through flow of subjacent blastemal cells (including rhabdite-forming cells) along the wound border; these cells subsequently occupy a peripheral position in the wound. This process is supplemented by cell immigration and insertion into the adjacent old epidermis and in the wound cell sheet. Rhabdite-forming cells contribute predominantly to this process. Eventually integration between old epidermal cells and the newly recruited cells which differentiate into epidermal cells results in final epithelization. Complete wound healing is based on interactions between the epidermal cell system and the regenerating subepidermal membrane-connective tissue filament-muscle cell system.     

Peptide map comparisons of epidermal and spleen H-2 molecules

Peptide map comparisons of molecules encoded in the mouseH-2 complex isolated from epidermal cell preparations have been carried out. We previously showed that the Ia molecules from both theI-A andI-E subregions are synthesized by nonlymphoid bone-marrow-derived cells, probably Langerhans cells. The K and D or transplantation molecules are synthesized by both true epidermal cells and nonlymphoid bone-marrow-derived cells. The tryptic maps generated by separating tryptic peptides by high pressure liquid chromatography (HPLC) of epidermal H-2 molecules are identical to their spleen-cell counterparts. The biological significance of this finding is discussed.     

Elektronenmikroskopische Untersuchungen über Struktur und Entwicklung der Epidermis von Trinchesia granosa (Gastr. Opisthobranchia)

Summary A special vacuolized epidermis is found — unique amongst Gastropoda in the strikingly-coloured group of marine snails: Nudibranchia. The developmental changes and the fine structure of the epidermal cells from hatching until sexual maturity (development without free swimming veliger) has been descibed in the Eolid Trinchesia granosa The present paper deals only with the main cells, i.e. the vacuolar cells of the epidermis, while the less numerous mucoid and pigmentary cells are omitted.While in the hatching stage the single-layered cells are separated by big intercellular clefts, they are closely interdigitated in the adult. With respect to differences in cell shape, position and structure of the nuclei, the fine structure of the cytoplasm, and the number of vacuoles, two main types of cells can be distinguished. They are characterized as A- and B-cells. A-cells and B-cells, as well as transitional forms can be observed in hatching animals, while in adult animals only A-cells are observed.The epidermal cells of all developmental stages are characterized by vacuole-bodies, special structured ellipsoid bodies, each lying within a vacuole. The cell in the hatching stage contains only few vacuole-bodies, whereas they form an extremely dense layer in the apical part of the adult epidermis. Each vacuole-body consists of an ellipsoid viscous mantle and a dumb-bell shaped cylindrical axis containing a more or less osmiophilic nucleus. The electron microscopic findings indicate — in correlation with observations of the living epidermis — that the vacuole-bodies have absorbtive, filtrative and mechanic functions.

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Mit Unterstützung durch den Schweizerischen Nationalfonds zur Förderung der Wissenschaften und die Deutsche Forschungsgemeinschaft.     

Changes in dye coupling of stomatal cells of Allium and Commelina demonstrated by microinjection of Lucifer yellow

Lucifer yellow has been microinjected into stomatal cells of Allium cepa L. epidermal slices and Commelina communis L. epidermal peels and the symplastic spread of dye to neighboring cells monitored by fluorescence microscopy. Dye does not move out of injected mature guard cells, nor does it spread into the guard cells when adjacent epidermal or subsidiary cells are injected. Dye does spread from injected subsidiary cells to other subsidiary cells. These results are consistent with the reported absence of plasmodesmata in the walls of mature guard cells. Microinjection was also used to ascertain when dye coupling ceases during stomatal differentiation in Allium. Dye rapidly moves into and out of guard mother cells and young guard cells. Hovewer, dye movement ceases midway through development as the guard cells begin to swell but well before a pore first opens. Since plasmodesmata are still present at this stage, the loss of symplastic transport may result from changes in these structures well in advance of their actual disappearance from the guard cell wall.Abbreviations DIC differential interference contrast - GMC guard mother cell - LY Lucifer yellow - Pd plasmodesmata You can observe a lot by watching Lawrence Berra, as quoted in Sports Illustrated, vol. 60 (No. 14), p. 94, 2 April 1984     

Development of a new transformation method using magnetite cationic liposomes and magnetic selection of transformed cells

A new method using magnetite cationic liposomes (MCLs)/DNA complex for transformation is proposed and efficient separation of transformed cells is reported. In our method, gene expression and antibodies as a marker are not required for separation. The specific activity of separated cells was the highest when the complex consisting of 25 g MCLs and 2 g plasmid DNA was used for transformation. The specific activity was about 5 times higher than that of the cells before the separation. © Rapid Science Ltd. 1998     

Spatial distribution of growth rates and of epidermal cell lengths in the elongation zone during leaf development in Lolium perenne L.

Relative elemental growth rates (REGR) and lengths of epidermal cells along the elongation zone of Lolium perenne L. leaves were determined at four developmental stages ranging from shortly after emergence of the leaf tip to shortly before cessation of leaf growth. Plants were grown at constant light and temperature. At all developmental stages the length of epidermal cells in the elongation zone of both the blade and sheath increased from 12 m at the leaf base to about 550 m at the distal end of the elongation zone, whereas the length of epidermal cells within the joint region only increased from 12 to 40 m. Throughout the developmental stages elongation was confined to the basal 20 to 30 mm of the leaf with maximum REGR occurring near the center of the elongation zone. Leaf elongation rate (LER) and the spatial distributions of REGR and epidermal cell lengths were steady to a first approximation between emergence of the leaf tip and transition from blade to sheath growth. Elongation of epidermal cells in the sheath started immediately after the onset of elongation of the most proximal blade epidermal cells. During transition from blade to sheath growth the length of the blade and sheath portion of the elongation zone decreased and increased, respectively, with the total length of the elongation zone and the spatial distribution of REGR staying near constant, with exception of the joint region which elongated little during displacement through the elongation zone. Leaf elongation rate decreased rapidly during the phase when only the sheath was growing. This was associated with decreasing REGR and only a small decrease in the length of the elongation zone. Data on the spatial distributions of growth rates and of epidermal cell lengths during blade elongation were used to derive the temporal pattern of epidermal cell elongation. These data demonstrate that the elongation rate of an epidermal cell increased for days and that cessation of epidermal cell elongation was an abrupt event with cell elongation rate declining from maximum to zero within less than 10 h.Abbreviations LER leaf elongation rate - REGR relative elemental growth rates     

Production and Emission of Volatile Compounds by Petal Cells

We localized the tissues and cells that contribute to scent biosynthesis in scented and non-scented Rosa × hybrida cultivars as part of a detailed cytological analysis of the rose petal. Adaxial petal epidermal cells have a typical conical, papillate shape whereas abaxial petal epidermal cells are flat. Using two different techniques, solid/liquid phase extraction and headspace collection of volatiles, we showed that, in roses, both epidermal layers are capable of producing and emitting scent volatiles, despite the different morphologies of the cells of these two tissues. Moreover, OOMT, an enzyme involved in scent molecule biosynthesis, was localized in both epidermal layers. These results are discussed in view of results found in others species such as Antirrhinum majus, where it has been shown that the adaxial epidermis is the preferential site of scent production and emission.Key Words: floral scent, petal epidermis, Rosa, terpenes, volatilesMany plant species produce volatile compounds and these molecules serve a range of purposes. For example, compounds that are emitted from leaves are generally required for the defence of the plant against insect predators. On the other hand, floral compounds attract beneficial insects, leading to pollination of the flower. In leaves, scent compounds are very often synthesised in specialized cells grouped in structures termed trichomes or secretory glands. In many flowers, it is well documented that floral fragrance is produced by the corolla,¹ although other flower parts, such as the stamens in Ranunculus acris,² sometimes play an important role in fragrance emission. In some flowers, in particular those belonging to the Orchidaceae family, scent is emitted by specialized areas of the petal, which have been termed osmophores by Vogel.³ However, in most flowers, when petals produce scent, it is thought to be emitted by all the cells of the petal in a diffusive manner.⁴ In many flowers, such as roses, the adaxial petal epidermal cells have a conical-papillate shape whereas the cells of the abaxial epidermis are flat (Fig. 1).⁵ The shape of these conical cells is controlled by a Myb-factor named MIXTA in Antirrhinum majus⁶ and their shape has been shown to play a role in the diffusion of light, thereby enhancing the attractiveness of the flower.⁷ Flowers of the mixta mutant have flat adaxial petal epidermal cells that reflect less light⁸ and as a consequence attract less insects.⁹ Along the same lines, Kolosova et al.¹⁰ demonstrated that S-adenosyl-L-methionine:benzoic acid carboxyl methyltransferase (BAMT), an enzyme involved in scent biosynthesis, was localized in the conical cells of the inner epidermal layer and to a much lesser extent, in the cells of the outer epidermis of the lobes of snapdragon petals. On the basis of these latter observations, some authors have proposed that the papillate cell shape could enhance the diffusion of scent molecules or influence its directionality and be of adaptive significance not only by enhancing light reflection but also by enhancing scent production.^11,12Open in a separate windowFigure 1Hand-made cross-section of Rosa × hybrida petal; Ad, adaxial epidermis; Ab, abaxial epidermis; P, spongy parenchyma. Bar = 20 µm.To test the hypothesis that the adaxial epidermis is a privileged site for the production and emission of scent, we chose a highly scented flower, the rose. Contrary to what was expected, we found that both the adaxial and abaxial epidermal layers of the petal were sites of scent production and emission. We were able to show that NaDi reagent stained purple droplets in both epidermal layers of the rose petal, indicating that they both contain terpenes. Several different techniques, including the analysis of epidermal peels and epidermal layer-specific headspace analysis failed to detect a strong difference between the production and emission of scent in the two epidermal layers. Moreover, the detection of OOMT protein, an enzyme involved in 3,5-dimethoxytoluene production, in both the abaxial and adaxial epidermis, indicated that biosynthesis of at least some phenolic scent compounds occurs in both tissues. It will be interesting to extend this approach using in situ hybridization or immunolocalization to determine whether other pathways such as terpene metabolism are also active in the abaxial epidermis.It is striking to note that in Clarkia breweri, which has actinomorphic flowers like the rose, expression of the S-adenosyl-L-methionine:(iso) eugenol O-methyltransferase (IEMT) gene seems to occur in both epidermal layers.¹³ A. majus flowers have a different structure, they are highly zygomorphic with a flower shape that is adapted for bee pollination and includes specialized cell types in different parts of the flower (the lobes and the tube). To determine whether emission of scent in highly specialized flowers such as A. majus is linked to cell shape, it would be very useful to know whether mixta mutant flowers which have flat epidermal cells are impaired in their capacity to emit scent. However, the explanation may not be as simple. A recent study of the synthesis and emission of methyl benzoate showed that in Nicotiana suaveolens, as in the rose, both epidermal layers of the petal lobes are involved in scent production, whereas in Stephanotis floribunda, SAMT, another enzyme involved scent biosynthesis, is localized only in the adaxial epidermis and subepidermal regions of the flower petal lobes.¹⁴ It is intriguing to note that N. suaveolens has bullate to rugose epidermal cell layers on both sides of the petal whereas S. floribunda has tight flat to bullate epidermal cells.The reasons for the differences in the potential for scent emission of the two petal epidermal layers in the rose and other species are not known. However, our results and a survey of the literature clearly indicate that, in petals, epidermal cells may have diverse shapes and that the shape of the cells is not necessarily a reliable indicator of the secretory potential of those cells. It will be interesting to see whether common structural features and/or molecular factors are responsible for the differences between these various cell types.     

Integrin-like protein at the invaginated plasma membrane of epidermal cells in mature leaves of the marine angiosperm<Emphasis Type="Italic"> Zostera marina</Emphasis> L.

By immunoblotting with anti-human integrin polyclonal antibodies (1, 3 or 5), a single distinct band of about 60 kDa was detected in total protein extracts from mature leaves of the seagrass Zostera marina L., but no band was detected in total protein extracts from immature seagrass leaves, freshwater grass leaves or Arabidopsis thaliana (L.) Heynh. leaves. This integrin-like protein was detected by indirect immunofluorescence microscopy on the surface of non-spherical protoplasts of epidermal cells isolated from mature seagrass leaves using an anti-integrin 3 polyclonal antibody. Electron-microscopic analyses with the same antibody indicated that this integrin-like protein was localized specifically in the invaginated plasma membrane of epidermal cells in mature seagrass leaves. Therefore, this integrin-like protein of about 60 kDa may be involved in the developmentally regulated invagination of the plasma membrane in epidermal cells of the seagrass Z. marina.     

Direct turgor pressure measurements in individual leaf cells of Tradescantia virginiana

The water relations of leaves of Tradescantia virginiana were studied using the miniaturized pressure probe (Hüsken, E. Steudle, Zimmermann, 1978 Plant Physiol. 61, 158–163). Under well-watered conditions cell turgor pressures, P _o, ranged from 2 to 8 bar in epidermal cells. In subsidiary cells P _o was about 1.5 to 4.5 bar and in mesophyll cells about 2 to 3.5 bar. From the turgor pressure, relaxation induced in individual cells by changing the turgor pressure directly by means of the pressure probe, the half-time of water exchange was measured to be between 3 and 100 s for the epidermal, subsidiary, and mesophyll cells. The volumetric elastic modulus, , of individual cells was determined by changing the cell volume by a defined amount and simultaneously measuring the corresponding change in cell turgor pressure. The values for the elastic modulus for epidermal, subsidiary, and mesophyll cells are in the range of 40 to 240 bar, 30 to 200 bar, and 6 to 14 bar, respectively. Using these values, the hydraulic conductivity, L _p, for the epidermal, subsidiary, and mesophyll cells is calculated from the turgor pressure relaxation process (on the basis of the thermodynamics of irreversible processes) to be between 1 and 55·10^-7 cm s^-1 bar^-1. The data for the volumetric elastic modulus of epidermal and subsidiary cells indicate that the corresponding elastic modulus for the guard cells should be considerably lower due to the large volume changes of these cells during opening or closing. Recalculation of experimental data obtained by K. Raschke (1979, Encycl. Plant Physiol. N.S., vol. 7, pp 383–441) on epidermal strips of Vicia faba indicates that the elastic modulus of guard cells of V. faba is in the order of 40–80 bar for closed stomata. However, with increasing stomatal opening, i.e., increasing guard cell volume, decreases. Therefore, in our opinion Raschke's results would indicate a relationship between guard cell volume and which would be inverse to that for plant cells known in the literature. assumes values between 20–40 bar when the guard cell colume is soubled.     

Biochemical identification and immunological localization of two non-keratin polypeptides associated with the terminal differentiation of avian scale epidermis

Summary The expression of two previously uncharacterized polypeptides produced in epidermal cells of chick reticulate and scutate scales during late embryonic scale histogenesis and in hatchling birds has been studied biochemically and immunologically. These polypeptides have been identified by two-dimensional pH gradient gel electrophoresis as basic in charge, with apparent molecular weights of 20 and 23 kD, and they have been characterized immunologically and by amino acid analysis as non-keratin in nature. Monoclonal antibodies which react with both polypeptides have been used for immunohistochemical and immunogold electron-microscopic localization. Immunoreactivity was observed in suprabasal cells of reticulate scale epidermis, where it codistributed with bundles of -type cytokeratins in the -keratin-rich layers of epidermis known as the alpha stratum and in suprabasal cells of the outer epidermal surface of scutate scales, where it codistributed with -and -type keratin filament bundles in the -keratin-rich layers of epidermis known as the beta stratum.     

Regulation and clinical implications of corneal epithelial stem cells

The corneal epithelium is known to have a rapid self-renewing capacity. The major advance in the field of cornead epithelial cell biology in the last decade is the establishment of the location of corneal epithelial stem cells at the limbus, i.e., the junctional zone between the cornea and the conjunctiva. This concept has helped explain several experimental and clinical paradoxes, produced a number of important clinical applications, and spawned many other research studies. This unique enrichment of epithelial stem cells at a site anatomically separated from their transient amplifying cells makes the ocular surface an ideal model to study the regulation of epithelial stem cells. The present review includes data from more recent studies and lays out other areas for future investigation, especially with respect to the role of apoptosis and cytokine dialogue between limbal epithelial stem cells and their stromal microenvironment.Abbreviations EGF epidermal growth factor - EGFR epidermal growth factor receptor - bFGF basic fibroblast growth factor - HGF hepatocyte growth factor - IGF-I insulin-like growth factor type I - IL-1 interleukin 1 - K3 or K12 keratin type 3 or 12 - KGF keratinocyte growth factor - LIF leukemia inhibitory factor - PDGF platelet-derived growth factor - PKC protein kinase C - TGF- transforming growth factor- - TGF- transforming growth factor- - TPA phorbol ester tumor promoting agents     

Pattern control in insect segments: superimposed features of the pattern may be subject to different control mechanisms

Summary The integument of an insect segment displays two distinct pattern features which are based on different properties of the constituent epidermal cells. Normally, the uniform orientation of epidermal cell polarities (polarity pattern) is strictly correlated with the sequence of differentiated cells (differentiation pattern). Here it is reported that in the integument of the cotton bug Dysdercus epidermal cells can adopt orientations that do not correlate with the pigmentation pattern and which are not compatible with the gradient model. The results indicate that different features of a composite pattern can be independently controlled.     

Plant small monomeric G-proteins (RAC/ROPs) of barley are common elements of susceptibility to fungal leaf pathogens,cell expansion and stomata development

Small monomeric RAC/ROP GTPases act as molecular switches in signal transduction processes of plant development and stress responses. They emerged as crucial players in plant-pathogen interactions either by supporting susceptibility or resistance. In a recent publication, we showed that constitutively activated (CA) mutants of different barley (Hordeum vulgare) RAC/ROPs regulate susceptibility to barley fungal leaf pathogens of different life style in a contrasting way. This illustrates the distinctive signalling roles of RAC/ROPs for different plant-pathogen combinations. We also reported the involvement of RAC/ROPs in plant epidermis development in a monocotyledonous plant. Here we further discuss a failure of CA HvRAC/ROP-expressing barley to normally develop stomata.Key words: Hordeum vulgare, G-proteins, RAC, ROP, cell expansion, stomata, transpirationMembers of the RHO family of small G-proteins in plants (RAC/ROPs) regulate signal transduction processes at the plasma membrane.¹ They act as multifunctional signalling switches in plant development and a variety of stress responses. RAC/ROP GTPases play regulatory roles in polar growth and cell morphogenesis in several cell systems including pollen tubes, developing root hairs and leaf pavement cells.²In a recent publication,³ we showed that constitutively activated (CA) mutants of different barley (Hordeum vulgare) RAC/ROPs support susceptibility to the barley powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh). CA HvRAC1 supported susceptibility to biotrophic Bgh but resistance to hemibiotrophic Magnaporthe oryzae in barley at the penetration level in both cases. Additionally, CA HvRAC1 supported local callose deposition at sites of attack from Bgh and a secondary H₂O₂ burst in whole non-penetrated epidermal cells. This supports a regulatory function of RAC/ROPs in plant defence¹ and the potential corruption of defence pathways in susceptibility to Bgh. Because the rice ortholog of HvRAC1, OsRAC1, can regulate an H₂O₂ burst via activation of the plasma membrane NADPH oxidase OsRBOHB,⁴ one can speculate that the secondary H₂O₂ burst in CA HvRAC1 barley could also be caused by over-activation of an NADPH oxidase. However, CA HvRAC1 barley was also more susceptible to fungal penetration, and penetrated cells did not show an H₂O₂ burst. Hence, CA HvRAC1 did not contribute to penetration resistance, and the H₂O₂ burst might have been suppressed by Bgh after successful penetration. Interestingly, Bgh secretes a catalase during interaction with the plant.⁵The involvement of RAC/ROPs in plant development has been widely studied in the dicots Arabidopsis and tobacco. In Arabidopsis, CA AtRAC/ROPs disturb root hair tip growth and epidermal cell morphogenesis.^6,7 We showed similar developmental aberrations as a result of CA HvRAC/ROP expression in monocotyledonous barley. Root hair polarity disruption and enhanced leaf epidermal cell expansion was observed in CA HvRAC/ROP expressing barley. Here, we further report on reduced or abnormal development of stomata as an effect of CA HvRAC/ROP expression.In barley, stomata and short epidermal cells alternate in a row of leaf epidermal cells (Fig. 1A). The number of stomata number was significantly reduced in three CA HvRAC/ROP (CA HvRACB, CAHvRAC3, CA HvRAC1) expressing barley genotypes when compared to azygous controls (barley siblings that lost the transgene due to segregation) (Fig. 1E). In part, this could be explained by enhanced length of epidermal cells intercalated between stomata (Fig. 1B). The presence of longer epidermal cells in all CA HvRAC/ROP-barleys further supports that RAC/ROPs are operating in epidermal cell expansion.³Open in a separate windowFigure 1Stomatal abnormalities observed in CA HvROPexpressing transgenic barley leaves. (A) Wild type leaf adaxial epidermis with alternating stomata complexes (arrows) and short epidermal cells (asterisk). (B) Presence of more than one short epidermal cell in between two stomata. Arrows point the stomata. Double headed arrows highlight intercalated cells with enhanced cell length (C) Two stomata lacking an intercalated short epidermal cell. (D) Stoma failed to develop and left an abnormal blank cell. (E) Average number of stomata present in 5 cm of a stomatal row in transgenic plants expressing distinct CA barley CA HvRAC/ROPs. For all samples, stomatal rows present on either side of the mid rib were counted in the leaf upper epidermis. Fully expanded leaves of 3-weeks-old barley plants were used for counting stomata. Error bars show 95% confidence intervals. Repetition of the experiment led to similar results. Scale bars = 50 µm.Previously, we carried out porometry experiments to measure stomata conductivity in CA HvRACB expressing barley leaves.⁸ The CA HvRACB leaves showed up to 50% less transpiration than azygous controls without any treatment. Additionally, CA HvRACB leaves were less responsive to abscisic acid (ABA) and subsequently they could not effectively reduce transpiration when treated with ABA or when cut-off from water supply.⁸ Our data on numbers of stomata per leaf segment could now explain the lower rates of transpiration in non-stressed CA HvRACB barley when compared to wild type.Apart from the stomata number, developmental abnormalities were observed in the arrangement of epidermal cells. Generally, the shape of epidermal cells was less regular in CA HvRAC/ROP barley.³ We also observed the presence of more than one short epidermal cell in between two stomata (Fig. 1B) or two stomata lacking an intercalated short epidermal cell (Fig. 1C), or stomata failed to develop, which ended up in an abnormally short epidermal cell (Fig. 1D). Although such abnormalities were also rarely observed in wild type plants, all three CA HvRAC/ROP-barley leaves exhibited a clearly higher frequency of abnormalities in a given length of a stomata row. Together, CA HvRAC/ROPs had an effect on both the number and development of stomata. These observations suggest that RAC/ROPs are not only operating in cell expansion but also in barley cell differentiation for stomata development.     

The separation of basal and differentiating cells from human epidermis for DNA cytofluorometry.

A method for the selective separation of human epidermal cells, in which the Feulgen-stainable material suffers minimal damage, was investigated. The principal stage involves alpha-chymotrypsin treatment of skin specimens as well as shaking in an isotonic solution. With respect to DNA distribution pattern, there was good agreement between that of epidermal cells separated with the microdissection-ultrasonic irradiation method, reported previously by us, and those separated by the present method.     

Neurulation in the anterior trunk region of the zebrafish Brachydanio rerio

We have studied the process of neurulation within the anterior trunk region of the zebrafish by means of serial sectioning of staged embryos and labelling cells by applications of the dye Dil and intracellular injections of fluoresceine dextran amine. The first morphological manifestation of the prospective neural plate is a dorsomedial ectodermal thickening which becomes visible immediately after gastrulation. Within 1–2 h, by the time somatogenesis begins, two bilaterally symmetrical thickenings have appeared more laterally, which eventually fuse with the medial thickening to form the neural keel. The central canal forms next by separation of the cells on either side of the midline of the neural keel, beginning ventrally at the 17-somite stage and progressing towards dorsal levels. By means of fluorescent dye labelling in the late gastrula, we found that both the medial and lateral thickenings contribute to the nerve cord. The medial thickening was found to contain, exclusively, neural progenitor cells from the 90–100% epiboly stage on, whereas the adjacent regions contained a mixture of neural and epidermal progenitor cells, as well as prospective neural crest cells. Between the 90–100% epiboly and 2-somite stages, this heterogeneity of developmental capabilities is resolved into territories, with epidermogenic and neurogenic cells clearly separated from each other. To achieve this segregation into neural and epidermal anlagen, cells from the lateral thickenings have to move over a distance of roughly 400 m within 1–2 h. Epidermal overgrowth of the nerve cord occurs during the morphogenetic movements that accompany nerve cord formation. Correspondence to: J.A. Campos-Ortega