Outer Epidermal Wall Research Articles

Strain rate does not affect cortical microtubule orientation in the isolated epidermis of sunflower hypocotyls

A hypothesis exists that external and internal factors affect the orientation of cortical microtubules in as much as these lead to changes in cell elongation rate. Factors that stimulate elongation are proposed to lead to transverse microtubule orientation, whereas factors that inhibit elongation lead to longitudinal orientation. The elongation rate is equal to the rate of longitudinal irreversible strain in cell walls. Incubated epidermis peeled from sunflower hypocotyls does not extend unless it is stretched by loading and the pH of the incubation medium is appropriately low. Thus, peels provide a convenient model to investigate the relationship between longitudinal strain rate and cortical microtubule orientation. In the present study, it was found that peeling affects microtubule orientation. Peels were incubated for several hours in Murashige & Skoog medium (both unbuffered and buffered) to attain a steady state of microtubule orientation before loading. The effects of loading and pH on strain rate and orientation of microtubules under the outer epidermal walls were examined in three portions of peels positioned with respect to the cotyledonary node. Appropriate loading caused longitudinal strain of peels at pH 4.5 but not at pH 6.5. However, no clear effect of strain rate on microtubule orientation in the peels was observed. Independent of applied load and pH of the incubation medium, the microtubule orientation remained unchanged, i.e. orientation was mainly oblique. Our results show that strain rate does not affect cortical microtubule orientation in isolated epidermis of the sunflower hypocotyl model system, although orientation could be changed by white light.

Floral nectary structure, nectar production, and carbohydrate composition in theLiliumAsiatic hybrid ‘Trésor’

Floral nectary structure, nectar production, and carbohydrate composition were compared from petals (“inner tepals”) and sepals (“outer tepals”) of Lilium Asiatic hybrid ‘Trésor’ (Liliaceae). The six nectaries each occupied a narrow furrow bordered by two convergent ridges extending adaxially from the petal and sepal base. Each sepal nectary furrow was shorter and more concealed. In both nectary types, many vascular bundles comprising xylem and phloem supplied 5.5–8 layers of nectariferous parenchyma cells below the epidermis, which lacked stomata. Transmission electron microscopy of sepal nectaries demonstrated that parts of the outer epidermal wall adhered to an intact but uplifted cuticle in nectar-secreting flowers. Both apoplastic and symplastic routes were continuous from the vascular bundles to the nectary epidermis. Starch breakdown from amyloplasts throughout the nectary likely augmented nectar production. Nectar solute concentration from another Asiatic hybrid, ‘Orange Pixie’, was also significantly higher in petals. In ‘Trésor’, significantly more nectar was available from sepals, possibly reflecting reduced evaporation from multiple nectar droplets within the covered nectary furrow. However, for both hybrids, the same quantity of nectar sugar was produced by petals and sepals. Nectar composition from petals and sepals also was alike, in ‘Orange Pixie’ averaging 67/19/14 (= sucrose/fructose/glucose) and 59/25/17, respectively, and in ‘Trésor’ averaging 68/23/10 and 62/27/12, respectively.

Onion epidermis as a new model to study the control of growth anisotropy in higher plants

To elucidate the role of cellulose microfibrils in the control of growth anisotropy, a link between their net orientation, in vitro cell wall extensibility, and anisotropic cell expansion was studied during development of the adaxial epidermis of onion (Allium cepa) bulb scales using polarization confocal microscopy, creep tests, and light microscopy. During growth the net cellulose alignment across the whole thickness of the outer epidermal wall changed from transverse through random to longitudinal and back to transverse relative to the bulb axis. Cell wall extension in vitro was always higher transverse than parallel to the net cellulose alignment. The direction of growth anisotropy was perpendicular to the net microfibril orientation and changed during development from longitudinal to transverse to the bulb axis. The correlation between the degree of growth anisotropy and the net cellulose alignment was poor. Thus the net cellulose microfibril orientation across the whole thickness of the outer periclinal epidermis wall defines the direction but not the degree of growth anisotropy. Strips isolated from the epidermis in the directions perpendicular and transverse to a net cellulose orientation can be used as an extensiometric model to prove a protein involvement in the control of growth anisotropy.

Developmental reorientation of transverse cortical microtubules to longitudinal directions: a role for actomyosin‐based streaming and partial microtubule‐membrane detachment

Transversely oriented cortical microtubules in elongating cells typically reorient themselves towards longitudinal directions at the end of cell elongation. We have investigated the reorientation mechanism along the outer epidermal wall in maturing leek (Allium porrum L.) leaves using a GFP-MBD microtubule reporter gene and fluorescence microscopy. Incubating leaf segments for 14-18 h with the anti-actin or anti-actomyosin agents, 20 microm cytochalasin D or 20 mM 2,3-butanedione monoxime, inhibited the normal developmental reorientation of microtubules to the longitudinal direction. Observation of living cells revealed a small subpopulation of microtubules with their free ends swinging into oblique or longitudinal directions, before continuing to assemble in the new direction. Electron microscopy confirmed that longitudinal microtubules are partly detached from the plasma membrane. Incubating leaf segments with 0.2% 1 degree-butanol, an activator of phospholipase D, which has been implicated in plasma membrane-microtubule anchoring, promoted the reorientation, presumably by promoting microtubule detachment from the membrane. Stabilizing microtubules with 10 microm taxol also promoted longitudinal orientation, even in the absence of cytoplasmic streaming. These results were consistent with confocal microscopy of live cells before and after drug treatments, which also revealed that the slow (days) global microtubule reorientation is superimposed over short-term (hours) regional cycling in a clockwise and an anti-clockwise direction. We propose that partial detachment of transverse microtubules from the plasma membrane in maturing cells exposes them to hydrodynamic forces of actomyosin-driven cytoplasmic streaming, which bends or shifts pivoting microtubules into longitudinal directions, and thus provides an impetus to push microtubule dynamics in the new direction.

The growing outer epidermal wall: design and physiological role of a composite structure.

The cells of growing plant organs secrete an extracellular fibrous composite (the primary wall) that allows the turgid protoplasts to expand irreversibly via wall-yielding events, which are regulated by processes within the cytoplasm. The role of the epidermis in the control of stem elongation is described with special reference to the outer epidermal wall (OEW), which forms a 'tensile skin'. The OEW is much thicker and less extensible than the walls of the inner tissues. Moreover, in the OEW the amount of cellulose per unit wall mass is considerably greater than in the inner tissues. Ultrastructural studies have shown that the expanding OEW is composed of a highly ordered internal and a diffuse outer half, with helicoidally organized cellulose microfibrils in the inner (load-bearing) region of this tension-stressed organ wall. The structural and mechanical backbone of the wall consists of helicoids, i.e. layers of parallel, inextensible cellulose microfibrils. These 'plywood laminates' contain crystalline 'cables' orientated in all directions with respect to the axis of elongation (isotropic material). Cessation of cell elongation is accompanied by a loss of order, i.e. the OEW is a dynamic structure. Helicoidally arranged extracellular polymers have also been found in certain bacteria, algae, fungi and animals. In the insect cuticle crystalline cutin nanofibrils form characteristic 'OEW-like' herringbone patterns. Theoretical considerations, in vitro studies and computer simulations suggest that extracellular biological helicoids form by directed self-assembly of the crystalline biopolymers. This spontaneous generation of complex design 'without an intelligent designer' evolved independently in the protective 'skin' of plants, animals and many other organisms.

Cytoskeleton and cell wall function in penetration resistance

Plants successfully repel the vast majority of potential pathogens that arrive on their surface, with most microorganisms failing to breach the outer epidermal wall. Resistance to penetration at the epidermis is a key component of basal defence against disease and critically depends on fortification of the cell wall at the site of attempted penetration through the development of specialised cell wall appositions rich in antimicrobial compounds. Formation of cell wall appositions is achieved by rapid reorganisation of actin microfilaments, actin-dependent transport of secretory products to the infection site and local activation of callose synthesis. Plants are finely tuned to detect the presence of pathogens on their surface, perceiving both chemical and physical signals of pathogen origin. In the on-going evolution of interaction strategies, plants must continually monitor and out manoeuvre pathogen avoidance or suppression of plant defences in order to preserve the effectiveness of penetration resistance.

Cell elongation in Arabidopsis hypocotyls involves dynamic changes in cell wall thickness

Field-emission scanning electron microscopy was used to measure wall thicknesses of different cell types in freeze-fractured hypocotyls of Arabidopsis thaliana. Measurements of uronic acid content, wall mass, and wall volume suggest that cell wall biosynthesis in this organ does not always keep pace with, and is not always tightly coupled to, elongation. In light-grown hypocotyls, walls thicken, maintain a constant thickness, or become thinner during elongation, depending upon the cell type and the stage of growth. In light-grown hypocotyls, exogenous gibberellic acid represses the extent of thickening and promotes cell elongation by both wall thinning and increased anisotropy during the early stages of hypocotyl elongation, and by increased wall deposition in the latter stages. Dark-grown hypocotyls, in the 48 h period between cold imbibition and seedling emergence, deposit very thick walls that subsequently thin in a narrow developmental window as the hypocotyl rapidly elongates. The rate of wall deposition is then maintained and keeps pace with cell elongation. The outer epidermal wall is always the thickest ( approximately 1 mum) whereas the thinnest walls, about 50 nm, are found in inner cell layers. It is concluded that control of wall thickness in different cell types is tightly regulated during hypocotyl development, and that wall deposition and cell elongation are not invariably coupled.

Adaptations and phytogeography in the ice-plant family (Aizoaceae) — the interaction of the genetic equipment and ecological parameters. II. Hide-and-seek: plants sunken in the ground

Plants with sunken growth never shed their leaves completely but keep their leaf tips level with the ground most of the time, being difficult to detect in habitat. In Aizoaceae, 73 species in the 12 genera Aloinopsis (1), Argyroderma (2), Conophytum (21), Dinteranthus (1), Fenestraria (1), Frithia (2), Gibbaeum (3), Lithops (36), Psammophora (1), Tanquana (2), Titanopsis (2), and Vanheerdea (1) possess this life form exclusively or predominantly; in Fenestraria (1), Frithia (2), and Lithops (36), only subterranean growth is known. Protection from predators and reduction of evaporation, supported by the development of mostly xeromorphic features in the outer epidermal wall, appear to be the main adaptive traits in all taxa, combined with the ability of the plants to inhabit small niches of soft soil in often stony terrain, making use of the lack of competition from bigger plants with more extensive root systems. Completely different strategies have been found, confirming that no single way of evolution for subterranean plants exists. In most genera, the sunken growth is found in few species only in marginal parts of the distribution area, but Lithops has spread over a very wide area with different climatic conditions, presenting all states of speciation and hence difficult to handle in the hierarchal taxonomic system. Conophytum, on the other hand, has evolved a number of species, too, but these are most often adapted to specific soils like quartz and seem to represent parallel evolutionary lines.

A Raman-scattering Study on the Net Orientation of Biomacromolecules in the Outer Epidermal Walls of Mature Wheat Stems (Triticum aestivum)

Raman spectroscopy can be used to examine the orientation of biomacromolecules using relatively thick samples of material, whereas more traditional means of analysing molecular structure require prior isolation of the components, which often destroys morphological features. In this study, Raman spectroscopy was used to examine the outer epidermal cell walls of wheat stems. Polarized Raman spectra from the epidermal cell walls of wheat stem were obtained using near-infrared-Fourier transform Raman scattering. By comparing spectra taken with Raman light polarized perpendicular or parallel to the longitudinal axis of the cell, the orientation of macromolecules in the cell wall was investigated. The net orientation of macromolecules varies in the epidermal cell walls of the different components of wheat stem. The net orientation of cellulose is parallel to the longitudinal axis of the cells, whereas the xylan and the phenylpropane units of lignin tend to lie perpendicular to the longitudinal axis of the cells, i.e. perpendicular to the net orientation of cellulose in the epidermal cell walls. The results imply that cellulose, lignin and xylan form a relatively ordered network that defines the mechanical and structural properties of the cell wall. Such results are likely to have a significant impact on the formulation of definitive models for the static and growing cell wall.

Study of Erwinia amylovora colonization and migration on blossoms of susceptible and tolerant apple cultivars

The stigmata of detached flowers of susceptible and tolerant apple cultivars were inoculated with about 104 gfp labeled Erwinia amylovora . There were no apparent differences in the colonization, multiplication and survival of the bacteria on the stigmatic surface of the culivars. Bacteria were washed down to the hypanthium surface 24 hours after inoculation. The visual symptoms of the infection were the discoloration and shrinkage of the floral parts. The gradual browning associated with the infection appeared first on the surface of the hypanthium followed by the discoloration of the style. The color of the filaments turned into brown only 120 hours after the inoculation. Bacterial cells were not detected in the tissues of the styles and filaments. The traits of the hypanthium surface are of prominent importance in the progression of the infection. The wrinkled surface, the convex shape of the outer epidermal cell walls with thin cuticle and the sunken stomata helped to preserve a water film for a longer period providing medium for the motility of the bacteria in the susceptible cultivar. Bacteria were restricted to small water droplets on the flat and waxy surface of the hypanthium of the tolerant cultivar and only a few were able to enter the tissues.
 Large bacterium aggregations were detected in the intercellular spaces of the parenchyma of the susceptible cultivar 48 hours after the inoculation. In the next period the Erwinia amylovora cells gradually invaded the intercellulars of the hypanthium wall, the wall of the ovary and the pedicel. Low level of bacterium aggregation was found in the intercellulars of the tolerant cultivars. It is suggested that the progression of the infection was inhibited also by physiological factors.

Compatible and Incompetent Paxillus involutus Isolates for Ectomycorrhiza Formation in vitro with Poplar (Populus×canescens) Differ in H2O2 Production

Isolates of Paxillus involutus (Batsch) Fr. collected from different hosts and environmental conditions were screened for their ability to form ectomycorrhizal symbiosis with hybrid poplar P. x canescens (= Populus tremula L. x P. alba) in vitro. The ability to form ectomycorrhiza varied between the fungal isolates and was not correlated with the growth rate of the fungi on agar-based medium. The isolate MAJ, which was capable of mycorrhiza synthesis under axenic conditions, and the incompetent isolate NAU were characterized morphologically and anatomically. MAJ formed a typical hyphal mantle and a Hartig net, whereas NAU was not able to penetrate the host cell walls and caused thickenings of the outer cell walls of the host. MAJ, but not NAU, displayed strong H2O2 accumulation in the outer hyphal mantle. Increases in H2O2 in the outer epidermal walls and adjacent hyphae of the incompetent isolate were moderate. No increases of H2O2 in response to the mycobionts were found inside roots. Suggested functions of H2O2 production in the outer hyphal mantle of the compatible interaction are: growth regulation of the host's roots, defence against other invading microbes, or increasing plant-innate immunity. The system established here for P. x canescens compatible and incompetent fungal associations will be useful to take advantage of genomic information now available for poplar to study tree-fungal interactions at the molecular and physiological level.

Cellulose orientation at the surface of the Arabidopsis seedling. Implications for the biomechanics in plant development

In diffuse growing cells the orientation of cellulose fibrils determines mechanical anisotropy in the cell wall and hence also the direction of plant and organ growth. This paper reports on the mean or net orientation of cellulose fibrils in the outer epidermal wall of the whole Arabidopsis plant. This outer epidermal wall is considered as the growth-limiting boundary between plant and environment. In the root a net transverse orientation of the cellulose fibrils occurs in the elongation zone, while net random and longitudinal orientations are found in subsequent older parts of the differentiation zone. The position and the size of the transverse zone is related with root growth rate. In the shoot the net orientation of cellulose fibrils is transverse in the elongating apical part of the hypocotyl, and longitudinal in the fully elongated basal part. Leaf primordia and very young leaves have a transverse orientation. Throughout further development the leaf epidermis builds a very complex pattern of cells with a random orientation and cells with a transverse or a longitudinal orientation of the cellulose fibrils. The patterns of net cellulose orientation correlate well with the cylindrical growth of roots and shoots and with the typical planar growth of the leaf blade. On both the shoot and the root surface very specific patterns of cellulose orientation occur at sites of specific cell differentiation: trichome–socket cells complexes on the shoot and root hairs on the root.

Femtosecond laser-induced-breakdown spectrometry for Ca2+ analysis of biological samples with high spatial resolution

We describe an analytical method for element-specific in situ investigations of biological samples with high spatial resolution, using laser-induced-breakdown spectroscopy (LIBS). The prerequisites for spatially highly resolved LIBS are an appropriate analytical performance in combination with precise micro-ablation conditions, both laterally and axially. In order to identify the best suitable laser source, the analytical performance and the ablation process were studied for ns-LIBS and fs-LIBS. The analytical performance was studied on an aqueous solution of CaCl2, where water served as a first substitute for biological material. The ablation process was investigated in the outer epidermal wall of a sunflower seedling stem. Besides direct measurements, the physiology of the sunflower seedling can be used in order to estimate the ablation depth. Our results show that analytical measurements with high spatial resolution can be performed on biological samples using fs-LIBS. This was demonstrated by in situ measurements of wall-associated calcium ion (Ca2+) distributions within the peripheral cell wall of the sunflower seedling (Helianthus annuus L.) stem. In this biological sample an axial resolution of about 100 nm was achieved.

Characterization of anatomical features and silica distribution in rice husk using microscopic and micro-analytical techniques

Rice husk is a by-product of rice milling process, and a great resource as a raw biomass material for manufacturing value-added composite products. One of the potential applications is to use rice husk as filler for manufacturing lignocellulosic fiber–thermoplastic composites. This study was conducted to examine the silica distribution in rice husk in preparation to use it as reinforcing filler for thermoplastic polymers. Microscopic techniques, such as light microscopy, scanning electron microscopy and field-emission SEM (FE-SEM) were used to observe the surface and internal structure of rice husk. Microscopic examination showed that two main components of husk, lemma and palea consisted of outer epidermis, layers of fibers, vascular bundles, parenchyma cells, and inner epidermis, in sequence from the outer to the inner surface. Histochemical staining showed that epidermal and fiber cell walls were lignified, and the walls of parenchyma and lower epidermal cells were not lignified. The outer epidermal walls were extremely thick, highly convoluted and lignified. The outer surface of both lemma and palea were conspicuously ridged. The energy dispersive X-ray micro-analysis attached to the FE-SEM provided information on the distribution of silica in the husk. Most of the silica was present in the outer epidermal cells, being particularly concentrated in the dome-shaped protrusions. These observations provided valuable background information on the organization of husk tissues and the distribution of silica, which will help optimize processes related to the use of rice husk for making lignocellulosic fiber–thermoplastic composites in our future work.

Cellulose orientation in the outer epidermal wall of angiosperm roots: implications for biosystematics.

The net orientation of cellulose fibrils in the outer epidermal wall of the root elongation zone of 57 angiosperm species belonging to 29 families was determined by means of Congo Red fluorescence and polarization confocal microscopy. The angiosperms can be divided in three groups. In all but four plant families, the net orientation of the cellulose fibrils is transverse to the root axis. Three families, the Poaceae, Juncaceae and Cyperaceae, have a totally different organization. In the root elongation zone of these plants, the net orientation of cellulose fibrils in the outer epidermal wall is parallel with the root axis. In roots of one family, the Arecaceae, an elongation zone in the literal sense of the word is absent and cellulose fibrils are randomly oriented.

Stem Elongation and Cell Wall Proteins in Flowering Plants

Abstract: The growth of stems (hypocotyls, epicotyls) and stem‐like organs (coleoptiles) in developing seedlings is largely due to the elongation of cells in the sub‐apical region of the corresponding organ. According to the organismal concept of plant development, the thick outer epidermal wall, which can be traced back to the peripheral cell wall of the zygote, creates a sturdy organ sheath that determines the rate of stem elongation. The cells of the inner tissues are the products of secondary partitioning of one large protoplast; these turgid, thin‐walled cells provide the driving force for organ growth. The structural differences between these types of cell walls are described (outer walls: thick, sturdy, helicoidal cellulose architecture; inner walls: thin, extensible, transversely‐oriented cellulose microfibrils). On the basis of these facts, current models of cell wall loosening (and wall stiffening) are discussed with special reference to the expansin, enzymatic polymer remodelling and osmiophilic particle hypothesis. It is concluded that the exact biochemical mechanism(s) responsible for the coordinated yielding of the growth‐controlling peripheral organ wall(s) have not yet been identified.

Lateral redistribution of auxin is not the means for gravitropic differential growth of coleoptiles: A new model.

Gravicurvature in water- and auxin (IAA)-incubated coleoptiles of rye (Secale cereale L.) is similar, despite a general strongly enhancing effect of exogenous IAA on the overall (cell) elongation of these organs. Longitudinally split coleoptiles or isolated longitudinally halved coleoptiles (horizontally positioned as upper or lower halves) respond gravitropically in the same way as water-incubated intact coleoptiles, irrespective of whether the halves are incubated in distilled water or IAA. A new model for the principal mechanism of regulation of gravitropic growth is proposed which depends on, yet does not involve, the redistribution of IAA as the means for gravistimulated differential growth, as postulated by the Cholodny-Went hypothesis (CWH). It is based on a gravimediated temporarily restrained infiltration of IAA-induced wall-loosening factors into the growth-limiting outer epidermal walls of the concave organ flank.

Cell biology and genetics of root hair formation in Arabidopsis thaliana.

In this review we integrate the information available on the cell biology of root hair formation with recent findings from the analysis of root hair mutants of Arabidopsis thaliana. The mature Arabidopsis root epidermis consists of root-hair-producing cells and non-root-hair-producing cells. Root hair growth begins with a swelling of the outer epidermal wall. It has been postulated that this is due to a pH-mediated localised cell wall loosening. From the bulge a single root hair emerges which grows by tip growth. The root hair tip consists of a vesicle-rich zone and an organelle-rich subapical zone. The vesicles supply new plasma membrane and cell wall material for elongation. The cytoskeleton and its associated regulatory proteins such as profilin and spectrin are proposed to be involved in the targeting of vesicles. Ca2+ influxes and gradients are present in hair tips, but their function is still unclear. Mutants have been isolated with lesions in various parts of the root hair developmental pathway from bulge identity and initiation to control of tip diameter and extent and polarity of elongation.

Gravitropism of axial organs in multicellular plants.

Gravitropism of plant organs such as roots, stems and coleoptiles can be separated into four distinct phases: 1. perception (gravity sensing), 2. transduction of a signal into the target region and 3. the response (differential growth). This last reaction is followed by a straightening of the curved organ (4.). The perception of the gravitropic stimulus upon horizontal positioning of the organ (1.) occurs via amyloplasts that sediment within the statocytes. This conclusion is supported by our finding that submerged rice coleoptiles that lack sedimentable amyloplasts show no graviresponse. The mode of signal transduction (2.) from the statocytes to the peripheral cell layers is still unknown. Differential growth (3.) consists of a cessation of cell expansion on the upper side and an enhancement of elongation on the lower side of the organ. Based on the facts that the sturdy outer epidermal wall (OEW) constitutes the growth-controlling structure of the coleoptile and that growth-related osmiophilic particles accumulate on the upper OEW, it is concluded that the differential incorporation of wall material (presumably glycoproteins) is causally involved. During gravitropic bending, electron-dense particles ('wall-loosening capacity') accumulate on the growth-inhibited upper OEW. It is proposed that the autotropic straightening response, which is in part due to an acceleration of cell elongation on the curved upper side, may be attributable to an incorporation of the accumulated particles ('release of wall-loosening capacity'). This novel mechanism of autotropic re-bending and its implications for the Cholodny-Went hypothesis are discussed.

The effects of UV‐B radiation on epidermal anatomy in loblolly pine (Pinus taeda L.) and Scots pine (Pinus sylvestris L.)

ABSTRACTThe effects of enhanced UV‐B radiation on the needle anatomy of loblolly pine (Pinus taeda L.) and Scots pine (Pinus sylvestris L.) were studied in the field under supplemental UV‐B radiation supplied by a modulated irradiation system. The supplemental UV‐B levels were designed to simulate either a 16 or 25% loss of stratospheric ozone over College Park, Maryland. Enhanced UV‐B radiation caused different responses in these two species. The needles of loblolly pine had larger amounts of tannin in the lumen of epidermal cells and more wall‐bound phenolics in the outer epidermal walls of UV‐B‐treated needles, whereas the most pronounced effect on Scots pine needles was increased cutinization. In both species, the outer epidermal cell walls thickened and the needle cross‐sectional and mesophyll areas decreased (statistically significantly only in Scots pine). This suggests that more carbon may have been allocated to the protection mechanisms at the expense of photosynthetic area. The difference in response between these species suggests that the response to UV‐B radiation is not mediated by a single mechanism and that no generalization with regard to the effects of UV‐B on conifers can be made.