Non-invasive LC-PolScope imaging of biominerals and cell wall anisotropy changes

The formation of defined shapes by cells is one of the challenging questions in biology. Due to the anisotropy of cell walls and of certain biominerals, the LC-PolScope represents a promising tool for tracking dynamic structural changes in vivo non-invasively and, to some extent, quantitatively. A complex three-dimensional biogenic system, the in vitro precipitation of calcium oxalate induced by cellulose stalks produced by Dictyostelium discoideum, was analyzed. Although the retardance values and orientation of the crystals with respect to the stalk were quickly and easily detected, this study raised a number of issues that were addressed in this work. The effect of the refractive index of the embedding medium was examined by taking advantage of the homogeneous size and shape distribution of kiwifruit raphides, a biologically controlled calcium oxalate biomineral and of cotton (Gossypium) seed fibers. The retardance remained consistent when embedding these samples in media with increasing refractive indices from 1.33 to 1.42 or 1.47 for sucrose or glycerol gradients, respectively. The general applicability of LC-PolScope image processing for biominerals and cell wall formation during development in vivo was demonstrated in a particular group of green algae, the Desmidiaceae. Various organization levels of the cell wall were identified, thus confirming earlier findings based on electron microscopy and immunostaining investigations. It can be concluded that LC-PolScope microscopy is an attractive tool for studying dynamic ordering of biomolecules, such as plant cell walls, when additional parameters regarding the structure, composition, and refractive indices of the specimen are available.     

Matric potentials of leaves

A pressure chamber was used to measure matric potentials of frozen and thawed leaves. Significant matric potentials were demonstrated in sunflower (Helianthus annuus L.), yew (Taxus cuspidata Sieb. and Zucc.), and rhododendron (Rhododendron roseum Rehd.). Matric potentials were particularly negative in rhododendron and were correlated with the amount of cell wall present and with the volume of water outside the leaf protoplasts at comparable matric potentials. It was concluded that matric forces in leaves are associated mainly with cell walls, at least within the physiological range of water contents. Calculations indicated that the water potential of the solution in the cell wall could be estimated for living tissue from the sum of matric and osmotic potentials acting on water outside the protoplasts.     

Callose content in cell walls of leaf epidermis and mesophyll in Alisma plantago-aquatica L. plants growing in different conditions of water supply

The influence of water regime on relative callose content in cell walls of A. plantago-aquatica leaf tissues has been studied at the phases of budding and flowering-fruiting. The callose content in cell walls was shown to vary depending on the type of tissue, phase of ontogenesis, and growing conditions.     

Ruminococcus flavefaciens Cell Coat and Adhesion to Cotton Cellulose and to Cell Walls in Leaves of Perennial Ryegrass (Lolium perenne)

Ruminococcus flavefaciens was shown to possess a prominent glycoprotein coat, which contained rhamnose, glucose, and galactose as its principal carbohydrates. Periodate-reactive carbohydrate occurred as a surface layer of the coat. The ruminococci adhered strongly by means of this coat to cotton cellulose and to cell walls in leaf sections of Lolium perenne L. (perennial ryegrass). The coat was diffuse at the point of contact so that the bacterial cell wall was in close contact with the substrate. Adhesion was influenced by the availability of damaged plant cell walls and by the cell wall type and occurred most rapidly to cell walls of the epidermis and sclerenchyma, followed by the phloem and mesophyll. Plaques of bacteria with filamentous coat extensions developed on all these tissues. The bacteria did not readily adhere to the walls of the bundle sheath cells or metaxylem or protoxylem vessels and did not adhere to the cuticle or chloroplasts. The epidermal and phloem cell walls were more rapidly digested than the walls of other cell types.     

Down-Regulation of ZmEXPB6 (Zea mays β-Expansin 6) Protein Is Correlated with Salt-mediated Growth Reduction in the Leaves of Z. mays L.

The salt-sensitive crop Zea mays L. shows a rapid leaf growth reduction upon NaCl stress. There is increasing evidence that salinity impairs the ability of the cell walls to expand, ultimately inhibiting growth. Wall-loosening is a prerequisite for cell wall expansion, a process that is under the control of cell wall-located expansin proteins. In this study the abundance of those proteins was analyzed against salt stress using gel-based two-dimensional proteomics and two-dimensional Western blotting. Results show that ZmEXPB6 (Z. mays β-expansin 6) protein is lacking in growth-inhibited leaves of salt-stressed maize. Of note, the exogenous application of heterologously expressed and metal-chelate-affinity chromatography-purified ZmEXPB6 on growth-reduced leaves that lack native ZmEXPB6 under NaCl stress partially restored leaf growth. In vitro assays on frozen-thawed leaf sections revealed that recombinant ZmEXPB6 acts on the capacity of the walls to extend. Our results identify expansins as a factor that partially restores leaf growth of maize in saline environments.     

Defects in leaf epidermis of Arabidopsis thaliana plants with CDKA;1 activity reduced in the shoot apical meristem

In Arabidopsis thaliana, like in other dicots, the shoot epidermis originates from protodermis, the outermost cell layer of shoot apical meristem. We examined leaf epidermis in transgenic A. thaliana plants in which CDKA;1.N146, a negative dominant allele of A-type cyclin-dependent kinase, was expressed from the SHOOTMERISTEMLESS promoter, i.e., in the shoot apical meristem. Using cleared whole mount preparations of expanding leaves and sequential in vivo replicas of expanding leaf surface, we show that dominant-negative CDKA;1 expression results in defects in epidermis continuity: loss of individual cells and occurrence of gaps between anticlinal walls of neighboring pavement cells. Another striking feature is ingrowth-like invaginations of anticlinal cell walls of pavement cells. Their formation is related to various processes: expansion of cells surrounding the sites of cell loss, defected cytokinesis, and presumably also, the actual ingrowth of an anticlinal cell wall. The mutant exhibits also increased variation in cell size and locally reduced waviness of anticlinal walls of pavement cells. These unusual features of leaf epidermis phenotype may shed a new light on our knowledge on morphogenesis of jigsaw puzzle-shaped pavement cells and on the CDKA;1 role in regulation of plant development via influence on cytoskeleton and plant cell wall.     

Accumulation and Speciation of Vanadium in Lycium Seedling

Lycium seedling was subjected to varying doses of V for 40–45 days to examine the effects on uptake, accumulation, and speciation of V in Lycium seedling by differential centrifugation and enzymolysis. V concentrations in Lycium seedling organs were in sequence as follows: root > leaf > stem. V uptake into stem and leaf were primarily combined with acid-soluble polar compounds, polysaccharide, and immobile materials on the cell walls. There were different speciations of V in root with different V stress levels. Enzymolysis results suggest that about 60 % of the V in Lycium seedling root was combined with pectin and cellulose. It is the antidotal effect of pectin and cellulose in the cell wall that reduced the V damage to Lycium seedling.     

Lorenz-Mie light scattering in cellular biology.

The Lorenz-Mie light scattering is discussed as a tool allowing living cell characterization. The scattered light carries information about the size, shape, internal structure and refractive index of the cell. The advantages of light scattering methods consist in high speed, nondestructive, sensitive and relatively easy measurements. Light scattering methods are compatible with other methods. In light scattering in both static and flow systems. For sphere-like cells reliable size and refractive index information can be extracted. On the empirical basis, light scattering pattern can be used for the cell identification and separation purposes. The full utilization of the light scattering information is limited due to the lack of theoretical knowledge about the complex scatterer properties and efficient inversion schemes. The rapid progress in computer technique and in single-particle scattering experiments may significantly improve the interpretation of light scattering patterns of the biological particles.     

Interaction of Pseudomonas solanacearum with Suspension-Cultured Tobacco Cells and Tobacco Leaf Cell Walls In Vitro

Attachment of radiolabeled Pseudomonas solanacearum cells to suspension-cultured tobacco cells and tobacco leaf cell walls was measured in vitro by a filtration technique that allowed separation of attached and unattached bacteria. An avirulent strain (B1) attached more rapidly to suspension-cultured cells than did the virulent parent strain (K60), and B1 attachment was less sensitive to inhibition by high ionic strength than was K60. Attachment of B1 bacteria to suspension-cultured cells and to leaf cell walls was comparable (50 to 70%), but only a small proportion (10 to 20%) of K60 bacteria attached to leaf cell walls under optimal conditions. With high bacterial populations (10⁸ bacteria per ml), attachment of K60 to suspension-cultured cells was greatly reduced. Attachment of both strains was completely inhibited by pretreating bacterial cells with heat (41°C) or azide and was partially inhibited by EDTA and kanamycin. The mechanism of attachment is not known, but ionic forces may be involved.     

Permeability of the suberized mestome sheath in winter rye

Mestome sheath cells of winter rye (Secale cereale L. cv Puma) deposit suberized lamellae in their secondary cell walls. Histochemical tests including acid digestion and staining with Sudan IV and Chelidonium majus root extract were used to detect the presence of suberin in the primary cell wall. There was no evidence of a Casparian band between adjacent mestome sheath cells. Fluorescent dye techniques were used to trace solute movement through the rye leaf apoplast. Calcofluor white M2R, a fluorescent dye which binds to cell walls as it moves apoplastically, proved to be too limited in its mobility in leaves to test mestome sheath permeability. Trisodium 3-hydroxy-5,8,10 pyrene trisulfonate, a fluorescent dye which is mobile in the apoplast, moved easily up the vascular bundles in the transpiration stream, and diffused outward from the veins to the epidermal cell walls within minutes of reaching a particular level in the leaf. We conclude that the suberized mestome sheath of rye leaves is freely permeable to solutes moving apoplastically through radial primary cell walls.     

Rumen bacterial degradation of forage cell walls investigated by electron microscopy

The association of rumen bacteria with specific leaf tissues of the forage grass Kentucky-31 tall fescue (Festuca arundinacea Schreb.) during in vitro degradation was investigated by transmission and scanning electron microscopy. Examination of degraded leaf cross-sections revealed differential rates of tissue degradation in that the cell walls of the mesophyll and pholem were degraded prior to those of the outer bundle sheath and epidermis. Rumen bacteria appeared to degrade the mesophyll, in some cases, and phloem without prior attachment to the plant cell walls. The degradation of bundle sheath and epidermal cell walls appeared to be preceded by attachment of bacteria to the plant cell wall. Ultrastructural features apparently involved in the adhesion of large cocci to plant cells were observed by transmission and scanning electron microscopy. The physical association between plant and rumen bacterial cells during degradation apparently varies with tissue types. Bacterial attachment, by extracellular features in some microorganisms, is required prior to degradation of the more resistant tissues.     

Subcellular localization of silicon and germanium in grass root and leaf tissues by SIMS: evidence for differential and active transport

Silicon transport and incorporation into plant tissue is important to both plant physiological function and to the influence plants have on ecosystem silica cycling. However, the mechanisms controlling this transport have only begun to be explored. In this study, we used secondary ion mass spectrometry (SIMS) to image concentrations of Si in root and shoot tissues of annual blue grass (Poa annua L.) and orchard grass (Dactylis glomerata L.) with the goal of identifying control points in the plant silica uptake pathway. In addition, we used SIMS to describe the distributions of germanium (Ge); the element used to trace Si in biogeochemical studies. Within root tissue, Si and Ge were localized in the suberized thick-walled region of endodermal cells, i.e. the proximal side of endodermal cells which is in close association to the casparian strip. In leaves, Si was present in the cell walls, but Ge was barely detectable. The selective localization of Si and Ge in the proximal side of endodermal cell walls of roots suggests transport control is exerted upon Si and Ge by the plant. The absence of Si in most root cell walls and its presence in the cell walls of leaves (in areas outside of the transpiration terminus) suggests modifications in the chemical form of Si to a form that favors Si complexation in the cell walls of leaf tissue. The low abundance of Ge in leaf tissue is consistent with previous studies that suggest preferential transport of Si relative to Ge.     

Carbohydrates and carbohydrate esters of ferulic acid released from cell walls of Lolium multiflorum by treatment with cellulolytic enzymes

41% of the cell walls from mature leaf blades of Lolium multiflorum were digested by treatment during 14 days with C₁ enzyme (cellulase) which had been purified by gel filtration and ion-exchange chromatography. Cellobiose was the main sugar released from the walls, together with some glucose and higher oligosaccharides. Considerable amounts of carbohydrate esters of ferulic and p-coumaric acids were also released. When the C₁ enzyme was further purified by isoelectric focusing, only 8% of the cell walls were digested. Purified C_x (CM-cellulase) containing β-glucosidase digested 51% of the cell walls in 16 hours: the major component detected in the soluble products was glucose together with some β (1 → 4)-xylobiose, xylose and arabinose. Higher oligosaccharides and carbohydrate esters of ferulic and p-coumaric acids were also present. It was shown that these acids were present in the cell walls mainly in the trans-configuration.     

叶子花表皮特征的研究及意义

对叶子花（Bougainvillea spectabilis）正常叶和变态叶上气孔密度、气孔指数和保卫细胞大小进行了研究。结果表明：正常叶上表皮的表皮细胞为多边形,垂周壁平直;下表皮的表皮细胞为不规则型,垂周壁浅波状;气孔类型为不规则型。变态叶上表皮没有发现气孔,变态叶下表皮的表皮细胞垂周壁则由浅波形逐渐变为深波形,气孔类型为不规则型和轮列型。随着变态叶的发育,变态叶下表皮的气孔密度降低,气孔指数升高;变态叶保卫细胞的长增大,宽减小。变态叶的平均气孔密度和平均气孔指数明显低于正常叶。正常叶和变态叶的保卫细胞均呈肾形。     

The localisation of pectin in Sphagnum moss leaves and its role in preservation

The localisation of pectin in Sphagnum moss leaves and its role in preservation has been investigated. Light microscopy using ruthenium red to detect pectin in whole and sectioned Sphagnum papillosum leaves revealed it is abundant in hyaline cell walls, fibrils, papillae, chlorophyllous cell walls and thickenings around hyaline cell pores. Transmission electron microscopy of ultrathin cell walls labelled with poly-l-lysine colloidal gold revealed pectin was distributed throughout the cell wall. The preservative/microbiocidal properties of these pectins are explained by the acid-dissociation properties of galacturonic acid carboxyls and their incorporation in the unique cell arrangement of the Sphagnum leaf. Liquid from a salmon fillet absorbed into S. papillosum leaves and incubated at room temperature for 22 h had a pH around 4.85, was dominated by Lactobacillus sp. and smelled fresh compared to experimental controls. Chlorite-treated Sphagnum leaves could have a potential as a food tray pad that absorbs liquid and prevents the growth of spoilage bacteria inside it.     

Visualization of cortex organization and dynamics in microorganisms, using total internal reflection fluorescence microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups. At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells. We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.     

Cell wall cementing materials of grass leaves

Treatment of grass leaves with either a purified pectin lyase of Aspergillus japonicus or a purified xylanase of Trichoderma viride could lead to the isolation of some single leaf cells. However, a mixture of pectin lyase and xylanase brought about more rapid isolation of single cells than did either of the two enzymes alone, indicating a synergistic effect. Analysis of the components released from oat cell walls by the enzymes indicated that both homogalacturonans with a high degree of esterification and a kind of glucuronoarabinoxylan with ferulic acid ester may play a role in cell wall cementing in grass leaves.     

Degradation of Perennial Ryegrass Leaf and Stem Cell Walls by the Anaerobic Fungus Neocallimastix sp. Strain CS3b

The degradation of cell walls isolated from stems and leaves of perennial ryegrass by the anaerobic fungus Neocallimastix sp. strain CS3b was studied in a defined medium. The combined cellulose and hemicellulose fraction represented 53.1 (wt/wt) and 63.3% (wt/wt) of the dry weight of control grass leaf and stem cell walls, respectively. In both leaf and stem cell walls, glucose was the major neutral monosaccharide, followed by xylose, arabinose, and galactose. After 2 days of fermentation with Neocallimastix sp. strain CS3b, treated cell walls contained smaller amounts of neutral sugars compared with those of undigested cell walls. These results were more evident for glucose, xylose, and arabinose than for galactose. Furthermore, the sugar content of leaf cell walls decreased before a decline in the sugar content of stem cell walls was observed. Data from formate and hydrogen production indicated that the growth of Neocallimastix sp. strain CS3b was completed in 4 days in the culture system used. During this period, the fungus liberated about 95% of the fermentable sugars in untreated material. On a percentage basis, no significant differences were found in final extent of degradation of glucose, xylose, and arabinose. Galactose, however, was degraded to a lesser extent.     

Altered matrix polysaccharides in cell walls of pocket galls formed by an aphid on Distylium racemosum leaves

The present work reports the results of a study on the isolation and characterization of matrix polysaccharides in the cell walls of galls formed by an aphid (Neothoracaphis yanonis) on Distylium racemosum leaves. Cell walls were isolated from both healthy Distylium leaf and gall tissues and then extracted sequentially with cyclohexane‐trans‐1,2‐diaminetetra‐acetate (CDTA), Na₂CO₃, 1 m KOH, and 4 m KOH. The amount of pectin solubilized from gall cell walls was approximately 2.6‐fold higher than the pectin solubilized from leaf cell walls, whereas the amount of hemicellulose solubilized from gall cell walls was 1.4‐fold higher than that from normal leaf cell walls. When the polysaccharides were fractionated by anion‐exchange chromatography, considerable increases in arabinose and galactose were observed in CDTA‐soluble pectic polymer (fraction PI‐1) from gall cell walls, whereas the gall cell walls had less xylose in 1 m KOH‐soluble hemicellulosic polymers (fractions HI‐2, HI‐3, and HI‐4) than did the cell walls from the healthy leaf. The hemicellulosic polymers of the gall cell walls exhibited distinctly different patterns of molecular mass, compared with the healthy leaf cell walls. These results suggest that an extensive change occurs in the matrix polysaccharide structure of the cell walls of Distylium galls formed by an aphid. In addition, many glycosylhydrolase activities were detected in the protein fraction solubilized with strong saline solution from the gall cell walls, and the activities of β‐galactosidase, β‐xylosidase and α‐l ‐arabinofuranosidase were considerably increased under gall formation.