Ultrastructural and immunochemical features of the cell wall sac formed in mulberry (Morus alba) idioblasts

A peculiar inward growth, named a “cell wall sac”, formed in mulberry (Morus alba) idioblasts, is a subcellular site for production of calcium carbonate crystals. On the basis of ultrastructural observations, a fully expanded cell wall sac could be divided into two parts—an amorphous complex consisting of multi-layered compartments with multiple fibers originating from the innermost cell wall layer, and a peripheral plain matrix with fiber aggregates. Immunofluorescent localization showed that low and highly esterified pectin epitopes were detected at the early stages of development of the cell wall sac, followed by complete disappearance from the both parts of fully enlarged mature sac. In contrast, the xyloglucan epitope remained in the compartment complex; this was supported by the observation that the xyloglucan epitope labeled with immuno-gold particles is found on fibers in the complex part.     

Calcium carbonate deposition in a cell wall sac formed in mulberry idioblasts

Summary. Although calcium carbonate is known to be a common biomineral in plants, very little attention has been given to the biological control of calcium carbonate deposition. In mulberry leaves, a subcellular structure is involved in mineral deposition and is described here by a variety of cytological techniques. Calcium carbonate was deposited in large, rounded idioblast cells located in the upper epidermal layer of mulberry leaves. Next to the outmost region (“cap”) of young idioblasts, we found that the inner cell wall layer expanded to form a peculiar outgrowth, named cell wall sac in this report. This sac grew and eventually occupied the entire apoplastic space of the idioblast. Inside the mature cell wall sac, various cellulosic membranes developed and became the major site of Ca carbonate deposition. Concentrated Ca²⁺ was pooled in the peripheral zone, where small Ca carbonate globules were present in large numbers. Large globules were tightly packed among multiple membranes in the central zone, especially in compartments formed by cellulosic membranes and in their neighboring membranes. The maximum Ca sink capacity of a single cell wall sac was quantified using enzymatically isolated idioblasts as approximately 48 ng. The newly formed outgrowth in idioblasts is not a pure calcareous body but a complex cell wall structure filled with substantial amounts of Ca carbonate crystals. Correspondence and reprints: Graduate School of Science and Technology, Kyoto Institute of Technology, Goshokaido, Matsugasaki, Sakyo, Kyoto 606-8585, Japan.     

Cell wall sheath surrounding calcium oxalate crystals in mulberry idioblasts

Summary. The distribution and ultrastructural features of idioblasts containing calcium oxalate crystals were studied in leaf tissues of mulberry, Morus alba L. In addition to the calcium carbonate crystals formed in epidermal idioblasts, large calcium oxalate crystals were deposited in cells adjacent to the veins and surrounded by a cell wall sheath which had immunoreactivity with an antibody recognizing a xyloglucan epitope. The wall sheath formation indicates exclusion of the mature crystal from the protoplast. Correspondence: Y. Sugimura, Graduate School of Science and Technology, Kyoto Institute of Technology, Goshokaido, Matsugasaki, Sakyo, Kyoto 606-8585, Japan.     

Composition and ultrastructure of the suberized cell wall of isolated crystal idioblasts from Agave americana L. leaves

Styloid-calcium-oxalate-crystal-containing idioblasts possess an interior cell-wall layer which has a lamellar ultrastructure. Idioblasts were isolated by centrifugation of an Agave americana leaf homogenate through 2M sucrose. The aliphatic monomers of the polymeric material from an idioblast fraction were primarily -hydroxy acids (32%) and dicarboxylic acids (35%), with C_18:1 dicarboxylic acid being the most dominant monomer (25%). Nitrobenzene oxidation of the idioblasts yielded syringaldehyde and vanillin in a ratio of 0.46:1. The major class of wax associated with the idioblasts was free fatty acids (34%). A major homologue of both the fatty acid and fatty alcohol fractions of this wax was C₂₂. The hydrocarbon fraction of the wax had a broad chainlength distribution with a large amount of even-numbered (47%) and shorter-chain homologues. The ultrastructure, the composition of the aliphatic and aromatic components of the polymeric material as well as the composition of the wax show that the idioblast cell wall is suberized. The wax and cutin polymer of the epidermis of A. americana leaves were chemically characterized for comparative purposes.Scientific paper No. 6115, Project 2001, College of Agriculture Research Center, Washington State University, Pullman, WA 99164, USA     

Induction characteristics and response of photosynthetic quantum conversion to changes in irradiance in mulberry plants

A study was conducted, using rapid time course of chlorophyll (Chl) fluorescence parameters, and light-response curves of Chl fluorescence parameters, to determine the induction requirements and response of photosystem II (PSII) photochemistry and non-photochemical reactions after changes in irradiance in greenhouse mulberry plants. The induction of PSII photochemistry rapidly approached to steady state after leaves were treated from darkness to low irradiance (LI). When irradiance of leaves changed from darkness to high irradiance (HI), a biphasic induction was observed. A slight photoinhibition occurred in the leaves exposed to sunlight coming to the greenhouse, whereas a chronic photoinhibition occurred in the leaves fully exposed to sunlight outside the greenhouse. The chronic photoinhibition was demonstrated by sustained reduction of maximal quantum yield of PSII photochemistry (Fv/Fm). Moreover, the leaves of mulberry plants in greenhouse were sensitive to abrupt changes in irradiance and the sensitivity of leaves suffered in a short-term (1h) high light treatment was reduced, based on the changes in photosynthetic quantum conversion. These results demonstrated an inducible response of photosynthetic quantum conversion to changes in irradiance in mulberry.     

Use of a fluorescent membrane probe to identify zooxanthellae in hospite among dissociated endoderm cell culture from coral

Summary. Preparation of homogeneous endoderm cells and culture is a prerequisite to understanding the cellular and molecular mechanism of endosymbiosis in the cnidarian-dinoflagellate association. During the cell isolation from the stony coral Euphyllia glabrescens, various amounts of symbiotic endoderm cells were found to release their symbionts (Symbiodinium spp., or zooxanthellae in generic usage) into the culture. Due to the bulky occupation by zooxanthellae inside the endoderm cell, the symbiotic endoderm cells, or zooxanthellae in hospite, are difficult to be distinguished from released zooxanthellae by microscopic examination. We now report a method for this identification using a fluorescent analogue of sphingomyelin, N-[5-(5,7-dimethyl boron dipyrromethene difluoride)-1-pentanoyl]-D-erythro-sphingosylphosphorylcholine (C₅-DMB-SM). Incubation of symbiotic endoderm cells with C₅-DMB-SM–defatted bovine serum albumin (DF-BSA) complex results in bright fluorescent membrane staining. Nevertheless, the membrane staining of free-living or released zooxanthellae by this complex is significantly decreased or even diminished. This method has provided a fast and reliable assay to identify symbiotic endoderm cells and will greatly accelerate the progress of endosymbiosis research. Correspondence and reprints: National Museum of Marine Biology and Aquarium, 2 Houwan Road, Checheng, Pingtung, 944, Taiwan, R.O.C.     

Cytochemical and biochemical observations on the cell wall of the spore ofGlomus epigaeum

Summary The cell wall of the spore ofGlomus epigaeum Daniels and Trappe, which has fibrillar subunits regularly arranged in arcs, was studied ultrastructurally and biochemically.The periodic acid/thiocarbohydrazide/silver proteinate (PATAg) reaction for polysaccharide location (Thiéry 1967) and the silver methenamine reaction for protein location (Swift 1968) were performed on whole spores, progressively alkaline-extracted and autoclaved spores, and untreated and alkaline-extracted cell wall fractions. The cytochemical results and those obtained from frozen sections indicated that the fibrils forming the main structure of the outer and inner wall consist of chitin. Quantitative determinations showed that chitin is the most important component (47%) of the alkali-insoluble residue and represents 27.2% of the whole cell wall fraction. It occurs predominantly as the acetylated form. Cytochemical and biochemical observations showed that the matrix surrounding the fibrils is made of alkali-soluble, PATAg positive polysaccharides (4.98% of the whole cell wall fraction). Monomers were identified by gas liquid chromatography as being -lactone of glucuronic acid, and glucose, rhamnose and mannose. Alkali-soluble proteins are an important part of the matrix, being spread mostly throughout the inner wall and constituting a large portion (55.1 %) of the alkali-soluble fraction.From the results we derive a model in which the chemical components are interconnected to build up a macromolecular network, in agreement with electron-microscopic observations.     

Cell wall formation in zoospores of Allomyces arbuscula

Carbohydrate composition was determined in isolated cell walls of meiospores of Allomyces arbuscula after incubation for 15 min (encysted meiospores: cysts), 150 min (germlings: cysts + rhizoids) and 24 h (cysts + rhizoids + hyphae). The principal constituent in all cell wall samples is chitin, accounting for about 75% of the recovered carbohydrates. In addition, cell walls of all stages examined contain polysaccharides which release galactose, glucose, mannose, arabinose, xylose, fucose, and rhamnose on acid hydrolysis. While different developmental stages show minor quantitative changes in chitin, the ratio of galactose to glucose decreases sharply during differentiation of ungerminated cysts into germlings with rhizoids and hyphae. The increase in glucose is accompanied by a decrease in the amount of xylose and/or fucose and of galactose.List of Abbreviation TFA trifluoroacetic acid     

Autonomous changes in the orientation of cortical microtubules underlying the helicoidal cell wall of the sunflower hypocotyl epidermis: spatial variation translated into temporal changes

Summary. Angles (λ) at which parallel cortical microtubules (cMTs) were oriented with respect to the longitudinal direction were measured in Helianthus annuus hypocotyl epidermal cells. Histograms showing λ frequencies in cell populations at the instant of epidermis fixation were obtained. Analysis of the histograms indicates that, in a particular position within a cell, the angle λ changes periodically with time, i.e., there is a cycle of λ change at that position. This cycle is most likely rotational rather than oscillatory, i.e., the change in λ has a defined chirality (clockwise or counterclockwise). The full diversity of histograms can be consistently explained by rotational cycles with a variable velocity of λ change, and with a cMT rebuilding stage taking place at a different phase of the cycle. The rotational cycles also provide the simplest explanation of cMT arrays in which the angle λ changes along a cell (fixed) and no parallel orientation of cMTs is apparent at a certain position. This explanation assumes a gradient in the phase of the rotational cycle along the cell. The symmetry of the angular characteristics of the rotational cycle, with respect to the morphological directions in cells, leads to the concept that these directions typically represent the principal directions of a certain tensor quantity, which may control the cycling. Possible interactions between the rotational cycle of cMT reorientation and the helicoidal cycle during cell wall formation are discussed. Correspondence and reprints: Department of Biophysics and Cell Biology, Silesian University, ulica Jagiellonska 28, 40-032 Katowice, Poland.     

Dynamics of limiting cell wall porosity in plant suspension cultures

Changes in the limiting porosity of cell walls, i.e. the size limit for permeation of neutral molecules through the wall, were studied in several higher-plant cell-suspension cultures. For this purpose, samples of biomass fixed at different cultivation times were investigated using a method based on size-exclusion chromatography of polydisperse dextrans before and after equilibration with the extracted cell clusters. In suspension cultures of Chenopodium album L., Dioscorea deltoidea Wall. and Medicago sativa L., the mean size limit (MSL; critical Stokes' radius for exclusion of neutral polymers from half of the intracellular space) was found to vary between 2.4 and 3.8 nm. It decreased significantly during transition from the growth phase to the stationary phase. In the case of the C. album culture this change was found to be irrespective of whether sucrose in the medium was completely depleted at the end of the growth phase or not. The MSL was kept constant for long periods of the stationary phase if cell viability was maintained by repeated sucrose supplement. In a suspension strain of Triticum aestivum L., the MSL of cell wall permeation was comparatively small (1.75 nm) and remained constant during all cultivation phases. Relations between limiting porosity and cell wall growth, loss of pectic compounds to the medium, cross-linking activities and cell wall stiffening are discussed. Received: 19 December 1996 / Accepted: 23 April 1997     

Preinfection cell wall formation in roots and developing nodules ofAlnus rubra Bong

Summary The establishment of actinorhizal root nodules involves penetration of host cell walls and intracellular colonization by the nitrogen-fixing endosymbiont,Frankia (Actinomycetales). In the early stages of the infection process inAlnus, unusual cell walls with undulate profiles were observed in root tip meristematic derivatives, and in early (preinfection) derivatives of the nodule lobe meristem, inFrankia-inoculated plants. The irregular cell walls attached obliquely to preexisting walls, but were not discontinuous. Serial sections revealed that the unusual walls divided two daughter cells. Microtubules in bundled arrays were abundant near the undulate walls, and radiated in several planes. In the root tips, the anomalous cell walls were observed within one day of inoculation withFrankia.     

Changes in the rate of synthesis of wall polysaccharides during the cell cycle of yeast

Reevaluation and comparison of seemingly contradictory literature data on the mode of synthesis of wall polysaccharides during the cell cycle ofSaccharomyces cerevisiae explained the source of discrepancies and demonstrated their general consonance in the following points: 1. The rate of synthesis of glucan and mannan is not constant and does not increase continuously throughout the entire cell cycle. 2. The rate of synthesis of both polysaccharides is considerably reduced at the time of cell division and in the prebudding phase.     

Changes in composition of the outer epidermal cell wall of pea stems during auxin-induced growth

The gross composition of the outer epidermal cell wall from third internodes of Pisum sativum L. cv. Alaska grown in dim red light, and the effect of auxin on that composition, was investigated using interference microscopy. Pea outer epidermal walls contain as much cellulose as typical secondary walls, but the proportion of pectin to hemicellulose resembles that found in primary walls. The pectin and hemicellulose fractions from epidermal peels, which are enriched for outer epidermal wall but contain internal tissue as well, are composed of a much higher percentage of glucose and glucose-related sugars than has been found previously for pea primary walls, similar to non-cellulosic carbohydrate fractions of secondary walls. The epidermal outer wall thus has a composition rather like that of secondary walls, while still being capable of elongation. Auxin induces a massive breakdown of hemicellulose in the outer epidermal wall; nearly half the hemicellulose present is lost during 4 h of growth in the absence of exogenous sugar. The percentage breakdown is much greater than has been seen previously for whole pea stems. It has been proposed that a breakdown of xyloglucan could be the basis for the mechanical loosening of the outer wall. This study provides the first evidence that such a breakdown could be occurring in the outer wall.M.S. Bret-Harte would like to thank Dr. Peter M. Ray, of Stanford University, for helpful discussions and for technical and editorial assistance, Dr. Winslow R. Briggs, of the Camegie Institude of Washington, for the use of experimental facilities and for helpful discussions, Dr. Wendy K. Silk, of the University of California, Davis, for helpful discussions and financial support, Dr. Paul B. Green for financial support, and Drs. John M. Labavitch and L.C. Greve, of the University of California, Davis, for performing the -cellulose analysis on short notice, in response to a request by an anonymous reviewer. This work was supported by a National Science Foundation Graduate Fellowship to M.S. B.-H., National Science Foundation Grant DCB8801493 to Paul B. Green, and the generosity of Wendy K. Silk (Department of Land, Air, and Water Resources, University of California, Davis) during the final writing.     

The change of pattern in microfibril arrangement on the inner surface of the cell wall of Closterium acerosum during cell growth

Closterium acerosum (Schrank) Ehrenberg cells cultured on cycles of 16 h light and 8 h dark, undergo cell division synchronously in the dark period. After cell division, the symmetry of the daughter semicells is restored by controlled expansion, the time required for this restoration, 3.5–4 h, being relatively constant. The restoration of the symmetry is achieved by highly oriented surface expansion occurring along the entire length of the new semicell. During early semicell expansion, for about 2.5 h, microfibrils are deposited parallel to one another and transversely to the cell axis on the inner surface of the new wall. Wall microtubules running parallel to the transversely oriented microfibrils are observed during this period. About 2.5 h after septum formation, preceding the cessation of cell elongation, bundles of 7–11 microfibrils running in various directions begin to overlay the parallel-arranged microfibrils already deposited. In the fully elongated cells, no wall microtubules are observed.     

High-speed pollen release in the white mulberry tree, Morus alba L

Anemophilous plants described as catapulting pollen explosively into the air have rarely attracted detailed examination. We investigated floral anthesis in a male mulberry tree with high-speed video and a force probe. The stamen was inflexed within the floral bud. Exposure to dry air initially resulted in a gradual movement of the stamen. This caused fine threads to tear at the stomium, ensuring dehiscence of the anther, and subsequently enabled the anther to slip off a restraining pistillode. The sudden release of stored elastic energy in the spring-like filament drove the stamen to straighten in less than 25 μs, and reflex the petals to velocities in excess of half the speed of sound. This is the fastest motion yet observed in biology, and approaches the theoretical physical limits for movements in plants. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. An erratum to this aticle is available at .     

Preparative laser capture microdissection and single-pot cell wall material preparation: a novel method for tissue-specific analysis

In adaptation to their function the walls of plant cell display tissue-specific variations of composition according to their developmental stage, cell type and stress of various origin. It is therefore important to obtain a precise analytical data describing the cell wall composition with respect to these different factors. In the present work, laser capture microdissection (LCM) was used for isolating different tissues from the stem of Urtica dioica L. at a semi-preparative scale. The technique was associated for the first time to a one-pot sequential cell wall preparation and hydrolysis for the carbohydrate analysis of each cell type. The results demonstrate that the combination of LCM and micro-analytical methods can provide individual cell type composition and should improve our knowledge of the biochemical diversity of cell walls in plants. This approach will be of potential interest for the understanding of the effects of stress or genetic engineering on the composition of the cell walls.     

Distribution of antigenic oligomannosyl side chains in the cell wall mannans of several strains of<Emphasis Type="Italic"> Candida tropicalis</Emphasis>

In order to clarify the distribution of antigenic oligomannosyl side chains in the cell wall mannans of the pathogenic yeast Candida tropicalis, the chemical structure of mannans isolated from four C. tropicalis strains was investigated using nuclear magnetic resonance, two-dimensional homonuclear Hartmann-Hahn (2D-HOHAHA) spectroscopy. Two-dimensional maps of the 2D-HOHAHA clearly showed the distribution of oligomannosyl side chains in the mannans. The linear side chain Manalpha1-3Manalpha1-(2Manalpha1-)(n)2Man [n> or =2] is present in the mannans from C. tropicalis IFO 0589 and IFO 1400, but not in the mannans from IFO 0199 and IFO 1647. The mannan of IFO 0589 is the only mannan with the branched side chains, Manalpha1-3[Manalpha1-6]Manalpha1-(2Manalpha1-)(n)2Man and Manalpha1-2Manalpha1-3[Manalpha1-6]Manalpha1-(2Manalpha1-)(n)2Man [n> or =2]. However, this mannan lacked the phosphate group and the beta-1,2-linked oligomannosyl side chain which are features of this group. The mannans of the C. tropicalis strains IFO 0589 and IFO 1400 possessed the side chains containing an alpha-1,3-linked mannose residue previously observed in Candida albicans.     

Composition of the cell wall of Chlorella fusca

Isolated cell walls of mature Chlorella fusca consisted of about 80% carbohydrate, 7% protein, and 13% unidentified material. Mannose and glucose were present in a ratio of about 2.7:1 and accounted for most of the carbohydrate. Minor components were glucuronic acid, rhamnose, and traces of other sugars; galactose was absent. After treatment with 2 M trifluoroacetic acid or with 80% acetic acid/HNO₃ (10/1, v/v), a residue with a mannose/glucose ratio of 0.3:1 was obtained, probably representing a structural polysaccharide. An X-ray diffraction diagram of the walls showed one diffuse reflection at 0.44 nm and no reflections characteristic of cellulose. Walls from young cells contained about 51% carbohydrate, 12% protein, and 37% unidentified material. Mannose and glucose were also the main sugars; their absolute amounts per wall increased 6–7 fold during cell growth. Walls isolated with omission of a dodecylsulphate/mercaptoethanol/urea extraction step had a higher protein content and, with young walls, a significantly higher glucose and fucose content. These data and other published cell wall analyses show a wide variability in cell wall composition of the members of the genus Chlorella.Abbreviations GLC gas liquid chromatography - TFA trifluoroacetic acid     

A novel type of teichoic acid from the cell wall of Bacillus subtilis VKM B-762

The cell wall of Bacillus subtilis VKM B-762 contains, along with 1,5-poly[4-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)ribitol phosphate], a novel type of glycopolymer involving three types of inter-monomeric bonds in the repeating unit, viz., amide, glycosidic and phosphodiester:Such a structural pattern of natural glycopolymers has been hitherto unknown. This polymer represents a novel type of teichoic acids.     

A dynamical model for plant cell wall architecture formation

We discuss a dynamical mathematical model to explain cell wall architecture in plant cells. The highly regular textures observed in cell walls reflect the spatial organisation of the cellulose microfibrils (CMFs), the most important structural component of cell walls. Based on a geometrical theory proposed earlier [A. M. C. Emons, Plant, Cell and Environment 17, 3–14 (1994)], the present model describes the space-time evolution of the density of the so-called rosettes, the CMF synthesizing complexes. The motion of these rosettes in the plasma membrane is assumed to be governed by an optimal packing constraint on the CMFs plus adherent matrix material, that couples the direction of motion, and hence the orientation of the CMF being deposited, to the local density of rosettes. The rosettes are created inside the cell in the endoplasmatic reticulum and reach the cell-membrane via vesicles derived from Golgi-bodies. After being inserted into the plasma membrane they are assumed to be operative for a fixed, finite lifetime. The plasma membrane domains within which rosettes are activated are themselves also supposed to be mobile. We propose a feedback mechanism that precludes the density of rosettes to rise beyond a maximum dictated by the geometry of the cell. The above ingredients lead to a quasi-linear first order PDE for the rosette-density. Using the method of characteristics this equation can be cast into a set of first order ODEs, one of which is retarded. We discuss the analytic solutions of the model that give rise to helicoidal, crossed polylamellate, helical, axial and random textures, since all cell walls are composed of (or combinations of) these textures. Received: 10 July 1999 / Revised version: 7 June 2000 / Published online: 16 February 2001