The involvement of hydrogen peroxide in the differentiation of secondary walls in cotton fibers

H₂O₂ is a widespread molecule in many biological systems. It is created enzymatically in living cells during various oxidation reactions and by leakage of electrons from the electron transport chains. Depending on the concentration H₂O₂ can induce cell protective responses, programmed cell death, or necrosis. Here we provide evidence that H₂O₂ may function as a developmental signal in the differentiation of secondary walls in cotton (Gossypium hirsutum) fibers. Three lines of evidence support this conclusion: (a) the period of H₂O₂ generation coincided with the onset of secondary wall deposition, (b) inhibition of H₂O₂ production or scavenging the available H₂O₂ from the system prevented the wall differentiation process, and (c) exogenous addition of H₂O₂ prematurely promoted secondary wall formation in young fibers. Furthermore, we provide support for the concept that H₂O₂ generation could be mediated by the expression of the small GTPase Rac, the accumulation of which was shown previously to be strongly induced during the onset of secondary wall differentiation. In support of Rac's role in the activation of NADPH oxidase and the generation of reactive oxygen species, we transformed soybean (Glycine max) and Arabidopsis cells with mutated Rac genes. Transformation with a dominantly activated cotton Rac13 gene resulted in constitutively higher levels of H₂O₂, whereas transformation with the antisense and especially with dominant-negative Rac constructs decreased the levels of H₂O₂.     

Chemical force microscopy of cellulosic fibers

Atomic force microscopy with chemically modified cantilever tips (chemical force microscopy) was used to study the pull-off forces (adhesion forces) on cellulose model surfaces and bleached softwood kraft pulp fibers in aqueous media. It was found that for the –COOH terminated tips, the adhesion forces are dependent on pH, whereas for the –CH₃ and –OH terminated tips adhesion is not strongly affected by pH. Comparison between the cellulose model surfaces and cellulosic fibers under our experimental conditions reveal that surface roughness does not affect adhesion strongly. X-ray photoelectron spectroscopy (XPS) and Fourier Transformed Infrared (FTIR) spectroscopy reveal that both substrate surfaces have homogeneous chemical composition. The results show that chemical force microscopy can be used for the chemical characterization of cellulose surfaces at a nano-level.     

Identification and partial characterization of proteins and proteoglycans encrusting the secondary cell walls of flax fibres

 Four proteins were isolated from depectinised elementary fibres of flax (Linum usitatissimum L.), using either alkali or cellulase digestion treatments. All the four proteins were characterized by a deficiency or low contents of hydroxyproline and by high levels of glutamic acid/glutamine and/or aspartic acid/asparagine. The two proteoglycans solubilized with cellulase strongly reacted with β-glucosyl Yariv reagent but not with α-glucosyl Yariv reagent and contained appreciable amounts of alanine, glycine, serine and threonine, suggesting a relationship with cell wall hydroxyproline-deficient arabinogalactan-proteins. The two alkali-extracted proteins did not show any reaction with β-glucosyl Yariv dye. Due to the harsh treatment, they might only partially represent the original proteins. Due to its high level of glycine (41%), one of these proteins might be classified as a glycine-rich protein. The latter polypeptide, of low molecular molar mass, contained 14.6% leucine and might consist of a domain related to leucine-rich proteins. The data show that these proteins and arabinogalactan-protein-like proteoglycans were strongly associated with the secondary walls of flax fibres. Their presence in small amounts (0.1–0.4%), raises the problem of their putative structural role. Received: 22 October 1999 / Accepted: 17 January 2000     

Homofusion of Golgi secretory vesicles in flax phloem fibers during formation of the gelatinous secondary cell wall

The gelatinous type of secondary cell wall is present in tension wood and in phloem fibers of many plants. It is characterized by the absence of xylan and lignin, a high cellulose content and axially orientated microfibrils in the huge S2 layer. In flax phloem fiber, the major non-cellulosic component of such cell walls is tissue-specific galactan, which is tightly bound to cellulose. Ultrastructural analysis of flax fiber revealed that initiation of gelatinous secondary cell wall formation was accompanied by the accumulation of specific Golgi vesicles, which had a characteristic bicolor (dark-light) appearance and were easily distinguishable from vesicles made in different tissues and during the other stages of fiber development. Many of the bicolor vesicles appeared to fuse with each other, forming large vacuoles. The largest observed was 4 mum in diameter. Bicolor vesicles and vacuoles fused with the plasma membrane and spread their content in a characteristic "syringe-like" manner, covering a significant area of periplasm and forming "dark" stripes on the inner wall surface. Both Golgi derivatives and cell wall layers were labeled by LM5 antibody, indicating the presence of tissue- and stage-specific (1-->4)-beta-galactan. We suggest that this specific type of galactan secretion, which allows coverage of a large area of periplasm, is designed to increase the chance of the galactan meeting the cellulose microfibrils while they are still in the process of construction. The membrane fusion machinery of flax fiber must possess special components, which may be crucial for the formation of the gelatinous type cell wall.     

Immunocytochemical characterization of early-developing flax fiber cell walls

Summary The deposition and formation of a thick secondary wall is a major event in the differentiation of flax (Linum usitatissimum) fibers. This wall is cellulose-rich; but it also contains significant amounts of other matrix polymers which are noncellulosic such as pectins. We have used immunocytochemical techniques with antibodies specific for various epitopes associated with either pectins or arabinogalactan proteins (AGPs) to investigate the distribution of these polymers within the walls of differentiating young fibers of 1- and 2-week-old plants. Our results show that different epitopes exhibit distinct distribution patterns within fiber walls. Unesterified pectins recognized by polygalacturonic acid-rhamnogalacturonan I (PGA/RG-I) antibodies and rhamnogalacturonan II recognized by anti-RG-II-borate complex antibodies are localized all over the secondary wall of fibers. PGA/RG-I epitopes, but not RG-II epitopes, are also present in the middle lamellae and cell junctions. In marked contrast, -(14) galactans recognized by the LM5 monoclonal antibody and AGP epitopes recognized by anti--(16) galactan and LM2 antibodies are primarily located in the half of the secondary wall nearest the plasma membrane. LM2 epitopes, present in 1-week-old fibers, are undetectable later in development, suggesting a regulation of the expression of certain AGP epitopes. In addition, localization of cellulose with the cellobiohydrolase I-gold probe reveals distinct subdomains within the secondary walls of young fibers. These findings indicate that, in addition to cellulose, early-developing flax fibers synthesize and secrete different pectin and AGP molecules.     

Cell wall proteins of flax phloem fibers

A cell wall has been isolated from single-type cells, phloem fibers of flax (Linum usitatissimum L.) being at the stage of the active formation of a thick secondary cell wall. Weakly bound proteins of the cell wall of phloem fibers were extracted and separated, and their mass spectra were recorded. The identification of the proteins and their assignment to a particular cell compartment were performed using a variety of bioinformatics methods. In all, 93 proteins were identified of which many proteins were defined as predicted, putative, and hypothetical. Twenty one proteins were identified as cell-wall proteins. The absence of the marker proteins of primary cell walls such as xyloglucan endotransglycosylase and expansins indirectly confirms the predominance of the secondary cell wall in a sample for protein extraction.     

Enzyme-retting of flax and characterization of processed fibers

Enzyme-retting formulations consisting of Viscozyme L, a pectinase-rich commercial enzyme product, and ethylenediaminetetraacetic acid (EDTA) were tested on Ariane fiber flax and North Dakota seed flax straw residue. Flax stems that were crimped to disrupt the outer layers were soaked with various proportions of Viscozyme-EDTA solutions, retted, and then cleaned and cottonized with commercial processing equipment. Fiber properties were determined and crude test yarns were made of raw and Shirley cleaned flax fibers and cotton in various blend levels. Cleaned fibers were obtained from both seed and fiber flax types, but with variations due to treatment. Retting formulations produced fibers having different properties, with enzyme levels of 0.3% (v/v as supplied) giving finer but weaker fibers than 0.05% regardless of EDTA level. Experimental yarns of blended flax and cotton fibers varied in mass coefficient of variation, single end strength, and nep imperfections due to sample and formulation. With cost and fiber and yarn quality as criteria, results established a range in the amounts of components comprising retting formulations as a basis for further studies to optimize enzyme-retting formulations for flax. Under conditions examined herein, Viscozyme L at 0.3% (v/v) plus 25 mM EDTA produced the best test yarns and, therefore, established a base for future studies to develop commercial-grade, short staple flax fibers for use in textiles.     

Taxonomic significance of cellulosic cell walls in the bangiales (Rhodophyta)

Cellulose has been characterized from isolated cell walls of the conchocelis phases of both Porphyra umbilicalis and P. leucostricta. Evidence for cellulose II (regenerated cellulose) in Schweitzer's reagent extracts was provided by X-ray powder analysis and paper chromatography of partial hydrolyzates. The presence of cellulose in the conchocelis phase of species of Porphyra provides evidence for the continuity of cell wall composition within the Rhodophyta. Adoption of a classification scheme incorporating consolidation of all red algal orders under the single class Rhodophyceae is proposed.     

Building flax fibres: more than one brick in the walls

The objective of the review is to provide fundamental knowledge on the chemical composition and structural characteristics of flax fibres. These are long and multinucleate cells without septum or partition (average length 2–5 cm) and have a secondary wall of very large thickness (5–15 μm). Fibres are gathered in bundles of one to three dozen cells that encircle the vascular cylinder. The bundle cohesion is insured by pectins, accumulating in the primary wall and cell junctions. In contrast, lignin, which is present in very low amount, does not seem to play a major role in bundle cohesion. At maturity, secondary wall is characterised by (i) a high level of cellulose with microfibrils locked into an almost axial direction and (ii) 5–15% non-cellulosic polysaccharides (NCPs). The chemical composition of NCPs depends on growth stage, indicating important cell wall remodelling, fibre position and variety. Despite the large disparity of the results reported in the literature, galactose appears to be the predominant sugar of NCPs, and β-1-4-galactan together with rhamnogalacturonan of type I (RG-I) and polygalacturonic acid (PGA) become, with fibre maturity, the most abundant tightly bound NCPs. Glycine-rich proteins (GRPs) and arabinogalactan-proteins (AGPs), also present in flax fibres, are both characterised by appreciable levels of glycine and acidic amino acid and are deficient in hydroxyproline, and may contribute to the cross-linking of pectins. (Galacto)glucomanans/glucans rather than xylans consist of cross-linking polymers in fibre secondary wall. A model is proposed where cellulose microfibrils, tethered by cross-linking (galacto)glucomanans/glucans, are embedded in a pectic matrix.     

Variability in the composition of tissue-specific galactan from flax fibers

Tissue-specific galactan of sclerenchyma fibers, with cell walls of the gelatinous type, was examined in flax plants (Linum usitatissimum L.) of 23 various genotypes. The content and average degree of polymerization of side chains of galactan were estimated before its deposition into the cell wall. The variability of the analyzed parameters of tissue-specific galactan from flax fibers was high; within the same genotype, the scope of paratypic variability between replicates and years of research was comparable to variability between different genotypes. The average length of side chains in the studied samples ranged from 5 to 41 galactose residues. The average degrees of polymerization of galactan side chains in flax fibers was found to be discrete, which could be explained by block assemblage of the polymer in the Golgi apparatus.     

Fibrillation tendency of cellulosic fibers—Part 4. Effects of alkali pretreatment of various cellulosic fibers

The effects of alkali type and the concentration in the alkali treatments on the weight loss in six cellulosic fibers and their influences on the fibrillation tendency were investigated. The fibril number of the cellulosic fibers pretreated with alkalis (FN_pre) increased with increasing the alkali concentrations as well as the weight loss of the fiber except in the lyocell fiber treated in NaOH and KOH solutions. The FN_pre in lyocell was reduced as the fibers were treated in 5 mol/l NaOH and KOH solutions. This result and the fact that the fibers were split in organic alkali such as tetramethylammonium hydroxide even at the low weight loss suggested that not only the loss of cellulose component but also reorganization of microfibril structure, inhomogeneous swelling of the fibers and other influences control the fibrillation tendency of cellulosic fibers.     

Characterization of cellulosic fibers and fabrics by sorption/desorption

Three cellulosic substrates: lyocell (CLY), viscose (CV), and modal (CMD) in the form of fibers and fabrics were subjected to wet/dry or wash/dry treatments. The accessibility of untreated and treated substrates to water and iodine was investigated using dynamic water-vapor sorption, moisture retention, and iodine sorption methods, to study the influence of treatments on sorption-desorption hysteresis, fraction of moisture sorbed as a monomolecular layer, water retention, and iodine sorption. It was found that the sorption properties of untreated and treated substrates differed with sorbate type as well as substrate type and form.     

Intrusive growth of flax phloem fibers is of intercalary type

Flax (Linum usitatissimum L.) phloem fibers elongate considerably during their development and intrude between existing cells. We questioned whether fiber elongation is caused by cell tip growth or intercalary growth. Cells with tip growth are characterized by having two specific zones of cytoplasm in the cell tip, one with vesicles and no large organelles at the very tip and one with various organelles amongst others longitudinally arranged cortical microtubules in the subapex. Such zones were not observed in elongating flax fibers. Instead, organelles moved into the very tip region, and cortical microtubules showed transversal and helical configurations as known for cells growing in intercalary way. In addition, pulse-chase experiments with Calcofluor White resulted in a spotted fluorescence in the cell wall all over the length of the fiber. Therefore, it is concluded that fiber elongation is not achieved by tip growth but by intercalary growth. The intrusively growing fiber is a coenocytic cell that has no plasmodesmata, making the fibers a symplastically isolated domain within the stem. Electronic Supplementary Material Supplementary material is available for this article at     

Collagen and elastin fibers in human pulmonary alveolar walls

The morphology and morphometric data of collagen and elastin fibers in the pulmonary alveolar walls are presented. Specimens were obtained from postmortem lungs quick-frozen at specified transpulmonary pressures. Collagen was stained by silver, and elastin was stained by orcein. Photomicrographs were composed by computer. Young lungs typically show small collagen fibers that radiate from the "posts," whereas larger fiber bundles traverse the septum irrespective of capillary blood vessels. In older lungs, rings of collagen around the posts appear enlarged. Elastin bundles do not show obvious variation in pattern with age and inflation pressure. Statistical frequency distributions of the fiber width and curvature are both skewed, but the square root of the width and the cube root of the curvature have approximate normal distributions. Typically, for young lungs at transpulmonary pressure of 4 cmH2O, the mean of (width)1/2 (in micron1/2) for collagen fibers is 0.952 +/- 0.242 (SD), that of (curvature)1/3 (in micron-1/3) is 0.349 +/- 0.094. The corresponding values for elastin are 0.986 +/- 0.255 and 0.395 +/- 0.094.     

Escherichia coli growth on lactose requires cycling of beta-galactosidase products into the medium

Growth on lactose was found to be restricted in an Escherichia coli strain deficient in its ability to transport glucose and galactose. If the latter sugars were removed from the medium as they were being produced, a wild-type strain grew only poorly, while the transport-deficient strain did not grow at all. These results suggested that all of the products of beta-galactosidase action on lactose are released into the medium before being metabolized. This contention was strongly supported by the finding that the appearance of products in the medium was equal to lactose disappearance at three limiting lactose concentrations and by an experiment which showed that essentially all of the label from added lactose ( [1-14C]glucose) was found in the medium as glucose when chased with unlabelled lactose.     

Engineering of PHB synthesis causes improved elastic properties of flax fibers

Flax stem is a source of fiber used by the textile industry. Flax fibers are separated from other parts of stems in the process called retting and are probably the first plant fibers used by man for textile purposes (1). Nowadays flax cultivation is often limited because of its lower elastic property compared to cotton fibers. Thus the goal of this study was to increase the flax fiber quality using a transgenic approach. Expression of three bacterial genes coding for beta-ketothiolase (phb A), acetoacetyl-CoA reductase (phb B), and PHB synthase (phb C) resulted in poly-beta-hydroxybutyrate (PHB) accumulation in the plant stem. PHB is known as a biodegradable thermoplastic displaying chemical and physical properties similar to those of conventional plastics (i.e., polypropylene). The fibers isolated from transgenic flax plants cultivated in the field and synthesizing PHB were then studied for biomechanical properties. All measured parameters, strength, Young's modulus, and energy for failure of flax fibers, were significantly increased. Thus the substantial improvement in elastic properties of fibers from the transgenic line has been achieved. Since the acetyl CoA, substrate for PHB synthesis, is involved not only for energy production but also for synthesis of many cellular constituents, the goal of this study was also the analysis of those metabolites, which interfere with plant physiology and thus fiber quality. The analyzed plants showed that reduction in lignin, pectin, and hemicellulose levels resulted in increased retting efficiency. A significant increase in phenolic acids was also detected, and this was the reason for improved plant resistance to pathogen infection. However, a slight decrease in crop production was detected.     

Heterogeneous acylation of flax fibers. Reaction kinetics and surface properties

Flax fibers composed mainly of cellulose were subjected to heterogeneous valerylation reaction. The progress of the chemical modification was assessed by transmission FTIR. The heterogeneous esterification reaction followed first-order kinetics, and a plateau was reached already after 30 min. The intensity of the FTIR hydroxyl absorption band (nu = 3400 cm(-1)) did not appreciably decrease during the acylation reaction, showing that only a small fraction of the fiber hydroxyls was involved in the reaction. The degree of valerate substitution (DS) at the fiber surface (50 A thick layer) was evaluated by means of ESCA. Surface valerylation increased with reaction time and leveled off at DS around 1 after 30 min, in agreement with the FTIR data. The chemically modified fibers maintain the Cellulose I crystal structure and the original crystallinity degree up to the longest reaction time investigated (180 min). Dynamic contact angle measurements showed that surface hydrophobicity as indicated by advancing contact angle rapidly increased upon valerylation reaching a plateau after about 10 min. Chemical modification does not appreciably alter fiber thermal stability (by TGA) and morphology (by SEM).     

Modification and characterization of cellulosic cotton fibers for efficient immobilization of urease

Cotton fibers were first grafted by polyacrylonitril in the presence of KMnO(4) and oxalic acid as a combined redox initiator. Moreover, modification of the grafted cotton fibers was done by changing the nitrile group (-CN) into hydrazidine group through the reaction with hydrazine hydrate, then the fibers were activated by glutaraldehyde to introduce free aldehyde groups which were able to react with amino groups of urease to form Schiff's base, and result in cotton fibers immobilized urease. The efficiency of the immobilization was evaluated by examining the relative enzymatic activity of enzyme before and after the immobilization of urease. The results showed that the optimum temperature of immobilized urease was 35°C, which was higher than that of the free enzyme (30°C), and the immobilized urease exhibited a higher relative activity than that of free urease over 35°C. The optimal pH for immobilized urease was 6.5, which was lower than that of the free urease (pH 7.0), and the immobilization resulted in stabilization of enzyme over a wider pH range. The kinetic constant value (K(m)) of immobilized urease was higher than that of the free urease. However, the thermal and operational stabilities of immobilized urease have been improved greatly.     

Processes of protoplast senescence and death in flax fibers: An ultrastructural analysis

Plant fibers represent specialized cells that perform a mechanical function. Their development includes the following phases, typical for the most plant cells: determination, extension growth, specialization, senescence, and death. Ultrastructural analysis of these cells has been carried out at the late phases of their development (senescence and dying off) using flax phloem fibers, a classical object for the analysis of sclerenchyma fiber formation. The results of the performed analysis show that flax fiber protoplasts remain viable until the end of a vegetation season. The ultrastructural analysis of flax phloem fibers has not revealed any typical apoptosis features. Gradual degradation of the cytoplasm starts during the active thickening of a secondary cell wall and occurs via the intensification of autolytic processes, causing a partial loss of cell content. The rupture of tonoplast is the final stage. The obtained data allow us to suppose that the protoplast dying off occurs during process of the senescence, and its program is similar to the cell death program realized in the xylem fibers of woody plants.