Pectinase treatments on technical fibres of flax: Effects on water sorption and mechanical properties

This study is focused on enzymatically upgrading the functional properties of flax fibres. Green flax fibres were treated with a polygalacturonase and a pectate lyase (PaL) and their properties were compared with dew-retted fibres. Morphological observations, vapour-sorption analyses and mechanical measurements showed that PaL-treatment was able not only to mime retting in terms of bundle division, but also to improve the mechanical properties of technical fibres. Conversely, these properties were shifted down after the polygalacturonase treatment, mainly due to the presence of contaminating glycanases. At the level of the elementary fibres, nanoindentation data indicated the highest stiffness of the secondary wall for PaL-treated fibres. The tensile properties exhibited equal, but moderate values of the Young's modulus (∼37 ± 14 GPa) and breaking strength (∼650 ± 300 MPa) for retted and PaL-treated fibres; we hypothesize an impact of the growth conditions on the fibre chemical structure with an excess of matrix pectins compared to the amount of glucomannan coating the cellulose microfibrils.     

Secondary cell-wall assembly in flax phloem fibres: role of galactans

Non-lignified fibre cells (named gelatinous fibres) are present in tension wood and the stems of fibre crops (such as flax and hemp). These cells develop a very thick S2 layer within the secondary cell wall, which is characterised by (1) cellulose microfibrils largely parallel to the longitudinal axis of the cell, and (2) a high proportion of galactose-containing polymers among the non-cellulosic polysaccharides. In this review, we focus on the role of these polymers in the assembly of gelatinous fibres of flax. At the different stages of fibre development, we analyse in detail data based on sugar composition, linkages of pectic polymers, and immunolocalisation of the β-(1→4)-galactans. These data indicate that high molecular-mass gelatinous galactans accumulate in specialised Golgi-derived vesicles during fibre cell-wall thickening. They consist of RG-I-like polymers with side chains of β-(1→4)-linked galactose. Most of them are short, but there are also long chains containing up to 28 galactosyl residues. At fibre maturity, two types of cross-linked galactans are identified, a C–L structure that resembles the part of soluble galactan with long side chains and a C–S structure with short chains. Different possibilities for soluble galactan to give rise to C–L and C–S are analysed. In addition, we discuss the prospect for the soluble galactan in preventing the newly formed cellulose chains from completing immediate crystallisation. This leads to a hypothesis that firstly the secretion of soluble galactans plays a role in the axial orientation of cellulose microfibrils, and secondly the remodelling and cross-linking of pectic galactans are linked to the dehydration and the assembly of S2 layer.     

Cytochemical localization and function of the 3-linked glucan callose in the developing cotton fibre cell wall

Summary Several recent biochemical studies concerning the hemicellulosic content of the developing cotton fibre wall have pointed to an important increase of 1,3-linked glucans at the onset of the secondary wall formation and their slow decrease until the end of fibre development (Meinert andDelmer 1977,Huwyler et al. 1978, 1979,Maltby et al. 1979). These almost insoluble glucans are extra-cellular and possibly associated with the S₁ or winding layer, but no other data on their exact localization were given.By means of a specific fluorescence method, using a 0.05% decolourized aniline blue solution, we show that one of these 3-linked glucans,callose, is always localized, independently of the fibre age, in the innermost wall layer bordering the cell lumen, from the onset of the secondary thickening up to the end of fibre development. Some possible roles assumed by these callose deposits are suggested and discussed. They may be involved in the normal mechanism of cellulose biosynthesis, as being effectively consumed by turnover or, more probably, as forming a permanently restored interface or matrix where cellulose microfibrils undergo a sort of maturation and are oriented before their definitive incorporation in the organized cell wall. They are not to be confused with the wound callose deposits which characterize damaged or immature fibres.     

Orientation of microfibrils and microtubules in developing tension-wood fibres of Japanese ash (Fraxinus mandshurica var. japonica)

The orientation of cellulose microfibrils (MFs) and the arrangement of cortical microtubules (MTs) in the developing tension-wood fibres of Japanese ash (Fraxinus mandshurica Rupr. var. japonica Maxim.) trees were investigated by electron and immunofluorescence microscopy. The MFs were deposited at an angle of about 45° to the longitudinal axis of the fibre in an S-helical orientation at the initiation of secondary wall thickening. The MFs changed their orientation progressively, with clockwise rotation (viewed from the lumen side), from the S-helix until they were oriented approximately parallel to the fibre axis. This configuration can be considered as a semihelicoidal pattern. With arresting of rotation, a thick gelatinous (G-) layer was developed as a result of the repeated deposition of parallel MFs with a consistent texture. Two types of gelatinous fibre were identified on the basis of the orientation of MFs at the later stage of G-layer deposition. Microfibrils of type 1 were oriented parallel to the fibre axis; MFs of type 2 were laid down with counterclockwise rotation. The counterclockwise rotation of MFs was associated with a variation in the angle of MFs with respect to the fibre axis that ranged from 5° to 25° with a Z-helical orientation among the fibres. The MFs showed a high degree of parallelism at all stages of deposition during G-layer formation. No MFs with an S-helical orientation were observed in the G-layer. Based on these results, a model for the orientation and deposition of MFs in the secondary wall of tension-wood fibres with an S₁ + G type of wall organization is proposed. The MT arrays changed progressively, with clockwise rotation (viewed from the lumen side), from an angle of about 35–40° in a Z-helical orientation to an angle of approximately 0° (parallel) to the fibre axis during G-layer formation. The parallelism between MTs and MFs was evident. The density of MTs in the developing tension-wood fibres during formation of the G-layer was about 17–18 per m of wall. It appears that MTs with a high density play a significant role in regulating the orientation of nascent MFs in the secondary walls of wood fibres. It also appears that the high degree of parallelism among MFs is closely related to the parallelism of MTs that are present at a high density.Abbreviations FE-SEM field emission scanning electron microscopy - G gelatinous layer - MF cellulose microfibril - MT cortical microtubule - S₁ outermost layer of the secondary wall - TEM transmission electron microscopy We thank Dr. Y. Akibayashi, Mr. Y. Sano and Mr. T. Itoh of the Faculty of Agriculture, Hokkaido University, for their experimental or technical assistance.     

Improving retting of fibre through genetic modification of flax to express pectinases

Flax (Linum usitatissimum L.) is a raw material used for important industrial products. Linen has very high quality textile properties, such as its strength, water absorption, comfort and feel. However, it occupies less than 1% of the total textile market. The major reason for this is the long and difficult retting process by which linen fibres are obtained. In retting, bast fibre bundles are separated from the core, the epidermis and the cuticle. This is accomplished by the cleavage of pectins and hemicellulose in the flax cell wall, a process mainly carried out by plant pathogens like filamentous fungi. The remaining bast fibres are mainly composed of cellulose and lignin. The aim of this study was to generate plants that could be retted more efficiently. To accomplish this, we employed the novel approach of transgenic flax plant generation with increased polygalacturonase (PGI ) and rhamnogalacturonase (RHA) activities. The constitutive expression of Aspergillus aculeatus genes resulted in a significant reduction in the pectin content in tissue-cultured and field-grown plants. This pectin content reduction was accompanied by a significantly higher (more than 2-fold) retting efficiency of the transgenic plant fibres as measured by a modified Fried’s test. No alteration in the lignin or cellulose content was observed in the transgenic plants relative to the control. This indicates that the over-expression of the two enzymes does not affect flax fibre composition. The growth rate and soluble sugar and starch contents were in the range of the control levels. It is interesting to note that the RHA and PGI plants showed higher resistance to Fusarium culmorum and F. oxysporum attack, which correlates with the increased phenolic acid level. In this report, we demonstrate for the first time that over-expression of the A. aculeatus genes results in flax plants more readily usable for fibre production. The biochemical parameters of the cell wall components indicated that the fibre quality remains similar to that of wild-type plants, which is an important pre-requisite for industrial applications. Magdalena Musialak and Magdalena Wróbel-Kwiatkowska participated equally in the preparation of this paper     

Polysaccharides, tightly bound to cellulose in cell wall of flax bast fibre: Isolation and identification

Part of matrix polymers of flax bast fibre cell wall is tightly bound to cellulose and can not be extracted by conventional methods. To analyze these polymers, the residue, remaining after cell wall treatment with chelators and alkali, was dissolved in solution of lithium chloride in N,N-dimethylacetamide. Cellulose was precipitated by water and completely degraded by cellulase, giving the possibility to separate matrix polysaccharides, which remained in polymeric form. The obtained polymers were fractionated by gel permeation chromatography and characterized by monosaccharide analysis, staining with LM5 antibody and Yariv reagent, ¹H and ¹³C NMR. The total yield of the polysaccharides that are tightly bound to cellulose in flax fibre, was 4.6%. The major fractions (molecular mass 100–400 kDa) were composed of galactose, accompanied by two other significant monomers, GalA and Rha, with the ratio 1.1–1.4. Composition and structure of the cellulose bound galactan permit to consider it as fragment of the high-molecular mass (2000 kDa) galactan, synthesized by the developing fibres, while forming the secondary cell wall of gelatinous type.     

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.     

Detection in situ and characterization of lignin in the<Emphasis Type="Italic"> G</Emphasis>-layer of tension wood fibres of<Emphasis Type="Italic"> Populus deltoides</Emphasis>

The occurrence of lignin in the additional gelatinous (G-) layer that differentiates in the secondary wall of hardwoods during tension wood formation has long been debated. In the present work, the ultrastructural distribution of lignin in the cell walls of normal and tension wood fibres from poplar (Populus deltoides Bartr. ex Marshall) was investigated by transmission electron microscopy using cryo-fixation–freeze-substitution in association with immunogold probes directed against typical structural motifs of lignin. The specificity of the immunological probes for condensed and non-condensed guaiacyl and syringyl interunit linkages of lignin, and their high sensitivity, allowed detection of lignin epitopes of definite chemical structures in the G-layer of tension wood fibres. Semi-quantitative distribution of the corresponding epitopes revealed the abundance of syringyl units in the G-layer. Predominating non-condensed lignin sub-structures appeared to be embedded in the crystalline cellulose matrix prevailing in the G-layer. The endwise mode of polymerization that is known to lead to these types of lignin structures appears consistent with such an organized cellulose environment. Immunochemical labelling provides the first visualization in planta of lignin structures within the G-layer of tension wood. The patterns of distribution of syringyl epitopes indicate that syringyl lignin is deposited more intensely in the later phase of fibre secondary wall assembly. The data also illustrate that syringyl lignin synthesis in tension wood fibres is under specific spatial and temporal regulation targeted differentially throughout cell wall layers.Abbreviations G-layer Gelatinous layer - G Guaiacyl monomeric unit - PATAg Periodic acid–thiocarbohydrazide–silver proteinate - S Syringyl monomeric unit     

Gibberellin signaling

A study of stem anatomy and the sclerenchyma fibre cells associated with the phloem tissues of hemp (Cannabis sativa L.) plants is of interest for both understanding the formation of secondary cell walls and for the enhancement of fibre utility as industrial fibres and textiles. Using a range of molecular probes for cell wall polysaccharides we have surveyed the presence of cell wall components in stems of hemp in conjunction with an anatomical survey of stem and phloem fibre development. The only polysaccharide detected to occur abundantly throughout the secondary cell walls of phloem fibres was cellulose. Pectic homogalacturonan epitopes were detected in the primary cell walls/intercellular matrices between the phloem fibres although these epitopes were present at a lower level than in the surrounding parenchyma cell walls. Arabinogalactan-protein glycan epitopes displayed a diversity of occurrence in relation to fibre development and the JIM14 epitope was specific to fibre cells, binding to the inner surface of secondary cell walls, throughout development. Xylan epitopes were found to be present in the fibre cells (and xylem secondary cell walls) and absent from adjacent parenchyma cell walls. Analysis of xylan occurrence in the phloem fibre cells of hemp and flax indicated that xylan epitopes were restricted to the primary cell walls of fibre cells and were not present in the secondary cell walls of these cells.     

Glucan synthesis by intact cotton fibres fed with different precursors at the stages of primary and secondary wall formation

Seed clusters of individual locules from fruit capsules of Gossypium arboreum L. with adhering intact fibres were fed with radioactive uridinediphosphoglucose (UDPG), guanosinediphosphoglucose (GDPG), glucose and sucrose. The incorporation into high molecular weight glucans of the fibres was studied. For primary wall fibres, UDPG at 1 mM was by far the best precursor, whereas sucrose was the best precursor for secondary wall fibres. No competition was observed between the incorporation of glucose from UDPG and from sucrose when the two were fed simultaneously to secondary wall fibres, indicating that their metabolic pathways are well separated when they are fed from the apoplast. Inhibitors of respiratory ATP-formation strongly inhibited incorporation of sucrose but not that of UDPG. Sucrose incorporation was studied at five different stages of development of the cotton fibres. At the stage of most intense secondary wall formation the incorporation rate was about 300 times that during primary wall formation (24 days post anthesis (DPA)). Incorporation from 1 mM UDPG or GDPG by secondary wall fibres (35 DPA) was less than twice that of primary wall fibres (22 DPA), indicating that the two sugar nucleotides are not readily used as precursors for secondary wall cellulose when they are fed to the exterior of intact cells. The high molecular weight non-cellulosic glucans formed from UDPG and sucrose at 5 and 1,000 M were solubilized in strongly alkaline solutions or dimethyl-sulfoxide (DMSO) and were partially characterized by degradation with an exo--1,3-glucanase. After feeding for one hour, at most 1/3 of the radioactivity in high molecular weight material was found in cellulose and at least 2/3 in -1,3-glucan. The proportions varied little for fibres in the age range of 30 to 48 DPA when sucrose was the precursor although the total incorporation varied by a factor of about four. The fact that at all stages of secondary wall formation -1,3-glucan is synthesized at a very high rate, but that the total amount in the cell wall does not exceed 2% in the later stages of wall formation, can be interpreted in terms of a high turnover of this polysaccharide if it is assumed that wound effects are negligible in the system under study.Abbreviations UDPG uridinediphosphoglucose - GDPG guanosinediphosphoglucose - HEPES N-2-hydroxyethylpiperazine-N-2-ethansulphonic acid - DMSO dimethyl-sulfoxide - DNP 2,4-dinitrophenol - DPA days post anthesis     

Cadmium-induced alterations of the structural features of pectins in flax hypocotyl

In the course of our studies on the putative role of pectins in the control of cell growth, we have investigated the effect of cadmium on their composition, remodelling and distribution within the epidermis and fibre tissues of flax hypocotyl (Linum usitatissimum L.). Cadmium-stressed seedlings showed a significant inhibition of growth whereas the hypocotyl volume did not significantly change, due to the swelling of most tissues. The structural alterations consisted of significant increase of the thickness of all cell walls and the marked collapse of the sub-epidermal layer. The pectic epitopes recognized by the anti-PGA/RGI and JIM5 antibodies increased in the outer parts of the epidermis (external tangential wall and junctions) and fibres (primary wall and junctions). Concomitantly, there was a remarkable decrease of JIM7 antibody labelling and consequently an increase of the ratio JIM5/JIM7. Conversely, the ratio JIM7/JIM5 increased in the wall domains closest to the plasmalemma, which would expel the cadmium ions from the cytoplasm. The hydrolysis of cell walls revealed a cadmium-induced increase of uronic acid in the pectic matrix. Sequential extractions showed a remodelling of both homogalacturonan and rhamnogalacturonan I. In fractions enriched in primary walls, the main part of the pectins became cross-linked and could be extracted only with alkali. In fractions enriched in secondary walls, the homogalacturonan moieties were found more abundantly in the calcium-chelator extract while the rhamnogacturonan level increased in the boiling water extract.     

A flax fibre proteome: identification of proteins enriched in bast fibres

Background  

Bast fibres from the phloem tissues of flax are scientifically interesting and economically useful due in part to a dynamic system of secondary cell wall deposition. To better understand the molecular mechanisms underlying the process of cell wall development in flax, we extracted proteins from individually dissected phloem fibres (i.e. individual cells) at an early stage of secondary cell wall development, and compared these extracts to protein extracts from surrounding, non-fibre cells of the cortex, using fluorescent (DiGE) labels and 2D-gel electrophoresis, with identities assigned to some proteins by mass spectrometry.     

Architecture of the cell wall of a green alga,Oocystis apiculata

Summary The cell wall of a green alga,Oocystis apiculata, was visualized by electron microscopy after preparation of samples by rapid-freezing and deep-etching techniques. The extracellular spaces clearly showed a random network of dense fibrils of approximately 6.4 nm in diameter. The cell wall was composed of three distinct layers: an outer layer with a smooth appearance and many protuberances on its outermost surface; a middle layer with criss-crossed cellulose microfibrils of approximately 15–17 nm in diameter; and an inner layer with many pores between anastomosing fibers of 8–10 nm in diameter. Both the outer and the inner layer seemed to be composed of amorphous material. Cross-bridges of approximately 4.2 nm in diameter were visualized between adjacent microfibrils by the same techniques. The cross-bridges were easily distinguished from cellulose microfibrils by differences in their dimensions.     

Membrane–wall attachments in plasmolysed plant cells

Summary. Field emission scanning electron microscopy of plasmolysed Tradescantia virginiana leaf epidermal cells gave novel insights into the three-dimensional architecture of Hechtian strands, Hechtian reticulum, and the inner surface of the cell wall without the need for extraction. At high magnification, we observed fibres that pin the plasma membrane to the cell wall after plasmolysis. Treatment with cellulase caused these connecting fibres to be lost and the pinned out plasma membrane of the Hechtian reticulum to disintegrate into vesicles with diameters of 100–250nm. This suggests that the fibres may be cellulose. After 4h of plasmolysis, a fibrous meshwork that labelled with anti-callose antibodies was observed within the space between the plasmolysed protoplast and the cell wall by field emission scanning electron microscopy. Interestingly, macerase-pectinase treatment resulted in the loss of this meshwork, suggesting that it was stabilised by pectins. We suggest that cellulose microfibrils extending from strands of the Hechtian reticulum and entwining into the cell wall matrix act as anchors for the plasma membrane as it moves away from the wall during plasmolysis.Correspondence and reprints: Institute of Ecology and Conservation Biology, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria.     

Immunolabelling of intervessel pits for polysaccharides and lignin helps in understanding their hydraulic properties in Populus tremula?×?alba

Background and Aims The efficiency and safety functions of xylem hydraulics are strongly dependent on the pits that connect the xylem vessels. However, little is known about their biochemical composition and thus about their hydraulic properties. In this study, the distribution of the epitopes of different wall components (cellulose, hemicelluloses, pectins and lignins) was analysed in intervessel pits of hybrid poplar (Populus tremula × alba).Methods Immunogold labelling with transmission electron microscopy was carried out with a set of antibodies raised against different epitopes for each wall polysaccharide type and for lignins. Analyses were performed on both immature and mature vessels. The effect of sap ionic strength on xylem conductance was also tested.Key Results In mature vessels, the pit membrane (PM) was composed of crystalline cellulose and lignins. None of the hemicellulose epitopes were found in the PM. Pectin epitopes in mature vessels were highly concentrated in the annulus, a restricted area of the PM, whereas they were initially found in the whole PM in immature vessels. The pit border also showed a specific labelling pattern, with higher cellulose labelling compared with the secondary wall of the vessel. Ion-mediated variation of 24 % was found for hydraulic conductance.Conclusions Cellulose microfibrils, lignins and annulus-restricted pectins have different physicochemical properties (rigidity, hydrophobicity, porosity) that have different effects on the hydraulic functions of the PM, and these influence both the hydraulic efficiency and vulnerability to cavitation of the pits, including ion-mediated control of hydraulic conductance. Impregnation of the cellulose microfibrils of the PM with lignins, which have low wettability, may result in lower cavitation pressure for a given pore size and thus help to explain the vulnerability of this species to cavitation.     

Ultrastructure and chemistry of soluble and polymeric lipids in cell walls from seed coats and fibres of Gossypium species

Electron-microscopic examination in conjunction with extraction procedures and chemical analysis have confirmed that a suberin-like lipid biopolymer is located within the concentric polylamellate layers found in the secondary cell walls of green cotton fibres (Gossypium hirsutum cv. green lint). A polymer of similar ultrastructure and chemical constitution also occurs mainly in the secondary seed-coat walls of the outer epidermis of both green and white varieties of G. hirsutum. The suberins composed of predominantly C₂₂ compounds are, however, markedly different from those present in the periderms of the same plants; these comprise mainly C₁₆ and C₁₈ compounds. Long-chain 1-alkanols (C₂₆–C₃₆) and alkanoic acids (C₁₆–C₃₆) are the principal components of the wax from white fibres but these lipid classes comprise a much smaller proportion of that from green fibres. unidentified highmolecular-weight compounds were the major constituents of the green-fibre was extract which also contains a number of yellow-green pigments, probably flavonoid in nature. These pigments are thought to be associated with the ultrahistochemical reaction with silver proteinate that was observed only in the green-fibre cell walls. A total of 16 wild and cultivated cotton species were examined with the electron microscope for the presence of suberin. The outer seed-coat epidermis of all the examined species but only the fibres of the wild ones were found to be suberized. Among the analysed mutants of fibre colour in G. hirsutum only the gene Lg (green lint) seemed to be associated with suberin.Abbreviations GLC gas-liquid chromatography - TLC thinlayer chromatography Fibres=fibre cells of the seed coat epidermis without fibre base; Seed coast=include the base of fibre cells, and short, so-called fuzz fibres     

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.