ARTICLE |
Correspondence to: Azeddine Driouich, UMR CNRS 6037, Centre Commun de Microscopie Electronique, IFRMP23, Université de Rouen, 76821 Mont Saint Aignan Cedex, France. E-mail: adriouic@crihan.fr
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Summary |
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Flax fibers have been the subject of many biochemical studies, which revealed that cellulose and pectins are the major constituents of their walls. In contrast, little is known about the location of these polymers within the walls of mature fibers by microscopic methods. This has been technically hampered by the very thick secondary wall of fibers, resulting in inadequate tissue preservation unsuitable for immunogold microscopy. In this study, we adapted the basic chemical fixation, dehydration and infiltration methods to achieve a good preservation of the cell structures of mature fibers and reduced damage to antigens. We were able to apply postembedding immunocytochemical techniques to map the location of various pectic epitopes within the walls of mature fibers. Our immunolabeling data show that homogalacturonans were exclusively found in the middle lamellae and the cell junctions but were not detectable in the secondary wall. In contrast, rhamnogalacturonan I (RG I)-associated epitopes, as well as galactan and arabinan epitopes, were abundantly distributed over the secondary wall of mature fibers.
(J Histochem Cytochem 49:15251535, 2001)
Key Words: immunocytochemistry, flax fibers, LR White, pectins, secondary walls
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Introduction |
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Natural fibers from jute, ramie, or flax are commonly used in the textile industry. They also have good potential as reinforcing agents in composite polymers. Flax fibers are composed of elongated cells, which are cemented together into bundles of filaments along the stem. Each cell produces a very thick, cellulose-rich secondary wall that fills the entire cell at maturity (
Recently, we have been able to perform ICC analyses on LR White-embedded immature flax fibers (1- to 2-week-old plants) using classical fixation and embedding techniques (
Our first task was to achieve a good preservation of the ultrastructure, particularly for the fiber cell tissue, and the second was to map the distribution of different epitopes within the greatly thickened secondary walls of mature fiber cells. To the best of our knowledge this represents the first attempt to obtain information on the occurrence and location of polysaccharides in mature fiber walls (capsulation stage) using immunogold microscopy.
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Materials and Methods |
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Plant Material
Flax plants (Linum usitatissimum L., variety Ariane) were harvested from a field near Rouen (Motteville, France) at different stages of their development.
Sample Preparation for Microscopy
Small fragments from the 18th internode were excised and immediately vacuum-fixed for 90 min in 2% (w/v) paraformaldehyde (Prolabo; Fontenay-Sous-Bois, France), 0.5% (v/v) glutaraldehyde (Oxford Agar), 0.1 M phosphate buffer (pH 7.2). The samples were rinsed in the same buffer, dehydrated in a graded aqueous ethanol series (10, 20, 35, 40, 50, 60, 70, 80, 90, 100%: 30 min each step) and further dehydrated with two washes in propylene oxide for 60 min each. They were then progressively infiltrated with LR White resin (LRW), according to the following schedule: 10, 30, 50, 70, 90% LRW/propylene oxide for at least 24 hr each step, and several incubations in 100% LRW. This last step was adapted to each sample depending on the age of the plant. It lasted about 90 days for mature samples and 30 days for younger ones. The first two infiltration steps were done under vacuum. The infiltrated samples were then embedded in gelatin capsules and polymerized for 24 hr at 60C.
Light Microscopy and Histochemical Staining
Staining of Fresh Samples with SafraninLight Green.
Hand-cut cross-sections from fresh stems were brought to 50% ethanol solution (v/v), then stained in safranin solution (Sigma; St Louis, MO) for 2 hr as described by
Staining of LRW-embedded Samples with Methylene BlueBasic Fuchsin.
The staining was performed according to the protocol of
Electron Microscopy and Immunogold Labeling
Ultrastructural Observations.
Ultrathin sections (90 nm) from LRW-embedded samples were submitted to PATAg (periodic acid thiocarbohydrazide silver proteinate) test, which allowed polysaccharide detection as previously described (
Detection of Anionic Sites with the Cationic Gold Probe.
Labeling with cationic gold probe was performed on ultrathin sections (90 nm) from LRW-embedded samples collected on gold grids and treated with 3% acetic acid at pH 2.6 for 15 min at room temperature. The sections were incubated on a drop of 10-nm cationic poly-L-lysine colloidal gold complex (Biocell; Cardiff, UK) diluted 1:100 (v/v) in 3% acetic acid for 1 hr as previously described (
Immunolabeling Procedure
The antibodies used in this study included: (a) the polyclonal antibodies anti-PGA/RG I, which recognize the unesterified polygalacturonic acids of the complex polygalacturonic acid (PGA)/rhamnogalacturonan I (RG I) (-1,5-arabinan-epitopes (
Thin sections (9095 nm) mounted on gold grids were incubated in a blocking solution of 3% non-fat dried milk in PBST 0.1% (10 mM Na-phosphate (Sigma), 500 mM NaCl (Sigma), 0.1% Tween-20 (Sigma) for 30 min. All incubations were carried out overnight at 4C, or for 4 hr at room temperature as previously described (
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Results |
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Fiber Differentiation
Fig 1 shows the average of stem length at different growth stages of flax plants. After a period of active growth (4280 days; 585 cm), the elongation of the stem slowed down at the onset of floral bud development (8098 days; 8590 cm; see also 100 days), the plants usually have a stem length of 90110 cm.
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Fiber differentiation, characterized by deposition of a cellulose-rich secondary wall, starts very early during flax development (
In this study, plants were field-harvested at two stages of development, i.e., 50 and 98 days, corresponding to two stages of fiber differentiation. Hand-cut transverse sections from fresh stems, stained with safraninlight green, showing the anatomic organization at both stages are seen in Fig 2. In 50-day-old plants, differentiating fibers (not mature) are arranged in one layer at the periphery of the stem (Fig 2A). They have a relatively thick secondary wall and a wide lumen. In 98-day-old plants, mature fibers (Fig 2B and Fig 2C), are organized into bundles, with up to 20 cells per bundle, located between the cortical parenchyma (cp) and the vascular cylinder, which consists mostly of xylem cells (x). The secondary wall is remarkably thick and the lumen width has undergone a pronounced reduction.
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Ultrastructure of LRW-embedded Stems
To improve the quality of ultrastructural preservation of mature fibers and to perform immunogold labeling, we devised a modified method based on a vacuum fixation combining both formaldehyde and glutaraldehyde to enhance the rate of penetration. Vacuum was also applied during early stages of resin infiltration, and LRW embedding duration was increased, especially in the later stages (see Discussion).
To check for the structural preservation of fibers at both stages of maturity after fixation and LRW-embedding, stem sections were examined using both light and transmission electron microscopy. As illustrated in Fig 3A and Fig 3B, transverse semithin sections stained with methylene bluebasic fuchsin reveal well-preserved tissues, including xylem and fiber cells. The good quality of preservation is confirmed by transmission electron microscopy (TEM) observations of 90-nm ultrathin sections (Fig 3C and Fig 3D). It is interesting to note that the cohesion of fiber bundles in chemically fixed and LRW-embedded stems is well maintained as in the native state (compare with hand-cut transverse sections of fresh, non-fixed tissue in Fig 2). The primary and secondary cell walls (Fig 3D), as well as cell junctions and the narrow lumen of fibers are clearly distinguishable (Fig 3D).
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After checking the preservation quality of fibers, we proceeded to gold labeling of pectic polysaccharides using different gold probes.
Cell wall Localization of Anionic Sites
The poly-L-lysinegold complex is a cationic probe that binds to the negatively charged groups associated with wall polymers such as pectins and xylans (
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Immunogold Labeling of Homogalacturonans
To check for the presence of homogalacturonans and to examine their distribution in fiber walls, we used the MAb JIM5, which recognizes pectin epitopes containing up to 40% of methylesterified PGA (
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Immunolocalization of Rhamnogalacturonan I-associated Epitopes
Complex polysaccharides such as RG I have been reported to be present in the primary walls of flax tissues including fibers (
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Labeling of fiber walls with LM5 and LM6, two MAbs that recognize (1-4)-ß-D-galactans and (1-5)--D-arabinans, respectively (
Control experiments performed with the omission of all the primary antibodies recognizing pectins show no labeling over fiber cell walls (data not shown).
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Discussion |
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The goal of the present study was to find a tissue preparation and embedding method that could be applied to mature flax fibers so that they could be used in immunogold microscopy. Working on stems harvested at two growth stages (i.e., rapid phase of growth and maturation) of flax plants made it possible to show that several polysaccharides of the pectic class are present in different cell wall domains and undergo developmental changes during bundle formation and fiber maturation.
Preservation of Fiber Cell Walls
In the past we have been able to achieve good preservation of mature fibers embedded in Spurr's resin (
Therefore, to circumvent the difficulties encountered, a modification of the classical method, based mainly on an extended vacuum infiltration/fixation step before embedding in LRW resin, was used. This enabled flax fibers, which are reputed to be difficult to infiltrate, to be successfully and homogeneously embedded in LRW. The fixation definitely yielded better preservation of fiber bundles than that obtained with the basic method used for young fibers, and enhanced their structural stability. Although some minor alterations such as cracks were apparent, the secondary walls were uniformly and reproducibly well preserved.
Location of Wall Polymers
By in situ localization using different gold probes, we show that there are differences in the distribution of polysaccharides within the cell walls of mature fibers (see diagram in Fig 7). A large amount of cationic gold labeling is seen within the secondary wall and all other wall domains of fibers at both stages of differentiation. The specificity of the cationic gold probe was previously characterized. It was shown to bind mainly to the COO- groups of galacturonic acid of un-esterified pectins, although it can also recognize other acidic molecules such as proteins or phenolics (
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The undefined RG I epitope recognized by CCRCM2 was not detected in the fiber walls during the rapid phase of growth, whereas it was abundantly present in the secondary wall at maturity. The absence of labeling in the cell walls of differentiating fibers might be due to the lack of RG I or to a substitution, such as acetyl-esterification, which is not recognized by the antibody. The fact that labeling is reproducibly detected in the cytoplasm indicates that the epitope is synthesized and possibly secreted into the cell wall. Therefore, RG I pectin is probably present in the wall but the epitope could not be detected. RG I polymers decorated with short side chains of galactosyl residues, which are partially acetylesterified, were found in flax fibers (-1-5-arabinans in their side chains. ß-1-4-galactans (recognized by LM5) may also be part of the side chain of RG I present in mature fiber secondary walls. However, the presence of free galactans and arabinans in the secondary wall cannot be excluded.
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Footnotes |
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1 These authors contributed equally to this work.
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Acknowledgments |
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We gratefully acknowledge the gift of some of the antibodies from Drs M. Hahn, P. Knox, and L.A. Staehelin. We also thank Drs M. Jarvis and M. Vicré for critical reading of the manuscript.
Received for publication February 13, 2001; accepted June 27, 2001.
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