The average thickness of cell wall layers in
Plant cell walls are typically described as complex macromolecular composites consisting of an ordered array of cellulose microfibrils embedded in a matrix of non-cellulosic polysaccharides and lignin. Generally, the plant cell wall can be divided into three major layers: middle lamella, primary cell wall, and secondary cell wall. Investigation of plant cell walls is complicated by the heterogeneous and complex hierarchical structure, as well as variable chemical composition between different sub-layers. Thus, a complete understanding of the ultrastructure of plant cell walls is necessary. Transmission electron microscopy (TEM) has proven to be a powerful tool in elucidating fine details of plant cell walls at nanoscale. The present chapter describes the layering structure and topochemistry of plant cell wall revealed by TEM.
- Plant cell wall
- Transmission electron microscopy
Determining the ultrastructural organization of plant cell walls represents one of the most challenging problems in plant biology. Although considerable progress has been made in understanding the basic organization and functions of plant cell wall components, due to the highly complex and dynamic nature of the plant cell wall, the variation in cell wall architecture of wood and gramineous species remains poorly understood. These structural features are associated with cell growth and morphogenesis, which are also crucial in determining the mechanical properties of plant cell walls [1, 2]. Given that one of the critical processing steps in biomass conversion involves systematic deconstruction of cell walls, this structural information is also pivotal for developing novel approaches to convert biomass into liquid biofuels. Therefore, a comprehensive investigation of the architecture of the plant cell wall will not only help us to understand the assembly and biosynthesis of the plant cell wall, but will also contribute to improving the efficiency of biomass deconstruction .
The plant cell wall is a layered construction composed mainly of stiff crystalline cellulose microfibrils (Mfs) embedded in an amorphous matrix of non-crystalline cellulose, hemicelluloses and pectin, as well as various aromatic compounds and proteins . Besides the varieties of chemical constituents, the ultrastructural organization of plant cell wall varies between species and cell types. Generally, the plant cell wall consists of three major layers: (i) the middle lamella (Ml), (ii) the primary wall (Pw), and (iii) the secondary wall (Sw). Due to the highest thickness, Sw accounts for the largest proportion of the plant cell wall. Sw in higher plants consists mainly of cellulose, lignin, and xylan and is the major component of biomass in many species. In hardwood fibers and softwood tracheids, the Sw is normally further differentiated into an outer layer (S1), a middle layer (S2), and an inner layer (S3), with the S2 having the largest thickness [5, 6]. By comparison, in gramineous species the lamellation of the Sw in fiber is generally described as alternating broad and narrow layers .
In the twentieth century, microscopic approaches began to offer high-resolution images to enhance our understanding of cell wall organization. Over the past few decades, atomic force microscopy (AFM) has been successfully applied to high-resolution architecture, assembly, and structure dynamic studies of a wide range of biological systems, which has enabled researchers to visualize the ultrastructure of the plant cell wall [8, 9]. More recently, confocal Raman microspectroscopy (CRM) has now also been successfully applied to acquire information on the preferential orientation of plant polymer functional groups and components distribution in situ [10, 11]. However, although these approaches have been comprehensively used to obtain new information on cell wall architecture, until now the highly complex and dynamic nature of the plant cell wall at nanoscale has limited our ability to generate detailed structural models.
By comparison, due to the higher spatial resolution (<1nm) and specificity when combined with chemical staining and immunolabeling approaches, transmission electron microscopy (TEM) can provide ultrastructural and topochemical information simultaneously and has been used to investigate plant cell wall [12-14]. In this chapter, we mainly discuss the application of TEM in detecting cell wall layering structure and cell wall topochemistry (lignin distribution and hemicelluloses distribution).
2. Cell wall layering structure
To get any information using transmitted electrons in the TEM, specimens have to be thin. “Thin” is a relative term, in this context it means electron transparent. For a specimen to be transparent to electrons, it must be thin enough to transmit sufficient electrons such that enough intensity falls on the screen, charge coupled device (CCD), or photographic plate to give an interpretable image in a reasonable time. Generally this requirement is a function of the electron energy and the average atomic number (Z) of the specimen. It is almost an axiom in TEM that thinner is better and specimens <100 nm should be used wherever possible. However, a too thin section would produce low-contrast TEM image, which hides the subtle structure. For plant cell wall, specimens are generally cut to a thickness of ~80 nm when they are silvery gold in color under ultramicrotome. In extreme cases such as doing high-resolution TEM (HRTEM) or electron spectrometry, specimen thicknesses <50 nm (even <10 nm) are essential.
2.1. Cell wall layering structure in hardwoods and softwoods
TEM examination showed that
In addition to fiber cell wall, the ultrastructural variation in vessel, axial parenchyma, and ray parenchyma was also investigated. As shown in Fig. 1b, the vessel wall was divided into three layers (V-S1, V-S2, and V-S3) of variable electron density. The width of V-S1 ranged from 0.18 μm to 0.32 μm, approximately equal to that of F-S1, while the V-S2 was much thinner, with width from 0.55 μm to 0.67 μm. For the axial parenchyma (AP), the secondary wall was clearly resolved into an outer layer (AP-S1), a middle layer (AP-S2), and an inner layer (AP-S3) (Fig. 1c). Unlike the widest F-S2 layer accounting for the largest proportion of the secondary wall, in axial parenchyma the AP-S3 was the major proportion of secondary wall with the thickness ranging from 0.60 μm to 1.67 μm. In ray parenchyma (RP), the secondary wall consisted of two well-defined layers (outer layer, RP-S1, and inner layer, RP-S2), which did not fit conventional S1, S2, and S3 classification (Fig. 1d). Measurements taken on TEM micrograph evidenced that the average width of the RP-S1 was 0.61 μm, while the average thickness of the RP-S2 was 0.80 μm.
The ultrastructure of pit membrane (PM) among various wood elements (inter-fiber, fiber-vessel, fiber-axial parenchyma, and fiber-ray parenchyma) was also investigated. The thickness of PM varied considerably, with PM between fiber and ray parenchyma having a mean thickness of 500 nm, while PM between fiber and vessel had an average thickness of 220 nm. Thin PM with an average thickness of 230 nm was also found between parenchyma cells. PM varied also in their electron density, with inter-fiber PM (Fig. 2a) appearing distinctly denser than fiber-vessel (Fig. 2b) and fiber-parenchyma (axial and ray parenchyma) PM (Fig. 2c and 2d), which may reflect textural and/or compositional differences. The electron density variations originated from the deposition of lignin that is directly and linearly proportional to lignin concentration . Thus, we can assume that the inter-fiber PMs have the highest lignin concentration, followed by fiber and parenchyma (axial and ray parenchyma) and fewest in the PM between fiber and vessel.
Compared to hardwood, the cell type of softwood is uniform, mainly containing tracheids. In normal wood of
2.2. Cell wall layering structure in gramineous species
The investigation of the
In addition to the layering features of Sf secondary wall, the ultrastructural variation in conductive tissue (xylem vessels) was also visualized using TEM images (Fig. 6a and 6b). The secondary wall of Mxv and Pxv could not be clearly divided into sub-layers. This is probably due to either the uniform electron density or the cellulose microfibrils (Mfs) orientation.
3. Lignification and lignin distribution
Next to cellulose, lignin is the most abundant and important polymeric organic substance in plant cell wall. It is a complex phenolic polymer formed by radical coupling reactions of three main monolignols:
The lignification of plant cell walls is generally known to last for a long period, from the S1 stage to the F stage. After the enlargement of cell size, the secondary wall is thickened with the formation of the S1, S2, and S3 layers. The outermost region of the cell wall, including the intercellular layer, the cell comers, and the primary wall, is lignified during the S1 stage when the surface enlargement of the cell is completed, and just before the S1 starts thickening. This lignification, which will be called “intercellular layer (I)-lignification,” may play an important role in stabilizing the cell size and adhering adjacent cells with one another. This I-lignification continues during the differentiation of the S1 and S2 layers, and even until the formation of the S3. On the other hand, the lignification of the secondary wall, which will be called “S-lignification”, proceeds mainly after the development of a secondary wall framework, that is, in the final (F) stage of differentiation, although its initiation can be detected already during the S2 stage.
To obtain more detailed information, the immunogold-labeling technique has been applied to differentiate between macromolecular features of condensed (mainly C–C bonds) and non-condensed (
3.2. Lignin distribution
3.2.1. Distribution of lignin in softwoods
A number of topochemical detections established that the compound middle lamella is more highly lignified than the secondary wall in typical softwood tracheids [15, 17, 19, 20, 39]. Moreover, the SEM-EDXA technique provided quantitative information of lignin distribution with relatively high accuracy. The distribution of lignin in loblolly pine (
3.2.2. Distribution of lignin in hardwoods
In contrast to the tracheid as the main cell in softwoods, hardwoods have a variety of cells, such as vessels, parenchyma, and fibers. The lignin distribution between secondary wall and middle lamella in hardwood fibers is similar to that in softwoods; however the secondary wall of hardwood fibers is often less lignified than the secondary wall of softwood tracheids. Figure 7 shows the distribution of lignin in
3.2.3. Distribution of lignin in reaction woods
Reaction woods appear on leaning stems or branches by any force such as a landslide or snowfall. In softwoods, the reaction wood forms at the lower side of leaning stems or branches, where the compression stress reacts on the xylem. Therefore, this reaction wood is generally called compression wood. Compression wood differs from normal wood in its anatomical appearance. Differences include a more lignified secondary cell wall (S2L) layer, absence of an S3 layer, and the presence of intercellular spaces in the cell corner region [42, 43]. The distribution of lignin in compression wood has been extensively investigated. Compression wood shows marked changes in the distribution of lignin across the cell wall with reduced lignification of the middle lamella and increased lignification of the S2L layer. In mild compression wood, the lignification of the CCML and the S2L regions is generally comparable, while the S1 and S2 layers were less lignified (Fig. 3) . In severe compression wood, intercellular spaces reduce the contribution of middle lamella lignin to overall lignin content, which is nevertheless increased by the greater lignification of the S2L layer.
On the contrary, reaction wood named as tension wood is formed at the upper side of leaning stems or branches in hardwoods where the xylem loads the tensile stress. Tension wood is characterized by the presence of a cellulose abundant gelatinous layer (GL) forming part of the secondary wall in fibers [44-46]. In maple and oak TW fibers, the GL was divided into concentric sub-layers that appeared either as single rings or as several concentric zones of high and low contrast. Weak staining with potassium permanganate was also visualized at the interface of adjoining concentric layers in maple but was more widespread across the GL in oak, indicating the deposition of aromatic compounds within the cellulose structure of the GL .
4. Hemicelluloses deposition
Hemicelluloses are a heterogeneous group of polysaccharides, including xyloglucans, xylans, mannans, and glucomannans. They form physical and chemical bonds to cellulose and lignin and therefore have an important role in building the three-dimensional structures of plant cell walls . The detailed structure of the hemicelluloses and their abundance vary widely among species and cell types. Combination of TEM and immunolabeling has provided detailed information about the deposition of main hemicelluloses related to tissue development and differentiation.
4.1. Hemicelluloses deposition in softwoods
Glucomannans (GMs) are the most abundant hemicelluloses found in softwoods. GMs are composed of a linear backbone of randomly β-(1,4)-linked D-glucosyl and D-mannosyl residues. The ratio of glucosyl and mannosyl units in softwood GMs is approximately 1:3, and D-galactosyl residues are occasionally attached to the backbone with α-(1,6)-glycosidic bonds. In addition to the galactosyl side chain of GMs, softwood GMs also contain partially substituted hydroxyl groups with
Many studies have reported the distribution of GMs in the softwood cell wall using various immunochemical probes specific to GMs in combination with TEM. Using the enzyme-gold complex method, Joseleau and Ruel (1984) have demonstrated that GMs of spruce (
In addition to GMs, the distribution of xylans in tracheid walls was also investigated. Xylans in wood cell walls are basically composed of a backbone of xylose units that are linked by β-(1-4)-glycosidic bonds. Softwood xylans that are called arabino-4-
Furthermore, to extend the understanding of distributional diversities of hemicelluloses among cells, the deposition of GMs and AGXs in ray cells and pits was investigated by immunolabeling . In comparison with tracheids, ray cells have different deposition processes of GMs and AGXs. GM labeling in ray cells began to be detected at the early stage of S1 formation in tracheids, whereas AGX labeling began to be detected in ray cells at the S2 formation stage in tracheids. In mature ray cells, GM labeling was absent in the innermost layer of ray cells, whereas AGXs were uniformly distributed in the entire ray cell walls. In pits, GM labeling was detected in pit membranes at an early stage of pit formation, but disappeared during pit maturation, indicating that enzymes capable of GM degradation may be involved in pit formation. In contrast to GM labeling, AGX labeling was not observed in pit membranes during the entire pit developmental process.
4.2. Hemicelluloses deposition in hardwoods
4.3. Hemicelluloses deposition in gramineous species
Most of the existing research about distribution of hemicelluloses in gramineous species concentrate on Arabidopsis, which is one of the most frequently used model plants in plant science. Several immunocytochemical studies have reported the distribution of xylans in Arabidopsis stem [64-66]. Xylan deposition in xylary fibers (fibers) was initiated at the cell corner of the S1 layer and the xylan labeling increased gradually during fiber maturation. Metaxylem vessels showed more developed stages of secondary cell wall formation than fibers, but revealed almost identical xylan labeling patterns to fibers during maturation. The consistency of the immunolabeling patterns between LM10 and LM11 in the cell wall of fibers, vessels, and protoxylem vessels indicated that vascular bundle cells may be chemically composed of a highly homogeneous xylan type. In contrast, interfascicular fibers showed different labeling patterns between the two antibodies and also between different developmental stages. Immunolocalization studies of mannans in Arabidopsis stems have shown that mannans are distributed in the various cell types with different concentrations [67-69]. Temporal and spatial variations in mannan labeling between cell types in the secondary xylem of Arabidopsis stems were examined using immunolocalization with mannan-specific monoclonal antibodies (LM21 and LM22). Mannan labeling in secondary xylem cells (except for protoxylem vessels) was initially detected in the cell wall during S2 formation and increased gradually during development. Labeling in metaxylem vessels (vessels) was detected earlier than that in xylary fibers (fibers), but was much weaker than fibers. The S1 layer of vessels and fibers showed much less labeling than the S2 layer. Some strong labeling was also detected in pit membranes of vessel pits.
The potential of TEM for investigation of plant cell walls has already been demonstrated on various plant tissues. The high spatial resolution allows detection of changes in the ultrastructure and cell wall polymer deposition on the cell and cell wall level. Nevertheless, complex sample preparation procedure will limit its extensive application, especially in living plant tissues. Thus, when combined with other in situ microscopic techniques (such as atom force microscopy, confocal laser microscopy, confocal Raman microscopy), much more information hidden in plant cell wall will be illustrated.
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