Localization of cell wall polysaccharides in normal and compression wood of radiata pine: relationships with lignification and microfibril orientation

Abstract

The distribution of noncellulosic polysaccharides in cell walls of tracheids and xylem parenchyma cells in normal and compression wood of Pinus radiata, was examined to determine the relationships with lignification and cellulose microfibril orientation. Using fluorescence microscopy combined with immunocytochemistry, monoclonal antibodies were used to detect xyloglucan (LM15), β(1,4)-galactan (LM5), heteroxylan (LM10 and LM11), and galactoglucomannan (LM21 and LM22). Lignin and crystalline cellulose were localized on the same sections used for immunocytochemistry by autofluorescence and polarized light microscopy, respectively. Changes in the distribution of noncellulosic polysaccharides between normal and compression wood were associated with changes in lignin distribution. Increased lignification of compression wood secondary walls was associated with novel deposition of β(1,4)-galactan and with reduced amounts of xylan and mannan in the outer S2 (S2L) region of tracheids. Xylan and mannan were detected in all lignified xylem cell types (tracheids, ray tracheids, and thick-walled ray parenchyma) but were not detected in unlignified cell types (thin-walled ray parenchyma and resin canal parenchyma). Mannan was absent from the highly lignified compound middle lamella, but xylan occurred throughout the cell walls of tracheids. Using colocalization measurements, we confirmed that polysaccharides containing galactose, mannose, and xylose have consistent correlations with lignification. Low or unsubstituted xylans were localized in cell wall layers characterized by transverse cellulose microfibril orientation in both normal and compression wood tracheids. Our results support the theory that the assembly of wood cell walls, including lignification and microfibril orientation, may be mediated by changes in the amount and distribution of noncellulosic polysaccharides.

Figure 1.

Comparison of bright-field and fluorescence images of normal and compression wood. Bright-field samples are stained with toluidine blue. Fluorescence images show lignin autofluorescence in all cell wall layers. A, Normal wood in transverse view showing earlywood cell walls. Bar = 60 μm. B, Normal wood in transverse view showing lignin autofluorescence (ML, middle lamella + primary wall, S1, S2, and S3 layers of the secondary cell wall). Bar = 20 μm. C, Compression wood in transverse view showing early wood cell walls. Bar = 60 μm. D, Compression wood in transverse view showing lignin autofluorescence at the same exposure as B (S2L, highly lignified outer S2 layer; S2i, less lignified inner S2 layer; ICS, intercellular space). Bar = 20 μm.

Figure 2.

Immunolocalization images of normal and compression wood experiments with and without primary LM21 (mannan) antibody. A, Normal wood in transverse view showing LM21 epitope localization. B, Normal wood negative control image lacking primary antibody (AB), at the same exposure as A. C, Compression wood in transverse view showing LM21 epitope localization. D, Compression wood negative control image lacking primary antibody, at the same exposure as C. Bar = 20 μm.

Figure 3.

Immunolocalization of polysaccharides in transverse sections of normal wood (A, C, E, G, I, and K) and compression wood (B, D, F, H, J, and L). A and B, LM15 (xyloglucan) epitope showing localization in the primary cell walls of tracheids in both wood types. C, LM10 (low and unsubstituted xylan) epitope showing localization in the S1 and S3 layers in normal wood tracheids and high abundance at pit borders (arrow). D, LM10 epitope in compression wood tracheids, showing localization in the middle lamella at the cell corners, in the S1 layer, and in the inner region of the S2 layer. E, LM11 (unsubstituted and highly substituted xylan and arabinoxylan) epitope in normal wood, showing localization throughout the primary and secondary cell walls of tracheids, with stronger label in the S1 and S3 layers of the secondary cell wall. F, LM11 epitope in compression wood showing distribution throughout the cell wall, with the exception of the outer S2 region (arrow). G and H, LM5 [β(1,4)-Galactan] epitope, showing sparse localization in the primary wall of normal wood tracheids (G) but abundant distribution in the outer secondary wall of compression wood tracheids (H). I, LM21 (galactoglucomannan) epitope in normal wood, showing localization in the secondary cell wall of tracheids with increased abundance in some areas of the S1 layer. J, LM21 epitope in compression wood tracheids, showing localization across the secondary wall, with increased abundance in the S1 region and reduced abundance in the outer S2L region. K and L, LM22 (galactoglucomannan) epitope, showing distribution in a similar pattern to the LM21 epitope. Bar = 20 μm.

Figure 4.

Immunolocalization of polysaccharides in rays, resin canals, and delignified tracheids. A, LM15 (xyloglucan) epitope is abundant in primary walls of unlignified ray parenchyma cells (RP) and slightly less abundant in primary walls of ray tracheids (RT) and axial tracheids (tangential longitudinal view). Bar = 20 μm. B, LM21 (galactoglucomannan) epitope is detected in secondary walls of ray tracheids (RT) and thick-walled ray parenchyma cells (TRP) both of which are lignified (tangential longitudinal view). Bar = 20 μm. C, LM10 (low or unsubstituted xylan) epitope is abundant in the secondary wall of ray tracheids (transverse view). Bar = 20 μm. D, LM5 [β(1,4)-galactan] epitope is abundant in the primary walls of resin canal (RC) parenchyma cells but sparse in the epithelial cells (transverse view). Bar = 40 μm. E, LM15 (xyloglucan) epitope is abundant in the primary walls of both resin canal (RC) parenchyma and epithelial cells but is less abundant in the primary wall of axial tracheids (transverse view). Bar = 80 μm. F, LM21 and G, LM22 (galactoglucomannan) epitope shows the same pattern of distribution in delignified compression wood tracheids as in untreated tracheids (transverse view). Bar = 20 μm.

Figure 5.

Detection of lignin autofluorescence and polysaccharide epitopes (Ab) in transverse sections of normal and compression wood shown as separate and overlay images. Colors in the overlay images show lignin as green and polysaccharide as magenta. Other than LM5, the distribution of most polysaccharide epitopes is an inverse image of the lignin autofluorescence intensity. A, LM10 epitope; B, LM11 epitope; C, LM21 epitope; D, LM22 epitope; E, LM5 epitope; F, LM10 epitope; G, LM11 epitope; H, LM21 epitope; I, LM22 epitope. Bar = 20 μm.

Figure 6.

Colocalization of lignin and polysaccharide epitopes in transverse sections of normal and compression wood represented by PDM images. In the PDM images white indicates colocalization and black indicates exclusion. A, LM10 epitope; B, LM11 epitope; C, LM21 epitope; D, LM22 epitope; E, LM5 epitope; F, LM10 epitope; G, LM11 epitope; H, LM21 epitope; I, LM22 epitope. Bar = 20 μm.

Figure 7.

Overlay images of normal and compression wood in transverse section showing details of polysaccharide/lignin colocalization and xylan colocalized with birefringence. A, In compression wood tracheids, LM22 epitope (magenta) is colocalized with reduced lignification (green) in the inner S2 region (S2i) and shows a distinct radial alignment in some locations (arrow). B, In normal wood tracheids, LM10 epitope (magenta) is colocalized with birefringence (green), confirming that the epitope is abundant in the S1 and S3 layers (arrows). C, In compression wood tracheids, LM11 epitope (magenta) is localized to the outer part of the S1 layer as detected by birefringence shown in bright green (arrow) and indicating a transverse cellulose microfibril angle. LM11 epitope is also localized in the inner S2 region, which shows a stronger birefringence suggesting a more transverse orientation of cellulose microfibrils than in the outer S2 region although this is less pronounced than in the S1 layer. Bar = 20 μm.