Aspen Tension Wood Fibers Contain β-(1---> 4)-Galactans and Acidic Arabinogalactans Retained by Cellulose Microfibrils in Gelatinous Walls

Abstract

Contractile cell walls are found in various plant organs and tissues such as tendrils, contractile roots, and tension wood. The tension-generating mechanism is not known but is thought to involve special cell wall architecture. We previously postulated that tension could result from the entrapment of certain matrix polymers within cellulose microfibrils. As reported here, this hypothesis was corroborated by sequential extraction and analysis of cell wall polymers that are retained by cellulose microfibrils in tension wood and normal wood of hybrid aspen (Populus tremula × Populus tremuloides). β-(1→4)-Galactan and type II arabinogalactan were the main large matrix polymers retained by cellulose microfibrils that were specifically found in tension wood. Xyloglucan was detected mostly in oligomeric form in the alkali-labile fraction and was enriched in tension wood. β-(1→4)-Galactan and rhamnogalacturonan I backbone epitopes were localized in the gelatinous cell wall layer. Type II arabinogalactans retained by cellulose microfibrils had a higher content of (methyl)glucuronic acid and galactose in tension wood than in normal wood. Thus, β-(1→4)-galactan and a specialized form of type II arabinogalactan are trapped by cellulose microfibrils specifically in tension wood and, thus, are the main candidate polymers for the generation of tensional stresses by the entrapment mechanism. We also found high β-galactosidase activity accompanying tension wood differentiation and propose a testable hypothesis that such activity might regulate galactan entrapment and, thus, mechanical properties of cell walls in tension wood.

Figure 1.

Anatomy, macroscopic appearance, and monosaccharide composition of NW and TW. A, Light microscopy images of NW from an upright tree and TW from a tilted tree stained with Alcian Blue-safranin. Note the prominent nonlignified G-layers stained blue in TW and the lignified compound middle lamella and S-layers stained red in both NW and TW. Bar = 50 μm. B, Appearance of NW and TW after milling. C, Monosaccharide composition (mol %) of TFA hydrolysates of NW and TW.

Figure 2.

Immunodot analysis of cell wall polysaccharides in buffer-extractable, KOH-extractable, and cellulose-retained (obtained after cellulase treatment) fractions of NW and TW with antibodies recognizing (1→4)-β-xylan (LM11; McCartney et al., 2005), (1→4)-β-galactan (LM5; Jones et al.1997), RG-I backbone (RU2; Ralet et al., 2010), the XXXG motif of xyloglucan (LM15; Marcus et al., 2008), and an unknown epitope of AGP (JIM14; Knox et al., 1991). Total fractions (aliquots taken before gel filtration) were used for analysis. Values (4 μg, 1 μg, and 0.25 μg) on top of the membrane images indicate the amount of carbohydrates spotted on each vertical line of dots.

Figure 3.

Analysis of two subfractions of buffer-extractable polysaccharides of NW and TW. A, Elution profile with designation of two subfractions. DW, Dry weight. B and C, Proportions of monosaccharides obtained after TFA hydrolysis of high- and low-molecular-weight fractions, respectively. Error bars show se (n = 3 biological repeats). D and E, Oligosaccharide fragments obtained after enzymatic digestion and separation by PACE on 8-aminonaphthalene-l,3,6-trisulfonic acid (ANTS) gels. For the AG-II analysis, the high-molecular-mass subfractions of NW and TW were adjusted to equalize their total Ara contents and digested with exo-β-(1→3)-galactanase alone or in combination with arabinofuranosidase (D). For β-(1→4)-galactan analysis, the same samples were adjusted to equalize total sugar amounts and digested with endo-β-(1→4)-galactanase (E). Controls are the samples without enzymatic digestion to check for background signals. Bands with differing yields from NW and TW samples are marked by arrowheads: red, released by exo-β-(1→3)-galactanase; blue, released by α-arabinanase followed by exo-β-(1→3)-galactanase; and green, released by endo-β-(1→4)-galactanase.

Figure 4.

Analysis of two subfractions of KOH-extractable cell wall polysaccharides of NW and TW. A, Elution profile with designation of two subfractions. DW, Dry weight. B and C, Proportions of monosaccharides obtained after TFA hydrolysis of high- and low-molecular-weight fractions, respectively. Error bars show se (n = 3 biological repeats). D, Immunodot analysis of the subfractions with antibodies recognizing (1→4)-β-xylan (LM11; McCartney et al., 2005), (1→4)-β-galactan (LM5; Jones et al., 1997), RG-I backbone (RU2; Ralet et al., 2010), (1→4)-β-mannan (LM21; Marcus et al., 2010), and the XXXG motif of xyloglucan (LM15; Marcus et al., 2008). Values (4 μg, 1 μg, and 0.25 μg) on top of the membranes indicate the amount of carbohydrates spotted on each vertical line of dots.

Figure 5.

Analysis of two subfractions of cellulose-retained polymers from samples of NW and TW. A, Elution profile with designation of two subfractions. DW, Dry weight. B and C, Proportion of monosaccharides obtained after TFA hydrolysis of high- and low-molecular-weight fractions, respectively. Error bars show se (n = 3 biological repeats). D and E, Oligosaccharide fragments obtained after enzymatic digestion and separation by PACE on ANTS or 2-aminoacridone (AMAC) gels. For the β-(1→4)-galactan, the high-molecular-mass subfractions of NW and TW were adjusted to equalize total sugar amounts and digested with endo-β-(1→4)-galactanase (D). For the RG-I backbone treatment experiments (Jensen et al., 2010), similar samples were digested with RG-I lyase (D). No standards were available for the RG-I fragments. For the AG-II analysis, the high-molecular-mass subfractions of NW and TW were adjusted to equalize amounts of total Ara and digested with exo-β-(1→3)-galactanase (E). Controls are the samples without enzymatic digestion to check for background signals. Bands with differing yields from NW and TW are marked by arrowheads: green, released with endo-β-(1→4)-galactanase; red, released with exo-β-(1→3)-galactanase; and orange, released with RG-I lyase.

Figure 6.

Immunolocalization of cell wall polysaccharides in fibers of NW (left) and TW (right) with LM5 antibody (A and B), RU2 antibody (C and D), LM11 antibody (E and F), LM15 antibody (G and H), and LM21 antibody (I and J). CML, Compound middle lamella; CW, cell wall; GL, G-layer, S1 and S2, secondary cell wall layers. Silver enhancement was applied for different times (2–5 min), leading to different sizes of particles. Bars = 1 µm.

Figure 7.

β-1,4-Galactosidase activity is present in cell walls of developing NW and TW and is highly increased in TW. Yellow coloration corresponds to the fluorescence channel signals from resorufin released by the action of β-1,4-galactosidase from the substrate. The control shown at right is the image of developing TW that was heated before the assay to denature proteins. No fluorescence signal was detected, and the image of the fluorescence channel was superimposed on the transmitted light channel to show the presence of TW tissue.

Figure 8.

A, Scheme of the main components in S-layers and G-layers of fibers in TW. In S-layers, cellulose microfibrils are oriented helicoidally with alternating MFA between layers; in thick G-layers, the orientation of all cellulose microfibrils is close to axial. XG+XET, XG and xyloglucan endotransglycosylase, which acts to staple S-layers and G-layers (Mellerowicz et al., 2008; Hayashi et al., 2010). Cellulose microfibrils in S-layers are separated by xylan and lignin and have poor chances to interact laterally; in highly cellulosic G-layers, microfibrils tend to interact laterally, thereby increasing crystallinity. RG-I+Galactan, RG-I and β-(1→4)-galactan trapped between laterally interacting G-layer cellulose microfibrils, creating tension in them. Galactan chains are modified in murо by a specific galactosidase (BGAL), making the polymer more compact and providing conditions for the lateral interaction of cellulose microfibrils (not shown). CML, Compound middle lamellae. B, Possible spatial structure of the RG-I and galactan (Mikshina et al., 2015): β-(1→4)-galactan side chains (galactan) attached to RG-I backbone. Molecules of RG-I are associated due to β-(1→4)-galactan chain interaction. Note that the backbone is located at the surface of such associated molecules.