Molecular Plant Advance Access originally published online on July 28, 2009
Molecular Plant 2009 2(5):893-903; doi:10.1093/mp/ssp054
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Xyloglucan for Generating Tensile Stress to Bend Tree Stem
a Kyoto University, RISH, Gokasho, Uji, Kyoto 611-0011, Japan
b Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan
c Forest Tree Breeding Center, Hitachi, Ibaraki 319-1301, Japan
d CBD Technologies, Rehovot 76100, Israel
e Division of Forest and Biomaterials Science, Kyoto University, Kyoto 606-8502, Japan
f Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan
g Institute of Sustainability Science, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan
1 To whom correspondence should be addressed. E-mail taka{at}rish.kyoto-u.ac.jp, fax and tel. +81 774 38 3618.
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Abstract |
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 TOP Abstract INTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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In response to environmental variation, angiosperm trees bend their stems by forming tension wood, which consists of a cellulose-rich G (gelatinous)-layer in the walls of fiber cells and generates abnormal tensile stress in the secondary xylem. We produced transgenic poplar plants overexpressing several endoglycanases to reduce each specific polysaccharide in the cell wall, as the secondary xylem consists of primary and secondary wall layers. When placed horizontally, the basal regions of stems of transgenic poplars overexpressing xyloglucanase alone could not bend upward due to low strain in the tension side of the xylem. In the wild-type plants, xyloglucan was found in the inner surface of G-layers during multiple layering. In situ xyloglucan endotransglucosylase (XET) activity showed that the incorporation of whole xyloglucan, potentially for wall tightening, began at the inner surface layers S1 and S2 and was retained throughout G-layer development, while the incorporation of xyloglucan heptasaccharide (XXXG) for wall loosening occurred in the primary wall of the expanding zone. We propose that the xyloglucan network is reinforced by XET to form a further connection between wall-bound and secreted xyloglucans in order to withstand the tensile stress created within the cellulose G-layer microfibrils.
Key Words: G-layer • tensile stress • xyloglucan • xyloglucan endotransglucosylase
Received for publication April 4, 2009. Accepted for publication June 20, 2009.
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INTRODUCTION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Higher plants control their posture by bending their stems to respond to environmental variation. Woody plants bend not only their apical stems, by differential growth of their elongating region, but also their secondary xylem, by forming tension wood at one side of the thickening region. Tension wood is a type of reaction wood formed on the upper side of the leaning stems of angiosperms. The other type of reaction wood is compression wood, which forms on the ground-facing sides of the leaning stems of gymnosperms. Both types of reaction wood are induced by gravitropical stimuli, mechanical stimuli, or both (Wilson and Archer, 1977). The stem bends by the mechanical action that is induced by the abnormal growth stress of the tensile or compression wood. Herbaceous plants such as Arabidopsis thaliana do not need to bend their secondary xylems because they can ‘move’ locations every few months or years by the transfer of seeds. Trees must bend their stems in response to environmental variation because they spike roots into the ground and live for many years. The bending is not caused by differential growth, such as in elongating apical stems, but rather by fiber cell shrink with strong tensile stress along the cell axis at one side of the stem, where strong tensile stress occurs in a non-lignified, cellulose-rich G-layer in the walls of fiber cells (Onaka, 1949; Wilson and Archer, 1977; Yoshida and Okuyama, 2002). Tension wood generates tensile stress by forming a non-lignified, cellulose-rich G-layer in the walls of fiber cells.
The cellulose microfibrils of the G-layer must align at zero to 2.5° angle to the axis of the fiber cells due to the abnormal growth stress, whereas the S2 layers, the main part of the normal secondary wall, have a 15–30° microfibril angle to the cell axis. The microfibrils could be stressed by interacting with other matrix components, such as xyloglucan in the primary cell wall (Hayashi, 1989a), because the poplar G-layer consists primarily of cellulose and xyloglucan together with a small amount of galactan and glucomannan (Nishikubo et al., 2007). Xyloglucan is now one of the most important functional components in the G-layer (Mellerowicz et al., 2008). Immunocytochemical analyses also showed that 1,4-β-galactan, arabinogalactan, and rhamnogalacturonanI-type pectin were bound to the G-layer (Arend, 2008; Bowling and Vaughn, 2008). Goswami et al. (2008) presented an alternate mechanism for the tension wood response, in which swelling of the G-layer caused bulging, consequent shortening of fibers, and producing stress in the wood. The question thus becomes how the secondary xylem generates and maintains such high growth stress in the G-layer during tension wood development.
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RESULTS |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Reduction of Specific Polysaccharides
In order to examine whether matrix polysaccharides are effective at generating tensile stress during a gravitropic response, we horizontally laid the stems of transgenic poplars overexpressing one of several endoglycanases, including xyloglucan endo-1,4-β-glucanse (xyloglucanase, XEG) (Park et al., 2004), endo-1,4-β-xylanase (xylanase, XYL) (Banik et al., 1996), arabinofuranosidase (Kotake et al., 2006) plus endo-3,6-β-galactanase (arabinofuranosidase and galactanase, AG) (Kotake et al., 2004), and cellulose endo-1,4-β-glucanse (cellulase, CEL) (Shani et al., 2004). We produced 40 independent clones of transgenic poplars overexpressing Aspergillus xyloglucanase (AaXEG2, AY160774) with the PaPopCel1 (D32166 [GenBank] ) signal peptide, eight clones of transgenic poplars overexpressing xylanase (HvXYL1, U59312 [GenBank] ), four clones of transgenic poplars overexpressing Arabidopsis cellulase (AtCel1, X98544 [GenBank] ) with its signal peptide, and eight clones of transgenic poplars overexpressing galactanase (Tv6GAL, AB104894) including arabinofuranosidase (RsAraf1, AB234292) with Arabidopsis arabinogalactan protein signal (AtAGP4, AF082301). To assess the expression of the transgene, we used a polyclonal antibody against the recombinant products of AaXEG2, HvXYL1, AtCel1, and Tv6GAL. The transgenic poplars overexpressing xyloglucanase gave a 28-kDa signal on a Western blot (Park et al., 2004). Eight independent plants overexpressing xylanase gave a 40-KDa signal, four overexpressing cellulase gave a 50-KDa signal, and eight overexpressing galactanase gave a 40-KDa signal.
The specific degradation of each polysaccharide was confirmed by methylation analyses of the matrix polysaccharides from the upper side of the stem (the tension side of the secondary xylem) compared with those from the normal woods (Table 1). A decrease in each glycosyl linkage should correspond to the action of each specific enzyme according to the following: the 4,6-linked glucosyl linkage was decreased for xyloglucanase, the 4-linked xylosyl linkage for xylanase, terminal arabinofuranoses for arabinofuranosidase, the 3,6-linked galactosyl linkage for galactanase, and noncrystal 4-linked glucosyl and mannosyl linkages for cellulase. It should be noted that Arabidopsis cellulase (AtCel9B1) hydrolyzes xyloglucan very slowly, as the reaction efficiency of plant cellulase for xyloglucan is very low compared with the efficiencies for carboxymethylcellulose, phosphor-swollen cellulose, and (1
3), (14)-β-glucan (Nakamura and Hayashi, 1993). It should also be noted that the overexpression of cellulase does not contribute to a lack of cellulose but could instead cause the degradation of glucomannan and the trimming of amorphous regions of cellulose microfibrils.
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Figure 1 shows their shapes at day 15 and the bending progression over the time course. The overexpression of xyloglucanase prevented the basal region of the stem from bending upward, whereas the expression of other endoglycanases had no effect on the upward bending of the basal regions. In addition, the transgenic poplar overexpressing xyloglucanase grew faster, but their stems twisted to the left or right side, while the stems of other poplars bent straight upward. Although the trimming of cellulose microfibrils by overexpressed cellulase caused the loosening of xyloglucan cross-linkages in Arabidopsis thaliana (Park et al., 2003), poplar plants overexpressing cellulase did not show the same response as those overexpressing xyloglucanase under gravitropical stress. When longitudinal strain was measured on the upper side of the stem by using a strain-gauge, there was a very small shrinkage in the transgenic poplar overexpressing xyloglucanase (Figure 2). However, the wild-type poplar shrank much more than the transgenic, with repeating diurnal alteration, shrinking during day time and relaxation in night time.
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That xyloglucan is required for the bending of poplar stems was further confirmed by the fact that 4,6-linked glucosyl residues, derived from xyloglucan 1,4-β-glucan backbones, were markedly increased in the tension side of all poplars tested except those overexpressing xyloglucanase (Table 1). However, its presence is not highly involved in the growth of the primary wall during differential growth between the upper and lower regions of the elongating stem, because the apical regions of all transgenic plants bent upward within a day after horizontal placement.
Comparison of Tension Wood between Wild-Type and Transgenic Stems Overexpressing Xyloglucanase
Upon analysis of the wood of the transgenic poplars and that of the wild-type plants, G-layer-like cellulose fibers had formed inside the S2 layer of the fiber cells of poplars overexpressing xyloglucanase. The upper side of the secondary xylem stained blue-purple with ZnCl2/KI/I2 in cross-sections of both wild-type and transgenic plants (Figure 3A). Enlarging these cross-sections revealed the strong blue-purple coloring reaction indicative of a marked increase in cellulose formation (Figure 3B) with no red staining of phloroglucinol/HCl for lignin (Krishnamurthy, 1999) (Figure 3C). Although both secondary xylems contained similar amounts of cellulose, the thickness of the G-layer at the transverse section was wider in the transgenic than in the wild-type plants (Figure 3D and Table 2). In addition, the cellulose microfibril angle was straighter along the axis of the fiber cells in the transgenic plants (Table 2). By scanning electron microscopy, immunogold labeling of xyloglucan using an anti-xyloglucan antibody was frequently observed at the inner surfaces of the growing S2 layers and G-layers in the wild-type plants (Figure 3E and 3F). Xyloglucan was always found at the inner surface in both immature and mature G-layers, as well as in the primary cell walls (Figure 3G). It should be noted that poplar xyloglucan is highly fucosylated (Hayashi and Takeda, 1994), allowing the antibody (CCRC-M1) to detect the polysaccharide (Puhlmann et al., 1994). The immunofluorescence labeling of xyloglucan decreased in the layered G-layers, probably because the layering might involve fixing microfibrils and become so tight that it becomes difficult for the antibody to access and bind xyloglucan between the microfibrils.
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When we measured the growth stress of the tension wood using the strain-gauge method (Yoshida and Okuyama, 2002), the upper side of the xylem revealed less tensile stress: the transgenic poplars overexpressing xyloglucanase exhibited over a 25% reduction of the growth strain of the wild-type plants during tension wood formation (Table 2). We conclude that xyloglucan probably functions in creating the tensile stress of the G-layer cellulose microfibrils.
Visualization of Xyloglucan, XET, and Its Activities in G-Layer
Immunofluorescence staining of poplar xyloglucan endotransglucosylase (XET) revealed strong labeling at the tension (wood) side and weak labeling at the opposite side (Supplemental Figure 1A and 1B). To examine whether XET and xyloglucan were present together, double-labeling experiments were further performed in the tension wood and G-layer using rabbit anti-poplar XET and mouse anti-xyloglucan antibodies. XET labeling was observed on the longitudinally and transversely sliced cut surface of G-layers shrunken, and xyloglucan labeling in those of the primary wall (Figure 4A and 4B). Then, the double labeling was more clearly visible in the G-layer-sliced ring, which was shrunken out from the thinly sliced section after the release of the strong tensile stress. XET labeling was apparent on the transversely sliced cut surface of the G-layer, and xyloglucan labeling became visible on the inner surface of the G-layer, along with very faint signals on the cut and outer surfaces (Figure 4C). Xyloglucan was more abundant at the inner surface than at the outer surface, probably because XET had endotransglucosylated xyloglucans to fix cellulose microfibrils to the inner surface during layering. The G-layer might be layered so tightly that antibodies could not pass through for staining. After removing the G-layers, a faint xyloglucan signal was detected in the inner surface of the S2 layer, despite its strong signal in the primary wall and the lack of XET signal (Figure 4D). Negative controls labeled with the secondary antibodies are shown in Supplemental Figure 2. Based on methylation analysis, the isolated G-layer yielded 4-linked glucose, 4,6-linked glucose, 3,6-linked galactose, 4-linked xylose, 4-linked mannose, T-xylose, T-fucose, and T-glucose in the ratio of 88:5:3:2:2:1:0.1:0.1. After extracting the G-layer with 24% KOH, 13% of the carbohydrate was solubilized, and the insoluble residue indicated only 4-linked glucose according to methylation analysis. Therefore, the G-layer contained xyloglucan at a level of 7.0 ± 2.7% of the total carbohydrates. We then extracted proteins from the G-layers and subjected them to two-dimensional gel electrophoresis. After Western blotting, one signal appeared at 
32 kDa in molecular weight with a pI of 8.1 (Figure 5). When the area corresponding to the signal was digested with trypsin and subjected to LC–MS/MS, the peptide signals matched one of the XET isozymes (pttXTH6) in Table 3. Based on the phylogenetic trees of the deduced amino acid sequences, pttXTH6 belongs to the XTH family group 1, which may not exhibit xyloglucan hydrolysis (Rose et al., 2002). Based on proteomic analysis, some spots corresponded to the gene products that are differentially regulated in tension wood (Andersson-Gunneras et al., 2006).
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Furthermore, XET activity was detected in situ using either whole xyloglucan (50 kDa) labeled with fluorescein or using xyloglucan heptasaccharide (XXXG) labeled with Texas red (Takeda et al., 2002). The incorporation of whole xyloglucan was observed more widely in immature to mature xylems at the tension side than was the incorporation of XXXG into the two to three cell rows from the cambial region (Figure 4E). The incorporation of whole xyloglucan began at the inner surface S1 and S2 layers and was retained at entire G-layers during layer formation, concurrent with its disappearance from the S1 and S2 layers of mature fiber cells. On the opposite side, where G-layers did not form, both whole xyloglucan and XXXG were incorporated into the same narrow region (Figure 4F). Nevertheless, whole xyloglucan was incorporated into the inner surfaces of the S1 and S2 layers in this region (Supplemental Figure 1C and 1D). These results show that the incorporation of whole xyloglucan occurs strongly at the inner surfaces of all wall layers, while that of XXXG occurs in the primary wall.
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DISCUSSION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Functional Role of Xyloglucan
The immunolocalization of xyloglucan to the inner surface of the G-layers (Figures 3 and 4) showed that, during tension wood development, the de novo synthesized cellulose G-layer microfibrils could be fixed by xyloglucan cross-bridges, in which secreted xyloglucans could be used in transglucosylation reactions and could potentially be linked to xyloglucans in the pre-existing wall. Xyloglucan would be expected to tighten the G-layer, probably by ‘stapling’ its microfibrils into the whole primary wall, with the S1 and S2 layers bearing the tensile stress. Since the 24% KOH-insoluble residues of the G-layer did not indicate a 4,6-linked glucose, it is unlikely that covalent bonding occurs between xyloglucan and cellulose, as suggested by Hrmova et al. (2007). A fiber cell should generate a strong growth strain towards the inside along its cell axis in the upper side of stem while xyloglucan withstands the tensile stress within the G-layer. Then, associated fiber cells could pull each other in the tension side to bend the stem upwards. However, the constitutive degradation of xyloglucan might cause the G-layer microfibrils to prevent binding to the pre-existing walls. This would explain why the overexpression of xyloglucanase induced the G-layer to orient at a smaller angle to the cell axis than the angle that occurs in wild-type plants despite maintaining the same cellulose content. If the cellulose microfibrils in the layer are not fixed to pre-existing walls in the fiber cells, stress relaxation should occur in the fiber cells.
Although the leaves and petioles protrude at right angles against the stem axis in the wild-type, the petioles of transgenic poplars lean downwards. This is probably because the petiole tensile stress is also lower due to constitutive degradation of xyloglucan in the transgenic poplars. Because endotransglucosylase activity at the inner surface of wall layers was found for whole xyloglucan but not for XXXG, the transferase type of the enzyme could potentially form an enzyme-accepting xyloglucan complex in the inner surface at the onset of integration of the secreted xyloglucan. This is in agreement with a pea-split test (Takeda et al., 2002), in which the integration of whole xyloglucan caused by the action of wall-bound xyloglucan endotransglycosylase suppressed cell elongation, while that of its fragment oligosaccharide (XXXG) accelerated elongation. When xyloglucan is secreted into the inner surface during wall layering, xyloglucan endotransglucosylase could bind the internal region of the secreted xyloglucan, cleave it, and transfer this newly generated reducing end to the non-reducing end of wall-bound xyloglucan. Thus, the xyloglucans in the walls should form a larger network during endotransglucosylation at the inner surface, wherein xyloglucan terminals remain more studded than at the outer surface (Figure 4C). We conclude that the endotransglucosylase should work not as a wall-loosening enzyme, but instead as a wall-tightening enzyme in fiber cells by fixing cellulose microfibrils. This agrees with a previous finding (Bourquin et al., 2002; Mellerowicz et al., 2008) that xyloglucan endotransglucosylation could reinforce the cross-linking of cellulose microfibrils between poplar primary wall and S1 layers. Since fluorescent XXXG was incorporated into the G-layers of non-fresh sections, XET might be diffused in the walls in the absence of tension stress.
Tensile Stress
Cellulose biosynthesis could occur in a loose manner on the inner surface of the G-layer under high transpiration during the day and in a tighter manner between the plasma membrane and the G-layer under high turgor pressure at night on the inner surface of the wall (Yoshida et al., 2003). As hemicelluloses are deposited between the cellulose microfibrils at the inner surface of the secondary wall in turgid cells at night (Yoshida et al., 2000; Hosoo et al., 2003), endotransglucosylation of xyloglucan probably occurs in the G-layer when cellulose microfibrils are pushed towards the pre-existing walls. Therefore, the microfibrils might be fixed to the walls at night, despite the fact that cellulose biosynthesis occurs during the day. Yoshida et al. (2000, 2003) stated that differentiating tension wood causes cell expansion by incorporating more water during the night than does differentiating normal wood. If the microfibrils can be fixed by xyloglucans when the cells expand at night, the microfibrils accumulated in the layer could support tensile stress. In fact, the strain due to the stress was increased gradually with diurnal changes, decreasing at night and increasing during day time in the wild-type poplar (Figure 2). For this reason, the thickness of the G-layer in the transverse section was thinner in the wild-type than in the transgenic plants, and the cellulose microfibrils were fixed at a specific angle along the fiber cell axis in the wild-type plants. The xyloglucans occur in the secondary wall, where the glucans could fix the microfibrils to the inner surface of the wall to withstand the tensile stress generated within the G-layer.
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METHODS |
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Production of Transgenic Poplars
Transgenic poplars overexpressing xyloglucanase (AtXEG, AY160774 [GenBank] ) with the signal peptide of PaPopCel1 (Park et al., 2004) and cellulase (AtCel1, X98544 [GenBank] : Shani et al., 2004), under control of the CaMV35S promoter, were reported previously. The signal peptide of PaPopCel1 (D32166 [GenBank] ) was used for secretion. The poplar overexpressing xylanase was generated using plasmids according to the following. The structural gene of endo-1,4-β-xylanase (HvXYL, U59312 [GenBank] ) was amplified by PCR using the first strand of Horderum vulgare cDNA pMX1 (1.6A) (Banik et al., 1996) as a template and using a forward primer containing a BamHI site (5'-GAGGATCCCAACGAGACCCTGGTG-3’) and a reverse primer containing an internal SacI site (5'-GAAGAGCTCACAACAATTATCCCTTGACG-3’), both of which are complementary to the DNA sequence of xylanase (accession number U59312). The amplified PCR product was excised with BamHI and SacI and inserted into the binary vector pBE2113–GUS under control of the CaMV35S promoter and E12-6
enhancer sequences. The poplar overexpressing both arabinofuranosidase and galactanase was generated using plasmids according to the following. The structural gene of 
-L-arabinofuranosidase was amplified by PCR using the first strand from RSAraf1 (AB234292
[GenBank]
) cDNA from Paphanus sativus (Kotake et al., 2006) as a template using a forward primer containing a BamHI site (5'-CGCGGATCCCAAGAAGAATACTG-3’) and a reverse primer containing an internal SacI site, both of which are complementary to the DNA sequence of arabinofuranosidase. The signal peptide gene of arabinogalactan protein was amplified by PCR using the first strand from AtAGP4 (AF082301
[GenBank]
) cDNA from Arabidopsis thaliana as a template using a forward primer containing a SpeI site (5'-ATGGGTTCCAAGATTGTCCAAG-3’) and a reverse primer containing an internal BamHI site (5'-GCGGATCCAGCGAGTGCTGAAGTGGCGA-3’), both of which are complementary to the signal peptide. The structural gene of 1,6-β-galactanase was amplified by PCR from the first strand from Tv6GAL (AB104898
[GenBank]
) cDNA from Trichoderma viride11 as a template using a forward primer containing a BamHI site (5'-GGATCCATGGACACCACGCTTACCATC-3’) and a reverse primer containing an internal SacI site (5'-GAGCTCATTGCAACACAACGCC-3’), both of which are complementary to the DNA sequence of galactanase. The amplified PCR products for arabinofuranosidase and the signal peptide with the galactanase were excised and inserted between the XbaI and SacI sites in the binary vector pBE2113–GUS under the control of the CaMV35S promoter E12-6 enhancer sequences (Mitsuhara et al., 1996). The plasmid harboring either xylanase or arabinofuranosidase with galactanase cDNA was electrophorated into Agrobacterium tumefaciens LBA4404 (Kotake et al., 2006, 2004).
Isolation of G-Layers
Approximately 5-cm diameter sections of poplar tension wood were used to isolate G-layers. The wood was transversely sliced into 14-µm sections that were immersed in 95% ethanol to shrink the layers (Norberg and Meier, 1966; Furuya et al., 1970). The layers, shrunken out of the sections by treatment with ultrasonic waves for 5 min, were obtained by successive filtration through 10 and 30-µm nylon meshes.
Antibodies
The anti-xyloglucan mouse monoclonal antibody (CCRC-M1) recognizing the terminal fucosyl residue of xyloglucan (Puhlmann et al., 1994) was kindly provided by Dr M. Hahn (University of Georgia, USA). The rabbit polyclonal antibody against poplar XET (pttXTH34 gene product; accession number AF515607, originally pttXET16A) (Kallas et al., 2005) was kindly provided by Dr H. Brumer (Royal Institute of Technology, Sweden). As secondary antibodies, Alexa Fluorâ-488 goat anti-mouse IgG and –568 goat anti-rabbit IgG (Invitrogen, USA) were used to detect anti-xyloglucan and anti-XET antibodies, respectively.
Wall Analysis
The dried xylem was ground in liquid nitrogen and the resulting powder was freeze-dried. Each sample was successively extracted four times with water and 24% KOH containing 0.1% NaBH4. The insoluble wall residue (cellulose fraction) was washed twice with water and solubilized with ice-cold 72% sulfuric acid. The amount of cellulose was also determined by measuring the acid-insoluble residue; the samples were extracted with acetic/nitric reagent (80% acetic acid/concentrated nitric acid, 10:1) in a boiling water bath for 30 min (Updegraff, 1969). The resulting insoluble material was washed in water and solubilized with ice-cold 72% sulfuric acid. Total sugar in each fraction was determined by the phenol–sulfuric acid method (Dubois et al., 1956). The alkali-soluble fraction was neutralized, dialyzed, and freeze-dried for use in methylation analysis (Hayashi, 1989b). Partially methylated alditol acetates were analyzed using an Agilent gas chromatography–mass spectrometer with a glass capillary column (0.25 x 15 m) of DB-225. Lignin content was determined by the Klason method (Chiang and Funaoka, 1990).
Strain Measurement
The growth stress of tension wood was applied by fixing the poplar plants (about 2 m in height) to poles inclined at 30°. They were then grown for 4 months in a greenhouse. The strain-gauge method (Yoshida et al., 2003) was used to measure the longitudinal released strain of growth stress, namely growth strain, at the outer surface of the secondary xylem after removal of 10-mm squares of outer bark. Strain gauges (2 mm long) were glued to the smooth surface of the xylem longitudinally against the stem axis. The growth stresses were then measured after releasing stress by cutting off the xylem with pruning shears.
Microscopy
The sections were observed by conventional light microscopy, during which cellulose and lignin were stained by the zinc–chloride–iodine (ZnCl2/KI/I2) and phloroglucinol–HCl methods, respectively (Krishnamurthy, 1999). Specimens were prepared for transmission electron microscopy by the conventional ultra-thin sectioning method. After fixation in 3.5% glutaraldehyde and 1% OsO4, dehydration with graded ethanol, and embedding in Spurr's resin, the sections were double stained with uranyl acetate and lead citrate prior to analysis. Statistical analyses were carried out using data from the micrographs of G-layers in the transverse sections of mature fiber cells. The microfibril angle of G-layers was determined by using the programmed polarizing microscopy. The innermost surface of the developing fiber wall was observed by field emission scanning electron microscopy (FE–SEM), as described (Hosoo et al., 2002). Radial sections were prepared using freezing/sliding microtome, and some of the sections were immunogold labeled with an anti-xyloglucan antibody. The sections freeze-dried in t-butyl alcohol were coated with 1-nm-thick osmium or 1.5-nm-thick platinum–palladium. The innermost surface was observed by FE–SEM (S-4500, Hitachi) at an accelerating voltage of 1.5 kV and a working distance of 5 nm.
The isolated G-layers were fixed with 4% paraformaldehyde in 10 mM PIPES buffer on glass slides for 60 min. After the layers were washed five times in PIPES buffer, we treated them with blocking solution containing 0.1 M glycine, 1.0% BSA, and 0.05% Triton X-100 in phosphate-buffered saline (PBS) for 60 min. Next, the layers were washed in PBS and incubated with anti-xyloglucan and anti-poplar XET (pttXTH34 gene product) antibodies for 90 min. After another wash in PBS, the samples were incubated with the secondary antibodies of Alexa Fluorâ-488 (green) goat anti-mouse IgG and –568 (red) goat anti-rabbit IgG for 90 min. The G-layers were examined using a confocal laser scanning microscope (Leica) equipped with an argon/krypton laser. The shape and thickness of the G-layer were checked by serial sections from poplar tension wood (Clair et al., 2005).
G-Layer Proteins
The freeze-dried G-layers were re-suspended in rehydration buffer (4% CHAPS containing 1% dithiothreitol (DTT)) and the proteins were extracted by ultrasonic waves for 3 min. The extract was purified with the 2-D Clean-Up Kit (Amersham, USA) and the resulting pellet was used for first-dimension electrophoresis on an IPGphor isoelectric focusing system according to the instructions provided by the manufacturer (Amersham). Proteins were focused using a Multiphor II electrophoresis system (Amersham) and then subjected to second-dimension SDS–PAGE. After electrophoresis, the two-dimensional gels were fixed and either stained with silver or Western blotted using an antibody against poplar XET.
Protein Identification by Mass Spectrometry
Protein spots were excised from silver-stained gels and subjected to trypsin digestion. The digests were separated and analyzed using Magic LC (Michrom Bioresources, USA) and Q-Tof2 (Micromass, UK) systems, in which mass data were processed using the Mascot search software (Matrix Science, UK). Searches were done with mass measurements of 0.5 Da in MS mode and 0.3 Da in MS/MS mode, in which the identities given for each sequence similarity, listed in Table 3, are defined as peptide sequence matches by comparison with peptide sequences from a particular database (Geisler-Lee et al., 2006).
Fluorescent Xyloglucans
Fluorescent xyloglucan (Takeda et al., 2002) was prepared by dissolving 10 mg of CNBr and 20 mg of pea xyloglucan (50 kDa) in 1 ml of water and adjusting the pH to 11.0 by adding NaOH. The activated polysaccharide was incubated with 4 mg fluorescein-amine overnight at room temperature. The fluorescein-labeled xyloglucan was purified by gel filtration on a Sephadex G-50 column. Calculations showed that 1 µmol of fluorescein incorporated into 110 µmol of sugar residues, corresponding to a substitution rate of 3.7 mol fluorescein per mol of xyloglucan. Fluorescent XXXG was prepared by conjugating Texas red-hydrazine to the XXXG (Fry, 1997). The reaction products were subjected to gel filtration on a Bio-Gel P-2 column, and the fluorescent carbohydrate fraction was further purified by paper chromatography using Whatman 3MM paper with 1-propanol/ethyl acetate/water, at a ratio of 3:2:1, and eluted with water from the excised paper.
In Situ Xyloglucan Endotransglucosylase Activity
The transverse sections (50 µm) of xylems with fluorescent derivatives were incubated for 30 min in 300 µl of 2 mM MES/KOH buffer (pH 6.2) containing 0.2 mM fluorescent whole xyloglucan or 9 mM fluorescent XXXG, while shaking in the dark at 23°C. The sections that were incubated with whole xyloglucan were washed three times in 0.01 M NaOH for 30 min (Takeda et al., 2002), and those incubated with XXXG were washed with 5% formic acid in 90% ethanol for 5 min followed by 5% formic acid for 5 min (Vissenberg et al., 2000). The sections were washed twice with water and examined using a Zeiss Axioscope microscope equipped with epifluorescence illumination.
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SUPPLEMENTARY DATA |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Supplementary Data are available at Molecular Plant Online.
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FUNDING |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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This work was supported by the Program for the Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN), Japan Society for the Promotion of Science, KAKENHI (No. 17380108, 19208016, 19580188 and 19405030), and Global Center of Excellence Program (E-04): In Search of Sustainable Humanosphere in Asia and Africa.
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Acknowledgements |
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We thank E. Mellerowicz, H. Brunner, B. Sundberg, and N. Nishikubo for supplying the antibody against poplar XET; H. Yano for numerous discussions about tensile stress; M. Hahn for supplying the antibody against xyloglucan; G.B. Fincher for supplying Hordeum vulgare cDNA for xylanase; and T. Kotake and K. Tsumuraya for the plasmid harboring cDNAs for arabinofuranosidase and galactanase. No conflict of interest declared.
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