The Structure of Plant Cell Walls: IV. A Structural Comparison of the Wall Hemicellulose of Cell Suspension Cultures of Sycamore (Acer PseudoPlatAnus) and of Red Kidney Bean (Phaseolus Vulgaris)

The molecular structure and chemical properties of the hemicellulose present in the isolated cell walls of suspension cultures of sycamore (Acer pseudoplatanus) cells has recently been described by Bauer et al. (Plant Physiol. 51: 174-187). The hemicellulose of the sycamore primary cell wall is a xyloglucan. This polymer functions as an important cross-link in the structure of the cell wall; the xyloglucan is hydrogen-bonded to cellulose and covalently attached to the pectic polymers.     

Increase in XET activity in bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) cells habituated to dichlobenil

Bean (Phaseolus vulgaris L.) cells have been habituated to grow in lethal concentrations of dichlobenil (DCB), a specific inhibitor of cellulose biosynthesis. Bean callus cells were successively cultured in increasing DCB concentrations up to 2 μM. The 2-μM DCB habituated cells were impoverished in cellulose and xyloglucan, had an increased xyloglucan endotransglucosylase (XET; EC 2.4.1.207) activity, together with an increased growth rate and a decreased molecular size of xyloglucan. However, the application of lethal concentrations of two different cellulose-biosynthesis inhibitors (DCB and isoxaben) for a short period of time produced little effect on XET activity and xyloglucan molecular size. We propose that the weakening of plant cell wall provoked by decrease in cellulose content might promote the xyloglucan tethers and increase the ability of xyloglucan to bind to cellulose in order to give rigidity to the wall.     

Hypergravity increases the molecular mass of xyloglucans by decreasing xyloglucan-degrading activity in azuki bean epicotyls.

Elongation growth of dark-grown azuki bean (Vigna angularis Ohwi et Ohashi cv. Takara) epicotyls was suppressed by hypergravity at 30 x g and above. Acceleration at 300 x g significantly decreased the mechanical extensibility of cell walls. The amounts of cell wall polysaccharides (pectin, hemicellulose-II and cellulose) per unit length of epicotyls increased under the hypergravity condition. Hypergravity also increased the amounts and the weight-average molecular mass of xyloglucans in the hemicellulose-II fraction, while decreasing the activity of xyloglucan-degrading enzymes extracted from epicotyl cell walls. These results suggest that hypergravity increases the amounts and the molecular mass of xyloglucans by decreasing xyloglucan-degrading activity. Modification of xyloglucan metabolism as well as the thickening of cell walls under hypergravity conditions seems to be involved in making the cell wall mechanically rigid, thereby inhibiting elongation growth of azuki bean epicotyls.     

Purification of xyloglucan endotransglycosylase based on affinity sorption of the active glycosyl-enzyme intermediate complex to cellulose.

Xyloglucan endotransglycosylase (XET) catalyzes the cleavage of xyloglucan (XG) molecules by a transglycosylation mechanism involving two steps: (a) endocleavage of the beta-(1,4)-linked polyglucosyl backbone of the xyloglucan molecule with formation of a glycosyl-enzyme intermediate; (b) transfer of the glycosyl residue from the intermediate to the C-4 position of the nonreducing end glucosyl unit of another molecule of XG or an XG-derived oligosaccharide with liberation of the enzyme (Z. Sulová et al., 1998, Biochem. J. 330, 1475-1480). The formation of a relatively stable active complex of XET with XG and the tendency of xyloglucan to bind tightly via hydrogen bonds to cellulose were exploited in the present method of purification of XET. Crude extracts from nasturtium (Tropaeolum majus) cotyledons and other plant sources containing the enzyme were mixed with XG in order to form the XET:XG complex, which was applied onto cellulose. Unadsorbed proteins were removed by washing and the XET was released from the adsorbed XET:XG complex by transglycosylation of its glycosyl moiety to added XG-derived oligosaccharides. The described procedure resulted in an over 100-fold increase in specific activity of XET in a single step. Further purification of the enzyme to homogeneity was achieved by gel-permeation chromatography on Bio-Gel P30. Similar procedure could be used for purification of XET from other plant sources, such as lentil (Lens culinaris) seeds, pea (Pisum sativum) epicotyls, and supernatant of suspension-cultured Catharanthus roseus cells.     

WAXS and 13C NMR study of Gluconoacetobacter xylinus cellulose in composites with tamarind xyloglucan

- Model composites, produced using cellulose from stationary cultures of the bacterium Gluconoacetobacter xylinus and tamarind xyloglucan, were examined by wide-angle X-ray scattering (WAXS) and CP/MAS solid-state (13)C NMR spectroscopy. The dominant crystallite allomorph of cellulose produced in culture media with or without xyloglucan was cellulose I(alpha) (triclinic). The presence of xyloglucan in the culture medium reduced the cross-section dimensions of the cellulose crystallites, but did not affect the crystallite allomorph. However, when the composites were refluxed in buffer, the proportion of cellulose I(beta) allomorph increased relative to that of cellulose I(alpha). In contrast, cellulose I(alpha) remained the dominant form when cellulose, produced in the absence of xyloglucan, was then heated in the buffer. Hence the presence of xyloglucan has a profound effect on the formation of the cellulose crystallites by G. xylinus.     

Enhancement of growth by expression of poplar cellulase in Arabidopsis thaliana

To study the role of cellulose and cellulase in plant growth, we expressed poplar cellulase (PaPopCel1) constitutively in Arabidopsis thaliana. Expression increased the size of the rosettes due to increased cell size. The change in growth was accompanied by changes in biomechanical properties due to cell wall structure indicative of decrease in xyloglucan cross-linked with cellulose microfibrils by chemical analysis and nuclear magnetic resonance (NMR) spectra. The result supports the concept that the paracrystalline sites of cellulose microfibrils are attacked by poplar cellulase to loosen xyloglucan intercalation and this irreversible wall modification promotes the enlargement of plant cells.     

Real-Time Imaging of Cellulose Reorientation during Cell Wall Expansion in Arabidopsis Roots

Cellulose forms the major load-bearing network of the plant cell wall, which simultaneously protects the cell and directs its growth. Although the process of cellulose synthesis has been observed, little is known about the behavior of cellulose in the wall after synthesis. Using Pontamine Fast Scarlet 4B, a dye that fluoresces preferentially in the presence of cellulose and has excitation and emission wavelengths suitable for confocal microscopy, we imaged the architecture and dynamics of cellulose in the cell walls of expanding root cells. We found that cellulose exists in Arabidopsis (Arabidopsis thaliana) cell walls in large fibrillar bundles that vary in orientation. During anisotropic wall expansion in wild-type plants, we observed that these cellulose bundles rotate in a transverse to longitudinal direction. We also found that cellulose organization is significantly altered in mutants lacking either a cellulose synthase subunit or two xyloglucan xylosyltransferase isoforms. Our results support a model in which cellulose is deposited transversely to accommodate longitudinal cell expansion and reoriented during expansion to generate a cell wall that is fortified against strain from any direction.The walls of growing plant cells must fulfill two simultaneous and seemingly contradictory requirements. First, they must expand to accommodate cell growth, which is anisotropic in many tissues and determines organ morphology. Second, they must maintain their structural integrity, both to constrain the turgor pressure that drives cell growth and to provide structural rigidity to the plant. These requirements are met by constructing primary cell walls that can expand along with growing cells, whereas secondary cell walls are deposited after cell growth has ceased and serve the latter function.One of the major constituents of both types of cell walls is cellulose, which exists as microfibrils composed of parallel β-1,4-linked glucan chains that are held together laterally by hydrogen bonds (Somerville, 2006). Microfibrils are 2 to 5 nm in diameter, can extend to several micrometers in length, and exhibit high tensile strength that allows cell walls to withstand turgor pressures of up to 1 MPa (Franks, 2003). In vascular plants, cellulose is synthesized by a multimeric cellulose synthase (CESA) complex composed of at least three types of glycosyl transferases arranged into a hexameric rosette (Somerville, 2006). After delivery to the plasma membrane, CESA initially moves in alignment with cortical microtubules (Paredez et al., 2006), but its trajectory can be maintained independently of microtubule orientation. For example, in older epidermal cells of the root elongation zone in Arabidopsis (Arabidopsis thaliana), cellulose microfibrils at the inner wall face are oriented transversely despite the fact that microtubules reorient from transverse to longitudinal along the elongation zone (Sugimoto et al., 2000), suggesting that microtubule orientation and cellulose deposition are independent in at least some cases.Depending on species, cell type, and developmental stage, cellulose microfibrils may be surrounded by additional networks of polymers, including hemicelluloses, pectins, lignin, and arabinogalactan proteins (Somerville et al., 2004). Hemicelluloses are composed of β-1,4-linked carbohydrate backbones with side branches and include xyloglucans, mannans, and arabinoxylans. Xyloglucan is thought to interact with the surface of cellulose and form cross-links between adjacent microfibrils (Vissenberg et al., 2005). In some cell types, pectin or lignin may also participate in cross-linking or entrapment of other cell wall polymers. It is unclear how the associations between networks of different cell wall components are relaxed to allow for cell wall expansion during growth.Several models have been proposed for the behavior of cell wall components during wall expansion. The passive reorientation hypothesis (also called the multinet growth hypothesis; Preston, 1982) postulates that in longitudinally expanding cells, cellulose microfibrils are synthesized in a transverse pattern and are then reoriented toward the longitudinal axis due to the strain generated by turgor pressure (Green, 1960). This phenomenon has been observed in the multicellular alga Nitella (Taiz, 1984). In higher plants, there is less direct evidence for passive reorientation, and another hypothesis holds that wall expansion involves active, local, and controlled remodeling of cellulose microfibrils along a diversity of orientations (Baskin, 2005). Such remodeling could be achieved by proteins such as xyloglucan endotransglycosylases (XETs), which break and rejoin xyloglucan chains, and expansins, which loosen cell walls in vitro in a pH-dependent manner (Cosgrove, 2005). Marga et al. measured cellulose microfibril orientation at the innermost layer of the cell wall before and after in vitro extension and did not observe reorientation (Marga et al., 2005). This suggests that processes other than microfibril reorientation might be involved in wall expansion, at least under certain circumstances or in some wall layers. Thus, the degree to which cellulose microfibrils are reoriented after their synthesis during wall expansion has remained unclear.One difficulty in resolving this problem has been the inability to directly image cellulose microfibrils in the growing cell wall. Existing methods to assess cellulose structure and orientation in plant cell walls are limited by the low contrast of cellulose in transmission electron microscopy, the ability to image only the surface of the wall using field emission scanning electron microscopy, and the use of polarized light microscopy in combination with dyes such as Congo red to measure only the bulk orientation of cellulose microfibrils (Baskin et al., 1999; Sugimoto et al., 2000; Verbelen and Kerstens, 2000; MacKinnon et al., 2006). In addition, the sample manipulation required for the former two methods has the potential to introduce artifacts (Marga et al., 2005). Although cellulose microfibril orientation differs at the inner and outer surfaces of the cell wall (Sugimoto et al., 2000) and presumably changes over time, the dynamics of cellulose reorientation during cell wall expansion have not been observed to date.In this study, we tested fluorescent dyes for their potential to allow imaging of cellulose distribution in the walls of Arabidopsis seedlings by confocal microscopy. We used one of these dyes to characterize the distribution of cellulose in wild-type root cells and in mutants with reduced cellulose or xyloglucan. By directly observing the fine structure of cellulose over time in growing wild-type root cells, we concluded that cellulose microfibrils in these cells reorient in a transverse to longitudinal direction as predicted by the passive reorientation hypothesis.     

Pea Xyloglucan and Cellulose: VI. Xyloglucan-Cellulose Interactions in Vitro and in Vivo

Since xyloglucan is believed to bind to cellulose microfibrils in the primary cell walls of higher plants and, when isolated from the walls, can also bind to cellulose in vitro, the binding mechanism of xyloglucan to cellulose was further investigated using radioiodinated pea xyloglucan. A time course for the binding showed that the radioiodinated xyloglucan continued to be bound for at least 4 hours at 40°C. Binding was inhibited above pH 6. Binding capacity was shown to vary for celluloses of different origin and was directly related to the relative surface area of the microfibrils. The binding of xyloglucan to cellulose was very specific and was not affected by the presence of a 10-fold excess of (1→2)-β-glucan, (1→3)-β-glucan, (1→6)-β-glucan, (1→3, 1→4)-β-glucan, arabinogalactan, or pectin. When xyloglucan (0.1%) was added to a cellulose-forming culture of Acetobacter xylinum, cellulose ribbon structure was partially disrupted indicating an association of xyloglucan with cellulose at the time of synthesis. Such a result suggests that the small size of primary wall microfibrils in higher plants may well be due to the binding of xyloglucan to cellulose during synthesis which prevents fasciation of small fibrils into larger bundles. Fluorescent xyloglucan was used to stain pea cell wall ghosts prepared to contain only the native xyloglucan:cellulose network or only cellulose. Ghosts containing only cellulose showed strong fluorescence when prepared before or after elongation; as predicted, the presence of native xyloglucan in the ghosts repressed binding of added fluorescent xyloglucan. Such ghosts, prepared after elongation when the ratio of native xyloglucan:cellulose is substantially reduced, still showed only faint fluorescence, indicating that microfibrils continue to be coated with xyloglucan throughout the growth period.     

Lack of α-Xylosidase Activity in Arabidopsis Alters Xyloglucan Composition and Results in Growth Defects

Xyloglucan is the main hemicellulose in the primary cell walls of most seed plants and is thought to play a role in regulating the separation of cellulose microfibrils during growth. Xylose side chains block the degradation of the backbone, and α-xylosidase activity is necessary to remove them. Two Arabidopsis (Arabidopsis thaliana) mutant lines with insertions in the α-xylosidase gene AtXYL1 were characterized in this work. Both lines showed a reduction to undetectable levels of α-xylosidase activity against xyloglucan oligosaccharides. This reduction resulted in the accumulation of XXXG and XXLG in the liquid growth medium of Atxyl1 seedlings. The presence of XXLG suggests that it is a poor substrate for xyloglucan β-galactosidase. In addition, the polymeric xyloglucan of Atxyl1 lines was found to be enriched in XXLG subunits, with a concomitant decrease in XXFG and XLFG. This change can be explained by extensive exoglycosidase activity at the nonreducing ends of xyloglucan chains. These enzymes could thus have a larger role than previously thought in the metabolism of xyloglucan. Finally, Atxyl1 lines showed a reduced ability to control the anisotropic growth pattern of different organs, pointing to the importance of xyloglucan in this process. The promoter of AtXYL1 was shown to direct expression to many different organs and cell types undergoing cell wall modifications, including trichomes, vasculature, stomata, and elongating anther filaments.The primary wall that surrounds the growing cells of plants has to be able to extend in response to turgor pressure. This process needs to be tightly regulated to avoid a mechanical failure of the wall. The direction of expansion also needs to be controlled so that different cell types can develop their particular morphology. In addition, the growth of the different tissues in an organ has to be tightly coordinated so that it can achieve its final shape (Baskin, 2005). The mechanical behavior of the expanding cell wall has been likened to a fiber-reinforced composite, with crystalline cellulose microfibrils embedded in an amorphous matrix of hemicellulose and pectin. How this works at the molecular level is still the subject of much research and speculation (Geitmann and Ortega, 2009).Xyloglucan is the main hemicellulose in the primary cell walls of gymnosperms and most angiosperm families and is present in all extant groups of land plants, although with some differences in structure (Peña et al., 2008; Scheller and Ulvskov, 2010). All these xyloglucans have a backbone of (1→4)-linked β-d-glucopyranosyl residues, many of which are substituted with α-d-xylopyranosyl residues at O6. In many vascular plants, including Arabidopsis (Arabidopsis thaliana), every fourth glucosyl residue of the xyloglucan backbone is unsubstituted (Vincken et al., 1997). In the standard nomenclature for xyloglucan structures, these residues are represented by G, while X, L, and F indicate Glc residues substituted, respectively, with α-d-Xylp, β-d-Galp-(1→2)-α-d-Xylp, and α-l-Fucp-(1→2)-β-d-Galp-(1→2)-α-d-Xylp side chains (Fry et al., 1993). Conventionally, the reducing end of the molecule is positioned to the right. Treatment of Arabidopsis xyloglucan with an endoglucanase that attacks unsubstituted residues results in oligosaccharide mixtures that include XXG, GXXG, XXXG, XXLG, XLXG, XLLG, XXFG, and XLFG, with some of the Gal residues O-acetylated (Madson et al., 2003; Obel et al., 2009).Although the detailed arrangement and possible connections of the different components of primary cell walls are still unclear, xyloglucan chains are long enough to attach simultaneously to neighboring microfibrils and thus could generate resistance to cell wall extension (Obel et al., 2007). There is also considerable evidence for the covalent linkage of xyloglucan to the pectic polysaccharide rhamnogalacturonan I (Popper and Fry, 2008). The attachment of xyloglucan to cellulose microfibrils is based on hydrogen bonds, and it might be controlled by expansin proteins (Cosgrove, 2005). Xyloglucan connections between microfibrils could also be broken or created by enzymes in the xyloglucan transglycosylase/hydrolase (XTH) family (Nishitani and Vissenberg, 2007). These enzymes cleave xyloglucan chains in front of unsubstituted Glc residues and stay covalently bound to this residue, forming an enzyme-donor complex (Johansson et al., 2004). They can later attach the Glc residue to the nonreducing end of another xyloglucan molecule, acting as xyloglucan endotransglucosylases (XETs). A group of XTHs can also use water as an acceptor, acting as xyloglucan endohydrolases, but they seem to be a minority (Baumann et al., 2007; Eklöf and Brumer, 2010). It is unclear at the moment if endoglucanases from other families are involved in xyloglucan metabolism (Lopez-Casado et al., 2008).The importance of xyloglucan as a regulator of cell wall extension has been thrown into doubt by the identification of an Arabidopsis mutant that has no detectable xyloglucan but still manages to develop normally (Cavalier et al., 2008). Apart from being slightly smaller, this mutant has defective root hairs, but it seems clear that Arabidopsis must have alternative ways of regulating the separation of cellulose microfibrils. It is interesting nonetheless that microfibrils seem to be more irregularly spaced in the xyloglucan-deficient mutant (Anderson et al., 2010).The end result of endoglucanase activity on xyloglucan is the release of oligosaccharides with an unsubstituted Glc at the reducing end. Specific exoglycosidase activities are then necessary to release each type of residue (Iglesias et al., 2006). α-Xylosidase activities in both pea (Pisum sativum) and Tropaeolum majus can only remove unsubstituted Xyl residues from the nonreducing end of the molecule (O’Neill et al., 1989; Fanutti et al., 1991). A β-glucosidase is then required to remove the unsubstituted Glc before α-xylosidase can act again (Crombie et al., 1998). β-Galactosidase and α-fucosidase activities are also required for the complete disassembly of the different Arabidopsis oligosaccharides (Edwards et al., 1988; Léonard et al., 2008). There is currently no information on the enzymes that might be involved in xyloglucan deacetylation or on how the presence of acetyl residues affects exoglycosidases.The Arabidopsis gene AtXYL1 (At1g68560) was identified as coding for an α-xylosidase activity against xyloglucan oligosaccharides by the similarity of its product to purified cabbage (Brassica oleracea var capitata) α-xylosidase (Sampedro et al., 2001). The identification was confirmed through heterologous expression in yeast. According to the Carbohydrate Active Enzymes database (http://www.cazy.org/), AtXYL1 is a member of glycosyde hydrolase family 31, which includes mainly α-glucosidases and α-xylosidases(Cantarel et al., 2009). A reduction of up to 70% of α-xylosidase activity was reported in antisense lines where AtXYL1 was silenced, but this reduction did not cause changes in morphology (Monroe et al., 2003). This article presents the characterization of two independent insertional mutants in AtXYL1 that have no detectable α-xylosidase activity and show remarkable changes in xyloglucan composition along with alterations in the growth pattern.     

Mechanical effects of plant cell wall enzymes on cellulose/xyloglucan composites

Xyloglucan-acting enzymes are believed to have effects on type I primary plant cell wall mechanical properties. In order to get a better understanding of these effects, a range of enzymes with different in vitro modes of action were tested against cell wall analogues (bio-composite materials based on Acetobacter xylinus cellulose and xyloglucan). Tomato pericarp xyloglucan endo transglycosylase (tXET) and nasturtium seed xyloglucanase (nXGase) were produced heterologously in Pichia pastoris. Their action against the cell wall analogues was compared with that of a commercial preparation of Trichoderma endo-glucanase (EndoGase). Both 'hydrolytic' enzymes (nXGase and EndoGase) were able to depolymerise not only the cross-link xyloglucan fraction but also the surface-bound fraction. Consequent major changes in cellulose fibril architecture were observed. In mechanical terms, removal of xyloglucan cross-links from composites resulted in increased stiffness (at high strain) and decreased visco-elasticity with similar extensibility. On the other hand, true transglycosylase activity (tXET) did not affect the cellulose/xyloglucan ratio. No change in composite stiffness or extensibility resulted, but a significant increase in creep behaviour was observed in the presence of active tXET. These results provide direct in vitro evidence for the involvement of cell wall xyloglucan-specific enzymes in mechanical changes underlying plant cell wall re-modelling and growth processes. Mechanical consequences of tXET action are shown to be complimentary to those of cucumber expansin.     

Further studies of the ability of xyloglucan oligosaccharides to inhibit auxin-stimulated growth

The structural features required for xyloglucan oligosaccharides to inhibit 2,4-dichlorophenoxyacetic acid-stimulated elongation of pea stem segments have been investigated. A nonasaccharide (XG9) containing one fucosyl-galactosyl side chain and an undecasaccharide (XG11) containing two fucosyl-galactosyl side chains were purified from endo-β-1,4-glucanase-treated xyloglucan, which had been isolated from soluble extracellular polysaccharides of suspension-cultured sycamore (Acerpseudoplatanus) cells and tested in the pea stem bioassay. A novel octasaccharide (XG8′) was prepared by treatment of XG9 with a xyloglucan oligosaccharide-specific α-xylosidase from pea seedlings. XG8′ was characterized and tested for its ability to inhibit auxin-induced growth. All three oligosaccharides, at a concentration of 0.1 microgram per milliliter, inhibited 2,4-dichlorophenoxyacetic acid-stimulated growth of pea stem segments. XG11 inhibited the growth to a greater extent than did XG9. Chemically synthesized nona- and pentasaccharides (XG9, XG5) inhibited 2,4-dichlorophenoxyacetic acid-stimulated elongation of pea stems to the same extent as the same oligosaccharides isolated from xyloglucan. A chemically synthesized structurally related heptasaccharide that lacked a fucosyl-galactosyl side chain did not, unlike the identical heptasaccharide isolated from xyloglucan, significantly inhibit 2,4-dichlorophenoxyacetic acid-stimulated growth.     

Effects of hypergravity on the elongation growth in radish and cucumber hypocotyls

The elongation growth of the hypocotyls of radish and cucumber seedlings was examined under hypergravity in a newly developed centrifuge (Kasahara et al. 1995). The effects of hypergravity on elongation growth differed between the two species. The rate of elongation of radish hypocotyls was reduced under basipetal hypergravity (H+2O g) but not under acropetal hypergravity (H-13 g), as compared to growth under the control conditions (C+1 g and C-1 g). In cucumber hypocotyls, elongation growth was inhibited not only by basipetal but also by acropetal hypergravity. Under these conditions, the reduction in the elongation growth of both radish and cucumber hypocotyls was accompanied by an increase in their thickness. Although no distinct differences in relative composition of neutral sugars were found, the amounts of cell-wall components (pectic substances, hemicelluloses and cellulose) per unit length of hypocotyls were increased by exposure to hypergravity.     

A revised architecture of primary cell walls based on biomechanical changes induced by substrate-specific endoglucanases

Xyloglucan is widely believed to function as a tether between cellulose microfibrils in the primary cell wall, limiting cell enlargement by restricting the ability of microfibrils to separate laterally. To test the biomechanical predictions of this "tethered network" model, we assessed the ability of cucumber (Cucumis sativus) hypocotyl walls to undergo creep (long-term, irreversible extension) in response to three family-12 endo-β-1,4-glucanases that can specifically hydrolyze xyloglucan, cellulose, or both. Xyloglucan-specific endoglucanase (XEG from Aspergillus aculeatus) failed to induce cell wall creep, whereas an endoglucanase that hydrolyzes both xyloglucan and cellulose (Cel12A from Hypocrea jecorina) induced a high creep rate. A cellulose-specific endoglucanase (CEG from Aspergillus niger) did not cause cell wall creep, either by itself or in combination with XEG. Tests with additional enzymes, including a family-5 endoglucanase, confirmed the conclusion that to cause creep, endoglucanases must cut both xyloglucan and cellulose. Similar results were obtained with measurements of elastic and plastic compliance. Both XEG and Cel12A hydrolyzed xyloglucan in intact walls, but Cel12A could hydrolyze a minor xyloglucan compartment recalcitrant to XEG digestion. Xyloglucan involvement in these enzyme responses was confirmed by experiments with Arabidopsis (Arabidopsis thaliana) hypocotyls, where Cel12A induced creep in wild-type but not in xyloglucan-deficient (xxt1/xxt2) walls. Our results are incompatible with the common depiction of xyloglucan as a load-bearing tether spanning the 20- to 40-nm spacing between cellulose microfibrils, but they do implicate a minor xyloglucan component in wall mechanics. The structurally important xyloglucan may be located in limited regions of tight contact between microfibrils.     

Fractionation of xyloglucan fragments and their interaction with cellulose.

Tamarind seed xyloglucan was partially degraded with a purified endoglucanase (endoV) from Trichoderma viride. Analysis by high-performance anion-exchange chromatography showed that this digest was composed of fragments consisting of 1 to 10 repeating oligosaccharide units ([xg]1-[xg]10). To study the adsorption of xyloglucan fragments to cellulose in detail, this digest was fractionated on BioGel P-6. Fragments were separated satisfactorily up to 5 repeating oligosaccharide units ([xg]5). The galactose substitution of the fragments increased with increasing molecular weight. The BioGel P-6 pools, as well as polymeric xyloglucan ([xg] infinity), were tested for their ability to interact with Avicel crystalline cellulose. Quantitative binding to cellulose occurred for sequences consisting of (at least) 4 repeating units. The adsorption of [xg]4 to Avicel was very high relative to that of [xg] infinity. The dimensions of these fragments were such that they could also penetrate the smaller pores of cellulose. Apparently, the effective surface area for the polymers is much smaller. Adsorption isotherms of [xg] infinity and [xg]4 showed a pattern that is typical for polydisperse systems. However, the mechanisms underlying these patterns were different. At high xyloglucan concentrations, this polydispersity resulted in preferential adsorption of the larger molecules in the case of [xg] infinity and a more extensive colonization of the smaller pores of cellulose in the case of [xg]4. The pH influenced the interaction between xyloglucan (fragments) and cellulose to only a small extent.     

Asparagine biosynthesis in soybean nodules

Two auxin-induced endo-1,4-β-glucanases (EC 3.2.1.4) were purified from pea (Pisum sativum L. var. Alaska) epicotyls and used to degrade purified pea xyloglucan. Hydrolysis yielded nonasaccharide (glucose/xylose/galactose/fucose, 4:3:1:1) and heptasaccharide (glucose/xylose, 4:3) as the products. The progress of hydrolysis, as monitored viscometrically (with amyloid xyloglucan) and by determination of residual xyloglucan-iodine complex (pea) confirmed that both pea glucanases acted as endohydrolases versus xyloglucan. K_m values for amyloid and pea xyloglucans were approximately the same as those for cellulose derivatives, but V_max values were lower for the xyloglucans. Auxin treatment of epicotyls in vivo resulted in increases in net deposits of xyloglucan and cellulose in spite of a great increase (induction) of endogenous 1,4-β-glucanase activity. However, the average degree of polymerization of the resulting xyloglucan was much lower than in controls, and the amount of soluble xyloglucan increased. When macromolecular complexes of xyloglucan and cellulose (cell wall ghosts) were treated in vitro with pea 1,4-β-glucanase, the xyloglucan component was preferentially hydrolyzed and solubilized. It is concluded that xyloglucan is the main cell wall substrate for pea endo-1,4-β-glucanase in growing tissue.     

Evidence that sucrose loaded into the phloem of a poplar leaf is used directly by sucrose synthase associated with various beta-glucan synthases in the stem

Sucrose (Suc) synthase (SuSy) is believed to function in channeling UDP-Glc from Suc to various beta-glucan synthases. We produced transgenic poplars (Populus alba) overexpressing a mutant form (S11E) of mung bean (Vigna radiata) SuSy, which appeared in part in the microsomal membranes of the stems. Expression of SuSy in these membranes enhanced the incorporation of radioactive Suc into cellulose, together with the metabolic recycling of fructose (Fru), when dual-labeled Suc was fed directly into the phloem of the leaf. This overexpression also enhanced the direct incorporation of the glucosyl moiety of Suc into the glucan backbone of xyloglucan and increased recycling of Fru, although the Fru recycling system for cellulose synthesis at the plasma membrane might differ from that for xyloglucan synthesis in the Golgi network. These findings suggest that some of the Suc loaded into the phloem of a poplar leaf is used directly by SuSys associated with xyloglucan and cellulose synthases in the stem. This may be a key function of SuSy because the high-energy bond between the Glc and Fru moieties of Suc is conserved and used for polysaccharide syntheses in this sink tissue.     

Overexpression of poplar cellulase accelerates growth and disturbs the closing movements of leaves in sengon

In this study, poplar (Populus alba) cellulase (PaPopCel1) was overexpressed in a tropical Leguminosae tree, sengon (Paraserianthes falcataria), by the Agrobacterium tumefaciens method. PaPopCel1 overexpression increased the length and width of stems with larger leaves, which showed a moderately higher density of green color than leaves of the wild type. The pairs of leaves on the transgenic plants closed more slowly during sunset than those on the wild-type plants. When main veins from each genotype were excised and placed on a paper towel, however, the leaves of the transgenic plants closed more rapidly than those of the wild-type plant. Based on carbohydrate analyses of cell walls, the leaves of the transgenic plants contained less wall-bound xyloglucan than those of the wild-type plants. In situ xyloglucan endotransglucosylase activity showed that the incorporation of whole xyloglucan, potentially for wall tightening, occurred in the parenchyma cells (motor cells) of the petiolule pulvinus attached to the main vein, although the transgenic plant incorporated less whole xyloglucan than the wild-type plant. These observations support the hypothesis that the paracrystalline sites of cellulose microfibrils are attacked by poplar cellulase, which loosens xyloglucan intercalation, resulting in an irreversible wall modification. This process could be the reason why the overexpression of poplar cellulase both promotes plant growth and disturbs the biological clock of the plant by altering the closing movements of the leaves of the plant.     

Molecular interactions in bacterial cellulose composites studied by 1D FT-IR and dynamic 2D FT-IR spectroscopy

Specific strain-induced orientation and interactions in three Acetobacter cellulose composites: cellulose (C), cellulose/pectin (CP) and cellulose/xyloglucan (CXG) were characterized by FT-IR and dynamic 2D FT-IR spectroscopies. On the molecular level, the reorientation of the cellulose fibrils occurred in the direction of the applied mechanical strain. The cellulose-network reorientation depends on the composition of the matrix, including the water content, which lubricates the motion of macromolecules in the network. At the submolecular level, dynamic 2D FT-IR data suggested that there was no interaction between cellulose and pectin in CP and that they responded independently to a small amplitude strain, while in CXG, cellulose and xyloglucan were uniformly strained along the sample length.     

A Xyloglucan-Specific Endo-1,4-[beta]-Glucanase Isolated from Auxin-Treated Pea Stems

A xyloglucan-specific endo-1,4-[beta]-glucanase was isolated from the apoplast fraction of auxin-treated pea (Pisum sativum) stems, in which both the rate of stem elongation and the amount of xyloglucan solubilized were high. The enzyme was purified to apparent homogeneity by sequential cation-exchange chromatographies, affinity chromatography, and gel filtration. The purified enzyme gave a single protein band on sodium dodecyi sulfate-polyacrylamide gel electrophoresis, and the molecular size was determined to be 77 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 70 kD by gel filtration. The isoelectric point was about 8.1. The enzyme specifically cleaved the 1,4-[beta]-glucosyl linkages of the xyloglucan backbone to yield mainly nona- and heptasaccharides but did not hydrolyze carboxymethylcellulose, swollen cellulose, and (1->3, 1->4)-[beta]-glucan. By hydrolysis, the average molecular size of xyloglucan was decreased from 50 to 20 kD with new reducing chain ends in the lower molecular size fractions. This suggests that the enzyme has endo-1,4-[beta]-glucanase activity against xyloglucan. In conclusion, a xyloglucan-specific endo-1,4-[beta]-glucanase with an activity that differs from the activities of cellulase and xyloglucan endotransglycosylase has been isolated from elongating pea stems.