Distribution of xyloglucan in Phaseolus aureus seeds

Phaseolus aureus seeds were separated into three parts: hulls,cotyledons and hypocotyls. After the polysaccharides in eachpart had been fractionated by successive extraction with water,0.25% ammonium oxalate-oxalic acid, 4% KOH and 24% KOH, thexyloglucan content in each fraction was determined by Kooiman'smethod for quantitative analysis of amyloid coupled with cellulasetreatment. In each part, most of the xyloglucans were localizedin the 24% KOH extracts and moreover, these xyloglucans fromthe three seed parts had almost identical properties, whichagreed well with those of xyloglucans from the cell walls ofetiolated bean hypocotyls. (Received April 18, 1977; )     

Modification of cell wall polysaccharides during cell elongation in Phaseolus vulgaris hypocotyls

The effect of gibberellic acid (GA) and naphthylacetic acid (NAA) on hypocotyl elongation and cell wall polysaccharides was studied using Phaseolus vulgaris seedlings grown in light condition. The hypocotyl was demarcated into two segments — one near the root was called lower and the one near the cotyledon was called upper. The upper segment showed a typical sigmoidal growth curve while lower segment did not show any growth at all. GA promoted the growth of upper segment while NAA showed clear inhibition in both the segments. Xyloglucan content showed a clear inverse correlation with growth. Pectic polysaccharides did not show a clear trend, though showed an initial inverse correlation with growth. It is concluded that degradation of low and high molecular weight xyloglucans are involved in cell wall loosening which in turn may be responsible for the elongation growth of Phaseolus hypocotyls in light.     

The relationship between xyloglucan endotransglycosylase and in-vitro cell wall extension in cucumber hypocotyls

It has been proposed that cell wall loosening during plant cell growth may be mediated by the endotransglycosylation of load-bearing polymers, specifically of xyloglucans, within the cell wall. A xyloglucan endotransglycosylase (XET) with such activity has recently been identified in several plant species. Two cell wall proteins capable of inducing the extension of plant cell walls have also recently been identified in cucumber hypocotyls. In this report we examine three questions: (1) Does XET induce the extension of isolated cell walls? (2) Do the extension-inducing proteins possess XET activity? (3) Is the activity of the extension-inducing proteins modulated by a xyloglucan nonasaccharide (Glc₄-Xyl₃-Gal₂)? We found that the soluble proteins from growing cucumber (cucumis sativum L.) hypocotyls contained high XET activity but did not induce wall extension. Highly purified wall-protein fractions from the same tissue had high extension-inducing activity but little or no XET activity. The XET activity was higher at pH 5.5 than at pH 4.5, while extension activity showed the opposite sensitivity to pH. Reconstituted wall extension was unaffected by the presence of a xyloglucan nonasaccharide (Glc₄-Xyl₃-Gal₂), an oligosaccharide previously shown to accelerate growth in pea stems and hypothesized to facilitate growth through an effect on XET-induced cell wall loosening. We conclude that XET activity alone is neither sufficient nor necessary for extension of isolated walls from cucumber hypocotyls.     

Changes in peroxidase and IAA oxidase activities during cell elongation in Phaseolus hypocotyls

Cytoplasmic and salt-extracted peroxidase and IAA oxidase activities were studied in Phaseolus vulgaris hypocotyls treated with gibberellic acid (GA, 200 μM), naphthyl acetic acid (NAA, 100 μM) and distilled water control (DW). Peroxidase activity was assayed with four hydrogen donors during the initial phase of hypocotyl elongation. Though peroxidase activity showed a decreasing trend with time in all the hydrogen donors studied; considerable variation with different hydrogen donors was observed. NAA had maximum peroxidase activity as compared to DW or GA treatment. The activity showed a clear inverse correlation with hypocotyl growth. IAA oxidase activity showed a similar trend with growth as peroxidase activity. A highly significant correlation was observed between peroxidase and IAA oxidase activities and high molecular weight xyloglucan content (P<0.001). Finally, the possible role of peroxidase and IAA oxidase activities in hypocotyl elongation growth is discussed.     

Glycoproteins from the cell wall of Phaseolus coccineus.

1. The use of a modified sodium chlorite/acetic acid delignification procedure for the solubilization of a hydroxyproline-rich glycoprotein fraction from the depectinated cell walls of Phaseolus coccineus is described. 2. The crude glycoprotein was associated with some pectic material; hydroxyproline and serine were the most abundant amino acids, and arabinose, galactose and galacturonic acid the predominant monosaccharides. 3. The bulk of the hydroxyproline is O-glycosidically substituted with tetra- and tri-arabinofuranosides. From methylation analysis the linkages in these arabinosides could be inferred. 4. Ion-exchange chromatography of the crude glycoprotein gave one major and two minor hydroxyproline-rich fractions, with similar amino acid but different monosaccharide composition. 5. In the major fraction, serine appears to be O-glycosidically substituted with a single galactopyranoside residue that can be removed by the action of alpha-galactosidase but not beta-galactosidase. Removal of arabinofuranoside residues by partial acid hydrolysis greatly enhanced the action of alpha-galactosidase. 6. Methylation followed by carboxy reduction with LiAl2H4 has shown the presence of (1 leads to 4)-linked galacturonic acid in the crude glycoprotein fraction but not in the major fraction from the ion-exchange column. Hence the bulk of the pectic material is not associated with the major glycoprotein component. It is suggested that the glycoprotein is held in the wall by phenolic cross-links. 7. Similarities with the glycopeptide moiety of potato lectin provides further evidence for a class of hydroxyproline-rich glycoproteins with common features.     

Competitive binding of pectin and xyloglucan with primary cell wall cellulose

Assemblies of pectin, xyloglucan and cellulose were studied in vitro using two ternary systems. In the first one, xyloglucan concentration varied, while pectin amount was kept constant. In the second one, pectin concentration varied, whereas xyloglucan amount was fixed. The use of ternary systems allowed to put forward the hypothesis that pectin/cellulose and xyloglucan/cellulose associations may exist together or separately, depending on the proportion of non-cellulosic polysaccharides in cell walls. It can be hypothesized that pectin plays a double role within primary cell walls: (i) pectin loosely bound to cellulose, in xyloglucan-rich cell walls, (ii) pectin associated with cellulose, in xyloglucan-poor cell walls.     

The role of wall calcium in the extension of cell walls of soybean hypocotyls

Calcium crosslinks are load-bearing bonds in soybean (Glycine max (L.) Merr.) hypocotyl cell walls, but they are not the same load-bearing bonds that are broken during acid-mediated cell elongation. This conclusion is reached by studying the relationship between wall calcium, pH and the facilitated creep of frozenthawed soybean hypocotyl sections. Supporting data include the following observations: 1) 2-[(2-bis-[carboxy-methyl]amino-5-methylphenoxy)methyl]-6-methoxy-8-bis[carboxymethyl]aminoquinoline (Quin 2) and ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) caused only limited facilitated creep as compared with acid, despite removal of comparable or larger amounts of wall calcium; 2) the pH-response curves for calcium removal and acid-facilitated creep were different; 3) reversible acid-extension occurred even after removal of almost all wall calcium with Quin 2; and 4) growth of abraded sections did not involve a proportional loss of wall calcium. Removal of wall calcium, however, increased the capacity of the walls to undergo acid-facilitated creep. These data indicate that breakage of calcium crosslinks is not a major mechanism of cell-wall loosening in soybean hypocotyl tissues. This research was supported by Department of Energy grant DE-FG06-88ER13830 and NASA grant NAGW 1394. The authors are grateful to Dr. David Rayle (San Diego State University, Cal.) for stimulating discussions and comments during the course of this work.     

Cell elongation in Arabidopsis hypocotyls involves dynamic changes in cell wall thickness

Field-emission scanning electron microscopy was used to measure wall thicknesses of different cell types in freeze-fractured hypocotyls of Arabidopsis thaliana. Measurements of uronic acid content, wall mass, and wall volume suggest that cell wall biosynthesis in this organ does not always keep pace with, and is not always tightly coupled to, elongation. In light-grown hypocotyls, walls thicken, maintain a constant thickness, or become thinner during elongation, depending upon the cell type and the stage of growth. In light-grown hypocotyls, exogenous gibberellic acid represses the extent of thickening and promotes cell elongation by both wall thinning and increased anisotropy during the early stages of hypocotyl elongation, and by increased wall deposition in the latter stages. Dark-grown hypocotyls, in the 48 h period between cold imbibition and seedling emergence, deposit very thick walls that subsequently thin in a narrow developmental window as the hypocotyl rapidly elongates. The rate of wall deposition is then maintained and keeps pace with cell elongation. The outer epidermal wall is always the thickest ( approximately 1 mum) whereas the thinnest walls, about 50 nm, are found in inner cell layers. It is concluded that control of wall thickness in different cell types is tightly regulated during hypocotyl development, and that wall deposition and cell elongation are not invariably coupled.     

Action of xyloglucan hydrolase within the native cell wall architecture and its effect on cell wall extensibility in azuki bean epicotyls

Xyloglucan hydrolase (XGH) has recently been purified from the cell wall of azuki bean (Vigna angularis Ohwi et Ohashi) epicotyls as a new type of xyloglucan-degrading enzyme [Tabuchi et al. (2001) Plant Cell Physiol. 42: 154]. In the present study, the effects of XGH on the mechanical properties of the cell wall and on the level and the molecular size of xyloglucans within the native wall architecture were examined in azuki bean epicotyls. When the epidermal tissue strips from the growing regions of azuki bean epicotyls were incubated with XGH, the mechanical extensibility of the cell wall dramatically increased. XGH exogenously applied to cell wall materials (homogenates) or epidermal tissue strips decreased the amount of xyloglucans via the solubilization of the polysaccharides. Also, XGH substantially decreased the molecular mass of xyloglucans in both materials. These results indicate that XGH is capable of hydrolyzing xyloglucans within the native cell wall architecture and thereby increasing the cell wall extensibility in azuki bean epicotyls.     

Role of transglycosylation in the integration of xyloglucan into the growing plant cell wall in vivo

Abstract

Xyloglucan endotransglycosylase (XET) activity is widespread in plant cell walls, but its action on xyloglucan in vivo has been difficult to prove because the reaction products are not expected to differ chemically from the reactants. By feeding of cultured Rosa cells with [¹³C]glucose and [³H]arabinose followed by [¹²-C]glucose, and isopyenic centrifugation of the extracted xyloglucan in caesium trifluoroacetate, we have obtained evidence for the annealing of segments of newly-secreted xyloglucan to xyloglucan chains that were already present in the cell wall. This is the first evidence for interpolymeric transglycosylation of xyloglucan in vivo.     

Effects of hypergravity on growth and cell wall properties of cress hypocotyls

Elongation growth of etiolated hypocotyls of cress (Lepidiumsativum L.) was suppressed when they were exposed to basipetalhypergravity at 35 g and above. Acceleration at 135 g causeda decrease in the mechanical extensibility and an increase inthe minimum stress-relaxation time of the cell wall. Such changesin the mechanical properties of the cell wall were prominentin the lower regions of hypocotyls. The amounts of cell wallpolysaccharides per unit length of hypocotyls increased underthe hypergravity condition and, in particular, the increasein the amount of cellulose in the lower regions was conspicuous.Hypergravity did not influence the neutral sugar compositionof either the pectin or the hemicellulose fraction. The amountof lignin was also increased by hypergravity treatment, althoughthe level was low. The data suggest that hypergravity modifiesthe metabolism of cell wall components and thus makes the cellwall thick and rigid, thereby inhibiting elongation growth ofcress hypocotyls. These changes may contribute to the plants'ability to sustain their structures against hypergravity. Key words: Cell wall extensibility, cellulose, hypergravity, Lepidium sativum L., lignin     

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.     

Quantitative and qualitative changes in cell wall polysaccharides in relation to growth and cell wall loosening in Lactuca sativa hypocotyls

The correlation between hypocotyl elongation, cell wall loosening and changes in cell wall polysaccharides was studied using intact lettuce seedlings grown in the dark or in light together with gibberellic acid (GA) and/or 5-fluorodeoxyuridine (FUDR). The following results were obtained:
1) The production of pectic, hemicellulosic and cellulosic polysaccharides look place in parallel with hypocotyl elongation, which was substantially affected by different growth conditions.
2) The mole percentage sugar composition of pectic and hemicellulosic polysaccharides changed in response to dark, light, GA, or FUDR treatments.
3) The amounts of xylose and glucose in hemicellulosic polysaccharides and those of galactosc, rhumnose and uronic acid in pectic polysaccharides increased in parallel with hypocotyl elongation.
4) Statistical analysis of the quantitative relationship between sugars composing polysaccharides revealed that the uronic acid content changed in parallel with those of rhamnose and galactose in pectic polysaccharides, and the content of xylose varied in parallel with those of fucose and glucose.
5) The content of hemicellulosic polysaccharides was correlated with cell wall loosening represented by a decrease in the minimum stress-relaxation time. Changes in the stress-relaxation time value were correlated with those in the content of araltinose and galactose in hemicellulosic polysaccharides.
Based on these results, the relationship between hypocotyl elongation, changes in cell wall polysaccharides, and cell wall loosening is discussed with respect to the effect of GA and FUDR on hypocotyl elongation.     

Effects of hypergravity on growth and cell wall properties of cress hypocotyls

Elongation growth of etiolated hypocotyls of cress (Lepidium sativum L.) was suppressed when they were exposed to basipetal hypergravity at 35 x g and above. Acceleration at 135 x g caused a decrease in the mechanical extensibility and an increase in the minimum stress-relaxation time of the cell wall. Such changes in the mechanical properties of the cell wall were prominent in the lower regions of hypocotyls. The amounts of cell wall polysaccharides per unit length of hypocotyls increased under the hypergravity condition and, in particular, the increase in the amount of cellulose in the lower regions was conspicuous. Hypergravity did not influence the neutral sugar composition of either the pectin or the hemicellulose fraction. The amount of lignin was also increased by hypergravity treatment, although the level was low. The data suggest that hypergravity modifies the metabolism of cell wall components and thus makes the cell wall thick and rigid, thereby inhibiting elongation growth of cress hypocotyls. These changes may contribute to the plants' ability to sustain their structures against hypergravity.     

Stimulation of elongation growth and xyloglucan breakdown in Arabidopsis hypocotyls under microgravity conditions in space

Seedlings of Arabidopsis thaliana (L.) Heynh. (ecotype Columbia and an ethylene-resistant mutant etr1-1) were cultivated for 68.5, 91.5 and 136 h on board during the Space Shuttle STS-95 mission, and changes in the elongation growth and the cell wall properties of hypocotyls were analyzed. Elongation growth of dark-grown hypocotyls of both Columbia and etr1-1 was stimulated under microgravity conditions in space. There were no clear differences in the degree of growth stimulation between Columbia and etr1-1, indicating that the ethylene level was not abnormally high in the cultural environment of this space experiment. Microgravity also increased the mechanical extensibility of cell walls in both cultivars, and such an increase was attributed to the increase in the apparent irreversible extensibility. The levels of cell wall polysaccharides per unit length of hypocotyls decreased in space. Microgravity also reduced the weight-average molecular mass of xyloglucans in the hemicellulose-II fraction. Also, the activity of xyloglucan-degrading enzymes extracted from hypocotyl cell walls increased under microgravity conditions. These results suggest that microgravity reduces the molecular mass of xyloglucans by increasing xyloglucan-degrading activity. Modifications of xyloglucan metabolism as well as the thickness of cell wall polysaccharides seem to be involved in an increase in the cell wall extensibility, leading to growth stimulation of Arabidopsis hypocotyls in space.