Inhibition of auxin-induced elongation and xyloglucan breakdown in azuki bean epicotyl segments by fucose-binding lectins

Auxin-induced elongation of epicotyl segments of azuki bean ( Vigna angularis Ohwi and Ohashi cv. Takara) was suppressed by fucose-binding lectins from Tetragonolobus purpureus Moench and Ulex europaeus L. These lectins also inhibited auxin-induced cell wall loosening (decrease in the minimum stress-relaxation time of the cell walls) of segments. Auxin caused a decrease in molecular mass of xyloglucans extracted with 24% KOH from the cell walls. The lectins inhibited auxin-induced changes in molecular mass of the xyloglucans. The autolytic release of xylose-containing products from the pectinase-treated cell walls was also suppressed by the lectins. Fucose-binding lectins pretreated with fucose exhibited little or no inhibitory effect on auxin-induced elongation, cell wall loosning, or breakdown of xyloglucans. These results support the view that the breakdown of xyloglucans is involved in the cell wall loosening responsible for auxin-induced elongation in dicotyledons.     

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.     

Differential Effect of Auxin on Molecular Weight Distributions of Xyloglucans in Cell Walls of Outer and Inner Tissues from Segments of Dark Grown Squash (Cucurbita maxima Duch.) Hypocotyls

Effects of indole-3-acetic acid (IAA) on the mechanical properties of cell walls and structures of cell wall polysaccharides in outer and inner tissues of segments of dark grown squash (Cucurbita maxima Duch.) hypocotyls were investigated. IAA induced the elongation of unpeeled, intact segments, but had no effect on the elongation of peeled segments. IAA induced the cell wall loosening in outer tissues as studied by the stress-relaxation analysis but not in inner tissues. IAA-induced changes in the net sugar content of cell wall fractions in outer and inner tissues were very small. Extracted hemicellulosic xyloglucans derived from outer tissues had a molecular weight about two times as large as in inner tissues, and the molecular weight of xyloglucans in both outer and inner tissues decreased during incubation. IAA substantially accelerated the depolymerization of xyloglucans in outer tissues, while it prevented that in inner tissues. These results suggest that IAA-induced growth in intact segments is due to the cell wall loosening in outer tissues, and that IAA-accelerated depolymerization of hemicellulosic xyloglucans in outer tissues is involved in the cell wall loosening processes.     

Xyloglucan oligosaccharides cause cell wall loosening by enhancing xyloglucan endotransglucosylase/hydrolase activity in azuki bean epicotyls

Addition of xyloglucan-derived oligosaccharides shifted the wall-bound xyloglucans to a lower molecular mass distribution and increased the cell wall extensibility of the native epidermal tissue strips isolated from azuki bean (Vigna angularis) epicotyls. To ascertain the mechanism of oligosaccharide function, we examined the action of a xyloglucan endotransglucosylase/hydrolase (XTH) showing both endotransglucosylase and endohydrolase activities, isolated from azuki bean epicotyl cell walls, in the presence of xyloglucan oligosaccharides. The addition of xyloglucan oligosaccharides enhanced the xyloglucan-degrading activity of XTH against isolated xyloglucan substrates. When the methanol-fixed epidermal tissue strips were incubated with XTH, the molecular mass of wall-bound xyloglucans was decreased and the cell wall extensibility increased markedly in the presence of the oligosaccharides. These results suggest that xyloglucan oligosaccharides stimulate the degradation of xyloglucans by enhancing the XTH activity within the cell wall architecture, thereby increasing the cell wall extensibility in azuki bean epicotyls.     

Use of an α-d-glucosidase for the specific cytochemical identification of lateral α-d-xylosyl end groups in plant xyloglucans

Summary α-Linked d-xylosyl side chains represent the typical feature common to all xyloglucans not shared by other cell wall polysaccharides. Since no easily available α-d-xyloxidase is known, advantage was taken of the conformational and configurational homologies between α-d-xylopyranose and α-d-glucopyranose to make an α-d-glucosidase-gold complex which was able to recognize α-d-xylosyl terminal residues of xyloglucans. This marker was used together with α-l-fucosidase gold complex for the double labeling on two different structural features of the same macromolecule in plant primary cell wall.     

Changes in wall-bound polysaccharidase activities during the culture cycle of a Rubus fruticosus cell suspension

Several hydrolytic enzyme activities were detected in the wall of developing cells of Rubus truticosus in suspension culture. The corresponding substrates of the enzymes are mostly polysaccharide wall constituents, except for chitinase activity. The activities measured when the enzymes were in the free state or wall-bound showed the positive influence of the cell wall micro-environment. Changes in the activities during a cell culture cycle demonstrated that those enzymes acting on xyloglucans behaved differently from the others, and suggest that xyloglucans undergo modifications in vivo over a longer period of time during the exponential growth phase. The same activities were identified in the culture medium. Endo-1,4-β-d-glucanase activities which depolymerized car☐ymethylcellulose (CMC) and xyloglucans (XG) were assayed viscosimetrically. It was found that XG oligosaccharides exhibited an inhibitory effect on the depolymerization of xyloglucans but not on that of CMC. This suggests that true xyloglucanases are present in the culture of Rubus cells.     

Dynamic Changes in Distribution of Lignin and Hemicelluloses in Cell Walls During Differentiation of Secondary Xylem in Eucommia ulmoides (in English)

The dynamic changes in the distribution of lignin and hemicelluloses (xylans and xyloglucans) in cell walls during the differentiation of secondary xylem in Eucommia ulmoides Oliv. were studied by means of ultraviolet light microscopy and transmission electron microscopy combined with immunogold labelling. In the cambial zone and cell expansion zone, xyloglucans were localized both in the tangential and radial walls, but no xylans or lignin were found in these regions. With the formation of secondary wall S1 layer, lignin occurred in the cell corners and middle lamella, while xylans appeared in S1 layer, and xyloglucans were localized in the primary walls and middle lamella. In pace with the formation of secondary wall S2 and S3 layer, lignification extended to S1, S2 and S3 layer in sequence, showing a patchy style of lignin deposition. Concurrently, xylans distributed in the whole secondary walls and xyloglucans, on the other hand, still localized in the primary walls and middle lamella. The results indicated that along with the formation and lignification of the secondary wall, great changes had taken place in the cell walls. Different parts of cell walls, such as cell corners, middle lamella, primary walls and various layers of secondary walls, had different kinds of hemicelluloses, which formed various cell wall architecture combined with lignin and other cell wall components.     

杜仲次生木质部分化过程中木质素与半纤维素组分在细胞壁中分布的动态变化

利用紫外光显微镜、透射电子显微镜结合免疫胶体金标记,研究了杜仲（Eucommia ulmoides Oliv.)次生木质部分化过程中木质素与半纤维素组分（木葡聚糖和木聚糖）在细胞壁分布的动态变化。在形成层及细胞伸展区域,细胞壁具有木葡聚糖的分布,而没有木聚糖和木质素沉积,随着次生壁S1层的形成,木质素出现在细胞角隅和胞间层,木聚糖开始出现在S1层中,此时木葡聚糖则分布在初生壁和胞间层;随着次生,壁S2层及S3层的形成和加厚,木质逐逐步由细胞角隅和胞间层扩展到S1、S2和S3层,其沉积呈现出不均匀的块状或片状沉积模式,在次生壁各层形成与其木质化的同时,木聚糖逐渐分布于整个次生壁中,而木糖聚糖仍局限分布于初生壁和胞间层。结果表明,随着细胞次生壁的形成与木质化,细胞壁结构发生较大变化。细胞壁的不同区域,如细胞角隅、胞间层、初生壁和次生壁各层,具有不同的半纤维素组成,其与木质等细胞壁组分结构构成不同的细胞壁分子结构。     

Role of xyloglucan in gravitropic bending of azuki bean epicotyl

The mechanism of the gravitropic bending was studied in azuki bean epicotyls. The cell wall extensibility of the lower side became higher than that of the upper side in the epicotyl bending upward. The contents of matrix polysaccharides of the cell wall (pectin and xyloglucan in hemicellulose-II) in the lower side became smaller than those in the upper side. The molecular mass of xyloglucans in the lower side decreased. After an epicotyl was fixed to a metal rod to prevent the bending, gravistimulation was applied. Fundamentally the same results were obtained with respect to rheological and chemical characteristics of the cell wall as those of epicotyls showing gravitropic bending. The present results suggested that the initial gravitropic bending was caused by the increase in extensibility of the lower side and the decrease in extensibility of the upper side via the change of the cell wall matrix, especially xyloglucans.     

Changes in Wall Polysaccharides of Squash (Cucurbita maxima Duch.) Hypocotyls under Water Stress Condition: II. Composition of Pectic and Hemicellulosic Polysaccharides

Hypocotyl growth of dark-grown squash (Cucurbita maxima Duch.)seedlings was greatly reduced by the addition of polyethyleneglycol (60 mM) to the hydroponic solution through inhibitionof cell elongation. Measurement of the mechanical propertiesof the cell walls revealed that the cell wall of stressed hypocotylswas loosened as much as that of the unstressed hypocotyls, suggestingthat the stressed hypocotyl could not elongate even though thecell wall loosened. Galactose and arabinose in the pectic fraction,which are probably attached to high mol wt rhamnogalacturonans,increased under stressed as well as under unstressed condition.Other polysaccharides including pectic low mol wt galacturonans,hemicellulosic xyloglucans, galactoglucomannans, arabinans,and glucuronoarabinoxylans increased more under unstressed condition.The mol wt of xyloglucans in the hemicellulosic fraction increasedunder unstressed but not under stressed condition. These results suggest that changes in wall structure, such asincreases in high mol wt rhamnogaracturonans rich in arabinoseand galactose residues, and the suppression of polymerizationof xyloglucans are involved in the process of cell wall loosening. (Received December 15, 1986; Accepted June 8, 1987)     

Changes in the apoplastic pH are involved in regulation of xyloglucan breakdown of azuki bean epicotyls under hypergravity conditions

Hypergravity inhibited elongation growth of azuki bean (Vigna angularis Ohwi et Ohashi) epicotyls by decreasing the mechanical extensibility of cell walls via the increase in the molecular mass of xyloglucans [Soga et al. (1999) Plant Cell Physiol. 40: 581]. Here, we report that the pH value of the apoplastic fluid in epicotyls increased from 5.8 to 6.6 by hypergravity (300 x g) treatment. When the xyloglucan-degrading enzymes extracted from cell walls of the 1 x g control epicotyls were assayed in buffer at pH 6.6 and 5.8, the activity at pH 6.6 was almost half of that at pH 5.8. In addition, when enzymically active cell wall preparations obtained from 1 x g control epicotyls were autolyzed in buffer at pH 5.8 and 6.6 and then xyloglucans were extracted from the autolyzed cell walls, the molecular mass of xyloglucans incubated at pH 5.8 decreased during the autolysis, while that at pH 6.6 did not change. Thus, the xyloglucans were not depolymerized by autolysis at the pH value (6.6) observed in the hypergravity-treated epicotyls. These findings suggest that in azuki bean epicotyls, hypergravity decreases the activities of xyloglucan-degrading enzymes by increasing the pH in the apoplastic fluid, which may be involved in the processes of the increase in the molecular mass of xyloglucans, leading to the decrease in the cell wall extensibility.     

Regulation of polysaccharide breakdown during auxin-induced cell wall loosening

Auxin induces cell elongation by increasing the extensibility of the cell wall. Biochemical modifications of wall constituents lead to such changes in the mechanical properties of the cell wall (wall loosening). The results obtained in the studies using antibodies and lectins as specific probes indicate that the breakdown of xyloglucans in dicotyledons and (1→3), (1→4)-β-glucans in Poaceae is involved in auxin-induced wall loosening. In dicotyledons, xyloglucans are degraded by the direct hydrolysis with an endoglucanase to oligosaccharides and by the two-step reaction via a product with intermediate size. (1→3), (1→4)-β-Glucan breakdown in Poaceae coleoptiles is mediated by the two-step reaction with endo-and exoglucanases. Although auxin inducesde novo synthesis of some hydrolases involved in breakdown of these polysaccharides, the breakdown activity is also regulated by the wall environment such as pH, by the mobility of hydrolases through wall networks, by the interaction of hydrolases with wall polysaccharide complex, and by the presence and the concentrations of different types of regulatory molecules. Recipient of the Botanical Society Award of Young Scientists, 1992.     

Xyloglucan antibodies inhibit auxin-induced elongation and cell wall loosening of azuki bean epicotyls but not of oat coleoptiles

Polyclonal antibodies were raised in rabbits against isoprimeverose (Xyl₁Glc₁), xyloglucan heptasaccharides (Xyl₃Glc₄), and octasaccharides (Gal₁Xyl₃Glc₄). Antibodies specific for hepta- and octasaccharides suppressed auxin-induced elongation of epicotyl segments of azuki bean (Vigna angularis Ohwi and Ohashi cv Takara). These antibodies also inhibited auxin-induced cell wall loosening (decrease in the minimum stress-relaxation time and the relaxation rate of the cell walls) of azuki segments. However, none of the antibodies influenced auxin-induced elongation or cell wall loosening of coleoptile segments of oat (Avena sativa L. cv Victory). Auxin caused a decrease in molecular mass of xyloglucans in the cell walls of azuki epicotyls and oat coleoptiles. The antibodies inhibited such a change in molecular mass of xyloglucans in both species. Preimmune serum exhibited little or no inhibitory effect on auxin-induced elongation, cell wall loosening, or breakdown of xyloglucans. The results support the view that the breakdown of xyloglucans is associated with the cell wall loosening responsible for auxin-induced elongation in dicotyledons. The view does not appear to be applicable to poaceae, because the inhibition of xyloglucan breakdown by the antibodies did not influence auxin-induced elongation or cell wall loosening of oat coleoptiles.     

Xyloglucan breakdown during cotton fiber development

Cotton (Gossypium herbaceum L.) fibers elongated almost linearly up to about 20 days post anthesis. The molecular mass of xyloglucans in fiber cell walls decreased gradually during the elongation stage. When enzymatically active (native) cell wall preparations of fibers were autolyzed, the molecular mass of xyloglucans decreased. The decrease was most prominent in wall preparations obtained from the rapidly elongating fibers. The xyloglucan-degrading activity was recovered from the fiber cell walls with 3 mol/L NaCl, and the activity was high at the stages in which fibers elongated vigorously. These results suggest the possible involvement of xyloglucan metabolism in the regulation of cotton fiber elongation.     

Evaluation of effectiveness of the methods for isolation of cell wall polysaccharides during cell elongation in <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> seedlings

Phaseolus vulgaris seedlings were grown in light with or without chromium. Changes in cell wall components i.e. pectic polysaccharides and xyloglucan contents were looked into during cell elongation, by two different methods in order to find the most suitable method for isolation of cell wall polysaccharides. The first method was short and easy. It made use of organic solvents for preparation of cell wall components and ammonium oxalate and oxalic acid buffer and high temperature for extracting pectic polysaccharides; 0.7 M and 4.3 M KOH was used for extracting low and high molecular weight xyloglucans respectively. On the other hand, in the second method, cell wall components were fractionated by sequential treatments with different inorganic solvents, chelating agents, sodium lauryl sulphate, etc. KOH (1 M and 4 M) was used for extracting xyloglucans. The advantage of using the second method for extracting cell wall polysaccharides especially pectic polysaccharides is discussed.     

Major changes in the cell wall during silique development in Arabidopsis thaliana

Fruit development is a highly complex process, which involves major changes in plant metabolism leading to cell growth and differentiation. Changes in cell wall composition and structure play a major role in modulating cell growth. We investigated the changes in cell wall composition and the activities of associated enzymes during the dry fruit development of the model plant Arabidopsis thaliana. Silique development is characterized by several specific phases leading to fruit dehiscence and seed dispersal. We showed that early phases of silique growth were characterized by specific changes in non-cellulosic sugar content (rhamnose, arabinose, xylose, galactose and galacturonic acid). Xyloglucan oligosaccharide mass profiling further showed a strong increase in O-acetylated xyloglucans over the course of silique development, which could suggest a decreased capacity of xyloglucans to be associated with each other or to cellulose. The degree of methylesterification, mediated by the activity of pectin methylesterases (PMEs), decreased over the course of silique growth and dehiscence. The major changes in cell wall composition revealed by our analysis suggest that it could be major determinants in modulating cell wall rheology leading to growth or growth arrest.     

Gravity resistance, another graviresponse in plants--function of anti-gravitational polysaccharides]

The involvement of anti-gravitational polysaccharides in gravity resistance, one of two major gravity responses in plants, was discussed. In dicotyledons, xyloglucans are the only cell wall polysaccharides, whose level, molecular size, and metabolic turnover were modified under both hypergravity and microgravity conditions, suggesting that xyloglucans act as anti-gravitational polysaccharides. In monocotyledonous Poaceae, (1-->3),(1-->4)-beta glucans, instead of xyloglucans, were shown to play a role as anti-gravitational polysaccharides. These polysaccharides are also involved in plant responses to other environmental factors, such as light and temperature, and to some phytohormones, such as auxin and ethylene. Thus, the type of anti-gravitational polysaccharides is different between dicotyledons and Poaceae, but such polysaccharides are universally involved in plant responses to environmental and hormonal signals. In gravity resistance, the gravity signal may be received by the plasma membrane mechanoreceptors, transformed and transduced within each cell, and then may modify the processes of synthesis and secretion of the anti-gravitational polysaccharides and the cell wall enzymes responsible for their degradation, as well as the apoplastic pH, leading to the cell wall reinforcement. A series of events inducing gravity resistance are quite independent of those leading to gravitropism.     

Structure of the Primary Cell Walls of Suspension-Cultured Rosa glauca Cells: I. Polysaccharides Associated with Cellulose

Cell walls of suspension-cultured cells of Rosa glauca were fractionated by two different extraction procedures. The first involved a stepwise fractionation scheme based on alkaline extraction. The second took advantage of the powerful cellulose solvent system N-methylmorpholine N-oxide/dimethyl sulfoxide which is capable of solubilizing whole cell walls. From the analytical composition of each solubilized fraction and of the corresponding residues, the fate of each type of cell wall polysaccharide constituent was followed at each step of the extraction scheme and the mode of action of the extractant was interpreted. Although the two fractionation procedures were very different, they yielded very similar cellulosic complex residues and extracts, thus delimiting two blocks of polysaccharides in the cell wall. The cellulose residues still comprised uronic acid-containing polysaccharides and hemicelluloses in association with cellulose. Graded acid hydrolysis provided evidence for the central role of a homogalacturonan core interconnecting xyloglucans and arabinogalactans. A tentative model showing the possible interaction existing between the constituent polysaccharides still associated to cellulose after alkaline extraction is presented. Hydrogen bonding between xyloglucan and cellulose is confirmed, and glycosidic linkages between xyloglucans and pectic polymers are suggested.     

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.     

Auxin-Induced Changes in the Molecular Weight Distribution of Cell Wall Xyloglucans in Avena Coleoptiles

The effect of auxin on the molecular weight (Mw) distributionof cell wall xyloglucans was investigated by gel permeationchromatography using coleoptile segments of Avena sativa L.cv. Victory, and the following results were obtained.

1. The water-insoluble hemicellulose (HC-A) mainly consisted ofxyloglucans. Iodine staining method revealed that relativelylarge amounts of xyloglucans were present in the water-solublehemicellulose (HC-B) and water-soluble polysaccharide (WS) fractions.
2. IAA did not cause remarkable changes in xyloglucan contentsin the hemicellulose, but significantly increased the xyloglucancontent in the WS fraction.
3. IAA substantially decreased theweight-average Mw of HC-A. Thiseffect became apparent within30 min of the incubation period,and was not affected by the0.15 M mannitol or 2% sucrose applied.Hydrogen ions also causeda decrease in the weight-average Mwof HC-A; its effect beingreversible.
4. Neither IAA nor hydrogen ions caused any remarkablechangesin the weightaverage Mw of water-soluble xyloglucansin theHC-B.

These results suggest that cell wall xyloglucans have an importantrole in auxininduced cell wall loosening in oat coleoptile cells. (Received May 10, 1984; Accepted August 20, 1984)