Turgor-dependent Changes in Avena Coleoptile Cell Wall Composition

The effects of reduced turgor pressure on growth, as measured by cell elongation, and on auxin-mediated changes in cell walls, as measured by analyses of wall composition, were examined using Avena coleoptile segments. Although moderate (1-4 bar) decreases in turgor resulted in a progressive decline in growth proportional to the decrease in turgor, the major auxin-induced change in wall composition, a decrease in noncellulosic wall glucose, was unaffected. Severe (5-8 bar) decreases, however, did inhibit this auxin effect on the wall, and with turgor decreases of 9 bars or more this auxin effect was no longer apparent. The results show that turgor pressure is required for this auxin-mediated wall modification and also that this modification of wall glucose occurs at turgor pressures less than those required for wall extension. Changes in other wall components were generally unaffected by altering turgor pressure.     

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.     

Cell expansion patterns and directionality of wall mechanical properties in nitella

As a means of assessing the extent to which deformation of isolated walls relates to in vivo cell expansion, the directionality of wall mechanical properties was examined in Nitella. Measurements were made of plastic and elastic deformation and creep under both uniaxial and multiaxial stress conditions. Walls of different structural characteristics were obtained from control, isopropyl N-phenylcarbamate (IPC)-treated and IPC recovery cells. Although microfibrils in the inner portion of the wall were transverse for control and recovery cells but random for IPC cells, all walls had similar over-all microfibrillar orientations. Consequently, differences in wall mechanical properties should reflect structural differences in the inner wall. It is the action of the prevailing stress pattern on the inner, not overall, wall microfibrillar organization which dictates the directionality of growth in Nitella. The results indicate that the directional character of expansion is preserved to a large extent in the mechanical properties of isolated walls, and that most, but not all, of the deformation is determined by the inner wall. In addition, directional differences in the threshold for acid-induced extension varied in accord with the pattern of inner wall microfibrils.     

The effect of auxin on stress relaxation in isolated Avena coleoptiles

In order to characterize further the mechanical properties of coleoptile cell walls, stress relaxation measurements were made on methanol-boiled sections of Avena coleoptiles. Relaxation was measured both in mechanically conditioned specimens and in specimens which had not been previously extended. In both cases the relaxation was proportional to log time. Mechanical conditioning increased the relaxation modules and decreased the relative rate of relaxation. In contrast, pretreatment of the live coleoptiles with indoleacetic acid reduced the relaxation modulus and the absolute rate of relaxation but did not affect the relative rate of relaxation. Essentially similar pictures of the mechanical properties of coleoptile walls are obtained from stress relaxation and creep tests; the wall behaves as a nonlinear viscoelastic material.     

Control of Cell Elongation in Nitella by Endogenous Cell Wall pH Gradients: MULTIAXIAL EXTENSIBILITY AND GROWTH STUDIES

The multiaxial stress of turgor pressure was stimulated in vitro by inflating isolated Nitella cell walls with mercury. The initial in vitro extension at pH 6.5, 5 atmospheres pressure, returned the wall approximately to the in vivo stressed length, and did not induce any additional extension during a 15-minute period. Upon release of pressure, a plastic deformation was observed which did not correlate with cell growth rates until the final stages of cell maturation. Since wall plasticity does not correlate with growth rate, a metabolic factor(s) is implicated. Walls at all stages of development exhibited a primary yield stress between 0 and 2 atmospheres, while rapidly growing cells (1-3% per hour) exhibited a secondary yield stress of 4 to 5 atmospheres. The creep rate and plastic deformation of young walls were markedly enhanced by acid buffers (10 millimolar, pH ≤ 5.3).     

Auxin-induced changes in cell wall extensibility of maize roots

The rheological properties of corn (Zea mays L. cv. Garant) root elongation zones were investigated by means of a computer-controlled extensiometer. Creep closely followed a logarithmic time function, which was used to quantify creep activity. Pretreatment with auxin, which inhibits extension growth in roots, lowered the creep activity and the apparent plastic extensibility. While the time course of the inhibition of apparent plastic extensibility lagged behind the cessation of elongation growth, the drop in creep activity matched the growth inhibition more closely. Creep activity and apparent plastic extensibility were not significantly affected by pH. These data support the view that the auxin-induced cell wall stiffening (e.g. by cross-linking processes), while causal for the growth inhibition, is not brought about by a cell wall alkalinization. Received: 10 December 1996 / Accepted: 19 August 1997     

Tensile characteristics of collenchyma cell walls at different calcium contents

Collenchyma fibres from celery ( Apium graveolens L.) were extracted with detergent and phenol-acetic acid-water to leave the intact cell walls, free from active enzymes. Under a small, constant stress the cell wall fibres showed elastic and plastic extension and viscoelastic deformation, but viscous flow was observed only at high stresses close to the breaking stress. After complete removal of calcium ions with cyclohex-anediamine tetraacetic acid (CDTA) and incubation for 18 h, comparable levels of these extensibility components were observed at much lower stresses. However, partial removal of calcium ions with citrate did not increase the plastic, elastic or viscoelastic components even when the residual calcium was reduced to 3.5% of the exchange capacity. The breaking stress of the fibres was rather more sensitive to calcium removal, being reduced by 50% at 7% calcium saturation. CDTA-extracted fibres broke by cell separation at very low stress. These characteristics did not appear compatible with removal of calcium ions, or their displacement by protons, as a mechanism for auxin-induced growth in this material: however such mechanisms are not excluded in other tissues or under other conditions. Strong chelating agents which remove enough calcium to weaken cell walls should be avoided in experiments on other mechanisms of auxin-induced growth.     

Inhibition of formation of protein-bound hydroxyproline by free hydroxyproline in Avena coleoptiles

Free hydroxyproline inhibits the formation of protein-bound hydroxyproline from proline to a considerably greater extent than it does the incorporation of proline into protein of auxin-treated Avena coleoptiles. This inhibition is greater in the wall than in the cytoplasmic fraction. In the absence of auxin, free hydroxyproline exerts little or no inhibition of hydroxyproline formation. Furthermore free hydroxyproline has no effect on respiration, RNA synthesis or the incorporation of leucine into protein. Hydroxyproline is not a general inhibitor of metabolism or protein synthesis in Avena coleoptiles.

These results suggest that free hydroxyproline may inhibit auxin-induced cell elongation by blocking the formation or utilization of a particular hydroxyproline-rich protein which must be incorporated into the cell wall during auxin-induced wall extension.

     

Stress relaxation property of the cell wall and auxin-induced cell elongation

A stress-relaxation method has been developed to measure the mechanical property of the plant cell wall, as a physically defined terms. In the method, the stress relaxation property of the cell wall is simulated with a Maxwell viscoelastic model whose character is represented by four parameters; the minimum relaxation time, To, the relaxation rate, b, the maximum relaxation time, Tm and the residual stress, c. Thus, the mechanical property of the cell wall is represented by the four parameters. Physical and physiological meanings of the parameters are discussed. Auxin effects on the parameters were also studied. The cell elongation is simply thought to be extension of the cell wall under a force. The extension of the cell wall can be simulated by the mechanical property of the cell wall. However, the calculated extension was found to be incomparable to the real cell growth, indicating that there has to be other factors limiting the rate of cell growth. Major factors governing cell growth are discussed to be the cell wall mechanical property, the osmotic potential and water movement in the apoplast. A possibility to predict cell expansion with the three factors was discussed and a novel equation representing cell growth was obtained: $$1/R = 1/R_w + 1/R_p $$ whereR is the rate of cell elongation,R _w is the rate of cell wall extension due to the osmotic pressure andR _p is the rate of cell elongation determined by water conductivity.     

Rapid and reversible modifications of extension capacity of cell walls in elongating maize leaf tissues responding to root addition and removal of NaCl

A creep extensiometer technique was used to provide direct evidence that short (20 min) and long-term (3d) exposures of roots to growth inhibitory levels of salinity (100mol m^-3 NaCl) induce reductions in the irreversible extension capacity of cell walls in the leaf elongation zone of intact maize seedlings (Zea mays L.). The long-term inhibition of cell wall extension capacity was reversed within 20 min of salt withdrawal from the root medium. Inhibited elongation of leaf epidermal tissues was also reversed after salt removal. The salt-induced changes in wall extension capacity were detected using in vivo and in vitro assays (shortly after localized freeze/thaw treatment of the basal elongation zone). The rapid reversal of the inhibition of wall extensibility and leaf growth after salt removal from root medium of long-term salinized plants, suggested that neither deficiencies in growth essential mineral nutrients nor toxic effects of NaCl on plasmamembrane viability were directly involved in the inhibition of leaf growth. There was consistent agreement between the scale, direction and timing of salinity-induced changes in leaf elongation growth and wall extension capacity. Rapid metabolically regulated changes in the physical properties of growing cell walls, caused by osmotic (or other) effects, appear to be a factor regulating maize leaf growth responses to root salinization.     

A fungal endoglucanase with plant cell wall extension activity

We have identified a wall hydrolytic enzyme from Trichoderma reesei with potent ability to induce extension of heat-inactivated type I cell walls. It is a small (23-kD) endo-1,4-beta-glucanase (Cel12A) belonging to glycoside hydrolase family 12. Extension of heat-inactivated walls from cucumber (Cucumis sativus cv Burpee Pickler) hypocotyls was induced by Cel12A after a distinct lag time and was accompanied by a large increase in wall plasticity and elasticity. Cel12A also increased the rate of stress relaxation of isolated walls at very short times (<200 ms; equivalent to reducing t(0), a parameter that estimates the minimum relaxation time). Similar changes in wall plasticity and elasticity were observed in wheat (Triticum aestivum cv Pennmore Winter) coleoptile (type II) walls, which showed only a negligible extension in response to Cel12A treatment. Thus, Cel12A modifies both type I and II walls, but substantial extension is found only in type I walls. Cel12A has strong endo-glucanase activity against xyloglucan and (1-->3,1-->4)-beta-glucan, but did not exhibit endo-xylanase, endo-mannase, or endo-galactanase activities. In terms of kinetics of action and effects on wall rheology, wall loosening by Cel12A differs qualitatively from the action by expansins, which induce wall extension by a non-hydrolytic polymer creep mechanism. The action by Cel12A mimics some of the changes in wall rheology found after auxin-induced growth. The strategy used here to identify Cel12A could be used to identify analogous plant enzymes that cause auxin-induced changes in cell wall rheology.     

Effect of anti-wall protein antibodies on auxin-induced elongation, cell wall loosening, and β-D-glucan degradation in maize coleoptile segments

Antiserum raised against the LiCl extract of maize shoot cell walls suppresses auxin-induced elongation of maize coleoptile segments. A series of polyclonal antibodies were raised against protein fractions separated from the LiCl extract of maize ( Zea mays L. cv. B73 x Mo17) coleoptiles by SP-Sephadex and Bio-Gel P-150 chromatography. To understand the role of cell wall proteins in growth regulation, the effect of these antibodies on auxin-induced elongation and changes in the cell walls of maize coleoptiles was examined. Four of the fractions prepared reacted with the antiserum raised against the total LiCl extract and effectively suppressed its growth-inhibiting activity. Only these fractions contained the proteins responsible for eliciting growthinhibiting antibodies. The antibodies capable of growth inhibition of auxin-induced elongation of segments also inhibited auxin-induced cell wall loosening (decrease in the minimum stress-relaxation time of the cell walls) of segments. The antibodies raised against one of the protein fractions separated by SP-Sephadex inhibited the autolytic reactions of isolated cell walls and the auxin-induced decrease in (1→3), (1→4)-β-D-glucans in the cell walls. Thus, the degradation of β-D-glucans by cell wall enzymes may be associated with the cell wall loosening that is responsible for cell elongation. Because the other antibodies did not influence the auxin-induced degradation of (1→3), (1→4)-β-D-glucanses, β-D-glucanases and other cell wall enzymes may cooperate in regulation of cell elongation in maize coleoptiles.     

beta-d-Glucan Antibodies Inhibit Auxin-Induced Cell Elongation and Changes in the Cell Wall of Zea Coleoptile Segments

Antiserum was raised against the Avena sativa L. caryopsis β-d-glucan fraction with an average molecular weight of 1.5 × 10⁴. Polyclonal antibodies recovered from the serum after Protein A-Sepharose column chromatography precipitated when cross-reacted with high molecular weight (1→3), (1→4)-β-d-glucans. These antibodies were effective in suppression of cell wall autohydrolytic reactions and auxin-induced decreases in noncellulosic glucose content of the cell wall of maize (Zea mays L.) coleoptiles. The results indicate antibody-mediated interference with in situ β-d-glucan degradation. The antibodies at a concentration of 200 micrograms per milliliter also suppress auxin-induced elongation by about 40% and cell wall loosening (measured by the minimum stress-relaxation time of the segments) of Zea coleoptiles. The suppression of elongation by antibodies was imposed without a lag period. Auxin-induced elongation, cell wall loosening, and chemical changes in the cell walls were near the levels of control tissues when segments were subjected to antibody preparation precipitated by a pretreatment with Avena caryopsis β-d-glucans. These results support the idea that the degradation of (1→3), (1→4)-β-d-glucans by cell wall enzymes is associated with the cell wall loosening responsible for auxin-induced elongation.     

A computer simulation of the creep process of the cell wall using stress relaxation parameters

The stress relaxation of the cell wall of pea plants was measured and viscoelastic parameters were obtained according to the stress-relaxation analysis developed by Yamamoto et al. (Plant & Cell Physiol. 1970). The creep process of the cell wall was simulated by a numerical integration using stress-relaxation parameters, because the direct conversion of the stress relaxation process to the creep is impractical. In the conversion, a personal computer was programmed for the Maxwell viscoelastic model with a compiler language. Cell wall creep was measured with a specially constituted apparatus and compared with that calculated by the simulation process. The results suggested that the creep can be reproduced by a computer simulation using the stress-relaxation parameters. Both creep and stress-relaxation properties of plant cell walls can be analyzed by using a single model.     

Inhibition of RNA Synthesis and Auxin-Induced Cell Wall Extensibility and Growth by Actinomycin D

A linear stress strain analyzer was used to determine the effects of inhibitors of RNA and protein synthesis on auxin-induced increases in cell wall extensibility. With etiolated soybean hypocotyl, maize mesocotyl and Avena coleoptile sections and light-grown pea internode sections, inhibition of RNA synthesis resulted in inhibition of auxin-induced extensibility changes and cell expansion. The results with both actinomycin D and cycloheximide support an earlier conclusion that unstable cell constituents, presumably enzymes, are essential for cell wall loosening induced by auxin as well as for cell elongation.     

Viscoelastic versus plastic cell wall extensibility in growing seedling organs: a contribution to avoid some misconceptions

In a recent publication (Kutschera, 1996), it was reported thatthe cell walls of growing rye coleoptiles exhibit irreversible(plastic) extensibility in a rheological extension test. Basicallysimilar measurements with cell walls of maize coleoptiles hadpreviously shown that the apparent plastic extensibility determinedin this material is in reality due to the slowly reversible(viscoelastic) extensibility of the walls. A recent reinvestigationof this discrepancy showed that rye coleoptile walls also behaveas a perfectly viscoelastic material if precautions are takento prevent measuring artefacts. Similar results were obtainedwith cell walls from the growing zone of various other seedlingorgans (maize mesocotyl, maize root, cucumber hypocotyl). Itis concluded that plastic extensibility has not yet been convincinglydemonstrated by rheological tests that determine the intrinsicmaterial properties of cell walls. Reported changes in mechanicalmaterial properties of cell walls produced by growth-controllingfactors such as auxin or light may generally be attributed tochanges in viscoelasticity which are not directly related tothe chemo-rheological processes controlling wall extension ofgrowing cells. Key words: Cell wall extensibility, extension growth, plastic cell wall extensibility, viscoelastic cell wall extensibility     

Wall-yielding properties of cell walls from elongating cucumber hypocotyls in relation to the action of expansin

The wall-yielding properties of cell walls were examined using frozen-thawed and pressed segments (FTPs) obtained from the elongation zones of cucumber hypocotyls with a newly developed programmable creep meter. The rate of wall extension characteristically changed depending on both tension and pH. By treatment of the FTPs with acid, the yield tension (y) was shifted downward and the extensibility (phi) was increased. However, the downward shift of y was greatly suppressed and the increase in phi was partly inhibited in boiled FTPs. The boiled FTPs reconstituted with expansin fully recovered the acid-induced downward y shift as well as the increase in phi. Even under the tension below y, wall extension took place pH dependently. Such extension was markedly slower (low-rate extension) than that under the tension above y (high-rate extension). At a higher concentration (8 M), urea markedly inhibited the creep ascribable to the inhibition of the acid-induced downward y shift and increase in phi. Moderate concentrations (2 M) of urea promoted wall creep pH dependently. The promotion was equivalent to a 0.5 decrease in pH. The promotion of creep by 2 M urea was observed in boiled FTPs reconstituted with expansin but not in boiled FTPs. These findings indicated that the acid-facilitated creep was controlled by y as well as in cucumber cell walls. However, y and phi might be inseparable and mutually related parameters because the curve of the stress extension rate (SER) showed a gradual change from the low-rate extension to the high-rate extension. Expansin played a role in pH-dependent regulation of both y and phi. The physiological meaning of the pH-dependent regulation of wall creep under different creep tensions is also discussed with reference to a performance chart obtained from the SER curves.     

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.     

Uptake and Metabolic Fate of Glucose, Arabinose, and Xylose by Zea mays Coleoptiles in Relation to Cell Wall Synthesis

According to the acid-growth hypothesis, auxin-induced secretion of hydrogen ions activate “wall loosening” enzymes that change the rheological properties of the cell wall. The wall loosening process may yield monosaccharides by the enzymic cleavage of load-bearing polysaccharides. Our study was initiated to determine the metabolic fate of such sugars when released from the major hemicellulosic polysaccharides of the cell walls of Zea mays coleoptiles.     

Nonlinear viscoelasticity of cheese

Cheese viscoelasticity is commonly measured using steady uniaxial compression, steady uniaxial extension, shear and compressive creep, and stress relaxation in shear and compression. Viscoelastic properties for many cheeses have also been studied using small amplitude oscillatory shear (SAOS). However, there is little on the measurement of nonlinear viscoelastic properties. The large deformation test usually conducted on cheese to study nonlinear viscoelasticity is uniaxial compression, but this test hardly departs from linear behavior. In this work, the nonlinear viscoelasticity of four cheese varieties was studied using large amplitude oscillatory shear (LAOS). A sliding plate rheometer incorporating a shear stress transducer was used. The data were evaluated using spectral analysis, and results are presented mainly in the form of shear stress versus shear rate loops.