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 mechanical behavior of isolated Avena coleoptile walls subjected to constant stress: properties and relation to cell elongation

In order to assess the role of the mechanical properties of the wall in auxin-induced cell elongation, a study has been made of the ability of isolated Avena coleoptile walls to extend (creep) when subjected to a constant applied stress. Creep occurs as a viscoelastic extension which has the following characteristics: the extension is proportional to log time and is partly reversible, and the extension rate has a Q₁₀ of about 1.05 and is markedly greater in auxin-pretreated walls. In nonconditioned walls the extension rate is proportional to applied stress, but pre-extension causes the appearance of an apparent yield strain. The similarity of creep and instantaneous plastic deformation in response to temperature or to pretreatment with auxin or KCN suggests that the instantaneous deformation is simply the viscoelastic extension which occurs at very short times. A comparison of these viscoelastic properties with the properties of auxin-induced cell elongation indicates that cell elongation requires more than just a physical extension of the wall. It is suggested that elongation occurs as a series of extension steps, each of which involves a viscoelastic extension preceded or accompanied by an auxin-dependent biochemical change in the wall properties.     

Extensibility of isolated cell walls in the giant tip-growing cells of the xanthophycean alga Vaucheria terrestris

Apical cell wall fragments isolated from the giant-cellular xanthophycean alga Vaucheria terrestris sensu Götz were inflated with silicone oil by applying internal pressure ranging from 0.1 to 0.7 MPa, and the time-course of cell wall deformation was recorded and analyzed by videomicroscopy. Cell wall extensibility in the tip-growing region was estimated by the pressure required for cell wall extension, the amount of total extension until cell wall rupture and the rate of cell wall extension. Apical cell walls exhibited gradual extension, or creep, during inflation, which was eventually followed by rupture at the apical portion, whereas no appreciable extension was found in the cylindrical basal portion of the cell wall fragment. Besides the largest extension observed around the tip, substantial extension was also observed along the subapical region of the cell wall. The wall extensibility was dependent on the buffer pH used for infiltration before inflation. The optimum pH for the extension was about 8.0, but the cell wall was much less extensible after infiltration with an acidic buffer. Cell wall extensibility was dependent on the pH of the buffer used before inflation, regardless of that used in the previous infiltration. Moreover, pretreatment of the cell wall with a protease caused considerable loosening of cell walls, but affected the pH dependence of cell wall extensibility little. These results indicate that the extensibility of the cell walls in the giant tip-growing cells of the alga is distinct from that of plant cells that exhibit "acid growth" in its dependence on environmental pH and the role of cell wall proteins.     

A Comparison of Acid-induced Cell Wall Loosening in Valonia ventricosa and in Oat Coleoptiles

The acid-induced loosening of cell walls of Valonia ventricosa has been compared to that of frozen-thawed oat coleoptiles. The two acid extension responses are similar in regard to the shape of the pH response curve and the increase in plastic compliance induced by acid treatment. In both systems the acid response can be inhibited by Ca²⁺ and in both the removal of the protons leads to a rapid termination of wall loosening. The two responses differ in several significant ways, however. The acid-induced extension of Valonia walls is more rapid than that of coleoptile walls, but of smaller total magnitude. Acid-induced loosening can occur in Valonia without the wall being under tension, but not in coleoptiles. The acid-induced extension of Valonia walls is not inhibited by 8 molar urea, whereas the response in oat coleoptiles is completely inhibited by this treatment. Ethylenediaminetetraacetate (EDTA) can cause wall loosening in Valonia comparable to that produced by low pH, whereas in coleoptiles EDTA causes a much smaller response. These results with Valonia are consistent with a mechanism of acid-induced wall loosening in which a central role is played by the displacement of Ca²⁺ from the wall, while the larger part of acid-induced wall loosening in oat coleoptiles appears to be via a different mechanism.     

Evidence Against the Involvement of Ionically Bound Cell Wall Proteins in Pea Epicotyl Growth

Ionically bound cell wall proteins were extracted from 7 day old etiolated pea (Pisum sativum L. cv Alaska) epicotyls with 3 molar LiCl. Polyclonal antiserum was raised in rabbits against the cell wall proteins. Growth assays showed that treatment of growing region segments (5-7 millimeters) of peas with either dialyzed serum, serum globulin fraction, affinity purified immunoglobulin, or papain-cleaved antibody fragments had no effect on growth. Immunofluorescence microscopy confirmed antibody binding to cell walls and penetration of the antibodies into the tissues. Western blot analysis, immunoassay results, and affinity chromatography utilizing Sepharose-bound antibodies confirmed recognition of the protein preparation by the antibodies. Experiments employing in vitro extension as a screening measure indicated no effect upon extension by antibodies, by 50 millimolar LiCl perfusion of the apoplast or by 3 molar LiCl extraction. Addition of cell wall protein to protease pretreated segments did not restore extension nor did addition of cell wall protein to untreated segments increase extension. It is concluded that, although evidence suggests that protein is responsible for the process of extension, the class(es) of proteins which are extracted from pea cell walls with 3 molar LiCl are probably not involved in this process.     

Measuring Plant Cell Wall Extension (Creep) Induced by Acidic pH and by Alpha-Expansin

Growing plant cell walls characteristically exhibit a property known as ''acid growth'', by which we mean they are more extensible at low pH (< 5) ¹. The plant hormone auxin rapidly stimulates cell elongation in young stems and similar tissues at least in part by an acid-growth mechanism ^{2, 3}. Auxin activates a H⁺ pump in the plasma membrane, causing acidification of the cell wall solution. Wall acidification activates expansins, which are endogenous cell wall-loosening proteins ⁴, causing the cell wall to yield to the wall tensions created by cell turgor pressure. As a result, the cell begins to enlarge rapidly. This ''acid growth'' phenomenon is readily measured in isolated (nonliving) cell wall specimens. The ability of cell walls to undergo acid-induced extension is not simply the result of the structural arrangement of the cell wall polysaccharides (e.g. pectins), but depends on the activity of expansins ⁵. Expansins do not have any known enzymatic activity and the only way to assay for expansin activity is to measure their induction of cell wall extension. This video report details the sources and preparation techniques for obtaining suitable wall materials for expansin assays and goes on to show acid-induced extension and expansin-induced extension of wall samples prepared from growing cucumber hypocotyls.To obtain suitable cell wall samples, cucumber seedlings are grown in the dark, the hypocotyls are cut and frozen at -80 °C. Frozen hypocotyls are abraded, flattened, and then clamped at constant tension in a special cuvette for extensometer measurements. To measure acid-induced extension, the walls are initially buffered at neutral pH, resulting in low activity of expansins that are components of the native cell walls. Upon buffer exchange to acidic pH, expansins are activated and the cell walls extend rapidly. We also demonstrate expansin activity in a reconstitution assay. For this part, we use a brief heat treatment to denature the native expansins in the cell wall samples. These inactivated cell walls do not extend even in acidic buffer, but addition of expansins to the cell walls rapidly restores their ability to extend.Open in a separate windowClick here to view.^{(58M, flv)}     

Physical Basis for Altered Stem Elongation Rates in Internode Length Mutants of Pisum

Biophysical parameters related to gibberellin (GA)-dependent stem elongation were examined in dark-grown stem-length genotypes of Pisum sativum L. The rate of internode expansion in these genotypes is altered due to recessive mutations which affect either the endogenous levels of, or response to, GA. The GA deficient dwarf L181 (ls), two GA insensitive semierectoides dwarfs NGB5865 and NGB5862 (Ika and Ikb, respectively) and the `slender' line L197 (la cry^[ill]), which is tall regardless of GA content, were compared to the wild-type tall cultivar, Torsdag. Osmotic pressure, estimated by vapor pressure osmometry, and turgor pressure, measured directly with a pressure probe, did not correlate with the differences in growth rate among the genotypes. Mechanical wall properties of frozen-thawed tissue were measured using a constant force assay. GA deficiency resulted in increased wall stiffness judged both on the basis of plastic compliance and plastic extensibility normalized for equal stem circumference. Plastic compliance was not reduced in the GA insensitive dwarfs, though Ika reduced circumference-normalized plasticity. In contrast, in vivo wall relaxation, determined by the pressure-block technique, differed among genotypes in a manner which did correlate with extension rates. The wall yield threshold was 1 bar or less in the tall lines, but ranged from 3 to 6 bars in the dwarf genotypes. The results with the ls mutant indicate that GA enhances stem elongation by both decreasing the wall yield threshold and increasing the wall yield coefficient. In the GA-insensitive mutants, Ika and Ikb, the wall yield threshold is substantially elevated. Plants possessing Ika may also possess a reduced wall yield coefficient.     

Tensile strength of cell walls of living cells

A gas decompression technique was used to determine the breaking strength of cell walls of single cells. Breaking strengths of the bacterium Salmonella typhimurium and the unicellular green alga Chlamydomonas eugametos were 100 and 95 atmospheres, respectively, while those of sporophytes of the water mold Blastocladiella emersonii were 65 atmospheres, and those of suspension cultured cells of carrot were only 30 atmospheres. Estimation of wall tensile stress based on breaking pressures, cell radii, and estimation of wall thickness, indicates that microfibrillar walls are not necessarily stronger than walls of primitive organisms. Hence, alternative hypotheses for their evolution must be considered.     

Autolysis Promotes the Extension Capacity of Zea mays Coleoptile Cell Walls in Response to Acid pH Solutions

The relationship between autolytic degradation of ß(1–3),(1–4)-D-glucanand acid pH-induced extension of isolated Zea mays cell wallshas been investigated using a constant-load extension technique.Acidic buffer (4.5) was able to induce an additional extension(E_a) on cell walls already extended at pH 6.8 buffer under a20 g-mass load, indicating that the additional extension (E_a)was the parameter that better represented the effect of thedifferent treatments on the mechanical properties of maize coleoptilecell walls. The additional extension in response to acidic pHwas higher when cell walls had been previously autolysed for24 h at pH 5.5. Furthermore, the acid-pH effect was dependenton the presence during the constant load extension of some thermo-labilefactors, suggesting the participation of expansins. Acid pHincreased E_a of native cell walls through an increase in theplastic extension (E_p) in agreement with a one step mechanismleading directly to irreversible (plastic) wall extension assuggested by Cosgrove (1977). The autolytic degradation of ß(1–3),(1–4)-D-glucan was also able to modify the mechanicalproperties of maize coleoptile cell walls increasing its elasticextension (E_e) in response to pH 4.5 buffer but that modificationonly leads to an increase in wall extension when expansins areactive, suggesting a cooperation between ß-glucanturnover and expansin action. (Received August 5, 1998; Accepted March 16, 1999)     

Photoinhibition of Stem Elongation by Blue and Red Light : Effects on Hydraulic and Cell Wall Properties

The underlying mechanism of photoinhibition of stem elongation by blue (BL) and red light (RL) was studied in etiolated seedlings of pea (Pisum sativum L. cv Alaska). Brief BL irradiations resulted in fast transient inhibition of elongation, while a delayed (lag approximately 60 minutes) but prolonged inhibition was observed after brief RL. Possible changes in the hydraulic and wall properties of the growing cells during photoinhibition were examined. Cell sap osmotic pressure was unaffected by BL and RL, but both irradiations increased turgor pressure by approximately 0.05 megapascal (pressure-probe technique). Cell wall yielding was analyzed by in vivo stress relaxation (pressure-block technique). BL and RL reduced the initial rate of relaxation by 38 and 54%, while the final amount of relaxation was decreased by 48 and 10%, respectively. These results indicate that RL inhibits elongation mainly by lowering the wall yield coefficient, while most of the inhibitory effect of BL was due to an increase of the yield threshold. Mechanical extensibility of cell walls (Instron technique) was decreased by BL and RL, mainly due to a reduction in the plastic component of extensibility. Thus, photoinhibitions of elongation by both BL and RL are achieved through changes in cell wall properties, and are not due to effects on the hydraulic properties of the cell.     

Enlargement in chara studied with a turgor clamp : growth rate is not determined by turgor

A new method, the turgor clamp, was developed to test the effects of turgor on cell enlargement. The method used a pressure probe to remove or inject cell solution and change the turgor without altering the external environment of the cell walls. After the injections, the cells were permanently at the new turgor and required no further manipulation. Internode cells of Chara corallina grew rapidly with the pressure probe in place when growth was monitored with a position transducer. Growth-induced water potentials were negligible and turgor effects could be studied simply. As turgor was decreased, there was a threshold below which no growth occurred, and only reversible elastic/viscoelastic changes could be seen. Above the threshold, growth was superimposed on the elastic/viscoelastic effects. The rate of growth did not depend on turgor. Instead, the rate was highly dependent on energy metabolism as shown by inhibitors that rapidly abolished growth without changing the turgor. However, turgors could be driven above the maximum normally attainable by the cell, and these caused growth to respond as though plastic deformation of the walls was beginning, but the deformation caused wounding. Growth was inhibited when turgor was changed with osmotica but not inhibited when similar changes were made with the turgor clamp. It was concluded that osmotica caused side effects that could be mistaken for turgor effects. The presence of a turgor threshold indicates that turgor was required for growth. However, because turgor did not control the rate, it appears incorrect to consider the rate to be determined by a turgor-dependent plastic deformation of wall polymers. Instead, above the turgor threshold, the rapid response to energy inhibitors suggests a control by metabolic reactions causing synthesis and/or extension of wall polymers.     

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.     

Gibberellic Acid stimulation of cucumber hypocotyl elongation : effects on growth, turgor, osmotic pressure, and cell wall properties

Recently developed techniques have been used to reinvestigate the mechanism by which gibberellic acid (GA₃) stimulates elongation of light-grown cucumber (Cucumis sativus L.) seedlings. Osmotic pressure and turgor pressure were slightly reduced in GA₃-treated seedlings, which elongated 3.5 times faster than control seedlings. This indicated that GA₃ enhancement of growth was not controlled by changes in the osmotic properties of the tissues. Stress/strain (Instron) analysis revealed that plastic extension of the cell walls of GA₃-treated seedlings increased by up to 35% above the control values. Stress-relaxation measurements on frozen-thawed tissue showed that T₀ the minimum relaxation time, was reduced following application of GA₃. In vivo wall relaxation (measured by the pressure block technique) showed that the wall yield coefficient was increased, and the yield threshold was slightly reduced. Thus GA₃ affected both the mechanical (viscoelastic) and biochemical (chemorheological) properties of the cell walls of light-grown cucumber. The previous hypothesis, that GA₃ stimulates cucumber hypocotyl growth by increasing osmotic pressure and cell turgor, is contradicted by our results.     

Mechanism of Gibberellin-Dependent Stem Elongation in Peas

Stem elongation in peas (Pisum sativum L.) is under partial control by gibberellins, yet the mechanism of such control is uncertain. In this study, we examined the cellular and physical properties that govern stem elongation, to determine how gibberellins influence pea stem growth. Stem elongation of etiolated seedlings was retarded with uniconozol, a gibberellin synthesis inhibitor, and the growth retardation was reversed by exogenous gibberellin. Using the pressure probe and vapor pressure osmometry, we found little effect of uniconozol and gibberellin on cell turgor pressure or osmotic pressure. In contrast, these treatments had major effects on in vivo stress relaxation, measured by turgor relaxation and pressure-block techniques. Uniconozol-treated plants exhibited reduced wall relaxation (both initial rate and total amount). The results show that growth retardation is effected via a reduction in the wall yield coefficient and an increase in the yield threshold. These effects were largely reversed by exogenous gibberellin. When we measured the mechanical characteristics of the wall by stress/strain (Instron) analysis, we found only minor effects of uniconozol and gibberellin on the plastic compliance. This observation indicates that these agents did not alter wall expansion through effects on the mechanical (viscoelastic) properties of the wall. Our results suggest that wall expansion in peas is better viewed as a chemorheological, rather than a viscoelastic, process.     

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.     

Cell wall extensibility during branch formation in the xanthophycean alga Vaucheria terrestris

In the tip-growing filamentous cell of the xanthophycean alga Vaucheria terrestris sensu Götz, a new growing tip develops in the non-growing, cylindrical region of the cell that was exposed by local illumination. The present study examined changes in the strength and extensibility of the cell wall of the new growing tip and in the matrix components of the inner surface of the cell wall. The internal pressure required to rupture the cell walls decreased remarkably during the early to middle stages of growing tip development, but the cell wall hardly extended before rupture. In contrast, during the middle and late stages of development, cell walls were extended by internal pressure. Atomic force microscopy revealed that protease-resistant, fine granular matrix components were present only at the apical portion of a normal growing tip, and were absent in the non-growing cylindrical region. In the early and middle stages of new growing tip development, these matrix components appeared in the cell walls in patches. These results suggest that first cell wall strength decreases and then cell wall extensibility increases in the development of new growing tips, and that protease-resistant, fine granular matrix components may be involved in rendering a cell wall extensible.     

Cell wall and cell growth characteristics of giant‐celled algae

Because of their large sizes and simple shapes, giant‐celled algae have been used to study how the structural and mechanical properties of cell walls influence cell growth. Here we review known relationships between cell wall and cell growth properties that are characteristic of three representative taxa of giant‐celled algae, namely, Valonia ventricosa, internodal cells of characean algae, and Vaucheria frigida. Tip‐growing cells of the genus Vaucheria differ from cells undergoing diffuse growth in V. ventricosa and characean algae in terms of their basic architectures (non‐lamellate vs. multilamellate) and their dependence upon pH and Ca²⁺ for cell wall extensibility. To further understand the mechanisms controlling cell growth by cell walls, comparative analyses of cell wall structures and/or associated growth modes will be useful. The giant‐celled algae potentially serve as good models for such investigations because of their wide variety of developmental processes and cell shapes exhibited.     

Ruminococcus flavefaciens Cell Coat and Adhesion to Cotton Cellulose and to Cell Walls in Leaves of Perennial Ryegrass (Lolium perenne)

Ruminococcus flavefaciens was shown to possess a prominent glycoprotein coat, which contained rhamnose, glucose, and galactose as its principal carbohydrates. Periodate-reactive carbohydrate occurred as a surface layer of the coat. The ruminococci adhered strongly by means of this coat to cotton cellulose and to cell walls in leaf sections of Lolium perenne L. (perennial ryegrass). The coat was diffuse at the point of contact so that the bacterial cell wall was in close contact with the substrate. Adhesion was influenced by the availability of damaged plant cell walls and by the cell wall type and occurred most rapidly to cell walls of the epidermis and sclerenchyma, followed by the phloem and mesophyll. Plaques of bacteria with filamentous coat extensions developed on all these tissues. The bacteria did not readily adhere to the walls of the bundle sheath cells or metaxylem or protoxylem vessels and did not adhere to the cuticle or chloroplasts. The epidermal and phloem cell walls were more rapidly digested than the walls of other cell types.     

Water Potential Modulates Extensibility of Rye Coleoptile Cell Walls

Extensibility of walls of frozen/thawed rye (Secale cereale) coleoptile segments as a function of the water potential of the incubation solution (Ψ₀) was analyzed employing the creep test method. Negative Ψ₀ exerts an inhibiting effect on extension of isolated walls. The lower the Ψ₀ of polyethylene glycol 6000 (PEG), the less the walls of frozen/thawed segments extended under load. This inhibiting effect of Ψ₀ on wall creep was reversible and independent of the preincubation temperature of the segments. An increase in Ψ₀ resulted in increased extension rate within 2–4 min, whereas a decrease in Ψ₀ resulted in gradually decreasing extension rate after 8–12 min. This finding implies that wall extension changes during growth induced by changes of Ψ₀ in vivo are not only due to changes of turgor pressure but also due to a direct influence by negative Ψ₀ on physical wall properties. The results are discussed with respect to the regulation of extension growth during conditions of water stress.     

Xyloglucan Endotransglycosylase Activity, Microfibril Orientation and the Profiles of Cell Wall Properties Along Growing Regions of Maize Roots

A series of physical and chemical analyses were made on theexpanding zone of maize seedling roots grown in hydroponics.Comparison of longitudinal profiles of local relative elementalgrowth rate and turgor pressure indicated that cell walls becomelooser in the apical 5 mm and then tighten 5–10 mm fromthe root tip. Immersion of roots in 200 mol m^–3 mannitol(an osmotic stress of 0·48 MPa) rapidly and evenly reducedturgor pressure along the whole growing region. Growth was reducedto a greater extent in the region 5–10 mm from the roottip than in the apical region. This indicated rapid wall-looseningin the root tip, but not in the more basal regions. Following 24 h immersion in 400 mol m^–3 mannitol (an osmoticstress of 0·96 MPa) turgor had recovered to pre-stressedvalues. Under this stress treatment, growth was reduced in theregion 4–10 mm from the root tip, despite the recoveryof turgor, indicating a tightening of the wall. In the rootapex, local relative elemental growth rate was unchanged incomparison to control tissue, showing that wall properties herewere similar to the control values. Cellulose microfibrils on the inner face of cortical cell wallsbecame increasingly more parallel to the root axis along thegrowth profile of both unstressed and stressed roots. Orientationdid not correlate with the wall loosening in the apical regionof unstressed roots, or with the tightening in the region 5–10mm from the root tip following 24 h of osmotic stress. Longitudinal profiles of the possible wall-loosening enzymexyloglucan endotransglycosylase (XET) had good correspondencewith an increase in wall loosening during development. In thezone of wall tightening following osmotic stress, XET activitywas decreased per unit dry weight (compared with the unstressedcontrol), but not per unit fresh weight. Key words: Osmotic stress, turgor, growth, cell wall properties, microfibrils, XET