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Cellulose orientation determines mechanical anisotropy in onion epidermis cell walls

Cellulose orientation determines mechanical anisotropy in onion epidermis cell walls Abstract The role of cellulose microfibril orientation in determining cell wall mechanical anisotropy and in the control of the wall plastic versus elastic properties was studied in the adaxial epidermis of onion bulb scales using the constant-load (creep) test. The mean or net cellulose orientation in the outer periclinal wall of the epidermis was parallel to the long axis of the cells. In vitro cell wall extensibility was 30–90% higher in the direction perpendicular to the net microfibril orientation than parallel to it. This was the case for the size of the initial deformation occurring just after the load application and for the rate of time-dependent creep. Loading/unloading experiments confirmed the presence of a real irreversible component in cell wall extension. The plastic component of the time-dependent deformation was higher perpendicular to the net cellulose orientation than parallel to it. An acid buffer (pH 4.5) increased the creep rate by 25–30% but this response was not related to cellulose orientation. The present data provide direct evidence that the net orientation of cellulose microfibrils confers mechanical anisotropy to the walls of seed plants, a characteristic that may be relevant to understanding anisotropic cell growth. Allium, cell wall extensibility, cellulose orientation, confocal microscopy, creep test Introduction Plant cells are surrounded by the stiff yet extensible cell walls that play a crucial role in determining the rate and direction of cell expansion and in the control of plant shape in general (Hernandez and Green, 1993; Cosgrove, 2000a). The link between the wall properties relevant for growth and the rate of steady-state plant cell expansion was established by Lockhart (1965) and expressed in the equation that holds true if water transport to the growing cell is not limiting:  r=(1/V)(dV/dt)=Φ(P−Y)where r is the relative growth rate measured as a change in cell volume (1/V)(dV/dt); Φ is the wall yielding coefficient; P is the turgor pressure; and Y is the yield threshold, the minimum turgor required for growth. According to Lockhart, cell expansion occurs because the effective turgor pressure (P–Y) irreversibly stretches the cell wall. The parameters Φ and Y depend on physical (mechanical) properties of the cell wall and on the metabolic reactions modifying the interactions between the wall constituents (Taiz, 1984; Cosgrove, 1993). The mechanics of a cell wall are determined by its structure that resembles that of composite materials (Kerstens et al., 2001). The walls consist of stiff semi-crystalline cellulose microfibrils cross-linked by hemicelluloses and embedded in a gel-like matrix of pectins (Cosgrove, 1997). Cellulose microfibrils have traditionally been considered as the main load-bearing components in the cell wall whose orientation determines the direction of preferential wall extension (Carpita and Gibeaut, 1993). This statement was mostly based on the observation that, in growing cells, cellulose microfibrils are usually transversely oriented to the vector of the cell extension (Baskin et al., 1999; Sugimoto et al., 2000). At the same time, there have been surprisingly few direct confirmations that a specific alignment of cellulose microfibrils confers to the cell walls a mechanical anisotropy which is then translated into a preferential direction of cell growth. Such evidence was reported in early work on cell walls of Nitella, a green alga (reviewed in Taiz, 1984). It is, however, not obvious that these data can be extrapolated to higher plants. Nitella walls are many times thicker than primary walls of higher plants and, accordingly, contain many more microfibril layers across the wall thickness (Baskin et al., 1999). Also this alga, to our knowledge, is not considered as ancestral to higher plants, and there is no reliable information on its wall architecture. Searching for appropriate seed plant models to study the relationship between microfibril orientation and mechanical cell wall anisotropy has led to the use of onion and Kalanchoë epidermal peels (Kerstens et al., 2001). With an Instron-like assay, stress–strain curves for these tissues were determined. It was demonstrated for the first time for higher plants that a net parallel orientation of cellulose microfibrils in the onion epidermis wall coincided with a high mechanical anisotropy. In contrast, the Kalanchoë epidermis had a random net orientation of microfibrils and was mechanically isotropic. The present study confirms and extends the data obtained in the previous work (Kerstens et al., 2001) using the creep (constant-load) method for measuring cell wall extensibility in vitro. Unlike the stress–strain assay, the creep method mimics the action of turgor on growing cell walls (Taiz, 1984). Due to this fact, data on creep analysis have been related to the parameters Φ and Y from the Lockhart equation (Okamoto and Okamoto, 1994; Büntemeyer et al., 1998; Thompson, 2001). A similar attempt in relation to the net microfibril orientation was undertaken in this work. Another advantage of the constant-load test is its ability to distinguish between irreversible (plastic) and reversible (elastic) components of cell wall deformation. About 70 years ago, it was postulated that the plastic component of cell wall extensibility in vitro correlates with the rate of plant cell expansion in vivo (Heyn, 1932). This point of view was questioned by Schopfer and co-workers, who claimed that the in vitro plastic extensibility of isolated cell walls in fact represents retarded elastic wall extension (Hohl and Schopfer, 1992; Nolte and Schopfer, 1997). The present study confirms the presence of a real irreversible component in the extension of onion epidermal cell walls. Using the advantages of the creep test, it was also possible to investigate for the first time the effect of net microfibril orientation on plastic versus elastic cell wall properties in higher plants. Materials and methods Plant material Onion (Allium cepa L. cv. Bonkajuin) bulbs were commercially acquired. The bulbs came from an early harvest at the beginning of June and had not reached their maximal mature size. In the field, they had some potential for further growth at the moment of harvesting. Strips of adaxial epidermis, 25–30 mm long and 4 mm wide, were peeled from the equatorial part of the adaxial side of the third (from the outside) live scale in directions parallel and perpendicular to the axis of the bulb. These ‘longitudinal’ and ‘transverse’ epidermal strips were immediately frozen in liquid nitrogen, stored at −20 °C, and used within 1 week after isolation. Analysis of the net cellulose microfibril orientation The net or mean orientation of cellulose microfibrils in the outer periclinal wall of the adaxial epidermis was determined using polarization confocal microscopy and fluorescent Congo Red staining as described previously (Verbelen and Stickens, 1995; Verbelen and Kerstens, 2000). Thawed epidermal strips were stained in a 1% (w/v) solution of Congo Red (Sigma) in water for 2 h. After rinsing in water, the Congo Red-mediated fluorescence of cell walls was studied using a confocal laser scanning microscope (Bio-Rad MRC 600 mounted on a Zeiss Axioskop) with a ×20 (NA 0.5) dry objective and a co-axial rotating object table. The dichroic fluorescent dye Congo Red preferentially absorbs light directed parallel to the dipole moment of its chromophoric group. It has an ability to bind specifically to β-1,4-linked glucan polymers, especially to cellulose fibrils, and the dipole moments of Congo Red chromophoric groups will be aligned in the same direction as the cellulose polymers. If the cellulose fibrils have some preferential orientation, Congo Red fluorescence will be maximal when the vector of the polarized exciting laser beam is parallel to the net cellulose orientation. When the vector is perpendicular to the net microfibril orientation, the fluorescence intensity will be minimal. In the case of a net random cellulose orientation, the wall fluorescence intensity will be equal for all orientations of the polarized light vector. The extent to which cellulose microfibrils were aligned with respect to the cell's long axis was expressed as the axiality ratio. This is the ratio between the wall fluorescence intensities with the vector of the excitation light parallel and perpendicular, respectively, to the cell longitudinal axis. The axiality ratio equals unity if the net cellulose microfibril orientation is random. It will have values above or below unity in walls with, respectively, a net cellulose orientation parallel or perpendicular to the cell axis. The fluorescence intensity of Congo Red-stained cell walls was quantified using Photoshop 4.0 software (Adobe). Extensiometry In vitro extensibility of onion epidermal cell walls was studied with a custom-built constant-load extensiometer similar to that described by Kutschera and Schopfer (1986). Wall extension was measured using a displacement transducer (model 200HR, Schaevitz, Hampton, VA, USA) coupled to an in-line amplifier (model MC-IP-15B-B10 from ALTHEN GmbH, Kelkheim, Germany) and a data acquisition system (model DI-700 from DATAQ, Akron, OH, USA) connected to a PC. The cell wall strain curves were recorded and analysed with, respectively, WinDaq/Lite and WinDaq Waveform Browser software (both from DATAQ, Akron, OH, USA). The constant loads used in the creep test should ideally induce the same stress in the isolated walls as that existing in vivo. It is very difficult, however, to measure the real force extending the epidermal cell wall in an onion bulb because of its complex nature. This force is summed from the turgor of epidermal cells, the tension between the epidermis and the underlying parenchymatic cells (Kutschera et al., 1987), and the pressure developing between the adjacent scales of the bulb as a result of their growth. Therefore, the strategy in this work was to use a maximally broad range of loads to model the effect of any possible magnitude of the unknown cumulative force on the extension of the epidermis. The lever of the extensiometer with the upper clamp attached to its left shoulder was pre-equilibrated before securing a cell wall sample. Thereafter, the lever was held in the horizontal position by a lock under its right shoulder while an initial load was placed on the scale of the extensiometer. The lower clamp consisted of two vertical plexiglass plates with two U-shaped rubber rims stuck on their internal surfaces opposite to each other. Closing the clamp gripped the lower end of the cell wall sample and formed a water-tight chamber around it. This chamber was filled with 10 mM sodium citrate buffer (pH 6.0, if not otherwise stated) so that the cell wall sample was completely immersed in the buffer during the measurement. A 10 mm segment of an epidermal strip was secured between the clamps. Cell wall extension was induced by unlocking the lever. A rapid, practically instantaneous cell wall extension referred to as ‘initial deformation’ was measured 1 s after the load application. It was followed by a slow, time-dependent deformation (creep) whose kinetics are represented as an average rate for the intervals 1 s–5 min, 5–10 min, and 10–15 min after the load application (Fig. 2). To study the reversibility of cell wall extension in vitro, 2 or 5 g loads were subsequently added onto and then removed from the scale of the extensiometer at 60 s intervals. On the basis of the recorded cell wall strain curves, total deformation, initial deformation, and creep were calculated for every step of loading and unloading. Total deformation (DT) is the change in wall length between subsequent increases or decreases in load. Initial deformation (DI) is the change in wall length measured 1 s after loading (unloading). Creep (C) is the amplitude of the time-dependent cell wall deformation from 1 s to 60 s after the load application (removal). Thus, DT=DI+C. To estimate total deformation reversibility, the sum of total deformations at all steps of unloading-induced cell wall contraction was divided by the sum of total deformations at all steps of loading-induced cell wall extension. The result was expressed as percentage reversibility (elasticity). A similar procedure was used to calculate separately the reversibilities of creeps and initial deformations. The sum of creeps (initial deformations) at all steps of unloading was divided by the sum of creeps (initial deformations) at all loading steps and expressed as a percentage of creep (initial deformation) reversibility. Results Quantification of the cellulose microfibril orientation in the outer epidermal cell wall The onion bulbs used in this study contained two or three dead dry scales and from four to seven live scales that surrounded several juvenile ‘bulblets’. The long axes of epidermal cells in all scales were parallel to the axis of the bulb. A confocal image of a typical Congo Red-stained cell from the adaxial epidermis of the third (from outside) live scale is illustrated in Fig. 1. Fluorescence intensity is coded in shades of grey. It is clear that when the vector of the laser beam is parallel to the cell's long axis (Fig. 1A), fluorescence in the cell wall is much stronger than when the vector is perpendicular to the cell axis (Fig. 1B). The cellulose microfibrils thus have a net longitudinal orientation in this tissue. Analysis of 100 epidermal cells selected at random from 10 different onion bulbs has established that the net cellulose alignment in the outer epidermal wall of the third scale was characterized by the axiality ratio of 1.52±0.04 (SE). Fig. 1 View largeDownload slide Mean cellulose microfibril orientation in the adaxial epidermis of the onion bulb scale. Polarization confocal micrographs of epidermal cell walls stained with Congo Red. The vector of polarization of the laser is indicated by arrows. The intensity of fluorescence is coded in shades of grey from low (black) to high (white). Its values for the outer periclinal wall of the cell in the centre of the figure are shown as numbers in the lower left corners of the micrographs. The fluorescence intensity is maximal (A) and minimal (B) with the vector of polarization parallel and perpendicular, respectively, to the cell long axis. This shows that the net orientation of cellulose microfibrils is parallel to the longitudinal axis of the cell. Scale bar=50 μm. Fig. 1 View largeDownload slide Mean cellulose microfibril orientation in the adaxial epidermis of the onion bulb scale. Polarization confocal micrographs of epidermal cell walls stained with Congo Red. The vector of polarization of the laser is indicated by arrows. The intensity of fluorescence is coded in shades of grey from low (black) to high (white). Its values for the outer periclinal wall of the cell in the centre of the figure are shown as numbers in the lower left corners of the micrographs. The fluorescence intensity is maximal (A) and minimal (B) with the vector of polarization parallel and perpendicular, respectively, to the cell long axis. This shows that the net orientation of cellulose microfibrils is parallel to the longitudinal axis of the cell. Scale bar=50 μm. All scales located inward from the third scale also had a clear longitudinal net orientation of cellulose microfibrils in the outer periclinal walls of their adaxial epidermis. The two outermost live scales had only a slightly longitudinal or even a random net orientation of microfibrils in the corresponding wall. The third scale was chosen for further analysis because it was the largest scale with a net longitudinal cellulose orientation. In the terminology used subsequently, ‘longitudinal strips’ refer to epidermis samples where the load is applied parallel to the net cellulose orientation and thus also parallel to the axis of the cell. In ‘transverse strips’, the load is applied perpendicular to the net cellulose orientation. The in vitro cell wall extensibility depends on the net cellulose microfibril orientation Applying a constant load to an epidermal strip resulted in an immediate high-amplitude initial deformation that was followed by a slow, time-dependent creep (Fig. 2). It is clear that extension is much larger transverse to the cellulose orientation than parallel to it. The rate of creep always decreases with time during 60 min of measurement (data not shown). Analysis of the deformation curves revealed that the whole range of the loads used (5–20 g) induced stronger extension in the transverse than in the longitudinal strips (Fig. 3). This difference was highly significant (P <0.01) for the initial deformations (Fig. 3A) and the creep rates during the intervals 5–10 and 10–15 min after load application (Fig. 3C, D), where a practically 2-fold difference was registered for the 5 g and 10 g loads. Thus the isolated epidermal cell walls are much more extensible in the direction perpendicular to the mean orientation of cellulose microfibrils than in the direction parallel to the net cellulose orientation. Fig. 2 View largeDownload slide Dynamics of extension of an isolated onion epidermis with longitudinal (thick line) and transverse (thin line) net orientation of cellulose microfibrils. Typical curves recorded after a 15 g load application (arrow) are represented. Vertical dashed lines indicate the time intervals (1 s–5 min, 5–10 min and 10–15 min after the load application) during which the average creep rate was determined. Fig. 2 View largeDownload slide Dynamics of extension of an isolated onion epidermis with longitudinal (thick line) and transverse (thin line) net orientation of cellulose microfibrils. Typical curves recorded after a 15 g load application (arrow) are represented. Vertical dashed lines indicate the time intervals (1 s–5 min, 5–10 min and 10–15 min after the load application) during which the average creep rate was determined. Fig. 3 View largeDownload slide Dependence of extension on the load in onion epidermal walls with longitudinal (thick lines, closed circles) and transverse (thin lines, open circles) net orientation of cellulose microfibrils. Comparison of initial deformations (A) and creep rates during the intervals 1 s–5 min (B), 5–10 min (C), and 10–15 min (D) after the load application. These values were calculated after analysis of experimental curves of the type indicated in Fig. 2. Data are means ±SE (n=13). Fig. 3 View largeDownload slide Dependence of extension on the load in onion epidermal walls with longitudinal (thick lines, closed circles) and transverse (thin lines, open circles) net orientation of cellulose microfibrils. Comparison of initial deformations (A) and creep rates during the intervals 1 s–5 min (B), 5–10 min (C), and 10–15 min (D) after the load application. These values were calculated after analysis of experimental curves of the type indicated in Fig. 2. Data are means ±SE (n=13). Interestingly, the difference between the initial creep rates (0–5 min) of longitudinal and transverse strips was smaller (compare Fig. 3B with C and D). At 20 g load, it was even statistically insignificant. The reason for this discrepancy became evident through analysis of creep during the first seconds after the load application (Fig. 4). The dynamics of creep of longitudinal and transverse strips were absolutely identical during the first 10 s after the 20 g load application. This was due to the non-linear behaviour of the stress–strain dependence in transverse strips (Fig. 4). For the later time intervals, the dependence of creep rates on the applied forces was linear in longitudinal as well as in transverse epidermal strips (Fig. 3B–D). Fig. 4 View largeDownload slide Initial creep rates (1–21 s after load application) of onion epidermal walls with net longitudinal (thick lines) and transverse (thin lines) orientation of cellulose microfibrils. Each curve is the average of 10–12 experiments with different epidermal strips at a given load. For convenience of comparison, the starting points of the curves are superimposed at time zero. Notice that for 20 s of measurement, 5 g increments in the load induced similar increments in extension in longitudinal strips but decreasing increments in transverse strips. This indicates that the dependence of the initial creep rate on the load is non-linear in the transverse orientation of cellulose microfibrils. Error bars are the SE. Fig. 4 View largeDownload slide Initial creep rates (1–21 s after load application) of onion epidermal walls with net longitudinal (thick lines) and transverse (thin lines) orientation of cellulose microfibrils. Each curve is the average of 10–12 experiments with different epidermal strips at a given load. For convenience of comparison, the starting points of the curves are superimposed at time zero. Notice that for 20 s of measurement, 5 g increments in the load induced similar increments in extension in longitudinal strips but decreasing increments in transverse strips. This indicates that the dependence of the initial creep rate on the load is non-linear in the transverse orientation of cellulose microfibrils. Error bars are the SE. A 25 g load caused rupture in ∼50% of the transverse strips but never in longitudinal strips. This fact confirms the higher tensile strength of the epidermal tissue in the direction parallel to the net orientation of cellulose microfibrils. Plastic and elastic components of in vitro extensibility depend on the net microfibril orientation Experiments were set up to find out if a true plastic component of in vitro extensibility exists in onion epidermal walls and, if so, to determine the effect of cellulose microfibril orientation on plastic versus elastic behaviour of isolated cell walls. In a first approach, cell wall deformation was recorded during a single 1 min loading step followed by complete unloading. Irrespective of the load used, the epidermal cell walls immediately returned to their original length after unloading (not shown). This phenomenon was, however, not due to the elastic behaviour of the cell wall. It just reflected the fact that the lever of the extensiometer (see Materials and methods) was pre-equilibrated before the measurement and fixed horizontally. After the complete unloading, the lever came back to its original horizontal equilibrium position, returning the secured cell wall sample to the point it occupied before stretching. A single pre-loading step allowed this artefact to be separated from the real elastic cell wall properties. In this approach, extension experiments start from epidermis samples that are kept under slight tension. As an example, Fig. 5 depicts an experiment performed with 2 g loads. Epidermal strips were pre-loaded with 2 g for 1 min. Then an additional 2 g load was applied for 1 min. After its removal, the extension was measured during the next 20 min (Fig. 5). For both orientations of the cellulose fibrils, the unloading resulted in an instant elastic shrinkage of the epidermal strips, followed by a limited (not more than 5 μm) retarded elastic contraction, that lasted 2–3 min. Thereafter, the length of the epidermal strips did not change any more (Fig. 5). Thus 30–40% of cell wall extension is irreversible. This indicates that real plastic deformation exists in onion epidermal cell walls. Using 200 or 500 mg for the pre-loading step as well as 1 g for the loading did not qualitatively change this cell wall behaviour. In all cases, a marked plastic component of the wall extension was detected (results not shown). Fig. 5 View largeDownload slide Plastic component in the extension of epidermal walls with net longitudinal (thick lines) and transverse (thin lines) orientations of cellulose microfibrils. Epidermal strips were pre-loaded with a 2 g load (+L) for 1 min. Thereafter an additional 2 g was applied (+L, loading) and removed 1 min later (–L, unloading). Changes in the subsequent tissue deformation were recorded for 20 min longer. After unloading, a significant part of tissue deformation remains, demonstrating the presence of a real plastic component (Ep) in onion epidermal wall deformation. Typical curves are shown. Fig. 5 View largeDownload slide Plastic component in the extension of epidermal walls with net longitudinal (thick lines) and transverse (thin lines) orientations of cellulose microfibrils. Epidermal strips were pre-loaded with a 2 g load (+L) for 1 min. Thereafter an additional 2 g was applied (+L, loading) and removed 1 min later (–L, unloading). Changes in the subsequent tissue deformation were recorded for 20 min longer. After unloading, a significant part of tissue deformation remains, demonstrating the presence of a real plastic component (Ep) in onion epidermal wall deformation. Typical curves are shown. To explore the effect of net cellulose microfibril orientation on plastic versus elastic components of cell wall extension, longitudinal and transverse epidermal strips were stretched in 1 min intervals by a series of equal loads (2 or 5 g) up to a maximum of 8 and 20 g, respectively. Thereafter, the loads were removed in 1 min intervals back to the final 2 or 5 g stretching force. Thus the initial 2 or 5 g served as pre-loading steps in the experiments. Such a spectrum of loads allowed estimation of the viscoelastic behaviour of onion cell walls subjected to a maximally broad range of forces. A typical experimental curve of a 2 g cycle is illustrated in Fig. 6. In both epidermal strips, a significant part of the wall deformation did not revert after unloading, which confirmed its partly plastic nature. Statistical analysis of many experimental curves of the type indicated in Fig. 6 has shown that loading induced much higher extensions in transverse than in longitudinal epidermal strips (Fig. 7A) and also that the elastic shrinkages during unloading were higher to the same extent. Therefore, total deformation reversibility (see Materials and methods) was statistically the same for longitudinal and transverse epidermal strips (Fig. 9A). Since the deformation at every step of loading (unloading) includes a rapid initial deformation followed by a time-dependent creep, the values of these components were also analysed (Fig. 7B, C). The reversibility of the initial deformation was similar (∼60%) in longitudinal and transverse strips (Fig. 9B). However, the two orientations differ specifically on the level of creep. On the one hand, creep was significantly higher in transverse than in longitudinal strips (Fig. 7C). On the other hand, the slow time-dependent contraction upon unloading was similar in both strips or even higher in longitudinal strips (Fig. 7C). Therefore, the reversibility of creep in longitudinal strips (14.2%) was significantly (P <0.01) higher than in transverse strips (8.9%). This means that the time-dependent extension of onion epidermal walls is proportionally more elastic parallel to the net cellulose orientation than perpendicular to the net cellulose orientation. The 5 g loading/unloading cycles gave qualitatively similar results (Figs 8,9). Longitudinal and transverse strips differed only in the percentage of their creep reversibility, which was significantly higher in the former (6.5% compared with 3.4%) (Fig. 9). Fig. 6 View largeDownload slide Extension of epidermal walls with net longitudinal (thick lines) and transverse (thin lines) orientations of cellulose microfibrils in the course of a 2 g loading/unloading cycle. Loads of 2 g were applied and subsequently removed in 1 min intervals, and the resulting cell wall deformation was recorded. Arrows indicate the time points at which the loads were applied or removed. Numbers above the curves show the total loads stretching the walls at the corresponding stages of the cycle. Typical curves are shown. Fig. 6 View largeDownload slide Extension of epidermal walls with net longitudinal (thick lines) and transverse (thin lines) orientations of cellulose microfibrils in the course of a 2 g loading/unloading cycle. Loads of 2 g were applied and subsequently removed in 1 min intervals, and the resulting cell wall deformation was recorded. Arrows indicate the time points at which the loads were applied or removed. Numbers above the curves show the total loads stretching the walls at the corresponding stages of the cycle. Typical curves are shown. Fig. 7 View largeDownload slide Results of statistical analysis of cell wall extension curves of the type indicated in Fig. 6 during 2 g loading/unloading cycles. Changes in the total deformation (A), the initial deformation (B), and the creep (C) of the epidermal walls with net longitudinal (black bars) and transverse (white bars) orientation of cellulose microfibrils. Negative values indicate contraction during unloading. The total deformation was calculated as the change in wall length during 60 s between the subsequent load applications and removals (see Fig. 6). The initial deformation is the change in wall length measured 1 s after loading (unloading). The creep is the amplitude of the time-dependent cell wall deformation during the interval 1–60 s after load application (removal). Data are means ±SE (n=21). Fig. 7 View largeDownload slide Results of statistical analysis of cell wall extension curves of the type indicated in Fig. 6 during 2 g loading/unloading cycles. Changes in the total deformation (A), the initial deformation (B), and the creep (C) of the epidermal walls with net longitudinal (black bars) and transverse (white bars) orientation of cellulose microfibrils. Negative values indicate contraction during unloading. The total deformation was calculated as the change in wall length during 60 s between the subsequent load applications and removals (see Fig. 6). The initial deformation is the change in wall length measured 1 s after loading (unloading). The creep is the amplitude of the time-dependent cell wall deformation during the interval 1–60 s after load application (removal). Data are means ±SE (n=21). Fig. 8 View largeDownload slide Results of statistical analysis of cell wall extension curves during 5 g loading/unloading cycles. Changes in the total deformation (A), the initial deformation (B), and the creep (C) of epidermal walls with net longitudinal (black bars) and transverse (white bars) orientation of cellulose microfibrils. Negative values indicate contraction during unloading. The total deformation was calculated as the change in wall length during 60 s between the subsequent load applications and removals. The initial deformation is the change in wall length measured 1 s after loading (unloading). The creep is the amplitude of the time-dependent cell wall deformation during the interval 1–60 s after the load application (removal). Data are means ±SE (n=10). Fig. 8 View largeDownload slide Results of statistical analysis of cell wall extension curves during 5 g loading/unloading cycles. Changes in the total deformation (A), the initial deformation (B), and the creep (C) of epidermal walls with net longitudinal (black bars) and transverse (white bars) orientation of cellulose microfibrils. Negative values indicate contraction during unloading. The total deformation was calculated as the change in wall length during 60 s between the subsequent load applications and removals. The initial deformation is the change in wall length measured 1 s after loading (unloading). The creep is the amplitude of the time-dependent cell wall deformation during the interval 1–60 s after the load application (removal). Data are means ±SE (n=10). Fig. 9 View largeDownload slide Reversibility (elasticity) of the total deformations (A), the initial deformations (B), and the creeps (C) of epidermal cell walls with net longitudinal (black bars) and transverse (white bars) orientation of cellulose microfibrils in the 2 g and the 5 g loading/unloading cycles. To estimate total deformation reversibility, the total deformations at all steps of unloading-induced cell wall contraction were summed and divided by the sum of the total deformation values at all steps of loading-induced cell wall extension. The result was expressed as percentage reversibility (elasticity). A similar procedure was used to calculate separately the reversibility of creep and of initial deformation. Data are means ±SE (n=21 for the 2 g loading/unloading cycle and n=10 for the 5 g loading/unloading cycle). Fig. 9 View largeDownload slide Reversibility (elasticity) of the total deformations (A), the initial deformations (B), and the creeps (C) of epidermal cell walls with net longitudinal (black bars) and transverse (white bars) orientation of cellulose microfibrils in the 2 g and the 5 g loading/unloading cycles. To estimate total deformation reversibility, the total deformations at all steps of unloading-induced cell wall contraction were summed and divided by the sum of the total deformation values at all steps of loading-induced cell wall extension. The result was expressed as percentage reversibility (elasticity). A similar procedure was used to calculate separately the reversibility of creep and of initial deformation. Data are means ±SE (n=21 for the 2 g loading/unloading cycle and n=10 for the 5 g loading/unloading cycle). When comparing the two loading/unloading cycles, it is clear that the total deformation (Fig. 9A) and its two components (Fig. 9B, C) were much less reversible in the 5 g than in the 2 g cycle. Higher loads thus increase the proportion of plastic deformation in onion epidermal walls. Prolonged unloading steps (up to 15 min) revealed in both cycles that the retarded elastic contractions described in Figs 7C and 8C did not last for longer than 10 min (results not shown). The dynamics and the amplitude of the late part of the elastic contraction presented only a negligible fraction of the total deformation during unloading. The effect of acid pH on cell wall extensibility The orientation of cellulose microfibrils in the cell wall seems to define the direction but not the rate of cell expansion (Baskin et al., 1999; Sugimoto et al., 2000). The latter is probably regulated by specific cell wall proteins breaking and/or establishing cross-links between different cell wall polymers (McQueen-Mason et al., 1992; Fry et al., 1992; Brownleader et al., 2000; Okamoto-Nakazato et al., 2000). Among these proteins, the expansins induce cell wall expansion at acid pH values by disrupting the hydrogen bonding between cellulose microfibrils and matrix hemicelluloses (Cosgrove, 2000b). It was hypothesized that different groups of factors regulate growth in the directions parallel and transverse to the mean cellulose orientation by catalysing shear and separation of microfibrils correspondingly (Baskin et al., 1999). To check whether lowering the pH could stimulate extension differentially depending on the cellulose orientation, the effect of an acid buffer on extensibility of transverse and longitudinal epidermal strips was studied. Lowering the pH to 4.5 markedly (P <0.05) stimulated the creep rate in longitudinal as well as in transverse epidermal strips, but only during the time intervals 10–15 and 5–15 min after the load application (Fig. 10C, D). Its effects on the initial deformations and the creep rates at the earlier stages were not statistically significant (Fig. 10A and B). The percentage of the creep rate stimulation (25–30%) was the same for longitudinal and transverse strips (Fig. 10C, D). As a consequence, the ratio of their extensibilities in the two directions is similar to that at neutral pH (Fig. 3). Preincubation of the tissue in the acid buffer for 30 min before stretching did not change the above-mentioned pH effect on in vitro extensibility, which indicates that tissue permeability was not a limiting factor (results not shown). Thus the acid-induced extension of the isolated cell walls does not depend on the mean cellulose microfibril orientation. Fig. 10 View largeDownload slide The effect of an acid pH buffer on the initial deformations (A) and the creep rates during the intervals 1 s–5 min (B), 5–10 min (C), and 10–15 min (D) of epidermal walls with net longitudinal and transverse orientation of cellulose microfibrils. Black bars, control treatment (10 mM sodium citrate buffer, pH 6.0); white bars, experimental treatment (10 mM sodium citrate buffer, pH 4.5). Data are means ±SE (n=15). Fig. 10 View largeDownload slide The effect of an acid pH buffer on the initial deformations (A) and the creep rates during the intervals 1 s–5 min (B), 5–10 min (C), and 10–15 min (D) of epidermal walls with net longitudinal and transverse orientation of cellulose microfibrils. Black bars, control treatment (10 mM sodium citrate buffer, pH 6.0); white bars, experimental treatment (10 mM sodium citrate buffer, pH 4.5). Data are means ±SE (n=15). Discussion Upon loading, the epidermis samples stretched in a biphasic way. The initial, practically instantaneous deformation was followed by a slow time-dependent creep. In the past, both parts of the extension have been considered as relevant wall mechanical properties that could help to understand processes involved in cell expansion (Kutschera and Schopfer, 1986; Cosgrove, 1989). The fact that acid pH stimulated the creep rate but did not increase the initial deformation (Fig. 10) suggests that creep measured in vitro might be a good indicator for the parameters Φ and Y that regulate the rate of plant cell expansion in vivo and that the initial deformation may characterize cell wall mechanical properties that have no relationship to in vivo growth regulation. Similar conclusions were drawn in work on maize roots (Büntemeyer et al., 1998). For all loads used, the initial deformation and the creep rate were higher perpendicular to the mean cellulose orientation (transverse strips) than parallel to it (longitudinal strips) (Fig. 3). This confirms previous findings that showed higher extensibility of onion epidermis walls in the direction perpendicular to the mean microfibril orientation using a stress–strain method (Kerstens et al., 2001). Okamoto and Okamoto (1994) have used the linear dependence of the creep rate on the load to estimate ϕ and y, the in vitro analogues of Φ (yield coefficient) and Y (yield treshold) parameters from the Lockhart equation. In a similar approach for our data, linear fits were calculated for the ratio between late creep rates and loads. The slopes of the lines obtained gave the values of ϕ, which was higher for transverse (0.41) than for longitudinal (0.36) epidermal strips. This indicates that the mean orientation of cellulose microfibrils has a moderate effect (14%) on ϕ. Loading/unloading experiments (Figs 5, 6) demonstrated the presence of a real plastic component in the extension of onion epidermal cell walls in vitro. The plastic component of time-dependent deformation is higher in the direction transverse to the net microfibril orientation than parallel to it (Fig. 9C). This difference is potentially involved in growth anisotropy. Indeed, in vivo, a higher proportion of the deformation could be transformed into irreversible cell wall extension in the direction transverse to the net cellulose orientation compared with the direction parallel to the net cellulose orientation. A real plastic component has been described for rye coleoptiles (Kutschera, 1996), onion abaxial epidermis (Wilson et al., 2000), and tomato fruit epidermis (Thompson, 2001). The common methodological feature of all these studies was the presence of a pre-loading step and consequently the absence of a fully unloaded stage. However, Schopfer and co-workers challenged the presence of an irreversible plastic component in cell wall extension in vitro. They came to the conclusion that apparent plastic deformation of the wall in fact represented its retarded elastic behaviour (Hohl and Schopfer, 1992; Nolte and Schopfer, 1997). Their conclusion is based exclusively on experiments where complete unloading took place. While being useful in material testing, this step is physiologically irrelevant. The creep test models the action of the turgor on the growing cell wall. In this respect, complete unloading is equivalent to the state of plasmolysis when the turgor equals zero and plant cells do not grow. Cell expansion is always associated with a state of stress inside the cell wall. That is why measurements aimed at distinguishing between reversible versus irreversible components of the cell wall extension in vitro should be based on comparison between different stress states of the cell wall. The magnitudes of these stresses should ideally be close to those existing in the wall in vivo. Cellulose and xyloglucans form the major load-bearing network in the cell wall (Shedletzky et al., 1992) that apparently plays a key role in determining the rate of the wall extension during loading. At the same time, pectins confer compression resistance to the cell wall (McCann and Roberts, 1994). It is known that wall deformation causes a certain microfibril realignment in the direction of stress and a concomitant compression in the matrix (Spatz et al., 1999; Köhler et al., 2000; Wilson et al., 2000). In the onion cell walls, the pectin network, compressed by realigned microfibrils during loading, will then return to its original shape after load removal. This process could substantially contribute to the wall contraction observed upon unloading. Thus the different amount of the retarded elasticity correlating with the mean microfibril orientation could result from the interaction between the two polymeric networks of the cell wall. Seemingly, a low pH, whose effect on the wall extension might be mediated through expansin activation, is not instrumental in defining the degree of wall mechanical anisotropy. The stimulatory effect of a pH 4.5 buffer on the creep rate of the epidermal walls was indeed similar parallel and transverse to their net microfibril orientation (Fig. 10C, D). Interestingly, isolated walls of Nitella were found to be 10 times more sensitive to low pH in the longitudinal (perpendicular to the microfibril orientation) than in the transverse direction (Métraux and Taiz, 1979). This difference between a seed plant and an alga might be due to the fact that expansins are absent in Characean algae that have a different mechanism of acid-induced wall extension (Cosgrove, 2000a). To sum up, this work provides direct evidence that the net or mean predominant orientation of cellulose microfibrils confers to the cell wall of higher plants a mechanical anisotropy that might be instrumental for growth anisotropy. Provided that extensibilities measured in vitro depend on the Φ and Y parameters from the Lockhart equation, this finding predicts that onion epidermal cells should grow predominantly in the direction perpendicular to the mean orientation of cellulose microfibrils in their cell walls. Not only the higher extensibility but also the lower proportion of retarded elasticity may contribute to the growth of a cell in the direction perpendicular to the alignment of microfibrils in its cell wall. The validity of this hypothesis is currently being studied on growing onion bulbs. References Baskin TI,  Meekes HTHM,  Liang BM,  Sharp RE.  Regulation of growth anisotropy in well-watered and water-stressed maize roots II. 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Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: [email protected] http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Experimental Botany Oxford University Press

Cellulose orientation determines mechanical anisotropy in onion epidermis cell walls

Suslov, D; Verbelen, J-P
Journal of Experimental Botany , Volume 57 (10) – May 23, 2006
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Oxford University Press
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© The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: [email protected]
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0022-0957
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1460-2431
DOI
10.1093/jxb/erj177
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16720609
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Abstract

Abstract The role of cellulose microfibril orientation in determining cell wall mechanical anisotropy and in the control of the wall plastic versus elastic properties was studied in the adaxial epidermis of onion bulb scales using the constant-load (creep) test. The mean or net cellulose orientation in the outer periclinal wall of the epidermis was parallel to the long axis of the cells. In vitro cell wall extensibility was 30–90% higher in the direction perpendicular to the net microfibril orientation than parallel to it. This was the case for the size of the initial deformation occurring just after the load application and for the rate of time-dependent creep. Loading/unloading experiments confirmed the presence of a real irreversible component in cell wall extension. The plastic component of the time-dependent deformation was higher perpendicular to the net cellulose orientation than parallel to it. An acid buffer (pH 4.5) increased the creep rate by 25–30% but this response was not related to cellulose orientation. The present data provide direct evidence that the net orientation of cellulose microfibrils confers mechanical anisotropy to the walls of seed plants, a characteristic that may be relevant to understanding anisotropic cell growth. Allium, cell wall extensibility, cellulose orientation, confocal microscopy, creep test Introduction Plant cells are surrounded by the stiff yet extensible cell walls that play a crucial role in determining the rate and direction of cell expansion and in the control of plant shape in general (Hernandez and Green, 1993; Cosgrove, 2000a). The link between the wall properties relevant for growth and the rate of steady-state plant cell expansion was established by Lockhart (1965) and expressed in the equation that holds true if water transport to the growing cell is not limiting:  r=(1/V)(dV/dt)=Φ(P−Y)where r is the relative growth rate measured as a change in cell volume (1/V)(dV/dt); Φ is the wall yielding coefficient; P is the turgor pressure; and Y is the yield threshold, the minimum turgor required for growth. According to Lockhart, cell expansion occurs because the effective turgor pressure (P–Y) irreversibly stretches the cell wall. The parameters Φ and Y depend on physical (mechanical) properties of the cell wall and on the metabolic reactions modifying the interactions between the wall constituents (Taiz, 1984; Cosgrove, 1993). The mechanics of a cell wall are determined by its structure that resembles that of composite materials (Kerstens et al., 2001). The walls consist of stiff semi-crystalline cellulose microfibrils cross-linked by hemicelluloses and embedded in a gel-like matrix of pectins (Cosgrove, 1997). Cellulose microfibrils have traditionally been considered as the main load-bearing components in the cell wall whose orientation determines the direction of preferential wall extension (Carpita and Gibeaut, 1993). This statement was mostly based on the observation that, in growing cells, cellulose microfibrils are usually transversely oriented to the vector of the cell extension (Baskin et al., 1999; Sugimoto et al., 2000). At the same time, there have been surprisingly few direct confirmations that a specific alignment of cellulose microfibrils confers to the cell walls a mechanical anisotropy which is then translated into a preferential direction of cell growth. Such evidence was reported in early work on cell walls of Nitella, a green alga (reviewed in Taiz, 1984). It is, however, not obvious that these data can be extrapolated to higher plants. Nitella walls are many times thicker than primary walls of higher plants and, accordingly, contain many more microfibril layers across the wall thickness (Baskin et al., 1999). Also this alga, to our knowledge, is not considered as ancestral to higher plants, and there is no reliable information on its wall architecture. Searching for appropriate seed plant models to study the relationship between microfibril orientation and mechanical cell wall anisotropy has led to the use of onion and Kalanchoë epidermal peels (Kerstens et al., 2001). With an Instron-like assay, stress–strain curves for these tissues were determined. It was demonstrated for the first time for higher plants that a net parallel orientation of cellulose microfibrils in the onion epidermis wall coincided with a high mechanical anisotropy. In contrast, the Kalanchoë epidermis had a random net orientation of microfibrils and was mechanically isotropic. The present study confirms and extends the data obtained in the previous work (Kerstens et al., 2001) using the creep (constant-load) method for measuring cell wall extensibility in vitro. Unlike the stress–strain assay, the creep method mimics the action of turgor on growing cell walls (Taiz, 1984). Due to this fact, data on creep analysis have been related to the parameters Φ and Y from the Lockhart equation (Okamoto and Okamoto, 1994; Büntemeyer et al., 1998; Thompson, 2001). A similar attempt in relation to the net microfibril orientation was undertaken in this work. Another advantage of the constant-load test is its ability to distinguish between irreversible (plastic) and reversible (elastic) components of cell wall deformation. About 70 years ago, it was postulated that the plastic component of cell wall extensibility in vitro correlates with the rate of plant cell expansion in vivo (Heyn, 1932). This point of view was questioned by Schopfer and co-workers, who claimed that the in vitro plastic extensibility of isolated cell walls in fact represents retarded elastic wall extension (Hohl and Schopfer, 1992; Nolte and Schopfer, 1997). The present study confirms the presence of a real irreversible component in the extension of onion epidermal cell walls. Using the advantages of the creep test, it was also possible to investigate for the first time the effect of net microfibril orientation on plastic versus elastic cell wall properties in higher plants. Materials and methods Plant material Onion (Allium cepa L. cv. Bonkajuin) bulbs were commercially acquired. The bulbs came from an early harvest at the beginning of June and had not reached their maximal mature size. In the field, they had some potential for further growth at the moment of harvesting. Strips of adaxial epidermis, 25–30 mm long and 4 mm wide, were peeled from the equatorial part of the adaxial side of the third (from the outside) live scale in directions parallel and perpendicular to the axis of the bulb. These ‘longitudinal’ and ‘transverse’ epidermal strips were immediately frozen in liquid nitrogen, stored at −20 °C, and used within 1 week after isolation. Analysis of the net cellulose microfibril orientation The net or mean orientation of cellulose microfibrils in the outer periclinal wall of the adaxial epidermis was determined using polarization confocal microscopy and fluorescent Congo Red staining as described previously (Verbelen and Stickens, 1995; Verbelen and Kerstens, 2000). Thawed epidermal strips were stained in a 1% (w/v) solution of Congo Red (Sigma) in water for 2 h. After rinsing in water, the Congo Red-mediated fluorescence of cell walls was studied using a confocal laser scanning microscope (Bio-Rad MRC 600 mounted on a Zeiss Axioskop) with a ×20 (NA 0.5) dry objective and a co-axial rotating object table. The dichroic fluorescent dye Congo Red preferentially absorbs light directed parallel to the dipole moment of its chromophoric group. It has an ability to bind specifically to β-1,4-linked glucan polymers, especially to cellulose fibrils, and the dipole moments of Congo Red chromophoric groups will be aligned in the same direction as the cellulose polymers. If the cellulose fibrils have some preferential orientation, Congo Red fluorescence will be maximal when the vector of the polarized exciting laser beam is parallel to the net cellulose orientation. When the vector is perpendicular to the net microfibril orientation, the fluorescence intensity will be minimal. In the case of a net random cellulose orientation, the wall fluorescence intensity will be equal for all orientations of the polarized light vector. The extent to which cellulose microfibrils were aligned with respect to the cell's long axis was expressed as the axiality ratio. This is the ratio between the wall fluorescence intensities with the vector of the excitation light parallel and perpendicular, respectively, to the cell longitudinal axis. The axiality ratio equals unity if the net cellulose microfibril orientation is random. It will have values above or below unity in walls with, respectively, a net cellulose orientation parallel or perpendicular to the cell axis. The fluorescence intensity of Congo Red-stained cell walls was quantified using Photoshop 4.0 software (Adobe). Extensiometry In vitro extensibility of onion epidermal cell walls was studied with a custom-built constant-load extensiometer similar to that described by Kutschera and Schopfer (1986). Wall extension was measured using a displacement transducer (model 200HR, Schaevitz, Hampton, VA, USA) coupled to an in-line amplifier (model MC-IP-15B-B10 from ALTHEN GmbH, Kelkheim, Germany) and a data acquisition system (model DI-700 from DATAQ, Akron, OH, USA) connected to a PC. The cell wall strain curves were recorded and analysed with, respectively, WinDaq/Lite and WinDaq Waveform Browser software (both from DATAQ, Akron, OH, USA). The constant loads used in the creep test should ideally induce the same stress in the isolated walls as that existing in vivo. It is very difficult, however, to measure the real force extending the epidermal cell wall in an onion bulb because of its complex nature. This force is summed from the turgor of epidermal cells, the tension between the epidermis and the underlying parenchymatic cells (Kutschera et al., 1987), and the pressure developing between the adjacent scales of the bulb as a result of their growth. Therefore, the strategy in this work was to use a maximally broad range of loads to model the effect of any possible magnitude of the unknown cumulative force on the extension of the epidermis. The lever of the extensiometer with the upper clamp attached to its left shoulder was pre-equilibrated before securing a cell wall sample. Thereafter, the lever was held in the horizontal position by a lock under its right shoulder while an initial load was placed on the scale of the extensiometer. The lower clamp consisted of two vertical plexiglass plates with two U-shaped rubber rims stuck on their internal surfaces opposite to each other. Closing the clamp gripped the lower end of the cell wall sample and formed a water-tight chamber around it. This chamber was filled with 10 mM sodium citrate buffer (pH 6.0, if not otherwise stated) so that the cell wall sample was completely immersed in the buffer during the measurement. A 10 mm segment of an epidermal strip was secured between the clamps. Cell wall extension was induced by unlocking the lever. A rapid, practically instantaneous cell wall extension referred to as ‘initial deformation’ was measured 1 s after the load application. It was followed by a slow, time-dependent deformation (creep) whose kinetics are represented as an average rate for the intervals 1 s–5 min, 5–10 min, and 10–15 min after the load application (Fig. 2). To study the reversibility of cell wall extension in vitro, 2 or 5 g loads were subsequently added onto and then removed from the scale of the extensiometer at 60 s intervals. On the basis of the recorded cell wall strain curves, total deformation, initial deformation, and creep were calculated for every step of loading and unloading. Total deformation (DT) is the change in wall length between subsequent increases or decreases in load. Initial deformation (DI) is the change in wall length measured 1 s after loading (unloading). Creep (C) is the amplitude of the time-dependent cell wall deformation from 1 s to 60 s after the load application (removal). Thus, DT=DI+C. To estimate total deformation reversibility, the sum of total deformations at all steps of unloading-induced cell wall contraction was divided by the sum of total deformations at all steps of loading-induced cell wall extension. The result was expressed as percentage reversibility (elasticity). A similar procedure was used to calculate separately the reversibilities of creeps and initial deformations. The sum of creeps (initial deformations) at all steps of unloading was divided by the sum of creeps (initial deformations) at all loading steps and expressed as a percentage of creep (initial deformation) reversibility. Results Quantification of the cellulose microfibril orientation in the outer epidermal cell wall The onion bulbs used in this study contained two or three dead dry scales and from four to seven live scales that surrounded several juvenile ‘bulblets’. The long axes of epidermal cells in all scales were parallel to the axis of the bulb. A confocal image of a typical Congo Red-stained cell from the adaxial epidermis of the third (from outside) live scale is illustrated in Fig. 1. Fluorescence intensity is coded in shades of grey. It is clear that when the vector of the laser beam is parallel to the cell's long axis (Fig. 1A), fluorescence in the cell wall is much stronger than when the vector is perpendicular to the cell axis (Fig. 1B). The cellulose microfibrils thus have a net longitudinal orientation in this tissue. Analysis of 100 epidermal cells selected at random from 10 different onion bulbs has established that the net cellulose alignment in the outer epidermal wall of the third scale was characterized by the axiality ratio of 1.52±0.04 (SE). Fig. 1 View largeDownload slide Mean cellulose microfibril orientation in the adaxial epidermis of the onion bulb scale. Polarization confocal micrographs of epidermal cell walls stained with Congo Red. The vector of polarization of the laser is indicated by arrows. The intensity of fluorescence is coded in shades of grey from low (black) to high (white). Its values for the outer periclinal wall of the cell in the centre of the figure are shown as numbers in the lower left corners of the micrographs. The fluorescence intensity is maximal (A) and minimal (B) with the vector of polarization parallel and perpendicular, respectively, to the cell long axis. This shows that the net orientation of cellulose microfibrils is parallel to the longitudinal axis of the cell. Scale bar=50 μm. Fig. 1 View largeDownload slide Mean cellulose microfibril orientation in the adaxial epidermis of the onion bulb scale. Polarization confocal micrographs of epidermal cell walls stained with Congo Red. The vector of polarization of the laser is indicated by arrows. The intensity of fluorescence is coded in shades of grey from low (black) to high (white). Its values for the outer periclinal wall of the cell in the centre of the figure are shown as numbers in the lower left corners of the micrographs. The fluorescence intensity is maximal (A) and minimal (B) with the vector of polarization parallel and perpendicular, respectively, to the cell long axis. This shows that the net orientation of cellulose microfibrils is parallel to the longitudinal axis of the cell. Scale bar=50 μm. All scales located inward from the third scale also had a clear longitudinal net orientation of cellulose microfibrils in the outer periclinal walls of their adaxial epidermis. The two outermost live scales had only a slightly longitudinal or even a random net orientation of microfibrils in the corresponding wall. The third scale was chosen for further analysis because it was the largest scale with a net longitudinal cellulose orientation. In the terminology used subsequently, ‘longitudinal strips’ refer to epidermis samples where the load is applied parallel to the net cellulose orientation and thus also parallel to the axis of the cell. In ‘transverse strips’, the load is applied perpendicular to the net cellulose orientation. The in vitro cell wall extensibility depends on the net cellulose microfibril orientation Applying a constant load to an epidermal strip resulted in an immediate high-amplitude initial deformation that was followed by a slow, time-dependent creep (Fig. 2). It is clear that extension is much larger transverse to the cellulose orientation than parallel to it. The rate of creep always decreases with time during 60 min of measurement (data not shown). Analysis of the deformation curves revealed that the whole range of the loads used (5–20 g) induced stronger extension in the transverse than in the longitudinal strips (Fig. 3). This difference was highly significant (P <0.01) for the initial deformations (Fig. 3A) and the creep rates during the intervals 5–10 and 10–15 min after load application (Fig. 3C, D), where a practically 2-fold difference was registered for the 5 g and 10 g loads. Thus the isolated epidermal cell walls are much more extensible in the direction perpendicular to the mean orientation of cellulose microfibrils than in the direction parallel to the net cellulose orientation. Fig. 2 View largeDownload slide Dynamics of extension of an isolated onion epidermis with longitudinal (thick line) and transverse (thin line) net orientation of cellulose microfibrils. Typical curves recorded after a 15 g load application (arrow) are represented. Vertical dashed lines indicate the time intervals (1 s–5 min, 5–10 min and 10–15 min after the load application) during which the average creep rate was determined. Fig. 2 View largeDownload slide Dynamics of extension of an isolated onion epidermis with longitudinal (thick line) and transverse (thin line) net orientation of cellulose microfibrils. Typical curves recorded after a 15 g load application (arrow) are represented. Vertical dashed lines indicate the time intervals (1 s–5 min, 5–10 min and 10–15 min after the load application) during which the average creep rate was determined. Fig. 3 View largeDownload slide Dependence of extension on the load in onion epidermal walls with longitudinal (thick lines, closed circles) and transverse (thin lines, open circles) net orientation of cellulose microfibrils. Comparison of initial deformations (A) and creep rates during the intervals 1 s–5 min (B), 5–10 min (C), and 10–15 min (D) after the load application. These values were calculated after analysis of experimental curves of the type indicated in Fig. 2. Data are means ±SE (n=13). Fig. 3 View largeDownload slide Dependence of extension on the load in onion epidermal walls with longitudinal (thick lines, closed circles) and transverse (thin lines, open circles) net orientation of cellulose microfibrils. Comparison of initial deformations (A) and creep rates during the intervals 1 s–5 min (B), 5–10 min (C), and 10–15 min (D) after the load application. These values were calculated after analysis of experimental curves of the type indicated in Fig. 2. Data are means ±SE (n=13). Interestingly, the difference between the initial creep rates (0–5 min) of longitudinal and transverse strips was smaller (compare Fig. 3B with C and D). At 20 g load, it was even statistically insignificant. The reason for this discrepancy became evident through analysis of creep during the first seconds after the load application (Fig. 4). The dynamics of creep of longitudinal and transverse strips were absolutely identical during the first 10 s after the 20 g load application. This was due to the non-linear behaviour of the stress–strain dependence in transverse strips (Fig. 4). For the later time intervals, the dependence of creep rates on the applied forces was linear in longitudinal as well as in transverse epidermal strips (Fig. 3B–D). Fig. 4 View largeDownload slide Initial creep rates (1–21 s after load application) of onion epidermal walls with net longitudinal (thick lines) and transverse (thin lines) orientation of cellulose microfibrils. Each curve is the average of 10–12 experiments with different epidermal strips at a given load. For convenience of comparison, the starting points of the curves are superimposed at time zero. Notice that for 20 s of measurement, 5 g increments in the load induced similar increments in extension in longitudinal strips but decreasing increments in transverse strips. This indicates that the dependence of the initial creep rate on the load is non-linear in the transverse orientation of cellulose microfibrils. Error bars are the SE. Fig. 4 View largeDownload slide Initial creep rates (1–21 s after load application) of onion epidermal walls with net longitudinal (thick lines) and transverse (thin lines) orientation of cellulose microfibrils. Each curve is the average of 10–12 experiments with different epidermal strips at a given load. For convenience of comparison, the starting points of the curves are superimposed at time zero. Notice that for 20 s of measurement, 5 g increments in the load induced similar increments in extension in longitudinal strips but decreasing increments in transverse strips. This indicates that the dependence of the initial creep rate on the load is non-linear in the transverse orientation of cellulose microfibrils. Error bars are the SE. A 25 g load caused rupture in ∼50% of the transverse strips but never in longitudinal strips. This fact confirms the higher tensile strength of the epidermal tissue in the direction parallel to the net orientation of cellulose microfibrils. Plastic and elastic components of in vitro extensibility depend on the net microfibril orientation Experiments were set up to find out if a true plastic component of in vitro extensibility exists in onion epidermal walls and, if so, to determine the effect of cellulose microfibril orientation on plastic versus elastic behaviour of isolated cell walls. In a first approach, cell wall deformation was recorded during a single 1 min loading step followed by complete unloading. Irrespective of the load used, the epidermal cell walls immediately returned to their original length after unloading (not shown). This phenomenon was, however, not due to the elastic behaviour of the cell wall. It just reflected the fact that the lever of the extensiometer (see Materials and methods) was pre-equilibrated before the measurement and fixed horizontally. After the complete unloading, the lever came back to its original horizontal equilibrium position, returning the secured cell wall sample to the point it occupied before stretching. A single pre-loading step allowed this artefact to be separated from the real elastic cell wall properties. In this approach, extension experiments start from epidermis samples that are kept under slight tension. As an example, Fig. 5 depicts an experiment performed with 2 g loads. Epidermal strips were pre-loaded with 2 g for 1 min. Then an additional 2 g load was applied for 1 min. After its removal, the extension was measured during the next 20 min (Fig. 5). For both orientations of the cellulose fibrils, the unloading resulted in an instant elastic shrinkage of the epidermal strips, followed by a limited (not more than 5 μm) retarded elastic contraction, that lasted 2–3 min. Thereafter, the length of the epidermal strips did not change any more (Fig. 5). Thus 30–40% of cell wall extension is irreversible. This indicates that real plastic deformation exists in onion epidermal cell walls. Using 200 or 500 mg for the pre-loading step as well as 1 g for the loading did not qualitatively change this cell wall behaviour. In all cases, a marked plastic component of the wall extension was detected (results not shown). Fig. 5 View largeDownload slide Plastic component in the extension of epidermal walls with net longitudinal (thick lines) and transverse (thin lines) orientations of cellulose microfibrils. Epidermal strips were pre-loaded with a 2 g load (+L) for 1 min. Thereafter an additional 2 g was applied (+L, loading) and removed 1 min later (–L, unloading). Changes in the subsequent tissue deformation were recorded for 20 min longer. After unloading, a significant part of tissue deformation remains, demonstrating the presence of a real plastic component (Ep) in onion epidermal wall deformation. Typical curves are shown. Fig. 5 View largeDownload slide Plastic component in the extension of epidermal walls with net longitudinal (thick lines) and transverse (thin lines) orientations of cellulose microfibrils. Epidermal strips were pre-loaded with a 2 g load (+L) for 1 min. Thereafter an additional 2 g was applied (+L, loading) and removed 1 min later (–L, unloading). Changes in the subsequent tissue deformation were recorded for 20 min longer. After unloading, a significant part of tissue deformation remains, demonstrating the presence of a real plastic component (Ep) in onion epidermal wall deformation. Typical curves are shown. To explore the effect of net cellulose microfibril orientation on plastic versus elastic components of cell wall extension, longitudinal and transverse epidermal strips were stretched in 1 min intervals by a series of equal loads (2 or 5 g) up to a maximum of 8 and 20 g, respectively. Thereafter, the loads were removed in 1 min intervals back to the final 2 or 5 g stretching force. Thus the initial 2 or 5 g served as pre-loading steps in the experiments. Such a spectrum of loads allowed estimation of the viscoelastic behaviour of onion cell walls subjected to a maximally broad range of forces. A typical experimental curve of a 2 g cycle is illustrated in Fig. 6. In both epidermal strips, a significant part of the wall deformation did not revert after unloading, which confirmed its partly plastic nature. Statistical analysis of many experimental curves of the type indicated in Fig. 6 has shown that loading induced much higher extensions in transverse than in longitudinal epidermal strips (Fig. 7A) and also that the elastic shrinkages during unloading were higher to the same extent. Therefore, total deformation reversibility (see Materials and methods) was statistically the same for longitudinal and transverse epidermal strips (Fig. 9A). Since the deformation at every step of loading (unloading) includes a rapid initial deformation followed by a time-dependent creep, the values of these components were also analysed (Fig. 7B, C). The reversibility of the initial deformation was similar (∼60%) in longitudinal and transverse strips (Fig. 9B). However, the two orientations differ specifically on the level of creep. On the one hand, creep was significantly higher in transverse than in longitudinal strips (Fig. 7C). On the other hand, the slow time-dependent contraction upon unloading was similar in both strips or even higher in longitudinal strips (Fig. 7C). Therefore, the reversibility of creep in longitudinal strips (14.2%) was significantly (P <0.01) higher than in transverse strips (8.9%). This means that the time-dependent extension of onion epidermal walls is proportionally more elastic parallel to the net cellulose orientation than perpendicular to the net cellulose orientation. The 5 g loading/unloading cycles gave qualitatively similar results (Figs 8,9). Longitudinal and transverse strips differed only in the percentage of their creep reversibility, which was significantly higher in the former (6.5% compared with 3.4%) (Fig. 9). Fig. 6 View largeDownload slide Extension of epidermal walls with net longitudinal (thick lines) and transverse (thin lines) orientations of cellulose microfibrils in the course of a 2 g loading/unloading cycle. Loads of 2 g were applied and subsequently removed in 1 min intervals, and the resulting cell wall deformation was recorded. Arrows indicate the time points at which the loads were applied or removed. Numbers above the curves show the total loads stretching the walls at the corresponding stages of the cycle. Typical curves are shown. Fig. 6 View largeDownload slide Extension of epidermal walls with net longitudinal (thick lines) and transverse (thin lines) orientations of cellulose microfibrils in the course of a 2 g loading/unloading cycle. Loads of 2 g were applied and subsequently removed in 1 min intervals, and the resulting cell wall deformation was recorded. Arrows indicate the time points at which the loads were applied or removed. Numbers above the curves show the total loads stretching the walls at the corresponding stages of the cycle. Typical curves are shown. Fig. 7 View largeDownload slide Results of statistical analysis of cell wall extension curves of the type indicated in Fig. 6 during 2 g loading/unloading cycles. Changes in the total deformation (A), the initial deformation (B), and the creep (C) of the epidermal walls with net longitudinal (black bars) and transverse (white bars) orientation of cellulose microfibrils. Negative values indicate contraction during unloading. The total deformation was calculated as the change in wall length during 60 s between the subsequent load applications and removals (see Fig. 6). The initial deformation is the change in wall length measured 1 s after loading (unloading). The creep is the amplitude of the time-dependent cell wall deformation during the interval 1–60 s after load application (removal). Data are means ±SE (n=21). Fig. 7 View largeDownload slide Results of statistical analysis of cell wall extension curves of the type indicated in Fig. 6 during 2 g loading/unloading cycles. Changes in the total deformation (A), the initial deformation (B), and the creep (C) of the epidermal walls with net longitudinal (black bars) and transverse (white bars) orientation of cellulose microfibrils. Negative values indicate contraction during unloading. The total deformation was calculated as the change in wall length during 60 s between the subsequent load applications and removals (see Fig. 6). The initial deformation is the change in wall length measured 1 s after loading (unloading). The creep is the amplitude of the time-dependent cell wall deformation during the interval 1–60 s after load application (removal). Data are means ±SE (n=21). Fig. 8 View largeDownload slide Results of statistical analysis of cell wall extension curves during 5 g loading/unloading cycles. Changes in the total deformation (A), the initial deformation (B), and the creep (C) of epidermal walls with net longitudinal (black bars) and transverse (white bars) orientation of cellulose microfibrils. Negative values indicate contraction during unloading. The total deformation was calculated as the change in wall length during 60 s between the subsequent load applications and removals. The initial deformation is the change in wall length measured 1 s after loading (unloading). The creep is the amplitude of the time-dependent cell wall deformation during the interval 1–60 s after the load application (removal). Data are means ±SE (n=10). Fig. 8 View largeDownload slide Results of statistical analysis of cell wall extension curves during 5 g loading/unloading cycles. Changes in the total deformation (A), the initial deformation (B), and the creep (C) of epidermal walls with net longitudinal (black bars) and transverse (white bars) orientation of cellulose microfibrils. Negative values indicate contraction during unloading. The total deformation was calculated as the change in wall length during 60 s between the subsequent load applications and removals. The initial deformation is the change in wall length measured 1 s after loading (unloading). The creep is the amplitude of the time-dependent cell wall deformation during the interval 1–60 s after the load application (removal). Data are means ±SE (n=10). Fig. 9 View largeDownload slide Reversibility (elasticity) of the total deformations (A), the initial deformations (B), and the creeps (C) of epidermal cell walls with net longitudinal (black bars) and transverse (white bars) orientation of cellulose microfibrils in the 2 g and the 5 g loading/unloading cycles. To estimate total deformation reversibility, the total deformations at all steps of unloading-induced cell wall contraction were summed and divided by the sum of the total deformation values at all steps of loading-induced cell wall extension. The result was expressed as percentage reversibility (elasticity). A similar procedure was used to calculate separately the reversibility of creep and of initial deformation. Data are means ±SE (n=21 for the 2 g loading/unloading cycle and n=10 for the 5 g loading/unloading cycle). Fig. 9 View largeDownload slide Reversibility (elasticity) of the total deformations (A), the initial deformations (B), and the creeps (C) of epidermal cell walls with net longitudinal (black bars) and transverse (white bars) orientation of cellulose microfibrils in the 2 g and the 5 g loading/unloading cycles. To estimate total deformation reversibility, the total deformations at all steps of unloading-induced cell wall contraction were summed and divided by the sum of the total deformation values at all steps of loading-induced cell wall extension. The result was expressed as percentage reversibility (elasticity). A similar procedure was used to calculate separately the reversibility of creep and of initial deformation. Data are means ±SE (n=21 for the 2 g loading/unloading cycle and n=10 for the 5 g loading/unloading cycle). When comparing the two loading/unloading cycles, it is clear that the total deformation (Fig. 9A) and its two components (Fig. 9B, C) were much less reversible in the 5 g than in the 2 g cycle. Higher loads thus increase the proportion of plastic deformation in onion epidermal walls. Prolonged unloading steps (up to 15 min) revealed in both cycles that the retarded elastic contractions described in Figs 7C and 8C did not last for longer than 10 min (results not shown). The dynamics and the amplitude of the late part of the elastic contraction presented only a negligible fraction of the total deformation during unloading. The effect of acid pH on cell wall extensibility The orientation of cellulose microfibrils in the cell wall seems to define the direction but not the rate of cell expansion (Baskin et al., 1999; Sugimoto et al., 2000). The latter is probably regulated by specific cell wall proteins breaking and/or establishing cross-links between different cell wall polymers (McQueen-Mason et al., 1992; Fry et al., 1992; Brownleader et al., 2000; Okamoto-Nakazato et al., 2000). Among these proteins, the expansins induce cell wall expansion at acid pH values by disrupting the hydrogen bonding between cellulose microfibrils and matrix hemicelluloses (Cosgrove, 2000b). It was hypothesized that different groups of factors regulate growth in the directions parallel and transverse to the mean cellulose orientation by catalysing shear and separation of microfibrils correspondingly (Baskin et al., 1999). To check whether lowering the pH could stimulate extension differentially depending on the cellulose orientation, the effect of an acid buffer on extensibility of transverse and longitudinal epidermal strips was studied. Lowering the pH to 4.5 markedly (P <0.05) stimulated the creep rate in longitudinal as well as in transverse epidermal strips, but only during the time intervals 10–15 and 5–15 min after the load application (Fig. 10C, D). Its effects on the initial deformations and the creep rates at the earlier stages were not statistically significant (Fig. 10A and B). The percentage of the creep rate stimulation (25–30%) was the same for longitudinal and transverse strips (Fig. 10C, D). As a consequence, the ratio of their extensibilities in the two directions is similar to that at neutral pH (Fig. 3). Preincubation of the tissue in the acid buffer for 30 min before stretching did not change the above-mentioned pH effect on in vitro extensibility, which indicates that tissue permeability was not a limiting factor (results not shown). Thus the acid-induced extension of the isolated cell walls does not depend on the mean cellulose microfibril orientation. Fig. 10 View largeDownload slide The effect of an acid pH buffer on the initial deformations (A) and the creep rates during the intervals 1 s–5 min (B), 5–10 min (C), and 10–15 min (D) of epidermal walls with net longitudinal and transverse orientation of cellulose microfibrils. Black bars, control treatment (10 mM sodium citrate buffer, pH 6.0); white bars, experimental treatment (10 mM sodium citrate buffer, pH 4.5). Data are means ±SE (n=15). Fig. 10 View largeDownload slide The effect of an acid pH buffer on the initial deformations (A) and the creep rates during the intervals 1 s–5 min (B), 5–10 min (C), and 10–15 min (D) of epidermal walls with net longitudinal and transverse orientation of cellulose microfibrils. Black bars, control treatment (10 mM sodium citrate buffer, pH 6.0); white bars, experimental treatment (10 mM sodium citrate buffer, pH 4.5). Data are means ±SE (n=15). Discussion Upon loading, the epidermis samples stretched in a biphasic way. The initial, practically instantaneous deformation was followed by a slow time-dependent creep. In the past, both parts of the extension have been considered as relevant wall mechanical properties that could help to understand processes involved in cell expansion (Kutschera and Schopfer, 1986; Cosgrove, 1989). The fact that acid pH stimulated the creep rate but did not increase the initial deformation (Fig. 10) suggests that creep measured in vitro might be a good indicator for the parameters Φ and Y that regulate the rate of plant cell expansion in vivo and that the initial deformation may characterize cell wall mechanical properties that have no relationship to in vivo growth regulation. Similar conclusions were drawn in work on maize roots (Büntemeyer et al., 1998). For all loads used, the initial deformation and the creep rate were higher perpendicular to the mean cellulose orientation (transverse strips) than parallel to it (longitudinal strips) (Fig. 3). This confirms previous findings that showed higher extensibility of onion epidermis walls in the direction perpendicular to the mean microfibril orientation using a stress–strain method (Kerstens et al., 2001). Okamoto and Okamoto (1994) have used the linear dependence of the creep rate on the load to estimate ϕ and y, the in vitro analogues of Φ (yield coefficient) and Y (yield treshold) parameters from the Lockhart equation. In a similar approach for our data, linear fits were calculated for the ratio between late creep rates and loads. The slopes of the lines obtained gave the values of ϕ, which was higher for transverse (0.41) than for longitudinal (0.36) epidermal strips. This indicates that the mean orientation of cellulose microfibrils has a moderate effect (14%) on ϕ. Loading/unloading experiments (Figs 5, 6) demonstrated the presence of a real plastic component in the extension of onion epidermal cell walls in vitro. The plastic component of time-dependent deformation is higher in the direction transverse to the net microfibril orientation than parallel to it (Fig. 9C). This difference is potentially involved in growth anisotropy. Indeed, in vivo, a higher proportion of the deformation could be transformed into irreversible cell wall extension in the direction transverse to the net cellulose orientation compared with the direction parallel to the net cellulose orientation. A real plastic component has been described for rye coleoptiles (Kutschera, 1996), onion abaxial epidermis (Wilson et al., 2000), and tomato fruit epidermis (Thompson, 2001). The common methodological feature of all these studies was the presence of a pre-loading step and consequently the absence of a fully unloaded stage. However, Schopfer and co-workers challenged the presence of an irreversible plastic component in cell wall extension in vitro. They came to the conclusion that apparent plastic deformation of the wall in fact represented its retarded elastic behaviour (Hohl and Schopfer, 1992; Nolte and Schopfer, 1997). Their conclusion is based exclusively on experiments where complete unloading took place. While being useful in material testing, this step is physiologically irrelevant. The creep test models the action of the turgor on the growing cell wall. In this respect, complete unloading is equivalent to the state of plasmolysis when the turgor equals zero and plant cells do not grow. Cell expansion is always associated with a state of stress inside the cell wall. That is why measurements aimed at distinguishing between reversible versus irreversible components of the cell wall extension in vitro should be based on comparison between different stress states of the cell wall. The magnitudes of these stresses should ideally be close to those existing in the wall in vivo. Cellulose and xyloglucans form the major load-bearing network in the cell wall (Shedletzky et al., 1992) that apparently plays a key role in determining the rate of the wall extension during loading. At the same time, pectins confer compression resistance to the cell wall (McCann and Roberts, 1994). It is known that wall deformation causes a certain microfibril realignment in the direction of stress and a concomitant compression in the matrix (Spatz et al., 1999; Köhler et al., 2000; Wilson et al., 2000). In the onion cell walls, the pectin network, compressed by realigned microfibrils during loading, will then return to its original shape after load removal. This process could substantially contribute to the wall contraction observed upon unloading. Thus the different amount of the retarded elasticity correlating with the mean microfibril orientation could result from the interaction between the two polymeric networks of the cell wall. Seemingly, a low pH, whose effect on the wall extension might be mediated through expansin activation, is not instrumental in defining the degree of wall mechanical anisotropy. The stimulatory effect of a pH 4.5 buffer on the creep rate of the epidermal walls was indeed similar parallel and transverse to their net microfibril orientation (Fig. 10C, D). Interestingly, isolated walls of Nitella were found to be 10 times more sensitive to low pH in the longitudinal (perpendicular to the microfibril orientation) than in the transverse direction (Métraux and Taiz, 1979). This difference between a seed plant and an alga might be due to the fact that expansins are absent in Characean algae that have a different mechanism of acid-induced wall extension (Cosgrove, 2000a). To sum up, this work provides direct evidence that the net or mean predominant orientation of cellulose microfibrils confers to the cell wall of higher plants a mechanical anisotropy that might be instrumental for growth anisotropy. Provided that extensibilities measured in vitro depend on the Φ and Y parameters from the Lockhart equation, this finding predicts that onion epidermal cells should grow predominantly in the direction perpendicular to the mean orientation of cellulose microfibrils in their cell walls. Not only the higher extensibility but also the lower proportion of retarded elasticity may contribute to the growth of a cell in the direction perpendicular to the alignment of microfibrils in its cell wall. The validity of this hypothesis is currently being studied on growing onion bulbs. References Baskin TI,  Meekes HTHM,  Liang BM,  Sharp RE.  Regulation of growth anisotropy in well-watered and water-stressed maize roots II. 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Journal

Journal of Experimental Botany – Oxford University Press

Published: May 23, 2006

Keywords: Allium cell wall extensibility cellulose orientation confocal microscopy creep test

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