Planta (2005) 220: 582–592 DOI 10.1007/s00425-004-1368-5
ORIGINAL ARTICLE
Elodie Parre Æ Anja Geitmann Pectin and the role of the physical properties of the cell wall in pollen tube growth of Solanum chacoense
Received: 23 March 2004 / Accepted: 20 July 2004 / Published online: 21 September 2004 Springer-Verlag 2004
Abstract The cell wall is one of the structural key players ical approach provides new insights concerning the regulating pollen tube growth, since plant cell expansion mechanics of pollen tube growth and the architecture of depends on an interplay between intracellular driving living plant cells. forces and the controlled yielding of the cell wall. Pectin is the main cell wall component at the growing pollen Keywords Cell expansion Æ Cell wall Æ tube apex. We therefore assessed its role in pollen tube Cytomechanics Æ Pectin Æ Pollen tube Æ Tip growth growth and cytomechanics using the enzymes pectinase and pectin methyl esterase (PME). Pectinase activity was Abbreviation PME: Pectin methyl esterase able to stimulate pollen germination and tube growth at moderate concentrations whereas higher concentrations caused apical swelling or bursting in Solanum chacoense Bitt. pollen tubes. This is consistent with a modification of the physical properties of the cell wall affecting its extensibility and thus the growth rate, as well as its Introduction capacity to withstand turgor. To prove that the enzyme- induced effects were due to the altered cell wall Pollen tubes are fast-growing cells exhibiting tip growth. mechanics, we subjected pollen tubes to micro-indenta- In flowering plants, pollen grains germinate on a tion experiments. We observed that cellular stiffness was receptive stigma and send pollen tubes down the style reduced and visco-elasticity increased in the presence of toward the ovary. This allows the delivery of the sperm pectinase. These are the first mechanical data that con- cells to the ovules in order to perform fertilization. The firm the influence of the amount of pectins in the pollen growth process in pollen tubes is confined to the dome- tube cell wall on the physical parameters characterizing shaped apex and occurs in one direction only (Derksen overall cellular architecture. Cytomechanical data were 1996; Hepler et al. 2001). Since the pollen tube grows also obtained to analyze the role of the degree of pectin rapidly and over long distances, the production of the methyl-esterification, which is known to exhibit a gra- cell wall, its composition and the configuration of its dient along the pollen tube axis. This feature has fre- components are important features that regulate the quently been suggested to result in a gradient of the behavior of pollen tube growth, and thus the fertilization physical properties characterizing the cell wall and our process (Taylor 1997). data provide, for the first time, mechanical support for In general, plant cell growth is driven by internal this concept. The gradient in cell wall composition from turgor pressure and restricted by the ability of the cell apical esterified to distal de-esterified pectins seems to be wall to extend under this pressure. To what extent this correlated with an increase in the degree of cell wall equilibrium between turgor and cell wall determines the rigidity and a decrease of visco-elasticity. Our mechan- rate of tip growth in cells exhibiting polar elongation, such as pollen tubes, root hairs or fungal hyphae, has been subject of vivid discussion (Money and Hill 1997, E. Parre Æ A. Geitmann (&) and references therein). The importance of additional Institut de Recherche en Biologie Ve´ge´tale, De´partement factors such as the putative propulsion force of the de Sciences Biologiques, Universite´de Montre´al, 4101, cytoskeletal elements and the regulation of insertion of Rue Sherbrooke Est, Montre´al, Que´bec, H1X 2B2, Canada E-mail: [email protected] new cell wall material is still largely unknown for tip- Tel.: +1-514-8728492 growing cells in general and for pollen tubes in partic- Fax: +1-514-8729406 ular. There seems to be no doubt, however, that the 583 force counteracting the internal driving force is provided outer withstanding forces in this cellular system; (ii) our by the cell wall. Even though the structure of the pollen data revealed that both the amount and the configura- tube cell wall has been described in detail, few ap- tion of pectins are key factors determining the physical proaches have been made to assess its mechanics in a properties of the apical cell wall, thus controlling pollen quantitative manner since Green (1969) stated that tip tube growth. growth is regulated by the tip-to-flank gradient in the rate of wall surface expansion. In the pollen tube cell wall, two or three layers can be distinguished depending on the plant species and the Materials and methods method of investigation (Heslop-Harrison 1987; Steer and Steer 1989). It consists of a primary pecto-cellulosic Pollen tube growth layer and a secondary callosic wall. In the tip of the pollen tube, the cell wall lacks the callosic inner lining Solanum chacoense Bitt. plants were grown in the (Heslop-Harrison 1987), and thus pectins appear to be Montreal Botanical Garden greenhouses. Pollen was major component of the cell wall in this region crucial collected after dehiscence, dehydrated on anhydrous for cell expansion (Roggen and Stanley 1969;Lietal. calcium sulfate overnight and stored at 20 C. On the 1994, 1997, 2002; Derksen et al. 1995; Holdaway-Clarke day of use pollen was re-hydrated in a humid atmo- and Hepler 2003). sphere and cultivated in drops of liquid or solidified Immunocytochemical studies have shown that pectins medium. The latter was obtained by addition of 20 or located at the tip of the growing pollen tube are highly 40 mg ml 1 low-melting agarose (Type I-B Low EEO; esterified, while they are de-esterified in areas away from Sigma, St. Louis, MO, USA) or 10 mg ml 1Gel-Gro the tip (Li et al. 1994, 1995, 1997, 2002; Jauh and Lord (Gellan gum; gel strength twice as high as that of aga- 1996; Lennon and Lord 2000). This gradient in the de- rose; ICN Biochemicals, Aurora, OH, USA). The 1 gree of esterification is the result of the tip-focused growth medium contained 100 lgml H3BO3, 1 1 growth process. The wall components are secreted at the 300 lgml Ca(NO3)2ÆH2O, 100 lgml KNO3, 1 1 extreme apex as esterified pectin residues produced by 200 lgml MgSO4Æ7H2O, 50 lgml sucrose (Brew- the Golgi apparatus (Levy and Staehelin 1992; Haseg- baker and Kwack 1963). Various concentrations of awa et al. 1998). Upon arrival in the cell wall, they be- pectinase (0.2 units/mg solid; USB, Cleveland, OH, come increasingly de-esterified during maturation USA) or PME (238 units/mg solid; Sigma) were added through the activity of the enzyme pectin methyl-ester- at the start of germination (to assess the effect on ger- ase (PME; Kauss and Hassid 1967; Li et al. 1997; mination) or after 30 min of germination (to assess the Geitmann 1999). De-esterification permits Ca2+ ions to effect on elongation). Controls were performed by add- cross-link the acidic pectins to form a semi rigid pectate ing enzyme that had been denatured by boiling for gel (Jarvis 1984; Carpita and Gibeaut 1993), which is 10 min. Germination rate and pollen tube length were presumed to provide mechanical support for the distal assessed in the light microscope after 2 h incubation on areas of the elongating tube by altering the physical microscope slides. properties of the cell wall. Esterified and thus non-gel- ated pectins, which are present predominantly at the apex, are presumed to allow turgor-driven expansion of the tube (Morris et al. 1982; McNeil et al. 1984; Li et al. Fluorescence label 1994, 1997; Geitmann et al. 1995; Derksen 1996; Jauh and Lord 1996; Geitmann and Cresti 1998; Franklin- For immuno-fluorescence labeling, pollen tubes were Tong 1999; Geitmann 1999). However, no experimental fixed after 2 h germination in 30 mg ml 1 freshly evidence has been brought forward to show that the prepared formaldehyde in Pipes buffer (1 mM EGTA, physical properties of the cell wall at the apex are indeed 0.5 mM MgCl2, 50 mM Pipes) for 30 min. To allow different from those in the distal part of a growing pollen for quantitative comparison of fluorescence intensity, tube. A gradient of the cytomechanical properties along pollen tubes grown in solidified medium (Gel-Gro) the longitudinal pollen tube axis measured by micro- were resuspended by adding 0.1 M citrate buffer indentation of Papaver pollen tubes is consistent with (55 mM citric acid, 125 mM sodium citrate, pH 6) at this concept (Geitmann and Parre 2004), but whether or 30 C, to assure that the gel did not limit the access of not it is due to the physical properties of the cell wall or antibodies to the cells (liquid medium controls were in particular to those of the pectin component remains treated similarly). Subsequently, cells were incubated to be shown. with monoclonal antibodies JIM5 and JIM7 (gener- Here we provide mechanical data that support two ously provided by Dr. Paul Knox) diluted 1:50 in PBS longstanding hypotheses that hitherto were largely based buffer, followed by an incubation with goat anti-rat on circumstantial evidence: (i) we show that pollen tube IgG–Alexa Fluor 594 (diluted 1:100 in buffer; growth and germination are governed by physical Molecular Probes, Eugene, OR, USA) over night at properties of the apical cell wall, thus supporting the 4 C. JIM5 and JIM7 recognize homogalacturonans importance of an equilibrium between inner driving and with a low and a high degree of esterification, 584 respectively (VandenBosch et al. 1989; Knox et al. 1990). Pollen tubes were mounted and observed in a Results fluorescence microscope using a Texas red filter set. Controls were performed by omitting incubation with Pectinase affects pollen tube growth and germination the primary or the secondary antibody. rates Decolorized aniline blue and calcofluor white (Fluo- rescent Brightener 28; Sigma) were used to stain callose To determine whether the abundance of pectins deter- and cellulose, respectively, in fixed pollen tubes (see mines cell wall extensibility we applied varying concen- above). After two washes, cells were incubated for trations of pectinase. This enzyme treatment was 15 min with the staining agent (1 mg ml 1 aniline blue supposed to digest 1,4-a-D-galactosiduronic acid, and 1 thus should affect both acidic and esterified pectins. in 0.15 M K2HPO4; 1 mg ml calcofluor white in Labeling pollen tubes treated with 6.4 mg ml 1 pectin- double-distilled H2O), mounted immediately and ob- served with UV light excitation. ase with monoclonal antibodies JIM5 (acidic pectins) and JIM7 (methyl-esterified pectins) confirmed this, since images were black at exposure times identical to the control (data not shown). Bright-field and fluorescence microscopy The distribution of other cell wall components such as cellulose and callose was not affected by this treat- Specimens labeled for cell wall components were ob- ment as revealed by fluorescence labeling (data not served in a fluorescence microscope (Nikon TE2000) shown). Figure 1 shows that high pectinase concentra- equipped with a Roper fx cooled CCD camera. tions in pollen inhibited germination (Fig. 1a) and tube Exposure times of images that had to be compared for growth (Fig. 1b) by causing bursting, whereas moderate fluorescence intensity were identical. These pictures were not corrected for brightness or contrast. Plots of the maximal fluorescence intensity along the longitu- dinal axis of the pollen tube were obtained by applying the Surface Plot function of ImagePro (Media Cybernetics).
Micro-indentation
Hydrated pollen was grown on coverslips coated with stigmatic exudate (to assure pollen adherence) and covered with liquid growth medium. After germina- tion had occurred, coverslips were submerged in the growth-medium-containing experimental chamber of the micro-indenter mounted on a Nikon TE2000 inverted microscope. The design and principles of operation of the micro-indenter have been described previously (Petersen et al. 1982; Elson et al. 1983). Briefly, the bending of a horizontal glass beam gauged the resistance to cellular deformation. A vertical glass stylus (tip diameter 10 lm) was mounted at the end of a 23-mm- long horizontal Vycor (Corning Inc., Corning, NY, USA) glass beam. The other end of the beam was mounted on a linear piezoelectric motor, which moves vertically according to a programmed waveform. Opti- cal sensors monitored the vertical positions of the stylus and the motor. The extent to which the beam was bent was proportional to the force exerted on the tip by the cell, and was determined by comparing tip displace- Fig. 1a,b Effect of different pectinase concentrations on Solanum chacoense pollen germination (a) and tube length after 2 h (b)of ments in the presence and absence of cell contact. The pollen grown in liquid medium ()) or medium solidified with force exerted by the cell on the stylus was determined 20 mg ml 1 (s)or40mgml 1 (m) agarose. Moderate enzyme with the help of the force constant of the beam, which concentrations stimulated both germination and tube length at 2 h had been obtained by prior calibration. In the experi- in all three media, whereas higher concentrations were inhibitory. Both germination and pollen tube length were reduced in stiffer ments reported in this paper, the motor was pro- media and optimal enzyme concentrations and relative stimulation grammed to execute a single triangular waveform with a had a tendency to differ from those observed in liquid medium, as velocity of 10 lms 1 and a total amplitude of 5 lm. summarized in Table 1 585 concentrations significantly increased both germination area, or to the presence of other cell wall polymers in and elongation rate. The highest increase (17%) in pol- the distal parts that keep resisting the internal forces len tube length measured after 2 h was accomplished by once pectin has been degenerated. To distinguish be- addition of pectinase at 0.5 mg ml 1. Denaturing the tween these two alternatives we identified the distribu- enzyme by boiling for 10 min eliminated both the tion of two putative load-bearing cell wall components stimulating and inhibiting effects (n=10). in the pollen tube cell wall: cellulose and callose. Fig- Since the enzyme was added from the beginning of ure 3a–c illustrates that callose was completely absent germination, an increase in pollen tube length could be from the pollen tube tip. Similarly, cellulose concen- due to accelerated germination and/or an increase in tration was significantly lower at the tip compared to pollen tube growth rate. In order to distinguish between the distal part of the tube (Fig. 3d–f). Both polymers these two variables, we applied growth-enhancing con- increased very gradually towards the distal parts of the centrations of the enzyme to Solanum pollen tubes tube. This is consistent with findings in other species. 30 min after they had germinated under control condi- Pectins on the other hand were present in distal and tions. After an additional 90 min of incubation in the apical regions. The antibodies JIM5 (Fig. 4a–c) and presence of 0.5 mg ml 1 pectinase, pollen tubes treated JIM7 (Fig. 4d–f) each label only subpopulations of with active enzyme were significantly longer pectin, but both labels taken together indicated that the (120±3 lm) than those treated with denatured enzyme amount of pectin was not reduced at the apex com- (100±4 lm; n=10). This indicates that the stimulating pared to the distal regions of Solanum pollen tubes. effect on pollen tube length was to a significant degree, This was confirmed by JIM5 and JIM7 labeling of albeit perhaps not exclusively, due to an effect on the pollen tubes that had been treated with PME. This pollen tube growth rate. enzyme treatment supposedly caused a transformation of methyl-esterified pectins into acidic pectins and should thus make the entire pectin population recog- Pollen tube apical swelling nizable to the JIM5 antibody, whereas it should reduce JIM7 labeling. Figure 4g–i shows that JIM5 label was We had observed that pectinase-induced bursting oc- significantly stronger at the apex than in distal parts curred exclusively at the pollen tube apex. To identify and thus indicates high total pectin concentration in the length of the region that seemed to be particularly this region. JIM7 label on the other hand was com- sensitive to pectinase treatment, we assessed the effect of pletely absent from the pollen tube (not shown), con- a moderately inhibitory concentration (8 mg ml 1)of firming that all esterified pectins were de-esterified by pectinase on pollen tube morphology. Pollen tubes PME treatment. The labeling patterns for the three cell grown in the presence of this enzyme concentration wall components, pectin, cellulose and callose, are showed swelling of the apical region starting 30 lm from consistent with the concept that pectin is the only cell the apex. The diameter of the distal part of the tube was wall component at the tip that resists the inner pro- unchanged (Fig. 2). pulsion forces.
Distribution of cell wall components Pectinase affects pollen tube penetration growth
The confinement of the observed swelling to the apex To investigate whether the equilibrium between pollen might be due to either a lower amount of pectins in this tube cell wall and the inner driving forces is influenced by the stiffness of the growth environment, we as- sessed to what extent pollen tube growth through a solidified medium was affected by pectinase. Solidifi- cation caused a reduction in germination and tube length at 2 h compared to the liquid control. At low enzyme concentrations, pectinase stimulated germina- tion and pollen tube elongation both in liquid and in solidified media (Fig. 1). Surprisingly, the stimulating effect was relatively bigger in solidified than in liquid medium, as summarized in Table 1. The enzyme con- centration necessary for optimal stimulation of both germination and pollen tube elongation had a ten- dency to be lower in solidified media than in liquid medium (n=9).
1 Interestingly, there was also a significant difference in Fig. 2a,b Effect of the presence of 8 mg ml pectinase in the the inhibitory pectinase concentration in the different germination medium on Solanum pollen tubes. The tube apex was 1 swollen at the apical 30 lm(a) compared to the untreated control media. Whereas 15 mg ml pectinase almost com- (b). Bar = 10 lm pletely inhibited pollen tube germination and growth in 586
Fig. 3a–f Fluorescent labeling of cell wall components in Solanum Local stiffness and visco-elasticity of the pollen tube pollen tubes and pollen grains. Pollen tubes were fixed and are affected by pectinase subsequently stained for callose with decolorized aniline blue (a– c) or for cellulose with calcofluor white (d–f). Bright-field images are provided for reference (a,d). Relative fluorescence intensity To assess the role of pectin in the mechanical properties along the longitudinal axis was plotted with the Surface Plot of the cell wall in a quantitative manner, local cellular function of ImagePro (c,f). The vertical axis represents unitless, stiffness and visco-elasticity were measured using a mi- relative grey values for the pixel with the highest brightness on the cro-indentation device. Local deformations were in- vertical line crossing the x-axis. The x-axis corresponds to the horizontal axis of the respective fluorescence micrograph. Label for duced at two positions on growing pollen tubes, at the both callose and cellulose was absent at the pollen tube apex. The apex and at a distal position between 20 and 30 lm from intensity increased very gradually towards the distal portion of the the apex. As shown earlier for other species (Geitmann tube. Bar = 10 lm and Parre 2004), normally growing Solanum pollen tubes were characterized by a difference in stiffness and medium solidified with 20 or 40 mg ml 1 agarose, this degree of visco-elasticity between the growing apex and concentration only had a partially inhibitory effect in the distal region. Distal stiffness was liquid medium. 4,760±417·10 5 Ncm 1 (mean ± SE) whereas apical stiffness amounted to 85±5% of the value for the distal part of the same tube (n=7). The apex generally showed The abundance of cell wall pectin is affected a bigger delay upon retraction of the deforming stylus by the stiffness of the growth medium (hysteresis) than the distal region, thus indicating an increased visco-elastic behavior. To find out whether the pectin content was the reason For pectinase treatment we used 6.4 mg ml 1, since for the difference in pectinase sensitivity between liquid this concentration neither stimulated nor inhibited and solidified media we compared pectin label of images pollen tube growth, but immunofluorescence label with taken at identical exposure times of pollen tubes grown JIM5 and JIM7 revealed that the amount of pectins in liquid medium and medium solidified by addition of was significantly reduced in the cell wall of pollen tubes 10 mg ml 1 Gel-Gro (corresponding to the gel strength grown in the presence of the enzyme. Images taken at of 20 mg ml 1 agarose). The use of Gel-Gro allowed us the same exposure time as control tubes were com- to re-suspend the pollen after fixation, thus ensuring that pletely black (not shown). Pollen tubes that were ger- antibody access to the cells was not limited by the minated in the presence of 6.4 mg ml 1 pectinase stiffened medium—an essential prerequisite for com- showed a decrease in the stiffness for both the apex and parison of fluorescence intensity. the distal parts of the tube. The distal stiffness was Label with JIM5 and JIM7 revealed that the reduced to 2,990±148·10 5 Ncm 1 whereas the ratio amount of both types of pectin decreased dramatically between apical and distal stiffness remained essentially for pollen tubes grown in stiffer germination medium the same at 82±5% (n=3). Both apical and distal (Fig. 5). The distribution pattern between apex and regions showed an increase in the hysteresis upon distal parts for esterified and non-esterified pectins re- retraction of the micro-indenter stylus, thus indicating mained approximately the same as that in cells grown an increase in the degree of viscosity of the cellular in liquid medium. structure. Figure 6a,b illustrates the differences in the 587
Fig. 4a–i Immunofluorescent label for pectins in Solanum pollen inhibit pollen tube growth. Our results indeed confirmed tubes and pollen grains. Pollen tubes were fixed and subsequently a negative effect on germination and tube growth for labeled for acidic pectins with JIM5 (a–c, g–i) or for methyl- 1 esterified pectins with JIM7 (d–f). Label for acidic pectins was concentrations of 0.1 mg ml and above (Fig. 7). absent from the pollen tube apex; the distribution in the distal To demonstrate that de-esterification indeed rigidi- regions was rather homogeneous. Label for methyl-esterified fied the cell wall, we assessed pollen tube stiffness of pectins was highest at the apex and decreased considerably towards PME-treated pollen tubes with the micro-indenter. We distal regions. The pollen tube in g–i was grown in medium 1 chose a PME concentration that was not completely containing 0.5 mg ml PME to convert methyl-esterified pectins into the acidic variety, thus presumably allowing JIM5 to label the inhibitory, but that resulted in an increase of immu- entire pectin population. Label intensity was highest at the apex, nolabel for acidic pectins at the apex. PME at thus indicating that the total amount of pectin is highest in this 0.5 mg ml 1 was an appropriate concentration that region. Bar = 10 lm fulfilled the requirement. JIM5 labeling of pollen tubes grown in the presence of 0.5 mg ml 1 PME revealed a force–distance graphs concerning stiffness and hystere- strong label for acidic pectins at the apex (Fig. 4g–i), sis for the apex. whereas JIM7 label for methyl-esterified pectins was almost completely absent (the image was black at an exposure time identical to that of the control—data not Pectin methyl-esterification influences the stiffness shown). We observed that the apex of pollen tubes of the pollen tube cell wall treated with 0.5 mg ml 1 PME showed a dramatic increase in cellular stiffness reaching 6,554± To show that pectin configuration is indeed a critical 936·10 5 Ncm 1 (n=6). This represents a 1.6-fold factor for pollen tube growth we applied PME at vary- increase compared to the control samples. Interestingly, ing concentrations. This enzyme is supposed to trans- the deformation at the apex of PME-treated pollen form methyl-esterified pectin into the acidic, gel-forming tubes also generally revealed low hysteresis, thus indi- variety. If a high degree of methyl-esterification were a cating a reduced viscous component in the visco-elastic prerequisite for cellular expansion at the pollen tube behavior of the cell (Fig. 6c). Unexpectedly, the distal apex, PME treatment would be expected to decrease or parts of pollen tubes treated with PME showed a
Table 1 Optimal pectinase concentrations that result in stimulation of Solanum chacoense pollen germination and tube elongation in liquid and solidified media
Medium stiffness Maximal germination rate Maximal pollen tube length at 2 h
Relative increase Optimal pectinase Relative increase Optimal pectinase compared to concentration compared to concentration untreated control untreated control (%) (mg ml 1) (%) (mg ml 1)
Liquid 17 0.5 17 0.5 Agarose 20 mg ml 1 50 0.1 34 0.1 40 mg ml 1 90 0.1 70 0.1 588
Fig. 5a–h Comparison of immunofluorescence label intensity for pectins in Solanum pollen tubes grown in liquid or solidified medium. Within a single experiment, exposure times were identical for all images. Both label with JIM5 for acidic pectins (a–d) and JIM7 for methyl-esterified pectins (e–h) evidenced that pollen tubes grown in liquid medium (a,b,e,f) were significantly more strongly labeled than those grown in solidified medium (c,d,g,h). Bar = 10 lm
reduction in cellular stiffness to 2,639± of hinge effect, facilitating deformation at 30 lm from 498·10 5 Ncm 1. This was puzzling, but we presume the apex. that the hardened apex might have allowed some sort
Discussion Fig. 6a–c Force–distance graphs for micro-indentation experi- ments performed at the apex of growing Solanum pollen tubes. Cell wall extensibility controls pollen tube growth The relative position of the stylus is given in positive values for downward movement. Zero position is approximately 1 lm above the pollen tube surface. The upper curve represents the deforming Tip growth is presumed to be regulated by an interplay movement of the stylus and the lower curve the retracting between various forces that counteract each other. The movement. The slope of the curve indicates the stiffness whereas force that drives cellular expansion is thought to be the surface area between the two curves expresses the delay upon provided by one or several features on the inside of the retraction of the deforming stylus (hysteresis), which is an indication for the dissipated energy and thus the viscosity of the cell: turgor, propulsive forces due to the cytoskeletal deformed object. The apex of pollen tubes grown in the presence of elements and possibly other unidentified features. The 6.4 mg ml 1 pectinase (b) showed an increase in hysteresis and a relative contributions of the individual factors are still lower stiffness compared to that of the control pollen tubes (a). elusive and they are likely to differ among cell types and This indicates a higher viscosity component in the visco-elastic behavior of the enzyme-treated cell. The apex of pollen tubes growth conditions (Money and Hill 1997). On the out- grown in the presence of 0.5 mg ml 1 PME (c) was stiffer than that side of the cell, the cell wall is the main structure that of the control and reacted as if it were nearly completely elastic withstands the internal forces. Both plastic and elastic 589
consistent with an increase in elastic deformability of the cell wall due to the enzyme activity. On the other hand, the enzyme induced an increase in hysteresis upon retraction of the indenting stylus, indicating that the viscosity component of the visco-elastic cell behavior had increased. These data suggest, for the first time based on the quantitative mechanical assessment of single cells, that the pectin component plays a role in the physical characteristics of the pollen tube cell wall. The observed increase in pollen tube growth rate indicated that reduction in the amount of pectins lowered the cell wall’s resistance to tensile stress, which in turn allowed the internal forces to push and expand the remaining apical cell wall more rapidly compared to the control conditions. On the other hand, softening the cell wall too much resulted in bursting of pollen grains and tubes as the internal forces exceeded the cell wall resistance. Together these data corroborate the hypothesis that the physical properties of the cell wall influence the rate of the initial formation and of the continuous extension of the api- cally growing pollen tube. This provides substantial support for the concept of a force equilibrium control- ling tip growth in pollen tubes.
Pectin is a key component in the apical cell wall extensibility Fig. 7a,b Effect of varying concentrations of PME on Solanum pollen germination rate (a) and tube length at 2 h (b). Concentra- tions above 0.1 mg ml 1 reduced both germination rate and tube We observed that addition of moderately inhibitory length and complete inhibition was achieved at an enzyme amounts of the digesting enzyme pectinase induced concentration of 20 mg ml 1. Data are means ± SE (n=4) apical swelling whereas higher amounts resulted in the bursting of the pollen tubes at the apex, causing a sud- extension of the cell wall are purported to enter the den release of the cytoplasm. At other cellular locations equation determining plant cell expansion (Lockhart on the pollen tube, swelling or bursting was never ob- 1965; Ortega 1985; Cosgrove 1997). Mechanical cell wall served, even though the pressure-induced circumferen- studies on individual living cells are few, however, as tial stress in the cylindrical tube is twice as high as the micromanipulation techniques have mostly been used on tension stress in the apex (Green 1962; Lockhart 1965). entire pieces of tissue or on in vitro-reconstituted cell This indicated that only at the apex was the digestion of wall analogues (Hiller et al. 1996; Davies et al. 1998; pectins sufficient to destabilize the cell wall in such a Chanliaud et al. 2002, 2004). manner that the turgor was able to expand the wall or Early studies using pectinase revealed an inhibitory break it open completely. Apparently, other regions of effect of this enzyme on pollen tube growth at higher the cell were protected from shape-changing or fatal concentrations (Brewbaker and Kwack 1964) and on pectinase effects. The protection could either be due to a root hair elongation (Cormack 1956; Ekdahl 1957; massive amount of pectins or to the presence of other Jackson 1959). Roggen and Stanley (1969), however, cell wall components that are responsible for resisting found that Pyrus communis pollen tube growth could be tensile stress upon digestion of the pectin component. To stimulated by addition of moderate pectinase concen- precisely relate the regions of swelling and those of trations. This observation encouraged us to look at the unaltered morphology to the distribution of cell wall effect of this enzyme in more detail and to use it to study components in Solanum, we used fluorescence labeling. the mechanical aspects of pectins in the cell wall of As expected, pectin was the major component at the growing pollen tubes. growing apex of the pollen tubes. Whereas the polymer Our data confirmed that moderate concentrations of was present in the distal regions as well, the amount was pectinase resulted in a stimulation of both pollen tube equal to or smaller than that surrounding the apex. germination and growth in Solanum pollen. Micro- Significant amounts of cellulose and callose, on the other indentation of pollen tubes grown in the presence of the hand, were present only in the distal parts of the cell that enzyme confirmed the softening effect on the entire corresponded to the region that did not swell upon pollen tube cell wall, since cellular stiffness was reduced pectinase application. Cellulose and/or callose are significantly in both the apical and distal regions. This is therefore likely to be responsible for the increased 590 stability of the distal parts. They presumably confer a whereas if the value attains a certain fatal threshold, mechanical stability against tension stress. The absence bursting occurs. Let us now assume that in a pollen tube of these cell wall components at the tip, on the other that penetrates a solidified medium, the inner driving hand, suggests that they play only a minor role in the force also has to act against the stiffened medium. We control of the cell wall extensibility governing the therefore include it in the equation: growth process at the apex. This is consistent with our own data that address the role of these polymers in more Inner driving force > Cell wall stiffness detail (not shown). Pectins are therefore clearly the þ Medium stiffness extension-controlling cell wall polymers in the growing pollen tube apex, which is consistent with observations Assuming that the inner driving force is unaffected by the made on cell wall analogues in vitro (Chanliaud 2002). substance responsible for the solidification of the med- Pectinase treatment at moderate enzyme concen- ium, the cell wall stiffness could thus be lowered without trations also resulted in an increase in the percentage leading to bursting upon replacement of the liquid by a of germinating pollen grains. This finding suggests stiff medium. Indeed, the cell wall stiffness might neces- that, similar to the pollen tube apex, the Solanum sarily have to be lowered to allow the inner driving force pollen grain aperture must be rich in pectins. This has to be higher than the sum of cell wall and medium stiff- been observed for various species (Geitmann et al. ness in order to allow apical expansion. To accomplish 1995; Aouali et al. 2001; Suarez-Cervera et al. 2002) the reduction in cell wall stiffness, one solution would be and our immunofluorescent label confirmed this to be to reduce the amount of pectins. As a consequence, lower the case for Solanum pollen as well (data not shown). amounts of pectinase would be sufficient to achieve either Surprisingly, even though the Solanum pollen grain stimulation or inhibition since the available pectins aperture also labels intensively for callose (data not would be digested more quickly. The observed shift in shown), pectinase treatment at high enzyme concen- sensitivity is consistent with this hypothesis. trations was sufficient to induce bursting of pollen The question is then how the reduction in the amount grains. Pectins do therefore seem to have a controlling of pectins is accomplished. Putative intracellular regu- function in withstanding the turgor within the grain lation processes include the reduction of the production while simultaneously allowing for controlled cell wall rate or a decrease in the secretion rate of the cell wall yielding upon pollen tube emergence. This is consistent precursors. Alternatively, once secreted, pectins could be with the observation that the earliest visible cell wall digested by the pollen tube’s own enzymes. Pollen tubes alteration upon emergence of the pollen tube is a loss are known to produce pectin-degrading enzymes of pectic material (Heslop-Harrison and Heslop-Har- (Dearnaley and Daggard 2001, and references therein), rison 1992; Taylor 1997). PME (Mu et al. 1994, and references therein; Li et al. 2002) and expansins (Darley et al. 2001)tobeableto soften the transmitting tissue. A stiffened medium might therefore cause the up-regulation of enzyme production The exertion of penetration forces requires a change in an active feedback mechanism. A third option would in the equilibrium between inner driving forces be a sort of passive feedback mechanism: the stiffened and the cell wall medium might reduce the diffusion of enzymes produced by the pollen tube and thus cause an increased local Pollen tube growth in solidified medium in the presence concentration around the cell, which would be respon- and absence of pectinase revealed two striking results. sible for reducing the pectin contents. The comparative Firstly, the stimulation of germination and pollen tube analysis of the immunofluorescence label for pectins on growth was relatively bigger in solidified medium, and pollen tubes grown in liquid and solidified media is not secondly, the optimal pectinase concentration for both conclusive in this regard. It revealed that the amount of stimulation and inhibition differed between solidified pectins was indeed dramatically lower in the tubes and liquid media. Both effects could be explained if the grown in stiff medium, but whether this was due to solidification of the medium is taken into the equation lowered production and/or secretion of cell wall com- formed by the equilibrium between inner propulsive ponents or a post-secretion degeneration by the pollen forces and the cell wall. tube’s own enzymes remains unclear. This topic most Without attempting to discuss the equations for plant certainly warrants further research. Nonetheless, our cell growth established by Lockhart (1965) and modified data provide an indication of the mechanism that allows later (as summarized by Money and Hill 1997), pollen tip-growing cells to penetrate a solid substrate: a tight tube expansion probably necessitates the following ra- control of the amount of driving force retaining cell wall ther simplified condition: polymers in the growth zone, localized either pre- or Inner driving force > Cell wall stiffness post-secretion. This necessity to be able to control the physical properties of the cell wall is not surprising since The difference between the two opposing forces is small, cell wall softening to allow tip growth upon lowered presumably, yet probably above a certain yield threshold turgor pressure has been observed previously in fungal (Taiz 1984; Cosgrove 1986) during normal growth, hyphae (Money and Harold 1992). 591
The pectin configuration determines the mechanical physiology and fertilization. Elsevier, North-Holland, Amster- properties of the pollen tube apex dam, pp 145–151 Carpita N, Gibeaut D (1993) Structural models of primary cell walls in flowering plants: consistency of molecular structure The physical properties of the cell wall are not only with the physical properties of the wall during growth. Plant J controlled by the total amount of pectins, but also by 3:1–30 their configuration. Since immunofluorescence localiza- Chanliaud E, Burrows KM, Jeronimidis G, Gidley MJ (2002) Mechanical properties of primary plant cell wall analogues. tion first revealed a high apical concentration of methyl- Planta 215:989–996 esterified pectin (Li et al. 1994) it has been suggested that Chanliaud E, De Silva J, Strongitharm B, Jeronimidis G, Gidley this pectin configuration is responsible for a higher MJ (2004) Mechanical effects of plant cell wall enzymes on extensibility of the apical cell wall. This concept is based cellulose/xyloglucan composites. Plant J 38:27–37 on the fact that in the esterified configuration the pectin Cormack RGH (1956) A further study of the growth of Brassica roots in solutions of pectic enzymes. Can J Bot 34:983–987 polymers are not gelated by calcium ions (Jarvis 1984;Li Cosgrove DJ (1986) Biophysical control of plant cell growth. Annu et al. 1994). In dicots, pectin gelation has been related to Rev Plant Physiol 37:377–405 developmental stages characterized by cell wall rigidifi- Cosgrove DJ (1997) Assembly and enlargement of the primary cell cation (Kim and Carpita 1992; McCann et al. 1993, wall in plants. Annu Rev Cell Devel Biol 13:171–201 Darley CP, Forrester AM, McQueen-Mason SJ (2001) The 1994). Here we provide for the first time mechanical molecular basis of plant cell wall extension. Plant Mol Biol evidence in individual living cells that shows that 47:179–195 transformation of methyl-esterified pectins into the de- Davies GC, Hiller S, Bruce DM (1998) A membrane model for esterified variety causes the cell wall to stiffen and to lose elastic deflexion of individual plant cell walls. J Texture Stud 29:645–667 the viscosity component of the visco-elastic behavior. In Dearnaley JDW, Daggard GA (2001) Expression of a polygalac- other words, both elastic and plastic deformability of the turonase enzyme in germinating pollen of Brassica napus. Sex cell wall seem to be reduced by the de-esterification of Plant Reprod 13:265–271 the pectin polymers. The micro-indentation experiments Derksen J (1996) Pollen tubes: a model system for plant cell therefore contributed important cytomechanical infor- growth. Bot Acta 109:341–345 Derksen J, Rutten T, Lichtscheidl I, de Win A, Pierson E, Rongen mation to the understanding of the cell wall rheology of G (1995) Quantitative analysis of the distribution of organelles growing plant cells. in tobacco pollen tubes: implications for exocytosis and endo- Consequently, the inhibitory effect of PME on pollen cytosis. Protoplasma 188:267–276 tube germination and pollen tube growth above Ekdahl I (1957) On the growth mechanism of roots hairs. Physiol 1 Plant 10:798 0.1 mg ml can be explained by hardening of the cell Elson EL, Daily BB, McConnaughey WB, Pasternak C, Petersen wall at the aperture and the pollen tube apex, respec- NO (1983) Measurement of forces which determine the shapes tively. This is consistent with observations made for of adherent cells in culture. In: Liu T-Y, Sakakibara S, other species (Geitmann 1997). Our data therefore pro- Schechter A, Yagi K, Yajima H, Yasunobu KT (eds) Frontiers in biochemical and biophysical studies of proteins and mem- vide strong mechanical evidence for the concept that a branes. Elsevier, New York, pp 399–411 low degree of pectin esterification is essential for pollen Franklin-Tong VE (1999) Signaling and the modulation of the tube expansion at the apex. Taken together, our data pollen tube growth. Plant Cell 11:727–738 provide, for the first time, mechanical evidence for the Geitmann A (1997) Growth and formation of the cell wall in pollen long-standing hypothesis that the amount of pectins and tubes of Nicotiana tabacum and Petunia hybrida. Egelsbach, Ha¨ nsel-Hohenhausen their degree of esterification are essential structural Geitmann A (1999) The rheological properties of the pollen tube factors in pollen tube cytomechanics. cell wall. In: Cresti M, Cai G, Moscatelli A (eds) Fertilization in higher plants. Springer, Berlin Heidelberg New York, pp 283– Acknowledgements This research was supported by grants from the 297 2+ Natural Sciences and Engineering Research Council of Canada Geitmann A, Cresti M (1998) Ca channels control the rapid (NSERC), the Canadian Foundation for Innovation (CFI), and the expansions in pulsating growth of Petunia hybrida pollen tubes. Fonds Que´becois de la Recherche sur la Nature et les Technologies J Plant Physiol 152:439–447 (FQRNT) to A. Geitmann. The generous gifts of monoclonal Geitmann A, Parre E (2004) The local cytomechanical properties of antibodies JIM5 and JIM7 from Keith Roberts, John Innes Centre, growing pollen tubes correspond to the axial distribution of Norwich, UK, and from Paul Knox, Leeds University, UK, are structural cellular elements. Sex Plant Reprod 17:9–16 gratefully acknowledged. We also thank William B. McConnaug- Geitmann A, Hudak J, Vennigerholz F, Walles B (1995) Immu- hey, Washington University, St. Louis, Missouri, for assistance nogold localization of pectin and callose in pollen grains and with the micro-indentation setup. pollen tubes of Brugmansia suaveolens—implication for the self- incompatibility reaction. J Plant Physiol 147:225–235 Green PB (1962) Mechanism for plant cellular morphogenesis. Science 138:1404–1405 Green PB (1969) Cell morphogenesis. Annu Rev Plant Physiol References 20:365–394 Hasegawa Y, Nakamura S, Kakizoe S, Sato M, Nakamura N Aouali N, Laporte P, Clement C (2001) Pectin secretion and dis- (1998) Immunocytochemical and chemical analyses of golgi tribution in the anther during pollen development in Lilium. vesicles isolated from the germinated pollen of Camellia Planta 213:71–79 japonica. J Plant Res 111:421–429 Brewbaker J, Kwack B (1963) The essential role of calcium ion in Hepler PK, Vidali L, Cheung AY (2001) Polarized cell growth in pollen germination and pollen tube growth. Am J Bot 50:859–865 higher plants. Annu Rev Cell Devel Biol 17:159–87 Brewbaker J, Kwack B (1964) The calcium ion and substances Heslop-Harrison J (1987) Pollen germination and pollen-tube influencing pollen growth. In: Linskens HF (ed) Pollen growth. Int Rev Cytol 107:1–78 592
Heslop-Harrison Y, Heslop-Harrison J (1992) Germination of McCann MC, Stacey NJ, Wilson R, Roberts K (1993) Orientation monocolpate angiosperm pollen: evolution of actin cytoskele- of macromolecules in the walls of elongating carrot cells. J Cell ton and wall during hydration, activation and tube emergence. Sci 106:1347–1356 Ann Bot 69:385–394 McCann MC, Shi J, Roberts K, Carpita NC (1994) Changes in Hiller S, Bruce DM, Jeronimidis G (1996) A micro-penetration pectin structure and localization during the growth of un- technique for mechanical testing of plant cell walls. J Texture adapted and NaCl-adapted tobacco cells. Plant J 5:773–785 Stud 27:559–587 McNeil M, Darvill AG, Fry SC, Albersheim P (1984) Structure and Holdaway-Clarke TL, Hepler PK (2003) Control of pollen tube function of the primary cell walls of plants. Ann Rev Biochem growth: role of ion gradients and fluxes. New Phytol 159:539– 53:625–63 563 Money NP, Harold FM (1992) Extension growth of the water mold Jackson WMT (1959) Effect of pectinase and cellulase preparations Achlya: interplay of turgor and wall strength. Cell Biol 89:4245– on the growth and development of root hairs. Physiol Plant 4249 12:502–510 Money NP, Hill TW (1997) Correlation between endoglucanase Jarvis MC (1984) Structure and properties of pectin gels in plant secretion and cell wall strength in oomycete hyphae: implica- cell walls. Plant Cell Environ 7:153–164 tions for growth and morphogenesis. Mycologia 89:777–785 Jauh GY, Lord EM (1996) Localization of pectins and arabino- Morris ER, Powell DA, Gidley MJ, Rees DA (1982) Conforma- galactan-protein in lily (Lilium longiflorum L.) pollen tube and tions and interactions of pectins. I. Polymorphism between gel style, and their possible roles in pollination. Planta 199:251–261 and solid states of calcium polygalacturonate. J Mol Biol Kauss H, Hassid WZ (1967) Enzymic introduction of the methyl 155:507–16 ester groups of pectins. J Biol Chem 242:3449–3453 Mu JH, Stains JP, Kao TH (1994) Characterization of a pollen- Kim J-B, Carpita NC (1992) Changes in esterification of the uronic expressed gene encoding a putative pectin esterase of Petunia acid groups of cell wall polysaccharides during elongation of inflata. Plant Mol Biol 25:539–544 maize coleoptiles. Plant Physiol 98:646–653 Ortega JKE (1985) Augmented equation for cell wall expansion. Knox JP, Linstead PJ, King J, Cooper C, Roberts K (1990) Pectin Plant Physiol 79:318–320 esterifications spatially regulated both within cell walls and Petersen NO, McConnaughey WB, Elson EL (1982) Dependence between developing tissues of root apices. Planta 181:512–521 of locally measured cellular deformability on position on the Lennon KA, Lord EM (2000) In vivo pollen tube cell of Arabid- cell, temperature, and cytochalasin B. Proc Nat Acad Sci opsis thaliana. Tube cell cytoplasm and wall. Protoplasma USA79:5327–5331 214:45–56 Roggen HPJR, Stanley RG (1969) Cell wall hydrolysing enzymes Levy S, Staehelin LA (1992) Synthesis, assembly and function of in wall formation as measured by pollen tube extension. Planta plant cell wall macromolecules. Curr Opin Cell Biol 4:856–862 84:295–303 Li YQ, Chen F, Linskens H, Cresti M (1994) Distribution of Steer MW, Steer JM (1989) Pollen tube tip growth. New Phytol unesterified and esterified pectins in cell walls of pollen tubes of 111:323–358 flowering plants. Sex Plant Reprod 7:145–152 Suarez-Cervera M, Arcalis E, Le Thomas A, Seoane-Camba JA Li YQ, Chen F, Faleri C, Ciampolini F, Linskens HF, Cresti M (2002) Pectin distribution pattern in the apertural intine of (1995) Presumed phylogenetic basis of the correlation of pectin Euphorbia peplus L. (Euphorbiaceae) pollen. Sex Plant Reprod deposition pattern in pollen tube walls and the stylar structure 14:291–298 of angiosperms. Proc K Ned Akad Wet 98:39–44 Taiz L (1984) Plant cell expansion: regulation of cell wall Li YQ, Moscatelli A, Cai G, Cresti M (1997) Functional interac- mechanical properties. Annu Rev Plant Physiol 35:585–657 tions among cytoskeleton membranes and cell wall in pollen Taylor LP (1997) Pollen germination and tube growth. Annu Rev tube of flowering plants. Int Rev Cytol 176:133–199 Plant Physiol Plant Mol Biol 48:461–491 Li YQ, Mareck A, Faleri C, Moscatelli A, Liu Q, Cresti M (2002) VandenBosch KA, Bradley DJ, Knox JP, Perotto S, Butcher GW, Detection and localization of pectin methylesterase isoforms in Brewin NJ (1989) Common components of the infection thread pollen tubes of Nicotiana tabacum L. Planta 214:734–740 matrix and the intercellular space identified by immunocyto- Lockhart JA (1965) An analysis of irreversible plant cell elonga- chemical analysis of pea nodules and uninfected roots. EMBO J tion. J Theor Biol 8:264–75 8:335–342