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First published online June 10, 2005; 10.1104/pp.105.059865 Plant Physiology 138:1334-1346 (2005) © 2005 American Society of Plant Biologists Pectin Methylesterase, a Regulator of Pollen Tube Growth1,[w]Biology Department (M.B., P.K.H.), Department of Biochemistry and Molecular Biology (A.Y.C.), and Plant Biology Graduate Program (A.Y.C., P.K.H.), University of Massachusetts, Amherst, Massachusetts 01003
The apical wall of growing pollen tubes must be strong enough to withstand the internal turgor pressure, but plastic enough to allow the incorporation of new membrane and cell wall material to support polarized tip growth. These essential rheological properties appear to be controlled by pectins, which constitute the principal component of the apical cell wall. Pectins are secreted as methylesters and subsequently deesterified by the enzyme pectin methylesterase (PME) in a process that exposes acidic residues. These carboxyls can be cross-linked by calcium, which structurally rigidifies the cell wall. Here, we examine the role of PME in cell elongation and the regulation of its secretion and enzymatic activity. Application of an exogenous PME induces thickening of the apical cell wall and inhibits pollen tube growth. Screening a Nicotiana tabacum pollen cDNA library yielded a pollen-specific PME, NtPPME1, containing a pre-region and a pro-region. Expression studies with green fluorescent protein fusion proteins show that the pro-region participates in the correct targeting of the mature PME. Results from in vitro growth analysis and immunolocalization studies using antipectin antibodies (JIM5 and JIM7) provide support for the idea that the pro-region acts as an intracellular inhibitor of PME activity, thereby preventing premature deesterification of pectins. In addition to providing experimental data that help resolve the significance and function of the pro-region, our results give insight into the mechanism by which PME and its pro-region regulate the cell wall dynamics of growing pollen tubes.
Pollen tube growth, which delivers the sperm cells to the female gametophyte in the ovule, is essential for plant reproduction. The elongation process is driven by the secretion of Golgi-derived vesicles that dock and fuse with the plasma membrane at the extreme apex of the tube, providing new plasma membrane and cell wall components necessary for polarized pollen tube growth. Their fast growth and relative ease of culture in vitro make pollen tubes a well-established model system for studying cell elongation in plants. In the search for cellular components that regulate pollen tube growth, most of the attention has been drawn to secretory membrane traffic, intracellular motility, ion activities, and turgor pressure, while the contribution of the cell wall has been somewhat neglected (Holdaway-Clarke and Hepler, 2003  ). Yet, the apical cell wall certainly is a key component since it has to be strong enough to withstand the internal turgor pressure, but, at the same time, provide enough plasticity to allow the incorporation of new membrane and cell wall material to support tip growth (Steer and Steer, 1989).
The wall in the tip region of the pollen tube is composed of a single pectin layer, where neither cellulose nor callose has been detected (Ferguson et al., 1998
Immunolocalizations have shown that in both Nicotiana tabacum and Lilium longiflorum, esterified pectins are almost exclusively present in the apical region of the wall while relatively unesterified pectins are present along the entire length of the pollen tubes (Li et al., 1994
It is known that many plant PME genes encode so-called pre-pro-proteins. The pre-region is required for protein targeting to the endoplasmic reticulum (ER), while only the mature part of the PME, without the pro-region, is extracted from the cell wall. Although several functions for the pro-region have been suggested (Micheli, 2001
Effect of Exogenous PME on Pollen Tube Growth and Morphology
To determine whether PMEs affect the average pollen tube growth rate, the culture medium was supplemented with PME from orange peel (Fig. 1). When applied before germination of the pollen grains, PME caused a dramatic decrease of the germination rate for both Lilium formosanum and N. tabacum (data not shown). To avoid this effect from influencing the growth rate calculations, pollen grains were first allowed to germinate in standard growth medium and only afterward cultured in PME-supplemented medium (Holdaway-Clarke et al., 2003
A closer look at the morphology of PME-treated tubes from both species showed a significant increase in the cell wall thickness over time, which is most prominent at the apex (Fig. 2, A and B). Application of boiled orange peel PME did not affect the pollen tube growth rate or morphology (data not shown). Pollen tubes possess an intracellular tip-focused Ca2+ gradient that has been related to cell elongation (Pierson et al., 1994 , 1996). Ratiometric Ca2+ imaging of L. formosanum tubes revealed that PME treatment triggers a dissipation of this gradient, most probably caused by the apical cell wall thickening and subsequent inhibition of cell elongation (Fig. 2C).
Characterization of a N. tabacum Pollen Tube PME
Knowing that exogenous PME induces apical cell wall thickening and growth inhibition of pollen tubes, we screened a N. tabacum pollen cDNA library for PMEs using the Petunia inflata pollen cDNA clone PPE1 (Mu et al., 1994
Although several roles for the pro-region have been suggested, including being an autoinhibitory domain, a targeting factor, and/or a chaperone (Micheli, 2001 ), the biological function of this region remains to be established. Figure 3B shows a schematic representation of the deduced pre-pro-PME protein. At this stage, we can only speculate about the exact borders of the pro-region and the PME domain. Based on alignments with fungal PMEs, which do not possess a pro-region, and the predictions from the Conserved Domain Search, it seems that the PME domain starts between amino acids 175 and 200, resulting in a mature protein of 39 to 41.5 kD with a predicted pI of 8.6. The pro-region is expected to run from amino acids 23 to 175 to 200, resulting in a protein domain that translates into 17 to 19.5 kD and an pI of 4.5. Although our PME was obtained from a N. tabacum pollen cDNA library, this does not necessarily imply that expression is confined to pollen or pollen tubes. The spatial expression pattern of the pre-pro-PME was examined using reverse transcription (RT)-PCR. Total RNA was isolated from all major N. tabacum tissues, and PCR was performed using cDNA from the first-strand reaction with primers specific for the PME clone. Primers specific for N. tabacum actin were included as an internal control. Expression of this pre-pro-PME was restricted to pollen and pollen tubes (Fig. 4), and, therefore, the isolated clone was named NtPPME1 (Nicotiana tabacum pollen tube PME1).
What Can We Learn from the Arabidopsis Transcriptome?
Several Arabidopsis (Arabidopsis thaliana) pollen transcriptome studies have shown that genes encoding for proteins related to cell wall biosynthesis and regulation are highly expressed in Arabidopsis pollen (Becker et al., 2003
The expression of genes encoding for invertase/PME inhibitors is very high in pollen compared to the other tissues (Table I). Here we note that, due to the sequence similarity between invertase inhibitors and PMEIs, it is impossible to separate the two from each other (Rausch and Greiner, 2004 ). Invertases, and thus also the regulation of their activity by invertase inhibitors, are likely to play an important role in pollen tube growth since these enzymes hydrolyze Suc to Glc and Fru, which are important metabolites for respiration and synthetic processes (Ylstra et al., 1998; Rausch and Greiner, 2004). It is known, however, that at least some of the Arabidopsis pollen-expressed inhibitor family members represent functional PMEIs (Wolf et al., 2003). Together, the transcriptome data underline the importance of PMEs and the regulation of their activity for pollen germination and/or tube growth.
We have seen that in Arabidopsis pollen many different PME isoforms are highly expressed. To establish how many PME isoforms are present in N. tabacum pollen tubes, proteins were extracted with a low- and high-salt buffer and stained for PME activity after electrophoretic separation. A single band with an apparent molecular mass of approximately 40 kD appeared upon activity staining after SDS-PAGE, which corresponds to the predicted molecular mass of the mature PME encoded by NtPPME1 (Fig. 5A). Also, after native acidic continuous PAGE, only one active PME band could be detected (Fig. 5B). The pollen tube proteins extracted with a low-salt buffer showed one major band, corresponding to a pI of about 8.5, after separation by isoelectric focusing (IEF) followed by activity staining. Upon reextraction of the remaining pellet with the high-salt buffer, two bands corresponding to pIs of approximately 8 and 9 appear (Fig. 5C), which may indicate the presence of more isoforms or may reflect posttranslational modifications of the same isoform. These data suggest that the isolated NtPPME1 clone encodes the principal PME expressed in N. tabacum pollen tubes since both the apparent molecular mass of approximately 40 kD and pI of 8.5 match closely with the values predicted for the mature NtPPME1 (respectively, 39–41.5 kD and pI 8.6; Figs. 3B and 4). However, further analysis of the PME isoforms expressed in Arabidopsis pollen (Table II) reveals that the predicted pI (8.7–10.0) and molecular mass (36.2–39.3 kD) of most deduced PME domains are very similar to those obtained upon PME activity staining of N. tabacum pollen tube protein extracts. This indicates that, as in Arabidopsis pollen, multiple PME isoforms might be expressed in N. tabacum pollen tubes.
The presence of multiple PME isoforms in N. tabacum pollen tubes (four acidic, one neutral, and two basic) is reported by Li et al. (2002) . The fact that we are unable to detect the acidic and neutral isoforms might be related to the different PME activity-staining methods that have been used. However, it is a concern that the heterologous anti-flax PME antibody used by Li et al. (2002) recognized two isoforms (pI 5.5 and 7.3) with molecular masses of about 160 kD after SDS-PAGE. By contrast, the molecular masses of PMEs purified from other plant species range between 22 and 62 kD, with most of them being in the 30- to 40-kD range (Gaffe et al., 1997; Ren and Kermode, 2000; Table II). We have been unable to confirm the presence of these high molecular mass (160 kD) PME isoforms upon activity staining after SDS-PAGE (Fig. 5A) with the sensitive assay described by Hou and Lin (1998).
N. tabacum pollen tubes were transformed by microprojectile bombardment with various chimeric DNA constructs containing NtPPME1 cDNA, or its deletion variants, and a C-terminal GFP under the control of the pollen-specific Lat52 promoter (Twell et al., 1989
In pollen tubes transformed with the complete NtPPME1 gene (pre-pro-PME-GFP), fluorescence labeling appeared to be associated with the ER network and Golgi dictyosomes, both of which are known components of the secretory pathway (Fig. 6B). Curiously, labeling is markedly reduced in the apical inverted cone, a domain previously thought to be an accumulation of secretory vesicles (Derksen et al., 1995 ). Importantly, a distinct fluorescent signal occurs at the pollen tube apical cell surface, which indicates that NtPPME1 has been correctly targeted via the secretory pathway to the apical cell wall. Note here that the fluorescence is brightest at the extreme apex of the tube, and that the intensity falls off within a few microns from the polar axis, such that no staining is observed along the shank of the tube. In marked contrast to the results above, the expression of NtPPME1 in which the PME domain was deleted (pre-pro-GFP) shows no label in the pollen tube wall (Fig. 6C). However, within the cytoplasm, there is a marked similarity between this construct and the full-length pre-pro-PME-GFP. Thus, the ER network and Golgi dictyosomes are strongly labeled, while the region of the inverted cone is relatively unlabeled. A similar fluorescence pattern has been obtained in pollen tubes transformed with NtPPME1 in which the pro-region was deleted (pre-PME-GFP; Fig. 6D; Supplemental Movie 1). These results show that exocytosis to the cell wall requires the presence of both the pro-region and the PME domain since deletion of either one disrupts the targeting process.
To determine whether overexpression of the whole protein as well as its separate domains has an effect on the pollen tube growth, pollen tubes were cotransformed with the gene of interest and Lat52-GFP, which served as a marker for transformation. Overexpression of NtPPME1, as well as the separate pro-region, did not significantly affect pollen tube growth (Fig. 7). However, overexpression of the PME domain dramatically reduced the pollen tube growth rate as the average pollen tube lengths reached only about one-half the size of the control tubes (Fig. 7). Interestingly, partial rescue of tube growth can be achieved by coexpression of the separate pro-region and PME domain (Fig. 7). These results suggest that the PME domain alone is sufficient for its hydrolytic activity and that the pro-region, directly or indirectly, can rescue the observed inhibitory effect caused by the PME domain.
Expression of the PME Domain Alters the Methylesterification Profile of Pectins
The distribution of methylesterified and deesterified pectins can be studied using the monoclonal antibodies JIM5 and JIM7. JIM5 has been shown to bind preferably to at least four contiguous unesterified GalUA residues with adjacent or flanking methylesterified residues. By contrast, JIM7 binds to a relatively highly methylesterified pectin epitope (Clausen et al., 2003 To determine whether expression of the PME domain fused to the SP of NtPPME1 affects the degree of pectin methylesterification, pollen tubes were cotransformed with Lat52-pre-PME and Lat52-GFP. Pollen tubes were grown for 6 h, chemically fixed, and labeled with the antipectin antibodies. In nontransformed pollen tubes, very few JIM5 epitopes are present in the apical cell wall (Fig. 8A), indicating that unesterified pectins are scarce in this region. The accumulation of unesterified pectins only starts abruptly in the subapical cell wall region and continues evenly along the more distal parts of the pollen tube (Fig. 8A). By contrast, in pollen tubes that express the PME domain (lacking the pro-region), JIM5 epitopes are present along the entire pollen tube wall including the apical cell wall (Fig. 8C).
The methylesterified epitopes recognized by JIM7 are abundantly present at the very apex of nontransformed pollen tubes, while labeling is significantly lower in the subapical and distal wall regions (Fig. 8F). The apical cell wall labeling by JIM7 is dramatically reduced in pollen tubes expressing the PME domain (Fig. 8H). Figure 8K, in which the arrow indicates the tip of a transformed tube while the arrowhead points to the tip of an untransformed tube, shows another example for this change in the apical accumulation of methylesterified pectins caused by the expression of the PME domain. These immunolocalization data show that expression of the PME domain, lacking the pro-region, results in the accumulation of deesterified pectins in the apical pollen tube wall and thus support our hypothesis that the PME domain alone is sufficient for exerting its enzymatic activity.
PME Affects Pollen Tube Growth
PMEs have been implicated in a number of processes, including cell wall extension, fruit maturation and senescence, pathogenesis, systemic movement of tobacco mosaic virus, cambial cell differentiation, and border cell separation from root caps (Moustacas et al., 1991
The demethylesterification of pectins by PME can have opposite consequences for the plasticity of the cell wall. On one hand, PMEs produce acidic pectins, which can bind Ca2+ and reinforce the wall structure. On the other hand, the protons produced by the hydrolysis decrease the local cell wall pH and, consequently, can promote the activity of cell wall hydrolases and result in cell extension and growth (Gaffe et al., 1994
Experiments in which L. formosanum and N. tabacum pollen tubes were exposed to exogenous orange peel PME resulted in a significant thickening of the apical cell wall (Fig. 2, A and B) and inhibition of tube growth (Fig. 1), indicating that PME can alter the apical cell wall dynamics. This is in agreement with recent data from Parre and Geitmann (2005)
Exogenous PME
Endogenous PME
Screening of a N. tabacum pollen cDNA library resulted in the isolation of NtPPME1, which is specifically expressed in pollen and pollen tubes (Fig. 4). Based on sequence analysis of the Arabidopsis genome, it has been proposed that PME genes may be divided into two classes. Type I genes contain only two or three introns and encode a long pro-region, whereas type II genes contain five or six introns and encode either a short or no pro-region (Micheli, 2001
The pro-region has been suggested to play a role in the targeting of PMEs toward the cell wall, to function as a chaperone in the correct conformational folding of the mature PME, and/or to act as an inhibitor of the enzyme activity carried out by the mature PME (Micheli, 2001
Targeting of PME
Inhibitor of PME Activity
Provisional support for the inhibitory activity of the pro-region derives from our studies showing that the blockage of tube growth by expression of the PME domain can be partially rescued by coexpression of the pro-region (Fig. 7). That the recovery of growth is not complete might be explained by the fact that intramolecular inhibition is more effective than the intermolecular inhibition seen in our experiment. At this stage, we cannot resolve whether this recovery of growth is the result of direct enzymatic inhibition, due to the interaction of the pro-region with the active site of the PME domain, as has been shown for the interaction between the kiwi PMEI and a PME from tomato (Lycopersicon esculentum; D'Avino et al., 2003
Chaperone Function
In this study, we confirm and extend the idea that PMEs contribute to the regulation of pollen tube growth. Both endogenous expression and exogenous application of PME inhibit pollen tube growth, probably through changes in the rheological properties of the apical pectinaceous tube wall. We also show that our pollen-specific NtPPME1 is encoded as a pre-pro-PME. However, we provide experimental data revealing that the pro-region participates in the correct targeting of PME and give support to the idea that this region can serve as an inhibitor of PME activity, possibly preventing the premature deesterification of pectins prior to secretion. Future studies need to address in more detail the mechanism by which correct targeting of PME is achieved, and how the inhibition as well as stimulation of PME activity is regulated.
Pollen Tube Growth Conditions Lilium formosanum pollen was germinated and cultured in growth medium consisting of 7% (w/v) Suc, 0.1 M KCl, 1.6 mM H3BO3, and 15 mM MES buffer adjusted to pH 5.7 with KOH. Nicotiana tabacum (cv Petit Havana SR1) pollen was germinated and cultured in medium consisting of 2% (w/v) Suc, 15% PEG-3350, 0.01% (w/v) H3BO3, 0.01% (w/v) HNO3, 0.02% (w/v) MgSO4, 0.07% (w/v) CaCl2, and 20 mM MES buffer, pH 6. Pollen tubes were germinated and grown on a rotor at room temperature. For microscopic observations, a pollen suspension was plated and immobilized with a growth medium solution containing a final concentration of 0.7% (w/v) low-melting agarose. The N. tabacum low-melting agarose solution contained 7% (w/v) Suc instead of PEG-3350. The immobilized pollen tubes were covered with 1 mL of liquid growth medium and placed in a moist chamber until observation.
Pollen tubes were grown as described before. After at least 1 h of germination, a pollen tube sample was taken and stored at 4°C to stop growth. The remaining pollen tube culture was then divided into smaller aliquots, the pollen tubes were allowed to settle, and the growth medium was replaced with media containing the experimental concentrations of orange (Citrus sinensis) peel PME (Sigma, St. Louis). Orange peel PME stock solutions (0.1 unit mL–1 or 1 unit mL–1) were prepared in 25 mM sodium phosphate, pH 7.5. Boiled preparations (10 min) of orange peel PME did not show any PME activity staining (Hou and Lin, 1998
A N. tabacum (cv Petit Havana SR1) pollen cDNA library (Chen et al., 2002
Total RNA from the different N. tabacum tissues was isolated using the RNeasy plant mini kit (Qiagen, Chatsworth, CA). First-strand cDNA synthesis was performed using AMV reverse transcriptase (Invitrogen, Carlsbad, CA), according to the manufacturer's instructions, using 2.5 µg RNA and oligo(dT) primers. Two microliters of cDNA from the first-strand reaction were used as template for PCR with NtPPME1 specific primers: forward, 5'-GAAATGCAATCATGAACACTGATTTG-3'; reverse, 5'-CATTTGTGGTTTTGCTGCTAGGAAC-3', giving a 1,081-bp product. As an internal control, N. tabacum actin was amplified in the same PCR reaction using the following primers: forward, 5'-GATGCCTATGTTGGTGATGAAGCTC-3'; reverse, 5'-CACCATCACCAGAGTCCAACACAAT-3', giving a 400-bp product.
The relative expression levels used for the Arabidopsis (Arabidopsis thaliana) transcriptome data in Tables I and II were provided by Pina et al. (2005)
N. tabacum pollen (100 mg) was germinated in 10-mL germination medium and cultured for 7 h at 25°C. After a mild centrifugation, the culture medium was removed and 0.5 mL low-salt extraction buffer (10 mM Tris buffer, pH 7.7, 0.1 M NaCl) was added to the pollen tube pellet and tubes were disrupted with a small Potter homogenizer. The homogenate was transferred into a new tube, and proteins were extracted by repeated vortexing (20 s) and incubation on ice (20 min). The extract was centrifuged (14,000g, 10 min), and the supernatant was collected (low-salt protein extract). The remaining proteins in the pellet were reextracted with 0.5-mL high-salt extraction buffer (10 mM Tris buffer, pH 7.7, 1 M NaCl) using the same procedure. The protein extracts were desalted using Microcon YM-10 centrifugal filter units (Millipore, Billerica, MA) into 10 mM Tris buffer, pH 7.7.
Protein samples were incubated overnight at room temperature with sample buffer in a 3:1 ratio (sample buffer: 60 mM Tris buffer [pH 6.8], 2% [w/v] SDS, 2% [v/v]
Protein samples were separated by acidic continuous native PAGE, as described by Ren and Kermode (2000)
IEF gel electrophoresis was performed using IEF Ready Gels (pH 3–pH 10) together with the Mini-Protean II apparatus, according to the manufacturer's instruction (Bio-Rad). Following electrophoresis, the staining procedure with ruthenium red was the same as described for the acidic continuous native gel, except that the incubation time with the esterified pectin substrate was only 30 min.
Standard recombinant DNA methodology was used in constructing all of the chimeric genes used in this study. All fusion genes were cloned behind the pollen-specific promoter Lat52 (Twell et al., 1990
Microprojectile bombardment was performed using the helium-driven PDS-1000/He biolistic system (Bio-Rad). Tungsten particles (1.1 µm) were coated with plasmid DNA, according to the manufacturer's recommendation (Bio-Rad). For the GFP localization studies, typically 1 to 3 µg DNA and, for the pollen tube growth experiments, 15 µg DNA, were used to coat 3 mg of tungsten particles, which were used to bombard an individual pollen sample (10 mg). For the growth experiments, Lat52-GUS was included in the DNA samples used for bombardment to ensure that the pollen grains were transformed with equivalent amounts of DNA. For immunofluorescence, individual pollen samples (10 mg) were bombarded with tungsten particles (3 mg) coated with a DNA mixture of 3 µg Lat52-pre-PME and 3 µg Lat52-GFP. The parameters for bombardment were as described by Chen et al. (2002)
Pollen was cobombarded with Lat52-pre-PME and Lat52-GFP, and pollen tubes were germinated and grown for 6 h in liquid growth medium under the conditions described. After mild centrifugation with a hand centrifuge, the growth medium was removed and the pollen tubes were fixed in 3% (w/v) formaldehyde in 50 mM PIPES buffer, pH 6.7, containing 1 mM EGTA and 0.5 mM MgCl2 for 15 min. Following three brief washes in phosphate-buffered saline (PBS), the samples were incubated with the monoclonal antibodies JIM5 and JIM7, diluted 1:50 in PBS containing 1% (w/v) bovine serum albumin overnight at 4°C. Subsequently, the samples were washed three times in PBS and incubated with Alexa Fluor 555 goat anti-rat IgG (Molecular Probes, Eugene, OR), diluted 1:400 in PBS containing 1% (w/v) bovine serum albumin for 1 h at 37°C. After three brief washes in PBS, the pollen tubes were transferred to a microscope slide, mounted in an antifade solution (10% [v/v] PBS, 90% [v/v] glycerol, and 4% [w/v] n-propyl gallate), and examined on a fluorescence microscope. Transformed and nontransformed pollen tubes could be distinguished based on the expression of GFP in the transformed tubes.
Differential interference contrast (DIC) and wide-field epi-fluorescence images were acquired using a Nikon Eclipse TE300 inverted microscope, 40xoil immersion lens, N.A. 1.3 (Nikon, Melville, NY), and a Cool Snap CCD camera (Photometrics, Tucson, AZ) driven by MetaMorph software (Universal Imaging, West Chester, PA). To test the effect of orange peel PME on pollen tube morphology, the growth medium in the microscope slide chamber was replaced by growth medium supplemented with the orange peel PME, and DIC images were taken at regular time intervals. Boiled preparations of orange peel PME were used as a control. Ratiometric imaging of [Ca2+]i was performed as previously described (Roy et al., 1999 For length measurements of GFP-expressing tubes, wide-field epi-fluorescence images were taken using a Nikon Diaphot 300 inverted microscope with a 10x lens (Nikon). Pollen tube lengths were measured using MetaMorph software (Universal Imaging). Sequence data of NtPPME1 have been deposited with the NCBI/GenBank data libraries under accession number AY772945.
We gratefully thank Teh-hui Kao (Department of Biochemistry and Molecular Biology, Penn State University), who generously provided the Petunia inflata PPE1 clone. José Feijó and Jörg Becker (Centro de Biologia do Desenvolvimento, Instituto Gulbenkian de Ciência, Oeiras, Portugal) are acknowledged for sharing their Arabidopsis ATH1 genome array data with us. Paul Knox (Centre for Plant Sciences, University of Leeds, UK) is thanked for his generous gift of the JIM5 and JIM7 antibodies. We also thank Hen-ming Wu for helpful suggestions and for sharing molecular tools and expertise, and Maura Cannon for the use of her biolistic microprojectile apparatus. Luis Cardenas is thanked for help with the ratiometric imaging. We thank Sheila McCormick for the Lat52 promoter. Confocal microscopy was performed at the Central Microscopy Facility of the University of Massachusetts. Received January 18, 2005; returned for revision March 15, 2005; accepted March 15, 2005.
1 This work was supported by the National Science Foundation (grant no. MCB–0077599 to P.K.H. and grant no. BBS–8714235 to the University of Massachusetts Central Microscope Facility), and by the U.S. Department of Agriculture (grant no. 0101936 to A.Y.C.). 
[w] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.059865. * Corresponding author; e-mail mbosch{at}bio.umass.edu; fax 413–545–3243.
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ABSTRACT
RESULTS
-D-galacturonic acid (GalUA) residues. Current evidence indicates that these pectins are synthesized and methylesterified in the Golgi and thereafter secreted into the wall in a highly methylesterified state (Li et al., 1995
100 each.
-mercaptoethanol, 25% [v/v] glycerol, and 0.05% [w/v] bromphenol blue). Samples were separated with SDS-PAGE, according to the discontinuous buffer system of Laemmli (1970)
