Cell-cell adhesion in fresh sugar-beet root parenchyma ... - CiteSeerX

Copyright ß Physiologia Plantarum 2006, ISSN 0031-9317

Physiologia Plantarum 126: 243–256. 2006

Cell-cell adhesion in fresh sugar-beet root parenchyma requires both pectin esters and calcium cross-links Mazz Marrya,b,1, Keith Robertsa, S. Juliet Jopsona, I. Max Huxhamc, Michael C. Jarvisb, Julia Corsara, Eoin Robertsonc and Maureen C. McCannd,* a

Department of Cell and Developmental Biology, John Innes Centre, Colney, Norwich, NR4 7UH, UK Environmental, Agricultural and Analytical Chemistry Section, Department of Chemistry, University of Glasgow, Glasgow, G12 8QQ, UK c Integrated Microscopy Facility, Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK d Department of Biological Sciences, Purdue University, West Lafayette, IN47907-1392, USA 1 Current address: Department of Biological Sciences, North Dakota State University, Fargo, ND 58105-5517, USA b

Correspondence *Corresponding author, e-mail: [email protected] Received 25 May 2005; revised 8 September 2005 doi: 10.1111/j.1399-3054.2005.00591.x

Multicellular plants depend for their integrity on effective adhesion between their component cells. This adhesion depends upon various cross-links; ionic, covalent or weak interactions between the macromolecules of the adjacent cell walls. In sugar-beet (Beta vulgaris L. Aztec) root parenchyma, cell-cell adhesion is disrupted by successive extractions with a calcium-chelating agent (imidazole) and a de-esterifying agent (sodium carbonate) but not by the calciumchelating agent or the de-esterifying agent alone. Cell-cell adhesion in sugarbeet parenchyma thus depends upon both ester and Ca2þ cross-linked polymers. Pectic polysaccharides are removed by these treatments. Both parallel-electron energy-loss spectroscopy (PEELS) and Image-EELS show that calcium-binding sites are removed from the wall by imidazole. Using a monoclonal antibody that recognizes a relatively unesterified epitope of homogalacturonan, JIM 5, we show that a subset of JIM 5-reactive antigens remain in the middle lamella after Ca2þ chelation and that this subset is removed by cold (4 C) Na2CO3-induced breakage of ester bonds. Fourier transform infrared, nuclear magnetic resonance, and spectrophotometric assays show that methyl and feruloyl esters are removed from the wall by Na2CO3 but acetyl esters remain. Sodium carbonate extraction at 20 C removes cell wall autofluorescence and most of the feruloylated moieties from the wall. We propose that the chelator-resistant subset of ester-linked JIM 5-reactive pectins are important for cell-cell adhesion.

Introduction The walls of neighbouring cells in multicellular plants are joined together to create an apoplastic continuum,

but the nature of the molecules involved and the crosslinking mechanisms are poorly understood. Electron microscopy shows the existence of a distinct layer of

Abbreviations – CDTA, cyclohexanediamine-N,N,N0 ,N0 -tetraacetate; CWM, cell wall material; FTIR, Fourier transform infrared; HGA, homogalacturonan; Image-EELS, image-electron energy-loss spectroscopy; NMR, nuclear magnetic resonance; PEELS, parallel-electron energy-loss spectroscopy; RG I, rhamnogalacturonan I. This article is dedicated in loving memory to Terry Marry, a wonderful father who inspired me so much. I am very proud to be your son.

Physiol. Plant. 126, 2006

243

material between primary walls of cells in parenchymatous tissues of many species called the middle lamella, that is enriched in pectic polysaccharides. Dissolution of middle lamella pectins to promote cell separation has been implicated in fruit ripening (Fischer and Bennett 1991), abscission (Patterson 2001), and in the thermal instability of foods to cooking (Waldron et al. 1997a). Diverse cross-links are implicated in creating a network of pectic polysaccharides within the primary wall and middle lamella. Homogalacturonan (HGA) chains can condense by cross-linking with Ca2þ to form junction zones, linking parallel or antiparallel chains (Jarvis 1984, Powell et al. 1982). Goldberg et al. (1996) have proposed a cable structure based on conformational analysis of galacturonans by solid-state 13C nuclear magnetic resonance (NMR) spectroscopy. This structure contains two levels of aggregation. First, single chains join in places to form dimeric junction zones. Second, in concentrated gels, the dimers also function as interjunction segments between junction zones with four or more chains in the 21 (180 rotation between GalA residues) and 31 (right-handed helix) conformations to form cables (Goldberg et al. 1996). In a wide range of dicotyledonous plant tissues, linear, low-ester HGAs are most abundant in the middle lamella and especially at tricellular junctions and the corners of intercellular spaces, which are key load-bearing locations (Bush et al. 2001, Huxham et al. 1999, Knox et al. 1990, Parker et al. 2001, Willats et al. 1999). In apples (Huxham et al. 1999) and potatoes (Bush et al. 2001), EELS microscopy shows that both the middle lamella and, in particular, the load-bearing locations at cell junctions show elevated levels of calcium-binding sites. Evidence for the in planta occurrence of Ca2þ bridges comes mostly from extraction of wall material by chelating agents such as cyclohexanediamineN,N,N0 ,N0 -tetraacetate (CDTA) or imidazole, both of which release pectin. Normally, much less than half of the total pectin present in the wall is removed by these agents (Goldberg et al. 1996), and only limited cell separation occurs under these conditions (McCartney and Knox 2002). However, calcium-chelating agents alone are sufficient to induce the separation of onion parenchyma cells (Ng et al. 2000). Although CDTA extraction releases significant amounts of pectin from cell walls, a fuller extraction of pectins requires cold alkali, indicating the presence of covalent bonds that anchor pectin in the wall (Jarvis 1982). In onion cell walls, HGA is primarily anchored within the wall by calcium bridges, whereas rhamnogalacturonan I (RG I) is mostly covalently cross-linked (Redgwell and Selvendran 1986). Recent work on (1!4)-b-galactan and (1!5)-a-arabinan epitopes 244

indicates extensive regulation of these neutral polysaccharides often in relation to the middle lamella (Bush et al. 2001, Jones et al. 1997, McCartney et al. 2000, Willats et al. 1999). Arabinans and galactans can occur as side chains of RG I (McCann and Roberts 1991). The arabinan side chains of RG I may be feruloylated, providing sites for oxidative cross-linking (Ishii 1997), and Clausen et al. (2004) have shown the presence of feruloylated galactan in sugar-beet roots. There is also some evidence for the natural occurrence of o-D-galacturonoyl ester cross-links, that is esters that involve the carboxyl group of a galacturonosyl residue and the hydroxyl group of some other wall component, such as a polysaccharide (Brown and Fry 1993a, Kim and Carpita 1992). In rose suspension culture cells, about one-third of the xyloglucan is covalently linked to RG I (Thompson and Fry 2000). Hydrophobic interactions between methoxyl groups and hydrogen bonds between un-dissociated carboxyl and secondary alcohol groups may also be involved in holding pectic polysaccharides within the plant cell wall. Extracted HGA with a high degree of methyl esterification form gels held together by such mechanisms (Morris et al. 1982, Oakenfull and Scott 1984). However, the mechanical properties and porosity of these gels depend strongly on whether the esterification pattern is random or blockwise, independent of the degree of esterification involved (Willats et al. 2001a). The monoclonal antibody LM7, that recognizes randomly methylated HGA with a non-blockwise distribution, binds to the points of cell contact at intercellular spaces (Willats et al. 2001b) indicating that in vivo esterification pattern is tightly regulated. Sugar-beet, a member of the Chenopodiaceae, has an unusual cell wall composition for a dicotyledonous plant. Renard et al. (1993) reported that, in sugar-beet pulp, the sugar moieties of pectic polysaccharides (arabinose, rhamnose, galactose and galacturonic acid) account for more than 50% (w/w) of the cell wall material (CWM), whilst Oosterveld et al. (1996) put this figure at 63%. Oosterveld et al. (1996) calculated that approximately 70% of the pectin in sugar-beet pulp consists of branched RG I, assuming a rhamnose to galacturonic acid ratio of 1:1. However, the in vivo proportion of RG I in sugar-beet may be lower, because the pulping process releases substantial amounts of galacturonic acid from the beet pectin. Sugar-beet HGA and RG I contain a high proportion of acetyl esters (Dea and Madden 1986). Sugar-beet cell walls contain significant levels of ferulic acid (Guillon and Thibault 1989, Micard et al. 1994, Rombouts and Thibault 1986). Feruloyl groups have been found ester linked to the arabinosyl and galactosyl residues of extracted sugar-beet pectin (Colquhoun et al. Physiol. Plant. 126, 2006

1994, Guillon and Thibault 1990, Ishii 1997). Such residues are found in highly exposed pectin domains, and therefore accessible to wall peroxidases or laccases, which could catalyze oxidative coupling to create diferulate (Fry 1983). Previously, we analysed pectic polysaccharides extracted from sugar-beet root parenchyma, the first data obtained on fresh cell walls rather than sugar-beet pulp, a heat-treated and mechanically damaged material (Marry et al. 2000). In this article, we investigate the nature of the cross-links involved in cell-cell adhesion in fresh sugar-beet root parenchyma using a combination of microscopic and spectroscopic techniques. We conclude that ester and Ca2þ cross-linked pectins are both required for cell-cell adhesion.

Materials and methods Plant material CWM was prepared from parenchyma from sugar-beet (Beta vulgaris L. Aztec) roots at maturity, that is after cessation of growth, as described previously (Marry et al. 2000). The CWM was not boiled in methanol, as this may alter the chemistry of cell-cell adhesion. Sequential extraction of polysaccharides from sugar-beet CWM Polymers were extracted from CWM with 2 M imidazole pH 7.0 at 20 C for 6 h followed by an overnight incubation in fresh 2 M imidazole (Mort et al. 1991). Both of these steps were repeated before an overnight incubation at 4 C in 0.05 M Na2CO3 and 20 mM NaBH4, followed by a 3-h incubation in fresh solution at 20 C and finally a 2-h incubation in 1 M KOH and 10 mM NaBH4 at 20 C (adapted from Redgwell and Selvendran 1986). In some experiments, either unextracted or imadazoleextracted CWM was treated with 0.34 M sodium chlorite in 65 mM acetic acid for 1 h at 20 C (adapted from Fry 1982). Each cell wall residue was washed five times with dH2O by centrifugation and re-suspension, and then, a portion was fixed for low-temperature embedding and the remainder frozen in liquid nitrogen and freezedried. Fourier transform infrared (FTIR) spectra showed no traces of the extractants contaminating the CWM as described previously (McCann et al. 1992). Determination of percentage of material extracted at each step Eight 2 g wet weight aliquots of CWM were placed into separate tubes and imidazole added to seven of them. Physiol. Plant. 126, 2006

The remaining aliquot was frozen in liquid nitrogen and stored at 20 C. The sequential extraction procedure was carried out, and one tube containing the CWM residue after each extraction step was frozen and stored. All CWM residues were then thawed, washed thoroughly with dH2O, and freeze-dried. The percentage of material removed in each extraction step relative to the dry weight of unextracted CWM was calculated. The assay was carried out in duplicate on the CWM residues of four separate sequential extractions.

Cell-cell adhesion assay Slices of mature sugar-beet root parenchyma (5 · 5 · 2 mm) were taken through four successive extractions in 20 ml of 2 M imidazole pH 7.0 at 20 C. A control using dH2O was used. Some slices were treated with two successive extractions in 20 ml of 0.05 M Na2CO3, some with the four imidazole extractions followed by the two Na2CO3 extractions, whilst others were treated with acidified sodium chlorite to break phenolic cross-links (Fry 1982). Following all incubations, the slices were subjected to shear forces using a Parr cell disruption bomb (Parr Instruments, Moline, IL). Each sample was pressurized to 150 bar in nitrogen gas for 1 min at 20 C, decompressed rapidly, and ejected through a ball valve and outlet tube (3.3 mm ID). This material was viewed in a plastic Petri dish using a Zeiss Axiovert 25 inverted light microscope.

Immunogold labelling for transmission electron microscopy Samples of unextracted CWM and residual CWM from the extraction steps were fixed overnight with 2.5% glutaraldehyde in 0.05 M sodium cacodylate, adjusted to pH 7.2, at room temperature, prior to embedding in agarose gel. The blocks of agarose-embedded CWM were cut into small cubes (2 · 2 · 2 mm) and fixed for 1 h as before. The fixed blocks were then lowtemperature embedded in LR White resin (Wells 1985). Resin-embedded sections were cut on a Reichert-Jung ultramicrotome to a thickness of 0.1 mm and picked up on carbon-filmed and plastic-coated gold grids. Immunogold labelling with the monoclonal antibodies JIM 5 and JIM 7 (Clausen et al. 2003, Knox et al. 1990, Willats et al. 2000) was carried out as described previously (Bush and McCann 1999). Micrographs were taken using a JEOL 1200 EX transmission electron microscope. 245

Fluorescence microscopy Samples of unextracted and extracted pieces of sugar-beet tissue were fixed in formaldehyde overnight, dehydrated in 100% ethanol for 4 h, infiltrated in LR white resin for 48 h and polymerized at 60 C overnight. Sections (2 mm) were cut using a glass knife on an LKB ultratome III and dried onto glass slides over a hot plate at 60 C. Unstained sections were imaged under oil immersion on a Zeiss Axioskop fluorescence microscope with an excitation filter between 340 and 380 nm and no emission filter and photographed using a digital camera and under constant conditions.

Parallel-electron energy-loss spectroscopy and image-EELS Parallel-electron energy-loss spectroscopy (PEELS) analysis was carried out using a Zeiss (LEO) TEM902 operating under the following conditions: an accelerating voltage of 80 kV, a 60 mm objective aperture, a beam current of 125 · 108 A and a vacuum of 7 · 107 Torr. Using an intermediate aperture of 50 mm at ·12 000 magnification, defined areas of 2.26 mm2 containing cell wall near the junction of cells and areas of resin immediately adjacent to the wall were analysed. PEELS spectra from five regions over a 100 eV energyloss range were collected from LR White resin-embedded sections on gold grids using a slow-scan cooled charge-coupled device camera and a 0.5-s exposure time. Comparable sections were incubated for 1 h at 20 C with 5 mM calcium acetate (calcium-doped), washed briefly in dH2O for 30 s, and examined to compare the distributions of the wall-associated calcium-binding sites. Image-EELS analysis was carried out using a Zeiss (LEO) TEM902 operating as for PEELS and at ·12 000. Electronspectroscopic image sequences containing 25 3-eV energy slices over the energy-loss range DE 5 310–380 eV for calcium and 24 3-eV energy slices over DE 5 250– 320 eV for carbon were collected from resin-embedded sections with the same exposure time as for PEELS. Duplicate calcium-doped sections were analysed. The electron energy-loss regions of organic compounds of interest in a plant cell wall have been previously characterized (Huxham et al. 1999), including the carbon K-edge (DE 5 285 eV) and the calcium L2, 3 edge (DE 5 345 eV). Elemental intensity ratios for defined regions of 100 · 100 nm were analysed from the image sequences using ESIVISION software. Integrated energy-loss values per pixel for carbon and calcium were determined using the least mean square fit of the 246

Ae–r power-law model of the immediate background of each spectrum using the intensities derived from each pre-edge energy window and extrapolating for an equal energy-loss range beyond the edge. In the case of calcium, the pre-edge energy-loss range used was 310–340 eV, and the integrated ionization edge spectra was from 345 to 380 eV. The carbon intensity was calculated by integration of the spectra from 280 to 320 eV with background subtraction using the power law, with additional linear extrapolation in Excel from 320 to 420 eV. This dual derivation improved the accuracy of the carbon integration value for analysis. Data for carbon and calcium intensities per unit cross-sectional area (pixel) were used to calculate the relative intensity (Ca/C, multiplied by 100 for convenience), to normalize differences in section thickness (Huxham et al. 1999). This assumes equal carbon mass per unit volume. Statistical analyses were performed using SPSS 9.0 (SPSS Inc., Chicago IL). FTIR spectroscopy Spectra were obtained on a Bio-Rad FTS40 FTIR spectrometer equipped with a Spectra-Tech IR-Plan microscope accessory. All spectra were obtained at a resolution of 8 cm1, with 256 co-added interferograms, and microscope aperture dimensions of 100 · 100 mm. Aliquots (40 ml) of each wall extract (suspended in a 1 mg ml1 w/v dH2O solution) were dried in a layer on the barium fluoride window (13 mm diameter · 2 mm thickness) at 37 C for 2 h. A single beam traversing each sample was collected in transmission mode and ratioed to a single beam of the corresponding background collected from a clear region of the barium fluoride window. Twenty absorbance spectra of each sample were collected and averaged. The averaged spectra were baseline-corrected and area-normalized to compensate for differences in sample thickness. Difference spectra were generated by digital subtraction of an average spectrum from an average reference spectrum, as described previously (McCann et al. 1992). NMR spectroscopy Freeze-dried CWM was hydrated with H2O (1 cm3 g1). Cross-polarization magic-angle spinning (CP-MAS) 13C NMR spectra were obtained with proton spin-lock fields of c. 40 kHz, using the method of setting the HartmannHahn condition described by Ha et al. (1997). CP-MAS spectra (Foster et al. 1996) with WALTZ-16 multiplepulse proton de-coupling were recorded as described by Renard and Jarvis (1999). Physiol. Plant. 126, 2006

Spectrophotometric assays The unextracted and extracted CWM residues were assayed for uronic acid, methyl ester, acetyl ester and feruloyl ester contents. Uronic acid assays were carried out as described previously (Filisetti-Cozzi and Carpita 1991, McCann et al. 1994). Methyl esterification was assayed by measuring release of methanol after saponification (Wood and Siddiqui 1971) with modifications to increase sensitivity and reproducibility (Carpita and McCann 1996). The feruloyl content of each CWM residue was determined by the method of Guillon and Thibault (1989) except that a standard curve of ferulic acid (Fluka) from 5 to 50 nmol was used to determine feruloyl content (Marry et al. 2000). o-Acetyl content was determined by the Hestrin method. Non-specific esters in the samples were corrected for by adding HCl before the alkaline hydroxylamine reagent to prevent the esters from forming any hydroxamic acid (Downs and Pigmann 1976).

Results A simple cell separation assay indicates which chemical bonds are involved in cell-cell adhesion Slices of fresh sugar-beet root parenchyma (5 · 5 · 2 mm) were incubated in various combinations of a Ca2þ chelating agent (2 M imidazole), a de-esterifying agent (50 mM Na2CO3 plus 20 mM NaBH4), reagents to break phenolic cross-links (0.34 M acidified chlorite) and hydrogen bonds (1 M KOH plus 10 mM NaBH4), and then inspected for resulting cell separation. We used imidazole as the chelating agent, as this can be removed entirely from the sample following water washes (Marry et al. 2000, Mort et al. 1991). The addition of NaBH4 reduced the possibility of belimination (Redgwell and Selvendran 1986). Agitation by vortexing of the sections did not induce cell separation as in the cell-cell adhesion assay developed by Ng et al. (2000), and hence, the samples were transferred to a Parr bomb, pressurized to 150 bar for 1 min, and then extruded through a ball valve. The extruded material was collected and viewed in the light microscope. Sections only incubated in water or imidazole (Fig. 1A, B) or Na2CO3 remained intact. Incubation with imidazole followed by incubation with either water or acid chlorite also released no cells from the sections. However, incubation with imidazole followed by incubation with Na2CO3 at 4 C resulted in disruption of the sections (Fig. 1C), to release large aggregates


A

B

C

Fig. 1. Photographs of sugar-beet root parenchyma after chemical extractions and extrusion through the ball valve of a Parr bomb. Slices of sugar-beet root parenchyma incubated in water (A) or 2 M imidazole (B) remained intact. (C) Extraction with 50 mM Na2CO3 at 4 C after extraction with 2 M imidazole disrupted the tissue slices, releasing large aggregates of cells and a few single cells.

of cells and a few single cells. The second Na2CO3 extraction further disrupted cell-cell adhesion releasing smaller cell aggregates and a larger number of single cells. Forty-one percentage of the dry weight of sugarbeet CWM is extracted by imidazole and Na2CO3 Previously, we showed that complex mixtures of pectic polysaccharides were extracted from sugar-beet CWM by imidazole and Na2CO3, whilst xylan was extractable only in 1 M KOH (Marry et al. 2000). Here, we quantify the proportion of dry weight of CWM represented by these extraction steps. CWM was prepared from fresh sugar-beet root parenchyma tissue and extracted four times with 2 M imidazole and then twice with 50 mM Na2CO3 at 4 C and then 20 C and then finally with 1 M KOH. The mean values of the percentage of material removed at each extraction step are summarized in Table 1 (with standard variance) and are expressed relative to the dry weight of unextracted CWM. The extraction procedure removed a total of 41% by weight of the CWM. About 17% of the CWM was chelator soluble, and 20% was soluble in weak alkali. Subsequent extraction with 1 M KOH removed only a further 4% of material from the cell wall. The highest proportion of CWM (12.7% by weight) was removed by the second Na2CO3 extraction (Table 1). PEELS and Image-EELS show that calcium-binding sites are removed by imidazole extraction Low-temperature embedding of sugar-beet CWM for electron microscopy does not preserve native levels of calcium ions. Doping sections with exogenous Ca2þ, however, permits sites of calcium-binding affinity to be detected by EELS (Bush et al. 2001). Following calcium doping, calcium ions were detected throughout unextracted cell walls by PEELS (Fig. 2A) and by Image-EELS

247

Table 1. Percentage of material removed after each extraction step, expressed relative to the dry weight of unextracted cell wall material (CWM). Values are the mean of duplicate assays from four separate sequential extractions with standard variance.

A

CWM after extraction with

% material removed

First imidazole Second imidazole Third imidazole Fourth imidazole First Na2CO3 Second Na2CO3 1 M KOH

7.0 4.8 2.3 2.8 7.6 12.7 3.8

Relative intensity

Total % removed

0.7 0.5 0.4 0.3 0.4 0.6 0.3

41 0.8

Calculated spectrum

248

344

Cell wall Resin 290 310 330 350 370 390 Energy loss (eV)

B (Fig. 2B). The PEELS spectra showed that calcium ions are bound to CWM (intensity at 344 eV) but not to the resin (Fig. 2A). Using Image-EELS, we calculated the average carbon content per unit area and relative intensity ratio of Ca/C of the walls for 10 sample areas of 100 · 100 nm, for regions of both the cell wall (yellow region shown as an example) and the middle lamella (blue region). The cell wall regions of the unextracted CWM contained a mean carbon intensity of 169963 8055 and a Ca/C intensity ratio of 0.54 0.04. The middle lamella regions of the unextracted CWM contained a mean carbon intensity of 178758 9133 and a Ca/C intensity ratio of 0.77 0.08. The difference in the Ca/C intensity ratios between the cell wall regions and the middle lamella regions of unextracted CWM were significantly different using a two-tailed t-test. The PEELS spectra of calcium-doped sections of the wall residue following four sequential imidazole extraction steps and resin background are shown in Fig. 2C. The calculated difference spectrum between cell wall and resin background has no features of Ca, suggesting that Ca2þ-binding domains have been removed from the CWM by sequential imidazole extraction. With ImageEELS (Fig. 2D), regions of the cell wall had a mean carbon intensity of 181897 8506 and a Ca/C intensity ratio of 0.10 0.07 (Fig. 2D, yellow). The middle lamella regions contained a mean carbon intensity of 176537 9314 and a Ca/C intensity ratio of 0.11 0.06 (Fig. 2D, blue). The difference in the Ca/C intensity ratios between the unextracted cell wall regions and the middle lamella regions and the corresponding regions following imidazole extraction were significantly different using a two-tailed t-test. We used PEELS and Image-EELS to map the distribution of elemental nitrogen (intensity at 397 eV) in both unextracted cell walls and the walls following imidazole extraction. However, we found no significant

Relative intensity

C

Ca

290 310 330 350 370 390 Energy loss (eV)

D

Fig. 2. Parallel- and Image-electron-energy loss spectroscopy (PEELS and image-EELS) of calcium-doped sections of resin-embedded sugarbeet cell wall material. PEELS analysis of defined areas of 2.26 mm2 containing cell wall near the junction of cells and areas of resin immediately adjacent to the wall were collected over a 100-eV energy-loss range. Spectroscopic image sequences contained 25 3-eV energy slices over the energy-loss range DE 5 310–380 eV for calcium and 24 3-eV energy slices over DE 5 250–320 eV for carbon. Following calcium doping, calcium ions were detected throughout unextracted cell walls by PEELS (A) and by Image-EELS (B). (A) The PEELS spectra show that calcium ions are present in the walls but not the resin. The calculated difference spectrum of the wall and the resin shows a peak of calcium ions (black line) at 344 eV. (B) Micrograph showing regions (100 · 100 nm) of cell wall (yellow box), middle lamella (blue box) and resin (red box) sampled for Image-EELS and the corresponding spectra. Calcium ions were present throughout the cell wall, with an increased intensity of calcium ions within the middle lamella. Neither the PEELS spectra (C) nor the Image-EELS (D) of the wall residue following imidazole extraction contained detectable levels of calcium ions. Scale bars in B and D represent 0.5 mm.

differences in the distribution of the low levels of nitrogen present between cell wall regions and middle lamella regions of the unextracted CWM. We also found no significant reduction of elemental nitrogen following imidazole extraction (data not shown). A subset of JIM 5-reactive antigens remain in the middle lamella after Ca2+ is removed Immunogold labelling was used to localize pectic polysaccharides in unextracted CWM and in wall residues


A

B

C

D

E CW ML CW

CW ML CW

Fig. 3. Electron micrographs of low-temperature-embedded cell wall material (CWM) isolated from sugar-beet roots and immunogoldlabelled with the monoclonal antibodies JIM 5 and JIM 7, using 10 nm colloidal gold-conjugated secondary antibody. (A) Unextracted CWM. JIM 5 epitopes are abundant throughout the cell wall (CW). (B) CWM after four sequential extractions with imidazole. JIM 5 epitopes are present only in the middle lamella (ML). (C) CWM after sequential extraction with imidazole and Na2CO3 at 4 C. Very few JIM 5 epitopes remain. Only single CWs are visible, as cell separation has occurred. (D) Unextracted CWM, in which JIM 7 epitopes are abundant throughout the CW. (E) CWM after imidazole extractions. JIM 7 epitopes are reduced throughout the CW and ML. Domains of primary CW and ML are indicated. Scale bar represents 500 nm.

remaining after chemical extractions. The unextracted CWM contained antigens recognized by both JIM 5 and JIM 7 antibodies that recognize relatively unesterified and methyl-esterified HGA, respectively (Clausen et al. 2003, Knox et al. 1990, Willats et al. 2000), throughout the cell wall (Fig. 3A, D). After four sequential imidazole extraction steps, the JIM 5 antigens have been

removed from the primary wall but not from the middle lamella (Fig. 3B). No JIM 5-reactive antigens remained after the first Na2CO3 extraction step (Fig. 3C). Only a small proportion of methyl-esterified pectic antigens, recognized by the JIM 7 antibody, were present in the wall residue following the imidazole extractions (Fig. 3E), whereas no JIM 7-reactive antigens were present after Na2CO3 extractions (data not shown). The unextracted CWM and wall residues remaining after chemical extractions were analysed for uronic acid content, methyl ester content and acetyl ester content (Table 2). The uronic acid values show a consistent reduction in pectic material remaining with successive extraction steps. Both methyl ester and acetyl ester contents were significantly reduced after the first two imidazole extractions, with the second Na2CO3-extracted wall residue samples containing no esters (Table 2).

Phenolic compounds are present throughout the cell wall Fluorescence images of sections of sugar-beet root parenchyma show that the walls are highly autofluorescent (Fig. 4A) with some regions of higher intensity at cell corners and in the primary cell wall and middle lamella. Tissue sections extracted with 2 M imidazole (Fig. 4B) showed the same pattern and intensity of fluorescence as the unextracted CWM. Subsequent treatment with acid chlorite at either 20 or 60 C did not affect the pattern and intensity of fluorescence (data not shown). Fluorescence was much reduced and restricted to some cell corners in the wall residue remaining after extraction with 50 mM Na2CO3 at 4 C (Fig. 4C), whilst a second 50 mM Na2CO3 extraction at 20 C (Fig. 4D) eliminated all autofluorescence. All samples were analysed under identical conditions.

Table 2. The uronic acid, % methyl ester and % acetyl ester content of the cell wall material (CWM) residues remaining at each step during sequential extraction. In each case, the values for the % methyl esterification are relative to the nmol uronic acid present in 1 mg dry weight CWM. The values for the % acetylation are calculated from nmol acetic acid released relative to the nmol uronic acid present. Values are the mean of duplicate assays from five separate sequential extractions. Uronic acid content (nmol)

% methyl esterification

% acetyl esterification

Unextracted CWM

2237 21

30 6

10 4

CWM after extraction with 1st Imidazole 2nd Imidazole 3rd Imidazole 4th Imidazole 1st Na2CO3 2nd Na2CO3 KOH

2005 1695 1180 1038 900 610 426


18 15 12 11 9 12 11

19 8 5 4 3 0 0

3 2 2 3 2

7 6 4 2 0 0 0

3 4 2 2

249

B

A

B

1060 1040

1.40

1740 0.70

Absorbance

1100 1600

1414 1140

1740 i iii ii 0.50

D

1000 800 1400 Wavenumbers (cm–1) 1720 1740

C 0.36

D

250

1740 0.14

1720

0.02 1780

FTIR spectra of the CWM residue following imidazole extraction and the first and the second Na2CO3 extractions are shown in Fig. 5A (i, ii and iii, respectively). All three residues show a carboxylic ester peak at 1740 cm1, carboxylate ion peaks at 1600 and 1414 cm1 together with carbohydrate peaks at 1140, 1100, 1060 and 1040 cm1. The spectra did not have amide peaks (1650 and 1550 cm1), indicative of low amounts of proteins in these CWM residues. Expansion of the ester region of the spectra (Fig. 5B) shows that both the carboxylic and phenolic esters are greatly reduced in the CWM residue of the two Na2CO3 extractions. A digital subtraction spectrum shows that methyl esters (1740 cm1) and phenolic esters (1720 and 1700 cm1) are present in imidazole-extracted CWM but are missing after extraction with Na2CO3 at 4 C (Fig. 5C). Fig. 5D shows a digital subtraction spectrum indicating that some methyl esters are present in the CWM residue after the first Na2CO3 extraction but not after the second Na2CO3 extraction.

1760 1740 1720 1700 Wavenumbers (cm–1)

Absorbance

Absorbance 0.32

FTIR and NMR spectroscopies confirm that both methyl and phenolic esters are removed by Na2CO3 extraction

iii

1780

1700

Fig. 4. Autofluorescence images of sections of sugar-beet root parenchyma after chemical extractions. (A) The sections of unextracted material show increased fluorescence at cell corners and, in particular, regions of the primary cell wall and middle lamella. (B) Material extracted with 2 M imidazole has the same pattern and intensity of fluorescence as unextracted tissue. (C) Fluorescence was much reduced and restricted to some cell corners in tissue after extraction with 50 mM Na2CO3 at 4 C. (D) After a second Na2CO3 extraction at 20 C, there was no autofluorescence. Scale bars represent 100 mm.

i ii

0.50 1800

C

1720

Absorbance

A

1740 1700 Wavenumbers (cm–1)

1800

1760 1720 1680 Wavenumbers (cm–1)

Fig. 5. Fourier transform infrared spectra of sugar-beet cell wall material (CWM) after sequential chemical extraction. (A) Spectra of sugarbeet CWM after four extraction steps with 2 M imidazole (i), then Na2CO3 at 4 C (ii) and then a second Na2CO3 extraction at 20 C (iii). The spectra show an ester peak at 1740 cm1, carboxylate ion peaks at 1600 and 1414 cm1 together with carbohydrate peaks at 1140, 1100, 1060 and 1040 cm1. Amide peaks (1650 and 1550 cm1), indicative of the presence of proteins, are absent from the spectra. (B) An enlargement of the ester region shows that both unsaturated (1720 cm1) and saturated ester (1740 cm1) peaks are greatly reduced in the CWM residue of the two Na2CO3 extractions. (C) A digital subtraction spectrum (spectrum I spectrum ii) shows that methyl esters (1740 cm1) and phenolic esters (1720 cm1) are removed from the CWM during extraction with Na2CO3 at 4 C. (D) A digital subtraction spectrum (spectrum ii spectrum iii) shows that some methyl esters are present in the CWM residue of the first but not the second Na2CO3 extraction.

CP-MAS NMR experiments indicate that the wall residues following the first and the fourth imidazole extractions (Fig. 6, spectra A and B, respectively) and the two Na2CO3 extractions (Fig. 6, spectra C and D) contain galacturonans (69 p.p.m), C-4 of surface chains of cellulose (84 p.p.m), C-4 of interior cellulose chains (89 p.p.m) and arabinans (109 p.p.m). The proportion of methyl (54 p.p.m) and acetyl (21 p.p.m) esters are all reduced from the wall residues from the first imidazole extraction to the second Na2CO3 extraction. After the second Na2CO3 extraction, methyl esters are absent, but acetyl esters are still detectable. Signals from phenolic esters could not be clearly assigned. There was a marked reduction in feruloyl content in the wall residue of the second Na2CO3 extraction (Table 3), which is consistent with the highest feruloyl content being observed in the polymers removed from the cell wall at this step (Marry et al. 2000). Treatment of


Relative signal intensity

A

B

C

D

130 120 110 100 90

80

70 60 p.p.m.

50

40

30

20

10

0

Fig. 6. Cross-polarization magic-angle spinning nuclear magnetic resonance spectra of cell wall material (CWM) following the first (A) and the fourth imidazole extractions (B) and the first (C) and the second Na2CO3 extractions (D). All of the CWMs contain galacturonans (69 p.p.m), C-4 of surface chains of cellulose (84 p.p.m), C-4 of interior cellulose chains (90 p.p.m) and arabinans (109 p.p.m). The proportion of methyl (54 p.p.m) (grey arrow) and acetyl (21 p.p.m) (black arrow) esters are all reduced from the wall residues from the first imidazole extraction to the second Na2CO3 extraction. After the second Na2CO3 extraction, no methyl esters are detected but some acetyl esters are still present (D).

the wall residues with acid chlorite also significantly reduced ferulic acid content of the CWM (Table 3).

Discussion Adhesion between plant cells is a fundamental feature of plant growth and development and an essential part of the strategy by which growing plants achieve and maintain mechanical strength. Turgor pressure within cells induces tensile and compressive stresses in the

Table 3. Percentage feruloylation of untreated cell wall material (CWM), acid chlorite-treated CWM and CWM residues remaining after extraction with imidazole and then Na2CO3 at 4 and 20 C. In each case, the values for the % feruloylation are calculated from nmoles ferulic acid relative to the nmoles of arabinose and galactose in each extract. Values are the mean of duplicate assays from five separate sequential extractions. % feruloylation Unextracted CWM 20 C Chlorite-treated CWM 60 C Chlorite-treated CWM CWM after extraction with Fourth Imidazole First Na2CO3 Second Na2CO3


0.181 0.02 0.096 0.08 0.091 0.06 0.136 0.05 0.131 0.06 0.063 0.02

plane of the cell wall and cell separation stresses perpendicular to the plasma membrane (Jarvis and McCann 2000). Turgor pressure, by distending cells towards a circular cross-section, tends to separate cells from their neighbours at the tri-cellular junctions. If this happens, it introduces an intercellular space, reducing the cell separation stresses and redistributing them to the corners of the three-way junctions (Jarvis 1998). In many tissues of most dicots that have been studied, calcium ions and the linear, low-ester pectins to which they bind are concentrated at the tri-cellular junctions and the corners of the intercellular spaces exactly where the stress is greatest. In this article, we show that, in fresh sugar-beet parenchyma, calcium cross-linked pectins are not sufficient for cell adhesion, but act in combination with ester-linked pectins to hold cells together. Pectic polymers are released when cell-cell adhesion is disrupted There is increasing evidence that pectic polysaccharides contribute to the mechanical strength and physical properties of primary walls (Jarvis 1984, Jarvis and McCann 2000, MacDougal and Ring 2003, Ryden et al. 2003, Toole et al. 2002, Wilson et al. 2000) and that changes in the structures of these polysaccharides are associated with different developmental stages of plant cells and tissues (Willats et al. 2001b). The use of monoclonal antibodies to defined pectic epitopes has shown the tight developmental regulation of pectins in zones of cell-cell adhesion: JIM 5, PAM 1, LM 7 and LM 8 antibodies all have distinctive labelling patterns in cell corners (Willats et al. 1999, 2000, 2001a, 2001b). For pectic polysaccharides to be effective in intercellular adhesion, they must be cross-linked into a three-dimensional network. It could be a purely pectic network, insoluble due to cross-linking (Goldberg et al. 1996) or due to a topology in which microfibrils are entangled. In principle, cell-cell adhesion could be mediated either through the cellulose and associated cross-linking glycan network (Carpita and Gibeaut 1993, McCann and Roberts 1991) or through the pectic polysaccharide network or through both. Cross-linking glycans are hydrogen bonded to the cellulose microfibrils along glucan chain surfaces (Levy et al. 1991) and are long enough to span between microfibrils (McCann and Roberts 1991). It is possible that microfibrils in adjacent cells could be cross-linked together by xyloglucans and xylans and released by the activities of endo-transglycosylases (Rose et al. 1998) and/or expansins (McQueenMason and Rochange 1999). Alternatively, the network might include pectic chains covalently linked to other insoluble polysaccharides on both sides of the middle 251

lamella. When sugar-beet cells are separated by imidazole and Na2CO3, only pectic polysaccharides and small amounts of soluble proteins are removed from the wall. Entanglement of xyloglucans or cellulose microfibrils is therefore unlikely to contribute to cellcell adhesion within this tissue. Previously, we showed that complex but distinct mixtures of HGA, RG I, branched arabinans and branched galactans are removed by extraction with imidazole and Na2CO3 (Marry et al. 2000). Linkage analysis of CWM residues (data not shown) is consistent with this, showing that the relative mole percent of rhamnose (t-, 2- and 2,4-linked) decreases from 6% in unextracted CWM to 1% after the second Na2CO3 extraction step, and similarly, 5-linked arabinan decreases from 16 to 6%. The two Na2CO3 steps represent over half of the 41% of CWM extracted during the sequential extraction (Table 1). Methyl, acetyl and feruloyl esters were detected in the Na2CO3-extractable polymer fractions (Marry et al. 2000). The second Na2CO3-extractable polymer fraction contained the highest proportion of feruloyl esters (Marry et al. 2000), consistent with the residual CWM having half of its feruloyl content after this extraction step (Table 3). Immunogold labelling using the JIM 5 antibody showed that imidazole removed relatively unesterified pectic antigens from throughout the cell wall but not from the middle lamella. These antigens were subsequently extracted by Na2CO3 treatment, concomitant with cell separation. The JIM 5 antibody binds weakly to completely de-esterified pectin, and binding is greatly increased by the presence of methyl-esterified GalA residues up to a level of about 40%, whilst the JIM 7 antibody binds over a range from about 15 to 80% methyl-esterification (Willats et al. 2000). Clausen et al. (2003) have provided evidence that JIM 5 binds to unesterified GalA residues with adjacent or flanking methyl-esterified residues, whilst JIM 7 binds to methylesterified residues with adjacent or flanking unesterified GalA residues. These data are consistent with a model of cell wall architecture in which the middle lamella is a domain of distinct composition (McCann and Roberts 1991). The colourless non-ripening tomato mutant has weaker cell-cell contacts in the pericarp (Thompson et al. 1999) and has absent or a low level of HGA/ calcium-based cell adhesion as indicated by epitope patterns. Interestingly, deposition of 5-linked arabinans is also disrupted (Orfila et al. 2001). In the tobacco nolac-H14 (non-organogenic callus with loosely attached constituent cells) culture cell line, pectic polysaccharides are secreted to the culture medium and not retained either in cell walls or middle 252

lamellae. Arabinosylated pectins found in a strong alkali-extractable fraction in a control tobacco culture line are absent in the nolac-H14 line (Iwai et al. 2001). However, there is no clear correlation between loss of a particular linkage of arabinose (data not shown), or amount of arabinose removed at each extraction step, and the failure of cell-cell adhesion in sugar-beet walls. Most of the 33 mole percent of arabinose in unextracted walls is extracted by chelator and de-esterifying agents, leaving 6 mole percent in KOH-extracted walls. A hierarchy of cross-links exist within the cell walls of sugar-beet parenchyma A polymer network can be disrupted by either breaking the cross-links between the chains or cleaving the chains themselves between cross-links. Immunogold labelling with the JIM 5 antibody shows that a subset of low-ester pectic epitopes remain within the middle lamella of the sugar-beet cell wall residue after four sequential extractions with imidazole. The EELS spectra of Ca2þ-doped sections clearly show that calcium-binding sites are present in unextracted cell walls but reduced in abundance after imidazole extraction and that there are relatively more calcium-binding sites within the middle lamella. We detected only low amounts of nitrogen throughout the unextracted walls and middle lamella, which did not alter during the extractions (data not shown). Furthermore, the FTIR spectra of the CWM residues (Fig. 5) did not have absorbances (1650 and 1550 cm1) characteristic of amide bonds of proteins. Thus, there is a subset of relatively unesterified pectin molecules that are held within the middle lamella by a mechanism other than calcium bridges or protein interactions. Using our cell separation assay, we found that CWM remaining after calcium chelation is resistant to mechanical disruption. Also, a subsequent treatment with acid chlorite did not induce further cell separation. This indicates that water-soluble molecules, ionic bonds, calcium bridges between HGA chains and acid-labile bonds are not sufficient for cell-cell adhesion. The separation of cells after extraction with Na2CO3 suggests that sugar-beet cell adhesion may be mediated by b-elimination of methyl-esterified HGA (Fry 1988, Waldron et al. 1997b), cleavage of ester cross-links, such as o-Dgalacturonoyl or diferulate bridges or both. As neither imidazole nor Na2CO3 treatment alone is sufficient to promote cell separation, calcium cross-links and ester bonds are both required for cell-cell adhesion in sugar-beet root parenchyma. Physiol. Plant. 126, 2006

Different populations of esters exist in sugar-beet walls FTIR spectroscopy showed that both methyl esters and phenolic esters are removed during Na2CO3 extraction of imidazole-extracted sugar-beet CWM. CP-MAS NMR spectra show that Na2CO3 extraction removes methyl esters but not all acetyl esters. Thus, both methyl and phenolic esters may be involved in cell-cell adhesion but acetyl esters are unlikely to contribute. In vitro analysis of calcium-mediated model pectin gels indicated that the compressive strength, elasticity, water-holding capacity and porosity of the gels are significantly influenced by the pattern as well as the degree of methyl esterification of HGA domains (Willats et al. 2001a). Methyl-esterified HGA may function in cell-cell adhesion by forming strong gels through biophysical interactions (Powell et al. 1982). However, immunogold-labelling with the JIM 7 antibody shows that the remaining methyl-esterified HGA antigens in the CWM after extraction with imidazole are distributed throughout the wall and are of low abundance. Both the methyl ester and the acetyl ester content was significantly reduced after the first two imidazole extractions, with the second Na2CO3-extracted wall residue samples containing no esters (Table 2). Cell separation induced by Na2CO3 is also correlated with removal of autofluorescence from the cell walls, a decrease in phenolic esters measured by FTIR spectroscopy and a significant decrease in the degree of feruloylation, methyl esterification and acetyl esterification of Na2CO3-extracted CWM. Diferulic acid cell-wall crosslinks have been implicated in the thermal stability of Chinese water chestnuts, maintaining cell adhesion during cooking (Waldron et al. 1997a). In sugar-beet, over 20% of ferulic acid is in dimer form (Waldron et al. 1997b). Ester-linked ferulate and diferulate can be hydrolysed by cold dilute alkali, but the lability of the feruloyl ester bond (Fry 1982) is much less than that of o-D-galacturonoyl esters (Brown and Fry 1993b) or of acetyl esters (Fry, personal communication). The linkages between feruloyl esters and neutral sugars associated with sugar-beet pectin have been characterized by GLC, HPLC and NMR (Colquhoun et al. 1994, Ishii 1994, Ishii and Tabita 1993), and observations by Guillon and Thibault (1990) suggest that about half of the feruloyl ester associated with sugarbeet pectin is bound to arabinose with the other half bound to galactan. Two feruloylated arabinoses, [(FAra)-(1–5)-Ara and Ara-(1–3)-(F-Ara)-(1–5)-Ara] have been isolated from sugar-beet pulp (Ishii 1997). The presence of a feruloylated galactan [(F-Gal)-(1–4)-] epitope in sugar-beet has recently been shown by the monoclonal antibody LM9 (Clausen et al. 2004). However, the linkages involving the arabinofuranosyl and


galactofuranosyl residues are acid-labile, which may account for the 50% reduction in feruloylation after extraction of CWM with acidified chlorite. As this treatment, following calcium chelation, does not effect cell separation, it seems unlikely that feruloylated arabinans and galactans are involved in cross-linking. Further, using solid state NMR, Renard and Jarvis (1999) have shown that the arabinans in sugar-beet are highly mobile. The borate di-diester cross-links of RG II dimers are labile only in acid (Ishii et al. 1999) and therefore unlikely to be broken by alkaline extraction. However, we cannot rule out the possibility that intact dimers were extracted by chelator or alkali treatments. The rare monosaccharides diagnostic for RG II were not detected in our sugar analyses. Other kinds of alkali-susceptible ester cross-linking may exist within walls but are as yet uncharacterized (Kim and Carpita 1992, McCann et al. 1994). We propose that the imidazole-unextractable pectin left in the middle lamella may be cross-linked to other components of the wall matrix by alkali-labile esters, such as o-D-galacturonoyl esters. In summary, we have used a combination of spectroscopic, microscopic and chemical analyses to investigate the nature of the cross-links involved in cell-cell adhesion in sugar-beet parenchyma tissue. Adhesion is regulated by pectic polysaccharides, while cellulose and cross-linking glycans do not seem to be involved. We have shown that Ca2þ or ester cross-linked pectins by themselves are not sufficient for cell adhesion but are both required to hold sugar-beet cells together. This evidence is consistent with the idea that intercellular adhesion depends on the crosslinking of polysaccharides into networks by a combinatorial hierarchy of cross-linking mechanisms. Acknowledgements – The authors gratefully acknowledge the assistance of Jan Peart and Anna Cullingford for production of JIM 5 and JIM 7 antibodies, Sue Bunnewell for photographic assistance, Stephen Fry, Max Bush, Grant Calder, Iain MacKinnon and Jordi Chan for useful discussions and Alan White and Nicholas Carpita for assistance with GC-MS and useful discussions. Financial support is gratefully acknowledged from Hercules Incorporated, USA (Mazz Marry), the Royal Society for a University Research Fellowship (Maureen McCann), the BBSRC (Mazz Marry, Maureen McCann, Mike Jarvis, Max Huxham, Julia Corsar, Eoin Robertson and Keith Roberts) and US Department of Energy DE-FG02-03ER15445 (Maureen McCann).

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