Molecular Plant Advance Access originally published online on July 15, 2009
Molecular Plant 2009 2(5):910-921; doi:10.1093/mp/ssp049
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Cell Wall Microstructure Analysis Implicates Hemicellulose Polysaccharides in Cell Adhesion in Tomato Fruit Pericarp Parenchyma
Centre for Plant Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom
1 To whom correspondence should be addressed. E-mail j.p.knox{at}leeds.ac.uk, fax +44-113-3433144, tel. +44-113-3433169.
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Abstract |
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Methods developed to isolate intact cells from both unripe and ripe tomato fruit pericarp parenchyma have allowed the cell biological analysis of polysaccharide epitopes at the surface of separated cells. The LM7 pectic homogalacturonan epitope is a marker of the junctions of adhesion planes and intercellular spaces in parenchyma systems. The LM7 epitope persistently marked the former edge of adhesion planes at the surface of cells separated from unripe and ripened tomato fruit and also from fruits with the Cnr mutation. The LM11 xylan epitope was associated, in sections, with cell walls lining intercellular space but the epitope was not detected at the surface of isolated cells, being lost during cell isolation. The LM15 xyloglucan epitope was present at the surface of cells isolated from unripe fruit in a pattern reflecting the former edge of cell adhesion planes/intercellular space but with gaps and apparent breaks. An equivalent pattern of LM15 epitope occurrence was revealed at the surface of cells isolated by pectate lyase action but was not present in cells isolated from ripe fruit or from Cnr fruit. In contrast to wild-type cells, the LM5 galactan and LM21 mannan epitopes occurred predominantly in positions reflecting intercellular space in Cnr, suggesting a concerted alteration in cell wall microstructure in response to this mutation. Galactanase and mannanase, along with pectic homogalacturonan-degrading enzymes, were capable of releasing cells from unripe fruit parenchyma. These observations indicate that hemicellulose polymers are present in architectural contexts reflecting cell adhesion and that several cell wall polysaccharide classes are likely to contribute to cell adhesion/cell separation in tomato fruit pericarp parenchyma.
Key Words: Cell walls • fruit development • tomato • cell adhesion • cell separation • polysaccharide
Received for publication April 21, 2009. Accepted for publication June 20, 2009.
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INTRODUCTION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
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Plant cell walls are sets of highly complex composites of structurally diverse polysaccharides that contribute to a range of cell processes. Cell walls both constrain and regulate cell expansion whilst maintaining adhesion between cells, thus allowing organs with robust mechanical properties to grow and develop. The polysaccharide components of the cell walls of all land plants fall broadly into three classes (Popper, 2008; O'Neill and York, 2003; Liepman et al., 2007). Cellulose molecules aggregated into microfibrils provide the tough, loading-bearing fibers of cell walls. Hemicellulose polysaccharides, also referred to as cross-linking glycans, are known to have the capacity to hydrogen bond to the surface of microfibrils and are proposed to tether the microfibrils, providing a load-bearing molecular framework for both primary and secondary cell walls (Cosgrove, 2005). Hemicellulose polysaccharides are structurally diverse and include xyloglucans, xylans, and mannans and each group can be structurally modulated by varied substitution of the backbone (O'Neill and York, 2003; Liepman et al., 2007). The third major class of polysaccharides are the pectins. This class is composed of diverse acidic polysaccharides of varying potential for structural modulation and, together, these polymers may be integrated into large interacting networks that modulate the properties of the apoplast and cell wall matrices (Willats et al., 2001b; Mohnen, 2008). Pectic homogalacturonan (HG, 1,4-galacturonan) can be variously methyl-esterified to potentiate cross-linking by calcium ions and other cations. HG is attached to rhamnogalacturonan-I (RG-I) polymers in which alternating rhamnosyl and galacturonosyl residues present a backbone in which rhamnosyl residues are substituted with neutral side chains with 1,4-galactan and 1,5-arabinan as abundant features (Mohnen, 2008). Other polymer domains include rhamnogalacturonan-II that can cross-link HG chains by boron-mediated dimers and xylogalacturonan domains comprising HG substitution with xylosyl residues. Such modifications of HG backbones will influence the capacity of HG-processing enzymes such as pectin methylesterases, polygalacturonases, and pectate lyases to act (Mohnen, 2008; Pelloux et al., 2007; Willats et al., 2001a; González-Carranza et al., 2007).
Hemicellulosic and pectic polysaccharides are two groups of cell wall polymers with varied sets of structural features and modifications that are often of uncertain or unknown function. The current broad view is that these polysaccharides are integrated into cell walls, where they contribute to sets of flexible materials that impart the wide multi-functionality to plant cell walls. Cell or organ homogenization is required for the isolation and structural analyses of cell wall polysaccharides with the loss of all developmental and cell-related information. Molecular probes such as monoclonal antibodies and carbohydrate-binding modules are used to place polysaccharide structures in context and to gain insight into the architecture of individual cell walls and the developmental regulation of cell wall structures (Willats and Knox, 2003; Knox, 2008). Several studies using monoclonal antibodies have indicated that cell wall microstructure varies at the level of individual cell walls. For example, the pectic epitopes have been detected in patterns relating to pit fields on the inner face of tomato parenchyma cell walls (Casero and Knox, 1995; Orfila and Knox, 2000). The LM7 pectic homogalacturonan epitope has been specifically detected at the junction of adhered and unadhered cell walls at the corners of intercellular space in a variety of systems (Willats et al., 2001a). Such studies indicate that cell walls are highly spatially organized and spatially varied structures, with a range of polymer configurations within individual cell walls. Several features of cell wall microstructures relate to the contacts between cells in plant organs, although the molecular mechanisms of the maintenance of cell adhesion and its control are not well understood. There is some indication that pectic HG is involved in maintaining cell junctions, but how HG is tethered across middle lamellae to primary cell walls and to plasma membranes is unknown (Jarvis et al., 2003; Waldron and Brett, 2007; González-Carranza et al., 2007). In addition to pectic HG, pectic arabinans have been implicated in maintaining cell adhesion in cultured cell clusters and in organs (Iwai et al., 2001; Orfila et al., 2001; Leboeuf et al., 2004; Peña and Carpita, 2004; Devaux et al., 2005). The presence of a xylogalacturonan-related structural feature has been correlated with cell detachment (Willats et al., 2004).
The development and ripening of the tomato fruit pericarp are useful systems for the study of intercellular contacts. The pericarp is predominantly a parenchyma system with large cells. Moreover, cell wall changes during tomato fruit ripening have been studied extensively (Giovannoni, 2007; Seymour et al., 2008). The Cnr (Colourless non-ripening) tomato is a pleiotropic dominant mutation that results in mature fruits displaying much reduced cell-to-cell adhesion (Thompson et al., 1999; Orfila et al., 2001; Eriksson et al., 2004). The Cnr mutation has been identified as an epigenetic mutation in a gene encoding an SBP-box transcription factor (Manning et al., 2006). In comparison with wild-type, ripe-stage Cnr fruits have stronger, non-swollen cell walls throughout the pericarp and extensive intercellular space in the inner pericarp and a low level of HG calcium-based cell adhesion and disrupted deposition of (1
5)-
-L-arabinan (Orfila et al., 2001). The expression and activity of a range of cell wall-modifying enzymes (including polygalacturonase, pectinmethylesterase, galactanase, and xyloglucan endotransglycosylase) are altered in Cnr during both development and ripening (Eriksson et al., 2004).
We have developed strategies to isolate intact parenchyma cells from tomato fruit pericarp for the analysis of cell wall microstructures and the occurrence of cell wall polysaccharide epitopes in outer cell wall regions that contribute to intercellular adhesion planes or the lining of intercellular space. In addition to the LM7 pectic HG epitope, we have observed distinct patterns and restricted occurrences of hemicellulosic epitopes (xylan, xyloglucan) at cell junctions. Enzyme intervention experiments suggest that polymers other than HG (galactan, mannan) have roles in the maintenance of cell adhesion. A study of these polymers in Cnr fruit cell walls indicated that the epimutation results in a large-scale concerted change to polysaccharide epitope patterning in outer cell wall/intercellular regions.
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RESULTS |
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Chemical/Mechanical Separation of Parenchyma Cells of the Tomato Fruit Pericarp
To explore the spatial distribution of polysaccharides within individual cell walls and to determine any heterogeneity in cell wall microstructures in relation to cell adhesion and intercellular spaces, we developed methods to separate intact cells from the parenchyma tissue of tomato fruit pericarp. The outer surfaces of separated cells could then be used to study the formerly adhered and unadhered regions of the cell walls. Two relevant stages in relation to cell wall changes associated with tomato fruit development and ripening are green mature at 40 d post-anthesis (dpa), which is before any ripening-related stages, and red ripe at 60 dpa.
Excised pieces of 40-dpa tomato fruit pericarp parenchyma tissue were subject to sequential chemical incubations known to selectively and sequentially solubilize sets of cell wall polysaccharides (Redgwell and Selvendran, 1986). These solvents are the cation-chelator CDTA (that will broadly release cation-cross-linked pectic polymers) and increasing strengths of alkali that would release hemicelluloses. Such sequential extractions are aimed at leaving a residue of cellulose (Redgwell and Selvendran, 1986). However, these steps do not solubilize polymer classes entirely and several pectic and other polysaccharide epitopes may be found in all fractions and residues (Orfila et al., 2002). Such sequential solubilization procedures can provide information on the complex populations of polysaccharides that underpin cell wall architectures and the links and interactions between polysaccharides. During a 10-step sequence of incubations of 40-dpa pieces of pericarp parenchyma with CDTA and a graded series of alkalis, all with gentle agitation, it was found that only a few cells became separated at each stage, but 
40% of parenchyma cells were released, in combination with agitation, with the final incubation with 4 M KOH. At the red ripe stage (60 dpa), 90 % of parenchyma cells were separated for analysis of cell surfaces by gentle agitation in water alone.
The Cnr mutation of tomato is a pleiotropic mutation that results in a suite of molecular and mechanical changes to pericarp parenchyma cell walls, including reduced cell-to-cell adhesion (Thompson et al., 1999; Orfila et al., 2001, 2002; Manning et al., 2006). In the case of Cnr fruit pericarp parenchyma tissue, 75% cells were effectively separated by the 10-step extraction sequence at the green mature 40-dpa stage. Pericarp parenchyma cells were loosely attached at the red ripe 60-dpa stage and 90 % of parenchyma cells were readily separated by incubation in water.
Separated pericarp parenchyma cells maintained their shapes and cell wall robustness and were suitable for the direct analysis of cell wall microstructures using immunofluorescence procedures. The accessible outer regions of separated cells comprised cell walls that had contributed to former adhesion planes between adjacent cells and also to cell wall regions lining former intercellular spaces.
The LM7 Pectic HG Epitope, Specifically Associated with Edges of Adhesion Planes/Linings of Intercellular Space, Persists during Alkali-Induced Separation of Green 40-dpa Fruit Parenchyma Cells, during Ripening and Is Present in Cnr Pericarp Parenchyma Cell Walls
The LM7 pectic HG epitope (Willats et al., 2001a) is the only cell wall polysaccharide epitope to date that has been detected universally at the junction of adhered and unadhered cells in parenchyma systems (i.e. at corners of intercellular spaces of all parenchyma systems of all species examined). This epitope has previously been reported to be sensitive to pectate lyase treatment and to bind to a partially methyl-esterified epitope of pectic HG requiring some methyl esters for recognition (Willats et al., 2001a; Clausen et al., 2003). We were surprised to observe that the LM7 HG epitope was readily and consistently detected at the surfaces of the 10-step chemically separated 40-dpa wild-type pericarp parenchyma cells. The LM7 epitope was found to be present and to mark the edge of former adhesion planes as shown in Figure 1. The LM7 epitope was always present in continuous lines fully encircling the edges of former adhesion planes as shown in Figure 1. Although these persisted through alkali treatments (indicating that the methyl esters required for recognition were stabilized to high pH), the binding of the LM7 antibody was sensitive to the action of pectate lyase (Figure 1). The use of the Calcofluor fluorochrome to label these cells indicated the presence of cellulose and no variations in fluorescence relating to adhesion planes could be readily seen in any case with Calcofluor.
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A similar pattern of occurrence of the LM7 epitope was observed at the surface of cells separated from 60-dpa parenchyma tissue (Figure 1) and also at the surface of Cnr parenchyma cells as shown for a 60-dpa Cnr cell in Figure 1. These observations indicate that the positioning of the LM7 HG epitope at the edge of adhesion planes is not modulated by changes associated with ripening or with the Cnr mutation and their known impacts on cell wall properties. This suggests that the LM7 HG epitope is not directly involved in maintaining cell-to-cell adhesion in the tomato fruit pericarp parenchyma system.
LM5 Galactan and LM6 Arabinan Epitopes Are Weakly Absent from Cell Walls in Linings of Intercellular Space at 60-dpa Parenchyma Cells and Show Altered Patterns in Cnr Fruit Parenchyma Cell Walls
The monoclonal antibodies LM5 and LM6 can be used to detect 1,4-galactan and 1,5-arabinan motifs of the pectic polymer rhamnogalacturonan-I. Previous work using sections of tomato pericarp parenchyma has indicated that these epitopes are distributed throughout cell walls but that the LM6 epitope is disrupted in Cnr cell walls (Jones et al., 1997; Orfila et al., 2001). Using these probes for the analysis of epitopes at the surface of separated cells, it was observed that both epitopes were abundant at the surface of 40 and 60-dpa separated cells (Figure 2). In the case of 60-dpa cells, both epitopes were notably less abundant in occurrence in regions lining intercellular spaces (Figure 2) and thus distinct to the occurrence of the LM7 HG epitope. At the surface of 60-dpa Cnr separated cells, the LM6 epitope was present only weakly, but appeared to be more abundant in regions formerly lining intercellular spaces. This altered pattern of occurrence of the surface distribution of epitope was more apparent for LM5, where the epitope was most abundant in regions that appeared to reflect the edge of adhesion planes/intercellular space. These observations indicated that a cell wall restructuring involving RG-I-related epitopes had taken place in the outer regions of cell walls of pericarp parenchyma cells as a consequence of the Cnr mutation.
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Patterns of Hemicellulose Epitopes in Cell Walls in Relation to Former Cell Adhesion Planes and Cell Walls Lining Intercellular Spaces
Equivalent analyses of hemicellulose polysaccharide epitopes indicated contrasting occurrences in relation to cell wall architectures. Immunodetection of xylan epitopes in sections of 40-dpa pericarp tissue indicated weak recognition of cell walls lining intercellular spaces and some intercellular regions at middle lamellae as shown in Figure 3 for the LM11 xylan monoclonal antibody. However, immunodetection of xylan epitopes at the surface of 40-dpa alkali-separated cells did not reveal any such pattern, the epitope having been solubilized by the procedures. At the surface of water-separated 60-dpa cells, xylan probes bound weakly in a punctuate manner but rarely related to adhesion plane/intercellular space features (Figure 3). In contrast, the LM15 xyloglucan epitope was revealed to be abundant at the lining of adhesion planes in the 10-step, chemically separated 40-dpa parenchyma cells as shown in Figure 3. The LM15 epitope was also detected at discrete regions of the adhesion planes themselves. Notably, the occurrence of the LM15 xyloglucan epitope at the lining of former adhesion planes/intercellular spaces was uneven, with varying fluorescence intensities and gaps. At the surface of 60-dpa separated cells, the LM15 epitope was not detected. In some cell walls, the presence of the LM15 xyloglucan epitope can be masked by HG and revealed by HG-degrading enzymes (Marcus et al., 2008). Pectate lyase treatment of 60-dpa separated cells revealed the LM15 xyloglucan epitope was present but distributed evenly across the cell surface (not shown). A rat monoclonal antibody directed to a mannohexaose oligosaccharide was developed during this project and designated LM21. The LM21 mannan epitope was detected relatively evenly at the surface of 40-dpa cells and in a punctuate manner at the surface of 60-dpa cells. In neither of these cases was the pattern of LM21 mannan epitope fluorescence associated with cell wall features such as former adhesion planes or intercellular space.
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At the surface of isolated Cnr cells, it was observed that the LM11 epitope was only present weakly and in association with former adhesion planes/linings of intercellular space in 60 dpa (Figure 3). The LM15 epitope was not associated with spaces at 40 dpa and only very weakly at 60 dpa. In contrast to the LM11 xylan and the LM15 xyloglucan epitopes and in contrast to wild-type cell walls, the LM21 mannan epitope at the surface of Cnr cells at both 40 and 60 dpa was clearly present in a pattern that reflected adhesion planes/intercellular space (Figure 3).
In summary, xylan epitopes associated with linings of intercellular space are not detected at the surface of separated cells. The LM15 epitope association with the edge of adhesion planes is lost both during ripening and as a result of the Cnr mutation. Moreover, the cell walls of Cnr pericarp appears to undergo a change in microstructures in which edge of adhesion plane/intercellular space-associated epitopes involve a loss of the LM15 xyloglucan and an occurrence of the LM21 mannan epitope. The occurrence of the LM21 mannan epitope in relation to microstructure of Cnr cells therefore reflected the occurrence of the LM5 galactan epitope.
These observations indicate that a suite of changes to cell walls are associated with the ripening process—loss of patterned LM15 xyloglucan epitope and increased patterning of LM5 galactan and LM21 mannan epitopes in Cnr. The patterning of the LM7 HG epitope persisted during ripening and in Cnr.
Pectic Homogalacturonan, Pectic Galactan, and Mannan Are Cell Wall Polysaccharides that Can Be Targeted for Enzyme-Induced Cell Separation
To explore the roles and contributions of individual cell wall polysaccharides that may act in maintaining cell adhesion in parenchyma systems before ripening-related changes, a range of enzymes targeting specific polysaccharide domains of pectic and hemicellulose polymers were applied in excess to excised pieces of 40-dpa tomato pericarp parenchyma. These enzymes were pectate lyase, endo-polygalacturonase, 1,4-galactanase, 1,5-arabinanase, xylanase, xyloglucanase, and mannanase. The results, shown in Figure 4, indicate that the pectate lyase, the polygalacturonase, the galactanase, and the mannanase were effective in releasing significant numbers of intact cells from parenchyma tissues from 40-dpa wild-type parenchyma cells, whereas the arabinanase, xylanase, and xyloglucanase had no impact.
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The LM15 Xyloglucan Epitope Association with Cell Walls Lining Intercellular Space Is Revealed by an HG-Degrading Enzyme but Not by a Mannan- or a Galactan-Degrading Enzyme
Immunodetection of cell wall polysaccharides at the surface of cells isolated by enzyme action indicated that the LM15 epitope was revealed by PL action to occur in a manner similar to that revealed by chemical/mechanical separation, namely to be lining cell walls at intercellular space (Figure 5). Again, the lines of LM15 epitope fluorescence surrounding former adhesion planes were not continuous, but often present in gaps and, in some instances, LM15-associated fluorescence was observed to be distinct from and loosely attached to the cell wall (Figure 5).
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In contrast, cell separation induced by mannanase and galactanase action did not result in equivalent appearances of the LM15 epitope. At the surface of mannanase-released cells, the LM15 epitope was more evenly distributed across the cell wall surface, although there was some indication of a slightly more abundant association with possible linings of intercellular space (Figure 5). At the surface of galactanase-released cells, the LM15 was evenly distributed. Further treatment of mannanase- and galactanse-separated cells with pectate lyase did not change these patterns of LM15 epitope detection (not shown).
The LM7 Pectic HG Epitope Is Disrupted by Enzyme-Induced Cell Separation of Tomato Pericarp Parenchyma
The patterned occurrence of the LM7 HG epitope was disrupted by both the mannanase and galactanase treatments to release cells from pericarp parenchyma tissue. In both cases, the LM7 HG epitope was only detected in punctuated regions that appeared to be evenly distributed across cell surfaces. The LM21 mannan epitope was revealed at the surface of PL-released cells but only very weakly at the surface of galactanase-released cell walls. The LM5 galactan epitope was evenly distributed across cell surfaces in pectate lyase- and mannanase-released cells and the LM21 mannan epitope was only weakly detected at the surface of pectate-lyase and galactanase-released cells (not shown).
Release of Polysaccharide Epitopes from Tomato Pericarp Cell Walls by Sequential Extractions
In a separate series of experiments, a preparation (alcohol-insoluble residue) of 40-dpa green mature tomato pericarp parenchyma cell walls was subject to sequential extraction with water, CDTA, sodium carbonate, and KOH. The relative occurrence of selected polysaccharide epitopes in the pooled fractions for each solvent was determined by ELISA. The results shown in Figure 6 indicate that the LM7 HG epitope was released in equal amounts in all four pools in contrast to the JIM5 and JIM7 HG epitopes that were most abundant in the water-released fraction. The LM5 and LM6 RG-I-related epitopes had similar patterns of occurrence and were abundant in all four fractions. In the case of the hemicellulose epitopes, the LM11 xylan epitope was mostly present in the KOH fraction. In contrast, the LM15 xyloglucan and LM21 mannan epitopes had a similar pattern of release from the cell walls with presence in all four fractions and relatively abundant occurrence of these epitopes from the water fraction in addition to the KOH-released fraction (Figure 6). The novel pattern of cell surface recognition by the LM15 epitope and the relative abundance of the epitope in the water extract led us to confirm that LM15 was binding to xyloglucan in these materials. The sensitivity of the water extract to pre-treatment with xyloglucanase is shown in Figure 6C and indicates a water-soluble population of xyloglucan in the tomato fruit.
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DISCUSSION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
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The observations presented here indicate that cell wall microstructure is more complex and varied within cell walls than previously envisaged. The use of probes for xylan and xyloglucan indicates that epitopes of these polymers can be detected, at outer and intercellular regions of cell walls, in patterns that reflect cell junctions. Moreover, the use of the Cnr mutant has indicated that the occurrence or positioning of different polymers within cell walls may be a highly orchestrated process, with the potential for sets of polymers to be coordinately deposited.
Cell Wall Polysaccharide Epitopes Associated with the Edge of Adhesion Planes and Vertices of Intercellular Space
We have shown that cells, isolated from a parenchyma system by the sequential selective release of sets of cell wall polysaccharides, can be studied to reveal patterns of cell wall polysaccharide epitopes relating to former middle lamellae and former linings of intercellular spaces. The observation that the LM7 pectic HG epitope is present in continuous lines at the junction of former adhesion planes and corners of intercellular space confirms the observations made on this epitope in sections of parenchyma systems (Willats et al., 2001a). The observation that the occurrence of the LM7 HG epitope persists both during fruit ripening and in the Cnr mutant suggests that this epitope is not directly involved in maintaining links, but perhaps functions in maintaining a cell wall microdomain or localized cell wall environment allowing the regulation and modulation of other factors that function more directly in the maintenance of cell adhesion and its loss. The junction between adhered and unadhered cell walls is the load-bearing point stabilizing parenchyma systems (Jarvis, 1998; Jarvis et al., 2003; Willats et al., 2001a; Waldron and Brett, 2007).
Hemicellulose polysaccharides have not been widely implicated in maintaining cell adhesion or cell separation processes. The LM11 xylan epitope is shown to occur specifically in cell wall regions lining intercellular space and some middle lamellae regions of tomato fruit parenchyma. The presence of the xylan epitope at this location did not survive cell separation for analysis of intact cells. The solubilization of the xylan epitope, most abundantly into the KOH-extracted pool of polymers (Figure 6), could therefore be a factor in cell release in this system. In contrast, the LM15 xyloglucan epitope was detected at the surface of 40-dpa parenchyma cells and at the edge of adhesion planes in a pattern equivalent to that observed for the LM7 HG epitope. However, there are two clear distinctions between the occurrences of these epitopes. First, the LM15 xyloglucan epitope was not present in continuous lines, but with gaps and apparent breaks. Second, this pattern of the LM15 epitope did not persist during ripening nor was it present in plants with the Cnr mutation. A similar disrupted, non-continuous pattern of the LM15 xyloglucan epitope at the edge of former adhesion planes was also seen in cells separated by pectate lyase action. These observations are suggestive for a role of xyloglucan in maintaining cell-to-cell links within the tomato fruit pericarp. It is of interest in this context that xyloglucan-modifying enzymes are expressed throughout tomato fruit growth and ripening (Saladié et al., 2006; Miedes and Lorences, 2009) and that xyloglucan can be covalently attached to pectic polysaccharides (Femenia et al., 1999; Popper and Fry, 2008). Therefore, it is possible that pectic polymers functioning in cell adhesion are tethered into cell wall structures by links through xyloglucan located in cell wall regions that are important for maintaining cell adhesion.
Previous work has indicated the specific occurrence of the LM15 xyloglucan epitope at the corners of intercellular space of pith parenchyma of tobacco stem sections when uncovered by a pectate lyase pre-treatment (Marcus et al., 2008). Xylan epitopes have also been detected at cell corners after pectate lyase treatment of tobacco stem sections—in this case, in the thickened primary cell walls of tightly adhered collenchyma cells with no intercellular space (Hervé et al., 2009). The extent to which these two classes of hemicellulose polymers occur in restricted regions of primary cell walls and how this relates to cell adhesion events in other organs and species remains to be determined. The failure of a xylanase or a xyloglucanase to effect cell release from tomato fruit pericarp slices—when a galactanase or a mannanase could—may reflect restricted access of exogenous enzymes to key intercellular regions of cell walls due to variations in cell wall porosity or specific in situ structural features of these polymers that resist hydrolysis.
Does the Cnr Mutation Result in a Large-Scale Reorganization of Cell Wall Microstructure?
A striking feature observed at the surface of Cnr cells is that the LM5 galactan and LM21 mannan epitopes were present in regions reflecting former intercellular space/edge of adhesion planes—whereas these epitopes were not found at these locations in the wild-type cells. In fact, these epitopes displayed relatively reduced occurrence in these regions of 60-dpa cells (Figure 2). Conversely, the LM15 xyloglucan epitope was lost from these regions in Cnr. It is known that the Cnr mutation is an epigenetic change resulting in modulation of a SBP-box transcription factor (Manning et al., 2006) with wide consequences for cell walls including significant changes to cell wall modifying enzymes (Eriksson et al., 2004). How the large numbers of enzymes that are known to impact on cell wall polysaccharides are orchestrated during cell development is not clear. It can be presumed that they contribute to restructuring to establish and maintain cell wall microdomains responsive to functional needs. The Cnr epimutation results in a profound change in cell wall microstructure that includes changes to cell adhesion patterning in the fruit parenchyma systems before any ripening-related changes as evidenced by analysis of 40-dpa cells. These observations therefore begin to provide insight into the mechanisms that may control cell wall microstructuring during cell development. The observations can lead to a hypothesis that modulation of groups of polymers such as, for example, pectic galactan and mannan are collectively modulated in terms of positioning within intercellular regions.
The observation that incubation of pericarp slices with a galactanase or a mannanase resulted in the release of cells from 40-dpa parenchyma in addition to pectic HG-degrading enzymes is also novel. However, these two polymers (that were together changed in response to Cnr) may also indicate a connected involvement in some aspect of cell wall microstructure that is linked into pectic HG occurrence at the middle lamellae. Galactanase and mannanase enzymes are well studied features of developing tomato fruit with the capacity to change wall properties (Smith and Gross, 2000; Smith et al., 2002; Schröder et al., 2004, 2006; Ishimaru et al., 2009) and it is of interest that the fruit mannanase has been identified to have transglycosylase activity (Schröder et al., 2006). These observations again highlight our lack of understanding of the fine details of how the diverse sets of polysaccharides found in cell walls are linked together and how they are spatially positioned. How are microdomains such as that identified by the LM7 pectic HG epitope orchestrated? The loss of the distinctive patterns of both the LM7 pectic HG and LM15 xyloglucan epitopes from cells released from the pericarp tissue by both the galactanase or mannanase action may suggest that the polymers targeted by these enzymes are involved in generating or maintaining the distinctive pattern of these epitopes seen in untreated cells. This may indicate that groups of polymers function together in, as yet, unknown ways in coordination of spatial aspects of cell wall architectures.
In summary, we have developed new methodologies for the examination of cell wall microstructures in relation to outer cell wall regions and cell walls that have formed the interface with neighboring cells. We present evidence that xylan and xyloglucan are restricted to cell wall regions associated with the edge of cell adhesion planes reflecting possible roles in maintenance of parenchyma anatomy. More widely distributed galactan and mannan polymers can be targeted to effect cell release from a parenchyma system. Insights provided by the Cnr mutation indicate that mechanisms may exist for the orchestration of groups of cell wall polymers and for large-scale remodeling and concerted changes to cell wall microstructures.
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METHODS |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
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Plant Material
Tomato (Solanum lycopersicum L. var. Ailsa Craig) plants and the mutant Cnr (Thompson et al., 1999; Manning et al., 2006) were grown under standard glasshouse conditions (day temperature 26°C, night temperature 18–19°C, supplementary lighting 16 h), and regular additions of N, P, K, fertilizer. Fruits were harvested at 40 d post anthesis (dpa) (green mature) and 60 dpa (red ripe). Seeds for the Cnr mutant were kindly provided by Ken Manning from Horticulture Research International (Warwick HRI, UK).
Separation of Intact Cells from Pericarp Parenchyma Systems
Cell separation by chemical treatment of 40-dpa parenchyma
Intact cells were obtained from tomato pericarp parenchyma at the green mature stage (40 dpa) by incubation in series of aqueous solvents aimed at the sequential solubilization of cell wall polymers (Redgwell and Selvendran, 1986). Five grams of tomato pericarp cubes (0.06 cm3) were successively suspended in 20 mL of the following: (1) deionized water 30 min at RT; (2) cyclohexanediamino-N,N,N',N',-tetraacetic acid (CDTA) 0.1 M, pH 6.5, 6 h, RT; (3) step 2 repeated; (4) CDTA 0.1 M, pH 6.5, overnight extraction, RT; (5) step 4 repeated; (6) 0.05 M Na2CO3 containing 20 mM NaBH4 overnight at 4°C; (7) 0.05 M Na2CO3 and 20 mM NaBH4, 3 h extraction at 20°C; (8) 0.5 M KOH with 20 mM NaBH4 at 4°C, 1 h; (9) second extraction with KOH, 1 M, with 20 mM NaBH4 at 20°C, 1 h; and (10) third extraction with KOH, 4 M, with 20 mM NaBH4 at 20°C, 1 h. All steps were performed with gentle rocking. Some intact cells were recovered in each step of the sequential extraction by sedimentation (except for the first extraction in deionized water), cells were washed several times with deionized water, and fixed in a solution containing 4% (w/v) paraformaldehyde in 50 mM PIPES and 5 mM EGTA buffer, pH 6.9, then stored at 4°C prior to immuno and cytochemical labeling for light microscopy.
Water extraction of 60-dpa parenchyma
Intact cells were recovered at the red ripe stage (60 dpa) by suspension of tomato pericarp cubes in deionized water for 3 h with gentle rocking. Separated cells were washed with deionized water and fixed as described above and stored at 4°C prior to analysis.
Release of intact cells by enzyme treatment
Two-and-a-half grams of tomato pericarp cubes (0.06 cm3) were incubated overnight at 40°C with agitation in 20 mL of appropriate buffer solution containing 50 µg mL–1 of the following enzymes: 1,5-β-arabinanase and 1,4-β-galactanase (from Aspergillus niger); 1,4-β-xylananse M2 (from Trichoderma longibrachiatum), xyloglucanase 74A (from Clostridium thermocellum, in 50 mM phosphate buffer, pH 6.5), 1,4-β-endomannanase (from Bacillus spp, in 0.1 M glycine–NaOH buffer, pH 8.8), 1,4-β-endopolygalacturonase M2 (from Aspergillus aculeatus), and a pectate lyase (from Cellvibrio japonicus, in N-cyclohexyl-3-aminopropane sulfonic acid (CAPS) buffer containing 2 mM CaCl2, pH 10). The buffer solution for enzymes when not specified was 50 mM NaOAc, pH 4.5. Intact cells recovered after incubation were extensively washed with deionized water and immediately fixed and stored at 4°C. When cell supernatants were recovered for analysis, enzyme activity was inactivated by boiling for 10 min. The arabinanase, galactanase, xylanase, mannanase, and endopolygalacturonase were obtained from Megazyme, Bray, Ireland. The xyloglucanase 74A was purchased from Nzytech Ltd (Lisbon, Portugal) and the use of the pectate lyase 10A has been described previously (Marcus et al., 2008).
Immunochemistry and Immunofluorescence Analysis
Formaldehyde-fixed separated parenchyma cells released by chemical/water extraction or by enzyme action were washed (3 x 10 min) with phosphate-buffered saline (PBS) prior to immunolabeling. Samples were incubated in PBS containing 3% (w/v) of bovine serum albumin (BSA, Sigma, UK) and a five-fold dilution of hybridoma supernatant of the following rat monoclonal antibodies: LM7 (Willats et al., 2001a), LM5 (Jones et al., 1997), LM6 (Willats et al., 1998), LM11 (McCartney et al., 2005), LM15 (Marcus et al., 2008) for 1.5 h at room temperature. A new rat monoclonal antibody (designated LM21), directed to 1,4-mannan, prepared by immunization of a manno-1,4-hexaose coupled to BSA (Marcus SE and Knox JP, unpublished) was also used in this study. After incubation with primary antibodies, cells were washed in PBS at least three times with a 100-fold dilution of anti-rat IgG (whole molecule) linked to fluorescein isothiocyanate (FITC, Sigma, UK) in BSA/PBS for 1.5 h in darkness. The samples were washed in PBS at least three times and incubated with Calcofluor White (25 µg mL–1) (Fluorescent Brightener 28, Sigma, UK) for 5 min in darkness. Samples were washed and mounted in a glycerol–PBS-based anti-fade solution (Citifluor AF1, Agar Scientific, UK). In some cases, tomato pericarp parenchyma cells were embedded in wax and epitopes examined in sections using procedures described elsewhere (Marcus et al., 2008). Monoclonal antibody-labeled materials were examined in a microscope equipped with epifluorescence irradiation (Olympus BX-61). Images were captured with a Hamamatsu ORCA285 camera and Improvision Volocity software.
Sequential Extraction and Epitope Analysis of Polysaccharides from Cell Wall Material
Cell walls were prepared as alcohol-insoluble residues from tomato fruits harvested at 40 dpa. Tomato fruits were peeled, chopped (seeds discarded), and homogenized in 80% (v/v) aqueous ethanol, followed by several washings with absolute ethanol and drying by solvent exchange with absolute ethanol and acetone. Recovered material was successively solubilized through the 10 steps of sequential extraction described above for 40-dpa fruit. Samples extracted with Na2CO3 and KOH were neutralized with glacial acetic acid, then dialysed (13 000 MW cut-off) against deionized water for 3 d at room temperature and freeze-dried. Prior to analysis, samples were re-suspended in deionized water at a concentration of 10 mg mL–1. ELISAs were used to determine the presence of polysaccharide epitopes present in four pooled fractions: water-extracted, CDTA-extracted, Na2CO3-extracted, and KOH-extracted. Microtitre plates (Maxisorp, Nunc, Denmark) were coated overnight at 4°C with 100 µL per well of fractions at 50 µg mL–1. The coating solution was removed, and plates were blocked with 200 µL per well with 3% (w/v) of milk protein in phosphate-buffered saline solution (MP/PBS) at room temperature for 1.5 h, followed by extensive washing. The plates were incubated with 100 µL per well of MP/PBS containing five-fold dilution of the antibody hybridoma supernatant for 1.5 h at RT; after the incubation time, the plates were washed extensively and incubated again with a secondary antibody (anti-rat-IgG horseradish peroxidase conjugate, Sigma, UK) diluted 1/1000 in MP/PBS. After washing, plates were developed with 150 µL per well of tetramethyl benzidine-based substrate, the reaction stopped with 30 µL of 2 M H2SO4, and absorbance read at 450 nm. Confirmation of LM15 binding to xyloglucan in the water extract was obtained by a 1-h pre-treatment of immobilized material with xyloglucanase 74A (described above in cell separation assays).
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We acknowledge funding from the UK Biotechnology and Biological Sciences Research Council (BBSRC).
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We are grateful to Ken Manning at the Warwick HRI for providing seeds of Cnr. No conflict of interest declared.
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