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Plant cell wall architecture: the role of pectins
J. Visserand A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996ElsevierScienceB.V.All rights reserved.
91
Plant cell wall architecture: the role of pectins
M. C. McCann and K. Roberts
Department of Cell Biology, John Innes Centre, Colney lane, Norwich, NR4 7UH, UK.
Abstract
In order to elucidate the role of pectin in cell wall architecture, we have established a suite of techniques applicable at the single cell wall level, including the fast-freeze deep-etch rotary-shadowed replica technique, the use of immunogold probes for specific pectic epitopes, and Fourier Transform Infrared microspectroscopy. We have used these techniques on both isolated pectic polymers and pectins in situ within the cell wall in a wide variety of biological systems. Pectins are chemically heterogeneous, localised in particular cell-wall domains and developmentally-regulated. Screening for appropriate pectin and pectinase mutants using FTIR microspectroscopy offers a possible approach to determine the structural and regulatory functions of these molecules during plant growth and differentiation.
1. PECTIN F O R M S A S T R U C T U R A L N E T W O R K W I T H I N THE CELL WALL
In order to understand how cell wall architecture translates into the mechanical and rheological properties of whole tissues and organs, a minimal requirement is to find out how the three relatively invariant cell wall polysaccharide classes, cellulose, cross-linking glycans, and pectins, are put together in space. Our initial structural studies relied on first developing rapid-freeze, deep-etch, rotary-shadowed replica methods and then applying these to a very simple primary wall system, onion parenchyma cells (1). By conventional EM (Electron Microscopy) fixation, dehydration and resinembedding, the cell wall appears as a fuzzy zone in the electron microscope with little defined structure, and walls of very different composition can have a remarkably similar appearance (2). The fast-freeze, deep-etch, rotaryshadowed replica technique has several important advantages over conventional EM techniques; no chemical fixatives or dehydrants, are used, so
92 the wall is as close to the in vivo state as possible; no specific stains are necessary, all of the molecules present are visualised at high resolution: the three-dimensional molecular arrangement of the wall is preserved and can be visualised by the use of stereo pairs of micrographs.
Fig 1. Electron micrograph of a platinum/carbon replica prepared by the fastfreeze, deep-etch, rotary-shadow replica technique printed in reverse contrast. Cell walls of onion parenchyma have an elaborate structure with many thin fibres bridging between thicker cellulosic microfibrils. Scale bar represents 200nm.
Isolated wall polymers can also be visualised by spraying the polymers in glycerol onto a freshly cleaved surface of mica, drying them down in vacuo, and then rotary-shadowing the preparation with platinum/palladium and carbon (3). It is also possible to adsorb polymers to plastic-filmed gold grids, to immunolabel with a colloidal-gold-conjugated secondary antibody and then to negatively stain to see if the distribution of particular epitopes is uniform along the polymer (3) (Fig 2). By combining our images of these walls and their constituent polymers during chemical extraction, with data from immunogold labelling of thinsectioned material, we were able to construct a simple structural model of the primary cell wall of onion (Fig 3).
93
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Fig 2 Immunogold negative staining, with a monoclonal antibody (JIM 5 (13)) that recognises a relatively unesterified pectic epitope, of rhamnogalacturonans extracted from onion cell walls. Arrows indicate 5 nm colloidal gold particles. Scale bar represents 200nm. Cellulosic microfibrils of 5 to 12 nanometres diameter are spaced 20 to 40 nanometres apart. Xyloglucans, which range up to 400 nanometres in length, hydrogen-bond to two or more cellulose microfibrils, cross-linking them to form a network. The onion wall is 100 nm thick and so there is only room to accommodate 3 or 4 layers of microfibrils between the plasma membrane and the middle lamella. The middle lamella is the region of interface of two walls from neighbouring cells and, in onion, is composed mainly of calcium-cross-linked pectins. A second network of pectins that is independent but coextensive with the cellulose/xyloglucan network, limits the porosity of the wall to about 10 nanometres. Immunogold labelling with monoclonal antibody probes shows the presence of pectic epitopes throughout the cell wall (3), and the relative independence of the pectin network is implied by the relative ease with which it is extracted by chemical agents which break Ca2+ and ester bonds. Using the fast-freeze, deep-etch, rotary-shadowed replica technique, we have demonstrated that the removal of the pectin network does not seem to' affect the structural integrity of the cellulose-hemicellulose network but does increase the pore size of the meshwork of fibrils (1). It has also been shown that the pectin matrix
94 determines wall porosity using FRAP (Fluorescence recovery after photobleaching) to examine macromolecular transport of fluorescentlyderivatised dextrans and proteins of graded sizes across soybean cell walls (5). There is evidence for the presence of a large proportion of galactose in the onion cell wall (6) and a recent NMR study (7) suggests that this neutral galactan may be further involved in limiting porosity, by forming short flexible rods, anchored at one end that protrude into the pores of the network. NMR spectroscopy obtained as either solution-state or solid-state spectra can be used to determine the relative mobilities of different wall polymers and side-chains within the wall: neutral pectins such as galactans are highly mobile within the wall environment and these may further restrict access of regulatory enzymes and molecules to the cellulose-xyloglucan network (7).
MIDDLE LAMELLA
PRIMARY CELL WALL
PLASMA MEMBRANE
Fig 3 An extremely simplified and schematic representation of how three broad classes of polymer are arranged in the onion cell wall (taken from McCann and Roberts 1991 (4)). Although simplistic, the sizes and spacings of the polymers are based on direct measurements of native walls (1) and are drawn to scale. Scale bar represents 50nm.
Our 'two network' model of the primary wall receives support from a variety of indirect observations. For example it has been shown that when a cell wall is regenerated by a carrot protoplast a h o m o g a l a c t u r o n a n / rhamnogalacturonan shell is laid down first, through which the cellulose/ hemicellulose network is later intercalated (8). Further evidence that pectin may form an independent network is seen in the fact that walls from suspension-cultured cells of totnato (Lycopersicon esculentum VF 36),
95 adapted to growth on 12~tM 2,6-dichlorobenzonitrile (DCB), a cellulose synthesis inhibitor, make a pectin-rich wall that virtually lacks a cellulosexyloglucan network (9). The formation of one network has been prevented, while the other is still able to provide a cell wall with at least some of its usual functions. The cells have a reduced growth rate and their unusual cell walls differ from those of non-adapted cells by having both reduced levels of hydroxyproline (about one-third that of non-adapted walls), and a much higher proportion of homogalacturonan and rhamnogalacturonan-like polymers. More recent studies (10) further support the notion that Ca2+bridged pectates comprise the major load-bearing network in DCB-adapted cells since both tomato and tobacco cells grown on DCB can be lysed by treatment with the divalent cation chelator, cyclohexanediaminetetraacetic acid (CDTA). In the absence of suitable cell wall mutants, DCB-adapted tomato cells provide an opportunity to characterise the pectin network of the plant cell wall. It should be noted that synthesis and secretion of hemicellulose is not inhibited but, in the absence of a cellulose framework for it to stick to, most of the xyloglucan secreted remains in soluble form in the cells' culture medium (9, 10) while other non-cellulosic polysaccharides and other uronic-acid-rich polymers predominate in the wall. Infrared spectroscopy is a well-established technique in the spectroscopist's arsenal which has only recently been applied to biological samples following the advent of rapid Fourier Transform data acquisition technology that permits subtraction of water absorbance. In the spectrometer an infrared source emits radiation with a range of frequencies which is then passed through the sample. Particular chemical bonds in the sample absorb radiation of specific frequencies from the beam, and this is plotted out as an absorbance spectrum against frequency (11). The real power of this technique is in the attachment of the microscope accessory where the beam is diverted to pass through a sample sitting on the microscope stage and this permits us to define precisely the area of a single cell wall as small as 10 by 10 microns from which infrared data can be obtained (12). In the infrared region 1200-900 cm-1, pectins have a similar profile to that of polygalacturonic acid, and can easily be distinguished from non-pectic polysaccharides. When the pectins are treated with base, the ester linkage will be destroyed, and in such cases, two absorptions may be seen at about 1600 and 1420 cm-1 (antisymmetric and symmetric COO- stretches): these two peaks are then diagnostic for pectin in salt form. The most diagnostic peak in the infrared spectra of cell walls is the peak centred at about 1745 cm-1, arising from the ester carbonyl stretching associated with pectin. Its exact frequency and bandshape reveal the type (saturated-alkyl or aryl esters) a n d / o r environment of the ester groups.
96
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Fig 4 FTIR spectra of walls of DCB-adapted and n o n - a d a p t e d tomato suspension cells, onion parenchyma cell walls, and polygalacturonic acid (Sigma). a = ester peak, b = free acid stretches from pectins, y axis is absorbance, x axis is wavenumber (frequency inverse). The Fourier Transform Infrared (FTIR) spectrum obtained from nonadapted tomato cell walls is very similar to that from the onion parenchyma cell wall (both contain cellulose, xyloglucan and pectin) although there is more protein in the tomato walls (amide stretches at 1550 and 1650 cm-1) (Fig 4). In DCB-adapted tomato cell walls, the spectrum more closely resembles that of either purified pectins or of a commercial polygalacturonic acid sample from Sigma with peaks in common at 1140, 1095, 1070, 1015 and 950 cm-1 in the carbohydrate region of the spectrum as well as the free acid stretches at 1600 and 1414 cm-1 and an ester peak at 1725 cmq. An ester band at 1740 cm-1 is evident in both onion parenchyma and non-adapted tomato cell wall samples. It is possible that this shift in the ester peak simply reflects the different local molecular environment of this bond, but it is also possible that a different ester is made in the DCB-adapted cell walls, as phenolic esters absorb around 1720 cm-1 whilst carboxylic esters absorb at 1740 cm-1. The
97 carboxylic acid stretches at 1600 and 1414 cm-1 are more prominent in the adapted walls, indicating a much higher proportion of unesterified pectin in these walls. These results are consistent with chemical analyses which indicate that unadapted walls contain about 20% uronic acid, whereas in adapted walls this component rises to about 40-45% of the dry weight. Using conventional electron microscopy techniques, the appearance and thickness of the adapted walls are very similar to unadapted walls (2). We infer from this that conventional EM stains such as uranyl acetate and lead citrate show some specificity for pectic polymers and that neutral molecules such as cellulose and xyloglucan are not well imaged by these stains. The ability of the adapted cells to regulate the thickness of the walls they make in the absence of a normal cellulose/xyloglucan network raises the question of what factors normally limit the thickness of a cell wall, which seems to be under relatively strict developmental control and is genotypespecific. Two possibilities exist: either the width is determined by an independent mechanism which acts on both networks or the width is determined by one network that in turn regulates the other. As wall thickness is conserved in the absence of the cellulose/xyloglucan network it seems very likely that, by default, the pectin ( a n d / o r other non-cellulosic wall components) are involved in its determination. Replicas of the tomato cell walls are very similar to those of onion parenchyma cell walls but replicas of the DCB-adapted walls did not show the structure of the walls clearly. The principle components of the adapted walls are shorter thinner fibres which seemed to form a gel-like structure with little evidence of long cellulosic microfibrils characteristic of the unadapted cells. It is possible that such a gel will bind water more strongly and reduce the amount of etching that takes place, resulting in a less well-defined replica (2).
2. PECTINS ARE SPATIALLY LOCALISED IN CELL WALL DOMAINS Even within the relatively simple onion cell wall, different pectin subpopulations can be distinguished by their sugar composition (6), FTIR spectra (12), antibody affinities and length distributions (Na2CO3-extractable pectins are larger molecules than CDTA-extractable pectins) (4). Using two monoclonal antibodies that recognise relatively methyl-esterified and unesterified pectic epitopes (13), we have shown the variation of pectic polysaccharide localisation between species, between tissues, and between domains within a single wall. In some species, onion (2), tomato, and sugar beet (13), the interface regions between cells, ie the middle lamella and the cell corners, are rich in relatively unesterified pectins which may function in cell-cell adhesion and play an important structural role in tissue integrity. Cell corners, in particular, may act as joists in the scaffolding function of the wall, bearing much of the mechanical load of the tissue (Jeronomidis, pers. comm.). In Zinnia leaves, although all of the cell-walls contain methyl-esterified pectin,
98 this is localised to an outer region of the wall in the mesophyll and palisade cells. The outer layer of esterified pectin may restrict cell-cell adhesion so that air spaces can develope (14). Pathogens also tend to attack cell corners, and the complex mixture of pectins and arabinogalactan proteins found there may have a role in enmeshing the invading organism as well as signalling defense mechanisms to operate through the release of small pectic fragments. Within a plant, different cell types such as cortex, epidermis, phloem sieve tubes or xylem each have different proportions of wall components and therefore presumably different architectures. Cell walls also display different surface markers, arabinogalactan proteins, that reflect early developmental cell patterning and cell positions within the plant (15). As well as tissuespecific differences that arise from the cell's developmental history, the wall is also locally modified in structure and composition at different regions of the cell surface. Even around a single cell, modifications occur that distinguish between transverse and longitudinal walls, for example in sieve tube elements there is a preferential digestion of end walls. We know that mechanisms for polarised secretion of wall components exist such that one wall of a cell may have a different composition from the wall on the opposite side of the cell. In carrot root cap cells, wall thickness, composition and distribution of pectic epitopes differ between the wall facing the soil and that facing the epidermal cell (4). Some substances such as waxes are only secreted to the outer epidermal face. Three times as much wall material must be deposited on the outer epidermal face to allow the same change in length for both surfaces of the cell as the cell wall on this surface is three times thicker. Within a single wall there are zones of different architecture; the middle lamella, plasmodesmata, casparian strips, thickenings, channels, pitfields and the cell corners, and there are also domains across a wall where the degree of pectin esterification is modified. Immunogold labelling with an antibody to unesterified pectin, showed that three zones could be defined across the width of even the simple onion parenchyma wall: a distinct layer of rigid rod-like polymers in the middle lamella, a fuzzy zone next to this also labelled, and then a region nearest to the plasma membrane which does not label (3). The size of micro-domains in the wall compared with the sizes of the polymers that must be accommodated in these regions implies that mechanisms must exist for packaging and positioning of large molecules. For example, unesterified pectins can range up to 700nm in length and yet, in some cell types, are accommodated in a middle lamella of 10 to 20 nm width, and so must at least be constrained to lie parallel to the plasma membrane. Pectic gels made from extracted pectins have hundred-fold greater volumes than the cell walls from which they are extracted. This micro-diversity within walls is now changing our view of the wall from a homogeneous and uniform building material to a mosaic of different wall architectures, a tesselated structure, in which the tiles have unique properties that each contribute to the multifunctional properties of the apoplast.
99 3. PECTINS ARE MODIFIED D U R I N G CELL G R O W T H
A major goal in cell wall research is to understand the molecular basis of cell elongation since it is the cell wall that places constraints on both the size and shape of the cell (16). ,Expansion can occur either isodiametrically or by elongation to generate a range of specialised cell shapes, and the rate of expansion must somehow be coupled to the rate of cell division. Given the spatial complexities of wall architecture, the problem is not simply how coordinate regulation of wall polysaccharide synthesis and deposition is effected in a single cell, but how two or three different architectures (eg at tissue boundaries where two longitudinal walls of different tissue type and one transverse wall meet and adhere) can be synchronously reorganised such that shearing between adjacent cells does not occur. Another major question is how the necessary re-orientation of cellulose and other wall polymers is achieved to establish the direction in which the cell will elongate. From our simple model, one prediction that can be made is that changes in both networks, the cellulose/xyloglucan network and the pectin network, are demanded during cell growth. Cell shape is essentially predetermined by the net orientation of cellulose microfibrils within the cell wall and this in turn appears to depend on the net orientation of cortical microtubules within the cell. In the switch from isodiametric to elongation growth, transversely oriented cortical microtubule arrays may determine the orientation of newly deposited cellulose microfibrils. What happens to other wall polymers when cellulose microfibrils become oriented? Polarisers can be inserted in the path of the IR beam to determine whether band frequencies of specific functional groups are oriented transversely or longitudinally with respect to the long axis of the cell (17). Polarised FTIR microspectroscopy shows that both cellulose and free acid stretches attributable to pectin are oriented transversely to the direction of cell elongation during growth of both carrot and tobacco suspension cells (17, 18). The polarised spectra of epidermal tissue in an elongating carrot stem shows that an extreme case of polarisation of molecules exists in the epidermis with virtually every peak polarised including those associated with pectic polysaccharides and proteins (17). This may reflect the putative role of epidermal tissue to act as the major constraint in organ growth. Supportive evidence for the idea of oriented pectin deposition in epidermal tissue comes from indirect immunofluorescence microscopy of glancing sections of pea stem epidermis using the antibody to unesterified pectin. Images show a clear banding pattern of the pectic epitope transverse to the long axis of the cell (17). Other tissue types have distinctive spectra in which only some components are polarised. In the elongating cortex of carrot stem, peaks in the carbohydrate region 1200 to 900 and from protein are polarised, while the stele has polarised phenolics, protein and a dramatically different carbohydrate profile (17). It seems that the organisation of cell wall architecture during elongation is tissue-specific and may be species-specific, reflecting tissuespecific and species-specific differences in composition. As the wall remains the same thickness and the microfibril spacing is
100 also maintained, it is clear that a cell that has elongated 20-fold will have only 1/20th of the original wall material remaining in the final elongated wall. It is therefore probable that the polarised peaks in the spectra arise from newly deposited wall components during growth. However, we cannot deduce the absolute orientation of these molecules with respect to the long axis of the cell without more detailed information on their conformations. One of the most common modifications to pectins is methyl esterification of the carboxylic acid functional groups resulting in screening of the negative charges used for calcium cross-linking. In walls of tobacco suspension cells, the proportion of total esters is initially about 50% in dividing cells, rises to 78% during maximal elongation and then drops to 68% at stationary phase after elongation has stopped (18). Methyl esters account for only one half of total ester in tobacco cells during the cell division phase. However, the proportion of methyl esters rises to about two-thirds during elongation and the increase in esterification is accounted for entirely by methyl esters. The proportion of methyl ester does not decline at stationary phase, so the decrease in total ester must be due to loss of a different ester. An increase in methyl ester accompanies cell elongation but loss of non-methyl esters is associated with cessation of expansion.
Table 1 Proportions of uronic acid and methyl and non-methyl esters in tobacco suspension cell walls (mol%)
Tobacco Cells
Uronic Acid
GalA
Total ester Methyl ester Other ester
Dividing Elongating Elongated
Day 5 9 16
26 19 21
53 78 68
25 51 48
28 27 20
The mole fraction of uronic acid in polymers in the walls of unadapted cells decreases slightly during the culture period. However, there is a large increase in the mass of all polymers during cell expansion and elongation. The proportion of total GalA esters also increases transiently from 65% to 80% during maximal elongation of maize coleoptiles (19). This transient increase is a result of formation of an unidentified ester, whereas methyl esters decreased slightly in proportion throughout elongation. GLC-MS analysis shows that only a proportion of the ester can be accounted for by
101 methyl esters in both cell types. In tobacco, the proportion of the unidentified esters remains unchanged as the cells transit from division to expansion, but, as in maize coeoptiles, decreases in these esters accompany culmination of growth events. Carrot suspension cells also show a decrease in esterification after elongation (17). The alterations in methyl and total esterified uronic acids measured by GLC-MS are consistent with results from immunolabelling of thin sections with monoclonal antibodies that recognise specific pectic epitopes. Immunolabelling density with JIM 5, a monoclonal antibody that recognises a relatively unesterified pectic epitope, is almost abolished in all walls during elongation of unadapted cells whilst labelling with JIM 7, a monoclonal antibody that recognises a relatively methylesterified pectic epitope, is increased (18). Although JIM 7 labels walls of cells at stationary phase reflecting the lack of change in methyl ester content, JIM 5 labelling density is substantially increased by only a 10% decrease in total ester (18). Hence the pectin in the wall is close to the limit of pectin esterification at which the antibody will still bind and de-esterification is due to loss of other esters implying the activity of a novel esterase. Methyl esterification during elongation must occur uniformly along newly synthesized pectin molecules as no JIM 5-reactive epitopes are available for antibody binding. It seems likely that fine control of gel matrix properties can only be achieved if positions of esterification on long pectin molecules (some range up to 700nm in length) (3) are very precisely defined. At maximum elongation, it appears that the unesterified pectin is secreted into the medium and that therefore an entire pectic network is being replaced (Fig 5). This raises some interesting questions. How does t h e n e w network of more methyl esterified pectin become inserted into the wall? Is the whole network replaced before elongation can occur or are there domains that are capable of wall loosening? What happens in tissues, where the 'older' unesterified pectin must move into the middle lamella? The pectic network in the wall is replaced by newly-synthesized highly-esterified pectins, and older un- or de-esterified pectins may contribute to the increase in surface area of the middle lamella region during growth. Modifications in the degree of esterification of polygalacturonic acid and the size, frequency and conformation of junction zones could influence the fixed-charge density and/or porosity of the pectin gel (20). Loosening of the pectin network concomitant with wall expansion may involve changes in pectin gel rheology w h i c h influence m e t a b o l i s m of the cellulose/xyloglucan network by the fine control of pectin methyl esterification, calcium ion and proton concentration, and the activity of pectin methylesterase (PME) (21). The enormous number of cell wall enzymes may include as yet uncharacterised transesterases and pectin transglycosylases.
102
Fig 5 Immunogold labelling on thin sections of low-temperature embedded cell walls from 9-day old tobacco cells with JIM 5, a monoclonal antibody that recognises a relatively unesterified pectic epitope. Cell walls of elongating cells label very weakly, but material that is being secreted into the culture medium labels strongly. The old part of the wall is labelled but new wall material is not. Measurements of yield stress on gels of isolated pectins have shown that the optimum degree of esterification (d.e.) for maximum gel strength is around 70% (22). The gel strength is not dependent on divalent cation concentration, and may be a result of non-covalent interactions with ester groups helping to stabilise interchain junctions. Morris et al (1980) note a sharp reduction in gel strength at around 80% d.e. which they speculate is due to disruption of these non-covalent interactions. Maximal elongation of both tobacco cells and maize coleoptiles occurs at 80% d.e. of pectin. Whilst isolated pectin gels are clearly abiotic systems, gelation characteristics of pectins in the wall may well be greatly modified by the presence of other components, and the isolated pectin gels were made under conditions of low water activity, nevertheless, aggregation of pectins by non-ionic interchain associations has also been documented by gel permeation chromatography (23) in aqueous pectin solutions, and as a contributing mechanism to network formation in calcium pectate gels (24). High-performance size exclusion chromatography measurements (25) and direct visualisation in
103
the electron microscope (3) have also demonstrated aggregation of isolated pectins. It is possible that disruption of the non-covalent interactions, which may be analogous to tertiary interactions in protein folding, allows increased accessibility of expansins (26) or transglycosylases (27) or hydrolases (28) to the cellulose-xyloglucan network in the cell wall as well as providing a suitable ionic environment for relevant enzymes. A 10% reduction in esterification results in a gel of optimum strength (22). By stationary phase, unadapted tobacco cells have 70% d.e. Further, the complexity and diversity in pectin structure and localisation in cell walls of different species, tissues, and in different wall domains around a single cell (3) may reflect a functional diversity in the fine control of gel rheology. Given the variety in cell wall composition between tissues, and even between walls bordering a single cell (4), it is important to demonstrate that data obtained from bulk chemical analysis is applicable to the walls of all cells in the population sampled. Using FTIR microspectroscopy and electron microscopy, two methodologies which permit analysis at the level of a single cell wall, we have shown that the observed changes in esterification at different growth stages occur, however, in the entire cell population, thus validating the conclusions from the bulk analysis (18).
4. PECTINS M A Y H A V E I M P O R T A N T ( U N K N O W N ) D E V E L O P M E N T A L FUNCTIONS
The classical picture of plant development consists of the production of new cells in specialized regions of cell proliferation, the subsequent coordinated expansion or elongation of these cells and finally, depending on their position with respect to neighbours, a process of cell specialization or differentiation. During growth, it seems that there is replacement or reorganisation of existing networks with newly synthesized polymers that may confer new rheological properties on the wall architecture, to produce architectures that are capable of elongation or expansion, and it seems probable, given the complex spatial localisation patterns of pectins and their heterogeneous nature, that specific changes in pectic composition are necessary for some differentiation events. The Z i n n i a mesophyll cell system is unique in higher plants, in offering controllable semi-synchronous cell differentiation (29). Gentle grinding of Zinnia leaves in a mortar and pestle releases isolated mesophyll and palisade cells leaving sheets of epidermis and the vasculature, that can be separated by centrifugation. If these single cells are incubated in a 1:1 ratio of auxin to cytokinin then first the microtubules bundle up and align, then cellulose is laid down in secondary cell wall thickenings, and finally, after about 4 days, lignin is deposited in the thickenings, and finally the cell autolyses to form a tracheary element (30). About 60% of the mesophyll cells will synchronously trans-differentiate to tracheary elements making the
104 Zinnia system very useful for looking at developmentally-regulated changes
in wall architecture (14). Changes in wall architecture occur during isodiametric cell expansion, cell elongation, and the thickening of a growing wall to its mature thickness, and so it is important to define whether the synthesis and secretion of particular cell-wall molecules correlates with differentiation events or with general cell expansion. Cells are stimulated into producing wall polysaccharides upon subculture, so we used a non-inductive medium, in which the cells only expand, for comparison with inductive medium at all times.
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Fig 6 Immunodot-blots of culture-medium aliquots sampled during the time-course of differentiation of Z i n n i a mesophyll cells to tracheary elements and dotted on to nitrocellulose. I = Inductive medium, N = Noninductive medium. The JIM 7 epitope dries in a series of concentric rings on the nitrocellulose, indicating a mixed population of pectins. During the timecourse, the rhamnose content of inductive culture medium increases dramatically compared with non-inductive medium.
105 Cells in liquid culture are known to secrete extracellular polysaccharides and proteins, and so analysis of the culture medium provides a convenient "snapshot" of which soluble polymers are newly synthesized (14). Aliquots of culture medium sampled during the time course of differentiation were analysed by immunodot-blots on nitrocellulose. The methyl-esterified pectic epitope, recognised by the JIM 7 antibody, is present in both inductive and non-inductive medium, but the pectins are synthesized and secreted at greater concentration in inductive medium than in non-inductive medium (14). However, pectins are a heterogeneous group of cell-wall polymers, and methyl esterification is a common modification to the newly-synthesized polysaccharides. The JIM 7-reactive epitope dries on dot-blots in a reproducible series of concentric rings with consistent marked differences between the rings present in the inductive and non-inductive medium suggesting that during the drying of the dots some chromatographic separation of a mixture of pectins occurs on the nitrocellulose. Sugar and linkage analysis shows that a branched rhamnogalacturonan (4-GalA, 2Rhm, 2,4-Rhm) with short side-chains (no Type I arabinogalactan detected) increases in amount during the time-course, with the rhamnogalacturonan being over ten-fold more abundant than in the non-inductive medium. Rhamnogalacturonan I has also been shown to be a developmental marker for the non-hair-forming cells in epidermal cells of Arabidopsis roots (Hahn, pers. comm.). In the meristematic region of the root, the epitope is present only in the epidermis and only in specific cells.
5. F U T U R E D I R E C T I O N S
International collaboration has allowed us access to a set of selected for by sugar analysis (31), that will allow the phenotypic effects of subtle changes in sugar and lignin monomer composition to be explored (collaboration with Dr W-D Reiter, Connecticut). We have recently used FTIR spectroscopy in conjunction with Principal Component Analysis to show that a rapid screen using FTIR spectroscopy (2 minutes per spectrum) is feasible to select for cell-wall mutants. Although many polysaccharides have absorbances in the so-called fingerprint region of the spectrum, where many complex vibrational modes overlap and peaks cannot be assigned uniquely, in this region the spectra constitute speciesspecific and tissue-specific fingerprints of cell walls, reflecting even subtle differences in composition. Ester and free acid peaks are well-resolved in the spectrum, making FTIR spectroscopy a possible approach to screen for pectin or pectinase mutants. The recent development and application of methodologies sensitive at the single cell wall level has shown that traditional bulk analytical techniques average out important intrinsic heterogeneity in sampled populations. By exploring the diversity of cell walls using novel cryopreservation techniques for electron microscopy and non-invasive
Arabidopsis mutants,
106
vibrational spectroscopies, it should become possible to correlate macroscopic properties with the presence of specific architectures, leading to predictive models for the macroscopic properties of cell wall-derived products. With a large spectral database, statistical methods such as principal component and cluster analysis can be used to determine if correlations exist between spectral features and, for example, taxonomic classification, rheological properties, or tensile strength. Many hundreds of different proteins are present in the cellwall, and many of these may interact with pectins. By using a mutant approach, we hope to address the functions of these proteins and the consequences for pectin structure and function.
6. R E F E R E N C E S
1. McCann, M.C., Wells, B. and Roberts, K. (1990) J. Cell Sci. 96, 323-34. 2. Wells, B., McCann, M.C., Shedletzky, E., Delmer, D. and Roberts, K. (1994) J. Microscopy, 173, 155-164. 3. McCann, M.C., Wells, B. and Roberts, K. (1992) J. Microscopy 166, 123-136. 4. McCann, M.C. and Roberts, K. (1991) In: The Cytoskeletal Basis of Plant Growth and Form (ed. C.W Lloyd), pp 109-129. Academic Press. 5. Baron-Epel, O., Gharyl, P. K. and Schindler, M. (1988) Planta 175, 389-395. 6. Redgwell RJ, Selvendran RR. (1986) Carbohydrate Research 157, 183-199. 7. Foster, T.J., Ablett, S., McCann, M.C. and Gidley, M.J. (1995) Mobilityresolved NMR spectroscopy of cell walls. Biopolymers, in press. 8. Shea, E.M., Gibeaut, D.M. and Carpita, N.C. (1989) Planta 179, 293-308. 9. Shedletzky, E., Shmuel, M., Delmer, D.P. and Lamport, D.T.A. (1990) Plant Physiol 94, 980-987. 10. Shedletzky, E., Shmuel, M., Trainin, T., Kalman, S. and Delmer, D.P. (1992) Plant Physiol. 100, 120-130. 11. Williams DH, Fleming I. (1980) In Spectroscopic Methods in Organic Chemistry, edn 3, pp. 35-73. McGraw Hill, New York. 12. McCann, M.C., Hammouri, M.K., Wilson, R.H., Belton, P.S. and Roberts, K. (1992)Plant Physiology 100, 1940-1947. 13. Knox, J.P., Linstead, P.J., King, J., Cooper, C. and Roberts, K. (1990) Planta 181,512-521. 14. Stacey, N.J., Roberts, K., Carpita, N.C., Wells, B. and McCann, M.C. (1995) Dynamic changes in cell surface molecules are very early events in the differentiation of mesophyll cells from Zinnia elegans into tracheary elements. The Plant Journal, in press. 15. Pennell, R.I., Janniche, L., Kjellbom, P., Scofield, G.N., Peart, J.M. and Roberts, K. (1991) The Plant Cell 3, 1317-1326. 16. McCann, M.C. and Roberts, K. (1994) J. Experimental Botany, 45, 1683-1691. 17. McCann, M.C., Stacey, N.J., Wilson, R. and Roberts, K. (1993) J. Cell Sci. 106, 1347-1356 18. McCann, M.C., Shi, J., Roberts, K. and Carpita, N.C. (1994) The Plant Journal, 5(6), 773-785.
107 19. Kim, J-B. and Carpita N.C. (1992) Plant Physiology 98, 646-653. 20. Carpita, N.C. and Gibeaut, D.M. (1993) The Plant Journal 3, 1-30. 21. Ricard, J. and Noat, G. (1986)Eur. J. Biochem. 155, 183-190. 22. Morris, E. R., Gidley, M. J., Murray, E. J., Powell, D. A., and Rees, D. A. (1980) Internatl. J. Biol. Macromolec. 2, 327-330. 23. Davis, M. A. F., Gidley, M. J., Morris, E. R., Powell, D. A. and Rees, D. A. (1980)Int. J. Biol. Macromol. 2, 330-332. 24. Gidley, M. J., Morris, E. R., Murray, E. J., Powell, D. A. and Rees, D. A. (1980) Int. J. Biol. Macromol. 2, 332-334. 25. Fishman, M. L., Gillespie, D. T., Sondey, S. M. and Barford, R. A. (1989) J. Agric. Food Chem. 37, 584-591 26. McQueen-Mason, S., Durachko, D. M. and Cosgrove, D.J. (1992) Plant Cell 4, 1425-1433. 27. Fry, S. C., Smith, R. C., Renwick, K. F., Martin, D. J., Hodge, S. K. and Matthews, K.J. (1991) Biochem. J. 282, 821-828. 28. Taiz, L. (1984) Annu. Rev. Plant Physiol. 35, 585-657. 29. Chasan, R. (1994) The Plant Cell 6, 917-919 30. Fukuda, H. and Komamine, A. (1980)Plant Physiol. 52, 57-60. 31. Reiter WD, Chapple CCS, Somerville CR. (1993) Science 261, 1032-1035.
91
Plant cell wall architecture: the role of pectins
M. C. McCann and K. Roberts
Department of Cell Biology, John Innes Centre, Colney lane, Norwich, NR4 7UH, UK.
Abstract
In order to elucidate the role of pectin in cell wall architecture, we have established a suite of techniques applicable at the single cell wall level, including the fast-freeze deep-etch rotary-shadowed replica technique, the use of immunogold probes for specific pectic epitopes, and Fourier Transform Infrared microspectroscopy. We have used these techniques on both isolated pectic polymers and pectins in situ within the cell wall in a wide variety of biological systems. Pectins are chemically heterogeneous, localised in particular cell-wall domains and developmentally-regulated. Screening for appropriate pectin and pectinase mutants using FTIR microspectroscopy offers a possible approach to determine the structural and regulatory functions of these molecules during plant growth and differentiation.
1. PECTIN F O R M S A S T R U C T U R A L N E T W O R K W I T H I N THE CELL WALL
In order to understand how cell wall architecture translates into the mechanical and rheological properties of whole tissues and organs, a minimal requirement is to find out how the three relatively invariant cell wall polysaccharide classes, cellulose, cross-linking glycans, and pectins, are put together in space. Our initial structural studies relied on first developing rapid-freeze, deep-etch, rotary-shadowed replica methods and then applying these to a very simple primary wall system, onion parenchyma cells (1). By conventional EM (Electron Microscopy) fixation, dehydration and resinembedding, the cell wall appears as a fuzzy zone in the electron microscope with little defined structure, and walls of very different composition can have a remarkably similar appearance (2). The fast-freeze, deep-etch, rotaryshadowed replica technique has several important advantages over conventional EM techniques; no chemical fixatives or dehydrants, are used, so
92 the wall is as close to the in vivo state as possible; no specific stains are necessary, all of the molecules present are visualised at high resolution: the three-dimensional molecular arrangement of the wall is preserved and can be visualised by the use of stereo pairs of micrographs.
Fig 1. Electron micrograph of a platinum/carbon replica prepared by the fastfreeze, deep-etch, rotary-shadow replica technique printed in reverse contrast. Cell walls of onion parenchyma have an elaborate structure with many thin fibres bridging between thicker cellulosic microfibrils. Scale bar represents 200nm.
Isolated wall polymers can also be visualised by spraying the polymers in glycerol onto a freshly cleaved surface of mica, drying them down in vacuo, and then rotary-shadowing the preparation with platinum/palladium and carbon (3). It is also possible to adsorb polymers to plastic-filmed gold grids, to immunolabel with a colloidal-gold-conjugated secondary antibody and then to negatively stain to see if the distribution of particular epitopes is uniform along the polymer (3) (Fig 2). By combining our images of these walls and their constituent polymers during chemical extraction, with data from immunogold labelling of thinsectioned material, we were able to construct a simple structural model of the primary cell wall of onion (Fig 3).
93
r
Fig 2 Immunogold negative staining, with a monoclonal antibody (JIM 5 (13)) that recognises a relatively unesterified pectic epitope, of rhamnogalacturonans extracted from onion cell walls. Arrows indicate 5 nm colloidal gold particles. Scale bar represents 200nm. Cellulosic microfibrils of 5 to 12 nanometres diameter are spaced 20 to 40 nanometres apart. Xyloglucans, which range up to 400 nanometres in length, hydrogen-bond to two or more cellulose microfibrils, cross-linking them to form a network. The onion wall is 100 nm thick and so there is only room to accommodate 3 or 4 layers of microfibrils between the plasma membrane and the middle lamella. The middle lamella is the region of interface of two walls from neighbouring cells and, in onion, is composed mainly of calcium-cross-linked pectins. A second network of pectins that is independent but coextensive with the cellulose/xyloglucan network, limits the porosity of the wall to about 10 nanometres. Immunogold labelling with monoclonal antibody probes shows the presence of pectic epitopes throughout the cell wall (3), and the relative independence of the pectin network is implied by the relative ease with which it is extracted by chemical agents which break Ca2+ and ester bonds. Using the fast-freeze, deep-etch, rotary-shadowed replica technique, we have demonstrated that the removal of the pectin network does not seem to' affect the structural integrity of the cellulose-hemicellulose network but does increase the pore size of the meshwork of fibrils (1). It has also been shown that the pectin matrix
94 determines wall porosity using FRAP (Fluorescence recovery after photobleaching) to examine macromolecular transport of fluorescentlyderivatised dextrans and proteins of graded sizes across soybean cell walls (5). There is evidence for the presence of a large proportion of galactose in the onion cell wall (6) and a recent NMR study (7) suggests that this neutral galactan may be further involved in limiting porosity, by forming short flexible rods, anchored at one end that protrude into the pores of the network. NMR spectroscopy obtained as either solution-state or solid-state spectra can be used to determine the relative mobilities of different wall polymers and side-chains within the wall: neutral pectins such as galactans are highly mobile within the wall environment and these may further restrict access of regulatory enzymes and molecules to the cellulose-xyloglucan network (7).
MIDDLE LAMELLA
PRIMARY CELL WALL
PLASMA MEMBRANE
Fig 3 An extremely simplified and schematic representation of how three broad classes of polymer are arranged in the onion cell wall (taken from McCann and Roberts 1991 (4)). Although simplistic, the sizes and spacings of the polymers are based on direct measurements of native walls (1) and are drawn to scale. Scale bar represents 50nm.
Our 'two network' model of the primary wall receives support from a variety of indirect observations. For example it has been shown that when a cell wall is regenerated by a carrot protoplast a h o m o g a l a c t u r o n a n / rhamnogalacturonan shell is laid down first, through which the cellulose/ hemicellulose network is later intercalated (8). Further evidence that pectin may form an independent network is seen in the fact that walls from suspension-cultured cells of totnato (Lycopersicon esculentum VF 36),
95 adapted to growth on 12~tM 2,6-dichlorobenzonitrile (DCB), a cellulose synthesis inhibitor, make a pectin-rich wall that virtually lacks a cellulosexyloglucan network (9). The formation of one network has been prevented, while the other is still able to provide a cell wall with at least some of its usual functions. The cells have a reduced growth rate and their unusual cell walls differ from those of non-adapted cells by having both reduced levels of hydroxyproline (about one-third that of non-adapted walls), and a much higher proportion of homogalacturonan and rhamnogalacturonan-like polymers. More recent studies (10) further support the notion that Ca2+bridged pectates comprise the major load-bearing network in DCB-adapted cells since both tomato and tobacco cells grown on DCB can be lysed by treatment with the divalent cation chelator, cyclohexanediaminetetraacetic acid (CDTA). In the absence of suitable cell wall mutants, DCB-adapted tomato cells provide an opportunity to characterise the pectin network of the plant cell wall. It should be noted that synthesis and secretion of hemicellulose is not inhibited but, in the absence of a cellulose framework for it to stick to, most of the xyloglucan secreted remains in soluble form in the cells' culture medium (9, 10) while other non-cellulosic polysaccharides and other uronic-acid-rich polymers predominate in the wall. Infrared spectroscopy is a well-established technique in the spectroscopist's arsenal which has only recently been applied to biological samples following the advent of rapid Fourier Transform data acquisition technology that permits subtraction of water absorbance. In the spectrometer an infrared source emits radiation with a range of frequencies which is then passed through the sample. Particular chemical bonds in the sample absorb radiation of specific frequencies from the beam, and this is plotted out as an absorbance spectrum against frequency (11). The real power of this technique is in the attachment of the microscope accessory where the beam is diverted to pass through a sample sitting on the microscope stage and this permits us to define precisely the area of a single cell wall as small as 10 by 10 microns from which infrared data can be obtained (12). In the infrared region 1200-900 cm-1, pectins have a similar profile to that of polygalacturonic acid, and can easily be distinguished from non-pectic polysaccharides. When the pectins are treated with base, the ester linkage will be destroyed, and in such cases, two absorptions may be seen at about 1600 and 1420 cm-1 (antisymmetric and symmetric COO- stretches): these two peaks are then diagnostic for pectin in salt form. The most diagnostic peak in the infrared spectra of cell walls is the peak centred at about 1745 cm-1, arising from the ester carbonyl stretching associated with pectin. Its exact frequency and bandshape reveal the type (saturated-alkyl or aryl esters) a n d / o r environment of the ester groups.
96
Tomato
DCB-adapted tomato b
0.3
0.25
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|
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,
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|
|
1300
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900
Polygalacturonic acid
Onion
0.16
1.0
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0.4 0.0
---____
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0.04-
,
1700'
'
' 1300'
'
' 960
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'
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Fig 4 FTIR spectra of walls of DCB-adapted and n o n - a d a p t e d tomato suspension cells, onion parenchyma cell walls, and polygalacturonic acid (Sigma). a = ester peak, b = free acid stretches from pectins, y axis is absorbance, x axis is wavenumber (frequency inverse). The Fourier Transform Infrared (FTIR) spectrum obtained from nonadapted tomato cell walls is very similar to that from the onion parenchyma cell wall (both contain cellulose, xyloglucan and pectin) although there is more protein in the tomato walls (amide stretches at 1550 and 1650 cm-1) (Fig 4). In DCB-adapted tomato cell walls, the spectrum more closely resembles that of either purified pectins or of a commercial polygalacturonic acid sample from Sigma with peaks in common at 1140, 1095, 1070, 1015 and 950 cm-1 in the carbohydrate region of the spectrum as well as the free acid stretches at 1600 and 1414 cm-1 and an ester peak at 1725 cmq. An ester band at 1740 cm-1 is evident in both onion parenchyma and non-adapted tomato cell wall samples. It is possible that this shift in the ester peak simply reflects the different local molecular environment of this bond, but it is also possible that a different ester is made in the DCB-adapted cell walls, as phenolic esters absorb around 1720 cm-1 whilst carboxylic esters absorb at 1740 cm-1. The
97 carboxylic acid stretches at 1600 and 1414 cm-1 are more prominent in the adapted walls, indicating a much higher proportion of unesterified pectin in these walls. These results are consistent with chemical analyses which indicate that unadapted walls contain about 20% uronic acid, whereas in adapted walls this component rises to about 40-45% of the dry weight. Using conventional electron microscopy techniques, the appearance and thickness of the adapted walls are very similar to unadapted walls (2). We infer from this that conventional EM stains such as uranyl acetate and lead citrate show some specificity for pectic polymers and that neutral molecules such as cellulose and xyloglucan are not well imaged by these stains. The ability of the adapted cells to regulate the thickness of the walls they make in the absence of a normal cellulose/xyloglucan network raises the question of what factors normally limit the thickness of a cell wall, which seems to be under relatively strict developmental control and is genotypespecific. Two possibilities exist: either the width is determined by an independent mechanism which acts on both networks or the width is determined by one network that in turn regulates the other. As wall thickness is conserved in the absence of the cellulose/xyloglucan network it seems very likely that, by default, the pectin ( a n d / o r other non-cellulosic wall components) are involved in its determination. Replicas of the tomato cell walls are very similar to those of onion parenchyma cell walls but replicas of the DCB-adapted walls did not show the structure of the walls clearly. The principle components of the adapted walls are shorter thinner fibres which seemed to form a gel-like structure with little evidence of long cellulosic microfibrils characteristic of the unadapted cells. It is possible that such a gel will bind water more strongly and reduce the amount of etching that takes place, resulting in a less well-defined replica (2).
2. PECTINS ARE SPATIALLY LOCALISED IN CELL WALL DOMAINS Even within the relatively simple onion cell wall, different pectin subpopulations can be distinguished by their sugar composition (6), FTIR spectra (12), antibody affinities and length distributions (Na2CO3-extractable pectins are larger molecules than CDTA-extractable pectins) (4). Using two monoclonal antibodies that recognise relatively methyl-esterified and unesterified pectic epitopes (13), we have shown the variation of pectic polysaccharide localisation between species, between tissues, and between domains within a single wall. In some species, onion (2), tomato, and sugar beet (13), the interface regions between cells, ie the middle lamella and the cell corners, are rich in relatively unesterified pectins which may function in cell-cell adhesion and play an important structural role in tissue integrity. Cell corners, in particular, may act as joists in the scaffolding function of the wall, bearing much of the mechanical load of the tissue (Jeronomidis, pers. comm.). In Zinnia leaves, although all of the cell-walls contain methyl-esterified pectin,
98 this is localised to an outer region of the wall in the mesophyll and palisade cells. The outer layer of esterified pectin may restrict cell-cell adhesion so that air spaces can develope (14). Pathogens also tend to attack cell corners, and the complex mixture of pectins and arabinogalactan proteins found there may have a role in enmeshing the invading organism as well as signalling defense mechanisms to operate through the release of small pectic fragments. Within a plant, different cell types such as cortex, epidermis, phloem sieve tubes or xylem each have different proportions of wall components and therefore presumably different architectures. Cell walls also display different surface markers, arabinogalactan proteins, that reflect early developmental cell patterning and cell positions within the plant (15). As well as tissuespecific differences that arise from the cell's developmental history, the wall is also locally modified in structure and composition at different regions of the cell surface. Even around a single cell, modifications occur that distinguish between transverse and longitudinal walls, for example in sieve tube elements there is a preferential digestion of end walls. We know that mechanisms for polarised secretion of wall components exist such that one wall of a cell may have a different composition from the wall on the opposite side of the cell. In carrot root cap cells, wall thickness, composition and distribution of pectic epitopes differ between the wall facing the soil and that facing the epidermal cell (4). Some substances such as waxes are only secreted to the outer epidermal face. Three times as much wall material must be deposited on the outer epidermal face to allow the same change in length for both surfaces of the cell as the cell wall on this surface is three times thicker. Within a single wall there are zones of different architecture; the middle lamella, plasmodesmata, casparian strips, thickenings, channels, pitfields and the cell corners, and there are also domains across a wall where the degree of pectin esterification is modified. Immunogold labelling with an antibody to unesterified pectin, showed that three zones could be defined across the width of even the simple onion parenchyma wall: a distinct layer of rigid rod-like polymers in the middle lamella, a fuzzy zone next to this also labelled, and then a region nearest to the plasma membrane which does not label (3). The size of micro-domains in the wall compared with the sizes of the polymers that must be accommodated in these regions implies that mechanisms must exist for packaging and positioning of large molecules. For example, unesterified pectins can range up to 700nm in length and yet, in some cell types, are accommodated in a middle lamella of 10 to 20 nm width, and so must at least be constrained to lie parallel to the plasma membrane. Pectic gels made from extracted pectins have hundred-fold greater volumes than the cell walls from which they are extracted. This micro-diversity within walls is now changing our view of the wall from a homogeneous and uniform building material to a mosaic of different wall architectures, a tesselated structure, in which the tiles have unique properties that each contribute to the multifunctional properties of the apoplast.
99 3. PECTINS ARE MODIFIED D U R I N G CELL G R O W T H
A major goal in cell wall research is to understand the molecular basis of cell elongation since it is the cell wall that places constraints on both the size and shape of the cell (16). ,Expansion can occur either isodiametrically or by elongation to generate a range of specialised cell shapes, and the rate of expansion must somehow be coupled to the rate of cell division. Given the spatial complexities of wall architecture, the problem is not simply how coordinate regulation of wall polysaccharide synthesis and deposition is effected in a single cell, but how two or three different architectures (eg at tissue boundaries where two longitudinal walls of different tissue type and one transverse wall meet and adhere) can be synchronously reorganised such that shearing between adjacent cells does not occur. Another major question is how the necessary re-orientation of cellulose and other wall polymers is achieved to establish the direction in which the cell will elongate. From our simple model, one prediction that can be made is that changes in both networks, the cellulose/xyloglucan network and the pectin network, are demanded during cell growth. Cell shape is essentially predetermined by the net orientation of cellulose microfibrils within the cell wall and this in turn appears to depend on the net orientation of cortical microtubules within the cell. In the switch from isodiametric to elongation growth, transversely oriented cortical microtubule arrays may determine the orientation of newly deposited cellulose microfibrils. What happens to other wall polymers when cellulose microfibrils become oriented? Polarisers can be inserted in the path of the IR beam to determine whether band frequencies of specific functional groups are oriented transversely or longitudinally with respect to the long axis of the cell (17). Polarised FTIR microspectroscopy shows that both cellulose and free acid stretches attributable to pectin are oriented transversely to the direction of cell elongation during growth of both carrot and tobacco suspension cells (17, 18). The polarised spectra of epidermal tissue in an elongating carrot stem shows that an extreme case of polarisation of molecules exists in the epidermis with virtually every peak polarised including those associated with pectic polysaccharides and proteins (17). This may reflect the putative role of epidermal tissue to act as the major constraint in organ growth. Supportive evidence for the idea of oriented pectin deposition in epidermal tissue comes from indirect immunofluorescence microscopy of glancing sections of pea stem epidermis using the antibody to unesterified pectin. Images show a clear banding pattern of the pectic epitope transverse to the long axis of the cell (17). Other tissue types have distinctive spectra in which only some components are polarised. In the elongating cortex of carrot stem, peaks in the carbohydrate region 1200 to 900 and from protein are polarised, while the stele has polarised phenolics, protein and a dramatically different carbohydrate profile (17). It seems that the organisation of cell wall architecture during elongation is tissue-specific and may be species-specific, reflecting tissuespecific and species-specific differences in composition. As the wall remains the same thickness and the microfibril spacing is
100 also maintained, it is clear that a cell that has elongated 20-fold will have only 1/20th of the original wall material remaining in the final elongated wall. It is therefore probable that the polarised peaks in the spectra arise from newly deposited wall components during growth. However, we cannot deduce the absolute orientation of these molecules with respect to the long axis of the cell without more detailed information on their conformations. One of the most common modifications to pectins is methyl esterification of the carboxylic acid functional groups resulting in screening of the negative charges used for calcium cross-linking. In walls of tobacco suspension cells, the proportion of total esters is initially about 50% in dividing cells, rises to 78% during maximal elongation and then drops to 68% at stationary phase after elongation has stopped (18). Methyl esters account for only one half of total ester in tobacco cells during the cell division phase. However, the proportion of methyl esters rises to about two-thirds during elongation and the increase in esterification is accounted for entirely by methyl esters. The proportion of methyl ester does not decline at stationary phase, so the decrease in total ester must be due to loss of a different ester. An increase in methyl ester accompanies cell elongation but loss of non-methyl esters is associated with cessation of expansion.
Table 1 Proportions of uronic acid and methyl and non-methyl esters in tobacco suspension cell walls (mol%)
Tobacco Cells
Uronic Acid
GalA
Total ester Methyl ester Other ester
Dividing Elongating Elongated
Day 5 9 16
26 19 21
53 78 68
25 51 48
28 27 20
The mole fraction of uronic acid in polymers in the walls of unadapted cells decreases slightly during the culture period. However, there is a large increase in the mass of all polymers during cell expansion and elongation. The proportion of total GalA esters also increases transiently from 65% to 80% during maximal elongation of maize coleoptiles (19). This transient increase is a result of formation of an unidentified ester, whereas methyl esters decreased slightly in proportion throughout elongation. GLC-MS analysis shows that only a proportion of the ester can be accounted for by
101 methyl esters in both cell types. In tobacco, the proportion of the unidentified esters remains unchanged as the cells transit from division to expansion, but, as in maize coeoptiles, decreases in these esters accompany culmination of growth events. Carrot suspension cells also show a decrease in esterification after elongation (17). The alterations in methyl and total esterified uronic acids measured by GLC-MS are consistent with results from immunolabelling of thin sections with monoclonal antibodies that recognise specific pectic epitopes. Immunolabelling density with JIM 5, a monoclonal antibody that recognises a relatively unesterified pectic epitope, is almost abolished in all walls during elongation of unadapted cells whilst labelling with JIM 7, a monoclonal antibody that recognises a relatively methylesterified pectic epitope, is increased (18). Although JIM 7 labels walls of cells at stationary phase reflecting the lack of change in methyl ester content, JIM 5 labelling density is substantially increased by only a 10% decrease in total ester (18). Hence the pectin in the wall is close to the limit of pectin esterification at which the antibody will still bind and de-esterification is due to loss of other esters implying the activity of a novel esterase. Methyl esterification during elongation must occur uniformly along newly synthesized pectin molecules as no JIM 5-reactive epitopes are available for antibody binding. It seems likely that fine control of gel matrix properties can only be achieved if positions of esterification on long pectin molecules (some range up to 700nm in length) (3) are very precisely defined. At maximum elongation, it appears that the unesterified pectin is secreted into the medium and that therefore an entire pectic network is being replaced (Fig 5). This raises some interesting questions. How does t h e n e w network of more methyl esterified pectin become inserted into the wall? Is the whole network replaced before elongation can occur or are there domains that are capable of wall loosening? What happens in tissues, where the 'older' unesterified pectin must move into the middle lamella? The pectic network in the wall is replaced by newly-synthesized highly-esterified pectins, and older un- or de-esterified pectins may contribute to the increase in surface area of the middle lamella region during growth. Modifications in the degree of esterification of polygalacturonic acid and the size, frequency and conformation of junction zones could influence the fixed-charge density and/or porosity of the pectin gel (20). Loosening of the pectin network concomitant with wall expansion may involve changes in pectin gel rheology w h i c h influence m e t a b o l i s m of the cellulose/xyloglucan network by the fine control of pectin methyl esterification, calcium ion and proton concentration, and the activity of pectin methylesterase (PME) (21). The enormous number of cell wall enzymes may include as yet uncharacterised transesterases and pectin transglycosylases.
102
Fig 5 Immunogold labelling on thin sections of low-temperature embedded cell walls from 9-day old tobacco cells with JIM 5, a monoclonal antibody that recognises a relatively unesterified pectic epitope. Cell walls of elongating cells label very weakly, but material that is being secreted into the culture medium labels strongly. The old part of the wall is labelled but new wall material is not. Measurements of yield stress on gels of isolated pectins have shown that the optimum degree of esterification (d.e.) for maximum gel strength is around 70% (22). The gel strength is not dependent on divalent cation concentration, and may be a result of non-covalent interactions with ester groups helping to stabilise interchain junctions. Morris et al (1980) note a sharp reduction in gel strength at around 80% d.e. which they speculate is due to disruption of these non-covalent interactions. Maximal elongation of both tobacco cells and maize coleoptiles occurs at 80% d.e. of pectin. Whilst isolated pectin gels are clearly abiotic systems, gelation characteristics of pectins in the wall may well be greatly modified by the presence of other components, and the isolated pectin gels were made under conditions of low water activity, nevertheless, aggregation of pectins by non-ionic interchain associations has also been documented by gel permeation chromatography (23) in aqueous pectin solutions, and as a contributing mechanism to network formation in calcium pectate gels (24). High-performance size exclusion chromatography measurements (25) and direct visualisation in
103
the electron microscope (3) have also demonstrated aggregation of isolated pectins. It is possible that disruption of the non-covalent interactions, which may be analogous to tertiary interactions in protein folding, allows increased accessibility of expansins (26) or transglycosylases (27) or hydrolases (28) to the cellulose-xyloglucan network in the cell wall as well as providing a suitable ionic environment for relevant enzymes. A 10% reduction in esterification results in a gel of optimum strength (22). By stationary phase, unadapted tobacco cells have 70% d.e. Further, the complexity and diversity in pectin structure and localisation in cell walls of different species, tissues, and in different wall domains around a single cell (3) may reflect a functional diversity in the fine control of gel rheology. Given the variety in cell wall composition between tissues, and even between walls bordering a single cell (4), it is important to demonstrate that data obtained from bulk chemical analysis is applicable to the walls of all cells in the population sampled. Using FTIR microspectroscopy and electron microscopy, two methodologies which permit analysis at the level of a single cell wall, we have shown that the observed changes in esterification at different growth stages occur, however, in the entire cell population, thus validating the conclusions from the bulk analysis (18).
4. PECTINS M A Y H A V E I M P O R T A N T ( U N K N O W N ) D E V E L O P M E N T A L FUNCTIONS
The classical picture of plant development consists of the production of new cells in specialized regions of cell proliferation, the subsequent coordinated expansion or elongation of these cells and finally, depending on their position with respect to neighbours, a process of cell specialization or differentiation. During growth, it seems that there is replacement or reorganisation of existing networks with newly synthesized polymers that may confer new rheological properties on the wall architecture, to produce architectures that are capable of elongation or expansion, and it seems probable, given the complex spatial localisation patterns of pectins and their heterogeneous nature, that specific changes in pectic composition are necessary for some differentiation events. The Z i n n i a mesophyll cell system is unique in higher plants, in offering controllable semi-synchronous cell differentiation (29). Gentle grinding of Zinnia leaves in a mortar and pestle releases isolated mesophyll and palisade cells leaving sheets of epidermis and the vasculature, that can be separated by centrifugation. If these single cells are incubated in a 1:1 ratio of auxin to cytokinin then first the microtubules bundle up and align, then cellulose is laid down in secondary cell wall thickenings, and finally, after about 4 days, lignin is deposited in the thickenings, and finally the cell autolyses to form a tracheary element (30). About 60% of the mesophyll cells will synchronously trans-differentiate to tracheary elements making the
104 Zinnia system very useful for looking at developmentally-regulated changes
in wall architecture (14). Changes in wall architecture occur during isodiametric cell expansion, cell elongation, and the thickening of a growing wall to its mature thickness, and so it is important to define whether the synthesis and secretion of particular cell-wall molecules correlates with differentiation events or with general cell expansion. Cells are stimulated into producing wall polysaccharides upon subculture, so we used a non-inductive medium, in which the cells only expand, for comparison with inductive medium at all times.
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162 JIM 7
Fig 6 Immunodot-blots of culture-medium aliquots sampled during the time-course of differentiation of Z i n n i a mesophyll cells to tracheary elements and dotted on to nitrocellulose. I = Inductive medium, N = Noninductive medium. The JIM 7 epitope dries in a series of concentric rings on the nitrocellulose, indicating a mixed population of pectins. During the timecourse, the rhamnose content of inductive culture medium increases dramatically compared with non-inductive medium.
105 Cells in liquid culture are known to secrete extracellular polysaccharides and proteins, and so analysis of the culture medium provides a convenient "snapshot" of which soluble polymers are newly synthesized (14). Aliquots of culture medium sampled during the time course of differentiation were analysed by immunodot-blots on nitrocellulose. The methyl-esterified pectic epitope, recognised by the JIM 7 antibody, is present in both inductive and non-inductive medium, but the pectins are synthesized and secreted at greater concentration in inductive medium than in non-inductive medium (14). However, pectins are a heterogeneous group of cell-wall polymers, and methyl esterification is a common modification to the newly-synthesized polysaccharides. The JIM 7-reactive epitope dries on dot-blots in a reproducible series of concentric rings with consistent marked differences between the rings present in the inductive and non-inductive medium suggesting that during the drying of the dots some chromatographic separation of a mixture of pectins occurs on the nitrocellulose. Sugar and linkage analysis shows that a branched rhamnogalacturonan (4-GalA, 2Rhm, 2,4-Rhm) with short side-chains (no Type I arabinogalactan detected) increases in amount during the time-course, with the rhamnogalacturonan being over ten-fold more abundant than in the non-inductive medium. Rhamnogalacturonan I has also been shown to be a developmental marker for the non-hair-forming cells in epidermal cells of Arabidopsis roots (Hahn, pers. comm.). In the meristematic region of the root, the epitope is present only in the epidermis and only in specific cells.
5. F U T U R E D I R E C T I O N S
International collaboration has allowed us access to a set of selected for by sugar analysis (31), that will allow the phenotypic effects of subtle changes in sugar and lignin monomer composition to be explored (collaboration with Dr W-D Reiter, Connecticut). We have recently used FTIR spectroscopy in conjunction with Principal Component Analysis to show that a rapid screen using FTIR spectroscopy (2 minutes per spectrum) is feasible to select for cell-wall mutants. Although many polysaccharides have absorbances in the so-called fingerprint region of the spectrum, where many complex vibrational modes overlap and peaks cannot be assigned uniquely, in this region the spectra constitute speciesspecific and tissue-specific fingerprints of cell walls, reflecting even subtle differences in composition. Ester and free acid peaks are well-resolved in the spectrum, making FTIR spectroscopy a possible approach to screen for pectin or pectinase mutants. The recent development and application of methodologies sensitive at the single cell wall level has shown that traditional bulk analytical techniques average out important intrinsic heterogeneity in sampled populations. By exploring the diversity of cell walls using novel cryopreservation techniques for electron microscopy and non-invasive
Arabidopsis mutants,
106
vibrational spectroscopies, it should become possible to correlate macroscopic properties with the presence of specific architectures, leading to predictive models for the macroscopic properties of cell wall-derived products. With a large spectral database, statistical methods such as principal component and cluster analysis can be used to determine if correlations exist between spectral features and, for example, taxonomic classification, rheological properties, or tensile strength. Many hundreds of different proteins are present in the cellwall, and many of these may interact with pectins. By using a mutant approach, we hope to address the functions of these proteins and the consequences for pectin structure and function.
6. R E F E R E N C E S
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