Mixed-linkage (1→3),(1→4)-β-d-glucan is not unique to the Poales and is an abundant component of Equisetum arvense cell walls

Authors

  • Iben Sørensen,

    1. Department of Biology, The University of Copenhagen, Ole Maaløes vej 5, Copenhagen DK-2200, Denmark,
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  • Filomena A. Pettolino,

    1. Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Melbourne, Vic. 3010, Australia,
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  • Sarah M. Wilson,

    1. Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Melbourne, Vic. 3010, Australia,
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  • Monika S. Doblin,

    1. Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Melbourne, Vic. 3010, Australia,
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  • Bo Johansen,

    1. Department of Biology, The University of Copenhagen, Østerfarimagsgade 2D, Copenhagen DK-1353, Denmark, and
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  • Antony Bacic,

    1. Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Melbourne, Vic. 3010, Australia,
    2. Australian Centre for Plant Functional Genomics, School of Botany, University of Melbourne, Melbourne, Vic. 3010, Australia
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  • William G. T. Willats

    Corresponding author
    1. Department of Biology, The University of Copenhagen, Ole Maaløes vej 5, Copenhagen DK-2200, Denmark,
      *(fax +45 35322128; e-mail willats@bio.ku.dk).
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*(fax +45 35322128; e-mail willats@bio.ku.dk).

Summary

Mixed-linkage (1→3),(1→4)-β-d-glucan (MLG) is widely considered to be a defining feature of the cell walls of plants in the Poales order. However, we conducted an extensive survey of cell-wall composition in diverse land plants and discovered that MLG is also abundant in the walls of the horsetail Equisetum arvense. MALDI-TOF MS and monosaccharide linkage analysis revealed that MLG in E. arvense is an unbranched homopolymer that consists of short blocks of contiguous 1,4-β-linked glucose residues joined by 1,3-β linkages. However, in contrast to Poaceae species, MLG in E. arvense consists mostly of cellotetraose rather than cellotetriose, and lacks long 1,4-β-linked glucan blocks. Monosaccharide linkage analyses and immunochemical profiling indicated that, in E. arvense, MLG is a component of cell walls that have a novel architecture that differs significantly from that of the generally recognized type I and II cell walls. Unlike in type II walls, MLG in E. arvense does not appear to be co-extensive with glucuroarabinoxylans but occurs in walls that are rich in pectin. Immunofluorescence and immunogold localization showed that MLG occurs in both young and old regions of E. arvense stems, and is present in most cell types apart from cells in the vascular tissues. These findings have important implications for our understanding of cell-wall evolution, and also demonstrate that plant cell walls can be constructed in a way not previously envisaged.

Introduction

Almost all plant cells are surrounded by a complex, multifunctional and glycan-rich cell wall (Carpita and Gibeaut, 1993; Fry, 2004). As well as providing mechanical support, cell walls act as defensive barriers against pathogens and the environment, and play important roles in determining cell fate and in modulating growth and development (Bacic et al., 1988; Carpita and Gibeaut, 1993; O’Neill et al., 1990; Ridley et al., 2001). Materials from cell walls are also commercially important, and are a source of functional food ingredients, industrial fibres, nutraceuticals and feedstocks for second-generation bio-fuels (Himmel et al., 2007; Kim and Triplett, 2001; Reyna-Villasmil et al., 2007; Willats et al., 2006). In the non-lignified primary walls of growing cells, the main load-bearing components are cellulose microfibrils that are crossed-linked by non-pectic, non-cellulosic polysaccharides (Carpita and Gibeaut, 1993; Cosgrove, 2005). This network is embedded in a complex hydrated matrix of pectins and glycoproteins. The structure of cellulose is conserved across the plant kingdom, but the structures and relative amounts of other cell-wall components are highly variable, not just between plant species but also between organs, within tissues and even within cell-wall microdomains (McCann et al., 2007; Willats et al., 2001).

The vast majority of angiosperms, including the dicots and non-commelinid monocots, as well as gymnosperms, have primary walls that are designated ‘type I’, in which xyloglucan (XG) is the principal cross-linking polysaccharide, and is present in roughly equal amounts to cellulose (Carpita and Gibeaut, 1993; Trethewey et al., 2005). Type I walls are also typically rich in pectin and some contain relatively high levels of protein. In contrast, the commelinid monocots have distinctly different ‘type II’ walls, in which pectin is usually much less abundant and the principal cross-linking polysaccharides are glucuronoarabinoxylans (GAXs) rather than XG (Carpita and Gibeaut, 1993). One group within the commelinid monocots, the Poales (including the cereals and grasses of the Poaceae family), have walls that contain a homopolymer of (1→3),(1→4)-β-d-glucan (also known as mixed-linkage glucan or MLG; Buckeridge et al., 2004; Carpita, 1996; Smith and Harris, 1999; Threthewey et al., 2005; Urbanowicz et al., 2004). MLG has roles both as a structural element within the walls of growing cells and as a storage polysaccharide within the endosperm, and has been the subject of extensive research, not least because of its considerable economic importance and health benefits (Lazaridou and Biliaderis, 2007; Stone and Clark, 1992; Wood, 2007). Most studies of Poaceae walls indicate that MLG is virtually absent from meristematic cells, and its synthesis starts at the onset of cell elongation and peaks when elongation rates are highest. Once elongation has ceased, MLG is hydrolyzed and is much reduced in the walls of non-growing cells (Buckeridge et al., 2004; Carpita et al., 2001; Gibeaut et al., 2005). Low levels are retained in vascular tissues (Threthewey et al., 2005).

It is widely reported that, within the plant kingdom, MLG is a unique feature of Poales cell walls, and the appearance of MLG is thought to be a pivotal event in the evolution and diversification of higher plants (Buckeridge et al., 2004; Carpita and Gibeaut, 1993; Carpita and McCann, 2000; Harris, 2005). Using a recently developed high-throughput microarray-based technique for cell-wall analysis (Moller et al., 2007), we conducted an extensive survey of cell-wall composition across the land plants. This led to the discovery that MLG is in fact not unique to the Poales but is also highly abundant in the walls of the horsetail Equisetum arvense. We describe here the structure and tissue location of MLG in E. arvense, and consider the evolutionary significance of the discovery of MLG in this ancient plant lineage.

Results

Discovery of MLG in E. arvense

Comprehensive microarray polymer profiling (CoMPP) is a high-throughput technique that enables the cell-wall composition of diverse plant species to be rapidly analyzed (Moller et al., 2007). CoMPP was performed on cell-wall material (alcohol-insoluble residue, AIR) produced from stems, leaves and roots from a range of land plant species (Figure S1). As expected, this analysis indicated that MLG was abundant in AIR from Poaceae species but was not detectable, or was detected at very low levels, in most other species (data not shown). However, CoMPP also revealed that MLG was present in the stems, roots and leaves of E. arvense, and at similar levels to that in the Poaceae species Zea diploperennis (data not shown). To verify the presence of MLG in E. arvense, AIR from the stems of two examples from different locations in Denmark together with Arabidopsis thaliana (negative control) and Z. diploperennis was analyzed using a standard assay for the detection of MLG, in which barley (Hordeum vulgare) flour was also used as a positive control (McCleary and Glennie-Holmes, 1985; Figure 1). As expected, MLG was abundant in barley flour and Z. diploperennis but was not detected in A. thaliana. The assay also confirmed the initial CoMPP finding that MLG was present in E. arvense, and was more abundant in both samples tested than in Z. diploperennis stem samples. Further analysis of AIR taken from the top, middle and base regions of E. arvense stems indicated that MLG was progressively less abundant up the stem, and the amounts (as a percentage of total cell-wall material) of MLG detected in the top, middle and base regions were 2.9, 3.9, and 4.6%, respectively (data not shown).

Figure 1.

 Licheninase-based assay for MLG.
Relative levels of glucose released from alcohol-insoluble residues (AIR) of plant stem samples following digestion with a (1→3),(1→4)-β-d-glucan-specific endoglucanase (licheninase) and subsequent digestion with β-glucosidase. Samples tested were E. arvense (two examples, #1 and #2, from two locations in Sjælland, Denmark), A. thaliana (negative control) and Z. diploperennis. Barley flour was used as a positive control. Numbers on the right are the glucose content (% w/w) of the AIR samples calculated by reference to a glucose standard curve.

MALDI-TOF MS analysis of the structure of MLG in E. arvense

To analyze the structure of MLG in E. arvense, oligosaccharides produced by licheninase digestion of AIR were permethylated and subjected to MALDI-TOF MS analysis (Figure 2a). As a control, a parallel analysis was also performed on MLG derived from barley flour by licheninase digestion (Figure 2b). Consistent with previous analyses, the most abundant hexose oligomers in barley MLG had degrees of polymerization (DP) of 3 and 4, with DP3 units being the most abundant. A range of other hexose oligomers with DPs up to 16 were also detected (Figure 2b). However, the spectrum obtained for E. arvense MLG (Figure 2a) was markedly different to that of barley. In E. arvense, DP4 oligomers predominated, and only relatively low levels of DP2 and DP3 oligomers were detected. Oligomers of DP5-7 were also present at low levels, but, in contrast to barley, oligomers of DP > 7 were not detected. HPAEC analysis indicated that the DP2 hexoses in E. arvense were laminaribiose (data not shown).

Figure 2.

 MALDI-TOF MS analysis of licheninase products.
MALDI-TOF MS analysis of the permethylated β-glucan oligosaccharide products released by licheninase digestion of AIR from E. arvense stems (a) and barley flour (b). Numbers above the peaks refer to the degree of polymerization (DP) of hexose oligomers. Note that the most abundant oligomer in E. arvense had a DP of 4, whilst oligomers of DP3 were most abundant in the barley sample. The numbers in parentheses are masses automatically generated by the spectrometer.

Monosaccharide linkage analysis of E. arvense MLG oligomers

All previous analyses have indicated that MLG in the Poaceae are homopolymers consisting only of glucose, and the MALDI-TOF MS analysis of E. arvense MLG revealed that only hexoses were present (Figure 2a). However, as MALDI-TOF MS indicated that E. arvense MLG had a somewhat different block structure to that found in the Poaceae, we were interested to determine whether there were also other structural differences. Linkage analysis was performed on the oligosaccharides produced by licheninase digestion of E. arvense cell walls (Figure 3). Terminal-, 1,3- and 1,4-Glc linkages were detected with a molar ratio of approximately 1:1:2, confirming that E. arvense MLG consists primarily of cellotetraose units linked by single (1→3)-β linkages. No other monosaccharides or linkage types were detected.

Figure 3.

 Linkage analysis of the oligosaccharide digestion products of licheninase-treated E. arvense AIR.
Glycosyl linkage composition of the products produced by licheninase digestion of AIR from E. arvense stems (aliquots of the same products as used in Figure 2a). Numbers above the bars are the ratios of terminal-, 3- and 4-Glc where the lowest mean value (3-Glc) was set to 1 and other mean values adjusted accordingly.

Comprehensive monosaccharide linkage analysis of E. arvense cell walls

In contrast to the cell walls of Poaceae species, relatively little is known about the structure and composition of E. arvense walls. Therefore, to better understand the presence of MLG in E. arvense within the wider context of cell-wall composition, a comprehensive monosaccharide linkage analysis of E. arvense cell walls from the top, middle and base regions of stems was performed (Table 1). All the samples analyzed were rich (up to 53.3%) in 4-Glcp, indicative primarily of cellulose, as there were only low levels (1.4% maximum) of 4,6-Glcp that would indicate the presence of XGs. High levels (up to 37.4%) of 4-GalAp indicated that homogalacturonan (HG) is also abundant in E. arvense cell walls, and in all samples the degree of methyl esterification of GalAp was approximately 37.9%. Only trace levels of 2-Rhap and low levels of 2,4-Rhap were detected, suggesting that the pectin in these walls is rich in HG rather than rhamnogalacturonan. The 3,6 Galp linkage that is typical of the type II arabinogalactans was present at only trace levels, but 4-Galp and 5-Araf, which are indicative of the galactan- and arabinan-containing side chains of pectin, were present at up to 2.5% and 1.8%, respectively. Only trace levels of terminal Fucp were present, suggesting that any XG is probably non-fucosylated. The 3,4-Xylp and 2,4-Xylp linkages that are indicative of GAXs, and are typically abundant in Poales, were not detected and detected at low levels (1.2%), respectively, in E. arvense. Differences in composition between the various regions of the stems were generally modest, but there was notably more 4-Manp in the top of stems, and the levels of both 3-Glcp and 4-Glcp were highest in stem bases.

Table 1.   Glycosyl linkage composition of AIR samples from the top, middle and base regions of E. arvense stems
MonosaccharideDeduced linkageStem topStem middleStem base
  1. Mol% levels of monosaccharide linkages. Terminal Rha(p) was deduced from 1,5-di-O-acetyl-6-deoxy-2,3,4-tri-O-methyl-hexitol and similarly for the other sugars. Uronic acids and their esters were reduced to 6,6′-dideuterio neutral sugars before methylation. Trace amounts are <0.5%. ND, not detected; p, pyranose; f, furanose. Numbers in parentheses for the GalA(p) fractions correspond to the degree of methyl esterification.

Linkage composition (mol%)
 Rha(p)TerminalTraceTraceTrace
1,2-TraceTraceTrace
1,2,4-1.20.90.9
 Ara(f)Terminal2.21.91.7
1,5-1.41.80.7
 Ara(p)TerminalTraceTraceTrace
1,2-TraceNDND
2,3,4-TraceTraceTrace
 Fuc(p)TerminalNDTraceTrace
 Xyl(p)Terminal0.70.80.5
1,2-TraceNDTrace
1,4-1.51.21.4
1,2,4-1.10.71.2
 Glc(p)Terminal0.90.50.8
1,3-1.82.02.9
1,4-49.246.253.3
1,3,4-TraceTraceTrace
1,2,4-TraceTraceTrace
1,4,6-1.40.90.7
2,3,4,6-1.31.9Trace
 Gal(p)Terminal2.32.22.5
1,4-2.30.92.5
1,6-TraceNDTrace
1,3,4-TraceTraceTrace
1,3,6-TraceTraceTrace
2,3,4,6-TraceNDND
 GalA(p)1,4-23.7 (37.9)37.4 (37.9)25.6 (37.9)
 Man(p)1,4-5.02.72.9
1,4,6-TraceTraceND
2,3,4,6-0.9TraceTrace

Based on the observed monosaccharide linkages, it is possible to infer levels of known polysaccharides in E. arvense cell walls (Table 1). Some caution is required as E. arvense cell walls appear to differ significantly from the typical type I and type II walls on which such predictions are based (Kim and Carpita, 1992; Sims and Bacic, 1995). Nevertheless, E. arvense cell walls can be broadly characterized as containing MLG, some arabinans and type I galactans, being rich in cellulose and pectin, and containing relatively low levels of XG, GAXs and galacto(gluco)mannan.

CoMPP profiling of E. arvense cell walls

The CoMPP technique (Moller et al., 2007) was used to obtain an overview of the polysaccharide composition of the cell walls of E. arvense stems. For comparison, the same amount of AIR from A. thaliana and Z. diploperennis stems was also analyzed (Figure S2). (1→4)-β-d-galactan, (1→5)-α-l-arabinan and HG epitopes (recognized by mAbs LM5, LM6, JIM5 and JIM7) that are associated with pectin polymers were relatively abundant in 1,2-diamino-cyclo-hexane-tetra-acetic acid (CDTA) extracts of E. arvense cell walls, and at levels generally similar to that found in A. thaliana. This was in agreement with the high levels of pectin-associated monosaccharide linkages observed for E. arvense (Table 1 and Table S1). Galactan and arabinan epitopes were also detected at relatively high levels in NaOH-extracted AIR samples in E. arvense. Consistent with previous analysis of type II angiosperm walls, pectic epitopes were present at low relative levels in Z. diploperennis. In agreement with the licheninase-based assay shown in Figure 1, MLG was relatively abundant in NaOH-extracted stem material of E. arvense. Poaceae cell walls are rich in xylan-containing polymers, and this was reflected in the high signals obtained for Z. diploperennis using the anti-xylan mAb LM11. Relative to Z. diploperennis, signals obtained for mAb LM11 binding to E. arvense samples were low, and this was consistent with the low levels of GAXs inferred from linkage analysis (Table S1). Except in A. thaliana, very low signals were obtained using the anti-fucosylated XG mAb CCRC-M1, and this is in accordance with the trace levels of terminal fucose and 4,6-Glcp residues in the linkage analysis from E. arvense walls (Table 1 and Table S1).

Analysis of the location of MLG in E. arvense stems by immunofluorescence labelling

The location of MLG in E. arvense stems was assessed by indirect immunofluorescence labelling using an anti-MLG mAb and visualized by confocal microscopy (Figure 4). The chemical processing steps required for resin embedding and sectioning can sometimes have unpredictable effects on cell-wall epitopes (Knox, 2006; Willats and Knox, 2003). For this reason, in an initial study, we examined the top and middle regions of E. arvense stems, which are amenable to sectioning in their fresh, unfixed state. As shown in Figure 4(a,b), a series of longitudinal ridges is a characteristic feature of E. arvense stems. In both the top and middle of stems, mAb labelling was strong in the epidermis but weak or absent from cortical parenchyma cells within the main body of ridges. In stem tops, this unlabelled cortical parenchyma represents a greater unit area of the total tissue and this labelling pattern is consistent with the lower level of MLG in stem tops observed using CoMPP analysis and in the licheninase assay. Labelling was strong in pith parenchyma in both stem regions, and in middle stem regions it was also strong in the cortical parenchyma between vallecular canals and in sclerenchyma cells at the apex of ridges. However, labelling was weak or absent in cells in the vascular bundles of both stem top and middle regions. Control sections, for which primary antibody was omitted, were essentially free from labelling (Figure 4c).

Figure 4.

 Tissue distribution of MLG in E. arvense stems by immunofluorescence imaging.
Indirect immunofluorescence labelling of fresh transverse sections through the top (a) and middle (b) of E. arvense stems. Sections were probed with an MLG mAb (Meikle et al., 1994). A negative control with no primary antibody is also shown (c). ep, epidermis; pp, pith parenchyma; cp, cortical parenchyma; vb, vascular bundle; sc, sclerenchyma; vc, vallecular canal; cc, carinal canal. Scale bars = 200 μm.

Immunogold labelling of MLG in E. arvense stems

A more detailed study of the location of MLG in E. arvense stems was performed using immunogold labelling with the same anti-MLG mAb as used for the immunofluorescence analysis (Figure 5). Sections from glutaraldehyde-fixed tissues were taken through the top, middle and base regions of E. arvense stems, and sclerenchyma, parenchyma and cells in the vascular tissue were examined in all three stem regions. The toluidine blue-stained sections in Figure 5(a–c) are included to show the positions within stem cross-sections of the sclerenchyma, cortical parenchyma and vascular bundles from which the images shown in Figure 5(d–l) are taken. The thick walls of sclerenchyma cells from all three stem regions were heavily labeled, and in all cases the labelling extended throughout the thickness of walls but was less abundant in the middle lamellae (Figure 5d–f). Some sclerenchyma walls, such as that shown from the stem top in Figure 5(d), had a striated appearance, but the gold labelling was of the same density throughout the wall thickness. The thinner walls of cortical parenchyma cells from all three stem regions were also extensively labelled, although the density of labelling in walls in stem base cells (Figure 5i) was somewhat less compared to the walls of top and middle regions (Figure 5g,h). In the middle and base regions of the stems, gold labelling extended throughout walls to a region of denser material corresponding to the position of the middle lamella (Figures 5h,i). In stem tops, there was less labelling in the region of walls facing the middle lamella (Figure 5g), and labelling was absent from middle lamellae between cortical parenchyma cells in all three stem regions. In striking contrast to the sclerenchyma and parenchyma cells, there was no or very little labelling in the walls of cells in the vascular bundles (Figures 5j–l). The images shown in Figure 5(j–l) are from the walls of xylem cells, but labelling was also absent from the walls of all other cells examined in vascular bundles (data not shown). The specificity of binding of the mAb used to detect MLG was verified by using two controls. First, in selected sections, the mAb was co-incubated with barley flour which is a commonly used source of MLG (Figure 5m). Second, selected sections were treated with licheninase prior to labelling (Figure 5n). In both cases, labelling was essentially abolished by these treatments.

Figure 5.

 Immunogold based localization of MLG in E. arvense stems.
Glutaraldehyde-fixed transverse sections through the top (a, d, g, j), middle (b, e, h, k) and base (c, f, i, l) of E. arvense stems were stained with toluidine blue (a–c) or immunogold-labelled using an MLG mAb (d–l). For each stem region, the distribution of gold labelling was observed in sclerenchyma (d–f), cortical parenchyma (g–i) and vascular bundle tissue (j–l). The location of these three tissue types within stem cross-sections is indicated in the toluidine blue-stained sections (a–c). Two controls for antibody labelling are shown. In (m), the section was co-incubated with MLG-rich barley flour, and in (n) the section was pre-treated with licheninase. The section shown in (m) was from the stem base and the image is of cortical parenchyma tissue. The image shown in (n) is from the stem middle and the image is of sclerenchyma tissue. sc, sclerenchyma; cp, cortical parenchyma; vb, vascular bundle; vc, vallecular canal; cc, carinal canal; ml, middle lamella. Scale bars = 200 μm (a–c) and 0.5 μm (d–n).

Immunofluorescence labelling of other cell-wall components in E. arvense stems

To place the presence of MLG within the wider context of other cell-wall components, unfixed stem sections were also labelled with a range of mAbs with specificities for other cell-wall polymers, including HG, galactan, arabinan, xylan, and XG (Figure 6). HG with a relatively high degree of methyl esterification (recognized by mAb JIM7) was present in all cell types in the top (Figure 6a) and middle (data not shown) regions of stems. The JIM5 mAb binds preferentially to an HG epitope with a lower degree of methyl esterification, and JIM5 binding was more restricted than that of JIM7. In stem tops, binding was strongest in the epi- and endodermal cells, but was weak or absent in cortical and pith parenchyma (data not shown). However, in the middle of stems, JIM5 labelling was more widespread and binding was especially strong in the cortical parenchyma between the vallecular canals, in the endodermis, in phloem cells and in the lining of the central cavity (Figure 6g). The labelling patterns observed with mAbs with specificity for (1→4)-β-d-galactan (LM5) and (1→5)-α-d-arabinan (LM6) were essentially identical. In stem tops, binding was strong in the epidermis but weak in other tissues (LM5 binding is shown in Figure 6b). In the middle of stems, both these mAbs bound to cell walls in all tissues, except the region of cortical parenchyma between ridges (LM6 binding is shown in Figure 6f). In contrast to the pectic epitopes, the XG epitope recognized by mAb LM15 had a highly restricted location in both stem top and middle regions. In stem tops, apart from some occasional labelling in the cortical parenchyma, LM15 binding was limited to a ring-like binding pattern corresponding to the position of the vascular bundles (Figure 6c). LM15 binding to vascular cells was clearly visible in the sections through stem middles in which the lining of the central cavity was also labelled (Figure 6h). The mAb LM11 has broad specificity for xylan-containing cell-wall polysaccharides, and binds to unsubstituted xylan and to a lesser extent to GAXs (McCartney et al., 2005). In stem tops, LM11 bound to a layer of cortical parenchyma cells directly under the epidermis (but not to the epidermis itself) and to cortical parenchyma just outside the endodermis. Labelling was weak in other cortical and pith parenchyma cells (Figure 6d). In stem middles, LM11 bound strongly to the cortical parenchyma between stem ridges, to cells in the vascular bundles and to the lining of carinal canals. However, binding was very weak in the cortical parenchyma between vallecular canals and in the pith parenchyma (Figure 6e). One observation of particular note was that there was almost no overlap of the locations of MLG and xylan-containing epitopes, as shown for example by comparing Figures 4(b) and 6(e). Closer examination of the binding of XG (mAb LM15) and xylan (mAb LM11) probes to the vascular bundles revealed a subtle difference in the location of these epitopes (Figure 6i,j). LM11 bound to certain metaxylem and phloem cells and also to cells in the inner endodermis (Figure 6j). In contrast, LM15 binding was more restricted, and labelling was observed in a limited population of xylem and phloem cells but was absent from endodermal and inner endodermal cells (Figure 6i). Higher-magnification imaging of MLG labelling in vascular bundles confirmed that MLG was essentially absent from all cell types (Figure 6k). Labelling was also essentially absent from negative control sections in which primary antibody was omitted (Figure 6l).

Figure 6.

 Tissue distribution of cell-wall polymers other than MLG in E. arvense stems by immunofluoresence imaging.
Indirect immunfluorescence labelling of fresh transverse sections through the top (a–d) and middle (e–l) of E. arvense stems. Sections were labelled with monoclonal antibodies with specificity for homogalacturonan (HG) with higher (mAb JIM7) (a) or lower (mAb JIM5) (g) degrees of methyl esterification, for xylan (mAb LM11) (d, e, j), for (1→4)-β-d-galactan (mAb LM5) (b), for (1→5)-α-l-arabinan (mAb LM6) (f), for xyloglucan (mAb LM15) (c, h, i) and for MLG (k). A negative control with no primary antibody is also shown (l). ep, epidermis; vb, vascular bundle; cp, cortical parenchyma; en, endodermis; vc, vallecular canal; cc, carinal canal; ce, central cavity. The inset in (d) shows the apex of a stem ridge and illustrates that epidermal cells are unlabelled by mAb LM11. Scale bars = 100 μm.

Discussion

Land plants probably evolved from the charophycean green algae, and the transition from an aqueous to a gaseous environment triggered profound physical and structural changes – including the diversification of cell-wall structures (Graham, 1996; Karol et al., 2001; Kenrick and Crane, 1997). In some cases, the appearance of specific cell-wall components can be placed in a clear phylogenic context (Harris, 2005). For example, charophycean green algae cell walls do not contain XG or hydroxyproline, whilst xylan epitopes are ubiquitous in tracheophytes but absent in liverworts and mosses (Carafa et al., 2005; Popper and Fry, 2003). Detailed cross-kingdom analysis has revealed more graded phylogenetic differences. For example, the abundance of borate cross-linked rhamnogalacturonan II appears to have broadly increased during plant evolution, whilst that of glucuronic acid has decreased (Matsunaga et al., 2004; Popper and Fry, 2003). In general, however, basal plants do not have notably simpler cell walls than more advanced plants, and cell-wall evolution appears to be driven primarily by elaboration rather than dramatic innovation of polysaccharides. Despite enormous variations in the size, shape, habitat and physiology of land plants, their cell walls have conventionally been classified into only two groups, type I and type II. Until relatively recently, MLG was thought to be a unique feature of the type II walls of the Poaceae (Carpita, 1996; Carpita and Gibeaut, 1993). However, a wider survey of the Poales revealed that MLG is actually present in several other families (Smith and Harris, 1999; Trethewey et al., 2005), and that the walls of these plants, in common with the Poaceae, contain relatively low levels of pectic polymers and high levels of GAXs.

The discovery that MLG is highly abundant in E. arvense cell walls is significant for two reasons: (i) it is the first conclusive demonstration of this polysaccharide in a land plant outside of the Poales, and (ii) in E. arvense, MLG is a component of a novel cell-wall architecture that is distinctly different from either type I or type II walls. Instead of co-existing with high levels of GAXs as in type II walls, in E. arvense MLG occurs together with high levels of pectin and cellulose that are comparable to those found in type I dicot walls, but with low levels of XG. These findings have implications for our understanding of the functional relationships that are possible between cell-wall polymers, the modulations in cell-wall structure that accompany growth, and also for cell-wall evolution. In a contemporary independent study, Fry et al. (2008) also demonstrated the presence of MLG in four other Equisetum species.

Many details of land plant evolution are disputed, but whatever model is considered, E. arvense and the Poales are phylogenetically distant. As angiosperms, the Poales are considered some of the most advanced, highly evolved plants. In contrast, the 15 extant Equisetum species are now the only link to a group of ancient land plants that were once ecologically prominent (Des Marais et al., 2003; Guillon, 2007). Our CoMPP survey indicated that MLG is not present in a range of land plants that are more basal than the Poales but less basal than Equisetum. Previous studies of a range of vascular plant taxa also indicated that MLG was present in the Poales but absent from other more basal species (Popper and Fry, 2004). These findings indicate that MLG is extremely rare in the plant kingdom, and the occurrence of MLG in E. arvense reflects the unusual and isolated phylogenetic status of the genus.

As in the Poaceae, MLG in E. arvense is a homopolymer consisting only of 1,3-β- and 1,4-β-linked glucose residues. However, there are differences in the glucan block structures of Poaceae and E. arvense MLG. In cereal species, the molar ratio of DP3:DP4 units in MLG ranges from approximately 1.5:1 to 4.5:1, and longer oligosaccharide units of up to DP >13 may also be present (Fincher and Stone, 2004; Lazaridou and Biliaderis, 2007; Wei et al., 2006). In contrast, DP4 units were the most abundant oligomers in MLG from E. arvense, and, based on MALDI-TOF analysis, occurred at up to 10 or 20 times the abundance of the DP3 units. Furthermore, oligomers with DPs >7 were not detected. A DP2 oligomer that did not co-elute with cellobiose by HPAEC is proposed to be laminaribiose. These differences in structure are likely to result in different structural properties (Li et al., 1996; Woodward et al., 1988), and may suggest that MLG does not necessarily perform the same role in cell walls of Equisetum as in Poales species. In the Poaceae, MLG is associated with growing cells and is largely absent in mature tissues where growth has ceased (Buckeridge et al., 2004; Carpita et al., 2001; Gibeaut et al., 2005). This is not the case in E. arvense; MLG was abundant in both young and old regions of stems and was somewhat more abundant in mature non-growing stem base regions. The cell walls and structural roles of MLG have been extensively characterized in Poaceae species. It has been suggested that MLG acts as a tether between cellulose microfibrils, in much the same way as XG does in type I walls (Huber and Nevins, 1982). In this model, it is thought that growth is mediated by cell-wall loosening resulting from cleavage of MLG by endo- and exoglucanases that appear in cell walls during growth (Inouhe et al., 2000; Thomas et al., 2000). More recently, however, this view has been challenged, and an alternative model has been suggested in which highly substituted GAXs rather than MLG are the principal cross-linking polysaccharides (Buckeridge et al., 2004). In this scenario, MLG and GAXs with a low degree of substitution are proposed to form a co-extensive network that tightly coats cellulose microfibrils. However, linkage analysis suggested that GAXs were not abundant in E. arvense cell walls, and immunolabelling with probes for MLG and xylan-containing polysaccharides suggested an almost complete spatial separation of these polymers. Whilst labelling of MLG was strong in all tissues apart from the parenchyma between longitudinal stem ridges and the vascular bundles (Figure 4b), the opposite was true for xylan-containing polysaccharides (Figure 6e). The anti-xylan mAb used binds to a range of xylan-containing polysaccharides with high and low degrees of substitution, but it is possible that an unusual GAX polymer exists in E. arvense that is not labelled. Linkage analysis also suggested that XG is not abundant in E. arvense walls. Furthermore, immunofluorescence labelling indicated that XG had a highly restricted location and was only detected in vascular cells and the lining of the central cavity. However, it is possible that a novel XG polymer is present in E. arvense walls that was unlabelled, or that levels of XG in other cell types were below detectable levels. Nevertheless, taken together, our findings suggest that, in E. arvense, MLG is not generally co-extensive with either GAXs or XG but is abundant in many primary and secondary walls of non-vascular cells.

Plant cell walls are one of the most abundant sources of biomass on earth, and provide a wealth of materials ranging from unrefined bulk products such as timber to highly defined oligosaccharides with specific pharmacological activities. However, the vast majority of plant cell walls have not been analyzed, and the unexpected presence of MLG in E. arvense was revealed only because the cell walls of a large number of phylogenetically diverse plants were sampled. This study indicates that large-scale surveying of cell walls across the plant kingdom may yield valuable new information that contributes to our understanding of cell-wall evolution and possibly also novel plant products.

Experimental procedures

Plant material

Stems, leaves and roots were harvested from living plants in the Copenhagen Botanical Gardens. An additional sample of E. arvense was collected from Farum (approximately 15 miles north of Copenhagen). All samples were stored at −80°C until processed. In the case of E. arvense stems, ‘top’ refers to the three uppermost internodes, ‘bottom’ to the three lowest internodes, and ‘middle’ to three internodes equidistant between the base and apex of stems.

Preparation of cell-wall material

Alcohol-insoluble residue (AIR) was prepared essentially as described previously (Fry, 1988).

Comprehensive microarray polymer profiling (CoMPP) analysis

CoMPP was performed as previously described (Moller et al., 2007) with minor modifications (see Appendix S1). MLG was detected using an anti-MLG mAb (Meikle et al., 1994).

Licheninase-based β-glucan assay

A standard β-glucan detection kit (Megazyme; http://www.megazyme.com) was used that is based on the release of glucan oligomers produced by digestion of AIR with licheninase (McCleary and Glennie-Holmes, 1985). The assay is based on the detection of glucose released by the digestion of cell-wall material with licheninase, and the weight of MLG as a percentage of total cell-wall material can be calculated by reference to a standard curve. The procedure was performed as described in the manufacturer’s protocol except that starting material was 5 mg AIR. The percentage of AIR (w/w) that was MLG was calculated as described.

Linkage analysis of MLG oligosaccharides

Methylated oligosaccharides were hydrolysed (2 m trifluroacetic acid), reduced and acetylated as described by Sims and Bacic (1995). The permethylated peracetylated alditols were then analyzed by GC-MS as previously described (Sims and Bacic, 1995).

Linkage analysis of total cell-wall material from E. arvense

Monosaccharide linkage analysis was performed as described previously (Kim and Carpita, 1992; Sims and Bacic, 1995). Briefly, 5 mg of AIR was prepared from the top, middle and base regions of E. arvense stems. Carboxyl reduction was carried out in order to analyze uronic acids, and samples were methylated prior to linkage analysis as described above.

Fluorescence microscopy

Transverse sections of fresh E. arvense stems were cut using a vibrating knife microtome (Vibratome; http://www.vibratome.com). Sections were incubated for 1 h in primary antibody diluted 1:10 in PBS (140 mm NaCl, 2.7 mm KCl, 10 mm Na2HPO4, 1.7 mm KH2PO4, pH 7.2) containing 5% w/v fat-free milk powder (MP/PBS). mAbs were used with specificity for XG (LM15, Leroux et al., 2007), HG (JIM5 and JIM7, Clausen et al., 2003), xylan-containing polymers (LM11, McCartney et al., 2005) and MLG (Meikle et al., 1994). After washing in PBS, sections were incubated for 1 h with fluorescein isothiocyanate (FITC)-conjugated secondary antibody (either anti-rat-FITC or anti-mouse-FITC as appropriate, both Sigma, http://www.sigmaaldrich.com/). After washing in PBS, sections were mounted in anti-fade agent (CitiFluor; http://www.citifluor.co.uk) and examined using a laser scanning confocal microscope (LSCM510, Carl Zeiss, http://www.zeiss.com/).

Preparation of Equisetum stems for light and electron microscopy

E. arvense stems were fixed in 2.5% v/v glutaraldehyde (EM grade) in PBS and stored at 4°C. Samples were washed three times in PBS and dehydrated in a graded ethanol series. The tissue was slowly infiltrated with LR White resin (London Resin Company; http://www.londonresin.com) over several days. Individual pieces of stem were placed in gelatin capsules, filled with fresh resin and polymerized overnight at 55°C.

Sectioning and toluidine blue staining

Transverse sections (800 nm) were cut from resin blocks containing stem pieces on a microtome (Ultracut R, Leica; http://www.leica-microsystems.com). Sections were collected and dried onto glass microscope slides and stained with a 2% v/v aqueous toluidine blue solution for 30 sec before washing and drying on a hotplate.

Transmission electron microscopy and immunogold labelling

Ultra-thin sections (80 nm) were cut from resin blocks (see above) on a microtome (Ultracut R) using a diamond knife and collected on 100 and 200 mesh gold grids. Sections were blocked in 1% w/v BSA in PBS for 30 min before treatment with an anti-MLG mAb (Meikle et al., 1994) in PBS for 1 h at room temperature and again overnight at 4°C. Sections were washed three times in PBS and twice in blocking buffer, and incubated for 1 h in a 1:20 dilution of goat anti-mouse secondary antibody conjugated to 18 nm gold in blocking buffer (Jackson ImmunoResearch; http://www.jacksonimmuno.com). All grids were washed twice with PBS and several times in milliQ water (Millipore; http://www.millipore.com) before staining in 2% w/v aqueous uranyl acetate. The sections were viewed on a BioTwin transmission electron microscope (FEI; http://www.fei.com) and images were captured on a multiscan digital camera (Gatan; http://www.gatan.com). As controls for MLG binding, either diluted primary antibody was mixed 1:1 with a 1 mg ml−1 solution of (1→3,1→4)-β-d-glucan from barley endosperm (Biosupplies; http://www.biosupplies.com.au) and left for 1 h before applying it to the sections or sections were treated with a specific (1→3),(1→4)-β-d-glucan endohydrolase prior to incubation with the primary antibody.

Other methods

The methodology for MALDI-TOF MS analysis of oligosaccharides produced by licheninase digestion of AIR and for estimation of the polysaccharide content of E. arvense cell walls are give in Appendix S1.

Acknowledgements

Our thanks to Ulla Christensen (Copenhagen University, Denmark) for help with licheninase assays, to the Danish Research Agency for funding for IS, to Paul Knox (University of Leeds, UK) for the LM15 antibody, and to Bruce Stone (La Trobe University, Australia), Peter Ulvskov (Århus University, Denmark) and Henrik Scheller (Copenhagen University, Denmark) for insightful discussions. SW, MSD and AB acknowledge the support of a Grains Research and Development Corporation Functional Genomics grant on the End-Use Quality of Cereals. FAP and AB acknowledge the support of an Australian Research Council-Linkage Projects grant (LP0667986).

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