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Physiology and Biochemistry |
3),(14)-ß-d-Glucans in the cell walls of the Poales (sensu lato): an immunogold labeling study using a monoclonal antibody1
2School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand; 3Plant Research Laboratory, New York Botanical Garden, Bronx, New York 10458 USA
Received for publication February 13, 2005. Accepted for publication June 30, 2005.
ABSTRACT
(1
3),(14)-ß-Glucans had previously been detected in nonlignified cell wall preparations of only the Poaceae and five other families in the graminoid clade of the Poales (s.l.). Cell walls of vegetative organs of 12 species in nine families of the Poales (s.l.) were examined by immunogold labeling using a monoclonal antibody to (13),(14)-ß-glucans. Three types of wall-labeling patterns were identified depending on the density of labeling of the nonlignified walls of epidermal and parenchyma cells and the lignified walls of sclerenchyma fibers and xylem tracheary elements: type 1 in Poaceae and Flagellariaceae, type 2 in Restionaceae and Xyridaceae, and type 3 in Cyperaceae and Juncaceae. Type 1 had the heaviest labeling of nonlignified walls and type 2 the heaviest labeling of lignified walls. Type 3 had the least wall labeling, with only very light labeling of nonlignified and lignified walls. No labeling was found over walls of Typhaceae, Sparganiaceae, or Bromeliaceae. The results are discussed in relation to Poales phylogeny.
Key Words: (13),(14)-ß-glucan • immunogold labeling • phylogenetics • plant cell walls • Poaceae • Poales • transmission electron microscopy
Cell walls of all angiosperms contain cellulosic microfibrils embedded in a matrix of noncellulosic polysaccharides. Proteins, glycoproteins, and phenolic components, including lignin, may also be present. The structures and proportions of the different noncellulosic polysaccharides vary with the taxon, particularly in nonlignified walls. This variation can be placed in the context of angiosperm phylogeny (Harris, 2000
, 2005
), which over the last decade has been inferred from DNA sequences such as the plastid genes rbcL and atpB and nuclear 18S rDNA (Soltis et al., 1999
; Savolainen and Chase, 2003
), and has resulted in a new classification of the angiosperms (APG, 1998
; APG II, 2003
). These phylogenetic studies have identified within the monocotyledons a major monophyletic group known as the commelinids (APG II, 2003
). This group comprises the unplaced Dasypogonaceae and three major clades: the Arecales (palms), the Zingiberales-Commelinales, and the Poales (Chase et al., 2000
; APG II, 2003
). The Poales is a broadly defined order of 17–20 families. Circumscription of the order varies with the recognition of three monogeneric families, Sparganiaceae, Lyginiaceae, and Hopkinsiaceae: Sparganiaceae is sometimes included in Typhaceae (APG II, 2003
) and Lyginiaceae and Hopkinsiaceae are segregates of Restionaceae (Briggs and Johnson, 2000
). The graminoid clade (GPWG, 2001
; Bremer, 2002
), comprising the Poaceae (grasses and cereals) and six related families, is identical to the Poales, as defined by Dahlgren et al. (1985)
.
The nonlignified walls of the commelinids, unlike those of the other monocotyledons (noncommelinids), contain ester-linked ferulic acid (Harris and Hartley, 1980
; Rudall and Caddick, 1994
; Harris, 2000
, 2005
). In the nonlignified walls of the Poaceae (Mueller-Harvey et al., 1986
) and of Ananas comosus (pineapple) (Bromeliaceae) (Smith and Harris, 2001
), the ferulic acid is ester-linked to glucuronoarabinoxylans (GAXs). These polysaccharides comprise only a minor component of the noncellulosic polysaccharides of the nonlignified walls of species in the basal Arecales (palms) clade (Carnachan and Harris, 2000
), but are a major component of the nonlignified walls of species in the other commelinid clades, particularly the Poales (Harris et al., 1997
; Smith and Harris, 1999
; Harris, 2000
, 2005
).
Within the Poales, the walls of the Poaceae have been extensively studied because of the economic importance of the grasses and cereals. In addition to feruloylated GAXs, the nonlignified walls of the Poaceae contain variable proportions of (13),(14)-ß-glucans and small proportions of xyloglucans and pectic polysaccharides (Carpita, 1996
; Smith and Harris, 1999
; Harris, 2000
, 2005
). In the lignified walls of the Poaceae, the major noncellulosic polysaccharides are GAXs, although the degree of substitution of the xylan main chain is less than in the GAXs of the primary cell walls.
Of the various polysaccharides in Poaceae walls, the (13),(14)-ß-glucans, unbranched ß-glucans containing (13) and (14) links, are particularly interesting for a variety of reasons. For example, the (13),(14)-ß-glucans in the walls of cereal grains, such as Hordeum vulgare (barley), are of considerable applied importance. In brewing, they affect the production and quality of beer, and when H. vulgare grain is used as feed for chickens, they affect their productivity (Stone and Clarke, 1992
). (13),(14)-ß-Glucans have been implicated in the control of cell expansion (Carpita, 1996
; Buckeridge et al., 2004
). (13),(14)-ß-Glucans are also interesting because they occur only in the walls of the Poaceae and related families of the graminoid clade of the Poales (Smith and Harris, 1999
). Smith and Harris (1999)
isolated wall preparations from organs containing mostly nonlignified walls and determined the concentrations of (13),(14)-ß-glucans in these preparations by a direct and specific enzymatic assay using the (13),(14)-ß-glucan endohydrolase from Bacillus subtilis (McCleary and Codd, 1991
). In addition to the Poaceae, (13),(14)-ß-glucans were found in the wall preparations from the Anarthriaceae, Centrolepidaceae, Ecdeiocoleaceae, Flagellariaceae, and Restionaceae; material from the Joinvilleaceae was not available for analysis. Popper and Fry (2004)
confirmed the presence of (13),(14)-ß-glucans in nonlignified walls of Flagellariaceae (Flagellaria guineensis).
Immunogold labeling using monoclonal antibodies is a very effective way of detecting and determining the location of specific wall polysaccharides, both within particular walls and among walls of different cell types (Willats et al., 2000
). A monoclonal antibody that binds specifically to (13),(14)-ß-glucans has been raised by Meikle et al. (1994)
and has been used in conjunction with immunogold electron microscopy to determine the occurrence and distribution of these polysaccharides in the walls of the Poaceae, including those of developing Oryza sativa (rice) endosperm (Brown et al., 1997
), Triticum aestivum (wheat) aleurone (Meikle et al., 1994
), Zea mays (maize) coleoptiles (Carpita et al., 2001
) and the first leaf, coleoptile and root tip of H. vulgare (barley) seedlings (Trethewey and Harris, 2002
). In the present study, we used this method to determine the occurrence and location of these polysaccharides in the walls of vegetative organs of species in nine families of the Poales (Chase et al., 2000
; APG II, 2003
), including the Poaceae.
MATERIALS AND METHODS
Plant material
The species that were used, their sources, and the organs examined are shown in Table 1. Seedlings of Lolium multiflorum were grown in seed mix in a glasshouse at the University of Auckland. Vouchers of Orectanthe sceptrum (L.M. Campbell et al. s.n.) and Aratitiyopea lopezii (L.M. Campbell et al. 813) are at the New York Botanical Garden.
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Samples of O. sceptrum and A. lopezii were fixed in the field and, for safety, formaldehyde was not used in the fixative solution. Instead, the samples were cut and fixed in 3% glutaraldehyde in 100 mM NaOH-PIPES buffer (pH 7.2), washed in buffer, and dehydrated to 80% ethanol for transport to New Zealand. Upon arrival, some of the samples were rehydrated by washing them successively in 70, 50, and 30% ethanol, followed by water. Some of these samples were used for histochemical studies (described later), and others were postfixed using osmium tetroxide, dehydrated, and embedded as described before. Other samples in 80% ethanol were washed successively in 90, 95, and 100% ethanol and were embedded as described, without postfixation.
To determine whether immunogold labeling was affected by the composition of the primary fixative, samples of immature stem of Leptocarpus similis were fixed in the solution containing both glutaraldehyde and formaldehyde and compared with samples fixed in the solution containing only glutaraldehyde. The samples were then postfixed with osmium tetroxide, before embedding, sectioning, and immunolabeling. To determine whether immunogold labeling was affected by postfixation in osmium tetroxide, samples of Lolium multiflorum and Restio tetraphyllus were first fixed in the glutaraldehyde-formaldehyde solution, and then either embedded directly or postfixed before embedding, sectioning, and immunogold labeling.
Light microscopy
Sections (1–1.5 µm thick) of plant material embedded in resin were cut with a diamond or glass knife using an ultramicrotome (LKB Produkter AB, Bromma, Sweden) and stained with 1% w/v toluidine blue O in 100 mM sodium phosphate buffer (pH 6.8) for 2 min, washed in water, and mounted in Entellan mounting medium (ProSciTech, Thuringowa, Queensland, Australia).
For histochemical studies, fresh transverse sections were cut, by hand using a razor blade, midway along the chosen organs of each species. Lignin was detected by the red color reaction given by phloroglucinol-HCl; sections were immersed in a freshly prepared solution obtained by mixing 1 mL of 2% (w/ v) phloroglucinol in 95% (v/v) aqueous ethanol with 2 mL of 10 M HCl (Harris et al., 1980
). Sections were also stained with toluidine blue O (0.05%, w/v, in 0.02 M sodium benzoate buffer, pH 4.4) (Feder and O'Brien, 1968
). Prepared in this way, toluidine blue O stains polychromatically: lignin and other phenolic components stain green or blue-green, whereas polyanions such as pectic homogalacturonans stain pink or purple (O'Brien and McCully, 1981
). Autofluorescence in UV radiation due to lignin and/or ester-linked ferulic acid in the cell walls was examined using sections mounted in H2O and in 0.1 M NH4OH (Harris and Hartley, 1976
, 1980
; Smith and Harris, 1995
).
Bright-field and fluorescence microscopy was carried out using a Carl Zeiss F2 microscope (Oberkochen, Germany) equipped for epifluorescence and fitted with a 20-W halogen quartz lamp and a high-pressure mercury-vapor lamp (HBO 50). For UV fluorescence microscopy, the following filters were used: a G365 excitation filter, a FT395 chromatic beam splitter, and a LP420 barrier filter (Zeiss filter set 48 77 02). Photomicrographs of bright-field images were recorded using a Leica DMRE microscope (Leica Microsystems, Wetzlar, Germany) fitted with a Leica DC500 digital camera.
Immunogold labeling
Ultrathin sections (70–100 nm thick) were cut and collected on Parlodion-coated, nickel grids (200 mesh). The sections were preincubated for 15 min at room temperature with phosphate-buffered saline (PBS) (50 mM sodium phosphate buffer, pH 7.2, 0.15 M NaCl) containing 1% (w/v) bovine serum albumin (BSA) (fraction V) (Sigma, St. Louis, Missouri, USA) (PBS/BSA) to block any nonspecific binding sites. The sections were then incubated for 1 h at room temperature on drops of the monoclonal antibody BG 1 (Biosupplies Australia, Parkville, Victoria, Australia) in PBS/ BSA; the antibody concentration was 20 µg · mL–1 as protein. The monoclonal antibody is specific for (13),(14)-ß-glucans; it had no cross-reactivity against (13)-ß-glucan or cellulose (Meikle et al., 1994
). After washing with PBS/BSA (5x, 1 min per wash), the sections were incubated for 1 h at room temperature with a goat antimouse IgG (H and L) conjugated to 15 nm colloidal gold (Electron Microscopy Sciences, Fort Washington, Pennsylvania, USA) at 1 : 25 dilution in PBS/BSA. The sections were then washed with water (5x, 2 min per wash), stained with uranyl acetate (5% w/v in 50% ethanol) for 20 min and again washed (once with 50% ethanol for 30 s, twice with water, 2 min per wash). Sections were then stained with a solution of lead citrate (Reynolds, 1963
) for 2 min and washed with water (5x, 2 min per wash). After drying, the sections were examined with a transmission electron microscope (either a Philips Tecnai 12 electron microscope operating at 120 kV, or a Philips CM12 operating at 100 kV; Philips, Eindhoven, Netherlands).
Controls were done as follows: (1) omitting the incubation with the monoclonal antibody, (2) omitting the incubation with the goat antimouse IgG, (3) replacing the monoclonal antibody with the same concentration of a mouse monoclonal antibody specific for the rotavirus nonstructural protein 4.
RESULTS
Cell types and wall histochemistry
Figure 1 (a–i) shows bright-field light micrographs of sections of organs of the species examined that showed immunogold labeling of the walls with the (13),(14)-ß-glucan antibody. The walls of the different cell types were classified as lignified if they stained red for lignin with phloroglucinol-HCl. Walls that were positive for lignin also fluoresced blue in UV radiation, when mounted in both water and 0.1 M ammonium hydroxide, and stained blue-green with toluidine blue O, which is also consistent for lignin. Walls that gave no color reaction with phloroglucinol-HCl stained pink-purple with toluidine blue O, consistent with them being nonlignified. These walls fluoresced blue in UV radiation, when mounted in water, but green when mounted in 0.1 M ammonium hydroxide, indicating the presence of ester-linked ferulic acid (Harris and Hartley, 1976
, 1980
; Smith and Harris, 1995
).
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The walls of sieve tube elements and their associated companion cells were all nonlignified, as were the thick walls of multicellular hairs seen in transverse sections of the stem of L. similis (Fig. 1e).
Immunogold labeling
The presence, density, and location of gold label over the walls of epidermal (outer periclinal walls) and particular types of parenchyma cells (see above), which were nonlignified, and the walls of xylem tracheary elements and sclerenchyma fibers, which were lignified, are summarized in Table 2. Species with labeled walls were divided into three types (1–3), depending on the density and location of the labeling (Table 2).
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The lignified walls of the xylem tracheary elements and sclerenchyma fibers in the F. indica and L. multiflorum leaves were all labeled in a similar way (Table 2). Labeling was light over the compound middle lamella (comprising the middle lamella and the primary walls) of two contiguous xylem tracheary elements (Fig. 2f) and two contiguous sclerenchyma fibers (Fig. 2g), but the secondary walls of these cells were only very lightly labeled. These secondary walls were also only very lightly labeled where the cells adjoined parenchyma cells (Fig. 2h).
Type 2 wall-labeling pattern
In the epidermal cells (both adaxial and abaxial in leaves), the thick outer periclinal walls of Leptocarpus similis and Restio tetraphyllus (Restionaceae), and Aratitiyopea lopezii (Xyridaceae) were lightly labeled (Table 2). These walls in O. sceptrum were only very lightly labeled (Fig. 4a). In L. similis (Fig. 3a) and A. lopezii (Fig. 4b), the light labeling of these walls was confined to two thirds of their widths, extending from the plasma membrane. In R. tetraphyllus, the light labeling of these walls was confined to a region adjacent to the plasma membrane, approximately one fifth of the width of the cell wall (Fig. 3b, arrow). In all species, except O. sceptrum, the anticlinal walls between adjoining epidermal cells (in leaves both abaxial and adaxial) were thin and only very lightly labeled adjacent to the plasma membrane (Fig. 3d). In O. sceptrum, these walls in both the abaxial and adaxial epidermis were extremely thick (
5 µm), but the labeling was also very light with particles scattered over the entire wall (Fig. 4c, arrow). The thick, nonlignified walls of the stem hair cells in L. similis were moderate to heavily labeled over about four fifths of the width of the wall (Fig. 3c); a band under the cuticle was unlabeled (Fig. 3c, arrow).
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In all the species, the nonlignified walls of xylem parenchyma, phloem parenchyma, phloem sieve tube elements and associated companion cells also had similar labeling. However, in all species where the nonlignified walls of parenchyma cells adjoined xylem tracheary elements (Fig. 4f) or sclerenchyma fibers (Fig. 4g), the parenchyma walls were unlabeled, except for the occasional gold particle.
In all type 2 species, adjoining sclerenchyma fibers and adjoining xylem tracheary elements had compound middle lamellae that were virtually unlabeled, with only an occasional gold particle (Figs. 3h–j; 4g–i). By contrast, the thick secondary walls of sclerenchyma fibers (Fig. 3h, i) and xylem tracheary elements (Fig. 3j) in L. similis and R. tetraphyllus were moderately labeled. However, in A. lopezii in both xylem tracheary elements (Fig. 4f) and sclerenchyma fibers (Fig. 4h), the secondary walls were light to moderately labeled. In O. sceptrum, the secondary walls of sclerenchyma fibers were also light to moderately labeled (Fig. 4g), but those of xylem tracheary elements were only lightly labeled (Fig. 4i).
Type 3 wall-labeling pattern
This was shown by the walls of Cyperus haspan and Baumea juncea (Cyperaceae), and Juncus inflexus (Juncaceae) (Table 2). In all of these species, the nonlignified, thick, outer periclinal walls of the epidermal cells were only very lightly labeled, with the label scattered over the walls (Fig. 5a–c). The thin, anticlinal walls between adjoining epidermal cells and the inner periclinal walls were unlabeled except for the occasional gold particle adjacent to the plasma membrane (Fig. 5d). In all species, the nonlignified walls of the chlorenchyma (Fig. 5e), central pith parenchyma (Fig. 5f, g), phloem parenchyma, xylem parenchyma, phloem sieve tube elements and associated companion cells, all showed only the occasional gold particle adjacent to the plasma membrane. Similar labeling was also found over the walls of the cortical parenchyma cells, including those of B. juncea adjacent to the sclerenchyma fibers, which were very weakly lignified (data not shown).
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Although in these type 3 species wall labeling was at the most only very light, this labeling was consistently present in all replicates examined, even with further washing after incubation with the primary and secondary antibodies.
No wall labeling
No labeling was found over any of the walls of the nonlignified or lignified walls of the other Poales species examined: Typha orientalis (Typhaceae), Sparganium subglobosum (Sparganiaceae), and Aechmea fasciata (Bromeliaceae). In addition, there was no labeling over any of the walls of the palm Phoenix canariensis (Arecaceae, order Arecales).
Controls
No labeling was found over any of the walls after control experiments in which the (13),(14)-ß-glucan antibody was omitted and in which this antibody was replaced by a monoclonal antibody for the rotavirus nonstructural protein 4. No differences in labeling location or density were found between sections from samples fixed only with the primary fixative and those with both the primary fixative and osmium tetroxide, or between samples fixed with a mixture containing 2% (w/v) formaldehyde and 1% (v/v) glutaraldehyde and those with only 3% (v/v) glutaraldehyde (both postfixed in osmium tetroxide).
DISCUSSION
As far as we are aware, this is the first report of the immunogold labeling of walls with an antibody to (13),(14)-ß-glucans in taxa outside the Poaceae. Our study showed the presence of (13),(14)-ß-glucans in both nonlignified and lignified walls of vegetative organs in six of the nine Poales families examined, extending the distribution from that previously found using an enzymatic assay (Smith and Harris, 1999
). However, the density and location of wall labeling varied, with three wall-labeling patterns identified (Table 2): the Poaceae and Flagellariaceae had the heaviest labeling of nonlignified walls (type 1); the Restionaceae and Xyridaceae had the heaviest labeling of lignified walls (type 2); and the Cyperaceae and Juncaceae had the least wall labeling, with only very light labeling of nonlignified and lignified walls (type 3).
Labeling of the nonlignified walls of the type 1 species Lolium multiflorum and Flagellaria indica was similar to that found in the nonlignified walls of the first leaf and coleoptile of Hordeum vulgare (Trethewey and Harris, 2002
) and the coleoptile of Zea mays (Carpita et al., 2001
); most of the walls were heavily and evenly labeled. However, in the outer periclinal walls of the epidermal cells of the leaves of F. indica and H. vulgare, and of the coleoptiles of both H. vulgare and Z. mays, there was an unlabeled band beneath the cuticle. Furthermore, in H. vulgare coleoptiles, the walls of the parenchyma cells immediately below the epidermis were labeled only adjacent to the plasma membrane (Trethewey and Harris, 2002
). In contrast to type 1, the labeling density of the nonlignified walls of species in type 2 was only very light or light, and in type 3 was either very light or virtually absent, with the labeling of the parenchyma walls being only adjacent to the plasma membrane. Interestingly, a number of authors have also reported labeling of walls adjacent to the plasma membrane by antibodies to various epitopes of pectic polysaccharides (Freshour et al., 1996
; Jones et al., 1997
; Matoh et al., 1998
; McCartney et al., 2000
).
The gold labeling of the nonlignified walls of the Poales species can be compared with the concentrations of (13),(14)-ß-glucans, determined enzymatically, in preparations of nonlignified walls from the same species and organs. The heavy labeling densities of the nonlignified walls of type 1 species corresponds to the finding of 1.8% or more of (13),(14)-ß-glucans in the wall preparations: Lolium multiflorum (leaf, 1.8%, Smith and Harris, 1999
); Hordeum vulgare (coleoptile, 6.4%, and first leaf, 4.8%, Trethewey and Harris, 2002
); Flagellaria indica (immature stem, 3.2%, Smith and Harris, 1999
) and (leaf, 3.0%, Trethewey and Harris, unpublished data). The concentrations of (13),(14)-ß-glucans in nonlignified wall preparations of Xyridaceae have not been reported, but they have for four species of Restionaceae, two of which (L. similis and R. tetraphyllus) were used in the present study (Smith and Harris, 1999
). The low labeling densities over nonlignified walls in these species correspond to low concentrations of (13),(14)-ß-glucans (0.1% or less) in wall preparations: L. similis (immature stem, 0.1%); R. tetraphyllus (immature stem, trace); Chondropetalum tectorum and Ischyrolepis subverticellata (seedling stems, 0%). The labeling densities over the nonlignified walls of the type 3 species, which are in Cyperaceae and Juncaceae, were, at most, only very light, indicating trace amounts of (13),(14)-ß-glucans. The (13),(14)-ß-glucan concentrations in nonlignified wall preparations of species in these families were not determined by Smith and Harris (1999)
. However, other researchers were unable to detect these polysaccharides in nonlignified wall preparations from Cyperus alternifolius (Cyperaceae) and Juncus greenei (Juncaceae) (Stinard and Nevins, 1980
), and from C. esculentus, C. papyrus, and J. effusus (Popper and Fry, 2004
). Not finding wall labeling in the other families of Poales (sensu APG II, 2003
) we examined (Bromeliaceae, Typhaceae, and Sparganiaceae) is consistent with the inability of Stinard and Nevins (1980)
to detect (13),(14)-ß-glucans in nonlignified wall preparations from Billbergia nutans (Bromeliaceae) and Typha latifolia (Typhaceae). Moreover, no (13),(14)-ß-glucans were detected by Smith and Harris (1995)
in a nonlignified wall preparation from Ananas comosus (pineapple) (Bromeliaceae). In comparing our densities of immunogold labeling in nonlignified walls and the concentrations of (13),(14)-ß-glucans determined enzymatically in other studies, it is clear that immunogold labeling with this antibody is a very sensitive method of detecting (13),(14)-ß-glucans in walls.
(13),(14)-ß-Glucans are not usually considered to be components of lignified walls, so our finding lignified walls of xylem tracheary elements and sclerenchyma fibers labeled with the (13),(14)-ß-glucan antibody is particularly interesting. However, linkage analyses of the wall polysaccharides of these cell types in vegetative organs of Lolium multiflorum and L. perenne have shown the presence of low concentrations of (13)-linked glucosyl residues, probably from (13),(14)-ß-glucans (Chesson et al., 1985
; Smith and Harris, 1999
). The labeling of the compound middle lamella and secondary wall of the sclerenchyma fibers and xylem tracheary elements in Lolium multiflorum and Flagellaria indica was similar to that previously reported for the walls of these cell types in the first leaf and coleoptile of H. vulgare (Trethewey and Harris, 2002
). The light labeling of the compound middle lamella probably results from (13),(14)-ß-glucans synthesized at an early stage of wall development, before the formation of the secondary wall. However, the very light labeling of the secondary wall indicates that at least some (13),(14)-ß-glucans are synthesized during the development of this wall. The light or moderate labeling of the secondary walls of these cell types in the type 2 species (Restionaceae and Xyridaceae) indicates an even greater synthesis of these glucans.
The monoclonal antibody used in the present study was raised against a conjugate of bovine serum albumin and (13),(14)-ß-glucan from barley grain (Meikle et al., 1994
). This glucan has a chemical structure typical of the (13),(14)-ß-glucans found in Poaceae walls. In these glucans, the proportions of (13)- and (14)-linkages vary with species, but the (14)-linkage predominates (70–75%). The (13)-linkages occur singly, and more than 90% of the (14)-linkages occur in groups of two and three, resulting in a polysaccharide composed mostly of ß-(13)-linked cellotriosyl (G4G4G) and cellotetraosyl (G4G4G4G) units; the ratio of these units vary with species (Wood et al., 1991
,1994
; Stone and Clarke, 1992
). The epitope for optimum binding of the monoclonal antibody consists of at least a hexasaccharide with the structure G4G4G3G4G4GR and could also include an additional glucose residue linked to the 3-position at the nonreducing terminus (Meikle et al., 1994
). It is therefore likely that the walls of species in families other than the Poaceae that are labeled with this antibody have (13),(14)-ß-glucans with structures typical of those found in the Poaceae. So far, this has been demonstrated analytically only for the (13),(14)-ß-glucans in the nonlignified walls of Flagellaria guineensis (Popper and Fry, 2004
). Nevertheless, small amounts of the antibody may bind to (13),(14)-ß-glucans with chemical structures different from those found in Poaceae walls. Thus in walls with light or very light labeling, it is possible, although unlikely, that much larger concentrations of (13),(14)-ß-glucans are present than is indicated by the density of labeling. However, if this were true for the nonlignified walls of L. similis and R. tetraphyllus, we would expect that the enzymically determined concentrations of (13),(14)-ß-glucans in wall preparations would be higher than were found. Further analytical studies are required of the chemical structures of (13),(14)-ß-glucans in the walls of families other than the Poaceae.
The distribution of the different types of wall-labeling patterns and the concentrations of (13),(14)-ß-glucans in nonlignified wall preparations can be related to hypotheses of the phylogenetic relationships among Poales families. A recent cladistic analysis of monocotyledons based on chloroplast (rbcL) and mitochondrial (atpA) sequence data presented a phylogeny for 17 of the 20 families included in Poales (Davis et al., 2004
) (Fig. 6). This analysis showed two clades, cyperoid and graminoid, that largely correspond to Dahlgren et al.'s (1985)
Cyperales and Poales, respectively. These clades were also resolved in other cladistic analyses with a broad sampling of Poales, although overall relationships varied (Bremer, 2002
; Michelangeli et al., 2003
). The cyperoid clade comprises the three families Cyperaceae, Juncaceae, and Thurniaceae (including Prionium), and the graminoid clade comprises the seven families Anarthriaceae, Centrolepidaceae, Ecdeiocoleaceae, Flagellariaceae, Joinvilleaceae, Poaceae and Restionaceae (Bremer, 2002
; Michelangeli et al., 2003
). In Davis et al. (2004)
, the graminoid clade differs from Dahlgren's circumscription of Poales in that Flagellariaceae, often considered sister to the remaining Poales (Davis, 1995
; Linder and Kellogg, 1995
; Stevenson and Loconte, 1995
; Givnish et al., 1999
; Rudall et al., 1999
; Chase et al., 2000
; Stevenson et al., 2000
; Michelangeli et al., 2003
), is not part of the graminoid clade, but was resolved as sister to a clade including the cyperoid clade, Mayacaceae, Hydatellaceae, and a portion of Xyridaceae (Fig. 6). A sister relationship of Flagellariaceae to Cyperales was found in an early analysis by Chase et al. (1993)
.
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3),(14)-ß-glucans (1.7%) to the wall preparation from leaves of L. multiflorum (Smith and Harris, 1999
). However, the walls of E. monostachya have not been examined using immunogold labeling. Flagellaria indica (Flagellariaceae) also has type 1 labeling. Whether the type 1 pattern is independently derived in Flagellariaceae and the Poaceae subclade or characterizes the Poaceae subclade including the Flagellariaceae (Fig. 7) is equivocal given the present state of knowledge of the distribution of the labeling patterns among taxa.
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3),(14)-ß-glucans in nonlignified wall preparations. Also assigned to this subclade are Centrolepis strigosa (Centrolepidaceae) (Bremer, 2002
; Michelangeli et al., 2003
) and Anarthria prolifera (Anarthriaceae). Although the walls of these two species have not been examined by immunogold labeling, nonlignified wall preparations from them contained much lower concentrations of (13),(14)-ß-glucans (0.2 and 0.5%, respectively) than species from the Poaceae subclade (Smith and Harris, 1999
).
Davis et al.'s (2004)
study included four of the five genera of Xyridaceae and resulted in two lineages of Xyridaceae that are more closely related to other families than to each other (see also Michelangeli et al., 2003
) (Fig. 6). These lineages correspond to two subfamilies (Suessenguth and Beyerle, 1935
; Takhtajan, 1997
; Thorne, 2000
) or families (APG, 1998
) comprising genera usually recognized for Xyridaceae. Xyris, for which the wall-labeling pattern is unknown, occurs in a clade that is sister to the cyperoid clade. A relationship of Xyridaceae to Cyperales was found in an analysis of one nuclear and two plastid genes (Chase et al., 2000
). The genera examined in the present study, Aratitiyopea lopezii and Orectanthe sceptrum, together with Abolboda have been treated as Abolbodaceae, and these taxa are resolved as sister to Eriocaulaceae. Aratitiyopea lopezii and O. sceptrum have a similar (type 2) wall-labeling pattern to the distantly related Restionaceae. It would be interesting to know if the type of immunogold labeling supports the recognition of two families for Xyridaceae (s.l.).
The cyperoid clade, which comprises the Cyperaceae, Juncaceae, and Thurniaceae contains those species with the type 3 wall-labeling pattern: Baumea juncea and Cyperus haspans (Cyperaceae), and Juncus inflexus (Juncaceae). Our evidence suggests that the nonlignified walls of these species contain only trace amounts of (13),(14)-ß-glucans.
The Poales species that showed no immunogold labeling of their walls belong to the Bromeliaceae (Aechmea fasinata), Typhaceae (Typha orientalis), and Sparganiaceae (Sparganium subglobosum), families that form a clade basal to the clade in which immunogold gold labeling is present (Fig. 6). To ascertain if the presence of immunogold labeling for (13),(14)-ß-glucans is synapomorphic for Poales and secondarily lost in the clade with Bromeliaceae, or is derived further within Poales, the earliest diverging lineage of Poalaes, Rapateaceae (Bremer, 2002
; Michelangeli et al., 2003
; Davis et al., 2004
), needs to be examined for this feature.
In the context of the Poales phylogeny presented by Davis et al. (2004
; see also Michelangeli et al., 2003)
, the immunogold labeling patterns are homoplasious. A more simplified scenario for the evolution of the labeling patterns can be derived from an alternative phylogenetic hypothesis for Poales (Bremer, 2002
) (Fig. 7). In this case, the unidentified genes that encode the glycosyltransferases involved in the biosynthesis of (13),(14)-ß-glucans (Buckeridge et al., 2004
) could have first appeared in extinct taxa that gave rise to the extant cyperoid clade and the graminoid plus Eriocaulaceae and a monophyletic Xyridaceae clade (Fig. 7). The concentrations of these glucans in nonlignified walls increased during evolution and reached their maximum in the Poaceae subclade of the graminoid clade.
The immunogold wall-labeling patterns obtained with the monoclonal antibody against (13),(14)-ß-glucans have the potential to be used as taxonomic characters within the Poales. This study calls attention to taxa that should be examined to further assess the distribution of these characters and our understanding of Poales phylogeny. However, care is required in the choice of plant material because the concentrations of (13),(14)-ß-glucans in nonlignified walls of vegetative organs of Poaceae alter during development (Carpita, 1996
). These glucans occur in only low concentrations in walls of tissues that are mostly meristematic, but are synthesized during cell expansion (Carpita, 1984
; Carpita et al., 1985
). A concentration of (13),(14)-ß-glucans of only 0.5% was found in the walls of meristematic cells in the root tips of seedling H. vulgare, and this corresponded to only light immunogold labeling adjacent to the plasma membrane (Trethewey and Harris, 2002
).
Another wall feature that also has the potential to be used as a taxonomic character in the Poales is the orientation of the cellulose microfibrils in the outer epidermal cell walls of the root elongation zone (Kerstens and Verbelen, 2002
). This can be determined by polarization confocal laser scanning microscopy using the fluorescence dichroism of Congo red. In species examined of the Poaceae, Cyperaceae, and Juncaceae, the net orientation of these cellulose microfibrils was parallel to the root axis. By contrast, in species examined of the Bromeliaceae, Typhaceae, and Sparganiaceae, the net orientation of these cellulose microfibrils was transverse to the root axis. Whether or not the orientation of cellulose microfibrils in root epidermal walls is correlated with the occurrence in these walls of (13),(14)-ß-glucans remains to be determined.
FOOTNOTES
1 The authors thank Dr. A. Turner for advice with the electron microscopy; Dr. J. A. Taylor for the gift of the monoclonal antibody specific for rotavirus nonstructural protein 4; M. Foster for technical help; P. DeLange for a specimen of Sparganium subglobosum; and Dr. G. A. Romero-González for generous assistance with fieldwork. Logistical support was provided by the government of Venezuela's Amazonas state, the local "Apoyo Aéreo" group of the National Guard (FAC), the Ministry of the Environment (MARNR), the National Institute of Parks (INPARQUES), and the local Civil Defense and SAR. The Huottuja (Piaroa) native community of Raudal de Danto provided logistic support and access to the Cuao-Sipapo Massif, for which we are grateful. 
4 Author for correspondence (e-mail: p.harris{at}auckland.ac.nz
)
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