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- Materials and Methods
Early cells evolved in an aqueous environment and the primary cell wall (PCW) evolved as a strategy for coping with the associated osmotic problems (Gerhart & Kirschner, 1997). The PCW has become one of the defining characteristics of plants, many of which now live in a terrestrial environment. The PCW is fundamentally involved in many plant processes, including tissue cohesion, defence (e.g. against microbes), ion-exchange, the production of oligosaccharins and the regulation of cell expansion (Goldberg et al., 1994; Brett & Waldron, 1996; Cassab, 1998; Dumville & Fry, 1999). Demands on the PCW may have changed during terrestrial plant evolution, thereby influencing its optimal composition.
Angiosperm crop species are still the most extensively studied and the PCW composition of all higher plants is assumed to be comparable though not identical, the major monosaccharide residues being d-glucose (Glc), d-galactose (Gal), d-mannose (Man), d-xylose (Xyl), l-arabinose (Ara), l-fucose (Fuc), l-rhamnose (Rha) and d-galacturonate (GalA) (Albersheim, 1976; McNeil et al., 1984; Fry, 2000). The PCW has been found to differ between vascular plants at both the monosaccharide and polysaccharide level. Gramineous monocot PCWs contain the same monosaccharide residues as the rest of the angiosperms (both non-gramineous monocots and dicots) but usually have more Xyl and less Gal and Fuc (Burke et al., 1974; Carpita, 1996). Gymnosperm PCWs are similar in composition to those of dicotyledonous angiosperms but contain more Man residues (Edashige & Ishii, 1996). At the polysaccharide level, mixed-linkage glucan (MLG) appears to occur uniquely in gramineous monocots and closely related members of the Poales (Smith & Harris, 1999).
Extant pteridophytes (= non-seed vascular plants, including ferns, horsetails and club-mosses) are the relatively few surviving progeny of a great diversity of extinct taxa that formerly were ecologically dominant (Raven, 1993; Kenrick & Crane, 1997). Few studies exist of the PCW composition of pteridophytes, although studies by Vissenberg et al. (2003) show the PCWs of lycopodiophytes (early diverging pteridophytes) to contain xyloglucan endotransglucosylase (XET), an enzyme activity capable of transglycosylating xyloglucan (a cell wall polysaccharide). Additionally, we previously reported (Popper et al., 2001) that lycopodiophytes, the earliest diverging extant vascular plants (Raubeson & Jansen, 1992; Manhart, 1994, 1995; Pryer et al., 1995; Wolf, 1997; Duff & Nickrent, 1999) uniquely contain high concentrations of 3-O-methyl-d-galactose (MeGal) in their PCWs. Homosporous lycopodiophytes, bryophytes and charophytes also contain 3-O-methyl-rhamnose (MeRha) in their PCWs (Popper et al., 2004). MeRha has been shown to occur as a modification of the non-reducing Rha residue present on the aceric acid-containing side chain of rhamnogalacturonan II (RG-II); the primary structure of RG-II appears otherwise to be conserved (Matsunaga et al., 2004). Matsunaga et al. (2004) also report that the sporophyte PCWs of lycopodiophytes, equisetophytes, psilotophytes and ferns contain amounts of RG-II comparable with those in angiosperm PCWs. By contrast, PCW material from the gametophyte generation of all bryophytes analysed contained only 1% of the amounts of an RG-II-like polysaccharide present in angiosperm PCWs (Matsunaga et al., 2004).
We are therefore documenting the evolution of the PCW composition of vascular plants. Vascular plants (tracheophytes) form a well-supported monophyletic group which originated around 420 million years ago (Judd et al., 1999). It is of interest to identify any changes in cell wall composition that have accompanied steps in their subsequent diversification. In the present paper we report that PCW composition varies within the tracheophytes as well as between vascular plants and bryophytes.
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- Materials and Methods
High XET action in all vascular plants tested, from lycopodiophytes to gramineous monocots (Vissenberg et al., 2003), correlates well with our finding that its substrate, the polysaccharide xyloglucan, is also present in all vascular plants tested. Xyloglucan has been characterized from dicot PCWs where it is the major hemicellulose that hydrogen-bonds to, and probably forms tethers between, adjacent cellulose microfibrils (Fry, 1989; Carpita & Gibeaut, 1993). XET activity can cut and rejoin tethering xyloglucan chains, thus allowing cell wall loosening and cell expansion (Fry, 1989, 1992, 1995). The importance of xyloglucan is suggested by the universality of its occurrence in vascular plants. Xyloglucan derived from angiosperms has been reported to vary in side-chain composition. Some storage xyloglucans contain no Fuc (Kooiman, 1961); those extracted from the Solanaceae have α-l-Ara linked to Xyl at position 2, contain little Fuc and are less substituted with Xyl (Eda & Kato, 1978; Akiyama & Kato, 1982; Ring & Selvendran, 1981). Xyloglucan has been found to occur at a lower concentration in the PCWs of bryophytes (Popper & Fry, 2003), where it may differ in composition. Bryophyte xyloglucan may lack the Fuc side-chain since xyloglucan was not detected in bryophyte water-conducting cells by CCRC-M1, an antibody which recognizes an epitope containing a terminal α-Fuc residue (12)-linked to a β-Gal residue (Ligrone et al., 2002). XET activity has been detected in sporophyte and gametophyte tissues from the liverwort Marchantia and gametophyte tissue from a moss, Mnium (Fry et al., 1992).
Edashige & Ishii (1996) reported glucomannan to be a major component (10.6% of total PCW, w/w) in suspension-cultured cells of the gymnosperm Cryptomeria japonica. This is consistent with our finding of a high concentration of Man residues in the acid hydrolysate of AIR from young tissue of some gymnosperms. Among the gymnosperms, we studied a cycad (Encephalartos alteinsteinii), a conifer (Pinus sylvestris) and three gnetophytes (Gnetum gnemon, Gnetum indicum, Gnetum montana). The cycad and conifer had similar, high concentrations of Man in their AIRs. Gnetophyte AIR contained a lower concentration of Man residues than the other gymnosperms. Molecular evidence provided by developmental genes suggested that the gnetophytes are more closely related to conifers than to flowering plants (Winter et al., 1999) but our Man data suggest that the gnetophytes are more similar to angiosperms than to the rest of the gymnosperms.
The mannan content revealed a pronounced segregation between eusporangiate and leptosporangiate pteridophytes. A eusporangium is one that originates from several initial cells, has a sporangial wall more than one cell thick and tends to produce numerous sporocytes; a leptosporangium originates from a single initial cell, has a one-cell-thick wall and produces few sporocytes (Foster & Gifford, 1959). Among the non-seed plants, the PCWs of leptosporangiate ferns (Table 1) contained far less Man than those of the bryophytes (Popper & Fry, 2003), the lycopodiophytes, an equisetophyte, a psilotophyte and a eusporangiate fern (Marattia fraxinea) (Table 1). Lycopodiophytes, psilotophytes and equisetophytes, like M. fraxinea, exhibit the eusporangiate condition; and gene sequencing studies have shown that the eusporangiate ferns are more closely related to the equisetophytes and psilotophytes than to the leptosporangiate ferns (Pryer et al., 2001). Leptosporangiate ferns appear to have diversified in an environment dominated by seed plants (Schneider et al., 2004) and may therefore have faced selective pressures similar to those experienced by the seed plants themselves. This circumstance could explain why the PCWs of leptosporangiate ferns, including Osmunda, one of the earliest diverging extant leptosporangiate ferns (Pryer et al., 2001; Schneider et al., 2004), are more similar to those of seed plants than to those of eusporangiate pteridophytes. It is possible that the PCW composition of the vegetative tissues of bryophytes, lycopodiophytes, equisetophytes, psilotophytes and eusporangiate ferns is similar to that of the secondary cell wall of leptosporangiate ferns, gymnosperms and angiosperms. The neoteny theory of plant evolution postulates that angiosperms arose from their ancestors by a modified and extended juvenile phase (Takhtajan, 1976).
Lycopodiophytes are a well-supported monophyletic group which diverged early during vascular plant evolution. Within the lycopodiophytes there are two distinct groups: homosporous and the more advanced heterosporous. All lycopodiophytes tested contain MeGal in their PCWs (Popper et al., 2001) but the homosporous genera (Lycopodium, Huperzia and Diphasiastrum) are the only lycopodiophytes that resemble bryophytes in having high concentrations of MeRha in their PCWs (Popper et al., 2004). Matsunaga et al. (2004) also isolated MeRha from PCW material of a homosporous lycopodiophyte and a psilotophyte and showed the MeRha to be a component of RG-II (an important structural pectic polysaccharide) in these taxa. Bryophyte and charophyte PCWs also contain MeRha (Popper et al., 2004). These results suggest that the earliest-diverging extant vascular plants share some PCW characters with bryophytes and that during vascular plant evolution there was a reduction in hexose methylation.
Tannins may not be strictly a PCW component but are often associated with PCW-rich material and some tannins may be deposited within the PCW. There are condensed and non-condensed tannins. Condensed tannins (proanthocyanidins) are specifically detectable by the butanol/HCl test (Fry, 2000). They are an important class of secondary plant metabolites; in many cases they are the active principles of medicinal plants (De Bruyne et al., 1999). However, it is likely they evolved as a deterrent to herbivory as they can have negative effects on feed digestibility (Schofield et al., 2001).
Proanthocyanidins are present in AIR of leptosporangiate ferns and absent from that of the lycopodiophytes, a psilotophyte, an equisetophyte and the eusporangiate fern M. fraxinea. This chemical demarcation between leptosporangiate and eusporangiate pteridophytes coincides with that found for mannan (see Table 1) and supports the molecular evidence that the eusporangiate ferns are more closely related to the equisetophytes and psilotophytes than to the leptosporangiate ferns (Pryer et al., 2001). Bate-Smith & Learner (1954) reported the presence of a ‘moderate[ly]’ strong positive reaction for the presence of proanthocyanidin in M. fraxinea. Vascular plants (tracheophytes) form a well-supported, monophyletic group including the lycopodiophytes, psilotophytes, equisetophytes and eusporangiate ferns as well as the leptosporangiate ferns, gymnosperms and angiosperms (Judd et al., 1999). Leptosporangiate ferns are the earliest diverging plants in which proanthocyanidins start to predominate over flavonols (De Bruyne et al., 1999). Proanthocyanidins remain important in early diverging angiosperms but synthesis decreases in more advanced orders (De Bruyne et al., 1999). It seems likely that the production of proanthocyanidins evolved at the same time as the leptosporangiate condition rather than the vascular condition as proposed by Bate-Smith (1977).
GalA was the most abundant PCW uronic acid in all land plants (including all bryophytes) tested. However, the GlcA : GalA ratio was relatively high in early diverging bryophytes (Popper & Fry, 2003; Popper et al., 2003). We did not find GlcA in high concentration in any vascular plants tested but it was present in higher concentration in earlier diverging angiosperms than the more recently diverged ones. Jarvis et al. (1988) also found the Commelinanae, more recently evolved monocots, to have a greatly reduced uronic acid residue concentration. 4-O-Methylglucuronic acid has been reported to be present in high concentration in some dicot secondary cell walls, but is present at a lower concentration of about 0.2–0.8% of the d. wt. in all gramineous monocot PCWs tested (Darvill et al., 1980; Harris et al., 1997).
Our results confirm the report by Smith & Harris (1999) that MLG is present in the AIR of Flagellaria (Poales, Flagellariaceae). We did not detect MLG in Elegia capensis AIR (Poales, Restionaceae). However, Smith & Harris (1999) found concentrations of MLG in various species of Restionaceae to vary from undetectable to 0.1% (w/w, MLG/total cell wall composition). The super-order Poanae, as described by Takhtajan (1996), appears to be well defined as all families (Flagellariaceae, Joinvilleaceae, Restionaceae, Anarthriaceae, Ecdeiocolaceae, Centrolepidaceae, Poaceae) contain MLG.
Our results suggest that throughout vascular plant evolution the PCW and associated tannins have been adapted and modified. Stebbins (1992) thought that alterations in cell wall composition may have played a leading role in the evolution of vascular plants. Within and between the three monophyletic groups of extant vascular plants: (1) lycopodiophytes; (2) equisetophytes; psilotophytes, eusporangiate and leptosporangiate ferns; and (3) seed plants (Pryer et al., 2001) there are alterations in PCW composition. In particular, there is a pronounced chemical demarcation between the eusporangiate pteridophytes (high mannan, low tannin) and the leptosporangiate pteridophytes (low mannan, high tannin). However, the major monosaccharide residue composition appears to be relatively stable and all vascular plants contain a cellulose–xyloglucan network. Therefore it seems likely that all vascular plants accomplish cell expansion in the same way despite differences in PCW composition.