Molecular Plant Advance Access originally published online on August 17, 2009
Molecular Plant 2009 2(5):873-882; doi:10.1093/mp/ssp063
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(1,3;1,4)-β-D-Glucans in Cell Walls of the Poaceae, Lower Plants, and Fungi: A Tale of Two Linkages
The Australian Centre for Plant Functional Genomics, School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, Glen Osmond, SA 5064, Australia
1 To whom correspondence should be addressed. E-mail geoff.fincher{at}adelaide.edu.au.
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Abstract |
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 TOP Abstract INTRODUCTIONSTRUCTURES OF (1,3;1,4)-β-D...FUNCTIONAL ROLES OF (1,3;1,4)...THE CslF AND CslH...WHERE IN THE CELL...CO-EXPRESSION ANALYSESCONCLUDING REMARKSFUNDING |
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(1,3;1,4)-β-D-Glucans consist of unbranched and unsubstituted chains of (1,3)- and (1,4)-β-glucosyl residues, in which the ratio of (1,4)-β-D-glucosyl residues to (1,3)-β-D-glucosyl residues appears to influence not only the physicochemical properties of the polysaccharide and therefore its functional properties in cell walls, but also its adoption by different plant species during evolution. The (1,3;1,4)-β-D-glucans are widely distributed as non-cellulosic matrix phase polysaccharides in cell walls of the Poaceae, which evolved relatively recently and consist of the grasses and commercially important cereal species, but they are less commonly found in lower vascular plants, such as the horsetails, in algae and in fungi. The (1,3;1,4)-β-D-glucans have often been considered to be components mainly of primary cell walls, but recent observations indicate that they can also be located in secondary walls of certain tissues. Enzymes involved in the depolymerisation of (1,3;1,4)-β-D-glucans have been well characterized. In contrast, initial difficulties in purifying the enzymes responsible for (1,3;1,4)-β-D-glucan biosynthesis slowed progress in the identification of the genes that encode (1,3;1,4)-β-D-glucan synthases, but emerging comparative genomics and associated techniques have allowed at least some of the genes that contribute to (1,3;1,4)-β-D-glucan synthesis in the Poaceae to be identified. Whether similar genes and enzymes also mediate (1,3;1,4)-β-D-glucan biosynthesis in lower plants and fungi is not yet known. Here, we compare the different fine structures of (1,3;1,4)-β-D-glucans across the plant kingdom, present current information on the genes that have been implicated recently in their biosynthesis, and consider aspects of the cell biology of (1,3;1,4)-β-D-glucan biosynthesis in the Poaceae.
Key Words: Cereals • chemical structure • glycosidic linkage analysis • Poaceae • polysaccharide biosynthesis • wall deposition
Received for publication June 9, 2009. Accepted for publication July 16, 2009.
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INTRODUCTION |
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TOPAbstractINTRODUCTIONSTRUCTURES OF (1,3;1,4)-β-D...FUNCTIONAL ROLES OF (1,3;1,4)...THE CslF AND CslH...WHERE IN THE CELL...CO-EXPRESSION ANALYSESCONCLUDING REMARKSFUNDING |
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The (1,3;1,4)-β-D-glucans are distributed asymmetrically across the plant kingdom. They are found in the cell walls of most, if not all, members of the Poaceae, of which the most economically important species to humankind are the cereals and the grasses. However, (1,3;1,4)-β-D-glucans are also found in some unusual niche locations, such as in the walls of Equisetum spp. (Fry et al., 2008; Sorensen et al., 2008), bryophytes and algae (Popper and Fry, 2003), in pathogenic fungi such as Rhynchosporium secalis (Pettolino et al., 2009), in lichen-forming ascomycete symbionts (Gorin et al., 1988; Stone and Clarke, 1992), and in a few other vascular plants outside the Poaceae (Smith and Harris, 1999; Trethewey et al., 2005). The (1,3;1,4)-β-D-glucans are most abundant in walls of the cereals, specifically in the starchy endosperm of grain, where they can contribute up to 70% by weight of the walls in barley, rye, and oats (Fincher and Stone, 2004).
The interesting distribution pattern of (1,3;1,4)-β-D-glucans in plant species gives rise to a number of questions about how the polysaccharide might have evolved in such diverse organisms. Did the (1,3;1,4)-β-D-glucans evolve independently in these diverse species or did they evolve very early as a wall component of fungi and primitive plants, to be progressively rejected by all but a small number of species in the lineage leading to the grasses? The sporadic and generally rare occurrence of (1,3;1,4)-β-D-glucans in plants might be taken as evidence for the former possibility, namely independent evolution in multiple species, which would be plausible in the light of suggestions that the (1,3;1,4)-β-D-glucans could appear through the evolution of a single new gene from similar, pre-existing genes that are widespread in plants (Fincher, 2009a, 2009b). Another question relates to the apparently widespread adoption of (1,3;1,4)-β-D-glucans in species of the Poaceae, which have appeared recently in evolutionary history but now cover about 20% of the land surface of our planet (Gaut, 2002). Have (1,3;1,4)-β-D-glucans in the walls of the Poaceae in any way contributed to this evolutionary success through the provision of any unidentified competitive advantage to the grasses in plant ecosystems? Finally, the (1,3;1,4)-β-D-glucans consist of unbranched chains with (1,3)- and (1,4)-β-glucosyl residues, but the ratios and distribution of these two linkage types along the (1,3;1,4)-β-D-glucan chain are quite variable. Are the different ratios and arrangements of (1,3)- and (1,4)-β-glucosyl residues in (1,3;1,4)-β-D-glucans from different species important determinants of function in the wall and hence evolutionary vigour, or do they simply reflect an imprecise biosynthetic mechanism that is of no functional consequence?
Against this background, the fine structural details of (1,3;1,4)-β-D-glucans from various lower and higher plant species are compared in the sections below, and recent advances in our understanding of the biosynthesis of (1,3;1,4)-β-D-glucans are discussed in relation to the development of a rationale for the different structural and functional features of the polysaccharide in walls.
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STRUCTURES OF (1,3;1,4)-β-D-GLUCANS |
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TOPAbstractINTRODUCTIONSTRUCTURES OF (1,3;1,4)-β-D...FUNCTIONAL ROLES OF (1,3;1,4)...THE CslF AND CslH...WHERE IN THE CELL...CO-EXPRESSION ANALYSESCONCLUDING REMARKSFUNDING |
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The abundance of (1,3;1,4)-β-D-glucans in cereal grains has enabled the extraction of sufficient material for detailed analyses and most information on (1,3;1,4)-β-D-glucan has been obtained from these sources. A combination of chemical and enzymic analyses has been used to define the structures of the polysaccharides. Methylation analysis has been used to demonstrate the presence of (1,4)-D-glucopyranosyl and (1,3)-D-glucopyranosyl residues, but will not allow one to conclude whether the linkages are in the β- or
-anomeric configuration. Similarly, methylation will not show if the (1,4)-D-glucopyranosyl and (1,3)-D-glucopyranosyl residues are constituents of a single polysaccharide. Thus, enzymic procedures with specific (1,3;1,4)-β-D-glucan endohydrolases, usually from Bacillus spp. or from barley, have been particularly important for the structural analysis of cereal (1,3;1,4)-β-D-glucans (Anderson and Stone, 1975; Woodward et al., 1983). The (1,3;1,4)-β-D-glucan endohydrolases hydrolyse a (1,4)-β-D-glucopyranosyl linkage, provided this is immediately adjacent to a (1,3)-β-D-glucopyranosyl residue (Fincher, 2009a). Thus, the constituent β-D-glucopyranosyl monomers in cereal (1,3;1,4)-β-D-glucans are linked either through their C(O)3 or C(O)4 atoms, with the (1,4)- linkage being more abundant. However, the ratio of (1,4)- to (1,3)-linkages is quite variable and it is this ratio, combined with the particular distribution of the linkages along the chain, that has a profound influence on the behaviour, and thus the functional properties of, the polysaccharide. For example, the ratio of (1,4)-β-D-glucopyranosyl to (1,3)-β-D-glucopyranosyl residues generally ranges from 2.2 to 2.6:1 in cereals, although there are notable exceptions, such as the (1,3;1,4)-β-D-glucan from sorghum endosperm, which has a ratio of 1.15:1 (Fincher and Stone, 2004). The two types of linkage are arranged neither at random nor with predictable regularity, although a (1,3)-linkage almost always exists in isolation, separated by two or more (1,4)-linkages from its nearest (1,3)-β-glucopyranosyl neighbor; two adjoining (1,3)-linkages seldom, if ever, occur.
In this regard, the polysaccharide can be considered as a series of (1,4)-β-linked oligoglucosides, or cellodextrins, linked by single (1,3)-β-linkages. Cellodextrin units of two (cellotriosyl) or three (cellotetraosyl) adjacent (1,4)-linkages predominate, although longer units of 5–20 adjacent (1,4)-linkages are also found; the latter make up about 10% of the total polysaccharide chain in the water-soluble (1,3;1,4)-β-D-glucan from barley starchy endosperm cell walls. The arrangement of these cellotriosyl and cellotetraosyl units in the same water-soluble barley endosperm (1,3;1,4)-β-D-glucan has been analyzed using Markov algorithms (Staudte et al., 1983), which indicate that the arrangement is essentially random. However, the diagnostic ratio of the two most abundant cellotriosyl and cellotetraosyl units varies according to the cereal of origin. For example, in walls from the starchy endosperm of wheat, the ratio may be 3.0–4.5:1, but, in oats, it ranges from 1.8 to 2.3:1 (Fincher and Stone, 2004). The net result of these structural features in the (1,3;1,4)-β-D-glucans from the Poaceae is that the polysaccharides have (1,3)-β-linkages inserted at irregular intervals along the (1,3;1,4)-β-D-glucan chain. These cause irregularly spaced molecular kinks to form in the polysaccharide and these not only prevent extensive intermolecular alignment of chains into well structured microfibrils, but they also result in polysaccharides that are capable of forming a gel-like matrix in walls of the cells and that are soluble in aqueous media, despite their very large molecular mass (Fincher, 2009a, 2009b).
In Equisetum arvense, one of the only land plants outside the Poales where (1,3;1,4)-β-D-glucans have been detected, the (1,3;1,4)-β-D-glucan contains a predominance of (1,3)-β-linked cellotetraosyl units, with low levels of cellobiosyl and cellotriosyl units, and no blocks of more than seven adjacent (1,4)-β-D-glucopyranosyl residues (Sørensen et al., 2008). Other, independent analyses of (1,3;1,4)-β-D-glucans from a range of Equisetum species in a study by Fry et al. (2008) did show some longer blocks of up to nine adjacent (1,4)-linked units, but blocks of 12 or more adjacent (1,4)-linked β-D-glucopyranosyl residues could not be detected.
(1,3;1,4)-β-D-Glucans have also been detected in fungi, where, again, they exhibit distinctive linkage characteristics (Fontaine et al., 2000). For example, the (1,3;1,4)-β-D-glucan known as lichenin from certain Ascomycete fungi that form symbiotic relationships with algae in lichens has a ratio of (1,4)-β-D-glucopyranosyl to (1,3)-β-D-glucopyranosyl residues of 2.3:1 in the case of Icelandic moss (Cetraria islandica) (Honegger and Haisch, 2001). Enzymic analyses show that lichenin consists mostly of (1,3)-β-linked cellotriosyl residues, with relatively low levels of cellotetraosyl units (Perlin and Suzuki, 1962). Most recently, (1,3;1,4)-β-D-glucans have been found in the cell walls of a significant plant pathogen, Rhynchosporium secalis, which causes barley leaf scald (Pettolino et al., 2009). Based on DNA analyses, this pathogen has also been assigned to the Ascomycota group of fungi (Goodwin, 2002; Linde et al., 2003). Close examination of the fungal hyphae reveals that there are distinct inner and outer wall layers (Figure 1). It is the inner hyphal wall that labels most heavily when probed with the monoclonal antibody specific to (1,3;1,4)-β-D-glucans, which could indicate that the (1,3;1,4)-β-D-glucan is more accessible in this layer rather than it being absent from the outer wall. The surrounding plant cell wall is also only lightly labeled, which is likely to reflect the lower amount of (1,3;1,4)-β-D-glucan found in the walls of epidermal cells as compared to walls of other cell types. As with (1,3;1,4)-β-D-glucans from other fungi, the Rhynchosporium secalis (1,3;1,4)-β-D-glucan consists mostly of (1,3)-β-linked cellotriosyl residues (Pettolino et al., 2009).
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FUNCTIONAL ROLES OF (1,3;1,4)-β-D-GLUCANS |
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TOPAbstractINTRODUCTIONSTRUCTURES OF (1,3;1,4)-β-D...FUNCTIONAL ROLES OF (1,3;1,4)...THE CslF AND CslH...WHERE IN THE CELL...CO-EXPRESSION ANALYSESCONCLUDING REMARKSFUNDING |
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The apparently modest differences in (1,3;1,4)-β-D-glucan fine structure discussed above might be relatively more important for overall (1,3;1,4)-β-D-glucan conformation and biological function. As the polysaccharide tends towards a predominance of (1,3)-β-linked cellotetraosyl units, as in the Equisetum spp. or towards a predominance of (1,3)-β-linked cellotriosyl units, as in the fungi, in both cases, the molecular kinks imposed by the (1,3)-β-glucosyl residues will become more regularly spaced in the polysaccharide, which, in turn, will become less soluble and less suitable for gel formation in the matrix phase of the wall. The conformational irregularity of cell wall (1,3;1,4)-β-D-glucans therefore appears to be a feature that is confined to the polysaccharides of the Poaceae, and raises the question as to whether that irregularity is the key property that has led to the broad adoption and retention of (1,3;1,4)-β-D-glucans in walls of the Poaceae. Even within the Poaceae, there appear to be significant differences in the fine structures of the (1,3;1,4)-β-D-glucans, where those in the starchy endosperm walls of wheat are considerably less soluble than those from equivalent walls from barley and oat grain (Fincher and Stone, 2004).
Accordingly, the conformational regularity or irregularity of (1,3;1,4)-β-D-glucans will define its properties and therefore its physicochemical behavior in a matrix such as the cell wall. As noted above, the irregularly spaced molecular kinks produce an asymmetrical shape that cannot easily align or aggregate into fibrils, but rather is soluble and, through limited intermolecular junction zone formation (Fincher, 2009a), is capable of producing a gel-like material offering some structural support for the wall, combined with flexibility and porosity. Such interactions in the context of the cell wall can be influenced by other factors, including associations with other polysaccharides or proteins.
The two types of vascular plants that have been shown to contain (1,3;1,4)-β-D-glucans, namely the Poaceae and the Equisetum spp., have very different cell walls. As an example of the former, cereals are referred to as having a type II cell wall that contains low levels of pectic polysaccharides and substantial amounts of glucuronoarabinoxylans, while plants with type I walls contain more pectin and xyloglucan (Carpita, 1996). However, Sørensen et al. (2008) noted that the Equisetum spp. do not seem to fit into either category, possessing a wall rich in pectins, cellulose, and (1,3;1,4)-β-D-glucans but low in xyloglucans.
It has been widely held that in the Poaceae, (1,3;1,4)-β-D-glucans are associated with growing cells and are essentially absent in mature tissues (Buckeridge et al., 2004; Carpita et al., 2001; Gibeaut et al., 2005). However, Sørensen et al. (2008) reported that this might not be the case in Equisetum arvense, where (1,3;1,4)-β-D-glucans are abundant in both young and old regions of stems and somewhat more abundant in mature non-growing stem base regions. In these plants, (1,3;1,4)-β-D-glucans were not present in the vascular tissues.
In cereals, (1,3;1,4)-β-D-glucans are generally found in expanding cells of organs such as the coleoptile (Gibeaut and Carpita, 1993; Gibeaut et al., 2005) but they can also be seen in some of the vascular and fiber cells of the leaf (Figure 2; Trethewey et al., 2005), suggesting a structural role in secondary cell walls. In the walls of the starchy endosperm, which do not exhibit secondary thickening, the (1,3;1,4)-β-D-glucans can be present at high concentrations and can contribute up to 18% of the total glucose stored in the grain (Morrall and Briggs, 1978). Thus, they clearly participate as storage polysaccharides in the grain and are mobilized when the grain germinates. (1,3;1,4)-β-D-glucans may also fulfill this role, providing an energy source in other growing tissues and Roulin et al. (2002) suggested that (1,3;1,4)-β-D-glucans in walls of barley leaves might be used as a readily available source of metabolizable glucose during periods of darkness, when glucanase levels rise to effect hydrolysis.
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Despite these differences in (1,3;1,4)-β-D-glucan distribution and in the fundamental structural composition of the walls themselves, there may be a common role for (1,3;1,4)-β-D-glucans in the architecture of both plant types through either microfibril tethering and/or silica deposition. In Equisetum, there is evidence that the (1,3;1,4)-β-D-glucan is firmly bound into the wall (Fry et al., 2008) and its predominance in the older sections of the stem might suggest a strengthening role (Sørensen et al., 2008). This could be achieved when (1,3;1,4)-β-D-glucans act as inter-fibrillar tethers, with the intrinsic difference between cereals and Equisetum lying in the nature of the scaffold polysaccharides that are held together by the (1,3;1,4)-β-D-glucans. This will only become clear as we gain more knowledge of the polysaccharide–polysaccharide interactions in cell walls in these plants, and in those other organisms that also contain (1,3;1,4)-β-D-glucans.
Another significant similarity between walls in the Poales and in Equisetum spp. is the accumulation of silica in the cell walls (Currie and Perry, 2007) but little is currently known about how the silica accumulates or why it is there. Fry et al. (2008) suggest that (1,3;1,4)-β-D-glucans may play a central role in wall silification, possibly influencing the amount and/or the location of deposition. This is yet another area that awaits further study.
The structures of the (1,3;1,4)-β-D-glucans, or lichenins, from symbiotic ascomycota renders them highly hydrophilic and, when fully hydrated, these polysaccharides are translucent and allow light transmission through to the photosymbiont (Honegger and Haisch, 2001). The lichenins are also thought to be important in maintaining water relations in the thallus of lichens during wetting and drying cycles (Honegger and Haisch, 2001).
Clearly, whether (1,3;1,4)-β-D-glucans function as storage or structural polysaccharides in plants and fungi remains to be fully explored, although it is likely that they function in both roles simultaneously (Fincher, 2009b). From an evolutionary standpoint, the unique structure of (1,3;1,4)-β-D-glucans has been exploited by cereals in particular and it is possible that the widespread adoption of this polysaccharide in the Poaceae confers other key advantages that we have not yet identified.
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THE CslF AND CslH GENE FAMILIES HAVE BEEN IMPLICATED IN (1,3;1,4)-β-D-GLUCAN BIOSYNTHESIS |
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TOPAbstractINTRODUCTIONSTRUCTURES OF (1,3;1,4)-β-D...FUNCTIONAL ROLES OF (1,3;1,4)...THE CslF AND CslH...WHERE IN THE CELL...CO-EXPRESSION ANALYSESCONCLUDING REMARKSFUNDING |
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The structure of (1,3;1,4)-β-D-glucan molecules must influence the way in which we consider how they are made. How, for example, are only single (1,3)-linkages inserted between the (1,4)-linked cellodextrin blocks? How are the variable lengths of the (1,4)-linked blocks achieved, and why are they mostly short with occasional longer stretches? Is the polysaccharide chain synthesized processively or is it assembled in short oligomeric pieces that are subsequently linked together (Fincher, 2009b)? Answering even one of these questions is difficult, since we have yet to define the biosynthetic machinery or many of the proteins that are likely to be involved. However, in the last few years, we have made some progress in defining at least some of the genes involved in (1,3;1,4)-β-D-glucan synthesis.
The application of comparative genomics techniques provided fundamental information that enabled the identification of a group of candidate genes that might participate in (1,3;1,4)-β-D-glucan synthesis. The identification of highly significant quantitative trait loci (QTLs) controlling mature barley grain (1,3;1,4)-β-D-glucan content (Han et al., 1995), when combined with the presence of a group of cellulose synthase-like CslF genes in the syntenic region of the rice genome, provided a strong hint that these monocot-specific CslF genes might be involved in making a monocot-specific polysaccharide. The transfer of OsCslF genes from rice into transgenic Arabidopsis, a eudicot that does not usually make (1,3;1,4)-β-D-glucans, proved to be a key experiment. Plants expressing various rice CslF genes were shown to produce and deposit (1,3;1,4)-β-D-glucan in their cell walls, as detected by a specific monoclonal antibody (Burton et al., 2006), and this indicated that the rice CslF genes were able to direct synthesis of this polysaccharide. Given that expression of the transgenes was driven by the constitutive 35S promoter, the low levels of (1,3;1,4)-β-D-glucan in the transgenic lines and the restriction of the polysaccharide to certain cell types implied that there is likely to be one or more proteins that combine with the CslF gene products to produce the (1,3;1,4)-β-D-glucans (Burton et al., 2006), much as there is likely to be for cellulose (Doblin et al., 2002), and also that the protein of monocot origin was able to recruit eudicot companions to form such a functional complex in the transgenic Arabidopsis lines.
The CslF gene family has now been defined in barley (Burton et al., 2008). It comprises seven members, four of which are located on chromosome 2H under the QTL originally identified by Han et al. (1995). Two of the remaining genes align with other QTLs, notably HvCslF9 on chromosome 1H, which is also close to the HvGlb1 gene, one of two (1,3;1,4)-β-D-glucan endohydrolases in barley, and HvCslF6, which is located on chromosome 7H and is associated with QTLs for barley grain (1,3;1,4)-β-D-glucan content identified by Molina-Cano et al. (2007) and Igartua et al. (2002). Transcript analyses in various tissues show that the family members display individual patterns of abundance (Burton et al., 2008). Transcript levels for many of the genes are low across a range of tissues but HvCslF6 mRNA is the notable exception. The transcript for this gene is found at high levels in many of the tissues examined, especially in the developing endosperm. There appear to be two dominant transcripts in this tissue during grain development, namely that of HvCslF9 in the early stages of endosperm development, and later that of HvCslF6 as the grain expands and matures. Transcripts for other family HvCslF genes are also found in this tissue at much lower levels (Burton et al., 2008), but these should not be discounted as functional members of a (1,3;1,4)-β-D-glucan synthase complex, since transcript level does not necessarily equate to protein level or enzyme activity.
At the protein level, the HvCslF enzymes appear to be similar in size and structure, although the HvCslF6 enzyme contains a unique 54 amino acid loop that distinguishes it from the rest of the family; the functional relevance of this loop has yet to be determined. In other cereals, such as wheat and rice, the orthologs of HvCslF6 also show higher levels of transcription than other family members (unpublished data). The CslF gene families in other cereals and grasses also consist of multiple members; there are eight CslF genes in rice, 11 in sorghum, and seven in Brachypodium (A.J. Harvey, R.A. Burton, and G.B. Fincher, unpublished data).
Recently, it has also been established that CslH genes are able to direct the synthesis of (1,3;1,4)-β-D-glucans (Doblin et al., 2009). Using an experimental approach similar to that used to define the function of the CslF genes, members of this gene family were expressed in transgenic Arabidopsis plants, giving rise to (1,3;1,4)-β-D-glucan that was detected in walls of the transgenic lines with the (1,3;1,4)-β-D-glucan-specific monoclonal antibody. Using sensitive high-performance anion-exchange chromatography (HPAEC) methods and relatively large amounts of tissue, it was possible to detect the (1,3;1,4)-β-D-glucan in these plants, not only through the immunocytochemical methods, but also with chemical and enzymic procedures, and this enabled analysis of the linkage ratio in the newly synthesized (1,3;1,4)-β-D-glucans following digestion with the specific Bacillus (1,3;1,4)-β-D-glucan endohydrolase mentioned in an earlier section. Comparison of the enzymic hydrolysate of the (1,3;1,4)-β-D-glucan synthesized in the walls of the transgenic Arabidopsis lines with the (1,3;1,4)-β-D-glucan from barley flour showed the existence of the diagnostic cellotriosyl and cellotetraosyl groups but, in addition, there was a significant but variable amount of laminaribiose (DP2, G3GR) in the transgenic lines (Figure 3). The presence of laminaribiose in the enzymic hydrolysate indicates the presence of single (1,4)-β-D-glucopyranosyl residues between two (1,3)-β-D-glucopyranosyl residues, as can be depicted in the structure G3G4G3G4, where G represents a β-D-glucopyranosyl residue and 3 and 4 represent the linkage types. Single (1,4)-β-D-glucopyranosyl residues have been detected in low abundance in cereal (1,3;1,4)-β-D-glucans (Perlin and Suzuki, 1962) and at somewhat higher levels in (1,3;1,4)-β-D-glucans from Equisetum (Fry et al., 2008; Sørensen et al., 2008) and fungi (Fontaine et al., 2000; Honegger and Haisch, 2001; Stone and Clarke, 1992). This type of structure may arise in a non-native host plant due to subtle differences in assembly processes (Doblin et al., 2009).
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).
). G4G3GR (3-O-β-cellobiosyl D-glucose, DP3) and G4G4G3GR (3-O-β-cellotriosyl D-glucose, DP4) peaks are indicated. Data from The CslH gene family in barley is smaller than the CslF gene family. There are three CslH genes in rice, but only one in barley. The barley HvCslH gene maps to chromosome 2H but is a significant distance proximal to the HvCslF gene cluster and the QTL already identified on that chromosome (Doblin et al., 2009). The transcript level of the gene is extremely low in all tissues, including the developing endosperm. In situ analysis indicates that the transcript is located in parts of the leaf that display secondary cell wall thickening, more precisely around the sclerenchyma fiber cells (Doblin et al., 2009). There appears to be no correlation between the presence of the CslH transcript and any of the CslF transcripts. Despite the apparently limited amounts of endogenous transcript and protein, the CslH gene is still able to direct the synthesis of (1,3;1,4)-β-D-glucans in both barley and a heterologous host. It remains to be seen whether this gene is conscripting the same ‘helper’ proteins as the CslF transgenic lines for the biosynthesis of (1,3;1,4)-β-D-glucan in the Arabidopsis cells.
Examination of the phylogenetic tree that comprises the cellulose synthase superfamily shows that there are three ‘Poaceae-specific’ gene families, two of which have now been implicated in (1,3;1,4)-β-D-glucan synthesis. It has also been suggested that the evolution of (1,3;1,4)-β-D-glucans in the Poaceae only required the evolution of a single new gene, namely a CslF or a CslH gene (Fincher, 2009b). The third family of Poaceae-specific Csl genes, designated CslJ, is found in certain cereals, including barley, wheat, sorghum, and maize, but not in rice or Brachypodium (Fincher, 2009a). Barley is currently believed to contain only a single CslJ gene and its function remains to be elucidated.
For those species outside the cereals where (1,3;1,4)-β-D-glucans have been found, there is currently no information as to whether CslF or CslH gene orthologs exist in their genomes, so it is not possible to conclude whether the ability to synthesize this polysaccharide shares a common genetic origin. Judging by fossil records, Equisetum diverged from progenitors of other plants around 380 million years ago, making it an extremely isolated genus (Bell and Hemsley, 2000). It therefore seems likely that the ability to make (1,3;1,4)-β-D-glucans has evolved independently and at least twice in vascular plants, and possibly several times in Bryophytes and fungi. This would be plausible in plants if the corresponding genes had evolved from other members of the widely distributed cellulose synthase gene superfamily (Hazen et al., 2002), but this possibility has not yet been confirmed because genome sequence information and EST databases for these organisms have not been readily available. This is likely to change in the immediate future. So far, our general database searches for orthologs of the CslF and CslH genes have not revealed any closely related genes in the lower plants and fungi, but more stringent searches will be possible as new genome sequences are deposited in public databases. A significant number of the fungi that synthesize (1,3;1,4)-β-D-glucan belong to the Ascochyta, which is the largest group of fungi and contains over 30 000 known species (Kirk et al., 2001). This group contains such diverse members as truffles, yeast, fungal symbionts of lichens, well known model species such as Penicillium and Neurospora, and a significant number of plant pathogenic fungi. However, synteny between related fungal species is often poor and this can complicate comparative genomics approaches. For example, Podospora anserina, a close relative of Neurospora crassa, has evolved new genes by duplication since the separation of the two species, leading to poor synteny now (Espagne et al., 2008). A more complete picture of the occurrence of (1,3;1,4)-β-D-glucans and other novel polysaccharides, together with the identification of the genes that mediate their biosynthesis, awaits more detailed bioinformatical methods and more genome sequences.
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WHERE IN THE CELL ARE (1,3;1,4)-β-D-GLUCANS MADE? |
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TOPAbstractINTRODUCTIONSTRUCTURES OF (1,3;1,4)-β-D...FUNCTIONAL ROLES OF (1,3;1,4)...THE CslF AND CslH...WHERE IN THE CELL...CO-EXPRESSION ANALYSESCONCLUDING REMARKSFUNDING |
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Now that we have identified at least some of the genes involved in the synthesis of (1,3;1,4)-β-D-glucans in cereals, where do we go from here? There is a large number of unanswered questions regarding the synthesis, deposition, and remodeling of cell wall polysaccharides in plants. Can we at least pinpoint where in the cell (1,3;1,4)-β-D-glucan synthesis takes place? For many years, a biochemical approach was pursued in attempts to purify and characterize (1,3;1,4)-β-D-glucan synthases. These generally relied on the isolation of cellular membrane fractions, followed by tracking the incorporation of [14C]-glucose from UDP-[14C]glucose into various polysaccharides. Such experiments consistently pointed to the presence of (1,3;1,4)-β-D-glucan synthase enzyme activity in the Golgi (Gibeaut and Carpita, 1993; Henry and Stone, 1982) and this is, itself, consistent with the detection of the HvCslH protein in the Golgi vesicles and ER, but not the plasma membrane, of transgenic Arabidopsis lines (Figure 4) (Doblin et al., 2009). However, it is difficult to reconcile these data with recent immunocytochemical data. In the latter, the extensive use of a (1,3;1,4)-β-D-glucan-specific monoclonal antibody developed by Meikle et al. (1994) by our group and others, and as exemplified by Wilson et al. (2006), has consistently failed to locate (1,3;1,4)-β-D-glucan in the Golgi, even when the cell wall itself contains high levels of (1,3;1,4)-β-D-glucan and is heavily labeled by the antibody. Based on this evidence, it has been suggested that (1,3;1,4)-β-D-glucans might be made in a two-phase process (Fincher, 2009a). Smaller building blocks, perhaps (1,4)-linked cellodextrins, may be synthesized either by CslF or CslH enzymes in the Golgi, possibly linked to a carrier molecule, such as a lipid, and are not detectable by the monoclonal antibody in this state. They could subsequently be transported to the plasma membrane, where other enzymes would assemble them into the final form of the polymeric (1,3;1,4)-β-D-glucan, which would be simultaneously deposited into the cell wall. At this stage, having acquired the two linkages, the correct epitope would be detectable by the monoclonal antibody. The identity of the second enzyme that might covalently link the cellodextrins together is unknown but there are a number of possible candidates, including callose synthases, xyloglucan endotransglycosylases, or some other as yet unidentified glycosyl transferase (Fincher, 2009a). A further possibility may be that the CslF and CslH enzymes themselves catalyze the polymerization of the putative (1,4)-linked cellodextrins, through a (1,3)-β-glucosyl linkage. The latter would not seem to fit with the presence of the CslH proteins in the Golgi (Doblin et al., 2009), but the sub-cellular locations of the individual CslF proteins are not yet known and until we are able to develop either specific antibodies or generate transgenic lines that carry all of the barley CslF genes, individually, in a non-cereal background, we will not be able to pinpoint their sub-cellular locations. The task of raising antibodies that specifically recognize each of the seven individual CslF proteins is likely to be complicated by the close homology of these proteins and others in the cellulose synthase superfamily, including, in particular, the proteins encoded by the CesA and CslD gene families. Nevertheless, the generation of unique non-cereal lines carrying individual HvCslF genes is currently underway (R.A. Burton and G.B. Fincher, unpublished data).
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An alternative molecular mechanism for the synthesis of (1,3;1,4)-β-D-glucans might involve the insertion of the two linkage types into the elongating polysaccharide chain by the same enzyme, at a location outside of the Golgi. Polysaccharide synthase enzymes that are multifunctional in that they can insert two linkage types into the same molecule are exemplified by hyaluronan synthases (Doblin et al., 2009). This analogy comes with the caveat that the resulting polysaccharide chain of hyaluronan contains strictly alternating residue and linkage types, which is something that does not occur in (1,3;1,4)-β-D-glucans. In summary, it is clear that we need more information and careful biochemical analyses to enable us to confirm both the site and the mechanism of (1,3;1,4)-β-D-glucan synthesis in plants.
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CO-EXPRESSION ANALYSES |
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TOPAbstractINTRODUCTIONSTRUCTURES OF (1,3;1,4)-β-D...FUNCTIONAL ROLES OF (1,3;1,4)...THE CslF AND CslH...WHERE IN THE CELL...CO-EXPRESSION ANALYSESCONCLUDING REMARKSFUNDING |
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To assist us in defining more precisely the cellular, biochemical, enzymic, and molecular aspects of (1,3;1,4)-β-D-glucan biosynthesis in the grasses, a complementary avenue of exploration would be to make use of the expanding genomic, transcriptional, and other bioinformatic resources that are becoming available. In particular, the identification of genes that might be co-expressed with CslF and/or CslH genes could provide testable hypotheses as to what other proteins, enzymes, or metabolites could be involved in the biosynthetic process. If co-expression were examined at the transcript profile level, as is usually the case, it must be noted that apparently related levels of transcripts of different genes in different tissues will in many cases be of little or no relevance to the biosynthetic process. Nevertheless, co-expression studies often reveal genes that might be involved in the same cellular or biochemical process and that might not have been obviously linked with the process previously. Because the extensive transcript information needed for this type of analysis is generally not available for many of the organisms in which (1,3;1,4)-β-D-glucans are found, such as fungal symbionts and Equisetum spp., co-expression analyses of the CslF and CslH genes must, of necessity, be focused on the cereals and associated model species of grasses, for which there are some complete genome sequences and, in most cases, large EST databases.
Through the use of quantitative real-time PCR (Q–PCR), we have been able to monitor transcript levels of many genes likely to be involved in cell wall biosynthesis across numerous different tissues and even as tissues develop (Burton et al., 2004, 2008). Co-expression analysis of these data has shown, for example, that groups of three cellulose synthase CesA genes appear to be co-regulated in barley (Burton et al., 2004), and this has been confirmed in a number of other plant species, including Arabidopsis (Taylor et al., 2003), tobacco, maize (Appenzeller et al., 2004), and rice.
A similar analysis applied to the barley CslF genes reveals no such coordinate expression, however, either between members of the same family (Burton et al., 2008) or between any of the CslF and CslH genes (Doblin et al., 2009). However, in barley, there is a very strong correlation coefficient of 0.89 (using the correlate function from the Microsoft Excel software) between the CesA transcripts that have been assigned to primary cell wall cellulose synthesis in barley, namely CesA1, CesA2, and CesA6, and transcript abundance of the CslF6 gene. This can be demonstrated by analyzing the Barley1 Affymetrix microarray reference dataset (Close et al., 2004), as shown in Figure 5. Such analyses may give clues as to which enzymes function in multi-enzyme complexes, or which may be using similar substrates, such as small (1,4)-linked cellodextrins, in the biosynthetic process. We are also able to combine transcriptional datasets through a method known as the generalized singular value decomposition (GSVD), which allows the transcription of a gene that is not present in a microarray dataset to be correlated with the transcription of genes that are present on the microarray. In this way, co-expressed genes can be identified across both datasets. By applying the GSVD to a Q–PCR dataset generated from various barley tissues and the Barley1 reference experiment data, Schreiber et al. (2008) were able to show that CslF3, a gene not present on the microarray, is co-expressed with a number of genes that are represented on the microarray. Notably, these genes included a ceramide glucosyl transferase that has been implicated in micro-domain formation in the plasma membrane (Borner et al., 2005), together with other putative glucosyltransferases (Schreiber et al., 2008). The application of methods such as the GSVD to rapidly increasing bioinformatic data from plants could provide valuable clues as to the identity of proteins that might exist in a complex synthesizing (1,3;1,4)-β-D-glucans.
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CONCLUDING REMARKS |
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TOPAbstractINTRODUCTIONSTRUCTURES OF (1,3;1,4)-β-D...FUNCTIONAL ROLES OF (1,3;1,4)...THE CslF AND CslH...WHERE IN THE CELL...CO-EXPRESSION ANALYSESCONCLUDING REMARKSFUNDING |
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While there is now quite strong evidence that products of the CslF and CslH genes are associated with the biosynthesis of (1,3;1,4)-β-D-glucans in the Poaceae, the precise cellular location or locations of polysaccharide synthesis and assembly have not been defined unequivocally. Further, it appears likely that proteins and enzymes other than the CslF and CslH proteins will be involved, but, at this stage, we have not been able to demonstrate their participation in (1,3;1,4)-β-D-glucan biosynthesis. Finally, at the molecular level, important and numerous questions are yet to be answered in relation to the mechanisms of chain termination and hence chain length, to the mechanisms of insertion of (1,4)-β-D-glucopyranosyl to (1,3)-β-D-glucopyranosyl residues in an irregular but non-random fashion and, hence, in the control of physicochemical properties, and to the possible participation of other enzymes in (1,3;1,4)-β-D-glucan synthesis. In addition to these biological questions, which are now being addressed using new and ever developing technologies, understanding the mechanisms of (1,3;1,4)-β-D-glucan biosynthesis will have applications in improving cereal and grass species for the food, feed, fuel, and fiber industries. For example, the solubility, high molecular mass, and the propensity of (1,3;1,4)-β-D-glucans to form solutions of high viscosity make this polysaccharide particularly important in human health and nutrition, as an anti-nutritive factor in animal feed formulations, in industrial downstream processing procedures such as malting and brewing, and in its potential as a component of grass-derived biomass residues for bioethanol production. Understanding the biosynthetic process at a detailed molecular level will undoubtedly provide opportunities for the manipulation and improvement of cereals and grasses for these economically, financially, and environmentally important industries.
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FUNDING |
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TOPAbstractINTRODUCTIONSTRUCTURES OF (1,3;1,4)-β-D...FUNCTIONAL ROLES OF (1,3;1,4)...THE CslF AND CslH...WHERE IN THE CELL...CO-EXPRESSION ANALYSESCONCLUDING REMARKSFUNDING |
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This work has been supported by grants from the Australian Research Council, the Grains Research and Development Corporation, the CSIRO Flagship Collaboration Fund, and the South Australian government.
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Acknowledgements |
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We also thank Neil Shirley, Filomena Pettolino, Natalie Kibble, Monika Doblin, Marilyn Henderson, and Sarah Wilson for providing data and electron micrographs.
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