Department of Biochemistry, La Trobe University, Melbourne, VIC, 3086, Australia
The mature cereal grain comprises the maternal tissues of the pericarp-seed coat and the nucellar remnants, which overlie the tissues of the progeny comprising the embryo, or primordial plant, which is separated from the starchy endosperm-aleurone layer by the scutellum. Each of these tissues is composed of cells whose protoplasts are enclosed by a cell wall. In the case of the tissues of pericarp-seed coat at maturity, the contents of the cells have disappeared and only the thick walls remain. The cells of the starchy endosperm, which are also dead, are packed with starch granules and storage protein and usually have quite thin walls. In contrast, the cells of the aleurone, which are still alive, have thick bilayered walls enclosing the protein and lipid-rich cell contents (Figure 1).
Figure 1. Scanning electron micrograph of a portion of the outer surface of a wheat grain, showing starchy endosperm, aleurone and overlying nucellar remnants, seed coat and inner pericarp. The outer pericarp has been lost in preparation (Joyner, 1985).
Impact of cell walls and their components on grain utilization
The cell walls of the various grain tissues and their components have important impacts on the end uses of grain. Thus in milling, water uptake in the conditioning step and the breakage pattern is influenced by the cell wall organization and composition in the pericarp-seed coat tissues. In the development of doughs in bread and pasta making the wall components directly affect water absorption, mixing behaviour, dough rheology and the final properties of the baked product. In malting (germination) the rate of water uptake and endosperm dissolution is critically determined by the physical structure and composition of the cell walls. In brewing, mashing and the wort and beer filtration steps and the character and stability of the final beer product are importantly affected by wall structure and composition. Cell walls in grains are the primary contributors to cereal dietary fibre with important implications in human nutrition and disease prevention. The robust walls of aleurone cells (Figure 1) are not broken in roller milling and because they are resistant to digestion in the upper alimentary tract their nutritious cell contents (high quality protein, lipids and B vitamins) remain unavailable until the aleurone particles reach the large intestine. In monogastric animal (pig and poultry) nutrition, cell walls of the grain are implicated in depression of feed quality and in compromised carcase and egg hygiene.
Composition and structure of cell walls in relation to biological functions of grain tissues
The compositions of endosperm, aleurone, outer pericarp (beeswing bran) are compared in Table 1. The walls of the starchy endosperm and the overlying aleurone cells are rich in polymers with polysaccharides as the most abundant component, together with smaller amounts of proteins. The walls of the pericarp-seed coat cells are basically similar in composition but some cell types contain, in addition, significant amounts of the hydrophobic, polyphenolic polymer, lignin, and the walls of the epidermal cells have an outer layer of water-repellant, polyester, cutin. These compositions reflect the different functions of the tissues to which the cells belong. The starchy endosperm is a transient food reserve and during germination the simple, thin walls are readily degraded by hydrolases synthesized in, and secreted from, the aleurone, allowing access to their starch and protein substrates. The bilayered walls of aleurone cells are themselves a barrier to the release of the hydrolytic enzymes and in the early stages of germination the thicker, outer aleurone wall layer is preferentially degraded but the inner layer persists until the cells die. The pericarp tissues which are, in fact, modified leaves protect the developing and mature endosperm-embryo. The closely associated seed coat that overlies the outer surface of the aleurone has a layer of suberin (cork) that forms an impermeable barrier preventing the penetration of water into the underlying endosperm.
The polysaccharide composition of walls of both the pericarp-seed coat and the tissues of the progeny characteristically comprise cellulose and two non-cellulosic polysaccharides, the arabinoxylans and the (1→3,1→4)-ß-glucans, although their proportions depend on the cell type (Table 2). These non-cellulosic polysaccharides are characteristic of the grass family among the monocotyledons and set them apart from the remainder of the monocots and the dicots whose major non-cellulosic wall polysaccharides are typically xyloglucans and pectins.
Organization of wall components
The working model of the cell wall suggests the non-cellulosic polysaccharides, in the case of grasses arabinoxylans and (1→3,1→4)-ß-glucans, and proteins constitute a gel-like matrix phase in which the microfibrillar cellulose and associated glucomannans are embedded (Figure 2).
Organization of polymeric components in cell walls. A simplifed schematic representation of the spatial arrangement of polymers in a primary cell wall of a cereal e.g. the wall of a starchy endosperm cell. Note the cellulosic microfibrils are embedded in a network of non-cellulosic matrix polysaccharides [arabinoxylans and (1→3,1→4)-ß-glucans and proteins (not shown)] which are associated with the surfaces of several microfibrils. The native primary wall may contain ~60% water but in walls of some cells in the pericarp-seed coat the water is replaced by lignin which encrusts the non-cellulosic polysaccharides and proteins and may be covalently bound to them (after McCann and Roberts 1991).
Transmission electron microscopy of the surfaces of unextracted wheat endosperm walls are without structural features (Mares and Stone 1973a) but after extraction of water-soluble polymers, chiefly arabinoxylans, profiles of cellulose microfibrils can be seen in an amorphous background. Further extraction of the water-insoluble arabinoxylan and (1→3,1→4)-ß-glucan with 1M NaOH, fully exposes a web microfibrils, as expected from the wall model. The cellulose microfibrils are themselves associated with 4M KOH-soluble glucomannans.
Significantly, there is little difference in the basic chemistry or molecular size distribution of water-extractable and water-unextractable (1M NaOH-soluble) arabinoxylans from the endosperm walls (see for example Mares and Stone 1973b). The different extractability of the non-cellulosic polysaccharides most likely depends on the nature and extent of non-covalent and/or covalent associations between wall polymers. Physical associations between arabinoxylans and (1→3,1→4)-ß-glucans themselves, with one another (Izydorczyk and Bilarderis 1995) and between these polymers and the surfaces of the microfibils is envisaged in the model (Figure 2).
The involvement of covalent bridges between arabinoxylans through dehydrodiferulic ester bridges (Figure 3) has also been proposed to play a role in wall integrity. The observation that dilute alkali, and more specifically, neutral hydroxamic acid, treatment liberates a substantial fraction of the arabinoxylan from water-extracted, endosperm walls (Mares and Stone, 1973b) might support this proposal. However dilute alkalis may also alter the physical form of the non-cellulosic polysaccharides causing them to dissociate from the wall. A comprehensive analysis of these interactions still remains to be provided. A physicochemical assessment of polymer interactions in the native wall using currently available physical methods may provide the needed information.
The availability of isolated walls of aleurone cells from wheat (Minifie and Stone 1988; Bacic and Stone 1981; Rhodes and Stone 2002) has allowed possible covalent associations between polymeric components to be explored. As in endosperm walls the non-cellulosic polysaccharides are chiefly (1→3;1→4)-β-glucans and arabinoxylans but the content of monomeric hydroxycinnamic acids esterified to the arabinoxylans is a much higher (1.8 % cf. 0.05 %) (Table 1). Consequently the aleurone walls are strongly autofluorescent (Bacic and Stone 1981; Rhodes et al. 2002). Very small amounts of esterified dehydrodiferulic acid are present so that bridges between arabinoxylan chains involving these structures are not important in aleurone wall organization. Proteins comprise 1% of the aleurone wall and are readily detected throughout the wall using the fluorescent dye Ponceau Red. At least three types proteins are present: glycine-rich (37-86%), proline-rich (11-39%) and serine-rich (up to 23%) (Rhodes and Stone 2002). Removal of arabinoxylan and (1→3;1→4)-β-glucan using specific hydrolases leaves a residue enriched in protein (4.5%) and a highly-branched arabinoxylan that retains its UV-induced autofluorescence even after alkali treatment. This led to the suggestion (Rhodes and Stone 2002) that the ferulic acid esterified to the arabinoxylan may be cross-linked to wall protein through ferulate-tyrosine bridges (Geissmann and Neukom 1973) (Figure 3).
Figure 3. Three modes of covalent cross-linking between wall polymers. Top. A diferulate cross-link between two arabinoxyan chains. Middle. A tyrosyl-ferulate cross-link between a protein and an arabinoxylan. Bottom. A dityrosyl cross-link between two protein chains.
Additionally some of the wall proteins themselves may be cross-linked through tyrosine-tyrosine dimmers (Figure 3). Small amounts of such dimers have been reported in endosperm proteins (Hanft and Koehler 2005). Although proteins are well-known wall components their potential for interaction and perhaps reinforcement of the predominant polysaccharide components needs to be further explored.
In contrast to the walls of the endosperm and aleurone cells many of the pericarp-seed coat walls are reinforced with lignin, which replaces the water in the gel-like matrix (Figure 2) and overlies the non-cellulosic polysaccharides and proteins found there. As a consequence the walls are physically strong and resistant to digestion by micro-organisms in the human and monogastric large intestine and in the rumen. Lignified walls of cells in stems (internodes) of grasses have compositions similar to those of the pericarp (Table 1) and there is a clear relationship between the decrease in vitro digestibility and the accumulation of wall-bound hydroxycinnamic acids during maturation (Lam et al. 1990). Detailed examination of the forms of wall-bound hydroxycinnamic acids showed that some of the ferulic acid esterified to wall polysaccharides (arabinoxylan) forms dehydrodiferulic acid bridges between arabinoxylan chains (Figure 3). In addition, some of the ferulic acid (and dehydrodiferulic acid) esterified to the arabinoxylan is also etherified to lignin forming ester-ether bridges between the two wall polymers (Iiyama et al. 1990). A correlation between ester-ether bridge content, but not lignin content, and in vitro digestibility was shown for internodes of the pasture grass, Phalaris aquatica (Lam et al. 2003). Proteins could also be involved in this cross-linking. The covalent cross-linking between the polymeric components in the lignified wall matrix walls would exclude polysaccharide hydrolases and so prevent access to their substrates.
Development of endosperm cell walls
Endosperm development in wheat begins with the cellularization of the multinucleate primary (3n) endosperm cell in the five or six days following anthesis (Mares et al. 1977). The periclinal walls of first-formed cells are deposited between nuclei lining the periphery of the endosperm mother cell without nuclear division or phragmoplast formation (Mares et al. 1977). This mode of wall initiation is quite unlike that in vegetative cells where the new wall is deposited centripetally at the cell plate situated in a phragmoplast between the daughter nuclei (Otegui et al. 2001). The endosperm cellularization process in cereals has now been described in detail by Brown et al. (1996). In the syncytial endosperm of rice, the, callose, was shown to be a major wall component in the developing periclinal walls using (1→3)-β-glucan specific monoclonal antibody (Brown et al. 1997). Extension of these observations to cellularizing barley endosperm using four specific monoclonal antibodies has shown that the initially deposited callose in periclinal walls is replaced by (1→3;1→4)-β-glucan, and later arabinoxylan and glucomannan are added to the wall (Doblin et al. 2004). Although the cytology of periclinal wall development in the nuclear endosperm is different from normal wall formation, a transient deposition of callose is common to both types (Otegui et al. 2001).
Approaches to manipulating wall structure and composition
The critical involvement of cell walls and their components in the end uses of cereal grains suggests that changing wall composition e.g. increase or decrease in (1→3;1→4)-β-glucan content, alteration of the intensity of cross-linking of polysaccharide components could be advantageous for certain end-uses. Already selection based on natural variation in wall composition (mutant cereals) is possible and marker-assisted selection for traits such as ara/xyl ratio, (1→3;1→4)-β-glucan, ferulic acid, ester/ether bridges, wall protein content is feasible and in some cases markers have already been identified. Generation of transgenic cereals with desirable cell wall compositions using current technology is not yet a possibility since apart from the synthases for cellulose and heteromannans, the genes for the xylan and (1→3;1→4)-β-glucan synthesis are yet to be identified. Chemical and enzymatic treatments have been applied to the walls of thee pericarp-seed coat of post-harvest grain to increase their fermentability by microorganisms.
Bacic, A. & Stone, B.A. (1980) Carbohydr. Res. 82: 372-377.
Bacic, A. & Stone, B.A. (1981) Aust. J. Pl. Physiol. 8: 475- 495.
Brown, R.C., Lemmon, B.E., & Olsen, O-A. (1996) J. Plant Res.109: 301-313.
Brown, R.C., Lemmon, B.E., Stone, B.A. Olsen, O.A. (1997) Planta 202: 414-426.
Doblin, M,, Wilson, S., Burton, R., Shirley, N., Stone, B., Fincher, G., Newbegin, E. & Bacic, A. (2004) In Xth Cell Wall Meeting, Sorrento, Italy, p. 178.
Geissmann, T. & Neukom, H. (1973) Cereal Chem. 50: 414-416.
Hanft, F.& Koehler, P.J. (2005) Agric. Food Chem. 53: 2418-2423.
Iiyama, K., Lam, T.B-T. & Stone BA (1990) Phytochem. 29: 733-737.
Izydorcyyk, M.S. & Bilarderis, C.G. (1995) Carbohydr. Polym. 29: 33-48.
Joyner, S.J. (1985). Masters Thesis. La Trobe University, Melbourne
Lam, T.B-T., Iiyama, K.& Stone, B.A. (1990). Phytochem. 29: 429-433.
Lam, T.B-T, Iiyama, K. & Stone, B.A. (2003). Phytochem. 64: 603-607.
Mares, D.J. & Stone, B.A. (1973a) Aust. J. Biol. Sci. 26: 793-812.
Mares, D.J. & Stone, B.A. (1973b) Aust. J. Biol. Sci. 26: 813-830.
Mares D.J., Stone, B.A., Jeffrey, C.& Norstog, K. (1977) Aust. J. Bot. 25: 599-613.
McCann, M.C. & Roberts, K. (1991) In: Lloyd CW (ed.) The Cytoskeletal Basis of Plant Growth and Form. London: Academic Press. pp. 109-129.
Minifie, J. & Stone, B.A. (1988) USP 4,746,073. )
Otegui, M.S., Mastronarde, D.N., Kang, B.H., Bednarek, S.Y. & Staehelin, L.A. (2001) Plant Cell 13: 2033-2051.
Rhodes, D.I., Sadek, M. & Stone B.A. (2002) J. Cereal Sci. 36: 67-81.
Rhodes D.I., Stone, B.A. (2002) J. Cereal Sci. 36: 83-101.
Ring, S.G. & Selevndran, R.R. (1980) Phytochem. 19:1723-1730.
Selvendran, R.R., Ring, S G., O'Neill, M.A. & Du Pont, M.S. (1980) Chem.& Ind. p.885-888.
Shibuya, N., Nakane, R., Yasui, A., Tanaka, K. & Iwasaki, T. (1985) Cereal Chem. 62: 252-258.