Revolutionary Times in Our Understanding of Cell Wall Biosynthesis and Remodeling in the Grasses

A major part of the daily caloric intake of human societies around the world is derived from a diverse range of foods prepared from members of the grass family, including wheat (Triticum aestivum), rice (Oryza sativa), sorghum (Sorghum bicolor), the millets (Panicum miliaceum and Pennisetum americanum), barley (Hordeum vulgare), and sugar cane (Saccharum officinarum). Grasses cover perhaps 20% or more of the earth's land surface (Gaut, 2002), and many of these are used as forage and fodder for the production of sheep, cattle, and other domesticated livestock. Maize (Zea mays) is also used widely in animal feed diets, while sorghum, switchgrass (Panicum virgatum), and several other perennial grasses are attracting considerable attention as future biomass energy crops (McLaren, 2005). The grasses are noteworthy for the unusual composition of their cell walls, because walls of grasses have less pectin and xyloglucan, but more heteroxylan, than walls from other higher plants. Most significantly, walls of the grasses contain as major constituents the (1,3;1,4)-β-d-glucans, which are not widely distributed outside the Poaceae. The compositions of walls from selected barley organs are shown in Table I Comparison of cell wall polysaccharide composition in various barley tissues Note the relatively low levels of xyloglucans and pectin in these walls, particularly those from grain. ND, Not detected. Comparison of cell wall polysaccharide composition in various barley tissues Note the relatively low levels of xyloglucans and pectin in these walls, particularly those from grain. ND, Not detected. Although there have been exciting new discoveries in the synthesis of cellulose, pectic polysaccharides, mannans, and xyloglucans in recent years, these discoveries have been made predominantly in dicotyledonous plants (Ye et al., 2006; Mohnen, 2008; Zabotina et al., 2008) and will not be covered here. This update, therefore, will be restricted to recent advances in our understanding of the biosynthesis of the characteristic and major wall polysaccharides of the grasses, namely the heteroxylans and (1,3;1,4)-β-d-glucans. So, why might we argue that we have come upon revolutionary times in our understanding of cell wall biosynthesis in the grasses? What new information has come to light in recent years? Progress in defining the genes and biological mechanisms underlying the synthesis of the major polysaccharides of walls in the grasses had remained painfully slow throughout the biochemical and molecular biological eras, mainly because the enzymes that catalyze the biosynthetic reactions are membrane proteins that usually lose activity quickly after cell disruption, before purification of the enzymes can be effected. Without even partially purified enzyme preparations, we were unable to obtain amino acid sequence information and hence could not identify the corresponding genes. However, emerging technologies of forward and reverse genetics and functional genomics have provided new tools to tackle these difficult problems and have yielded spectacular results. Thus, comparative genomics and forward genetics have been used to identify candidate genes that encode polysaccharide synthases involved in (1,3;1,4)-β-d-glucan biosynthesis in the grasses, while powerful bioinformatic techniques are providing important clues and candidates for the enzymes that mediate in the biosynthesis of the other key wall polysaccharide of the grasses, namely the heteroxylans. Data generated in these studies have raised ancillary but fundamental questions about the subcellular location of wall polysaccharide synthesis in the grasses. Is the Golgi apparatus the only site for the complete synthesis of matrix phase polysaccharides of the wall, including the (1,3;1,4)-β-d-glucans and the heteroxylans, or are there other possibilities? Functional genomics analyses have also pointed to previously unsuspected roles for hydrolytic enzymes and transglycosylases in wall polysaccharide synthesis and remodeling in the grasses. In particular, the highly abundant xyloglucan transglycosylases/hydrolases (XTHs) might act not only as modulators of xyloglucan structure but also as heterotransglycosylating enzymes that covalently link different classes of polysaccharides in the wall. There have been hints in the literature for some time that wall polysaccharides might be covalently linked, but experimental evidence that had not previously been available is now starting to build. While much of the new data have been highly informative with respect to the genes and enzymes involved in the biosynthesis of wall polysaccharides in the grasses, many of the additional questions mentioned above challenge the way in which wall polysaccharide biosynthesis and remodeling have traditionally been viewed. In this brief update, recent breakthroughs in the identification of genes that mediate wall synthesis in the grasses are discussed in the context of the complexities of chemical structures of the synthesized polysaccharides, their heterogeneity in structure and size, their physicochemical properties and functional imperatives, and their remodeling during growth and development. To fully appreciate the complexity of biochemical and cellular processes that lead to wall biosynthesis in the grasses, it is important to understand the chemical structures of the wall polysaccharides and the changes that occur to them during plant cell growth and development as well as how these structures are tailored to the functional needs of the wall. Therefore, structural characteristics of the two major wall polysaccharides of the grasses are briefly outlined below. The heteroxylans that are abundant in walls of the grasses can be classified into two main types, namely the arabinoxylans and the glucuronoarabinoxylans. The arabinoxylans are the major noncellulosic polysaccharides in walls of starchy endosperm and aleurone layer cells in cereal grains, whereas the glucuronoarabinoxylans are characteristically found in walls of the pericarp seed coat tissues (Fincher and Stone, 2004). Structural features of some common noncellulosic polysaccharides from cell walls of higher plants. A, Structure of a portion of a (1,4)-β-d-xylan backbone with a xylosyl residue substituted at C(O)3 with an α-l-arabinofuranosyl residue, which in turn is substituted at C(O)5 with a ferulic acid residue (Fincher and Stone, 2004). B, Substitution patterns of heteroxylans, which consist of a (1,4)-β-d-xylan backbone (blue) substituted at C(O)3 and C(O)2, and sometimes at both carbon atoms, with α-l-arabinofuranosyl residues (cyan). Some α-l-arabinofuranosyl residues are substituted with ferulic acid (red). In addition, the (1,4)-β-d-xylan backbone can be substituted with α-d-glucuronosyl residues and their methyl esters, while some xylosyl residues of cereal heteroxylans can be acetylated (Fincher and Stone, 2004). Some regions of the (1,4)-β-d-xylan backbone are unsubstituted. More details of heteroxylan structures are provided by York and O'Neill (2008). C, Diagrammatic representation of a xyloglucan, showing the (1,4)-β-d-glucan backbone (blue) substituted at C(O)6 with α-d-xylopyranosyl residues (red) or with short oligosaccharide chains of α-d-xylopyranosyl, β-d-galactopyranosyl (green), and α-l-fucopyranosyl residues. D, Diagrammatic representation of a (1,3;1,4)-β-d-glucan, showing (1,4)-β-d-glucosyl residues (blue) and (1,3)-β-d-glucosyl residues (red). The (1,3)-β-d-glucosyl residues introduce molecular kinks into the chain, and the irregular spacing of the (1,3)-β-d-glucosyl residues results in an asymmetric polysaccharide that can be soluble at high degrees of polymerization. It should be noted that while blocks of two or three adjacent (1,4)-β-d-glucosyl residues predominate, up to 10% of the polysaccharide chain consists of longer blocks of five to 20 adjacent (1,4)-β-d-glucosyl residues (Fincher and Stone, 2004). The glucuronoarabinoxylans also contain substituents of d-GlcUA (GlcAp) and its 4-O-methyl ester, linked to the C(O)2 of Xylp units of the xylan backbone. A number of the Araf units in the arabinoxylans of grasses can be esterified with the hydroxycinnamic acids, ferulic acid and, to a lesser extent, its nonmethoxylated analog p-coumaric acid. The hydroxycinnamates are found at C(O)5 of Araf units that are linked to C(O)3 of the Xylp units (Fig. 1A). Pena et al. (2007) demonstrated recently that glucuronoarabinoxylans from various dicotyledonous plants have an oligosaccharide consisting of 4-β-d-Xylp-(1,4)-β-d-Xylp-(1,3)-α-l-Rhap-(1,2)-α-d-GalpA-(1,4)-d-Xylp at their reducing termini, but attempts to identify a similar structure in arabinoxylans from grasses has so far been unsuccessful (W. York, personal communication). The Araf and other substituents sterically inhibit aggregation of the (1,4)-β-d-xylan chains and lead to the formation of an extended, asymmetrical polysaccharide that has physicochemical properties suited to its function as a major matrix phase component of walls in grasses. The physicochemical properties of the arabinoxylans are similar to those of the xyloglucans and other wall polysaccharides, but they are achieved through different chemistries (Fig. 1C). In summary, the heteroxylans from walls of the grasses have diverse chemical structures that are likely to be modified in response to changing functional requirements of the wall during growth and development. It is highly probable that synthesis of the heteroxylans involves the action of multiple polysaccharide synthase and/or glycosyl transferase enzymes. Progress toward the identification of these enzymes and the genes that encode them is summarized in the following sections. (1,3;1,4)-β-d-Glucans are unsubstituted, unbranched polysaccharides containing β-d-glucopyranosyl monomers polymerized through both (1,3)- and (1,4)-linkages. Within the grasses, barley, oat (Avena sativa), and rye (Secale cereale) grains are rich sources of (1,3;1,4)-β-d-glucans, while wheat, rice, and maize have lower concentrations of the polysaccharide (Fincher and Stone, 2004). The (1,3;1,4)-β-d-glucans are relatively minor components of walls in vegetative tissues of cereals and grasses. While it is often assumed that (1,3;1,4)-β-d-glucans are found only in the Poaceae, molecules with similar structures have been reported in species of angiosperms that are members of a major clade of the enlarged order Poales, which is referred to as the “core Poales” and includes the Poaceae and closely related families (Trethewey et al., 2005). In addition, (1,3;1,4)-β-d-glucans are found in lichens such as Iceland moss (Cetraria islandica; Olafsdottir and Ingolfsdottir, 2001), where they are located in walls of the mycobiont or fungal partner of the symbiosis (Honegger and Haisch, 2001), in certain fungal cell walls (Fontaine et al., 2000), in various algal species (Nevo and Sharon, 1969; Popper and Fry, 2003), in bryophytes (Popper and Fry, 2003), and in the cell walls of the horsetail (Equisetum species; Fry et al., 2008b; Sørensen et al., 2008). The ratio of cellotriosyl to cellotetraosyl units varies between species. In wheat, they range from 3.0:1 to 4.5:1, in barley from 2.9:1 to 3.4:1; in rye, the ratio is about 2.7:1, and in oats it is 1.8:1 to 2.3:1 (Fincher and Stone, 2004). The net effect of these linkage arrangements is the irregular distribution of (1,3)-β-d-glucosyl residues along what would otherwise be a regular, “cellulosic” chain. The (1,3)-β-d-glucosyl residues cause molecular “kinks” in the chain, and the irregular occurrence of these kinks means not only that the overall shape of the polysaccharide is irregular but also that the molecules will not align over extended regions. (1,3;1,4)-β-d-Glucans with these types of structure, therefore, will remain in solution even when their degree of polymerization exceeds 1,000 (Woodward et al., 1983b). Within the wall, however, the longer blocks of adjacent (1,4)-β-d-glucosyl residues might provide the potential for alignment over limited regions of the polysaccharide and hence for junction zone formation between (1,3;1,4)-β-d-glucan chains and with other wall polysaccharides such as cellulose or arabinoxylans (Carpita et al., 2001; Fincher and Stone, 2004). In aqueous media, barley (1,3;1,4)-β-d-glucans an extended with an ratio of about (Woodward et al., 1983b). This asymmetrical has to the polysaccharide to function as the matrix phase component of the wall. The structure the polysaccharide to provide some degree of structural for the wall but to remain and to the of and other molecules the wall during growth and development. The is for the high of (1,3;1,4)-β-d-glucans in aqueous and hence for the characteristics to (1,3;1,4)-β-d-glucans in animal feed and in and the other the of wall polysaccharides in grasses is for the of barley and other (1,3;1,4)-β-d-glucans human and and 2005). mentioned the why such of (1,3;1,4)-β-d-glucan structure are important is that molecular mechanisms for their synthesis into the chemical and physicochemical that are in the of the polysaccharide in the cell wall. The therefore, how it is that only (1,3)-β-d-glucosyl residues are between blocks of adjacent (1,4)-β-d-glucosyl residues that are three or residues in but be up to (1,4)-β-d-glucosyl residues and how it is that the cellotriosyl and cellotetraosyl units are distributed along the chain. many years, biochemical were in attempts to the properties of enzymes for and (1,3;1,4)-β-d-glucan synthesis in walls of the grasses, and these usually involved the of from of selected tissues and, in many the of these for as the by the membrane of from into and Stone, and However, the biosynthetic enzymes activity quickly following of a of polysaccharides with the including and (1,3;1,4)-β-d-glucan, and overall of into polysaccharide usually a there were of purified (1,3;1,4)-β-d-glucan synthases or xylan synthases from the grasses. amino acid sequence information could be generated this and the of the genes the therefore, remained of cellulose synthase and cellulose families in higher plants. The cellulose synthase and families from higher plants contain to members and have been classified into a number of and et al., these the and genes were shown to be for the Poaceae, while the and to be found only in et al., The is found in certain including barley, wheat, sorghum, and but not in rice or The for the sorghum is and barley for the genes are and This prepared by and is from a similar previously et al., It should be noted that the of can and The experimental data that the genes as potential in (1,3;1,4)-β-d-glucan synthesis were comparative genomics in rice and a major for (1,3;1,4)-β-d-glucan in barley et al., the to be located the rice In that of rice, a of rice genes and these genes candidates for a in (1,3;1,4)-β-d-glucan synthesis et al., The rice genes were into which not have genes or (1,3;1,4)-β-d-glucans in its (1,3;1,4)-β-d-Glucans were in the walls of the et al., This that the rice genes were to the synthesis of (1,3;1,4)-β-d-glucans, but it also that other genes would be for (1,3;1,4)-β-d-glucans synthesis et al., it demonstrated that there are at genes in these have been and their patterns et al., 2008). It is noteworthy that there to be only wall polysaccharide that is for the grasses, namely the (1,3;1,4)-β-d-glucans, there are three of namely the and and et al., The briefly mentioned by et al. but we would now that this be as a new of genes and provide and in of this new (Fig. The as the in the et al., 2002), after the that the I can be difficult to from the The is only found in certain grasses, including barley, wheat, sorghum, and but not in rice or have to the potential roles of and genes in (1,3;1,4)-β-d-glucan synthesis in similar to those used for the functional of the rice genes in it has been demonstrated that the can also mediate (1,3;1,4)-β-d-glucan synthesis in and and our data that the genes could also be involved and the biosynthesis of (1,3;1,4)-β-d-glucan in grasses the action of multiple enzymes or involves a in which an we are a way from the biosynthetic in et al., 2008). and as noted the mechanisms for the arrangements of (1,3)-β-d-glucosyl and (1,4)-β-d-glucosyl residues along the chain have not been at the biosynthetic is or enzymes catalyze the synthesis of (1,3;1,4)-β-d-glucans with different will be the of the which has a acid in the et al., 2008). The complexity of the (1,3;1,4)-β-d-glucan structure would be to be in a similar complexity in the for (1,3;1,4)-β-d-glucan et al. data in of an to the mechanisms for the structural of (1,3;1,4)-β-d-glucans in et al. that a cellulose up component of a (1,3;1,4)-β-d-glucan synthase and the cellotetraosyl units and while a glycosyl transferase is with the component to the residues that complete the cellotriosyl and et al. that the hydrolytic might also in (1,3;1,4)-β-d-glucan synthesis and that the (1,3;1,4)-β-d-glucan that is in barley where there is the net synthesis of of et al. also (1,3;1,4)-β-d-glucan during of barley grain. The could function to or (1,3;1,4)-β-d-glucan chains et al., or to synthesized chains from the biosynthetic enzymes et al., In some the levels of (1,3;1,4)-β-d-glucans in walls during growth and development. (1,3;1,4)-β-d-glucans to of walls during the phase of growth in barley but following the of growth at about (1,3;1,4)-β-d-glucan to et al., 2005). et al. (2007) also reported the of (1,3;1,4)-β-d-glucans in maize In related et al. reported that when barley were into the (1,3;1,4)-β-d-glucan of cell walls by about and that this with levels of (1,3;1,4)-β-d-glucan and The that cell wall (1,3;1,4)-β-d-glucans might be in and the so generated could as an energy of sugar et al., with the biosynthesis of (1,3;1,4)-β-d-glucans, (1,4)-β-d-xylan synthase activity has been in membrane from grasses over many and et al., but the enzymes have not been purified to the that amino acid sequence information can be The genes and enzymes that are for synthesis in the grasses have not been other polysaccharide biosynthetic it might be that an I polysaccharide synthase with multiple would mediate the synthesis of the (1,4)-β-d-xylan backbone. A transferase with a would be to be involved in the of α-l-arabinofuranosyl along with a different transferase for the of the substituents to the (1,4)-β-d-xylan backbone et al., the of the chemical between (1,4)-β-d-xylan and (1,4)-β-d-glucan it might be that a enzyme or enzymes would mediate the synthesis of the (1,4)-β-d-xylan backbone. However, analyses of candidate genes from the various have not evidence that these genes are involved et al., recent analyses of and bioinformatic information during of synthesis an of various I glycosyl from families and in the synthesis of heteroxylans, and it has been that these I glycosyl transferase enzymes might be for the synthesis of the (1,4)-β-d-xylan backbone et al., et al., Pena et al., et al., More and upon data available at the time for members of the Poaceae, et al. (2007) that genes in the families might encode (1,4)-β-d-xylan genes in the encode xylan or and genes in the encode The of a reducing oligosaccharide consisting of 4-β-d-Xylp-(1,4)-β-d-Xylp-(1,3)-α-l-Rhap-(1,2)-α-d-GalpA-(1,4)-d-Xylp et al., that additional glycosyl would be for the synthesis of this portion of the but so far there is evidence that such an oligosaccharide is in arabinoxylans from the grasses (W. York, personal communication). York and O'Neill that the potential of enzymes and these other recent challenge how heteroxylans in plants are and they a number of new that are with the with the (1,3;1,4)-β-d-glucans, hydrolytic enzymes could in the biosynthesis of arabinoxylans in the grasses. There is evidence that the structure of arabinoxylans changes after the of the polysaccharide into in barley the ratio of substituted to xylosyl units changes from about to over about et al., 2005). This that about of backbone residues in the synthesized are substituted with residues but that the substituents are during growth et al., in a that might be by the action of et al., changes have been reported in the arabinoxylans of maize of to (1,3;1,4)-β-d-glucans and arabinoxylans in sections. of barley were with (1,3;1,4)-β-d-glucans and In the high levels of (1,3;1,4)-β-d-glucans can be in the walls but could be in the or with the Golgi apparatus In in the the that the polysaccharide is located both in the Golgi and in the wall. This of polysaccharide distribution is in and barley cells et al., provided by To these et al. that the (1,3;1,4)-β-d-glucan the Golgi or in the barley cells might have been by substituents such as which would these substituents were in the wall, would is that the (1,3;1,4)-β-d-glucans are synthesized at different in the in the phase of were synthesized in the the the (1,3;1,4)-β-d-glucan would not to these et al., This would why we have been unable to (1,3;1,4)-β-d-glucans in the Golgi et al., In the the synthesized might be to the where they could be into (1,3;1,4)-β-d-glucans, they would be by the this the of might be through the a or It is noteworthy that there have been that cellulose synthesis might occur through a et al., of the into the polysaccharide would the action of a transferase xyloglucan or synthases this the synthesized in the Golgi were mainly cellotriosyl units but longer units at of the the of the membrane would the structure of the (1,3;1,4)-β-d-glucan found in the walls of grasses. It be that while such a of of the (1,3;1,4)-β-d-glucan would be with of our it is also a that has not been of action of The (blue) is to the enzyme and a (1,4)-β-d-glucosyl linkage is by amino acid residues at the The reducing portion of the from the enzyme while the portion of the covalently linked to the through the The is to the by the of the reducing portion of the The residue of the is with the covalently reducing residue of the The results in the of the portion to the The polysaccharide from the There is evidence that can a range of and in to xyloglucan et al., a more function for the of grasses, namely that could link different polysaccharides in and hence cell wall and et al. that some of the from which are by families of genes that were often at high levels in both and might be the more abundant matrix phase polysaccharides of cell walls in barley, namely the arabinoxylans and the (1,3;1,4)-β-d-glucans, than xyloglucans et al. (2007) that highly purified barley a of the of the formation of between xyloglucans and at and between xyloglucans and (1,3;1,4)-β-d-glucans at relatively low at in et al., The activity a of because the energy for the formation of the new linkage is provided from an linkage than from an sugar The of et al. (2007) that the of barley might (1,3;1,4)-β-d-glucans and/or arabinoxylans with the of and demonstrated activity in from and with the recent that from and from contain a enzyme that molecules from (1,3;1,4)-β-d-glucans to et al., The that the from species has a for (1,3;1,4)-β-d-glucans to but they also that cereals this activity et al., of fungal cell walls during formation and has been to a of transferase enzymes of which might link different polysaccharides such as and in the fungal wall and the walls et al., 2004). There have also been that pectic polysaccharides might be covalently linked with xyloglucans in plant cell walls et al., and Fry, et al., Popper and Fry, 2005). However, the enzyme purified by et al. (2007) not link polysaccharides such as or to xyloglucan, it link arabinoxylans to molecular et al., that this a It that other might different and including arabinoxylans and (1,3;1,4)-β-d-glucans. the of the families in the grasses, this will the and/or purification of different by the of and between different classes of wall polysaccharides in higher plants have been assumed to be in The summarized above at evidence that between different classes of wall polysaccharides might occur in including the grasses, in evidence for this has to be to our understanding of cell wall in the grasses are by recent that while genes are involved in the biosynthesis of some noncellulosic wall polysaccharides, it is not (1,3;1,4)-β-d-glucans with different with respect to the and distribution of the units along the chain. It is not enzymes in heteroxylan and of function is for the various candidate glycosyl transferase genes and enzymes that have recently been for the synthesis of the (1,4)-β-d-xylan backbone of wall arabinoxylans in grasses. it has been that the (1,3;1,4)-β-d-glucans of walls of grasses might be synthesized through a previously of cellular and and that enzymes might catalyze the formation of between different types of wall polysaccharides in the grasses. To and these it will be to both biochemical and cell biological to these it will be to the subcellular and mechanisms that are with (1,3;1,4)-β-d-glucan It will also be to highly purified from grasses to their and, at the to chemical evidence for the of junction regions between covalently linked polysaccharides of different types the cell wall such between different polysaccharides in plant cell walls, this will not only the way we cell wall in plants in but will also have important for wall and A understanding of between wall polysaccharides would also provide to processes such as and and and of the by an of including in and