Update on Mechanisms of Plant Cell Wall Biosynthesis: How Plants Make Cellulose and 1 Other (1/4)-B-D-Glycans

UpdateonMechanismsofPlantCellWallBiosynthesis

Update on Mechanisms of Plant Cell Wall Biosynthesis: How Plants Make Cellulose and 1 Other (1/4)-b-D-Glycans

Nicholas C. Carpita* Department of Botany and Plant Pathology, and Bindley Bioscience Center, Purdue University, West Lafayette, Indiana 47907–2054

The discovery of a gene that encodes a cotton ences between plants with type I walls and those of the (Gossypium hirsutum) cellulose synthase (Pear et al., grasses with type II walls (Fig. 1A; Penning et al., 1996) revolutionized and invigorated the plant cell 2009). For CesA genes and certain Csl genes, establish- wall community to find the genes that encode the ment of specific function for the synthases they en- machinery of cell wall polysaccharide synthesis. The code comes from the analysis of mutants lacking a landscape was framed by the completion of the ge- particular function and, in some specific examples, nome sequence of Arabidopsis (Arabidopsis thaliana; by heterologous expression. However, genetic ap- Arabidopsis Genome Initiative, 2000), which gave a proaches alone do not inform us about the biological complete gene inventory for a model plant species, but mechanism of synthesis. The knowledge gained from one with many genes yet to be annotated for function. molecular genetic approaches now needs to be aug- An estimated 10% of the plant genome, about 2,500 mented by biochemical and cell biological approaches genes, is devoted to construction, dynamic architec- to achieve a greater understanding of proteins and ture, sensing functions, and metabolism of the plant their interactions within a synthase complex, their cell wall. Based largely on prior discoveries of function organization at membranes, and their dynamics. This in prokaryotic organisms, most of the tentatively an- Update focuses on the biochemical mechanisms of the notated genes are organized into gene families for synthesis of a single type of linkage, the (1/4)-b-D substrate generation, glycosyl transfer, targeting and linkage, in which one sugar is inverted nearly 180° trafficking, cell wall rearrangement, and modification with respect to each neighboring sugar in the chain. by hydrolases, esterases, and lyases (Yong et al., 2005; This linkage presents a unique steric problem for Penning et al., 2009). However, the biochemical activ- processive catalysis that all living organisms have ities of most enzymes involved in glycosyl transfer solved but we are still struggling to understand. within these families remain to be verified, and an This Update reviews our present state of knowledge additional 40% of the genome encodes genes whose of the biochemical mechanisms of polysaccharide functions are not known. As many of these proteins synthesis, including some classic discoveries, and contain secretory signal peptides (Arabidopsis Ge- presents an alternative hypothesis on the biochemical nome Initiative, 2000), it is reasonable to infer that mechanisms and organization of complexes involved some have roles in cell wall construction. in synthase reactions that yield (1/4)-b-D linkages. The Arabidopsis cellulose synthase/cellulose synthase- like (CesA/Csl) gene superfamily, which includes 10 CesA genes and 29 Csl genes in six distinct groups, was one CELLULOSE SYNTHESIS of the first large families to be described (Richmond and Somerville, 2000), and comparative analyses of a In flowering plants, cellulose is a para-crystalline reference dicot, Arabidopsis, with a reference grass, array of about two to three dozen (1/4)-b-D-glucan rice (Oryza sativa), revealed substantive differences in chains. Microfibrils of 36 glucan chains have a theo- family structures, adding two groups not seen in the retical diameter of 3.8 nm, but x-ray scattering and dicot genome (Hazen et al., 2002). Extension of these NMR spectroscopy indicate that some microfibril di- annotations to compare all cell wall-related gene fam- ameters could be as small as 2.4 nm, or about two ilies of the grasses with those of the dicots reveals dozen chains (Kennedy et al., 2007). The microfibrils some correlation of family structure with the differ- are synthesized at the plasma membrane by terminal complexes of six-membered “particle rosettes” that produce a single microfibril (Giddings et al., 1980; 1 This work supported by the Center for Direct Catalytic Conver- sion of Biomass to Biofuels, an Energy Frontier Research Center Mueller and Brown, 1980). Thus, each of the six funded by the U.S. Department of Energy, Office of Science, Office of components of the particle rosette is expected to Basic Energy Sciences (award no. DE–SC0000997). synthesize four to six of the glucan chains, and 24 to * E-mail [email protected]. 36 chains are then assembled into a functional micro- www.plantphysiol.org/cgi/doi/10.1104/pp.110.163360 fibril (Doblin et al., 2002). In freeze fracture, the par-

Ò Plant Physiology , January 2011, Vol. 155, pp. 171–184, www.plantphysiol.org Ó 2010 American Society of Plant Biologists 171 Carpita

estimated to be 50 nm wide and extend 35 nm into the cytoplasm (Bowling and Brown, 2008), a feature that has escaped consideration in many published models of the rosette structure (Fig. 2, B and C). Cellulose synthase is an ancient enzyme (Nobles et al., 2001), and cellulose synthase genes in green algae are homologous to those of flowering plants (Roberts et al., 2002). The deduced amino acid sequences of CesAs share regions of similarity with the bacterial CesA proteins, namely the four catalytic motifs containing the D, DxD, D, Q/RxxRW that are highly conserved among those that synthesize several kinds of (1/4)-b-D-glycans (Saxena et al., 1995). The higher plant CesA genes are predicted to encode polypeptides of about 110 kD, each with a large, cytoplasmic N-terminal region containing zinc-finger (ZnF) domains, and eight membrane spans sandwiching the four U motifs of the catalytic domain (Fig. 1B; Delmer, 1999). Further evidence for the functions of plant CesA genes in cellulose synthesis came from Arabidopsis mutants of three of the CesA genes involved in primary wall synthesis: the temperature-sensitive radial Figure 1. The CesA/Csl gene superfamily. A, Of the 10 Arabidopsis swelling mutant rsw1 (AtCesA1; Arioli et al., 1998), CesA genes, at least three are coexpressed during primary wall forma- the dwarf-hypocotyl procuste mutant prc1 (AtCesA6; tion, and mutations in each of them, AtCesA1 (RSW1; At4g32410), Fagard et al., 2000), and a stunted root phenotype AtCesA6 (PRC1; At5g64740), and AtCesA3 (CEV1 and ELI1; with altered jasmonate and ethylene signaling (cev1) At5g05170), result in cellulose deficiencies, indicating that each is and ectopic lignification (eli1) mutant alleles (AtCesA3; essential for cellulose synthesis. The irx mutants AtCesA8 (IRX1; Ellis and Turner, 2001; Canõ-Delgado et al., 2003). At4g18780), AtCesA7 (IRX3; At5g17420), and AtCesA4 (IRX5; Despite coexpression in the same cells and an expec- At5g44030) are deficient in cellulose synthesis specifically in secondary tation of redundancy, cellulose synthesis is impaired in walls. Seven additional subgroups were identified that are the likely / each mutant. The same was observed with the irregular synthases for noncellulosic polysaccharides with backbones of (1 4)- xylem mutants irx1, irx3, and irx5, which display a b-D-glycans. Whereas the CesA genes of Arabidopsis, rice, and maize appear to be orthologous, the Csl genes are divergent between dicots phenotype of collapsed mature xylem cells as a result and grasses, species that make two distinct kinds of walls. From mutants of lowered cellulose content during secondary cell and heterologous expression studies, members of the CslA group encode wall deposition (Taylor et al., 2000, 2003). A widely the synthases of (gluco)mannans, members of the CslC group are likely accepted hypothesis is that the AtCesA1, AtCesA3, to encode the glucan backbone of xyloglucans, and the rice- and maize- and AtCesA6 proteins assemble to function in primary only members of CslH and CslF encode the synthases of the mixed- wall cellulose synthesis, while the AtCesA4, AtCesA7, linkage (1/3),(1/4)-b-D-glucans found only in grasses (after Penning and AtCesA8 proteins assemble to make secondary et al., 2009). B, Domain model and class-specific regions (CSRs) for wall cellulose (Fig. 1A), with each member of the trio three CesAs known to function in primary cell wall cellulose synthesis. performing a nonredundant function in the complex Two ZnF domains (in yellow) are found in the N terminus before the first (Taylor, 2008). Lack of one CesA prevents incorpora- membrane-spanning domain (in blue). Eight transmembrane helices, two upstream and six downstream of the catalytic domain, are predicted tion of the other two into the plasma membrane to interact to form a channel through which a single b-glucan chain is (Gardiner et al., 2003). However, at least some of the secreted to the cell wall. The large central catalytic domain contains four subunits are potentially interchangeable, as inferred highly conserved “U motifs” of D, DxD, D, and QxxRW, important for by the dominant-negative inhibition of growth and substrate binding and catalysis. Once thought to be a hypervariable primary wall thickness caused by constitutive expres- region (Pear et al., 1996), the class-specific regions are conserved among sion of a mutated fra5 (irx3 allele) transgene (Zhong orthologs of the same subclade and vary in the number of upstream et al., 2003) and by the semi-dominant-negative phe- conserved Cys residues, the number of consecutive basic amino acids, notype observed in the heterozygous AtCesA3 mutant Lys and Arg, and the number of consecutive acidic amino acids, Asp and (Daras et al., 2009). AtCesA1 is essential for cellulose Glu, downstream from the basic residues (after Carpita and Vergara, synthesis (Beeckman et al., 2002), whereas knockouts 1998; Vergara and Carpita, 2001). of AtCesA3 (Ellis and Turner, 2001; Canõ-Delgado et al., 2003) and AtCes6 (Fagard et al., 2000) result in ticle rosettes, found only on the P-face of the mem- partially impaired synthesis but not in total inhibition. brane, are about 25 nm in diameter, but this size Desprez et al. (2007) indicated that the AtCesA2 and represents only the membrane-spanning and short AtCesA5 proteins have partially redundant functions exterior domains (Fig. 2A). Hidden in surface views of with AtCesA6. rosette structures in the plasma membrane, the much A direct association of three distinct CesA polypep- larger catalytic domains of the cellulose synthases are tides was demonstrated in vitro and by colocalization

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rations subjected to native PAGE gave an 840-kD complex and that null mutants, but not missense mutations, gave smaller 420-kD complexes (Wang et al., 2008). Atanassov et al. (2009) afﬁnity trapped a ladder of complexes of CesA oligomers to about 700 to 730 kD. Consistent with the observations of Wang et al. (2008), only smaller oligomeric complexes of two of the CesAs are detected when the third is missing (Atanassov et al., 2009). Such an association of CesAs was indicated independently in yeast two-hybrid studies(Timmersetal.,2009).

DOES SYNTHESIS OF EACH (1/4)-b-D-GLUCAN CHAIN REQUIRE ONE OR TWO CATALYTIC POLYPEPTIDES? After over four decades of study, the biochemical mechanism by which cellulose is made remains a mystery (Delmer, 1999; Saxena and Brown, 2005; Somerville, 2006; Guerriero et al., 2010), with only a few reports of cellulose synthesis in vitro with isolated membranes (Kudlicka and Brown, 1997; Lai-Kee-Him et al., 2002). For both cellulose and the related (1/4)- b-D-glycan, chitin, synthesis proceeds by the attachment of glucosyl residues to the nonreducing terminus of the acceptor glucan chain (Koyama et al., 1997; Imai Figure 2. Particle rosette structures associated with cellulose synthesis et al., 2003). The simplest hypothesis is that each CesA in angiosperms. A, Freeze-etch images of the P-face of the plasma polypeptide synthesizes a single glucan chain. In the membrane showing clusters of rosettes associated with the developing Delmer (1999) model, the eight membrane spans form of secondary wall spiral thickenings of a Lepidium tracheary element a channel through which a single glucan chain is (from Herth, 1985). The inset shows the 6-fold symmetry of a single extruded (Fig. 3A). This mode of synthesis comes with particle rosette from a Zinnia tracheary element developing in vitro a very big steric problem for synthesis. To make a (1/ (from C. Haigler, unpublished data, as seen in Delmer, 1999). A 4)-b-D linkage means that each glucosyl residue is substructure can be observed in each of the particles. In these freeze- turned 180° with respect to each neighbor. Thus, the etch images, only the membrane-spanning domains and extracellular loops of the CesA proteins can be observed. B, Cytoplasmic structure O-4 position of nonreducing terminal sugar of the (circled) underlying the rosettes in plasma membrane footprints (from acceptor chain is displaced several angstroms upon Bowling and Brown, 2008). These structures always are at the terminus addition of each successive unit (Fig. 3B). For the next of a microfibril (arrow). Bar = 200 nm. C, A Markham rotational glycosyl transfer to occur, the site of catalysis must analysis of one of these shows the reinforcement of hexagonal shape move several angstroms within the protein, the accep- with 60° rotational steps. All other angles of rotation cancel to circular tor chain must swivel 180°, or the catalytic or acid-base (Bowling and Brown, 2008). amino acids must toggle between two forms to ac- count for the displacement. To overcome this steric problem, several models have proposed that two sites in vivo by Taylor et al. (2003). Domain-swap experi- or modes of glycosyl transfer reside within the cata- ments with wild-type and mutant AtCesA1 and lytic complex, so that disaccharide units are added AtCesA3 proteins in their respective mutants resulted iteratively (Carpita et al., 1996; Koyama et al., 1997; in dominant-positive and dominant-negative effects, Carpita and Vergara, 1998; Buckeridge et al., 1999, 2001; consistent with both catalytic and C-terminal domains Saxena et al., 2001) or that two polypeptides associate being important for function (Wang et al., 2006). Direct to form two opposing catalytic sites (Buckeridge et al., interactions of three distinct CesA polypeptides in 2001; Vergara and Carpita, 2001). In either model, vivo were shown by bimolecular fluorescence com- glycobiosyl units, or any even-numbered oligomeric plementation (Desprez et al., 2007). Although some units, are added to the nonreducing end to ensure that complementary pairs gave stronger fluorescence the (1/4)-b-D linkages are strictly preserved without than others, both homodimers and heterodimers of inversion of substrate, active site, or terminus of the AtCesA1, AtCesA3, and AtCesA6 are inferred. Wang growing chain (Fig. 3C). et al. (2008) used pull-down experiments similar to Despite the rationale for a two-site model of ca- those of Taylor et al. (2003) to show that these three talysis, biochemical evidence from other types of primary wall CesA proteins interact. Furthermore, polysaccharide synthases indicate that a single they showed that Triton-soluble microsomal prepa- polypeptide is sufficient. Hyaluronan (HA) is an un-

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Figure 3. Models for cellulose synthase and the steric problem of making a (1/4)-b-D-glycosyl linkage. A, The first model of conformation for a single CesA protein subunit was proposed by Delmer (1999). Each CesA subunit must interact with other such subunits to form the synthase complex. The ZnFs, plant-specific conserved region (P-CR), and class-specific region (CSR) are potential interaction sites (figure modified from Delmer, 1999). B, The steric problem of synthesis is illustrated in top view and end view. Addition of a single glycosyl residue in a (1/4)-b-D linkage without rotation of the end of the chain or the active site of the synthase would result in movement of the O-4 several angstroms. C, The conceptual solution to the steric problem is a catalytic dimer of simultaneous glycosyl transfer to form a cellobiosyl residue to the O-4 position of the terminal glucosyl residue of the chain. Synthesis of an even number of units always maintains the acceptor position as the O-4 position as the chain is extruded. In the catalytic dimer model, if one of the sites is damaged, then the point of attachment becomes the O-3 position, which would maintain the point of attachment as the O-3, hence producing callose thereafter (after Buckeridge et al., 1999, 2001). branched polysaccharide composed of repeating synthesis of a repeating (1/4)-b-D-glucosyl linkage units of (1/3)-b-D-GlcNAc and (1/4)-b-D-GlcUA of the cellulose glucan chains. In fact, two recent (DeAngelis and Weigel, 1994). HA synthases exist in studies demonstrate unequivocally that at least some three distinct classes, with class I containing integral HA synthases and homologs of SpsA synthase form membrane proteins to transport the HA across the dimers. An identical structure to the SpsA synthase is membrane (Fig. 4). Bacterial and mammalian HA the 3BCV polypeptide from Bacteroides fragilis, which synthases have been shown to contain both transferase is also predicted to contain a single substrate-binding activities in a single polypeptide (DeAngelis and site. In contrast to SpsA synthase, the 3BCV protein Weigel, 1994; Yoshida et al., 2000; Williams et al., crystallizes as a dimer, and each monomer possesses 2006). Such a finding argues that the synthesis of a a bound UDP (Fig. 5B; http://www.pdb.org/pdb/ single HA polymer requires only a single polypeptide. explore/explore.do?structureId=3BCV). The dimer- The crystal structure of a nonprocessive family 2 ization occurs through the C-terminal regions, which glycosyltransferase (GTs) sharing sequence similar- appear to be flexible and, for this reason, were deleted ity with a portion of the catalytic domain of a CesA, the Bacillus subtilis SpsA synthase, provided the first conformationoftheactivesiteandtheroleofthe aspartyl residues in the positioning of the uridinyl group of a UDP-sugar (Fig. 5A; Charnock and Davies, 1999; http://www.pdb.org/pdb/explore/explore.do? structureId=1QGS). Charnock and colleagues (2001) argued that only a single site for a nucleotide-sugar substrate is accommodated within a single polypeptide of SpsA.

A CATALYTIC DIMER HYPOTHESIS From the studies of the class I HA synthases and the SpsA crystal structure, it is inferred that a single polypeptide alone has all the features needed for synthesis. However, these features still do not address Figure 4. Models of HA synthases. A, Class I synthases. B, Class II mechanistically the fundamental steric problem of synthases (after Weigel and DeAngelis, 2007).

174 Plant Physiol. Vol. 155, 2011 Synthesis of (1/4)-b-D-Glycans

Figure 5. Crystal structures of type 2 glycosyl transferases. A, The SpsA synthase crystallizes as a monomer with a single binding site for UDP (Charnock and Davies, 1999; http://www.pdb.org/pdb/explore/ explore.do?structureId=1QGS). B, The B. fragilis SpsA homologous polypeptide crystallizes as a dimer (http://www.pdb.org/pdb/explore/explore.do? structureId=3BCV), each with a single binding domain for UDP. C, Each member of the crystal dimer E. coli chondroitin polymerase has two UDP-GlcA- or UDP-binding domains (http://www.pdb.org/pdb/ explore/explore.do?structureId=2Z86).

from the crystal structure of the SpsA synthase cellulose synthase is of sufficient size to extrude a (1/ (Charnock and Davies, 1999). This, to our knowledge, 4)-b-D-glucan chain. The question about sufficient is the first direct evidence by crystal structure of a channel size was raised also with respect to the HA homodimer formed by GT2 proteins, but even more synthases by Weigel and DeAngelis (2007), who sug- complicated structures are also found. For example, gested that certain phospholipids required for activity an Escherichia coli strain K4 chondroitin polymerase possibly integrate with the membrane spans to widen contains two “Rossmann fold-like” domains within the channel for extrusion. However, whether lipid a single polypeptide, each binding a UDP-GlcA or interactions with a small number of domains would be UDP, and it also crystallizes as a dimer, giving a total of significant is still in question. Callose synthases are four nucleotide-binding domains (Fig. 5C; http:// about twice the size of CesAs and contain 16 mem- www.pdb.org/pdb/explore/explore.do?structureId= brane spans (i.e. double those of a CesA; Hong et al., 2Z86). Although the fit is not exceptionally good, the 2001). Plasma membrane hexose and maltose trans- CesA catalytic domain threads through the E. coli porters of prokaryotes and eukaryotes are homolo- chondroitin polymerase best of all for known crystal gous (Maiden et al., 1987), and virtually all of them structures of glycosyl transferases involving nucleo- contain a minimum of 11 to as many as 18 transmem- tide sugars (D. Kihara, personal communication), and brane spans per functional unit (Reifenberger et al., it is consistent with the suggestion by Brown and 1995; Pao et al., 1998; Sherson et al., 2000; Klepek et al., Saxena (2000) and Saxena et al. (2001) of a conforma- 2010). tion within which the catalytic domain of a single The sensitivity of detergent-solubilized CesA com- CesA would allow the synthesis of cellobiose units of plexes to dithiothreitol suggested to Atanassov et al. the chain within a single polypeptide. (2009) that disulfide bonds are involved in the cou- The class II synthases contain two different types of pling into larger complexes. Other features of the GT2 modules but not the membrane-spanning do- protein outside the region of catalysis, such as the mains (Fig. 4). One type of HA synthase possesses two ZnFs, which show high similarity to RING-finger repeats of the UDP-Glc and acceptor-binding do- domains that bind zinc in a “cross-brace” manner mains, so the synthesis of the characteristic disaccha- (Freemont, 2000), might function in the organization of ride of HA by a single synthase is rationalized (Jing these into the larger rosette structure. Kurek et al. and DeAngelis, 2000). However, a direct interaction of (2002) proposed that CesAs are coupled through the two synthases is inferred for HA synthesis to explain ZnF domain in a redox-dependent manner, constitut- the finding that host cells harboring constructs in ing the first step in the clustering of CesAs into which each site is independently disrupted are still rosettes. Moreover, the discovery that a thioredoxin- able to make HA (Jing and DeAngelis, 2000; Weigel like protein associates with the CesA ZnF domain in and DeAngelis, 2007). Because the class I HA syn- a yeast two-hybrid screen led to the suggestion thases both have activities on a single peptide does not that reduction of the domains by oxidoreductases preclude the possibility that the formation of homo- returns the CesAs to monomeric forms, which are di- dimers of single isoforms of HA synthase is necessary rected to the ubiquitin-dependent turnover pathway for function, which, like the chrondroitin polymerase, (Kurek et al., 2002). The experimental herbicide CGA would give four nucleotide sugar-binding sites per 325#615 blocks crystallization of the b-D-glucan chains dimer. into cellulose microfibrils, phenocopying the rsw1 Solving the steric problem aside, one must also ask if swollen root tip (Peng et al., 2001). This phenotype a channel of eight membrane spans proposed for the can be abrogated completely in the presence of hydro-

Plant Physiol. Vol. 155, 2011 175 Carpita gen peroxide, suggesting that the inhibitor blocks rosette assembly by enzymatic oxidation (Kurek et al., 2002). If ZnF domains of two CesAs couple as part of the recruitment into rosette particles, the question remaining is how all the others interact to form a complete complex. Although not discussed specifically, the study by Kurek et al. (2002) presented data that full-length CesA proteins formed tetramers and even higher ordered pairings, while the ZnF domains were limited to coupling of a single pair. Timmers et al. (2009) showed that heterodimer interactions indicated by yeast two-hybrid analysis do not require the ZnF domains. Taken together, these data provide evidence that domains other than the ZnFs of the CesA participate in coupling reactions if two to three dozen CesAs or more are aggregated to form a rosette complex. Comparison of CesA sequences suggests potential heterodimeric interaction domains within the catalytic domain. The initial scarcity of CesA protein sequences and the apparent variability within the so-called “hypervariable region” led to the assumption that this region was probably not essential in catalysis (Pear et al., 1996). However, it is now understood that these regions are well conserved across grass and dicot species with a distinct subclade structure. Potential protein-protein interactions through subdomains of this region containing conserved Cys residues, clusters of consecutive basic Lys and Arg residues, and clusters of acidic Asp and Glu residues form the basis of a class-specific region (Fig. 1B; Vergara and Carpita, 2001). To test the catalytic dimer hypothesis, we expressed Figure 6. A catalytic dimer hypothesis for cellulose synthase. A, A fusion proteins containing only the catalytic domain of catalytic dimer model of two CesAs to form a complex that synthesizes Arabidopsis and maize (Zea mays) CesAs with affinity a single (1/4)-b-D-glucan chain. Homodimerization or heterodimer- tags and observed that dimers and higher order ag- ization of CesAs gives mirrored active sites that generate cellobiosyl gregates collapse reversibly to monomeric forms by units, which are then attached to the nonreducing end of the extruded thiol-reducing agents (C. Rayon, A. Olek, L. Paul, and glucan chain. Dimerization also results in a channel composed of 16 S. Ghosh, unpublished data). Because the ZnF was membrane-spanning domains, equivalent to that of callose synthase absent in these constructs, dimerization must occur and consistent with eukaryotic monosaccharide transporters. B, Di- merization results in two ZnF domains that are now able to couple two through thiol-sensitive sequences in the catalytic neighbors instead of just one. C, Six such complexes interact to domain. Such an interaction of CesAs to form constitute one particle of the six-particle rosette. homodimers or heterodimers solves the three basic problems of the single polypeptide-single polymer conundrum: (1) the steric problem is solved by coor- just one. Further experiments are needed to establish dinate synthesis and attachment of cellobiose units preferred heterodimer interactions, the stoichiome- instead of monomers, preserving the integrity of the try of UDP-Glc binding, and the role of the ZnF in O-4 site of attachment at the nonreducing terminus of recruitment of the catalytic dimers into larger com- the chain; (2) a channel of 16 membrane-spanning plexes. domains is consistent with sugar transport and callose extrusion; and (3) the interaction produces two ZnF domains for recruitment of the catalytic dimer into THE BIOLOGICAL SYNTHESIS OF CELLULOSE rosette particles (Fig. 6). An exciting prospect is that conservation of space would be maintained if CesAs Cellulose synthase has a half-life of less than 30 min, turn out to have a structure like the chondroitin remarkably short for a membrane protein (Jacob-Wilk polymerase dimers and function like class II HA et al., 2006). Assembly of rosettes occurs in the Golgi synthases, because a CesA catalytic dimer with four stacks, and they must be continually secreted to the nucleotide-binding domains would be capable of plasma membrane to maintain cellulose synthesis generating two (1/4)-b-D-glucan chains instead of (Haigler and Brown, 1986). Additional proteins are

176 Plant Physiol. Vol. 155, 2011 Synthesis of (1/4)-b-D-Glycans suspected to be necessary for the formation of primers participants in protein-protein interactions within a of polymer synthesis, metabolic channeling of sub- complex in vivo. Fluorescence tagging has also al- strates, crystallization of the chains, and termination of lowed visualization of the movement of cellulose chains (Doblin et al., 2002; Somerville, 2006; Guerriero synthase complexes at the plasma membrane (Paredez et al., 2010). Furthermore, a proteomics survey of et al., 2006; Wightman and Turner, 2008; Gutierrez plasma membrane proteins shows that certain CesAs et al., 2009). These studies also established the dynam- are phosphorylated at several locations within the ics of the relationships with the cortical microtubule catalytic and N-terminal domains (Nu¨ hse et al., 2004). network in real time. As reviewed by Baskin (2001) Modification of some potential phosphorylation sites and Szymanski and Cosgrove (2009), these studies with amino acids that either prevent (Ala) or mimic bring resolution to ideas on the alignment of cortical (Glu) phosphorylation has multiple effects that reduce microtubules and cellulose microfibrils generated long either synthesis rates or interactions with the micro- ago through observations with inhibitors (Green, 1962) tubule cytoskeleton independently (Chen et al., 2010). and in the electron microscope (Ledbetter and Porter, In affinity-labeling experiments with [32P]UDP-Glc, 1963). Improvements in imaging tools are still needed an 84-kD polypeptide was found to be associated with that permit visualization of the delivery of Golgi- a plasma membrane fraction containing the highest derived vesicles to the sites of cellulose synthesis, and activity of callose synthase (Delmer et al., 1991) and much progress has already been made along these subsequently was identified as Suc synthase (SuSy). lines (Held et al., 2008; Konopka and Bednarek, 2008; Confirmation of plasma membrane association was Crowell et al., 2009). made immunocytochemically (Amor et al., 1995), and Delmer and Amor (1995) proposed that the association of SuSy represented a UDP-Glc delivery mechanism to THE SYNTHESIS OF NONCELLULOSIC cellulose synthase. b-Glucan microfibrils are synthe- POLYSACCHARIDES WITH sized from Suc and UDP on immobilized tobacco (1/4)-b-D-GLYCAN BACKBONES (Nicotiana tabacum) plasma membrane sheets (Hirai Because of the same conserved domains of nucleo- et al., 1998). More recently, SuSy was immunologically associated with CesA proteins in the rosette structures tide-sugar binding and catalysis as those encoding Csl (Fujii et al., 2010), strengthening the idea of an asso- CesAs, subfamilies of genes were predicted to encode the synthases of noncellulosic polymers with ciation of SuSy directly with cellulose synthases for / b metabolic channeling of Glc through a localized pool (1 4)- -D-glycan backbones, primarily (gluco)man- of UDP-Glc. However, a quadruple mutant that elim- nans and galacto(gluco)mannans, xyloglucans, glucu- ronoarabinoxylans (GAX), and the grass-specific (1/ inates all detectable SuSy in vegetative tissue does not / b impair cellulose synthesis (Barratt et al., 2009). Over- 3),(1 4)- -D-glucans (Delmer, 1999; Richmond and expression of SuSy in developing vascular tissue of Somerville, 2000; Hazen et al., 2002). For the most part, this has turned out to be true, but GAXs are a clear transgenic poplar yields small but significant increases exception. There is still an incomplete knowledge of in cellulose content (Coleman et al., 2009). Taken together, SuSy does not appear to be required for most of the gene products and interactions among them to make specific b-D-glycans, even among fam- cellulose synthesis but may enhance rates by concen- ilies where at least one member has a confirmed trating substrate at the site of synthesis. Peng et al. (2002) provided evidence that sitosterol- glycosyl transferase activity. There is also a growing cellodextrins synthesized from sitosterol-b-glycoside disconnection between the classic studies on the synthesis of these polysaccharides in vitro and the dis- serve as primers of glucan chain initiation, with the KORRIGAN glucanohydrolase trimming the sitosterol covery of genes encoding the machinery that warrants from the growing chain. DeBolt and colleagues (2009) a revisit. question this role, as they found that double mutants of two major sterol-b-glucoside synthases result in CslF AND CslH: MIXED-LINKAGE severe defects in cuticle formation but not in cellulose / / b synthesis. However, the sterol-glucosides are substan- (1 3),(1 4)- -D-GLUCANSYNTHASEISTHE TOPOLOGICAL EQUIVALENT OF tially reduced in the double mutant but not entirely CELLULOSE SYNTHASE eliminated, leaving open the question. Outside the Golgi stacks, a membrane compartment The mixed-linkage (1/3),(1/4)-b-D-glucan is also containing KORRIGAN (Robert et al., 2005) might made in the grasses (Poales; Carpita, 1996; Buckeridge represent a dynamic factory associated with both the et al., 2004), certain lichens (Wood et al., 1994), and microtubule network and the plasma membrane that Equisetum (Fry et al., 2008; Sørensen et al., 2008), but aligns and directs the cellulose synthase complex and differences in the distribution of their cellodextrin coordinate that function with the deposition of the many oligomers indicate that they probably arose by con- polysaccharides directed to it from packaged Golgi vergent evolution of synthases. For the grasses, this vesicles. Bimolecular fluorescence complementation glucan is not a random mixture of (1/3)-b-D- and techniques, as shown for CesA interactions (Desprez (1/4)-b-D-glucosyl linkages but is composed primar- et al., 2007), can give important clues to selected ily of cellotriosyl and cellotetraosyl units in a ratio of

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about 2.5:1 connected by single (1/3)-b-D linkages generating system to a single site of glycosyl transfer (Wood et al., 1994). Upon cleavage with a Trichoderma would thereafter make only callose (Buckeridge et al., cellulase, smaller amounts of higher cellodextrin series 2001), whose synthesis does not require turning the are observed, with the odd-numbered cellodextrin catalytic site or acceptor 180° (Fig. 3B). While it can be 2-fold higher in abundance than the next even-num- argued that membrane disruption activates callose bered unit in the series. The synthesis of the (1/3), synthase in vitro in plasma membrane preparations (1/4)-b-D-glucan has been demonstrated in vitro of all angiosperms (Nishimura et al., 2003), only Golgi with isolated, intact Golgi membranes and UDP-Glc membranes from grasses, the only angiosperms that (Gibeaut and Carpita, 1993; Buckeridge et al., 1999, make the (1/3),(1/4)-b-D-glucan, make callose when 2001; Urbanowicz et al., 2004). Whereas micromolar damaged (Gibeaut and Carpita, 1993; Buckeridge et al., concentrations of UDP-[14C]Glc result in much shorter 1999). The most direct evidence for the default synthe- oligomers and polymers enriched in cellotetraosyl sis of callose from a damaged cellulose synthase comes units rather than cellotriosyl units (Buckeridge et al., from the experiments of Blanton et al. (2000), who 1999), much larger polysaccharides enriched in cello- showed that isolated membranes of a cellulose syn- triosyl units are observed with higher concentrations thase mutant of the cellular slime mold Dictyostelium of substrate (Buckeridge et al., 1999, 2001). The ratios discoideum also lost the ability to make callose in vitro. of cellotriosyl-cellotetraosyl and cellopentosyl-cello- Whereas some cellulose is made in vitro with wild-type hexaosyl are increased proportionally with substrate membrane preparations, callose linkages predominate. concentrations higher than 250 mM (Buckeridge et al., Because cellulose synthase is a single gene in Dictyos- 1999), indicating that the mechanism of synthesis of telium, loss of the ability to make (1/3)-b-D-glucan in the odd-numbered cellodextrin unit is fundamentally vitro as well as (1/4)-b-D-glucan in membranes from different from synthesis of the even-numbered units. the mutant strongly suggests that the single polypep- Proteolysis protection assays show further that the tide is responsible for both activities. active site of catalysis is on the outward-facing Golgi Two groups of Csl genes, CslF and CslH, which are membrane (Urbanowicz et al., 2004). Golgi mem- found only in grasses (Hazen et al., 2002), have been branes treated with proteinase K specifically lost their shown to catalyze (1/3),(1/4)-b-D-glucan biosyn- ability to make the odd-numbered cellodextrin units, thesis. Heterologous expression of a rice CslF in whereas the synthesis of the cellotetraosyl and higher Arabidopsis, a species that does not make (1/3), order even-numbered units was unaffected. Again, (1/4)-b-D-glucan, results in small amounts of the b-D- loss of the ability to make cellotriosyl units is corre- glucan in the cell walls (Burton et al., 2006). However, lated with significant loss in size of the (1/3),(1/4)- considerably greater amounts of the (1/3),(1/4)-b- b-D-glucan product (Urbanowicz et al., 2004). We D-glucan result when a CslH is coexpressed with CslF, proposed a similar catalytic dimer model wherein suggesting a synergistic role for both CslH and CslF in even-numbered units are synthesized by core cellulose the synthesis of the polysaccharide and that a catalytic synthase-like proteins and the odd-numbered units heterodimer enhances the activity (Doblin et al., 2009). arise by an additional GT that has yet to be identified If an accessory glycosyl transferase is necessary to (Buckeridge et al., 2001, 2004). make the odd-numbered cellodextrin unit, then Arab- Limited proteolysis and detergent reconstitution idopsis must produce a related isoform. This finding experiments with the mixed-linkage (1/3),(1/4)-b- of concerted action by two distinct group members D-glucan of grass species provides kinetic evidence for highlights the possibility that synthases of other cross- three sites of glycosyl transfer within the catalytic linking glycans might be encoded by Csl genes of domain: two from the cellulose synthase-like core different groups. domain and a third, separable activity (Urbanowicz et al., 2004). (1/3),(1/4)-b-D-Glucan is the topological equivalent of cellulose synthase at the Golgi CslA: MANNAN AND membrane. Limited proteolysis or detergent treatment GLUCOMANNAN BIOSYNTHESIS causes loss of the ability to make the diagnostic odd- numbered cellotriose units for synthesis without af- One of the first cell wall polysaccharides to be fecting the ability to generate the even-numbered synthesized in vitro was glucomannan. An early con- cellotetraosyl unit (Urbanowicz et al., 2004). clusion that GDP-Glc is the substrate for (1/4)-b-D As the topologic equivalent of cellulose synthase at linkages of cellulose and that UDP-Glc is the substrate the Golgi membrane, the (1/3),(1/4)-b-D-glucan for (1/3)-b-D-glucans (Chambers and Elbein, 1970) synthase shares another feature with cellulose syn- had already been disproven, yet GDP-Glc is still listed thase. When its resident membranes are damaged, erroneously as the substrate for cellulose synthesis on cellulose synthase (Delmer, 1977) and the (1/3),(1/ most wall charts of biochemical pathways. Kinetic 4)-b-D-glucan synthase (Buckeridge et al., 2001) “de- evidence obtained with cotton fiber cells cultured in fault” to synthesis of the (1/3)-b-D-glucan, callose, vitro showed unequivocally that UDP-Glc is the sub- possibly by disruption of the complete active site to a strate for cellulose synthesis (Carpita and Delmer, single glycosyl transferase activity (Buckeridge et al., 1981). Addition of both GDP-Glc and GDP-Man to 2001; Urbanowicz et al., 2004). Loss of the cellobiosyl- membrane preparations resulted in marked stimula-

178 Plant Physiol. Vol. 155, 2011 Synthesis of (1/4)-b-D-Glycans tion of incorporation into a glucomannan product XXXG, rather than a random distribution of XXXXG (Elbein and Hassid, 1966; Piro et al., 1993). and XXXG units (Tine´ et al., 2006). This is an intriguing The knowledge of GDP-nucleotide sugars as sub- result for two reasons: (1) the 4-5-5-4 framework of strates was instrumental in the discovery that a CslA these types of xyloglucans preserves the even-num- gene encodes a mannan synthase by expression pro- bered unit symmetry of the backbone; and (2) to make filing of guar (Cyamopsis tetragonolobus) seed develop- such a framework, as many as 18 glucosyl residues ment, a species that accumulates large amounts of might be contained within the complex in order to be galactomannan as a cell wall storage carbohydrate “read” properly to preserve the unit structure, making (Dhugga et al., 2004). Liepman et al. (2005, 2007) the complex much larger than expected. confirmed that at least four members of the CslA The topology of xyloglucan synthesis at the Golgi group function in mannan and/or glucomannan syn- membrane is still uncertain. Several lines of evidence thesis. However, mixed substrates of GDP-Glc and suggest that synthesis relies on transporters for UDP- GDP-Man in the heterologous expression system Glc for backbone synthesis of (1/4)-b-D-glucans in (Liepman et al., 2007) do not give the marked en- vitro (Orellana, 2005), and UDP-GlcA is transported hancement of glucomannan synthesis long ago ob- to provide UDP-Xyl for transfer within the lumen served in vitro (Elbein and Hassid, 1966). While the (Hayashi et al., 1988). Lerouxel et al. (2006) proposed CesA genes appear orthologous across several species, that synthesis of the backbone is the topological the Csl genes are not (Penning et al., 2009). In fact, the equivalent of cellulose synthase, but the addition of CslA group is resolved into three subgroups that either all subtending sugars of Xyl, Gal, and Fuc are within are Arabidopsis dominated, grass dominated, or mixed. the lumen of the Golgi. The CslA members defined as mannan synthase genes Based on heterologous expression in Pichia, Cocuron (Liepman et al., 2007) fall into both the Arabidopsis- et al. (2007) provide evidence that CslC genes encode dominated and the mixed subgroups (Penning et al., the synthases of the xyloglucan backbone. Although 2009). The functions of these other subgroup members Pichia is unable to make UDP-Xyl, coexpression of the of the grasses need to be defined. xylosyl transferase with CslC is sufficient to induce the extension of (1/4)-b-D-glucan chains, indicating that CslC: XYLOGLUCAN BIOSYNTHESIS a close interaction of these proteins might stabilize the synthase to allow extension of the backbone. A com- Xyloglucans were among the first complex cell wall plication to unequivocal annotation of function of this polysaccharides whose synthesis was demonstrated in subfamily is the finding of a CslC at the plasma vitro. Early studies showed that labeled sugars from membrane instead of the Golgi membrane (Dwivany UDP-Glc and UDP-Xyl are incorporated into several et al., 2009). Three members of the GT34 family are polysaccharides using microsomal membranes and established as the xylosyl transferases involved in were later refined by isolation of Golgi membranes xyloglucan synthesis, and these Golgi-resident pro- (Ray et al., 1969; Ray, 1980). Small amounts of xylo- teins are predicted to face the lumen (Cavalier et al., glucan-like oligomers with the characteristic a-D-Xyl- 2008; Zabotina et al., 2008). (1/6)-D-glucosyl unit, isoprimeverose, are made Xyloglucan is decorated in various ways in a spe- with small amounts of UDP-Glc and UDP-Xyl, but cies-specific manner (Hoffman et al., 2005; Pen˜ aetal., Gordon and Maclachlan (1989) found that when con- 2008). Most are primarily galactosylated, with a char- centrations of each nucleotide-sugar are increased to acteristic a-L-Fuc-(1/2)-b-D-Gal-(1/2)-a-D-Xyl tri- millimolar levels, large polymers containing the char- saccharide extension (Bauer et al., 1973), but others, acteristic heptasaccharide XXXG (for nomenclature, such as those of solanaceous species, have a truncated see Table I) unit structure are synthesized. The tetra- unit structure substituted with a-L-Ara-(1/2)-a-D- glucosyl unit of the xyloglucan backbone and the Xyl extensions instead of Gal (Sims et al., 1996), and precisely repeated three xylosyl units added to make the Asteridae and Oleales species have mixtures of the XXXG structure are consistent with an even-num- these two forms of xyloglucan substitution (Hoffman bered cellobiose unit synthase reaction for the glucan et al., 2005). Furthermore, the xyloglucans synthe- backbone. Even in the structural variant of solana- sized in the Golgi are modified in ways that make ceous xyloglucan, where just two xylosyl units are them structurally different from those that are as- added, a tetraglucosyl unit backbone is preserved by sembled onto the cellulose microfibrils in the wall the replacement of the third xylosyl group with an (Obel et al., 2009). acetate (Sims et al., 1996). An apparent exception is the The purification of a xyloglucan-specific fucosyl ability of certain tree legumes, such as jatoba´ (Hyme- transferase led to the discovery of a GT37 gene encod- naea courbaril), to make XXXXG units in addition to ing it (Perrin et al., 1999). While the synthase complex XXXG (Buckeridge et al., 2000). Curiously, partial must involve a close interaction between the glucan digestion of this polymer with a Trichoderma cellulase, synthases and the xylosyl transferases that decorate it which cleaves only at unbranched positions, yields (Cavalier et al., 2008), the association of the galactosyl octomer, nonamer, and decamer backbone oligomers and fucosyl transferases might be more transient. whose ratios predict that the polymer consists of 4-5- Transferases extracted from the membrane are able 5-4 frameworks separated by variable amounts of to add Gal from UDP-Gal (Madson et al., 2003) and

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Table I. Six possible xyloglucan oligomers are produced by digestion made GAXs, in part employing a nascent C-4 epimer- with a Trichoderma endoglucanase ase that interconverts UDP-Xyl and UDP-Ara (Porchia Because of characteristic side group construction of these complex and Scheller, 2000; Kuroyama and Tsumuraya, 2001; glucans, a single-letter code was devised to denote the nonreducing Porchia et al., 2002; Zeng et al., 2008). terminal sugar of the side chain, with other constituents understood Apart from a hint that the AtCslD5 may be involved (Fry et al., 1993). Thus, XXXG signifies a tetraglucosyl backbone with (Bernal et al., 2007) in the synthesis of (1/4)-b-D- three residues bearing a xylosyl side group. For many angiosperms, this xylans, it has yet to be directly demonstrated that any heptasaccharide is decorated further with variable amounts of Gal at Csl gene plays a role. In fact, informatics approaches the two Xyl residues closer to the reducing end, and if Gal is added to yield non-Csl genes as more likely candidates for en- the first Xyl residue, a Fuc residue is usually added. In addition to the coding the machinery for xylan synthesis (Mitchell glucan backbone synthase, at least three types of glycosyl transferases, et al., 2007), and several mutants with deficiencies in and as many as six, are needed to construct all the side groups. normal xylan synthesis, such as parvus (Lao et al., 2003; Oligomer Notation Lee et al., 2007), irx8 (Brown et al., 2005), irx7/fra8 (Zhong et al., 2005; Brown et al., 2007), irx9 and irx14 Xyl Xyl (Brown et al., 2007), and irx10 and irx10-L (Brown et al., Glc 2 Glc 2 Glc 2 Glc XXXG 2009), do not include a member of the Csl gene family. Xyl (1/4)-b-D-Glucuronoxylan (GX) synthesis appears to involve a complex initiation sequence. Penã et al. Xyl Xyl 2 2 2 (2007) discovered that collapsed xylem mutants defi- Glc Glc Glc Glc XLXG cient in xylan, irx8 and fra8, were essentially devoid of Xyl b / a Gal a complex tetrasaccharide, -D-Xyl-(1 3)- -L-Rha- (1/2)-a-D-GalA-(1/4)-D-Xyl, located at the reducing Gal end of the xylan polymer, whereas an irx9 mutant also Xyl Xyl severely deficient in xylan contained an overabun- Glc 2 Glc 2 Glc 2 Glc XXLG dance of the tetrasaccharide. The presence or absence Xyl of this tetrasaccharide greatly affected the size distribution of the xylans. In irx8 and fra8, a broader Fuc distribution is observed, with some polymers longer Gal than observed in the wild type. Xylans of irx9 have Xyl Xyl 2 2 2 short chains, with nearly all of them containing the Glc Glc Glc Glc XXFG tetrasaccharide (Penã et al., 2007). These results sug- Xyl gested a model whereby short chains of (1/4)-b-D- xylan are primed by the tetrasaccharide, and these are Gal Xyl Xyl spliced, cleaving the primer, to make the long poly- Glc 2 Glc 2 Glc 2 Glc XLLG saccharides (York and O’Neill, 2008). Xyl The IRX8 (GAUT12) and PARVUS (GATL1) genes Gal encode members of the GT8 group C, and IRX7/FRA8 genes encode members of the GT47 group E, that Fuc synthesize the primer tetrasaccharide, whereas IRX9 Gal and IRX14 encode members of GT43 that are likely to Xyl Xyl encode the synthases of the (1/4)-b-D-xylan oligo- 2 2 2 Glc Glc Glc Glc XLFG meric backbones that are stitched together by a yet Xyl Gal unidentified glycosyl transferase (Brown et al., 2007; Penã et al., 2007). GT8 family members encode retain- ing-type transferases, and the GT47 members encode inverting-type transferases with respect to the anome- Fuc from GDP-Fuc (Perrin et al., 1999; Vanzin et al., ric linkages formed compared with the anomeric link- 2002) to exogenous xyloglucan in vitro. age in the nucleotide sugar, so GT8 members make a-D linkages, whereas the GT47 members make b-D or a-L a XYLAN BIOSYNTHESIS linkages. While an -D-GalA is found in the tetrasaccharide, (1/2)-a-D-GlcA (4-O-Me-GlcA) side groups The synthesis of grass (1/4)-b-D-xylans from UDP- are also attached at precise intervals along the xylan Xyl with microsomal membranes was first demon- chain (Nishitani and Nevins, 1991) and a general strated by Bailey and Hassid (1966). Cooperative blockwise synthesis of six consecutive branched xy- action of two nucleotide-sugar substrates, in this in- losyl residues in grass xylans is observed (Carpita and stance UDP-Xyl and UDP-GlcA, resulted in the syn- Whittern, 1986). IRX10 and IRX10-L appear to encode thesis of (1/4)-b-D-xylans with subtending GlcA xylosyl transferases also from GT47, but double mu- units (Waldron and Brett, 1983; Baydoun et al., 1989). tants of these genes have greatly reduced GlcA sub- Similar studies have shown that membrane prepara- stitutions along the shorter chains (Brown et al., 2009). tions from grasses and mixtures of nucleotide sugars It will be interesting to determine if the addition of

180 Plant Physiol. Vol. 155, 2011 Synthesis of (1/4)-b-D-Glycans these GlcA side groups plays a role as an attachment living cells under conditions that did not impair or recognition point where short xylan chains are metabolism (Delmer et al., 1982). Such gradients exist grafted together to make a long chain, a suggestion across other compartments, and, in contrast to cellu- foretold by the original work on the cooperative action lose synthesis at the plasma membrane, it is mainte- of UDP-GlcA and UDP-Xyl in the in vitro synthesis of nance of a pH gradient that prolongs the synthesis of glucuronoxylan (Waldron and Brett, 1983; Baydoun the mixed-linkage (1/3),(1/4)-b-D-glucan in vitro in et al., 1989). isolated maize Golgi membranes (Gibeaut and Carpita, York and O’Neill (2008) take a broader perspective 1993). The physiological and biochemical bases for the of xylan synthesis and have suggested that reducing effect of these potentials and gradients on polysac- end addition should not be ruled out. In their model, a charide synthesis are still not understood. They do type of alternating “pendulum” mechanism is pro- serve to illustrate that, beyond the biochemical mech- posed for the introduction of the (1/4)-b-D-xylan anism of synthesis, technologies that preserve not only linkages, a mechanism analogous to the dimer syn- the protein complexes but also their cellular context thesis described here for (1/4)-b-D linkages in gen- need to be developed to truly understand the synthesis eral. We are just learning the identities of the GlcA and of macromolecules across membrane surfaces. Araf transferases of the GX and GAX polymers and the distinctions between the GX devoid of Ara that is abundant in the secondary xylem and the GAX with its ACKNOWLEDGMENTS rich Ara substitution that is the major polymer of the I thank Maureen McCann (Purdue University) and Peter Ulvskov (Uni- primary cell walls of grasses (Scheller and Ulvskov, versity of Copenhagen) for their review of the manuscript and their many 2010). Recently, proteomic approaches of isolated GAX helpful suggestions. I also thank Daisuke Kihara (Purdue University) for his synthase complexes from wheat membranes demon- contributions to the discussion on protein structure and modeling. The strated a close interaction of GT43 and GT47 family artwork in Figure 6 is by Pamela Burroff-Murr (Purdue University). members with a GT75 UDP-Ara mutase, an enzyme Received July 24, 2010; accepted November 2, 2010; published November 4, that interconverts the UDP-arabinopyranose and 2010. 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