Xylan, a polymer of p-(1-4)-linked D-xylose is one of the main components of woody plants. Xylans are usually substituted by side chains of arabinose or glucuronic acid and may be acetylated. Thus, glucuronoxylan (GX) is composed of a linear backbone of p — (1-4)-linked D-xylosyl (Xyl) residues, some of which bear a single a-D-glucuronic acid (GlcA) or 4-0-methyl-a-D-glucuronic acid (MeGlcA) residue at O-2. The Xyl residues can also be substituted with arabinosyl and acetyl residues (128). Xylosyltransferase and glucuronyltransferase activities have been detected in numerous plants (129). However, none of the genes encoding these enzymes has been identified, nor have any of the enzymes been purified to homogeneity and biochemically characterized.

Mutations in three glycosyltransferases, FRAGILEFIBER8 (FRA8), IRREGULARXYLEM8 (IRX8), and IRX9, have been shown to be required for normal vessel morphology and wall thickness and for normal amounts of xylose and cellulose in cell walls (73, 74, 130, 131). These genes are specifically expressed in cells undergoing secondary wall thickening. Plants carrying mutations in these genes have reduced amounts of wall GX and a decreased ratio of GlcA to MeGlcA residues in the GX(129, 130, 132).

IRX8, IRX9, and FRA8 are specifically expressed in fibers and vessels and their encoded proteins are localized in the Golgi (129,130,132). Thus, they have the properties expected of enzymes involved in glucuronoxylan synthesis. However, it has not been possible to directly associate enzyme activity with the proteins. Thus, it is not clear how they participate in the synthesis of xylan. Pena and coworkers (129) showed that the glycosyl sequence 4-P-D-Xylp — (1 ^ 4)-p-D-Xylp-(1 ^3)-a-b-Rhap-(1 ^2)-a-D-GalpA-(1 ^4)-D-Xylp was present at the reducing end of Arabidopsis GX, as previously noted for birch (Betula verrucosa) and spruce (Picea abies) wood. They further noted that mutations in IRX8 and IRX9, and by inference FRA8, lead to reductions in the amount of the GXreducing end sequence suggesting that these genes participate in the synthesis of the GX reducing end sequence and suggest that IRX9 has an essential role in the elongation of the xylan backbone.

The fra8 gene encodes a GT47 family enzyme and expression of the poplar (Populus alba x tremula) GT47C gene in fra8 plants rescues the defects in secondary wall thickness and GX synthesis, suggesting that GT47C is a functional homolog of FRA8 (133). The FRA8 gene encodes a putative GT in family GT47 (130). This family includes enzymes with an inverting mechanism, which usually leads to p-glycosidic linkages (when typical a-linked donor substrates are used). Thus, if UDP-a-D-Xyl is the donor substrate, it is possible that FRA8 catalyzes the formation of the p-linkage of xylose to either O-3 of the rhamnose or O-4 of the penultimate xylose of the GX reducing end glycosyl sequence (129). However, in plants, the addition of a-Rha residues is catalyzed by inverting GTs that use UDP-p-L-Rha as the donor substrate (134). Therefore, it is also possible that FRA8 catalyzes the addition of rhamnose during the biosynthesis of the GX reducing end sequence.

The IRX8 (GAUT12) gene encodes a putative GT in family GT8 (73,74,129,132). Several members of the GT8 family catalyze the transfer of uronic acids to glycans. For example, three Arabidopsis GT8 proteins, QUASIMODO1 (QUA1) (135), PARVUS (136), and GALAC- TURONOSYLTRANSFERASE1 (GAUT1) (137), have been identified and are believed to have a role in pectin biosynthesis. Of these three, only GAUT1 has been biochemically char­acterized and shown to have galacturonosyltransferase activity (137). Family GT8 enzymes are retaining glycosyl transferases that catalyze the formation of a-glycosidic bonds when using a-linked donor substrates such as UDP-a-D-GalA. Thus, it is possible that IRX8 cat­alyzes the addition of an a-D-GalA residue to O-4 of the reducing Xyl residue present in the GX reducing end sequence described above.

Mutation of the IRX9 gene, which encodes a putative GT in family GT43 (138), was shown to result in plants with decreased amounts of wall GX, suggesting that this gene is required for GX synthesis (131). The poplar (Populus tremulax tremuloides) GT43A and Ptt GT43B genes, which are homologs of IRX9, have been shown to be highly expressed during wood formation (139). In addition, a cotton (Gossypium hirsutum) gene, which resides in the same phylogenetic subgroup as Ptt GT43A, Ptt GT43B, and IRX9, is highly expressed during cotton fiber development (140). Together, these findings suggest that family 43 GTs have an important role in secondary wall synthesis. Enzymes in this family are distinguished by an inverting mechanism, typically catalyzing the formation of p-glycosidic bonds using a-linked glycosyl donors. Our demonstration that the irx9 mutation leads to a decrease in the chain length of GX suggests that IRX9 encodes a xylan synthase responsible for adding p — xylosyl residues to the nascent GX. This hypothesis is consistent with our results indicating that IRX9 is highly expressed in cells undergoing secondary wall biogenesis and that IRX9 is localized in the Golgi, where GX synthesis occurs (141, 142). However, it is also possible, as we discussed previously (130), that an inverting GT can catalyze the formation of an a-linkage when a p-linked substrate (such as a glycosyl phospholipid) is used as the donor. Such inverting enzymes can also catalyze the formation of high-energy p-linked glycosides (such as glycosyl phospholipids) that are subsequently used as glycosyl donors. Thus, an alternative interpretation is that IRX9 is directly or indirectly involved in the transfer of а-linked GlcA residues to the GX backbone. Additional studies are required to determine whether IRX9 catalyzes the addition of xylose or GlcA to the GX backbone.

In the lignified walls of the Poaceae, the major non-cellulosic polysaccharides are glu — curonoarabinoxylans (GAXs), although the degree of substitution of the xylan main chain is less than in the GAXs of the primary cell walls. In the non-lignified walls of the Poaceae and other species such as pineapple (Ananas comosus), ferulic acid is ester-linked to GAXs. These polysaccharides comprise only a minor component of the non-cellulosic polysaccharides of the non-lignified walls of species in the basal Arecales (palms) clade (143), but are a major component of the non-lignified walls of species in the other commelinid clades, particularly the Poales (144-146).