Homogalacturonan (HG) is the most abundant pectic polysaccharide. It is a homopolymer of 1,4-linked a-D-galactosyluronic acid that is partially methylesterified and may be O — acetylated at O-2 or O-3. As mentioned above, the distribution of methylesters in HG during synthesis or in the wall is not known (157), although there is evidence that the distribution of non-esterified galacturonosyl residues in the wall is not random (195). The degree of polymerization (DP) of HG, as well as the question of whether it is linear, or may be branched or cross-linked (204) remains a matter of debate. However, the DP has been estimated to range from 72 to 100 (304) or more (214). The synthesis of HG requires at least one HG:a1,4-galacturonosyltransferase (HG-GalAT) (Table 5.2), at least one HG- methyltransferase (HG-MT) (also referred to as pectin methyltransferase), and at least one HG:O-acetyltransferase (HG-AT).

5.4.6.1 HG:galacturonosyltransferase (HG:GalAT)

The only HG biosynthetic glycosyltransferase for which enzymatic function of the encoded gene has been established is the HG:GalAT called GAUT1 (Galacturonosyltransferase 1;

At3g61130) (137). The identification of GAUT1 followed an extensive study of HG:a1,4GalAT activity in multiple plant species including in mungbean (305-308), tomato (306), turnip (306), sycamore (309), tobacco suspension (310-312), radish roots (205), pea (230, 313), Azuki bean (Vigna angularis) (314), petunia (315), and Arabidopsis (137, 316) (see Table 5.4). This GalAT activity was shown to be particulate (i. e., membrane bound) in all species studied and was measured as the transfer of GalA from UDP-GalA onto endoge­nous acceptors. The GalAT activity in pea was localized to the Golgi (230) with its catalytic site facing the lumenal side of the Golgi (230), providing the first direct enzymatic evidence that the synthesis of HG occurs in the Golgi.

HG:GalAT activity in microsomal membranes, measured using UDP-[14C]GalA (317, 318), incorporates the GalA moiety onto endogenous acceptors to yield relatively large molecular mass labeled products of ~ 105 kDa in tobacco microsomal membranes (310) and >500 kDa in pea Golgi (230). Cleavage of the radiolabeled product into GalA, di — galacturonic acid (diGalA), and trigalacturonic acid (triGalA) by a purified endopolygalac — turonase demonstrated that the product was HG (310). In tobacco, the product produced in vitro in microsomes was ~50% esterified (310) while the product produced in pea Golgi was less esterified (230), suggesting that the degree of methyl esterification of newly synthesized HG may be species-specific and that methylesterification may occur after the synthesis of at least a short stretch of HG. GalAT activity can also be studied in detergent — permeabilized microsomes to allow access of the enzymes to exogenous pectic acceptors. For example, detergent-permeabilized microsomes from etiolated azuki bean seedlings trans­fer [14C]GalAfrom UDP-[14C]GalA onto acid-soluble polygalacturonate (PGA) exogenous acceptors (314). The azuki bean enzyme exhibited a broad pH range of 6.8-7.8 and a surprisingly high-specific activity of 1300-2000 pmol mg-1 min-1, considering the large amount (3.1-4.1 nmol mg-1 min-1) of polygalacturonase activity that was also present in the microsomal preparations.

Success in identifying the gene encoding a GalAT required solubilizing GalAT activity from membrane preparations so as to facilitate purification of the enzyme. The first solubilization of an HG:GalAT was achieved with tobacco GalAT (311). Detergent-solubilized GalAT adds GalA onto the non-reducing end (312) of exogenous HG with a preference for HG oligosaccharides (oligogalacturonides; OGAs) of a DP of greater than 9 (311,315), although OGA acceptors as small as a trimer can be used (315, 319). Although detergent-solubilized GalAT can use polymeric pectin substrates such a polygalacturonic acid and pectin, such polymers are less favorable substrates (314).

Studies carried out under conditions that provide information regarding the mode of elongation of the OGAs by GalAT, i. e., with excess OGA acceptor to UDP-GalA ratios (320), suggest that solubilized tobacco, radish and Arabidosis enzymes, and permeabilized pea Golgi galacturonosyltransferase, have a distributive (non-processive) mode of action in vitro (230, 311, 321). Under these conditions, the bulk of the HGA elongated in vitro by solubilized GalAT from tobacco membranes (311), or detergent-permeabilized Golgi from pea (230), is elongated by a single GalA residue. As expected, as the UDP-GalA:OGA ratio is increased, the OGA products become progressively longer (137), but it is important to note that this, in itself, does not denote processivity, it simply means that the enzyme can use the product of a previous catalytic event as a substrate for a subsequent catalytic event. Interestingly, the membrane-permeabilized galacturonosyltransferase activity reported from pumpkin may represent a processive mode of elongation since reactions containing approximately equimolar amounts of UDP-GalA and acceptor yielded a population OGAs elongated by up

to five galacturonosyl residues (319). However, further information on the size distribution of the OGAs produced in reactions containing excess acceptor will be required to confirm this. Solubilized petunia galacturonosyltransferase, in reactions containing ~60-fold excess UDP-GalA to OGA acceptor, added up to 27 galacturonosyl residues onto the OGA acceptors (315), indicating that the enzyme can elongate OGA products from a previous reaction, but not specifically addressing the mode of elongation of the enzyme (320). The apparent lack of in vitro processivity of the solubilized GalAT suggests either that enzyme does not synthesize HG in a processive manner in vivo, or that the characteristics of HG-GalAT measured in vitro may be an artifact due to the dissociation of a required biosynthetic complex or loss of cofactor(s) or substrate(s) during solubilization of the enzyme. We have been unable to obtain evidence for processive in vitro solubilized GalAT activity (i. e., the production of extensively elongated acceptors under reaction conditions with excess OGA acceptor to UDP-GalA) (Quigley and Mohnen, unpublished results) [see (192)]. We also obtained no evidence that the inclusion of the methyl donor S-adenosylmethionine (308, 310, 314) and/or the acetyl donor acetylCoA promote the processivity of GalAT (192) (unpublished results). Clarification of the mode of action of GalAT and the mechanism of HG synthesis should be aidedby access to purified or recombinantly expressed enzyme(s), and may require isolation of enzyme complexes (see below).

Efforts to purify GalAT activity from tobacco proved unsuccessful due to loss of activity. Therefore, a partial purification and tandem mass spectrometry approach was used to iden­tify a gene encoding GalAT activity. The detergent-solubilized HG:GalAT from Arabidopsis suspension cells was partially purified by column chromatography to yield an enriched fraction containing approximately 20 protein bands. The proteins were trypsinized, the polypeptides analyzed for amino acid sequence by tandem mass spectrometry (137), and the sequences compared against the Arabidopsis gene database. The partially purified active

GalAT fraction contained two proteins with sequences that identified them as putative glyco — syltransferases which were eventually named GAUT1 and GAUT7 (137). Evaluation of their amino acid sequences indicated that both GAUT1 and GAUT7 had characteristics consistent with the biochemical properties of HG:GalAT: a basic PI and an apparent Type II mem­brane protein topology. The encoded proteins consisted of three domains: a short N-terminal region, a single membrane spanning region, and a larger C-terminal domain. Transient ex­pression of N-terminal truncated forms of the coding regions of GAUT1 and GAUT7 in human HEK293 cells indicated that GAUT1 had GalAT activity. Thus, GAUT1 became the first enzymatically verified HG:galacturonosyltransferase (GAlacatU ronosylT ransferase 1, GAUT1). The transiently expressed truncated from of GAUT7 did not have GalAT activity.

Sequence comparison of GAUT1 against the Arabidopsis genome identified 24 additional Arabidopsis genes with high sequence similarity to GAUT1. We named this 25-member group of related genes the GAUT1-related gene family (Table 5.5). The GAUT1-related genes represent a subclass of the Arabidopsis CAZy family GT-8 genes. Fifteen of the GAUT1- related genes have high sequence similarity to GAUT1 (37-100% identity/56-100% simi­larity) and we named these genes GAUT1-GAUT15. The 15 GAUT proteins have predicted masses of 61-78 kDa and most encode proteins with a predicted membrane anchor (GAUTs 1,6-15) or with a signal peptide (GAUTs 3-5) consistent with a Type II membrane topology or with an intramicrosomal membrane location, respectively. GAUT2 is the only GAUT not predicted to be present in, or pass through, the intracellular membrane transport system (i. e., the ER/Golgi system). The remaining 10 GAUT1-related genes have somewhat lower sequence similarity to GAUT1 (39-44% identity/43-53% similarity) and we named these the GAUT1-Like (GATL) genes. The GATL genes encode predicted 33-44 kDa proteins with predicted signal peptides. The proven location of GAUT1 and GAUTs 3, 7, 8, and 9 is in the Golgi (323, 324).

Multiple T-DNA insert mutants of many of the GAUT and GATL genes are available (e. g., see http://signal. salk. edu/cgi-bin/tdnaexpress) and several mutants have described phenotypes. The qua1/gaut8 mutant has ~25% reduced amounts of GalA in the walls of Arabidopsis rosette leaves or total plants (135), ~30% reduced GalA levels in walls of stem (260), and modest reductions in the GalA content of suspension cultured cells (325), sugges­tive of a role of the mutated gene as a putative GalAT involved in pectin synthesis. However, the mutant walls are also reduced in Xyl (at least stem walls) and protein extracts from mu­tant stems have both reduced HG:a-1,4-GalAT activity and (3-1,4-xylosyltransferase activity (260). The lack of a confirmed enzyme activity of the recombinantly expressed or purified GAUT8 protein, along with the pleiotropic effects of the mutant, have made a conclusive identification of the function of the GAUT8 protein elusive.

The parvus/glzl/gatll mutants (136,326) also have characteristics consistent with a defect in pectin synthesis. The parvus/glzl/gatll mutants grown under low humidity are semi­sterile dwarfs that have reduced anther dehiscence. The parvus mutants also have slightly elevated Rha, Ara, and Gal and reduced Xyl compared to WT (136). These changes in neutral sugar compositions are consistent with a role of the parvus gene in the synthesis of the pectic polysaccharide RG-I. However, since the levels of GalA in the mutant walls were not determined, it is not known if the mutant walls are altered in GalA content.

We PCR-tested 57 Arabidopsis GAUT1-related gene family T-DNA insertion mutant lines from the T-DNA mutant collection (http://signal. salk. edu/cgi-bin/tdnaexpress) and identified 36 homozygous mutant lines (Caffall and Mohnen, unpublished). Glycosyl residue

composition analyses were carried out on amylase-treated walls from 82 unique tissue samples from homozygous mutants of 13 ofthe 15 GAUT family members by combined gas chromatography/mass spectrometry (GC/MS) of per-О-trimethylsilyl (TMS) derivatives of the monosaccharide methyl glycosides (327). Tissues from mutant lines of 8 of the 13 GAUT1-related family genes tested had >15% reduction in the amount of galacturonic acid in their walls, consistent with a function of the mutated genes as GalATs. However, a detailed biochemical analysis of the enzyme activity of each of the GAUTs will be required to establish gene function. Interestingly, some mutants also accumulated higher amounts of arabinose, rhamnose, fucose, and xylose and reduced amounts of mannose, galactose and/or glucose. The significance of these correlative changes with reduced levels of GalA is not yet clear; however, we speculate that the reduction of one pectic polysaccharide (e. g., HG) may be associated with, or compensated for, by a change in the amount of another polysaccharide (e. g., loss of HG may lead to an increase in RG-I which has Rha, Ara and/or Gal; alternatively, loss of an RG-I biosynthetic GalAT could lead to loss of RG-I and to an increase in HG with a resulting increase in GalA and a reduction in Rha, Ara and and/or Gal). Such a compensation of one wall polymer for another (328,329) could occur via several mechanisms including sensing of polysaccharide or nucleotide-sugar levels, or the covalent or non­covalent association of one type of polysaccharide with another (e. g., it has been reported that pectin may be covalently linked to the hemicellulose xyloglucan (107).

Although GAUT1 encodes a GalAT, its function in pectin synthesis is far from clear. For example, it is not yet understood at what stage of pectin synthesis, i. e. initiation or elongation, GAUT1 has its primary role. Also, it is not clear whether GAUT1 functions alone or in a complex. Our preliminary results suggest that, at least in vitro, GAUT1 can function in a complex with at least one other GAUT (Atmodjo and Mohnen, unpublished). The enzyme function of the other GAUT and GATL members of the GAUT1-related gene family remains to be determined. QUA1/GAUT8 is a good candidate for a GalAT, but the reduced levels of Xyl and xylosyltransferase activity of the qua1 mutants, at least in stem tissue, raises the question of what the connection is between pectin (e. g., HG) and hemicellulose (i. e., xylan) synthesis). The pectins and hemicelluloses are traditionally considered to be two different classes of wall polysaccharides, although there is some evidence that these two classes maybe tightly, and possibly covalently, linked in the wall (258, 332, 333). Thus, the characteristics of the qual mutant raise several questions. Is xylan synthesis dependent upon pectin synthesis? The characteristics of the parvus mutant and the GATL proteins are also intriguing. What is the function of the GATLs? The characteristics of the parvus mutant are consistent with a function in pectin synthesis; however, these proteins have no apparent membrane spanning domain but rather a signal peptide. If the GATLs are involved in pectin synthesis, do they interact with other lumenal pectin biosynthetic enzymes in the form of Golgi-localized complexes? The identification of GAUT1 as a functional GalAT and of the GAUT1-related gene family provides the gene/protein tools required to address some these questions.

Recently, characterization of mutants of GAUT12/IRX8 have provided evidence support­ing a possible role of GAUT12/IRX8 as a HG:GalAT involved in the synthesis of a subfraction of HG to which (3-1,4-xylan is attached (132) or as a putative a1,4-GalAT that adds a GalA onto a xylose at the reducing end of a xylan primer (129). Proof of the function of GAUT12, however, requires confirmation of enzyme activity.