Pectin is likely the most structurally complex family of polysaccharides in nature (Figures 5.2 and 5.3). Pectin is particularly abundant in primary walls, i. e. those walls surrounding

Homogalacturonan

4)aGalA-( 1,4)-aGalA-(1,4)-aGalA-(1,4)-aGalA-( 1,4)-aGalA-(1,4)-aGalA-( 1,4)-aGalA-(1,

Rhamnogalacturonan I

Ara — and/or Gal-containing Ara — and/or Gal-containing

side chains GlcA side chains

4 4 4

4 44

4)-aGalA-(1,2)-aLRha-(1,4)-aGalA-(1,2)-aLRha-( 1,4)-aGalA-(1,2)-aLRha-( 1,4)-aGalA-( 1,2)-

growing and dividing cells and the terminal wall in many cells of the soft parts of the plant. Pectin is also abundant in the middle lamella which is the junction between adjacent cells. Pectin comprises ~35% of the polysaccharides in dicot and non-graminaceous monocot primary walls, and 2-10% of the wall in the grasses (157, 158). Pectin is also present in the walls of gymnosperms, pteridophytes, and bryophytes as well as Chara, a charophycean alga, which is believed to be the closest extant relative of land plants (159). Although pectin is not a major component of secondary walls, it is present as the outer layer of secondary walls and can represent ~5% of harvested tree wood. Thus, depending on the plant and tissue used, pectin will be present in the biomass used for biofuel production and, since it comprises a complex interconnected matrix in the wall, likely affects the recalcitrance of biomass to deconstruction for biofuel production.

Pectins have multiple roles in plant defense, growth, and development (158, 160). They provide wall structure (161), bind and exchange apoplastic anions and macromolecules (162, 163), influence cell-cell adhesion (164, 165), and are involved in cell signaling (166,

о = Acetyl groups о = Methyl groups

Figure 5.3 Schematic structure of pectin showing the three main pectic polysaccharides homogalac — turonan (HG), rhamnogalacturonan I (RG-I), and rhamnogalacturonan II (RG-II) linked to each other. A region of substituted galacturonan known as xylogalacturonan is also shown (XGA). The representative pectin structure shown is not quantitatively accurate, HG should be increased 12.5-fold and RG-I increased 2.5-fold to approximate the amounts of these polysaccharides relative to each other plant walls. The monosaccharide symbols used are either from the Symbol and Text Nomenclature for Representation of Glycan Structure. Nomenclature Committee Consortium for Functional Glycomics (http://www. functionalglycomics. org/glycomics/molecule/jsp/carbohydrate/carbMoleculeHome. jsp) or from D. Mohnen. (The figure is modified from http://www. uk. plbio. kvl. dk/plbio/cellwall. htm.) (Reproduced in color as Plate 4.) 167). Pectins have roles in pollen tube growth (168), seed hydration (169-171), leaf ab­scission (172), guard cell function (173), organ formation (174, 175), fruit development (158), and possibly water movement (176). Pectic oligosaccharides are intercellular sig­nal molecules (177) in plant development (166) and defense responses (178, 179). Mutant plants with altered pectin structure maybe dwarfed (161,180), have brittle leaves (164), re­duced numbers of side shoots and flowers (175), and reduced cell-cell adhesion (135,181).

Pectin is defined as a family of plant cell wall polysaccharides that contain 1,4-linked galac­turonic acid (157). Galacturonic acid (GalA) comprises roughly70% oftotal cell wall pectin and is a major component of the three major types of pectic polysaccharides: homogalac — turonan (HG), rhamnogalacturonan I (RG-I), and the substituted galacturonans for which rhamnogalacturonan II (RG-II) is the most ubiquitous and structurally invariant member (157). In addition, pectin includes the less abundant substituted galacturonan xylogalactur­onan (XGA) (182-187) and apiogalacturonan (AG) (158,188-191). The complex structure of the pectic polysaccharides makes the study of pectin synthesis challenging. It is estimated that at least 58 enzymes are required to synthesize pectins, including methyltransferases, acetyltransferases and numerous glycosyltransferases (192).

HG accounts for ~65% of pectin (193, 194) and is a homopolymer of a-D-1,4-linked GalA residues (Figures 5.2 and 5.3) that is partially methylesterified at the C-6 carboxyl (157, 195), may be O-acetylated at O-2 or O-3 (196-199), and may contain other esters
whose structure remains unclear (200-204). RG-I accounts for 20-35% of pectin (194) and is a family of polysaccharides with an alternating [^4)-a-D-GalA-(1^2)-a-L-Rha — (1^] backbone (Figures 5.2 and 5.3). Between 20 and 80% of the rhamnosyl residues are substituted with side chains composed predominantly of linear and branched а-L-Ara/ and (3-D-Galp (157, 205). The main types of side chains include a-1,5-linked L-arabinan with some 2- and 3-linked arabinose or arabinan branching, p-1,4-linked D-galactans with some 3-linked L-arabinose or arabinan branching and p-1,3-linked D-galactan with p-6-linked galactan or arabinogalactan branching (205). RG-I side branches may also contain a-L-Fucp, p-D-GlcpA, and 4- O-Me p-D-GlcpA residues (206). The composition and length of RG-I side chains varies between cell types and in different plant species (158,160). RG-II accounts for ~10% of pectin (158, 194) and contains 12 different types of sugars in over 20 different linkages. The HG backbone of RG-II is substituted at O-2 and O-3 with four structurally complex oligosaccharides A-D (159) (Figures 5.2 and 5.3). RG-II in the plant generally occurs as a RG-II dimers crosslinked by borate diesters (159). The 4-linked galacturonans that are substituted at O-3 with D-xylose (the xylogalacturonans, XGA) are often found in reproductive tissues (157, 186, 193, 207) whereas galacturonans substituted at O-2 or O-3 by D-apiofuranose (188, 189) (the apiogalacturonans, AG) are restricted to selected aquatic monocots (e. g., Lemna).

When walls are isolated from the plant, the pectic polysaccharides appear to be covalently cross-linked since harsh chemical treatments or digestion by pectin-degrading enzymes is required to isolate HG, RG-I, and RG-II separately from each other. It is not known, however, how the pectic polysaccharides are covalently linked to each other or to other polymers in the wall. It is also not clear where and how that crosslinking occurs, i. e. via the action of glycosyltransferases in the Golgi or by transglycosylases or other enzymes in the wall. The available data (208) support a model whereby HG, RG-I, and RG-II are linked via their backbones. However, due in part to the uncertainty of how and where the pectic polysaccharides are cross-linked, it is currently not possible to predict the complete reper­toire of biosynthetic enzymes that are needed to synthesize pectin. Furthermore, although the general types of pectic polysaccharides are similar in different plant species, there is a growing body of evidence showing that species-, cell-type-, and developmental state-specific differences in pectin structure exist, thereby making it likely that the number and types of enzymes required to synthesize pectin will depend on the plant, tissue and developmental state of the cells of interest.

Finally, a knowledge of the structure of the “mature” polysaccharides in the wall, or at least those that can be isolated from the wall and characterized, does not necessarily reflect the structures as they are synthesized, but rather the structures as they are inserted into the wall and after they have been modified by wall-localized enzyme catalyzed (and chemical) reactions. In the following discussion ofpectin, there is no attempt to define species-specific differences in pectin synthesis, since our understanding of the species-specific tailoring of pectin structures and synthesis is only just beginning to be studied. Rather, this review em­phasizes our current understanding of biosynthetic enzymes required for the basic pectin structures that appear to be common in all species. Although only few of the genes encoding pectin biosynthetic enzymes have been confirmed by demonstration of enzymatic activ­ity of the encoded proteins, recent progress in identifying genes encoding putative pectin biosynthetic enzymes make it likely that more pectin biosynthetic genes will be functionally identified in the near future. The availability of such genes should facilitate the elucidation of how diverse pectin biosynthetic enzymes work together, likely within protein complexes (Atmodjo and Mohnen, unpublished results) to synthesize the multifunctional family of pectic polysaccharides.

Several comprehensive reviews on pectin biosynthesis (158,192,205,209), as well as more general reviews on plant wall biosynthesis (72, 99, 126, 155, 210-213, 214) and strategies to identify wall biosynthetic glycosyltransferases (118, 215-218) and regulation of cell wall synthesis (219-221) have previously been published. This review will attempt to merge recent advances in pectin synthesis with the prior studies so as to reflect our current understanding of pectin synthesis.