Pectic Substances

Malcolm A ONeill, The University of Georgia, Athens, Georgia, USA Alan G Darvill, The University of Georgia, Athens, Georgia, USA Peter Albersheim, The University of Georgia, Athens, Georgia, USA
Pectins are plant polysaccharides comprising mainly 1,4-linked a-D-galactosyluronic acid. They are found in the primary cell walls of all seed-bearing plants and in the junction between cells.

Secondary article
Article Contents
. Overview . Molecular Structure . Biosynthesis of Pectin . Enzymic Degradation of Pectins . Functions of Pectin in the Cell Wall . Pectin Gels . Acknowledgements

Overview
Pectins are a group of plant polysaccharides whose principal component is 1,4-linked a-d -galactosyluronic acid (ONeill et al., 1990). Pectins are present in the primary cell walls of all seed-bearing plants and in the junction between cells called the middle lamella. The primary wall surrounds growing plant cells, meristematic cells and cells in succulent tissue such as fruit. The secondary walls of dierentiated plant cells (e.g. xylem tracheids) are often lignied and cellulose-rich, and contain little pectin. Pectins are major components (2030%) of the primary walls of dicotyledons (e.g. tomato, soybean and apple), nongraminaceous monocotyledons (e.g. onion and garlic) and gymnosperms (e.g. Douglas Fir). In contrast, pectin accounts for 5 10% of the primary wall of the Poaceae (e.g. wheat, maize, and rice). The limited data available suggest that pectins are present in the walls of lower plants (e.g. ferns) and some green algae (e.g. Nitella and Coloechaete). Some plants also produce water-soluble polysaccharides that contain galactosyluronic acid residues. These polysaccharides are structurally related to pectins but are usually referred to as gums and mucilages in the literature. Recent studies of cell wall pectins have provided evidence that the pectic polysaccharides are structurally modied during the growth and development of plant cells. Pectin fragments are signal molecules that can activate plant defence responses to pathogens and modify the growth and development of plant tissue. Pectins are a major component of dietary bre and have been reported to lower serum cholesterol levels to bind toxic metals and to have immunostimulating and antiulcer activities. Pectins eect the texture and processing characteristics of fruits and vegetables. The ability of pectins to form gels (pectin is derived from the Greek pektos coagulated) has been exploited by man for centuries and these polysaccharides have been used in various commercial applications, especially in the food industry (Visser and Voragen, 1996).

Molecular Structure
Pectins have been isolated from various plant tissues and organs (ONeill et al., 1990). Immature tissues or suspension-cultured cells are used as a source of primary walls to elucidate the structure and function of pectins in plants, whereas pectin for commercial use is typically extracted from citrus peel and from the insoluble material after the juices have been expressed from sugar beets and apples. Pectins are solubilized by treating primary walls or tissues with hot water, hot dilute acid (1% nitric acid for 3 h at 708C), cold dilute alkali (pH 10 for 4 h at 48C), chelating agents and/or enzymes (e.g. endo-a-1,4-polygalacturonase; EPGase). Pectins solubilized with dilute acid are often structurally modied owing to the hydrolysis of acid-labile substituents, whereas alkaline extraction may cause partial demethyl-esterication and degradation of the polymer by b-elimination reactions. Such structural modications are minimized by using chelating agents or homogeneous enzymes to solubilize pectins. Nevertheless, the chelatorand enzyme-solubilized pectins are unlikely to have identical structures to pectins in the wall. Pectins may be contaminated with other polysaccharides irrespective of how they are solubilized and can be puried by a combination of anion-exchange and size-exclusion chromatographies. A pectin is considered chemically homogeneous when it elutes from a chromatography column as a peak with a constant chemical composition, even though it may be heterogeneous with respect to its molecular mass. The primary structure of a pectic polysaccharide is known only when the following characteristics have been determined: (1) the glycosyl-residue composition; (2) the glycosyl-linkage composition; (3) the absolute congurations (d or l ), the ring forms (pyranose [p] or furanose [f]), and the anomeric congurations (a or b) of the glycosyl residues; (4) the sequence of glycosyl residues; and, (5) the location of non-carbohydrate substituents (e.g. O-methyl and O-acetyl groups). To date only three pectic polysaccharides (homogalacturonan, rhamnogalacturonan and substituted galacturonans) have been isolated from primary cell walls and

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

Pectic Substances

structurally characterized (ONeill et al., 1990; Visser and Voragen, 1996). The glycosyl residues that are present in pectins are shown in Figure 1. Homogalacturonan (HG) is widely accepted to be a linear chain of 1,4-linked a-d -galactopyranosyluronic acid (GalpA) residues in which some of the carboxyl groups are methyl-esteried (Figure 2). HGs may, depending on the plant source, be partially O-acetylated and contain other, as yet, unidentied esters. Rhamnogalacturonans are a group of pectic polysaccharides that contain a backbone of the repeating disaccharide !4)-a-d -GalpA-(1!2)-a-l-Rhap-(1!. These polysaccharides are usually referred to as rhamnogalacturonan I (RG-I) in the literature, although other terms such as hairy regions are used. The backbone GalpA residues may be O-acetylated on C2 and/or C3, but there is no evidence that the GalpA residues are methylCOOH HO OH OH OH DGal pA HO OH DGlcpA O H OH H COOH O OH

esteried. Between 20% and 80% of the rhamnosyl (Rhap) residues are, depending on the plant source and the method of isolation, substituted at C4 with neutral or acidic oligosaccharide side-chains (Figure 3). The length of these side-chains may range from a single glycosyl residue to more than 20 glycosyl residues. The sidechains predominantly contain linear and branched a-l-arabinofuranosyl (Araf), and b-d -galactopyranosyl (Galp) residues, although their relative proportions may dier depending on the plant source. Other glycosyl residues including a-l fucosyl (Fucp), b-d -glucuronosyl (GlcpA), 4-O-methyl-bd -glucuronosyl (4-O-Me-GlcpA) and phenolic acids such as ferulic acid may also be present in some of the sidechains (Figure 3). Substituted galacturonans (SGs) are a group of polysaccharides that contain a backbone of linear 1,4-linked ad -GalpA residues (ONeill et al., 1990). Xylogalacturo-

HO CH 3

OH CH 3 H HO OH

O HO

OH

H LFucp

OH

OH

L Rhap

O OH HOH2C OH LAraf

H OH OH HOH2C

OH

HO OH

OH OH H HO

OH

H OH LAraf OH LArap

H OH DXlyp

CH 2OH HO OH H OH DGal p HO O OH OH OH OCH 3

2O Me D Xyl p

COOH O H O CH 3 CH 3O HO OH
2 O Me LFucp

OH OH H CH 3O

OH

H OH

4 O Me D GlcpA

CH 2 OH HO HO OH COOH CH O OH HO OH COOH H 3C OH Kdo Dha L Aceric acid COOH O OH O COOH OH H OH OH OH O CH 2OH H OH

D Api f

Figure 1 The glycosyl residues that are known to be present in the three pectic polysaccharides that have been isolated from plant cell walls.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

Pectic Substances

O Unesterified GalpA C OH O Carbon 1

HO

HO

O C OH O

Carbon 4

HO HO

O C OH O Methyl-esterified GalpA

-1,4-linkage

HO
HO

O C OCH 3 O

HO
HO

O C OCH 3 O

D-Galactosyluronic

acid (GalpA) residue

HO
HO

C OCH 3
CH 3CO O Acetyl ester
HO

Figure 2 The structure of homogalacturonan. Homogalacturonan is composed of 1,4-linked a-D-galactopyranosyluronic acid residues (GalpA). The carboxyl groups of the GalpA residues are often methyl-esterified. Some of the hydroxyl groups may be O-acetylated.

nans, which are present in the walls of reproductive plant tissues (e.g. apple and pine pollen), contain b-d -xylosyl (Xylp) residues attached to C3 of the backbone. Apiogalacturonans, which are present in the walls of some aquatic monocotyledons (e.g. Lemna and Zostera), contain b-d apiofuranosyl (Apif) residues attached to C2 of the backbone either as a single Apif residue or as the disaccharide b-d -Apif-(1!3)-b-d -Apif-(1!. A third substituted galacturonan, which is referred to as rhamnogalacturonan II (RG-II) in the literature, is present in the walls of all higher plants(ONeill et al., 1990). RG-II is not structurally related to RG-I, which has a backbone composed of the repeating disaccharide !4)-a-d -GalpA(1!2)- a-l -Rhap-(1!. RG-II contains 11 dierent glycosyl residues including the unusual sugars 3-C-carboxy-5deoxy-b-l -xylose (aceric acid; AcefA), 2-keto-3-deoxy-d manno-octulosonic acid (Kdo), 2-keto-3-deoxy-d -lyxoheptulosaric acid (Dha), Apif, 2-O-Me-Xylp, and 2-OMe-Fucp (see Figure 1). The RG-II backbone contains at least seven 1,4-linked a-d -GalpA residues, some of which

may be methyl-esteried. Two structurally dierent octasaccharides are attached to C2 of the RG-II backbone (see A and D in Figure 4) and two structurally dierent disaccharides are attached to C3 of the backbone (see B and C in Figure 4). The locations on the backbone of the side-chains with respect to the particular GalpA residue to which the side-chains are attached have not been established. Recent studies have shown that RG-II is present in the primary wall predominantly as a dimer that is crosslinked by a borate ester (Figure 5). Despite the complexity of RG-II, its structure appears to be conserved in the walls of all higher plants.

Biosynthesis of Pectin
Pectin biosynthetic studies have not been particularly revealing, perhaps in part owing to the structural complexity of these polysaccharides (Mohnen, 1998). The in vivo
3

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

Pectic Substances

--D-Galp-(1

6)- -D-Galp-(1

6)- -D-Galp-(1 [A]

4)- -L-Rhap-(1 2

--L-Araf -(1

5)- -L-Ara f-(1

2)- -L-Ara f-(1 [B]

3)- -D-Gal p-(1

4)- -L-Rhap-(1 2

--L-Araf-(1

5)- -L-Ara f-(1 3

1 -L-Ara f

5)- -L-Ara f-(1 [C]

4)- -L-Rha p-(1 2

O O 2-L-Ara f-(1 5)- -L-Ara f-(1 [E] HO OCH 3

4-O-Me--D-Glc pA-(1 [D]

6)-D-Gal p -(1

Figure 3 Selected examples of the structures of oligosaccharides that are attached to the backbone of RG-I. The rhamnosyl residue in A C originates from the RG-I backbone. Structure E is O-(2-O-trans-feruloyl)-a-L-Araf-(1,5)-L-Ara.

synthesis of pectic polysaccharides in the Golgi apparatus has been demonstrated using autoradiography and immunocytochemical localization. The synthesized pectins are transported from the Golgi in vesicles that migrate to, and fuse with, the plasma membrane. The polysaccharides are released into the extracellular space and may then be incorporated into the wall. In some plant tissues and cells a portion of the pectin is not incorporated into the wall but is secreted as a water-soluble polysaccharide. For example, pectic polysaccharides are present in the growth media of some suspension-cultured plant cells. Pectin, in addition to other polysaccharides, may also be secreted by the cells of mucilage-secreting tissue (e.g. root caps). The factors that control the incorporation of pectins into the wall have not been determined, nor is it known whether pectins are structurally modied prior to or after their incorporation into the wall. Pectins are believed to be synthesized from nucleoside diphosphate (NDP)-monosaccharides (e.g. UDP-GalpA). In growing tissues, the NDP-monosaccharides (e.g. NDPGalpA, -Rhap, -Galp, -Araf, -GlcpA, -Fucp, -Xylp, -Apif) are predominantly formed from UDP-Glc (or UDP-Man) via nucleotide-sugar interconversion pathways. For example, the glucosyl residue of UDP-Glc is enzymically oxidized at C6 to generate UDP-GlcpA, which is itself enzymically epimerized at C4 to generate UDP-GalpA. Kdo is probably formed by the condensation of phosphoenolpyruvate (PEP) and d -arabinose and then converted to its CMP-derivative by CMP-Kdo synthase. A precursor of Dha may be synthesized by condensation of PEP and d -threose, or Dha may be formed from CMPKdo. The formation of 2-O-Me-Xylp and 2-O-Me-Fucp has not been investigated. It is not known whether O4

acetylation or O-feruloylation of a monosaccharide occurs at the NDP-monosaccharide level or whether esterication occurs after a glycosyl residue has been attached to pectin. The synthesis and interconversion of NDP-monosaccharides is catalysed by enzymes located predominantly in the cytosol. The NDP-monosaccharides are transported by NDP-monosaccharide translocators into the Golgi apparatus, where they become available to glycosyltransferases. Glycosyltransferases catalyse the transfer of a glycosyl residue from an NDP-monosaccharide, in an anomeric- and linkage-specic manner, to a mono-, oligoor polysaccharide. Pectin biosynthesis requires many dierent glycosyltransferases ( 4 50), but only a few of these enzymes have been even partially characterized. For example, a preparation of membrane proteins contains a putative galacturonosyltransferase (polygalacturonate1,4-a-galacturonosyl transferase; PGA-GalAT). Membrane-bound PGA-GalAT transfers GalpA from UDPGalpA to an endogenous primer and generates a highmolecular mass product. In contrast, a solubilized PGAGalAT transfers only a single GalpA residue onto the nonreducing end of an exogenous acceptor containing at least nine 1,4-linked a-d -GalpA residues. This has led to the suggestion that PGA-GalAT is a multicomponent, membrane-bound synthase complex and that solubilization disrupts the complex. Some of the putative transferases (e.g. galactosyltransferase, arabinosyltransferase, and apiosyltransferase) required for the synthesis of the oligosaccharide side-chains of pectins have also been partially characterized. These side-chains may be synthesized directly on the backbone since there is no evidence that they are rst assembled on lipid intermediates, as are bacterial polysaccharides and

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

Pectic Substances

[B]

Rhap 5 Kdo

Araf 5 Dha

[C]

3 3 ? ? 4GalpA-4GalpA-4GalpA-4GalpA-4GalpA-4GalpA-4GalpA-4GalpA? ? 2 2 Apif 3 GalpA 3Rhap2 4 3Fucp 4 GlcpA 2 [A] Galp Rhap ?Araf GalpA Apif 3 Rhap 3 AcO Acefa 2 2Galp 4 2Arap [D]

2MeXylp

2MeFucp AcO 2Rhap

Figure 4 The glycosyl sequence of rhamnogalacturonan II (RG-II). The structures of the side-chains (A D) have been determined. The positions of these side-chains relative to each other along the backbone are not known and their positions have been assigned arbitrarily (denoted by ?). In the plant cell wall, two RG-II molecules are crosslinked together by a single borate-diol ester to form a dimer (see Figure 5). OAc 5 O-acetyl ester, Me 5 Omethyl ether.

the N-linked oligosaccharides of plant and animal glycoproteins. Enzymes that catalyse the methyl-esterication of HG (HG methyltransferase, HGA-MT) have been partially characterized. HGA-MT catalyses the transfer of CH3 from S-adenosylmethionine to the carboxyl group of a 1,4linked a-d -GalpA residue. HG is hypothesized to be secreted into the wall in a highly esteried form where it may then be partially de-esteried in a developmentally controlled manner by pectin methylesterases or other as yet unidentied enzymes. Methyltransferases that catalyse the methylesterication of the 1,4-linked a-d -GalpA residues of RG-I and RG-II backbones have been reported, but additional evidence is required to substantiate these claims. No O-acetyltransferases or O-feruloyltransferases involved in pectin biosynthesis have been characterized.

Enzymic Degradation of Pectins

Enzymes that fragment and modify pectins are produced by numerous organisms including plants, fungi and bacteria (Visser and Voragen, 1996). The pectolytic enzymes produced by plants are believed to modify the properties of the wall during cell growth and tissue development, while those produced by phytopathogenic fungi and bacteria facilitate penetration and colonization of plant tissue. Microbial pectolytic enzymes are also involved in the breakdown of plant biomass in soil, aquatic

environments and the lower digestive tracts of animals. Pectin-degrading enzymes are used commercially in many processes, including the production of wines and for the clarication of fruit juices. Pectins are fragmented by hydrolases and lyases. Hydrolases (exo- and endoglycanases) cleave glycosidic bonds by hydrolysis. Exoglycanases (e.g. exopolygalacturonase, EC 3.2.1.67) typically release a glycosyl residue from the terminal nonreducing end of a polymer (see (a) in Figure 6) whereas endoglycanases (e.g. EPGase, EC 3.2.1.15) hydrolyse internal glycosidic bonds, thereby generating oligosaccharide fragments (see (b) in Figure 6). Lyases fragment acidic polysaccharides by a b-elimination reaction that generates oligosaccharides containing a D4,5unsaturated residue at the terminal nonreducing end. Pectin lyases (EC 4.2.2.10) and pectate lyases (EC 4.2.2.2) fragment regions of methyl-esteried HG and unesteried HG, respectively (Figure 7), while rhamnogalacturonan lyase fragments the sterically unhindered regions of the RG-I backbone (Figure 8). Pectin structure is modied by pectin methylesterases (EC 3.1.1.11) that release methanol from methyl-esteried GalpA (e.g. COC(O)H3! COOH 1 CH3OH), by O-acetyl esterases (e.g. rhamnogalacturonan acetylesterase) which release acetic acid from an O-acetylated GalpA residue (COC(O)CH3! C OH 1 CH3COOH), and by O-feruloylesterases that release ferulic acid from the side-chains of RG-I.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

Pectic Substances

Backbone of one RG-II molecule

OH O

COOH O

OH O

COOH O

OH O O

OH O O COOH

OH

OH O O O COOH

OH

OH O O COOH

O R2O CH 2

O RO
1

CH 2

Two unesterified 3-linked Apif residues R2O

HO HO

OH OH RO
1

O O

_ B

O O

Two 3-linked Apif residues crosslinked by a borate ester

CH 2 COOH O O OH O O OH COOH OH OH O OH O O O COOH O

CH 2 O O O OH O COOH OH O COOH O OH

Backbone of a second RG-II molecule

Figure 5 Borate ester crosslinking of rhamnogalacturonan II (RG-II). In the plant cell wall, two RG-II molecules are crosslinked together by a single boratediol ester to form a dimer. The borate ester is located on C2 and C3 of two of the four 3-linked apiofuranosyl (Apif) residues of the dimer. The borate ester is believed to crosslink the Apif residue in each of the two 2-O-Me-Xyl-containing side-chains (R1, see A in Figure 4) but not the Apif residue in each of the two aceric acid-containing side-chains (R2, see D in Figure 4).

Functions of Pectin in the Cell Wall

The walls of growing plant cells were for many years considered to provide mechanical strength by being rigid and inert structures (0.11 mm thick). This view has gradually changed following the rst detailed model of the primary wall proposed in 1973 (Keegstra et al., 1973). The primary walls of dicots and non-graminaceous monocots are now believed to consist of a rigid, rod-like cellulose/xyloglucan load-bearing network that is embedded in, and interacts with, a compression-resistant, hydrated pectin network (Carpita and Gibeaut, 1993). Small quantities of structural glycoprotein are often intercalated into these networks, as are enzymes and phenolic esters. The walls of the Poaceae also consist of these two interacting polysaccharide networks. However, these walls contain considerably less pectin, much of the xyloglucan is replaced by glucuronoarabinoxylan, and phenolic esters are present to a much greater extent (Carpita and Gibeaut, 1993). The reader should not, however, be left with the impression that a plant cell is imprisoned by its wall. Cell-to-cell contact is, with the

exception of stomatal guard cells, maintained by plasma membrane-derived pores (plasmodesmata) that pass through the walls. These complex intercellular organelles are believed to be important in cellular communication as they regulate the intercellular movement of low-molecular mass compounds, proteins and RNA. Information on how HG, RG-I and RG-II are linked together in the wall is largely lost when they are solubilized by chemical or enzymic treatments. However, the formerly held notion that HG chains are interrupted by the insertion of a single Rhap residue between regions containing 20 to 30 GalpA residues is almost certainly incorrect. HG and RG-I may be covalently linked together since they are both solubilized by treating walls with EPGase, although no oligosaccharides containing the disaccharide repeating unit of RG-I attached to fragments of HG (e.g. GalA-RhaGalA-Rha-GalA-GalA-GalA) have been isolated and structurally characterized. Moreover, there is no evidence that HG is glycosidically linked to the side-chains of RG-I. On the other hand, HG and RG-II are likely to be covalently linked since they both have 1,4-linked a-d GalpA backbones. The backbone of EPGase-solubilized

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

Pectic Substances

exo -Polygalacturonase

endo-Polygalacturonase

COOH OH

OH O O OH O COOH

COOH O OH O OH OH

OH O O COOH

COOH O OH O OH OH

OH O O COOH

OH

OH

(a) Products of cleavage by exo -polygalacturonase

COOH OH O OH H~OH +

OH O OH O HO COOH

COOH O OH O OH OH

OH O O COOH

COOH O OH O OH OH

OH O O COOH

OH

(b) Products of cleavage by endo -polygalacturonase

COOH OH

OH O O OH O COOH

COOH O OH O OH OH

OH H~OH O COOH +

OH

COOH OH O OH O OH

OH O OH O COOH

OH

Figure 6 Mode of action of exo- and endopolygalacturonases. Exopolygalacturonases cleave the glycosidic bond of a terminal nonreducing GalpA residue (a), whereas endopolygalacturonases cleave the glycosidic bonds of internal GalpA residues (b).

RG-II has been shown to contain up to fteen 1,4-linked ad -GalpA residues, providing indirect evidence that RG-II is linked to HG. HG has been reported to contain, in addition to methylesters, other as yet unidentied esters that may crosslink HG to other wall polymers. HG and RG-I have also been reported to be covalently linked to cellulose, xyloglucan, and/or structural glycoproteins, but additional evidence is required to substantiate these claims. The notion that HG, RG-I and RG-II are covalently linked together in the cell wall, if correct, raises several questions about how such a complex is synthesized. Is the complex synthesized directly, or are HG, RG-I and RG-II synthesized separately and then linked together in the Golgi? Are HG, RG-I and RG-II synthesized separately and then secreted into the wall where polymerases link them together? Evidence for wall-located polymerizing enzymes is for the most part lacking, although phenolic ester crosslinking of pectins is believed to be catalysed by wall-bound peroxidases. A crosslinked pectic network may be generated in the wall by borate ester crosslinking of RGII (see Figure 5), although there is no evidence that the ester is formed enzymically (ONeill et al., 1996). The distribution within growing walls of pectins has been studied in some tissues and cells. Cytochemical and

immunochemical localization techniques have provided evidence that the middle lamella is enriched with HG and RG-I. RG-I and HG with a low degree of methylesterication are particularly enriched in corner junctions and regions of wall surrounding air spaces. RG-II is distributed throughout the wall, although it may be enriched at sites proximal to the plasma membrane. Fourier-transform infrared spectroscopic analysis of tissues and single cells (McCann et al., 1995) and immunocytochemical localization studies suggest that the distribution and the structure of pectin may dier in a celland tissue-specic manner and that the pectin structure may be developmentally regulated. Plant cell expansion and growth, which is driven by internal turgor pressure, must be accompanied by the controlled expansion of the wall. Walls extend rapidly and irreversibly by a process of polymer creep that is believed to involve the breaking and reforming of load-bearing bonds, although the nature of these bonds is not known. In order for the wall not to lose strength or to become much thinner during elongation growth, the existing wall architecture must also be modied to allow the incorporation of newly synthesized polymers. Numerous mechanisms to explain wall expansion have been proposed. These
7

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

Pectic Substances

endo-Pectate lyase

endo-Pectin lyase

COOH O O OH O OH OH

OH O O COOH

COOH O OH O OH OH

OH O O COOCH 3

COOCH 3 O OH O OH OH

OH O O COOCH 3

(a) Products of cleavage by endo-pectate lyase COOH O O OH O OH OH O COOH HO OH H~OH + HO OH O C O O OH O COOCH 3 OH OH O COOCH 3 O OH O OH O COOCH 3 OH O

(b) Products of cleavage by endo-pectin lyase COOH O O OH O OH OH O COOH OH OH O COOH O OH O OH O COOCH 3 HO OH H~OH + H 3CO OH OH O O OH O COOCH 3 O

O C

Figure 7 Mode of action of endopectate lyases and endopectin lyases. Endopectate lyases cleave glycosidic bonds adjacent to an internal non-methylesterified GalpA residue (a). Endopectin lyases cleave internal glycosidic bonds adjacent to a methyl-esterified GalpA residue (b). Both enzymes cleave the glycosidic bond by b-elimination and generate oligosaccharides terminated at their nonreducing end with a D4,5 unsaturated residue (4-deoxy-b-L-threoenopyranosyluronic acid).

include the hydrolysis of pectin, xyloglucan and cellulose by wall glycanases; a decrease in the amount of Ca2 1 crosslinked pectin; a change in the orientation of pectin molecules within the wall and an increase in their degree of methyl-esterication; the activity of PME; the activity of wall transglycosylases (e.g. xyloglucan endotransglycosylase); the presence of proteins (e.g. expansins) that have been proposed to promote growth; and the hydrolysis and reformation of borate ester-crosslinked RG-II. However, none of these proposals provides an unequivocal explanation for wall expansion. The middle lamella is particularly enriched in pectins where the ability of pectins to form crosslinks between themselves and possibly other wall polymers is important for cellcell adhesion. Plant developmental processes such as fruit ripening, leaf and fruit abscission, and pod dehiscence all require a reduction in cellcell adhesion. This may be regulated in part by the enzymic modication and fragmentation of pectin (Hadeld and Bennett, 1998). For example, ripening and softening of tomato fruit is correlated with the production of EPGase. EPGases, however, are not responsible for ripening-associated soft-

ening since transgenic tomato fruits containing 5 1% of the normal EPGase activity ripen normally. Nevertheless, these fruits have improved storage characteristics since tissue integrity is maintained during fruit senescence. Normal ripening also occurs in transgenic tomato plants with reduced PME activity, but there is a signicant decrease in tissue integrity during fruit senescence, and the fruits are more susceptible to microbial attack (Hadeld and Bennett, 1998). The role of other pectin-modifying enzymes (e.g. endo- and exogalactanases, and acetylesterases) in fruit ripening has not been determined. Pectins are the predominant polyanion in primary cell walls and aect the ionic environment of the wall. HG has an anity for divalent cations such as Ca2 1 , although other cations such as Mg2 1 also bind to HG. HG also has an anity for Pb2 1 , Ba2 1 , Sr2 1 , La3 1 and Al3 1 , and the toxic eects of these cations may be due in part to the displacement of Ca2 1 from HG and the subsequent alteration of wall pectin properties. The borate estercrosslinked dimer of RG-II has an unusually high anity for large divalent (e.g. Sr2 1 , Pb2 1 , Ba2 1 ) and trivalent (La3 1 , Pr3 1 , Ce3 1 and Eu3 1 ) cations, although it is not

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

Pectic Substances

O HO H
HO
+

RG Lyase

O HO O CH 3
O

2-linked Rhap

CH 3

OR

OR

COOH

O
Cleavage by -elimination

COOH

O OH O HO O

4-linked GalpA

HO
-carbon

HO OH
4-Deoxy--L-threo-hex4-enopyranosyl uronic acid

O HO O CH 3 O OH O OR

-carbon

CH 3 O COOH HO O OH O

OR

O COOH

HO

Products

RG-I substrate

Figure 8 Mode of action of rhamnogalacturonan lyase. The enzyme cleaves the glycosidic bond between a 2-linked rhamnosyl residue and a 4-linked galactosyluronic acid residue by a b-elimination reaction and generates oligosaccharides terminated at their nonreducing end with a 4-deoxy-b-L-threoenopyranosyluronic acid residue. R 5 H or an oligosaccharide side-chain.

known whether this cation binding ability has a function in the wall. It is not known whether RG-I has specic cationbinding properties. Pectins have an anity for polyamines (e.g. spermidine and spermine) that are present in cell walls, although the biological signicance of their interactions, if they occur, has not been established. The size of molecules that can diuse through the wall may be regulated by controlling the pore size of the wall. The size of these pores (49 nm) has been proposed to be correlated with pectin structure and with the formation of borate ester-crosslinked RG-II, although other factors such as the pH and ionic status of the wall are also likely to be important. Enzymic modication of pre-existing wall pectins has a major eect on calciumpectin interactions. For example, the walls of young, rapidly growing tissue and cells contain highly esteried HG that does not gel readily. In the walls of older, nongrowing tissues, calcium crosslinks may contribute to the overall increase in wall rigidity since the HG is less esteried, presumably owing to the action of pectin methylesterase. Numerous phytopathogenic fungi and bacteria produce EPGases and pectin/pectate lyases that facilitate the penetration and colonization of their plant hosts. These enzymes fragment cell wall pectins and generate lowmolecular mass oligosaccharides including those composed of 1,4-linked a-d-GalpA residues (oligogalacturonides, OGAs). OGAs are a carbon source for the pathogen but they are also signal molecules that are perceived by,

and elicit host defence responses in, plant cells (Darvill et al., 1992). Plants are known to produce proteins that specically inhibit fungal EPGases and promote the accumulation of biologically active OGAs. The mechanisms by which plant cells perceive OGAs are not known, although biological activity is correlated with the presence of an unmodied reducing terminus and with the degree of polymerization (DP) of the OGA. For example, OGAs with DPs between 10 and 15 induce rapid and long-term defence-related responses in plant cells and tissues (Table 1). The rapid (12 min) responses, which include membrane H 1 /K 1 /Ca2 1 ux, membrane depolarization, membrane protein phosphorylation and an oxidative burst, are transient (14 h) and may initiate long-term responses. The ability of OGAs to induce rapid membrane responses has led to the suggestion that plant membranes contain OGA receptors. Evidence for a putative membraneassociated OGA-binding protein has been obtained in potato. This protein may have homology with viral movement proteins that interact with plasmodesmata, although the biological signicance of this homology is not known. OGAs with DPs between 10 and 15 also induce morphogenetic changes in plant tissues (Table 1). This has led to the suggestion that OGAs are a class of endogenous plant growth regulators (Darvill et al., 1992). OGAs are the only pectic-derived fragments that have been shown unequivocally to have biological activity. Fragments of the RG-I backbone and side-chains, and RG-II, may also
9

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

Pectic Substances

Table 1 Biological activities of oligosaccharides composed of 1,4-linked a-d galactopyranosyluronic acid residues Activity A. Plant defense response Induction of phytoalexins Induction of b-1,3-glucanases Induction of chitinases Induction of lignin synthesis Induction of proteinase inhibitors Induction of peroxidases Induction of hypersensitive response Elicitation of necrosis B. Rapid cell surface responses Eux of K 1 and Ca2 1 Protein phosphorylation Membrane depolarization Oxidative burst C. Development and growth Induction of ower formation Inhibition of root formation Inhibition of auxin-induced elongation Induction of ethylene synthesis
a b

DPa 812 915 NDb ND 811 220 ND ND ND 1215 1420 1215 ND 1014 1014 48 48

Plant Soybean Castor bean Parsley Tobacco Wheat Tomato Castor bean Tobacco Cowpea Tobacco Tomato Tobacco Soybean Tobacco Tobacco Pea Tomato

DP 5 degree of polymerization. For example, a DP of 8 corresponds to an oligogalacturonide that contains 8 galactosyluronic acid residues. ND 5 not determined.

have biological activity, although no compelling evidence has been obtained to substantiate such a claim.

Pectin Gels
The ability of pectins to form gels or increase the viscosity of liquids is the basis for their commercial use (Thakur et al., 1997). Pectins containing HG with a low degree of methyl-esterication (low-methoxy pectins) gel in the presence of calcium, whereas high-methoxy (HM) pectins gel at low pH in the presence of high concentrations of a cosolute such as sucrose. High-methoxy pectins are of considerable importance in the food industry since processed fruit products (e.g. jams and jellies) are acidic and contain a high concentration of sucrose. HG chains in solutions and gels are believed to adopt a 21 helix conformation (two GalpA residues per 3608 turn of the helix), while a 31 helix (three GalpA residues per 3608 turn of the helix) may predominate in the dry state. The HG chains do not intertwine, as in DNA, but line up alongside one another. In the presence of Ca2 1 , regions of HG become ionically crosslinked (junction zones) to form a network in which water molecules are entrapped. At low

Ca2 1 concentrations, two HG chains are linked by tightly bound Ca2 1 . Cooperative binding of Ca2 1 occurs in regions containing more than ten contiguous nonesteried GalpA residues and further stabilizes the junction zones. In the presence of excess Ca2 1 , the junction zones may consist of multiple crosslinked HG chains. Gel formation by HM pectins also involves the formation of junction zones, although the mechanisms are less understood. The formation of these junction zones, which is promoted by a sucrose-induced decrease in water activity, most likely involves hydrophobic interactions between methyl ester groups and hydrogen bonding between the glycosyl residues of the HG chains. HM pectin gels are weaker than LM pectin gels and their stability decreases with increasing temperature. The properties of pectin gels are also aected by the charge distribution along the HG backbone, the average molecular mass of HG, the ionic strength of the solution, and the nature of the crosslinking cation. Gel formation is inhibited by O-acetylation of HG and by the presence of RG-I regions attached to the HG. The properties of pectins are altered by their interaction with other ionic polymers. For example, thermoreversible gels are formed from mixtures of HM pectin and alginate (an acidic algal polysaccharide). In fruit juices, pectins and soluble proteins interact to generate colloidal aggregates that aect the processing characteristics and the texture of

10

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

Pectic Substances

the product. These eects are minimized by treating the juices with pectin-degrading enzymes. Pectins are potentially useful as pharmaceutical products since they have been shown to inuence serum cholesterol levels and to induce various immune responses, and because they can bind toxic heavy metals (Visser and Voragen, 1996). For example, most if not all of the Pb2 1 present in wines is tightly bound to the borate estercrosslinked RG-II dimer (Pellerin et al., 1997); the dimer is a major polysaccharide component of wine. Pectin sulfate prolongs blood clotting and can be used in place of heparin, although its toxicity prevents its extended use. Pectin gels may also have use as slow-release drug carriers. The last 20 years have seen considerable progress in our understanding of the roles of pectin in the growth and development of plants and of their value as food and pharmaceutical products. Nevertheless, there is still much to be learned about these complex plant polysaccharides and future research will no doubt provide information that is both useful and intriguing.

McCann MC, Roberts K, Wilson RH et al. (1995) Old and new ways to probe plant cell-wall architecture. Canadian Journal of Botany 73 (supplement 1): S103S113. Mohnen D (1998) The biosynthesis of pectins and galactomannans. In: Pinto BM (ed.) Comprehensive Natural Products Chemistry, vol. 3, pp. 497527. Oxford: Elsevier. ONeill M, Albersheim P and Darvill AG (1990) The pectic polysaccharides of primary cell walls. In: Dey PM (ed.) Methods in Plant Biochemistry, vol. 2, pp. 415441. London: Academic Press. ONeill MA, Warrenfeltz D, Kates K et al. (1996) Rhamnogalacturonan II, a pectic polysaccharide in the walls of growing plant cells forms a dimer that is covalently cross-linked by a borate ester. In vitro conditions for the hydrolysis and formation of the dimer. Journal of Biological Chemistry 271: 2292322930. Pellerin P, ONeill MA, Pierre C et al. (1997) Complexation du plomb ` re de rhamnogalacturonane II, un dans les vins par les dime polysaccharide pectique du raisin. Journal International des Sciences de la Vigne et du Vin 31: 3341. Thakur BR, Singh RK and Handa AK (1997) Chemistry and uses of pectin A review. Critical Reviews in Food Science and Nutrition 37: 4773. Visser J and Voragen AGJ (eds) (1996) Pectins and Pectinases, Progress in Biotechnology, vol. 14. Amsterdam: Elsevier.

Acknowledgements
The nancial support of the United States Department of Energy (Grant Nos. DE FG02-96ER20220 and DE-FG0593ER20097) is gratefully acknowledged.

Further Reading
Cosgrove DJ (1997) Creeping walls, softening fruit, and penetrating pollen tubes. The growing role of expansins. Proceedings of the National Academy of Sciences of the USA 94: 55045505. Fry SC (1995) Polysaccharide-modifying enzymes in the plant cell wall. Annual Review of Plant Physiology and Molecular Biology 46: 497520. Ishii T (1997) Structure and function of feruloylated polysaccharides. Plant Science 127: 111127. Jarvis MC and Appleby DC (1995) Chain conformation in concentrated pectic gels: evidence from 13C NMR. Carbohydrate Research 275: 131 145. Kohn R (1975) Ion binding on polyuronates alginates and pectin. Pure and Applied Chemistry 42: 371397. Linskens H-F and Jackson JF (eds) (1996) Modern Methods of Plant Analysis vol. 17, Plant Cell Wall Analysis. Berlin: Springer-Verlag. Matoh T (1997) Boron in plant cell walls. Plant and Soil 193: 5970. Mezitt LA and Lucas WJ (1996) Plasmodesmal cell-to-cell transport of proteins and nucleic acids. Plant Molecular Biology 32: 251273. Sakai T, Sakamoto T, Hallaert J and Vandamme EJ (1993) Pectin, pectinases, and protopectinase: production, properties, and application. Advances in Applied Microbiology 39: 213294.

References
Carpita NC and Gibeaut DM (1993) Structural models of primary cell walls in owering plants: consistency of molecular structure with the physical properties of the walls during growth. The Plant Journal 3: 1 30. Darvill A, Augur C, Bergmann C et al. (1992) Oligosaccharins: oligosaccharides that regulate the growth, development and defence responses in plants. Glycobiology 2: 181198. Hadeld KA and Bennett AB (1998) Polygalacturonases: many genes in search of a function. Plant Physiology 117: 337343. Keegstra K, Talmadge KW, Bauer WD and Albersheim P (1973) Structure of plant cell walls III. A model of the walls of suspensioncultured sycamore cells based on the interconnections of the macromolecular components. Plant Physiology 51: 188197.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

11