Plant Physiol. (1980) 66, 1128-11340032-0889/80/66/1 128/07/$00.50/0

Structure of Plant Cell WallsX. RHAMNOGALACTURONAN I, A STRUCTURALLY COMPLEX PECTIC POLYSACCHARIDE IN THEWALLS OF SUSPENSION-CULTURED SYCAMORE CELLS'

Received for publication April 14, 1980 and in revised form July 10, 1980

MICHAEL MCNEIL, ALAN G. DARVILL, AND PETER ALBERSHEIM2Department of Chemistry, University of Colorado, Boulder, Colorado 80309

ABSTRACT

The purification and characterization of a pectic polymer, rhamnogalac-turonan I, present in the primary cell walls of dicots is described. Rham-nogalacturonan I accounts for approximately 7% of the mass of the wallsisolated from suspension-cultured sycamore cells. As purified, rhamnoga-lacturonan I has a molecular weight of approximately 200,000 and iscomposed primarily of L-rhamnosyl, D-galacturonosyl, L-arabinosyl, and D-galactosyl residues. The backbone of rhamnogalacturonan I is thought tobe composed predominantly of D-galacturonosyl and L-rhamnosyl residuesin a ratio of approximately 2:1. About half of the L-rhamnosyl residues are2-linked and are glycosidically attached to C4 of a D-galacturonosyl residue.The other half of the L-rhamnosyl residues are 2,4-inked and have a D-galacturonosyl residue glycosidically attached at C2. Sidechains averaging6 residues in length are attached to C4 of the L-rhamnosyl residues. Thereare many different sidechains, containing variously linked L-arabinosyl,and/or n-galactosyl residues.

Pectic polysaccharides are defined as those containing 4-linked-a-D-galacturonosyl residues (25). Pectic polysaccharides are im-portant components of the primary cell walls of dicots, accountingfor approximately 30% of the walls (8, 20, 25, 28). RG-11,3 a pecticpolysaccharide containing many unusually linked glycosyl resi-dues and accounting for 4% of the cell walls of suspension-culturedsycamore cells, has recently been described (11). Here, the isolationand partial characterization of another pectic polysaccharide, RG-I, are reported. Isolated RG-I accounts for 7% of the cell wall massor approximately 23% of the cell wall pectic polysaccharides. RG-I contains the glycosyl residues historically associated with cellwall pectic polymers (25). However, the structural characteristicsof RG-I are different from those previously reported for any otherpectic polysaccharide (25).

MATERIALS AND METHODS

Preparation of Solvents. DMSO was distilled from calciumhydride at a pressure of 12 mm Hg. All other solvents were reagentgrade.

Isolation of Cell Walls. Cell walls were isolated from suspen-sion-cultured sycamore (Acer pseudoplatanus) cells as described(28).

' This work was supported by United States Department of EnergyContract EY-76-S-02-1426.2To whom correspondence should be addressed.'Abbreviations: RG-I and RG-II, rhamnogalacturoan I and II, respec-

tively; NaBD4, sodium borodeuteride, DMSO, dimethyl sulfoxide.

Enzyme Purification. Endo-a- 1,4-polygalacturonase was puri-fied from Colletotrichum lindemuthianum (15) and checked forpurity as described (I 1).

Enzymic Extraction of Pectic Polysaccharides. The pectic poly-saccharides were extracted from isolated sycamore cell walls by C.lindemuthianum endopolygalacturonase by the procedures de-scribed (I 1).

Gel Filtration Chromatography. Gel filtration chromatographywas performed on a 1.4- x 40-cm agarose A-1.Sm column or ona 3- x 50-cm agarose A-Sm column. The columns were equili-brated and eluted with 50 mm Na-acetate (pH 5.2). The void andincluded volumes of the columns were determined with bluedextran (Sigma) and NaCl, respectively. In some cases (as notedin the text), the agarose A-1.5m column was equilibrated andeluted with 50 mm Na-acetate (pH 5.2) containing 10 mm EDTAor with 50 mm Na acetate (pH 5.2) containing 50 nmM EDTA and1.0 M NaCl.Colorimetric Assays. Neutral sugar (hexose) concentrations

were estimated by the anthrone method of Dische (14), and uronicacid concentrations were estimated by the m-hydroxydiphenylmethod of Blumenkrantz and Asboe-Hansen (10). Protein wasassayed by the method of Lowry et al. (24).

Determination of Absolute Configuration. Analysis of absoluteconfiguration of the sugars of RG-I was performed as describedby Gerwig et al. (16, 17) using (-)-2- butanol glycosides and asdescribed by Leontein et al. (21) using (+)-2-octanol glycosides.

Analysis of Glycosyl and Glycosyl-linkage Compositions. Theneutral and amino glycosyl compositions were determined by thealditol acetate method (1, I 1). Glycosyl-linkage compositions weredetermined by GC and GC-MS analysis of the partially methyl-ated alditol acetate derivatives (9, 28). Polysaccharide methyla-tions were performed using modifications (11, 28, 30) of theprocedure reported by Hakomori (18.)Some samples were ethylated or trideuteromethylated. In these

cases, ethylation or trideuteromethylation was performed in ex-actly the same way as with methylation, except that ethyl iodideor trideuteromethyl iodide (Stohler Isotope Chemicals) were usedin place of methyl iodide.The ring forms, pyranosyl or furanosyl, of the glycosyl residues

of RG-I, whose ring form could not be determined by methylationanalysis, were determined by the method of Darvill et al. (12).

Reduction of Uronosyl Residues. The glycosyl linkage of theuronosyl residues of RG-I was determined following their reduc-tion to the corresponding 6,6-di-deuterohexosyl residues. Thereduction was accomplished by the method of Taylor and Conrad(29) using sodium borodeuteride and deuterium oxide instead ofsodium borohydride and H20. The samples then were analyzedfor glycosyl and glycosyl-linkage composition as described above.The quantities of unlabeled hexose and of dideutero-labeled hex-ose (resulting from the reduction of the corresponding uronosylresidue) derivatives were determined by quantitation of the ap-

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STRUCTURE OF PLANT CELL WALLS. X

propriate fragment ions obtained during mass spectrometric anal-ysis.GC-MS. GC analysis of the alditol acetates was performed on

Column A which contained 0.2% ethylene glycol succinate, 0.2%ethylene glycol adipate, and 0.4% XF- 1 150 on Gas Chrom P (1).GC analysis of the partially methylated alditol acetates was per-formed on Column A or on Column B, which contained 0.3%OV275 and 0.4% XF-I 150 on Gas Chrom Q (13), and on ColumnC, a 25-m open tubular glass capillary column containing SE 30(LKB, Broma, Sweden). All GC-MS analyses were carried out ona Hewlett-Packard GC-MS system (model 5980A) coupled to aHewlett-Packard (model 5933A) data system.

All of the GC flame-ionization responses to partially methylatedalditol acetates were corrected to mole responses as described bySweet et al. (27).DMSO-Ion Treatment of Methylated RG-I. Fully methylated

non-uronosyl-reduced RG-I was treated with DMSO ion usingthe method of Lindberg et al. (22) to achieve fl-elimination fromC4 of the uronosyl residues. In a typical experiment, 10 mgmethylated RG-I was dried overnight in a vacuum oven. One mlDMSO (freshly distilled from calcium hydride at 12 mm Hg)containing 0.05 ml 2,2-dimethoxy propane (as a H20 scavenger)was added to the sample. The sample was made 0.9 M with respectto DMSO ion and stirred for 24 h at room temperature. After 24h, 5% of the sample was removed, treated with excess ethyl iodide,and fractionated on a LH-20 column as described below, and theglycosyl-linkage composition of the aliquot was determined. Theremainder of the sample was neutralized with 50% acetic acid andpartitioned between H20 and chloroform, and the chloroformlayer was applied to a 10-ml LH-20 column. The 0.4-ml columnfractions were assayed by the anthrone method and the anthronepositive fractions were combined. The glycosyl-linkage composi-tion of the sample then was determined.

Isolation of Disaccharides from Methylated RG-I. Fully meth-ylated non-uronosyl-reduced RG-I (3 mg) was heated for 3 h at100 C in 1 ml 96% formic acid. The sample then was reduced withsodium borodeuteride and remethylated with trideuteromethyliodide, using a single addition of DMSO ion followed by a singleaddition of trideuteromethyl iodide. The sample then was sus-pended in 0.5 ml H20 and this aqueous layer was extracted threetimes with 0.5 ml chloroform. The combined chloroform extractwas itself extracted once with 0.5 ml H20 and the chloroform-soluble fraction then was blown to dryness. The sample then wasanalyzed by GC-MS using column C isothermally at 260 C. Theeluting disaccharides were identified by their electron impact massspectral fragmentations (23).

RESULTS

Purification of RG-I. Sycamore cell walls were prepared (28)and treated with endopolygalacturonase (1 1). The endopolygalac-turonase-solubilized cell wall material was applied to a 1.5- x 12-cm DEAE-Sephadex A-25 ion-exchange column that had beenpre-equilibrated with 10 mm K-phosphate (pH 7.0). The columnwas washed with 2 column volumes of phosphate buffer. Theabsorbed polysaccharide was eluted with a 300-ml linear 0 to 0.5M NaCl gradient in the phosphate buffer (Fig. 1). Three pecticpolysaccharide-containing fractions, labeled A, B, and C, elutedin the salt gradient. The column fractions containing the secondcarbohydrate peak (pectic fraction B) and those containing thethird carbohydrate-containing peak (pectic fraction C) werepooled separately, dialyzed against distilled H20, and lyophilized.The yields of fractions A, B, and C were approximately 20, 70,and 50 mg, respectively,/g cell walls.The pectic polysaccharide in fraction C was dissolved in 0.5 ml

distilled H20 and further fractionated by gel filtration on agaroseA-5m yielding the two pectic fractions illustrated in Figure 2.Agarose A-Sm column fractions 41 to 49 contained rhamnogalac-

* 3.,Ec0

la 2.

4

z4 2,iEC I.0

4 1,

z

cr0nco

0 10 20 30 40

r.:0

r-

F RACTION NNUMBER

FIG. 1. Chromatography of endopolygalacturonase-solubilized cellwall material on a DEAE-Sephadex column (1.5 x 12 cm). After sampleloading, the column was washed with 2 column volumes of 10 mM K-phosphate (pH 7.0). Material that absorbed to the column was elutedusing a linear NaCl gradient (0 to 0.5 M) in the phosphate buffer. Collectedfraction volume was 4 ml. Column fractions were assayed for neutralglycosyl residues by the anthrone method (50) (A at 620 nm) and fordetection of uronosyl residues by the m-hydroxydiphenyl method ofBlumenkrantz and Asboe-Hansen (10) (A at 520 nm).

i 0.4-E

0

,0A0.3

E0NF0T2

0

4

0

20 30 40 50

FRACT IO0N NUMBER

FIG. 2. Chromatography, on agarose A-Sm column (3 x 50 cm), ofmaterial in fraction C of the DEAE-Sephadex column (Fig. 1). Collectedfraction volume was 2.5 ml. Column fractions were assayed as describedin the legend of Figure 1.

turonan II, which has been described previously (11) and whichis not further considered here. Agarose A-5m column fractions 30to 40 (RG-I fraction C) were combined, dialyzed, lyophilized, andreapplied to the same agarose A-Sm column (Fig. 3). Columnfractions 19 to 32 from this second agarose A-Sm column werepooled, dialyzed, and lyophilized to give a yield of about 20 mg/g cell walls of what is referred to as purified RG-I fraction C.

Fraction B of the DEAE-Sephadex column (Fig. 1) was purifiedby a procedure similar to that used to purify fraction C. Thisprocedure yielded about 50 mg/g cell walls of purified RG-Ifraction B. Purified RG-I fractions B and C together constituteRG-I. Fractions B and C have similar glycosyl compositions,although fraction C is richer in rhamnosyl and galacturonosylresidues and fraction B is richer in arabinosyl residues. Peak A(Fig. 1), which is not considered here, is composed almost entirelyof galacturonosyl residues. The results reported below were ob-

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McNEIL, DARVILL, AND ALBERSHEIM

E 1.5

0

(0

91.0

0

w 0.5-z

0

5 20 25 30 35

FRACTION NUMBER

FIG. 3. Rechromatography, on agarose A-Sm column (3 x 50 cm), ofmaterial in fractions 30 to 40 from the first agarose A-Sm column (Fig. 2).Collected fraction volume was 3.0 ml. Column was assayed as describedin legend of Figure 1. Various column fractions were individually assayedby the alditol acetate method (1) for their neutral glycosyl residue com-

positions (Table I).

tained with purified RG-I fraction C. However, most of theexperiments were repeated with similar results with RG-I fractionB.Mol Wt of RG-1. The apparent mol wt of RG-I, as determined

by gel filtration on the Agarose A-Sm column (Fig. 3), is approx-imately 3 x 105 (compared to globular proteins) or 1 x 105(compared to linear dextrans). This suggests that, since RG-I is abranched polysaccharide, it has a mol wt of approximately 2 xl0o.Experiments were performed to determine whether the apparent

mol wt of RG-I resulted from aggregation of smaller polysaccha-rides. One experiment involved gel filtration chromatography inthe presence of chelating agents and in concentrated salt solutions.RG-I was chromatographed on an agarose A-1.5m column in 50mm Na-acetate (pH 5.2). Under these conditions, some of thepolysaccharide voided the column and some of the polysaccharidewas partially included in the gel. The addition of either 10 mmEDTA or both 50 mm EDTA and 1 M NaCl to the 50 mm Na-acetate elution buffer did not detectably change the elution profileof RG-I on the agarose A-1.5m column.A second approach to investigate the possibility of aggregation

in RG-I involved conversion of the carboxyl groups of the uron-osyl residues ofRG-I to primary alcohols. The gel filtration profileof RG-I on agarose A-5m was examined after reduction of morethan 97% of the uronosyl carboxyl groups of RG-I. This reductiondid not detectably change the elution profile of RG-I on theagarose A-Sm column.The large average size of RG-I is also suggested by an inability

to detect reducing terminal glycose residues in the molecule. Thiswas shown by the fact that, after reduction ofthe uronosyl residuesand concurrent reduction of the glycose residues at the reducingend of the polysaccharide, no permethylated alditol acetates cor-responding to a prereduced glycose residue could be detected bymethylation analysis, i.e. no alditol acetate containing 0-methylgroups on carbons I and 5 (or 4) were detected. Since theprereduced residue would be present in amounts too small todetect for a polymer consisting of 1000 or more glycosyl residues,this is the expected result. Prereduction of RG-II, which is amolecule containing approximately 50 glycosyl residues, yieldedreadily detectable amounts, between 1.0 and 1.5%, of the prere-duced and carboxyl-reduced 4-linked galacturonic acid derivative(1 1). Hence, it is concluded that an average mol wt of 200,000 is

a reasonable estimate for RG-I.Heterogeneity of RG-I. As purified, RG-I is not a structurally

homogeneous polymer. The colorimetric assay elution profile ofRG-I on the agarose A-5m column (Fig. 3) shows that there arefractions relatively enriched in uronosyl residues and fractionsenriched in neutral glycosyl residues. Selected fractions from theagarose A-5m column (Fig. 3) were analyzed by the alditol acetatemethod for neutral glycosyl residues (Table I). The data (Table Iand Fig. 3) show that the early eluted fractions are rich inarabinosyl residues, whereas the later eluted fractions are rich inrhamnosyl and galacturonosyl residues.

Absolute Configuration of the Sugars of RG-1. The absoluteconfiguration of the sugars of RG- 1 was determined by preparingthe (-)-2-butanol glycosides (16, 17) and the (+)-2-octanol gly-cosides (21). The galactosyl and galacturonosyl residues of RG-Iare in the D configuration, whereas the arabinosyl, rhamnosyl,and fucosyl residues are in the L configuration. The presence ofsmall amounts (<2%) of the opposite configurations cannot beruled out using these methods.

Composition of RG-I. The glycosyl composition of purified RG-I is presented in Table II. The polysaccharide contains no aminosugars or protein. As indicated in Table II, 97% (± 7%) by weightof RG-I has been accounted for. The glycosyl-linkage composi-tion, determined by methylation analysis of uronosyl carboxyl-reduced RG-I, is presented in Table III. The ring forms of theglycosyl residues that could not be determined from the methyl-ation analysis were determined as described (12).

Distribution of Galacturonosyl Residues in RG-I. RG-I doesnot have long uninterrupted regions of 4-linked a-D-galacturon-osyl residues. This was shown by the following experiment. Theendopolygalacturonase used to extract RG-I from the plant cellwall is known to require as a substrate 4 or more contiguous 4-linked a-D-galacturonosyl residues (15). However, this endopoly-galacturonase does not hydrolyze linear galacturonan regions in

Table I. Comparison of Neutral Glycosyl Residue Compositions ofSelected Fractions in Peak of RG-I Elutingfrom agarose A-Sm (see Fig. 3)

Column FractionGlycosyl Residues

22 25 29 31a 33a

mol %L-Rhamnosyl 17 19 23 24 27L-Fucosyl 2 2 2 3 3L-Arabinosyl 41 38 32 30 30Xylosyl 2 2 2 2 4D-Galactosyl 32 31 31 31 26Glucosyl 6 7 9 9 9

a These fractions also contained small amounts (less than 2 mol %) of 2-O-methylfucosyl, 2-0-methylxylosyl and apiosyl residues, residues char-acteristic of RG-II (1 1).

Table II. Glycosyl Residue Composition ofRG-IThese residues account for 97% (±7%) by weight of RG-I.

Glycosyl Residue Amountamol %

D-Galacturonosyl 36L-Arabinosyl 24D-Galactosyl 20L-Rhamnosyl 15L-Fucosyl 2Glucosyl 1Xylosyl I

a The galacturonosyl content of RG-I varies ±20%o from preparation topreparation.

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STRUCTURE OF PLANT CELL WALLS. X

Table III. Glycosyl-linkage Composition of RG-I

Deter-mined Po- Deduced

Glycosyl Residue sition of Glycosidic Amount0-methyl LinkageGroups

mol %D-Galacturonopyranosyl 2, 3, 4, 6 Terminal 1.8

2,3,6 4 33.02,6 3,4 1.03, 6 2, 4 Trace

L-Arabinofuranosyl 2, 3, 5 Terminal 9.43,5 2 0.42,5 3 0.82,3 5 8.02 3,5 2.03 2,5 1.6

2, 3, 5a 0.6D-Galactopyranosyl 2, 3, 4, 6 Terminal 4.0

3,4,6 2 0.82,4,6 3 1.62,3,6 4 9.02,3,4 6 4.03, 6 2, 4 Trace3,4 2,6 0.82,4 3,6 0.82, 3 4, 6 Trace

L-Rhamnopyranosyl 2, 3, 4 Terminal 0.53,4 2 7.03 2,4 6.0

2, 3, 4 TraceL-Fucopyranosyl 2, 3, 4 Terminal 1.6Glucopyranosylh 2, 3, 6 4a 3.2Xylopyranosylb 2, 3 4a 1.0

3,4 2 0.5a These glycosyl residues are present in varying amounts in different

preparations of RG-1 and are thought to originate from polysaccharidecontaminants.

b The ring form of these residues is assumed; all other ring forms weredetermined by the position of 0-methyl groups or by the method ofDarvill et al. (12).

which many of the uronosyl residues are methyl esterified (15).To examine the possibility that RG-I contains uninterruptedregions of galacturonosyl residues sufficiently methyl-esterified toinhibit the endopolygalacturonase, the polysaccharide was de-esterified at pH 12 for 2 h at 4 C (15) prior to retreatment withendopolygalacturonase. The elution profile of de-esterified, en-dopolygalacturonase-retreated RG-I on agarose A-Sm is virtuallyidentical with the elution profile of untreated RG-I, indicatingthat de-esterification of RG-I did not provide any new substratesites for the endopolygalacturonase. Therefore, RG-I is unlikelyto possess long uninterrupted regions of contiguous 4-linked a-D-galacturonosyl residues.

Identification of Neutral Glycosyl Residues Attached to C4 ofSome of the 4-linked Galacturonosyl Residues of RG-I. Themethods (22) used in this analysis are summarized in Figure 4.RG-I was methylated using only a single addition of DMSO ionfollowed by a single addition of methyl iodide. Such a single-stepmethylation of RG-I results in the formation of methyl ethers ofall the free hydroxyl groups and methyl esters of the carboxygroups of the galacturonosyl residues. The methylated RG-I thenwas treated with potassium DMSO ion in DMSO. This additionof strong base causes ,8-elimination from C4 of the galacturonosylresidues, thereby releasing the glycosyl residue attached to C4 and

MeOuM e,~~OO, C=OMe

R/

MOO OR

OMe

REACTIONDMSO ION

MeO O-

MeHtpO

Oe R

46-

MeOA-o

MeO OR

REACTION 2DMSO ION

MeO OMeO + H O

OR

DMSO ION

SEE FIG 5

FIG. 4. Reaction sequence for identifying the neutral glycosyl residuesglycosidically attached to C4 of some of the 4-linked D-galacturonosylresidues. The reaction sequence is illustrated for a 2-linked L-rhamnosylresidue glycosidically attached to C4 of a D-galacturonosyl residue. Thefinal product is not detected by the GLC analysis used and, thus, theglycosyl residues attached to C4 of the 4-linked galacturonosyl residuesare identified by their disappearance after treatment with DMSO ion. Rand R', unspecified neutral glycosyl residues.

Table IV. Glycosyl-linkage Composition of RG-I (Not Including UronosylResidues) Both Before and After Base-catalyzed Elimination of Uronosyl

ResiduesThe data are an average of three experiments.

Deter-mined Po- Deduced Before After

Glycosyl Residue sition of Glycosidic Elimi- Elimi-0-Methyl Linkage nation nationGroups

mol % mol %

L-Arabinofuranosyl 2, 3, 5 Terminal 20 183,5 2 3 22,5 3 1 22,3 5 17 262 3,5 6 43 2,5 3 4

2,3,5 1 1D-Galactopyranosyl 2, 3, 4, 6 Terminal 7 5

3,4,6 2 2 22,4,6 3 3 32,3,6 4 10 112,3,4 6 5 53,6 2,4 1 13,4 2,6 1 12,4 3,6 1 1

L-Rhamnopyranosyl 2, 3, 4 Terminal 2 13,4 2 8 14 2,4 6 7

2,3,4 1 1L-Fucopyranosyl 2, 3, 4 Terminal 3 2

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McNEIL, DARVILL, AND ALBERSHEIM

IREACTION

DMSO ION

PRODUCT +

REACTION 2ETHYL IODIDE

RO>

MoO OEt

FIG. 5. Reaction sequence for identifying the neutral glycosyl residuesto which some of the D-galacturonosyl residues are glycosidically attached.The reaction sequence is illustrated with a 2,4-linked L-rhamnosyl residueto which a 4-linked D-galacturonosyl residue is glycosidically attached toC2. The starting material was produced by treatment of methylated RG-Iwith base as described in Fig. 4. R and R' unspecified neutral glycosylresidues.

producing an unsaturated uronosyl residue (Reaction 1 in Fig. 4).The glycose residue, released by elimination from C4 of the

galacturonosyl residue, has an unsubstituted reducing group at C1.This reducing group, in the open-chain form, possesses a carbonylgroup which, in the presence of the strong base, leads to a secondelimination reaction. This second elimination releases the sub-stituent on C3 of the glycose (whether the substituent is an 0-methyl group or another glycosyl residue), producing an unsatu-rated glycose derivative (Reaction 2 in Fig. 4). Thus, glycosylresidues glycosidically attached to the C4 of galacturonosyl resi-dues are converted, by base-catalyzed elimination, to unsaturatedglycoses.The fl-eliminated sample containing the unsaturated glycose

residues is hydrolyzed, reduced, and acetylated. Using this pro-

cedure, the unsaturated glycose residues do not form derivativesdetectable by GC and, thus, are lost. The disappearance of selectedglycosyl residues is the method by which the glycosyl residuesattached to C4 of galacturonosyl residues in RG-I have beenidentified.The partially methylated alditol acetates formed by hydrolysis,

reduction and acetylation of methylated RG-I are compared inTable IV with the partially methylated alditol acetates formed byhydrolysis, reduction, and acetylation of methylated RG-I whichhad been eliminated with DMSO ion. The neutral glycosyl resi-dues most noticeably degraded by treatment of methylated RG-Iwith the DMSO ion (_90% loss) are 2-linked L-rhamnosyl resi-dues. It is concluded, therefore, that 90% or more of the 2-linkedL-rhamnosyl residues are glycosidically attached to C4 of D-gal-acturonosyl residues (Fig. 6, structure a).The base-catalyzed degradation of methylated RG-I yields one

inexplicable result. The amount of the 5-linked L-arabinosyl resi-

dues is reproducibly increased from 17 to 26 mol % (Table IV).Determination of Neutral Glycosyl Residues to Which Galac-

turonosyl Residues Are Glycosidically Attached. The experimentsjust described determined that nearly all of the 2-linked L-rham-nosyl residues are glycosidically attached to C4 of D-galacturonosylresidues. The following experiments were designed to determinethe identity of the neutral glycosyl residues which have D-galac-turonosyl residues glycosidically attached to them. The experi-ments also determine the carbon atoms of the neutral glycosylresidues to which the galacturonosyl residues are attached. Thereactions used in these experiments are summarized in Figures 4and 5 (22).RG-I was methylated in a single step and treated with potassium

DMSO ion. fl-Elimination of the methyl-esterified D-galacturon-osyl residues proceeded as in Reaction I of Figure 4, yielding theA4:5 unsaturated D-galacturonosyl derivatives. The unsaturatedD-galacturonosyl residues undergo a second reaction in the pres-ence of the strongly basic DMSO ion in which the glycosidic bondof the unsaturated galacturonosyl residue is cleaved by an unde-termined mechanism (Reaction 1 in Fig. 5). This results in theformation of an unsubstituted hydroxyl group on the glycosylresidue to which an unsaturated galacturonosyl residue had beenattached (7). These unsubstituted hydroxyl groups were ethylated(Reaction 2 in Fig. 5), thereby labeling the carbon atoms of neutralglycosyl residues to which galacturonosyl residues had been gly-cosidically attached. The resulting methylated, DMSO-ion-treated, and ethylated RG-I was hydrolyzed, reduced, and acety-lated. The resulting partially alkylated, partially acetylated alditolswere identified and quantitated by GC and GC-MS. The partiallymethylated, partially acetylated alditols that contained an O-ethylgroup are presented in Table V.

O-Ethyl groups were detected on several glycosyl residues butpredominantly on C2 of 2,4-linked L-rhamnosyl residues. In fact,all of the 2,4-linked L-rhamnosyl residues have galacturonosylresidues glycosidically attached to C2 because all of the 2,4-linkedL-rhamnosyl residues recovered after the DMSO-ion eliminationof methylated RG-I were converted to 2 O-ethyl rhamnosylderivatives (Fig. 6, structure b).RG-I apparently also contains L-arabinosyl residues with D-

galacturonosyl residues attached to C2 or C3, and RG-I containsD-galactosyl residues with D-galacturonosyl residues attached toC4, and other D-galactosyl residues with D-galacturonosyl residuesattached to C6 (Table V).The ethyl labeling could not determine whether D-galacturon-

osyl residues are glycosidically attached to C2 of the 2-linked L-rhamnosyl residues. This is because the 2-linked L-rhamnosylresidues are degraded by the DMSO-ion treatment (Table IV andReaction 2 in Fig. 4). The glycosidic attachment of D-galacturon-

Table V. Partially Methylated, Partially Ethylated, Acetylated AlditolsDetected in Amounts 20.5 mol % in Methylated RG-I after DMSO-ion

Elimination and EthylationThe data are an average of three experiments.

Deter- Deter- Deter- De- Yield ofmined mined mined duced Alkyl-

Glycosyl Residue Position position Positionof 0- of 0- of O Glyco- atedMethyl Ethyl Acetyl sidic AlditolGroups Groups Groups Linkage Acetate

mol %L-Arabinofurano- 2, 5 3 1,4 3142

syl 3,5 2 1,4 2D-Galactopyrano- 2, 3, 6 4 1, 5 4 0.5

syl 2,3,4 6 1,5 6 1.9L-Rhamnopyrano- 3 2 1, 4, 5 2, 4 6.0

syl

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STRUCTURE OF PLANT CELL WALLS. X

(a) + 2 Rha + 4 Gal A +

4(b) - 4 Ga1 A +2 Rhaa

(c) 4 Ga1 A 2 Rha

FIG. 6. Structural characteristics of the backbone chain of RG-1.represents a glycosidic bond; -_ represents point of attachment of otherglycosyl residues in RG-I.

osyl residues to C2 of at least some of the 2-linked L-rhamnosylresidues has been established by isolation and characterization ofthat disaccharide (see below).

Isolation and Characterization of Disaccharides from Methyl-ated RG-I. Some of the disaccharides produced by partial for-molysis of methylated RG-I have been identified. MethylatedRG-I was partially formolyzed, reduced, and trideuteromethy-lated. Alkylation with trideuteromethyl iodide, following partialformolysis and reduction, labeled the carbon atoms of the disac-charides to which other glycosyl residues had been attached in theintact polysaccharide. The alkylation with trideuteromethyl iodideenabled alkylated disaccharide alditols containing L-rhamnitolresidues that were originally 2-linked to be distinguished fromalkylated disaccharide alditols containing L-rhamnitol residuesthat were originally 2,4-linked, as the latter (but not the former)contain an O-trideuteromethyl group at C4.A variety of alkylated disaccharide alditols were separated and

identified by combined capillary column GC and electron impactMS (23). The quantitatively major alkylated disaccharide alditolobtained was a 4-linked galacturonosyl residue attached to C2 ofrhamnitol. Location of the trideuteromethyl groups demonstratedthis disaccharide alditol originated in RG-I from two differentlylinked L-rhamnosyl residues, those which were originally 2,4-linked (Fig. 6, disaccharide b) and those which were originally 2-linked (Fig. 6, disaccharide c). The D-galacturonosyl residues wereoriginally 4-linked in both disaccharides. The quantitatively majoralkylated disaccharide alditols b and c (Fig. 6) were each isolatedin two forms: in one form, the carboxyl group had been reducedand dideutero-labeled by sodium borodeuteride by an unknownmechanism; in the second form, the carboxyl group was trideuter-omethyl-esterified. These results confirm (see above) that C2 ofthe 2,4-linked L-rhamnosyl residues have 4-linked galacturonosylresidues glycosidically attached to them. These results show, too,that some of the 2-linked L-rhamnosyl residues have 4-linkedgalacturonosyl residues glycosidically attached at C2, although theproportion of the 2-linked L-rhamnosyl residues that have 4-linkedgalacturonosyl residues attached could not be determined by thisexperiment.

DISCUSSION

The presence of pectic polysaccharides in the primary cell wallsof dicots has been known for a long time (19, 31). RG-II, a pecticpolymer accounting for 4% of suspension cultured sycamore cellwalls, has been shown to contain at least 15 differently linkedglycosyl residues. The study here shows that RG-I, which accountsfor at least 7% of suspension-cultured sycamore cell walls andwhich is also structurally complex, is unrelated in structure to RG-II; for example, the L-rhamnosyl residues of RG-I are 2- and 2,4-linked, whereas the L-rhamnosyl residues of RG-II are predomi-nantly terminal and 3-linked (l1). RG-I and RG-II are bothreleased from the cell wall by the same highly purified endopol-

ygalacturonase, which indicates that both of these pectic polysac-charides are attached to other wall components through at leastshort linear chains of a-4-linked D-galacturonosyl residues. Theidentity of these other wall components is not known; in fact, RG-I and RG-II could be connected to each other in the wall, butRG-I and RG-II are clearly different and distinct molecules.The backbone of RG-I is rich in L-rhamnosyl and D-galactu-

ronosyl. Sidechains rich in L-arabinosyl and D-galactosyl residuesare attached to the backbone. This gross structural picture of RG-I is consistent with that which has been proposed for the neutralsugar rich regions of the pectic polymers (2-6, 26, 28). However,by taking into account the glycosyl residue composition andglycosyl-linkage composition of RG-I, it is concluded that the D-galacturonosyl and L-rhamnosyl backbone of RG-I could consistof as many as 500 glycosyl residues if there is only a singleuninterrupted rhamnogalacturonan chain/molecule (Tables IIand III). The length of the uninterrupted rhamnogalacturonanchains remains to be ascertained. It is also concluded that, onaverage, the sidechains are quite short, with an average length ofapproximately 6 (see below) glycosyl residues. This is in markedcontrast to earlier postulated structure for the neutral sugar richareas of pectic polymers (28). The results of the earlier studieswere misleading because the fractions examined were contami-nated with other pectic polymers, making the average length ofthe sidechains longer.The exact glycosyl residue sequence of the backbone of RG-I

remains unknown. However, the results of the investigation pre-sented here have elucidated some very interesting structural fea-tures of the backbone. The galacturonosyl residues are 4-linkedand the L-rhamnosyl residues are 2-linked with approximatelyhalf of the rhamnosyl residues further substituted at C4. The L-rhamnosyl and D-galacturonosyl residues appear to be arrangedin an orderly fashion. Most, if not all, of the unbranched 2-linkedL-rhamnosyl residues are glycosidically attached to C4 of D-gal-acturonosyl residues, and at least some of the 2-linked L-rham-nosyl residues have D-galacturonosyl residues attached to C2. Onthe other hand, most, if not all, of the branched 2,4-linked L-rhamnosyl residues are not located in RG-I in the same relativeposition as the 2-linked L-rhamnosyl residues, as the 2,4-linkedL-rhamnosyl residues are not destroyed by base-catalyzed elimi-nation of uronosyl residues (Fig. 4; Table IV). All of the branched2,4-linked L-rhamnosyl residues have D-galacturonosyl residuesattached at C2.The results presented here also give some information about the

sidechains attached to the RG-I backbone. It is concluded thatthe average length of the sidechains is small by comparing thenumber of sidechain glycosyl residues (arabinosyl and galactosyl)to the number of branched 2,4-linked rhamnosyl residues in thebackbone (Tables II and III). The 2,4-linked L-rhamnosyl residuesaccount for about 60% of the branchpoints of RG-I (Table III).The only other branched points of consequence are 3,5- and 2,5-linked L-arabinosyl residues (Tables III).The ratio of the total of L-arabinosyl and D-galactosyl residues

to 2,4-linked L-rhamnosyl residues is 6. If it is assumed that theL-arabinosyl and D-galactosyl residues are in the sidechains andthat the 2,4-linked L-rhamnosyl residues are in the backbone, thenthe average sidechain contains 6 glycosyl residues (clearly somesidechains could contain more than 6 residues and some, fewer).Furthermore, the fact that at least 14 differently linked L-arabi-nosyl and D-galactosyl residues are present in RG-I (Table III)suggests the presence of many structurally distinct sidechainsattached to C4 of the 2,4-linked rhamnosyl residues. The possiblenumber of structurally unique sidechains is increased by thepresence of three differently branched arabinosyl and at least twodifferently branched galactosyl residues (Table III). This com-plexity can be increased further by the presence of significantamounts of terminal and branched 3,4-linked galacturonosyl res-

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McNEIL, DARVILL, AND ALBERSHEIM

idues, some or all of which may be in the sidechains.The possibility that galacturonosyl residues are in the sidechains

of RG-I is indicated by the detection of 1.8 mol % terminalgalacturonosyl residues in the methylation analysis of RG-I (TableIII). Many of these terminal galacturonosyl residues must be inthe sidechains since RG-I, as isolated, contains about 500 galac-turonosyl and rhamnosyl backbone residues, whereas there existsabout 1 terminal galacturonosyl residue for every 50 backboneresidues. The presence of galacturonosyl residuesin the sidechainsof RG-I is supported further by the labeling of arabinosyl andgalactosyl residues withO-ethyl groups after removal of galactu-ronosyl residues from methylated RG-I by,B-elimination (TableV). The carbon atoms labeled withO-ethyl groups by this proce-dure indicate positions of attachment of galacturonosyl residuesin the original polysaccharide. The existence of a galacturonosylresidue on C6 of galactosyl residues (Table V) has been substan-tiated by the isolation, from methylated RG-I, of a disaccharidewith a galacturonosyl residue attached to C6 of a galactosyl residue(unpublished results).The two pectic polysaccharides of dicot primary cell walls which

have been isolated and partially characterized, RG-I and RG-II,are structurally extremely complex molecules. It is interesting tothink about why such molecules, whose function in the wall hasbeen considered purely structural, should be so complex.

Acknowledgment The expert technical assistance of David (Gollin is gratefullyacknowledged.

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1134 Plant Physiol. Vol. 66, 1980

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