A comparison of the polysaccharides extracted from dried and non-dried walls of suspension-cultured sycamore cells

A comparison of the polysaccharides extracted from dried and non-dried walls of suspension-cultured sycamore cells

Vol. 30, No. 12, pp. 3903-3908. 1991 Printedin Great Britain. 0031-9422fH 53.00+0.00 Q 1991 PergamonPress plc Phytochemisrry, A COMPARISON OF THE P...

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Vol. 30, No. 12, pp. 3903-3908. 1991 Printedin Great Britain.

0031-9422fH 53.00+0.00 Q 1991 PergamonPress plc

Phytochemisrry,

A COMPARISON OF THE POLYSACCHARIDES EXTRACTED FROM DRIED AND NON-DRIED WALLS OF SUSPENSION-CULTURED SYCAMORE CELLS* ALAN KOLLER,~

MALCOLM

A. O’NEILL,

ALAN G. DARVILL

and

PETER ALBERSHEIM~

Complex Carbohydrate Research Center and Department of Biochemistry, University of Georgia, 220 Riverbend Rd, Athens, GA30602, U.S.A. (Received in revisedfirm 7 June 1991)

Key Word Index--deer pseudoplatanus; glycosyl residue composition.

Aceraccae;

sycamore; cell walls; dehydration; extraction; polysaccharides;

Abstract-Suspension-cultured sycamore cells (Acer pseudoplotanus) were disrupted in aqueous K-Pi buffer and the insoluble residue (the cell wall) purified by extraction with organic solvents and air-dried (dry cell walls) or by washing with aqueous sodium dodecyl sulphate and stored frozen (wet cell walls). Polysaccharides solubilized from the purified wet and dry cell walls by enzymatic digestion and chemical extraction were isolated and their glycosyl-residue compositions compared. No significant differences were found in the types or yields of the polysaccharides solubilized by enzymatic digestion and chemical extraction of the wet and dry cell wall preparations. Moreover, the glycosylresidue compositions of the so-called ‘a-cellulose’ fraction that remains after extraction of the wet and dry cell wall preparations with alkali was indistinguishable from the glycosyl-residue compositions of the walls prior to extraction.

INTRODUCllON

RESULTS AND DISCU!S!3ION

The primary cell walls of plants are composed of a mixture of polysaccharides and glycoproteins [ 1,2]. It is important for cell wall structural studies that the cell wall components isolated by enzymatic digestion and chemical extraction reflect the composition of the walls in situ. In our laboratory, cell walls isolated from suspensioncultured sycamore cells have been used extensively to investigate the structures of the polysaccharides of the primary cell walls of higher plants [l J. The sycamore walls have normally been prepared by rupture of the cells followed by washing of the insoluble walls with aqueous buffers, extraction and dehydration with organic solvents, and subsequent air-drying of the walls [ 11. Alternatively, cell walls can be prepared by extracting homogenized plant tissues with aqueous buffers and detergents [2]. Isolated cell walls and extracted polysaccharides have generally been freeze-dried in our laboratory. However, we have observed that dried polysaccharides are difficult to completely re-solubilize, which suggests that dehydration of cell walls may reduce the amount and type of polysaccharides that can be solubilized by enzymatic digestion or chemical extraction. Therefore, we designed experiments to determine if the amount or type of polysaccharides that can be solubilized from cell walls is affected by dehydrating or drying cell walls.

*This is Part 33 of the series ‘The Structure of Plant Cell Walls’. For Part 32 see Puvanesarajah, V. et al. (1991) Curb. Res. (submitted). TPresent address: Franklin Pierce Law Center, 2 White Street, Concord, NH 03301, U.S.A. SAuthor to whom correspondence should be addressed.

The walls from suspension-cultured sycamore cells were either prepared by extraction of the aqueous K-Pi buffer-insoluble residue with organic solvents and airdried (dry cell wall) [l] or by washing the aqueous K-Pi buffer-insoluble residue with aqueous sodium dodecyl sulphate (SDS) [Z] and stored at -20” (wet cell wall). Approximately 2.2 g of air-dried walls were obtained from 3 1of suspension-cultured sycamore cells. A portion (1 ml) of the wet cell wall suspension (130 ml) was lyophilized yielding 18 mg of dry cell wall or the equivalent of a total of 2.3 g of dry cell wall. The glycosyl-residue compositions of the two cell wall preparations were not significantly different (Fig. 1). The wet and dry cell wall preparations also contained protein and hydroxyproline (Fig. 2). The dry cell wall preparation contained ca 50% more protein than the wet cell wall preparation. The amino acid composition of the dry cell wall showed that the additional protein was not enriched with hydroxyproline (Fig. 2). These results suggest that the dry cell wall preparation contained some cytoplasmic protein. The wet cell wall had been washed with aqueous 1% SDS, a procedure that solubilizes cytoplasmic protein that has adhered to the wall [2]. The wet cell wall suspension (27 ml, equivalent to 0.5 g dry weight of wall) and dried cell walls (0.5 g) were digested with a purified a-l,4-endopolygalacturonase (EPG) and subsequently with a j?-1,Cendoglucanase (EG), or the untreated walls (1.0 g) were extracted sequentially with trans-1,2-diaminocyclohexane-N,N,N’,N’tetraacetic acid (CDTA, an aqueous chelating reagent), then with sodium carbonate at 4” and then 23”, followed by 1M KOH at 4” and then 23”, then 4 M KOH, and finally 4 M KOH containing 4% borate (Fig. 3).

3903

3904

A.

ENDOaLUCANME

KOLLER

8OLUBlLKED I

-t

I

COTA SOLUBILIZED w

1

Fig. 1. Glycosyl-residue compositions of wet and dry cell wall preparations and polysaccharide fractions released by enzymatic treatment and chemical extraction. m =wet cell wall preparation. 0 =dry cell wall preparation. ARA =arabinosyl, RHA = rhamnosyl, FUC = fucosyl, XYL = xylosyl, MAN = mannosyl, GAL =galactosyl, GLC = glucosyl, GALA = galactosyluronic acid, and GLCA = glucosyluronic acid residues.

Digestion of the wet cell walls and dry cell wall by EPG and then EG solubilized polymeric material that accounted for 10 and 8.2% of the wall, respectively (Fig. 3). Some oligometric material would have been lost during dialysis of the EPG and EC extracts. It was assumed that these losses would be comparable for both wet and dry walls. The polysaccharides solubilized from the wet and dry cell walls by treatment with EPG were indistinguishable by glycosyl residue composition analysis (Fig. I). The glycosyl-residue composition of the EPG-solubilized ma-

et al.

terial is consistent with the presence of rhamnogalacturonan- (RG-I), a pectic polysaccharide present in the cell walls of sycamore [3] and other plants 143 that is known to be solubilized from cell walls by treatment with EPG. The material solubilized from the wet and dry cell walls by treatment with EPG also contained apiosyl, 2-Omethylfucosyl, and 2-0-methylxylosyl residues (results not shown). These glycosyl residues are diagnostic for rhamnogalacturonan-II, a pectic polysaccharide present in the cell walls of sycamore [S] and other plants [43, that is also known to be solubilized from cell walls by EPG. The polysaccharides solubilized from the EPG-treated wet and dry cell walls by treatment with EG were shown to be similar, although more arabinogalactan was released by EG treatment of wet cell walls and more xyloglucan by EG treatment of dry cell walls. The glycosyl residue composition of the EG-solubilized material is consistent with the presence of xyloglucans and arabinogalactan [I, 23, both known components of primary cell walls [6, 23. Small amounts of the pectic polysaccharide RG-I are also solubilized by treatment of the wet and dry walls with EG. The wet and dry cell wall residues remaining after treatment with EPG and EG still contained pectic polysaccharides. The non-cellulosic cell wall polysaccharides in the wet and dry cell wall residues were shown by glycosyl-residue composition analysis to contain arabinosyl (-40 mol%), galactosyl (= 20 mol%), rhamnosy1 (z 10 mol%), glucosyl (8 mol%), xylosyl(7 mol%), and galactosyluronic acid (= IS mol%) residues. These results are consistent with previous studies [3, 73 that have shown that treatment of the sycamore cell wall with EPG does not release all of the RG-I. The arabinosyl content of the wet and dry cell wall residues that had been treated with EPG and EG was enriched by ca 50% compared to unextracted cell walls (data not presented). This may be attributed to an enrichment in the cell wall residue of arabinosyl-rich hydroxyproline-rich glycoproteins [S] and of RG-I that was not solubilized by treatment with EPG. A portion of the RG-I not solubilized by treatment of the cell wall with EPG is known to contain a high proportion of branched rhamnosyl residues that are substituted with oligosaccharides composed of arabinosyl and galactosyl residues [7]. Sequential extraction of other samples of the wet cell walls and of the dry cell walls with CDTA and Na,CO, [2, 4, 1l] solubilized ca 10% of the wall (Fig. 3). These results are in contrast to studies with onion [9] and Kiwi fruit [IO] where it has been reported that 20-30% of the cell wall was solubilized by CDTA and Na,COJ. The relatively small amount of material solubilized by extracting the sycamore cell wall with CDTA and Na,CO, may be partly attributed to the fact that a portion of the polysaccharides associated with the middle lamella of these suspension-cultured cells may diffuse into the culture medium [I]. Glycosyl-residue composition analysis showed that galactosyluronic acid residues accounted for > 40 mol% of the material extracted from the wet and dry cell walls with CDTA (Fig. 1). The glycosyl-residue composition of the material extracted from wet and dry cell walls with CDTA, by Na,CO, at 4’, and by Na,CO, at 23” were similar. Sequential extraction of the CDTA-and Na,CO,treated cell wall residues with 1 M KOH at 4” and 23”. 4 M KOH, and 4 M KOH containing 4% borate solubilized similar amounts of material from the wet and from

Polysaccharides from sycamore cells PROTLIN

CONTRNT

-WET

YLL

CDTA

OC CtLL

WLL

=

-LL

WET YYILL

EXTMCTO

DRY VnLL

NaPCOS

HVOROKYPROLINE

1

m

3905

CONTENT

mDRY

Y KOtl

4U KOH

OC PROTEIN

4Y KOl4*KORATE

IN CELL mLL

ALPNA-CELLULOIC

EXTRACTI

WLL

60 -

40 -

30 -

20 -

10 -

OL I*LL

CDTA

NaPCOS

Y KOM

4U KOH

4”

KOH*BORATC

ALPHA-CELLIJLOOC

Fig. 2. Protein and hydroxyproline content of the wet and dry cell wall preparations and polysaccharide fractions released by chemical extraction. The protein content of the cell walls and cell wall extracts were obtained from the total amino acid composition determined by GC. The hydroxyproline content was determined by GC and expressed as normalized mol%. Hemi-B corresponds to the material that remained in solution when the alkaline extracts were adjusted to pH 5 with glacial HOAc.

the dry cell wall residues (Fig. 3). A precipitate (hemicellulose A) [23 was obtained when the KOH extracts of both types of walls were adjusted

to pH 5 with acetic acid.

The material that remained in solution (hemicellulose B) [2] generally accounted for >80% of the material extracted by base (Fig. 3). Glycosyl-residue composition analysis of the hemicellulose A fractions showed that the polysaccharides from the wet and dry cell wall residues were similar (Fig. 4). Glucosyl and arabinosyl residues combined to account for > 70 mol% of the carbohydrate solubilized by base that was insoluble at pH 5. Glycosyl-residue composition analysis of the hemicellulose B fractions solubilized with M KOH at 4” and 23” showed that these polysaccharides were similar to those solubilized by 4 M KOH and 4 M KOH plus borate. Xylosyl, glucosyl, arabinosyl and galactosyl residues accounted for > 85 mol% of the hemicellulose B fractions (Fig. 4), which

is consistent with the presence of xyloglucan and pectic arabinogalactan [6]. Hemicellulose B isolated from the M KOH extract of wet and dry cell walls also contained small amounts of glucosyluronic acid residues (Fig. 4) which suggests that the 1 M KOH hemicellulose B extract contained acidic arabinoxylans, another known component of primary cell walls [12]. The 1 M KOH (4 and 23”) and 4 M KOH treatments solubilized protein (Fig. 2). The protein in the hemicellulose B fractions solubilized with M KOH was not rich in hydroxyproline (Fig. 2). Hydroxyproline-poor proteins have also been isolated from the 1 M KOH extract of the cell walls of bean and partially characterized [13]. Hydroxyproline-rich proteins were present in the hemicellulose B fraction solubihzed from wet and dry cell walls with 4 M KOH (Fig. 2). The walls of suspension-cultured tobacco cells [14] and the walls of leaves and stems of red clover [15]

A. KOLLER et al.

3906

8

-WET

WILL

m

DRY WLL

7

J EPB

EG

CDTA

Fig. 3. Yields of polysaccharide

chemical extraction.

Na2COS

U KOH (4’)

M KOH (2s’)

fractions solubihzed from the wet and

4U KOH-BORATE

4U KOH

dry cell walls by enzymatic treatment and

The yields are expressed as % dry WI of the actual wt of the dry walls or calculated WI of the wet walls.

contain galactoglucomannans that can be solubilized with 4 MKOH or 4 M KOH containing 4% borate, respectively. Glycosyl composition analysis of the material solubilized from the wet and dry cell wall residues of sycamore with 4 M KOH and with 4 M KOH containing 4% borate (Fig. 4) showed that mannosyl residues accounted for < 5 mol% of the carbohydrate. The walls of suspension-cultured sycamore cells either do not contain galactoglucomannan or are not rich in this polysaccharide. Arabinosyl and galactosyl residues accounted for >55mol% of the carbohydrate solubilized with 4 M KOH containing 4% borate (Fig. 4). In addition, the material solubilized with 4 M KOH and 4% borate contained significant amounts of protein and hydroxyproline (Fig. 2). which suggests that a portion of the hydroxyproline-rich glycoprotein (HRGP) is solubilized by extraction with alkaline borate. Hydroxyprolinc-rich glycoproteins have also been shown to be present in the alkaline-borate extract of the cell walls of potato [16]. The HRGP solubiliLed with 4M KOH and with 4 M KOH containing 4% borate may correspond to the so-called salt-extractable HRGP that has been proposed not to have been cross-linked within the cell wall matrix [ 171. The mechanism by which HRGP is solubilized by alkali and by alkaline borate is not known. The non-cellulosic glycosyl residue compositions of the residue that remained after extraction of both the wet and dry cell walls with CDTA and alkali (‘a-cellulose’ residue) were virtually identical to the glycosyl residue compositions of the walls prior to chemical extraction (cf. Figs 1 and 4). The wet and dry walls contained 31.6 and 29.4% cellulose, respectively, before chemical extraction. The wet and dry ‘z-cellulose’ residues contained 36.1 and 33.2% cellulose, respectively. Glucosyl residues accounted for >40mol% of the carbohydrate in the hemicellulose A fractions isolated from the M KOH extract (Fig. 4) and the 4 M KOH extract (data not shown). This

result suggests that only a portion of the non-cellulosic cell wall polysaccharides can be solubilized with the chemical reagents used regardless of whether the walls were prepared ‘dry’ or ‘wet’. The resistance of cell wall polysaccharides and glycoproteins to complete extraction with aqueous chelating reagents and alkali is consistent with a model of the cell wall where the various non-cellulosic wall polymers are physically entrapped in the cellulose fibers [ 18, 193. This study has shown that there are no qualitative differences in the types of polysaccharides and glycoproteins released by enzymatic treatment or chemical extraction of wet and dry walls. Furthermore, the amounts of polysaccharides and glycoproteins that can be solubilized from wet cell walls are quantitatively similar to the amount of polysaccharides and glycoproteins that can be solubilized from dry ceil walls so that either method of preparation can be used for cell wall studies.

EXPERIMENTAI.

Preparation

of cell wull marerfal.

suspension-cultured were maintained M-6 medium [I].

Cell walls were pfepd from

sycamore (Acer pseudoplatanus) cells that

at 28’ in the dark in shake culture on modified The sycamore cells (6 1) were harvested after IO

days of culture and washed sequentially pH7.0

(5 I), and cold 0.5 M K-Pi,

suspended in cold 0.5

M K-Pi

with cold 0.1 M K-Pi,

pH 7.0, (5 I). The cells were

(I vol.) and subjected to 1000 psi

nitrogen for 30 mm in a Parr pressure bomb (Parr Instrument Co, Moline,

IL). The suspension was rapidly

conical flask

(1 1).The disrupted

relcascd into a

cells were centrifuged at 1000 g

for I5 min at 4’ and the pellet was washed with cold 0.5 The pellet containing

M K-PI.

the cell walls was resuspended in cold

water (4 vol.) and divided mto two equal vols, both of which wcrc centrifuged. One pellet was suspended in CHCI,-MeOH vol.) and

stirred

for 30 min.

(I

: I,

5

The solvent was removed by

Polysaccharides from sycamore cells

Fig. 4. Glycosyl-residue compositions of the polysaccharides solubilized from CDTA- and Na,CO,-treated wet and dry cell wall residues by treatment with 1 M KOH and with 4 M KOH containing 4% borate and of the remaining ‘z-cellulose’ residue.

filtration through scintered-glass, the residue washed with Me&O (5 vol.), and the cell walls air dried (yield 4.98g). The second cell wall pellet was resuspended in cold aq. 1.O% SDS (1 vol.) and stirred for I6 hr. The walls were collected by centrifugation, washed with (5 x 500 ml) water, dialysed (12ooOmol wt cutoff membrane), and the suspension stored frozen at - 20”. Starch was removed from the organic solvent- and SDSwashed cell walls by treatment, for 48 hr at 23”. with a-amylase (20000 units, Bacillus subtilis Type Ha, Sigma). The cell wall material gave no reaction for starch when treated with Iz/K1. The organic solvent-washed cell wall preparation was then washed sequentially with H,O (5 x 700 ml), with MeGH-CHCI,

3907

(I : 1) (5 x 400 ml), and with Me&O (3 x 300 ml). The residue was air-dried (final yield of dry wall 2.2 g). The SDS-treated cell walls were washed with H,O (4 x I I) and centrifuged. The pellet was resuspended in H,O (130 ml) and a portion (I ml) lyophilized (yield I8 mg of dry cell wall). The wet cell wall suspension contained the equivalent of 2.34 g of dry wall. The wet cell wall suspension was stored frozen at - 20”. Enzynurtic digestion of the cell walls. Dry cell walls (0.5 g) and an aliquot (27.1 ml, equivalent to 0.5 g dry wt) of wet cell wall slurry were separately suspended in 50mM NaOAc, pH 5.2 (50 ml), containing 0.01% thimerosal. The suspensions were treated for 24 hr at 33” with an a-1.4endopolygalacturonase (EPG, 3.8 units; 1 unit of EPG releases 1 pmol reducing sugar min- ’ from a 1% soln of polygalacturonic acid at pH 5.2 and 30”) purified from Aspergillus niger (Gollin, D. G.. unpublished results of this laboratory). The suspensions were centrifuged and the pellets washed with deionized water (50 ml), which was pooled with the supematants. The cell wall pellets were retreated with EPG for 24 hr. The combined EPG-solubilized components were pooled, filtered (GF/A, Whatman), coned to 50 ml. dialysed (I2 000 mol wt cutoff membrane) at 4” for 72 hr. and lyophilized. The EPG-treated wet and dry cell wall residues were suspended in 50 mM NaOAc, pH 5.2 (50 ml), containing 0.01% thimerosal and treated for 24 hr at 33” with 6.7 units of F-1,4endoglucanase (EG) purified from Trichoderm uiride [20]. [Note: 1 unit of EG releases I pmol reducing sugar hr _ ’ from a 0.002% solution of carboxymethyl cellulose at pH 5.2 and 30”.] The suspensions were centrifuged and the pellets washed with deionized Ha0 (50 ml), which was pooled with the supernatants. The residues were re-treated with EG for 24 hr. The combined EG-solubilized components were pooled, filtered (GF/A), coned to 10 ml, and passed through a DEAE-Sephadex column (I.1 x 17 cm) equilibrated with 20 mM K-Pi. pH 7.3. Frs containing anthrone-positive material were pooled, coned to 3 ml, desalted on a G-15 Sephadex column (1.5 x 26 cm), dialysed, and lyophiliaed. Chemical extraction ofrhe cell walls. Cell walls were extracted chemically according to published procedures [Z]. Wet cell wall material (54.2 ml, equivalent to I g of dry cell wall material) was pelleted by centrifugation. The wet pellet and dry cell wall material (I g) were separately extracted for 6 hr at 23’ with 0.05 M CDTA, pH 6.5 (100 ml). The cell wall residues were pelleted and the supernatants stored at 4”. The cell wall residues were re-extracted for 3 hr at 23” with 0.05 h4 CDTA, pH 6.5, and the supematants collected and pooled with the first CDTA extracts. The CDTA sol frs were filtered (GF/A), coned to 30 ml, dialysed (12000 mol wt cutoff membrane) at 4” for 72 hr. and lyophilized. Wet and dry cell wall residues from the CDTA extractions were extracted for I6 hr at 4’ with 50 mM Na,CO, containing 10 mM NaBH,, pH 10.8 (lOOmI). The wall residues were pelleted and the supematants filtered (GF/A). The filtrates were adjusted to pH 5 with glacial HOAc, coned to 30 ml, dialysed for 72 hr at 4”. and lyophilized. The remaining residues of the wet and dry cell wall preparations were then extracted for 3 hr at 23” with 50mM Na,CO, containing 10 mM NaBH,, pH 10.8 (100 ml). The wall components extracted with CDTA, Na,C03 at 4”, and Na,CO, at 23” were kept separate. The wet and dry cell wall residues remaining after Na,CO, extraction were extracted under Ar with M KOH (100 ml) containing 10 mM NaBH, for 2 hr at 4”. The wall residues were pelleted, the supernatants filtered (GF/A), adjusted to pH 5 with glacial HOAc, and coned to 30 ml. The extracts were dialysed for 72 hr at 4”, and lyophilized. The wall residues were re-extracted under Ar with M KOH (100 ml) containing IO mM NaBH, for

A. KOLLER et al.

3908

2 hr at 23”. The solubilized material was filtered, neutralized. coned, dialysed, and lyophilized. The 4 and 23” KOH extracts were kept separate. The remaining wet and dry cell wall residues were then extracted under Ar with 4 M KOH (100 ml) containing 10 mM NaBH, for 2 hr at 23”. The solubilized material was filtered, neutralized, coned, dialysed, and lyophilized. Finally, the wet and dry cell wall residues were extracted under Ar with 4 M KOH (100 ml) containing 4% boric acid (w/v) for 2 hr at 23”. The solubilized material was filtered, neutralized, coned, dialysed, and lyophilized. The cell wall residues remaining after chemical extraction (‘z-cellulose’) were suspended in 50 ml H,O, neutralized with glacial HOAc, dialysed for 72 hr. and lyophilized. Estimation of cellulose content in unextracted cell walls and in the ‘z-cellulose’ residue. Suspensions of the cell wall material (3 mg) in 2 M TFA (2 ml) were heated at 121” for 1 hr. The suspension was pelleted by centrifugation, the supernatant removed, and the pellet washed with MeGH (3 x 2 ml). The pellets were suspended in 67% HzSO, (2 ml), and stirred for 18 hr at 23,‘. The hexose content of the soln was determined colorimetritally [21] using D-glucose as standard. Determination

of the t~lycosyl

compositions

of the cell

wall

The glycosyl residue compositions of the cell wall extracts were determined by analysis of the trimethysilyl derivatives of the methyl ester methyl glycosides by GC Cl]. preparations

and cell wall extracts.

Determination cell wall extracts.

ofthe amino acid composition

of the cell walls and

The amino acid compositions of wet and dry cell walls and cell wall extracts were determined by GC using a modification (M. McNeil, Colorado State University, pen. comm.) of a published procedure [22]. Cell walls and their extracts (1 mg) were suspended in 6 M HCl(300 ~1) and heated under Ar for 18 hr at 110”. Each sample was coned to dryness and the residue dissolved in 0.01 M HCI (1 ml) before being passed through a column (0.5 x 5 cm) containing Dowex H + resin. The column was washed with 0.01 M HCI (3 ml) before the amino acids were eluted with 3.5 M NH,OH (3 ml). The eluate was coned to dryness, and the residue treated for 20 min at 120’ with isobutanol containing 3 M HCI (300 ~1). The isobutyl esters were coned to dryness and a soln of the residue in a mixt. of EtOAc (50 ~1) and heptafluorobutyric anhydride (25 ~1. HFBA, Aldrich Chemical Co.) was heated for 5 min at I SO”.The n-heptafluorobutyryl isobutyl esters were coned to a syrup. The syrup was dispersed in EtOAc (25 ~1) and Hc,O (25 ~1) and the mixt. heated for 3 min at 150” to acetylate histidine residues. The samples were cooled and EtOAc (200 ~1) was added. Portions (1 ~1) of this soln were analysed by CC on a 30 m x 0.25 mm fused silica DB-I column using the following oven temp. program: 100’ for 4 min. followed by a temp. increase of 8”min _ ’ to 240”. The R, and the molar response factors of the individual amino acids were determined by GC analysis of a derivatized mixt. of ammo acid standards (Pierce). The order of elution of the amino acid denvatives was as described [223.

Acknowledgements-This research was supported in part by Department of Energy grant no. DE-FG-09-87ER13810, as part of the USDA/DOE/NSF Plant Science Centers Program. This research was also supported by Department of Energy grant no. DE-FG-09-85ER13426. The authors thank Karen Howard for editorial assistance.

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