Synthesis Extracellular Polysaccharide Suspensions Acer ...Plant wall polysaccharides are intimately con-nected with maintenance of cellular form and con-trol of cell growth (6). Manypathogenic

Synthesis of Extracellular Polysaccharide by Suspensionsof Acer Pseudoplatanus Cells 1.2

Gretchen E. Becker, Paul A. Hui,3 and Peter Albersheim 4The Biological Laboratories, Harvard University, Cambridge, Massachusetts

Introduction

Plant wall polysaccharides are intimately con-nected with maintenance of cellular form and con-trol of cell growth (6). Many pathogenic fungi andbacteria attack plants with enzymes that degrade thewall polysaccharides. Yet little is known about thestructure and synthesis of the noncellulosic poly-saccharides, which constitute as much as 75 % ofthe cell wall (10).

Study of wall polysaccharide has been hinderedby the use of heterogeneous wall extracts. For ex-ample, pectin is solubilized with boiling water, pro-topectin with versene or dilute acid, and hemicellulosewith alkali. These extraction procedures reduce thedegree of polymerization of the polysaccharides andalso alter the structure in various ways. It wouldbe advantageous to isolate and characterize plantwall polysaccharides without the necessity of ex-traction.

Sycamore cambial cells grown in suspension possessmany desirable attributes for the study of polysac-charide structure and synthesis. Thornber andNorthcote have published data on the compositionof cambium cells in the intact sycamore (27) andLamport has studied a wall-specific hydroxyproline-rich protein in sycamore cell suspensions (15). Oneof the chief assets of this system is its friability; thecells in suspensions can be handled almost like bac-terial systems. The sycamore cells grow rapidlyin large amounts and can be plated or transferredby pipette. Another advantage is that by continuoustransfer one selects for rapidly growing cells so thatthe population in the logarithmic phase of growthis more uniform than cells of plant tissues. Further-more the supply of metabolites is readily controlled,and every cell has direct access to the externalmedium containing these metabolites. When desir-able the cells can be grown on a defined medium.

Although various wall polysaccharides may turnout to be important in controlling cell growth, we

Received Mar. 4, 1964.2 Supported in part by United States Public Health

Service Grants GM 07694 and GM 09758.3 Present Address: Department of Agricultural Chem-

istry, Swiss Federal Institute of Technology, Zurich,Switzerland.

4 Present Address: Department of Chemistry, Uni-versity of Colorado, Boulder, Colorado.

913

have concentrated on the galacturonic acid containingpolymers, since it is known that the carboxyl groups ofgalacturonic acid possess the ability to control cellgrowth (6). This paper reports the presence of ex-ternal uronide containing polysaccharides in the cul-ture fluid of sycamore cambial cells growing in sus-pension. The composition of these polysaccharides isdescribed, as well as studies on the incorporation ofglucose-C14 and various methyl donors into the methylgalacturonate residues. These polysaccharides arecompared to normal wall polysaccharides.

Materials and Methods

Growth of the Cells. The sycamore cambial cells(Acer pseudoplatanus, the English sycamore, is aspecies of American maple) were kindly supplied byDerek T. A. Lamport of RIAS, Baltimore, Md. Thecells have been cultured in a modification of the M-6medium of Torrey and Shigemura (28) with 2,4-dichlorophenoxyacetic acid (2,4-D) at a concentra-tion of 9 X 10-6 M. The medium contains sucrose(40 g/liter), yeast extract (1 g/liter), 2,4-D, andthe following minerals at pH 5.5; the final concentra-tions in mg/liter are given in brackets: Ca(NO3)2.4H20 [242], KNO3 [85], KCl [61], MgSO4. 7H20[42], KH2PO4 [20] and freshly prepared FeCl3 [25].

The cells were grown at 23° with mild rotation(80 cycles/min) under conditions of interrupted dark-ness. Fernbach flasks (2800 ml) containing 1100 mlof medium were used. The cells were subculturedevery 11 to 12 days by transferring with a sterilegraduate 100 ml of cells from the old medium to 1000ml of fresh medium.

For the growth curve 10 ml samples were removedaseptically on appropriate days, filtered with the aidof a small conical buchner funnel and washed with5 ml of distilled water. The cells were transferred toaluminum planchets and weighed, then dried to con-stant weight in an oven and the dry weight deter-mined. The filtrate was made 70 % with respect toethanol and allowed to stand overnight at -20°.The ethanol insoluble material was collected bycentrifugation, washed once with 80 % ethanol anddissolved in 2 ml distilled water. The solution wasrecentrifuged and the soluble portion decanted.Aliquots of the water soluble portion were tested forgalacturonic acid by a modified carbazole method (5).

Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

PLANT PHYSIOLOGY

Isolation of Polysaccharide. The cells from 12-day-old cultures were removed from the culture me-dium with the aid of a coarse sintered glass funnel.Microscopic examination showed that no cells passthrough the funnel. The filtrate was concentratedunder reduced pressure, made 70 % with respect toethanol, and allowed to precipitate overnight at -20'.The 70 % ethanol insoluble material was collectedwith the aid of a buchner funnel, redissolved in hotwater and reprecipitated with ethanol (70 %). Thisprocedure was repeated a third time, after which theprecipitate was washed with acetone and dried underreduced pressure over P205. The cells were homog-enized in 1.5 N sodium acetate buffer pH 4.4, andthe walls separated by centrifugation for 5 minutes ina clinical centrifuge. The walls were then groundtwice more with distilled water, 3 times with acetone,and finally dried under reduced pressure over P20O.The dried walls were treated in the same manner asthe dried external polysaccharide.

Analysis of Polysaccharide. The polysaccharidewas hydrolyzed in 1 N H2SO4 in a sealed tube (25mg material per 2 ml acid) by heating for 1 hour at1000. The sulfate ions were then removed by passingthe solution through a Dowex-1 column (20-50 mesh,carbonate form). The hydrolysates were concen-trated by distillation under reduced pressure to 2 to4 ml, and transferred into volumetric flasks, aliquotsof which were removed for analysis.

The neutral sugars were separated by descendingchromatography for 30 hours on Whatman No. 1 pa-per with ethyl acetate-pyridine-water (8: 2: 1) assolvent. All quantitative chromatogranms were spot-ted with micropipettes. Sugar test spots on qualita-tive chromatograms were detected by dipping in theamino-biphenyl reagent of Gordon et al (11). Afterchromatographic separation the indlividual sugarswvere located on the paper, cut out, and eluted withwater. Hexoses were estimated by the method ofMtorris (19), pentoses by a modifiedI anthrone testdescribed by Bailey (4).

In order to avoid the contribution of neutral sugarsto the test for uronic acid, a separate portion of theacid hydrolysate was analyzed. Such hydrolysateswere adjusted to pH 10 with NaOH and absorbedon Dowex-1 (20-50 mesh, preequilibrated with 0.02 Mformic acid). Neutral sugars were washed throughwith 0.01 M formic acid, and the galacturonic acideluted with concentrated formic acid. The eluatewas then concentrated under reduced pressure and theamount of uronic acid estimated bv the carbazolemethod of Bitter (5).

The methyl ester content was obtained directlyfroml the dried polysaccharide by measuring theamount of methanol liberated upon saponification for30 minutes with NaOH, as described by Boos (7).

The nitrogen and phosphorous determination wascarried out by Stephen M. Nagy of the MicrochemicalLaboratory of the Massachusetts Institute of Tech-nology.

In Vivo Experimizenits. Cells for the in vivo ex-

periments were collected in the logarithmic phase ofgrowth with the aid of a coarse sintered glass funnel.Care was taken that the cells were not allowed todry. The M-6 medium was washed from the cellson the funnel with a sterile salt solution containingthe M-6 salts, 2,4-D and 0.1 M KCl to bring thesolution to an appropriate osmotic concentration.The implements and solutions were sterile to reducecontamination. The cells were incubated at roomtemperature with shaking, at a concentration of 1 gcells/ml of solution containing the salts plus thevarious substrates and radioactive compounds men-tioned below. At the end of the incubation the cellswere collected by filtration or by centrifugation andlfractionated as below.

The external polysaccharide was precipitated witlhalcohol, collected by centrifugation and washed oncewith 80 % alcohol, redissolved in water and(l made toa given volume.

The cells were dlisrupted with a Fisher hanclhomogenizer. The internal cold water soluble, hotwater soluble and residual fractions were obtained,and in the case of the experiments with glucose-C14the fractions hydrolyzed and the galacturonic aci(lpurified by a modification (3) of the procedlure ofJansen et al (13). For the purposes of simplicitvthe data from the cold water soluble fractiIon havenot been use(l and the data from the hot vxatersoluble and versene extracts (13) have been combinedand presentedl together as wall polysaccharides.

Radioactive Comtpouinds and Determninationis.Radioactivity of samples dried on aluminum planclhetswas determined on a Tracerlab thin windowv Geiger-Miuller counter wvith automatic sample changer.Determination of amount of formaldehyde fed wN-ascarrie(d out by scintillation counting. Half mlaqueous samples kvere counted in 15 ml of a solventcontaining dlioxane, anisole, and dimethoxyethane(6: 1: 1) (8). The solvent included 1 part in 25of Liquifluor, a commercial phosphor purchase(d fromlPilot Chemical Company, Watertown, Mass. Radio-activity was determined in a Tri-Carb Liquid Scintil-lation Spectrophotometer Model 500 D (Packard In-strument Company).

The specific activity of the galacturonide resi(luesof the fractions wvas obtained by determining boththe amount of purifiecl galacturonic acid present [bya modified carbazole method (5)], ancl the amount ofradioactivity in a similar sample. All carbazole deter-minations andi measurements of radioactivity x-erecarried out in triplicate. When the incorporationof radioactivity into saponifiable methylI esters wasbeing measured, the galacturonic acid Nvas not purified,but the radioactivity of a sample was determinied oIn aplanchet, allowed to saponify for at least 5 hoursin the presence of ammonia vapors, and the radio-activity again determined. Saponification by am-mionia vapors is sufficient to hydrolyze the methylesters of galacturonic acid (13).

Uniformly labeled glucose-C14 with specific activ-ity 30 mc/mni (Calbiochem) xvas used in experiment

914


BECKER ET AL.-SYNTHESIS OF EXTRACELLULAR POLYSACCHARIDE

1, with specific activity 200 mc/mM (New EnglandNuclear) in experiment 2. Formaldehyde-C14H3(New England Nuclear) with specific activity 1.0was used in experiment 3. S-Adenosylmethionine-C14H3 of specific activity 10.0 mc/mM (Tracerlab),C14H3-Methionine of specific activity 10 mc/mM(New England Nuclear), and C14H,O with specificactivity 10 mc/mM (New England Nuclear) wereused in experiments 4 and 5.

Results

Growth of Cells. Lamport has described thegrowth of the sycamore cells in a coconut milk me-dium (16). Growth in the M-6 medium is similarand need not be discussed in detail. The cells growrapidly and in friable suspension. A growth curveis illustrated in figure 1. The cells have a doublingtime of around 3 days. Different samples vary from2.5 to 3.5 days. Figure 1 also shows that during the

1615 0x14

8~~~~~~~~/

1312 0

10 1

.7U,

Z6/wp5 s

4I

50 m

x

2/

* ~~~oDRY WEIGH4T0 WET WEIGHT

I K~~~~~POLYSACCHARIDE

I 3 5 7 9 11 13 15 17 19 21DAIS

FIG. 1. Growth curve. Polysaccharide was estimatedby measuring the amount of anhydrouronic acid (AUA)per sample. One relative unit equals 5 mg dry weight,50 mg wet weight, 100 jtg AUA, all per 10 ml sample ofsuspension.

Table I. Relative Composition of Externaland Wall Polysaccharide

The external and wall polysaccharide was hydrolyzed1 hour at 100° in H2SO4.

Component External Wallpolysaccharide* polysaccharide**Galacturonic acid 1.0 1.0Methanol 1.0 1.0Degree of

esterification 97 %o 98 %Galactose 0.9 0.7Glucose 1.0 1.1Mannose 2.1 0.4Xylose 1.0 0.7Arabinose 3.1 2.3Yield of hydrolysis 50 % 30 %* Relative molar ratio. Galacturonic acid = 32 AM/

100 mg polysaccharide.** Relative molar ratio. Galacturonic acid = 16 AM/

100 mg polysaccharide.

logarithmic phase of growth the production of theextracellular polysaccharide parallels the growth ofthe cells. At the end of the logarithmic phase thewhole picture of growth becomes more complex; thecells tend to differentiate or enlarge.

Composition of External Polysaccharide. Theexternal polysaccharide contains only traces of phos-phorus and 2 % nitrogen. The analysis is summar-ized in the data of table I. The amount of galactose,glucose and xylose is equivalent to that of galactur-onic acid. About twice as much mannose is presentand about 3 times as much arabinose. These resultswere obtained by acid hydrolysis. Enzymatic diges-tion by partially purified Pectinase (Nutritional Bio-chemical Company) followed by acid hydrolysis ofthe residue yields essentially the same results.

lS0

E

10

0

E

4

49

06

E

MINUTESFIG. 2. Incorporation of formaldehyde-C14 into methyl

esters of external and wall galacturonides.

915


~~~~~~~PLANTPHYSIOLOGY

The walls of the sycamore cells were hydrolyzedin the same manner as the external polysaccharide andanalyzed for sugars. The data of table I include thesugar composition of the walls. It can be seen thatthe composition of the external polysacchari-de is quitesimiilar to the normial cell wall polysaccharides ofthese cells. Both polysaccharides contain the samearray -of sugars, with a predominance of arabinose.The wall hiowever yields less manno,se. The yieldof hydrolysis of the walls is only 30 % as opposed to50 % for the external polysaccharide, but this dif-ference can be accounted for by the wall cellulose,wvh icli remiains unhydrolyzed under these conditions.The unhydrolyzed portions have not been analyzed in(leta.il, but are known to contain a significant amiountof proteini.

Preliminary evidence obtained by chromatographyon DEAL cellulose, using the method of Rosik et al.(22). indicates that the external polysaccharide isheterogeneous and that it can be separatedl intoa frac-tionis essentially free of uronic acid and those eni-richied1 in uroniile.

During active growth the cells synthiesize externalpolysaccharide equivalent to approximiately 50 % ofthe dry weight of the cell walls. Of this, 6.2 % isgalacturonic acid. The amounts of galacturonic acidin the external polysaccharide andI in the wall frac-ti-ons of the sycamore cells are presented in the dataof table II. The galacturonic acid content is com-l)aredl to that of oat shoots and of bindweed callustissue, also growing in the modified M-6 mediumi con-taining 10-6 At 2,4-D. The amount of galacturonicacidl in the wall is similar in all cases. There isabout one-third as much internal cold water solubleg.alacturonide in the cell cultures, but this is morethan miade up for by the amount of the external galac-turonide residues in the sycamiore cultures. Thebincdweed callus was not tested for the extracellularpolysaccharide, as the tissue was highly compactand unsuitable for fu-rther analysis.

Highly purified pectin transeliminase (2) readilyaittacks the polysaccharide. Because purified pectintranseliminase is highly specific for methylatedg-:)oalacturonides and will not attack unesterified galac-turonic acid, the polysaccharide must contain esteri-fled galacturonic acid. Analysis of the polysac-chiaride does indeed show that up to 97 C0 of thecarhoxyl groups are methyl esterified (table I).

In Vivo Synthesis of Polysaccharide. Glucose-

Table II. Anhydrouronic Acid Contenit

InternalWall water Externalpoly- soluble poly-

saccharides poly- saccharidessaccharides

Percent of wall dry wtOat seedlings 3.2 .21 ..Bindweed callus 3.8 .07Sycamore suspension 3.0 .06 23'

C14 is known to be a precursor of the polysaccharidegalacturonide residues in various whole plants (1, 25,26). Sycamore cells in suspension were incubated

for 5 h-ours in the presence of uniformly labeled glu-cose-C14, the cells were collected, fractionated, and

the activity in the galacturonic acid residues deter-mined. Laheled glucose is taken up very rapidly bythe sycamore cells. In experiment 1, 1.7 /.tmole ofgluc,ose was fedI to 40 g (wvet) of cells. After 5hiours 95 % of the counts were removed from themiedium. In experiment 2, 0.25 tLmole of glucose wvasfed to 200 g of cells, and 100 % of the counts wvere

taken up in 5 hours.

The label of the glucose fed is found in the galac-turonic acid residues obtained from the wall and ex-

ternal polysaccharides (table III). The galac-turonicacid residues of the external polysaccharides hiave a

specific activity 200 times that of the wall polysac-charides. Thus the external polysaccharides areeither synthiesized fromi a precursor pool (lifferentfrom that of the wall polysaccharides, or from the

same p-ool and are simiply not diluted by col-d polysac-chiaridles present in the wvall at the beginining of theexperimient.

A similar (lifference in specific activi-ty betweenithe galacturoinic acidI residues isolated fromi the ex-ternal and wall polysacchiarides is found when theincorporation of formaldehyde is mieasured. For-

mialdehiyde is kniown to be a good precursor of themiethyl ester of galacturonic acid residues (29). Theexperimients with formaldehyde were similar to theones utilizinig glucose, except that the galacturonicacid was not extensively purified, as the amount of

saponifiable counts wNas measured instead of totalradioactivity. Fifty g of cells were incubated insalt solutioni in the presence of 37 mic of formialde-

hyde-C'4. Five g samiples were taken at 30-miinuteintervals andc analyzed. The curves of figure 2 shiowthe results of such a typical time course experimient(experimient 3). It is clear that although the wallpolysaccharides contain the bulk of the activity, theexternal polysacchiaride hias a miuchi higher specificactivity (cf. also table V).

Souirce of the Methyl Ester of the GalacturonideResidues. In an attempt to ascertain the imimediate

precursor of the nmethiyl ester of pectin, experimientswere carried out using formaldehyde-C1', methionine-C14H3, known to he good precursors, anid S-adenosyl-

Table III. Specific Activity of Galacturonic AcidResiduies fromt Polysaccharides of Cell Sus-

pensoions Inicubated with Glucose-C14

Cells were washed and incubated for 5 hours in a saltsolution with glucose-C14 ; 51 Atc were fed to 40 g cells inExp. 1 and 50 juc were fed to 200 g cells in Exp. 2.

Wall

polysaccharideExternal

polysaccharide

dpm/mg anhydrouronic acidExp. 1 675 178,000Exp. 2 370 79,000

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Table IV. Incorporation of the Methyl Group from Various Donors into Galacturonide Methyl Esters (Experiment 3)The cells were incubated for 2 hours in a salt solution containing labeled precursor.

External galacturonide Wall galacturonideresidues* residues*

Methyl donor Total Saponifiable Total Saponifiableactivity activity activity activity

incorporated incorporated incorporated incorporated

dpm dpm dpm dpmS-adenosylmethionine-C14H, 16,000 730 14,000 240Methionine-C14H3 20,000 18,000 44,000 27,000Formaldehyde-C14 16,000 15,000 49,000 28,000* Data are corrected for variations in the number of counts fed, and are based on a theoretical 106 dpm introduced into

each flask.

methionine-C14H3, known to mediate between me-thione and methyl acceptors in some systems (12).

In these experiments 5 g of cells were washed andincubated for 2 hours with the labeled compounds.After reaction the cells were collected and the saponi-fiable activity in the various fractions determined.

The results of one experiment, experiment 4, canbe seen by the data of table IV. In this experimentthe following amounts of radioactive precursors werefed: 1,700,000 dpm of CH9O, 1,300,000 dpm of S-adenosylmethionine, and 4,900,000 dpm of methionine.The data of table IV are corrected for variationsin the number of counts fed, and are based on atheoretical 106 dpm fed to each of the 3 flasks. Itis clear from the data that although the label ofS-adenosylmethionine is incorporated into both thewall (16,000 dpm) and external (14,000 dpm) poly-saccharides, essentially none of the activity is saponi-fiable, and must therefore reside in some compoundsother than galacturonide methyl esters. Methionineand formaldehyde, on the other hand, are good pre-cursors of both the wall and external polysaccharidemethyl esters. Not only do the cells take up a largeproportion of the counts, but an equally large shareis saponifiable in ammonia vapors.

The data of table IV illustrate another point. Inthe external polysaccharide the methyl donors arevery specific precursors of the methyl ester groups.In the case of methionine 89 % and formaldehyde93 % of the total activity in the external polysac-charide is methyl ester. In other experiments usingformaldehyde as methyl donor up to 97 % of theactivity is saponifiable. In the wall, however, lessthan 60 % of the activity incorporated is found inthe form of methyl esters. Thus the methyl groupsare transformed into a greater variety of compoundsin the wall.A competition experiment (experiment 5) was

conducted in order to obtain further information con-cerning the precursor of the methyl ester group. Me-thionine and formaldehyde were tested for their abilityto inhibit the incorporation of each other. The washedcells were first incubated for 30 minutes in an excess(4 X 10-3 M) of the unlabeled compound, and then foran additional 2 hours in the presence of both excess un-

labeled compound and 2 X 10-5 M labeled precursor.When methionine was the cold diluent, 8 X 10-3 MD-L-methionine was used, so that the proper concen-tration of the L-isomer was present. The inhibitionof incorporation of the label by the unlabeled com-pound was measured after 2 hours. The results indi-cate that both methionine and formaldehyde inhibitthe incorporation of each other into the methyl estersof galacturonic acid, methionine being a slightly moreeffective inhibitor (table V).

Discussion

The amount of galacturonic acid in the walls ofthe sycamore cells is comparable to the amount foundin walls of bindweed callus tissue and oat shoots,3 to 4 % of the dry weight (table II). This amountdiffers from that reported for similar sycamore cellcultures by Lamport and Northcote (14). They re-ported 15 % wall pectin. They estimated, however,the weight of 70 % alcohol insoluble material afterextraction from the walls with versene, that is, thetotal polysaccharide plus any other substances pres-ent in the fraction (27). The data of table II rep-resent an estimate for the amount of uronic acid ineach fraction. It has been found in Avena coleoptilesthat 23 % of the hot water soluble fraction is actuallygalacturonic acid (1). We find that only 6.2 % ofthe external polysaccharide of sycamore cells isgalacturonic acid. Considering this difference indefinition, Lamport and Northcote's and our own re-sults are quite comparable.

The sycamore cells produce extracellular polysac-charides which can be isolated from the medium bysimple ethanol precipitation. The relative composi-tion is shown by the data of table I. It is qualita-tively similar to the composition of the wall polysac-charides obtained from the cambial region of a syca-more tree in a study by Thornber and Northcote(27). However, cambial cells of the intact treeyield a predominance of xylose (27), while the ex-ternal and wall polysaccharides from the cells insuspension possess a predominance of arabinose.The proportions of the other sugars are similar in allcases, except for a somewhat greater amount of

917


918 PLANT PHYSIOLOGY

Table V. Cotmipetittoio Between Mlethionine and Formaldehyde as Precursors of GalacturonidcMethyl Esters (Experiment 5)

Cells were preincubated for 30 minutes in salts ± 4 X 10-3 M cold precursor, then 2 x 10- M labeled precursor wasadded to the cells incubated for an additional 120 minutes.

Wall polysaccharide External polysaccharidePreincubation Labeled precursor Specific Reduction Specific Reduction

activity activity

dpm/mg dpm/mgSalts 8,400 200,000

Methionine-C14H3 41 % 75 %oSalts + formaldehyde 5,000 51,000

Salts 8,800 180,000Formaldehyde-C14 34 % 42 %

Salts + methionine 5,900 110,000

mannose in the external polysaccharides than in theother 2.A comparison of the actual amounts of the various

sugars shows that the external polysaccharides aremuch richer in noncellulosic polysaccharides thanthe walls of either the intact tree or the cells in sus-pension. The recovery of monosaccharides afteracid hydrolysis is greater from the external than fromthe wall polysaccharides of the cells in suspension.Both facts indicate that the external polysaccharidesare enriched in hemicellulose-like components andpoor in cellulose.

The amount and degree of esterification of thegalacturonide residues in the external polysaccharidesare similar to those of normal wall polysaccharides.We feel that this is important. Future studies ofthis system with regard to the structure and synthesiswill be more significant if the external polysaccaridesare normal unattached wall polysaccharides.

The high degree of esterification found in boththe wall and external galacturonic acid polysaccharideresidues (table I) is interesting in view of the ex-treme friability of the cells. The galacturonates areconsidered the cement which holds cells together.If the carboxyl groups of the uronic acids are highlyesterified, there would be less possibility of ionicallycross-linking the polysaccharide chains which con-tain such units. The absence of cross-linking is likelyto account in part for the tendency of these cells toseparate in solution.

Determination of the degree of esterification wasdone directly either on the external polysaccharides orthe cell walls, that is, without prior extraction. Anyoverestimation of the galacturonic acid content due toneutral sugars would tend to reduce the value ob-tained for percent esterification. Thus the high de-gree of esterification probably represents an accuratepicture of the native polysaccharicles.A comparison of the composition of the external

and wall polysaccharides seems to indicate that theexternal polysaccharides are synthesized in a normalmanner within the wall and then excreted into themedium. The results give no indication whether

the polysaccharides are synthesized internal, exter-nal, or within the cell membrane. This point couldhe important. If growth is controlled in some waythrough control of the synthesis of cell wall poly-saccharides, it would be of interest to know wherethe synthetic enzymes are located with regard to thecell membrane. The site of the enzymes will alsoinfluence attempts to isolate them. An in vitro syn-thesis of polysaccharide is required for meaningfulstudies of the control of polysaccharide synthesis.Proteins and enzymes (20) have been found in thecell wall, so that polysaccharide synthetic enzymescould be external to the cell membrane. On the otherhand, wall polysaccharides could, in a manner anal-ogous to the synthesis of extracellular protein inmany animal glands, be synthesized in membrane sys-tems within the cell, concentrated and excreted inbulk, without individual transport through the cellmembrane.

The labeling experiments show that the externalpolysaccharide, although similar in composition tothe wvall, has a much higher specific activity afterthe cells metabolize labeled precursors. This couldbe due to either of 2 factors. The external polysac-charide could come from a different precursor pool,or from newly synthesized wall polysaccharide. Thepresent experiments do not distinguish between the2 possibilities; yet the similarities of the polysac-carides andl calculations basedI on the estimatedI un-labeled polysaccharides in the 2 fractions suggestthat the latter is the miiore likely. The newly synthe-sizecl wall material probably escapes into the me(liumbefore it is firmly attached to or incorporated intothe wall.

The direct precursor of the methyl ester groups ofgalacturonidle residues has not been ascertainedl. S-adenosylmethionine is probably not the direct pre-cursor, since the methyl from this compound is incor-porated only slightly into the methyl esters of galac-turonic aci(l. Mlethionine and fornmaldehy(le, on theother hand, are very good precursors. and just aboutequal in their effectiveness (table IV).

Folic aci(d has been shown to be an intermediate



in several transmethylation reactions (9), and in sev-eral cases the N5-methyl derivative has been shownto be the active form (17, 23). Reduced folic acid(FH4) will react with formaldehyde to form a com-pound which can methylate homocysteine to formmethionine as follows (17, 18, 21): [1] FH4 + CH,O

-) N5N10 methylene-FH4 > N5-methyl-FH4;[2] N5-methyl-FH4 + homocysteine - methio-nine + FH4. The authors do not say if the lastreaction is reversible. If it is, the above schemecould provide a mechanism for conversion of bothformaldehyde and methionine into N5-methyl-tetra-hydrofolic acid, which could then serve as the directprecursor of the methyl esters of pectin. If this istrue, one would expect S-adenosylmethionine to bea poor precursor, as it would first have to be con-verted to free methionine, a reaction which would bemetabolically inefficient. The above scheme wouldalso explain why formaldehyde and methionine areequally good precursors; they would be convertedinto a common intermediate. The rate limiting stepmust occur after ithis common intermediate (3).This scheme would also explain the reciprocal inhibi-tion of the incorporation of methyl from methionineand formaldehyde.

Another important point about this pathway isthat it would not require the methyl of methionine tobe oxidized and rereduced before transference topectin. This agrees with the results of Sato et al.who have shown that the methyl of methionine istransferred intact to the pectic carboxyl groups inradish (24). Formaldehyde was shown to be a bet-ter source of methyl groups than methionine, whilewe found both sources to be equally effective. Thiscould be explained if reaction [2] in the above schemetends toward the synthesis of methionine in somesystems, or under certain conditions, so that theformation of N5-methyl-tetrahydrofolate would bemore rapid from methionine than formaldehyde.Alternatively, radishes might take up the formalde-hyde more readily than methionine. Of course, de-finitive answers can be obtained only through in vitrostudies, which we are attempting.

Summary

Sycamore (Acer pseudoplatanus) cambial cells insuspension grow rapidly in complex yeast extractmedium.

These cells contain galacturonic acid residuessimilar in amount to those found in bindweed callusand oat seedlings.

The cells secrete polysaccharides into the culturemedium. The polysaccharides are similar in com-position to the noncellulosic wall polysaccharides ofthese cells, and slightly different from the compositionreported for sycamore cambial cells in the intact tree.

Glucose-C14 and formaldehyde-C14 are taken uprapidly by the cells, and the label from each is foundin the galacturonic acid residues of both wall andexternal polysaccharides. The galacturonide residues

from external polysaccharides have a higher specificactivity than those of the wall polysaccharides.

S-adenosylmethionine is apparently not a pre-cursor of the methyl esters of galacturonic acid. Me-thionine and formaldehyde are equally good pre-cursors of these esters. The immediate precursorhas not been identified. It is proposed that thiscould be N5-methyl-tetrahydrofolic acid.

Literature Cited

1. ALBERSHEIM, P. AND J. BONNER. 1959. Metabol-ism and hormonal control of pectic substances.J. Biol. Chem. 234: 3105-08.

2. ALBERSHEIM, P. AND U. KILLIAS. 1962. Studiesrelating to the purification and properties of pectintranseliminase. Arch. Biochem. Biophys. 97: 107-15.

3. ALBERSHEIM, P. 1963. Hormonal control of myo-inositol incorporation into pectin. J. Biol. Chem.238: 1608-10.

4. BAILEY, R. W. 1953. The reaction of pentoseswith anthrone. Biochem. J. 68: 669-72.

5. BiTrER, T. AND H. M. MUIR. 1962. A modifieduronic acid carbazole reaction. Anal. Biochem. 4:330-34.

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Comparison of the Uptake of P32 and K42 by Intact Alfalfa and Oat Roots 1, 2.3Roger G. Lambert4 and A. J. Linck

Department of Plant Pathology and Physiology, University of Minnesota, St. Paul

The growth of a legume grown as a companionwith a grass has been shown by many workers(3, 4, 5, 8, 10, 11, 15) to he less than when the legumeis grown in pure stand.

Explanations for this growth reduction of thelegume include competition for light (5, 6), soilmoisture (11, 15), nutrients (1, 4, 7, 9, 10, 13), ex-cretion of toxic materials (2, 12, 14), and effects ofthe microflora (12).

Although all of these factors may influence thegrowtlh of both plants grown in close association,this paper reports investigation of the relative effi-ciency withl which intact roots of alfalfa and oat plantsabsorb phosphorus-32 (P32) and potassium-42 (K142)when placed in a common nutrient solution or whentreated separately.

1 Revised manuscript received July 7, 1964.2 This investigation was supported in part by a grant

from the Rockefeller Foundation.3 Paper 5210, Scientific Journal Series, Minnesota

Agricultural Experiment Station.4 Present address: Department of Biology, University

of Louisville, Louisville, Kentucky.

Materials and Methods

Alfalfa (Medicago sativca L., va. Ranger) an(loats (Avena sativa L., var. Ajax) were grown in acontrolled environment room maintained at 240 dur-ing a 12-hour photoperiod followed by a 12-hournyctoperiod maintained at 180. Seeds were ger-minated and the plants were supported on nylon screenheld over a solution containing 2 mmoles of KH2PO4,KNO3, and MIgSO4*7H2O; 3 mmoles of Ca (NO3)224H2O; 2.5 ml of versenol iron chelate solution (24g/liter); and 1 ml of a micronutrient solution (con-taining 2.5 g H,Bo4: 1.5 g MnCl2*4H2O; 0.1 gZnCI2; 0.05 g CuCl H.,O; and 0.05 g MoO3 per liter)in 1-liter polystyrene containers. The solution wasaerated by forcing air through glass wool attached toa piece of glass tubing. After 7 or 15 days, the plantswere removed and their roots either immersed inanother container with the nutrient solution to whichthe radioisotope had been added or placed in a specialapparatus designed to study the uptake of the mineralsby part of one root. The solution used to study up-take by whole root systems was aerated during a 6-hour uptake period in the light at 750 ft-c. After theuptake period, the roots were rinsed 3 times in nutrient

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