f-glucan synthetases plasma membraneand golgi apparatus ... · sis products was determined by...

Plant Physiol. (1974) 54, 333-340

f-Glucan Synthetases of Plasma Membrane and Golgi Apparatusfrom Onion Stem1

Received for publication December 27, 1973 and in revised form March 26, 1974

WILLIAM J. VAN DER WOUDE,2 CAROLE A. LEMBI, AND D. JAMES MORREDepartment of Botany and Plant Pathology and Department of Biological Sciences, Purdue University, WestLafayette, Indiana 47907

JAUNITA I. KINDINGER AND LAWRENCE ORDINDepartment of Biochemistry, University of California, Riverside, California 92502

ABSTRACT

Biosynthesis of glucans occurred in cell-free fractions iso-lated from onion stem (Allium cepa L.) enriched in eitherdictyosomes or plasma membranes. l8-1,3- and p-1,4-Glucanswere synthesized in differing proportions and at different ratesas the concentration of uridine diphosphoglucose or the pro-portion of dictyosomes or plasma membrane varied. At low(1.5 uM) UDP-glucose concentrations synthesis of alkali-insoluble glucan was correlated with abundance of dicyto-somes; most of the substrate utilized by plasma membrane wasfor glycolipid synthesis. At high (1 mM) UDP-glucose concen-tration, the synthesis of alkali-insoluble glucans correlated withthe abundance of plasma membrane. Substrate enhancementof g-1,4-glucan synthesis in dictyosome fractions was less thanproportional to increases in substrate concentration. In con-trast, ,-1,4-glucan synthesis by plasma membrane was morethan proportionately increased. At high substrate concentra-tions the synthesis of 6-1 ,3-glucans predominated in bothdictyosome and plasma membrane fractions. The results showthat the capacity to synthesize glucans resides in both Golgiapparatus and plasma membranes of onion stem, but thatthe plasma membrane has the greatest capacity for synthesis ofalkali-insoluble glucans at high UDP-glucose concentrations.

In vivo studies of cell wall biogenesis indicate that cellulosesynthesis occurs at the cell surface, possibly at the surface ofthe plasma membrane (28, 34, 39, 44, 52), while pectic andhemicellulosic polysaccharides are synthesized by the Golgiapparatus (1, 5, 10, 18, 27, 40). In contrast, Ray et al. (36)found UDP-glucose: /3-1,4-glucan glucosyltransferase (3-1,4-glucan synthetase) activity to be localized in a membrane frac-

'This work was supported by National Science Foundation GrantGB 23183; the Purdue University Joint Highway Research Projectto D.J.M.; Environmental Protection Agency, Office of AirPrograms Grant R 800870 (formerly AP 00213) to L.O.; a NationalScience Foundation Postdoctoral Fellowship to C.A.L.; and aNational Science Foundation Traineeship to W.J.V. Journal PaperNo. 5305 of the Purdue University Agricultural ExperimentStation.

2Present address: Department of Biology, University of Califor-nia, Riverside, Calif. 92502.

tion containing dictyosome cisternae. Other studies suggestedthe in vitro capability of plasma membranes for glucan synthe-sis (38, 51), but this cell component was not identified in thecell-free preparations. Phosphotungstic acid-chromic acid(PTA-CrO,), an electron-dense stain which specifically andcharacteristically stains plant plasma membranes (41-43), al-lows the identification and quantitation of plasma membranesin cell fractions (9, 11, 14, 26, 42, 48). The present work em-ploys this staining procedure and concerns an investigationof the /3-glucan synthetase activities of Golgi apparatus andplasma membranes from onion stems.

MATERIALS AND METHODSCell fractions were isolated from stem and meristematic re-

gions of green onions (scallions) by low shear homogenizationand differential and sucrose gradient centrifugation. The dic-tyosome and plasma membrane content of each fraction wasdetermined by electron microscopy and compared with its ac-tivity for incorporation of radioactivity from UDP-D-C'C)-glucose into products of varying solubility.

Plant Material. Green onions (Allium cepa) were obtainedcommercially, stored at S C, and used within 5 days of pur-chase. Stem explants, 0.5 to 1 cm diameter at the base and0.5 to 1 cm high, were removed from the central portions ofthe bulbs (19). Before homogenization, the explants were in-cubated at 26 C on wet filter paper in Petri plates for 16 to 18hr.

Preparation of Fractions. Membrane isolations were at 0to 4 C. Tissues were homogenized using a motorized razorblade chopper (20). The homogenization medium contained0.5 M sucrose in coconut milk medium (14, 20, 48) (100 mmK2HPO4, 20 mM Na2EDTA, and 1 mm dithiothreitol in coco-nut liquid endosperm which had been centrifuged at 100,000gfor 90 min to remove nuclei and other endogenous mem-branous components and adjusted to pH 6.5). Sucrose gradientsolutions were similar except for sucrose concentration. Finaldensities were determined by refractometry to account for theadded density of the centrifuged coconut milk (1.035 g/cc).Sucrose concentrations of the gradient solutions were 0.5, 0.65,0.8, 1.0, 1.2, and 1.3 M while their respective densities were1.087, 1.116, 1.125, 1.148, 1.172, and 1.185 g/cc.

Cell fractions were isolated by one of two procedures. Inprocedure I, based on the method of Lembi et al. (14), 20 gof tissue were homogenized in 40 ml of homogenization me-dium and filtered through Miracloth (Chicopee Mills, N.Y.).An aliquot was saved, and the remainder was centrifuged at10,000g for 15 min. The supernatant of density equivalent to

333

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VAN DER WOUDE ET AL.

0.32 M sucrose was layered onto discontinuous gradients con-sisting of 0.65, 0.80, 1.0, 1.2, and 1.3 M sucrose prepared incoconut milk. After centrifugation at 80,000g for 60 min,fractions from the density interfaces were diluted with 0.25 Msucrose in coconut milk medium and centrifuged in 2 aliquots at100,000g for 30 min. One pellet was prepared for electron mi-croscopy, and the other was assayed for glucan and glycolipidsynthetases. Portions of the filtered homogenate and resus-pended 10,000g pellet were sedimented at 80,000g for 60 minand also assayed.

Procedure II was based on the differential sedimentationmethod of Morre (19, 22, 25). Twelve grams of tissue werehomogenized in 25 ml of coconut milk medium and centri-fuged at 10,000g for 30 min. The supernatant was placed intubes above a cushion consisting of 2.0 and 1.5 M sucrose (pre-pared in coconut milk medium). After centrifugation at35,000g for 30 min, the supernatant overlying the materialwhich had sedimented onto the cushion was removed and re-placed with layers of 1.3, 1.2, 1.0, 0.8, and 0.5 M sucrose incoconut milk medium. The gradients were centrifuged at100,000g for 90 min, and the fractions were prepared forelectron microscopy and assayed as for procedure I.

Glucan Synthetase Assay. Glucan synthetase activity wasdetermined by the procedures of Ordin and Hall (31) andPinsky and Ordin (33). Fractions were suspended in 0.1 M tris-HC1 containing 4 mm Na2EDTA and 1 mm dithiothreitol, ata concentration of 1 to 4 mg protein/ml, pH 8.0. The com-plete assay mixture contained 0.3 nmole UDP-D-(1C)-glucose(New England Nuclear, specific radioactivity 227 ,uc/,umole),0 to 400 nmoles of UDP-D-(Q2C)-glucose (Sigma), 10 ,umoles oftris-HCl, 0.4 Mmole of Na2EDTA, 0.1 Mumole of dithiothreitol,4,umoles of MgCl2, 2 ,moles of cellobiose, and 1 aliquot of thefraction to provide 50 to 200 ,ug of protein in a final volume of200 M', pH 8.0. Assays were in 10 x 75 mm tubes and incu-bated at 25 C in a shaking water bath for 15 min. Reactionswere terminated by heating the tubes for 5 min in a bath ofboiling water.The assay mixture was fractionated into products of vary-

ing solubility as follows. Powdered cellulose, 1 to 2 mg, wasadded to each tube and the mixtures were extracted threetimes with 1 ml of hot water (boiling water bath) for 5 min.Tubes were centrifuged at 25,000g for 12 min to complete eachextraction and recover insoluble residues. The hot water ex-tracts were combined, and 0.5 ml of 20 mg/ml Ficoll (Phar-macia, mol wt 400,000) was added. The extracts were made70% in ethanol and after 2 hr at 0 to 5 C were centrifuged at25,000g for 30 min to collect the precipitate. The precipitatewas washed once with cold 70% ethanol and solubilized in hotwater. The hot water-insoluble residue was extracted once with1 ml of methanol-chloroform (2:1, v/v), and once with 1 mlof absolute methanol, and the extracts were combined to es-

timate the synthesis of lipid-soluble material (glycolipid) (33).Finally, the residues were extracted twice with 0.25 ml of hotI N NaOH for 5 min and once with 0.75 ml of cold water.

These two extracts were combined for measurement of hotwater-insoluble, alkali-soluble material. The alkali-, lipid-, andhot water-soluble extracts and the alkali-insoluble residueswere dried on planchets and their radioactivity was determinedusing a Nuclear-Chicago gas-flow detector system. Correctionswere made for self-absorption, and the results were expressedas the amount of glucose incorporated/hr-mg protein deter-mined by the Lowry (16) method. The presence of coconutmilk protein in resuspended cell fractions was less than 1 ,g/assay for which corrections were not made. Activities of zero

time assays and boiled controls were negligible. Average de-viation of duplicate determinations was less than ±+5%.

Characterization of the Alkali-insoluble Product. The natureof the alkali-insoluble products was examined using Strepto-myces sp. QM B 814 cellulase by the method of Pinsky andOrdin (33). This cellulase hydrolyzes both /-1,3- and /3-1,4-glucosidic bonds. The distribution of radioactivity in hydroly-sis products was determined by liquid scintillation counting ofsectioned paper chromatograms.

Electron Microscopy. Pellets of membrane fractions werefixed in 2% glutaraldehyde buffered with 0.1 M sodium phos-phate, pH 7.3, for 1 to 1.5 hr at 26 C and postfixed in buff-ered, 1% osmium tetroxide at 26 C for 1 to 1.5 hr (9). Speci-mens were dehydrated through a graded acetone series andembedded in Epon (17). Thin sections were stained either withlead citrate (37) or, to identify plasma membranes, by thePTA-CrO3 procedure (42). Sections were viewed with a PhilipsEM-200 electron microscope.The amounts of dictyosomes and plasma membranes in pro-

portion to the total amount of membrane present was deter-mined for each fraction. Electron micrographs from fourequidistant positions along the centrifugal force vector ofmembrane pellets were analyzed to obtain results representa-tive of the entire fraction. By the method of Loud (15), a trans-parent overlay of parallel lines spaced 1 cm apart was placedon prints enlarged to 19 X 24 cm at magnifications of 20,000.Points of line-membrane intersect were quantitated for eachcomponent. Data from the four micrographs were summed.The ratio of the number of intersects for a cell component tothe total number of intersects was expressed as a percentageto estimate the abundance of dictyosomes and plasma mem-

branes.

RESULTS

Distribution of Membrauies. Examples of electron micro-

graphs used to characterize cell fractions are shown in Fig-ures 1, 2, and 3. Fractions containing dictyosome membraneshad numerous cisternal profiles which were organized intostacks (Fig. 1). Generally, dictyosome membranes stained more

intensely with osmium tetroxide than the bulk of the smoothmembrane fragments other than dictyosomes which comprisethe A and B fractions (Fig. 1). These latter membrane frag-ments accounted for more than 50% of the A and B fractions,did not react with PTA-CrO3 and had dimensions and cyto-chemical properties (nonspecific inosine diphosphatase) com-patible with an origin from tonoplast (vacuole) membranes.Plasma membranes were distinguished by the PTA-CrO3 stain

(Figs. 2 and 3). When isolated by procedure I. dictyosomeswere most abundant in the fraction which equilibrated at the0.65/0.80 M sucrose interface (Table I). Plasma membraneswere in all fractions but were most abundant in the 1.01/1.2 Msucrose fraction in which few dictyosome membranes couldbe identified. The bulk of the mitochondria, plastids and nu-

clei were in the 0 to 1 0,000g fraction as were cell wall frag-ments.Glucan Synthetase Activities. At 1.5 Mm UDP-glucose, al-

kali-soluble and alkali-insoluble glucans were most activelysynthesized on a protein basis, by fractions containing dictyo-some membranes (Table I), while the plasma membrane-richfraction synthesized these products more slowly. Lipid-solublematerials contained most of the radioactivity incorporated byeach fraction with the rate of synthesis of lipid-soluble productseemingly proportional to the plasma membrane content.

In contrast to the results at 1.5 uM substrate (Table I), theplasma membrane fraction most actively synthesized alkali-soluble and -insoluble products at 1 mm substrate (Table II).However, this activity for all fractions was related to the sum

Plant Physiol. Vol. 54, 1974334



Plant Physiol. Vol. 54, 1974 GLUCAN SYNTHETASES OF PLASMA MEMBRANES

4..-.

N

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A f_se a- wrJ,s .#_E)'*I".N. " . S

¼- S- -,iror'<z ,

e4 / PMiPM

v4W .PFIG. 1-3. Electron micrographs of dictyosome-rich and plasma membrane-rich fractions from onion stem. The fractions were isolated by pro-

cedure I in the experiment of Table I and were fixed in glutaraldehyde and osmium. Fig. 1: a fraction containing 37%-( dictyosome membrane (D)collected at the 0.65,/0.80 M sucrose interface of the density gradient. Section was stained with lead citrate. X 35,000. Fig. 2: a fraction collected atthe 1.0/1.2 M sucrose interface which was enriched in plasma membrane (67%). Plasma membranes are not identifiable in this preparation whichwas stained with lead. X 20,000. Fig. 3: same fraction as in Fig. 2 in which plasma membranes (PM) have been identified by staining with PTA-CrO:. Arrows indicate unstained membranes. X 20,000.

of both dictyosome and plasma membrane in each fraction.The plasma membrane fraction synthesized lipid-soluble prod-ucts most actively. This activity correlated with the percentageof plasma membrane in all but the fraction of lowest density.

The enrichment of glycolipid synthetase activity in the plasmamembrane fraction over that of the total homogenate (0-80,000g fraction) was 2.5 when assayed at 1.5 Mm substrate and2.2 at 1 mm. The 0 to 80,000g fraction and the plasma mem-

335

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VAN DER WOUDE ET AL. Plant Physiol. Vol. 54, 1974

Table I. Distributioni of Total auid Specific Glucani anid Glycolipid Syntthetase Activities amontg Membranie Fractionts Assayed at ani InzitialSubstrate Co,icenitrationi of 1.5 ,sr UDP-Glhcose

Fractions were prepared by procedure I. With the exception of Figure 6, assay incubations of all experiments were for 15 mi.

Aembrane Specific Activity Total ActivityFraction ~~~Gradient MmrnFraction ~~~~Interface

Dictyo- Plasma Water- Lipid- Alkali- Alkali- Water- Lipid- Alkali- Alkali-somes imembrane soluble soluble soluble insoluble soluble soluble soluble insoluble

X sucrose % total sntenibrante nmoles glucose incorporated/lrrng protein mwtoles glutcose incorporated/hr

Totalparticulate(0-80,000g) 0.044 1.495 0.047 0.011 1.508 51.13 1.621 0.373Heavy particulate (0-10,OOOg) 0.027 1.635 0.021 0.004 0.682 140.71 0.530 0.102Gradient fractions l

A 0.32/0.65 23 11 0.052 0.609 0.084 0.018 0.060 0.710 0.097 0.021B 0.65/0.80 37 29 0.043 1.184 0.099 0.047 0.055 1.490 0.125 0.059C 0.80/1.00 22 37 0.109 1.776 0.060 0.024 0.138 2.260 0.076 0.031D 1.00/1.20 3 67 0.070 3.742 0.033 0.007 I 0.099 5.250 0.047 0.010

Table II. Distribuitioni of Total anid Specific Glhcan and Glycolipid Synthetase Activities anmong Membrane Fr-actionls Ilnlcubated Usinig aniIniitial Suibstrate Conicentrationi of I m.u UDP-Gliicose

These values are from the same experiment as Table I. The alkali-soluble and alkali-insoluble fractions were not separated. Fractionswere prepared by procedure I.

Abundance ofMembrane

Gradient InterfaceDictyo- Plasmasomes membrane

Specific Activity

I5~ater Lipid Alkali-solubleW1ater- Lipid-

soluble soluble -inslb-insolublel

Total Activitv

WN'ater-soluble

Lipid- Alkali-solublesoluble and

I -insoluble

Total particulate (0-80,000g)Heavy particulate (0-10,000g)Gradient fractions

ABCD

.u sucrose

0.32/0.650.65/0.800.80/1.001.00/1.20

% total tnenmbrane rnoles glutcose incorporatedprotein

188235

23 11 17137 29 18422 37 1463 67 146

5586

436280124

l/h'r-inzg

12991777

1134212719372620

ntnztoles gluecose incorporated/lhr

64305852

199232186205

19812144

5078102174

44,42644,247

1319268024643678

brane fraction contained 29 and 67% plasma membrane, re-spectively, an enrichment factor of 2.3.The distribution of total glucan synthetase activities among

gradient fractions (Tables I and II) paralleled the distributionof specific activity for these fractions since total protein ineach fraction was similar in amount. From specific and totalglucan synthetase activities of fractions isolated by differentialcentrifugation, synthesis of alkali-soluble and -insoluble glu-cans was associated primarily with slowly sedimenting particlesat 1.5 guM UDP-glucose and with rapidly sedimenting particlesat 1 mM UDP-glucose (compare activities of the 0-80,000gand the 0-l0,OOOg fractions in Tables I and II). Recoverieswere: protein, 86%; total glucan synthetase activity at 1.5u.M, 96%; total glucan synthetase activity at 1 mM, 120%. Anapparent small activation of glucan and glycolipid synthesisat 1 mM UDP-glucose occurred during fractionation.

Influence of Substrate Concentration. The effect of UDP-glucose concentration was examined using the particulatefraction of a 10,OOOg, 30-min supernatant (Fig. 4). Greaterthan proportional increases in the synthesis of alkali-solubleand -insoluble products occurred at substrate concentrationsas high as 140 ,uM. In contrast, the synthesis of lipid-solublematerials was less than proportionately enhanced by increasedsubstrate. The incorporation of radioactivity into alkali-solubleand -insoluble product was enhanced by increases in sub-strate concentration, even though equal amounts of radioac-tivity were supplied to each assay (specific radioactivity of thesubstrate was reduced).

2401.

5-

> 2200

w ' 160sn -

I-LO U

Z ._c

_) ,rn p

3

(4

40

'v5 - 20 40 60 80 100 120 140pM UDP-GLUCOSE

FIG. 4. Influence of UDP-glucose concentration on glucan and

glycolipid synthetase activities of a particulate fraction from onion

stem. The assays were performed at the indicated substrate con-

centrations on a 100,000g, 30-min particulate fraction of a 10,000g,30-min supernatant. Assays contained equal amounts of radio-

activity (30,000 cpm). Activities assayed at 1.5 sM UDP-glucosefor the synthesis of lipid-soluble and alkali-soluble and -insoluble

products were, respectively, 1.37 and 0.28 pmoles glucose in-

corporated/hr-,ug protein.

336

Fraction

NaOH- soluble and insoluble

p moles -

CPM

," /

\ , i~~~~~~~~~~-

--~~*~(CHC CH OH--soluble

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GLUCAN SYNTHETASES OF PLASMA MEMBRANES

£750 X *-._

500

U Hot water soluble250

LU Va) CHCI -CH OH--soluble

Il.z 2 50 DICTYOSOME NaOH -solubleLI)

z t01 water- soluble

H200-XOHinsoluble0 150

E

100

'°° L .zCHCICH3OH-soluble

0'0 0.25 0.5 1.0 2.0

mM UDP-GLUCOSE

FIG. 5. Influence of substrate concentration on glucan andglycolipid synthetase activities of dictyosome- and plasma mem-brane-rich fractions. Dictyosome and plasma membrane fractionswere isolated by procedure II and collected, respectively, from the0.5/0.8 and 1.0/1.2 M sucrose interfaces of the gradient. Anexample of the content of dictyosome and plasma membrane ingradient fractions obtained by procedure II is given in Fig. 7.

Dictyosome and plasma membranes were also assayed overa range of substrate concentrations (Fig. 5). At 0.25 to 2 mM,the plasma membrane fraction synthesized alkali-soluble andalkali-insoluble products more actively than the dictyosomefraction.When plasma membrane fractions were assayed at 1 mM

UDP-glucose, alkali-soluble and alkali-insoluble products wereformed approximately linearly with time (Fig. 6). However,the rate of synthesis of lipid-soluble and water-soluble prod-ucts decreased during the 15-min. incubation.

Identity of the Alkali-insoluble product. Cellulase hydrol-yzates of alkali-insoluble products contained /3-1,3- and /3-1,4-linked di- and triglucosides (Table III). No /3-1, 3: /3-1, 4-triglucoside was formed. At 1.5 aM UDP-glucose, the linkageof glucan products of dictyosome and plasma membrane frac-tions was primarily /3-1,4. However, at 250 ,uM and 1 mm UPD-glucose, these products were linked primarily /-1,3. At allconcentrations, the alkali-insoluble products of the dictyosomefraction contained the greater proportion of /3-1 4-glucan.Even so, calculations from the data of Figure 5 and Table IIIshow that at 250 ,M and 1 mm substrate. /3-1,4-linked, alkali-insoluble glucans were synthesized most actively by the plasmamembr2ne fractions (Table IV). The /3-1,3-glucan synthetaseactivities of both fractions increased disproportionately to sub-strate concentration. Increases in /-1, 4-glucan synthetaseactivity of the dictyosome fraction were less than proportionalto substrate increases while /3-1,4-glucan synthetases of theplasma membrane fraction responded greater than proportion-ally (Table IV).

Association of Glucan Synthetases Assayed at 1 mm UDP-Glucose with Plasma Membrane. When fractions were isolatedby procedure II and assayed at 1 mm UDP-glucose, plasma

membrane composition and glucan synthetase activity werecorrelated (Fig. 7). Least square regression lines for all butthe water-soluble activities were characterized by correlationcoefficients near unity (Fig. 7, legend). However, the fractioncontaining 23% dictyosome membrane and only 4% plasmamembrane synthesized alkali-soluble and -insoluble glucansat a rate greater than that expected if plasma membranesalone were responsible. A similar relationship was alreadydiscussed for data of Table II from fractions prepared byProcedure I. The correlation between plasma membrane con-tent and glucan synthetase was evident with fractions preparedby either procedure although the fractions prepared by pro-cedure I were of consistently higher specific activity (Table II;Fig. 8) than those prepared by procedure II (Figs. 5, 6, and 7).To identify the source of glucan synthetase activity in the

a I'00,

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0

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0

c 25

0 20

0

5

E0 2 5

25

20

15

1 0

5

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)O

10 15 0 2 5 10 15

TIME, MIN

FIG. 6. Time course for glucan and glycolipid synthetase re-actions catalyzed by a plasma membrane fraction assayed at 1mM UDP-glucose. The fraction was collected from the 1.0/1.2 Msucrose interface of the gradient using isolation procedure II. Dataare given for two separate experiments.

Table III. Distributionz of Radioactivity ini Cellulase HydrolysisProducts of Alkali-intsoluble Materials formed by Dictyosome-

and Plasma Membrane-rich FractioznsThe alkali-insoluble materials were produced in the experiment

of Figure 5 in which equal amounts of radioactivity (0.083 ,uCi)were supplied to each assay. Parentheses give radioactivity as percent of the total present in laminaritriose, laminaribiose (,B-1,3-linked glucosides), and cellobiose (,B-1,4-linked). The cellodextrinwas a glucose oligosaccharide of unknown linkage having a degreeof polymerization of about 4.

Total Recovered RadioactivityUDP-Glucose

Concn Cello- Lamrin- Cellobiose Lamin- Glu-dextrin aritriose aribiose cose

MM ~~~~~~~dpmDictyosome mem-brane fraction

1.5 40 5 (5.3)1 86 (90.5) 4 (4.2) 13250 106 351 (44.1) 105 (13.2) 339 (42.7) 2981000 64 115 (45.5), 32 (12.6) 106 (41.9) 118

Plasma membranefraction

1.5 91 50 (25.0) 114 (57.0)1 36 (18.0) 63250 98 167 (46.6) 41 (11.4),151 (42.0) 971000 81 173 (55.6)1 28 (9.0) 110 (35.4) 75

5 --

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10 - I'Y7 CHC13-CH30H

5 - SOLUBLE

I

o -

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NaOH SOLUBLEi0

0iO_~~~~~~

O - - -

-UA

i/NaOH -INSOLU BLE

337Plant Physiol. Vol. 54, 1974

20



VAN DER WOUDE ET AL.

Table IV. ,3-1,4 and fl-1,3 Alkali-inisoluble Glucant SynthetaseActivities of Dictyosome-rich anid Plasma

Membrane-rich FractionisAlkali-insoluble ,B-1,4- and ,3-1,3-glucan synthetase activities

were derived from rates for the synthesis of total alkali-insolubleglucan in the experiment of Figure 5 and the proportions of j-1,4and d-1,3 glucosidic linkages in the products (Table III). Paren-theses indicate relative increases in substrate concentration or inglucan synthetase activity.

Alkali-insoluble Glucan Synthetase ActivityUDP-Glucose _______________________

ConcnTotal #-1,4 d-1,3

,A.M nomoles glucose incorporated/ hr-mg protein

Dictyosome membranefraction

1.5 (1) I 0.091 (1) 0.082 (1) 0.009 (1)250 (167) 62.4 (686) 8.24 (100) 54.2 (6020)1000 (667) 135 (1480) 17.0 (207) 118 (13,100)

Plasma membrane frac-tion

1.5 (1) 0.053 (1) 0.030 (1) 0.023 (1)250 (167) 169 (3190) 19.3 (643) 150 (6520)1000 (667) 742 (14,000)66.8 (2230) 675 (29,300)

OD> E

u

en X

< 8

en 8z -C

J u

cs w

E

0 10 20 30 40 50 60PLASMA MEMBRANE, % TOTAL MEMBRANE

FIG. 7. Correlation of plasma membrane content and glucansynthetase activities of fractions assayed at 1 mM UDP-glucose.Arrows indicate, from left to right, fractions collected at the 0.5/

0.8, 0.8/1.0, 1.0/1.2, and 1.2/1.3 M sucrose gradient interfacesusing isolation procedure II. Least square regression lines of ac-tivity (Y) on percentage of plasma membrane (X) and theircorrelation coefficients were as follows.

Activity Least Square Regression Line Coefficient

Lipid-soluble Y = 12.74 + 0.80 X 0.9949Alkali-soluble Y = 61.87 + 8.92 X 0.9920Alkali-insoluble Y = -12.94 + 10.96 X 0.9920Alkali-soluble and -in- Y = 48.92 + 19.87 X 0.9991

soluble

0 to 1 0,OOOg pellet, a plasma membrane fraction was comparedwith various fractions which sedimented at low centrifugalforces and accumulated at the 1.0/1.2 M sucrose interfaceduring subsequent gradient centrifugation (Fig. 8). The rapidly

sedimenting fractions contained fragmented protoplasts, mito-chondria and proplastids in addition to plasma membranesand other smooth membranes, but dictyosome membranes wereexcluded. Glucan synthetase activities again correlated wellwith the plasma membrane content of each fraction (Fig. 8).

DISCUSSION

The results demonstrate synthesis of /3-1,3- and /-1,4-glucans by plasma membranes from onion stem. They showan association of glycolipid synthetases with plasma membranefractions and indicate a more limited capability for glycolipidsynthesis by dictyosome membranes. Additionally, they confirmthe association of /B-1,4-glucan synthetases with dictyosomemembranes (36 and show that dictyosomes may also synthesize/3-1,3-glucans.The influence of substrate concentration on the synthesis of

glucans by dictyosome and plasma membrane fractions is acritical factor in reconciling our results with those of Rayet al. (36). Dictyosome fractions were most active when thesubstrate was 1.5 /iM. At 1 mM UDP-glucose, plasma membranefractions were most active. A similar relationship was seenin the synthesis of alkali-insoluble glucans by membrane frac-tions from soybean hypocotyl (9). A membrane fraction of oat

2.0 Fract ion 2 3 PME2.'WE 1. 8 o Ne

c 1.6 Hot water-< soluble-|

1.4-

011.2

0

u 1.0

zfato0.8 as

z 0.6- k

0.4 -

(PM) 0.2 one

0 10 20 30 40 50 60

PLASMA MEMBRANE. % TOTAL MEMBRANE

FIG. 8. Correlation of plasma membrane content and glucan

synthetase activities of rapidly sedimenting and plasma membrane

fractions assayed at mms UDP-glucose. The plasma membrane

(PM) fraction was isolated by procedure I from one-half of a

total homogenate. The remainder of the homogenate was diluted1:1 with homogenization medium, centrifuged at 40g for 5 minand the pellet discarded. Next, three 15-min centrifugations wereperformed to produce fractions 1 (40-370g), 2 (370-lOOOg), and3 (1000-3300g). Particles in these fractions which subsequentlyequilibrated at a 1.0/1.2 M sucrose interface were isolated byprocedure I, quantitated by electron microscopic morphometry,and assayed for glucan synthetase activity. Regression lines ofactivity (Y) on percentage of plasma membrane (X) and their cor-relation coefficients were calculated by the method of leastsquares.

Activity Least Square Regression Line Coeaicient

Lipid-soluble Y = -12.41 + 2.33 X 0.9929Alkali-soluble Y = 11.26 + 10.89 X 0.9891Alkali-insoluble Y = -15.23 + 19.46 X 0.9943Alkali-soluble and -in- Y = -4.07 + 30.36 X 0.9979

soluble

338 Plant Physiol. Vol. 54, 1974



GLUCAN SYNTHETASES OF PLASMA MEMBRANES

root which was enriched in K-stimulated adenosine triphos-phatase activity and contained primarily plasma membranes byPTA-CrO3 staining was found by Hodges and co-workers (11)to occur at the 1.15/1.17 g/cc interface of a discontinuoussucrose density gradient. Maximum specific activity for alkali-insoluble glucan synthesis occurred at this same interface whenassayed at 1 mm UDP-glucose. In parallel assays at 1.5 uM,the activity peak occurred at the 1.13/1.15 g/ cc interface(W. J. Van Der Woude, R. T. Leonard, and T. K. Hodges,unpublished data). Low substrate concentrations may be re-sponsible for previous findings of glucan synthetase activityassociated primarily with dictyosome membranes (36). Invivo levels of UDP-glucose as high as 30 Mm during growth ofsuspension cultures of Lolium multiflorum endosperm havebeen reported (45). The present work indicates that higher sub-strate concentrations than those usually used (1-5 Mm) may bemore appropriate for the in vitro localization of glucan syn-thetases.As in other studies (31, 32, 46, 47), increasing the sub-

strate concentration from 1.5 Mm to 1 mm greatly increasedglucan synthesis and the proportion of /3-1,3-glucan formed.By fractionating solubilized cell particles from oat coleoptiles,Tsai and Hassid (46) found evidence for the synthesis of/3-1,3- and /3-1,4-glucans by separate enzymes. They sug-gested that substrate activation of /3-1, 3-glucan synthetasesincreased /3-1,3-glucan synthesis at high substrate concentra-tions (47). However, Ray (35) found particulate fractions ofpea epicotyl to synthesize principally /3-1,4-glucans at both 1Mm and 0.5 mm UDP-glucose.The synthesis of alkali-insoluble glucan by plasma membrane

fractions from onion was significantly stimulated by the addi-tion of 5 ,uM 2,4-D to assays containing 1 mm UDP-glucose(48). As in the present study, the product was linked principally,B-1,3.

/3-1 4-Glucan synthetase activities of dictyosome and plasmamembrane fractions indicate a capability of these membranesfor cellulose synthesis. However, as cautioned by Robinsonand Preston (38), it is presently impossible to equate the invitro synthesis of /3-1 ,4-glucans with the synthesis of cellulose.Although /3-1,3-glucans (callose) is not a usual component ofcell walls of higher plants, it is formed by plants in responseto wounding (4). The in vitro synthesis of /3-1,3-glucans mayresult in part from the rigors of cell fractionation. Excisionand preincubation of the onion stem explants may also bepartially responsible. A plasma membrane fraction fromfreshly excised onion tissues was found to synthesize only/3-1,4-glucans at 6.5 uM UDP-glucose (L. Ordin and C. A.Lembi, unpublished data).

Accumulating evidence suggests that glycolipids functionas cofactors or intermediates in the synthesis of cellulose cndother cell wall polysaccharides in plants (3, 6, 33, 50). Ourresults associate the synthesis of glycolipids with plasma mem-branes. Ongun and Mudd (30) indicate that membranes ofmitochondria and chloroplasts may also synthesize glycolipids,particularly steryl glucosides, the major lipid-soluble productsynthesized by particulate fractions of plants using UDP-glu-cose (12). In comparison with other cell fractions, plasmamembranes are rich in sterols (9, 11, 13, 26) which may serveas acceptors for the in vitro synthesis of steryl glycosides byisolated plasma membrane. The synthesis of glycolipids byisolated plasma membrane may mean that lipids of plasmamembranes are not fully glycosylated in vivo or, alternatively,that incorporation is via an exchange reaction with alreadyglycosylated lipids. Glycolipid synthesis by plasma membranefractions utilized most of the substrate in assays at 1.5 pMUDP-glucose. At low substrate concentrations, competition

for substrate by glycolipid synthetases may account for therelatively low glucan synthetase activities of plasma mem-branes.The in vivo function of glucan synthetases of dictyosome

membranes is enigmatic. Cellulosic scales are produced bydictyosomes in the marine alga Pleurochrysis (2). However,the higher plant Golgi apparatus does not appear to be aprimary site of in vivo cellulose synthesis but does function inthe synthesis of pectic and hemicellulosic polysaccharides (seeintroduction). The /3-1,3- and /-1 ,4-glucan synthetases ofdictyosome membranes may function in the synthesis of gly-coproteins (8) or hemicellulosic polysaccharides by perform-ing in concert with other glycosyl transferases. Alternatively, ifthe /-1,4-glucan synthetase of dictyosome membranes iscellulose synthetase, its activity in higher plants must be regu-lated. Its activity in vivo may remain potential until after aflow of the membranes to the cell surface (23, 29, 49). Otherevidence supports membrane flow from the Golgi apparatusto the plasma membrane (7, 21, 23, 24, 27). In higher plants,in vivo ("4C)-glucose-labeling studies combined with cell frac-tionation (5, 27, 40) provide kinetic evidence in support of thetransport, within membrane vesicles, of polysaccharides fromthe Golgi apparatus to the cell surface. Our results suggestthat dictyosomes and plasma membranes carry similar comple-ments of glucan synthetases. The Golgi apparatus may supplythe glucan synthetases to the plasma membrane.

Acknou ledgnicnts-We thank Catalina 'Montecillo and Dorothy Werderitshfor technical assistance in part of this work. Streptomyces sp. OM B 814 cellulasewas a gift from Dr. E. T. Reese.

LITERATURE CITED

1. BOWLES, D. J. AN-D D. H. 'NORTHCOTE. 1972. The sites of synthesis and trans-

port of extracellular polysaccharides in the root tissties of maize. Biochem.J. 130: 1133-1145.

2. BROWN, R. 'M., JR., W. W. FRANKE, H. KLEINIG, H. FALK, AND P. SITTE.1970. Scale formation in chrysophycean alga. I. Cellulosic and non-cellulosicwall components made by the Golgi apparatus. J. Cell Biol. 45: 246-271.

3. COLVIN, J. R. 1961. Synthesis of celltilose from ethanol-soluble precursors ingreen plants. Can. J. BioAiem. Phys:ol. 39: 1921-1926.

4. CURRIER, H. B. 1957. Callose substances in plant cells. Amer. J. Bot. 44:478-488.

5. EISI-NGER, W. AND P. 'M. RAY. 1972. Ftunction of Golgi apparatus in syntlhesisand transport of cell wall polysaccharides. Plant Physiol. 49: S-2.

6. ELBEIN, A. D. AND WV. T. FORSEE. 1973. Studies on the biosynthesis ofcellulose. In: F. Lorwtws. ed., Biogenesis of Plant Cell Wall Polysaccharides.Academic Press. New%-York. pp. 259-295.

7. FRANKE, WV. WV., D. J. 'MORRE. B. DEUMLING, R. D. CHEETHAM, J. KARTEN-BECK, E.-D. JARASCH, AND H.-W. ZENTRAF. 1971. Synthesis and turnoverof memiibrane proteins in rat liver: An examination of the membrane flowhypothlesis. Z. ANaturforsch. 26b: 1031-1039.

8. GARDINER, -M. G. ANXD M. J. CHRISPEELS. 1973. Involvement of the Golgiapparatus in the synthesis and secretion of hydroxyproline-rich cell wallglycoproteins. Plant Physiol. 51: S-60.

9. HARDIN, J. W., J. H. CHERRY, D. J. 'MORR:, AN-D C. A. LEMBI. 1972. Enhance-ment of RNA polymnerase activity by a factor released by auxin from plasmamembrane. Proc. Nat. Acad. Sri. U.S.A. 69: 3146-3150.

10. HARRIS, P. J. AND D. H. NORTHCOTE. 1971. Polysaccharide formation inplant Golgi bocies. Biochim. Biophys. Acta 237: 56-64.

11. HODGES, T. K., R. T. LEONARD, C. E. BRACKER, AND T. lV. KEENAN. 1972.Purification of an ion-stimulated adenosine triphosphatase from plantroots: association with plasma membranes. Proc. Nat. Acad. Sci. U.S.A.69: 3307-3311.

12. KAUSS, H. 1968. Enzymatische Glucosylierung von pflanzlichen Sterinen. Z.Natutforsch. R. 23: 1522-1526.

13. KEEN-AN-, T. W., R. T. LEONARD, AND T. K. HODGES. 1973. Lipid composi-tion of plasma membranes from Arena sativa roots. Cytobios 7: 103-112.

14. LE'MBI. C. A., D. J. MORRE, K. ST.-THOMSON, AND R. HERTEL. 1971. N-1-Napthylphthalamic acid-binding activity of a plasma membrane-rich frac-tion from maize coleoptiles. Planta 99: 37-45.

15. LoUD, A. V. 1962. A method for the quantitative estimation of cytoplasmicstructures. J. Cell Biol. 15: 481-487.

16. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, AND R. J. RAN-DALL. 1951.Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275.

Plant Physiol. Vol. 54, 1974 339



340 VAN DER W

17. LuFr, J. MI. 1961. Improvements in epoxy resin embedding methods. J. Bio-phys. Biochem. Cytol. 9: 409-414.

18. 'MOLLENHAUER, H. H. AND D. J. AIORRE. 1966. Golgi apparatus and plantsecretion. Annu. Rev. Plant Physiol. 17: 27-46.

19. MORRE, D. J. 1970. In vivo incorporation of radioactive metabolites by Golgiapparatus and other cell fractions of onion stem. Plant Physiol. 45: 791-799.

20. MORRE, D. J. 1971. Isolation of Golgi apparatus. Methods Enzymol. 22: 130-148.

21. MIORRE, D. J., WA. W. FRANKE, B. DEUIMLING, S. E. NYQUIST, AND L.OVTRACHT. 1971. Golgi apparatus function in membrane flow and differ-entiation: origin of plasma membrane from endoplasmic reticulum. Bio-membranes 2: 95-104.

22. MORRE, D. J., AND H. H. MIOLLENHAUER. 1964. Isolation of the Golgiapparatus from plant cells. J. Cell Biol. 23: 295-305.

23. 'MORRL, D. J. AND H. H. MOLLENHAUER. 1973. The endomembrane concept:a functional integration of endoplasmic reticulum and Golgi apparatus.In: A. WV. Robards, ed. Dynamics of Plant Ultrastructure. McGraw-Hill,New York. pp. 84-137.

24. MORRE, D. J., H. H. MOLLENHAUER, AND C. E. BRACKER. 1970. Origin andcontinuity of Golgi apparatus. In: J. Reinert and H. Ursprung, eds.,Results and Problems in Cell Differentiation. II. Origin and Continuity ofCell Organelles. Springer-Verlag, New York. pp. 82-126.

25. MORRfE, D. J., H. H. MOLLENHATER, AN-D J. E. CHAMBERS. 1965. Glutaralde-hyde stabilization as an aid to Golgi apparatus isolation. Exp. Cell Research38: 672-675.

26. MORRfE, D. J., J.-C. ROLAND, AND C. A. LEMBI. 1970. Comparisons of iso-lated plasma membranes from plant stems and rat liver. Proc. Indiana Acad.Sci. 1969. 79: 96-106.

27. 'MORRE, D. J., AND W. J. VAN DEnR WOUDE. 1974. Origin andl growthl of cellsurface components. In: E. D. Hay, T. J. King, and J. Papaconstantinou,eds., Macromolecules Regulating Growth andl Development. 30th Symp.Soc. Dev. Biol. Academic Press, New York. pp. 81-111.

28. MUHLETHALER, K. 1967. Ultrastrtucture and formation of plant cell walls.Annu. Rev. Plant Physiol. 18: 1-24.

29. NORTHCOTE, D. H. 1969. Fine structure of cytoplasm in relation to synthesisand secretion in plant cells. Proc. Roy. Soc. B. 173: 21-30.

30. ONGUN, A. AND J. B. MUDD. 1970. The biosynthesis of steryl glucosides inplants. Plant Physiol. 45: 255-262.

31. ORDIN-, L. AND M. A. HALL. 1968. Cellulose synthesis in hiiglher plants fromUDP glucose. Plant Physiol. 43: 473-476.

32. PEAUD-LENXEL, C. AND M. AXELOS. 1970. Structural features of the /B-glucans enzymatically synthesized from uridine diphosphate glucose bywlheat seedlings. FEBS Lett. 8: 224-228.

33. PINSKY, A. AND L. ORDIN. 1969. Role of lipid in the cellulose synthetaseenzyme system from oat seedlings. Plant Cell Physiol. 10: 771-785.

34. RAY, P. M. 1967. Radioautographic study of cell wall deposition in growingplant cells. J. Cell Biol. 35: 659-674.

'C)UDE ET AL. Plant Physiol. Vol. 54, 1974

35. RAY, P. M. 1973. Regulation of /-glucan synthetase activity by auxin inpea stem tissue. I. Kinetic aspects. Plant Physiol. 51: 601-608.

36. RAY, P. MI., T. L. SHIsN-NGER, AND MI. TM. RAY. 1969. Isolation of j8-glucansynthetase particles from plant cells and identification with Golgi mem-branes. Proc. Nat. Acad. Sci. U.S.A. 64: 605-612.

37. REY-NOLDS, E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17: 208-212.

38. ROBINSON, D. G. AND R. D. PRESTON. 1972. Polysaccharide synthesis in Mungbean roots-an X-ray investigation. Biochim. Biophys. Acta 273: 336-345.

39. ROBINSON, D. G. AND R. D. PRESTON. 1972. Plasmalemma structure in rela-tion to microfibril biosynthesis in Oocystis. Planta 104: 234-246.

40. ROBINSON, D. G. AND P. M. RAY. 1973. Separation of the synthesis ofcellulose and non-cellulosic wall polysaccharides. Plant Physiol. 51: S-59.

41. ROLAND, J.-C. 1969. Mise en evidence sur coupes ultrafines de formationspolysaccharidiques directement associees au plasmalemme. C. R. Acad. Sci.Paris 269: 939-942.

42. ROLAND, J.-C., C. A. LEMBI, AND D. J. MIORRL. 1972. Phosphotungstic acid-chromic acid as a selective electron-dense stain for plasma membranes ofplant cells. Stain Technol. 47: 195-200.

43. ROLAND, J.-C. AND B. VIAN. 1971. La reactivite du plasmalemme. Etudecytochemique. Protoplasma 73: 121-137.

44. SHAFIZADEH, F. AND G. D. McGINNIs. 1971. MIorphology and biogenesisof cellulose and plant cell-walls. Adv. Carbohydrate Chem. 26: 297-349.

45. SMITH, M. M. AND B. A. STONE. 1973. /3-Glucan synthesis by cell-freeextracts from Lolium mztltiflorum endosperm. Biochim. Biophys. Acta 313:72-94.

46. TSAI, C. M. AND W. Z. HASSID. 1971. Solubilization of uridine diphospho-D-glucose :'- (1-4) -glucan and uridine diphospho-D-glucose :/3- (1-+3) -glu-can glucosyltransferases from coleoptiles of Avena sativa. Plant Physiol.47: 740-744.

47. TSAI, C. M. AND WV. Z. HASSID. 1973. Substrate activation of /3- (13) -glucan synthetase and its effect on the structure of ,B-glucan obtained fromUDP-D-glucose and particulate enzyme of oat coleoptiles. Plant Physiol.51: 998-1001.

48. VAN DER WOUDE, XV. J., C. A. LEMBI, AND D. J. MORRE. 1972. Auxin (2,4-D)stimulation (in viro and in vitro) of polysaccharide synthesis in plasmamembrane fragments isolated fromn onion stems. Biochem. Biophys. Res.Commun. 46: 245-253.

49. VAN DER WOUDE, WV. J., D. J. 'MORRE, AND C. E. BRACKER. 1971. Isolationand characterization of secretory vesicles in germinated pollen of Liliumlongiflorum. J. Cell Sci. 8: 331-351.

50. VILLEMEZ, C. L. 1970. Characterization of intermediates in plant cell wallbiosynthesis. Bioclhem. Biophys. Res. Commun. 40: 630641.

51. VILLEMEZ, C. L., J. M. MICNAB, AND P. ALBERSHEIM. 1968. Formation ofplant cell wall polysaccharides. Nature 218: 878-880.

52. WOODING, F. B. P. 1968. Radioautographic and chemical studies of incorpora-tion into sycamore vascular tissue walls. J. Cell Sci. 3: 71-80.



f-glucan synthetases plasma membraneand golgi apparatus ... · sis products was determined by...

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