Plant Physiol. (1975) 56, 34-38

The Site of Cellulose SynthesisCELL SURFACE AND INTRACELLULAR 3-1,4-GLUCAN (CELLULOSE) SYNTHETASE ACTIVITIES INRELATION TO THE STAGE AND DIRECTION OF CELL GROWTH'

Received for publication September 11, 1974 and in revised form February 3, 1975

GORDON SHORE,2 YVES RAYMOND, AND GORDON A. MACLACHLANDepartment qf Biology, McGill University, Montreal, Quebec, Canada

ABSTRACT

,B-1,4-Glucan (cellulose) synthetase activity (UDP-glucose:,3-1 ,4-glucan-glucosyl transferase) present at cell surfaces ofgrowing regions of Pisum sativum epicotyl was assayed bysupplying UDP-'4C-glucose directly to thin slices of tissue.Initial rates of glucosyl transfer under these conditions ap-proached the rates of cellulose deposition observed in vivo inintact tissue at various stages of growth. Normal tissue homo-genization procedures destroyed the high surface activity,although a small amount of residual activity (3-10% of total)could be detected in particulate fractions. In homogenatesfrom elongating tissue, the residual activity was almost entirelyassociated with Golgi membrane. In homogenates of tissuewhich had ceased elongating, whether because of normalmaturation or treatment with ethylene (or high levels ofauxin), the activity was present in Golgi plus a membranefraction rich in smooth endoplasmic reticulum vesicles. It issuggested that cellulose synthetase activity associated with thesetwo organelles represents intracellular enzyme in transit tospecific sites of cellulose synthesis and microfibrillar orienta-tion at the cell surface.

In higher plants, alkali-insoluble ft-1 ,4-glucan (cellulose)formation is probably catalyzed by enzyme complexes locatedat the cell surface. These can be assayed by supplying sugarnucleotide substrates directly to intact cells or tissue slices(5, 7, 21). Surface enzyme must be extremely labile, however,because standard homogenization procedures result in almostcomplete destruction of its activity (21). Thus, cell-free systemscontain membrane-bound cellulose synthetase activities whichtogether account for a very small fraction of either cell-surface activity (21) or the normal rates of cellulose depositionobserved in vivo (1, 2, 9, 21). In homogenates derived fromelongating or swelling tissues, synthetase activity is foundapparently associated with Golgi membrane or ER3 vesicles(e.g., in peas; see refs. 15, 21), or Golgi plus plasma membrane(e.g., in onion; see ref. 23). The small amount of plasma mem-

'This study was financed by operating grants to G.A.M. andscholarships to G.S. and Y.R. awarded by the National ResearchCouncil of Canada.

Present address: National Institute for Medical Research, TheRidgeway, Mill Hill, London NW7 1AA England.

'Abbreviation: ER: endoplasmic reticulum.

brane synthetase in onion presumably represents residualsurface enzyme which has survived homogenization. Golgiand ER vesicles, however, are organelles which function inthe transport of products, including extracellular proteins, andin higher plant tissues neither of these organelles forms cellu-lose in vivo (3, 21, 24). Accordingly, it has been suggested (21)that such intracellular membrane-bound cellulose synthetase(s)are not active in vivo but are enzymes in transit to sites ofaction at the cell surface.The present work extends these observations to show that

cell-surface ft-1,4-glucan synthetase activity levels are com-parable in magnitude and correlated with rates of cellulosedeposition in vivo under a variety of conditions. Synthetaseactivities associated with ER seem to develop during cessationof elongation, whether this is attributable to normal cell matu-ration or to treatment of intact pea epicotyls with swellingagents.

MATERIALS AND METHODS

Plant Material. Pea seeds (Pisum sativum L. var. Alaska)were surface-sterilized with 0.5% NaOCl (20 min), soaked for8 hr in tap water at 25 C, and grown on vermiculite in 10-literglass jars at room temperature in darkness, until third inter-nodes reached 3 to 5 cm in length (7-8 days). Routinely, inexperiments involving ethylene treatment, a region 5 mm longimmediately beneath the hook was marked at zero time todelineate a "segment" of tissue (see also refs. 20, 21). Jarswere then sealed and epicotyls were subjected to continuousflow of air + ethylene (100 /d/l, 75 l/hr). Tissue developingfrom apical segments was analyzed after a further 12 or 48hr in darkness at room temperature.

Separation of Total Cytoplasmic Membranes and Cell WailFractions. Segments were excised, and fresh weight and lengthwere determined. The segments were homogenized vigorouslyat 2 C using a mortar and pestle in 2 volumes of a solutioncontaining 0.1 M tris, pH 8, 5 mm dithiothreitol, and 0.4 Msucrose. The homogenate was squeezed through nylon andthe residue was rehomogenized and combined with the filtrate.This total homogenate was centrifuged at 5OOg for 20 min tocollect the cell-wall fraction. The supernatant was centrifugedagain at 130,000g for 20 min to yield a particulate fractionwhich contained cytoplasmic membranes and most (>80%)of the alkali-insoluble ft-1,4-glucan synthetase activities whichcan be detected after thorough homogenization of this tissue.Such synthetase activities are negligible in supernatant frac-tions and relatively very low in washed cell walls. Cytoplasmicmembrane fractions were resuspended at 2 C using a Teflonpestle in 0.5 to 1 ml of 0.1 M tris, pH 8, and 5 mm dithiothre-itol.

34

SITE OF CELLULOSE SYNTHESIS

In order to estimate cellulose levels in epicotyl segments,

cell-wall fractions (from 25-50 segments) were washed for

three 10-min periods with hot (85 C) H20 (30 ml total) fol-

lowed by 10% NaOH (10 ml for 4 hr, at 25 C) and two 1-hr

periods with 85 C 1 N NaOH (20 ml total). Alkali-insolubleresidues were neutralized, dissolved in 72% (w/w) H2SO,, and

cellulose was measured as the anthrone-positive component

(10).Sucrose Density Gradient Proffles. Particulate fractions from

tissue segments were prepared for sucrose gradient analysis

(21) by a chopping technique (12) which avoids vigorous

homogenization. Segments (3-6 gm) were chopped with

razor blades on a cold (2 C) surface in 0.3 volumes of a

medium containing 0.1 M tris, pH 8, 0.1 mm MgCl2, 5 mM

dithiothreitol, and 0.4M sucrose. The brei was passed through

nylon and the filtrate (2.5-3 ml) was centrifuged at 5OOg for

20 min. An aliquot of the supernatant (2 ml, containing 2-7

mg of particulate protein) was layered on a 10-ml, 25 to 55%

(w/v) linear sucrose gradient and centrifuged (2 C) in an IEC

SB283 rotor at 20,000 rpm for 2 hr, by which time isopycnic

sedimentation of cellulose synthetase activity had been

achieved. A total of 20 fractions (0.5 ml) were collected and

used for measuring synthetase activity, refractive index, and

protein (1 1)./8-1,4-Glucan (Cellulose) Synthetase Assays. For assays of

synthetase activity in isolated membrane preparations, aliquots

of 100 gl, containing up to 500,ag of particulate protein,

from either sucrose gradient fractions, or resuspended 500 to

1 30,000g pellets were incubated in a standard reaction mixture

(275ul total) containing 0.1M tris, pH 8, 11 mm MgCl2, and

5 mM cellobiose, and relatively high (600 uM) or low (6 gM)substrate concentrations of either UDP-14C-glucose or GDP-

4C-glucose. Reaction mixtures were shaken for 7 min at 35

C and reactions were terminated by boiling for5 to10 min.Whatman cellulose powder (20 mg) was added as carrier for

radioactive products and the mixtures were washed three times

(10 min each) with hot (85 C) H20 (15 ml total). Insoluble

products were further extracted for two 15-min periods with

hot (85 C) 1 N NaOH (16 ml total). Alkali-insoluble residues

were neutralized, collected on glass fiber filters, dried, and

radioactivity was measured in scintillation fluid with a Beck-

man CPM-100. One unit of activity is equivalent to one pmole

of '4C-glucose incorporated into alkali-insoluble glucan/7 min.

Because very little (<5%) of the synthetase activity recovered

in these fractions was found to be associated with plasma

membrane under any of the conditions used in this or previous

(20, 21) studies, it is referred to here as intracellular synthetase.

It has been demonstrated (14, 20, 21) that >90% of the "C-

glucose transferred to alkali-insoluble product by such intra-

cellular synthetases, using either substrate at 6 or 600 jM, is

present in /3-1,4-linked glucan, and the composition of this

product was not altered by changes in the intracellular location

of enzyme (data not shown).For measurements of cell-surface synthetase activity (21),

thin transverse slices of epicotyl (0.2-0.4 mm thick, containing

mostly intact cells) were obtained from 2 to 4 segments and

collected in a graduated conical tube. Extraction medium

(2 C) was added until slices plus medium reached a volume of

100,ud. Synthetase activity was assayed using 600 uM UDP-

glucose as described above except that a thin glass rod was

inserted into reaction mixtures to keep slices suspended while

agitated during incubation. It has been repeatedly observed

that formation of alkali-insoluble glucan proceeded at a

linear rate for at least 10 min in these preparations with no

detectable lag period. All reactions were performed in dupli-

cate or triplicate (average deviation about 5%). Control ex-periments using sucrose-"C as substrate (21) indicated thattransfer of radioactive glucose from UDP-glucose by tissueslices occurred much more rapidly than synthesis from sugarsor their phosphorylated derivatives, as would be expected ifUDP-glucose were utilized at the cell surface without enteringintracellular pools.

Prolonged hydrolysis of the alkali-insoluble glucan formedin these reactions using high concentration of purified /-1,4-endoglucanase (5 mg/ml of Streptomyces cellulase for 5 daysat 50 C) produced only glucose and cellobiose (no lami-naribiose) as the major products, plus minor componentschromatographing with cellotriose and cellotetraose. Brieferincubations (1-2 days) produced these same products plusorigin material which was presumably undigested cellulose.None of the products was soluble in lipid solvents or washydrolyzed by Rhizopus /3-1, 3-glucanase.

Chemicals. GDP-14C-glucose and UDP-"C-glucose werepurchased from International Chemical and Nuclear Corp.and New England Nuclear Corp., respectively, and were usedat specific radioactivities of 1.5 mCi/mM (600 pLM substrate)or 150 mCi/mM (6 FLM substrate). Unlabeled glucose-diP-nucleosides were from Sigma Chemical Co. Ethylene waspurchased from Fisher Scientific.

RESULTS AND DISCUSSION

Growth and Cellulose Deposition. Figure 1 illustrates thechanges which occur in rates of growth and cellulose depositionduring development of the hook region into stem tissue ofintact pea epicotyls between 7 and 10 days of age. Rates ofelongation increased exponentially reaching approximately 10times the initial levels by 30 hr, and then declined to near zerogrowth by 60 hr. Rates of alkali-insoluble glucan (cellulose)deposition also increased, but after a lag period of severalhours. The deposition levels reached about 14 times initiallevels by 44 hr, before beginning to decrease. Both UDP-glucose- and GDP-glucose-dependent synthetase activities (as-saying only alkali-insoluble glucan formed by intracellularmembranes) also reached peak levels at about 2 days, i.e., ata time when growth rate was declining rapidly but cellulosedeposition was maximal.

Effects of ethylene on growth and cellulose deposition inintact pea epicotyls are shown in Table I. At zero time, a5-mm segment of epicotyl was delineated in the region im-mediately below the hook. These zero-time segments wereequivalent to tissue which had developed from the hook atapproximately 12 hr (Fig. IA), i.e., tissue at the start of thestage of most rapid cell elongation. Two days later, controlsegments had increased 5-fold in length and fresh weight (noswelling), while cellulose per segment had increased 6- to 7-fold. These major changes were accompanied by modest (50%)increases in particulate protein. Exposure to ethylene (100ul/l) prevented elongation of segments, and caused somelateral expansion, albeit not as much swelling as that resultingfrom treatment with high levels of auxin (cf. 20, 21). Ethylenealso prevented net increases in particulate protein and severelyreduced cellulose deposition per segment (increment in 48hr was only about one-third of control increment). In contrast,ethylene had no effect on the cellulose content per unit freshweight of tissue segments. This implies that average cellulosecontent per unit area of cell wall (i.e., wall thickness) remainedunaffected by ethylene treatment because segments+ ethylenecontained approximately the same number of cells.

Synthetase Levels in Elongating versus Swelling Tissue.Table I also compares changes in levels of recovered particu-

35Plant Physiol. Vol. 56, 1975

SHORE, RAYMOND, AND MACLACHLAN

12~-

6

CD1

CA)

0LJ

0

-c:

1--

6

0t ,10 24 48 7 2

TIME (HOURS)FIG. 1. Changes in levels of intracellular alkali-insoluble B-1,4-

glucan synthetase activities and rates of elongation and cellulosedeposition during growth of the hook region of intact pea epicotyls.Hooks of 7-day-old epicotyls were delineated with ink and tissuedeveloping from this segment was analyzed at various times forlength, cellulose content, and synthetase activity. In 72 hr, thehook increased in length from 2.7 to 46.1 mm, cellulose levelsincreased from 22 to 984 usg/segment and particulate protein rose

from 48 to 97 Ag/segment. Points on the graph for rates ofcellulose deposition and growth represent values for increments,plotted at the midpoint between sampling times relative to initialincrements. Values for the initial increments were: elongation =0.1 mm/hr-segment, measured between zero and 6 hr; cellulosedeposition = 1.9 ueg/hr segment, increment to 12 hr. Synthetaseactivities are plotted relative to those recovered from particulatefractions of the hook at zero time, i.e., 34 units using 600 ,uMGDP-glucose as substrate, (-*-) and 27 units using 600 ,sMUDP-glucose (-X-). In the experiments described in Table Iand Figure 2, subapical 5-mm segments at zero time are approxi-mately equivalent to tissue which develops from the hook at Ain this figure, and segments at 12 and 48 hr are equivalent to tissueat B and C, respectively.

late (intracellular) and tissue-slice (cell-surface) componentsof total UDP-glucose-dependent alkali-insoluble glucan syn-thetase activity. At all times, intracellular synthetase activityrepresented only a small fraction (3-10%) of that which couldbe assayed at the surface. In control (elongating) tissue, intra-cellular synthetase levels increased, then declined (see alsoFig. 1). The magnitudes of these changes were not large, how-ever, compared to the changes in synthetase activity at thecell surface. The latter continued to accumulate for at least48 hr, eventually reaching 30 times intracellular levels at thetime when the rate of cellulose deposition was greatest. Whenthe tissue was induced to expand laterally rather than elongate,intracellular synthetase levels did not change but cell-surfaceactivity declined, and this was accompanied by a marked re-

duction in cellulose deposition. It is evident that under all ofthese conditions, the rates of cellulose deposition in vivo weremuch more closely correlated with the levels of synthetaseactivities assayed at the surface than with levels recovered inintracellular membranes.

It was previously shown (21) that disruption of cell structurein pea epicotyl tissue slices (as during normal homogenizationprocedures) results in almost complete loss of the high levelsof UDP-glucose-dependent synthetase activity which aredetectable at intact cell surfaces, and there is little recovery inplasma membrane-rich fractions. This could be attributable-to physical damage or disruption (e.g., dissociation of enzymefrom primer) of surface enzyme complexes or to release ofintracellular inactivators (22). In either event, present datademonstrates that initial rates of cellulose synthetase activityassayed with UDP-glucose in tissue slices (up to 5.6 ,ug glucoseincorporated/hr. segment, Table I) are within the range ofrates recorded for cellulose deposition in these same tissuesin vivo (3-9 ,ug glucose incorporated/hr segment, Table I;2-25 ,ug/hr * seg, Fig. 1).The high levels of cell-surface synthetase activity achieved

with 600 gM UDP-'4C-glucose as substrate are not attained intests using GDP-'4C-glucose (data not shown here), despitethe fact that intracellular membrane sites utilize both of thesesubstrates approximately equally well for synthesis of /3-1 , 4-glucan in vitro (refs. 20, 21, and Fig. 1). In similar experi-ments using semi-intact cotton hairs (5, 7), it has been demon-strated that both GDP-glucose and UDP-glucose are acceptedas substrates for glucan synthesis, although relative rates oftheir utilization vary greatly with age and development. Avariety of developmental tests with pea epicotyl slices haveyielded preparations which, at best, utilize GDP-glucose atone-quarter of the rate of utilization of UDP-glucose; thisrepresents no more than three times the total GDP-glucose-

Table I. Chaniges in Cellulose anid Levels of Initracelluilar anidCell-suirface Compontents of Alkali-inisoluble d-1, 4-gluscan

Synithetase Activity in Elongatinig (Conitrol) versutsSwelling (+ Ethylenie) Pea Epicotyls

At zero time, subapical segments (5.0 mm) were marked im-mediately below the hook of intact epicotyls. Tissue which de-veloped from these segments in darkness + 100 1/l of ethylenewas analyzed at the times indicated for length, swelling (increasein fresh wti'length), particulate protein (500-130,000g), and 600/AM UDP-glucose-dependent synthetase activity present either inthe particulate fraction (intracellular membranes, see Fig. 2) orat the cell surface in tissue slices (see "Materials and Methods").One unit of synthetase activity = 1 pmole of glucose transferredto alkali-insoluble glucan/7 min. It requires approximately 12 hrfor the hook region to develop into a 5-mm subapical segment oftissue; accordingly, the segment above at zero time has propertiescomparable to the tissue assayed in Figure 1 at point A.

Cellulose

Treatment of Parti- SnhtsIntact Seedlings Length Swelling culate Cellulosei Protein Intra- Cell

cellular surface

In,,,/' m?Zg/mm A1g/ Ag/ f g/ng units/segmpientsegment segment segment fresh wet.

Zero time 5.0 2.7 60 60 3.7 85 132512 hr control 9.5 2.8 68 98 3.7 145 2010+ Ethylene 5.5 3.5 61 74 3.9 85 122048 hr controll 24.5 3.1 85 408 5.3 115 3615+ Ethylene 6.0 5.3 64 175 5.6 75 755

cel lulosedeposition -

growtho

A BI I

synthetase- activity

3k

36 Plant Physiol. Vol. 56, 1975

I

SITE OF CELLULOSE SYNTHESIS

1.1gm/cc

or It~~~~~~~~~~~~~~~~~~~~~~~~I

S 4 8 12 16 20 S 4 8 12 16 20 S 4 8 12 16 20FRACT ON

FIG. 2. Cnanges during growth in the isopycnic sedimentation profile of alkali-insoluble B-1 , 4-glucan synthetase adtivity of particulate frac-tions czntrifuged through sucrose gradients. Profiles derived from pea epicotyl tissue which had developed from 60 to 180 subapical segments(5 mm) at the times indicated (other properties of these segments are shown in Table I). Synthetase activity in gradient fractions was assayedusing 6 AM GDP-glucose as substrate and are plotted relative to values for the most active fraction (set at 10). Similar results were obtainedusing 600 ,zM GDP-glucose and 6 or 600 ,uM UDP-glucose. Peaks of synthetase activity occurred in fractions 2 to 4 (1.11 gm/cc), which alsocontains ER vesicles (21), and/or fractions 8 to 10 (1.15 gm/cc) which contains Golgi membranes (15, 21).

dependent synthetase activity recovered in intracellular mem-branes.

Intracellular Localization. Figure 2 shows the distribution ofintracellular cellulose synthetase activity in isopycnic sedi-mentation profiles of membrane-bound material obtained fromsegments at zero time (A) and again after 12 hr (B) and 48 hr(C) in the presence or absence of ethylene. Typical rates oftissue elongation, cellulose deposition, and synthetase activityin control segments at these times are shown in Figure 1 (i.e.,by arrows at A, B, and C, respectively) and in Table I. Inelongating intact tissue (zero time and 12 hr controls), mostof the activity which was recovered in the gradient sedimentedas a single band (fractions 8-10) at density 1.15 gm/cc, whichrepresents Golgi-bound synthetase (15, 21). In our experience,nearly all alkali-insoluble glucan synthetase activity, measuredusing either substrate at high or low concentration, is re-covered in this one fraction whenever pea tissue which isactively elongating is used as source of membrane (e.g., intactepicotyls at zero time or decapitated epicotyls supplied withgibberellic acid). However, when the tissue is induced to swellby application of auxin (21), or elongation is prematurelyterminated by ethylene treatment (Fig. 2B), or normal elonga-tion is completed as in 48-hr controls (Fig. 2C), a distinct newpeak of synthetase activity is observed (fractions 2-4) atdensity 1.11 gm/cc. This is coincident with the location ofsmooth ER vesicles (21). The appearance of cellulose synthe-tases in such low-density membrane evidently accompanies thetransition from elongating to nonelongating tissue.

Golgi- and ER-derived vesicles are capable of transportingmembrane components and secretory products, includingenzymic protein, to the cell surface in plants (3, 13, 18).Radioautographic (3, 24) and biochemical (3, 21) studies ofhigher plant tissues indicate that neither of these organellesforms cellulose or low mol wt cellodextrins in vivo, despitethe /8-1,4-glucan synthetase associated with them. Since rela-tively much higher levels of cellulose synthetase activity couldbe detected at undisturbed cell surfaces and these were wellcorrelated with actual rates of cellulose deposition in vivo(Table I), the suggestion (21) is reiterated that the intracellularB-1,4-glucan synthetase represents normally inactive enzymewhich is in the process of being transported to the surface.The alkali-insoluble product formed by enzyme at the sur-

face, or formed in vitro by enzyme associated with Golgi or

ER, was characterized as consisting primarily of /8-1 ,4-glucan(see "Materials and Methods," and ref. 21). The possibility,however, that this enzyme may contribute to the formationof other noncellulosic products (e.g., xyloglucan) while locatedin endocytoplasmic membrane should not be ruled out. Forexample, membrane fractions from pea (14, 21) and maize (3)form soluble, noncellodextrin polysaccharides from glucosein vivo. Moreover, the membrane fractions containing suchnewly-synthesized polysaccharides co-sediment in sucrosegradients with either Golgi (14, 21) or, depending upon stageof cell growth, Golgi plus ER vesicles (21).The differences observed during growth in density of par-

ticles bearing glucan synthetase activity (Fig. 2, and ref. 21),as well as the results of histochemical studies (e.g., of esteraseactivity [8]), demonstrate that the principal intracellular locusof an enzyme may change during differentiation. This couldresult from altered rates of transfer and/or accumulation ofenzyme in different particles which have a precursor (e.g.,ER) to product (e.g., Golgi) relationship. It could also indicatethe existence of two distinct organelles or pathways for trans-porting enzyme to the cell surface.

In particular, the development, following the termination ofelongation or during swelling, of cellulose synthetase activityin ER-rich fractions (Fig. 2) seems to be related to concurrentchanges in the direction in which cellulose microfibrils arelaid down in lateral cell-walls. When elongation ceases, thereis a shift from mainly transversely-oriented microfibrils tomicrofibrils which are oriented primarily in a longitudinaldirection (4, 6, 16, 19). It is possible that ER-bound cellulosesynthetase becomes established at the cell surface in such away as to function in the synthesis of longitudinal microfibrils,which limit the rate of cell elongation but not necessarilylateral expansion. In contrast, Golgi-bound synthetases may bedestined to form transverse microfibrils, because this is themajor orientation in cells in which elongation is rapid butwhere lateral expansion is restricted (17), and no other cyto-plasmic synthetase locus is observed in the tissue at that time(Fig. 2, and refs. 15, 21).

Finally, it should be pointed out that studies of effects ofhormones and antibiotics on levels of particulate glucansynthetase activity (14, 22) have generally assumed that thechanges observed result from altered biosynthesis or metabolicturnover of essential components of the active enzyme com-

Plant Physiol. Vol. 56, 1975 37

38 SHORE, RAYMOND, AND MACLACHLAN

plex. These studies should be reevaluated to entertain the

possibility that increments and decay kinetics of these activitiesreflect accumulation and secretion by intracellular transport

organelles.

Acknowledgment-We are very grateful to Dr. E. T. Reese for samples of

purified glucanases.

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