changes molecular weight of cellttlose in pea epicotyl during growth1 · aliquots were used for...

Plant Physiol. (1972) 49, 58-63

Changes in Molecular Weight of Cellttlose in the PeaEpicotyl during Growth1

Received for publication June 4, 197

F. S. SPENCER2 AND G. A. MACLACHLANDepartment of Biology, McGill University, Montreal, Quebec, Catada.

ABSTRACT

A procedure is described for preparing cellulose nitrate frompea tissues (Pisum sativum L. var. Alaska) in quantitativeyield, undegraded and uncontaminated by other polysac-charides. The average degree of polymerization of this product,estimated from viscosity measurements, increased during cellgrowth and development from a value of about 5000 glucoseunits in the apical meristem (plumule plus hook) to valuesnear 8000 in fully grown maturing tissues (>20 mm fromapex). The cellulose content per cell also increased (approxi-mately 10-fold) during growth in these tissues, as did par-ticulate glucan synthetase activity (3-fold rise). Since theyield of soluble celiulase activity is known to decrease fromhigh values in the meristem to barely detectable amounts inmature tissues, it is suggested that the relative levels andproperties of these hydrolytic and synthetic enzyme activitiescontrol the amount and degree of polymerization of celluloseformed during cell expansion in the pea epicotyl.Degree of polymerization distribution patterns showed that

a low molecular weight component of cellulose (degree of po-lymerization < 500) was prominent in young tissues whereashigh molecular weight components (degree of polymerization> 7000) predominated in mature tissues. Also, cellulose whichwas formed from radioactive sucrose during 30 minutes ofincubation showed a remarkably similar degree of polym-erization distribution to cellulose which was present in thetissue at the time of synthesis. It is concluded that new and oldparts of the epicotyl cellulose framework are subject to con-stant modification and equilibration by cellulose-metabolizingenzymes.

The microfibrillar network of cellulose which encloses theprotoplasts of plant cells is extensive and interwoven even inthe youngest meristematic cells, and it becomes more massiveand complex during growth and maturation (24). The wallframework appears to be so restrictive that biochemical reac-tions which have the potentiality for controlling its depositionand "loosening" have often been proposed as regulators for cellexpansion (12, 15, 25). The problem has been to identify criti-cal reactions and define circumstances under which they aregrowth limiting.

1 This study was supported by the National Research Council ofCanada and a McConnell Fellowship to F. S. S.

2Present address: Ontario Hydro, Research Division, Toronto,Ont., Canada.

In the present study, estimates were made of the amount anddegree of polymerization and the DP3 distribution of cellulosein pea epicotyl tissues at different stages of development. As-says were taken or are available of levels of cellulase and cellu-lose synthetase activities in enzyme preparations from thesesame tissues. The intention was to look for evidence, i.e.. cor-relations, to help assess the likelihood that hydrolytic andsynthetic enzymes actually operate in vivo to control the massand chain lengths of the cellulose deposited during growth.Such correlations have been observed in Acetobacter xylinlumcultures (11). In the pea epicotyl, it is established (2, 23, 26)that enzymes that use activated glucose donors to form glucanwhich is f8-l ,4-linked and alkali-insoluble are localized inmembranes. These may be expected to retain their synthesizingcapacities during tissue development, because excised epicotylsegments continue to form cellulose long after they have ceasedgrowing (16). In contrast, enzymes which hydrolyze celluloseor its derivatives are present both in buffer-soluble and mem-brane fractions of peas (5, 8, 26). Almost all such activity isconfined to the youngest meristematic regions (4, 17). whichare the only parts where turnover of pea wall glucan has beendetected (16). Obviously, if cellulose metabolism in vivo can beattributed to these enzymes, and the ratio of synthetic to hydro-lytic activities increases during growth and maturation, themean cellulose chain length may rise because of it. Presentresults show that pea cellulose DP does indeed rise duringgrowth.

Marx-Figini working with cotton hairs (18, 21) also foundthat cellulose DP increases during cell expansion, i.e., whilethe primary wall is deposited. But during the whole period ofhair maturation, while most of the final cellulose content ofcotton cells is laid down, cellulose DP is maintained at a re-markably constant and very high level (close to 13,000 glucoseunits). Similar results were reported for cellulose in developingValonia cells (20). Marx-Figini (19) interpreted these findingsas meaning that cellulose for the secondary wall is formed ator near the protoplast surface on a genetically controlled tem-plate of the appropriate dimensions, but that cellulose in grow-ing cells is generated by a different mechanism which is "time-controlled," i.e., resulting in a gradual chain lengthening.

Applying these considerations to the pea epicotyl, it may besupposed that either (a) cellulose is deposited initially at a highDP in all cells and becomes subject to partial hydrolytic degra-dation in young cells only, or (b) chain lengthening of primarywall cellulose is a slow process requiring the whole of thegrowing period for individual molecules to reach maximumDP. In an effort to choose between these explanations. radio-active sugar was supplied briefly to living pea tissue sectionsand cellulose was isolated and fractionated to compare the DP

'Abbreviation: DP: degree of polymerization.58

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MOLECULAR WEIGHT OF CELLULOSE

distribution of pre-existing species with that of new (labeled)species. It was anticipated that the relative specific radioactivi-ties (cpm per unit weight or per mole) of various cellulosechain lengths would indicate which was precursor to the rest.

MATERIALS AND METHODS

Nitration and Extraction of Pea Epicotyl Cellulose. Peaseeds (Pisum sativum L. var Alaska) were surface-sterilizedwith 0.5% NaOCl, soaked in water for 8 to 10 hr, planted intrays of vermiculite, and grown in darkness for 8 days. Wholeepicotyls, internodes, or sections were harvested when thirdinternodes were 3 to 5 cm in length.

Cellulose was isolated using a nitration procedure (28),which yields a derivative convenient for molecular weight de-termination by viscometry (27). It has been shown that /3-1,4-glucosidic bonds are not ruptured by nitration using this tech-nique (30) and that cellulose is almost completely extracted(>95%) in 5 hr at 17 C from tissues containing variousamounts of lignin up to 23% (31). The etiolated pea seedlingsused in this study contain only 1 to 3% lignin (16), an amounttoo small to exert "protective" effects and alter cellulose ni-trate yields. By using pea cell walls and nitrating at 5 C, maxi-mum yields of cellulose nitrate, DP, and degree of nitrationwere obtained within 5 hr; further nitration for up to 20 hr un-der anhydrous conditions caused no change in these properties.Pea epicotyl tissue (60 g fresh weight) was extracted with

cold (+5 C) 20 mm sodium phosphate buffer, pH 7.0, followedby 90% ethanol. Conventional alkali treatment was avoidedbecause it can degrade cellulose (32). For some nitrations, awater wash was used in place of 90% ethanol, and the resultantcrude walls were freeze-dried. Completely dried crude wallswere nitrated at +5 C in an anhydrous mixture of HNO3-H3PO-P205 (64:26: 10, w/w) (1).

Nitration reactions were terminated by diluting and washingon a coarse sintered funnel with cold 50% (v/v) aqueous aceticacid and water. The product was neutralized at room tempera-ture with saturated NaHCO3 and 10% acetic acid, and finallywashed with copious amounts of water, then methanol. Theresidue was suspended in acetone which dissolved most of it;insoluble impurities were removed by centrifugation. The ace-tone solution was poured into a large excess of water (e.g., 10volumes) in order to precipitate the cellulose nitrate. The finalproduct was freeze-dried and weighed. The yield was approxi-mately 1 g. Aliquots were used for viscosity measurement andnitrogen assay.

Viscosity Measurement and Molecular Weight Calculation.Cellulose nitrate was dissolved in anhydrous acetone, and theviscous solution was clarified by filtration through a nylon filter(14 ,u). The initial concentration in the filtrate was determinedby evaporating and weighing duplicate aliquots. Another ali-quot was added to a Cannon-Ubbelohde semimicro dilutionviscometer, size 100 (Cannon Instrument Co., State College,Pa.). Efflux times were measured to the nearest 0.1 sec at 25.0C. For each sample, values were obtained for the solvent and atleast 5 concentrations of cellulose nitrate, diluting in the range0.01 to 0.1% (w/v). The specific viscosity, 77sp, was calculatedfrom the efflux times and extrapolation to zero of a plot of -g.p/concentration versus concentration gave the intrinsic viscosity(27). Since the conformation of cellulose nitrate molecules insolution changes with extent of nitration, it was necessary toestimate the nitrogen content of each sample and then apply anappropriate correction to viscosity values (13).The corrected intrinsic viscosity, [77]T, was used to calculate

the viscosity-average molecular weight M, using the Mark-Houwink equation [?f]T = KMVa (27). The values for constantsemployed through this study (K = 5.96 x 10-5, a = 0.91) were

determined by light scattering (10). They were applicable tointrinsic viscosities adjusted to a shear rate of up to 500 sec'.

Fractionation of Cellulose Nitrate (27). Fractional precipita-tion was carried out by dissolving 1 g of cellulose nitrate in820 ml of anhydrous acetone and stirring rapidly while 180 mlof 1:1 acetone-water were added. Cellulose nitrate of high DP(>10,000 glucose units) is barely soluble in this concentrationof aqueous acetone at 25 C. Rapid stirring was continued whilea filtered air stream was directed at the surface of the solutionin order to reduce the acetone content and slightly cool thesolution (by 3-4 C). This was sufficient to bring about precipi-tation of the cellulose nitrate with highest DP. The cloudymixture was brought back to 25 C and equilibrated for about30 min and then centrifuged to collect the first fraction. Thisprocedure was repeated with successive supernatants in orderto collect a number of fractions, each containing approximately50 mg of cellulose nitrate.

Chemicals and Assays. Cellulose in crude walls was esti-mated after extraction with 1 N NaOH at room temperaturefor 48 hr followed by extraction with hot (85 C) 1 N NaOH for2 hr. The anthrone method (9) was used with glucose as stand-ard. DNA was measured with diphenylamine (3). Glucansynthetase activity was assayed by a standard procedure (2) inwhich the initial velocity of transfer of labeled glucose fromguanosine diphosphate glucose (GDP-glucose-"C) to alkali-in-soluble glucan was measured in the presence of a particulatefraction (microsomes). Synthetase reaction rate was propor-tional to the concentration of microsomal protein. It was notcatalyzed by soluble fractions, it was activated by cellobioseand able to transfer glucose from either guanosine or uridinesugar nucleotides to exogenously supplied /3-1,4-linked primers(26). The assay is referred to here as a measure of cellulosesynthetase activity, with recognition that the alkali-insolubleproduct may not have been completely cellulose and not all,B-1 , 4-linked products were necessarily recovered in this frac-tion. Cellulase activity was assayed viscosimetrically (6, 7)with a soluble substrate (carboxymethylcellulose). The pea en-zyme also hydrolyzes cellulose powder, but the viscosimetricassay is much more sensitive (17).

Nitrogen content of cellulose nitrate (duplicate samples,10-15 mg) was measured after digestion with H2SO-salicylicacid with spectrophotometry and a modified Nessler's tech-nique (22). Nitrogen analyses were checked by a microcombus-tion method (Organic Microanalyses, Montreal, Que.). Proteinwas estimated by the Lowry method (14).

Radioactive cellulose was prepared by incubating 30 to 50 gfresh weight of excised apical or basal pea epicotyl sections in1 volume of 1 mm sucrose-U-14C (5 jsc/,umole). Three suchincubations were carried out in darkness at 25 C for 30 min.Sections were then washed with cold water, extracted, andnitrated. Radioactivity in aliquots of dried cellulose nitrate wasdetermined with a standard gas flow detector and counter.

Polysaccharide sources were: Koch-Light (Colnbrook,Bucks., England) for laminarin, lichenan, and galactan andFisher Scientific for potato starch and Whatman No. 1 cellu-lose. A pectin-protein fraction was obtained from crude peawalls by extracting with hot 0.1 M EDTA, pH 7.0, and precipi-tating with ethanol. Pea hemicellulose was prepared from crudewalls by extracting with 10% NaOH and collecting the precipi-tate which appeared upon adjusting the extract to pH 5.0 withacetic acid at 25 C. In order to separate sugar components,polysaccharides were dissolved in 72% (w/w) H2S04, diluted to1 N acid and hydrolyzed in an autoclave. The hydrolysate wasneutralized with Ba(OH)2 and chromatographed on paper in asolvent which was particularly effective at separating hexoses(7:1:2 ethyl acetate-pyridine-water, v/v).

59Plant Physiol. Vol. 49, 1972



SPENCER AND MACLACHLAN

Table I. Recovery and DP of Celluilose Nitrate Prepared fromMixtures of Cellutlose plus Other Natutral Polymers

DP is calculated by dividing M, (viscosity-average mol wt) bythe molecular weight of the repeating unit in cellulose nitrate(corrected for degree of nitration). The mean nitrogen content ofproducts was 12.70%F + 0.24 (SE, 14 observations). The mean yieldof cellulose as calculated from the yield of cellulose nitrate was

98' of supplied. Mean DP = 5980 4 210 (SE, 7 observations).

Mixture -Nitrated(1 g of cellulose plus

1 g of additive)

Cellulose only+ Potato Starch+ Laminarin+ Lichenan+ Galactan+ Pea pectin + protein+ Pea hemicellulose

YieldIntrinsiciscosity DP

Nitrated Cellulose l7u]product content

dl,/gg

1.731.771.751.801.501.671.82

0.99 19.0 65701.01 17.5 60001.00 15.8 53701.03 16.3 55400.86 17.1 58400.96 19.3 66701.04 15.5 5240

Table II. Recovery anzd Initrinisic Viscosity of Cellullose NitrateDerived from Differenit Sectionis of the Pea Epicotyl

Third internodes from approximately 13,000 seedlings wereexcised and the plumule and hook (P + H) together with 5- X5-mm sections (numbered consecutively from apex) were col-lected. Representative sections were extracted with 1 N NaOHand alkali-insoluble hexosan (cellulose) was estimated withanthrone. Remaining sections were extracted and nitrated asoutlined in Figure 1 (mean N content of samples = 12.64%' + 0.17;SE, 19 observations). The products were dissolved in acetone andviscosities were determined as described in "Materials and Meth-ods." Data for first and second internodes and whole epicotylswere obtained in a separate experiment.

YieldIntrinsic

Region of Epicotyl Alkali- Cellulose Viscosityinsoluble content of ihexosan cellulose nitrate

g per 100 g fresh wt dl/g

Third internodeP + H

234S

Second internodeFirst internodeWhole epicotyls

0.650.490.440.330.370.350.560.550.67

0.630.480.430.290.370.370.510.510.61

15.2

17.7

18.7

20.2

22.1

21.8

24.4

25.2

20.6

RESULTS AND DISCUSSION

Cellulose Nitrate Recovery. Nitrogen, sugar, and viscosityassays of the final product obtained from nitrated etiolated peatissues were consistent with the conclusion that it was indeedcellulose nitrate of high molecular weight. The evidence is as

follows.Firstly, nitration of native cellulose by present methods

should yield an acetone-soluble water-insoluble product whichis somewhat less than fully nitrated (theoretical nitrogen =

14.14% for cellulose trinitrate). In the present study the mean

nitrogen content of 18 samples which were precipitated bywater from acetone was 12.67%. Similar values have been re-

ported for cellulose nitrates from a variety of other plants (29).Secondly, several samples from peas were denitrated by themethod of Timell (28) to yield demonstrably nitrogen-free ma-terial which, following hydrolysis and chromatography, showedglucose as the only benzidine-positive component. Thirdly,samples of products which were derived from whole epicotylsand dissolved in acetone had high viscosities (e.g., [yj]T > 30dl/g) which indicates that the glucan must have been highlypolymerized. Similar viscosities obtained from solutions ofauthentic cellulose nitrate (29) have a DP of about 8000 (i.e.,equivalent to a cellulose molecular weight of 1.3 million). Theonly other glucan known to exist in higher plants with such ahigh DP is starch, and the epicotyl contains very little of it(16).

Finally, direct evidence was obtained to show that polymersother than cellulose did not interfere with or contribute to theyield of product nitrated and isolated as described above.Authentic cellulose was recovered quantitatively as cellulosenitrate, but various other glucans, cell wall components andprotein were not carried through the procedure (Table I). Theintrinsic viscosity and hence DP of the product was notchanged significantly by such additives (all values were withintwice the standard deviation of the mean). In further tests (Ta-ble II), it was observed that the amounts of alkali-insolublehexosan (cellulose) which were assayed with anthrone in vari-ous regions of the pea epicotyl corresponded closely to calcu-lated yields of hexosan in nitrated products which were pre-pared from these same regions.

Accordingly, the product isolated after nitration from peasby present methods is referred to henceforth as cellulose ni-trate.

Cellulose DP during Development. Sections were excisedfrom third internodes of 8-day-old pea epicotyls in such a wayas to delineate regions of meristematic tissue (plumule andhook), elongation (mainly apical 10 mm of epicotyl), and re-gions of increasing maturation (10-25 mm). Older parts of theepicotyl (second and first internodes) were also harvested.The amount of cellulose per unit fresh weight which was re-covered from these tissues either as alkali-insoluble material oras cellulose nitrate decreased during growth (from about 0.65-0.3%) but increased again (to about 0.5%) during maturation(Table II). This does not mean that cellulose was only synthe-sized during maturation but, rather, that the rate of deposition

Table III. Distributtiont of Alkali-ilnsoluble Glucant (Cellulose) Syll-thetase Activity Compared to Other Componients in the

Pea EpicotylSections of tissue (labeled consecutively as in Table II) were

homogenized in 0.1 M tris buffer, pH 8, and a particulate fraction(10,000-140,000g) was used as source of synthetase activity. Thereaction system contained in a total volume of 275 ,ul: 0.1 to 1.0mg protein, 1 nmole GDP-glucose-14C, 25,moles tris, pH 8, 2.5,umoles MgCl,, 2,g cellobiose (see ref. 26). Units of synthetaseactivity = pmole glucose-14C incorporated into glucan insolublein hot (85C) 1 N NaOH in 10 min per mg particulate protein. Othercomponents of the tissue were estimated as described in "Mate-rials and Methods."

Sections Cellulose Fresh DNA Particulate CelluloseSynthetase Weight IProtein

iprotett mg/sect jAg/ sect

P + H 10.5 18.5 35.0 245 981 + 2 29.1 20.8 8.1 70 1023 +4 28.3 25.5 3.1 35 935 + 6 26.5 27.3 2.7 23 98

60 Plant Physiol. Vol. 49, 1972




did not keep pace with the great increase in fresh weight duringthe period of most rapid cell expansion. Indeed, the averagepea meristematic cell volume increased at least 20-fold by thetime it reached the level of maturation found in basal sectionsof the third internode (estimated by relative values for freshwt/DNA, see Table III and ref. 4). Cellulose levels per unitDNA increased about 10-fold during this period of develop-ment.

Values for intrinsic viscosity of the cellulose nitrate isolatedfrom these sections are given in Table II and DP calculatedfrom these values is shown in Figure 1. Cellulose in the mostapical region had the lowest observed DP (5200). Elongationand maturation in the third internode was accompanied by asteady increase in DP. The older tissues in the second and firstinternodes contained cellulose with the highest DP (8400-8700). Cellulose extracted from whole epicotyls also had arelatively high DP (8200) because most of it (>80%) was de-rived from the first two internodes.

Relative Hydrolase and Synthetase Activities during Devel-opment. Levels of cellulase activity were assayed earlier (17)in enzyme extracts from the same sections of the third inter-node which were used for measuring cellulose DP (Fig. 1,Table II). Expressed on a fresh weight, protein, DNA, or cellu-lose basis, the plumule and hook region was found to be richin this enzyme activity, but its concentration fell rapidly as cellsexpanded until it was barely detectable in more mature regionsof the epicotyl. There is evidence (6-8, 15) that pea cellulase issubject to turnover within hours in the epicotyl, and it is in-ducible there by the auxin type of growth hormone. Thus, it isnot surprising that cellulase activity is concentrated at the apexof the epicotyl since natural auxin is well known to be gener-ated and retained at relatively high levels in such meristematicregions.

In contrast, alkali-insoluble glucan (cellulose) synthetase ac-tivity was easily detected in particulate fractions isolated fromall parts of the epicotyl (Table III). Calculated on a particulateprotein or DNA basis, activity increased (about 3-fold) duringelongation and continued high during maturation. Celluloselevels per unit DNA also rose throughout this development.

Clearly, these data are consistent with the conclusion thatthe gradual increase in amount of high-DP cellulose which oc-curs during development in the epicotyl is due to improved

In

6

mC:)0--4cPli

LAJm-i

C:oCl-

L,C)

L'iLLJ

C-D.

m

6

4

2

0

100

80 F

C2-

5 60

< 40CD

20 F

0 2 4 6 8DEGREE OF POLYMERIZATION x10-3

0

FIG. 2. DP distribution of total and radioactive cellulose nitratein samples extracted from apical and basal regions of the pea epi-cotyl. Fractions were collected and analyzed as described in TableIV; weights and cpm of each fraction were calculated as a per-centage of total values and summed in sequence beginning withthe lowest DP fraction. These values were plotted versus DP togive cumulative (integral) distribution curves. The inserted graphwas obtained by differentiation of the cumulative percentage weightcurve.

Table IV. Cellulose Nitrate Fractionis from Apicaland Basal Regions of the Pea Epicotyl

Sections from approximately 4000 third internodes were sup-plied with dilute sucrose-14C (2.76 cpm/mAmole) for 30 min andthen extracted and nitrated and fractionated as described in "Ma-terials and Methods." A total of 1.39 g of cellulose nitrate(13.41% N, mean DP 4600) was obtained from 121 g fresh weightof apical tissue (3 sections: plumule + hook + apical 5 mm ofepicotyl, cf. Fig. 2). Basal tissue (2 sections cut 20-25 mm and25-30 mm from apex) yielded 0.97 g of cellulose nitrate (13.34%N, mean DP 6200) from 131 g fresh weight. Cellulose contents ofthe nitrates were 0.64 g and 0.41 g per 100 g fresh weight respec-tively; alkali-insoluble hexosan contents measured in the origi-nal crude walls were 0.64 and 0.45 g per 100 g fresh weight.

Fraction

INTERNODAL SECTIONS

FIG. 1. Mean DP of cellulose in different regions of the peaepicotyl. DP was calculated from intrinsic viscosity values (TableII) corrected for degree of nitration.

1

2345

67891011121314

Apical Region

! DP

753060705690375026501830149012407303751001059595

Basal Region

Weight

mg

114.366.242.632.345.424.419.029.015.6

Radio-activity

cpm X 103

114.170.072.830.548.120.915.025.012.8

DP

92307300601045001710675375590300

Radio-Wveight activity

cpm X 10'3

105.922.518.245.635.924.820.562.320.235.129.415.743.122.2

mg

199.835.236.468.736.729.522.080.223.032.042.535.593.153.5

8H

1St

In

AI

61Plant Physiol. Vol. 49, 1972



62

12

8

4

0

0 2 4 6 8

24

24

L.L-

E

\

CL-

DEGREE OF POLYMERIZATICFIG. 3. Specific radioactivities of variouso

calculated on a weight basis (A) and on a mc(B). Original data are recorded in Table IV.

chances for survival of the long chains fori.e., a reduced turnover rate with age.

DP Distribution of Cellulose Nitrate.tissue sections were excised from apical a

third internodes of pea epicotyls and inculradioactive sucrose in order to synthesizelabeled cellulose. The total cellulose in thnitrated and dissolved in acetone and fractiDP. Weights, cpm, and DP of the fracticTable IV. Cumulative values are graphdifferential distribution curves in Figure 2.ties of the fractions are shown in Figure 3.

In this experiment, apical sections yielcmean DP which was less by about one-thicellulose from basal sections (Table IV).due partly to the fact that apical sectionsDP cellulose than basal sections (e.g., 3515% of basal cellulose had a DP below 50tions contained less high DP cellulose than15% versus 35% respectively with DP >development from meristematic to matureproportion of cellulose with intermediate(50% remained with DP between 500 andof high DP-low DP cellulose increased ma

This does not necessarily mean that 1

converted to high DP cellulose during grow

are relative proportions only, they refer tosis and survival of various cellulose spec

ages. It is clear, particularly from differ(Fig. 2), that the major cellulose compone

500 which dominates young tissues is stiltissues. Since the latter contain much mo

than young tissues (Fig. 1), the absolute z

DP component per cell actually increase (Pea seedlings are probably not unique

Insofar as other studies have been made, ce

relatively low in young tissues, e.g., growi19, 21) and high in mature tissues, e.g.,

Moreover, auxin-induced cellulase activ(generated) in growing tissues (7, 15), althoisis continues in plant cells after growthuntil senescence begins (16, 19, 25, 26). V

tionships hold, there is no need to invoke fent mechanisms (e.g., time-controlled versu

lating cellulose deposition at different stag(

Cellulose Synthesis in Vivo. The newly swhich was derived in 30 min from sucro

SPENCER AND MACLACHLAN Plant Physiol. Vol. 49, 1972

basal tissue sections (Table IV) accounted for only a small frac-B/ tion of the total cellulose(less than 0.02%). Nevertheless, asshown in Figure 2, even in this short time period the labelbecame located throughout the DP range of cellulose, and the

/BASAl DP distribution curves for total and labeled cellulose weresimilar. Obviously, pea cells form cellulose of maximum DPin much less time than the cells take to reach maximum size.

APICAL Examination of the relative specific radioactivity of variouscellulose fractions (Fig. 3) shows that, in apical sections only,cellulose with low DP was somewhat more radioactive per unit

1/ weight than cellulose with high DP. Expressed on a molarI I I (chain end) basis, the data also indicate that apical sections

0 2 4 6 8 were a little deficient in their ability to produce and retain new

cellulose chains with high DP. These differences were small,)N X 10-3 and it is impossible to deduce from the data whether an excess

ellulose chain lengths of low DP cellulose was generated as such or derived from highDlar (chain end) basis DP cellulose by hydrolysis. Evidently, if one particular chain

length of cellulose were formed first and acted as precursor tothe rest, a biosynthetic period of less than 30 min would be

med in older tissue, needed in order to demonstrate its existence unequivocably.It is difficult to imagine how the observed DP distribution

A large number of patterns of newly formed and pre-existing cellulose could be so

ind basal regions of similar unless new and old parts of the cellulose frameworkbated for 30 min in were susceptible to constant modification and equilibration bya small amount of the particular levels of cellulose-metabolizing enzymes which

te sections was then are found in the wall at different developmental stages. It

ionated according to should be noted that if synthesis occurs solely by elongationans are recorded in of pre-existing chains, then long chain primers would have toed as integral and be correspondingly more available for addition than short chainSpecific radioactivi- primers in order for final specific radioactivities to be compara-

ble on a weight basis. Alternatively, representative samples ofled cellulose with a pre-existing chains could act either as templates for de novo

ird than the DP for synthesis of a population of chains of the same length, or asThe difference was targets for the rupture of chains at regular intervals followedcontained more low by insertion of new glucose units. An insertion and repairi% of apical versus mechanism for cellulose synthesis could explain the fact that'0). Also, apical sec- naturally occurring microfibril ends are rarely observed inbasal sections (e.g., electron micrographs of the growing wall framework (24). On7000). Thus during the basis of present evidence, none of these alternatives can betissue in peas, the eliminated from discussions of the manner in which cellulose isDP did not change deposited in the primary wall.7000) but the ratio1rkedly. LITERATURE CITEDow DP cellulose isrth; the above figures 1. ALEXANDER, W. J. AND F. L. MITCHELL. 1949. Rapid measurement of cellulose

net rates of synthe- 'viscosity in the nitration method. Anal. Chem. 21:1497-1500.2. BARBER, G. A.. A. D. ELBEIN, AND W. Z. HASSID. 1964. The synthesis of

ies at different cell cellu-lose lv enzyme systems from higher plants. J. Biol. Chem. 239: 4056-ential curves (inset, 4061.nt with DP around 3. BIURTON, K. 1936. A study of the conditions and mechanism of the diphenyl-1 present in mature amine reaction for the colorimetric estimation of deox-'ribonucleic acid.

1ioclieiii. J. 62: 315-323.Ire cellulose per cell 4. DATKO. A. H. AND G. A. IMACLACHLAN. 1970. Patterns of developnment of

amounts of this low gis-cosidase activities in the pea epicotyl. Can. J. Bot. 48: 1165-1169.

during development. 5. DAVIES. E. VND G. A. 'MACLACHLAN. 1968. Effects of indoleacetic acid on in-tracellular distribution of /3-glucanase activities in the pea epicotyl. Arch.in these propertles. 3Bioeliemii. Biophys. 128: 595-600.

1llulose DP is always 6. D1) VIES, E. AND G. A. -MACLACHLAN. 1969. Generation of cellulase activity

Lng cotton hairs (18, (utriing protein synthesis by pea microsomes in vitro. Arch. Biochemii. Bio-

wood (28, 29, 31) py.129: 581-587.iso concentrat1)d 7. FAN,. D. F. AND G. A. MACLACHLAN. 1966. Control of cellulase activity by'ity iS concentrated incloleacetic acicl. Can. J. Bot. 44: 1025-1034.

ugh cellulose synthe- 8. FAN, D. F. AN'D G. A. MACLACHLAN. 1967. Studies on the regulation of cellu-

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Plant Physiol. Vol. 49, 1972



changes molecular weight of cellttlose in pea epicotyl during growth1 · aliquots were used for...

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