Plant Physiol. (1968) 43, 1075-1082

Invertase Activity and Cell Growth in Lentil Epicotyls1Konrad Seitz2 and Anton Lang

M:SU/AEC Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48823

Received February 23, 1968.

Abstract. The activity of invertase and its relation to growth were studied in the epicotylsof lentil seedling,s incubated in the presence and absence of gibberelflic acid (GA).

Invertase activity per epicotyl increases relatively more rapidly than does length, reachesa maximum during most active elongation, and declines upon cessation of growth.

GA3 enhanoes both growth and increase in invertase activity, without altering the kineticsof the 2 processes. If GA3 is added during incubation invertase activity increases more rapidlythan does elongation rate.

Incubation of the seedlings in solutions of polyethyleneglycol inhibits the increase of bothgrowtih and invertese sotivity, the latter actually undergoin.g a decline, but coauses no greatchange in the relative effect of GA3* In presence of polyethyleneglycol GA3 has howevera relatively greater effect on invertase activity than on growth.

Sugars in the incubation medium have no significant effect on growth and invertase activityin the epicotyl, except inhilbition at relatively high concentrations.

Cycloheximide, actinomycin D, and 5-fluorodeoxyuridine (FUDR) inhibit both growth andthe increase in invertase activity. Added during incubation cycloheximide oauses oompleteinhibition of growth and a decrease in invertase activity with no appreciable lag phase. Withactinomycin D and FUDR the inhibition oocurs after lag periods of 2 to 3 and of at least10 hours, respectively. Thus the increase in enzyme activity is very probably based on de-novosynthesis, and the enzyme is in a state of turnover during growth.

The enzyme is present in soluble form in the cytoplasm, not firmly bound to any cellstructures.

Expansion growth of plant cells and the actionof plant growth hormones in this process dependupon synthesis of RNA and protein, and in somecases also on DNA synthesis (e.g. 2, 11, 13, 14).It seems possible that among the proteins which aresynthesized during cell growth are enzymes whichin turn play a regulatory function in growth, e.g.by determining the availability of substrates. Onesuch enzyme could be invertase. Invertase can beenvisaged to control cell growth by regulating thehydrolysis of sucrose an,d also its transport to cellsas well as between different compartments of thecell. It is known to have its maximal activity inthe most rapidly elongating part of growing roots(9, 15) and stems (6), and the activity has beenfound to increase when the growth of the tissue waspromoted by applied gibberellin (8, 10, 16).

In an effort to learn more about the possiblefunction of invertase in the growth of plant cellsand especially in cell expansion the relationshipbetween the activity of this enzyme and growtlh wasstudied both in presence and absence of exogenousgibberellic acid (,GA,). The experimental systemconsisted of epicotyls of etiolated lentil seedlings.

'Supported by the United States Atomic EnergyCommission under Contract No. AT(11-1)-1338.2 Present address: Botanisches Institut der Universi-tat, D-63 Giessen, Germany.

It was chosen because the lentil epicotyl, i.e. theinternode between the points of attachment of thecotyledons and of the first scale leaf, is morpho-logically a clearly defined organ, is known to elon-gate predominantly by cell expansion, and is re-sponsive to gibberellin (13).

Materials and Methods

According to the methods of Nitsan and Lang(13), lentil seeds (Lens culinaris Medik. = L.esculen7ta Mloench, cv. Persian; Washington StateUniversity, Pullman, Washington) were soaked for8 hours in distilled water and then germinated for4C0 hours in the dark on moist filter paper. Afterthis time the seed coats were removed and the radiclecut back. Seedlings in this condition are referredto as seedlings in the initial state. The seedlingswere then incubated for the experimental treatmentsunder shaking in the dark in 0.1 Hoagland's nutrientsolution containing 1000 units of penicillin GK perml. For incubation with sugars the seedlings weresurface sterilized and chloramphenicol (20 ug/ml)was added as an additional bacteriostatic agent. Theantibiotics had, at the concentrations used, no effecton growth or on invertase activity. Usually therewere 20 seedlings per 20 ml of solution. Allhandling and transfer operations were carried outin daylight.

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PLANT PIIYSIOLOGY

Elongation and the activity of invertase weremeasured only in the epicotyls. The invertase ac-tivity was measured in the following wav. Fortyor 60 epicotyls were ground in a mortar with sandand 10 ml of phosphate buffer (0.02 M; pH 5.3).The extract was then centrifuged for 8 minutes at1200 X g to clear it from sand, uinbroken cells and\xrall debris. The activity of the cell-wall fractioncan be neglected, as will be shown below. To analiquot of the supernatant an equal volume of 0.1 Msucrose, dissolved in buffer, was added. The sugarconcentration was determined polarimetrically at thebeginning and the end of a 2-hour period. Underthese conditions the enzyme activity was well withinthe linear range with regard to both enzyme con-centration and duration of assay. The only productsof sucrose hydrolysis were 2 substances which co-chromatographed on paper (ethyl acetate-pyridine-wvater, 8 :2 :1 ) with fructose and glucose, and therewas no hydrolytic action on maltose. There is no(loubt that the lentil-epicotyl enzyme is a typical plantinvertase (beta-fructofuranosidase). The sucroseconcentration used, while resulting in maximal rateof hydrolysis and thus optimal for the activity test,was too low to observe formation of trisaccharides.

Protein was measured in the extract bv theLowry method, using bovine serum albumin as astandard. The temperature for germination, incu-bation and enzyme assay was 220. All experimentswere run in duplicate and repeated at least 3 times.

Results

Timte Course of Growth and Invertase Activity.Length, invertase activity, and protein content inlentil epicotyls at the start of the experimental treat-ment are given in table I. For a quantitative com-

parison of the changes in invertase activitv andlength during growth the values of these parameters

are given as percent of this initial state.Upon incubation of the seedlings in basal medium

the epicotyls grow to about 6 times their originallength, growth being completed after approximately48 houirs (fig 1, top). The increase in invertaseactivity per epicotyl is greater, to about 10 timesthe initial value, whilst invertase per milg proteinincreases parallel to length during the first 34 hoursof growtlh, to a maximum of about 4 times theiniitial value. The most rapid increase in invertaseactivity coincides closely with the period of mostraapid elongation, actually preceding it slightlv. Assooni as growth ceases, the activity of the eiinzvie

declines, dropping, after 96 hours of incubation, toless thcan half the miaxinmal level.

Effect of Gibberellic Acid. Exogenous GA,added at the start of incubation results in a greatincrease in the rate of elongation and in invertase(fig 1. bottom). Length increases to 12, invertaseper epicotvl to 18 and invertase per mig protein to7 timles the initial valuie. The kinetics of growtland invertase inlcrease remain, however, essentiallythe same as in basal meedium.

Measured as percent of the untreated controlsGA, increases growth and invertase activity to thesame extent. The optimal concentration for eithereffect is also the same, namely 30 nim. However,the maximunm of the GA3 effect on invertase and onelongation is reached after different times of incuba-tion (table II). The effect on invertase is greatestafter 24 hours, being about 200 % for invertaseper milg l)rotein, and even more for invertase perepicotvl since the protein content in the GA:,-treatedepicotyls is also increased by about 10 %. Thereis also an increase of invertase per mllg fresh weightof the epicotyls but it is less pronounced. On theother hand the effect of GA,, on elongation isgreatest after longer times of incubation. reachingalso about 200 %, but only at 72 hours.

Table II. Effect of GA on Grolze/1i and InvertaseA ctivitiv After Different Timces of InctbationValues calculated from the data of figure 1, and ex-

pressed as percent of control. GA3 concentration was30 mnl.

Houirs of incubationLengthInvertase/mg protein

9119151

24170196

34171154

48188145

7219395

WN'hen GA3 is added to the samples after 24 hoursof incubation in basal medium, that is, during thetime of approximately constant and maximal rateof elongation (compare fig 1, top), there is no lagfor the increase in invertase activity but there is anoticeable lag for the effect on growth (fig 2).

If added during the extraction prociedures G(\Ahas no effect on the activity of the enzyme.

Osmnotic Inhibitioni of Growkth. Invertase activi tvbeing evidently closely correlated with growth itseen3ed interesting to determine whether the activityof the enzyime could be enhanced independentlv fromgrowth. An attempt to aniswver this question wvasmade by inhibiting elongation osmotically by meanisof polvethyleneglycol (MW 1000).

Tncreasing concentrations of polvethyvleneglvcol

Table I. Values for Lenglthl anzd Invrtlase Activity at the, Initial State

LengthProteinIlnvertaseNlvertase/mg protein

2.9 mm0.08 mg/epicotyl

: 0.12 ,umole sucrose hy.drolyzed/epicotyl hr-1.5 mole sucrose hydrolyzed mg protein'- hr-I

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SETTZ AND LANG-INVERTASE AND GROWTH IN LENTIL EPICOTYLS

BASAL MEDIUM

/X X

/

Ol

x

/#*

- GA

0*.-INVERTASE PER EPICOTYL

,LENGTH _%

PROTEIN

\I-INVERTASE PER EPICOTYLI\/ \\ soLENGTH

t/ "IK-I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.

I / INVERTASE PER Nb/ a,Xmm., <X` PROTEIN

x.xp.rPROTE IN

a , . ,I I

0 9 24 34 48 72 96

INCUBATION TIME (HOURS)FIG. 1. Kinetics of elongation and invertase activity during growth. Top, seedlings incubated in basal medium;

bottom, seedlings incubated in presence of GA3 (30 mM).

1200

1000

800

600

400

200"4

"4I..

'4.

i800r

1600 _

14001_

1200 _

1000 -

800 .

600 -

400 -

200

a

1077

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_ ,~~~~~~

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PLANT PHYSIOLOGY

z'600 - 0

s 400

k /200

X 0 9 24 34 48

Boo ~INVERTASE x

X 600 -

400 - 0 0

200

I

0 9 24 34 48

INCUBATION TIME (HOURS)

FIG. 2. Effect of GA3 (30 mM ), added in the course

of incubation, on growth (top) and invertase activity,measured per mg protein (bottom). Upper curves =

control, GA3 added at start of incubation; middle curves

(starting at arrow) = GA3 added after 24 hours incu-bation; bottom curves = control, basal medium.

resulted in an increasing inhibition of both growthand invertase activity approximately parallel to oneanother when measured after 24 hours of incubation.WVith a concentration of 0.22 M, growth in absence

of GA, was completely inhibited and invertase ac-tivitv underwent actually a decrease (table III).But the effect of GA3, measured as percent of thecontrols, was nearly the same as in the absence ofpolyethyleneglycol, beinig slightly lower for growthand higher for invertase. Thus, it was not possibleto separate invertase activity from growth by thisapproach.

If, after 24 hours incubation in polyethyleneglycolin the absence of GA3, the tissue was transferred tobasal medium growth and invertase activity bothrecovered during another 24 hours of incubation.This recovery was relatively less when the tissuehad been exposed to higher polyethyleneglycol con-

centrations, but the recovery in growth and inenzyme activity were similar. In presence of GA3,in contrast, recovery in invertase activity was con-

siderably greater than that in growth. Thus, GA,in the presence of polyethyleneglycol has a greatereffect on invertase than on length (table III).

Effect of Sugars. Fructose, glucose and sucrosein the medium had no influence on growth or inver-tase activity during 24 and 48 hours of incubation.At higher concentrations (0.1 M and above) theyresulted in an inhibition which was not significantlydifferent from the inhibition caused by the sameconcentrations of polyethyleneglycol or mannitol.

Effect of Inhibitors. When added at the startof incubation and at a concentration of 10 ug/mlcycloheximide inhibited any increase in length andin invertase activity during 24 hours of incubation,the invertase activity showing in fact a slight decline.A cycloheximide concentration of 1 jug/ml was suf-ficient to block the promotion caused by GA3(table IV).

Actinomycin D (50 ,ug/ml) also inhibited elon-gation and invertase increase (table IV).

FUDR (10 mM) added at 0 hours resulted, after24 hours of incubation, only in a relatively small

Table III. Effect of Polyethylenegilycol on Grozcth andInvertase Activity in Basal Medium and GA3Values in percent of the initial state.

InvertaseLength per mg protein

Basal 30 mM Basal 30 mmTime Conditions medium GA3 medium GA.,

After 24 hr Control 426 691 418 676incubation Polyethyleneglycol, 0.22 M 104 129 46 85

Recovery afteran additional Control 695 1455 646 88424 hr of Polyethyleneglycol 0.22 M presentincu-bation during the first 24 hr of incubation 279 613 92 477

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SEITZ AND LANG-INVERTASE AND GROWTH IN LENTIL EPICOTYLS

Table IV. Effect of Inhibitors on Growth and Inzcrtasc .4ctizity in Basal Mecdium and GA3Values in percent of the initial state, after 24 hours of incubation.

InvertaseLength per mg protein

Basal 30 mat Basal 30 mmCompound and conc medium GA medium GA

Control (110 addendia)Cycloheximide 1 ,g/mlCyclohexim-ide 10Og/mlActinomycin D 50 jug/milFUDR (10 miM)FUDR 1 nmM thyrmidine

328173103167251332

500

194106184338532

36;518388180283359

59417788203385572

inhibition, both growth and invertase activity beingreduced to about 70 % of the control. Thymidinefully reversed the effect of FUDR (table IV).

Preincubation in FUDR strongly reduced theeffect of GA3 subsequently added. This cannot beachieved by preincubation in an equally inhibitoryconcentration of cycloheximide; with this inhibitor,the action of subsequently added GA3. measured as

percent of control, is unchanged (table V).The effect of inhibitors added after 24 hours of

incubation, when elongation is proceeding at a near-

constant rate, is showli in figure 3. The data referto GA-treated samples only but the effects in basalnmedium were the same.

Cycloheximide added after 24 hours caused a

complete stol) in elongation within a very short time.Invertase activity also stopped increasing and thendecreased markedly, wvith a half-life of approximately14 hours (fig 3, top).

Actiniomiiycin D became effective only after a

considerably longer lag period of 2 to 3 hours, andthere was no change in the pattern of the decreasein enzyme activity (fig 3, center).

FUDR added at 24 hours caused, after a lagperiod of at least 10 hours, only a small inhibition

of growth and invertase synthesis (fig 3, bottom).Localization of the Enzymle Inside the Cell. For

an understanding of the function of iinvertase inplant cell growth it is important to know where inthe cell the enzyme is localized, and especiallywhether it is bound to some organelle or other cellstructure. However, the centrifuged extract (8 min,1200 X g) had about the same activity per ml as

the crude, unfiltered and uncentrifuged extract con-

taining cytoplasm as well as wall debris and un-

broken cells. There was no difference in this regardbetween younger (24 hr incubation) and older(72 hr incubation) epicotyls, and the entire activitystayed in the supernatant even after centrifugationfor 2 hours at 165,000 X g. When 0.3 % glutaral-dehyde, which is known to stabilize some of the cellmembranes and to increase the sedimentation rateof somiie organelles, was added to t-he medium either3 hours prior to, or during, or after extraction, therewas also no significant effect on the tendency of theenzyme to remain in the supernatant. Thus, theinvertase of lentil epicotyl cells appears not to befirmly bound to the cell wall or to any of the cellorganelles, but to exist in a free or easiily solublecondition in the "ground cytoplasm."

Table V. Effect of Preincubation in Equally Inhibitory Concentrations of Cvcloheximide and FUDRon the Response to GA 3

Values showv the effect of GA3, expressed as percent of control. GA3 (30 mM) was added after 24 hours of atotal incubation period of 48 hours.

InvertaseInhibitor Tim.e of addition Length per mg protein

Control (no inhibitors) 138 145

Cycloheximide (0.15 ,ug/ml) Simultaneouslywith GA 132 139

24 Hrbefore GA 147 141

FUDR (10 mM) Simultaneouslywith GA 142 136

24 Hrbefore GA 114 113

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1200r_CONTROL INHIBITOR x

1000[

800

6001

4001

200

a1)

a

_ 10000

.C800'4-

0

c 600a)

0

400Ck

I--

a)a) 200

0

L-

oc L

0

800

600

400

x- x LENGTH o-ox---x INVERTASE o---o

CYCLOHEXIMIDE X

v --x

vX-- o\

- 4 ".

I II

ACTINOMYCIN D

-o 0

0.~..%t"" fI,--o__~

F U D R

0 9 24 34 48INCUBATION TIME (HOURS)

FIG. 3. Effect of inhibitors added during growth.Top, cycloheximide, 10 ug/ml; middle, actinomycin D,100 ag/ml; bottom, FUDR, 10 mm, all added after 24hiours incubation (arrow!). Values iIi percent of theinitial state for length and for invertase per mg protein.Seedlings were incubated in presence of GA3 (30 mM).

Kinietics of Invertase Activity and Growth. Ascan be seen from the time course studies (fig 1)invertase activity in lentil epicotyls increases maxi-mallv duriiig most active elongation, and dropsrapid1v once groxvth is completed. This agrees very

well with miieasurements of invertase activitv in

growving roots (9, 14) and in suigarcane stems (6).A careful analysis of the data in figure 1 shows

that first there is a maximum in the rate of invertaseincrease; this is followed closely by a peak in growthrate (both rates measured as slope of the curves infig 1). But the maximum in invertase activity isreached a considerable time after the maximum ingrowth rate. This discrepancy might be caused byinvertase synthesis proceeding so vigorously thatthere is an overslhoot when growth is beginning tolevel off.

The increase in invertase activity is ncot a simplefunction of an overall increase in protein as theactivity of the enzyme, on a protein basis, increased4-fold in the absence of GA and 7-fold in itspresence.

The inlhibitor studies indicate that the increase is(lependent on protein, RNA and DNA svnthesis(table IV). This, and the very rapid effect ofcycloheximide (fig 3, top). which is known to be a

ipowerful inllibitor of protein synthesis, suggest th.tthe increase in invertase atctivity is due to de tlovosy!nthesis.

AIctinoiuvcin 1), conisidered to lie an inhibitor ofI)NA-dependent RNA synthesis, causes less inhibi-tion of invertase synthesis than does cycloheximideand, if added during the course of enzyme synthesis,becomes effective only after a distinct lag period of2 to 3 hours (table IN' and fig 3, center). Thismay be interpreted as indicating that sufficientamounts of a relatively stable imiessenger RNA are

present in the cells to maintaini a certaini level ofprotein svnthesis. However, onie should keep inmind that such interpretations are based on general-izations of findings made in microorganisms. and

that it is not known whether and to what extentthese results are valid with respect to higherorganisms.

FUDR inhibited inivertase increase eveni less,and with a longer lag period, than did actinomycin D(table IN, and fig 3, bottom). This and the more

pronounced inhibition when FUDR was added beforeGA, (table V) indicate that its effect is more

indirect.All inhibitors used caused also complete or par-

tial inhibition of growth of the epicotyls. Thisresult is in full agreement with the now well-knownfact that growth, even if based on cell enlargement,is dependent on RNA and protein svnthesis (e.g.11, 14). The results with FUDR agree with thoseof Nitsan and Lang (13) and Bopp (2). Thedegree of inhibition was less than that obtained bythe former authors in the same plant material, but

Discussion

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SEITZ AND LANG-lNVERTASE AND GROWTH IN LENTIL EPICOTYLS

this is probably because of the shorter incubationtime used in the present studies. It is worth n-otingthat while cycloheximide, applied 24 hours beforeGA3, had no selective effect on GA3-promotedgrowth and invertase activity, FUDR did, a con-centration of 10 mM reducing the difference withGA3-free controls from ca. 40 to ca. 10 % (table V).This is in agreement with the idea that gibberellin-regulated growth is often dependent, in some director indirect manner, on DNA synthlesis (compare 12).

It was not possible to detect any significanteffect of sugars added to the incubation medium,either on invertase activity or on growth. In thisconnection one sihould not forget that the seedlingswere incubated in presence of the cotyledons whichare undoubtedly providing the epicotyl with sugars,thus rendering it independent of exogenous sugarsupply. However, these results are quite in contrastto those obtained by Glasziou et al. (5) in sugarcane.

Relationship of Invertase and Growth in LentilEpicotyls. Invertase activity and growth exhibit, ingeneral, very similar kinetics. This is evident bothin normal growth and in experiments with inhibitors.Presence of GA3 results in increases of both growthrate and invertase level, as also reported by severalother authors (8,10,16), but here as well as in theinihibitor experiments the kinetic pattern of the 2processes remains unaltered (fig 1). This lattersituation is in contrast to the results of Kaufmanet al. (10) in Azvena internodes where presence ofGA3 did cause a change in the kinetics of bothelongation and invertase activity.

In some cases, however, the close agreementbetween growth and invertase is disrupted. Theincrease in invertase per epicotyl during the first34 hours of incubation is relatively greater than theincrease in length (fig 1). Under the influence ofan osmotic inhibitor (polyethyleneglycol) GA, hasa greater effect on invertase activity than on length(table III). Furthermore, when GA3 is added inmidcourse of growth, the gibberellin-dependent in-crease in invertase precedes that in growth rate(fig 2), which corresponds to the sequence of themiaxima in the effects of GA3 on invertase and ongrowth during incubation (table II). This resultfully agrees with the concept that growth promotionby hormones involves an increase in the synthesisof enzymes (11, 14).

The invertase in lentil epicotyl cells appears notto be associated with the cell walls and cannot besedimented by centrifugation, even if glutaraldehydeis added before the extraction. The ineffectivenessof glutaraldehyde may be due to the fact that itinactivates invertase before increasing its sedimen-tation rate. However, on the whole the resultssuggest that the invertase of the lentil epicotyl cellsis present in a free or easily soluble state. Thus, itwould not seem to be associated with the tonoplastwhere it could supply osmotically active sugars intothe expanding vacuole, as postulated by Cook (3).The situation is in contrast to that in storage tissues

where the enzyme is insolubly bound to the cell walls(1, 4). In sugarcane stems a soluble invertase wasfound only in immature tissues while in matureparts the enzyme was associated with the cell walls(7). It is however quite unlikely that a similarchange, which would simulate a decrease of activityin the soluble fraction, occurs in the lentil epicotylsas there was no difference in invertase activity inthe walls of younger and more mature epicotyls.

Control of the Invertase Level. Whenevergrowth ceases, whether spontaneously after 48 hoursof incubation or under the influence of inhibitors,invertase activity decreases. The half-life of thelentil epicotyl enzyme is about 14 hours, considerablylonger than the 2 hours reported by Glasziou et al.(5) in sugarcane. Thus, the enzyme is not onlysynthesized but is also degraded during growth,meaning that the invertase of lentil epicotyls isundergoing continuous turnover. This turnover pro-vides the cells with a more effective control systemfor the actual level of enzyme activity and thus forthe level of available sugars than would be providedby only turning on and off the synthesis of theenzyme.

The results indicate that invertase is closelycorrelated with growth, and suggest that the enzvmeis in some way essential for growth, making sugarsavailable for cell expansion.

Acknowledgments

Tlhaniks are due to Hoffman-La Roche for a generoussupply of 5-fluorodeoxyuridine; to Merck, Sharp, anIdDohme for the actinomycin D; and to Parke, Davis, andCompany for the chloramphenicol used in the experi-ments. We are inidebted to Dr. V. E. Youngman, Wash-ington State University, Pullman, for a supply of Persianlentils. The assistance of Mrs. Evelyn Lamb, RobertU. Byerrum, and Eberhard Mahn during this study isalso greatly appreciated.

Literature Cited

1. BACON, J. S. D., I. R. MACDONALD, AND A. H.KNIGHT. 1965. The development of invertase inslices of the root of Beta vulgaris L. washed underaseptic conditions. Biochem. J. 94: 175-82.

2. Bopp, M. 1967. Hemmung des Streckungswachs-tum,s etiolierter Sprossachsen durch FUDR. Z.Pflanzenphysiol. 57: 173-87.

3. COOK, F. S. 1959. Generative cycles of proteinand transanminase in the growing radicle. Can. J.Botany 37: 621-39.

4. EDELMAN, J. AND M. A. HALL. 1965. Enzymeformation in higher-plant tissues. Development ofinvertase and ascorbate-oxidase activities in maturestorage tissue of Helianthus tuberosu.s. Biochem.J. 95: 403-10.

5. GLAsziou, K. T., I. C. WALDRON, AND T. A. BULL.1966. Control of invertase synthesis in sugarcanie.Loci of auxin and glucose effects. Plant Physiol.41: 282-88.

6. HATCH, M. D. AND K. T. GLASZIOU. 1963. Sugaraccumulation cycle in sugarcane. I I. Relation-ship of invertase activity to sugar content and

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PLANT PHYSIOLOGY

g,rowth rate in storage tissue of plants in con-

trolled eoxvironmiients. Plant Phy siol. 38: 344-48.7. H.x\VKER, J. S. AND M. D. HATCH. 1965. Mech-

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8. H.\YASHI, T., Y. MIURAKA.II, .\ND S. MATSUNR\KA.1956. Bioch]emical studies; on "bakaniae" fungus.XXXVI. The physiological action of gibberellini.VIII. Chaniges in the actixities of various enymlllesiM leaf-sheaths of rice pilants treated xvith gib-l)erellilH. Bull. \gr. C(hem. Soc. Japani 20: 159-4.

9. HE.LLEBUST, J. A. .\Nl) 1). F. 1FOR\\ARD. 1962. Theinvertase of the corii radicle and its activity in,Liccessive stages of growx th. Cani. J. Botanm 40:113-26.

10. K.\AFMIAN, P., N. (GHOSHEII, AND H. IKUMxA. 1968.Promotion of growtlh and invertase activity by gib-berellic acid in developing A4zcna internodes. PlantPh\xsiol. 43: 29-34.

11. KFY, J. L. 1964. Ribonucleic acid and protein

synthesis as essential processes for cell elongationi.Plant Phy siol. 39: 365-70.

12. LANG, A. AND J. NITSAN. 1967. Relations among

cell growth, DNA synthesis, and gibberellin actioni.Annii. N. Y. Acad. Sci. 144(1): 180-90.

13. NITSAN, J. AND A. LANG. 1965. Inhlilbitionl of celldiv isilon and cell elongation inIhigher planlts byilnhibitoi-s of 1)NA svinthesis. Develop. Biol. 12:358-76.

14. NOOI)of:N, L. D. ANK \. THIMANN. 1966. A\tiolnof inhibitors of RNA anid lproteill synthesis on

cell enlargemnent. Plant Ph, siol. 41: 157--4.15. ROBINSON, E. AND R. BROxVNN. 1952. The develop-

mlenit of the enzyme complement in grow-ing rootcells. J. Exptl. Botany 3: 356-74.

16. SCHAEVERBEKE, J. 1966. Augmentation de Ilacti-vite sacoharasique danis les filets staminaux duZca mays au moment de l'anth&se. Interventionprobable de l'acide gibberellique dans la synthesed'une saccharase. C. R. Acad. Sci. Paris 262:277-81.

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