dna multiplication as a requirement for expression of ... · the experiments described in this...

DNA Multiplication as a Requirement for Expression ofFloral Stimulus in Pharbitis nil 1, 2

Jan A. D. ZeevaartDivision of Biology, California Institute of Technology, Pasadena

It has been shown previously ( 15) that photo-periodic induction in Xanthium is inhibited by ap-plication of the pyrimidine, 5-fluorouracil (5-FU).More recently (1) it has been demonstrated that theprocess which is inhibited by 5-FU and which isrelated to the process of flowering, is the synthesis ofribonucleic acid (RNA) in the Xanthium bud duringthe first part of the inductive dark period.

The experiments described in this paper were de-signed to determine if 5-FU also inhibits flowering inanother short-day plant which also requires but oneinductive long night, namely, Pharbitis nil, and if so,by what mechanism 5-FU exerts such inhibition.

It will be shown that 5-FU completely inhibitsflowering in Pharbitis by suppressing deoxyribo-nucleic acid (DNA) multiplication in the shoot apex.

Materials & MethodsPlants of Pharbitis nil Chois., strain Violet were

grown in the Earhart Plant Research Laboratory(18). Seedlings of this strain respond to one singledark period of 16 hours by the subsequent initiationof flower buds (8). The original seeds were gener-ously supplied by Prof. S. Imamura, Kyoto Univer-sity, Kyoto, Japan.

Seeds were treated with concentrated sulfuric acidfor 30 to 45 minutes and were then washed overnightwith tap water. The seeds were next planted in a50-50 volume mixture of vermiculite and crushedgranite in darkness at 31 C. After approximately 30hours the germinating seeds were transferred to agreenhouse with a day temperature (0800-1600 hr)of 35 C and a night temperature (1600-0800 hr) of27 C in order to prevent etiolation. Two days afterplanting, the seedlings were transplanted, one seedlingper container, to 6 ounce plastic beakers containingthe same mixture as before. Care was taken thatseed coats were removed from the cotyledons. Theseedlings were then grown for another 2 days at 23 C

1 Received Sept. 18, 1961.2 Supported in part by grants from National Science

Foundation (G-7129) and Frasch Foundation. Duringpart of the period in which this work was conducted, theauthor held a stipend from the Netherlands Organizationfor the Advancement of Pure Research (Z.W.O.). Theexperiments were done in the Earhart and Campbell PlantResearch Laboratories.

under continuous light from fluorescent and incandes-cent tubes yielding approximately 700 ft-c at plantlevel. The plants were wateredl with Hoagland'snutrient solution daily.

Four days after the seeds had been planted, theseedlings were ready for experimentation. At thistime the plumules were 1 to 2 mm long. Before eachexperiment seedlings were selected for uniformity ofcotyledons. From 9 to 18 seedlings were included ineach treatment. They were exposed to one singledark period of 16 hours at 23 C or 27 C. After theinductive treatment the seedlings were grown for atleast another 3 days under continuous light at 23 C.They were then transferred to a greenhouse (temp.31/23 C) in which the photoperiod was extended to16 hours with incandescent lamps. After sufficientseedling development had taken place (generally ca.20 days from planting) the number of flower budsper plant was determined, and if necessary the tipswere dissected under a binocular microscope. Thesame quantitative criteria are used as were intro-duced by the Japanese workers (8): A, Percentageof plants with flower primordia; B, percentage ofplants with terminal flower buds, and C, the averagenumber of flower buds per plant.

5-Fluorouracil (5-FU) and 5-fluorodeoxyuridine(5-FDU) (kindly supplied by Hoffmann-La Roche,Nutley, N.J.) and other nucleic acid precursors andinhibitors were applied in aqueous solution contain-ing Tween 20 (ca. 0.1 %), and with the aid of amicrosyringe, to either cotyledons or to plumules;0.1 cc per seedling was applied to the cotyledons;0.01 cc was applied per plumule.

Labeled 5-FU (California Corp. for BiochemicalResearch, Spec. act. 5 mC/mmole) was applied asdescribed above.

Before treatment with the metabolites, the seed-lings were transferred to a room kept at a constanttemperature of 23 C or 27 C and of high relativehunmidity to prevent rapid drying of applied solu-tions.

During treatment and before the long night theseedlings were exposed to red light provided byfiltering the radiation from four 8-foot fluorescenttubes through red cellulose acetate.

For chemical analysis cotyledons and plumuleswere harvested separately and RNA and DNA ex-tracted and determined following the procedures(lescribed by Bonner and Zeevaart (1).

296

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ZEEVAART-DNA MULTIPLICATION & FLORAL STIMULUS

For cytological work, the plumules were excised,fixed in Randolph's modification of Navashin's fixa-tive, dehydrated with tertiary butanol (11), and im-bedded in Tissuemat. Serial longitudinal sections,81 thick, were cut and stained using Harris' hae-matoxylin (6), and counterstained with erythrosin.Slides were mounted with Permount synthetic resin.The microscopic observations were confined to theeight most median sections, comprising a slice tissue64 / thick, the values given being averages fromfive or six tips. Only meta-, ana-, and telophasenuclei were counted, but in some tips prophases were

counted separately. Numbers of cell divisions wererecorded in: A, the apex; B, the subapical regionbetween the procambium and 1.5 mm below the apex;

c7_Control

a6

5 _

4 is-Cotyledon

applicationPlumul\e 3 application

0

0

C, the procambium; D, the two highest axillarybuds visible in the median section.

Results

Pharbitis seedlings were grown under the experi-mental conditions as described above until they were4 days old. Exposure of such seedlings to one16 hour dark period at 27 C resulted in the subse-quent formation of six to seven flower buds perplant. All axillary buds on the second to third nodeand higher as well as the terminal bud developedinto flower primordia. The optimum night tempera-ture for such an inductive dark period lay between

c 7aCi 60.60

0.50

34.013

0

f-20

o Iz

1o2 lo-, lo 6 lo 5 10-45-FU:t mole/plant 5 FDU: F mole/plont

-0-C 6

S0.5WI

4

.30

_ 20

z

-UFU to 3,/ cotyledons

srl I1

- 7C

0

6

00 5

0

= 4D

h.33

0

= 24-0

ZA

4

C*~~~~~I --I l0-~~~~~~~~ ..I ..

0 20 40 60 80 -0 16 20 24 30 41 " DHours after beginning of dark period Hours after beginning of dark period

Fig. 1. Inhibition of floral initiation in Pharbitis as a function of amount of 5-FU applied per plant. A singleapplication was made either to the cotyledons alone (0.1 cc) or to the plumule alone (0.01 cc) before one 16 hour darkperiod at 27 C. Seventeen plants per treatment.

Fig. 2. Inhibition of floral initiation in Pharbitis as a function of amount of 5-FDU applied per plant. A singleapplication was made either to the cotyledons alone or to the plumule alone before one 16 hour dark period at 27 C.Nine plants per treatment.

Fig. 3. Inhibition of floral initiation by 5-FU and 5-FDU in Pharbitis as a function of time of application. 5-FUapplied either to cotyledons (0.1lurmole per plant) or to plumules (0.015 ,umole per plant), 5-FDU to plumules (2 X10-5 ,umole per plant). 16 hour dark period at 27 C. Nine plants per treatment.

Fig. 4. Translocation of floral stimulus from Pharbitis cotyledons as demonstrated by removal of both cotvledonsat varied times after one 16 hour dark period at 27 C. Fifteen plants per treatment.

297

I



PLANT PHYSIOLOGY

25 and( 30 C. althotuglh 23 C also resulted in a goodresponse. Night temperatures belou- 20 C failed topermit pro(luction of anyv flower budls. The criticaldark period at 27 C was ca. 13 hours.

In preliminary experiments S-FU x-xas a potentinhibitol- of flowering in Pharbitis if applie(d eitherto the cotyledon or to the plumule. Data of a

reDresentative experiment in w-hich 5-FU w-as ap-plied over a wide range of concentrations, are slhow-in figure 1. In this experiment the samle concen-

trations of 5-FU were applied either to cotyledonsor to plumlules, so that the total amount of 5-E-U ap-pliedl per plant via the plumules was only a tenththat applied to the cotyledons. Applying 0.1 /jLmlole5-FU to the cotyledons caused almost complete in-hibitioni of flowering. The same degree of inhibitionwtas caused by applying 0.03 gmole to the plumiiules.Thlus, less 5-FU is requiredl to elicit a given (legreeof inhibition if the inhibitor is applied to the plu-mules than is required if application is made to thecotyledons. In certain experiments application tothe plumlules was even more effective than indlicatedin figure 1; 0.02 ;mole 5-FU occasionally resuiltedin comiiplete inhibition of floral initiation.

\Vhen untreated and 5-FU treated plants were

allowed to grow si(le by sidle under long-day condi-tions andCi beyond( the stage at which they were

normally (lissected, untreate(l plants prodlucedl flowersafter approximately 40 (lays, w-hereas those treatedNwith 5-FU at the beginninig of the single (lark periodcontinuedl a strictly vegetative growth and exhibiteda climbing habit.

The inhibitor, in addition to suppressing floralinitiation. redlucedl groxvth of the plumules consi(ler-ably (luring the first 2 or- 3 (laxys after application.This resuilte(d in somewhat smiialler an(d somiietimiesmisslhapeni anId wrinkle(d leaves oIn the first anlsecon(l nio(le. \VN1hen the in1hibitor \-as applie l inconcentrationis as hiigh as 5 X 10-3 A, the growthof the plumlule was often completely suppressed allit was replaced by the two cotyledonarv bu(ds.

In the experiment of figuri-e 2. 5-FDU was ap-

plied to either the cotyledoons or to the plumules over

a range of concentrations. It is clear that plumuleapplication on the basis of amiiount of inhibitor ap-

plied per plant was again mtuclh more effectixe thanapplication to the cotyledonis. Comparisoin of theeffectiveness of 5-FU (fig 1) with that of 5-FDU(fig 2) shows that 5-EDU is about one thousandtimes more effective than 5-FU in suppressing floralinitiation in Pharbitis, e.g., 2 X 10-5 /mole 5-EDUapplied to the plumule always resulted in almost com-

plete flower inhibition, whereas to obtain the samiieeffect with 5-FU, at least 0.02 ymole wvas neededper plumilule.

Cotyledon applicatiolns of ;-FDU. highly inef-fectixve. ielded variable results. In all further ex-

periments here reportedl. 5-FDUI x-vas applie(d to theplumules. With 5-FU, on the contrary. best resultswere obtainedl with cotyledon applications (0.1Lmole per plant). so that in most experiments 5-FUwas applie(d via the cotyledons.

Table ITransport of C14-Labeled 5-Fluorouracil (5-FU)*

Radioactivity detected, Spec. act.CQ4-5-FU cpml cpm X 10-3 (cpm/mg

apidapied RNA)applied ai In plumule In cotyledon of

extract RNA extract RNA pRNAuPlumule 450 57.1 3.0 0 0 4,140Cotyledons 2,997 4.1 2.0 1,509 10.0 2,440

* From cotyledons to plumule in Pharbitis and absenceof transport from plumule to cotyledons during andafter one inductive dark period. 0.1 /mole 5-F1 ap-plied to cotyledons, 0.015 Amole to plumule. Applica-tions made at beginning of 16-hour dark period at 25 C.Cotyledons and plumules harvested 29 hours later.Each number average of duplicate, each of 18 plants.

The effects of 5-FDU on growvth of the plumllulceand(I the symptoms exhibitedl by the first two leave,w-ere similar to those caused by 5-FU.

From the results of the experiments of figures1 and 2 it can be suggested that the inhibitors exerttheir effect in the plumule, and not in the cotyledons.In order to obtain more direct evidence regardingthe site of inhibition, experiments witlh labeled 5-FUwere carried out. Applications to the cotyledonsor to the plumules were made at the beginning (0fthe 16-hour dark period . The cotyledlons an(l plu-mules were harvested 29 hours later, extractecl, and(label distribution determined. The (lata of tableI show that labeled 5-FU applied to the plhlmulewas recovered in large amounts in the plumllule and(in the RNA of the plumule, but that no detectableactivity was transported to the cotyle(loni during the29-hour period. No detectable amounts of labele(d5-FU were detected in DNA. Labeled 5-FU ap-plied to the cotyledon, on the other hand, could notonly be recovered in the cotyledon. but also in con-sidlerable amounts in the plumule. The specificactivity of plumule RNA after cotyledon applicationwas somewhat lower than that found after directapplication to the plumules, although the inhibitionof flowering was approximately the same in the twvocases. From the data obtained with labeled 5-FUit can clearly be demonstrated that 5-FU exerts itsflower inhibiting effect (lirectly in the plumule.

The fact that 5-FU applied to the cotyledon isvery effective in suppressing flowering is clearly dlueto rapid translocation of the substance to the plumule.This will be further shown in the experinment offigure 6.

Since the process of plhotoperiodic induction canbe separated into a number of sequential steps (e.g.10) the effectiveness of an inhibitor as a functioniof time of application should give an indicationas to which process (es) in the chain of events is(are) affected. Results of such an experiment areshown in figure 3. In this experiment 5-FU wasapplied either to the cotyledons or to the plumulesat different times before and after the inductive dark

2983




Table IIAbility of Different Nucleic Acid PrecursorsTo Antidote 5-Fluorouracil Inhibition of

Floral Initiation in Pharbitis

% Plants % Plants Avg.Antidoting with with No.substance flower terminal flower

primordia buds buds

Control, no 5-FU 100 % 100 % 6.6Water 6 % 6% 0.2Thymine 0 % 0 % 0.0Thymidine 100 % 100 % 5.5Thymidylic acid 100 % 100 % 6.3

Uridine 6 % 6 %o 0.2Deoxyuridine 94 % 94 % 6.4Cytidine 12 % 6 % 0.5Deoxycytidine 94 % 94 % 5.55-Methyldeoxy-

cytidine 100 % 100 % 5.9

5-Fluorouracil: 0.1 ,umole applied to one cotyledon atbeginning of 16-hour dark period at 27 C. Antidotingsubstances: 0.1 ,umole per plumule applied before andafter dark period. 16 plants per treatment.

period. 5-FDU was similarly applied, but only tothe plumules. Applications of 5-FDU inhibitflowering completely if made up to 40 hours afterthe beginning of the dark period. Inhibition of5-FU exhibits a more gradual decrease in effective-ness with later application than the inhibition by5-FDU. The effect on flowering of both substancesbecomes negligible when applied later than approxi-mately 50 hours after the beginning of the darkperiod. This result has been confirmed in foursimilar experiments.

Translocation of the floral stimulus from coty-ledons or leaves to buds is a well defined partialprocess of photoperiodic induction. It has beenstudied under the present experimental conditions byexposing seedlings to one 16-hour dark period andthen removing both cotyledons at various intervalsafter the end of the dark period. Although suchtreatment resulted in slow growth of these seedlingsas compared with intact ones, most plants survived(fig 4). If cotyledons are removed 4 hours afterthe end of the dark period the flowering responseis almost saturated. The stimulus, therefore, moves

out of Pharbitis cotyledons much more rapidly thanit does from Xanthium leaves (e.g. 10, 14). Thepresent results also seem to indicate earlier trans-location than reported for Pharbitis by Kujirai andImamura (8), who darkened and removed only one

of the two cotyledons on each seedling; however,their procedure did not result in optimal response.

The inhibitors, 5-FU and 5-FDU, then, whichinhibit flowering only in the plumule, are fully ef-fective even if applied many hours after the floralstimulus has arrived in the apex. From this itmust be concluded that the process which is in-hibited by these substances, is the actual inductionof flower primordia.

The possibility of reversing the inhibitor effectsof 5-FU and 5-FDU by the application of variousnucleic acid precursors is indicated by the data intable II. 5-FU was applied to the seedlings via thecotyledons and before the dark period in an amount(0.1 /Amole per plant) known to give full inhibitionof flowering. The antidoting material (0.1 umole)was applied to the plumule before or after the in-ductive dark period. Applications of thymidine,thymidylic acid, deoxyuridine, deoxycytidine, and5-methyldeoxycytidine very effectively restoreflowering, but thymine, uridine, and cytidine do not.In additional experiments uracil and orotic acid alsoproved to be completely ineffective as antidotingmaterials. All five of the substances which over-come the inhibition are precursors of DNA. Itseems, therefore, that DNA multiplication is theprocess which is suppressed by the inhibitors andwhich is at the same time necessary for effectiveflower induction.

5-Fluorodeoxyuridylic acid blocks methylation ofdeoxyuridylic acid to thymidylic acid by combiningirreversibly with the enzyme thymidylate synthetase,resulting in deficiency of thymidylic acid, one of thebuilding blocks of DNA (3, 5). Also, 5-FDU in-hibition (12) is completely reversed by thymidinein a noncompetitive manner. These conclusions ap-pear to apply equally well to Pharbitis. Applyingeither of the inhibitors apparently results in defi-ciency of thymidylic acid in the plumule. Addingthis substance or thymidine overcomes the deficiency,but the base thymine does not. That the deoxy-nucleosides deoxyuridine and deoxycytidine are alsoeffective in overcoming the inhibition in the con-centrations used (table II) is probably due tocompetitive inhibition of the effect of 5-FU anc5-FDU (fig 5). In this experiment 2 X 10-6Amole 5-FDU per plant was applied via the plumuleand different amounts of thymidine or of deoxyuri-dine were also applied to the plumule at the sametime. Over the range of concentrations used thymi-

Table IIIGrowth Inhibition & Synthesis of RNA & DNA

by 5-Fluorouracil*

Applied to Fr wt RNA DNA

Cotyledon Plumule mg Rel. mg Rel. mg Rel.

Water Water 202.0 100 1.71 100 0.11 1005-FU Water 121.5 60 0.97 57 0.06 56Water Thymidine 204.5 101 1.66 97 0.11 985-FU Thymidine 196.5 97 1.55 91 0.10 91

* Inhibition of growth (fr wt) and synthesis of RNAand DNA by 5-fluorouracil (5-FU) in plumules ofPharbitis and reversion by thymidine. 0.125 tmole5-FU applied to one cotyledon of each seedling before17 hour dark period at 25 C. First leaflet removedafter 20 hours.

0.1 ,mole thymidine applied to plumules after 20and 29 hours. Plumules harvested after 53 hours.Each number, average of 2 replicates, each of 36 plants.

299



PLANT PHYSIOLOGY

7

- \Control

- ....Thymidine

I ff~IC

- /

0'o 50

O 1 13

v~~~~~~~~~~~~-- I

,..- L

I~~~~l

0

.oxyuridine L 3

0

0

; 2

o I

6I I I z "

0.02 0.04 0.06 0.08IL mole/plant

0.1

H20, 27° C

`_++KY -+ - - -/--,-

a- X-11

H20,23°C

5- FU,23°C

) 16 24 ' C

Hours after beginning of dark period

16 24 3-2 40

beginning of dark period

-70fi 6 o ° Control

66

cl 5- \.Thymid ine0 to plumule

D0 5-FDU \

0) 3- opplication0

-:2 8 | 7 H20 to0

6 1 - lumulez

0-5 0 16 20Hours after beginning of dark period

Fig. 5. Reversion of 5-FDU inhibition by thymidine and deoxyuridine. 2x10-5Amole 5-FDU per plant appliedto plumule before 16 hour dark period at 27 C. Different amounts of thymidine and deoxyuridine applied to plumulesalso before dark period. Nine plants per treatment.

Fig. 6. Translocation of 5-FU from one cotyledon during and after 16 hour inductive dark period. A single applica-tion of 0.1 umole 5-FU or water + Tween 20 (as a control) was made to one cotyledon at the beginning of one 16 hourdark period at 23 or 27 C. The treated cotyledon was removed at various intervals. Seventeen plants per treatment.

Fig. 7. Reversion of 5-FU inhibition by thymidine as a function of time of thymidine application. 5-FU: 0.1,umole applied to one cotyledon at beginning of 16 hour dark period at 23 C. Thymidine: 0.1 ,umole applied per plu-mule at varied times. O.Olcc of water + Tween 20 applied to plumules as control. Sixteen plants per treatment.

Fig. 8. Reversion of 5-FDU inhibition by thymidine as a function of time of thymidine application. 5-FDU:5 X 10-5 mole per plant applied to plumule at beginning of 16 hour dark period at 27 C. Thymidine: 0.1 ,umole ap-plied to plumule at varied times. Water + Tween 20 applied as a control. Seventeen plants per treatment.

300

0..c 7

6a

45

to

.04

0

o; I 4z

0

-4c

t-

o04n10

.0

L.0

00

z00

Hours after

O -

De




dine is more effective than deoxyuridine in reversinginhibition of flowering.

Thymidine can reverse inhibition of floweringcaused by 5-FU, as well as overcome the associatedgrowth inhibition (table III). 5-FU was appliedbefore the dark period and via the cotyledons andthymidine applied later to the plumule. The plu-mules were harvested 53 hours after the beginningof the dark period and fresh weights, RNA, andDNA contents determined. In table III are bothabsolute and relative values. 5-FU suppressed bothfresh weight and RNA and DNA contents of theplumules by approximately 40 %. Applying thy-midine to the plumules restored these almost tothe control values. In all cases fresh weight, RNA,and DNA content are closely correlated.

Labeled 5-FU is rapidly translocated from thecotyledon to the plumule (table I). The same con-

clusion was reached by a somewhat different ap-

proach. 0.1 ,umole 5-FU (or water containingTween 20 as a control) was applied to one cotyledonof each seedling before a 16-hour dark period at 23or 27 C. At various times before, during, and afterthe dark period the treated cotyledon was cut off,thus removing the source which supplied 5-FU to theplumule (Results plotted in fig 6). At a nighttemperature of 23 C essentially no 5-FU was trans-ported from the cotyledon during the dark period as

indicated by the flowering response and by the lackof symptoms in the first leaves. Translocation tookplace rapidly during the following light period, so

that removal of the 5-FU treated cotyledon after24 hours left behind almost complete inhibition offlowering.

The results at a night temperature of 27 C were

quite different. Complete flower inhibition was ob-tained even if the cotyledon containing 5-FU was

cut off at the end of the dark period. Some 5-FUwas, indeed, transported to the plumule even after8 hours of darkness. The different rates of 5-FUtranslocation at night temperatures of 23 and 27 Cwere obtained in four different experiments.

Removal of the cotyledon treated with water as

a control at the beginning of the long night did notresult in a decreased flowering response. Apparent-ly a single cotyledon is able to produce sufficientfloral stimulus for a maximal flowering response.

The data of figure 7 concern the reversibilityof 5-FU inhibition as a function of time of thymidineapplication. 5-FU was applied via the cotyledonbefore the dark period and thymidine applied to theplumule at various times thereafter. The nighttemperature was 23 C so that 5-FU was not translo-cated during the first 16 hours (see fig 6). Thymi-dine was most effective in overcoming flower in-hibition when applied after 16 to 20 hours. Fromfigures 4 and 6 it appears that this is the periodduring which the floral stimulus and 5-FU, respec-tively, are most rapidly translocated from the coty-ledons. This suggests two alternative explanations.First, it might be that thymidine must be applied by

Table IVEffect of 5-FDU on Mitotic Figures in

Pharbitis Seedling Tips*Number of mitotic figures per 64,

tissue sliceTreatment

Apex Subapical Pro- Axillaryregion cambium buds

After 24 hrWater 21 63 98 242X1O-6M 5-FDU 2 1 7 35X10-6M 5-FDU 1 0 4 2

After 48 hrWater 29 94 173 302X1O-6M 5-FDU 29 32 95 275X1O-6M 5-FDU 1 6 29 11* Number and distribution of mitotic figures in the

median 64,u of the tips of Pharbitis seedlings asaffected by 5 FDU. 0.01 cc of solutions applied before16 hour dark period at 27 C. Tips fixed after 24 and48 hours. Averages of five or six plants.

the time the floral stimulus arrives at the apex, or,second, thymidine must be applied to the plumulebefore 5-FU can exert its inhibition. The secondalternative seems to be the correct one. An experi-ment similar to that shown in figure 7 and carriedout at a night temperature of 27 C showed irreversi-ble inhibition after 16 hours (compare figure 6 for5-FU translocation). Results of another experi-ment which also support this conclusion are shownin figure 8. 5-FDU was applied directly to theplumule and thymidine earlier, simultaneously, orlater. It is evident that thymidine overcomes theinhibition only if applied shortly before or at thesame time as the inhibitor. Similar results wereobtained with 5-FU applied directly to the plumule.

From the fact that the inhibitions caused by 5-FUand by 5-FDU can be reversed by thymidine it hasbeen concluded above that the inhibitors suppressDNA multiplication. The normal increase in DNAcontent of the plumule is, indeed, inhibited by 5-FUapplication (table III). Inhibition of DNA multi-plication should also be reflected in a decrease inthe number of cell divisions in the tip. The fre-quency of mitotic figures in growing points treatedwith 5-FDU was, therefore, determined and com-pared with that in untreated tips. The results ofsuch an experiment are given in table IV. 5-FDUin both concentrations applied resulted in almostcomplete suppression of cell division 24 hours later.48 hours after application of 5-FDU tips treatedwith the lower concentration showed a remarkablerecovery in number of cell divisions in the apex andin a part of the subapical region. However, hardlyany cell divisions were found in the region fromca. 0.5 to 1 mm below the apex. The higher con-centration of 5-FDU did not permit any recoveryin the apex after 48 hours. These data coincidewith observations made on other plants treatedsimultaneously with those used for fixation. 5-FDU

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PLANT PHYSIOLOGY

in a concenitration of 2 x 10-6 M alnmost comiipletelyinhibited flowering, although the plumules developedfurther normally. With a concentration of 5 X10-6 M 5-FDU on the other hand, growth of theplumules was stopped completely and they were over-grown completely by the cotyledonary buds.

In a portion of the experiments the nunmber ofprophases was counted in as far as these could bedistinguishedl fromi interplhase nuclei. These countsindicated that even in tips whiclh exhibited no laterstages of cell dlivision, a considerable number of pro-phases were always present althouigh less in numberthan in the controls. Apparently (lividing cells in

Table V'Effects of Aminopterin & Colchicine on

Floral Initiation in Pharbitis*

Conc No. of % Plants %o Plants Avg. No.Of plants with with with ofterminal flower terminal flowerinhibitors shoots primordia buds buds

Control 9 100 %c 100 6%o 6.8Aminopterini0.0025 % 9 44 % 44 %G 1.30.005 % 7 14 to 14 % 0.10.01 % 8 25% 25 %o 0.9

Colchicine0.005% 9 100% 100% 6.60.01 % 9 100 %o 100 % 6.70.02 % 6 100 % 100 % 6.70.04% 7 100 % 100 %o 6.60.08 %o 5 100 % 100 % 5.4

* A single application was made to the plumules(0.01 cc) of the concentrations indicated at the endof one 16 hour dark period at 27 C. Nine plants pertreatment. Data only for plants with terminal shoots.

Pharbitis tips treatedc witlh 5-FDU are arrested inprophase. Tencer (17) has reported that in am-phibian embryos grown in the presence of 5-FDU,mitoses are also blocked at prophase.

As 5-FDU inhibits DNA multiplication as wellas cell division, the question arises whether botheffects are necessary for inhibition of floral initia-tion. This was further investigated by applyingtwo other inhibitors to Pharbitis plumules: A,aminopterin (4-amino pteroylglutamic acid) which,like 5-FDU, is known to block the synthesis ofthymiiidylic acid and hence DNA synthesis (e.g. 16)and B, colchicine which permits DNA multiplica-tiOnl, but blocks cell (livision (table V). Applica-tioIn of concentrations higher than those in table Vconmpletely suppressed growth of the plumules andpermitted the cotyledonary buds to grow out. Theresults indicate that aminopterin is a potent inhibitorof floral initiation as would be expected if its modeof action is silmilar to that of 5-FDU. Applicationof colchicine, however, did not result in flower in-hibition if the terminal shoots survived the treat-

ment. Similar results were obtained wlhen the in-hibitors were applied before the 16-hour dark period.It seems, therefore, that DNA multiplication in theapex, rather than cell division, is the process wlhichis essential for floral initiation.

Discussion

Since 5-FU is rapidly translocated from Pharbitiscotyledon to plumule, but not in the reverse (lirec-tion, it is clear that 5-FU does not affect those in-ductive processes in the cotyledon which lead to pro-duction of the floral stimulus. A similar conclu-sion has been reached for the short-day plant,Xanthium (1). Other experimenits (unpublished)show that 5-FU also does not affect photoperiodicinduction in leaves of the short-day plant. Perilla.This seems to indicate then, quite generally. thatproduction of the floral stimulus in the leaves ofshort-day plants dloes not involve nucleic aci(d mie-tabolism. [See also discussion in (1)].

The present results clearly in(licate that all floralinduction inhibitory effects of 5-FU and 5-FDU areexerted in the apices. Unlike the case of Xanthium(1, 15), however, 5-FU is still fully effective inPharbitis when applied to the plumules many hloursafter the dark period. 5-FDU is effective untilabout 40 hours after the beginning of the dark period.Most probably the flowering state is fixed in thegrowing point by this timle.

It has been shown for a variety of cells that both5-FU and 5-FDU after they are metabolized to 5-fluorodeoxyuridylic acid (e.g. 3, 5) cause deficiencyof thymidylic acid. If the same moode of action isassumed for Pharbitis, the conversion of 5-FU to thedeoxyribotide must occur at only ca. 0.1 of theefficiency with whiclh 5-FDU is converted.

5-FU inhibits both RNA an(d DNA syntlhesis.It is, moreover, incorporated into RNA. But sinceflower inhibition by 5-FU can be completely allevi-ated by thymidine, it must be concluded that RNAsynthesis per se is not a limiting factor for floralinitiation in Pharbitis. The mechanism bv whiichthe inhibitors, 5-FU and 5-FDU, suppress floweringis rather by blocking DNA nmultiplication and celldivision in the apex. The results obtained withaminopterin and colchicine inclicate that DNA mlul-tiplication in the apex is the essential process forthe floral stimulus to become effective.

If thymidine is added after the inhibitor ha.exerted its effect in the plumule, no reversal offlower inhibition is possible. In Xanthiuml (1).reversal of 5-FDU inhibition is possible if thymidineis applied 16 hours later. In other systems (7. 12)reversal is only possible if thymidine is appliedsimultaneously with or shortly after the inilibitor.Apparently in most cases deficiency of thynlividlicacid leads to irreparable (lamage in a few lhours andthis cannot be overcome simply by adding this suhb-stance.

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Gibberellic acid, a known stimulator of cell di-vision in the subapical region (13) has been foundin the course of the present experiments to be fullyineffective as an antidote for 5-FU inhibition. Thisis as would be expected if thymidylic acid is thelimiting factor. Gibberellic acid did result in con-siderable increase in length of the first internodes.

The bulk of the floral stimulus moves out of thecotyledons very quickly after the end of the darkperiod (fig 4). The stimulus apparently can ex-press itself in the initiation of flower primordia onlyif there is multiplying DNA in the apex or subapicalregion of the bud. Blocking of DNA multiplicationby 5-FDU keeps the plant vegetative. One can,therefore, conclude that the genes for floral dif-ferentiation can only be activated while DNA ismultiplying. The mechanism by which the floralstimulus starts developmental processes in the apexis apparently similar to the way in which ecdysoneinitiates metamorphosis in insects (2).

The present studies also permit an estimate ofthe lifetime of the floral stimulus in Pharbitis tipsunder the prevailing experimental conditions. Al-though 48 hours after applying 5-FDU to theplumule. cell division has again started, no flowerbuds are initiated. The floral stimulus must there-fore have become dissipated by this time. Thesedata together with those of figure 4 suggest a life-time of the stimulus in the tips (in amounts suffi-cient to cause flowering) of not more than ca. 24hours.

Salisbury (14) and Lincoln et al. (9) have previ-ously demonstrated that in Xanthium only activelygrowing buds are able to receive and act on thefloral stimulus before it becomes dissipated. Dor-mant buds are incapable of responding to the stimu-lus. The above authors have not reported any ob-servations as to whether cell divisions are presentin dormant buds. The present paper shows thatthe floral stimulus cannot become effective if theapex is rendered inactive experimentally.

Summary

Four day old seedlings of the short-day plant,Phlarbitis nil Chois, strain Violet, can be inducedto initiate six or seven flower buds by one 16-hourdark period at ca. 25 C.

5-Fluorouracil completely inhibits flowering whenapplied either to the cotyledon or to the plumule.A given amount of 5-fluorouracil is much moreeffective in inhibiting flowering when applied tothe plumule than when applied to the cotyledons.Moreover, labeled 5-fluorouracil is rapidly translo-cated from cotyledons to plumule, but not in the re-verse direction. 5-Fluorouracil, therefore, exerts itsinhibiting effect on flowering only in the growingpoint. 5-Fluorodeoxyuridine applied to the plumuleis about one thousand times more effective as aninhibitor of flowering than is 5-fluorouracil. Both

substances are still fully effective as inhibitors offlowering when applied after the end of the inductivedark period.

The floral stimulus is rapidly translocated fromPharbitis cotyledons to the plumule during the firstfew hours after the inductive dark period.

Only thymidine, thymidylic acid, deoxyuridine,deoxycytidine, and 5-methyldeoxycytidine can com-pletely overcoimie flower inhibition by 5-fluorouracilor 5-fluorodeoxyuridine if applied to the plumule be-fore or simultaneously with the inhibitor. It is con-cluded that both 5-fluorouracil and 5-fluorodeoxy-uridine inhibit flowering by causing a deficiency ofthymidylic acid which results in suppression of DNAmultiplication.

5-Fluorouracil inhibits growth of the plumulesand simultaneously suppresses RNA and DNAsynthesis. All these effects can be reversed by ap-plication of thymidine.

The translocation of 5-FU from cotyledon toplumule is strongly dependent upon the temperatureduring the inductive dark period. At 23 C no meas-urable amounts of 5-fluorouracil move out of thecotyledon, but at 27 C enough inhibitor is translo-cated during the long night to cause complete in-hibition of flowering.

Microscopic examination of sectioned growingpoints shows that 5-fluorodeoxyuridine completelyblocks cell division in the apex and subapical regionfor more than 24 hours but less than 48 hours afterapplication to the plumule.

Applying aminopterin to the plumule results inalmost complete inhibition of flowering, but applyingcolchicine has no effect on floral initiation.

It is concluded that the floral stimulus can ex-press itself in the initiation of floral primordia onlyin an apex with multiplying DNA. Apparently thegenes for flowering must be in the process of multi-plication in order to become activated by the floralstimulus.

Acknowledgments

The author gratefully acknowledges Dr. Moshe Negbi'sadvice and cooperation in the cytological work and CarlRovainen's technical assistance in this part of the work.

Literature Cited

1. BONINER, J. & J.A.D. ZEEVAART. 1962. Ribonucleicacid synthesis in the bud an essential component offloral induction in Xanthium. Plant Physiol. 37:43-49.

2. CLEVER, U. & P. KARLSON. 1960. Induktion vonPuff-Veranderungen in den Speicheldrusenchromo-somen von Chironomus tentans durch Ecdyson.Exptl. Cell Res. 20: 623-626.

3. COHEN, S. S., J. G. FLAKS, H. D. BARNER, M. R.LOEB & J. LICHTENSTEIN. 1958. The mode ofaction of 5-fluorouracil & its derivatives. Proc.Natl. Acad. Sci. 44: 1004-1012.

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4. EIDINOFF, M. L. & M. A. RICH. 1959. Growth in-hibition of a human tumor cell strain by 5-fluoro-2'-deoxyuridine: Time parameters for subsequentreversal by thymidine. Cancer Res. 19: 521-524.

5. HARBERS, E., N. K. CHAUDHURI & C. HEIDELBERGER.1959. Studies on fluorinated pyrimidines. VIII.Further biochemical & metabolic investigations. J.Biol. Chem. 234: 1255-1262.

6. JOHANSEN, D. A. 1940. Plant MIicrotechnique.McGraw-Hill, New York.

7. KARNOFSKY, D. A. & R. S. BASCH. 1960. Effectsof 5-fluorodeoxyuridine & related halogenatedpyrimidines on the sand-dollar embryo. J. Bio-chem. Biophys. Cytol. 7: 61-71.

8. KUJIRAI, C. & S. IMAMURA. 1958. tfber die photo-periodische Empfindlichkeit der Kotyledonen vonPharbitis Nil Chois. Botan. Mlag. Tokyo 71: 408-416.

9. LINCOLN, R. G., K. A. RAVEN & K. C. HANINER.1958. Certain factors influencing expression ofthe flowering stimulus in Xanthium. Part II.Relative contribution of buds & leaves to effective-ness of inductive treatment. Botan. Gaz. 119:179-185.

10. LIVERMAN, J. L. 1955. The physiology of flower-ing. Ann. Rev. Plant Physiol. 6: 177-210.

11. RANDOLPH, L. F. 1935. A new fixing fluid & arevised schedule for the paraffin method in plantcytology. Stain Technology 10: 95-96.

12. RICH, M. A., J. L. BOLAFFI, J. E. KNOLL, L. CHEONG,& M. L. EIDINOFF. 1958. Growth inhibition of ahuman tumor cell strain by 5-fluorouracil, 5-fluorouridine, & 5-fluoro-2'-deoxyuridine-Reversalstudies. Cancer Res. 18: 730-735.

13. SACHS, R. M., C. BRETZ, & A. LANG. 1959. Celldivision & gibberellic acid. Exptl. Cell Res. 18:230-244.

14. SALISBURY, F. B. 1955. The dual role of auxini inflowering. Plant Physiol. 30: 327-334.

15. SALISBURY, F. B. & J. BONNER. 1960. Inhibition ofphotoperiodic induction by 5-fluorouracil. PlantPhysiol. 35: 173-177.

16. SIMON, E. H. 1961. Evidence for the nonparticipa-tion of DNA in viral RNA synthesis. Virology13: 105-118.

17. TENCER, R. 1961. The effect of 5-fluorodeoxyuri-dine on amphibian embryos. Exptl. Cell Res. 23:418-419.

18. WENT, F. W. 1957. Experimental Control ofPlant Growth. Chronica Botanica, \Valtham,Mlass.

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