SEPARATION OF MECHANISMS INITIATING CELL DIVISION AND CELLEXPANSION IN LETTUCE SEED GERMINATION'

ALAN H. HABER AND HELEN J. LUIPPOLDBIOLOGY DIvISION, OAK RIDGE NATIONAl. LABORATORY,- OAK RIDGE, TENN.

INTRODUCTION

After imbibition of water by seeds under favorablecircumstances, the growth by which the embryo be-comes a young seedling occurs by both expansion ofcells originally present in the dormant embryo andmitotic divisions resulting in an increase in cell num-ber (15). Germination of lettuce seed has been ofspecial interest to plant physiologists because it canbe stimulated by gibberellins, kinetin, or light (8).The sequence of the onset of cell division and elonga-tion in germinating lettuce seed has been studied byEvenari et al (4), who found that the beginnings ofmitotic activity and of cell elongation seemed to coin-cide in time. No mitotic activity was found in seedsprevented from germinating by withholding favorablelight treatments. Their data suggested that, "Thestart of mitoses, the beginning of cell elongation andthe protrusion of the rootlet are correlated events dur-ing germination" (4). Cell division and expansiondo not begin simultaneously during germination ofmany other kin(ds of seeds. Cellular expansion pre-cedes mitosis by nIany hours (luring germination ofcorn (14) or barley (2), andl by several days in thebroad bean (16). On the other hand, mitotic ac-tivity occurs several days before germination in pineseeds (5). Similarly, the number of cells per em-bryonic axis of after-ripening cherry seeds increasedlweeks before there was a detectable increase in axislength, which suggests that mitosis also precedes cellexpansion in cherry seeds (11). Because divisionand expansion (lo not begin simultaneously (luringgermination of many species of seeds, it is possiblethat lettuce might behave similarly under selectedconditions. We therefore attempted to separate intime the inception of cell division from that of ex-pansion during lettuce seed germination. The resultsshow not only that the beginning of mitotic activitycan be made either to precede or to follow cell ex-pansion, but also that mitotic activity can be com-pletely separated from germination. These resultsare of general importance in understanding the mech-anisms of germination for all types of seeds.

MATERIALS AND METHODSAll experiments were performed with seeds of

Lactuca sati7'a, var. New York, from the same batch

' Received for publication June 3, 1959.2 Operated by Union Carbide Corporation for the

U. S. Atomic Energy Commission.

168

of seeds previouslv studied in connection with lightsensitivity (6) and dornmancy resulting from gammaradiation (7). Cobalt 60 gamma rays were ad-ministered to the air-dry seeds as described previous-ly (7). Seeds were then sown upon two pieces ofWhatman No. 1 filter paper moistened with 5 ml ofsolution in covere(d 9 cm Petri dishes. Dishes at260 C were under continuous white fluorescent light-ing of 35 ft-c; dishes at 100 or 300 C were in dark-ness.

Seeds were (lesignated as germinated with the firstvisible sign of rootlet expansion, namely protrusionthrough the seed coats. Such rootlets are hereaftercalled roots. Unexpanded rootlets excised from em-bryos of nongerminated seeds are hereafter designatedradicles.

Meristems of roots or radicles were fixed inethanol-acetic acid (3: 1), hydrolyzed in N HCl for8 min at 600 C, and stained with Feulgen's reagent.The material was squashed upon slides in such a waythat essentially all the cells from the apical 0.5 mm ofthe given root or radicle could be examined for mi-totic figures.

RESULTS

It vas found that, at 260 C, the beginning of mi-tosis and rootlet protrusion roughly coincided in time,as was observed by Evenari et al (4). Under theseconditions, the time from sowing to germination wasapproximately 16 hrs. During germination, we sel-dom saw mitotic figures before protrusion, althoughwe did observe one or two cells in division when weexamined a dozen nongerminated seeds before radicleprotrusion was visible. By 20 hrs (i.e., after 4 hrsgrowth) the roots had protruded approximately 1.5mm through the seed coats and contained an averageof several hundred mitotic figures. Experimentswere designed to try to separate in time the inceptionof division and of expansion. For this purpose, seedswere irradiated with Co60 gamma rays before mois-tening. Although ionizing radiation affects all partsof a cell, one might expect to observe more detectableinjury to mechanisms controlling cell division than tothose controlling cell expansion (9, 12). Thereforeseeds were given 641 kr of gamma radiation and sownon 10-5 M kinetin at 260 C. Kinetin was used to re-verse the inhibition of germination percentages effect-ed by irradiation with 641 kr (7). Table I illustratesthat, when seeds were given 641 kr gamma radiationand sown on kinetin, cell expansion (as indicated byrootlet protrusion) and mitosis were both greatly de-

HABER AND LUIPPOLD-LETTUCE SEED GERMINATION

layed. However, the beginning of mitosis was de-layed more than that of cell expansion. No mitosiswas detected in meristems from expanded roots aftertwo, three, and four days. Even more striking is thefact that mitosis was not observed in any of the rootsexamined until two days after germination was firstobserved. Mficronuclei were abundant in interphasecells from root tips of New York lettuce seeds afterthese seeds were given 214 kr from the same gamma

source (7). However, all root meristems that lackedmitotic figures also lacked micronuclei. Whereas theabsence of mitotic figures in the entire root tip showsthat no cells were undergoing mitosis at the time offixation, the absence of micronuclei further suggeststhat no mitotic divisions occurred before fixation.For comparison, when unirradiated seeds were germi-nated in water at 260 C and roots had grown 1.5 mm(20 hrs after sowing, i.e., approximately 4 hrs afterrootlet protrusion) 55, 60, 233, 348, 435, 524, 540, 592,and 692 figures were found in nine root tips. Al-though we thus conclude that gamma irradiation per-

mits cellular expansion to proceed before mitosis be-gins (table I), it is clear from our previous studiesthat no dose of gamma radiation can be found thatpermits germination that is not eventually accom-

panie(l by mitotic activity (7).

The results described in the last paragraph raisethe question as to whether other treatments might giveconverse results, namely, mitotic activity before ger-mination. Several investigators have used mannitolto prevent cellular expansion by interfering withwater uptake (1. 3). Consequently, one might expectthat mannitol wvould interfere more with cell expan-

sion than with cell division. As was found with ir-radiated seeds in kinetin (table I), seeds germinatedmuch more slowly in the presence of various concen-

trations of mannitol at 260 C than did untreated seedsin water (table II). In contrast to the results withgamma radiation, however, mitosis occurred beforeprotrusion when see(ds were in mannitol., since mitotic

figures were frequently found in nongerminated seeds(table II). For seeds in 0.5 M mannitol, mitoticfigures were detected after one day, even thoughgermination was not detected until a week later.Only 2.5 % of the seeds in 0.5 M mannitol had germi-nated after 16 days, and no further germination oc-curred thereafter. The presence of mitotic activityafter one day was compared with the percentagegermination after 16 days by calculating X2 by themethod of the 2 X 2 table (13). The correspondingvalue of p was 1.2 X 10-3. Thus we can conclude

TABLE ITIME COURSE OF GERMINATION AND MITOTIc ACTIVITY

IN SEEDs GINTEN 641 KR OF GAMMA RAYS ANDSOWN ON 10-5 M KINETIN AT 26' C

GERMINATED POPULATION ONLY

AVERAGE FRACTIONDAYS OFAVERAGEFROOTSAFTER GERMINA- ROOT WITHSOWING TION OF LENGTH MITOTIC

ALL SEEDS* (MM) FIGURES**

1 02 6 1.00 0/43 50 1.66 0/104 68 2.72 5/10

* Germination percentages for each day were calculatedfrom independent samples of 100 seeds each.

** Number of roots with one or more mitotic figures/number of roots examined.

not only that mannitol delays germination more thanit delays mitosis, but that it can prevent germinationof seeds that nonetheless undergo mitotic activity.

Further experiments were conducted to see iftreatments other than gamma radiation or mannitolalso could alter the sequence of the beginnings ofmitosis and germination. Results parallel to thosefound with irradiated seeds were found for untreated

LE IITIME COURSE OF GERMINATION AND MITOTIc ACTIVITY IN SEEDS SOWN ON SOLUTIONS OF MANNITOL AT 26° C

MANNITOL CONCENTRATION0.3 M 0.4 M 0.5 M

DAYS AFTER % No. OF 10 % No. OF 10 No. OF 10SOWING GERMINATION* RADICLES WITH GEILSIH RMINATION* AILSWT

MITOTIC FIGURES** GERMINATION* MITOTICFIGURESL GE MITOTICFIGURESW

1 0.0 3 0.0 4 0.0 32 0.3 8 0.0 6 0.0 23 2.0 6 0.0 4 0.0 24 4.6 1 0.3 0 0.0 05 9.1 2 0.6 2 0.0 06 ~~9.3 1.0 &0.0..7 9.3 31.3 0. 1

8 10.3 . 1.8 0.010 14.3 3 33 4 05 016 29.3 ..28.3 ..2.5..

* Calctllated from 100 to 350 seeds.**Radicles selected only from the populatiolns of seeds that had n1ot germinated at times indicated.

Data refer to radicles with one or more mitotic figures.

169

PLANT PHYSIOLOGY

TABLE IIITIME COURSE OF GERMINATION AND MITOTIC

ACTIVITY AT 100 C

GERMINATED POPULATION ONLY

DAYSAFTERSOWING

34567

GERMINA-TION OF

ALL SEEDS*

01

447183

AVERAGE FRACTIONROOT OF ROOTS

LENGTH WITH(MM) MITOTICFIGURES"*

0.51.522

. .

0/14/20***8/119/11

* Germination percentages for each day were calculatedfrom inidependent samples of 88 to 92 seeds.

** Nulmber of roots with mitotic figures/niumber ofroots examined.

*** See table IV for frequency distributioin of mitoticfigures.

see(ls germinated at 100 C (table III). Again, thetimes of cell division and of germination were greatly(lelaye(l in comparison to the times observed with un-treate(l seedls germinating at 260 C. Beginning ofmitosis seemed, however, to be more delayed than thebeginning of germination. Thus after five days, 16of 20 protruded roots had no mitotic figures. Of thefour root tips that did have mitotic figures after fiv,e(lays, three lhad only one figure, and one had twofigures. These data should be compared with thehuindreds of mitotic figures found in roots of the samelength when seeds were germinated at 260 C (see datain first paragraph of this section). The frequency(listributioni of mitotic figures among the 20 root tipsexamine(l after five days at 100 C is shown in tableIV. The frequencies expected from a Poisson dis-tributicon with the same mean number of mitoticfigures per root were calculated for comparison. Theagreement between the observed frequencies of mitoticfigures per tip and the frequencies calculated from aPoisson distribution suggests that soon after rootletshlave protru(led, mitosis occurs randomly.

Experiments were performed also at 300 C, juston the upper limit of the temlperature range that per-imits ger-miiination. Table 'Vr summilarizes an experi-

TABLE IVDISTRIBUTIGN 01o A:IiOrIC FIGURES AMiONG ROOT TIPS

FRO-M GER-MINATED SEEDS 5 DAYS AFTERSOWING AT 100 C

MITOIIC FIGURES OBSERVED FREQUENCY EXPECTED FROMPER ROOT TIP FREQUENCY POISSON DISTRIBUTION

0

19

3

Total

1631

0

?o

15.583.890.490.04

20.00

ment in wlhich separate dishes of seeds at 300 C wereexamined after various times for germination per-centages, and radicles were excised from 20 of thenongerminated seeds and examined for mitotic ac-tivity. It has been our experience that, at thistemperature, all seeds that will germinate do so with-in one or two days. Consequently, we do not considerthe 5 % germination after eight days to be a biologi-cally significant increase over the germlination per-centages at earlier times. Mitotic activity occurs innongerminated seeds at 300 C (table V) just as itdoes in mannitol at 260 C (table II). The appearanceof mitotic activity after one day was compared withthe percentage germination after eight days by themethod described. The difference was significant atthe level of 10-4 . Thus we can conclude that the 300 Ctemperature, like treatment with 0.5 M mannitol at260 C, prevents germination of seeds that undergomitotic activity. Figure 1 illustrates the frequencydistribution of mitotic figures among the same groupsof 20 radicles listed in table V. The data in figure 1suggest more readily than does table V that the mitoticactivitv in the nongerminating seeds tends to decreasewith increasing time. More important, however,

TABLE VTIME COURSE OF GERMINATION AND MITOTIC ACTIVITY IN

SEEDS AT 300 C

DAYS AFTER 51 No. OF 20 RADICLES WITHSOWING GERMINATION* MIITOTIC FIGURES**

12358

0.80.03.30.85.0

1313642

* Percentage of 120 seeds4* Radicles selected from noiigerminated seeds. See

fig 1 for frequenicy distribution of mitotic figures.

figure 1 shows a marked deviation of the observeddistributions from those calculated for the correspond-ing Poisson distributions with the same mean numberof mitotic figures per group of radicles. The ob-served frequencies are higher than the calculated fre-quencies for those radicles having no mitotic figures,as well as for classes having many more mitoticfigures than expected from the Poisson distributions.This type of (leviation from randomness suggests atendency for an all-or-none characteristic (i.e., nomitoses vs. nmany) in the occurrence of mitoses in Inon1-germiniated seeds. Such an all-or-none characteristicis analogous to the normal germination process inlettuce, for which it is often found that under a givenset of conditions a single batch of seeds will consistalmost entirely of two populations: one population inwhich none of the seedls germinate, and one popula-tion in which all of the seeds germinate and( b)ecomeseedlings capable of growth to maturity.

170

HABER AND LUIPPOLD-LETTUCE SEED GERMINATION

UNCLASSIFIEDORNL-LR-DWG 38417

84 DAY__III1I4II IIIDAY~IH08

2 DAYS

016

12 --__3DAYSc8

C24

4o-a]12 -|||r 5 DAYS

_.* T T 1

16

12IIII l I I IT Ill

4i \I 11111 1 111111111I 1111LII IIT10

0 4 8 12 16 20 24 28MITOTIC FIGURES PER RADICLE

Fic. 1. Distribution of mitotic figures among radiclesexcised from nongerminiated seeds at 300 C. Bars repre-sent observed frequencies. Curves connect points calcu-lated from Poisson distributions.

At 400 C. see(ds neither germinated nor did theyundergo any detectable mitotic activity.

DISCUSSION

These results indicate that the beginning of cell,division and the beginning of cell expansion are notcorrelatedl events during germination. Gamma radia-tion or low temperatures delay the beginning of celldivision until after cell expansion has resulted in root-let protrusion. Whereas root tips from irradiatedseeds germinated at 260 C or from unirradiated seedsgerminated at 100 C had no mitotic figures, roots ofcomparable length from untreated seeds germinated at26° C had several hundred mitotic figures. Converse-ly, nmannitol or high temperatures tend to permit somecell division before cell expansion can be detected byrootlet protusion. At 300 C in water or 260 C in 0.5W\I mannitol, mitoses occur in seeds that do not sub-sequently germinate under these conditions. It ispertinent that these seeds that can be prevented fromgerminating by holding at 300 C do germinate whentransferred back to 260 C (Haber, unpublished).Consequently, the failure of seeds having mitotic ac-tivity at 300 C to germinate cannot be explained bysaying that loss of viability intervened between thetime that mitotic activity began and the time that ex-pansion wouldlhave begun. Moreover, the appear-

ance of mitotic figures in nongerminating seeds at300 C cannot be explained by saying that mitoseswere initiated but not completed, since the number ofmitotic figures per radicle tendedl to decrease witltime (fig 1).

The data of Evenari et al (4) apparently resultedfrom the use of conditions under which the beginningsof cell division and cell elongation happened to coin-ci(le. In comparing their results with our own, an-other factor to consider is the different cytologicaltechniques used. In our studies, we used squashedpreparations, which enabled us to examine many cellsof a root tip including essentially all the thousands ofcells in the meristematic region. Evenari et al ex-amined 12-A-thick longitudinal sections instead of thewhole root tip. It should be emphasized that ourstudies are concerned with the initiation, and not theextent, of mitosis. The data do show that the extentof cell (livision is generally greater after rootlet pro-trusion than before protrusion. Such increased mito-tic activity, lhowever, is probably a result of growth,since it is neither correlated with nor necessary forthe initiation of cell expansion. Thus our results arean extension of, an(d not necessarily in contradictionto, the earlier work of Evenari et al (4).

Protrusion of the rootlet during lettuce seed gernmi-nation is caused by cell expansion, whereas mitoticdivision contributes little or nothing. This conclusionfollows from the fact that the radicle of a nongermi-nated seed can have as many as 29 mitotic figures atone time (fig 1), whereas a germinated seed can havea root that has expanded more than 1.6 mm throughthe seed coat without un(lergoing any mitoses (as in-(licated by the absence of mitotic figures and micro-nuclei after gamma irradiation, table I). This con-clusion is consistent with the facts that corn (14),barley (2), and broad bean (16) seedls normallyundergo rootlet protrusion before mitosis begins,whereas pine ( 5) and, presumably, cherry ( 1 1 ) seedsundergo mitotic activity before rootlet protrusion.Thus the mechanism of rootlet protrusion in seeds isparallel to the mechanism of root extension in matureplants, which is caused chiefly by expansion of cellsin the zone of elongation. Mitotic activity functionsin root elongation indirectly, i.e., by proliferating newcells which, in turn, are capable of contributing toroot extension by their own expansion.

Although gamma radiation can cause germinationto precede mitotic activity, we have shown that anydose of gamma radiation that will permit lettuce see(dgermination also will permit nmitotic activity (7).Consistent with this is our present observation that at100 C, wlhere germination also precedes mitotic ac-tivity, mitoses seemed to occur ranclomly among pro-truded roots (table IV). By way of contrast, at 300in water or at 260 C in 0.5 M mannitol, mitotic activitycould be completely separated from germination, sinceseeds undergoing mitotic activity dlid not germinateat all. Analysis of the frequency distribution ofmitoses among radicles of nongerminating seed at300 C shoved that mitoses were not occurring ran-

171

PLANT PHYSIOLOGY

domly, but tended to conform to an all-or-none pat-tern (fig 1). This suggests that there are mecha-nisms preventing mitotic activity in some and permit-ting mitotic activity in other nongerminating seeds at300 C. This is analogous to the findings that thereare mechanisms that prevent cell expansion in some

and permit cell expansion in other seeds duringnormal germination at 22 to 260 C. Clearly, themechanisms by which mitotic activity is blocked mustbe different from the mechanisms by which cell ex-

pansion is blocked, since the seeds with mitotic ac-

tivity at 300 C (fig 1) were nongerminating.IThese results also have implications for studies

of the mechanisms of action of germination stimula-tors. Since rootlet protrusion results from cell ex-

pansion and since kinetin stimulates germination ofgamma-irradliated seeds under conditions where cellexpansion precedes mitosis by several days (7, tableI), it is obvious that the germtination-stimulating ac-

tivity of kinetin must be attributed to an initiation ofcell expansion and not to any possible stimulation ofcell (livision. These considerations are especially in-teresting because kinetin was first studied in connec-

tion with its activity as a cell-division factor (10).Presumably other germination-stimulating agentsmust similarly function by stimulating cell expansion(7). This last statement is not intended to suggestthat the mechanisms of action of different germina-tion stimuilators are identical, since we have previous-ly shown that effects of gibberellic acid, kinetin, andred light could be separated from one another (8).Nor (lo we wish to imply that germination-stimulatingagents may not also affect mitotic activity in seedsapart from their effects on cell expansion. An in-teresting problem for future research will be to de-ternline whether the light and chemicals that stimulatenornmal gernmination can stimulate mitotic activity innongerminating seedls.

The different seqiuences of the inceptions of celldivision and cell expansion at extremely high and ex-

tremely low temperatures indicate different physi-ological properties of germination at the differenttemperatures. These considerations are reminiscentof earlier work fronm our laboratory, performed withGrand Rapids lettuce seeds, which showed that kinetinstimulates germination at superoptimal temperatureswhere gibberellic acidl is ineffective, whereas thereverse is true at low temperatures (8).

SUM MARYI. At 260 C lettuce seed germinates rapidly, and

the beginnings of cell division and cell expansionroughly coincide in time.

II. WVihen gamnma-irradiated seeds are sown on a

solution of kinetin at 260 C, germination, althoughgreatly delayed compared to that of unirradiated con-

trols, precedes cell division by several days.III. Results similar to those with irradiated seeds

were found with unirradiated seeds germinating at100 C. The distribution of mitoses among root tips

of newly germinated seeds at 100 C suggested thatmitoses occurred randomly among them.

IV. When seeds were germinated at 26° C insolutions of mannitol, cell divisions preceded germi-nation by many days. High concentrations of man-nitol prevented germination of seeds that neverthe-less underwent mitotic activity.

V. At 300 C, only a very small percentage of seedsgerminate. Most seeds, however, undergo cell divi-sion. The distribution of mitoses among radiclemeristems of nongerminating seeds showed that mi-toses were not occurring randomly among the un-expanded radicles. Rather, there was a tendency foran all-or-none characteristic in the occurrence ofmitoses within individual radicles. As time pro-gressed, the mitotic activity in these nongerminatingseedls diminished.

These results indicate that A. The initiation ofcell expansion an(l that of cell division are controlledby separate mechanisms during germination. B.Rootlet protrusion during germination results fromcellular expansion, whereas cell division plays littleor no role. C. The mechanisms by which kinetinand other agents stimulate lettuce seed germinationare related to the initiation of cellular expansion andnot to cell (livision.

LITERATURE CITED

1. BURSTR63,, H. 1954. Studies oni growth and meta-bolism of roots. XI. The influence of auxin andcoumarin derivatives on the cell wall. Physiol.Plantarum. 7: 548-559.

2. CALDECOTT, R. S. and L. SMITH 1952. A study ofX-ray-induced chromosomal aberrations in barley.Cytologia (Japan) 17: 224-242.

3. CLELAND, R. and J. BONNER 1956. The residualeffect of auxin oIn the cell wall. Plant Physiol.31: 350-354.

4. EVENARI Al., S. KLEIN, H. ANCHORI and N. FEIN-BRUN 1957. The beginning of cell division andcell elongation in germinating lettuce seed. Bull.Res. Council Israel 6D: 33-37.

5. Goo, M. 1952. When cell division begins in thegerminatinig seeds of Pinns th1tnbergii Parl. Jour.Jap. Forestry Soc. 34: 3-4.

6. HABER, A. H. 1959. Rendering the germination oflight-inisensitive lettuce seeds sensitive to light.Physiol. Planitarum. 12: 456-464.

7. HABER, A. H. and H. J. LUIPPOLD 1959. Dormancyresulting from gamma-irradiation of lettuce seed.Int. Jour. Radiation Biol. 1: 317-327.

8. HABER, A. H. and N. E. TOLBERT 1950. Effects ofgibberellic acid, kinetin, and light on the germina-tion of lettuce seed. In: Photoperiodism and Re-lated Phenomena in Plants and Animals. R. B.WVithrow, ed. AAAS, Washington, D. C. Pp.197-206.

9. HASKINs, F., M. DAVIDSON and R. BEERS 1958.Influence of seed irradiation with X-rays andthermal neutrons upon cell size and mitotic activityin root tips of maize. Amer. Naturalist 92: 365-369.

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HABER AND LUIPPOLD-LETTUCE SEED GERMINATION1

10. MILLER, C., F. SKOOG, F. OKUMURA, M. VON SALTZAand F. STRONG 1956. Isolation, structure andsynthesis of kinetin, a substance promoting celldivision. Jour. Amer. Chem. Soc. 78: 1375-1380.

11. POLLOCK, B. M. and H. 0. OLNEY 1959. Studiesof the rest period. I. Growth, translocation, andrespiratory changes in the embryonic organs of theafter-ripening cherry seed. Plant Physiol. 34:131-142.

12. SCHWARTZ, D. and C. E. BAY 1956. Furtherstudies on the reversal in the seedling height dosecurve at very high levels of ionizing radiations.

Amer. Naturalist 90: 323-327.13. SNEDECOR, G. W. 1956. Statistical Methods. (5th

ed.) Iowa State College Press, Ames14. TOOILE, E. H. 1924. The transformations and

course of dev._lopment of germinating maize.Amer. Jour. Bot. 11: 325-350.

15. TOOLF, E. H., S. B. HENDRICKS, H. A. BORTHWICKand V. K. TOOLE 1956. Physiology of seedgermination. Ann. Rev. Plant Physiol. 7: 299-324.

16. WOLFF, S. 1954. Some aspects of the chemical pro-tection against radiation damage to Vicia fabachromosomes. Genetics 39: 356-364.

INHIBITION OF PHOTOPERIODIC INDUCTION BY 5-FLUOROURACIL 1, 2FRANK B. SALISBURY AND JAMES BONNER

DEPARTMENT OF BOTANY AND PLANT PATHOLOGY, COLORADO STATE UNIVERSITY, FORT COLLINS;DIVISION OF BIOLOGY, CALIFORNIA INSTITUTE OF TECHNOTLCGY, PASADENA

In this paper it will be shown that photoperiodicinduction of the cocklebur, a short day plant, is in-hibited by the pyrimidine 5-fluorouracil (5-FU).The studies of other workers have shown that 5-FUinhibits the growth of various kinds of cells and tissuesby suppressing the formation of thymidine, and thatapplication of thymidine relieves the inhibitory effectsof 5-FU. In these cases 5-FU is an inhibitor ofDNA synthesis (2, 4,5). In other cases, however,5-FU inhibits RNA synthesis. Thus 5-FU inhibitsthe production of tobacco mosaic viral RNA by to-bacco leaves (1). This inhibition, which is relievedneither by thymidine nor by uracil, is associated withincorporation of 5-FU into the viral RNA (3). In.hibition of photoperiodic induction by 5-FU is notreversed by either thymidine or uracil but is reversedby orotic acid (6) which is known to be an intermedi-ate in the biogenesis both of uridine and cytidine andof deoxycytidine and thymidine. Reversal of 5-FUinhibition by orotic acid suggests the hypothesis thatthe inhibition is related in some way to suppressionof nucleic acid metabolism.

The experiments here reported were further de-signed to determine which component process of floralinduction is inhibited by 5-FU. It will be shownthat one effect of 5-FU is upon the inductive act bywhich vegetative buds are so changed that they sub-sequently develop into floral primordia.

IReceived June 3, 1959.2 Supported in part by grants from the Frasch Founda-

tion, National Science Foundation, and the Colorado StateUniversity Research Foundation. This work was re-ported at the Annual Meeting, American Society of PlantPhysiologists, Indiana University, August, 1958.

MIETHODS

The general procedures used in this investigation,which have been described in detail elsewhere (8, 9),were as follows: Cocklebur plants (Xanthium penn-sylvanicum Wall.3) were grown from seed of ourstandard inbred line. They were maintained in thevegetative condition in the greenhouse by the use ofsupplementary low intensity light to extend the naturalday length to approximately 20 hours per day. Theplants were used 60 days or more after planting and,therefore, after the appearance of the first typicallymature leaves. One day before each experiment, theplants to be used were sorted according to size of thehalf-expanded (most sensitive) leaf. One leaf abovethe half-expanded leaf and all leaves below this wereremoved from each plant. The plants were then dis-tributed into groups; all groups contained equal repre-sentations of each size class of leaf. In general, onegroup of 10 to 20 plants was used for each treatmentof each experiment; each experiment was repeatedthree to eight times as noted below.

Chemical treatments were applied by dipping theleaf or tip or both into a solution of the chemical inquestion. Dipping of the apical bud alone in thisway results in the retention by the bud of approximate-ly 0.05 cc of treatment solution. Treatment of the

3 Plants used have been classified by H. D. Harring-ton, taxonomist, Colorado State University, using recentmanuals of the Illinois region from which the plantsoriginated, as Xatthium italicum Moretti. We will con-tinue to use X. pennsylvaflicUtn Wall. until the taxonomyof Xanthium has been clarified. Specimens of the plantstypical of those used in our experiments are on file inthe Colorado State University Herbarium.

173