Plant Physiol. (1972) 50, 585-590

The Level of Phytohormones in Monoecions and GynoeciousCucumbers as Affected by Photoperiod and Ethephon1

Received for publication January 28, 1972

J. RUDICH, A. H. HALEVY, AND N. KEDARThe Hebrew University, Faculty of Agriculture, Rehovot, Israel

ABSTRACT

The endogenous levels of auxin, gibberellin, and inhibitorswere followed in monoecious and gynoecious cucumber (Cu-cumis sativus L.) plants, and in plants treated with the ethyl-ene-releasing compound Ethephon (2-chloroethyl phosphonicacid). Higher auxin inhibitor and lower gibberellin levels wereassociated with female tendency. The endogenous level of gib-berellin and auxin decreased in Ethephon-treated plants. Appli-cation of Ethephon induced a rise in abscisic acid. Rootapplication of abscisic acid promoted female tendency of gyno-ecious cucumbers grown under conditions which increase male-ness. High C02 levels, which are known to antagonize ethylene,increased maleness of gynoecious cucumbers. The possibilityof interrelationship between gibbereilin, auxin, ethylene, andabscisic acid on sex expression are discussed.

Studies of hormone systems involved in the regulation of sexexpression in cucurbits have been confined mostly to auxinsand gibberellins. Femaleness of cucumbers has been increasedby application of auxin (9, 19). When homologous sections ofhermaphrodite and andromonoecious cucumber plants werecompared, higher levels of auxin were found in the hermaphro-dite type (10). The role of auxin in the development of femaleflowers in cucumbers has been demonstrated (11) by growing,in tissue culture, flower buds of cucumber plants from nodeswhich produce only male flowers. Addition of IAA to thesecultures produced ovaries and stigmas. Short days and lowtemperatures enhanced the femaleness of certain cucumbercultivars (20) and of squash (23). Nitsch et al. (23) assumethat this was due to high levels of endogenous auxin foundunder short day conditions.

Treatments with exogenous gibberellin increased maleness incucumbers (25) or delayed female flower formation (4). Moredirect evidence for the participation of gibberellin influencingdifferentiation of male flowers was reported (2, 16). Theyfound higher levels of gibberellin in monoecious than ingynoecious varieties using the diffusion and exudation methods.Femaleness has been enhanced in muskmelon by treatmentwith growth retardants (14) which affect endogenous gibberel-lin levels (27). Cucumber, squash, and muskmelon plantsbearing male flowers have been found to produce femaleflowers after treatments with Ethephon (17, 22, 26) which re-lease ethylene and enhance ethylene production in plant tissue

I This paper represents a part of the Ph.D. thesis of J. Rudich.

(33). When ethylene evolution from cucumber plants was ex-amined, more ethylene was evolved from apices of the gynoe-cious than from those of monoecious type (28).The interactions between auxin and ethylene are complex.

Many studies have shown that auxins hasten the production ofethylene (1, 6, 8), that ethylene enhances the decomposition ofauxin (3), and that it also inhibits the movement of auxin in theplant (3). A number of effects, in the past attributed to highlevels of auxin, are now considered to be a result of ethyleneproduction under the influence of auxin (5, 7).

Little is known about the interaction between ethylene andgibberellin. Antagonism between them has been found ingermination of wheat and the production of both a-amylaseand invertase (31). Their action is also antagonistic in fruitripening and in sex expression of flowers (13, 26). We are notaware of any publication on the effect of ethylene on endoge-nous gibberellin levels. Kang et al. (18) found that gibberellinhad no effect on ethylene production in bean seedlings. Theyconcluded that GA and ethylene effects in the development ofthe seedlings were independent.

Since sex expression in cucumbers is influenced by Ethephonas well as by auxin and gibberellin, we tried to determine inthe present work the effect of Ethephon on the level of the abovephytohormones as well as on ABA and other native inhibitors,in an attempt to gain additional knowledge on the relation ofthese phytohormones to sex expression.

MATERIALS AND METHODSPlant Material. Endogenous levels of growth substances were

determined in the monoecious and gynoecious 'Bet Alpha' 2lines of cucumber (Cucuinis sativus L.) plants. The gynoeciousline was bred from the monoecious one and differs from it inthe gene for femaleness (2). Under short day conditions itproduces female flowers from the first or second node, underlong day conditions male flowers are produced at the first sixnodes, all the rest being female. In one experiment andromo-noecious muskmelon plants (cv. Ananas PMR) were used.

In most experiments plants were grown in growth chambersat 8-hr photoperiod under mixed fluorescent and incandescentlamp light of 3500 ft-c. Long day treatments were given byincandescent lamps of 70 to 80 ft-c. Temperature during theday, 8 hr, was 28 C, at all other times it was 18 C. In someexperiments CO2 level was also controlled in the chambers.

In a few experiments plants were grown in a phytotronunder natural light intensities. Long days were given by ex-tending the natural day to 16 hr by incandescent lamps of 100ft-c. Temperature during the day was 27 C and at night 22 C.

2 Seeds originating from a line bred at the Department of PlantGenetics of the Weizmann Institute of Science, Rehovot weregenerously supplied by ZRAIM Gedera Seed Co.

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RUDICH, HALEVY, AND KEDAR

External Application of Growth Regulators. ABA was ap-plied to plants by two ways: (a) by daily spraying at 1 to 250mg/I for 2 weeks starting at the first leat stage; (b) by rootapplication through nutrient solution. Plants were grown withhalf-strength Hoagland solution which was supplemented fivetimes at 4-day intervals with solutions containing various con-centrations of ABA. First application was at the first leaf stage.

Ethephon was sprayed on seedlings at the first leaf stage(26). For bioassay, plants with two to three leaves and anelongated first internode were used. The plants were cut justbelow the youngest leaf, the tip and youngest leaf were usedfor extraction or diffusion into agar.

Diffusion into Agar. Twenty apices were planted in each offour Petri dishes containing 1.5% agar. The dishes were kept at25 C in moist chamber for 24 hr and illuminated with 600 ft-cof fluorescent light. The agar was deep-frozen at -20 C over-night and was then thawed and washed with 100% methanol.

Extraction. Approximately 3 g fresh material consisting ofthe apex and youngest leaf of three-leaved seedlings were ex-tracted in 150 ml of 80% methanol. An identical number ofseedlings was used for each assay. The mixture was shakenovernight in a cold room at 3 to 4 C. After decanting, thesolids were further extracted by shaking with 150 ml of 100%methanol for half an hour. The methanol fractions were unitedand evaporated.

Fractionation. Several fractionation methods were tried andadapted for use with cucumber plants. The method used is indi-cated in the legend to figures.

Bicarbonate and Ether Fractionation. After ether extraction(10), the ether was washed five times with 0.2 M sodium bicar-bonate at pH 8.4. The bicarbonate was acidified to pH 3.0with HCI and was washed five times with peroxide-free ether.The acid ether fraction was used for determination of auxinactivity. Since auxin and inhibitor activity were found in allfractions, other methods in which auxin activity would concen-trate in one fraction were sought. In some experiments thecrude extract was acidified to pH 3.0 and partitioned five timesinto ether.

Ethyl Acetate Fractionation for Auxins. Extracting methanolwas evaporated, the aqueous residue was acidified to pH 3 withHCl and shaken six times with petroleum ether and then fivetimes with ethyl acetate or ether. Both petroleum ether andethyl acetate (or ether) fractions were tested for activity. Mostauxin activity was found in the ether or ethyl acetate fraction.

Methylene Chloride Fractionation for ABA. The pH of theaqueous residue was raised to 8.3 with NH4OH, centrifuged at10,000g for 15 min, and partitioned four times with methylenechloride. The aqueous phase was acidified to pH 3.0 with HCIand again partitioned four times with methylene chloride. Theacidic methylene chloride fraction was evaporated to dryness,dissolved in distilled absolute methanol, and used for ABAdeterminations.

Fractionation for Gibberellin Activity. Fractionation method,as described by Halevy and Shilo (15), was used, as well as thepetroleum ether, ethyl acetate method described for auxins.

Chromatography. Two-cm wide Whatman No. 2 strips wereloaded with 1.5-ml aliquots, equivalent to 500 mg fresh weight.Isopropanol: ammonia: water (8: 1: 1, v/v) was used as solvent.

Thin Layer Chromatography was carried out with platescoated with 250 ,u layer of Silica Gel G (Merck). The plateswere developed in two different solvent systems: (1) n-propa-nol: n-butanol: water: ammonium hydroxide 28% (6:2:2:1,v/v): (2) benzene:ethvl acetate:acetic acid (50:5:2, v/v). Theplates were developed by ascending chromatography to a dis-tance of 15 cm and dried at room temperature in a forced aircabinet.

Detection of ABA by Gas Liquid Chromaography. Metha-

nolic extract was partitioned with methylene chloride andchromatographed by thin layer chromatography in the twosolvent systems as above. The zone corresponding to authenticABA, detected under an ultraviolet lamp at z54 nm, wasscraped off and eluted with methanol. After methylation withdiazomethane dissolved in hexane, a 1-,ul portion ot the extractwas injected into a Packard 7400 gas chromatograph, using aglass spiral column 1.8 m long X 3.2 mm inner diameter,packed with 1.5% QFI on Gas-chrom Q 60 to 80 mesh. Thecolumn temperature was 200 C; injection and detector temper-ature were 225 C and 195 C, respectively. Nitrogen gas flowof 42 ml/min served as carrier. An electron capture detectorwas used (29) with radioactive tritium foil. Calibration curverelating the amount of cis-ABA methyl ester to the computedarea of the recorded peak was used for quantitative estimationof ABA. Retention time of ABA was 5.14 min. The results ofABA content are expressed in ,ug ABA per 100 g fresh weight.

Bioassays. The wheat coleoptile test based on Nitsch andNitsch (24) was used for testing auxins and auxin inhibitors.The wheat variety used was M-852 (supplied by Hazera Co.,Haifa). Tests of each RF were replicated three times, each repli-cate including three coleoptiles, i.e., nine per test.

Barley Endosperm Test for Gibberellins and Inhibitors. Halfseeds of the variety Omer were used. Tests of each RF were rep-licated three times, each replicate including two half seeds.Standards were replicated five times. The method used was asdescribed earlier (12, 15). Incubation at 25 C on the rotator (1rpm) lasted 32 hr. Reducing sugars in paper chromatographedwith the extractants were found to be negligible and no a-amylase activity was found in paper tested with the extractantsand solvents separately.

Rice Growth Test for Gibberellins. Rice seeds of the varietyTan ginbozu (32) were soaked and germinated in water. On the5th day seven germinated seeds (with radicles only) were trans-ferred to each 3- X 10-cm tube containing 5 ml of 1.5% agaron which a chromatogram section equivalent to one RF unitwas placed and which was covered with 1 ml of water. Theseedlings were grown at 25 C at light intensity of 600 ft-c. Oneweek after transfer the length of the second leaf sheath of thefive most developed seedlings in each tube was measured.

Cucumber Hypocotyl Test. Thirty to fifty g of fresh materialwere extracted as described above. One and one-half ml ofether extract were loaded on each strip of paper. Chromatog-raphy was ascending using isopropanol: ammonia: water(8:1:1) as the solvent system. Equal RF values were combinedand eluted with 80% ethanol. The resulting extract waspoured into a 250-ml beaker containing two layers of filterpaper. The ethanol and water were evaporated to dryness, 3 mlof water were added, and 15 cucumber seeds of the variety BetAlpha were sown in each beaker. After 5 to 7 days of continu-ous illumination at 600 ft-c, the hypocotyl of the 10 largestseedlings was measured. Each test was replicated three times,as were the standards of 50% GA, and 50% GA7 at concentra-tions of 10 nM, 0.1 ,tM. and 1 riM.

RESULTS

Effect of Daylength and Ethephon on Endogenous AuxinLevel. Under long day conditions endogenous auxin levels inthe gynoecious type were higher than in the monoecious one(Fig. 1). A relatively greater activity of inhibitors was found inthe monoecious variety at RF 0.9 to 1.

Greater auxin activity was found in monoecious plantsgrown under short day conditions enhancing femaleness thanin those grown under long days (Fig. 2). Auxin levels in theformer were approximately 10 times greater than in the latter.In all cases auxin activity focused between RF 0.3 and 0.5,

Plant Physiol. Vol. 50, 1972586

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Plant Physiol. Vol. 50, 1972 ETHEPHON AND PHYTOHORMONES IN_SEX EXPRESSION

IAA(M)25r

EE 23

-C

C 21

a._

IV 190 Monoecious Gynoecious

17k-U1 I I I I0 0.5 1.0 0 0.5 1.0 Rf

FIG. 1. Chromatograms of auxin-like substances and inhibitorsin monoecious and gynoecious cultivars of cucumbers. Acidic ethylacetate fraction (pH 3.0) of apices of 18-day-old seedlings. Biologi-cal activity was determined by the wheat coleoptile bioassay. Eachchromatogram represents 500 mg fresh weight. Values are means ofthree replicates.

25 r_ S.D.

IAA (M)7-10 7

showed gibberellin activity in control plants when tested bythe rice assay. GA activity in all three regions disappeared fol-lowing Ethephon treatment (Fig. 5). High levels of inhibitorwere found in the ether fraction at RF 0.4 to 0.9.

24-

E 22K [In

-~18

c, 16 Untreated 2 days 7 days

K oafter treatment after treatment

IAA (M)710--

Ii--110-5

-810-

13 daysafter treatment

L0 ._ 1.0 0 0.5 1.0 0 05 1.0 0 0.5 1.0 Rf

FIG. 3. Chromatograms of auxin-like substances and inhibitorsof an andromonoecious cultivar of muskmelon (Ananas PMR), atvarious times after a treatment with Ethephon (250 mg/l). Acidicethyl acetate fraction (pH 2.5), wheat coleoptile bioassay. Eachchromatogram represents 500 mg fresh weight.

E 2

Ec

c 21a,

c-

4,1c

10-810-9

3 0.5 1. I i0 0.5 1.0 0 0.5 1.ORf

FIG. 2. Chromatograms of auxin-like substances and inhibitorsin apices of a monoecious cultivar of cucumber seedlings grownunder long and short day conditions. Acidic ether fraction (pH 3.0),wheat coleoptile bioassay. Each chromatogram represents 500 mgfresh weight.

while IAA in the same solvent system has an R1 between 0.4and 0.5. The activity of an inhibitor found at R, 0.9 and 1.0 inthe monoecious variety was greater under short day than underlong day conditions. Auxin activity decreased during the firstfew days after Ethephon treatment of both cucumber andmuskmelon and returned to the previous activity levels approxi-mately 10 days after treatment (Fig. 3). Concurrent with thedecrease in auxin level, the activity of the inhibitor at R, 0.9to 1.0 increased and remained above its former level even 13days after Ethephon treatment (Fig. 3). It should be empha-sized that all bioassays were performed with apices of seedlingswhich had developed three leaves. The apex did not includeleaves or cotyledons which had been treated with Ethephon.

Gibberellin and Gibberellin Inhibitor. Diffusates from themonoecious variety had greater gibberellin-like activity thandiffusates from the gynoecious one. High gibberellin activitywas especially noted in the acid fraction (Fig. 4). The barleyand a-amylase test also facilitates the determination of endoge-nous inhibitors. At RF values 0.8 and 1.0 in the neutral fractionhigher levels of such inhibitors were found in the gynoeciouscucumber variety than in the monoecious one. The aqueousfraction of the diffusate generally showed no gibberellin-likeor inhibitor activity.

Treating the monoecious line with Ethephon resulted in dis-appearance of gibberellin activity from the acid fraction. Theseresults, however, were not consistent in the barley endospermtest.

Three main regions, at RF 0.1 to 0.2, 0.4 to 0.5, and 0.7,

Ethyl acetate fraction (pH 6)600-

Ethyt acetate acidic fraction(pH 3)

V]

GA3(M)

400-

200-

0- C3zciXza1C -W

-200-

- Manoecious Gynoecious Monoecious Gynoecious-600-

-800

0 05 1.0 0 0.5 1.0 0 0.5 1.0 0 0.5 1.0 Rf

FIG. 4. Chromatograms of gibberellin-like substances and in-hibitors in diffusates of apices from monoecious and gynoecious cu-cumber seedlings. Activity was tested in the barley endosperm bio-assary. Each chromatogram represents 40 apices placed on agar for24 hr. Values are means of three replicates.

24

22

E 20-Ca)

18

-Co 16LO)

Untreated

:tEthephon

GA3 (M)-10-7

w

14 _

0 0.5 1.0 0 0.5 1.0RfFIG. 5. Chromatograms showing the effect of Ethephon on

gibberellin-like substances and inhibitor levels in diffusates ofapices from monoecious cucumber seedlings. Activity was tested inthe rice seedling bioassay. Each chromatogram represents 40 apicesplaced on agar in three replicates. Acidic ether fraction (pH 3.0).Ethephon (250 mg/l) was applied to cucumber seedlings at thefirst leaf stage, and apices were cut from plants at the third leafstage.

587

'1

12.-

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588

GA3(M)--10 3

36+

32E

c

28

_ Untested Ethephon

240

16

120 ffi,0

0 0.5 1.0 0 0.5 1.0 f

FIG. 6. Chromatograms showing the effect of Ethephon ongibberellin-like substances and inhibitors in diffusates from cucum-ber seedling apices. Activity was tested in the cucumber hypocotylbioassay. Treatment diffusion and fractionation procedure was asin Figure 5.

2E

21

EE 21

LI)

' 2-C

QS 2C

1E

Untreated8_

6

2

O _

1J

GA (M)0o-7

0 0.5 1.0 0 0.5 10 Pf

FIG. 7. Effect of Ethephon on gibberellin-like substances andinhibitors in extracts of monoecious Bet Alpha cucumber leaves, as

tested in the rice seedling bioassay. Ethephon (500 mg/i) was

applied to 10-day-old seedlings, plants were harvested and extracted6 days after treatment. Acidic methylene chloride fraction (pH 3.0).Each chromatogram represents 3.0 g fresh weight. Plant was grownunder long day conditions in the phytotron.

This effect of Ethephon on the level of gibberellins andinhibitors in diffusates from cucumber was found also withthe cucumber test (Fig. 6). In this test most of the gibberellinactivity found in control plants appeared at RF 0.5, whereastreated plants showed no gibberellin activity. In contrast,strong inhibition was evident at RF 0.5 and 0.6 and also RF 0.7and 0.8 (Fig. 6).

High inhibitor activity was found in extracts of apices frommonoecious cucumber seedlings sprayed with Ethephon as

tested in the rice growth bioassay (Fig.7).ABA Level as Affected by Ethephon and Sex Type. ABA

content of the gynoecious line was twice that of the monoeciousone. Ethephon increased the level of ABA several times inboth lines (Table I).

Effect of ABA on Sex Expression. ABA applied as a dailyfoliar spray of 1 to 250 mg/l did not affect growth or sex

expression of plants in both monoecious and gynoeciouscucumbers. Root application of ABA (1-10 mg/l) to monoe-

cious line had no effect on type of flowers formed up to thetenth node. However, in a gynoecious line grown under con-

ditions which promote maleness, root applied ABA markedlyenhanced femaleness. This effect was manifested by decreasingthe number of male flowers, increasing the number of pistillateflowers, advancing their formation at a lower node, and also

Plant Physiol. Vol. 50, 1972

promoting the growth of the ovaries (Table II). The same ABAtreatments had no effect on elongation but slightly decreasednode numbers.

Effect of CO, Level on Sex Expression. Higher ethyleneevolution was associated with female tendency of cucumberbuds (28). In the present study, high CO2 level, which is knownto antagonize ethylene, markedly increased maleness by induc-ing the formation of male flowers in gynoecious plants (TableIII). An average of 2.2 male flowers per plant were formed inthe gynoecious plants. The high CO2 level also delayed theappearance of the pistillated flowers in the gynoecious line.

Table I. Effect of Ethephoni anid Sex Type onI ABA levels asdeterminied by GLC

Leaves (100 g fresh weight) of four to five leaf stage plants grownunder long day in a phytotron were extracted with methanol andpartitioned with methylene chloride (pH 3.0). A single spray withEthephon (500 mg/1) was applied at the two leaf stage, 10 daysprior to ABA extraction.

ABA ContentSex Type

Untreated Ethephon

jpg '100 g freslz wt

Monoecious 1.00 10.75Gynoecious 2.23 6.43

Table II. Effect oJ Root-applied ABA oni Sex Expressioni ofa Gynoecious Linle of Bet Alp/ha Cucutmber

Plants were grown in a growth chamber; day temperature was28 C; (12 hr); night temperature was 18 C. Average was 10 plantsper treatment. Values followed by different letters within acolumn are significant at the 5' level (Student-Newman-Keulsmultiple range test).

No. of Flower Budsi Node of-Node Length of _________ First O-r

Treatment NoO. First Five Pistillate OvgarNodes Stain. Pistil late Flw

inate Bud

CM,

Control 6.9 a 20.4 1.7 a 1.5 b 6.1 a 1.0 bABA, I mg 'l 6.3 ab 21.9 1.0 ab 1.9 b 5.0 b 1.4 bABA, 5 mg/i 6.1 b 24.7 0.5 b 4.3 a 3.8 c 3.3 aABA, 10 mg/I 6.0 b 21.5 0.0 b 4.1 a 2.5 d 3.6a

SE 0.1971 1.232 NS2 0.2653 0.3811 0.2981 0.331'

' Significant at 1 %0 level.2 NS: not significant.3 Significant at 5 c, level.

Table III. Effect of CO2 Level oni Sex Expression of Monioecioiusantd Gynioeciouts Linies of Bet Alpha Cucumiibers

Plants were grown in a growth chamber, day temperature (10hr) was 26 C; night temperature was 15 C. CO2 was applied for 15days from the first leaf stage. There were six plants per treatment.

Node of First Pistillate -Node of First AlaleFlower Flower

CO2 Level

Monoecious Gynoecious Mlonoecious Gynoecious

tnl/l300 -1 3.3 4.3 -1

3000 - 5.5 3.0 2.3

'No flowers of respective type appeared within the experimentalperiod.

RUDICH, HALEVY, AND KEDAR

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Plant Physiol. Vol. 50, 1972 ETHEPHON AND PHYTOHORMONES IN SEX EXPRESSION

Plants grown under high CO2 level were more developed thanthose grown at normal Co2 level. This was expressed also inearlier formation of male flowers in the monoecious plants.

DISCUSSION

We have demonstrated earlier a promotion of femaleness incucurbits by external application of the ethylene-releasingcompound Ethephon (26). Higher ethylene evolution wasfound in apices of gynoecious plants as compared to those ofmonoecious ones and from female buds as compared to maleones. Plants grown under short day conditions which promotefemaleness evolved more ethylene than those grown under longday conditions (28). We have suggested (28) that ethyleneparticipates in the endogenous regulation of sex expression,promoting femaleness. A further support to this suggestionis presented here (Table III). High CO2 levels which areknown to antagonize ethylene action (18) strongly enhancedmaleness in gynoecious plants. The gynoecious cucumbershows greater endogenous auxin activity than the monoeciousone (Fig. 1). This and the relatively higher auxin levels foundin cucumber plants grown under short day conditions whichpromote femaleness (Fig. 2) confirm earlier reports (9) andthe hypothesis of Nitsch et al. (23) that higher auxin levelis associated with female sex expression.

Auxin treatments induce higher ethylene evolution of manyplants (1, 5, 6, 8) including cucumbers (30), while ethylenelowered auxin level (6, and Fig. 3). It thus seems that ethyleneis a more direct regulator of femaleness.

Inhibitors levels seem also to be related to female sex ex-

pression. ABA content of gynoecious plants is higher thanof monoecious ones (Table I). Higher GA-inhibitor levels were

found in the neutral fraction of diffusates from gynoeciousas compared to monoecious plants (Fig. 4). Higher inhibitorlevels in the monoecious plants were also found in the auxinassays of cucumber extracts from plants growing under shortdays (Fig. 2). It has been suggested (9) that the ratio betweenauxin and inhibitor levels arising in mature leaves determinesthe sex of the flower bud. We should like to point out thatin our experiments apices and young leaves were extracted andthe inhibitor found here may not be identical with that de-scribed in previous studies (9).

Higher gibberellin content was found in monoecious plantsas compared to gynoecious ones (Fig. 4). This confirms earlierresults (2). Ethephon treatments reduced gibberellin activityin diffusates as found by the cucumber and rice assays (Figs.5, 6). Concurrent with the reduction in gibberellin levels, an

increase in native inhibitor levels was found in the gynoeciousline. Bioassay detection of inhibition after Ethephon treatmentmay. however, indicate that Ethephon residues themselvesare the inhibiting agent in the growth of the rice and cucumberhypocotyls. We have checked this possibility. Chromatographyof Ethephon and subsequent bioassay do not confirm this as-

sumption. Furthermore, Ethephon greatly increased ABA con-

tent of cucumber leaves (Table I). The presence of high levelof ABA and the neutral inhibitor in gynoecious plants and thelow inhibitors level in the monoecious plants (Fig. 4 and TableI) may support the assumption that Ethephon induces also theformation of ABA as was also found in orange fruits (13) androse petals (21) following treatment with Ethephon andethylene.ABA may participate in the regulation of female sex ex-

pression in cucumbers by interacting with gibberellin. Thishypothesis is supported by the increased femaleness of ABA-treated plants (Table II). The data presented here and inearlier paper may strengthen the view that four phytohormonesparticipate in the regulation of sex expression in cucumber:ethylene, auxin, GA, and ABA. Ethylene may promote fe-

maleness directly, by reducing endogenous GA level or bypromoting the production of ABA.

It may of course also be that each hormone stimulates theproduction of enzymes affecting stages in flower (and sex)development, there being no direct correlation between one

growth regulator and the increase or decrease in the levelof other hormones. At this stage we have not proved the causeand effect relationship between the different hormones andthus this second hypothesis cannot be ruled out.

Acknouwledgments-The expert technical assistance of Mrs. Hana Jackman isgratefully acknowledged. Etheplion (Amchem 62-240) was kindly supplied byAgan Chemicals, Tel Aviv. ABA (RO-08-0095) is a gift of Hoffman La Roche.Dwarf rice seeds w-er e kindly suppled by Prof. Y. 'Murakami, Tokyo.

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11. GALU-N, E., Y. YOUN-G. AND A. LANG. 1963. 'Morphogenesis of floral buds ofcucumber cultured in titro. Develop. Biol. 6: 370-387.

12. GOLDSCH'MIDT, E. E. AN-D S. P. MIONSELISE. 1968. Native growth inhibitorsfrom citrus shoots: partition, bioassay and characterization. Plant Physiol.43: 113-116.

13. GOLDSCHIMIDT, E. E., S. K. EILATI, AND R. GOREN. 1972. Increase in ABA-likegrowth inhibitors and decrease in gibberellin-like substances during ripen-ing and senescence of citrus fruits. In: D. G. Carr, ed., Seventh Interna-

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