kinetin, iaa, and gibberellin ethylene production ... · (kinetin trcatment). each group was grown...

Plant Physiol. (1968) 43. 2029-2036

Effects of Kinetin, IAA, and Gibberellin on Ethylene Production,and Their Interactions in Growth of Seedlings" 2

Yoram Fuchs3 and Morris LiebermanMarket Quality Research Division, Agricultural Research Service, Plant Industry Station,

Beltsville, Maryland 20705

Received August 15, 1968.

A bstract. Kinetin in concentrations of 10-e to 10-4 M, stimuated ethylene production in3 and 4-day old etiolated seedlings of Alaska pea (Pisucm sativum L. var. Ala-ka). Seedlingsof other species responded similarly. The response to kinetin depended on the age of theseedlings.

Kinetin alone did not influence ethylene production in 6-day old stem sections, but it greatlyincreased the enhancing effect of IAA.

Gibberellic acid had no effect on ethylene production by pea seedlings during the first6 days of growth. Ethylene and gibberellic acid are antagonistic in their effects on growth ofthe seedlings; ethylene interfered severely with the action of gibberellic acid but did notcompletely suppress it.

The inhibitors cycloheximide, cupferron, and N-ethylmaleimid?, caused considerable inhibi-tion of kinetin-induced ethylene production but were much less effective in the endogenousethylene-forming system.

The influence of auxins on ethvlene productionin several plants was first reported bv Zimmermanand Wilcoxon in 1935 (22). Recently several work-ers confirmed and extended this observation (1. 5)and currently there is an hypothesis which attributesmany of the effects of auxin (IAA) to its influenceon ethvlene production (7).

Gibberellins have also been associated withethylene production. For example, Abeles andRubenstein (1) and Lewis, Palmer, and Hield (13)respectively, reported an increase of ethylene evolu-tion by bean explants and parthenocarpic navaloranges, after treatment with gibberellins. Scott andLeopold (19), however, reported that the actions ofethylene and gibberellic acid are antagonistic in theelongation process of lettuce seedlings.

In contrast to IAA and gibberellins the relationof cytokinins to ethvlene production and action hasbeen studied mtuch less. In a recent report on leafabscission of bean explants Abeles, Holm, andGahagan (3) noted that explants treated with highconcentrations of kinetin and 6-benzyladenine doubled

1 Most of this material was included in a doctoralthesis submitted by Yoram Fuchs to the Graduate Schoolof the University of Maryland, College Park, Maryland.

2 Supported in part by National Science FoundationGrant GB-1094 and cooperative agreement between Mar-ket Quality Research Division, ARS, USDA, and De-partment of Botany, Agricultural Experiment Station,

University of Maryland.a Present address: The Volcani Institute of Agricul-

tural Research, Rehovot, Israel.

their ethylene production. We now present a studyof the effects of kinetin on ethvlene produiction, andthe interactions between kinetin, IAA, and gibberellinon ethylene production and growth of seedlings.

Materials and Methods

Plant Materials. Alaska pea seeds (Pisunmsativum L. var. Alaska) were soaked in a beaker inthe dark, in running tap water bubbled with air.The soaking was carried out for 48 hr at 250, atreatment which was found to produce uniform,disease-free seedlings. Thev were then planted onwet "Kimpack" (an absorbent of one-eighth inchthick packing paper) and grown at 25° and about95 % relative humidity. The seedlings were grownin darkness up to 6 days with occazional dim "safegreen" light. Seedlings were transferred to 50 mlflasks in the dark, at different ages, for the experi-ments. Age of seedlings was measured from thetime the soaking started. In some experiments 2-cmapical sections were cut from 6-day old seedlings andused as experimental material. In experiments inwhich 1 day old seedlings were tested, the dry seedswere placed directly into 50 ml flasks, containing2 ml of water or the test solution. Dwarf pea seeds(Pisumn sativuins L. var. Progress No. 9) weregerminated in the same way.

In other experiments seeds of radish (Raphantussativus L. var. Early Scarlet Globe), bean (Phase-olius vulgaris L. var. Tendergreen Improved), cu-cumber (Citrumis sativus L. var. Boston Pickling),

2029 www.plantphysiol.orgon March 25, 2020 - Published by Downloaded from

Copyright © 1968 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org

PLANT PHYSIOLOGY

wheat (Triticurn aestizvnn durum L. em. Thell.)oat (Aveiia sativa L. var. Victoria), barle) (Hor-deumiii vulgar-e L. var. Himalaya), and corn (Zeainias L. var. Golden Bantam), were grown on wetfilter paper in petri dishes for 48 hr, before beingplaced in 50 ml flasks for subsequent study ofethylene production.

Growcth Regulators and Inhibitors. Kinetin (6-furfuirvlamino purine). 6-benzvl adenosine, adenine,IAA (3-indole acetic acid), gibberellin X (gibberellicacid potassium salt) and chloramphenicol. wereplurchlased from Calbiochem4. N-etlhylmaleimide(NEMI) was purclhased from Baker Chemical Com-panv, 2.3,5-triiodobenzoic acid (TIBA) from East-man Kodak, cupferron (N-nitrosophenyl hvdroxylamine) from Malinckrodt Chemical Companv, andcvcloheximide from Sigma Chemical Company. Allchemicals were used as received without fur.herpturification.

M1ethods. All growth regulators and inhibitorswere prepared in 10-3 M phosphate buffer, pH 68,and adjustei with 0.1 N KOH to pH 6.8 wheinnecessary. 'The same buffer was used for controls.

In most experiments 2 ml of solution and 4 wholeseedlings were placed in a 50 ml flask which wasthen sealed with a i-hole rubber stopper, fitted witha clam-ped capillary tube. In some experiments 4pltumul1les, radicles. pairs of cotyledons or apical stemsections, 2 cm long, were used instead of wholeseedlings. In all experiments the sealed flasks wereheld for 24 hr in the dark at 250. The flasks werenot shaken. At the end of the holding period theatmosphere in the flask was sample(d with a syringefor ethylene determination.

Etlhylene was determined by gas chromatography(16). A glass column, 75 cm long and 3 mm indiameter, containing activated alumina (70-80 mesh),to whiclh 4 % water (by weight) was added, wasused. Rates of ethylene production are presentedon a fresh weight basis.

Unless otherwise indicated, at least 3 replicationiswere run for each experiment. Statistical analysiswvas carried otut using the analysis of variance andthe Duncan Multiple Range Test (12) at the 1 %or 5 % level of significance.

Additional details of experimeintal methods aredescribed in the text and in the legends of table,;cand figures wlhere applicable.

Results

Effect of Kinietin Concentration owI EthyleneProductiion. Kinetin in concentrations of 10-8 to10n-- i stimulated ethylene production in 3-day oldpea seedlings (fig 1). The rate of ethylene pro-

4 Mention of a specific trade name or company is madefol identification only and does not imply endorsementbv United States Department of Agriculture.

0-5)

N

-J

z0

C)

0001

LUz

J

I

-(L

40

4

I0~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

0~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

so30 els0 9 8 7 6 5 4 3

KINETIN (-LOG MOLARITY)FIG. 1. Ethylene production induced by kinetin. Two-

day old Alaska pea seedlings wvere grown at 250 in 50ml sealed flasks containing 2 ml of 10-3 M phosphate buf-fer, pH 6.8, and various concentrations of kinetin. Eachflask contained 4 seedlings. Ethylene production wasmeasured after a growth period of 24 hr. Seedlings with-out kinetin, grown under similar conditions, served ascontrols. Range of results of 3 experiments are indicatedby the double arrows at each poinit, which representsan average of the 3 experiments.

duction increased wvith increased concentration ofkinetin in the range of 10-7 to 10-4 M. Concentra-tions of kinetin greater than 10-4 M were not usedbecause of solubility limitations at neutral pH.Hovever, at pH 2.5, 10-3 M kinetin did not stimulateethylene production in 3-day old pea seedlings.Therefore 10-4 M kinetin was the standard concen-tration tused in subsequent experiments to prodtuce

40 j

20

- /~~~~~~~4

-20

0 3 6 9 12 15 18 -

TIME HOURS

FIG. 2. Effect of 10-4 M kinetin on ethylene produc-tion. Groups of 4 etiolated 2-day old Alaska peaseedlings were held for different times up to 24 hr in2 ml of 10-33 M phosphate buffer, pH 6.8. (control) or in10-4 M kinetin in 10-3 M phosphate buffer, pH 6.8,(kinetin trcatment). Each group was grown in 50 mlsealed flasks. The flasks were kept in the dark at 250.Each flask was sampled only once. Range of resultsof 3 experiments are indicated by the double arrows ateach point, which represents an average of 3 experiments.

2030

www.plantphysiol.orgon March 25, 2020 - Published by Downloaded from Copyright © 1968 American Society of Plant Biologists. All rights reserved.


FUCHS ANI) LIEBERMAN-KINETIN. IAA. AND GIBBERELLIN ON ILH\LrNI,N.I-PROM)l'T(ON 2031

maximum ethylene production by seedlings. IAA-induced ethvlene production in 6-day old pea-stemsections showed a similar "concentration-activity"curve (5).

Stimulation by Kinetin and Rate of EthyleneProduction. Hourly production of ethylene in 2-davold pea seedlings, in the presence and absence of10-4 M kinetin, is presented in figure 2. Threephases of ethylene production were ob-erved; a slowphase (0-5 hr) in which production was about thesame in both kinetin-treated and control seedlings;and ascending phase (5-8 hr) in which the greatestrate of increase was obtained, in both kinetin-treatedand control seedlings; and a quasi steady-state phase(8-24 hr) in which ethylene production continuedat a lowered steady rate in kinetin-treated seedlings,while ethylene production approached zero in con-trols. The rate of ethylene production of kinetin-treated seedlings was approximately 3 times and5 times greater than controls in phases II and IIIrespectively. The rate of ethylene production inphase III was considerably lowered in both kinetintreated and control seedlings. Part of this reductionin rate may be attributed to the accumulation of

KINETIN _

CONTROL F-7:::220

200

o 180

CN 160co

- 140z0°- 120D

0o 100

zi 80zLu

2:60LU

40, In20 H 1Hi

PLUMULES RADICLES COTYLEDONS WHOLE SEEDLINGS

FIG. 3. Kinetin-induced ethylene production in rad-icles, plumules and cotyledons of Alaska pea seedlings.Three-day etiolated Alaska pea seedlings were separatedby razor blade into radicles, plumules, and cotyledons.Four radicles, plumules, or cotyledons were incubatedin the dark for 24 hr at 250 in 50 ml flasks with 2 nilof 10-4 M kinetin in 10- m phosphate buffer, pH 6.8, o02 ml of 0-3 M phosphate buffer, pH 6.8 as the controltreatment. Ethylene production is expressed on a freshweight basis.

CO., in the sealed flasks. Carbon dioxi(le reached aconcentration of 8 % during the 24-hr period, whlichinhibited kinetin-induced ethvlene production about12%.

Effects of Kinetin on Ethylene Produictioni byRodicles, Plunwtiles, anid Cotyledons. Studies ofethylene production by the 3 major morphologicalregions of 3-day old pea seedlings show-ed thatethvlene production is different ill each of theseregions (fig 3). Plumules and radicles producenmost of the ethylene while the cotyledons fornivirtually no ethylene. Addition of kinetini to thesemorphological regions of the seedlings resulted illconsiderable increase in ethvlene production in radi-cles and plumules, but had no effect oni cotyledons.Kinetin induced about an 80 % stimulationl ofethylene production by radicals and about a 300 %increase in plumules. Thus only the actively grow-ing regionis which produce ethylene in relativelylarge amounts, are stimulated by kinetin.

Most of the weight of a 3-day old pea seedlingis found in the cotyledons (400 mg) while theplumules and radicle contribute very little (25 mgsand 50 mgs, respectively). Therefore the specificethvlene production of the plumules and radicalsappears very high, relative to the intact 3-day oldseedling, which is made up mostly of non-ethylleneforming cotyledons The amount of ethylene pro-duced on a per seedling basis by the 3 separatedregions. was approximately the same as that pro-duced by the intact seedling.

Effect of Kinietin on Ethylene Production inSeedlings of Several Species. Two-day old seed-ling! of radish, bean, cucumber, and corn werestimulated in ethylene production by 10-4 M kinetin(table I). XVheat. oat, barley, and Progress No. 9(a dwarf pea variety) seedlings, however, were notsignificantly affected by the same kinetin treatment.These data suggest that kinetin does not influenceethylene production in monocots to the same extenitas dicots. The lack of stimulation of ethylene pro-

Table I. Ethylene Produiction by Seedlinigs of SeveralSpecies as Affected by Kinetin

Groups of 4 seedlings, 2-days old, were grown in 50ml flasks in 2 ml solution of 10-3 M phosphate buffer, pH6.8, for the control, or 10-4 M kinetin in the same bufferfor 24 hr at 250 in the dark. All seedlings were pre-vi-usly germinated on wet filter paper in petri dishesfor 4?I hr.

Species

RadishBeanCucumberPea var. AlaskaPea var. Progress No. 9WheatOatsBarleyCorn

Ethylene productionControl Kinetin

(ag per g per 24 hr) 10:29.0 59.07.4 14.8

24.2 81.77.7 28.0

10.5 10.26.8 9.64.3 4.35.0 7.16.1 15.1



Table II. Effect of Adeenine, 6-Benvyladeniosine, antd Kilnetin ont Ethylen/e Productionz by Pea SeedlingsTwo-day old etiolated Alaska pea seedlings were growxn in the dark iil 2 ml solutians of adenine, 6-benzyladenio-

sine, and kiiielin at various concenitrations in 10 3 M phosphate buffer pH 6.8, for 24 hi- at 230.

Cytokinin None

Kinetin6-BenzyladenosineAdenine

11.6

11.611.6

10-4 M

29.836.015.5

Ethylene producti3iiConc of cytokinins

10---,* AI 10 6 At

(ALl Per' 9 pe1-21.025.213.7

24 lir) 10:;12.116.015.5

ductioni in the dwarf seedlings (Progress No. 9)suggests that either kinetin does not infltuenceethylene production in the dwarf variety, or maxi-munm ethylene production is occurring and additionalkinetin cannot further influence the system alreadyoperating at its peak. The reactioin of corn seed-lings to kinetin is also not within the pattern shownby the other monocot seedlings tested.

Effect of Olher Cytokinins. The cytokinin 6-benzvl adenosine stimulated ethylene production in2-daiy old pea seedlings, mtlch the same as kinetin(table II). However, adenine had no significantstimultlaLory effect on eihylene production of the peaseedlings. This agrees generally wvitlh the wvork ofSkoog et al. (20) wlho assayed 69 purine derivativesand related comnpounds. for their cvtokinin-like ac-

tivitv in the tobacco bioassay, and found adeninethe least active of the stubstances tested.

Me/hionine as a Sutbstrate for Kinctini-IniduicedEthylen1e Pr-oduictioni. Methionine is known to be a

suibstrate for ethylene production in apples, bananas(6, 14), and pea seedling3 treated wvith IAA (6).When 10-3 m ethionine was added to 2-day old pea

seedlings no increase in evolved ethylene waws ob-served. However, when 10-4 M kinetin was addedalong- witlh 10-3 AI methioninie, ethylene productionincreased about 250 % over controls and 60 % over

the kinetin-treated peas (table III). The amlount of

Table III. Kinietin-Dependent Stimulation of EthyleneProdutction by Methioniine in Alaska Pea SeedlingsGroups of 4 etiolated pea seedlings, 2-days old, were

grown in the dark for 24 hr at 250 in 50 ml flasks. Theflasks contained 2 ml of solutions of kinetin, methionine,or IAA in 10- m phosphate buffer, pH 6.8, as indicated.The actual amount of ethylene produced by the controlwas 0.013 Al per g per 24 hr. Numbers superscribed bynO letters in commilon are significantly different at the; % level.

Incubation solution

10-3 M Phosphate buffer (control)10-4 M Kinetin5 X 10-3 M MIethionine5 X 10-3 AI Methionine

+10-4 AI KinletiiiX 10-3 M Methioinine4 10-4 M IAA

Ethylene production% of control

ethylene fornmed by thle pea seedlings treated witheither kinetin plus nmethioniiie or IAA plus methi-onine, was about the saame. Since methioniine wasshown to convert to ethylene in pea seedlings treatedwith IAA (6), it is probable that metlhionine isconverted inlto ethylene in seedlings stimulated bykinetin.

Effect of Iinhibitors on Kinctin-Induced EthyleneProduction. Enzymes involved in ethylene produc-tion appear to contain both a metal and sulfhvdrylgroup in their active centers (14). NEM andcupferron, inhibitors of SH groups and metal ionsrespectively, significantly reduced kinetin-stimulatedethvlene production in seedlings (table IV). Tri-iodobenzoic acid (TIBA), ani inhibitor of IAAtransport, and cycloheximide, an inhibitor of proteinsyn.hesis, also strongly inhibited kinetin-inducedethylene production. Chloramphenicol, an inhibitor

Table IV. Effect of Inhibitors on Kinetin-InducedEthylene Production

Two-day old pea seedlings, in 2 ml of 10-3 Alphosphate buffer pH 6.8, containing 10-4 M inhibitors,held for 24 hr in the dark in sealed flasks.

Inhibitor

TIBA'CupferroiNEMCycloheximideChloramphenicol

Inhibition ofendogenous system

23

32810

0

Inhibitionof kinetin-

stimulated system

6571861000

I TIBA =2,3,5-Triiodobenzoic acid.

of protein synthesis in bacterial systems but not inplant ribosomal systems (15), had no effect on

ethylene production.The inhibitors of ethylene production were con-

siderably less effective against endogenous ethylene-forming systems than the kinetin-stimulated systems.This suggests that the kinetin-stimulated system isdifferent from the endogenous system. In addition,the relative ineffectiveness of cycloheximide againstthe endogenous ethylene-forming system, and itscomiplete inhibition of the kinetin-stimulated systemsupports the possibility that kinetin-induced ethylene

10-7 M

13.117.115.5

10-8 M

15.510.815.5

2()32 PLANT PHlIYSIOLOGY'



FUCHS AND LIEBERMAN-KINETIN, IAA, AND GIBBERELLIN ON ETHYLENE PRODUCTION 2033

Table V. Ethylene Production by Etiolated Pea Seedlings of Different Ages, in Presence of IAA, Kinetin, andGibberellin, Alonse and in Combinations

Seedlings were grown and treated with growth substances as described in the text. In all treatments the finalconcentration of each of the participating growth substance was 10-4 M and pH 6.8. Kinetin, IAA, and gibberellinwere dissolved in 10-3 M prosphate buffer pH 6.8. The experiments were replicated 4 times on 4 different days.

Ethylene productionKn + GA

Days Control Knl GA2 IAA Kn + GA GA + IAA Kn + IAA + IAA

(,4 per g per 24 hr) 1032 15 22 20 13 22 19 22 333 16 45 18 29 52 41 90 1004 7 23 8 41 24 40 63 755 5 12 6 57 14 59 95 104

1 Kinetin = Kn.2 Gibberellin = GA.

production is dependent on new enzyme formation in2-day old pea seedlings.

Ethylene Production in Relation to Growth Sub-stances and Age. Kinetin and IAA stimulatedethylene production in 3 to 5 day old pea seedlings,while gibberellin had virtually no effect (table V).During the first 2 days of growth there was littleeffect of any of the growth regulators on ethyleneproduc-tion by seedlings. On the third day the effectof kinetin was maximal and thereafter sharply de-clined. The stimulation by IAA, however, continuedto increase linearly throughout the 5-day period.Thus the age of the seedlings was shown to be thecontrolling factor in eliciting increased ethylene pro-duction after treatment with IAA or kinetin.

Kinetin and IAA-treated seedlings were shorterand thicker than the controls, whereas gibberellin-treated seedlings were longer and somewhat thinnerthan the controls. This is to be expected from theeffects of kinetin, IAA, and gibberellin on ethyleneproduction of etiolated pea seedlings, and the knowneffects of gibberellin on elongation of seedlings.The increased ethylene production by seedlingstreated with kinetin or IAA would tend to causedwarfing, while treatment of the pea seedlings withgibberellin, which does not induce ethylene produc-tion, causes elongation.

When both kinetin and IAA were added to seed-lings at the various ages, there was even greaterethylene production by the 3 to 5-day old seedlings(table V). However, when gibberellin was addedto kinetin or IAA, or to combinations of kinetin andIAA, it did not significantly (statistically) enhancethe stimulatory action of kinetin or IAA, or thecombined stimulatory action of kinetin and IAA.Therefore, gibberellins had little or no effect onethylene production by 3 to 5-day old etiolated peaseedlings, either alone or in various combinationswith IAA and kinetin. On the other hand, kinetinand IAA significantly stimulated ethylene productionin 3 to 5-day old etiolated pea seedlings when addedalone, and their stimulatory action was enhanced by

40-

0

r 32

-.28zO 24

3 2000x"li 16Ulz

> 12II--LU

8

Fr

II.

4

0

a

zz

a-

aLL~~~~~~~~~c a a b b c c b a a

FIG. 4. Ethylene produced by 6-day old etiolatedpea stem sections treated with kinetin, gibberellin, andIAA. Six-day old etiolated Alaska pea seedlings weregrown as described in the Materials and Methods.Groups of 4 apical stem sections, 2 cm long, were cutwith a razor blade and incubated in 50 ml sealed flasksfor 24 hr, at 250 in the dark. Ethylene was determinedat the end of a 24-hr period of growth. The flaskscontained 2 ml of solutions of the different growth sub-stances, each in final concentration of 10-4 M (X) or2 X 10-4 M (2X) in 10-3 M phosphate buffer, pH 6.8.In the control treatment the stem sections were incubatedwith 2 ml of buffer only. Letters under columns ongraph indicate Duncan Multiple Range Test (12).Columns on graph ascribed by no letters in commonare significantly different at 1% level.

bm,l6=,bo= . .- -

,iL



comiibining tlhese 2 growth regulators in 1 treatment.The comiibined action of growtli regulators on

eLlivlene production wvas also studied wvith 6-day oldpea stem sec.ions (fig 4). As previously noted(tible V), kinetini did not greatly stimulate ethyleneI)roduction in 5-day old pea seedlings, and also hadlittle effect on 6-day old pea seedling stem sections(fig 4). However, wNlhen IAA was combined withkinetin considerable stimulation of ethyilene produc-tion was obtained. IAA and kinetin interacted toincrease ethylene production in stem sections frometiolated peal seedlings, even at an age (6-day oldseedlings) when kinetin by it elf had little effect onethylene produiction. Gibberellin on the other handhIad little or no signiificant effect on ethylene pro-dtuction. alone or in combination with IAA andkinetin, in either whole pea seedlings or their stem.scctions.

El/iylene-Gibbcrellin Initeraction in Alaska PeaSec(llings. Inhibitioni of elongation of young peaseedlings is a well known effect of ethylene (5, 8, 17)iand it lhas beenl shown that kinetin causes an increasein etlhylene production in these eedlings. Thereforekinetini may catuse inhibiLion of growth. On theo-llhr h'and gibberelin stimulates elongation in Alaskapea seedlings in early stages of their growth (18),ai(il wve have already slhown (fig 4, table V) thatgibberellin has lit.le or no influlence on ethylenepro(ldc,ionl. The in .eraction between etlhylene andgbbCr-lli was ihel-efore investigated.

Pea scedlings gro\n in tlle (lark for 4 days (15mm leiglt) Were div.ded in:o 4 groups: 1. non-treatcd seedlings (cointr-ol) II, ehylene-treated seed-higs; III, gibberebin-trea.ed seedlilngs: and IV,see(lling2s treated wvith gibberellin onl tlle forll.h dayfollo\vcd by addilioii of etlhlene onl tlle fif.h day.At the ead of tlle sevenitlh da'y of growtlh tlle jars ofall treatmiients Nvere opened (ven.ed), to allow theesc:il)e of etlhylene froom the jars and the plantscon,inued to grow in the dark for 48 lhr, in normalroom atmosplhere. until the nintlh day. Measure-meicnlts of lheighlt (stemn lengLli) were made on thefiftl.h sevenxh, and niinth days of growth (table VI).

On the nintlh day plants treated wi.h gibberellin

PHYSIOLOGY

Table VII. (,Growth Rate of Seedlings Treated and Not-treated *,mith Eth vlene (Cibberellin, and Gibberellin,

k thlente,

TreatmientRate of growth (stem length)4th-5th 5th-7th 7th-9th

dav day day

I N o treatnment(control)

II Ethylene-added onfifth day, vented onseventh day

III Gibberellin-addedon fourth day

IV Gibberellin-addedon fourth day, thenethylene added onfifth day, andvented on seventh day

111/11n mom/h;- nun/hr0.6 1.3 1.8

0.6 0.0 1.3

0.6 1.9 2.3

O.6 0.3 1.6

1 Data derived from table VI.

and ethylene (IV) were slhorter thani conitrols (I)and those treated with gibberellin alone (III), buttaller than those treated only with ethylene (II)(table VI). Similar results were obtained on theseventh day when the stunting-effect of ethylene waseven more obvious (table VI). On the seventh daythe plan s in treatment IV, containing gibberellinand ethylene, elongated about 50 % while those oftrea.ment II. containing ethylene showed no elonga-tion. The gibberellin treated seedlings (treatmentIIT), however, were almost 3 times as tall as thosein treatment IV.

After release from the ethylene atmospheres, thegrowlh rates of treatments II and IV were notmarkedly different (table VII). However, treat-ment IV showed a somewhat higher growth rate,stuggesting a residual effect of gibberellin, even 5days after treatment. The grow.h rate of treatmentIV, between the seven.h and ninth days, was farbelow the rate for treatment III, which had beentreated only with gibberellin. The action of gib-berellin thus appears to be antagonistic to that of

Table VI. Ethylene-Gibberellin Ilteraction in Etiolated Alaska Pea Seedlings`unl)Tsrs in the t. ble represent the average height of at least 10 plants. Pea seeds were germinated in small plastic

baslkets in moist "Perlite" 16 seeds per basket. All baskets were placed in 4-liter jars to enclose the atmosphere.The expo-riments were done in the dark, vith occasional "safe green" light for necessary nmanipulations.

'' reatmiientNo treatment (control)Ethylene'-added on fifth day,vented on sevenitlh dayGibberellin2-added on fourth dav( ribbr-reIlin2-edded on fourth daV,then ethylene'-added on fifth lay

vented on seventh daxv.

Fourth

1515I515

Stem length (mm)Day of growthFifth Seventh

30 9530 30

30 12030 45

Ninth

18090

230120

2 u.l per liter of ethylene was added.

41 20 mgl/iter- gibberellic acid soluitioni was added to the apices of the seedlings.

I.II.

111.V.

20t34 PLANT



FUCHS AND LIEBERMAN-KINETIN, IAA, AND GIBBERELLIN ON ETHYLENE PRODUCTION 2035

ethylene on elongation of pea seedlings. Ethyleneseverely limits but does not completely suppress theaction of gibberellin.

Discussion

Ethylene production in 2 to 5 day old seedlingsis influenced by cytokinins as well as by IAA. Italso appears that there is an interaction betweenkinetin and IAA which regulates ethylene productionin the very young seedlings. Since growth ofmeristematic tissue is known to be influenced bycytokinins (20), one may now wonder whether ornot the ethylene induced by cytokinins plays a rolein the action of cytokinins in young tissues. Forexample, the inhibition of elongation in seedlings bykinetin (4, 10, 11, 21) may be attributed to its stimu-lation of ethylene production.

Gibberellin on the other hlandlihas no significanteffect on ethylene production either alone or incombinations with IAA and kinetin, in 1 to 6 dayold pea seedlings. In fact, the well-known action ofgibberellin, which results in stem elongation, appearsto be antagonized by the action of ethylene whichinduces dwarfing of seedlings. It is possible thatinteraction between kinetin, IAA, ethylene, and gib-berellin, control elongation in seedlings. Antagoniismiibetween the elongation and dwarfing effects of gib-berellin and ethylene was also reported by Scott andLeopold (19). In addition, another ethylene effect,the ripening of tomatoes, can also be antagonized bygibberellin (9). These data suggest that the actionsof gibberellin and ethylene are mutually antagonisticin their effects on growth and development of tisstues.

The inicreased production of ethylene induced bykinetin in these experiments is apparently not dueto conversion of kinetin to ethylene, since greaterstimulation of ethylene production was obtained whenkinetin was added in the presence of methionine.This suggests that kinetin acts to stimulate thesystem which converts precursors to ethylene, ratherthan being itself converted to ethylene. Burg, andClagett (6) suggested that IAA, which also stimu-lates ethylene production in seedlings, operates onthe precursor system.

The fact that both IAA-stimulated ethylene pro-duction (2) and kinetin-stimulated ethylene produc-tion are inhibited by inhibitors of protein synthesis(table IV), suggests that these growth regulatorsinduce ethylene production by affecting systems forprotein synthesis. This may mean that new enzymesare needed to implement the production of the in-duced ethylene. Since the endogenous ethylene-forming system and the kinetin-induced system areinhibited differently by cupferron, NEM, TIBA, andcycloheximide, we may suspect that the inducedethylene-forming system differs from the endogenoussystem. However, before concluding that 2 kindsof ethylene-forming systems exist in young seedlings,we should consider that the inhibitors may react with

kinetin or the kinetin-induced system causing inter-ruption of the stimulatory effect.

It appears that ethylene formiiationl in seedliingsis directly interrelated with the importanit growthregulators kinetin aind IAA, and is also indirectlyrelated, in a complementary way, with gibberellin.A proper balance of these interrelationships mayform the basis for regulatory control of growth anddevelopment in plant cells.

Acknowledgment

We thank H. W. Hruschka for advice on statisticalanalysis.

Literature Cited

1. ABELES, F. B. AND B. RUBINSTEIN. 1964. Regu-lation of ethylene evolution and leaf abscissionby auxin. Plant Physiol. 39: 963-69.

2. ABELES, F. B. 1966. Auxin stimulation of ethyleneevolution. Plant Physiol. 41: 58558".

3. ABELES, F. B., R. E. HOLM, AND H. i.. GAHAGAN.1967. Abscission: The role of aging. PlantPhysiol. 42: 1351-57.

4. BRIAN, P. W. AND H. G. HEMMING. 1957. Effectsof gibberellic acid and kinetin on growth of peastem sections. Naturwissenschaften 44: 594.

5. BURG, S. P. AND E. A. BURG. 196)'. The inter-action between auxin and ethylene ; ld its role inplant growth. Proc. Natl. Acad. &ic. 55: 262-69.

(. BURG, S. P. AND C. 0. CLAGETT. 1967. Conversionof methionine to ethylene in vegetative tissue andfruits. Biochem. Biophys. Res. Commuin. 27: 125--30.

7. BURG, S. P. AND E. A. BURG. 1967. Auxin stimu-lated ethylene formation: Its relationship to auxin-inhibited growth, root geotropism and other plantprocesses. Sixth Intern. Conf. Plant Growth Sub-stances, Carlton University Book of Abstracts.p 11.

8. CROCKER, W., A. E. HITCHCOCK, AND P. W. ZIM-MERMAN. 1935. Similarities in the effects ofethylene and plant auxins. Contrib. Boyce Thomp-son Inst. 7: 23148.

9. DOSTAL, H. C. AND A. C. LEOPOLD. 1967. Gib-berellin delays ripening of tomatoes. Science 158:1579.

10. KATSUMI, M. 1962. Physiological effects of kine-tin. Effect on the thickening of etiolated pea stemsections. Physiol. Plantarum 15: 115-21.

11. KATSUMI, M. 1963. Physiological effects of kin-etin. Effect of kinetin on the elongation, wateruptake and oxygen uptake of etiolated pea stemsections. Physiol. Plantarum 16: 66-72.

12. LE CLERG, E. L. 1957. Mean separation by thefunctional analysis of variance and multiple com-parisons. U.S.D.A., ARS Publication 20-23.

13. LEWIs, L. N., R. L. PALMER, AND H. Z. HiEL.1967. Interactions of auxins, abscission accelera-tors, and ethylene on abscission of citrus fruits.Sixth Intern. Conf. Plant Growth Substances,Carlton University Book of Abstracts, p 48.

14. LIEBERMAN, M., A. KUNISHI, L. W. MAPSON,AND D. A. WARDALE. 1966. Stimulation of ethy-lene production in apple tissue slices by methionine.Plant Physiol. 41: 376-82.



PLANT PHYSIOLOGY

15. MARCUS, A. AND J. FEELEY. 1965. Protein syn-

thesis in imbibed seeds. II. Polysome formationduring imbibition. J. Biol. Chem. 240: 1675-80.

16. MEIGH, D. F., K. H. NORRIS, C. C. CRAFT, AND M.LIEBERMAN. 1960. Ethylene production by to-mato and apple fruits. Nature 186: 902.

17. MICIIENER, H. D. 1938. The action of ethyleneon plant growth. Am. J. Botany 25: 711-20.

18. MOORE, T. C. 1967. Gibberellin relationships inthe Alaska pea (Pisttii sativmio). Am. J. Botany54(2) : 262-69.

19. SCorT, P. C. AND A. C. LEOPOLD. 1967. Opposingeffects of gibberellin and ethylene. Plant Physiol.42: 1021-22.

20. SKOOG, F., H. Q. HAMZI, A. M. SZWEYKOWSKA, N.J. LEIONARD, K. L. CARRAWAY, T. Fujii, J. P.HELGESON, AND R. N. LOEPPKY. 1967. Cyto-kinins: Structure/activity relationships. Phyto-chemistry 6: 1169-92.

21. SOMMER, N. F. 1961. Longitudinal and lateral re-

sponse of etiolated pea sections to indoleaceticacid, gibberellin, kinetin, sucrose and cobaltouschloride. Physiol. Plantarum 14: 74149.

22. ZIMMERMAN, P. W. AND F. WILCOXON. 1935. Sev-eral chemical growth substances which cause in-itiation of roots and other responses in plants.Contrib. Boyce Thompson Inst. 7: 209.

2036



kinetin, iaa, and gibberellin ethylene production ... · (kinetin trcatment). each group was grown...

Documents