Role of Ethylene in the Senescence of Detached Rice Leaves

0032-0889/83/73/0881/05/$00.50/0

Role

of

Ethylene

in the

Senescence of Detached Rice

Leaves'

Receivedfor publicationApril 15, 1983 and inrevised form July 21, 1983

CHING HUEI KAO AND SHANG FA YANG

Department

of

Agronomy,

National

Taiwan University, Taipei, Taiwan,

Republic

of China

(C. H. K.);

and

Department

of Vegetable

Crops, University

of California,

Davis,

California

95616

(S.

F. Y.)

ABSTRACT

The role of ethylene in the senescence of detached rice leaves in relation to their changes in 1-aminocyclopropane-1-carboxylic acid (ACC) content and ethyleneproduction wasstudied. Infreshlyexcised riceleaf segments, ACC level andethylene production rates were very low.Following incubation, theratesof ethyleneproductionincreased and reached a maximum in 12 h, and subsequently declined. The rise of

ethyleneproductionwasassociated with a 20- to30-foldincrease inACC

level.

Ethylene seems to beinvolved in theregulationof the senescenceof

detachedriceleaves.This conclusionwasbased on theobservationsthat

(a) maximum ethyleneproduction preceded chlorophyll degradation,(b) ACC application promoted chlorophyll degradation, (c) inhibitors of

ethyleneproductionandethyleneaction retardedchlorophylldegradation,

and (d) various treatments such aslight, cycloheximide, a,a-dipyridyl,

Ni2+,andcold temperature, which retardedchlorophylldegradation,also

inhibitedethyleneproduction.

Abscisic acid promoted senescence butsignificantlydecreasedethylene

production, whereas benzyladenine retarded senescence but promoted

ethylene production. This is interpreted to indicate that abscisic acid treatmentincreased thetissuesensitivitytoethylene,whereas

benzylad-enine treatment decreased it.

The senescence ofdetached riceleaves is characterized by a

decreasein Chl andproteincontentsandanincreaseina-amino nitrogen content (12). Senescence ofdetached rice leaves has beenreportedtoberetardedbyAg+,Co2+, Ni2+,orby anaerobic conditions(11, 15, 24). Sincethediscovery ofACC2as thekey intermediatein thepathwayofethylenebiosynthesis,our

under-standing oftheregulationof ethyleneproductionhas beengreatly clarified (1, 23). Cobalt ion and anaerobiosis are known to interfere withtheconversionof ACCtoethylene (23).Like

Co2+,

Ni2+inhibitsethyleneproduction in manyplanttissues (16). In contrast, Ag+ isa potent inhibitor of ethylene action in plants

(4, 5). Recent workof Aharoni andLieberman (3)andGepstein

and Thimann (9)suggestedthatendogenous ethyleneplayed a significant role in the senescence process of detached tobacco

andoatleaves.

Inthepresent investigation,weexamined the roleof ethylene ininducing senescence of detached rice leaves and the changes 'Supported byaresearchgrant from the National ScienceFoundation (PCM-8 114933). This work was conductedwhileC. H. K.held a Repub-licof China National Science Council Fellowship and S. F. Y. held a

GuggenheimFellowship.

2Abbreviations:ACC, I-aminocyclopropane-l-carboxylicacid; AOA, aminooxyaceticacid; AVG, aminoethoxyvinylglycine; CHI,

cyclohexi-mide;DP,aa-dipyridyl.

of ethylene production in relationto theirsenescence as influ-enced by various environmental and external factors, such as

light, C02, cytokinin, and ABA treatments, which may either

accelerate or retard senescence. Since ACC is the immediate

precursorofethylene anditslevelregulates ethyleneproduction

rate (1), the changes ofendogenous ACC level in relation to

ethyleneproductionratesduringsenescence werealsostudied.

MATERIALS AND METHODS

Plant Materials and Incubation Conditions.Rice (Orvza sativa

L. 'Taichung' Native 1) seedlings were cultured as previously described (14). Theapical 3-cmsegmentsexcised fromthethird

leavesof8-d-old seedlings were used. A group of 10 segments was floated in a 50-ml flask containing 10 ml test solution. Incubation wascarriedout at 30°Ceitherunder light (80

4mol

quanta m-2s-')provided byamixture of cool-whiteandGrolux

lamps orin darkness. In experiments with Ag+ treatment, leaf

segmentswerepretreated with 10mg/l silver nitratefor 30 min,

and then transferred to 50-ml flasks containing test solution

without Ag+. For the experiments in which various

concentra-tionsofCO2 wereused,all flaskswereflushed withC02-free air

and sealedwithrubberserumcaps. Toobtain desired

concentra-tions of C02, known amount ofCO2 was injected into each sealedflask;aC02-free atmospherewasachieved byhanging in

theflaska centerwellcontainingafilterpaperwickwettedwith 0.2mlof 20% KOH.Allexperimentswererepeatedatleast three

times. Similar results and identical trends were obtained each

time.The datareported herewerefromasingleexperiment.

Chl Determination. Chl was extracted anddetermined as

de-scribed before(12). ChlwasexpressedasA665 per 10 segments in 10 ml 80% ethanol.

Determination ofEthylene. Theflaskswereflushed withfresh air, sealed with rubberserum capsand incubated at 30C. For thetime-course experiment(Fig. 1), l-ml gassamplewas

with-drawn from the headspace ofthe flask at specified times. The

flaskswerethenflushed with fresh airandresealeduntil thenext

ethylene determination. Forthose experimentswith treatments

thatincludedABA,ACC, BA, CHI, DP,low temperature, inhib-itors of ethylene production or inhibitors of ethylene action, ethylene thataccumulated in the first 6 h ofincubation under

lightor dark condition was determined. Ethylene was assayed usinga gaschromatograph equippedwith an alumina column andaflame ionizationdetector.

Determination of ACC. ACCwasextractedanddetermined as

describedpreviously( 14).

Determination of Respiration Rates. Leaf segments were placed into 14-mltesttubescontaining0.5mlofdeionizedH20, and sealedwith rubberserumcapsafterflushingwith fresh air.

Respiration rates weredetermined bymeasuring theCO2 accu-mulated in darknessduringa 2-h period; respiration rateswere linear for4h.Gassamplesweretakenasdescribed for ethylene and determined with a gas chromatograph (model 800, Carle 881

Instruments, Inc.) equipped with a silica-gelcolumn and a ther-moconductivitydetector.

RESULTS

Changes in Ethylene Production, Respiration Rate, andChl and ACC Contents of Detached Leaves

during

Senescence in

Light or Darkness. The senescenceof rice leaves was followed bymeasuring the decrease ofChl. Figure1 showsthe time courses of Chl and ACC contents and ethylene and C02 production rates of leafsegments floating on water in the light or dark. The decreaseof Chl contentwasevidentat24hafter leaf detachment under bothlight and dark conditions. It is also clear from Figure

1 that light was effective in retarding senescence of rice leaf segments. Under both light and dark conditions, the rates of

ethylene production increased immediately after excision and reached amaximum in 12 h, and subsequently declined. Light substantially inhibited the endogenous ethylene production. The increaseof ACC level in the leaf segments coincided closely with the increase of ethylene production under both light and dark conditions. The endogenous level of ACC rapidly increased and peaked at 9 and 12 h after excision in the light and dark, respectively, and thendeclined. Therise of ethylene production wasassociated with a 20- and 30-fold increase in ACC content inlight and darkness,respectively. During the first 9 h incuba-tion, no significant difference could be found in ACC level betweenlight and dark treatments, but subsequently ACC

con-tentsinthelightwerelower.

,2 0.8_-.-. 0. Y _ o 0.4_ 0 -J 10_ 5-1008 _ DARK ----LIGHT

7500--,3

500___

C7,

Q

w'~~~~~~0

Therelationship between leaf senescence andrespiration rate

is not a simple one. Ifsenescence is viewed as ageneral decline in function (20), a steady decline of respiration rate would be

expected.On the other hand,some senescence-related changes, such as thesynthesis ofdegradative enzymes (20),seem torequire higher respiratory activity. Indetached leaves, therise of respi-rationrateis usually preceded bya steepfallor a constant rate

(2, 17, 18). Itis also true in rice leaf segments as shown in Figure 1. Respiration rate decreases during early stage of incubation, butit started to increase after about 12 and 48 hincubation in the light and dark, respectively. Under both light and dark

conditions, respiration peaked at a later stage (about 72 hafter incubation), during which time leaf segments had low Chl level. It seems thattheriseof respiration in the presentsystem isthe

result rather than the cause of leaf senescence. In climacteric fruits, the onset of respiratory increase is accompanied by the

onsetofethylene production, whereas theethylene production in rice leaves occurs considerably earlier than maximal respira-tion rate. Thus, it is unlikely that ethylene plays any causative roleininducing the rise ofrespirationrateduring the senescence of rice leaf segments. Figure 1 also shows that light substantially promotedrespiration rate throughout the course of senescence. Since respiration was determined under dark condition, the higherrespiration rate is unlikely due to photorespiration.

Effects of Inhibitors of Ethylene Production and ACC on the Senescence of Detached Leaves. If ethylene plays a regulatory role in senescence, it is expected that inhibitors of ethylene

DARK ---- LIGHT o* C2H4 40 A ACC 30 T E 20 '. \ Q~~~ - 0 24 48 72 96 TIME(h)

Table I. EffectsofACC,AVG,andAOA onEthyleneProduction and Chl Content ofDetached Rice Leaves in Light and Darkness Chlwasdeterminedafter3 and4din darknessandlight, respectively.

Darkness Light Treatment C2H Chl C2H4 Chl nlg-'h- A"5 nlg-'h-' A"s Control 7.88 0.383±0.021 3.62 0.403±0.034 ACC, 1 mM 15.95 0.334±0.002 9.37 0.292±0.008 AVG,0.2mM 1.22 0.452 ±0.031 1.23 0.521 ±0.006 AOA, 1 mM 2.22 0.458±0.019

biosynthesis wouldretardsenescence.AVGandAOA,whichare

known to inhibit ethyleneproduction by blocking ACC forma-tion (23),significantly retardedthesenescenceof detachedleaves incubated inthe lightordark(Table I).Table I also shows the effect of ACC, the immediate precursor ofethylene (1), which significantly promoted ethylene production, and accelerated

se-nescenceofdetached leaves under both dark andlightconditions.

C02+,

an inhibitorofethyleneproduction (5), which blocks the

conversion of ACC to ethylene (25), also retarded senescence

andethyleneproduction in rice leaves(Fig.2).

TheeffectsofCo2'aremorepronounced in darknessthan in

thelight. Itshould be notedthat theeffective concentration of

Co2+toretardsenescenceandtoinhibitethyleneproduction in thelight ismuchlowerthan that indarkness.

Effects of Inhibitors of Ethylene ActionontheSenescence of

DetachedLeaves.

Age

andCO2 are knownto inhibitethylene action in many physiological responses(4, 5, 7, 24). Both

Age

andCO2effectively retardedsenescenceofdetachedriceleaves, although they promoted ethylene production (Table II). The

promotiveeffect ofAg+andCO2onethyleneproduction inother plant systems has been well documented (3, 14). Ag+ was, however,lesseffective than CO2onretardation of Chl degrada-tion. Theseobservationsareincontrast tothoseof Aharoniand

Lieberman (3), who reported that Ag+was moreeffective than CO2 inretardingsenescenceintobaccoleaves.

Effects ofSenescenceRetardantsonEthylene Productionand

Senescence ofDetached Leaves. CHI and Ni2+ have been

re-portedtoretardsenescenceindetachedrice leaves(1 1, 15).The

8 6 0-'- 4 %S4 C., 2F 0 0

FIG. 2. Effects ofthe concentrations ofCo2+, determined after3 d.

10 100

chelator, DP, and low temperature (5C) were also known to

delaysenescenceinoatleaf tissues in thedark (19, 20).All these treatments areexpectedtoinhibit ethylene production, if ethyl-eneplaysarole inregulating senescence. Indeed, itis thecase. Table III shows theeffects ofCHI,DP,Ni2+,and lowtemperature

(5C) on leaf senescence and ethylene production. All these treatments notonlyeffectively retarded leafsenescence,but also,

inhibitedethyleneproduction.

Effects ofBA and ABA on Ethylene Production and Senes-cence of Detached Leaves. It has long been recognized that

cytokininsare effective in retarding the senescenceofmost, if

notall, leaves.Theeffect of cytokinins in retardingsenescenceis species-orvariety-specific; fortherice varietyused inthis inves-tigation, BAhasbeen foundtobe the mostactivecytokinin in retardingsenescencein thedark(11). The effect ofBA on leaf

senescence in relation to ethylene production is presented in Figure 3. Although BA effectively retarded senescence in the

light,aswell asinthe dark, BAsignificantlypromoted ethylene

production. Among the known promotersofsenescence, ABA

hasbeenstudiedmostwidely. Recently, Gepstein and Thimann (8) claimed thatABAisanendogenousfactor inleafsenescence.

Figure 4 showsthe effect ofABA on ethylene production and

leaf senescence in the light. Ethylene production was greatly decreasedbyABA. In contrast,leafsenescenceincreasedasthe

concentration ofABA were increased. In dark, ABA also

pro-motedleafsenescence anddecreasedethyleneproduction(data notshown).

DISCUSSION

The changes ofACC content and ethylene production have beenstudied in ripening fruitand senescing flowers (6, 10). In

senescing rice leaves,therelationshipbetweenACCandethylene

production isgenerally similar tothat found in ripening fruits andsenescing flowers.Infreshly excised rice leafsegments,both

ACCcontentand ethyleneproduction rate were verylow.

Sub-sequently, ethyleneproductionrateincreased whichwas

accom-panied byan increase in ACCcontent, suggestingthat ACC is an intermediatein thebiosynthesis of ethylene throughout the

senescenceprocess.Thus, thebiosyntheticpathway insenescing rice leaves appears to be the same asthat established in pple tissue(1). 0 io0' I10 161 0.8 0.6 ^-J 0.4 to 4

I-0,

-I -i 0.4 x 0 0 -J 0.2 0 0 [Co2 ) (mM)

appliedas COCI2, on Chl content and ethylene production ofdetached rice leaves. Chl was

DARK LIGHT \n-- C21-4 ._- -_ -.- 1L.Chl .____ L-- + \ g C2H4 Chi U I l l l l

Table II. EffectsofAgNO3orCO2 TreatmentonEthylene Production

andChlContentofDetached Rice Leaves in Light and Darkness

Chl was determined after 3 d in darkness or 4 d in the light in

experiment I and after3 d in experiment 2. For

AgC

treatment, the

detachedleaveswerepretreatedfor 30minin 10mg/l AgNO3 solution; forCO2 treatment, the leaves were incubated in 5% CO2. Ethylene

accumulated in the first 6hof incubationwasdetermined.

Darkness Light Treatment C2H4 Chi C2H4 Chi nl g-' h-' A"s nlg-'h' A"s Exp. I Control 7.04 0.348±0.004 2.40 0.385±0.005 Ag+ 15.58 0.491±0.031 9.75 0.544±0.055 Exp. 2 Control 5.81 0.216±0.021 4.64 0.494±0.034 CO2 8.03 0.570±0.022 16.76 0.650±0.009

Table III. Effects ofCHI, DP,Ni2+, and Low TemperatureonEthylene Production and Chi Content ofDetached Rice Leaves in Darkness Chlwasdetermined after 3 d in darkness. The concentrations ofCHI,

DP, and Ni2+ employed were0.5, 0.1, and 10 mM, respectively. Low temperature treatmentwasat5°C.

Treatment C2H4 Chl nlg-' h-' A"s Control 6.42 0.374±0.007 CHI 3.40 0.752 ±0.002 DP 5.44 0.810±0.004 Ni2+ 1.80 0.739±0.014 Lowtemperature 3.87 0.842±0.024 9 8 7 *l% 6 C) 2 DARK _0. 0

,/11/0--

/

Chl #,.. le00 -0 .00 .01II~ ~ - / C2H4 _04 0 -1 1I0 LIGHT

/Af

Chi *-/ t C2H4 I I l I .. .9 .6 .3 0.9 0.6 uN D 2 oti 0 0 Q 0.3 10% GO 6 5 4 -LOG[BAJ(M)

FIG. 3. Effects of BA concentrationsonethylene productionand Chl

contentof detachedriceleaves.Chlwasdetermined after3 d.

3 .C - 2 1 co 6 5 4 0.8 e e 0 0.6 < IL 0 0.4 ° 0 0. 0.2

-LOG(ABA]

(M)

FIG. 4. Effects ofABAconcentrationson ethylene production and

Chlcontentof detachedriceleavesinlight. Chlwasdetermined after2 d inlight.

Light substantially inhibited ethylene production throughout

the senescence process. During the first 9-h incubation, there

was nosignificant difference in ACCcontentbetween light and

dark conditions(Fig. 1), indicatingthat the inhibition ofethylene production by lightisexertednot atthe level of ACC butatthe conversionof ACCtoethylene. Using detachedrice and tobacco

leaves, Kao and Yang (14) found that inhibition of ethylene production by lightwas mediatedthrough the internal level of

C02, which directlymodulated theactivityof the enzyme

con-verting ACCtoethylene.

Sincethe increase ofethylene productioninrice leaves is rapid following excision,one mayarguethattheethylene production is probably a wound orother stress response. When detached

leaves were used to study senescence and determine ethylene production, wounding is always a problem. However, in the

present study, each long and narrow rice leafwas cut once

transversely, thearea ofwounding wasverysmall. It has been

reported that there was a slight but significant increase in the

freshweight of rice leafsegmentsfloatedonwater 13). Therefore,

therapidincrease ofethylene productioncouldnot resultfrom

water stress. The onset of ethylene production is therefore thoughttobeanexcision-relatedresponse.

In the study of hormonal control of leaf senescence, two approachesarecommonly employed.The first andmostwidely

used is external application ofplant hormones or chemicals

whichareknown toaffectthe synthesisortoexert an effecton

the action of the hormone. The second istocorrelatechangesin

endogenoushormonecontentwithsenescence.Thepresentstudy

indicated thatethylene participatesintheregulationof rice leaf

senescence. This conclusion was based on observations(under

both light and dark conditions) that (a) endogenous ethylene production preceded Chl degradation (Fig. 1); (b)inhibitors of

ethylene productionsuchasAVG and

CO2"

retardedsenescence

(Table I; Fig. 2);(c)ACC,the immediateprecursorofethylene, promotedsenescence(Table I); (d)inhibitorsofethyleneaction

as Ag+ andCO2 retarded senescence (Table II); and (e) other treatments suchaslight, CHI, DP, Ni2+, and lowtemperature,

which retarded senescence, also inhibited ethylene production (Fig. 1; Table III). Recently, the importance of ethylene in regulatingsenescenceof detached leaveshas also been reported byAharoni and Lieberman(3)andGepsteinand Thimann(9).

Usingoatleaves, Gepsteinand Thimann(9)found kinetindid notaffectethylene production under darkconditions, but

pro-moted ethylene production under light conditions. They also

found that ABA promoted ethylene production, which is in contrasttoourresults (Fig. 4). The effect ofABA onethylene

productionfoundbythemwasprobablyawoundresponse,since

0

'11*

*.. Chi %

*11

.

~~~%0

C2H4 '-S ' 1 I I' I

-theyused fineabrasivetoscrub leaftissues in ordertoenhance

the entry of ABA. Wright (22), however, reported that ABA

significantly inhibited ethylene production from both excised wiltedandnonstressedwheatleaves.

WhenABAandcytokininsareappliedexogenously,theyare

effective in promoting and retarding, respectively, leaf

senes-cence.Furthermore,theendogenouslevelofABA and cytokinins

increases and decreases, respectively, before theonsetof senes-cencesymptoms(20). Thus, it is probablethatleafsenescenceis

regulated bymanyfactorsincluding interactionsamong

cytoki-nins,ABA, and ethylene. Recently, Trewavas (21) developeda

concept ofusing variations of quantitative tissue sensitivity to

explain the developmental phenomena. It seemsthat ethylene

effect is governednot only bythe ethylene production rate of thetissue, but alsoby the tissuesensitivity toethylene. In the present system,ABApromotedsenescencebutinhibitedethylene

production, whereasBAretardedsenescencebutpromoted

eth-yleneproduction.Asimpleexplanationwhichaccountsforthese

seemingly contradictoryobservations is thatABAincreases tissue

sensitivity toethylene,whereas BAdecreases it. Consequently,

eventhough ABA inhibits and BA promotes ethylene produc-tion, the overall ethylene effects may not parallel with their

ethylene productionrates.

Acknowledgment-The assistance of DaoHungLiu isgratefullyacknowledged. LITERATURE CITED

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