An increase in ethylene sensitivity is associated with jasmonate-promoted senescence of detached rice leaves

J Plant Growth Regul (1996) 15:197-200 _{J o u r n a l o f}

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9 1996 Springer-Verlag New York Inc.

An Increase in Ethylene Sensitivity Is Associated with Jasmonate-Promoted

Senescence of Detached Rice Leaves

F.-Y. Tsai, K. T. Hung, and C. H. Kao

Department of Agronomy, National Taiwan University, Taipei, Taiwan, Republic of China

Received March 11, 1996; accepted October 24, 1996

Abstract. The role of ethylene in jasmonate-promoted

senescence of detached rice leaves was investigated. Eth-

ylene production in methyl jasmonate-treated leaf seg-

ments of rice was lower than in the control leaves. Treat-

ment of leaf segments with silver nitrate or/and silver

thiosulfate, inhibitors of ethylene action, inhibited meth-

yl jasmonate-, jasmonic acid-, linolenic acid-, and absci-

sic acid-promoted senescence of detached leaves. We

suggest that an increase in ethylene sensitivity, but not

ethylene level, is the initial event triggering the enhanced

senescence by jasmonates of detached rice leaves.

Key Words.

Ethylene--Jasmonates--Oryza sativa--

Senescene

Jasmonic acid (JA) and its methyl ester, methyl jas-

monate (M J), are naturally occurring growth regulators

found in higher plants (Meyer et al. 1984, Sembdner and

Parthier 1993). Jasmonates have been shown to be pow-

erful promoters of leaf senescence (Chou and Kao 1992,

Cuello et al. 1990, Sembdner and Parthier 1993, Ueda

and Kato 1981, Weidhase et al. 1987). However, the

mechanism whereby jasmonates promote leaf senes-

cence remains unclear. Ethylene was found to play a

significant role in promoting senescence of leaves (Mat-

too and Aharoni 1988). Using cucumber cotyledons,

Abeles et al. (1989) found that MJ was as effective as

ethylene in inducing senescence. In addition to this ob-

servation, silver thiosulfate (STS) had no effect on the

ability of MJ to promote senescence. Based on these

results they suggested that the action of MJ was inde-

A b b r e v i a t i o n s : JA, jasmonic acid; MJ, methyl jasmonate; STS, silver thiosulfate; ABA, abscisic acid.

* Author for correspondence.

pendent of ethylene. Cuello et al. (1990) also reported

that MJ and ethylene had different mechanisms in pro-

moting the senescence of detached barley leaves. In this

study the possible involvement of ethylene in the regu-

lation of jasmonate-promoted senescence of detached

rice leaves was investigated.

Materials and Methods

Rice (Oryza sativa L. cv. Taichung Native 1) was cultured as described previously (Kao 1980). The apical 3-cm segments excised from the third leaves of 12-day-old seedlings were used. A group of 10 segments was floated in a Petri dish containing 10 mL of test solutions. Incuba- tion was carried out at 27~ in darkness.

Chlorophyll was determined according to Wintermans and De Mots (1965) after extraction in 96% (v/v) ethanol. For protein extraction, leaf segments were homogenized in 50 m m sodium phosphate buffer (pH 6.8). The extracts were centrifugated at 17,600 xg for 20 min, and the supernatants were used for determination of protein by the method of Bradford (1976).

For determination of ethylene, leaf segments were placed vertically in test tubes that were plugged with rubber stoppers and incubated in darkness at 27~ for 1 h. The ethylene in the gas phase of the plugged tubes was determined by analysis of a 1-mL sample withdrawn with a hypodermic syringe as described elsewhere (Kao and Yang 1983).

In experiments with Ag § treatments, leaf segments were pretreated with 10 mg/mL silver nitrate for 30 min and then transferred to a Petri dish containing test solution. When STS was used as a source of Ag § leaf segments were treated with STS and test solution. A stock solution of STS was prepared by mixing equal volumes of 0.01 M AgNO 3 and 0.04 M Na2S203 (Liu et al. 1990).

All experiments were repeated at least twice. Data are presented as the results of a single experiment typical of the trends seen in the repeated experiments.

Results and Discussion

The senescence of detached rice leaves was followed by

measuring the decrease of chlorophyll and protein. Fig. 1

shows the time courses of protein and chlorophyll levels

of detached rice leaves floating on water or MJ (45 p,M)

in the dark. It is clear that the MJ significantly promotes

198 F.-Y. Tsai et al.

7 0

f I I i

5 o

0 .o

~n 4 -

i

e-

3 -

2

0 L. 2 - 0 "; O - M J U 1 - O + M J I I

6O

'7 m 5 0 01 E 4 0 r,

~.

3o

0

o. 2O

1 0

i

0

I I I I ! I

_ O ~

~ ) ~

-

0

I

I

I

I

0

1

2

3

D a y s

Fig. l. Time course of chlorophyll and protein levels in detached rice leaves floating on water or MJ (45 IxM) in darkness. Bars indicate S.E.

(n = 4).

1 5 '7

.c 10

'7 01 C I1

1"

5 W I I i I O - M J e + M J

0

I

I

I

I

0

1

2

3

D a y s

Fig. 2. Time course of ethylene production in detached rice leaves floating on water or MJ (45 IxM) in darkness. Bars indicate S.E.

(n = 4).

T 01 01

E

m r D. 0 iJ 0

U

7

01

E

E

o. _ B

1 -

0

6 0 - 4 0 - 2 0 - 0 "1" H 2 0

F

MJ ""I'- MJ § A g N 0 3

Fig. 3. Effect of silver nitrate pretreatment on the MJ-promoted senes- cence of detached rice leaves. Leaf segments were pretreated with silver nitrate (10 mg/mL) for 30 min in darkness and then transferred to distilled water or MJ (45 p~M) for 3 days. Bars indicate S.E. (n = 4).

the senescence of detached rice leaves. These results are

in agreement with earlier reports of other investigators

(Chou and Kao 1992, Cuello et al. 1990, Ueda and Kato

1981, Weidhase et al. 1987).

Ethylene production in water-treated leaf segments in-

creased significantly during the first 24 h and decreased

subsequently (Fig. 2). This result is consistent with our

early finding that ethylene production precedes the se-

nescence of detached rice leaves (Kao and Yang 1983),

indicating that ethylene production participates in the

regulation of rice leaf senescence. If MJ-promoted se-

nescence of detached rice leaves is mediated through

ethylene production, then ethylene production in M J-

treated leaf segments is expected to be higher than that in

water-treated leaf segments. However, as indicated in

Fig. 2, this does not seem to be the case.

If a change in ethylene production is excluded as an

explanation for the MJ-promoted senescence of detached

rice leaves, a change in sensitivity to ethylene is an al-

ternate possibility. This possibility was tested by using

Ethylene Sensitivity and Jasmonate-Promoted Senescence 7 o~ o}

E

P o o JZ 7 o~

E

o o D 1 - 0 6 0 4 0 2 0 " 0

l

I-I- MJ

~ + M J

0

0

5 0 2 0 0 8 0 0

STS, .uM

Fig. 4. Effect of STS on the MJ-promoted senescence of detached rice leaves. The concentration of MJ was 55 p,M. Chlorophyll and protein levels were determined after 3 days in darkness. Bars indicate S.E. (n

= 4).

the inhibitor of ethylene action, Ag § (Beyer 1976). Leaf

segments were pretreated with silver nitrate for 30 min

and then transferred to distilled water or MJ for 3 days in

the dark. As indicated in Fig. 3, silver nitrate pretreat-

ment resulted in an inhibition of MJ-promoted senes-

cence of detached rice leaves. Toxic effects of silver

nitrate have been reported (Lis et al. 1984, Liu et al.

1990). The advantages of using STS have been described

by others and are related to lower free Ag § in STS so-

lutions and a lower phytotoxicity of this compound (Liu

et al. 1990). Thus, a change in ethylene sensitivity in

MJ-treated leaf segments was tested further by using

STS. STS was also found to be effective in inhibit-

ing MJ-promoted senescence of detached rice leaves

Fig. 4).

JA was also observed to be effective, although to a

l e s s e r e x t e n t , in p r o m o t i n g t h e s e n e s c e n c e o f d e t a c h e d r i c e l e a v e s (Fig. 5). S i m i l a r to M J , t h e r e s p o n s e o f J A w a s also i n h i b i t e d b y S T S (Fig. 5). L i n o l e n i c a c i d (18:3) is k n o w n to b e t h e p r e c u r s o r in the p r o p o s e d b i o s y n t h e t i c p a t h w a y o f J A ( S e m b d n e r a n d P a r t h i e r 1993) a n d h a s b e e n s h o w n to b e a s e n e s c e n c e - 199

I-I-JA ~I+JA

5

E

,~

':r

..:.~

N

3

e - ,

o

1

~

~

~:?'~

-....:.,

o

=

6 0

'7

E 4 0

.r -r

2

a. 2 0

o

0

0

50

2 0 0 8 0 0

S T S , .gl M

Fig. 5. Effect of STS on the JA-promoted senescence of detached rice leaves. The concentration of JA was 45 IXM. Chlorophyll and protein levels were determined after 3 days in darkness. Bars indicate S.E. (n

= 4). O } E >=

|

Z o 3 2 1 0 I-1-18:3 ~+18:3

,lllm

4O t o 0 0 1 5 10 20 H=O 18:3 18:3 + STS,pM AgNO3

Fig. 6. Effect of STS and silver nitrate on linolenic acid-promoted senescence of detached rice leaves. Linolenic acid (18:3, 5 mm) was dissolved in 0.1% Tween 20. Water treatment included 0.1% Tween 20. For silver nitrate experiments, leaf segments were pretreated with silver nitrate (10 mg/mL) for 30 min in darkness. Chlorophyll and protein levels were determined after 3 days in darkness. Bars indicate S.E. (n = 4).

5

[ ] - A B A

I~I+ABA

"1"

0

0

6 0

o~ o~

E 40

t - O L .

a. 20

5 0

2 0 0 8 0 0

E

9 -/ 3 ..c D . o 0 o

1

i

0

0

200 F.-Y. Tsai et al.

STS, ju M

Fig. 7. Effect of STS on ABA-promoted senescence of detached rice leaves. The concentration of ABA was 45 I~M. Chlorophyll and protein levels were determined after 3 days in darkness. Bars indicate S.E. (n

= 4).

promoting substance (Ueda and Kato 1982). Our unpub-

lished data also show that the promotive effect of linole-

nic acid on the senescence of detached rice leaves may be

the result of an effect of JA. Thus, it is of great interest

to know whether the promotive response of linolenic

acid on the senescence of detached rice leaves is medi-

ated through a change in ethylene sensitivity. As indi-

cated in Fig. 6, linolenic acid indeed changes the sensi-

tivity of ethylene, as judged by the effect of silver nitrate

and STS.

In our earlier work we found that ABA promoted the

senescence of detached rice leaves but inhibited ethylene

production (Kao and Yang 1983). We proposed, but did

not prove, that ABA increases tissue sensitivity to eth-

ylene. Since jasmonates and ABA have both chemical

and physiological similarity (Anderson et al. 1989), we

investigated the effect of STS on ABA-promoted senes-

cence of detached rice leaves. The results in Fig. 7 indi-

cate that our early proposal appears to be correct.

Although it has been shown that the action of JAs was

independent of ethylene in other cases (Abeles et al.

1989, Cuello et al. 1990), our results strongly support the

notion that ethylene plays a regulatory role in the JA-

promoted senescence of detached rice leaves. It is eth-

ylene sensitivity rather than ethylene level which JAs

affect during the course of the senescence of detached

rice leaves.

Acknowledgments. This work was supported by National Science Council of Republic of China Grant (NSC 85-2321-B002-091). This paper is part 24 of the series Senescence of Rice Leaves.

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