Role of Ethylene in Plant Growth, Development, and Senescence

6 Role of Ethylene in Plant Growth, Development, and Senescence MORRIS LIEBERMAN

Downloaded by UNIV LAVAL on September 24, 2015 | http://pubs.acs.org Publication Date: September 27, 1979 | doi: 10.1021/bk-1979-0111.ch006

Post Harvest Plant Physiology Laboratory, Beltsville Agricultural Research Center, USDA, Beltsville, MD 20705 The simplest unsaturated carbon compound, ethylene, exerts a major influence on many i f not a l l aspects of plant growth and development. Although ethylene is a gas at physiological temperatures and pressures, it is now recognized as a plant hormone because it is a natural product of metabolism, acts in trace amounts and is neither a substrate nor cofactor in reactions which are associated with major developmental plant processes. Whether or not ethylene meets a l l the standard criteria established for hormones, there is no question that this gas is a powerful natural regulating substance in plant metabolism, and that it acts and interacts with other recognized plant hormones. With the advent of gas chromatography, ethylene has become the simplest plant hormone to assay since it is evolved from the tissues and requires no extraction or purification prior to analysis. An important advance in understanding ethylene action was realized with the rediscovery that auxin influences ethylene biosynthesis in juvenile tissues (1,2,3). These studies have led to an appreciation of the general nature of the hormonal action of ethylene, an action which extends beyond fruit ripening and senescence (its classical role) to seed germination (4),seedling growth (5), root growth (6), stress phenomena (7) and other physiological processes (8) that may be considered to be under hormonal control. Ethylene is therefore an important component in the mix of hormones that control plant metabolism. Ethylene

Biosynthesis

Methionine i s the major precursor i n the biochemical p a t h way to ethylene (9). Ethylene i s formed from carbons 3 and 4 of methionine which i s degraded i n r e a c t i o n s p o s s i b l y i n v o l v i n g f r e e r a d i c a l s and oxygen (9). Recently Adams and Yang (10,11) i d e n t i f i e d S-adenosylmethionine (SAM) and 1-aminocyclopropane-lc a r b o x y l i c a c i d (ACC) as intermediates i n the pathway from methionine to ethylene. The sequence of r e a c t i o n s i n the pathway

This chapter not subject to U.S. copyright. Published 1979 American Chemical Society

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.


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PLANT GROWTH

SUBSTANCES

from methionine to ethylene, i n c l u d i n g i n t e r m e d i a t e s , i s shown i n F i g . 1. Recognition of SAM as an intermediate i n d i c a t e s that ATP i s involved i n ethylene b i o s y n t h e s i s . ATP may thus provide a locus for r e g u l a t o r y c o n t r o l . I n h i b i t o r s are known f o r every step i n the pathway from methionine to ethylene. The r e a c t i o n from methionine to SAM i s i n h i b i t e d by L-2-amino-4-hexynoic a c i d (AHA) or L-2-amino-4-trans-hexenoic a c i d and r e l a t e d methionine analogues (13), and the step from SAM to ACC i s i n h i b i t e d by aminoethoxy v i n y l g l y c i n e (AVG) and c a n a l i n e (12,14). The f i n a l step from ACC to ethylene probably i n v o l v e s oxygen, perhaps i n a f r e e - r a d i c a l c h a i n r e a c t i o n , because i t i s i n h i b i t e d by anaerobiosis as w e l l as a number of a n t i o x i d a n t r a d i c a l - q u e n c h ing agents, such as η - p r o p y l g a l l a t e and 3 , 4 , 5 - t r i c h l o r o p h e n o l . Some of the enzymes i n v o l v e d i n t h i s r e a c t i o n pathway have been i d e n t i f i e d . The enzyme of the f i r s t step i n the r e a c t i o n pathway, SAM synthetase, i s a known enzyme i n p l a n t t i s s u e s (15). The enzyme converting SAM to ACC has been i s o l a t e d from tomato t i s s u e s (14) and appears to be a p y r i d o x a l phosphate-mediated enzyme. However, the enzyme c o n v e r t i n g ACC to ethylene has not been i s o l a t e d as y e t , although i n d i c a t i o n s are that i t r e a c t s with oxygen by a complex mechanism, perhaps to form f r e e - r a d i c a l intermediates. Adams and Yang (10) have suggested that the S atom of methionine i s r e c y c l e d i n the ethylene r e a c t i o n pathway, as shown i n F i g . 2. In t h i s scheme, 5 - m e t h y l t h i o a d e n o s i n e , the r e s i d u a l molecule which d e r i v e s from the r e a c t i o n converting SAM to ACC, i s f u r t h e r metabolized to 5 - m e t h y l t h i o r i b o s e , which then t r a n s f e r s the S-methyl group to homoserine to form methionine. This scheme i s h y p o t h e t i c a l , and the enzymes neces sary f o r a l l these r e a c t i o n s have not as yet been demonstrated. Knowledge of the complete r e a c t i o n pathway f o r ethylene production and the c h a r a c t e r i s t i c s of the enzymes systems i n v o l v e d , should shed l i g h t on the c o n t r o l and r e g u l a t i o n of ethylene p r o d u c t i o n and perhaps a l s o i t s r e l a t i o n s h i p to other hormones. 1

f

Ethylene A c t i o n A c t i o n of ethylene i n r i p e n i n g and senescence. H i s t o r i c a l l y the a c t i o n of ethylene i s a s s o c i a t e d with r i p e n i n g f r u i t . Ethylene p r o d u c t i o n i n mature f r u i t c o i n c i d e s w i t h the onset of the r i p e n i n g process and the c l i m a c t e r i c r i s e i n r e s p i r a t i o n . Exogenously a p p l i e d ethylene can induce mature unripe f r u i t to r i p e n and senesce as they would n a t u r a l l y , but at an a c c e l e r a t e d r a t e . Ethylene was t h e r e f o r e considered a r i p e n i n g or aging h o r mone a s s o c i a t e d e s p e c i a l l y with senescent f r u i t metabolism. How ever other p l a n t hormones a l s o p l a y a r o l e i n f r u i t r i p e n i n g and senescence. For example c y t o k i n i n s can suppress ethylene p r o d u c t i o n i n s l i c e s of r i p e avocado f r u i t suggesting an i n t e r a c t i o n



6.

ΜΕΊΉΙΟΝΙΝΕ-

ATP

SAM-

AHA

AHA= PP= AVG= PG=

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Role of Ethylene in Plant Growth

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PP

ACC-

AVG

PG

L~ 2-Amino- 4-hexynoic a c i d Pyridoxal phosphate Aminoethoxy v i n y l g l y c i n e Propyl g a l l a t e

Figure 1. Reactions from methionine to ethylene showing intermediates and inhibitors of each step in the pathway and the possible direct conversion of methionine to ethylene


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P L A N T G R O W T H SUBSTANCES

GLUCOSE HO-CHVCH CH-COOH r


RIBOSE

^sNH

2

(HOMOSERINE)

CHJ-S-CHJ-CHJ-CH-COOH NH

2

(METHIONINE)

(5—METHYLTHIORIBOSE) CH—S C H ^ ° \ ^

OH

O

ATP\

H

OH

CH3-S-CH5-CH -CK-COOH 2

NH ADENINE

2

-AD OH O H (S—ADENOSYLMETHIONINE)

(5'METHYLTHIOADENOSINE) C H

3

— S - C H ^ O v ^

OH

A

D

OH

( l - A M I N O C Y C L O P R O P A N E - 1 - C A R B O X Y L I C ACID)

Plant Physiology

Figure 2. Proposed pathway from methionine to ethylene indicating recycling o the S atom according to Adams and Yang (10)


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119

c y t o k i n i n s with the ethylene-forming system (Fig* 3) (16) . The p r o d u c t i o n and a c t i o n of ethylene a r e , however, not confined to mature f r u i t and senescent metabolism* Ethylene a l s o i n f l u e n c e s many f a c e t s of p l a n t growth and development,. The i n f l u e n c e of ethylene on growth of young t i s s u e s i s observed v i v i d l y i n i t s e f f e c t on e t i o l a t e d pea s e e d l i n g s to cause the well-known t r i p l e response—stunting, s u b a p i c a l s w e l l i n g and diageotropism—which i n v o l v e s a l l aspects of growth* Evidence of the t r i p l e response to exogenously a p p l i e d ethylene suggest i n t e r a c t i o n s of the gas with the t o t a l spectrum of p l a n t hormones, and i t i s these i n t e r a c t i o n s that r e q u i r e e l u c i d a t i o n .


of

Ethylene and a u x i n . The i n t e r - r e l a t i o n s h i p between auxin and ethylene was suggested by the d i s c o v e r y that supraoptimal l e v e l s of auxin (10~"*-10"~%) stimulated ethylene production i n a number of p l a n t t i s s u e s (17,18,19). Auxin-induced ethylene production i n s u b a p i c a l stem s e c t i o n s of pea s e e d l i n g s occurs a f t e r a l a g p e r i o d of about 1 to 3 hours, r e q u i r e s continuous presence of auxin and i s i n h i b i t e d by i n h i b i t o r s of RNA and p r o t e i n synthesis ( F i g . 4) (20,21). These data suggest that the ethylene-forming system i s induced by high l e v e l s of auxin and may i n v o l v e RNA to p r o t e i n s y n t h e s i s . In some t i s s u e s auxin can a c t i v a t e ethylene production i n 15 minutes or l e s s (22), which i s too short a time span f o r p r o t e i n s y n t h e s i s . Pea root t i p s a l s o appear to have an ethylene-forming system which can respond to low l e v e l s of IAA (1 μΜ) without a l a g p e r i o d , and t h i s system i s not i n h i b i t e d by cycloheximide (23). However, higher l e v e l s of IAA-induced ethylene production by pea root t i p s (10-100 uM) i n v o l v e s a l a g p e r i o d , i s i n h i b i t e d by cycloheximide and probably requires protein synthesis. The i n d u c t i o n of ethylene i n p l a n t t i s s u e s by supraoptimal concentrations of auxin i s w e l l e s t a b l i s h e d , but the reverse e f f e c t , that i s , the i n f l u e n c e of ethylene on auxin c o n c e n t r a t i o n i s l e s s w e l l known. There are r e p o r t s which i n d i c a t e that l e v e l s of exogenous ethylene (10-36 ppm) cause s i g n i f i c a n t r e d u c t i o n s i n endogenous l e v e l s of IAA (Table I) (24,J25,26) . These i n fluences of auxin on ethylene p r o d u c t i o n and ethylene on auxin l e v e l s suggest feedback r e l a t i o n s h i p s between these hormones which r e g u l a t e the l e v e l s of auxins and ethylene to cause spe c i f i c growth phenomena (27) a s , f o r example, the r e g u l a t i o n of c e l l shape and s i z e (28). Goldwin and Wain (29) showed that auxin-induced ethylene p r o d u c t i o n was r e l a t e d e x p o n e n t i a l l y to growth, and i s a consequence of c e l l u l a r growth processes induced by a u x i n . T h i r t y - f o u r compounds which were considered to be analogues of auxin were t e s t e d f o r t h e i r a b i l i t y to induce ethylene p r o d u c t i o n . Only those compounds which promoted c o n s i d e r a b l e extension growth were e f f e c t i v e inducers.. T h i s f i n d i n g suggests a r e l a t i o n between growth r a t e and ethylene p r o d u c t i o n » Perhaps v i a a feedback mechanism ethylene serves to slow down excessive growth i n p l a n t s e x c e s s i v e l y stimulated by high c o n -


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C 110

•

-

/ .—.

90

_c

80

-

70

—

60

-

/ I A A

per

φ

/ /

Kinetin

//

Ο Δ

r

c E t h y l1 e η e


100

50

-

40

30

20

10 1

1

Time

1

1

1

1

1

(hr) Plant Physiology

Figure 3.

Influence of IAA and cytokinins on ethylene production by post climacteric avocado tissue slices (16)



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Role of Ethylene in Plant Groioth

HOURS Plant Physiology

Figure 4. Effect of inhibitors of RNA and protein synthesis on IAA-induced ethylene production in subhook sections of etiolated pea seedlings. Arrows indicate time inhibitors were applied (20).


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PLANT GROWTH

SUBSTANCES


c e n t r a t i o n s of growth substances. The u l t i m a t e s i z e and shape of c e l l s , t i s s u e s and organs of a p l a n t may represent the r e s u l t of i n t e r a c t i o n of ethylene, as the modulating or braking r e g u l a t o r , with the mix of g r o w t h - a c c e l e r a t i n g hormones, such as auxins, g i b b e r e l l i n s and c y t o k i n i n s . Ethylene and GA. Scott and Leopold (30) noted the opposing a c t i o n s of GA^ and ethylene i n the l e t t u c e hypocotyl e l o n g a t i o n assay f o r GA^, i n the α - a m y l a s e i n d u c t i o n assay f o r GA, and i n i n d u c t i o n of i n v e r t a s e i n sugar beet t i s s u e . The opposing a c t i o n s of GA3 and ethylene i n the subhook r e g i o n of e t i o l a t e d pea seedlings were a l s o observed. T h i s r e g i o n , where c e l l elongation occurs most r a p i d l y , elongates abnormally when treated GA3 (10~5M) but thickens and does not elongate when treated with 1 ppm ethylene. Pretreatment of seedlings w i t h GA3 before treatment with ethylene prevents the s t u n t i n g and s w e l l i n g of the s u b - a p i c a l stem t i s s u e (31). F i g u r e 5 shows the appearance of c e l l s i n the s u b a p i c a l r e g i o n of pea s e e d l i n g s treated with GA3, ethylene, and a combination of GA3 and ethylene ( F i g . 5 ) . Treatment with GA3 elongated the c e l l s e x c e s s i v e l y , and treatment with ethylene caused the development of i s o d i a m e t r i c swollen cells. When treated with both GA3 and ethylene, the c e l l s were very s i m i l a r to c o n t r o l c e l l s i n s i z e and shape. These data i l l u s t r a t e the i n t e r a c t i o n s between GA3 and ethylene i n d e t e r mining s i z e and shape of c e l l s . C y t o k i n i n s and ethylene. C y t o k i n i n s can synergize IAAinduced ethylene production (32) i n e t i o l a t e d pea s e e d l i n g s , probably by i n c r e a s i n g the c o n c e n t r a t i o n of f r e e IAA v i a both suppression of IA4 conjugation and enhancement of IAA uptake (33). However, the i n f l u e n c e of c y t o k i n i n s on ethylene produc t i o n cannot be s o l e l y r e l a t e d to p r e s e r v i n g f r e e IAA, because k i n e t i n (10""%) i s much more e f f e c t i v e i n s t i m u l a t i n g ethylene production i n very young pea seedlings (2-day old) than i s IAA (10"%) (32) « Ir-aseki et a l . (34) a l s o noted that c y t o k i n i n can i n f l u e n c e ethylene p r o d u c t i o n i n the presence of IAA by some metabolic process unrelated to maintaining the l e v e l of f r e e IAA. Although c y t o k i n i n s appear to enhance ethylene p r o d u c t i o n i n s e e d l i n g s and excised segments of s e e d l i n g , e s p e c i a l l y i n conjunction with a u x i n , they tend to suppress ethylene p r o d u c t i o n i n c l i m a c t e r i c and p o s t c l i m a c t e r i c apple and avocado f r u i t s (16). T h i s tendency may r e l a t e to the known a c t i o n of c y t o k i n i n s i n suppressing l o s s of c h l o r o p h y l l and senescence i n aging leaves (35). Ethylene and ABA. A b s c i s i c a c i d (ABA), l i k e ethylene, i n h i b i t s growth of e t i o l a t e d s e e d l i n g s ; but the s e e d l i n g s do not show the t r i p l e response c h a r a c t e r i s t i c of e t h y l e n e - t r e a t e d s e e d l i n g s (36). A B A - i n h i b i t e d pea seedlings produce l e s s ethylene than s e e d l i n g s not treated with ABA i n response to h i g h


6.


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l e v e l s of IAA (10"%) (37). A p p a r e n t l y , ABA and ethylene i n h i b i t growth and i n t e r a c t with auxins by d i f f e r e n t mechanisms. However, ABA s t i m u l a t e s ethylene production i n mature f r u i t (16,37) and appears to act l i k e ethylene i n hastening senescence. There appears to be a c l o s e r e l a t i o n s h i p between ABA and ethylene i n inducing and a c c e l e r a t i n g the aging process i n p l a n t s . Ethylene as a s t i m u l a t o r of growth and development. The most observed a c t i o n s of ethylene on growing p l a n t s i n v o l v e s growth i n h i b i t i o n , or a c c e l e r a t i o n of senescence. These a c t i o n s are e s p e c i a l l y evident i n the antagonism or o p p o s i t i o n of ethylene to a u x i n s , g i b b e r e l l i n s and c y t o k i n i n s (27), as a l r e a d y o u t l i n e d above. A c t u a l l y ethylene stimulates growth i n many types of c e l l s , e s p e c i a l l y i n water p l a n t s (Table I I ) . When ethylene a c t s to s t i m u l a t e c e l l e l o n g a t i o n , as i n water p l a n t s , auxins and C 0 enhance the ethylene e f f e c t (38,39). This i n t e r a c t i o n i s the reverse of that observed on land p l a n t s wherein ethylene opposes the e f f e c t s of a u x i n , GA3 and c y t o k i n i n s . Considering ethylene a growth i n h i b i t o r may be i n a c c u r a t e i n l i g h t of the n a t u r a l p h y s i o l o g i c a l r o l e of t h i s gas i n growth, development and senescence. Whether or not ethylene a c t s to i n h i b i t growth depends on i t s c o n c e n t r a t i o n l e v e l , the stage of growth of the t i s s u e s on which i t a c t s , and the type of c e l l s and t i s s u e s to which i t i s a p p l i e d . The tomato mutant d i a g e o t r o p i c a , which i s c h a r a c t e r i z e d by h o r i z o n t a l growth of shoots and r o o t s , assumes a normal growth h a b i t when subjected to very low concentrations of ethylene, i n the order of 5 ppb, or h i g h l e v e l s of auxin (10""%) (40,41) . These s t u d i e s suggest that the morphological development of t h i s mutant may be c o n t r o l l e d by very low l e v e l s of endogenous ethylene p r o d u c t i o n r e s u l t i n g from an auxin-ethylene feedback mechanism. The i n h i b i t i o n of growth by ethylene i n subhook regions of e t i o l a t e d pea seedlings i s l a r g e l y due to r e d u c t i o n i n the p o l a r auxin t r a n s p o r t system which s u p p l i e s auxin to the c e l l s (42, 43). In the presence of ethylene the subhook r e g i o n of the pea stem does not grow i n l e n g t h but does continue to grow i n diameter, and there i s l i t t l e d i f f e r e n c e i n i n c r e a s e i n f r e s h weight between the c o n t r o l and e t h y l e n e - t r e a t e d seedlings i n the f i r s t 24 h r . During the next 2 days (72 hr a f t e r continuous treatment with e t h y l e n e ) , the i n c r e a s e i n f r e s h weight i s g r e a t er f o r the ethylene treatment than f o r the c o n t r o l i n s p i t e of the n o t i c e a b l e d i f f e r e n c e i n e l o n g a t i o n of the subhook. Ethylene at concentrations of 1 ppm and higher i n h i b i t e d root e l o n g a t i o n i n tomato, peas and r i c e . However, s t i m u l a t i o n of root e l o n g a t i o n was obtained with l e s s than 0.02 ppm i n tomatoes, l e s s than 0.15 ppm i n peas and l e s s than 1 ppm i n r i c e (44). Extension of r o o t s i n a l l three s p e c i e s could be i n creased by ethylene, but d i f f e r e n t concentrations were r e q u i r e d for each s p e c i e s . These data allow s p e c u l a t i o n that dynamic low l e v e l s of ethylene may be r e q u i r e d f o r normal growth and 2


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P L A N T G R O W T H SUBSTANCES

Table I .

IAA Content of E p i c o t y l s from C o n t r o l and EthyleneTreated (24 hr) E t i o l a t e d Pea Seedlings

Seedlings


'Alaska

IAA ng/seedling

Percent Difference

1

Control

6.5 + 0.7

Ethylene-treated (10-20 ppm)

2.7+0.3

-58.5

Sweet Eminent Control Ethylene-treated (19-36 ppm)

Table I I .

1.4+0.2

0.7+0.1

-50.0

E f f e c t of Ethylene and Other Hormones on Growth of Land and Water P l a n t s

P l a n t or T i s s u e

Auxin

GA

Ethylene

Land P l a n t s Pea Seedling E p i c o t y l s Pea Seedling Roots

+ +

++ +

+ +

+ +

Water P l a n t s Rice Seedlings Ranunculus A c l e r a t u s


+ +

6.

development of


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roots.

Wound or s t r e s s ethylene p r o d u c t i o n . The s i g n i f i c a n c e of ethylene i n p l a n t metabolism may be i n d i c a t e d by the changes i n ethylene p r o d u c t i o n that occur when t i s s u e i s wounded or placed under s t r e s s . T i s s u e which normally evolve l i t t l e or no ethylene show a surge i n ethylene p r o d u c t i o n 3 to 10 times the b a s a l l e v e l upon p h y s i c a l wounding (45), b r u i s i n g (46), f r e e z i n g (47), i r r a d i a t i o n (48), a t t a c k by microorganisms (49) and other stresses. The p r o f i l e of the surge i n ethylene p r o d u c t i o n , a f t e r a short l a g , i s shown i n F i g . 6 and suggests a dampened o s c i l l a t i o n , perhaps r e l a t e d to a negative feedback system i n the t i s s u e s (50,51). P l a n t s under water s t r e s s are known to produce increased amounts of ethylene, show a r i s e i n ABA and a d e c l i n e i n endogenous c y t o k i n i n s (52,53). Other p l a n t hormones are a l s o p r o b ably i n v o l v e d i n the response to water s t r e s s and other s t r e s s and wounding a c t i o n s . The surge of ethylene p r o d u c t i o n upon s t r e s s may therefore represent a response to a d i s t u r b a n c e of the hormonal balance i n t i s s u e s . The dampened o s c i l l a t i o n curve for wound ethylene production may r e f l e c t the dynamic r e t u r n of the d i s t u r b e d hormonal system to a proper hormonal balance under the new t i s s u e c o n d i t i o n s , and thus may a l s o r e f l e c t a h e a l i n g phenomenon. Ethylene r e c e p t o r s and r e g u l a t o r y c o n t r o l . The mode of a c t i o n of ethylene at the molecular l e v e l i s unknown. Some attempts, however, have been made to determine the r e c e p t o r s i t e s f o r ethylene (54) as w e l l as t h e i r c h a r a c t e r i s t i c s (55). There appears to be very l i t t l e i n c o r p o r a t i o n of ethylene a p p l i e d to t i s s u e s (only about 0.05%). The ethylene i n c o r p o r a t e d i n t o pea s e e d l i n g t i s s u e s which responded p h y s i o l o g i c a l l y to the gas was metabolized to CO2 and w a t e r - s o l u b l e metabolites (55). Metabolism of the i n c o r p o r a t e d - ^ C ethylene by pea seedlings and other t i s s u e s was i n h i b i t e d by h i g h l e v e l s of CO2 (7-10%) and Ag+ ions (10-500 ppm) (56). A g ions prevented the i n c o r p o r a t i o n of l^C ethylene i n t o w a t e r - s o l u b l e t i s s u e metabolites and counteracted the p h y s i o l o g i c a l e f f e c t s of ethylene i n r e t a r d i n g e p i c o t y l growth i n pea s e e d l i n g s , a b s c i s s i o n i n cotton and senescence of o r c h i d s (57). A g ions had l i t t l e e f f e c t on the metabolism of ethylene to - ^ C 0 . On the other hand, high l e v e l s of C 0 (7-10%) i n h i b i t e d the o x i d a t i o n of ethylene to "^C0 without a f f e c t i n g i n c o r p o r a t i o n of -^C H4 i n t o waters o l u b l e l a b e l e d compounds i n the same t i s s u e . These r e s u l t s suggest two s i t e s at which ethylene may be m e t a b o l i z e d . One s i t e i s blocked by A g ions and the other by C0 . High l e v e l s of ethylene could overcome the i n h i b i t i o n due to Ag ions and C 0 . The i n f l u e n c e of A g and CO^ on ethylene a c t i o n was observed i n senescing tobacco l e a f d i s k s i n which l o s s of c h l o r o p h y l l was taken as an index of senescence ( F i g . 7 ) . +

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Plant Physiology

Figure 5. Subapical cells in cortical region of etiolated pea seedlings held for 24 hr in water exposed to air (water-air) (4), GA-air (5), GA-ethylene (6), and ethylene (7) from Stewart et al. (SI)

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ι 40

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Plant Physiology

Figure 6. Profile of ethylene production by wounded stems of etiolated pea seedlings. The profile shows kinetic changes suggesting dampened oscillation (from Saltveit and Dilley (50))




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Figure 7. Influence of Ag (10 ppm), C0 (10%), and AVG (O.lmM) on chloro phyll retention in aging tobacco leaf disks senesing in the dark for six days 2


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+

Carbon d i o x i d e and A g ions c l e a r l y suppressed senescence, a s determined by c h l o r o p h y l l l o s s . Aminoethoxy v i n y l g l y c i n e (AVG}, the i n h i b i t o r of ethylene b i o s y n t h e s i s , a l s o s i g n i f i c a n t l y suppressed senescence, as determined by p r e s e r v a t i o n of c h l o r o p h y l l i n the l e a f d i s k s aging i n the dark.Combinations of C0 , Ag ions and AVG were e s p e c i a l l y e f f e c t i v e on p r e s e r v i n g c h l o r o p h y l l , presumably by suppressing both ethylene b i o s y n t h e s i s and a c t i o n at the two receptor s i t e s . A f t e r 6 days* aging at 2 5 ° i n the dark, the c o n t r o l s contained only 7% of the c h l o r o p h y l l present at the s t a r t , whereas 84% of the c h l o r o p h y l l was r e t a i n e d by the l e a f d i s k s treated with a combination of C0«, Ag and AVG. Leaf d i s k s which had been t r e a t e d with Ag (10 ppm) and C 0 (10%) to prevent ethylene a c t i o n , a c t u a l l y showed c o n s i d e r able increase i n ethylene production ( F i g . 8 ) . In the absence of auxin or c y t o k i n i n s , which increase ethylene p r o d u c t i o n , ethylene b i o s y n t h e s i s increased 2.5 times i n t h e presence of 5-15% CO2 and pretreatment with 10 ppm o £ Ag i o n s . On the a d d i t i o n of IAA or IAA and k i n e t i n to Ag and C 0 - treated l e a f d i s k s , ethylene production increased 8 to 9 times. This o b s e r v a t i o n suggests the p o s s i b i l i t y that the b i o s y n t h e s i s of ethylene depends on i t s u t i l i z a t i o n , perhaps through some negative feedback s i g n a l . Presumably the b i n d i n g of ethylene to i t s receptor or the metabolism of ethylene tends to dampen i t s biosynthesis. Auxin and combinations of auxin and c y t o k i n i n s c o n s i d e r a b l y augment ethylene p r o d u c t i o n i n Ag and CO - t r e a t e d l e a v e s . Once again t h i s points out the i n t e r a c t i o n between auxins, c y t o k i n i n s and ethylene. Production of ethylene appears to be dependent on auxin and a l s o on the degree to which ethylene i s metabolized. 2


1

2

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2

+

L o c a l i z a t i o n of the ethylene forming system. The ethylene-forming system i n p l a n t s has never been i s o l a t e d i n v i t r o because i t does not s u r v i v e d e s t r u c t i o n of the c e l l . In recent s t u d i e s p r o t o p l a s t s prepared from apple t i s s u e d i d not produce ethylene. The l o s s of ethylene-producing a b i l i t y by t i s s u e s l i c e s incubated i n c e l l w a l l - d i g e s t i n g enzymes during p r e p a r a t i o n of p r o t o p l a s t s i s shown i n F i g . 9. Methionine, the precursor of ethylene, delayed the l o s s of ethylene p r o d u c t i o n somewhat during p r e p a r a t i o n of the p r o t o p l a s t s . Ethylene production was r e s t o r e d to some extent when the p r o t o p l a s t s were c u l t u r e d f o r 3 or more days ( F i g . 10) (58). R e s t o r a t i o n of ethylene producing a b i l i t y by c u l t u r i n g was c o r r e l a t e d with regeneration of some c e l l - w a l l m a t e r i a l , as shown by i n c o r p o r a t i o n of m y o i n o s i t o l i n the e t h a n o l - i n s o l u b l e f r a c t i o n of the p r o t o p l a s t s and by increased fluorescence with c a l c a f l u o r - w h i t e (58). Regeneration of c e l l - w a l l m a t e r i a l was c o r r e l a t e d with ethylene production i n response to methionine added to the c u l t u r e d p r o t o p l a s t s . Production of ethylene by these c u l t u r e d p r o t o p l a s t s was not only dependent on a d d i t i o n of



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90-


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IAA+Kin+Ag

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7060-

Figure 8. Ethylene production of aging tobacco disks in the presence of Ag\ C0 , IAA, and kinetin in various concentrations 2

co

2

n(%)



PLANT GROWTH

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Figure 9. Loss of ethylene-producing ability in apple slices treated with a mixture of cell-wall-digesting enzymes in presence and absence of methionine (58): (Ο,Φ), preclimacteric; (A, A), climacteric.


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6.

TIME IN CULTURE (HOURS) Plant Physiology

Figure 10. Evidence for restoring ethylene-producing ability in cultured protoplasts, as correlated with myoinositol incorporation and increasedfluorescenceof EIS components of cultured protoplasts (58)



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methionine but was a l s o i n h i b i t e d by AVG and η - p r o p y l g a l l a t e , the i n h i b i t o r s of ethylene b i o s y n t h e s i s . A more r e c e n t l y used technique of i s o l a t i n g p r o t o p l a s t s from apple f r u i t i s l e s s d r a s t i c and i n v o l v e s the use of cellulysin. The p r o t o p l a s t s so i s o l a t e d appear to have t h e i r plasma membrane i n t a c t and produce ethylene from methionine and 1 - a m i n o - c y c l o p r o p a n e - l - c a r b o x y l i c a c i d (ACC), without p r i o r culturing. These p r o t o p l a s t s are very s e n s i t i v e to osmotic shock and suggest the importance of i n t a c t membrane s t r u c t u r e for ethylene b i o s y n t h e s i s . Once the membrane system i s damaged, the e t h y l e n e - s y n t h e s i z i n g c a p a b i l i t y i s l o s t . I t therefore appears that the system f o r ethylene b i o s y n t h e s i s , and perhaps a l s o the receptor s i t e s f o r ethylene, i s l o c a l i z e d i n membranes, p o s s i b l y the plasma membrane. This could account f o r a w e l l i n t e g r a t e d h i g h l y s t r u c t u r e d system l i n k i n g p r o d u c t i o n to a c t i o n and to other hormonal systems, whose receptor s i t e s have a l s o been a s s o c i a t e d with membranes (59). Conclusions Advances i n ethylene biochemistry and physiology have preceded along a number of f r o n t s . F i r s t l y the b i o s y n t h e t i c pathway from methionine to ethylene has been f u r t h e r c l a r i f i e d and intermediates i d e n t i f i e d . Secondly some progress has been made i n r e c o g n i s i n g two p o s s i b l e receptor s i t e s which are i n h i b i t e d by Ag ions and CO , r e s p e c t i v e l y . T h i r d l y the l o c a l i z a t i o n of ethylene production has been shown to be a s s o c i a t e d w i t h membranes i n s t u d i e s with p r o t o p l a s t s . There a l s o have been c l e a r i n d i c a t i o n s that i n t e r a c t i o n s of ethylene w i t h a u x i n s , c y t o k i n i n s , g i b b e r e l l i n s and ABA are i n v o l v e d i n both ethylene production and a c t i o n . G e n e r a l l y the e f f e c t s of ethylene tend to antagonize those of a u x i n s , c y t o k i n i n s and g i b b e r e l l i n s , and tend to r e i n f o r c e those of ABA, depending, however, on t i s s u e systems i n v o l v e d . Reinforcement of ethylene by ABA and v i c e v e r s a occurs more f r e q u e n t l y i n senescence. Although ethylene has been recognized mostly as an i n h i b i t o r of growth, e s p e c i a l l y of young t i s s u e s , there are examples that i t a c t s i n c o n j u n c t i o n with auxins and g i b b e r e l l i n s to enhance growth, e s p e c i a l l y i n water p l a n t s . Some, other experiments suggest that ethylene r e g u l a t e s morphogenesis at extremely low c o n c e n t r a t i o n s , i n the ppb range, which may i n f a c t be the n a t u r a l p h y s i o l o g i c a l concentrations i n many t i s s u e s . At higher c o n c e n t r a t i o n s , i n the ppm range, ethylene appears to be inhibitory. Because ethylene can e i t h e r i n h i b i t or s t i m u l a t e growth and development, depending on the t i s s u e type (18) and c o n c e n t r a t i o n , I suggest that ethylene modulates the a c t i o n of the s o - c a l l e d growth hormones—auxins, g i b b e r e l l i n s and cytokinins. Conversely a u x i n s , g i b b e r e l l i n s and c y t o k i n i n s appear to i n f l u e n c e ethylene p r o d u c t i o n and a c t i o n . These


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o b s e r v a t i o n s , which are f o r the most part o n l y at the d e s c r i p t i v e l e v e l of s o p h i s t i c a t i o n , i n d i c a t e that an under standing of i n t e g r a t e d i n t e r a c t i o n s between a l l hormones at the molecular l e v e l i s necessary i f the growth and development of crops i s to be f u l l y c o n t r o l l e d .


Literature Cited 1. Morgan, P.W.; Hall, W.C. Plant Physiol., 1962, 15, 420. 2. Abeles, F.B. Plant Physiol.,1966, 41, 585. 3. Burg, S.P.; Burg, E.A. Proc. Natl. Acad. Sci. USA, 1966, 55, 262. 4. Ketring, D.L.; Morgan, P.W. Plant Physiol.,1972, 50, 382. 5. Burg, S.P.; Burg, E.A. Plant Physiol.,1968, 43, 1069. 6. Chadwick, A.V.; Burg, S.P. Plant Physiol., 1970, 45, 192 7. Yang, S.F.; Pratt, H.K. In Biochemistry of Wounded Plant Storage Tissues, ed G. Kahl, 1978, de Gruyter, Berlin. In press. 8. Osborne, D.J. Sci. Prog., 1977, 64, 51. 9. Lieberman, M. Ann. Rev. Plant Physiol., 1979, 30, 533. 10. Adams, D.O.; Yang, S.F. Plant Physiol., 1977, 60, 892. 11. Adams, D.O.; Yang, S.F. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 170. 12. Rahiala, E . L . ; Kekomaki, M.; Janne, J.; Raina, Α.; Raiha, N.C.R. Biochim. Biophys. Acta 1971, 227, 337. 13. Lombardini, J . B . ; Coulter, A.W.; Talalay, P. Molec. Pharm., 1970, 6, 481. 14. Boller, T.; Herner, R.C.; Kende, H. Planta, 1979 (in press). 15. Mudd, S.H.; Biochim. Biophys. Acta., 1960, 38, 354. 16. Lieberman, M.; Baker, J . E . ; Sloger, M. Plant Physiol., 1977, 60, 214. 17. Zimmerman, P.W.; Wilcoxon, F. Contrib. Boyce Thompson Inst. 1935, 7, 209. 18. Morgan, P.W.; Hall, W.C.; Nature, 1964, 201, 91. 19. Abeles, F.B.Ann. Rev. Plant Physiol., 1972, 23, 259. 20. Lieberman, M.; Kunishi, A.T.; Plant Physiol., 1975, 55, 1074. 21. Kang, B.G.; Newcomb, W.; Burg, S.P. Plant Physiol., 1971, 47, 504. 22. Franklin, D.; Morgan, P.W.; Plant Physiol., 1978, 62, 161. 23. Steen, D.A.; Chadwick, A.V. Plant Physiol., 1973, 52, 171. 24. Lieberman, M.; Knegt, E. Plant Physiol., 1977, 60, 475 25. Michener, H.D.; Amer. J . Bot., 1938, 25, 711. 26. Valdovinos, J . G . ; Ernest, L . C . ; Henry, E.W. Plant Physiol., 1967, 42, 1803. 27. Lieberman, M.; Kunishi, A.T. In Plant Growth Substances, 1970, ed. D.J. Carr, 1972, Springer-Verlag, Berlin, New York, 549. 28. Osborne, D . J . , in Perspectives in Experimental Biology, ed N. Sutherland, 1976, 2: 89, Pergamon, Oxford. 29. Goldwin, G.K.; Wain, R.L. Ann. Appl. Biol. 1973, 75, 71. 30. Scott, P.C.; Leopold, A.C. Plant Physiol., 1967, 42, 1021.


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31. Stewart, R.N.; Lieberman, M.; Kunishi, A.T. Plant Physiol. 1974, 54, 1. 32. Fuchs, Y.; Lieberman, M. Plant Physiol., 1968, 43, 2029 33. Lau, O.L.; Yang, S.F. Plant Physiol., 1973,51,1011 34. Imaseki, H.; Kondo, K.; Watanabe, A. Plant Cell Physiol., 1975, 16, 777. 35. Richmond, A.E.; Lang, A. Science, 1957, 125, 650. 36. Lieberman, M.; Kunishi, A.T. Plant Physiol. Suppl. 1971, 47 22. 37. Gertman, E.; and Fuchs, Y. Plant Physiol. 1972, 50, 194. 38. Imaseki, H.; Pjon, C.J. Plant and Cell Physiol., 1970, 11, 827. 39. Ku, H.S.; Suge, H.; Rappaport, L; Pratt, H.K. Planta 1970, 90, 333. 40. Zobel, R.W.; Plant Physiol. 1973, 52, 385. 41. Zobel, R.W.; Roberts, L.W. Can. J. Bot., 1974, 32, 735. 42. Apelbaum, Α.; Burg, S.P. Plant Physiol., 1972, 50, 125. 43. Morgan, P.W.; Beyer, E.; Gausman, H.E. In Biochemistry and Physiology of Plant Growth Substances ed. F. Wightman and G. Setterfield, 1968, Runge, Ottawa, 1255-73. 44. Konings, H.; Jackson, M.B. Ann. Rep. Agric. Res. Council, Letcomb Lab., 1974, pp 23-24. 45. Meigh, D.F.; Norris, K.H.; Craft, C.C.; Lieberman, M. Nature, 1960, 186, 902. 46. Hanson, A.D.; Kende, H. Plant Physiol., 1976, 57, 538. 47. Elstner, E.F.; Konze, J.R. Nature, 1976, 263, 351. 48. Abdel-Kader, A.S.; Morris, L.M.; Maxie, E.C.Proc. Am. Soc. Hortic. Sci., 1968, 92, 553. 49. Imaseki, H.; Uritani, I; Stahmann, M.A. Plant Cell Physiol., 1968, 9, 757. 50. Saltveit, M.E.; Dilley, D.R. Plant Physiol., 1978, 61, 447. 51. Saltveit, M.E.; Dilley, D.R. Plant Physiol., 1978, 61, 675. 52. Aharoni, N.; Richmond, A. Plant Physiol., 1978, 61, 658. 53. Hall, M.A.; Kapuya, J.A.; Sivakumaran, S.; John, A. Pestic. Sci., 1977, 8, 217. 54. Sisler, E.; Wylie, P.A.; Plant Physiol. Suppl. 1978, 61, 91. 55. Beyer, E.M. Plant Physiol., 1975, 56, 273 56. Beyer, E.M. Plant Physiol., 1979 (In press). 57. Beyer, E.M. Plant Physiol., 1976, 58, 268 58. Mattoo, A.K.; Lieberman, M. Plant Physiol.,1977, 60, 794. 59. Hertel, R.; St. Thomson, K.; Russo, V.E.A. Planta, 1972, 107,325. RECEIVED

June

19,

1979.


Role of Ethylene in Plant Growth, Development, and Senescence

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