Shoot inversion-induced ethylene in pharbitis nil induces the release of apical dominance by restricting shoot elongation

Plant Science, 38 (1985) 163--172 Elsevier Scientific Publishers Ireland Ltd.

163

SHOOT INVERSION-INDUCED E T H Y L E N E IN P H A R B I T I S NIL INDUCES THE RELEASE OF APICAL DOMINANCE BY R E S T R I C T I N G SHOOT E L O N G A T I O N

T.K. PRASAD and M.G. CLINE

D e p a r t m e n t o f Botany, The Ohio State University, Columbus, OH 43210 (U.S.A.) {Received October 17th, 1984) (Revision received January 4th, 1985) (Accepted January 14th, 1985) Shoot inversion induces outgrowth of the highest lateral bud (HLB) adjacent to the bend in the stem in Pharbitis nil. In order to determine whether or not ethylene produced by shoot inversion plays a direct role in promoting or inhibiting bud outgrowth, comparisons were made of endogenous levels of ethylene in the HLB and HLB node of plants with and without inverted shoots. That no changes were found suggests that the control of apical dominance does not involve the direct action of ethylene. This conclusion is further supported by evidence that the direct application of ethylene inhibitors or ethrel to inactive or induced lateral buds has no significant effect on bud outgrowth. The hypothesis that ethylene evolved during shoot inversion indirectly promotes the outgrowth of the highest lateral bud (HLB) by restricting terminal bud (TB) growth is found to be supported by the following observations: (1) the restriction of TB growth appears to occur before the beginning of HLB outgrowth; (2) the treatment of the inverted portion of the shoot with AgNO3, an inhibitor of ethylene action, dramatically eliminates both the restriction of TB growth and the promotion of HLB outgrowth which usually accompany shoot inversion; and (3) the treatment of the upper shoot of an upright plant with ethrel mimics shoot inversion by retarding upper shoot growth and inducing outgrowth of the lateral bud basipetal to the treated region.

Key words: Pharbitis nil; apical dominance; shoot inversion; ethylene; bud outgrowth

Introduction Inversion of t h e upper shoot of P. nil results in the vigorous o u t g r o w t h o f the HLB adjacent to the bend in the stem (Fig. 1). The underlying mechanisms responsible for this release o f apical dominance are u n k n o w n [1] but it is possible that ethylene produced from shoot inversion could play a direct role either as a p r o m o t e r or as an inhibitor o f bud outgrowth. In 1968 it was suggested that auxin originating from t h e Pisum shoot apex might induce the nodal synthesis of ethylene which functions as an inhibitor of lateral bud growth in Abbreviations: ACC, 1-aminocyclopropane-l-carboxylic acid; AOA, (aminooxy)acetic acid; AVG, aminoethoxyvinyl glycine; HLB, highest lateral bud; TB, terminal bud.

apical dominance of upright plants [ 2 ] . However, this conclusion was seriously questioned when no decrease in ethylene production in de-scaled nodes was found following decapitation and when no release in apical dominance occurred in hypobaric conditions even though other s y m p t o m s of ethylene depletion were present [3,4]. Furthermore, it was claimed that direct applications of auxin to lateral buds o f Phaseolus resulted in growth rather than in inhibition [5]. However, on the basis o f recent determinations of endogenous ethylene levels in Pisum as well as inhibitor studies demonstrating the retarding effects of ethylene on bud growth, it has been argued that the auxin-induced ethylene inhibitor hypothesis should be re-examined [6]. In petunias [4] and p o t a t o e s [ 7 ] , ethylene

0168-9452/85/$03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

164

~G 'AL BUD HLB N (4th:

UPF RE,

(TB)

Fig. 1. T h e highest lateral bud of P. nil elongates and b e c o m e s t h e lead shoot due to t h e inversion of t h e upper p o r t i o n of t h e stem. The lowered t e r m i n a l bud is s h o w n near the base o f t h e plant.

treatments have been found to cause a release of apical dominance following ethylene removal. Applications of ethephon (an ethylene releasing agent) to ornamentals stimulates lateral bud outgrowth [8]. These and other similar findings have led to a proposal for a promotive role for ethylene in apical dominance [9,10]. It is well k n o w n that any chemical or physical treatment which causes a significant restriction of growth in a terminal (apical) bud (TB) will p r o m o t e the release of apical dominance in one of the lower lateral buds [ 1 1 , 1 2 ] . Inversion of the upper shoot of P. nil results in a restriction of growth in the terminal bud which appears to occur before the beginning of the lateral bud outgrowth [1,13]. Hence, a cause and effect relationship is possible. Since ethylene production is k n o w n to increase in tissue which is stressed [14] or gravistimulated [15--17] and ethylene treatment is k n o w n to inhibit growth, it is possible that shoot inversion induces

ethylene production which inhibits shoot elongation. The restriction of shoot growth m a y then bring a b o u t the release of apical dominance in the HLB adjacent to the bend in the stem. In Phaseolus it has been demonstrated that shoot enclosure b y glass results in an increase in ethylene evolution, a restriction of growth, and the subsequent outgrowth of the lateral bud basipetal to the enclosure [11 ]. Among possible explanations, an indirect role for ethylene has been suggested which involves ethylene-induced restriction o f growth in the terminal bud causing a diversion of nutrients from the terminal bud to the basipetal lateral bud which results in the o u t g r o w t h of the latter bud. In the present study we have carried out experiments to determine whether ethylene might play a direct inhibitory or promotive role in shoot-inversion release of apical dominance in t h e HLB of P. nil. We have also carried o u t experiments to determine whether ethylene might act indirectly to p r o m o t e outgrowth of the HLB by restricting TB growth. It should be noted that the term 'terminal bud' or 'TB' as is used here refers not only to the apex o f the main shoot but also to the region of growth, 10--15 cm behind the apex. Materials and m e t h o d s

Seeds of P. nil (L.) Choisy (strain Violet) from Marutane Co. Ltd., K y o t o , Japan were germinated in sandy loam soil at 26--31°C. The plants were grown under continuous light (cool white fluorescent and incandescent lamps; 90--850 E m -2 s-'). Shoot inversion experiments were started with 19- to 28day-old plants. After a treatment was initiated by inverting or decapitating the upper portion of the shoot {usually above node 4), daily measurements o f bud and/or shoot lengths (between HLB node and shoot apex) were made with a ruler. The diameter of the bend in the stem near the HLB was about 3 or 4 cm. In order to keep the elongating inverted

165

shoot apex from growing back up the main shoot, it was necessary, on nearly a daily basis, to retie the shoot apex to the stake adjacent to the lower portion of the shoot. Ethrel (2-chloroethyl phosphonic acid) dissolved in 0.05% (v/v) Tween 20 at 5 to 100 p.p.m., was applied in 0 . 2 - o r 0.3-ml portions to the HLB with a c o t t o n wad [5]. Aminoethoxyvinyl glycine (AVG) (0.05 or 4mM) was applied similarly. In other experiments ethrel (5 or 100 ppm), 1-aminocyclo propane-l-carboxylic acid (ACC) (0.5 mM), COC12 (0.2 raM), AgNO3 (2 mM), (aminooxy)acetic acid {AOA) (1 m M ) a n d L-canaline (1 mM) were applied in 4-~1 drops directly to the HLB twice (on the 1st day and 3rd day). Ethrel (25 ppm), ACC (0.5 mM), AgNO3 (0.5 mM), AVG (0.1 mM), L-canaline (1 mM}, CoC12 (0.2 mM), AOA (1 mM), and mimosine (1 mM) in 0.05% Tween 20 were also applied once as a spray or with a small brush on 3 successive days to the surface of all tissues in the upper shoot (above the 4th node). Most experiments were repeated at least twice. Ethylene determinations o f 1-ml samples were made using a Hewlett-Packard gas chromatograph with a flame ionization detector as described [16] except that ethylene was allowed to accumulate in a 10-ml vial for 2 h instead of 15 min. Moist filter paper was enclosed with tissue to prevent drying. Stem segments (2.5 cm), whole leaves and other plant parts were enclosed in 10-ml vials for analyses. Initially, determinations o f ethylene emanation o f HLB nodal sections were carried o u t only after the axillary bud (HLB) had been removed in order to avoid possible ethylene contribution by the HLB to the nodal section [5]. However, after it had been determined that the presence of the HLB had no such effect, the remaining nodal determinations were done w i t h o u t HLB removal. All determinations were repeated at least twice. Results If ethylene acts to directly p r o m o t e lateral

bud outgrowth, then the endogenous concentration of ethylene in the HLB node and the HLB should increase following shoot inversion. The results in Fig. 2, however, show that ethylene evolution did not increase in these tissues over a 48-h period following shoot inversion or decapitation. Moreover, when ethrel (an ethylene releasing c o m p o u n d ) is added directly to inactive lateral buds of upright plants, there is no induction of bud outgrowth (Table I). Furthermore when a variety of ethylene inhibitors are applied directly to an induced HLB (i.e. HLB is induced to elongate b y shoot inversion or by decapitation), the bud outgrowth is not significantly inhibited except in the case of AVG [18] at the high concentration of 4 mM (Table I) which may inhibit protein synthesis [ 19]. If auxin-induced ethylene maintains apical dominance b y directly inhibiting lateral b u d outgrowth and if such outgrowth following shoot inversion is due to a depletion of ethylene in the HLB node and in the HLB, it should be observed that ethylene evolution decreases in these tissues under these conditions. However, the results clearly demonstrate that this does not occur (Fig. 2). The

'.c 'E

SHOOT

INVERTED

v

Z

CONTROL 0 4l INVERTED

o I-_J

o

~> LLI w Z LLI _1 >"r I-LLI

~

HL B ~ z ~ - ~ C O N T R O L

SHOOT

DECAPITATED

~ r - - ~ = ~ = ~ HLB NODE

C 0 NT R0 L DECAP

' ,'2 ' 2',, ;6 HR AFTER INVERSION/DECAP

Fig. 2. E f f e c t o f s h o o t inversion o r d e c a p i t a t i o n o n e t h y l e n e p r o d u c t i o n in t h e HLB and H L B n o d e . T h e HLB was l o c a t e d at the 4th node. Each point repres e n t s an average o f 29 b u d s (HLB) or 9 HLB n o d e s .

Control

UR H20 7 3 0.1-+0

Control

DC H20 7 3 31.3±5.9

Control

DC H~O 6 4 9.1 +_ 1.9

T

O C D N G

T

O C D N G

T

O C D N G

DC 0.05 m M 6 4 6.8 +_ 0 . 8

AVG

DC 100 p p m 7 3 7.6_+12.3

Ethrel

UR 5 ppm 7 3 0

Ethrel

DC H20 7 3 31.3 + 5.9

Control

UR H20 6 4 0

Control

IV H~O 6 4 3.2+_1.6

Control

DC 4 mM 7 3 10.4 +_ 1.8

AVG

UR 0.05 m M 6 4 0

AVG

IV 5 ppm 6 4 2.8_+0.8

Ethrel

IV H20 9 4 25.0 + 2.1

Control

UR H20 7 3 0.1+-0

Control

IV 25 p p m 6 4 2.2_+2.1

Ethrel

IV 1 mM 9 4 23.0 + 3.1

Canaline

UR 4 mM 7 3 0.1 +-0

AVG

DC H~O 6 4 7.2+1.9

Control

IV 2 mM 9 4 24.5 + 3.0

AgNO3

IV H~O 6 4 2.3_+0.8

Control

DC 5 ppm 6 4 6.5+2.1

Ethrel

IV H20 9 4 33.0 + 4.1

Control

IV 0.05 m M 6 4 2.1+_0.2

AVG

DC 25 p p m 6 4 7.0+_2.2

Ethrel

IV 0.2 m M 9 4 31.1 +_ 3.3

CoCI 2

IV H20 7 3 3.6+0.6

Control

IV H20 7 3 3.6+_0.6

Control

IV 1 mM 9 4 32.5 + 3.0

AOA

IV 4 mM 7 3 2.1-+1.1

AVG

I~/ 100 p p m 7 3 0.8+_0.1

Ethrel

T, t r e a t m e n t ; O, s h o o t o r i e n t a t i o n ; C, c o n c e n t r a t i o n ; D, days; N, n o d e ; G, b u d g r o w t h ; UR, u p r i g h t p l a n t ; IV, p l a n t w i t h i n v e r t e d u p p e r s h o o t ; DC, d e c a p i t a t e d p l a n t .

T a b l e I. Mean g r o w t h (cm _+ S.D.) of HLB 6--9 days (as i n d i c a t e d ) following s h o o t i n v e r s i o n or d e c a p i t a t i o n . HLB was l o c a t e d at n o d e as specified. T h e s h o o t o f t h e u p r i g h t p l a n t was n o t i n v e r t e d or d e c a p i t a t e d . Chemicals were a p p l i e d in 0.2- o r 0 . 3 - m l p o r t i o n s w i t h a c o t t o n wad or in 4-ul d r o p s as described in Materials a n d m e t h o d s .

O~

167

rate of ethylene production in the buds and nodes remains essentially unchanged over a 48--96-h period following shoot inversion or decapitation. The direct application of the ethylene synthesis inhibitor, AVG, to inactive lateral buds of upright plants does not promote lateral bud outgrowth as might be expected if ethylene were functioning in an inhibitory role (Table I). The treatment of induced HLB's by ethrel does not inhibit bud outgrowth in Pharbitis except at high and possibly toxic concentrations (>25 ppm). ACC (0.5 raM), the precursor of ethylene, had no effect (data not shown). Following shoot inversion, ethylene production significantly increases in the bent and in the inverted regions of the shoot within 12 h (Fig. 3). The peak is reached within 48 h and the rate declines thereafter. Ethylene production in various plant parts of the inverted shoot shows a similar trend: the buds are highest, the leaves and nodes somewhat less, and the internodes, the least (Table II). The ethylene emanation of the plant parts in upright plants follows the same order but is always less than that of the corresponding parts from inverted shoots. From the top to the bottom of the shoot there is a gradual decrease in ethylene emitted from nodes and internodes (Fig. 4). It is probable that ethylene production occurring in the bent region of the inverted stem results from a different kind of stress than does ethylene evolution occurring in the inverted region of the shoot. Since the bent region of the stem consists of upward curving and downward curving sides, these two parts were separated as equal lengths and ethylene production was determined (Fig. 3B) to see if the downward side might contribute disproportionately to the total amount of ethylene evolved in the whole bent region of the stem. The ethylene production in the downward side was 50% greater than that in the upward side. The removal of ethylene from the inverted region of the shoot should eliminate the

2:: Iol

4

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o I-..J

I

o

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6

i

l

I

I

l

I

I

1

l

I

, I

I

I

L

B.

2

l

0

24

48

HR. A F T E R S H O O T

72

l

96

INVERSION

Fig. 3. E t h y l e n e e v o l u t i o n in (A) inverted region and (B) b e n t region of stem (see Fig. 1). Upright c o n t r o l , o - - - - - o ; inverted region, o o ; b e n t region (total), • *; b e n t d o w n w a r d s , ~ ~; b e n t upwards, A A S t e m segments used for e t h y l e n e d e t e r m i n a t i o n s in (A) consisted of segments taken f r o m 15-cm inverted region just below b e n t region excluding apex. 2.5-cm segments were used in (B) for the 5-cm b e n t region. Data points are means +- S.D.

presumed TB growth-inhibiting effect of ethylene. If restriction of shoot elongation is somehow causing HLB outgrowth, then the elimination of this restriction should also eliminate HLB outgrowth. Various ethylene inhibitors were applied with a brush to the entire inverted portion of the shoot at the time of inversion. AVG and L-canaline [20], ethylene synthesis inhibitors, slightly promoted the TB growth rate over that of the control (Fig. 5A) and partially inhibited HLB outgrowth (Fig. 5B). Other ethylene synthesis inhibitors (COC12 [21], AOA [22], and mimosine) had little or no effect (data not shown}. AgNO3 (0.5 mM) which inhibits

168 Table II. Ethylene production (nl g-~ h -~) in various plant parts. UR, upright shoot; IV, inverted shoot.

Odentation

h following shoot inversion ethylene production 0

12

24

48

96

UR IV

3.6 3.7

3.8 7.1

3.4 7.1

3.7 6.9

4.0 6.8

UR IV UR IV

2.3 2.5 2.1 2.1

2.2 4,7 2,1 3.5

2.4 6.2 1.8 4.1

1.9 6.8 1.9 4.3

1.4 4.9 1.4 3.1

Buds

Node 5

Nodes

Node 6 Node 5

ethylene action [23], dramatically promoted TB growth and almost completely inhibited H L B o u t g r o w t h ( F i g . 5). E t h r e l a n d A C C a c c e n t u a t e d t h e i n h i b i t i o n o f T B g r o w t h in the inverted shoot and promoted outgrowth of the HLB over that of the controls. When the upper portion of the shoot (above the 4th node} of an upright plant was coated or painted with ethrel (an ethylenereleasing compound) at a concentration of 25 ppm or higher, the growth of the upright TB was significantly inhibited (Fig. 6A) and

24 1

Leaves

Node 6 Node 5

UR IV UR IV

2.1 2.3 1.7 1.9

2.8 5.2 1.5 3.5

2.8 6.4 1.3 3.6

2.4 6.6 1.4 3.8

2.5 6.1 1.4 2.8

UR

1.3

1.4

1.2

1.0

0.8

IV

1.3

2.5

2.7

4.4

3.6

UR IV

1.8 1.7

1.6 2.4

1.8 2.6

1.7 2.9

1.9 3.1

"~

A.

18 ~

AgN03

Internodes

Between Nodes 5 and 6 Shoot apex

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~: rr

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NODES r-] INTERNODES I~1

2

j

30

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TROL

10

,,_J

> WT LLI ~ z~

"x~A NA L I N"-F""-A V G

ACC~X'~O _ N~

0

.3ANAL IN E AgNOa

X

9 DAYS AFTER SHOOT INVERSION

1

LU'-" ..J >"r" LU

,

L , ~ ~3 ~ ; ~6~ ~

¢,

ibl

;~.c,, ,,d I ,e, If,

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Fig. 4. Ethylene production in nodes (a, c, e, g, h, i, j) (open bars) and internodes (b, d, f) (hatched bars) of upright plant, j is the cotyledonary node.

Fig. 5. Growth rate of (A) lowered terminal bud and (B) HLB at various times following shoot inversion. Upright c o n t r o l , • *; inverted control, o ~; AgNO~ (0.5 raM), × × ; AVG (0.1 raM), .3 :,; L-canaline (1 raM), • *; ethrel (25 ppm), • • and ACC (0.5 mM), ~ c. The entire i,verted portion of the shoot was treated with ~b~, : , . ~ , i~,J, chemical.

169

TB

¢/)3 0

GROWTH

HLB

e.

1o

GROWTH

loooo=/ /

¢o

v t,.)

20 d

ppm

I.u I-

30

20

/

n3I-

10

10

0 n-

/jr,0

0 0

3

6

9

0

3

6

9

DAYS AFTER ETHREL APPLICATION

Fig. 6. Growth rate of (A) upright terminal bud and (B) highest lateral bud (at 4th node) following treatment of upper shoot (above 4th node) with various concentrations of ethrel. Control, G o; 5 p.p.m., × ×; 25 ppm, ~, A; 50 ppm~ • =; 100 ppm, • •.

t h e lateral b u d basipetal t o t h e e t h r e l - t r e a t e d p o r t i o n o f t h e s h o o t grew o u t in a m a n n e r very similar t o t h a t o f H L B g r o w t h f o l l o w i n g s h o o t inversion (Fig. 6B). A n analysis o f e t h y l e n e p r o d u c t i o n in s t e m s e g m e n t s o f l o o p e d plants s h o w s n o signific a n t c h a n g e in t h e H L B o r H L B n o d e b e f o r e o r a f t e r l o o p i n g (Table III, Fig. 7). T h e t o t a l l o o p e d p o r t i o n o f t h e stem tissue d o e s e m i t

m o r e e t h y l e n e on a p e r gram o f fresh tissue basis t h a n does t h e e q u i v a l e n t p o r t i o n o f an upright stem b u t less t h a n t h a t o f t h e b e n t region o f an inverted s h o o t . T h e u p w a r d o r i e n t e d TB

Table III. Ethylene production (nl g-~ h - ' ) in buds and stems of upright, inverted and looped plants 24 h following shoot manipulation (see Fig. 7). The inverted shoot contains the bent region of the stem. TB region of stem: top 15 cm of shoot, above loop.

HL=

TB TB

iX

BEND i

H,B ,( HER

LOOP

Shoots et.hylene production Upright

HLB HLB Node Upright/bent/looped region of stem TB region of stem

5.3 + 1.2 1.9 +_0.3

Inverted

---

Looped

5.1 +_0.9 1.7 +- 0.4

1.0+_0.1 4.4+_0.4 2.4+0.3 1.9 +_0.2 -2.1 "!"-0.4

A.

B.

C.

Fig, 7. A, upright plant; B, plant with inverted upper shoot; C, looped plant; X, control for bent or looped regions of stem. See Table III.

170

of the looped plant evolves only slightly more ethylene from the t o p 15 cm of the shoot than does the TB of the non-looped upright control. Discussion The fact that the endogenous levels of ethylene in the HLB node and in the HLB of Pharbitis do not change following shoot inversion together with the fact that ethylene inhibitors and ethrel have essentially no effects when applied directly to inactive or induced lateral buds demonstrate that ethylene functions neither as a direct p r o m o t o r or as a direct inhibitor of HLB o u t g r o w t h . That AgNO3, the remarkably effective inhibitor of ethylene action, does not inhibit HLB outgrowth when added directly to the bud, provides convincing evidence that ethylene given o f f during shoot inversion does not directly cause HLB outgrowth (Table I}. The argument that ethylene acts as a direct inhibitor to bud outgrowth in Pisum based on data that ethylene inhibitors double bud o u t g r o w t h in decapitated plants [6] would be more persuasive if it could be demonstrated that these inhibitors also would p r o m o t e outgrowth of inactive lateral buds in intact plants. Since the effects of the various treatments on Pharbitis with regard to apical dominance were generally the same for decapitated plants as for inverted shoots, it would seem that the control mechanisms for the maintainance and release o f apical dominance in these t w o treatments also would be similar in m a n y respects. In other plant systems, there have been t w o reports (Pisum, [6] ; Phaseolus, [5] ) of decreases of ethylene production in nodes following decapitation, one report of no change (Pisum, [3] ) and no reports of increases. However, in Phaseolus, even though decapitation causes a general decline in ethylene evolution which is consistent with an inhibitory role for ethylene, the addition of auxin to the stump does n o t increase subsequent ethylene production in the node which should occur if the ethylene is auxin-

induced [5]. Although the horizontal positioning o f oat shoots induces both ethylene production and titler rekease, IAAinduced ethylene is not thought to control apical dominance in this system [16]. The question also has been raised as to whether there is sufficient auxin in the Pisum shoot to induce the synthesis of enough ethylene to inhibit bud overgrowth as required in apical dominance [3]. The results of the present study appear to support the hypothesis that ethylene evolved during shoot inversion indirectly releases apical dominance b y inhibiting terminal bud growth in Pharbitis. Data are consistent with the following sequence of events: shoot inversion -* ethylene production -~ restriction o f terminal bud g r o w t h ~ outgrowth of highest lateral bud. The evidence presented here for significantly increased ethylene production in the inverted portion of the Pharbitis shoot during the first 12--24 h after shoot inversion is strong. In etiolated pea stems, ethylene strongly inhibits shoot elongation within 15 min [4]. The fact that there is a rapid drop in terminal bud elongation and vigorous outgrowth of the highest lateral bud in Pharbitis following shoot inversion are well d o c u m e n t e d [1]. Shoot elongation occurs as far back as 10--15 cm behind the TB in the upright plant (data not shown}. Preliminary data which indicate that terminal bud growth decreases within a 24-h period and that the presentation time for HLB outgrowth is 24--36 h [13] are consistent with, but do not prove, the hypothesis that restriction of TB growth causes HLB outgrowth. A more precise determination of the kinetics of these t w o responses would be very helpful in the analysis o f possible cause and effect relationships. The dramatic effect of AgNOa, the ethylene action inhibitor, on eliminating b o t h the restriction of terminal bud growth and HLB outgrowth which normally accompany shoot inversion is strongly supportive of the indirect ethylene hypothesis. Why AVG and L-canaline

171

have only partial effects and the other ethylene synthesis inhibitors appear to have no effects could be due to a variety of causes. Inadequate penetration could be a problem. Some inhibitors simply may be ineffective in causing total inhibition of ethylene synthesis. Perhaps sufficiently high concentrations of the inhibitor were not employed. When high concentrations of AVG (0.5 mM), COC12 (1 raM), AOA (2 mM), and mimosine (10 mM) were used, toxic effects on the plants were observed. However, the fact that ethrel application to the upper portion of an upright plant mimics so precisely the shoot inversion response (i.e. in restricting TB growth and promoting lateral bud outgrowth) also provides strong support for the hypothesis of indirect ethylene promotion of bud outgrowth. The fact that apical dominance is not usually released by the looping of the upper shoot [ 1] and the fact that there is no change in ethylene emanation in the HLB or HLB node demonstrate quite clearly that ethylene emitted from the stress of bending (i.e. from the bent region) in shoot inversion cannot be responsible for direct promotion of HLB outgrowth. Also, the fact that the high rate of elongation of the upright TB above the loop is correlated with low ethylene production from this region of the shoot provide additional support for the indirect ethylene promotion hypothesis. Many questions remain. How might shoot inversion stimulate ethylene synthesis? Perhaps statoliths falling on cytoplasmic membranes may indirectly activate the ACC synthase system via deformation of membrane structure. How does the ethyleneinduced restriction of TB growth result in HLB outgrowth? Does the restriction of TB growth cause a diversion of nutrients from the TB to the HLB thus triggering its outgrowth [10]? Or could HLB development be induced by the depletion of auxin in the shoot via inhibiting effects of ethyleneinduced restriction of growth on auxin synthesis and/or transport [10]? Or might

ethylene be inhibiting auxin synthesis and/or transport independent of its effects on growth? Perhaps gravity may act synergisticaUy with ethylene to inhibit auxin transport and thereby accentuate the depletion of auxin in the HLB. On the other hand, gravity inhibition of auxin transport from the TB might cause auxin to accumulate so as to induce ethylene synthesis which may then inhibit TB growth. Our present data do not discriminate between the nutrient diversion and auxin depletion hypotheses. In any case the shoot inversion-induced ethylene exposure of the TB appears to be equivalent to a slow decapitation of the TB in as much as both the metabolic sink and the source of auxin in the TB would seem to be diminished when the shoot is inverted [4]. The conclusions of the foregoing discussion must be tempered by our recent knowledge that plant responses to gravity can no longer be interpreted on the basis of a simple redistribution of auxin. Other factors such as tissue sensitivity, conjugation of the auxin molecule, interactions with other growth substances, and asymmetric movement of calcium and hydrogen ions may be of considerable influence. It should also be pointed out that although there often seems to be an inverse correlation between TB and HLB growth, this is not always the case (unpublished data and Ref. 11). Alterations in nutrient supply and in the levels of photosynthates (as influenced by variation in light i~ntensity) can affect this relationship. In summary, no evidence was found to support the hypothesis that shoot inversioninduced ethylene controls HLB outgrowth in P. nil by acting directly as a promoter or as an inhibitor. Strong circumstantial evidence has been presented to demonstrate that shoot inversion-produced ethylene in Pharbitis does promote the release of apical dominance by restriction of TB growth. However, unequivocal evidence has not been obtained to prove that growth restriction is actually due to ethylene action. More importantly, the

172

precise mechanisms by which ethylene and/or ethylene induced growth restriction trigger HLB outgrowth have yet to be elucidated.

Acknowledgement This work was supported in part by a grant from the National Aeronautics and Space Administration.

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