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Initiation of response in aleurone layers by gibberellic acid
BIOCHIMICA ET BIOPHYSICA ACIA
501
BBA 26443
I N I T I A T I O N OF RESPONSES IN A L E U R O N E L A Y E R S BY G I B B E R E L L I C ACID C L I F F O R D J. P O L L A R D
Department of Botany and Plant Pathology, Michigan State University, East Lansing, Mich. 48823
(u.s.A.) (Received J u n e 9th, 197 o)
SUMMARY
A new procedure for studying the action of gibberellic acid in barley (Hordeum vulgare cv. Betzes) aleurone layers was utilized. Exposure of the layers to gibberellic acid for short periods of time followed by thorough washing and subsequent incubation in the absence of the phytohormone resulted in increased secretion of enzymes and metabolites. Increased secretion of soluble sugar (a typical hormonal response) b y such treated and washed layers was linearly proportional to the time of exposure as well as to the concentration of hormone ill the exposure medium. It was decreased b y exposure under anaerobic conditions and was affected by the temperature of exposure. The hormone disappears from the medium during exposure but the results suggest that some of it can be removed from the layers by washing. Since cycloheximide and abscisic acid inhibited hormone-induced secretion when they were applied simultaneously and continuously with gibberellic acid but had no effect on pretreated layers whereas secretion was inhibited by anaerobiosis in both instances, at least two steps appear to be involved in hormonal action on secretion.
INTRODUCTION
The major findings from earlier studies on the action of gibberellic acid in cereal aleurone layers have been reviewed 1-3. The identity of m a n y of the metabolites and enzymes whose secretion is increased after application of the hormone to the layers, the sequence of these effects and their relationship to certain other aspects of cellular metabolism of the tissue were reported recently*, 5. Briefly, the earliest response it was possible to detect, increased secretion of soluble carbohydrate, was affected as early as 2 h after application of gibberellic acid; the latest, increased synthesis and secretion of amylase, was realized after 7 h of incubation. A variety of phosphatases and carbohydrases were affected at intermediate periods. Aside from their intrinsic value the results of the latter studies emphasize the complexity of the action of gibberellic acid in these tissues. It would appear axiomatic therefore that no single approach will likely lead to discerning the mode of action of the hormone; the desirability of having a variety of approaches available for the conduct of such studies is clear. This paper reports a novel procedure for studying some aspects of the mode of action of gibberellic acid. Experimentally this involves Biochim. Biophys. Acta, 222 (197 o) 5Ol-5O 7
502
c . J . POLLARD
exposing barley (Hordeum vulgate ev. Betzes) aleurone layers to the growth factor for short periods, extensive washing to remove excess hormone followed by incubation of the tissue in the absence of gibberellic acid and subsequent observation of the responses. The effects of certain inhibitors when incubated simultaneously and continuously with gibberellic acid, in contrast to their action on tissue pretreated with the hormone, will also be given. M A T E R I A L S AND METHODS
Since the conditions for the preparation of the layers, the materials as well as the methods used here have been described 4, 5, only the procedure used for washing will be given in detail. The layers were exposed to gibberellic acid (i #g/ml unless otherwise noted) with gentle shaking. They were then washed with ice cold 0.02 M CaCI~ although in more recent experiments cold distilled water has been used with equal apparent success. Three washing procedures have been used: (a) layers were shaken vigorously by hand in 250 ml glass stoppered erlenmeyer flasks with 50-200 ml of solution for 3 min, the solution decanted and the washing repeated for more than 20 times (b) layers were shaken for 2 h in a mechanical shaker in flasks as above, the solution being changed every 15 min and (c) layers were placed in 15oo ml of cold washing medium contained in a 2 1 flask in a cold room at 3 ° and stirred with a magnetic stirrer for 12-14 h, the medium being changed occasionally. The tissue was then incubated in 0.02 M CaC12 containing 250 big streptomycin sulfate per ml while being shaken on a mechanical shaker at 200 strokes per min. I n general, assays were done on aliquots of the incubation medium as given previously. Amylase activity was measured here, however, as given by CHRISPEELS AND VARNER 2. The bioassay for gibberellic acid was conducted as given by COOMBE et aL e except that total sugar secreted was measured rather than reducing sugar. All experiments were repeated to establish their validity. RESULTS
Evidence that a continuous supply of gibberellic acid is not needed to elicit responses Ordinarily, increased sugar secretion is observed in tissue treated continuously with the hormone after a lag of 2-4 h. An indication that tissues do not require continuous treatment with gibberellic acid is given in Table I. It can be observed that layers that were exposed to gibberellic acid for 4 h, washed thoroughly and incubated subsequently in the absence of the hormone secreted as much sugar over a 7 h period as tissue that was so treated and reincubated with gibberellic acid. After 21 h, however, more sugar was secreted b y the latter tissue. The data in Table I I illustrate the effect of washing on layers exposed to gibberellic acid for only 6 min. Note that this exposure (o washes) caused the layers to secrete 3-4 times as much soluble carbohydrate and the phosphatases as controls. It also caused the layers to secrete amylase. Washing the layers caused a progressive reduction in the amounts of carbohydrate and phosphatases secreted but 40 washes were no more effective than 20. It was shown in other experiments, however, that IO washes were less effective than 20. Of singular interest is the finding that no amylase was produced in layers that were washed more than 5 times. Note also that the layers Biochim. Biophys. Acta, 222 (197 o) 5 O l - 5 O 7
INITIATION OF RESPONSES BY GIBBERELLIC ACID
503
TABLE I EFFECT OF TRANSFER TO G I B B E R E L L I C
OF GIBBERELLIC
ACID TREATED
ALEURONE
L A Y E R S TO C O N T R O L S O L U T I O N OR
ACID SOLUTION ON THE SECRETION OF SOLUBLE CARBOHYDRATE
Treated layers were exposed to gibberellic acid for 4 h, removed and washed for 2 h with cold o.o2 M CaClz and subsequently placed into 7.5 ml of solutions. Groups contained 15 layers. Values for soluble carbohydrate are/zg glucose equivalent (anthrone) per 0. 5 ml. Incubation time
(h)
I 4 7 21
Soluble carbohydrate secreted Control to control
Treated to control
Treated to gibberellic acid
22 45 -168o
61 41° 2ioo 8470
49 384 218o 112o0
T A B L E II EFFECT
OF NUMBER
OF W A S H E S G I V E N G I B B E R E L L I C
ACID TREATED
L A Y E R S ON R E S P O N S E S
Fifty layers were exposed to 5 ° ml of gibberellic acid solution for 6 rain. They were then washed with 200 ml portions of cold 0.02 M CaCI~ by shaking by hand for 3 rain each time. At the n u m b e r of washes indicated io of the layers were removed. Controls were not treated with gibberellic acid b u t were washed for 4 ° times. All layers were then incubated in io ml CaC12 solution containing streptomycin sulfate for 16 h. Carbohydrate is total /~g glucose equivalents; amylase activity is ~de~0mt, x lO3 per io rain per o.2 ml. Phosphodiesterase activity (with bis-p-nitrophenyl phosphate) is Z]d410m~ × lOs per IO mill per O.2 ml and ATPase activity is dds00mv × lO3 per h per o. 5 ml. Washes
Control (no gibberellic acid) o 5 X 2o X 4° × 4 ° X + gibberellic acid
Amount or activity secreted Carbohydrate
Amylase
Phosphodiesterase
A TPase
2650 I 1 300 8640 6750 7000 12ooo
o 74 ° o o o 860
215 700 37 ° 24o 25 ° 77 °
18o 61o 485 44 ° 455 680
e x p o s e d t o g i b b e r e l l i c a c i d f o r 6 m i n a n d w e r e n o t w a s h e d s e c r e t e d m o r e t h a n 85 % a s m u c h of t h e m a t e r i a l s as w a s s e c r e t e d b y l a y e r s w a s h e d 40 t i m e s a n d t h e n t r e a t e d w i t h t h e h o r m o n e c o n t i n u o u s l y . T h e r e s p o n s e of t h e l a t t e r t i s s u e s u g g e s t s t h a t w a s h i n g d i d n o t i n t e r f e r e w i t h t h e c a p a c i t y of t h e t i s s u e t o r e s p o n d , as f o r e x a m p l e b y d e n a t u r a t i o n of a c t i v e p r o t e i n s o r b y r e m o v a l of o t h e r f a c t o r s . A n e x t e n s i o n of t h i s line of r e a s o n i n g a l l o w s for t h e s u g g e s t i o n t h a t s o m e h o r m o n e is r e m o v e d p r o g r e s s i v e l y f r o m t h e layers by the first few washes. That a linear relationship existed between the time layers were exposed to g i b b e r e l l i c a c i d a n d t h e a m o u n t of s o l u b l e s u g a r s e c r e t e d s u b s e q u e n t l y is i l l u s t r a t e d i n Fig. i . T h e c o n t r a s t i n g p a t t e r n of loss of h o r m o n e f r o m t h e m e d i u m , w h e r e i n a b o u t h a l f of t h e h o r m o n e h a d b e e n l o s t w i t h i n 30 m i n r e s e m b l e s a s a t u r a t i o n c u r v e . S i m i l a r l y , a r a p i d loss of t r i t i a t e d g i b b e r e l l i n A 1 ( k i n d l y s u p p l i e d b y D r . H a n s K e n d e ) 9 f r o m t h e m e d i u m h a s also b e e n o b s e r v e d . F o r e x a m p l e , 64 a l e u r o n e l a y e r s w e r e i n c u b a t e d w i t h 32 m l of t r i t i a t e d g i b b e r e l l i n A 1 of f i n a l c o n c e n t r a t i o n of I . I O - e M . Biochim. Biophys. Acta, 222 (197 o) 5Ol-5O7
504
c . J . POLLARD
T h e r a d i o a c t i v i t y of t h e solution initially was 126oo c o u n t s / m i n per ml. A t intervals 2 layers an d I ml of solution were r e m o v e d and an aliquot counted. A f t e r I, 2, 5, IO, 30 an d 12o m i n i n c u b a t i o n 3800, 4000, 4400, 5400, 7000 and 734 ° counts/rain per ml, r e s p e c t i v e l y h a d disappeared from t h e m e d i u m .
5.0
suG.R sEc 4.0
~=3.o
I
, .....- / . . . . . . . . .
:,~/ i
]5.0
"y i x
....
GA LOSSFROMMEOIUM
1.21 lib 111|,25°
~
4.0
l=
3.0!
~ a.l=
2.0
5.11 .17 o °iiAENIIIC,2} ~
_.
2.50
2.0
~ hO
SUGARSECRETI ' ON-CONTROL I
i
I
i
tl.0 i
30 60 90 120 EXPOSURE TIME (minutes)
1.25
oi,,
,'.I
I
¢,
ii,
6A CONCEHI|ITilB (aitfilraat/ill
Fig. I. Effect of exposure time to gibberellic acid (GA) on the secretion of sugar by layers and on the loss of gibberellic acid from the medium. Forty layers that were exposed to 20 ml of control and of gibberellic acid solutions were shaken and io layers and 5 ml portions removed at the intervals noted. All layers were then washed for 2 h in cold CaC12 solution. They were then incubated in io ml solution for 6 h. The gibberellic acid solutions used for exposures were bioassayed for gibberellic acid remaining by measuring sugar secreted by barley half seeds. Allowances were made for sugar secreted by layers during exposure. Fig. 2. Effect of gibberellic acid (GA) concentration of the medium, of temperature and of anaerobiosis during exposure on subsequent secretion of soluble sugar. Points on the line obtained by exposing io layers to 20 ml of gibberellic acid of concentration indicated for I h, Other points represent values obtained from layers exposed to solutions containing i /~g gibberellic acid per ml at the temperatures indicated or under reduced pressure in a desiccator at 25 ° (anaerobic). Washing of layers and incubation same as Fig. I.
Fig. 2 shows t h a t t h e a m o u n t of sugar secreted was d e p e n d e n t on t h e concent r a t i o n of gibberellic acid in t h e exposure m e d i u m , IOO/zg/ml being inhibitory. Th e t e m p e r a t u r e during exposure was critical; layers exposed to I # g gibberellic acid per ml at 37 ° an d o ° secreted sugar in a m o u n t s e q u i v a l e n t to c o n c e n t r a t i o n s of exposure of o.I a n d o . o i /zg/ml, r e s p e c t i v e l y at 25 °. Also, layers exposed u n d e r anaerobic conditions to I /zg/ml secreted sugar in an a m o u n t e q u i v a l e n t to w h a t w o u l d h a v e been secreted b y layers exposed to approx. 0.075/zg gibberellic acid per ml, aerobically.
Effect of inhibitors T h e actions of a c t i n o m y c i n D, c y c lo h e x i m id e and abscisic acid, when i n c u b a t e d c o n t i n u o u s l y w i t h t h e hormone, on a m y la s e synthesis h a v e been r e p o r t e d 4. Tab l e I I I gives t h e effect of these on gibberellic acid in d u ced sugar secretion when applied at t h e beginning of th e e x p e r i m e n t . U n d e r these conditions c y c l o h e x i m i d e is a p o t e n t i n h i b i t o r ; the a m o u n t of sugar secreted being below t h a t of control. A c t i n o m y c i n D does n o t inhibit u n ti l a f te r 7 h of i n c u b a t i o n a n d even after 30 h gave only a b o u t 5o % inhibition. Abscisic acid appears to i n h ib it t he gibberellic acid-induced secretion
Biochim. Biophys. Acta, 222 (197o) 5Ol-5O7
INITIATION OF RESPONSES BY GIBBERELLIC ACID
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TABLE III EFFECT OF INHIBITORS APPLIED SIMULTANEOUSLY AND CONTINUOUSLY WITH GIBBERELLIC ACID ON SUGAR SECRETION E a c h t r e a t m e n t c o m p r i s e d 12 l a y e r s in i o m l solutions. A t t h e i n t e r v a l s i n d i c a t e d a l i q u o t s w e re r e m o v e d a n d a s s a y e d for soluble s u g a r w i t h a n t h r o n e r e a g e n t . V a l u e s are /~g glucose e q u i v a l e n t s per ml. C o n c e n t r a t i o n s of abscisic acid, a c t i n o m y c i n D a n d c y c l o h e x i m i d e were 2, i o o a n d 2o p g / m l , respectively.
Treatment
Control + Gibberellic + Gibberellic + Gibberellic + Gibberellic
Incubation time (h)
acid acid + c y c l o h e x i m i d e acid + a c t i n o m y c i n D acid + abscisic a c i d
4.5
5.5
7.5
I2
30
33 41 16 46 41
39 53 28 49 39
49 63 23 61 42
55 495 33 179 55
526 2815 390 138o 490
T A B L E IV EFFECT OF INHIBITORS AND ANAEROBIOSIS ON SECRETION BY LAYERS PRETREATED WITH GIBBERELLIC ACID L a y e r s were e x p o s e d to g i b b e r e l l i c acid for 2 h. T h e y we re w a s h e d t h o r o u g h l y in cold o.o2 M CaCI 2 w i t h a m a g n e t i c s t i r r e r for i2 h. U n t r e a t e d l a y e r s also w a s h e d . E i g h t l a y e r s pe r g r o u p t h e n s h a k e n a t r o o m t e m p e r a t u r e for 3 h in T h u n b e r g t u b e s in 5 m l m e d i u m , c e n t r i f u g e d a n d a l i q u o t s a s s a y e d . V a l u e s for soluble c a r b o h y d r a t e are m i c r o g r a m s glucose e q u i v a l e n t s e c r e t e d p e r o . i m l w h e r e a s v a l u e s for A T P a s e a c t i v i t y a r e / ~ m o l e s p h o s p h a t e p r o d u c e d p e r h p e r 0. 5 m l a l i q u o t .
Treatment
Untreated + Gibberellic + Gibberellic + Gibberellic (10 -4 M) + Gibberellic
.4 mount or activity secreted
acid acid + abscisic acid (2 /2g/ml) acid + p-chloromercuribenzoate acid, a n a e r o b i c
Soluble carbohydrate
A TPase
8 129 144
o.o 3 1.54
12 4 87
1.52
1.69 I.I0
TABLE V EFFECT OF CYCLOHEXIMIDE ON THE SECRETION OF SOLUBLE CARBOHYDRATE BY LAYERS PRETREATED WITH GIBBERELLIC ACID L a y e r s t r e a t e d a n d w a s h e d as in T a b l e IV. G r o u p s of 9 l a y e r s t h e n s h a k e n a t r o o m t e m p e r a t u r e in 4.5 m l CaC12 a n d a l i q u o t s a n a l y z e d a t t i m e s i n d i c a t e d . V a l u e s a re # g glucose e q u i v a l e n t s p e r o . i ml.
Treatment
Untreated + Gib berellic acid + G i b b e r e l l i c acid + c y c l o h e x i m i d e
Soluble sugar secreted Incubation time (h) z
2
6
24
4 51 62
13 95 113
38 314 29o
i 19 146o lO6O
Biochim. Biophys. Acta, 222 (197 o) 5Ol-5O7
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C . J . POLLARD
mainly, since unlike cycloheximide, it does not inhibit to a level below that of untreated tissue. Also, it usually had no effect on control tissue. Abscisic acid does not inhibit the secretion of soluble carbohydrate or ATPase by layers pretreated with gibberellic acid (Table IV), a contrast to its effectiveness noted in Table I I I . This same disparity held for cycloheximide (Tables I I I and V). The apparent anomalous effects of abscisic acid and of cycloheximide suggest that at least 2 distinct steps are involved in the gibberellic acid responses; one in which the hormone commences the secretion, which is inhibitable by abscisic acid and cycloheximide and the other, possibly the act of secretion itself, which is not inhibited by these compounds but is affected by anaerobiosis. DISCUSSION
Perhaps the most fundamental finding reported here is that layers develop the potential for increased secretion of certain metabolites and enzymes after short exposure to gibberellic acid. This appears to be mediated through a process which requires oxygen and is affected by temperature as well as concentration of the hormone present during exposure. Aggregately, the data suggest that either the hormone commences the effects irreversibly during the exposure period or that it is taken up rapidly by active metabolic processes and cannot be removed by extensive washing. Conceivably, it could effect the responses then at a later time. Regardless of the mechanism involved, the action of the hormone during the critical exposure period m a y be thought of as initiation, the first action of the hormone in layers. The results obtained with cycloheximide and actinomycin D were not assessed here in terms of protein synthesis and RNA synthesis as is usually done since for the purpose of this work the manner in which they inhibit is not important. The data probably should not be compared with the effects of the inhibitors on gibberellic acid-induced amylase synthesis and secretion reported earlier by C~RISPEELS AND VARNER3 since our results indicate that some of the gibberellin responses that we have studied m a y be distinctly different from the effect of the hormone on amylase. However, data comparing effects of varied concentrations of abscisic acid and of eycloheximide on several gibberellic acid-induced responses under the same experimental conditions will be contained in a forthcoming publication. The significant point for this paper appears to be tile fact that through the use of cycloheximide and abscisic acid it has been possible to gain an indication that at least 2 steps are involved in the gibberellic acid responses. Some of the results noted here are not completely without precedence. For example, PALEG7 incubated barley endosperm in gibberellic acid for varying lengths of time, transferred them to water and assayed for reducing sugar (an indicator of amylase formation) later. Incubations in gibberellic acid for as little as 15 rain caused increased production of reducing sugar; 4 h being enough for m a x i m u m sugar production. Of indirect interest to the present work are studies on the fate of gibberellins in barley half seeds. PALEG AND COOMBE8 were unable to detect the loss of appreciable amounts of gibberellic acid after incubation for 24 h; KENDE 9 was unable to detect metabolites of ~Hjgibberellin A 1 after incubation of the labelled material with half seeds. In this regard, it appears that the findings reported here m a y be quite instrucBiochim. Biophys. Aata, 222 (197 o) 5Ol-5O 7
INITIATION OF RESPONSES BY GIBBERELLIC ACID
507
t i v e : studies in the future on the metabolic fate of gibberellic acid in such tissues should take into account the r a p i d i t y in which the responses are initiated. Several observations appear to validate the procedure i n t r o d u c e d here as a legitimate one for s t u d y i n g aspects of i n i t i a t i o n of gibberellic acid responses. These i n c l u d e : (a) the p a t t e r n of response given b y the layers after successive washes a n d the evidence t h a t the effect of washing on the responses is p r i m a r i l y the removal of h o r m o n e (since the full m a g n i t u d e of the responses are realized u p o n the a d d i t i o n of gibberellic acid to t h o r o u g h l y washed layers) a n d (b) the d e m o n s t r a t i o n of the l i n e a r i t y of the responses with time of exposure. So, h a v i n g identified a v a r i e t y of gibberellic acid responses a n d the sequence in which t h e y are affected in aleurone layers in earlier reports from this l a b o r a t o r y a n d h a v i n g d o c u m e n t e d here a procedure for observing the i n i t i a t i o n of the responses, the n e x t p u b l i c a t i o n will describe exp e r i m e n t s utilizing the procedure to s t u d y the relationship of the responses. REFERENCES J. E. VARNER, G. R. CHANDRAAND M. J. CHRISPEELS,J. Cell Comp. Physiol., 66 (1965) 55M. J. CHRISPEELSAND J. E. VARNER,Plant Physiol., 42 (1967) 398~. J. CHRISPEELSAND J. E. VARNER, Plant Physiol., 42 (1967) lOO8. C. J. POLLARDAND N. B. SINGH,Biochem. Biophys. Res. Commun., 33 (1968) 321. C. J. POLLARD,Plant Physiol., 44 (1969) 1227. 13. G. COOMBE,D. COHEN AND L. G. PALEG, Plant Physiol., 42 (1967) 113. L. G. PALEG, Rdgulateurs Naturels de la Croissance Vdgdtale, Centre National de la Recherche Scientifique, Paris, 1964, p. 303 . 8 L. G. PALEGAND B . G-. COOMBE,Plant Physiol., 42 (1967) 445. 9 I-I. KENDE, Plant Physiol., 42 (1967) 1612.
I 2 3 4 5 6 7
Biochim. Biophys. Acta, 222 (197o) 5Ol-5O7
501
BBA 26443
I N I T I A T I O N OF RESPONSES IN A L E U R O N E L A Y E R S BY G I B B E R E L L I C ACID C L I F F O R D J. P O L L A R D
Department of Botany and Plant Pathology, Michigan State University, East Lansing, Mich. 48823
(u.s.A.) (Received J u n e 9th, 197 o)
SUMMARY
A new procedure for studying the action of gibberellic acid in barley (Hordeum vulgare cv. Betzes) aleurone layers was utilized. Exposure of the layers to gibberellic acid for short periods of time followed by thorough washing and subsequent incubation in the absence of the phytohormone resulted in increased secretion of enzymes and metabolites. Increased secretion of soluble sugar (a typical hormonal response) b y such treated and washed layers was linearly proportional to the time of exposure as well as to the concentration of hormone ill the exposure medium. It was decreased b y exposure under anaerobic conditions and was affected by the temperature of exposure. The hormone disappears from the medium during exposure but the results suggest that some of it can be removed from the layers by washing. Since cycloheximide and abscisic acid inhibited hormone-induced secretion when they were applied simultaneously and continuously with gibberellic acid but had no effect on pretreated layers whereas secretion was inhibited by anaerobiosis in both instances, at least two steps appear to be involved in hormonal action on secretion.
INTRODUCTION
The major findings from earlier studies on the action of gibberellic acid in cereal aleurone layers have been reviewed 1-3. The identity of m a n y of the metabolites and enzymes whose secretion is increased after application of the hormone to the layers, the sequence of these effects and their relationship to certain other aspects of cellular metabolism of the tissue were reported recently*, 5. Briefly, the earliest response it was possible to detect, increased secretion of soluble carbohydrate, was affected as early as 2 h after application of gibberellic acid; the latest, increased synthesis and secretion of amylase, was realized after 7 h of incubation. A variety of phosphatases and carbohydrases were affected at intermediate periods. Aside from their intrinsic value the results of the latter studies emphasize the complexity of the action of gibberellic acid in these tissues. It would appear axiomatic therefore that no single approach will likely lead to discerning the mode of action of the hormone; the desirability of having a variety of approaches available for the conduct of such studies is clear. This paper reports a novel procedure for studying some aspects of the mode of action of gibberellic acid. Experimentally this involves Biochim. Biophys. Acta, 222 (197 o) 5Ol-5O 7
502
c . J . POLLARD
exposing barley (Hordeum vulgate ev. Betzes) aleurone layers to the growth factor for short periods, extensive washing to remove excess hormone followed by incubation of the tissue in the absence of gibberellic acid and subsequent observation of the responses. The effects of certain inhibitors when incubated simultaneously and continuously with gibberellic acid, in contrast to their action on tissue pretreated with the hormone, will also be given. M A T E R I A L S AND METHODS
Since the conditions for the preparation of the layers, the materials as well as the methods used here have been described 4, 5, only the procedure used for washing will be given in detail. The layers were exposed to gibberellic acid (i #g/ml unless otherwise noted) with gentle shaking. They were then washed with ice cold 0.02 M CaCI~ although in more recent experiments cold distilled water has been used with equal apparent success. Three washing procedures have been used: (a) layers were shaken vigorously by hand in 250 ml glass stoppered erlenmeyer flasks with 50-200 ml of solution for 3 min, the solution decanted and the washing repeated for more than 20 times (b) layers were shaken for 2 h in a mechanical shaker in flasks as above, the solution being changed every 15 min and (c) layers were placed in 15oo ml of cold washing medium contained in a 2 1 flask in a cold room at 3 ° and stirred with a magnetic stirrer for 12-14 h, the medium being changed occasionally. The tissue was then incubated in 0.02 M CaC12 containing 250 big streptomycin sulfate per ml while being shaken on a mechanical shaker at 200 strokes per min. I n general, assays were done on aliquots of the incubation medium as given previously. Amylase activity was measured here, however, as given by CHRISPEELS AND VARNER 2. The bioassay for gibberellic acid was conducted as given by COOMBE et aL e except that total sugar secreted was measured rather than reducing sugar. All experiments were repeated to establish their validity. RESULTS
Evidence that a continuous supply of gibberellic acid is not needed to elicit responses Ordinarily, increased sugar secretion is observed in tissue treated continuously with the hormone after a lag of 2-4 h. An indication that tissues do not require continuous treatment with gibberellic acid is given in Table I. It can be observed that layers that were exposed to gibberellic acid for 4 h, washed thoroughly and incubated subsequently in the absence of the hormone secreted as much sugar over a 7 h period as tissue that was so treated and reincubated with gibberellic acid. After 21 h, however, more sugar was secreted b y the latter tissue. The data in Table I I illustrate the effect of washing on layers exposed to gibberellic acid for only 6 min. Note that this exposure (o washes) caused the layers to secrete 3-4 times as much soluble carbohydrate and the phosphatases as controls. It also caused the layers to secrete amylase. Washing the layers caused a progressive reduction in the amounts of carbohydrate and phosphatases secreted but 40 washes were no more effective than 20. It was shown in other experiments, however, that IO washes were less effective than 20. Of singular interest is the finding that no amylase was produced in layers that were washed more than 5 times. Note also that the layers Biochim. Biophys. Acta, 222 (197 o) 5 O l - 5 O 7
INITIATION OF RESPONSES BY GIBBERELLIC ACID
503
TABLE I EFFECT OF TRANSFER TO G I B B E R E L L I C
OF GIBBERELLIC
ACID TREATED
ALEURONE
L A Y E R S TO C O N T R O L S O L U T I O N OR
ACID SOLUTION ON THE SECRETION OF SOLUBLE CARBOHYDRATE
Treated layers were exposed to gibberellic acid for 4 h, removed and washed for 2 h with cold o.o2 M CaClz and subsequently placed into 7.5 ml of solutions. Groups contained 15 layers. Values for soluble carbohydrate are/zg glucose equivalent (anthrone) per 0. 5 ml. Incubation time
(h)
I 4 7 21
Soluble carbohydrate secreted Control to control
Treated to control
Treated to gibberellic acid
22 45 -168o
61 41° 2ioo 8470
49 384 218o 112o0
T A B L E II EFFECT
OF NUMBER
OF W A S H E S G I V E N G I B B E R E L L I C
ACID TREATED
L A Y E R S ON R E S P O N S E S
Fifty layers were exposed to 5 ° ml of gibberellic acid solution for 6 rain. They were then washed with 200 ml portions of cold 0.02 M CaCI~ by shaking by hand for 3 rain each time. At the n u m b e r of washes indicated io of the layers were removed. Controls were not treated with gibberellic acid b u t were washed for 4 ° times. All layers were then incubated in io ml CaC12 solution containing streptomycin sulfate for 16 h. Carbohydrate is total /~g glucose equivalents; amylase activity is ~de~0mt, x lO3 per io rain per o.2 ml. Phosphodiesterase activity (with bis-p-nitrophenyl phosphate) is Z]d410m~ × lOs per IO mill per O.2 ml and ATPase activity is dds00mv × lO3 per h per o. 5 ml. Washes
Control (no gibberellic acid) o 5 X 2o X 4° × 4 ° X + gibberellic acid
Amount or activity secreted Carbohydrate
Amylase
Phosphodiesterase
A TPase
2650 I 1 300 8640 6750 7000 12ooo
o 74 ° o o o 860
215 700 37 ° 24o 25 ° 77 °
18o 61o 485 44 ° 455 680
e x p o s e d t o g i b b e r e l l i c a c i d f o r 6 m i n a n d w e r e n o t w a s h e d s e c r e t e d m o r e t h a n 85 % a s m u c h of t h e m a t e r i a l s as w a s s e c r e t e d b y l a y e r s w a s h e d 40 t i m e s a n d t h e n t r e a t e d w i t h t h e h o r m o n e c o n t i n u o u s l y . T h e r e s p o n s e of t h e l a t t e r t i s s u e s u g g e s t s t h a t w a s h i n g d i d n o t i n t e r f e r e w i t h t h e c a p a c i t y of t h e t i s s u e t o r e s p o n d , as f o r e x a m p l e b y d e n a t u r a t i o n of a c t i v e p r o t e i n s o r b y r e m o v a l of o t h e r f a c t o r s . A n e x t e n s i o n of t h i s line of r e a s o n i n g a l l o w s for t h e s u g g e s t i o n t h a t s o m e h o r m o n e is r e m o v e d p r o g r e s s i v e l y f r o m t h e layers by the first few washes. That a linear relationship existed between the time layers were exposed to g i b b e r e l l i c a c i d a n d t h e a m o u n t of s o l u b l e s u g a r s e c r e t e d s u b s e q u e n t l y is i l l u s t r a t e d i n Fig. i . T h e c o n t r a s t i n g p a t t e r n of loss of h o r m o n e f r o m t h e m e d i u m , w h e r e i n a b o u t h a l f of t h e h o r m o n e h a d b e e n l o s t w i t h i n 30 m i n r e s e m b l e s a s a t u r a t i o n c u r v e . S i m i l a r l y , a r a p i d loss of t r i t i a t e d g i b b e r e l l i n A 1 ( k i n d l y s u p p l i e d b y D r . H a n s K e n d e ) 9 f r o m t h e m e d i u m h a s also b e e n o b s e r v e d . F o r e x a m p l e , 64 a l e u r o n e l a y e r s w e r e i n c u b a t e d w i t h 32 m l of t r i t i a t e d g i b b e r e l l i n A 1 of f i n a l c o n c e n t r a t i o n of I . I O - e M . Biochim. Biophys. Acta, 222 (197 o) 5Ol-5O7
504
c . J . POLLARD
T h e r a d i o a c t i v i t y of t h e solution initially was 126oo c o u n t s / m i n per ml. A t intervals 2 layers an d I ml of solution were r e m o v e d and an aliquot counted. A f t e r I, 2, 5, IO, 30 an d 12o m i n i n c u b a t i o n 3800, 4000, 4400, 5400, 7000 and 734 ° counts/rain per ml, r e s p e c t i v e l y h a d disappeared from t h e m e d i u m .
5.0
suG.R sEc 4.0
~=3.o
I
, .....- / . . . . . . . . .
:,~/ i
]5.0
"y i x
....
GA LOSSFROMMEOIUM
1.21 lib 111|,25°
~
4.0
l=
3.0!
~ a.l=
2.0
5.11 .17 o °iiAENIIIC,2} ~
_.
2.50
2.0
~ hO
SUGARSECRETI ' ON-CONTROL I
i
I
i
tl.0 i
30 60 90 120 EXPOSURE TIME (minutes)
1.25
oi,,
,'.I
I
¢,
ii,
6A CONCEHI|ITilB (aitfilraat/ill
Fig. I. Effect of exposure time to gibberellic acid (GA) on the secretion of sugar by layers and on the loss of gibberellic acid from the medium. Forty layers that were exposed to 20 ml of control and of gibberellic acid solutions were shaken and io layers and 5 ml portions removed at the intervals noted. All layers were then washed for 2 h in cold CaC12 solution. They were then incubated in io ml solution for 6 h. The gibberellic acid solutions used for exposures were bioassayed for gibberellic acid remaining by measuring sugar secreted by barley half seeds. Allowances were made for sugar secreted by layers during exposure. Fig. 2. Effect of gibberellic acid (GA) concentration of the medium, of temperature and of anaerobiosis during exposure on subsequent secretion of soluble sugar. Points on the line obtained by exposing io layers to 20 ml of gibberellic acid of concentration indicated for I h, Other points represent values obtained from layers exposed to solutions containing i /~g gibberellic acid per ml at the temperatures indicated or under reduced pressure in a desiccator at 25 ° (anaerobic). Washing of layers and incubation same as Fig. I.
Fig. 2 shows t h a t t h e a m o u n t of sugar secreted was d e p e n d e n t on t h e concent r a t i o n of gibberellic acid in t h e exposure m e d i u m , IOO/zg/ml being inhibitory. Th e t e m p e r a t u r e during exposure was critical; layers exposed to I # g gibberellic acid per ml at 37 ° an d o ° secreted sugar in a m o u n t s e q u i v a l e n t to c o n c e n t r a t i o n s of exposure of o.I a n d o . o i /zg/ml, r e s p e c t i v e l y at 25 °. Also, layers exposed u n d e r anaerobic conditions to I /zg/ml secreted sugar in an a m o u n t e q u i v a l e n t to w h a t w o u l d h a v e been secreted b y layers exposed to approx. 0.075/zg gibberellic acid per ml, aerobically.
Effect of inhibitors T h e actions of a c t i n o m y c i n D, c y c lo h e x i m id e and abscisic acid, when i n c u b a t e d c o n t i n u o u s l y w i t h t h e hormone, on a m y la s e synthesis h a v e been r e p o r t e d 4. Tab l e I I I gives t h e effect of these on gibberellic acid in d u ced sugar secretion when applied at t h e beginning of th e e x p e r i m e n t . U n d e r these conditions c y c l o h e x i m i d e is a p o t e n t i n h i b i t o r ; the a m o u n t of sugar secreted being below t h a t of control. A c t i n o m y c i n D does n o t inhibit u n ti l a f te r 7 h of i n c u b a t i o n a n d even after 30 h gave only a b o u t 5o % inhibition. Abscisic acid appears to i n h ib it t he gibberellic acid-induced secretion
Biochim. Biophys. Acta, 222 (197o) 5Ol-5O7
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TABLE III EFFECT OF INHIBITORS APPLIED SIMULTANEOUSLY AND CONTINUOUSLY WITH GIBBERELLIC ACID ON SUGAR SECRETION E a c h t r e a t m e n t c o m p r i s e d 12 l a y e r s in i o m l solutions. A t t h e i n t e r v a l s i n d i c a t e d a l i q u o t s w e re r e m o v e d a n d a s s a y e d for soluble s u g a r w i t h a n t h r o n e r e a g e n t . V a l u e s are /~g glucose e q u i v a l e n t s per ml. C o n c e n t r a t i o n s of abscisic acid, a c t i n o m y c i n D a n d c y c l o h e x i m i d e were 2, i o o a n d 2o p g / m l , respectively.
Treatment
Control + Gibberellic + Gibberellic + Gibberellic + Gibberellic
Incubation time (h)
acid acid + c y c l o h e x i m i d e acid + a c t i n o m y c i n D acid + abscisic a c i d
4.5
5.5
7.5
I2
30
33 41 16 46 41
39 53 28 49 39
49 63 23 61 42
55 495 33 179 55
526 2815 390 138o 490
T A B L E IV EFFECT OF INHIBITORS AND ANAEROBIOSIS ON SECRETION BY LAYERS PRETREATED WITH GIBBERELLIC ACID L a y e r s were e x p o s e d to g i b b e r e l l i c acid for 2 h. T h e y we re w a s h e d t h o r o u g h l y in cold o.o2 M CaCI 2 w i t h a m a g n e t i c s t i r r e r for i2 h. U n t r e a t e d l a y e r s also w a s h e d . E i g h t l a y e r s pe r g r o u p t h e n s h a k e n a t r o o m t e m p e r a t u r e for 3 h in T h u n b e r g t u b e s in 5 m l m e d i u m , c e n t r i f u g e d a n d a l i q u o t s a s s a y e d . V a l u e s for soluble c a r b o h y d r a t e are m i c r o g r a m s glucose e q u i v a l e n t s e c r e t e d p e r o . i m l w h e r e a s v a l u e s for A T P a s e a c t i v i t y a r e / ~ m o l e s p h o s p h a t e p r o d u c e d p e r h p e r 0. 5 m l a l i q u o t .
Treatment
Untreated + Gibberellic + Gibberellic + Gibberellic (10 -4 M) + Gibberellic
.4 mount or activity secreted
acid acid + abscisic acid (2 /2g/ml) acid + p-chloromercuribenzoate acid, a n a e r o b i c
Soluble carbohydrate
A TPase
8 129 144
o.o 3 1.54
12 4 87
1.52
1.69 I.I0
TABLE V EFFECT OF CYCLOHEXIMIDE ON THE SECRETION OF SOLUBLE CARBOHYDRATE BY LAYERS PRETREATED WITH GIBBERELLIC ACID L a y e r s t r e a t e d a n d w a s h e d as in T a b l e IV. G r o u p s of 9 l a y e r s t h e n s h a k e n a t r o o m t e m p e r a t u r e in 4.5 m l CaC12 a n d a l i q u o t s a n a l y z e d a t t i m e s i n d i c a t e d . V a l u e s a re # g glucose e q u i v a l e n t s p e r o . i ml.
Treatment
Untreated + Gib berellic acid + G i b b e r e l l i c acid + c y c l o h e x i m i d e
Soluble sugar secreted Incubation time (h) z
2
6
24
4 51 62
13 95 113
38 314 29o
i 19 146o lO6O
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C . J . POLLARD
mainly, since unlike cycloheximide, it does not inhibit to a level below that of untreated tissue. Also, it usually had no effect on control tissue. Abscisic acid does not inhibit the secretion of soluble carbohydrate or ATPase by layers pretreated with gibberellic acid (Table IV), a contrast to its effectiveness noted in Table I I I . This same disparity held for cycloheximide (Tables I I I and V). The apparent anomalous effects of abscisic acid and of cycloheximide suggest that at least 2 distinct steps are involved in the gibberellic acid responses; one in which the hormone commences the secretion, which is inhibitable by abscisic acid and cycloheximide and the other, possibly the act of secretion itself, which is not inhibited by these compounds but is affected by anaerobiosis. DISCUSSION
Perhaps the most fundamental finding reported here is that layers develop the potential for increased secretion of certain metabolites and enzymes after short exposure to gibberellic acid. This appears to be mediated through a process which requires oxygen and is affected by temperature as well as concentration of the hormone present during exposure. Aggregately, the data suggest that either the hormone commences the effects irreversibly during the exposure period or that it is taken up rapidly by active metabolic processes and cannot be removed by extensive washing. Conceivably, it could effect the responses then at a later time. Regardless of the mechanism involved, the action of the hormone during the critical exposure period m a y be thought of as initiation, the first action of the hormone in layers. The results obtained with cycloheximide and actinomycin D were not assessed here in terms of protein synthesis and RNA synthesis as is usually done since for the purpose of this work the manner in which they inhibit is not important. The data probably should not be compared with the effects of the inhibitors on gibberellic acid-induced amylase synthesis and secretion reported earlier by C~RISPEELS AND VARNER3 since our results indicate that some of the gibberellin responses that we have studied m a y be distinctly different from the effect of the hormone on amylase. However, data comparing effects of varied concentrations of abscisic acid and of eycloheximide on several gibberellic acid-induced responses under the same experimental conditions will be contained in a forthcoming publication. The significant point for this paper appears to be tile fact that through the use of cycloheximide and abscisic acid it has been possible to gain an indication that at least 2 steps are involved in the gibberellic acid responses. Some of the results noted here are not completely without precedence. For example, PALEG7 incubated barley endosperm in gibberellic acid for varying lengths of time, transferred them to water and assayed for reducing sugar (an indicator of amylase formation) later. Incubations in gibberellic acid for as little as 15 rain caused increased production of reducing sugar; 4 h being enough for m a x i m u m sugar production. Of indirect interest to the present work are studies on the fate of gibberellins in barley half seeds. PALEG AND COOMBE8 were unable to detect the loss of appreciable amounts of gibberellic acid after incubation for 24 h; KENDE 9 was unable to detect metabolites of ~Hjgibberellin A 1 after incubation of the labelled material with half seeds. In this regard, it appears that the findings reported here m a y be quite instrucBiochim. Biophys. Aata, 222 (197 o) 5Ol-5O 7
INITIATION OF RESPONSES BY GIBBERELLIC ACID
507
t i v e : studies in the future on the metabolic fate of gibberellic acid in such tissues should take into account the r a p i d i t y in which the responses are initiated. Several observations appear to validate the procedure i n t r o d u c e d here as a legitimate one for s t u d y i n g aspects of i n i t i a t i o n of gibberellic acid responses. These i n c l u d e : (a) the p a t t e r n of response given b y the layers after successive washes a n d the evidence t h a t the effect of washing on the responses is p r i m a r i l y the removal of h o r m o n e (since the full m a g n i t u d e of the responses are realized u p o n the a d d i t i o n of gibberellic acid to t h o r o u g h l y washed layers) a n d (b) the d e m o n s t r a t i o n of the l i n e a r i t y of the responses with time of exposure. So, h a v i n g identified a v a r i e t y of gibberellic acid responses a n d the sequence in which t h e y are affected in aleurone layers in earlier reports from this l a b o r a t o r y a n d h a v i n g d o c u m e n t e d here a procedure for observing the i n i t i a t i o n of the responses, the n e x t p u b l i c a t i o n will describe exp e r i m e n t s utilizing the procedure to s t u d y the relationship of the responses. REFERENCES J. E. VARNER, G. R. CHANDRAAND M. J. CHRISPEELS,J. Cell Comp. Physiol., 66 (1965) 55M. J. CHRISPEELSAND J. E. VARNER,Plant Physiol., 42 (1967) 398~. J. CHRISPEELSAND J. E. VARNER, Plant Physiol., 42 (1967) lOO8. C. J. POLLARDAND N. B. SINGH,Biochem. Biophys. Res. Commun., 33 (1968) 321. C. J. POLLARD,Plant Physiol., 44 (1969) 1227. 13. G. COOMBE,D. COHEN AND L. G. PALEG, Plant Physiol., 42 (1967) 113. L. G. PALEG, Rdgulateurs Naturels de la Croissance Vdgdtale, Centre National de la Recherche Scientifique, Paris, 1964, p. 303 . 8 L. G. PALEGAND B . G-. COOMBE,Plant Physiol., 42 (1967) 445. 9 I-I. KENDE, Plant Physiol., 42 (1967) 1612.
I 2 3 4 5 6 7
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