Planta 9 Springer-Verlag 198l
Planta (1981) 152:481-486
Effects of two growth retardants on tissue permeability in Pisum sativum and Beta vulgaris Dorothy M. Fabijan, Pamela Plumb Dhindsa*, and David M. Reid BiologyDepartment, Universityof Calgary, Calgary,Alta. T2N 1N4, Canada
Abstract. The effects of growth retardants, 4-hydroxy5-isopropyl-2-methylphenyltrimethylammonium
chloride-l-piperidine carboxylate (AMO-1618 or AMO) and 2-chloroethyltrimethylammonium chloride (CCC), applied with and without gibberellic acid (GA3), on ]%[3H]alanine uptake and leakage from pea (Pisum sativum L.) and betacyanin efflux from beetroot (Beta vulgaris L.) tissue were examined. Both compounds decreased the amount of fi-[3H]alanine taken up into pea leaf discs, and increased the quantity of radioactive label that subsequently leaked out of this tissue. Efflux of betacyanin from slices of beetroot was also found to be promoted by treatment with CCC or AMO-1618. In no case were these effects reversed by application of GA3. It is concluded that the growth retardants may be altering tissue permeability by an interaction with the cell membranes, and this may account for some of the side effects of the retardants which cannot be explained on the basis of their inhibiting action on gibberellin synthesis. Key words: AMO-1618 - Beta - CCC - Growth retardants - Permeability and growth retardants - Pisum.
Introduction The group of compounds given the name "plant growth retardants" inhibit stem growth and some other growth processes in a wide range of species of higher plants. A major mode of action of these compounds is the ability to block the synthesis of the gibberellins (GA) (Kende et al. 1963; Zeevaart * Permanent address. Faculdadede Agronomomia,Universidade
Eduardo Mondlane,Maputo, Mozambique Abbreviations: AMO-1618 =4-hydroxy-5-isopropyl-2-methylphen-
yltrimethylammoniumchloride-l-piperidine carboxylate; CCC= 2-chloroethyltrimethylammoniumchloride; GA3=gibberellic acid
1966; Barnes et al. 1969) at one or two steps in their biosynthetic pathway (Shechter and West 1969; Robinson and West 1970). The growth retardants are thus useful tools in investigations of the biosynthesis of GA and their physiological role in plant growth. Growth retardants may also affect plant metabolism at a number of points other than GA biosynthesis. They are inhibitors of the in-vivo and in-vitro production of certain sterols (Douglas and Paleg 1974, 1978), can reduce the rates of chlorophyll, RNA and protein synthesis (Berry and Smith 1970; Knypl 1971), and can have a number of effects on plant growth that cannot be explained solely on the basis of a reduced production of GA (Wiinsche 1969; Crozier et al. 1973). In some species the retardants can even result in an increase of extractable GA-like substances (van Bragt 1969; Reid and Crozier 1970). Little is known of the mechanisms of action of the growth retardants in causing these side effects. While chemically quite diverse, many of the growth retardants have one particular structural feature; they possess a di- or trimethylammonium group. We were also struck by the structural similarity between these compounds and some of the precursors of the phospholipid components of the cell membranes. For example consider the similarity between choline and 2-chloroethyltrimethylammonium chloride (CCC); CCC is a chlorinated derivative of choline. If some of the non-GA effects of the growth retardants are related to this similarity in structure, the retardants might be expected to modify membrane permeability. We have also found that CCC and other growth retardants inhibit the nyctinastic and seismonastic movements of M i m o s a leaflets and that this response was not overcome by application of GA3 (unpublished observations). On the basis of these observations we conducted experiments designed to investigate the possibility that CCC and 4-hydroxy-5-isopropyl-2methylphenyltrimethylammonium chloride-l-piperi-
0032-0935/81/0152/0481 / $01.20
482
D.M. Fabijan et al. : Growth retardants and tissue permeability
d i n e c a r b o x y l a t e ( A M O - 1 6 1 8 ) m i g h t h a v e , as o n e o f t h e i r side effects, a n i n f l u e n c e o n t h e p e r m e a b i l i t y o f Pisurn sativurn l e a f a n d Beta vulgaris r o o t tissues. The interaction of gibberellic acid (GA3) with the g r o w t h r e t a r d a n t s w a s also s t u d i e d .
Materials and methods
Uptake and leakage of ~-[3]alanine in pea leaf discs. Pisum sativum L. cv. Alaska (Ferry Morse, Mountain View, Cal., USA,) seeds were planted in moist granite grit and grown in a growth cabinet in days of 18 h light (150 Win-z; using F 72T10/CW fluorescent tubes, Sylvania, Drummondville, Quel, Canada) at 23 ~ C, and 6 h of darkness at 18~ C. When the plants were 14 d old they were carefully removed from the granite grit, the roots gently washed in a stream of distilled water, and the plants placed with their roots in the test solution of CCC, AMO-1618 or GA3. On the next morning (18 h later) discs 1 cm in diameter were cut from mature (neither damaged nor senescent) leaves. Ten discs were floated in 5-cm Petri dishes on 10 ml of 10 mM maleate-KOH buffer, pH 5.8, with the appropriate quantity of/3-[3-3H]alanine. The dishes were placed for 1 or 2 h in a reciprocating water bath (Blue M; Blue Island, Ill., USA) set at 50 cycles min- 1 and held at 25 ~ C. After this period the discs were rinsed in a Buchner funnel with 250 ml of nonradioactive/~-alanine (1 mg/mI) for 5 min. This time should have been sufficient to remove the majority of the label from the apparent free space (Liittge and Higinbotham 1979, chpt. 6, and references therein). The uptake and leakage experiments were carried out on the same day. Uptake of label was estimated by freezing the discs on dry ice, thawing, and then homogenizing them with 2 ml of 0.1 M tris(hydroxymethyl)aminomethane (Tris; Fisher Scientific Co., Fairlawn, N.J., USA) at pH 8.0, in a small glass homogenizer. The liquid was decanted and the grinding procedure repeated three times with 1 ml of buffer each. The four volumes of buffer were combined and centrifuged at 1,000 g for 20 min. Finally 0.5-ml aliquots were taken for scintillation counting (Nuclear Chicago ISOCAP 300 scintillation counter; Searle Analytic, Des Plaines, Ill., USA) in 15 ml of Biofluor (New England Nuclear, Boston, Mass., USA). All this work has also been carried out solubilizing the discs in NCS tissue solubilizer (Amersham Corp., Arlington Heights, II1., USA). The results, while more variable, showed the same trends. For estimations of leakage of radioactivity from the tissue another set of rinsed discs, treated and allowed to take up the /?-[3H]alanine at the same time and in exactly the same manner as the discs used in the uptake studies, was placed in 10 ml of distilled water (conditions same as those for uptake). At intervals, samples (100 gl) of liquid were taken and the amount of radioactivity was measured by scintillation counting. The amount of "leakage" was calculated as : (Total counts leaked out into l0 ml of solution in a specific treatment) (Total counts taken up by 10 discs of an identical treatment carried out at the same time)
x 100=%leakage
Leakage of betaeyanin from beetroot. Beetroots (Beta vulgaris L. cv. Detroit; Western Canada Horticultural Specialists, Calgary) were grown in the same conditions as the peas except that they were in a peat moss:sand mixture (50:50, v/v), were regularly watered with Hoagland's nutrient solution, and were harvested when the roots had reached a diameter of 6-10 cm. Cylindrical
sections of the root were removed using a 1-cm cork borer and sections 1.5 mm thick were sliced from these cylinders. The sections were washed for 1 h in running distilled water, and batches of 10 sections each were transferred to 10 ml of test solution, held at 25 or 35 ~C under fluorescent light (Sylvania Gro-lux; 46 W m- 2), and 1 h later the quantity of pigment that had leaked out was determined spectrophotometrically at 540 nm. In the betacyanin efflux experiments the concentrations of test substances were of the same order as, and the conditions similar to those described by Poovaiah and Leopold (1967) and Naik and Srivastava (1968). In the pea and beetroot experiments each treatment had five replicates. The phrase "significantly different" as used in the text, indicates that the means under discussion are shown by Student's t-test to be significantly different at a 5% level of probability. CCC was purchased fi'om the American Cyanamid Co., Princeton, N.J., USA; AMO-1618 from Calbiochem-Behring, La Jolla, Cal., USA; and GA3 from Pfizer Co., New York, USA; glycine betaine, candicine iodide, fagaramide, hordenine hemisulphate, N,N-dimethyl-p-phenyldiamine and hypaphorine from ICN Pharmaceticals, K & K Laboratories Division, Plainsview, N.Y., USA; fi-[3-3H]alanine (1850 GBq/mmol) from NEN, Lachine, Que., Canada.
Results
Uptake and leakage of ~-[3H]alanine from pea leaf discs. T h e e x p e r i m e n t s w i t h t h e discs f r o m p e a leaves w e r e all d o n e f o l l o w i n g t h e s a m e g e n e r a l p r o c e d u r e . The roots were supplied with a range of concentrat i o n s o f A M O - 1 6 1 8 , C C C o r G A 3 a n d 18 h l a t e r t w o sets o f l e a f discs w e r e f l o a t e d o n /~-[3H]alanine f o r 1 o r 2 h. A f t e r w a s h i n g w i t h n o n r a d i o a c t i v e /~a l a n i n e , o n e set o f discs w a s h a r v e s t e d f o r e s t i m a t i o n o f t h e q u a n t i t y o f l a b e l t a k e n up. T h e r e m a i n d e r o f t h e discs w e r e t r a n s f e r r e d to f r e s h w a t e r a n d the a m o u n t o f r a d i o a c t i v i t y t h a t h a d l e a k e d o u t after v a r i o u s t i m e s was m e a s u r e d . Pretreatment of the plants with CCC reduced the u p t a k e o f / ~ - [ 3 H ] a l a n i n e (Fig. 1 A ) a n d i n c r e a s e d the a m o u n t o f l e a k a g e (Fig. 1 B). G i b b e r e l l i c acid s u p p l i e d to t h e p l a n t s at a w i d e r a n g e o f c o n c e n t r a t i o n s , h a d n o s t a t i s t i c a l l y s i g n i f i c a n t effect o n u p t a k e or l e a k a g e o f label. F o r e x a m p l e , in t h e e x p e r i m e n t s h o w n in F i g . 2, G A 3 w a s s u p p l i e d o n its o w n or t o g e t h e r w i t h a n d at an e q u i m o l a r c o n c e n t r a t i o n to C C C . O n its o w n G A 3 h a d n o effect o n u p t a k e o r leakage, and when added with CCC did not overcome the inhibitory influence of the growth retardant on u p t a k e . H o w e v e r , G A 3 t r e a t m e n t s i g n i f i c a n t l y enh a n c e d t h e C C C - i n d u c e d e f f l u x o f label. T h e results o f the A M O - 1 6 1 8 e x p e r i m e n t s w e r e very similar. Treatment of the plants with the retard a n t d e c r e a s e d the q u a n t i t y o f /~-[3H]alanine t h a t m o v e d i n t o the l e a f discs (Fig. 37), b u t was less effect i v e in t h a t t h e r e was n o effect b e l o w 10 . 3 M . A p r o m o t i o n o f t h e r a t e o f l e a k a g e was also o b s e r v e d a f t e r 1, 3 a n d 5 h, b u t a g a i n A M O - 1 6 1 8 w a s n o t as p o t e n t as was C C C (Fig. 3 B). F o r e x a m p l e , t r e a t m e n t w i t h 10 - z M C C C r e s u l t e d in a 4 6 % i n c r e a s e
D.M. Fabijan et al. : Growth retardants and tissue permeability
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Planta (1981) 152:481-486
Effects of two growth retardants on tissue permeability in Pisum sativum and Beta vulgaris Dorothy M. Fabijan, Pamela Plumb Dhindsa*, and David M. Reid BiologyDepartment, Universityof Calgary, Calgary,Alta. T2N 1N4, Canada
Abstract. The effects of growth retardants, 4-hydroxy5-isopropyl-2-methylphenyltrimethylammonium
chloride-l-piperidine carboxylate (AMO-1618 or AMO) and 2-chloroethyltrimethylammonium chloride (CCC), applied with and without gibberellic acid (GA3), on ]%[3H]alanine uptake and leakage from pea (Pisum sativum L.) and betacyanin efflux from beetroot (Beta vulgaris L.) tissue were examined. Both compounds decreased the amount of fi-[3H]alanine taken up into pea leaf discs, and increased the quantity of radioactive label that subsequently leaked out of this tissue. Efflux of betacyanin from slices of beetroot was also found to be promoted by treatment with CCC or AMO-1618. In no case were these effects reversed by application of GA3. It is concluded that the growth retardants may be altering tissue permeability by an interaction with the cell membranes, and this may account for some of the side effects of the retardants which cannot be explained on the basis of their inhibiting action on gibberellin synthesis. Key words: AMO-1618 - Beta - CCC - Growth retardants - Permeability and growth retardants - Pisum.
Introduction The group of compounds given the name "plant growth retardants" inhibit stem growth and some other growth processes in a wide range of species of higher plants. A major mode of action of these compounds is the ability to block the synthesis of the gibberellins (GA) (Kende et al. 1963; Zeevaart * Permanent address. Faculdadede Agronomomia,Universidade
Eduardo Mondlane,Maputo, Mozambique Abbreviations: AMO-1618 =4-hydroxy-5-isopropyl-2-methylphen-
yltrimethylammoniumchloride-l-piperidine carboxylate; CCC= 2-chloroethyltrimethylammoniumchloride; GA3=gibberellic acid
1966; Barnes et al. 1969) at one or two steps in their biosynthetic pathway (Shechter and West 1969; Robinson and West 1970). The growth retardants are thus useful tools in investigations of the biosynthesis of GA and their physiological role in plant growth. Growth retardants may also affect plant metabolism at a number of points other than GA biosynthesis. They are inhibitors of the in-vivo and in-vitro production of certain sterols (Douglas and Paleg 1974, 1978), can reduce the rates of chlorophyll, RNA and protein synthesis (Berry and Smith 1970; Knypl 1971), and can have a number of effects on plant growth that cannot be explained solely on the basis of a reduced production of GA (Wiinsche 1969; Crozier et al. 1973). In some species the retardants can even result in an increase of extractable GA-like substances (van Bragt 1969; Reid and Crozier 1970). Little is known of the mechanisms of action of the growth retardants in causing these side effects. While chemically quite diverse, many of the growth retardants have one particular structural feature; they possess a di- or trimethylammonium group. We were also struck by the structural similarity between these compounds and some of the precursors of the phospholipid components of the cell membranes. For example consider the similarity between choline and 2-chloroethyltrimethylammonium chloride (CCC); CCC is a chlorinated derivative of choline. If some of the non-GA effects of the growth retardants are related to this similarity in structure, the retardants might be expected to modify membrane permeability. We have also found that CCC and other growth retardants inhibit the nyctinastic and seismonastic movements of M i m o s a leaflets and that this response was not overcome by application of GA3 (unpublished observations). On the basis of these observations we conducted experiments designed to investigate the possibility that CCC and 4-hydroxy-5-isopropyl-2methylphenyltrimethylammonium chloride-l-piperi-
0032-0935/81/0152/0481 / $01.20
482
D.M. Fabijan et al. : Growth retardants and tissue permeability
d i n e c a r b o x y l a t e ( A M O - 1 6 1 8 ) m i g h t h a v e , as o n e o f t h e i r side effects, a n i n f l u e n c e o n t h e p e r m e a b i l i t y o f Pisurn sativurn l e a f a n d Beta vulgaris r o o t tissues. The interaction of gibberellic acid (GA3) with the g r o w t h r e t a r d a n t s w a s also s t u d i e d .
Materials and methods
Uptake and leakage of ~-[3]alanine in pea leaf discs. Pisum sativum L. cv. Alaska (Ferry Morse, Mountain View, Cal., USA,) seeds were planted in moist granite grit and grown in a growth cabinet in days of 18 h light (150 Win-z; using F 72T10/CW fluorescent tubes, Sylvania, Drummondville, Quel, Canada) at 23 ~ C, and 6 h of darkness at 18~ C. When the plants were 14 d old they were carefully removed from the granite grit, the roots gently washed in a stream of distilled water, and the plants placed with their roots in the test solution of CCC, AMO-1618 or GA3. On the next morning (18 h later) discs 1 cm in diameter were cut from mature (neither damaged nor senescent) leaves. Ten discs were floated in 5-cm Petri dishes on 10 ml of 10 mM maleate-KOH buffer, pH 5.8, with the appropriate quantity of/3-[3-3H]alanine. The dishes were placed for 1 or 2 h in a reciprocating water bath (Blue M; Blue Island, Ill., USA) set at 50 cycles min- 1 and held at 25 ~ C. After this period the discs were rinsed in a Buchner funnel with 250 ml of nonradioactive/~-alanine (1 mg/mI) for 5 min. This time should have been sufficient to remove the majority of the label from the apparent free space (Liittge and Higinbotham 1979, chpt. 6, and references therein). The uptake and leakage experiments were carried out on the same day. Uptake of label was estimated by freezing the discs on dry ice, thawing, and then homogenizing them with 2 ml of 0.1 M tris(hydroxymethyl)aminomethane (Tris; Fisher Scientific Co., Fairlawn, N.J., USA) at pH 8.0, in a small glass homogenizer. The liquid was decanted and the grinding procedure repeated three times with 1 ml of buffer each. The four volumes of buffer were combined and centrifuged at 1,000 g for 20 min. Finally 0.5-ml aliquots were taken for scintillation counting (Nuclear Chicago ISOCAP 300 scintillation counter; Searle Analytic, Des Plaines, Ill., USA) in 15 ml of Biofluor (New England Nuclear, Boston, Mass., USA). All this work has also been carried out solubilizing the discs in NCS tissue solubilizer (Amersham Corp., Arlington Heights, II1., USA). The results, while more variable, showed the same trends. For estimations of leakage of radioactivity from the tissue another set of rinsed discs, treated and allowed to take up the /?-[3H]alanine at the same time and in exactly the same manner as the discs used in the uptake studies, was placed in 10 ml of distilled water (conditions same as those for uptake). At intervals, samples (100 gl) of liquid were taken and the amount of radioactivity was measured by scintillation counting. The amount of "leakage" was calculated as : (Total counts leaked out into l0 ml of solution in a specific treatment) (Total counts taken up by 10 discs of an identical treatment carried out at the same time)
x 100=%leakage
Leakage of betaeyanin from beetroot. Beetroots (Beta vulgaris L. cv. Detroit; Western Canada Horticultural Specialists, Calgary) were grown in the same conditions as the peas except that they were in a peat moss:sand mixture (50:50, v/v), were regularly watered with Hoagland's nutrient solution, and were harvested when the roots had reached a diameter of 6-10 cm. Cylindrical
sections of the root were removed using a 1-cm cork borer and sections 1.5 mm thick were sliced from these cylinders. The sections were washed for 1 h in running distilled water, and batches of 10 sections each were transferred to 10 ml of test solution, held at 25 or 35 ~C under fluorescent light (Sylvania Gro-lux; 46 W m- 2), and 1 h later the quantity of pigment that had leaked out was determined spectrophotometrically at 540 nm. In the betacyanin efflux experiments the concentrations of test substances were of the same order as, and the conditions similar to those described by Poovaiah and Leopold (1967) and Naik and Srivastava (1968). In the pea and beetroot experiments each treatment had five replicates. The phrase "significantly different" as used in the text, indicates that the means under discussion are shown by Student's t-test to be significantly different at a 5% level of probability. CCC was purchased fi'om the American Cyanamid Co., Princeton, N.J., USA; AMO-1618 from Calbiochem-Behring, La Jolla, Cal., USA; and GA3 from Pfizer Co., New York, USA; glycine betaine, candicine iodide, fagaramide, hordenine hemisulphate, N,N-dimethyl-p-phenyldiamine and hypaphorine from ICN Pharmaceticals, K & K Laboratories Division, Plainsview, N.Y., USA; fi-[3-3H]alanine (1850 GBq/mmol) from NEN, Lachine, Que., Canada.
Results
Uptake and leakage of ~-[3H]alanine from pea leaf discs. T h e e x p e r i m e n t s w i t h t h e discs f r o m p e a leaves w e r e all d o n e f o l l o w i n g t h e s a m e g e n e r a l p r o c e d u r e . The roots were supplied with a range of concentrat i o n s o f A M O - 1 6 1 8 , C C C o r G A 3 a n d 18 h l a t e r t w o sets o f l e a f discs w e r e f l o a t e d o n /~-[3H]alanine f o r 1 o r 2 h. A f t e r w a s h i n g w i t h n o n r a d i o a c t i v e /~a l a n i n e , o n e set o f discs w a s h a r v e s t e d f o r e s t i m a t i o n o f t h e q u a n t i t y o f l a b e l t a k e n up. T h e r e m a i n d e r o f t h e discs w e r e t r a n s f e r r e d to f r e s h w a t e r a n d the a m o u n t o f r a d i o a c t i v i t y t h a t h a d l e a k e d o u t after v a r i o u s t i m e s was m e a s u r e d . Pretreatment of the plants with CCC reduced the u p t a k e o f / ~ - [ 3 H ] a l a n i n e (Fig. 1 A ) a n d i n c r e a s e d the a m o u n t o f l e a k a g e (Fig. 1 B). G i b b e r e l l i c acid s u p p l i e d to t h e p l a n t s at a w i d e r a n g e o f c o n c e n t r a t i o n s , h a d n o s t a t i s t i c a l l y s i g n i f i c a n t effect o n u p t a k e or l e a k a g e o f label. F o r e x a m p l e , in t h e e x p e r i m e n t s h o w n in F i g . 2, G A 3 w a s s u p p l i e d o n its o w n or t o g e t h e r w i t h a n d at an e q u i m o l a r c o n c e n t r a t i o n to C C C . O n its o w n G A 3 h a d n o effect o n u p t a k e o r leakage, and when added with CCC did not overcome the inhibitory influence of the growth retardant on u p t a k e . H o w e v e r , G A 3 t r e a t m e n t s i g n i f i c a n t l y enh a n c e d t h e C C C - i n d u c e d e f f l u x o f label. T h e results o f the A M O - 1 6 1 8 e x p e r i m e n t s w e r e very similar. Treatment of the plants with the retard a n t d e c r e a s e d the q u a n t i t y o f /~-[3H]alanine t h a t m o v e d i n t o the l e a f discs (Fig. 37), b u t was less effect i v e in t h a t t h e r e was n o effect b e l o w 10 . 3 M . A p r o m o t i o n o f t h e r a t e o f l e a k a g e was also o b s e r v e d a f t e r 1, 3 a n d 5 h, b u t a g a i n A M O - 1 6 1 8 w a s n o t as p o t e n t as was C C C (Fig. 3 B). F o r e x a m p l e , t r e a t m e n t w i t h 10 - z M C C C r e s u l t e d in a 4 6 % i n c r e a s e
D.M. Fabijan et al. : Growth retardants and tissue permeability
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