Planta (Berl.) 107, 261--268 (1972) 9 by Springer-Verlag 1972
Cuticular Penetration of Abscisic Acid A m o s B l u m e n f e l d a n d M a r t i n J. B u k o v a c Department of Horticulture, Michigan State University, East Lansing, Michigan, U.S.A. Received May 5, 1972 Summary. Penetration of 2-14C abscisic acid (ABA) through enzymatically isolated cuticles from tomato fruit and from the upper epidermis of apricot, pear and orange leaves was assessed. Penetration was linear with time, greater as the undissociated than the dissociated ion, and greater through dewaxed than nondewaxed cuticles. Significantly less (3-6 times) (2-14C)ABA penetrated the tomato fruit cuticle than NAA or 2,4-D. The leaf cuticles were less permeable than the tomato fruit cuticle. There was no evidence that the ABA was altered during transfer across the cuticle. Introduction
M a x i m u m p l a n t responses to abscisic acid (ABA) g e n e r a l l y h a v e been o b t a i n e d o n l y a f t e r t h e r e g u l a t o r h a d been i n t r o d u c e d t h r o u g h cut surfaces, or following r e p e a t e d foliar a p p l i c a t i o n s (E1-Antably et al., 1967; S m i t h et al., 1968; A d d i c o t t a n d Lyon, 1969). Two e x p l a n a t i o n s h a v e been offered for these observations. F i r s t l y , t h a t A B A does n o t p e n e t r a t e or p e n e t r a t e s v e r y p o o r l y into the p l a n t (E1-Antably et al., 1967; Milborrow, 1969b). Secondly, t h a t A B A is r a p i d l y m e t a b o l i z e d in t h e p l a n t to substances with lower or no biological a c t i v i t y (Milborrow, 1969a; Sondh e i m e r et al., 1971). I n this s t u d y we h a v e d i r e c t l y assessed t h e p e n e t r a t i o n of A B A t h r o u g h selected leaf (apricot, orange a n d pear) a n d fruit (tomato) cuticles a n d c o m p a r e d its p e n e t r a t i o n to t h a t of other c o m m o n p l a n t g r o w t h substances. These cuticles were selected because t h e y are a s t o m a t o u s , similar in m o r p h o l o g y a n d c h e m i s t r y to those of n u m e r o u s economic plants, can be effectively isolated a n d d a t a are a v a i l a b l e on t h e i r p e r m e a b i l i t y to various substances. Materials and Methods Preparation o] Cutieular Membranes Cnticular membranes were enzymatically separated from the underlying epidermal cells of mature greenhouse-grown tomato (Lycopersion esculentum Mill. 'Mich.-Ohio Hybrid'), and from the upper epidermis of fully expanded apricot ( Prunus armeniaca L. ' Goldcot'), pear (Pyrus c~rmmunis L. ' Bartlett ') and orange (Citrus sinensis L. Osbeck) leaves by methods previously described (Norris and
262
A. Blumenfeld and M. J. Bukovac:
GLASS TUBE
i
SCREW
g
VIAL
z
DONOR SOLUTION - rr
-
-
RECEIVER SOLUTION
Fig. 1. Diagram of the apparatus used for penetration studies
Bukovac, 1968). Wax-free cuticular membranes were prepared by extraction with chloroform for 24 h at 21 ~ C. The extracting solvent was renewed 5 times during the extraction period.
Penetration Apparatus Penetration was measured with diffusion units described by Morris and Bukovae (1969) or as detailed below. Thick-walled glass tubes (10 cm long) with isolated cuticle affixed to one end (morphological outer surface oriented toward the tube) served as donor units. Such units were fitted through a screw cap and inserted into a receiver solution (distilled water) contained in a glass scintillation vial (Fig. 1).
Measurement o] Penetration A donor solution, consisting of 0.4 or 0.5 ml of racemic ABA, at a predetermined concentration and pH, was added to the donor tube after equilibrating the affixed cuticle with water in the receiver for 2 h. The donor tube was then adjusted to equate the levels of the donor and receiver solutions. All units were held in a water b a t h at 25 ~ C. At predetermined times the receiver units were exchanged and the ABA in the receiver determined. a) Spectrophotometrie Determination. Non-buffered ABA solution (2000 rag/l) was added to the donor tube. The receiver was collected after 12, 24 and 48 h. The p H of the receiver solution was adjusted to 9.0 with NaOH and partitioned against petroleum ether 3 times. This step was necessary to remove interfering substances, which absorbed over a broad range (240-263 nm), masking the absorbance of ABA. We consider these data of relative value only, since complete separation was difficult. The receiver solution was then acidified to p H 2.8 and extracted with ether. The ether residue was dissolved in 0.005 N sulfuric acid-methanol and absorbance read at 263 n m with a Beckman D U Spectrophotometer (Cornforth et al., 1966).
Cuticular Penetration of Abscisic Acid
263
b) Radioisotopic Determination. Racemic (2-1tC)ABA was synthesized by Drs. C. M. Asmundson and O. E. Smith (University of California, Riverside). Unless otherwise stated, the donor solution was buffered at pI-I 4.8 with phosphate-citrate, and (2-14C)ABA(spec. act., 26 me/mmole) was present at 100 rag/1. For time-course studies, the receiver solution was changed after 3, 9, 21, 45 and 69 h and assayed for radioactivity. The effect of pH was determined at the pK (pH 4.8) and one pH unit below (pH 3.8) and above (pH 5.8). A comparison of the penetration of ABA with that of other plant growth substances, namely, 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthaleneacetic acid (NAA) was made using tomato fruit cuticle. Equimolar concentrations (5 • 10-aM) of the three compounds were buffered at a pH equal to the pK, i. e., pII 2.8 for 2,4-D, pH 4.2 for NAA and pH 4.8 for ABA. Radioactivity was determined by lyophilizing the receiver solution, adding 10 ml of scintillationfluid (2,5-diphenyloxazoh naphthalene: dioxane), and counting at 5% error or less in a Beckman Liquid Scintillation Spectrometer (Model 100). Biological Assay Aliquots from the experiment measuring penetration speetrophotometrieally were reduced to dryness. The residue was taken up with buffer and assayed following essentially the procedure of Nitsch and Nitsch (1956), using wheat (Triticum aestivum L.) coleoptiles.
Chromatography The purity of the (2-1aC)ABAand confirmation that the molecule was not altered during transfer across the cuticle was established with thin-layer chromatography. Silica gel was used as the solid phase and toluene:ethyl acetate:acetic acid (15:3:1, v/v), chloroform:ethyl acetate: acetic acid (60:40 : 5, v/v), or chloroform: ethyl acetate: formic acid (60: 40: 5, v/v) as the developing solvent.
Results
Penetration Determined Spectrophotometrically A B A p e n e t r a t e d the wax-free t o m a t o fruit cuticle, a n d greater quantities were detected i n the receiver with time (Table 1).
Penetration Determined with (2-14C) A B A a) Time-Course and Concentration. (2-14C)ABA p e n e t r a t i o n across the t o m a t o fruit cuticle was linear with time (Figs. 2, 3). The a m o u n t penet r a t i n g i n 45 h was a p p r o x i m a t e l y 2.5 % of the t o t a l in the donor solution. A 10-fold increase i n c o n c e n t r a t i o n resulted i n a p p r o x i m a t e l y a 75% increase i n p e n e t r a t i o n . b) E[ject o] pH. Greater quantities of (2-14C)A B A p e n e t r a t e d from the donor a t a p H below ( p i t 3.8) t h a n above (pH 5.8) or a t the p K (pH 4.8). P e n e t r a t i o n was linear with time for each h y d r o g e n - i o n c o n c e n t r a t i o n (Fig. 3). c) Ellect o/Cuticular Waxes. (2-14C)ABA p e n e t r a t i o n t h r o u g h t o m a t o fruit cuticle was m a r k e d l y e n h a n c e d b y r e m o v a l of cuticular waxes, being a p p r o x i m a t e l y 2-fold greater t h a n t h r o u g h cuticle with wax (Table 2).
264
A. Blumenfeld and M. J. Bukovac: 1.4
o > c~ E
1.0
"5 .-e
0.6
g 0.2
TIME
(h)
Fig. 2. Time-course of (2J4C)ABA penetration through isolated tomato fruit cuticle
Table i. Penetration of ABA through dewaxed tomato fruit cuticle Donor solution was not buffered. Penetration determined speetrophotometrically and b y inhibition of Triticum coleoptile section extension Measurement
Time 12 h
24 h
48 h
Absorbance a t 263 n m
0.038
0.081
0.188
Coleoptile extension ( % inhibition)
5
32
47
Table 2. Effect of cuticular waxes on penetration of (2-14C)ABA through isolated tomato fruit cuticle Radioactivity (cpm) in receiver solution after Measurement
24 h
48 h
+ W a x (as isolated) - - W a x (dewaxed) Ratio ( - - w a x / + wax)
83 158 1.90
173 315 1.82
Cuticular Penetration of Abscisic Acid
265
2.8
2.4
~.
2.0
z
1.6
pH 3.8
6
A
v
(z: Iw z
m <
Cuticular Penetration of Abscisic Acid A m o s B l u m e n f e l d a n d M a r t i n J. B u k o v a c Department of Horticulture, Michigan State University, East Lansing, Michigan, U.S.A. Received May 5, 1972 Summary. Penetration of 2-14C abscisic acid (ABA) through enzymatically isolated cuticles from tomato fruit and from the upper epidermis of apricot, pear and orange leaves was assessed. Penetration was linear with time, greater as the undissociated than the dissociated ion, and greater through dewaxed than nondewaxed cuticles. Significantly less (3-6 times) (2-14C)ABA penetrated the tomato fruit cuticle than NAA or 2,4-D. The leaf cuticles were less permeable than the tomato fruit cuticle. There was no evidence that the ABA was altered during transfer across the cuticle. Introduction
M a x i m u m p l a n t responses to abscisic acid (ABA) g e n e r a l l y h a v e been o b t a i n e d o n l y a f t e r t h e r e g u l a t o r h a d been i n t r o d u c e d t h r o u g h cut surfaces, or following r e p e a t e d foliar a p p l i c a t i o n s (E1-Antably et al., 1967; S m i t h et al., 1968; A d d i c o t t a n d Lyon, 1969). Two e x p l a n a t i o n s h a v e been offered for these observations. F i r s t l y , t h a t A B A does n o t p e n e t r a t e or p e n e t r a t e s v e r y p o o r l y into the p l a n t (E1-Antably et al., 1967; Milborrow, 1969b). Secondly, t h a t A B A is r a p i d l y m e t a b o l i z e d in t h e p l a n t to substances with lower or no biological a c t i v i t y (Milborrow, 1969a; Sondh e i m e r et al., 1971). I n this s t u d y we h a v e d i r e c t l y assessed t h e p e n e t r a t i o n of A B A t h r o u g h selected leaf (apricot, orange a n d pear) a n d fruit (tomato) cuticles a n d c o m p a r e d its p e n e t r a t i o n to t h a t of other c o m m o n p l a n t g r o w t h substances. These cuticles were selected because t h e y are a s t o m a t o u s , similar in m o r p h o l o g y a n d c h e m i s t r y to those of n u m e r o u s economic plants, can be effectively isolated a n d d a t a are a v a i l a b l e on t h e i r p e r m e a b i l i t y to various substances. Materials and Methods Preparation o] Cutieular Membranes Cnticular membranes were enzymatically separated from the underlying epidermal cells of mature greenhouse-grown tomato (Lycopersion esculentum Mill. 'Mich.-Ohio Hybrid'), and from the upper epidermis of fully expanded apricot ( Prunus armeniaca L. ' Goldcot'), pear (Pyrus c~rmmunis L. ' Bartlett ') and orange (Citrus sinensis L. Osbeck) leaves by methods previously described (Norris and
262
A. Blumenfeld and M. J. Bukovac:
GLASS TUBE
i
SCREW
g
VIAL
z
DONOR SOLUTION - rr
-
-
RECEIVER SOLUTION
Fig. 1. Diagram of the apparatus used for penetration studies
Bukovac, 1968). Wax-free cuticular membranes were prepared by extraction with chloroform for 24 h at 21 ~ C. The extracting solvent was renewed 5 times during the extraction period.
Penetration Apparatus Penetration was measured with diffusion units described by Morris and Bukovae (1969) or as detailed below. Thick-walled glass tubes (10 cm long) with isolated cuticle affixed to one end (morphological outer surface oriented toward the tube) served as donor units. Such units were fitted through a screw cap and inserted into a receiver solution (distilled water) contained in a glass scintillation vial (Fig. 1).
Measurement o] Penetration A donor solution, consisting of 0.4 or 0.5 ml of racemic ABA, at a predetermined concentration and pH, was added to the donor tube after equilibrating the affixed cuticle with water in the receiver for 2 h. The donor tube was then adjusted to equate the levels of the donor and receiver solutions. All units were held in a water b a t h at 25 ~ C. At predetermined times the receiver units were exchanged and the ABA in the receiver determined. a) Spectrophotometrie Determination. Non-buffered ABA solution (2000 rag/l) was added to the donor tube. The receiver was collected after 12, 24 and 48 h. The p H of the receiver solution was adjusted to 9.0 with NaOH and partitioned against petroleum ether 3 times. This step was necessary to remove interfering substances, which absorbed over a broad range (240-263 nm), masking the absorbance of ABA. We consider these data of relative value only, since complete separation was difficult. The receiver solution was then acidified to p H 2.8 and extracted with ether. The ether residue was dissolved in 0.005 N sulfuric acid-methanol and absorbance read at 263 n m with a Beckman D U Spectrophotometer (Cornforth et al., 1966).
Cuticular Penetration of Abscisic Acid
263
b) Radioisotopic Determination. Racemic (2-1tC)ABA was synthesized by Drs. C. M. Asmundson and O. E. Smith (University of California, Riverside). Unless otherwise stated, the donor solution was buffered at pI-I 4.8 with phosphate-citrate, and (2-14C)ABA(spec. act., 26 me/mmole) was present at 100 rag/1. For time-course studies, the receiver solution was changed after 3, 9, 21, 45 and 69 h and assayed for radioactivity. The effect of pH was determined at the pK (pH 4.8) and one pH unit below (pH 3.8) and above (pH 5.8). A comparison of the penetration of ABA with that of other plant growth substances, namely, 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthaleneacetic acid (NAA) was made using tomato fruit cuticle. Equimolar concentrations (5 • 10-aM) of the three compounds were buffered at a pH equal to the pK, i. e., pII 2.8 for 2,4-D, pH 4.2 for NAA and pH 4.8 for ABA. Radioactivity was determined by lyophilizing the receiver solution, adding 10 ml of scintillationfluid (2,5-diphenyloxazoh naphthalene: dioxane), and counting at 5% error or less in a Beckman Liquid Scintillation Spectrometer (Model 100). Biological Assay Aliquots from the experiment measuring penetration speetrophotometrieally were reduced to dryness. The residue was taken up with buffer and assayed following essentially the procedure of Nitsch and Nitsch (1956), using wheat (Triticum aestivum L.) coleoptiles.
Chromatography The purity of the (2-1aC)ABAand confirmation that the molecule was not altered during transfer across the cuticle was established with thin-layer chromatography. Silica gel was used as the solid phase and toluene:ethyl acetate:acetic acid (15:3:1, v/v), chloroform:ethyl acetate: acetic acid (60:40 : 5, v/v), or chloroform: ethyl acetate: formic acid (60: 40: 5, v/v) as the developing solvent.
Results
Penetration Determined Spectrophotometrically A B A p e n e t r a t e d the wax-free t o m a t o fruit cuticle, a n d greater quantities were detected i n the receiver with time (Table 1).
Penetration Determined with (2-14C) A B A a) Time-Course and Concentration. (2-14C)ABA p e n e t r a t i o n across the t o m a t o fruit cuticle was linear with time (Figs. 2, 3). The a m o u n t penet r a t i n g i n 45 h was a p p r o x i m a t e l y 2.5 % of the t o t a l in the donor solution. A 10-fold increase i n c o n c e n t r a t i o n resulted i n a p p r o x i m a t e l y a 75% increase i n p e n e t r a t i o n . b) E[ject o] pH. Greater quantities of (2-14C)A B A p e n e t r a t e d from the donor a t a p H below ( p i t 3.8) t h a n above (pH 5.8) or a t the p K (pH 4.8). P e n e t r a t i o n was linear with time for each h y d r o g e n - i o n c o n c e n t r a t i o n (Fig. 3). c) Ellect o/Cuticular Waxes. (2-14C)ABA p e n e t r a t i o n t h r o u g h t o m a t o fruit cuticle was m a r k e d l y e n h a n c e d b y r e m o v a l of cuticular waxes, being a p p r o x i m a t e l y 2-fold greater t h a n t h r o u g h cuticle with wax (Table 2).
264
A. Blumenfeld and M. J. Bukovac: 1.4
o > c~ E
1.0
"5 .-e
0.6
g 0.2
TIME
(h)
Fig. 2. Time-course of (2J4C)ABA penetration through isolated tomato fruit cuticle
Table i. Penetration of ABA through dewaxed tomato fruit cuticle Donor solution was not buffered. Penetration determined speetrophotometrically and b y inhibition of Triticum coleoptile section extension Measurement
Time 12 h
24 h
48 h
Absorbance a t 263 n m
0.038
0.081
0.188
Coleoptile extension ( % inhibition)
5
32
47
Table 2. Effect of cuticular waxes on penetration of (2-14C)ABA through isolated tomato fruit cuticle Radioactivity (cpm) in receiver solution after Measurement
24 h
48 h
+ W a x (as isolated) - - W a x (dewaxed) Ratio ( - - w a x / + wax)
83 158 1.90
173 315 1.82
Cuticular Penetration of Abscisic Acid
265
2.8
2.4
~.
2.0
z
1.6
pH 3.8
6
A
v
(z: Iw z
m <
