Factors affecting the absorption of gibberellin A3 by sour cherry leaves

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Factors affecting the absorption of gibberellin by sour cherry leaves

A3

Moritz Knoche, Norman K. Lownds and M a r t i n J. Bukovac* Department of Horticulture, Michigan State University, East Lansing, MI 48824, USA

Abstract

Keywords

The effects of leaf surface, light, temperature, time, concentration, and pH on absorption of[ 14C]gibberellin A3 (GA3) into newly expanded sour cherry (Prunus cerasus L., cv. Montmorency) leaves were investigated. GA3 penetration was sevenfold greater into the abaxial (7.8%) than adaxial (1.1%) surface. Light markedly enhanced penetration into the abaxial but not the adaxial surface. Penetration was greater at 25°C than at 15 ° or 35°C. GA3 absorption by the adaxial surface was rapid initially (0-1 h), followed by a slower, almost constant rate of uptake after the droplets dried (1 24h). Rate of GA3 uptake by the abaxial surface approached zero after 3h. Uptake by the tissue fraction of the adaxial surface was linearly related (r 2 = 0.99**) with GA3 concentration from 0.05 to 5 raM, whereas uptake by the abaxial surface was saturated between 1 and 5 mM GA3. Decreasing pH from 5.0 to 3.0 resulted in increased GA3 uptake. Gibberellin A3; foliar absorption; pH; temperature; light; Prunus

Introduction P e r f o r m a n c e o f foliar-applied a g r o c h e m i c a l s d e p e n d s on c o m p l e x interactions b e t w e e n the s p r a y solution and the plant surface (Baker, 1980). T h e effectiveness o f a s p r a y can be further influenced by e n v i r o n m e n t a l c o m p o n e n t s d u r i n g a n d after s p r a y a p p l i c a t i o n ( B u k o v a c , R e i c h a r d and W h i t m o y e r , 1986a). The.. dose o f the biologically active ingredient retained on the leaf, p e n e t r a t i n g the cuticle and reaching the site o f action, d e t e r m i n e s the biological response for systemic c o m p o u n d s ( G r e e n e a n d B u k o v a c , 1971; K i r k w o o d , M c K a y and Livingstone, 1982). Consistency in u p t a k e is o f p a r t i c u l a r i m p o r t a n c e in the p e r f o r m ance o f g r o w t h - r e g u l a t i n g ,;ubstances because o p t i m u m response is often o b t a i n e d over a n a r r o w dose range. I n c o n s i s t e n t p e r f o r m a n c e , a severe limitation to field use of g r o w t h regulators, m a y be ]'elated to differences in penet r a t i o n (Bukovacetal., 1986a; G r e e n e and Bukovac, 1971). A better u n d e r s t a n d i n g o f factors affecting m o v e m e n t o f the active ingredient f r o m deposit on the surface to site o f action is, therefore, a prerequisite for optimizing spray p e r f o r m a n c e and increasing consistency. Foliar application o f gibberellic acid to sour cherry trees has been s h o w n to inhibit flower initiation and to p r o m o t e spur f o r m a t i o n . G A 3 application has b e c o m e a r e c o m m e n d e d practice o f increasirLg fruiting potential and cropping efficiency ( B u k o v a c et al., 1986b); however, no data are available on factors affecting p e n e t r a t i o n o f G A 3 into sour cherry leaves. F o r this reason, the effects o f several selected factors on the a b s o r p t i o n o f G A 3 by sour cherry leaves that m a y lead to m o r e efficient spray application and *To whom correspondence shouh:l be addressed

cerasus

p e r f o r m a n c e were investigated. The effects o f spray application (leaf surface, time, light, t e m p e r a t u r e ) and spray solution factors (concentration, p H ) were particularly examined.

Materials and methods Plants

Sour cherry (Prunus cerasus L., cv. M o n t m o r e n c y ) trees growing at the H o r t i c u l t u r e Research Center in East Lansing, MI, were not sprayed with pesticides during shoot extension to provide a source o f pesticide-free leaf material. E x p e r i m e n t s were c o n d u c t e d between 1 June and 30 July, 1988. Young, fully e x p a n d e d leaves f r o m the third t h r o u g h fifth nodes ( f r o m the apex) o f current season's shoots were detached 3-4 h after d a y b r e a k . Chemicals

The spray solution was p r e p a r e d at a c o n c e n t r a t i o n o f 0.5 mM G A 3 unless specified otherwise. Specific activity o f the [1, 7, 12, 18JgC]gibberellin A3 ( A m e r s h a m Corp., Arlington Heights, IL, 9 9 % pure) was 259 M B q m m o l 1 Radiolabelled G A 3 was diluted with cold G A 3 (Sigma Chemical C o m p a n y , St Louis, M O , > 9 9 % pure) only for the highest level of the c o n c e n t r a t i o n response study. Solutions were buffered with 20raM citric acid and p H adjusted to 3.0 with N a O H unless otherwise stated. T r e a t m e n t and m e a s u r e m e n t of foliar absorption

T w o discs (21 m m diameter) were p u n c h e d f r o m each leaf, one from either side o f the midrib, and floated on distilled

0261-2194/92/01/0057-07 © 1992 Butterworth-HeinemannI..td CROP PROTECTION Vol. 11 February 1992

58 Absorption of GA3 by sour cherry leaves: M. Knoche et al.

water in Petri dishes held in a water bath at 25°C. Environmental conditions were as follows: photoperiod, 16 h; photosynthetically active radiation (PAR), 230 lamol s- 1 m - 2 except for the temperature study where PAR was 140 gmmol s- i m - 2 The GA3 solution was applied as 15 (0.24gl each) droplets per disc using a microsyringe fitted with a Tefloncoated needle mounted in an automatic dispenser (Hamilton, Reno, NV). GAg application was initiated within 2 h of collection of leaves. Uptake was determined 24 h after treatment except for the time-course study. Droplet residues were removed from the leaf surface by rinsing with 5 ml acetone : water (3 + 2 by volume). A 1 ml aliquot of the rinse was added to 10ml Safety Solve scintillation cocktail (Research Products International Inc., Mount Prospect, IL). Subsequently, the epicuticular wax was removed by stripping with cellulose acetate (Price, 1982; Silcox and Holloway, 1986) and dissolved in 10ml of a dioxane-based scintillation cocktail [1,4-dioxane containing naphthalene (100 g l ~) and 2,5-di-phenoloxazole (5g l-~)]. The leaf discs were oxidized at 900°C in a biological oxidizer (Model OX-400 Harvey Inc., Hillsdale, NJ) and the evolved 14CO2 trapped in a toluene-based scintillation cocktail containing CarboSorb. Corrections for background, efficiency and quenching were made where necessary. Samples were counted for 5 rain in a scintillation spectrometer (Model 1211, Rackbeta, LKB Wallac, 20101 Turku 10, Finland). GAg absorption, calculated as percentage of applied activity, was determined for the following fractions (Stevens and Bukovac, 1987): surface deposit (14C in acetone :water surface rinse); wax (t4C in cellulose acetate wax stripping); tissue (14C in oxidized leaf tissue after stripping); wax + tissue (sum of radioactivity in wax and tissue fractions). Average recovery of radioactivity was 97.6%.

Effects of spray application factors The time course of GA3 (0.5 mM, pH 3.0) uptake through adaxial and abaxial surfaces was determined over a 24 h period (1, 3, 6, 12 and 24h after treatment). The effect of light on GA3 (1.0mM, pH3.0) uptake through adaxial and abaxial leaf surfaces was determined by transferring one set of leaf discs to the dark after droplet drying. For comparison a second set was retained in the light for the entire 24 h absorption period. Relative humidity in the dark approached 100%, and was 60 + 10% in the light. Effects of temperature on GA 3 (0.5 raM, pH 3.0) absorption were measured by holding leaf discs in Petri dishes in water baths at temperatures of 15°, 25 ° and 35°i0.5°C. Absorption was followed through the abaxial surface only.

Effect of spray solution pH on GA3 (0.5 mM) uptake was examined at pH 3.0, 4.0 and 5.0 using the abaxial surface only. A citrate buffer stock solution was prepared at pH3.0. Aliquots were titrated to pH4.0 and 5.0 using NaOH. The degree of GA 3 dissociation was calculated for each pH according to the Henderson-Hasselbach equation using a pKa value of 4.0 (Tidd, 1964; Durley and Pharis, 1972). Contact a n g l e and surface tension m e a s u r e m e n t s

Wettability of sour cherry leaf surfaces was characterized by determining the contact angles of 1.0 I,tl droplets of distilled water on each surface. Base width and height of droplets were measured and contact angles calculated according to Mack's equation (Mack, 1936). Each determination consisted of five droplets per leaf surface replicated ten times. Surface tension of the spray solution (0.5mM GA3, pH 3.0) was measured using a Fisher surface tensiometer (Model 20, Fisher Scientific, Pittsburgh, PA). Each determination was repeated five times. Scanning electron microscopy (SEM) Leaf material for SEM studies was collected from the field, frozen immediately after excising discs and lyophilized. Samples were then mounted on aluminium stubs, coated with gold and observed with a JEOL JSM-35CF SEM. Specimens were photographed at accelerating potentials of 15 kV using positive/negative 665 Polaroid film (Polaroid, Cambridge, MA). Data analysis Except for the temperature and light studies, a completely randomized experimental design was used. Data were subjected to analysis of variance where appropriate. Comparison of treatment means was made using Duncan's multiple range test. Ten replications were used for most experiments; six replications were used in the light experiment. Standard errors were included in all graphs. Where not shown, they were smaller than data symbols. Curves were fitted using treatment means and the non-linear regression model Y= (Amax'X)/(k + X). The choice of this model was based on the assumption that GA3 absorption into leaf tissue is a saturation process approaching an asymptote with time. In this model Amax represents the asymptote value and k the time after treatment when Amax/2 is attained. Hence, k gives an estimate of the velocity of absorption.

Results C h a r a c t e r i z a t i o n of the leaf surface

Effects of spray solution factors Concentration response (pH3.0) was established on adaxial and abaxial surfaces at GAg concentrations of 0.05, 0.1, 0.5, I and 5mM.

CROP PROTECTION Vol. 11 February 1992

Scanning electron microscopy revealed that the adaxial surface was astomatous (Figure la). The interveinal area of the adaxial lamina was characterized by two types of ridges. First-order ridges formed a

A b s o r p t i o n of GA 3 by s o u r c h e r r y leaves: M. K n o c h e et al.

59

tion by the tissue fraction, no significant difference in GA 3 partitioning into the epicuticular wax fraction was found between the two surfaces. GA 3 had a low affinity for sour cherry leaf wax (Table 1). GA3 absorption into the tissue fraction was influenced by light (Table2). Penetration of GA3 into the abaxial surface in the light was greater than in the dark, whereas light did not enhance GA3 penetration into the adaxial surface. Temperature had a marked effect on GA3 uptake (wax+tissue fraction) through the abaxial surface (Table3). GA3 uptake was greatest at 25°C; an increase in temperature to 35°C or a decrease to 15°C reduced uptake.

Spray solution factors Penetration of GA3 into the adaxial tissue fraction was rapid during the initial 1 h period (Figure2, Table4). This rapid-uptake phase was followed by a second phase (1 24h) with an almost constant uptake rate, 0.36ng GA 3 h ~ (r 2 =0.97**). In contrast, penetration into the abaxial surface followed an asymptotic curve. Three hours after treatment, the slope of the GA 3 uptake curve (tissue fraction) approached zero. GA 3 partitioning into the wax fraction followed a similar pattern. Radioactivity in the Figure 1. Scanning electron micrographs of adaxial and abaxial sour cherry leaf surfaces: overview of veinal and interveinal area el adaxial (a) and abaxial (b) surfaces; adaxial surface over vein and interveinal area (c); stomata on abaxial surface and area adjacent to vein (d); surface fine structure over veins of the adaxial (e) and abaxial (f) surfaces

regular net (Figure la). Second-order ridges were present on the leaf surface surrounded by the first-order ridges. Although first-order ridges were not present on the abaxial surface, fine structure was generally similar to that on the adaxial surface (Figure la, b, c, d). The leaf surface in the vicinity of stomata appeared to be lacking in fine structure (Figure ld). First-order ridges were generally running parallel above the veins (Figure la, b, e, f). There was no evidence of crystalline epicuticular wax on either surface

(Figure l a-f). Wettability, as measured by the contact angle formed with distilled water, was significantly different between the two surfaces, 57 + 2.2 degrees on the adaxial and 76 4- 1.8 degrees on the abaxial surface, respectively.

Spray application factors Based on a summary of all experiments conducted (80 observations for the adaxial and 100 for the abaxial surface) under similar conditions (i.e. 0.5 mM GA3, 25°C, 230 gmoi s- 1 m - z, pH 3.0), there was sevenfold greater absorption of GA3 by the abaxial surface (tissue fraction) than the adaxial surface (Tablel). GA3 absorption through the adaxial surface averaged 1.1% of that applied, whereas uptake by the abaxial surface averaged 7.8 %. The range of means and coefficients of variation indicated high variability for GA 3 absorption. In contrast to GA3 absorp-

Table 1. Effect of leaf surface on GA 3 penetration into the epicuticular wax and tissue fractions of sour cherry leaves" G A 3 absorption (percentage of applied) Leaf surface

Fraction

Mean

Range

Coefficient of variation (%)

Adaxial

Wax Tissue

0,5 1,1

0.2-1. I 0.2 1.9

57.8 58.6

Abaxial

Wax Tissue

0.6 7.8

0.3-0.7 3.3-13.6

62.4 40.8

"Data represent average of all experiments conducted under comparable conditions (0.5 mM GA 3, pH 3.0, 230 gmol s ~m z, 25oc) with n = 80 for the adaxial and n = 100 for the abaxial surface, respectively

Table 2. Effect of light on GA 3 penetration (mean 4- s.c.) into the tissue fraction of sour cherry leaves G A 3 recovered in tissue fraction (percentage of applied) Leaf surface

Light

Dar k

Adaxial Abaxial

0.9 ± 0.2 13.7 5:2.4

3.3 + 0.3 8.8 4- 1.3

Table 3. Effect of temperature on GA s penetration (mean + s.c.) into the abaxial surface of sour cherry leaves Temperature (°C) 15 25 35

G A 3 in wax + tissue fractions (percentage of applied) 6.3+ 1.3 13.44- 1.7 8.34- 1.7

CROP PROTECTION Vol. 11 F e b r u a r y 1992

60

A b s o r p t i o n of GA 3 by s o u r c h e r r y leaves: M. K n o c h e et al. Light

Dark

Light

between 1 and 5mM, GA3 absorption was saturated. A significant surface effect was observed at all GA3 concentrations used (Figure 3). Solution p H had a marked effect on GA3 penetration (Figure4). When p H was increased from 3.0 to 4.0, the amount of GA3 in the tissue fraction was closely related to the presence of non-dissociated ion. However, at pH 5.0, this close relationship was less evident.

a

Discussion "6 g I

r--

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I

8 b tO

o

6

i

<

I T

'

T

2 ¢) 0 0

4

8

i 16

12

i

2t4

20

Time after treatment (h)

Differences in uptake between leaf surfaces, similar to those observed in our study, have been reported for other compounds and species (Sargent and Blackman, 1962; Sargent, 1968; Greene and Bukovac, 1971, 1974; Sch6nherr and Bukovac, 1972). Regardless of GA3 concentration, uptake by the abaxial surface was always greater. As the adaxial surface was more easily wetted than the abaxial, differences in absorption between leaf surfaces cannot be attributed to differential wettability. The study of leaf surface morphology revealed that the adaxial surface was astomatous whereas the abaxial surface contained stomata (Figure 1). Mass flow of an aqueous spray solution into the substomatal chamber is unlikely unless the surface tension is reduced to < 30 m N m - 1 (Sch6nherr

Figure 2. Time course of GAg penetration into the wax (©), tissue (O) and w a x + t i s s u e (El) fractions of the (a) adaxial and (b) abaxial surface of sour cherry leaves 20

Table4. Parameters Amax and k of regression equations for time

15

course of GA 3 penetration into different fractions of sour cherry leaves a

Regression coefficient 4-s.e.

Leaf surface and fraction

Area×

k

Coefficientof determination (r2)

5.66:t:0.45 2.58±0.45 3.45+0.43

0.94 0.93 0.94

a

10

5

Adaxial Wax Tissue Wax+tissue

1.20±0.16 1.894-0.12 3.064-0.13

Abaxial Wax Tissue Wax+tissue

0.554-0.10 4.984-0.13 5.38+0.12

0.414-0.86 1.114-0.65 0.964-0.67

0.89 0.87 0.89

~(Absorptionin %) [Amax× (timein h)]/[k+ (timein h)]

A C r. 0

o ,.0 ,<

0 80

I

I

I

I

I

I

I

1

2

3

4

I

b

6O

=

wax fraction of the adaxial surface increased rapidly at first (0-1h) then increased slowly with time (1-24h). The amount o f G A 3 sorbed into the wax fraction on the abaxial surface levelled off within I h. GA3 uptake into the adaxial tissue fraction increased almost linearly with an increase in GA3 concentration (Figure 3). Partitioning into the wax fraction levelled off at 1 mM. GA3 uptake into the abaxial tissue fraction was linear only between 0.05 and 1 mM. At concentrations

CROP PROTECTION Vol. 11 F e b r u a r y 1992

2O

0

,

I

5

Concentration {mM ) Figure3. Effect of concentration on GA 3 penetration into wax (©), tissue ( 0 ) and w a x + t i s s u e (E3) fractions of the (a) adaxial and (b) abaxial surface of sour cherry leaves

A b s o r p t i o n of GA 3 by s o u r c h e r r y leaves: M. K n o c h e etal. 7 A

20

"6

5

A

40 $ e-

.9 o

3

60

g o C3

2 80

1

..Q

<

® I

I

3.0

4.0

100 5.0

pH Figure4. Effect of spray solution pH on GA 3 penetration into the tissue ( • ) and wax (©) fractions; of the abaxial surface of sour cherry leaves. The degree of dissociation of GA 3 molecules (ram) was calculated according to the Henderson-Hasselbach equation using a pKa of 4.0

and Bukovac, 1972; Greene and Bukovac, 1974, 1977). In our studies, the surface tension of the spray solution (0.5 mM GA3, 20 mM citrate buffer) was 71.0 m N m 1 Other factors, including cuticle thickness, density and polarity of the epicuticular wax over the guard cells and around the stomatal pore (Turrell, 1947; Jyung, Wittwer and Bukovac, 1965; Taylor, Davies and Cobb, 1981) and a favourable microclimate (i~igh humidity) around open stoma, may affect penetration through stomatous surfaces. An important factor may be the higher energy status of guard and accessory cells (Martin and Juniper, 1970; Greene and Bukovac, 1971, 1977), which are the only epidermal cells with chloroplasts. To our knowledge, no information is available on cuticle thickness and density and on polarity of the wax i:a these localized areas for sour cherry. It is clear that the fine structure of the cuticle over guard and accessory cells differed from the surrounding interveinal tissue (Figure 1). No ridge-type structures were found over the guard cells in close proximity to the stomatal pore. The importance of these morphological differences in relation to upl:ake is unknown. Time course and concen~Lration responses between the adaxial and abaxial surfaces differed both in level and kinetics of uptake (Figures 2 and 3). The rapid initial phase (0-1 h) of GA3 absorption into the adaxial surface was expected because diffusion occurs primarily during the liquid phase of the droplet (Hull, 1970; Stevens, Baker and Anderson, 1988). Once the droplet dries, the concentration gradient, and therefore the flux across the cuticular membrane, would be expected to remain constant, resulting in a constant rate of uptake, as was observed [(uptake into tissue in ng)= 0.36 x (time in h) + 6.29; r 2 = 0.97**]. Owing to the apparent low permeability of the adaxial cuticle to GA3, the concentration at the physiological inner side would be expected to be veJry low. Molecules that diffuse across the cuticle would be removed quickly from below the droplet application zone, either by uptake into the symplast or by diffusion within the apoplast. Hence, within the time period studied, no .significant decrease in the rate of uptake occurred from the deposit residue. Furthermore,

61

GA 3 uptake through the adaxial surface into the tissue fraction (Figure3) increased linearly with increasing concentration [(uptake into tissue in rig)=2.07 x (concn in raM) + 0.98; r 2 = 0.99***]. These data suggest that diffusion through the cuticle was the rate-limiting process in uptake by the adaxial surface. Similar relationships were found for naphthaleneacetamide uptake by the adaxial pear leaf surface (Greene and Bukovac, 1971). GA3 uptake into the abaxial tissue fraction as a function of time and concentration showed a different relationship. Three hours after treatment the amount absorbed was --~4.5-fold greater than for the adaxial surface and no significant additional radiolabel accumulated in the tissue fraction over the remainder of the experimental period (Figure2, tissue fraction). As cuticular penetration is a diffusion process (Franke, 1967; Sargent, 1968; Price, 1982), the concentration gradient (Co-C0 across the cuticle determines the rate of penetration for a given membrane (Sargent, 1968; Price, 1982). Thus, if one assumes that after the droplets dry, the concentration outside the cuticle (Co) remains constant, the rate of uptake would decrease and approach zero only if the internal concentration (Ci) increases and approaches that of the external concentration (Co). Employing the calculations proposed by Sch6nherr and Bukovac (1978), it is evident that the internal concentration (Ci) could not have increased sufficiently to cause a decrease in the rate of penetration if a cylindrical-shaped volume of 10 p.m depth at the cell side a n d 106 gm 2 cross-sectional area (droplet/cuticle interface area) was used to calculate Ci. This calculation is based on the assumption that the molecules are equally distributed in this compartment. Horizontal and vertical transport and diffusion would also be expected to occur, yielding an even lower internal tissue concentration than predicted by this simplified model. It is clear from the above, that the rate of uptake can approach zero only if the driving force for diffusion is reduced by either an increase in Ci by an accumulation of GA3 in the apoplast or by a decrease in Co in the deposit residue on the cuticular surface. A decrease in the concentration gradient may result if a boundary layer is formed at the residue/cuticle interface or at the cuticle/cell-wall interface. The residue/cuticle interface of a macroscopically dry deposit can be visualized as a saturated solution of GA3 and buffer salts hydrated by cuticular and stomatal transpiration. This interface may become depleted of the active ingredient (a.i.) during the course of penetration, hence the formation of a boundary layer. Similarly, although less likely, a boundary layer may form at the cuticle/cell-wall interface, particularly in leaf disc systems where export of the penetrated a.i. may be limited. As boundary layers are more likely to form under conditions of high uptake, this hypothesis would explain the difference in level and kinetics of GA 3 uptake observed between the adaxial and abaxial surface. Accumulation of GA3 in the apoplast on the cell-wall side of the cuticle may also result from limited metabolic uptake into the symplast. Enhancement of uptake by light has been reported for other growth substances and species (Sargent, 1968; Greene and Bukovac, 1971, 1977; Sch6nherr and Bukovac, 1978) including our results with

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62

Absorption of GA 3 by sour cherry leaves: M. Knoche et al.

GA3 uptake into the abaxial surface of sour cherry ( T a b l e 2 ) . As stomata are present only on the abaxial surface, and the guard and accessory cells are the only epidermal cells containing chloroplasts, the stimulating effect of light on the lower surface indicates involvement of a metabolic process (Greene and Bukovac, 1971, 1977). It should be noted, however, that the effect of light on the energy status of guard and accessory cells is confounded with the light effect on stomatal opening. Because stomatal infusion can be excluded, the higher permeability of cuticular ledges in open stomata could also contribute to light enhancement of uptake (Sch6nherr and Bukovac, 1978). Temperature coefficients (Q10) were used in earlier studies as evidence for a metabolic or a physical component limiting the penetration process (Sargent and Blackman, 1962). Coefficients between 2 and 3 indicate metabolic processes, whereas coefficients between 1 "and 1.5 indicate physical processes including diffusion (Franke, 1967). The Q10 values in our study were 2.1 and 0.6 for the temperature increase from 15° to 25°C and 25 ° to 35°C, respectively. Norris and Bukovac (1969), however, reported temperature coefficients of up to 8.6 studying N A A penetration through isolated pear cuticles. They pointed out that the lipophilic nature of the cuticle might be a limitation for the use of Q10 values in cuticular penetration studies. In our study, the decrease in GA3 uptake from 25 ° to 35°C (Table 3 ) might be the result of rapid droplet drying at 35°C. Droplet drying times were 18.5, 13 and 5min at 15°, 25 ° and 35°C, respectively. Hence, the possibility of limited cuticle hydration and a shorter period of rapid uptake may account for the decrease at 35°C. The effect of pH on penetration of GA3 ( F i g u r e 4 ) is similar to that observed with weak organic acids (Sargent, 1968; Hull, 1970; Norris and Bukovac, 1972; Greenberg et al., 1984; Shulman, Fanberstein and Bazak, 1987). Two mechanisms are involved: first, increased polarity of the ingredient as a consequence of dissociation is regarded as the most important factor (Norris and Bukovac, 1972) and second, the cuticle has been shown to behave as a negatively charged polyelectrolyte (Sch6nherr and Bukovac, 1973). These two changes most probably account for the decrease in penetration as pH was increased from 3.0 to 4.0. The lack of a close agreement at pH 5.0 may be the result of penetration of some dissociated molecules. This may also be due to the relatively small contribution of the carboxyl group to the polarity of the GA3 molecule. Alternatively, there may be uneven distribution of charge in the cuticle and thus incomplete exclusion of charged molecules (Sch6nherr and Bukovac, 1978). Furthermore, the cuticle is not homogeneous and there may be domains that accommodate polar molecules (Sch6nherr and Bukovac, 1970). Interestingly, our observations are consistent with GA3 penetration into grapefruit peels (Greenberg et al., 1984). The similarity in GA3 partitioning into the epicuticular wax fraction of the adaxial and abaxial surfaces (Table 1) is most probably the result of similar wax deposition and chemistry. As GA3 is a comparatively polar compound [at pH 3.0, octanol/water partition coefficient koct/w= 4.41 (W.

CROP PROTECTION Vol. 11 February 1992

E. Shafer, personal communication)], partitioning into the epicuticular wax would be expected to be low. The wax fraction is a more efficient sorbent for highly apolar compounds (Stevens et al., 1988).

Acknowledgements The authors are grateful to Dr Peter D. Petracek for the scanning electron microscopy, and Drs Warren E. Shafer and Robert J. Cibulsky, Abbott Laboratories, Agricultural Research Center, Long Grove, IL 60047, for the gift of the radiolabelled GA3 and financial support. This study was supported in part by the Deutsche Akademische Austauschdienst (DAAD), the Michigan Agricultural Experiment Station and by a grant (SCA No 58-5114-7-1002) from the Agricultural Research Service, United States Department of Agriculture.

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Received 20 March 1991 Revised 13 June 1991 Accepted 13 June 1991

CROP PROTECTION Vol. 11 February 1992