Naphthaleneacetic Acid Permeability of Citrus Leaf Cuticle

Biochem. Physiol. Pflanzen 170, S. 309-319 (1976)

N aphthaleneacetic Acid Permeability of Oitrus Leaf Cuticle JORG SCHONHERR Institut fiir Botanik und Mikrobiologie Technische Universitat Miinchen Key Term Index: Permeability coefficients, distribution coefficients, diffusion coefficients, cuticular membranes, cuticular waxes, naphthaleneacetic acid; Citrus aurantium, Brassaia spec., Nerium oleander, Lycopersicon esculentum.

Summary The fluxes of naphthalene acetic acid (NAA) across astomatous isolated cuticular membranes (OM) and cutin matrices (MX) (cuticular membranes from which cuticular waxes has been extracted with lipid solvents) of Citrus aurantium L. leaves were analyzed on the basis of distribution (A), permeability (P) and diffusion coefficients (D). NAA distribution coefficients (A)([NAA] in the solid in moles kg-I/[NAA] in buffer solution in moles kg-I) were determined for cuticular membranes and the cutin matrices from leaves of Brassaia spec., Citrus aurantium L., Nerium oleander L., from tomato fruit (Lycopersicon esculentum L.) and for the cuticular waxes triac on tan, hexacosanol and hexadecanoic acid. A (OM) values (133 to 194) were smaller than A (MX) values (167 to 212). At pH values above three (the isoelectric point of the cuticles) distribution coefficients were a function of the degree of dissociation of NAA. The anionic form of NAA could not enter the cutin matrix because of Donnan exclusion. Below pH 3 the NAA anion entered th,e cutin matrix by counter ion exchange. Sorption of the nondissociated form of NAA by the cutin matrix was not influenced by the nature or density of the charges fixed to the cutin matrix. Distribution coefficients for the three cuticular waxes amounted to 5 to 7. Permeability coefficients for diffusion of NAA across the cutin matrix were 170 to 300 times greater than the values measured for cuticular membranes. The data are explained by treating the lipid components of the cuticle (wax and cutin matrix) as two resistances acting in series. NAA permeability of Citrus leaf cuticular membranes is shown to be completely determined by cuticular waxes, because of the very low solubility of NAA in the waxes. All the evidence presented is consistent with the movement of NAA within the lipid components of the membranes and not in aqueous pores.

Introduction

Plant cuticles are lipid membranes made up mainly of cutin (a hydroxyfatty acid polymer) and cuticular waxes (MARTIN and JUNIPER 1970). Consequently, permeability of cuticles was generally found to be positively correlated to lipid solubility of the permeating species (BUKOVAC et al. 1971; DARLINGTON and CIRULIS 1963; SARGENT et al. 1969; WHITE CROSS and MERCER 1972). Lipid solubility was expressed as partition coefficients between chloroform/water (SARGENT et al. 1969; DARLINGTON and CIRULIS 1963), olive oil/water (WHITE CROSS and MERCER 1972) or octanol/water (SARGENT et al. 1969). Cutin/water or cuticular wax/water distribution coefficients appear not to have been determined. Since the dissociated form of weak electrolytes is quite polar, a characteristic pH dependence of penetration of cuticles by weak electrolytes (such as

au"

310

A

J. SCHONHERR

abscisic acid, 2,4-dichlorphenoxyacetic acid and naphthalene acetic acid) was observed (BLUMENFELD and BUKOV AC 1972; BUKovAc et al. 1971). The permeability of cuticles was not related to thickness of the membranes (NORRIS 1974) and permeability to weak acids was generally increased by extraction of cuticular waxes (BuKovAc et al. 1971; BLUMENFELD and BUKovAc 1972; NORRIS 1974). This has led to speculations concerning the role of cuticular waxes in cuticular penetration of weak acids. These results indicate that penetration of cuticles by weak electrolytes cannot be adequately described by a homogeneous membrane model, using only one permeability coefficient. By means of a composite membrane model with two resistances in series it becomes possible to quantitatively separate the effects of cutin and cuticular waxes on penetration. Introduction of diffusion and distribution coefficients helps to analyze the nature of the two resistances. Material and Methods Isolation of cuticles. - Astomatous cuticles from the upper surfaces of leaves of Citrus aurantium L., Brassaia spec., Nerium oleander L., and from tomato (Lycopersicon esculentum L.) fruit were isolated enzymatically by incubation at 38°C in a mixture of 4 % (w/v) pectinase and 0.4 % w/v) cellulase (ION Pharmaceuticals, Inc., Cleveland, Ohio) at pH 3.8 (ORGELL 1955). Exchangeable cations were removed by a treatment with 1 N HCl (SCHONHERR and BUKovAc 1973) followed by washing with deionized water. Isolated cuticles were air dried and stored for further use. Cuticular waxes were extracted by successively placing the cuticles at room temperature in 5 changes each of methanol, chloroform and hexane (30 min each). The amounts of cuticular waxes removed were determined by weighing the cuticles prior to and after extraction. For brevity cuticles from which waxes had been extracted will b,e refered to as cutin matrix (MX). Cuticles which were left unaltered (except for isolation), that is cuticles which still contain all cuticular waxes, will be called cuticular membraues (CM). Distribution and permeability coefficients will be followed by the indices (MX) or (CM), depending on the material used for their determination. Distri bu tion c oeffi cien ts. - Pieces of cuticular membrane, cutin matrix or wax granules (20 mg) were equilibrated with 2 ml of 14C-NAA solutions (6 x 104 dpm ml-l, specific activity 61 mCi mmol- 1; Amersham Buchler). Solutions were buffered with either succinic acid 2,2-dimethylhydrazide (pH 2 to 5) (SCHONHERR and BUKovAL 1972), MES (pH 5 and 6) or HEPEs (pH 7) at a concentration of 0.02 l\£. Equilibrium was reached quickly, but the final sample was taken only 3 to 5 days after starting the experiment. Equilibrium distribution is expressed as distribution coefficient (J.) [NAA] in CM, MX or wax (dpm/g)

J.=

[NAA] in buffer (dpm/g)

(1)

The amount of NAA sorbed within the lipids was calculated from the change in radioactivity of the buffer [initial radioactivity (dpm) - final radioactivity (dpm)]. Perme abili ty c oefficien ts. - Details concerning the transport apparatus, tests for membrane integrity and experimental procedures have previously been described (SCHONHERR 1976). Briefly, the membranes (CM or MX) were inserted between the two compartments of a transport apparatus and 5 ml of identical buffer solution were pipetted into each compartment. The apparatus was placed into a water bath maintained at 25 ± 0.2 °c. The solutions were stirred at 800 rpm by stirring magnets driven by a magnetic stirrer positioned under the water bath. When thermal equilibrium had been reached, 14C-NAA dissolved in 10,u1 methanol was added to the outer solution (solution facing the morphological outer side of the cuticle) resulting in an specific activity of 2 x 107 dpm ml- 1. At zero time and in 30 min. intervals a 1 ml sample was withdrawn from the inner solution (receiver)

N aphthaleneacetic Acid Permeability of Citrus Leaf Cuticle

311

and its radioactivity determined by scintillation counting. Each time a sample had been taken "cold" buffer was added to the receiver to maintain constant volume. At least six successive samples were taken and the rate of diffusion (dnjdt in dpm cm- 2 S-l) under steady state conditions (Co-Ci) = constant) was· determined by the method of least squares. The linear regression coefficient was always better than 0.99. The permeability coefficient of diffusion (P) was determined from equ. (2) dn

1

(2)

P=----

dt Co -

Ci

where Co and Ci are the concentrations of 14C-NAA in the outer and inner solutions, respectively Additional details will be given in the legends. Diffusion coefficients. - NAA diffusion coefficients in the membrane (D) were calculated from the hold up time (te) and the thickness of the membranes (,1 x). According to Helfferich (1962) D = (,1 X)2 (3) 6 te where te is defined as the intersection of the extension of the steady state line with the time axis in a plot of total amount diffused vs. time. Membrane thickness was calculated from the weight of the membranes (g cm- 2 ), assuming a specific gravity of 1.1 g cm- 3 •

Results

Distribution coefficients NAA was strongly sorbed by the cuticular membranes and the cutin matrices. At equilibrium the NAA concentration in the membranes (expressed as mmoles NAA per gram membrane) was 133 to 194 times higher than the NAA concentration in the surrounding buffer solution (expressed as mmoles NAA per gram solution). Extraction of waxes from the cuticular membranes increased the distribution coefficients by 9 to 40% (Table 1). Solubility of NAA in octanol, triacontan, hexacosanol and hexadecanoic acid was much lower (Table 2). Distribution coefficients decreased with increasing pH of the buffer solutions. Experimental A (MX) values coincide with the dissociation curve for NAA, except at pH 2.3 and above pH 6, wehre they were slightly higher (Fig. 1). Sorption of NAA was always directly proportional to the NAA concentration in the buffer solution, that is, the distribution coefficients were independent of NAA concentration within the concentration rage tested (Fig. 2). Table 1. N AA distribution coefficients for various cuticular membranes and cutin matrices Values are averages of duplicate determinations at pH 3.0 and 25°C. Deviations from the mean were smaller than 5 % Species

Distribution coefficient A (CM)

Brassaia spec. Citrus aurantium Lycopersicon esculent. N erium oleander

133 142

174 167

194

212 191

21

137

Bioehem. Physiol. Pflanzen, Bd. 170

Amount of wax A(MX) mg wax(g cuticle 83.9

79.4 26.6 282.8

312

J. SCHONJlERR

Table 2. N AA distribution coefficients for octanol and cuticular waxes Values are averages of duplicate determinations at pH 3.0 and 25°C. Deviations from the mean were smaller than 10 % Compound

Distribution coefficient

octanol triacontan hexacosanol hexadecanoic acid

65.0 7.5 5.5 5.4

300

0

A.(MX)

0

0.02 M butter

.!I

0.02 M buffer, 0.001 M "'aCI

x

0.02 M butter, 0.01 M NaCI

•

Percent NAA ionized

200

25

."

m :;u 0 m z ~

50

Z

» » 0

z

N

100

m

0 75

o

100 6

7

8

9

pH Fig. 1. N AA distribution coefficients;' (MX) as a function of pH for the system tomato fruit cutin matrix/buffer solution. Values are averages of two determinations. Deviations from the mean were smaller than 5 %. Percent ionized was calculated from the equation 100/1 + 10(pKa-PH), (pKa = 4.24), as determined by titration of the sodium salt of NAA with HCI.

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313

Acid Permeability of Citrus Leaf Cutiele

Permeability coefficients Stirring the solutions increased permeability coefficients by 40 % (Table 3). Above 200 rpm there was no effect of the rate of stirring. P(MX) values were 170 to 300 times higher than P(CM) values (Table 3 and 4). P(MX) values decreased with increasing pH. The NAA flux was a function of the pH of the donor solution (Fig. 3).

10-2

.

en

.:.c

til II)

0

E

Z

Q

10- 3

t-

Il... 0:::

0

(/)

« q: z

10- 4

I

I

I

I

, I II

I

I I II

10-2

NAA EQUILIBRIUM CONCENTRATION (M) Fig. 2. Sorption of N AA by tomato fruit cutin matrix as function of pH and N AA concentration in the buffer solution. Values are averages of two determinations. Deviations from the mean were smaller than 5 'Yo. [NAAl higher than 10- 6 M were obtained by addition of nonradioactive NAA. 21*

J. SCHONHERR

314

Table 3. The effect of the rate of stirring all N AA permeability of isolated Citrus leaf cuticular membranes and cutin matrices Values are averages of 3 determinations at pH 4.24. Standard deviation given in parenthesis Rate of stirring

Permeability coefficients P (CM) P (l\IX)

rpm

X 107 cm S-l 0.93 (0.04) 1.54 (0.07) 1.57 (0.06) 1.52 (0.08) 1.50 (0.06)

o 200 400 600 800

x10 5 cms- 1 2.70 (0.17) 4.46 (0.18) 4.48 (0.24) 4.48 (0.19) 4.46 (0.21)

Diffusion coefficients NAA diffusion coefficients in the membrane were low and practically identical for cuticular membranes and cutin matrices (Table 4). Table 4. The effect of cuticular waxes on N AA transport parameters for isolated Citrus cuticles Values are averages of determinations from 6 membranes at pH 3.0. The coefficient for WAX was calculated according to eq. (6) Membrane

Coefficients

CM

MX WAX

P

D

3.69 X 10- 7 6.37 X 10- 5 3.71 X 10- 7

6.35 X 10-11 6.87 X 10- 11

4.0

0

NAA dissociation curve

''''E

3.0

o

donor and receiver pH equal

II>

pH of donor as shown, pH of

52

m z -4

receiver = 4.24

u

L/)

~

25 ' ~

2.0

50

1.0

75

x

0

X

~

a...

Z

» » z N m

0 100

0 2

3

4

5

6

7

8

9

pH Fig. 3. The effect of pH on diffusion of N AA across isolated Citrus leaf cutin matrix. Values are averages of 3 membranes. The vertical bars represent the standard deviation.

N aphthaleneacetic Acid Permeability of Citrus Leaf Cuticle

315

Discussion

The flux of a non-electrolyte across a homogeneous membrane is (HELFFERICR 1962) -DA -DAA J = - - (Co - Ci ) = - - (Co - Ci ) = - P (Co - Ci ) Llx

xV

(4)

where D = diffusion coefficient in the membrane, Ll x = membrane thickness, Co and Ci = concentrations of NAA in the membrane at the outer and inner membrane interface respectively, Co and Ci = NAA concentrations of the solutions at the membranej solution interface and A = CjC, as defined in equation (1). According to equation (4) the flux across a membrane can be analyzed on the basis of three parameters: the diffusion coefficient in the membrane, which reflects the mobility of the permeating species in the membrane; the distribution coefficient which reflects the concentration of the permeating species in the membrane; and the permeability coefficient (P) in which D and A appear as products. Of the three parameters, P is the most difficult to analyze, because of its dependence on A and membrane thickness. Equation (4) is applicable only if transport is determined by the permeability of the membrane and is unaffected by non-stirred liquid films on the membrane surfaces. Under the condition of film diffusion control the concentrations of the solutions at the solution/ membrane interface will be lower on the donor side and higher on the receiver side than the concentrations in the respective bulk solutions. Thus, the actual driving force (Ll C) will be lower than the concentration difference between the bulk solutions. In non-stirred solutions P values will therefore be underestimates, the error will be greater the larger P. The problem of film diffusion control has generally been overlooked in the past (BUKOVAC et al. 1971; BLUMENFELD and BUKOVAC 1972; DARLINGTON and CIRULIS 1963; DARLINGTON and BARRY 1963; NORRIS 1974). In the present study partial or complete film diffusion prevailed if solutions were not stirred At 200 rpm and above membrane diffusion control was obtained (Table 3). Both A (CM) and A (MX) are very high (Table 1, Figs. 1 and 2). Between pH 3 and 6 experimental distribution coefficients completely coincide with the dissociation curve of NAA. It must be concluded that only the non-dissociated form of NAA is sorbed by the cutin matrix. The distribution coefficient for the non-dissociated form of NAA (AO) and the distribution coefficient at a given pH (APR) are related by the equation

AO

=

1

+ lOpR-4.24

(5)

Thus, the distribution coefficient for any pH can be calculated from one determination at a given pH. Since A was independent of NAA concentration (between 3 x10- 6 to 3 X10- 3 M) the estimate will be valid for a wide concentration range (Fig. 2). Plant cuticles are polyelectrolytes with isoelectric points around 3 (unpublished results). Below pH 3 cuticles carry a net positive charge and the NAA anion can enter the cuticle by counter ion exchange. At pH 2.25 approximately 1 % of the total NAA molecules are present as anions. The higher value at pH 2.3 (Fig. 1) than that predicted

3a 316

Ii

J.

ii9"P

4

SCHONHERR

from eq. (5) is thus readily explained. Above pH 3 cuticles carry a net negative charge and the dissociated form of NAA cannot enter the membrane because of Donnan exclusion of the co-ion. This is the reason for the validity of equation (5). Above pH 6 the experimental distribution coefficients are also slightly above the dissociation curve. As the 14C-NAA was only guaranteed to be 99% pure the deviation at high pH values is probably due to a contamination (Fig. 1). A(CM) values were not affected by addition of NaCl known to increase the density of fixed charges in the cutin matrix (SCHONHERR and BUKOVAC 1973). The effect of pH on A(MX) can therefore be completely attributed to the effect of pH on dissociation of NAA (Fig. 1). Distribution coefficients varied between species (Table 1) and also between different lots of tomato fruit cutin matrix (Fig. 1 and 2, Table 1). This may be due to differences in the composition of cuticular lipids (cutin and waxes). Some of the deviations will be due to differences in the composition of isolated cuticular membranes, which always contain varying amounts of non-lipid components such as cellulose, polyuronic acids and proteins (MARTIN and JUNIPER 1970) not likely to sorb very much NAA.

Extraction of cuticular waxes resulted in higher distribution coefficients (Table 1). It is concluded, that the extracted waxes exhibit much lower distribution coefficients than the cutin matrix. This is confirmed, as the values for the three selected cuticular waxes (BAKER et a1. 1975; BAKER and PROCOPIOU 1975) vary only between 5 and 7 (Table 2). Even the value for octanol is quite low compared to the cutin matrix. Using octanol to assess lipid solubility of a permeating species in an attempt to predict permeability of cuticles appears to be of limited usefulness; it would unterestimate permeability if the limiting barrier was the cutin matrix and it would overestimate permeability if the limiting barrier was made up to triacontan, hexacosanol or hexadecanoic acid. Attempts to correlate cuticular permeability and solubility of the permeating species in the cutin matrix of cuticular membranes have been made before (BAKER and BUKOVAC 1971; BUKOVAC et a1. 1971; NORRIS and BUKOVAC 1969a) by studying sorption of 2,4-D, NAA or chlorinated phenoxyacetic acids in relation to penetration. Since only the initial concentration of the solutions were stated (not the final concentration when equilibrium had been reached) and sorption was always followed by prolonged washes with water, the data do not represent equilibrium values and cannot be converted to distribution coefficients. Permeability coefficients for diffusion of NAA across Citrus leaf cutin matrix were dependent on pH in a similar fashion as the distribution coefficients. Since only the non-dissociated NAA molecules can enter the cutin matrix (Fig. 1) they are the only ones that can cross the membranes. Also, the distribution coefficient depends on pH only via the pKa of NAA (eq. 5). Therefore, the steady state flux of NAA across the membranes should depend only on the pH of the donor and not on that of the receiver. This was observed to be the case (Fig. 3). This is of practical importance, because in applications of growth regulators and hormones (most of which are weak acids) to

"

•

N aphthaleneacetic Acid Permeability of Citrus Leaf Cuticle

317

cutinized plant surfaces, only the pH of the donor can be adjusted, but not that of the receiver (the epidermal cell wall). Since only the non-dissociated form can penetrate, there should be no or a negligible flux at pH values above 6.24, when practically all NAA molecules are dissociated. However, a small flux of NAA is observed. It is much larger than would be expected from the concentration of the nondissociated NAA, nor is it related to the concentration of the NAA anion. Since the 14C-NAA was only 99% pure the fluxes at high pH values are most likely due to a contamination. Extraction of waxes from cuticular membranes increased permeability by a factor of 170 to 300 (Table 4). Apparently, the cuticular waxes represent a formidable barrier to the movement of NAA molecules. This phenomenon has been observed before, even though the differences were much smaller (BuKov AC et al. 1971; BLUMENFELD and BUKovAc 1972; NORRIS 1974). However, their data are not suitable for a quantitative comparison because of the likelyhood of film diffusion control in their experiments. What is the nature of this wax barrier? If cuticular membranes were structurally homogenous, the increase in P due to wax extraction should only be proportional to the increase in the distribution coefficient (eq. (4)), that is about 18 %. Since this is not the case is it concluded that cuticular membranes are heterogeneous barriers composed of alternating layers differing in composition and structure. Investigations of cross-sections of cuticles with polarized light show a more or less contineous layer of birefringence near and parallel to the surface. Birefringence disappears on heating or extraction and is generally taken to be due a crystalline wax layer (N ORRIS and BUKoVAC 1969; ROELOFSEB 1952; SITTE and RENNIER 1963). The simplest model that would be consistent with the data is one having only two resistances in series. In this case 1 1 P(CM) = P(MX)

+

1 P(WAX)

(6)

and the permeability of the wax layer can be calculated. P (WAX) practically determines the permeability of the entire cuticular membrane (Table 4). As defined in eq. (4) P contains a mobility term (D) and a concentration term (A). The diffusion coefficients of NAA in the cutin matrix and in cuticular membranes are very similar (Table 4). Differences are not significant, as in the calculation of D the estimated membrane thickness enteres as the square. Still, D values obtained from eq. (3) can serve as order of magnitude estimates. Since D (CM) and D (MX) are identical, D (WAX) will be of similar magnitude, and difference in P (CM) and P (MX) can not be due to differences in the mobility of NAA in the two resistances. The low value of P (WAX) must therefore be attributed to a low value of .Ie (WAX) as shown directly for three cuticular waxes (Table 2). The alternative model (resistances in parallel) leads to a meaningless negative value for P (WAX) and would be in conflict with morphological evidence. It must be rejected. Since the permeability of cuticular membranes to moderately lipophilic molecules (such as NAA) is determined by the permeability of the wax layer, no inverse relation

I

318

J. SCHONHERR

between flux and thickness of cuticles can be expected (NORRIS 1974). As cuticular waxes are made up of numerous components (MARTIN and JUNIPER 1970) which may have quite different distribution coefficients and which may be localized at different sites within the membrane, no simple inverse relationship between the flux and the amount of wax can be expected either. It is often attempted to separate epicuticular from intracuticular waxes (BAKER and PROCOPIOU 1975; BAKER et al. 1975). The definition is only an operational one and not very satisfactory because it is difficult to proof that the lipids removed by a single short dip of a leaf in solvent are really only epicuticular waxes. As the whole concept lacks rigor it appears not very meaningful at the present tim"e to speculate on the location of the wax barrier within the cuticle. The problem requires further investigation. Water permeability of Citrus leaf cuticular membrane (SCHONHERR 1976 a) and cutin matrix has recently been investigated (SCHONHERR 1976). Permeability coefficients for the diffusion of tritiated water were numerically very similar to those presented here. The waxes were the sole barrier to water diffusion as well. In contrast to the results with NAA reported here, water permeability of Citrus leaf cutin matrix increased strongly with increasing pH (SCHONHERR 1976). Since the diameter of water filled pores in these membranes did not change with pH, it was concluded, that the increase in water permeability with increasing pH must be due to an inr;rease in the number of pores, a decrease in pore tortuosity or a combination of both. Since the cutin matrix is slightly permeable to glucose and the molecular weights of glucose (180) and NAA (186) are similar, the possibility of NAA penetration of the cutin matrix via the water filled pores exists, at least theoretically. However, all the evidence points to penetration by dissolution of NAA molecules within the lipid matrix of the membranes: (a) NAA is much more soluble in the cutin matrix or in the cuticular membrane than in water. (b) NAA flux was a function of the concentration of the non-dissociated form of NAA, while the more polar dissociated species could not enter the membranes because of Donnan exclusion. (c) NAA flux decreased with increasing pH while the water flux increased. There can be little doubt that most of the NAA molecules penetrate the cuticular matrix on a route different from that used by water molecules. Acknowledgement I gratefully acknowledge the skillful technical assistance of Miss E. HOFSCHUSTER. I am indebted to Dr. K. LENDZIAN, W. SCHMIDT and Prof. W. ZIEGLER for critically reviewing the manuscript. This study was supported by a grant from the Deutsche Forschungsgemeinschaft.

References BAKER, E. A., PROCOPIOU, J., and HUNT, G. M., The cuticles of Citrus species. Composition of leaf and fruit waxes. J. Sci. Food Chern. 26, 1093-1101 (1975). - - The cuticle of Citrus species. II. Composition of intracuticular lipids of leaves and fruits. J. Sci. Food Chern. 26, 1347-1352 (1975). and BUKOVAC, M. J., Characterization of components of plant cuticles in relation to penetration of 2,4-D. Ann. apvI. BioI. 67, 243-253 (1971).

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Acid Permeability of Citrus Leaf Cuticle

319

BUKOVAC, M. J., SARGENT, J. A., POWELL, R. G., and BLACKMAN, G. E., Studies on foliar penetration. VIII. Effect of chlorination on the movement of phenoxyacetic and benzoic acids through cuticle isolated from the fruits of Lycopersicon esculentum L. J. Exp. Bot. 22, 598-612 (1971). BLUMENFELD, A., and BUKOVAC, M. J., Cuticular penetration of abscisic acid. Planta (Berl.) 107, 261-268 (1972). DARLINGTON, W. A., and CIRULIS, N., Permeability of apricot leaf cuticle. Plant Physiol. 38,462-467 (1963). _ and BARRY, J. B., Effects of chloroform and surfactants on the permeability of apricot leaf cuticle. J. Arg. Fd. Chern. 13, 76-78 (1963). HELFFERICH, F., Ion exchange. McGraw-Hill, New York-San Francisco-Toronto-London: 1962. MARTIN, J. T., and JUNIPER, B. E., The cuticle of plants. Edward Arnold, 1970. NORRIS, R. F., Penetration of 2,4-D in relation to cuticle thickness. Amer. J. Bot. 61,74-79 (1974). - BUKOVAC, M. J., Structure of the pear leaf cuticle with special reference to cuticular penetration. Amer. J. Bot. 00, 975-983 (1969). _ _ Some phyaical-kinetic considerations in penetration of naphthaleneacetic acid through isolated pear leaf cuticle. Physiol. Plant. 22, 701-712 (1969 a). ORGELL, W. H., The isolation of plant cuticle with pectic enzymes. Plant Physiol. 30, 78-80 (1955). ROELOFS EN, P. A., The microscopic structure of cell walls. Acta Bot. Neerland. 1, 99-114 (1952). SARGENT, J. A., POWELL, R. G., and BLACKMAN, G. E., Studies on foliar penetration. The effects of chlorination on the rate of penetration of phenoxyacetic and benzoic acid into leaves of Phaseolus vulgaris. J. Exp. Bot. 20, 426-450 (1969). SCHONHERR, J., Water permeability of isolated cuticular membranes: The effect of pH and cations on diffusion, hydrodynamic permeability and size of polar pores in the cutin matrix. Planta (Berl.) 128, 113-126 (1976). Water permeability of isolated cuticular membranes. II. The effect of cuticular waxes on diffusion of water. Planta (Berl.) 131, 159-164 (1976) and BUKovAc, M. J., Dissociation constants of succinic acid 2,2-dimethylhydrazine. Agr. Food Chern. 20, 1263-1265 (1972). _ - Ion exchange properties of isolated tomato fruit cuticular membrane: Exchange capacity, nature of fixed charges and cation selectivity. Planta (Berl.) 109, 73-93 (1973). SITTE, P., und RENNIER, R., Untersuchungen an cuticularen Zellschichten. Planta (Berl.) 60, 19-40 (1963). WHITECROSS, M. I., MERCER, F. V., Permeability of isolated Eucalyptus gummifera cuticle towards alcohols and amides. Austr. J. Bot. 20, 1-7 (1972). Received June 2, 1976. Author's address: JORG SCHONHERR, Institut fUr Botanik und Mikrobiologie, Technische Universitat Miinchen, D-8 Miinchen.