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Effects of surfactants on calcium penetration of cuticles isolated from apple fruit
Scientia: Horticulturae, 46 ( 1991 ) 225-233
225
Elsevier Science Publishers B.V., A m s t e r d a m
Effects of surfactants on calcium penetration of cuticles isolated from apple fruit F.R. Harker and I.B. Ferguson DSIR Fruit and Trees, Mt. Albert Research Centre, Private Bag, Auckland (New Zealand) (Accepted for publication 10 August 1990)
ABSTRACT Harker, F.R. and Ferguson, I.B., 1991. Effects of surfactants on calcium penetration of cuticles isolated from apple fruit. Scientia Hortic., 46: 225-233. Transport of 45Ca2+ across enzymically isolated cuticles from apple fruit (Malus domestica Borkh. cultivar 'Granny Smith') was followed in an apparatus where the cuticle was clamped between two solutions, and the appearance of 45Ca2 + recorded in the sampling solution. 45Ca2 + transport from the outer to the inner surface of the cuticle could be increased by some surfactants when they were exposed to the inner, non-waxy surface. Addition of Armoblen T25 and Tween 20 increased the transport rate, Agral LN had little effect and Armoblen NPX inhibited transport. When the surfactants were applied to the outer surface there was little effect, except with Armoblen NPX where transport declined. As the cuticles did not contain any cracks or functional lenticels these results suggest that the surfzctants increased Ca 2+ transport by altering the permeability of the cuticle as a result of having direct access to the cutin and polysaccharide matrix. Keywords: apple; bitter pit; cuticle; calcium; surfactants.
INTRODUCTION
Bitter pit is a Ca2+-deficiency disorder in apple fruit. It can be controlled by treatments which involve the penetration of Ca 2+ across the skin of the fruit and subsequent movement of C a 2+ within the fruit flesh. Such treatments, whether as sprays of Ca 2+ salts applied regularly during fruit growth or as postharvest dips, increase the Ca 2 + content in the fruit flesh and reduce the incidence of bitter pit (Ferguson and Watkins, 1989). The primary barrier to penetration of the fruit by Ca 2+ is the fruit cuticle. Most Ca 2+ moves into the fruit through cracks and natural pores such as lenticels, but there is also a residual transport capacity for Ca 2+ in the cuticle membrane (Glenn et al., 1985; Harker and Ferguson, 1988 ). Chamel ( 1989 ) found little lenticel transport, however, possibly due to a failure to remove all of the cell wall material, or from suberization blocking the lenticel aperture.
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When applying sprays, adjuvants are often used to facilitate transcuticular transport. These usually reduce surface tension of spray droplets, allowing better spread over the cuticle surface and perhaps spontaneous infiltration of stomata (Jansen, 1964; Smith et al., 1966; Bland and Brian, 1975 ). The wetting abilities of some surfactants, however, are often insufficient to increase stomatal penetration (Schonherr and Bukovac, 1972 ). Some surfactants may directly affect cuticle structure, perhaps causing the dilation of hydrophilic cuticular pores and increasing solute permeability (Jansen, 1964). The effect of surfactants on uptake of Ca 2+ by apples seems to vary according to the type and concentration of surfactant used (Hanekom and De Villiers, 1977 ) and the variety of apple (Lidster and Porritt, 1978 ). Generally they appear to decrease or have no effect on Ca 2+ uptake into fruit, although a few studies have demonstrated that surfactants increase uptake (De Villiers and Hanekom, 1977 ), especially when used in conjunction with thickeners (Mason et al., 1974). Where surfactants reduce Ca 2+ uptake it is probably the result of decreased Ca R+ deposition on the fruit, presumably because of an increase in run-off and a thinner surface film of liquid being retained (Hanekom and De Villiers, 1977 ). A major concern in the use of surfactants is whether they facilitate transport of calcium across the cuticle proper, and not just through pores. This might occur by forming a lipophilic complex with Ca 2+ which is then able to penetrate the extracellular wax, or by modifying the internal cuticle structure so that the permeability of the cuticle is increased. Such effects of surfactants have been recorded in relation to herbicide applications (Smith et al., 1966). We therefore investigated the effect of a selected group of surfactants on the transport of Ca 2+ across isolated apple fruit cuticles. Those which increased transport appeared to do so by modifying the structure of the cutin and polysaccharide matrix. MATERIALS A N D M E T H O D S
F r u i t a n d cuticles. - Apple fruit ( M a l u s d o m e s t i c a Borkh. cultivar 'Granny
Smith') weighing about 200 g were harvested towards the end of the commercial harvest period from trees growing at Oratia, Auckland. Cuticles were isolated from discs of fruit peel by an enzymic method (Harker and Ferguson, 1988) using 4% ~ectinase and 0.4% cellulase in acetate buffer (0.2 M, pH 3.8), and incubation at 38°C for 4-6 days. This treatment produced clean cuticle membranes (18 m m diameter) with no adhering cellular debris (Harker and Ferguson, 1988). The cuticles were examined by light microscopy to determine whether any open lenticels were present. Open lenticels are clearly visible as elliptical pores surrounded by the impressions of guard cells, and provide a major pathway for calcium transport across isolated cuticles (Harker and Ferguson, 1988 ). Although the isolated cuticles contained sub-
EFFECTS OF SURFACTANTS ON Ca PENETRATION OF CUTICLES
227
erized lenticels and cracks, no open lenticels were detected. Transport would therefi~re not occur through large natural pores. C a 2+ p e n e t r a t i o n . - Cuticles were sandwiched between rubber O-rings ( I 1 m m diameter) and cemented with a fast-setting cement (cold-cure Silastomer 9161, Hopkins & Williams). The cuticle was then clamped between two chambers which allowed both faces of the cuticle to be exposed to separate solutions, with the outer surface of the cuticle exposed to the loading solution. The chambers (denoted as loading and sampling) contained 8 ml of solution which was stirred with magnetic stirrers. Details of the apparatus are given in Harker and Ferguson ( 1988 ). The loading solution was 1.5 × 10 - 6 M 45CAC12 (specific activity 32 MBq, ~tmol -I ) and the sampling solution was deionized water. Samples (300/~1) were taken from the sampling solution hourly, each sample being replaced with fi'esh water to maintain a constant volume. After 5 h, when the steady state rate of transport across the cuticle had been established, the surfactant was added to the loading, sampling or both chambers as appropriate. When the surfactant was added to one chamber only, an equivalent volume of water was ad[ded to the other to maintain constant volumes. Hourly sampling was continued for a further 5 h, and rates of transcuticular transport were compared before and after surfactant addition. The 300/tl sample was combined with ACSII scintillant (Amersham), and the activity of 45Ca measured in a Beckman LS2800 scintillation counter. Surfactants were used at concentrations of 0.05, 0.25 and 2.5% ( v / v ) , and new cuticles were used in each triplicated experiment. C a 2+ b i n d i n g . - Isolated cuticles ( 13 m m diameter) were placed in 8 ml of 0.1 M 45CAC12 (specific activity 44 kBq,/~mol - l ) with or without various surfactants. They were incubated at room temperature overnight, blotted dry to remove the surface film and washed for 5 min by vigorous stirring in 1 1 of deionized water then placed in scintillation vials. The cuticles were solubilized by incubation at 40°C in 0.5 ml of 0.06 N NCS tissue solubilizer (Amersham) for about 6 h. Then 3 ml ACSII scintillant was added and the samples were left in the dark for 2 days to allow chemical excitation to decay. 45Ca was measured by scintillation counting. Results are presented as nmo145Ca2+ c m - 2 cuticle, assuming 45Ca2+ had displaced all cations previously bound to the cuticle in an exchangeable form. This assumption is based on the length of incubation time and the high affinity of Ca 2+ for binding sites (Chamel, 1983). In some cases, waxes were extracted from the cuticle before measuring the Ca 2+ binding capacity. This was achieved by washing the cuticles in seven successive volumes (5 ml) of chloroform over a 24-h period followed by air drying. This resulted in a 37% reduction in cuticle weight.
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TABLE 1 Description of adjuvants used in cuticle experiments Trade name
Source
Description
Armoblen T25 Armoblen NPX Tween 20
Akzo Chemie Akzo Chemie J.T. Baker
Agral LN
I.C.I.
Cationic; Polyoxyethylene ( 15 ) tallow amine. Weak cationic; Polyoxyethylene ( > 15 ) fatty amine. Non-ionic; Polyoxyethylene (20) sorbitan monolaurate, Non-ionic; contains nonyl-phenyl ethoxyates.
- Ethoxylate surfactants were obtained from commercial sources. Consequently, the chemicals used were not in a pure state, but consisted of a group of similar molecules which varied in the length of the fatty alkyl chain. Trade names, sources and descriptions are given in Table 1. Surfactants.
RESULTS C a 2+ p e n e t r a t i o n . - After the first hour 45Ca2+ transport rates were linear. Changes in rate after the addition of the various surfactants are given as ratios of the linear rates before and after surfactant addition. Where the inhibition of transport increased with time so that the rate achieved after the addition of the surfactant was not linear, rates were calculated over the last hour. Ratios were used because rates of transport through individual cuticles varied for any one treatment due to natural variation in cuticle permeability (Harker and Ferguson, 1988; Chamel, 1989 ). However, the proportional rate changes for any one treatment were the same. Typical kinetics of Ca 2+ transport are shown in Fig. 1, where each curve represents transport across a different cuticle. When the surfactants were added to both chambers, and thus had access to both faces of the cuticle, Armoblen T25 and Tween 20 increased the rate of Ca 2+ transport, Agral LN had little effect and Armoblen N P X reduced the rate (Table 2 ). The influence of the surfactants on transport rates was consistent for all concentrations of surfactant, although the magnitude of the effect differed. A feature of these experiments was the sudden increase or decrease in the 45Ca2+ content of the sampling chamber on addition of some of the surfactants, before the new transport rate was established (Fig. 1 ). 45Ca 2+ content increased considerably after the addition of Armoblen NPX, and sometimes after the addition of Tween 20 and high concentrations of Agral LN. However, the addition of Armoblen T25 resulted in a sudden decrease in 45Ca2+ content of the sampling solution (Fig. 1 ). To determine what effect surfactants have when in contact only with the outer surface of the cuticle, the surfactants were added to the loading solutions at 2.5% concentrations. These high concentrations were used to maxi-
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EFFECTS OF SURFACTANTS ON Ca P E N E T R A T I O N OF CUTICLES
l 0
3
x 7
2
E
o_ o
rY w 0[3
< I C)
/ 0 4
c
Z n < (/9
3
Z
1
2
4
6
8
10
2
4
6
8
10
TIME (h)
Fig. 1. ?enetration of 45Ca 2+ a c r o s s isolated cuticles before and after addition of adjuvants to both loading and sampling solutions. The adjuvants Armoblen N P X (A), Tween 20 (B), Armoblen T25 (C) and Agral LN ( D ) were added after 5 h (4). Curves are the results of a typical experiment, and adjuvants were present at 0.25% concentration. TABLE2 Change.,; in transcuticular transport of 45Ca2+ after the addition of 0.05, 0.25 and 2.5% concentrations of adjuvant to both sampling and loading chambers (both inner and outer cuticle faces). Values represent the mean ratio of rates (R2/R,) +SE where R, is the initial rate (cpm h - ' ) and R2 is the rate after adj uvant addition. Ratios were calculated from three replicated experiments Adj uvant
Armoblcn NPX Armoblen T25 Tween 20 Agral LN
Concentration 0.05% (R2/Rt)
0.25% (Re~R,)
2.5% (Rz/R,)
0.28 _+0.13 3.57_+0.69 1.60_+ 0.53 1.03-+ 0.27
0.16 _+0.10 2.16_+0.14 1.44_+ 0.85 1.20_+ 0.11
- 0.08 _+0.13 2.13_+0.35 2.36_+ 0.41 1.12_+ 0.02
mize responses. Armoblen T25 and Agral LN had little effect on transport (Table 3 ). However, Armoblen NPX produced a gradual decrease in transport until the rate was 24% of the initial rate. Tween 20 caused a transient
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F.R. HARKERAND I.B. FERGUSON
TABLE3 Changes in transcuticular transport of 45Ca2+ after addition of adjuvants to the loading chamber (outer cuticle face). Values represent the mean ratio of rates (R2/RI) + SE where Rl is the initial rate (cpm h - ' ) and R2 is the rate after adjuvant addition. Ratios were calculated from three replicate experiments, and rates were determined in the linear phase, or over the last hour when not linear (*) Adjuvant
R2/RI
None Armoblen NPX Armoblen T25 Tween 20 Agral LN
1.00-+ 0.03 0.24 -+0.08* 1.06 _+0.03 0.72 _+0.18* 0.80 ± 0.46
TABLE 4
45Ca2+ adsorption onto waxy or dewaxed cuticles after immersion in 0.1 M 45CAC12 with or without various adjuvants (2.5%). Values are the mean adsorption ± SE for a minimum of four replicate experiments. Results are different from the control treatments as indicated (*, **, *** P = 1, 0.5 and 0.1, respectively) Experiment
Cuticle
Adjuvant
Adsorbed 45Ca 2+ (nmol cm 2)
A
Waxy Dewaxed Waxy Waxy Dewaxed Waxy Waxy Waxy
None None Armoblen NPX Armoblen T25 Armoblen T25 None Tween 20 Agral LN
50_+ 3 47 -+ 6 36_+ 4 (NS) 183 ± 8*** 231 -+ 16** 67 ± 3 27_+ 2** 44-+ 2*
B
increase in rate, but then the rate declined to less than the initial rate. The results suggested that the greatest effects of the surfactants were on the inner surface of the cuticle. This was tested with 2.5% Armoblen T25 by adding the compound to the sampling chamber only. The kinetics of transport were similar to those observed when the surfactant was given to both sides of the cuticle (Fig. 1 ), although the ratio of rates before and after addition was 3.59+0.35.
The separate influence of pH on penetration was investigated by buffering the sampling and loading solutions at pH 6 (5 mM N a i l maleate/KOH) for the first 5 h, then adding 100/tl of 0.6 N HC1 which lowered the pH to about 2.7. The drop in pH resulted in a gradual decrease in transport rate to 18% of the original. The pHs of the solutions containing 2.5% of the surfactants were: Armoblen T25, 9.5; Armoblen NPX, 5.9; Tween 20, 4.5; Agral LN, 6.3. C a 2+ a d s o r p t i o n .
-
In the absence of surfactants, the adsorption of
4 5 C a 2+
EFFECTS OF SURFACTANTS ON Ca PENETRATION OF CUTICLES
231
onto waxy or dewaxed cuticles was similar (Table 4), indicating that little Ca 2+ was bound by the wax on the surface or in the matrix. Addition of Armoblen T25 substantially increased adsorption. Tween 20 and Agral LN both decreased adsorption, and Armoblen NPX had no significant effect. DISCUSSION
The cuticles used in the present study contained no functional lenticels, and microscopic study showed that any cracks or other irregularities were suberized. Thus, Ca 2+ transport, where it occurred, was by necessity through the cuticle proper. This means that any property of the surfactants associated with ease of ion transport through macropores did not apply to the present results. Some surfactants may affect cuticle structure, by diffusing into the cuticle along hydrophilic-lipophilic interfaces, causing dilation of hydrophilic pores and a subsequent increase in permeability of the cuticle to polar solutes (Jansen, 1964; Smith et al., 1966 ). Surfactants have also been shown to damage and extract cuticle wax (Robertson and Kirkwood, 1969; Hunt and Baker, 1983 ), and any such action is likely to increase solute transport since removal of wa~: increases permeability (Harker and Ferguson, 1988). Surfactants may increase the mobility of solutes in the cuticle. At low concentrations, most surfactants dissolve in aqueous solutions. However, as concentrations increase, the surfactant molecules aggregate into micelles. Organic and inorganic compounds may then be absorbed by the micelles (Elwerthy et al., 1968) and transported into and across cuticles to an extent dependent on the partition behaviour of the micelle. The ability of surfacrants to diffuse into or become dissolved in the cuticle is reduced as they become more concentrated, and as the ethoxylate content of the molecules increases (Shafer and Bukovac, 1986). Where Ca 2+ transport was increased by a surfactant in our results, it is likely ~Lohave been from a modification of the cuticle permeability rather than a direct effect on Ca 2+ mobility resulting from micellar sequestration. This would particularly be the case with Armoblen T25 and Tween 20, where Ca z+ transport increased only when the surfactant was in contact with the inner surface of the cuticle. If surfactant-promoted transport required 45Ca2+ absorption into micelles before diffusion into and across the cuticle, the addition of Armoblen T25 and Tween 20 to the loading chamber should result in a similar increase in the rates of transport as was observed following addition to the sampling and loading chambers. However this did not happen (cf. Tables 2 and 3). The inhibitory effects of Armoblen NPX are remarkable since the chemical structures of the two Armoblen compounds are similar. A1t h o u ~ we do not have specific information, the NPX compound appears to differ only in having a greater number of ethoxylate groups. This may have a
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F.R. HARKER AND I.B. FERGUSON
significant effect on the partitioning behaviour of the molecule. Perhaps the greater chain length results in physical blockage of the transport pathways. The sudden changes in 45Ca2 + concentrations of the sampling solutions after the addition of surfactants (Fig. 1 ) may be due to changes in the binding of Ca 2+ to the cuticle. Presumably, Armoblen T25 increased the binding capacity of the cuticle for C a 2+, such that it removed Ca 2+ from the solution (Fig. 1 ). Results from the binding experiments support this (Table 4). Conversely, the increase in solution 45Ca2 + as a result of addition of the other compounds is probably due to release of bound 45Ca 2+, again confirmed by the binding studies. These effects may be attributed in part to the pH of the surfactant solutions, since C a 2+ adsorption to apple fruit cuticles increases when the pH increases from 3 to 8 (Charnel, 1983). The effect of the surfactants on Ca 2+ transport, however, is likely to be independent of pH, since compounds with similar pH, e.g. Armoblen NPX and Agral LN, have different effects on transport. C o m p o u n d s with similar effects on transport, e.g. 2.5% Armoblen T25 and 2.5% Tween 20, have widely different pH values. As well, apple fruit cuticles have an isoelectric point of pH 2.1 (Charnel, 1989 ). At pHs above the point, cuticles carry a net negative charge and are permselective to cations, and below, are positive and permselective to anions (Schonherr and Huber, 1977). Thus, the pH of the NPX solution would need to be less than 2.1 for it to account for the inhibition of C a 2+ transport; it was 5.9. One might expect that the cationic nature of some surfactants would cause interference with cation transport. However, no evidence in the present study supported a relationship between the cationic or non-ionic nature of the compounds and transport. These results seem to support the suggestion that transcuticular transport of cations occurs along polysaccharide fibrils which are embedded in the cutin matrix (Hoch, 1979). In the intact skin, these fibrils have some continuity with the pectic polysaccharides of the cell wall, and extend into the cuticle, probably becoming increasingly cutinized as they reach the surface (Holloway, 1982 ). The outermost surface of the cuticle, however, is covered by a layer of epicuticular wax. This may explain why surfactants were more effective in our experiments when included in the sampling solution where they had direct access to the polysaccharide fibrils. Presumably, surfactants in the loading solution were unable to gain access to the fibrils due to the epicuticular wax and cutinized nature of the polysaccharides. The characteristics of binding of C a 2 + to cuticles is reminiscent of binding to cell walls (Harker, 1986), perhaps supporting the suggestion that polysaccharides are major binding sites for cations in cuticles. Attention paid to those properties of surfactants which modify cuticle structure might benefit current commercial practices of spraying and dipping fruit in Ca 2+ salts. Surfactants partition into cuticles depending on their molecular size and critical micelle concentration (Shafer and Bukovac, 1986 ). Improved calcium penetration requires a surfactant which can diffuse across
EFFECTS OF SURFACTANTS ON Ca PENETRATION OF CUTICLES
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the layer of epicuticular wax and into the cuticle. Once in the cuticle, some surfactants may be able to improve the transport characteristics of transcuticular pathways associated with the polysaccharide fibrils. REFERENCES Bland, P.D. and Brian, R.C., 1975. Surfactants and the uptake and movement of paraquat in plants. Pestic. Sci., 6: 419-427. Charnel, A., 1983. Utilization of isolated apple fruit cuticles to study the behaviour of calcium supplied directly to the fruit. Acta Hortic., 138: 23-34. Chamel, A., 1989. Permeability characteristics of isolated "Golden Delicious" apple fruit cuticles with regard to calcium. J. Am. Soc. Hortic. Sci., 114: 804-809. De Villi ers, J.F. and Hanekom, A.N., 1977. Factors by which the post-harvest uptake of calcium by Golden Delicious apples is influenced. The Deciduous Fruit Grower, 27:85-91. Elworthy, P.H., Florence, A.T. and Macfarlane, C.B., 1968. Solubilization by Surface-active Agents and its Applications in Chemistry and the Biological Sciences. Chapman and Hall, London, 335 pp. Ferguson, I.B. and Watkins, C.B., 1989. Bitter pit in apple fruit. Hortic. Rev., 11: 289-355. Glenn, G.M., Poovaiah, B.W. and Rasmussen, H.P., 1985. Pathways of calcium penetration through isolated cuticles of 'Golden Delicious' apple fruit. J. Am. Soc. Hortic. Sci., 110: 166--171. Hanekom, A.N. and De Villiers, J.F., 1977. Factors by which pre-harvest uptake of calcium by Golden Delicious apples is influenced. The Deciduous Fruit Grower, 27:166-169. Harker, F.R., 1986. Studies of calcium transport in apple fruit. Ph.D. thesis, University of Aucldand, 157 pp. Harker, F.R. and Ferguson, I.B., 1988. Transport of calcium across cuticles isolated from apple fruit. Sci. Hortic., 36:205-217. Hoch, H.C., 1979. Penetration of chemicals into the Malus leaf cuticle - an ultrastructural analysis. Planta, 147: 186-195. Holloway, P.J., 1982. Structure and histochemistry of plant cuticular membranes: an overview. In: D.F. Cutler, K.L. Alvin and C.E. Price (Editors), The Plant Cuticle. Academic Press, London, pp. 1-32. Hunt, G.M. and Baker, E.A., 1983. Penetration ofchlormequat chloride into cereal leaves. Long Ashton Res. Stn. Univ. Bristol Rep. for 1982, pp. 80-81. Jansen, L.L., 1964. Surfactant enhancement of herbicide entry. Weeds, 12:251-255. Lidster, P.D. and Porritt, S.W., 1978. Some factors affecting uptake of calcium by apples dipped after harvest in calcium chloride solution. Can. J. Plant Sci., 58: 35-40. Mason, J.L., McDougald, J.M. and Drought, B.G., 1974. Calcium concentration in apple fruit resulting from calcium chloride dips modified by surfactants and thickeners. HortScience, 9: 122-123. Robertson, M.M. and Kirkwood, R.C., 1969. The mode of action of foliage-applied translocared herbicides with particular reference to the phenoxy-acid compounds I. The mechanism and :factors influencing herbicide absorption. Weed Res., 9: 224-240. Schonherr, J. and Bukovac, M.J., 1972. Penetration of stomata by liquids: dependence on surface tension, wettability and stomatal morphology. Plant Physiol., 49:813-819. Schonherr, J. and Huber, R., 1977. Plant cuticles are polyelectrolytes with isoelectric points around three. Plant Physiol., 59:145-150. Shafer, W.E. and Bukovac, M.J., 1986. Partition behaviour of surfactants, and their effects on NAA sorption, in enzymatically isolated tomato fruit cuticles. HortScience, 21:810. Smith, L.W., Foy, C.L. and Bayer, D.E., 1966. Structure-activity relationships of alkyl-phenol ethylene oxide ether non-ionic surfactants and three water soluble herbicides. Weed Res., 6: 233-242.
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Effects of surfactants on calcium penetration of cuticles isolated from apple fruit F.R. Harker and I.B. Ferguson DSIR Fruit and Trees, Mt. Albert Research Centre, Private Bag, Auckland (New Zealand) (Accepted for publication 10 August 1990)
ABSTRACT Harker, F.R. and Ferguson, I.B., 1991. Effects of surfactants on calcium penetration of cuticles isolated from apple fruit. Scientia Hortic., 46: 225-233. Transport of 45Ca2+ across enzymically isolated cuticles from apple fruit (Malus domestica Borkh. cultivar 'Granny Smith') was followed in an apparatus where the cuticle was clamped between two solutions, and the appearance of 45Ca2 + recorded in the sampling solution. 45Ca2 + transport from the outer to the inner surface of the cuticle could be increased by some surfactants when they were exposed to the inner, non-waxy surface. Addition of Armoblen T25 and Tween 20 increased the transport rate, Agral LN had little effect and Armoblen NPX inhibited transport. When the surfactants were applied to the outer surface there was little effect, except with Armoblen NPX where transport declined. As the cuticles did not contain any cracks or functional lenticels these results suggest that the surfzctants increased Ca 2+ transport by altering the permeability of the cuticle as a result of having direct access to the cutin and polysaccharide matrix. Keywords: apple; bitter pit; cuticle; calcium; surfactants.
INTRODUCTION
Bitter pit is a Ca2+-deficiency disorder in apple fruit. It can be controlled by treatments which involve the penetration of Ca 2+ across the skin of the fruit and subsequent movement of C a 2+ within the fruit flesh. Such treatments, whether as sprays of Ca 2+ salts applied regularly during fruit growth or as postharvest dips, increase the Ca 2 + content in the fruit flesh and reduce the incidence of bitter pit (Ferguson and Watkins, 1989). The primary barrier to penetration of the fruit by Ca 2+ is the fruit cuticle. Most Ca 2+ moves into the fruit through cracks and natural pores such as lenticels, but there is also a residual transport capacity for Ca 2+ in the cuticle membrane (Glenn et al., 1985; Harker and Ferguson, 1988 ). Chamel ( 1989 ) found little lenticel transport, however, possibly due to a failure to remove all of the cell wall material, or from suberization blocking the lenticel aperture.
0304-4238/91/$03.50
© 1991 - - Elsevier Science Publishers B.V.
226
F.R. H A R K E R A N D I.B. F F R G U S O N
When applying sprays, adjuvants are often used to facilitate transcuticular transport. These usually reduce surface tension of spray droplets, allowing better spread over the cuticle surface and perhaps spontaneous infiltration of stomata (Jansen, 1964; Smith et al., 1966; Bland and Brian, 1975 ). The wetting abilities of some surfactants, however, are often insufficient to increase stomatal penetration (Schonherr and Bukovac, 1972 ). Some surfactants may directly affect cuticle structure, perhaps causing the dilation of hydrophilic cuticular pores and increasing solute permeability (Jansen, 1964). The effect of surfactants on uptake of Ca 2+ by apples seems to vary according to the type and concentration of surfactant used (Hanekom and De Villiers, 1977 ) and the variety of apple (Lidster and Porritt, 1978 ). Generally they appear to decrease or have no effect on Ca 2+ uptake into fruit, although a few studies have demonstrated that surfactants increase uptake (De Villiers and Hanekom, 1977 ), especially when used in conjunction with thickeners (Mason et al., 1974). Where surfactants reduce Ca 2+ uptake it is probably the result of decreased Ca R+ deposition on the fruit, presumably because of an increase in run-off and a thinner surface film of liquid being retained (Hanekom and De Villiers, 1977 ). A major concern in the use of surfactants is whether they facilitate transport of calcium across the cuticle proper, and not just through pores. This might occur by forming a lipophilic complex with Ca 2+ which is then able to penetrate the extracellular wax, or by modifying the internal cuticle structure so that the permeability of the cuticle is increased. Such effects of surfactants have been recorded in relation to herbicide applications (Smith et al., 1966). We therefore investigated the effect of a selected group of surfactants on the transport of Ca 2+ across isolated apple fruit cuticles. Those which increased transport appeared to do so by modifying the structure of the cutin and polysaccharide matrix. MATERIALS A N D M E T H O D S
F r u i t a n d cuticles. - Apple fruit ( M a l u s d o m e s t i c a Borkh. cultivar 'Granny
Smith') weighing about 200 g were harvested towards the end of the commercial harvest period from trees growing at Oratia, Auckland. Cuticles were isolated from discs of fruit peel by an enzymic method (Harker and Ferguson, 1988) using 4% ~ectinase and 0.4% cellulase in acetate buffer (0.2 M, pH 3.8), and incubation at 38°C for 4-6 days. This treatment produced clean cuticle membranes (18 m m diameter) with no adhering cellular debris (Harker and Ferguson, 1988). The cuticles were examined by light microscopy to determine whether any open lenticels were present. Open lenticels are clearly visible as elliptical pores surrounded by the impressions of guard cells, and provide a major pathway for calcium transport across isolated cuticles (Harker and Ferguson, 1988 ). Although the isolated cuticles contained sub-
EFFECTS OF SURFACTANTS ON Ca PENETRATION OF CUTICLES
227
erized lenticels and cracks, no open lenticels were detected. Transport would therefi~re not occur through large natural pores. C a 2+ p e n e t r a t i o n . - Cuticles were sandwiched between rubber O-rings ( I 1 m m diameter) and cemented with a fast-setting cement (cold-cure Silastomer 9161, Hopkins & Williams). The cuticle was then clamped between two chambers which allowed both faces of the cuticle to be exposed to separate solutions, with the outer surface of the cuticle exposed to the loading solution. The chambers (denoted as loading and sampling) contained 8 ml of solution which was stirred with magnetic stirrers. Details of the apparatus are given in Harker and Ferguson ( 1988 ). The loading solution was 1.5 × 10 - 6 M 45CAC12 (specific activity 32 MBq, ~tmol -I ) and the sampling solution was deionized water. Samples (300/~1) were taken from the sampling solution hourly, each sample being replaced with fi'esh water to maintain a constant volume. After 5 h, when the steady state rate of transport across the cuticle had been established, the surfactant was added to the loading, sampling or both chambers as appropriate. When the surfactant was added to one chamber only, an equivalent volume of water was ad[ded to the other to maintain constant volumes. Hourly sampling was continued for a further 5 h, and rates of transcuticular transport were compared before and after surfactant addition. The 300/tl sample was combined with ACSII scintillant (Amersham), and the activity of 45Ca measured in a Beckman LS2800 scintillation counter. Surfactants were used at concentrations of 0.05, 0.25 and 2.5% ( v / v ) , and new cuticles were used in each triplicated experiment. C a 2+ b i n d i n g . - Isolated cuticles ( 13 m m diameter) were placed in 8 ml of 0.1 M 45CAC12 (specific activity 44 kBq,/~mol - l ) with or without various surfactants. They were incubated at room temperature overnight, blotted dry to remove the surface film and washed for 5 min by vigorous stirring in 1 1 of deionized water then placed in scintillation vials. The cuticles were solubilized by incubation at 40°C in 0.5 ml of 0.06 N NCS tissue solubilizer (Amersham) for about 6 h. Then 3 ml ACSII scintillant was added and the samples were left in the dark for 2 days to allow chemical excitation to decay. 45Ca was measured by scintillation counting. Results are presented as nmo145Ca2+ c m - 2 cuticle, assuming 45Ca2+ had displaced all cations previously bound to the cuticle in an exchangeable form. This assumption is based on the length of incubation time and the high affinity of Ca 2+ for binding sites (Chamel, 1983). In some cases, waxes were extracted from the cuticle before measuring the Ca 2+ binding capacity. This was achieved by washing the cuticles in seven successive volumes (5 ml) of chloroform over a 24-h period followed by air drying. This resulted in a 37% reduction in cuticle weight.
228
F.R. HARKER AND l.B. FERGUSON
TABLE 1 Description of adjuvants used in cuticle experiments Trade name
Source
Description
Armoblen T25 Armoblen NPX Tween 20
Akzo Chemie Akzo Chemie J.T. Baker
Agral LN
I.C.I.
Cationic; Polyoxyethylene ( 15 ) tallow amine. Weak cationic; Polyoxyethylene ( > 15 ) fatty amine. Non-ionic; Polyoxyethylene (20) sorbitan monolaurate, Non-ionic; contains nonyl-phenyl ethoxyates.
- Ethoxylate surfactants were obtained from commercial sources. Consequently, the chemicals used were not in a pure state, but consisted of a group of similar molecules which varied in the length of the fatty alkyl chain. Trade names, sources and descriptions are given in Table 1. Surfactants.
RESULTS C a 2+ p e n e t r a t i o n . - After the first hour 45Ca2+ transport rates were linear. Changes in rate after the addition of the various surfactants are given as ratios of the linear rates before and after surfactant addition. Where the inhibition of transport increased with time so that the rate achieved after the addition of the surfactant was not linear, rates were calculated over the last hour. Ratios were used because rates of transport through individual cuticles varied for any one treatment due to natural variation in cuticle permeability (Harker and Ferguson, 1988; Chamel, 1989 ). However, the proportional rate changes for any one treatment were the same. Typical kinetics of Ca 2+ transport are shown in Fig. 1, where each curve represents transport across a different cuticle. When the surfactants were added to both chambers, and thus had access to both faces of the cuticle, Armoblen T25 and Tween 20 increased the rate of Ca 2+ transport, Agral LN had little effect and Armoblen N P X reduced the rate (Table 2 ). The influence of the surfactants on transport rates was consistent for all concentrations of surfactant, although the magnitude of the effect differed. A feature of these experiments was the sudden increase or decrease in the 45Ca2+ content of the sampling chamber on addition of some of the surfactants, before the new transport rate was established (Fig. 1 ). 45Ca 2+ content increased considerably after the addition of Armoblen NPX, and sometimes after the addition of Tween 20 and high concentrations of Agral LN. However, the addition of Armoblen T25 resulted in a sudden decrease in 45Ca2+ content of the sampling solution (Fig. 1 ). To determine what effect surfactants have when in contact only with the outer surface of the cuticle, the surfactants were added to the loading solutions at 2.5% concentrations. These high concentrations were used to maxi-
229
EFFECTS OF SURFACTANTS ON Ca P E N E T R A T I O N OF CUTICLES
l 0
3
x 7
2
E
o_ o
rY w 0[3
< I C)
/ 0 4
c
Z n < (/9
3
Z
1
2
4
6
8
10
2
4
6
8
10
TIME (h)
Fig. 1. ?enetration of 45Ca 2+ a c r o s s isolated cuticles before and after addition of adjuvants to both loading and sampling solutions. The adjuvants Armoblen N P X (A), Tween 20 (B), Armoblen T25 (C) and Agral LN ( D ) were added after 5 h (4). Curves are the results of a typical experiment, and adjuvants were present at 0.25% concentration. TABLE2 Change.,; in transcuticular transport of 45Ca2+ after the addition of 0.05, 0.25 and 2.5% concentrations of adjuvant to both sampling and loading chambers (both inner and outer cuticle faces). Values represent the mean ratio of rates (R2/R,) +SE where R, is the initial rate (cpm h - ' ) and R2 is the rate after adj uvant addition. Ratios were calculated from three replicated experiments Adj uvant
Armoblcn NPX Armoblen T25 Tween 20 Agral LN
Concentration 0.05% (R2/Rt)
0.25% (Re~R,)
2.5% (Rz/R,)
0.28 _+0.13 3.57_+0.69 1.60_+ 0.53 1.03-+ 0.27
0.16 _+0.10 2.16_+0.14 1.44_+ 0.85 1.20_+ 0.11
- 0.08 _+0.13 2.13_+0.35 2.36_+ 0.41 1.12_+ 0.02
mize responses. Armoblen T25 and Agral LN had little effect on transport (Table 3 ). However, Armoblen NPX produced a gradual decrease in transport until the rate was 24% of the initial rate. Tween 20 caused a transient
230
F.R. HARKERAND I.B. FERGUSON
TABLE3 Changes in transcuticular transport of 45Ca2+ after addition of adjuvants to the loading chamber (outer cuticle face). Values represent the mean ratio of rates (R2/RI) + SE where Rl is the initial rate (cpm h - ' ) and R2 is the rate after adjuvant addition. Ratios were calculated from three replicate experiments, and rates were determined in the linear phase, or over the last hour when not linear (*) Adjuvant
R2/RI
None Armoblen NPX Armoblen T25 Tween 20 Agral LN
1.00-+ 0.03 0.24 -+0.08* 1.06 _+0.03 0.72 _+0.18* 0.80 ± 0.46
TABLE 4
45Ca2+ adsorption onto waxy or dewaxed cuticles after immersion in 0.1 M 45CAC12 with or without various adjuvants (2.5%). Values are the mean adsorption ± SE for a minimum of four replicate experiments. Results are different from the control treatments as indicated (*, **, *** P = 1, 0.5 and 0.1, respectively) Experiment
Cuticle
Adjuvant
Adsorbed 45Ca 2+ (nmol cm 2)
A
Waxy Dewaxed Waxy Waxy Dewaxed Waxy Waxy Waxy
None None Armoblen NPX Armoblen T25 Armoblen T25 None Tween 20 Agral LN
50_+ 3 47 -+ 6 36_+ 4 (NS) 183 ± 8*** 231 -+ 16** 67 ± 3 27_+ 2** 44-+ 2*
B
increase in rate, but then the rate declined to less than the initial rate. The results suggested that the greatest effects of the surfactants were on the inner surface of the cuticle. This was tested with 2.5% Armoblen T25 by adding the compound to the sampling chamber only. The kinetics of transport were similar to those observed when the surfactant was given to both sides of the cuticle (Fig. 1 ), although the ratio of rates before and after addition was 3.59+0.35.
The separate influence of pH on penetration was investigated by buffering the sampling and loading solutions at pH 6 (5 mM N a i l maleate/KOH) for the first 5 h, then adding 100/tl of 0.6 N HC1 which lowered the pH to about 2.7. The drop in pH resulted in a gradual decrease in transport rate to 18% of the original. The pHs of the solutions containing 2.5% of the surfactants were: Armoblen T25, 9.5; Armoblen NPX, 5.9; Tween 20, 4.5; Agral LN, 6.3. C a 2+ a d s o r p t i o n .
-
In the absence of surfactants, the adsorption of
4 5 C a 2+
EFFECTS OF SURFACTANTS ON Ca PENETRATION OF CUTICLES
231
onto waxy or dewaxed cuticles was similar (Table 4), indicating that little Ca 2+ was bound by the wax on the surface or in the matrix. Addition of Armoblen T25 substantially increased adsorption. Tween 20 and Agral LN both decreased adsorption, and Armoblen NPX had no significant effect. DISCUSSION
The cuticles used in the present study contained no functional lenticels, and microscopic study showed that any cracks or other irregularities were suberized. Thus, Ca 2+ transport, where it occurred, was by necessity through the cuticle proper. This means that any property of the surfactants associated with ease of ion transport through macropores did not apply to the present results. Some surfactants may affect cuticle structure, by diffusing into the cuticle along hydrophilic-lipophilic interfaces, causing dilation of hydrophilic pores and a subsequent increase in permeability of the cuticle to polar solutes (Jansen, 1964; Smith et al., 1966 ). Surfactants have also been shown to damage and extract cuticle wax (Robertson and Kirkwood, 1969; Hunt and Baker, 1983 ), and any such action is likely to increase solute transport since removal of wa~: increases permeability (Harker and Ferguson, 1988). Surfactants may increase the mobility of solutes in the cuticle. At low concentrations, most surfactants dissolve in aqueous solutions. However, as concentrations increase, the surfactant molecules aggregate into micelles. Organic and inorganic compounds may then be absorbed by the micelles (Elwerthy et al., 1968) and transported into and across cuticles to an extent dependent on the partition behaviour of the micelle. The ability of surfacrants to diffuse into or become dissolved in the cuticle is reduced as they become more concentrated, and as the ethoxylate content of the molecules increases (Shafer and Bukovac, 1986). Where Ca 2+ transport was increased by a surfactant in our results, it is likely ~Lohave been from a modification of the cuticle permeability rather than a direct effect on Ca 2+ mobility resulting from micellar sequestration. This would particularly be the case with Armoblen T25 and Tween 20, where Ca z+ transport increased only when the surfactant was in contact with the inner surface of the cuticle. If surfactant-promoted transport required 45Ca2+ absorption into micelles before diffusion into and across the cuticle, the addition of Armoblen T25 and Tween 20 to the loading chamber should result in a similar increase in the rates of transport as was observed following addition to the sampling and loading chambers. However this did not happen (cf. Tables 2 and 3). The inhibitory effects of Armoblen NPX are remarkable since the chemical structures of the two Armoblen compounds are similar. A1t h o u ~ we do not have specific information, the NPX compound appears to differ only in having a greater number of ethoxylate groups. This may have a
232
F.R. HARKER AND I.B. FERGUSON
significant effect on the partitioning behaviour of the molecule. Perhaps the greater chain length results in physical blockage of the transport pathways. The sudden changes in 45Ca2 + concentrations of the sampling solutions after the addition of surfactants (Fig. 1 ) may be due to changes in the binding of Ca 2+ to the cuticle. Presumably, Armoblen T25 increased the binding capacity of the cuticle for C a 2+, such that it removed Ca 2+ from the solution (Fig. 1 ). Results from the binding experiments support this (Table 4). Conversely, the increase in solution 45Ca2 + as a result of addition of the other compounds is probably due to release of bound 45Ca 2+, again confirmed by the binding studies. These effects may be attributed in part to the pH of the surfactant solutions, since C a 2+ adsorption to apple fruit cuticles increases when the pH increases from 3 to 8 (Charnel, 1983). The effect of the surfactants on Ca 2+ transport, however, is likely to be independent of pH, since compounds with similar pH, e.g. Armoblen NPX and Agral LN, have different effects on transport. C o m p o u n d s with similar effects on transport, e.g. 2.5% Armoblen T25 and 2.5% Tween 20, have widely different pH values. As well, apple fruit cuticles have an isoelectric point of pH 2.1 (Charnel, 1989 ). At pHs above the point, cuticles carry a net negative charge and are permselective to cations, and below, are positive and permselective to anions (Schonherr and Huber, 1977). Thus, the pH of the NPX solution would need to be less than 2.1 for it to account for the inhibition of C a 2+ transport; it was 5.9. One might expect that the cationic nature of some surfactants would cause interference with cation transport. However, no evidence in the present study supported a relationship between the cationic or non-ionic nature of the compounds and transport. These results seem to support the suggestion that transcuticular transport of cations occurs along polysaccharide fibrils which are embedded in the cutin matrix (Hoch, 1979). In the intact skin, these fibrils have some continuity with the pectic polysaccharides of the cell wall, and extend into the cuticle, probably becoming increasingly cutinized as they reach the surface (Holloway, 1982 ). The outermost surface of the cuticle, however, is covered by a layer of epicuticular wax. This may explain why surfactants were more effective in our experiments when included in the sampling solution where they had direct access to the polysaccharide fibrils. Presumably, surfactants in the loading solution were unable to gain access to the fibrils due to the epicuticular wax and cutinized nature of the polysaccharides. The characteristics of binding of C a 2 + to cuticles is reminiscent of binding to cell walls (Harker, 1986), perhaps supporting the suggestion that polysaccharides are major binding sites for cations in cuticles. Attention paid to those properties of surfactants which modify cuticle structure might benefit current commercial practices of spraying and dipping fruit in Ca 2+ salts. Surfactants partition into cuticles depending on their molecular size and critical micelle concentration (Shafer and Bukovac, 1986 ). Improved calcium penetration requires a surfactant which can diffuse across
EFFECTS OF SURFACTANTS ON Ca PENETRATION OF CUTICLES
233
the layer of epicuticular wax and into the cuticle. Once in the cuticle, some surfactants may be able to improve the transport characteristics of transcuticular pathways associated with the polysaccharide fibrils. REFERENCES Bland, P.D. and Brian, R.C., 1975. Surfactants and the uptake and movement of paraquat in plants. Pestic. Sci., 6: 419-427. Charnel, A., 1983. Utilization of isolated apple fruit cuticles to study the behaviour of calcium supplied directly to the fruit. Acta Hortic., 138: 23-34. Chamel, A., 1989. Permeability characteristics of isolated "Golden Delicious" apple fruit cuticles with regard to calcium. J. Am. Soc. Hortic. Sci., 114: 804-809. De Villi ers, J.F. and Hanekom, A.N., 1977. Factors by which the post-harvest uptake of calcium by Golden Delicious apples is influenced. The Deciduous Fruit Grower, 27:85-91. Elworthy, P.H., Florence, A.T. and Macfarlane, C.B., 1968. Solubilization by Surface-active Agents and its Applications in Chemistry and the Biological Sciences. Chapman and Hall, London, 335 pp. Ferguson, I.B. and Watkins, C.B., 1989. Bitter pit in apple fruit. Hortic. Rev., 11: 289-355. Glenn, G.M., Poovaiah, B.W. and Rasmussen, H.P., 1985. Pathways of calcium penetration through isolated cuticles of 'Golden Delicious' apple fruit. J. Am. Soc. Hortic. Sci., 110: 166--171. Hanekom, A.N. and De Villiers, J.F., 1977. Factors by which pre-harvest uptake of calcium by Golden Delicious apples is influenced. The Deciduous Fruit Grower, 27:166-169. Harker, F.R., 1986. Studies of calcium transport in apple fruit. Ph.D. thesis, University of Aucldand, 157 pp. Harker, F.R. and Ferguson, I.B., 1988. Transport of calcium across cuticles isolated from apple fruit. Sci. Hortic., 36:205-217. Hoch, H.C., 1979. Penetration of chemicals into the Malus leaf cuticle - an ultrastructural analysis. Planta, 147: 186-195. Holloway, P.J., 1982. Structure and histochemistry of plant cuticular membranes: an overview. In: D.F. Cutler, K.L. Alvin and C.E. Price (Editors), The Plant Cuticle. Academic Press, London, pp. 1-32. Hunt, G.M. and Baker, E.A., 1983. Penetration ofchlormequat chloride into cereal leaves. Long Ashton Res. Stn. Univ. Bristol Rep. for 1982, pp. 80-81. Jansen, L.L., 1964. Surfactant enhancement of herbicide entry. Weeds, 12:251-255. Lidster, P.D. and Porritt, S.W., 1978. Some factors affecting uptake of calcium by apples dipped after harvest in calcium chloride solution. Can. J. Plant Sci., 58: 35-40. Mason, J.L., McDougald, J.M. and Drought, B.G., 1974. Calcium concentration in apple fruit resulting from calcium chloride dips modified by surfactants and thickeners. HortScience, 9: 122-123. Robertson, M.M. and Kirkwood, R.C., 1969. The mode of action of foliage-applied translocared herbicides with particular reference to the phenoxy-acid compounds I. The mechanism and :factors influencing herbicide absorption. Weed Res., 9: 224-240. Schonherr, J. and Bukovac, M.J., 1972. Penetration of stomata by liquids: dependence on surface tension, wettability and stomatal morphology. Plant Physiol., 49:813-819. Schonherr, J. and Huber, R., 1977. Plant cuticles are polyelectrolytes with isoelectric points around three. Plant Physiol., 59:145-150. Shafer, W.E. and Bukovac, M.J., 1986. Partition behaviour of surfactants, and their effects on NAA sorption, in enzymatically isolated tomato fruit cuticles. HortScience, 21:810. Smith, L.W., Foy, C.L. and Bayer, D.E., 1966. Structure-activity relationships of alkyl-phenol ethylene oxide ether non-ionic surfactants and three water soluble herbicides. Weed Res., 6: 233-242.