Usefulness of Fluorescent Probe Prodan To Gain Insight into the

Article pubs.acs.org/JAFC

Usefulness of Fluorescent Probe Prodan To Gain Insight into the Polarity of Plant Cuticles Malgorzata Stawinoga,†,§ Mohamad Sleiman,†,# Jeoffrey Chastain,†,§ and Claire Richard*,†,§ †

Institut de Chimie de Clermont-Ferrand, Université Clermont Auvergne, Université Blaise Pascal, B.P. 10448, F-63000 Clermont-Ferrand, France § CNRS, UMR 6296, ICCF, F-63171 Aubiere, France # ENSCCF, Institut de Chimie de Clermont-Ferrand, Université Clermont Auvergne, B.P. 10448, F-63000 Clermont-Ferrand, France S Supporting Information *

ABSTRACT: Plant cuticle is a complex mixture of hydrophobic components that controls the uptake of pesticides by plants. Although the transport of lipophilic molecules across the cuticle has been intensively studied, development of tools to measure the cuticle polarity has received little attention. We developed a rapid and simple analytical method to evaluate the polarity of cuticles in situ. This method uses Prodan, 6-propionyl-2-(dimethylaminonaphthalene), a medium-sensitive fluorescent probe. Tests on model surfaces with varied polarity (i.e., wax paraffin, polyethylene, C18) were carried out to test the feasibility of the measurement and to optimize the application of Prodan. Moreover, on the basis of the Kamlet−Taft solvatochromic comparison method, a relationship between the emission characteristics of Prodan and the number of carbon atoms in primary alcohols mimicking the solid medium was established. After optimization, the method was validated on three natural plant cuticles (leaf of Zamiifolia, skin of green pepper, and skin of white grape). KEYWORDS: methodology, Prodan, fluorescence, plant cuticle polarity

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INTRODUCTION Knowledge of the surface properties of biological and chemical systems is essential to predict their role, but these properties are difficult to assess due to the heterogeneity and complexity of natural samples. There is a growing interest in developing simple and reliable methods for measuring surface properties, such as polarity and hydration. In biology, these two properties are key physicochemical characteristics of the lipid bilayer, and they control many processes.1 In polymer science, hydrophobicity and surface hydration govern many applications.2 In agrochemistry, the efficacy of foliage-applied agrochemicals depends on the amount of active ingredients delivered into the target species and/or tissues across the cuticle layer, which forms a natural interface between the plant and the environment.3,4 Plant cuticle is a thin (98%) was purchased from Sigma and used as received. Polyethylene PE-LD was obtained from SODIPRO, and wax paraffin (melting point = 53−57 °C, analysis given in the Supporting Information, Figure S1 and Table S1) and docosanol (>98%) were from Aldrich. The other chemicals and solvents were of the highest grade available. Preparation of Solutions and Model Films Containing Prodan. Films of wax paraffin, mixtures of wax paraffin and ndocosanol, and mixtures of wax paraffin and were prepared as follows. Powder or pellets of the long-chain alkanes were mixed in the chosen proportions and heated until melting. Prodan was added to the melted mixture. Then the mixture was poured on aluminum foil to obtain a thin film after cooling. Isolation of Cuticles. Leaves of Zamioculcas zamiifolia were refrigerated at −18 °C for several hours until frozen. Then, cuticles could be detached from the breakable leaves. Cuticles were first cleaned by gentle scraping of remaining flesh with a scalpel. Green peppers were refrigerated at −18 °C for several hours until frozen. Then, cuticles were detached with a scalpel and sonicated for 10 min to eliminate most of the epidermal remnants. Peels of white grapes were cleaned by gentle scraping of remaining flesh with a scalpel. Then cuticles were sonicated for 10 min to eliminate the epidermal remnants. All of the cuticles were finally washed with Milli-Q water. B

DOI: 10.1021/acs.jafc.5b02779 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Table 1. KTSCM Parameters for Four Alcohol Solvents and for Docosanola solvent

π*

α

β

ν/103 (cm−1)

methanol ethanol n-butanol n-octanol docosanol

0.6 0.54 0.47 0.4 0.035

0.98 0.86 0.84 0.77 0.374

0.66 0.75 0.84 0.81 0.81

19.92 20.35 20.73 21.19 23.47 (23.62)

a π* is polarity−polarizability, α represents the hydrogen bond donor ability, and β represents the hydrogen bond acceptor ability.

em νobsvd /10−3 = 25.2 ± 0 − (2.6 ± 0.2)π * − (2.5 ± 0.2)α

− (1.0 ± 0.1)β

These results showed that the excited state of Prodan experiences a bathochromic shift with the π*, α, and β parameters. Thus, the emission frequency shift of Prodan can be explained by the change in polarity and hydrogen-bonding interaction with the environment. To confirm if this equation applies also in our experimental conditions, we compared the measured values of ν for three alcohols (methanol, ethanol, octanol) with those reported by Moyano et al.31 The results were almost identical, indicating that the model presented in eq 1 is valid. After validation of the model, we explored the applicability of the same model for solid media such as paraffin and docosanol. However, because the values of π*, α, and β are not reported for these molecules, an estimation was made by plotting the variation of π* and α as a function of the number of carbon atoms (nC) for four primary alcohols (methanol, ethanol, butanol, octanol). As shown in Figure 2A, a linear relationship is found between π* and nC, which, if extrapolated to C22, allows the estimation of the parameter π* for docosanol. Similarly, we applied the same method in the case of α, which resulted in values of π* and α of 0.035 and 0.374, respectively. On the basis of data reported by Moyano et al.,31 it was found that β variation with the length of the carbon chain in a primary alcohol is very small and rangse between 0.81 and 0.84.32 Therefore, we chose a value of 0.82 for docosanol. Using these three KTSCM parameters, we estimated the predicted average value of ν for docosanol to be 23.37 × 103 cm−1 (minimum, 23.00 × 103; maximum, 23.73 × 103). Interestingly, this value is very close to that measured when Prodan is embedded in 100% docosanol (νobs = 23.47 × 103 cm−1). This finding suggests that the KTSCM can be extrapolated to explain the shift in emission frequency of Prodan in heterogeneous solid media, as corroborated by the excellent linear correlation between nC and the ν (Figure 2B). In addition, given that the emission frequency for wax paraffin was identical to that of heptane, we assumed that the KTSCM parameters of paraffin are identical to those of heptane, especially knowing that α and β are negligible and are thus not involved in the equation. Hence, we calculated the values of ν for various compositions of heptane/docosanol taking into account the relative contribution of each chemical, assuming that the overall value of ν of Prodan in the mixture is the sum of specific ν of each chemical (νheptane, νdocosanol) multiplied by their relative mass fraction. Table 2 presents the calculated values of ν and compares them with those measured experimentally for various compositions heptane/docosanol (paraffin/docosanol). As shown in Figure 3, the predicted values of emission maximum of Prodan (νcalcd) are in very good agreement with those measured (νobsd) when the percentage of

Figure 1. (A) Normalized emission spectra of Prodan in wax paraffin and in different solvents: 1, wax paraffin; 2, n-heptane; 3, docosanol; 4, acetonitrile; 5, 2-propanol; 6, methanol; 7, suspension in water. (B) Fluorescence of Prodan in heptane: 1, 2 × 10−6 M; 2, 5 × 10−5 M; 3, 10−3 M.

does not form aggregates that should give an emission around 430 nm, (2) the emission of Prodan is not influenced by the physical state of the medium, but by its chemical nature, and (3) a small band broadening occurs in solid state, which is attributed to interactions of Prodan with the solid environment.10 Relationship between the Emission Characteristics of Prodan and Properties of the Medium. To explain the experimental shift of emission maximum of Prodan embedded in different solid media, we attempted to establish a correlation between emission frequency and the properties of the heterogeneous media (docosanol and wax paraffin). One of the useful approaches for elucidating and quantifying different solute−solvent interactions is the Kamlet−Taft solvatochromic comparison method (KTSCM). According to the KTSCM, emission band frequency can be correlated with three parameters (Table 1), polarity−polarizability (π*), hydrogen bond donor ability (α), and hydrogen bond acceptor ability (β), using eq 1: ν = ν0 + sπ * + aα + bβ

(1)

The s, a, and b coefficients measure the relative sensitivity of ν to the indicated solvent property. In recent work, the values of these coefficients have been determined by Moyano et al.,31 on the basis of comparison with experimental data of Prodan absorption and emission in various common solvents.32 C


Article


Figure 3. Shift of the maximum emission frequency of Prodan with the percentage in weight of docosanol in mixture with wax paraffin: (○) docosanol, experimental values (νobsd); (×) docosanol, calculated data (νcalcd) (see text).

Impregnating Films with Prodan. In a second approach, we tested the possibility of impregnating films with Prodan. In the previous section, Prodan was introduced in films by hot mixing. This technique is not applicable in the case of real samples, especially when the objective is to work on untouched cuticles. Therefore, two methods of infiltration were tested: filtration of a Prodan solution in a water/ethanol, 50:50, mixture; and free diffusion of Prodan applied as 10 drops of 5 μL or as a single 200 μL drop. These experiments were carried out using a film of polyethylene that has the great advantage of being transparent. The two methods of infiltration give the same emission maximum at 405 ± 4 nm, indicating that in both cases the solvent has been fully eliminated and does not interfere in the result (Figure 4A). The intensity of fluorescence is, however, larger in the case of the single drop or infiltration under vacuum than in the case of small drops. To confirm that the method allows embedding Prodan in the polymer, we recorded the emission spectrum of Prodan deposited on a quartz plate in crystallized form. In this case, the emission maximum occurs at 427 nm (Figure 4A). Thus, the emission of Prodan in the polyethylene films is governed by the interaction of the single molecules with polymer chains and informs on the polarity of the medium. The maximum occurs at a wavelength slightly higher than in wax paraffin or n-heptane. This shift may be due to the presence of some polar additives in the polymer. Alternatively, it could be explained by a concentration effect. Indeed, the averaged concentration of Prodan is 2 × 10−3 M (see below), and in this case the maximum of emission is expected to be a little above 404 nm as experimentally observed. To clear up this question, it would be necessary to work on a polymer free of additives. Emission of Prodan in Cuticles. Three samples were chosen to validate the analytical method: the leaf of Zamioculcas zamiifolia, a succulent plant, and the skins of white grape berries and green pepper. In all cases, the extracted cuticles were transparent enough for enabling Prodan to absorb light and thus generate measurable fluorescence. Figure 4A depicts emission spectra obtained for model supports to illustrate the broad range of emission wavelength. Films of wax paraffin and polyethylene show thin bands centered around 400 nm indicating that the medium is homogeneous and that there is likely one type of interaction. Emission of Prodan deposited on a thin layer plate of chromatography made of C18 was also

Figure 2. Correlations of the number of carbon atoms in an alcohol with the polarity factor, π* (A), and with the emission wavenumber of Prodan (B).

Table 2. Estimates of KTSCM Parameters for Various Compositions of Paraffin/Docosanol and Comparison of Observed and Calculated Maximum Emission Wavelength of Prodan solid medium paraffin (heptane) 90:10 paraffin/ docosanol 80:20 paraffin/ docosanol 70:30 paraffin/ docosanol 50:50 parafin/ docosanol 25:75 paraffin/ docosanol 100 docosanol

π*

α

β

ν/103 (cm−1)

λcalcd (nm)

λobsd (nm)

−0.08 −0.069

0 0.037

0 0.081

25.57 25.20

NA 397

391 401

−0.057

0.075

0.162

25.00

400

406

−0.046

0.112

0.243

24.80

403

406

−0.023

0.187

0.405

24.39

410

410

0.006

0.280

0.608

23.88

419

419

0.035

0.374

0.81

23.37

428

426

docosanol in the mixture is significant (>30%). On the other hand, for low docosanol percentage, the measured values of emission maximum are higher than those predicted by the KTCSM model, suggesting that the docosanol is not evenly distributed in the wax paraffin but forms microdomains attributed to its self-association. The formation of microdomains in the mixture of long-chain alkanes was already demonstrated by a thermodynamic analysis.34 Despite this, these findings indicate clearly the feasibility of using the KTSCM commonly used for solvents to characterize the solvatochromic properties of heterogeneous solid media such as those reported in this work. Moreover, it appears that Prodan is sensitive enough to detect such a medium heterogeneity. D


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Figure 4. (A) Emission spectra of Prodan in model support: (1) film of wax paraffin; (2) film of polyethylene impregnated with a solution of Prodan 10−3 M in water/ethanol, 50:50 v/v added as a drop of 200 μL; (3) Prodan deposited on a quartz plate; (4) plate of thin layer chromatography C18 on which Prodan was deposited in heptane. (B) Emission spectra of Prodan in cuticles: Zamioculcas zamiifolia; white grape; green pepper.

reported in the literature. In the case of grape berries cuticle, alcohols (n-C20−n-C34) are reported to account for 40−67% of the soft wax fraction with predominating C26.34 Embedded in such a medium, Prodan should emit around 414 nm. Even though such an emission seems present in the blue part of the spectrum of Figure 4B, the maximum is significantly red-shifted, indicating that Prodan is mainly located in a more polar zone, probably the cutin. Pepper fruit waxes contain many components, among which long-chain alkanes C29 and C31 and fatty acids C24 and C26 are the most abundant, representing around 35 and 25%, respectively, of the constituents on average.35 The former would give a maximum of emission around 390 nm and the latter around 420−425 nm if we refer to methyl tetracosanoate data (see Figure S2). Both are far from the observed maximum, indicating that Prodan is not located in the pepper fruit waxes unless polar minor constituents shift significantly the emission maximum. The other compartment in which Prodan can be inserted is the cutin. The pepper cutin is a mixture of C16 and C18 monomers and their oxygenated derivatives, dominated by 10,16dihydroxyhexadecanoic acid that represents >50% of the total monomers.35 These constituents are expected to give a polarity higher than corresponding primary C16 and C18 alcohols. This is not far from what we found. This analysis points out two conclusions. First, it appears difficult to rationalize the data of Figure 4B using the reported cuticle composition because of the presence of a great variety of constituents in the cuticles, the possible great effect of some minor compounds on polarity, and the possible partitioning of constituents creating specific zones. The emission spectra of Prodan in these complex media bring, however, important information on the average polarities, and Prodan can be considered as a polarity indicator. Second, Prodan is probably mainly embedded in the cutin. The presence of Prodan in the cutin seems logical, first, because this inner compartment is larger than the outer one consisting of waxes36 and, second, because the solubility of organic compounds can be 10-fold higher in cutin than in cuticular waxes.36 Experimental Proofs of the Diffusion of Prodan into the Cuticles. The amount of Prodan included in the polyethylene film was directly obtained by measuring the film transmittance at 340 nm by taking into account that the thickness is 50 μm and the extinction coefficient of Prodan is equal to 2.3 × 104 L mol−1 cm−1 in n-heptane at 340 nm. Accordingly, a film measuring an area of 6 cm2 contained 7.1

measured. The emission maximum is at 491 nm, indicating that Prodan is located in the vicinity of free silanol groups. The emission spectra measured in the cuticles are shown in Figure 4B. The following characteristics appear: • The main emission maximum varies from 423 nm for Z. zamiifolia to 436 nm for the white grape and to 448 nm for the green pepper. • The results of the preliminary experiments strongly support that Prodan is embedded in the cuticles, and therefore these emissions are related to the chemical characteristics of the different cuticles. • The emission intensities vary with the cuticle in the order green pepper > white grape ≫ Z. zamiifolia. They are expected to depend on several parameters, in particular the capacity of Prodan to enter the cuticle and the cuticle transparency between 340 and 400 nm, the wavelength range where Prodan absorbs. Transparency of cuticles depends on the cuticle constituents and especially on the presence of absorbing secondary metabolites (polyphenols or carotenoids for instance) and can vary drastically from one species to another.35 • Emission bands are broad compared to those measured in the model supports, and detailed examination of the spectra reveals the presence of shoulders and broad longwavelength emissions on both sides of the maximum. For Z. zamiifolia, shoulders are observed at 408, 444, and around 460 nm. For white grape, there is a small shoulder at 426 nm and a broad emission extending up to 525 nm. For green pepper, one can see a clear shoulder at 433 nm and the same broad emission at long wavelength as for white grape. These fingerprints indicate that Prodan is located in zones of different polarity. In a first approach, these data can be analyzed using the relationship established between the emission characteristics of Prodan and the number of carbon atoms in primary alcohols constituting the solid medium of Figure 3B. For Z. zamiifolia, the maximum at 423 nm (23640 cm−1) would correspond to primary alcohols in C23; for the white grape, the maximum at 436 nm would correspond to primary alcohols in C19; and for the green pepper, the maximum at 448 nm would correspond to primary alcohols in C15. In a second approach, we examined whether these data match the chemical composition of cuticules (wax and cutin) as E


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Prodan and other alternative fluorescent probes to monitor the diffusion and reactivity of polar pesticides through cuticles.

μg. Prodan was extracted from the film by sonication for 30 min in 1 mL of ethanol. Using fluorescence, we could estimate that 7.5 μg of Prodan had been extracted, in full agreement with the result obtained by UV absorption measurements. For the cuticles that are much less transparent, the amount of Prodan was evaluated by extraction through sonication in ethanol. In the case of Z. zamiifolia cuticle, we found that the film of 6 cm2 and 19 μm thickness contained 2.4 μg. In the case of green pepper, we found 40.2 μg for a thickness of 59 μm. Results for polyethylene film and Z. zamiifolia cuticle are thus similar if we take into account that the polyethylene film is 2.6fold thicker than the Z. zamiifolia cuticle. They both absorbed 16.3% of the deposited Prodan (45.4 μg). Green pepper took much more, 88% of the deposited Prodan being embedded in the green pepper. To gain insights into the surface distribution of Prodan in cuticles and polyethylene films, images of Prodan fluorescence were recorded using a fluorescence microscopy. As shown in Figure 5, Prodan is clearly embedded into the polyethylene

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b02779. GC analysis of the paraffinic wax and shift of the maximum emission frequency of Prodan with the percentage of methyl tetracosanoate (in weight) in mixture with wax paraffin (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(C.R.) E-mail: [email protected]. Phone: +33 (0)4 73 40 71 42. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the Confocal Microscopy Facility ICCF (Imagerie Confocale Clermont-Ferrand) at Clermont University (France).

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REFERENCES

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Figure 5. Fluorescence images of Prodan impregnated into polyethylene film (left panel) and into green pepper cuticle (right panel). Prodan is mainly dissolved in polyethylene, even though small microcrystals are observed at the surface. In green pepper cuticle, Prodan diffuses until it reaches the residual epidermal cells, so that emission is a result of interactions within the inner cuticle.

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Usefulness of Fluorescent Probe Prodan To Gain Insight into the

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