Short Paper

Monitoring and visualising plant cuticles by confocal laser scanningmicroscopy

Sara Fernández, Sonia Osorio, Antonio Heredia*

Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias, Universidad de Málaga, 29071 Málaga, Spain

(Received May 11, 1999; accepted July 5, 1999)

Abstract — The present work shows the visualisation of phenolics and flavonoids of plant cuticles by confocal laser scanningmicroscopy (CLSM). Selected isolated fruit and leaf cuticles were monitored on the basis of autofluorescent phenolics andflavonoids which, in most cases, permitted us to obtain three-dimensional images of the cuticular membranes. The utility of thistechnique in investigations of cuticular translocation and diffusion of exogenous applied chemicals and cuticle degradation hasalso been explored. © 1999 Éditions scientifiques et médicales Elsevier SAS

Confocal laser scanning microscopy / cuticle / cutin / flavonoids / phenolic compounds

CLSM, confocal laser scanning microscopy

1. INTRODUCTION

Aerial surfaces of higher plants are covered withcuticle, an extracellular, non-living, lipid coveringforming the interface between the plant and its envi-ronment [6]. The cuticles of most plants have anintrinsic fluorescence emission that is mainly due tothe presence in this complex biopolymer of flavonoidsand cinnamic acids. Thus, the presence of flavonoidsin cuticles of tomato and pepper fruits has beenpreviously reported [1, 7, 11, 13]. Flavonoids are aclass of phenolic compounds of low molecular weightthat are widely distributed in the plant kingdom. Theyexhibit a diverse spectrum of biological functions andplay an important role in the interaction betweenplants and the environment [15].

During fruit ripening, specific flavonoids are bio-synthesised and transported from epidermal cells tothe different components or parts of the cuticularmembrane [9]. In fact, a major difference betweenmature green and ripe growth stages in tomato fruitsis the presence of the flavonoids naringenin andchalconaringenin in the cuticular membrane of ripefruits [10, 11]. Both phenolics are present in cutin(5–10 % of total weight) and epicuticular waxes(10–40 % of total weight) of tomato fruits [1, 7]. Onthe other hand, variable quantities of simple phenolic

acids, commonly coumaric (theiro-, m- andp-derivatives) and ferulic acids, are often releasedfrom other isolated plant cuticles during the de-esterification of cutin [7, 12]. These compounds arebound to the cutin polyester, being liberated andidentified only following treatment with reagents thatcleave ester linkages.

Recent developments in microscopic techniques,such as confocal laser scanning microscopy (CLSM),are providing the opportunity to study tissue localisa-tion of phenolic compounds in plant tissues in a moreprecise way [8]. CLSM can serve in the visualisationof chemicals and macromolecular structures usingtheir specific fluorescence properties on the basis oftheir molecular absorption and emission behaviour [4].

The ability of CLSM to create three-dimensionalimages is an important feature for plant biologists.Hutzler et al. [8] have recently demonstrated that thistechnique can give information on the subcellularlocalisation of phenolic compounds, in particularwhether or not the signal is derived from the vacuoleor the cell wall. In this short paper, we presentthree-dimensional images of plant cuticles isolatedfrom selected fruits and leaves using the autofluores-cence present in the plant cuticular membranes. Pos-sible and future applications of this technique, taking

* Author to whom correspondence should be addressed (fax +34 952132000; e-mail heredia@uma.es)

Plant Physiol. Biochem.,1999,37 (10), 789−794 / © 1999 Editions scientifiques et médicales Elsevier SAS. All rights reserved.

Plant Physiol. Biochem., 0981-9428/99/10/© 1999 E´ ditions scientifiques et médicales Elsevier SAS. All rights reserved.

into account the molecular barrier properties of thislipophilic membrane, are also presented.

2. RESULTS AND DISCUSSION

2.1. Autofluorescence and tri-dimensional imagesof isolated cuticles

Some authors [3, 5] have observed under the ultra-violet microscope a strong fluorescence of some plantcuticles, especially localised on the cell wall of theepidermal cells.

Figure 1shows a set of micrographs correspondingto the autofluorescence of some isolated fruit cuticlesafter excitation at 488 nm. All pictures were takenfrom the inner surface of the corresponding cuticlesample. Phenolics present strong autofluorescence insolution after UV excitation, whereas in solid samplesand because of their molecular self-aggregation, theyshow a low quantum yield of fluorescence when thesamples are excited at higher wavelengths. However,the fluorescence emission is sufficient to obtain goodimages.Figure 1 AandB shows the autofluorescenceof cuticles isolated from mature and green fruit pep-pers, respectively. Fluorescence of the green pepperisolate is confined to the cutinised cell wall of thiscuticle (figure 1 B), whereas the matured isolated fruitcuticle showed a weak additional autofluorescencelocalised around the cuticular region delimited by thecell wall, i.e. the cuticular matrix, mainly the cutin(figure 1 A).

Figure 1 C and D demonstrates one of the mostimportant advantages of CLSM compared to conven-tional optical and fluorescence microscopy. The abilityto scan in thez direction perpendicular to the systemaxis allows the study of the distribution of the fluo-rescence in a three-dimensional manner. Using thisapproach, spatial information on the cuticle samplecan be obtained from the fluorescence signal. Thesefacts have permitted the obtention of new images ofplant cuticles. From these images, it can be deducedthat, since thex-z scans of the isolated cuticle passesacross the thickness of the sample, the weak homoge-neously distributed fluorescence recorded between the

remaining cutinised cell walls demonstrates the pres-ence of phenolics in the cutin of ripe pepper cuticle(figure 1 C). This particular fluorescence was practi-cally zero in the case of the green pepper isolatedcuticle (figure 1 D) indicating the absence of phenolicsin the cutin and cuticular matrix of this cuticle.

CLSM also provided real images of the thickness ofthe isolated cuticles.Figure 1 E and F shows theautofluorescence of transversal sections of the isolates.In spite of the fact that only the fluorescence of thesample was recorded, the average thickness measuredfor both types of cuticles agree well (approximately30 µm) with that obtained by transmission electronmicroscopy and optical microscopy [6]. In ripe pepperfruit cuticle, the fluorescence, which indicates thedistribution of phenolics, forms a thick layer withoutdiscontinuities (figure 1 E), whereas the green pepperfruit cuticle sample shows severe discontinuities loca-lised between the region marked by the remaining cellwalls encrusted into the cuticular matrix (figure 1 F).This observations confirm what is evident infigure 1 CandD.

In order to extend the study to other types of plantcuticles, we carried out similar studies on cuticlesisolated from mature apple and tomato fruits.Figure 1 GandH shows the spatial distribution of theautofluorescence of these cuticles. They indicate twocuticles with a fluorescence pattern similar to thatdescribed infigure 1 C. Both types of fruit cuticlescontain significant amounts of phenolics in their cutinmatrices [7] that can justify their respective autofluo-rescence.

Isolated cuticles from leaves were also studied. Forthis communication, we have selected two classes ofleaf cuticles with different autofluorescence patterns.Figure 2 Ashows the autofluorescence of an isolatedcuticle from expanded leaves ofClivia miniata. This isa type of cuticle with a very weak intrinsic fluores-cence. The images indicate that the presence of phe-nolic compounds was restricted to the cutinised cellwall. The three-dimensional image of this leaf cuticleyielded a very dark picture (data not shown). On theother hand, the CLSM analysis obtained for theCitrusaurantiumisolates was completely different as is seen

Figure 1. Autofluorescence of selected isolated cuticles obtained by CLSM, viewed from the inner surface. The two-dimensional projections from20–24 sections of mature (A) and green (B) isolated pepper fruit cuticles in the red-colour mode after blue laser excitation at 488 nm.Three-dimensional representations of fluorescence intensity of the mature and green cuticle isolates are shown inC andD, respectively. Thesepictures are three-dimensional projections from 20–24 sections.E and F, Fluorescence intensity of a view corresponding to the thickness (seelocalisation of the different axis) at the front of the same isolated pepper fruit cuticles. Three-dimensional images corresponding to cuticles isolatedfrom apple fruit and ripe tomato fruit are showed inG andH, respectively.

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Figure 2. Autofluorescence of selected isolated cu-ticles obtained by CLSM after different treatments,viewed from the inner surface. Autofluorescence ofisolatedC. miniata (A) and C. aurantium (B) leafcuticles.C, Three-dimensional image of the isolatedCitrus cuticle. Experimental conditions as infigure 1.The autofluorescence and the corresponding three-dimensional image of an isolated green pepper fruitcuticle after paraquat deposition and translocation areshown in D and E, respectively. The effect of hy-droxyl radicals generated by a Fenton chemical reac-tion (see Methods) on an isolated mature fruit peppercuticle is shown inF (an optical section of autofluo-rescence) andG (three-dimensional reconstruction).

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in figure 2 B. This cuticle, isolated from the adaxialpart of the leaf, presented numerous stomata whichshowed a high intensity of fluorescence, in conjunctionwith a fluorescence pattern distributed across thecuticular membrane (figure 2 B). This is particularlyevident in the corresponding three-dimensional image(figure 2 C).

2.2. CLSM as a tool for monitoring cuticledeposition and degradation

Foliar penetration through the cuticle is a wide fieldof study including research in foliar uptake of nutri-ents, growth regulators and pesticides [2, 14], but themechanisms by which substances penetrate plant cu-ticles has not yet been well documented. In this sense,CLSM can help to understand the preferential move-ment and diffusion of certain substances. Cuticulartranslocation might be achieved using chemicals witha strong or medium intrinsic fluorescence, in a spectralregion distinct from the autofluorescence of the corre-sponding cuticle, and working with isolated cuticleswith very low or very localised fluorescence. In orderto test this approach, small drops of a micromolarparaquat aqueous solution were deposited on themorphological outer surface of isolated green peppercuticles. Paraquat, a powerful herbicide, has a strongfluorescence under blue light excitation (maximumwavelength absorption in methanol, 510 nm; in a morehydrophobic environment, the absorption shifts toshorter wavelengths) which could be monitored duringits cuticular translocation. After 2–3 h of deposition ofthe chemical on the cuticle surface, the summedfluorescence (endogenous compounds plus paraquat)of the pepper fruit cuticle appeared as is shown infigure 2 DandE. If we compare these pictures with themicrographs represented infigure 1 BandD, respec-tively, we can conclude that the chemical diffusesacross the cuticular membrane showing a homoge-neous ground fluorescence covering the whole surface.These data indicate that plant cuticles behave likehomogeneous membranes towards paraquat. There isno indication of the existence of preferential pathwaysor sites for the diffusion of the herbicide investigatedhere.

Another application of CLSM concerning plantcuticles involves environmental factors producing cu-ticular degradation. Specifically, our laboratory isstudying the effect of reactive oxygen species on plantcuticular components during pathogen-cuticle interac-tions. One of the more reactive chemical speciesgenerated during the first events of the infectionprocess is the hydroxyl radical (OH⋅). It has been

postulated that this radical can produce a set ofdifferent reactions including lipid oxidation, and thatcertain phenolics, mainly flavonoids, can act as mo-lecular scavengers of this reactive ion [16, 17]. Theeffect that this radical species has on the differentcuticular components [16], mainly waxes and cutin, isunknown.Figure 2 F and G shows, respectively, theautofluorescence and the three-dimensional image af-ter the treatment of mature isolated pepper fruit cuticlewith an OH⋅ generating solution. Pictures clearly showthat cuticular morphology appeared very degraded, thethickness of the cuticle was reduced and that theautofluorescence suffered a strong quenching. The firstobservation can be a direct effect of cutin cleavage viaradical reactions and the second, cuticle thinning,could be a structural consequence of the reaction ofphenolics and flavonoids with the OH⋅ radical.

In combination with ultrastructural studies andstructural analysis of the phenolic compounds, CLSMcan help to improve our understanding of this uniqueand fascinating biopolymer, the cuticle, by giving aglobal and even dynamic view of its processes.

3. CONCLUSION

CLSM has been shown to be an useful tool to obtainthree-dimensional images of isolated fruit and leafcuticles on the basis of autofluorescent phenolics andflavonoids. Despite the need of further research, thetechnique may be used to investigate the cuticularuptake of selected chemicals deposited on the outersurface of plant cuticles and to obtain importantqualitative information on cuticle reactivity and deg-radation.

4. METHODS

4.1. Plant material and cuticular isolation

Cuticles of ripe tomato (Lycopersicon esculentumL.), green and mature pepper (Capsicum annuumL.)and apple (Malus pumilaL.) fruits and bitter orange(Citrus aurantiumL.) and Clivia minata Reg. leaveswere enzymatically isolated using standard proceduresafter incubations of the plant tissues in a mixture ofcellulase and pectinase [13].

4.2. Cuticular treatments

Small drops (0.5µL) of freshly prepared 10µMparaquat (Sigma) aqueous solution were put on the

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outer surface of green pepper isolated cuticles. Thecuticles were kept at 25 °C for 15 h in a small closedchamber (relative humidity 95 %) until imaging. Onthe other hand, small pieces of isolated mature peppercuticles were exposed to a Fenton reagent mixtureconsisting of 10 mM H2O2 and 0.1 mM FeCl3 aqueoussolutions. This mixture produces hydroxyl radicals.

4.3. Confocal laser scanning microscopy andimage acquisition and processing

The CLSM analyses were performed on a LeicaTCS NT confocal laser scanning microscope (Leica,Heidelberg, Germany) and images were processedusing the Solid algorithm which is a direct composi-tion with diffuse gradient shading (Leica TCS soft-ware). In this work, small pieces of isolated fruitcuticles were put on microscope slides under a cover-slip and the autofluorescence of the tissue was studiedby CLSM. Generally, an excitation beam splitterDD488/568 was used. For visualisation of multispec-tral image data, the selected channel was associatedwith a colour value on the display system, red in thiscase, using a band pass filter (BP) of 515–565 nm.

Acknowledgments

This research has been supported by project PB94-1492, ‘Ministerio de Educación y Ciencia’, Spain. Theauthors thank Dr J. Santamaría, ‘Servicios Centralesde Investigación, Universidad de Málaga’, for techni-cal assistance.

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