Localization of production and emission of pollinator attractant on whole leaves of Chamaerops...

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American Journal of Botany 91(8): 1190–1199. 2004.

LOCALIZATION OF PRODUCTION AND EMISSION OF

POLLINATOR ATTRACTANT ON WHOLE LEAVES OF

CHAMAEROPS HUMILIS (ARECACEAE)1

JEAN-CLAUDE CAISSARD,2 AROONRAT MEEKIJJIRONENROJ,3

SYLVIE BAUDINO,2 AND MARIE-CHARLOTTE ANSTETT3,4

2Laboratoire BVpam (Biotechnologies Vegetales, plantes aromatiques et medicinales) EA 3061, Universite Jean Monnet,23 rue du Docteur Paul Michelon, F-42023 Saint-Etienne Cedex 02, France; and 3Centre d’Ecologie Fonctionnelle et Evolutive,

CNRS, 1919 Route de Mende, F-34 293 Montpellier Cedex 5, France

Volatile compounds, which frequently play important roles in plant–insect interaction, can be produced either by flowers to attractpollinators or by leaves to deter herbivores. The specialized structures associated with odor production differ in these two organs. TheEuropean dwarf palm Chamaerops humilis represents a unique intermediate between these two. In previous work, its leaves wereshown to produce volatile organic compounds (VOCs) that attract pollinators only during flowering. Because the leaf sinuses look likea gland, the sinus was examined histologically and with environmental scanning electron microscopy (ESEM) for evidence that thesinus emits VOCs. Volatile compounds emitted by the different parts of the leaf were extracted by washes and headspace then analyzedby gas chromatograph-mass spectrometer (GC-MS). The sinus does not have the expected gland-like structure; the VOCs are actuallyproduced by the whole leaf, even if the composition of the VOCs emitted by the sinus slightly differs. Thus, attraction of pollinatorsdoes not result from specialized secreting cells in leaves of flowering European dwarf palms. The results are discussed in the contextof a convergent evolution of leaves toward petals.

Key words: Arecaceae; floral scent; idioblasts; leaf scent; palm; pollination; terpenes; volatile organic compounds.

Plants produce many secondary compounds (or naturalproducts), many functioning as mediators of plant–insect in-teractions. These compounds may either repel herbivores orattract potential benefactors such as pollinators (e.g., Rodri-guez and Levin, 1976; Pichersky and Gershenzon, 2002). Re-pellent molecules typically act by contact and are produced bytrichomes, idioblasts, osmophores, or other secreting tissues,with stocks of secondary compounds kept in specialized cells.Some of these compounds are highly volatile and are alsoproduced by flowers to form floral perfumes that act at a dis-tance to attract pollinators. Like other floral parts (e.g., sepals,carpels, stamens) petals are homologous with leaves and cer-tainly evolved from leaves (Gutierrez-Cortines and Davies,2000; Pellmyr, 2002).

Secondary metabolites of leaves and floral elements thusmay have a common evolutionary origin and a shared devel-opmental pathway. For example, the chemical attractants ofcycad cones are supposed to have evolved from herbivore de-terrents (Pellmyr et al., 1991). However the detailed devel-opmental pathway connecting organ to molecular levels of at-tractants is still poorly known, even though MADS transcrip-tion factors seem to be involved in the evolution of flowers(Lawton-Rauh et al., 2000; Albert et al., 2002). Since flowersattract pollinators from a distance, only volatile organic com-pounds (VOCs) have been retained through natural selectionand are found in floral scents. The VOCs are all derived fromfatty acids, benzenoids, or isoprenoids (Knudsen et al., 1993).

1 Manuscript received 23 October 2003; revision accepted 30 March 2004.The authors thank Jean-Marie Bessiere for his invaluable help for com-

pound identification, Bruno Buatois for technical help with the GC-MS, DoyleMcKey for correcting our English, and Celine Mir and Martine Hossaert forhelp in collecting samples. Environmental scanning electron microscopy wasdone at the Centre de Microscopie Electronique Stephanois with the help ofIsabelle Anselme-Bertrand. We also thank J. M. Bessiere, F. Stauffer, and twoanonymous reviewers for helpful suggestions.

4 E-mail: [email protected].

Many terpenes are present in both leaf and floral VOCs andmay either attract or repel insects (Pichersky and Gershenzon,2002). For example, more than 600 articles have been pub-lished on the diverse roles of (E)-b-farnesene. Among diversefunctions, this sesquiterpene can be a plant (and insect) defen-sive allomone, an attractant pheromone, or a kairomone thatstimulates oviposition (reviewed by Crock et al., 1997). Thestructures producing the VOCs differ between leaves and flow-ers. Leaves produce their repellent odors in a variety of dif-ferent structures (such as trichomes, idioblasts, cavities, andducts) depending on the species. In contrast, flowers usuallyproduce their attractive fragrance in osmophores or in conicalcells located on the petals. These cells do not stock VOCs butrelease them into the air. Even though the importance of VOCsin species interactions have been recently investigated at themolecular level (Dudareva and Pichersky, 2000; Baldwin etal., 2001; Kolosova et al., 2001a; Pichersky and Gershenzon,2002), surprisingly little information is available on the exactcellular location of transport and emission of VOCs into thesurrounding air (e.g., Hudak and Thompson, 1997; Turner etal., 1999; Bouvier et al., 2000; Jasinski et al., 2001; Goodwinet al., 2003).

Recently, Dufay et al. (2003) found an interesting inter-mediate between these two paradigms of odor production (i.e.,repellent VOCs are produced on leaves and attractive VOCson flowers by different structures). Leaves of Chamaerops hu-milis L. (Arecaceae), the Mediterranean dwarf palm, producevolatile compounds that attract their species-specific pollinat-ing weevil (Derelomus chamaeropsis), whereas the flowers arealmost scentless. This odor production is limited to anthesisand thus may have a function similar to that of floral scents.The scent is more perceptible near the sinuses of the leaf (i.e.,between the leaflets), and Dufay et al. (2003) suggested thatscents could be produced at this location. This example pro-vides a unique opportunity to study the transition between fo-

August 2004] 1191CAISSARD ET AL.—LOCALIZATION OF ODOR EMISSION IN C. HUMILIS

liar and floral scent production. Indeed, some other species ofpalms, relatively close to C. humilis in phylogenetic trees (As-mussen and Chase, 2001), produce scents only in flowers (e.g.,Guihaia grossefibrosa, Arecaceae; Dufay, 2003) or in bothleaves and flowers (Serenoa repens, Arecaceae; Dufay, 2003).

In this paper, we describe the structure of the sinus at thehistological level. We show that terpenes and VOCs are lo-calized not only in the sinuses, but also in the rest of the leaf.We also show that the proportion of some molecules is higherin the VOCs emitted by the sinuses. Finally, we attempt tounderstand why VOCs seems to be produced by the leaf butare emitted by the sinus.

MATERIALS AND METHODS

Material—All leaves of flowering Chamaerops humilis and Trachycarpusfortunei used in the following experiments were collected from ornamentalpalms planted in Montpellier, France. Leaves of young, nonflowering C. hu-milis were collected from palms grown from seed in the greenhouse of theCentre d’Ecologie Fonctionelle et Evolutive (CEFE) in Montpellier.

Extraction of VOCs—To localize the site of odor production on the leaf,we extracted the VOCs by washing different parts of leaves: sinus, veins, andblade in dichloromethane. First, we dissected the leaves with a razor bladeseparating the sinus, veins, and blade. Care was taken not to include evensmall pieces of blade in the sinus and veins samples. Leaves with a perceptiblescent of flowering C. humilis (a total of six leaves from three different maleindividuals) and leaves without a perceptible scent (as controls) of young C.humilis before their first flowering (a total of 18 leaves, one for each of 18individuals) and of flowering Trachycarpus fortunei, a closely related speciesof the same subtribe (Asmussen and Chase, 2001; total of eight leaves, onefor each of four males and four females plants), were dissected. The numberof leaves dissected varied because we dissected the number of leaves neededto obtain 0.5 g (all fresh mass) of sinus. From the same palms, we kept 0.5g of veins and 2.5 g of blade from the different leaves. All these leaf samplesplus some staminate (2.5 g) and pistillate (2.5 g) flowers of both species werewashed for 36 h in 1 mL of dichloromethane (CH2Cl2) for samples of 0.5 gor 5 mL of CH2Cl2 for samples of 2.5 g. The washes were then analyzedquantitatively and qualitatively with a Varian CP3800 gas chromatograph(GC) coupled with a Varian Saturn 2000 mass spectrometer (MS) (Varian,Palo Alto, California, USA). We injected 1 mL in a CP sil 8 CB column (30m, 0.25 mm inner diameter, 0.25 mm film thickness) with helium as the carriergas. The temperature was kept at 508C for the first 3 min, then programmedto increase 38C/min to 1008C, 2.78C/min to 1408C, 2.48C/min to 1808C, andthen 68C/min to 2508C. Volatiles were identified by comparing their massspectra with those of the NIST98 library and with GC retention times andMS spectra of the authentic compounds when possible. For quantification, weadded 4 mg of each of two internal standards (nonane and dodecane) to eachsample before analysis. We then calculated the peak area of internal standardcorresponding to 1 mg in the sample and used this area per mass relationshipto estimate roughly the quantity of each compound present in the samples.

Headspace collection of VOCs—The location of odor emission is not nec-essarily the same as the location of odor production or storage. Thus, we alsoanalyzed VOCs emitted by the different parts of the leaf (sinus, veins, andblade dissected from a total of eight leaves, one for each of four males andfour females plants) of flowering C. humilis after dynamic headspace collec-tion. Each sample was placed in a nalophan bag (Kalle Nalto, Wursthullen,Germany). Pure air was blown into the bags at 400 mL/min and pulled outat 300 mL/min (Dufay et al., 2003) through an adsorption tube containing 30mg of Alltech SuperQ (ARS, Gainesville, Florida, USA) during 2 h. Thisdifference in flux avoids contamination with outside air through possibleleaks. The adsorption tubes were then eluted with 150 mL CH2Cl2 and ana-lyzed by GC-MS, using the same method as described earlier.

Environmental scanning electron microscopy—Fresh samples of leaveswere directly pasted onto a stage in a special low-pressure chamber of an S-3000N Hitachi microscope (Tokyo, Japan). Samples were then cooled from148C to a minimum of 2208C by the Pelletier effect. Pressure was then setto 110 Pa and tension to 15 kV for observation. Micrographs were interpretedautomatically by the hardware and software supplied with the microscope.

Light microscopy and histochemistry—Observations of freehand sectionsof leaves of flowering and nonflowering C. humilis and leaves of floweringT. fortunei were made with a Leitz DMRB microscope with standard or No-marski differential interference contrast (DIC) optics. Topographic histochem-istry was done with the RT reagent (Rawlins and Takahashi reagent; Jensen,1962) by soaking sections in a bath of 10% sodium hypochloride until theybecame yellow to white. They were then briefly rinsed in water and stainedwith the RT reagent for 5 s. Iodine water (1% potassium iodure and 0.5%iodine) was used to reveal starch grains and carbohydrates. Sections weredirectly stained in iodine water for 15–45 min, then observed. To reveal es-sential oils, three standard procedures for lipid staining (Sudan black, Sudanred IV, and fat red 7B) and one terpene staining (naphthol and diamine[NADI] reaction) were used. Sections were rinsed in 50% ethanol, stained for20 min in saturated Sudan black or Sudan red IV in 70% ethanol, rinsed againin 50% ethanol, and observed (Jensen, 1962). Staining with fat red 7B (Turneret al., 2000) was done in 50% ethanol after fixation in an aqueous mix of10% formaldehyde, 5% acetic acid, and 50% ethanol. For the NADI reaction(David and Carde, 1964), fresh sections were placed for 30 min to 1 h in afreshly made mixture of 0.001% 1-naphtol, 0.001% N,N-dimethyl-p-phenyl-enediamine dihydrochloride and 0.4% ethanol in 100 mmol/L sodium caco-dylate-HCl buffer (pH 7.2) and then directly observed.

RESULTS

Palm anatomy and localization of VOCs—Chamaeropshumilis leaves are palmate (Fig. 1). Leaflets (Fig. 2) have veinswith a non-chlorophyllous parenchyma (Fig. 3) and smallerveins. Veins can be localized in either reduplicate or indupli-cate folds (Fig. 2) and are marked by a stitching line (Figs. 4,5). All these veins end with a sinus, which looks like a glandswollen on the addaxial side (Fig. 6). The sinus is more orless swollen depending on the individual, but there is no cleartrend of swelling with the maturity or the flowering stage ofthe plant. The sinus appears slightly wet and shiny duringflowering and waxy the rest of the time.

To learn more about the histology and histochemistry of theleaf tissue of flowering C. humilis palm, topographic and car-bohydrate staining were used (see Materials and Methods).The RT reagent reveals cellulose and lignin and shows thatveins and smaller veins are not vascular bundles. Veins (Fig.7) are made of the same ‘‘unfolding parenchyma’’ as sinus.This parenchyma is thought to open the leaflet at a young stage(J. Dransfield, Royal Botanical Garden, Kew, UK, personalcommunication). Smaller veins are made of sclerenchyma(Fig. 7). Vascular bundles (Fig. 8) are dispersed all over thechlorophyllous parenchyma. Iodine water reveals starch grainsin all the tissues (Fig. 9) as well as circular structures withcarbohydrates in some cells (Figs. 10, 11). Such special cellsdispersed in parenchyma are idioblasts. In flowering C. hu-milis, they are often found in the unfolding parenchyma ofveins and sinuses and in the blade.

To localize scent production, we used several stains classi-cally employed for lipids (Sudan black, Sudan red IV, fat red7B) on leaves collected on flowering plants. No specific stain-ing was observed, except for the fat red 7B that revealed id-ioblasts in the unfolding parenchyma, the sinus, and the blade(Fig. 12), suggesting lipids within the idioblasts.

1192 [Vol. 91AMERICAN JOURNAL OF BOTANY

Figs. 1–7. Fresh leaf from Chamaerops humilis at flowering. 1. Entire palmate leaf (#0.2). 2. Detail of the junctions between leaflets (#1). 3. Free-handsection of a sinus in a reduplicate fold (#70). 4. Environmental scanning electron micrograph (ESEM) of the vein of a reduplicate fold (#60). 5. An ESEMdetail of the ziplike structure of the vein of a reduplicate fold (#1000). 6. ESEM of the sinus in a reduplicate folding (#60). 7. Three photonic micrographs(assembled with Adobe Photoshop software) of only one freehand section of a fresh leaflet with induplicate folding. Note that the vein does not correspond toa vascular bundle but to unfolding parenchyma (RT staining, #50). Figure Abbreviations: ab, abaxial epidermis; ad, adaxial epidermis; cp, chlorophyllousparenchyma; gl, globoid structure; id, idioblast; IF, induplicate folding; L, leaflet; P, petiole; ph, phloem; pl, plasmodesma; RF, reduplicate folding; S, sinus; sc,sclerenchyma; st, starch grain; sV, small vein; up, unfolded parenchyma; V, (large) vein; vb, vascular bundle; xy, xylem; Z, ziplike structure.

To test the importance of these structures in scent produc-tion, we also observed leaves of young plants grown fromseeds, which had never flowered (Fig. 13) and that producedonly a very small amount of volatile compounds (see nextsection). We also observed leaves of T. fortunei, a closely re-lated species with a similar leaf shape and anatomy (data notshown) but without scent (see next section). All these leavespresented the same kind of idioblasts. Because idioblasts arepresent in both scented and unscented leaves, they are prob-ably not directly involved in scent production, even if we can-not exclude that they only function in leaves of flowering C.humilis.

Lipid staining and the RT reagent also revealed thick cuti-cles for the two species at every phenological stage. However,in C. humilis sinuses, the cells of the epidermis have a conicalshape (Figs. 14, 16). This kind of anatomy does not seem aspronounced in T. fortunei (Fig. 15).

Terpenes, known to be components of VOCs (see next sec-tion), can be localized histochemically by the NADI reagent.Unfortunately, the other components of VOCs (benzenoids andfatty acid derivatives) cannot be observed with histochemistry.We then tested the NADI reagent on the three kinds of leaves(of flowering and nonflowering C. humilis and leaves of flow-

ering T. fortunei). In leaves of flowering C. humilis, manypurple droplets were apparent in the epidermis of the blade,the sinus (e.g., Figs. 16 and 17), the unfolding parenchyma ofboth sinus and blade and, in a lower quantity, in deeper layersof cells. The presence of these droplets, virtually absent in theother two kinds of leaves, was thus linked with scent produc-tion. In conclusion, production of terpenes is likely to occurin the whole leaf (blade, sinus, and veins), and their concen-tration is greatest near the epidermis.

Extraction of VOCs—To determine the location of scentproduction in the leaf, we dissected palm leaves into sinus,veins, and blade and also extracted the odoriferous volatilesfrom male and female flowers (Fig. 18). For leaves of flow-ering C. humilis, washes of all leaf parts contained largeamounts of volatile compounds. Blades had a higher quantitythan veins and sinus. This differs strikingly from the low con-tent of VOCs of C. humilis flowers, about a hundred timesless than blades. Different leaf parts of young C. humilis andof T. fortunei also contained volatile compounds but in lowerquantities. Male flowers of T. fortunei produced comparativelymore VOCs than leaves of the same species.

The main compounds in washes were roughly the same in


Figs. 8–17. Freehand sections with idioblasts and terpene-secreting cells of a leaf of a flowering Chamaerops humilis, a very young C. humilis that hasnever flowered (Fig. 13), and a flowering Trachicarpus fortunei (Fig. 15). All sections are from a sinus with chlorophyllous tissues that stain with greatercontrast (except in Fig. 8 in which RT staining was preceded by a clearing bath). 8. Section of a leaf showing a vascular bundle (RT staining, #100). 9. Starchgranules stained with iodine water (#1000). 10. Idioblasts stained with iodine water (#1000). 11. Idioblast stained with iodine water (DIC optics, #1000). 12.Section stained with Fat red 7B (#100). 13. Idioblast of very young C. humilis stained with iodine water (#400). 14. Section in the abaxial epidermis stainedwith Sudan red (#400). 15. Section in the abaxial epidermis of T. fortunei stained with Sudan red (#400). 16. Terpenes droplets (arrow head) stained withNADI near the abaxial epidermis (#400). 17. Terpenes droplets stained with NADI near the adaxial epidermis; note the strong staining of the cuticle on thisside of the sinus (#400).


Fig. 18. Volatile organic compounds concentration (washes with dichloromethane) in different plant parts of Chamaerops humilis and Trachicarpus fortunei.

different parts of the leaf for palms of flowering C. humilis(Fig. 19), palms of young, nonflowering C. humilis (Fig. 20),and palms of T. fortunei (Fig. 21). Thus, the different volatilecomponents are produced and/or stored in all leaf parts witha higher concentration in the blade (Fig. 18). Compoundsfound in the washes of the leaves of flowering C. humilis wereprincipally terpenoids and benzenoids, molecules that are typ-ical of floral scents, while the most abundant compounds ofleaves of young C. humilis and of T. fortunei were fatty acidderivatives, the typical ‘‘green leaf volatiles’’ (Figs. 19–21).Blades of both young C. humilis and T. fortunei contained ahigher percentage of (Z)-3-hexen-1-ol, a typical leaf alcohol.Leaves of flowering C. humilis were rich in methyl benzoate,linalool, and several terpenoids. This composition differs fromthe one found by Dufay et al. (2003) with headspace extraction(see next section). Leaves of young C. humilis produced com-paratively less methyl benzoate. They also contained a largeproportion of the terpenes (E)-b-ocimene and a-farnesene(Fig. 20) but in a small absolute quantity compared with leavesof flowering C. humilis. Figures 19 and 20 illustrate the im-portant qualitative differences in the composition of VOCsproduced by leaves of different ages and the quite uniquelyhigh terpene content of VOCs produced by leaves of floweringChamaerops.

Trachycarpus fortunei produced some benzenoids, surpris-ingly with some vanilin (also found in the flowers), but notterpenoids. Flowers of C. humilis produced fewer VOCs thandid leaves (Fig. 18). The main compound was (Z)-3-hexen-ol,a typical leaf alcohol (Fig. 22) also found in young leaves ofC. humilis and T. fortunei. Benzyl alcohol is also found in theflowers. Female flowers also produce (E)-b-ocimene but in asmall absolute quantity. The main compounds of flowers of T.fortunei were nonanal, benzene acetaldehyde, and an uniden-tified compound (Fig. 22). Male and female flowers had dif-ferent proportions of these compounds. The VOCs need to becollected from other individuals to determine if this was due

to individual variation (for flowers, a single individual of eachsex was sampled) or to sexual dimorphism.

Headspace collection of VOCs—To determine from whichleaf part the fragrances are emitted, we examined the quantityand the composition of scents collected from different parts ofthe leaf by dynamic headspace collection (Table 1). Main com-pounds found in all leaf parts are methyl benzoate, (E)-b-oci-mene, an unknown sesquiterpene (sesquiterpene 7 of Dufay etal., 2003) and a-Farnesene (Table 1). This overall compositionof the headspace resembles that found by Dufay et al. (2003)in which headspace extracts of leaves still on the plant alsoprimarily contained (E)-b-ocimene and the unknown sesqui-terpene (sesquiterpene 7 of Dufay et al., 2003). Methyl ben-zoate was not identified by Dufay et al. (2003), but it is usuallyfound in other C. humilis extracts as 5–10% of the total blend(Gaillard, 2003). a-Farnesene is common in our headspaceresults but is not commonly found in C. humilis extracts (Du-fay et al., 2003; M.-C. Anstett, unpublished data); this mole-cule could have been produced from dissection of the leavessince plants often react to damage through emission of volatilecompounds (e.g., Baldwin et al., 2002). We did not find (E)-b-farnesene as did Dufay et al. (2003), but it is only presentin some plants of some populations (Gaillard, 2003).

The different compounds in the bouquet of C. humilis weredetected in all leaf parts (Table 1), with the sinus emittingproportionally twice as much (E)-b-ocimene and half as muchmethyl benzoate than did the blade. The proportion of othercompounds was roughly identical in the three parts of leaves.This shows that all parts of the leaf emitted scents, but thatsome compounds are differentially emitted by different partsof the leaves.

Leaf parts were cut in small pieces, which could possiblyaffect odor composition. However, since the same treatmentwas used for both kinds of odor extraction, the comparison isstill valid. However, this treatment probably substantially de-


creases the quantity of VOC emission and thus the quantityof volatiles collected from the headspace. We therefore did nottry to quantify the production of VOCs in the headspace ex-tracts. Table 1 shows the differences in the percentages of themain compounds (present at more than 5% in at least one ofthe samples) for the two kinds of extractions. Even though thecompounds present are identical, their proportions differ great-ly. Among the major compounds in the headspace extract, the(E)-b-ocimene, the unknown sesquiterpene (sesquiterpene 7 ofDufay et al., 2003), and the a-farnesene were present in verysmall proportion in the washes (Table 1). On the other hand,methyl benzoate and linalool were major compounds of thewashes, but their proportion decreased in the headspace ex-tract. This could be due to different volatilities, but is alsosuggestive of a possible differential excretion of VOCs, es-pecially in the sinus.

DISCUSSION

Localization and emission of VOCs—The human nose isable to perceive the odors emitted by the palm leaf in thesinuses of flowering C. humilis (Dufay et al., 2003; M.-C.Anstett and A. Meekijjironenroj, personal observation). Thisobservation could be due to either the highest level of emis-sion of volatile compounds in the sinus or to the highest pro-portion of (E)-b-ocimene in these emissions. Our results clear-ly show that terpenes and other VOCs are present in the wholeleaf of flowering C. humilis. This apparent contradiction sug-gests that VOCs migrate from the whole leaf to the sinuswhere they could be principally emitted.

However, we cannot exclude the possibility that the humansense of smell used by Dufay et al. (2003) to locate the emis-sion of VOCs may be unable to detect some compounds. In-deed, there exist a polymorphism of sensitivity to some com-pounds. The most famous example concerns (1) 5 a-androst-16-en-3-one, which is smelled by 70.5% of women and 62.8%of men, but not perceived by other people (cited in Ohloff,1994). Furthermore, the human nose can be very sensitive totrace compounds but not to major compounds: e.g., the b-damascenone at 0.14% in rose oil contributes 70% of the scentperceived by humans (Ohloff, 1994). In C. humilis, headspaceextracts of sinus, blade, and veins differ in their proportionsof VOCs and thus may be perceived differently by the nose.Unfortunately, the very small size of the sinuses and veinsobliged us to dissect them before headspace collection. Thisdissection probably modifies the emission process and releasescompounds stocked beneath the cuticle. It is thus difficult toascertain where the maximum emission of VOCs takes place.

The localization of terpenes and VOC production in thewhole leaf of flowering C. humilis suggests a convergent evo-lution of the pollinator attraction function of leaves and petalsin this species. We will discuss further the possibilities for aleaf to emit attractive compounds and finally to act as a petal.

Production of VOCs in leaves—Volatile compounds cannotusually be localized by histochemistry, and terpenes are usu-ally difficult to distinguish from lipids in histological studies.NADI staining is one of the few specific stains for terpenes(David and Carde, 1964). Used in many studies (e.g., Ascen-sao and Pais, 1988; Ascensao et al., 1999; Platt and Thomson,1992), it always gives a purple stain, slightly different fromthe colors described by David and Carde (1964). As shownby our GC results, NADI staining was linked with VOCs and

terpene production by leaves. Terpene droplets were detectedin the epidermis and in deeper cell layers. However, the actualpath of transport from cell to cell and from cell to the plantsurface is still poorly known. Several secretion pathways havebeen described (Fahn, 1988), but their actual importance innatural secretion has not been established. In eccrine secretion,molecules travel directly through the plasma membrane, eitherpassively or actively through some specific transporter. Re-cently, an ATP-dependent transporter was cloned in tobacco(Jasinski et al., 2001), demonstrating the possible occurrenceof active transmembrane transport. In granulocrine transport,a very different mechanism, membranes of lipid vesicles or ofoil bodies fuse with the plasma membrane, releasing VOCs tothe outside of the cell (Fahn, 1988; Hudak and Thompson,1997; Suire et al., 2000). In C. humilis, terpenes are in drop-lets, which are required for granulocrine secretion, but that isnot sufficient to exclude eccrine secretion. However, it is im-portant to consider whether terpenes are produced and emittedexactly where the NADI reaction localized them.

Both histological and chemical results show that terpenesand other VOCs are present in the whole leaf of flowering C.humilis where idioblasts are also abundant. Our two controls(leaves of young, nonflowering C. humilis and of T. fortunei)produced only very small amounts of VOCs but containedabundant idioblasts. Moreover, NADI localized terpenes nearthe epidermis while idioblasts are dispersed in the parenchy-ma. Thus, VOC production is probably not the function ofidioblasts or, at least, not their only function.

Idioblasts can have very diverse functions. They are ofteninvolved in defense against herbivores or pathogens (e.g., Pell-myr et al., 1991; Vovides, 1991; Vovides et al., 1993) but, toour knowledge, they have never been found to be involved inattracting pollinators. They can produce alkaloids (e.g., SaintPierre et al., 1999), glucosinolates (e.g., Andreasson et al.,2001), lipids (e.g., Read and Menary, 2000), calcium oxalatecrystals (e.g., Nakata, 2003), neurotoxins (e.g., Vovides et al.,1993), or other compounds commonly associated with plantprotection and/or detoxification. Terpene production by idio-blasts was suggested in avocado, Persea americana (Laura-ceae; Platt and Thomson, 1992). Idioblasts are also known inpalm flowers (Geonoma interrupta, Arecaceae) in which theyare usually thought to produce tannins (e.g., Stauffer et al.,2002). In the palm Aphandra natalia (Arecaceae), some raph-ide-containing idioblasts are released at the same time as pol-len and could possibly deter pollen-feeding or ovipositing in-sects (Barfod and Uhl, 2001). Here we have shown that C.humilis idioblasts are present in all parts of the leaf and thattheir density is highly variable from leaf to leaf, but not as-sociated with scent production.

In C. humilis, the cuticle is quite thick on all parts of theleaves, representing a layer of more than one-half of the heightof an epidermal cell, whilst VOCs are probably emittedthrough this cuticle. In a first study of the permeability ofcuticular waxes of petal to VOCs, Goodwin et al. (2003)showed that the quite thick cuticle of snapdragon (Antirrhinummajus, Scrophulariaceae) provides little resistance to diffusionof methyl benzoate and that the thickness of the cuticle hasno apparent effect on emission of VOCs. Thus, the thick cu-ticle of C. humilis is not necessarily a barrier to the diffusionof VOCs.

Volatile attractants of palms—In leaves of C. humilis, thetotal amount of VOCs extracted from the blade was 23- to


Figs. 19–22. Composition of the volatile blend collected by washing dissected plant parts of Chamaerops humilis and Trachycarpus fortunei. 19. Leavesof flowering C. humilis (left to right, sinus, vein, and blade). 20. Leaves of young, nonflowering C. humilis (left to right, with sinus, vein, and blade). 21.Leaves of flowering T. fortunei (left to right, sinus, vein, and blade). 22. Male and female flowers of C. humilis and T. fortunei. Terpenoid compounds aregreen, benzenoid compounds are blue, and fatty acid derivatives are red. 1, unknown sesquiterpene; 2, a-farnesene; 3, (E)-b-ocimene; 4, benzyl alcohol; 5, (Z)-3-hexen-ol; 6, methyl benzoate; 7, hexenyl acetate; 8, benzene acetaldehyde; 9, indole; 10, linalool; 11, vanillin; 12, nonanal; 13, 3-hexanol.

107-fold greater (52.6 mg/g) than the total in pistillate (2.27mg/g) or staminate (0.49 mg/g) flowers of this species. This isa striking difference from the usual pattern in angiosperms.For example, in different Nicotiana species, volatile produc-

tion is 30–100 times lower in leaves than in flowers (Loughrinet al., 1990). Among the volatile compounds found in Cha-maerops, linalool and benzyl alcohol are among the most com-mon VOCs found in flower scent (Knudsen et al., 1993). Lin-


TABLE 1. Percentage of main compounds in headspace and wash collections for each leaf part of Chamaerops humilis.

Compound name

Headspace

Sinus Vein Blade

Wash

Sinus Vein Blade

Benzyl alcohol(E)-b-OcimeneMethyl benzoateLinalool

0.9330.7518.03

1.27

0.6921.4218.64

1.20

1.5112.1434.00

0.16

5.161.93

50.8612.72

6.482.11

48.239.89

2.851.77

38.7023.33

IndoleUnknown sesquiterpenea-Farnesene

2.0516.6512.75

1.8220.8717.09

1.8918.5718.91

5.986.223.34

6.895.303.07

1.563.917.59

alool is only present in leaves of flowering C. humilis whilstbenzyl alcohol is found in leaves of both flowering and non-flowering C. humilis and only in trace amounts in male flow-ers. Linalool is known to attract pollinators to flowers (Ragusoand Pichersky, 1995; Dudareva et al., 1996; Borg-Karlson etal., 2003). Furthermore, there is a marked modification ofVOC production by C. humilis leaves according to the flow-ering state of the plant. In leaves of young, preflowering C.humilis, 3-hexen-1-ol and (Z)-3-hexenyl acetate are the majorcompounds. They are also found at a low percentage, but atthe same absolute quantity, in leaves of flowering C. humilisand are also found in the unscented T. fortunei. These mole-cules are known to be released just after damage by herbivores(Baldwin et al., 2001, 2002) and could have resulted from thedissection.

Methyl benzoate is the most abundant compound in thewashes and is also one of the most abundant compounds inheadspace extracts of flowering C. humilis. This molecule isknown to attract insects. Trachicarpus fortunei does not pro-duce methyl benzoate but does produce small amounts of van-illin, a benzenoid derivative that is less effective in insect at-traction (Raguso and Pichersky, 1995). The other main com-pounds found in headspace extracts are (E)-b-ocimene and theunknown sesquiterpene. They are both present at a higher per-centage in headspace extracts than in washes, which couldindicate a role in pollinator attraction. Furthermore, a-farne-sene, only found in flowering C. humilis leaves, is also knownto be part of the floral scent of deceptive or sphingophilousflowers (Knudsen and Tollsten, 1993).

Nevertheless, plant scent in insect interactions cannot usu-ally be reduced to the effect of one major compound. Theneuronal pathway of olfactory information is finely tuned andits integration depends also on the memory and the experienceof the insect (Matsumoto and Mizunami, 2000; Tronson, 2001;Mustaparta, 2002). Furthermore, some compounds can be de-tected by insects at a very low concentration, acting as essen-tial components for pollinator attraction in a complex scent orsometimes even as the most informative molecule (Knudsenand Tollsten, 1993). Dufay et al. (2003) has already shownthat Derelomus chamaeropsis is attracted by the whole scentof C. humilis leaves. The activities of the individual com-pounds here still have to be tested in behavioral tests.

Convergent evolution of leaves toward petals—Many pub-lications show that leaves and petals are fundamentally similarorgans, with few divergent developmental pathways at theirorigin (Baum and Whitlock, 1999; Dilcher, 2000; Guttierrez-Cortines and Davies, 2000; Albert et al., 2002; Pellmyr, 2002).For example, the flowers of the triple ABC mutant apetala 2–1 apetala 3–1 agamous-1 of A. thaliana are replaced by

whorls of hairy leaflike organs (Coen and Meyerowitz, 1991).In the case of C. humilis, our data strongly suggest a conver-gent evolution of leaves toward petals for the pollinator at-traction function and for the structure of scent emission. Suchevolution is known for other secondary products, like thoseinvolved in coloration of plant organs. For example, the leavesof Dalbergaria florida (Gesneriacee) present red-spottedleaves known to attract hummingbirds for pollination (cited inProctor et al., 1996). Many other species can be observed withthese kind of adaptive traits (e.g., in Euphorbiaceae, Brome-liaceae). In C. humilis, the secondary products involved in thisconvergent evolution are not pigments but volatiles. For ex-ample, methyl benzoate is quite abundant in leaves of flow-ering C. humilis but, in Clarkia breweri (Onagraceae), thegene encoding for the benzoic acid carboxyl methyl transfer-ase that produces methyl benzoate is highly specific to theepidermal cells of petals (Kolosova et al., 2001b). It would beinteresting to determine whether this gene is expressed in themesophyll or in the foliage epidermis of flowering Chama-erops humilis. Furthermore, the same experiments could bedone with linalool synthase and the enzymes catalyzing thesynthesis of (E)-b-ocimene. These enzymes are also highlyspecific to flowers in C. breweri and A. majus (Dudareva etal., 1996, 2003).

Secondary metabolic pathways could have evolved as theyhave done in petals of other species. Furthermore, from a de-velopmental point of view, the developmental pathway of cellssecreting attractive VOCs is quite similar to the one of cellssecreting repulsive VOCs. Indeed, in transgenic 35S::MIXTANicotiana tabacum (Solanaceae), the over-expression of MIX-TA, normally involved in the differentiation of papillate cellsof A. majus petals, does not result in ectopic papillate cells inthe whole plant but results in numerous secreting trichomesnormally involved in plant defense (Glover et al., 1998).

In conclusion, we have shown that the pattern of terpeneproduction and emission in the leaves of C. humilis resemblesin many ways the same processes expressed in petals of manyangiosperms, suggesting a convergent evolution of leaves to-ward petals. The similarities at the functional, anatomical, andbiochemical levels could be due to common underlying de-velopmental and secondary metabolic machineries in these se-rially homologous organs.

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Localization of production and emission of pollinator attractant on whole leaves of Chamaerops...

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