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(American Journal of Botany. 2004;91:1190-1199.)
© 2004 Botanical Society of America, Inc.

Reproductive Biology

Localization of production and emission of pollinator attractant on whole leaves of Chamaerops humilis (Arecaceae)1

Jean-Claude Caissard2, Aroonrat Meekijjironenroj3, Sylvie Baudino2 and Marie-Charlotte Anstett3,4

2Laboratoire BVpam (Biotechnologies Végétales, plantes aromatiques et médicinales) EA 3061, Université Jean Monnet, 23 rue du Docteur Paul Michelon, F-42023 Saint-Etienne Cédex 02, France; 3Centre d'Ecologie Fonctionnelle et Evolutive, CNRS, 1919 Route de Mende, F-34 293 Montpellier Cédex 5, France

Received for publication October 23, 2003. Accepted for publication March 30, 2004.


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

Key Words: Arecaceae • floral scent • idioblasts • leaf scent • palm • pollination • terpenes • volatile organic compounds


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Plants produce many secondary compounds (or natural products), many functioning as mediators of plant–insect interactions. These compounds may either repel herbivores or attract potential benefactors such as pollinators (e.g., Rodriguez and Levin, 1976 ; Pichersky and Gershenzon, 2002 ). Repellent molecules typically act by contact and are produced by trichomes, idioblasts, osmophores, or other secreting tissues, with stocks of secondary compounds kept in specialized cells. Some of these compounds are highly volatile and are also produced by flowers to form floral perfumes that act at a distance to attract pollinators. Like other floral parts (e.g., sepals, carpels, stamens) petals are homologous with leaves and certainly evolved from leaves (Gutierrez-Cortines and Davies, 2000 ; Pellmyr, 2002 ).

Secondary metabolites of leaves and floral elements thus may have a common evolutionary origin and a shared developmental pathway. For example, the chemical attractants of cycad cones are supposed to have evolved from herbivore deterrents (Pellmyr et al., 1991 ). However the detailed developmental pathway connecting organ to molecular levels of attractants is still poorly known, even though MADS transcription factors seem to be involved in the evolution of flowers (Lawton-Rauh et al., 2000 ; Albert et al., 2002 ). Since flowers attract pollinators from a distance, only volatile organic compounds (VOCs) have been retained through natural selection and are found in floral scents. The VOCs are all derived from fatty acids, benzenoids, or isoprenoids (Knudsen et al., 1993 ).

Many terpenes are present in both leaf and floral VOCs and may either attract or repel insects (Pichersky and Gershenzon, 2002 ). For example, more than 600 articles have been published on the diverse roles of (E)-ß-farnesene. Among diverse functions, this sesquiterpene can be a plant (and insect) defensive allomone, an attractant pheromone, or a kairomone that stimulates oviposition (reviewed by Crock et al., 1997 ). The structures producing the VOCs differ between leaves and flowers. Leaves produce their repellent odors in a variety of different structures (such as trichomes, idioblasts, cavities, and ducts) depending on the species. In contrast, flowers usually produce their attractive fragrance in osmophores or in conical cells located on the petals. These cells do not stock VOCs but release them into the air. Even though the importance of VOCs in species interactions have been recently investigated at the molecular level (Dudareva and Pichersky, 2000 ; Baldwin et al., 2001 ; Kolosova et al., 2001a ; Pichersky and Gershenzon, 2002 ), surprisingly little information is available on the exact cellular location of transport and emission of VOCs into the surrounding air (e.g., Hudak and Thompson, 1997 ; Turner et al., 1999 ; Bouvier et al., 2000 ; Jasinski et al., 2001 ; Goodwin et al., 2003 ).

Recently, Dufaÿ et al. (2003) found an interesting intermediate between these two paradigms of odor production (i.e., repellent VOCs are produced on leaves and attractive VOCs on flowers by different structures). Leaves of Chamaerops humilis L. (Arecaceae), the Mediterranean dwarf palm, produce volatile compounds that attract their species-specific pollinating weevil (Derelomus chamaeropsis), whereas the flowers are almost scentless. This odor production is limited to anthesis and 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 Dufaÿ et al. (2003) suggested that scents could be produced at this location. This example provides a unique opportunity to study the transition between foliar and floral scent production. Indeed, some other species of palms, relatively close to C. humilis in phylogenetic trees (Asmussen and Chase, 2001 ), produce scents only in flowers (e.g., Guihaia grossefibrosa, Arecaceae; Dufaÿ, 2003 ) or in both leaves and flowers (Serenoa repens, Arecaceae; Dufaÿ, 2003 ).

In this paper, we describe the structure of the sinus at the histological level. We show that terpenes and VOCs are localized not only in the sinuses, but also in the rest of the leaf. We also show that the proportion of some molecules is higher in the VOCs emitted by the sinuses. Finally, we attempt to understand why VOCs seems to be produced by the leaf but are emitted by the sinus.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Material
All leaves of flowering Chamaerops humilis and Trachycarpus fortunei used in the following experiments were collected from ornamental palms planted in Montpellier, France. Leaves of young, nonflowering C. humilis were collected from palms grown from seed in the greenhouse of the Centre 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, and blade in dichloromethane. First, we dissected the leaves with a razor blade separating the sinus, veins, and blade. Care was taken not to include even small pieces of blade in the sinus and veins samples. Leaves with a perceptible scent of flowering C. humilis (a total of six leaves from three different male individuals) 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 18 individuals) and of flowering Trachycarpus fortunei, a closely related species of the same subtribe (Asmussen and Chase, 2001 ; total of eight leaves, one for each of four males and four females plants), were dissected. The number of leaves dissected varied because we dissected the number of leaves needed to obtain 0.5 g (all fresh mass) of sinus. From the same palms, we kept 0.5 g of veins and 2.5 g of blade from the different leaves. All these leaf samples plus some staminate (2.5 g) and pistillate (2.5 g) flowers of both species were washed for 36 h in 1 mL of dichloromethane (CH2Cl2) for samples of 0.5 g or 5 mL of CH2Cl2 for samples of 2.5 g. The washes were then analyzed quantitatively 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 µL in a CP sil 8 CB column (30 m, 0.25 mm inner diameter, 0.25 µm film thickness) with helium as the carrier gas. The temperature was kept at 50°C for the first 3 min, then programmed to increase 3°C/min to 100°C, 2.7°C/min to 140°C, 2.4°C/min to 180°C, and then 6°C/min to 250°C. Volatiles were identified by comparing their mass spectra with those of the NIST98 library and with GC retention times and MS spectra of the authentic compounds when possible. For quantification, we added 4 µg of each of two internal standards (nonane and dodecane) to each sample before analysis. We then calculated the peak area of internal standard corresponding to 1 µg in the sample and used this area per mass relationship to estimate roughly the quantity of each compound present in the samples.

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

Environmental scanning electron microscopy
Fresh samples of leaves were directly pasted onto a stage in a special low-pressure chamber of an S-3000N Hitachi microscope (Tokyo, Japan). Samples were then cooled from +4°C to a minimum of –20°C by the Pelletier effect. Pressure was then set to 110 Pa and tension to 15 kV for observation. Micrographs were interpreted automatically by the hardware and software supplied with the microscope.

Light microscopy and histochemistry
Observations of freehand sections of leaves of flowering and nonflowering C. humilis and leaves of flowering T. fortunei were made with a Leitz DMRB microscope with standard or Nomarski differential interference contrast (DIC) optics. Topographic histochemistry was done with the RT reagent (Rawlins and Takahashi reagent; Jensen, 1962 ) by soaking sections in a bath of 10% sodium hypochloride until they became yellow to white. They were then briefly rinsed in water and stained with the RT reagent for 5 s. Iodine water (1% potassium iodure and 0.5% iodine) was used to reveal starch grains and carbohydrates. Sections were directly stained in iodine water for 15–45 min, then observed. To reveal essential oils, three standard procedures for lipid staining (Sudan black, Sudan red IV, and fat red 7B) and one terpene staining (naphthol and diamine [NADI] reaction) were used. Sections were rinsed in 50% ethanol, stained for 20 min in saturated Sudan black or Sudan red IV in 70% ethanol, rinsed again in 50% ethanol, and observed (Jensen, 1962 ). Staining with fat red 7B (Turner et al., 2000 ) was done in 50% ethanol after fixation in an aqueous mix of 10% 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 a freshly made mixture of 0.001% 1-naphtol, 0.001% N,N-dimethyl-p-phenylenediamine dihydrochloride and 0.4% ethanol in 100 mmol/L sodium cacodylate-HCl buffer (pH 7.2) and then directly observed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Palm anatomy and localization of VOCs
Chamaerops humilis leaves are palmate (Fig. 1). Leaflets (Fig. 2) have veins with a non-chlorophyllous parenchyma (Fig. 3) and smaller veins. Veins can be localized in either reduplicate or induplicate folds (Fig. 2) and are marked by a stitching line (Figs. 4, 5). All these veins end with a sinus, which looks like a gland swollen on the addaxial side (Fig. 6). The sinus is more or less swollen depending on the individual, but there is no clear trend of swelling with the maturity or the flowering stage of the plant. The sinus appears slightly wet and shiny during flowering and waxy the rest of the time.



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  		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-hand section of a sinus in a reduplicate fold (≤70). 4. Environmental scanning electron micrograph (ESEM) of the vein of a reduplicate fold (≤60). 5. An ESEM detail 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 to a vascular bundle but to unfolding parenchyma (RT staining, ≤50). Figure Abbreviations: ab, abaxial epidermis; ad, adaxial epidermis; cp, chlorophyllous parenchyma; 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 learn more about the histology and histochemistry of the leaf tissue of flowering C. humilis palm, topographic and carbohydrate staining were used (see Materials and Methods). The RT reagent reveals cellulose and lignin and shows that veins 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, personal communication). Smaller veins are made of sclerenchyma (Fig. 7). Vascular bundles (Fig. 8) are dispersed all over the chlorophyllous parenchyma. Iodine water reveals starch grains in all the tissues (Fig. 9) as well as circular structures with carbohydrates in some cells (Figs. 10, 11). Such special cells dispersed in parenchyma are idioblasts. In flowering C. humilis, they are often found in the unfolding parenchyma of veins and sinuses and in the blade.



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  		 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 has never flowered (Fig. 13), and a flowering Trachicarpus fortunei (Fig. 15). All sections are from a sinus with chlorophyllous tissues that stain with greater contrast (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. Starch granules 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 stained with Sudan red (≤400). 15. Section in the abaxial epidermis of T. fortunei stained with Sudan red (≤400). 16. Terpenes droplets (arrow head) stained with NADI near the abaxial epidermis (≤400). 17. Terpenes droplets stained with NADI near the adaxial epidermis; note the strong staining of the cuticle on this side of the sinus (≤400)

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

To test the importance of these structures in scent production, we also observed leaves of young plants grown from seeds, which had never flowered (Fig. 13) and that produced only a very small amount of volatile compounds (see next section). We also observed leaves of T. fortunei, a closely related species with a similar leaf shape and anatomy (data not shown) but without scent (see next section). All these leaves presented the same kind of idioblasts. Because idioblasts are present in both scented and unscented leaves, they are probably not directly involved in scent production, even if we cannot exclude that they only function in leaves of flowering C. humilis.

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

Terpenes, known to be components of VOCs (see next section), can be localized histochemically by the NADI reagent. Unfortunately, the other components of VOCs (benzenoids and fatty 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 flowering T. fortunei). In leaves of flowering C. humilis, many purple droplets were apparent in the epidermis of the blade, the sinus (e.g., Figs. 16 and 17), the unfolding parenchyma of both sinus and blade and, in a lower quantity, in deeper layers of cells. The presence of these droplets, virtually absent in the other two kinds of leaves, was thus linked with scent production. In conclusion, production of terpenes is likely to occur in the whole leaf (blade, sinus, and veins), and their concentration is greatest near the epidermis.

Extraction of VOCs
To determine the location of scent production in the leaf, we dissected palm leaves into sinus, veins, and blade and also extracted the odoriferous volatiles from male and female flowers (Fig. 18). For leaves of flowering C. humilis, washes of all leaf parts contained large amounts of volatile compounds. Blades had a higher quantity than veins and sinus. This differs strikingly from the low content of VOCs of C. humilis flowers, about a hundred times less than blades. Different leaf parts of young C. humilis and of T. fortunei also contained volatile compounds but in lower quantities. Male flowers of T. fortunei produced comparatively more VOCs than leaves of the same species.



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

 
The main compounds in washes were roughly the same in 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 volatile components are produced and/or stored in all leaf parts with a higher concentration in the blade (Fig. 18). Compounds found in the washes of the leaves of flowering C. humilis were principally terpenoids and benzenoids, molecules that are typical of floral scents, while the most abundant compounds of leaves of young C. humilis and of T. fortunei were fatty acid derivatives, the typical "green leaf volatiles" (Figs. 19–21). Blades of both young C. humilis and T. fortunei contained a higher 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 from the one found by Dufaÿ et al. (2003) with headspace extraction (see next section). Leaves of young C. humilis produced comparatively less methyl benzoate. They also contained a large proportion of the terpenes (E)-ß-ocimene and {alpha}-farnesene (Fig. 20) but in a small absolute quantity compared with leaves of flowering C. humilis. Figures 19 and 20 illustrate the important qualitative differences in the composition of VOCs produced by leaves of different ages and the quite uniquely high terpene content of VOCs produced by leaves of flowering Chamaerops.



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  		 Figs. 19–22. Composition of the volatile blend collected by washing dissected plant parts of Chamaerops humilis and Trachycarpus fortunei. 19. Leaves of 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 are green, benzenoid compounds are blue, and fatty acid derivatives are red. 1, unknown sesquiterpene; 2, {alpha}-farnesene; 3, (E)-ß-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

 
Trachycarpus fortunei produced some benzenoids, surprisingly with some vanilin (also found in the flowers), but not terpenoids. Flowers of C. humilis produced fewer VOCs than did leaves (Fig. 18). The main compound was (Z)-3-hexen-ol, a typical leaf alcohol (Fig. 22) also found in young leaves of C. humilis and T. fortunei. Benzyl alcohol is also found in the flowers. Female flowers also produce (E)-ß-ocimene but in a small absolute quantity. The main compounds of flowers of T. fortunei were nonanal, benzene acetaldehyde, and an unidentified compound (Fig. 22). Male and female flowers had different proportions of these compounds. The VOCs need to be collected from other individuals to determine if this was due to individual variation (for flowers, a single individual of each sex was sampled) or to sexual dimorphism.

Headspace collection of VOCs
To determine from which leaf part the fragrances are emitted, we examined the quantity and the composition of scents collected from different parts of the leaf by dynamic headspace collection (Table 1). Main compounds found in all leaf parts are methyl benzoate, (E)-ß-ocimene, an unknown sesquiterpene (sesquiterpene 7 of Dufaÿ et al., 2003 ) and {alpha}-Farnesene (Table 1). This overall composition of the headspace resembles that found by Dufaÿ et al. (2003) in which headspace extracts of leaves still on the plant also primarily contained (E)-ß-ocimene and the unknown sesquiterpene (sesquiterpene 7 of Dufaÿ et al., 2003 ). Methyl benzoate was not identified by Dufaÿ et al. (2003) , but it is usually found in other C. humilis extracts as 5–10% of the total blend (Gaillard, 2003 ). {alpha}-Farnesene is common in our headspace results but is not commonly found in C. humilis extracts (Dufaÿ et al., 2003 ; M.-C. Anstett, unpublished data); this molecule could have been produced from dissection of the leaves since plants often react to damage through emission of volatile compounds (e.g., Baldwin et al., 2002 ). We did not find (E)-ß-farnesene as did Dufaÿ et al. (2003) , but it is only present in some plants of some populations (Gaillard, 2003 ).


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

 
The different compounds in the bouquet of C. humilis were detected in all leaf parts (Table 1), with the sinus emitting proportionally twice as much (E)-ß-ocimene and half as much methyl benzoate than did the blade. The proportion of other compounds was roughly identical in the three parts of leaves. This shows that all parts of the leaf emitted scents, but that some compounds are differentially emitted by different parts of the leaves.

Leaf parts were cut in small pieces, which could possibly affect odor composition. However, since the same treatment was used for both kinds of odor extraction, the comparison is still valid. However, this treatment probably substantially decreases the quantity of VOC emission and thus the quantity of volatiles collected from the headspace. We therefore did not try to quantify the production of VOCs in the headspace extracts. Table 1 shows the differences in the percentages of the main compounds (present at more than 5% in at least one of the samples) for the two kinds of extractions. Even though the compounds present are identical, their proportions differ greatly. Among the major compounds in the headspace extract, the (E)-ß-ocimene, the unknown sesquiterpene (sesquiterpene 7 of Dufaÿ et al., 2003 ), and the {alpha}-farnesene were present in very small proportion in the washes (Table 1). On the other hand, methyl benzoate and linalool were major compounds of the washes, but their proportion decreased in the headspace extract. This could be due to different volatilities, but is also suggestive of a possible differential excretion of VOCs, especially in the sinus.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Localization and emission of VOCs
The human nose is able to perceive the odors emitted by the palm leaf in the sinuses of flowering C. humilis (Dufaÿ et al., 2003 ; M.-C. Anstett and A. Meekijjironenroj, personal observation). This observation could be due to either the highest level of emission of volatile compounds in the sinus or to the highest proportion of (E)-ß-ocimene in these emissions. Our results clearly show that terpenes and other VOCs are present in the whole leaf of flowering C. humilis. This apparent contradiction suggests that VOCs migrate from the whole leaf to the sinus where they could be principally emitted.

However, we cannot exclude the possibility that the human sense of smell used by Dufaÿ et al. (2003) to locate the emission of VOCs may be unable to detect some compounds. Indeed, there exist a polymorphism of sensitivity to some compounds. The most famous example concerns (+) 5 {alpha}-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 to trace compounds but not to major compounds: e.g., the ß-damascenone at 0.14% in rose oil contributes 70% of the scent perceived by humans (Ohloff, 1994 ). In C. humilis, headspace extracts of sinus, blade, and veins differ in their proportions of VOCs and thus may be perceived differently by the nose. Unfortunately, the very small size of the sinuses and veins obliged us to dissect them before headspace collection. This dissection probably modifies the emission process and releases compounds stocked beneath the cuticle. It is thus difficult to ascertain where the maximum emission of VOCs takes place.

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

Production of VOCs in leaves
Volatile compounds cannot usually be localized by histochemistry, and terpenes are usually 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., Ascensão and Pais, 1988 ; Ascensão et al., 1999 ; Platt and Thomson, 1992 ), it always gives a purple stain, slightly different from the colors described by David and Carde (1964) . As shown by our GC results, NADI staining was linked with VOCs and terpene production by leaves. Terpene droplets were detected in the epidermis and in deeper cell layers. However, the actual path of transport from cell to cell and from cell to the plant surface is still poorly known. Several secretion pathways have been described (Fahn, 1988 ), but their actual importance in natural secretion has not been established. In eccrine secretion, molecules travel directly through the plasma membrane, either passively or actively through some specific transporter. Recently, an ATP-dependent transporter was cloned in tobacco (Jasinski et al., 2001 ), demonstrating the possible occurrence of active transmembrane transport. In granulocrine transport, a very different mechanism, membranes of lipid vesicles or of oil bodies fuse with the plasma membrane, releasing VOCs to the outside of the cell (Fahn, 1988 ; Hudak and Thompson, 1997 ; Suire et al., 2000 ). In C. humilis, terpenes are in droplets, which are required for granulocrine secretion, but that is not sufficient to exclude eccrine secretion. However, it is important to consider whether terpenes are produced and emitted exactly where the NADI reaction localized them.

Both histological and chemical results show that terpenes and 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 contained abundant idioblasts. Moreover, NADI localized terpenes near the epidermis while idioblasts are dispersed in the parenchyma. Thus, VOC production is probably not the function of idioblasts or, at least, not their only function.

Idioblasts can have very diverse functions. They are often involved in defense against herbivores or pathogens (e.g., Pellmyr et al., 1991 ; Vovides, 1991 ; Vovides et al., 1993 ) but, to our knowledge, they have never been found to be involved in attracting pollinators. They can produce alkaloids (e.g., Saint Pierre et al., 1999 ), glucosinolates (e.g., Andréasson et al., 2001 ), lipids (e.g., Read and Menary, 2000 ), calcium oxalate crystals (e.g., Nakata, 2003 ), neurotoxins (e.g., Vovides et al., 1993 ), or other compounds commonly associated with plant protection and/or detoxification. Terpene production by idioblasts was suggested in avocado, Persea americana (Lauraceae; Platt and Thomson, 1992 ). Idioblasts are also known in palm flowers (Geonoma interrupta, Arecaceae) in which they are usually thought to produce tannins (e.g., Stauffer et al., 2002 ). In the palm Aphandra natalia (Arecaceae), some raphide-containing idioblasts are released at the same time as pollen and could possibly deter pollen-feeding or ovipositing insects (Barfod and Uhl, 2001 ). Here we have shown that C. humilis idioblasts are present in all parts of the leaf and that their density is highly variable from leaf to leaf, but not associated with scent production.

In C. humilis, the cuticle is quite thick on all parts of the leaves, representing a layer of more than one-half of the height of an epidermal cell, whilst VOCs are probably emitted through this cuticle. In a first study of the permeability of cuticular waxes of petal to VOCs, Goodwin et al. (2003) showed that the quite thick cuticle of snapdragon (Antirrhinum majus, Scrophulariaceae) provides little resistance to diffusion of methyl benzoate and that the thickness of the cuticle has no apparent effect on emission of VOCs. Thus, the thick cuticle of C. humilis is not necessarily a barrier to the diffusion of VOCs.

Volatile attractants of palms
In leaves of C. humilis, the total amount of VOCs extracted from the blade was 23- to 107-fold greater (52.6 µg/g) than the total in pistillate (2.27 µg/g) or staminate (0.49 µg/g) flowers of this species. This is a striking difference from the usual pattern in angiosperms. For example, in different Nicotiana species, volatile production is 30–100 times lower in leaves than in flowers (Loughrin et al., 1990 ). Among the volatile compounds found in Chamaerops, linalool and benzyl alcohol are among the most common VOCs found in flower scent (Knudsen et al., 1993 ). Linalool is only present in leaves of flowering C. humilis whilst benzyl alcohol is found in leaves of both flowering and nonflowering C. humilis and only in trace amounts in male flowers. Linalool is known to attract pollinators to flowers (Raguso and Pichersky, 1995 ; Dudareva et al., 1996 ; Borg-Karlson et al., 2003 ). Furthermore, there is a marked modification of VOC production by C. humilis leaves according to the flowering state of the plant. In leaves of young, preflowering C. humilis, 3-hexen-1-ol and (Z)-3-hexenyl acetate are the major compounds. They are also found at a low percentage, but at the same absolute quantity, in leaves of flowering C. humilis and are also found in the unscented T. fortunei. These molecules are known to be released just after damage by herbivores (Baldwin et al., 2001 , 2002 ) and could have resulted from the dissection.

Methyl benzoate is the most abundant compound in the washes and is also one of the most abundant compounds in headspace extracts of flowering C. humilis. This molecule is known to attract insects. Trachicarpus fortunei does not produce methyl benzoate but does produce small amounts of vanillin, a benzenoid derivative that is less effective in insect attraction (Raguso and Pichersky, 1995 ). The other main compounds found in headspace extracts are (E)-ß-ocimene and the unknown sesquiterpene. They are both present at a higher percentage in headspace extracts than in washes, which could indicate a role in pollinator attraction. Furthermore, {alpha}-farnesene, only found in flowering C. humilis leaves, is also known to be part of the floral scent of deceptive or sphingophilous flowers (Knudsen and Tollsten, 1993 ).

Nevertheless, plant scent in insect interactions cannot usually be reduced to the effect of one major compound. The neuronal pathway of olfactory information is finely tuned and its integration depends also on the memory and the experience of the insect (Matsumoto and Mizunami, 2000 ; Tronson, 2001 ; Mustaparta, 2002 ). Furthermore, some compounds can be detected by insects at a very low concentration, acting as essential components for pollinator attraction in a complex scent or sometimes even as the most informative molecule (Knudsen and Tollsten, 1993 ). Dufaÿ et al. (2003) has already shown that Derelomus chamaeropsis is attracted by the whole scent of C. humilis leaves. The activities of the individual compounds here still have to be tested in behavioral tests.

Convergent evolution of leaves toward petals
Many publications show that leaves and petals are fundamentally similar organs, with few divergent developmental pathways at their origin (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 convergent evolution of leaves toward petals for the pollinator attraction function and for the structure of scent emission. Such evolution is known for other secondary products, like those involved in coloration of plant organs. For example, the leaves of Dalbergaria florida (Gesneriacee) present red-spotted leaves known to attract hummingbirds for pollination (cited in Proctor et al., 1996 ). Many other species can be observed with these kind of adaptive traits (e.g., in Euphorbiaceae, Bromeliaceae). In C. humilis, the secondary products involved in this convergent evolution are not pigments but volatiles. For example, methyl benzoate is quite abundant in leaves of flowering C. humilis but, in Clarkia breweri (Onagraceae), the gene encoding for the benzoic acid carboxyl methyl transferase that produces methyl benzoate is highly specific to the epidermal cells of petals (Kolosova et al., 2001b ). It would be interesting to determine whether this gene is expressed in the mesophyll or in the foliage epidermis of flowering Chamaerops humilis. Furthermore, the same experiments could be done with linalool synthase and the enzymes catalyzing the synthesis of (E)-ß-ocimene. These enzymes are also highly specific to flowers in C. breweri and A. majus (Dudareva et al., 1996 , 2003 ).

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

In conclusion, we have shown that the pattern of terpene production and emission in the leaves of C. humilis resembles in many ways the same processes expressed in petals of many angiosperms, suggesting a convergent evolution of leaves toward petals. The similarities at the functional, anatomical, and biochemical levels could be due to common underlying developmental and secondary metabolic machineries in these serially homologous organs.


   FOOTNOTES
 
1 The authors thank Jean-Marie Bessière for his invaluable help for compound identification, Bruno Buatois for technical help with the GC-MS, Doyle McKey for correcting our English, and Céline Mir and Martine Hossaert for help in collecting samples. Environmental scanning electron microscopy was done at the Centre de Microscopie Electronique Stéphanois with the help of Isabelle Anselme-Bertrand. We also thank J. M. Bessière, F. Stauffer, and two anonymous reviewers for helpful suggestions. Back

4 E-mail: anstett{at}cefe.cnrs-mop.fr Back


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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