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Anatomical and chemical analyses of leaf secretory cavities of Rustia formosa (Rubiaceae)¹

²Departamento de Botânica, Universidade Federal do Rio de Janeiro, CCS, Bloco H, 21.941-590, Rio de Janeiro, Brazil; ³Institute of Systematic Botany, The New York Botanical Garden, Bronx, New York 10458-5126 USA; ⁴Núcleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro, CCS, Bloco H, 21.941-590, Rio de Janeiro, Brazil; and ⁵Departamento de Produtos Naturais e Alimentos, Faculdade de Farmácia, CCS, Bloco A, 21.941-590, Rio de Janeiro, Brazil

Received for publication October 31, 2000. Accepted for publication June 29, 2001.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Foliar secretory cavities, commonly called leaf pellucid glands, have been reported in many families of vascular plants. In the Rubiaceae, these structures have only been found in the sister genera Rustia and Tresanthera, which are also anomalous within the family because they have poricidal anthers, and in the distantly related Heterophyllaea. General leaf anatomy, with particular attention to secretory cavities, as well as the chemical analysis of the secreted substances of Rustia formosa, is presented here for the first time. The secretory structures have been found in the lamina between the palisade and spongy parenchymas and in the cortical region of the petiole. The chemical analysis showed that the essential oil secreted is a complex mixture of at least 75 components, mostly of sesquiterpenoid composition. Illustrations of the leaf anatomy, details of the secretory structures of Rustia formosa, a gas chromatogram, and a table of the principal components of the leaf essential oil are included.

Key Words: anatomy • essential oil • Rondeletieae • Rubiaceae • Rustia • secretory cavities • Tresanthera

	INTRODUCTION

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Secretory cavities in the leaves of vascular plants are relevant taxonomic characters and important anatomical features that have originated many times in distantly and closely related families. Supporting the early origin of these structures, the primitive families of the basal Ranalean complex have idioblastic secretory cells and secretory cavities (West, 1969 ). Both characters have been reported to occur in the microsporophylls of the family Ginkgoaceae (Ameele, 1980 ) and have been recently discovered in fossil members of the Gigantopteridales (Li et al., 1994 ). In a few cases, the presence of foliar secretory cavities is the paramount anatomical feature and good field character that identifies certain families, as it is, for example, in the Myrtaceae, Rutaceae, Flacourtiaceae, and Theaceae (Gentry, 1993 , p. 51; Ribeiro et al., 1999 , pp. 70–71).

Metcalfe and Chalk (1950, pp. 1346–1348) listed 40 families with "sacs or cavities with unspecified contents," 16 families with "cavities with mucilaginous contents," and 14 families with "cavities with tanniferous contents," with the Rubiaceae listed in the first two categories. In a note before these lists, they warn that these "lists have been compiled because of the proven taxonomic value of secretory structures, but it must always be remembered, when using them, that the precise nature of their contents has been accurately determined in only a few plants." Considerable progress has been made since the publication of this milestone work, and the foliar secretory cavities have recently been studied in selected genera of the families Rutaceae, Myrtaceae, Myoporaceae, Fabaceae, Hypericaceae, Anacardiaceae, Asteraceae, Bombacaceae, Olacaceae, Scrophulariaceae, Menispermaceae, Ebenaceae, Myrsinaceae, and Cactaceae (Langenheim, 1967 ; Langenheim, Lee, and Martin, 1973 ; Fahn, 1979 ; Baas, van Oosterhoud, and Scholtes, 1982 ; Karrfalt and Tomb, 1983 ; Curtis and Lersten, 1986 ; Turner, 1986 ; Russin et al., 1988 ; Roth and Lindorf, 1991 ; Jordaan and Kruger, 1992 ; Fontelle, Costa, and Machado, 1994; Monteiro et al., 1995 ; Poli, Sacchetti, and Bruni, 1995 ; Otegui et al., 1998 ; Lersten and Beaman, 1998 ; Niklas et al., 2000 ). Nevertheless, in the second edition of their Anatomy of the Dicotyledons, Metcalfe and Chalk (1983 , pp. 223–224; Metcalfe, 1983, pp. 64–67) listed only 18 families with mucilage cavities and 24 families that have at least some genera with nonmucilaginous cavities in which the Rubiaceae were included. It is quite astounding that, considering the taxonomic, anatomical, ecological, and economic importance of foliar secretory structures, these features and their content remain so incompletely known.

Within the Rubiaceae, a predominantly tropical family of 650 genera and 13 000 species, only three genera are known to have foliar secretory cavities: the sister taxa Rustia Klotzsch and Tresanthera H. Karst. (tribe Rondeletieae sensu Delprete, 1999 ) and the distantly related Heterophyllaea J. D. Hook. (Tourn, 1981 ; tribe Spemacoceae sensu Bremer and Manen, 2000 ). A comparative study on the anatomy and content of the foliar secretory cavities of Heterophyllaea will be published in the near future (P. G. Delprete et al., unpublished manuscript). Rustia and Tresanthera are also peculiar in having poricidal anthers, opening by two apical pores in Rustia and by a common lateral pore in Tresanthera. Rustia is a genus of 14 species, represented by shrubs 3–4 m tall to trees up to 15 m tall, ranging from Nicaragua to southern Brazil, and with two main centers of diversity, one in the Andean cloud forests of northwestern South America (Colombia, Ecuador, and Peru), and the other in the Brazilian Atlantic forests in the state of Rio de Janeiro. Tresanthera is a monotypic genus of shrubs and trees 15–20 m tall, endemic of the Caribbean coastal forests of Venezuela and Trinidad and Tobago. These two genera have been recently monographed by Delprete (1999) .

Secretory cavities present in the leaves of Rustia and Tresanthera have been previously reported by several botanists and variably called "internal druses," "intercellular secretion-containers," "secretory cells," and "characteristic druses" ("inneren drusen," "intercellulare Secretbahälter," "secretführende," and "eingenthümliche Drusen," respectively; cf. Solereder, 1893) , "pellucid glands" (Steyermark, 1974; Dwyer, 1980), "translucent dots" (Robbrecht, 1988 ), and "pellucid dots" (Burger and Taylor, 1993). A brief history on the observation of secretory cavities of these two genera has recently been published by Delprete (1999 : pp. 24–25). Although foliar cavities of Rustia and Tresanthera have been reported for more than a century (Karsten, 1858, 1861 ; Solereder, 1890, 1893, 1908 ; Metcalfe and Chalk, 1950 ; Verdcourt, 1958 ; Robbrecht, 1988 ), no detailed study on the anatomy of these structures and their chemical content has ever been made.

Because of recently developed collecting programs, fresh material of Rustia formosa (Cham. and Schltdl. ex DC.) Klotzsch, the type species of the genus, has become available. Rustia formosa, the species with the widest distribution range of its genus, is a shrub 4–6 m tall (exceptionally up to 12 m tall) occurring in the gallery forest of the cerrado biome of the Brazilian states of Goiás, Minas Gerais, Distrito Federal, Rio de Janeiro, and São Paulo; further information about this species can be found in Delprete (1999) .

	MATERIALS AND METHODS

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Study site and plant selection
Plant material of Rustia formosa was collected in January 1999 in the Reserva Biológica Angelo Rizzo, Serra Dourada, Município de Mossâmendes, 16°04' S, 50°11' W, 500 m above sea level, in the state of Goiás, Brazil. The plant was a shrub 5–6 m tall with semi-leathery leaves and white corolla tubes with pink lobes; it was identified by P. G. Delprete, and voucher specimens (Delprete and Rizzo 7060-A) are deposited at the herbarium (UFG) of the Federal University of Goiás, Goiânia, Goiás, Brazil.

Anatomical protocol
For the anatomical analysis, mature leaves were fixed in ethanol 70% and dehydrated with ascending alcohol series and embedded in paraffin (Sass, 1951 ). Transversal sections 12 µm thick were made with a rotary microtome. Tissues were stained with Astra Blue + Basic Fuchsin (Roeser, 1962 ). For the study of the paradermal view of the epidermis, small rectangular areas of epidermis were removed from the medial portion of the leaf blade, preceded by its dissociation following the Jeffrey method (Johansen, 1940 ). Determination of stomata per square millimeter was calculated by averaging 30 1-mm square areas of ten leaves of the same individual. Lipidic substances were detected using Sudan III in free-hand sections (Johansen, 1940 ). The anatomical photographs were taken with an optical photomicroscope Zeiss Axiolab (Carl Zeiss, Microscopy and Imaging Systems, Thornwood, New York, USA).

Essential oil extraction and analysis
The essential oil from the fresh leaves of this plant was extracted by hydrodistillation in a Clevenger type apparatus for 3–4 h; this process yielded a colorless oil. The essential oil from Rustia formosa was analyzed by capillary gas chromatography (GC) and gas chromatography combined with mass spectroscopy technique (GC/MS). The GC analysis was performed in a Varian Star 3400 CX gas chromatograph (Varian, Palo Alto, California, USA), fused capillary column (DB-5; 25 m x 0.2 mm), hydrogen as carrier gas, temperature programming 60°–240°C (3°C/min). Retention times (RT) were measured in minutes. The GC/MS analysis was performed using a Hewlett-Packard HP5890 SII gas chromatograph (Agilent, Palo Alto, California, USA) coupled with a VG Autospec mass spectrometer (70 eV), using fused silica capillary column (DB-1; 25 m x 0.20 mm), helium as carrier gas, and temperature programming 60°–240°C (3°C/min). Identification of the substances was achieved by comparing the mass spectra with a computer library and by visual comparison with spectra reported in literature (Adams, 1995 ). The retention index (RI) of each sample was calculated using data of a homologous series of saturated aliphatic hydrocarbons (C₈ to C₂₂) in the same column and conditions as in the GC analysis of the oils.

	RESULTS

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Anatomical results
Epidermis
In paradermal view, both the adaxial and abaxial epidermises are composed of polygonal cells, with rectilinear anticlinal walls and an ornamented cuticle (Figs. 1 and 2). In the abaxial epidermis, some of the epidermal cells with small coniform papillae are present, surrounded by epidermal cells in radial disposition (Fig. 3). In transverse section, the adaxial epidermis is composed of two layers of cells, while the abaxial one is single layered (Fig. 5).

View larger version (163K): [in this window] [in a new window]   Figs. 1–4. Leaf epidermis of Rustia formosa. 1. Adaxial epidermis, frontal view. Bar = 50 µm. 2. Abaxial epidermis, frontal view. Bar = 50 µm. 3. Detail of one papilla of the abaxial epidermis, frontal view. Bar = 20 µm. 4. Abaxial epidermis, frontal view. Bar = 20 µm

View larger version (140K): [in this window] [in a new window]   Figs. 5–9. Leaf cross section of Rustia formosa. 5. Cross section of leaf blade with one secretory cavity. Bar = 50 µm. 6. Cross section of leaf blade margin with one secretory cavity. Bar = 50 µm. 7. Cross section of petiole with two secretory cavities in the parenchyma tissue. Bar = 50 µm. 8. Cross section of petiole with secretory cavity near the vascular bundle. Bar = 50 µm. 9. Cross section of the mesophyll; detail of a secretory cavity. Bar = 20 µm

Mesophyll
The mesophyll is made of chlorophyllous parenchyma differentiated into palisade and spongy parenchyma typical of a dorsiventral leaf blade (Fig. 5). The palisade parenchyma consists of two cellular layers, and the spongy parenchyma is comprised of several layers of thin-walled, irregularly placed and sparsely arranged cells, forming large intercellular spaces (Fig. 5).

Leaf margin
The leaf margin is lightly bent toward the abaxial face (Fig. 6). The epidermis is single layered and covered by a thick cuticle. The cells of the chlorophyllous parenchyma diminish in size toward the margin, and the number of chloroplasts decrease toward the margins.

Secretory cavities
In Rustia formosa, secretory cavities are distributed throughout the whole leaf. In the leaf lamina, cavities occur near the palisade parenchyma (Fig. 5), throughout the lamina to near the margin (Fig. 6), and near the parenchyma of the medial portions. In the petiole, they occur in the cortical region, surrounded by angular collenchyma (Fig. 8), and in the internal region of the cortex near the collateral vascular bundles (Fig. 7). They were not found in the vascular region of the petiole.

Each secretory cavity is a large intercellular space surrounded by epithelial cells, which are responsible for secretion into the cavity (Fig. 9). The histochemical test performed with Sudan III indicated the presence of lipid droplets in the epithelial cells.

Chemical results
Essential oil
The gas chromatogram of Rustia formosa leaf essential oil is shown in Fig. 10, and the identified terpenoid compounds are listed in Table 1. The essential oil from Rustia formosa is a complex mixture of at least 75 components, mostly of sesquiterpenoid composition. The relative percentages of the components in the mixture varied as follows: 31 components were present in a relative concentration that ranged from 0.1 to 0.5%, 32 components showed relative concentrations ranging from 0.5 to 2.0%, and 12 components showed relative concentrations varying from 2.0 to 13.0%, of which -muurolene (4.18%), -cadinene (4.49%), ß-caryophyllene (8.09%), germacrene B (12.24%), and curzerenone (12.40%) are the most abundant. Among the identified compounds, 12 were nonoxygenated sesquiterpenes [m/z (mass/charge of a fragment generated at the mass spectra) 204] while six were alcohols, ketones, or oxides.

View larger version (19K): [in this window] [in a new window]   Fig. 10. Gas chromatogram of the essential oil from Rustia formosa (temperature program 60°–240°C, 3°C/min; column DB-1; injector temperature 220°C)

	DISCUSSION

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The secretory cavities ("pellucid dots") are mostly spherical and widespread throughout the leaf, with the exception of the vascular bundles. They are present throughout the blade to near the margin (and are visible when holding the leaf against a light source), usually in the medial area between the palisade and spongy parenchymas. They were also found in the petiole, in the internal portion of the cortex, and in the vicinities of the vascular bundles (but never inside them). The epithelial cells are tangentially flattened and usually organized in two or three layers.

According to Solereder's (1893) , Metcalfe and Chalk's (1950) , and Delprete's (1990) hypotheses, it is most likely that the foliar secretory cavities of Rustia (and Tresanthera) are of schizogenous origin. In support of this, Turner (1999) and Turner, Berry, and Gifford (1998) showed that most probably the cavities and canals thought to be of lysigenous and schizo-lysigenous origins are instead artifacts of inappropriate sample preparation. More specifically, Turner, Berry, and Gifford (1998 , p. 75) presented "evidence that the lysigenous appearance of Citrus limon glands [often cited as the textbook case of lysigenous cavities] is a fixation artifact caused by osmotic swelling of glandular cells immersed in hypotonic solutions [...] This variation in disruptive swelling creates a false impression that autolysis follows maturation and may account for the reports of lysigeny by some investigators." Although the ontogeny of the leaf cavities of Rustia was not studied, their probable schizogenous origin remains to be proved.

Chemistry of cavity contents
The use of Sudan III to detect the presence of lipid substances also gives a positive reaction with essential oils (Cutter, 1986 ; Oliveira and Saito, 1991 ). The presence of lipid droplets in the secretory cavities and in the epithelial cells may indicate that the essential oil is produced by the epithelial cells and secreted into the cavities (for explanation on mechanisms of secretion, see Mauseth, 1988 , pp. 141–166), since the leaves release a pleasant fragrance only upon crushing.

Phylogenetic and ecological implications
We speculate that the essential oil contained in the secretory cavities of Rustia (and Tresanthera) function as a mechanism of protection against herbivory. The pleasant fragrance (to the human senses) that is liberated upon leaf crushing is due to the essential oils, which may function as a deterrent or as a poison for insect larvae or other potential herbivores (Rosenthal and Janzen, 1979 ; Gottlieb and Salatino, 1987 ; Rosenthal and Berenbaum, 1991 ).

According to phylogenetic analyses based on molecular data, Rustia and Tresanthera (not included in the molecular analysis, but treated as a sister taxon because of its morphological affinities) are shown to be highly advanced taxa (Rova et al., in press ). Therefore, the secretory cavities (and the poricidal anthers) of these genera should be treated as derived features and as synapomorphic characters. Most plant families that produce large quantities of essential oil usually do not produce alkaloids and vice versa (Gottlieb and Salatino, 1987 ). This may be explained by the assumption that both alkaloids and essential oils are produced by plants for protection against herbivory; therefore, it is unlikely to find both kinds of substances in the same taxon or in the same organs of the same species (i.e., alkaloids might be present in the bark but not in the leaves, where essential oils are usually stored).

As a second hypothesis, it is possible that these taxa might be one of the few cases where both alkaloids and essential oils are produced. If alkaloids are present in the leaves or in the bark of these genera, these might have evolved a double mechanism of protection against herbivory. Unfortunately, isolation of alkaloids from the leaves and the bark of these two genera has not been attempted; future research testing the presence of alkaloids in the various organs of these and related genera (P. G. Delprete et al., unpublished data) will test these hypotheses.

	FOOTNOTES

 
¹ The authors thank Angelo Rizzo for accompanying Delprete in the collection of fresh material; the CNPq for granting permission to collect Rubiaceae in the state of Goiás; and John Curtis, Dennis Stevenson, and two anonymous reviewers for helpful comments on the manuscript.

⁶ Author for reprint request (phone: 718 817 8819; FAX 718 817 8648; pdelprete{at}nybg.org ).

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This Article Abstract Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via ISI Web of Science (1) Citing Articles via Google Scholar Google Scholar Articles by Vieira, R. C. Articles by Leitão, S. G. Search for Related Content PubMed Articles by Vieira, R. C. Articles by Leitão, S. G. Agricola Articles by Vieira, R. C. Articles by Leitão, S. G.