Anatomical and Chemical Analyses of Leaf Secretory Cavities of Rustia formosa (Rubiaceae)

by Ricardo Cardoso Vieira, Piero G. Delprete, Suzana Guimarães Leitão, Gilda Guimarães Leitão
Anatomical and Chemical Analyses of Leaf Secretory Cavities of Rustia formosa (Rubiaceae)
Ricardo Cardoso Vieira, Piero G. Delprete, Suzana Guimarães Leitão, Gilda Guimarães Leitão
American Journal of Botany
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>Departamento de Botlnica, Universidade Federal do Rio de Janeiro, CCS, Bloco H, 21.941-590,
Rio de Janeiro, Brazil;
'Institute of Systematic Botany, The New York Botanical Garden, Bronx, New York 10458-5126 USA;
4Ndcleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro, CCS, Bloco H, 21.941-590, Rio de Janeiro,
Brazil; and
'Departamento de Produtos Naturais e Alimentos, Faculdade de FarmBcia, CCS, Bloco A, 21.94 1-590, Rio de Janeiro, Brazil

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.

Secretory cavities in the leaves of vascular plants are rele- vant 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 idio- blastic secretory cells and secretory cavities (West, 1969). Both characters have been reported to occur in the microspo- rophylls of the family Ginkgoaceae (Ameele, 1980) and have been recently discovered in fossil members of the Gigantop- teridales (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 fami- lies with "sacs or cavities with unspecified contents," 16 fam- ilies with "cavities with mucilaginous contents," and 14 fam- ilies with "cavities with tanniferous contents," with the Ru- biaceae 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, Hy- pericaceae, Anacardiaceae, Asteraceae, Bombacaceae, Olaca-

' Manuscript received 31 October 2000; revision accepted 29 June 2001.

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

Author for reprint request (phone: 718 817 8819; FAX 718 817 8648; e- mail:

ceae, Scrophulariaceae, Menispermaceae, Ebenaceae, Myrsi- naceae, 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 Macha- do, 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 gen- era with nonmucilaginous cavities in which the Rubiaceae were included. It is quite astounding that, considering the tax- onomic, anatomical, ecological, and economic importance of foliar secretory structures, these features and their content re- main 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, 198 1; tribe Spemacoceae sensu Bremer and Manen, 2000). A comparative study on the anatomy and content of the foliar secretory cavities of Heterophyllaea will be pub- lished in the near future (F? G. Delprete et al., unpublished manuscript). Rustia and Tresanthera are also peculiar in hav- ing poricidal anthers, opening by two apical pores in Rustia and by a common lateral pore in Tresanthera. Rustia is a ge- nus 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, Ec- uador, 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 Carib- bean coastal forests of Venezuela and Trinidad and Tobago. These two genera have been recently monographed by Del- prete (1 999).

Secretory cavities present in the leaves of Rustia and Tre- santhera have been previously reported by several botanists and variably called "internal druses," "intercellular secretion- containers," "secretory cells," and "characteristic druses" ("inneren drusen," "intercellulare Secretbahalter," "secretfiihrende," and "eingenthiimliche 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; Soler- eder, 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 GoiBs, Minas Gerais, Distrito Federal, Rio de Janeiro, and SBo Paulo; further information about this spe- cies can be found in Delprete (1999).


Study site and plant selection-Plant material of Rustia formosa was col- lected in January 1999 in the Reserva Biol6gica Angelo Rizzo. Sena Dourada, Municipio de Mossbmendes, 16"04' S, 50°1 1' W, -500 m above sea level, in the state of Goiis, 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 de- posited at the herbarium (UFG) of the Federal University of Goiis. GoiLnia, Goiis, Brazil.

Anafomical protocol-For the anatomical analysis, mature leaves were fixed in ethanol 70% and dehydrated with ascending alcohol series and em- bedded in paraffin (Sass, 1951). Transversal sections -12 p,m 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 cal- culated by averaging 30 l-mm square areas of ten leaves of the same indi- vidual. Lipidic substances were detected using Sudan 111 in free-hand sections (Johansen, 1940). The anatomical photographs were taken with an optical photomicroscope Zeiss Axiolab (Carl Zeiss, Microscopy and Imaging Sys- tems, 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 R~lsriaformosa was analyzed by capillary gas chromatography (GC) and gas chromatography combined with mass spectroscopy technique (GCIMS). 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"Cl min). Retention times (RT) were measured in minutes. The GCIMS analysis was performed using a Hewlett-Packard HP5890 SII gas chromatograph (Agi- lent, Palo Alto, California, USA) coupled with a VG Autospec mass spec- trometer (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"Cl min). Identification of the substances was achieved by comparing the mass spectra with a computer library and by visual comparison with spectra re- ported in literature (Adams, 1995). The retention index (RI) of each sample was calculated using data of a homologous series of saturated aliphatic hy- drocarbons (C, to C,,) in the same column and conditions as in the GC anal- ysis of the oils.


Anatomical results-Epidermis-In paradermal view, both the adaxial and abaxial epidermises are composed of polygo- nal 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, sur- rounded 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).

Stomata-Stomata are present only in the abaxial surface with the calculated average number of 133 stomata/mm2. They are predominantly paracytic and characterized by the occur- rence of three to six subsidiary cells of various shapes (Figs. 2 and 4). Some stomata have subsidiary cells with cuticular striations (Fig. 4).

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

Leaf margin-The leaf margin is lightly bent toward the abaxial face (Fig. 6). The epidermis is single layered and cov- ered by a thick cuticle. The cells of the chlorophyllous paren- chyma 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, cav- ities occur near the palisade parenchyma (Fig. 5), throughout the lamina to near the margin (Fig. 6), and near the parenchy- ma 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 vas- cular bundles (Fig. 7). They were not found in the vascular region of the petiole.

Each secretory cavity is a large intercellular space surround- ed by epithelial cells, which are responsible for secretion into the cavity (Fig. 9). The histochemical test performed with Su- dan I11 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 es- sential 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 var- ied as follows: 31 components were present in a relative con-

Retention time (min)
Fig. 10. Gas chromatogram of the essential oil from Rustia formosa (temperature program 60"-24O0C, 3"CImin; column DB-1; injector temperature 220°C).

TABLE1. Identified compounds in Rustia formosa essential oil contained in the secretory cavities (compounds with highest area percentage are indicated in boldface type). KI = Kovat's index (calculated using the retention time and used to identify the compounds), KI lit. = KI from literature (Adams, 1995).

Retention time  time from  Area W from  
from DB-1  DB-5 column  DB-5 chromato  
Com~ound  column (mm)  (mln)  eram  KI  KI lit.  
Germacrene D       
Germacrene B       
Caryophyllene oxide       

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G. Delprete et al., unpublished data) will test these hypotheses.


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