Trichomes and chemical composition of the volatile oil of Trichogonia cinerea (Gardner) R. M. King & H. Rob. (Eupatorieae, Asteraceae)

Trichogonia cinerea is endemic to Brazil and occurs in areas of cerrado and campo rupestre. In this study, we characterized the glandular and non-glandular trichomes on the aerial parts of this species, determined the principal events in the development of the former, and identifi ed the main constituents of the volatile oil produced in its aerial organs. Fully expanded leaves, internodes, fl orets, involucral bracts, and stem apices were used for the characterization of trichomes. Leaves, internodes, fl orets, and involucral bracts were examined by light microscopy and scanning electron microscopy, whereas stem apices were examined only by light microscopy. Branches in the reproductive phase were used for the extraction and determination of the composition of the volatile oil. The species has three types of glandular trichomes, biseriate vesicular, biseriate pedunculate, and multicellular uniseriate, which secrete volatile oils and phenolic compounds. The major components identifi ed in the volatile oil were 3,5-muuroladiene (39.56%) and butylated hydroxytoluene (13.07%).


INTRODUCTION
Trichomes are unicellular or multicellular appendages, which originate only from epidermal cells, develop outwards on the surface of various plant organs, and can be found on all parts of the plant. Their major distinction is between glandular and non-glandular trichomes. The trichomes that differ in type and/or location probably also differ in other, not always investigated, properties and in function (Werker 2000). Plants may need protection from various external factors, such as herbivores, pathogens, extensive light, and extreme temperatures. Therefore, densely spread nonglandular trichomes may serve as a mechanical barrier against most of the aforementioned factors (Werker 2000). Moreover, glandular and nonglandular trichomes are an important fi rst line of defense against herbivorous insects (Werker 2000, Glas et al. 2012) and the glandular trichomes may serve as a chemical protection against pathogens (Blakeman andAtkinson 1979, Werker 2000).
In addition to the ecological importance of trichomes, their characterization may also contribute to taxonomic studies, and the morphology of glandular and non-glandular trichomes is of great importance for systematic studies, at different taxonomic levels, of the Asteraceae. Narayana (1979) suggested that trichomes can be a diagnostic character in Vernonia Schreb.; whereas Wagner et al. (2014) used the diversity of non-glandular trichomes to make taxonomic differentiations in subtribe Lychnophorinae (Vernonieae). Despite the wide variety of glandular trichomes, biseriate glandular trichomes are typical of the Asteraceae (Metcalfe and Chalk 1950) and biseriate trichomes are present in several species of the tribes Astereae, Eupatorieae, Heliantheae, Inuleae, Mutisieae, and Vernonieae (Cortadi and Gattuso 1994, Castro et al. 1997, Cortadi et al. 1999. Furthermore, species of the tribes Eupatorieae and Heliantheae frequently have uniseriate glandular trichomes (Castro et al. 1997).
Chemically complex mixtures, composed almost entirely of terpenes and phenylpropenes, called volatile oils (Waterman 1993), are commonly found in species of Asteraceae. It is noteworthy that the volatile oils produced by many species of this family have proven to exhibit antimicrobial (Neerman 2003) and insecticide activity (Campos et al. 2014). Bohlmann et al. (1981 conducted chemosystematic studies of the tribe Eupatorieae and analyzed the sesquiterpene lactones and other constituents of Trichogonia species to determine if their chemical composition could give indications of the relationships among the species of this diverse genus. The name of the genus Trichogonia refers to the characteristic setulae on the angles of the fruits (King and Robinson 1987), and the presence of glandular stipitate trichomes on the corolla tube is an important feature in the taxonomy of this genus (Roque et al. 2012). Among the 20 recognized species of Trichogonia, 17 occur in Brazil. The species can be classifi ed into two groups, one has heads presenting convex receptacles and glandular stipitate trichomes on the corolla tube, and the other, which includes most species, is characterized by fl at receptacles and glabrous corolla tube (Roque et al. 2012).
Trichogonia cinerea (Gardner) R. M. King & H. Rob. is endemic to Brazil and belongs to the fi rst group described above, with convex receptacles and glandular stipitate trichomes on the corolla tubes. The species is found in the states of Bahia and Goiás, as well as in the Distrito Federal, where has been collected in areas of cerrado and campo rupestre (Roque et al. 2012). Although the trichomes are clearly visible on the aerial organs (reproductive and vegetative), their characterization has not been performed yet. In the current study, we aimed to characterize the glandular and nonglandular trichomes on the aerial parts of T. cinerea using light microscopy and scanning electron microscopy (SEM) to identify the main events in their development. Additionally, the composition of the volatile oil produced in the aerial organs of this species was determined.

MATERIALS AND METHODS
Samples of T. cinerea were collected in areas of cerrado rupestre (16º04.613' S, 50º11.318' W and 16º04.712' S, 50º11.481' W) in the state of Goiás, Brazil. Plants in the vegetative and reproductive phases as well as seeds were collected in April 2012 for the study of trichomes. For the volatile oil analyses, fertile branches were collected at 11 am in April 2013, with registered precipitation of 96 mm, minimum and maximum temperatures of 21ºC and 33ºC, respectively, and relative humidity of 71% (INMET 2013). A voucher specimen was deposited at the UFG herbarium (accession number 3994), acronyms according to Thiers 2013 (continuously updated).
For the characterization of trichomes, fully expanded leaves from the third to the fi fth node (from apex to base), samples from the second to the fifth internodes (from apex to base), stem apices, disc florets, and involucral bracts were used. Fragments from the middle third of both the petiole and the leaf blade were collected. For the ontogenetic study of glandular trichomes, stem apices were harvested from young plants obtained by germinating seeds. Some leaf and internode samples were free-hand cut transversely, some were mounted in historesin, and some others were examined by scanning electron microscopy (SEM). Florets and involucral bracts were analyzed using SEM, and stem apices were mounted in paraffi n. The samples used for histochemical analyses were processed immediately after collection and those used for light microscopy were fi xed in FAA 50 and stored in 70% ethanol (Johansen 1940), while samples used for SEM were fi xed by immersion in a modifi ed Karnovsky's solution (Karnovsky 1965). Free-hand cut leaf and internode sections were double-stained with a 1:3 solution of 0.1% (w/v) basic fuchsin and 0.3% (w/v) astra blue, modifi ed from Roeser (1972), and the clarifi ed samples were stained with 1% (w/v) aqueous safranin solution. The material was then mounted on slides in 50% (v/v) aqueous solution of glycerin. For inclusion in historesin samples were dehydrated in a graded ethyl alcohol series, kept in a pre-infi ltration solution for three days, and in an infi ltration solution for seven days. Samples were infi ltrated with hydroxyethyl methacrylate historesin (HistoResin Plus, Reichert-Jung, Heidelberg, Germany). The resulting blocks were cut into 10-μm thick sections using a rotary microtome Leica RM2245 (Leica Biosystems Nussloch GmbH, Nußloch, Germany) and stained with toluidine blue (O'Brien et al. 1964).
Seeds were germinated in plastic pots with sandy soil collected from areas naturally colonized by T. cinerea and watered periodically. Stem apices were collected 70 days after seed germination, from plants around 2.5 to 3.0 cm tall and with two pairs of leaves. Apices were dehydrated in an ethylalcohol to xylol series and embedded in paraffi n (Johansen 1940). The resulting blocks were cut into 12-μm thick longitudinal sections using a rotary microtome Leica RM2245, deparaffinized, and stained with ferric hematoxylin (Sass 1951) and a 1:3 solution of 0.1% (w/v) basic fuchsin and 0.3% (w/v) astra blue (modifi ed from Roeser 1972).
Historesin and paraffi n sections were mounted in colorless glass varnish Acrilex ® 500® (Acrilex Tintas Especiais S/A, São Bernardo do Campo, SP, Brazil, according to Paiva et al. (2006). In the histochemical analyses we used Lugol's iodine solution to detect starch, acidifi ed phloroglucinol to reveal the presence of lignin in cellular walls, sudan III for total lipids, ferrous sulphate, ferric chloride (Johansen 1940), and potassium dichromate (Gabe 1968) for phenolic compounds, bromophenol blue (Mazia et al. 1953) and coomassie blue (Fisher 1968) for proteins, Dittmar's reagent (Furr and Mahlberg 1981) for alkaloids, and NADI reagent (David and Carde 1964) for volatile oil. Photomicrographs of the sections were taken using a Leica ICC50 HD microscope camera attached to a Leica DM500 optical microscope and the software LAS EZ for image acquisition (Leica Microsystems GmbH, Heerbrugg, Switzerland).
The samples for SEM were dehydrated in an ethyl alcohol series, critical-point dried with liquid carbon dioxide (CO 2 ), and coated with gold in a Denton Vacuum Sputter Coater (Denton Vacuum, LLC, Moorestown, NJ, USA). The samples were analyzed using a scanning electron microscope JSM-6610 (JEOL Ltd, Tokyo, Japan), operated at 4 Kv, equipped with a Thermo Scientifi c NSS spectral imaging system (Thermo Fischer Scientifi c Inc., Madison, WI, USA) for energy-dispersive X-ray spectroscopy (EDS) at the Laboratório Multiusuário de Microscopia de Alta Resolução (LabMic), in the Instituto de Física of the Universidade Federal de Goiás (UFG), in Goiânia, GO, Brazil.
The branches used for oil analysis were stored in plastic containers under refrigeration for 12 h. After this period, the material was fragmented, gathered in a sample of 110 g, and submitted to hydrodistillation using a Clevenger-type apparatus for 3 h (Brasil 1988). The volume of oil obtained was determined using the volumetric tube of the equipment. The determination of the chemical constituents of the oil was performed by gas chromatography-mass spectrometry (GC/MS), using a Shimadzu QP5050A (Shimadzu Co., Kyoto, Japan), a fused-silica capillary column (CBP -5; 30 m × 0.25 mm; 0.25 μm fi lm thickness), at a fl ow rate of 1 mL/min of helium as the carrier gas, heating at pre-programmed temperatures (60ºC for 2 min, increasing to 240ºC at a rate of 3ºC/min, followed by heating up to 280ºC at a rate of 10ºC/min, and maintained at this temperature for 10 min), and an ionization voltage of 70 eV. The chemical compounds were identifi ed using the NIST/EPA/NIH Mass Spectral Library 2011 and by comparison of their retention indices and mass spectra with those of authentic compounds (Adams 2007). The retention indices were calculated by co-injecting a commercial C8-C32 aliphatic hydrocarbons mixture (Sigma-Aldrich Co. LLC, St. Louis, MO, USA) and the equation of van Den Dool and Kratz (1963).

RESULTS
Cross-sections of leaves, stems, involucral bracts, and fl orets (Figs. 1-3) allowed the identifi cation of glandular trichomes, herein classifi ed into three types: biseriate vesicular (Figs. 1a, 2a), formed by four pairs of cells; biseriate pedunculate (Figs. 1b,2b), with two to fi ve pairs of cells forming the stalk and four to fi ve pairs of secretory cells on the head; and multicellular uniseriate, curved towards the epidermis (Figs. 1c, 2c), with four to six cells forming the stalk and a unicellular head. In biseriate vesicular glandular trichomes, the cuticle over the secretory head expands (Figs. 1a, 2a), forming a large subcuticular space that corresponds to the storage site of secreted material. This space was not observed in the other types of trichome. In addition to the glandular trichomes, we found non-glandular trichomes on all the structures analyzed (Fig 1d  and Figs 2d-e), which are multicellular uniseriate, with a variable number of cells (2-5), voluminous cells at the base, and a tapering apical cell. All trichome types are found of the leaf blade, petiole, and stem (Fig. 2f), where the biseriate vesicular glandular trichomes are found in small depressions of the epidermis. The fl orets are densely covered by glandular and non-glandular trichomes (Fig. 3a),   3f) and involucral bracts. The secretions of glandular trichomes of T. cinerea are naturally yellow, and chloroplasts can be observed inside the secretory cells. The results of the histochemical analyses are shown in Table  I. Sudan III test revealed total lipids within the subcuticular space of biseriate vesicular glandular trichomes, in the secretory cells of biseriate pedunculate, and in the stalk and secretory cells of multicellular uniseriate trichomes. Phenolic compounds were detected in all three types of glandular trichomes using ferrous sulphate and potassium dichromate, and in biseriate vesicular trichomes using ferric chloride. The presence of volatile oil was confirmed using Nadi reagent, which formed blue droplets in the subcuticular space of biseriate vesicular trichomes, in the secretory cells of biseriate pedunculate trichomes, and in the stalk and secretory cells of multicellular uniseriate trichomes.
Glandular and non-glandular trichomes are present on the hypocotyl, epicotyl, and cotyledons of T. cinerea. Glandular trichomes in various stages of development can be observed on the stem apex (Fig. 4a). The fi rst stage of differentiation, with the expansion of a protodermal cell, is shared by all three types of glandular trichomes. In biseriate vesicular trichomes, the protodermal cell expands and increases in volume (Fig. 4b), then undergoes an anticlinal division to originate two cells (Fig.  4c). These cells expand synchronously, and the resulting cells undergo successive periclinal divisions (Figs. 4d-e). In multicellular uniseriate trichomes, the protodermal cell becomes curved as it expands (Fig. 4f), which is followed by successive periclinal divisions (Figs. 4g-i).
Differentiation of the biseriate pedunculate glandular trichome begins with a single expanded protodermal cell, which is voluminous and has dense contents (Fig. 5a). This cell undergoes anticlinal division, originating two cells (Fig. 5b) that undergo periclinal divisions (Fig. 5c). This is followed by successive non-synchronized periclinal divisions of the cells in each of the two series (Figs. 5d-f). Since the divisions are not synchronized, at the end of the differentiation phase the cells may occupy different heights (Fig. 5g).
The yield of the volatile oil isolated from the fertile branches of T. cinerea was 0.09% (v/w). The volatile oil was composed of 19 constituents, representing 73.93% of the total oil, and their percentages are listed in Table II. The sesquiterpenes were the most abundant (60.19%) compound,   with 54.22% sesquiterpene hydrocarbons, 5.97% oxygenated sesquiterpenes, 3.99% sesquiterpene alcohols and 13.07% phenolic alcohol (butylated hydroxytoluene). The major component was 3,5-muuroladiene, in a total of 39.56%. The second and the third most abundant components were butylated hydroxytoluene (13.07%) and E-caryophillene (5.63%), respectively, whereas oxygenated monoterpene represented only 0.67% of the total oil content.

DISCUSSION
Trichogonia cinerea presents biseriate and uniseriate glandular trichomes, which results in dense indumentum on the leaves. Some evidence suggests that this dense indumentum formed by nonglandular and/or glandular trichomes represents an adaptation to habitats with low water availability and high temperatures (Werker 2000). Furthermore, Manetas (2003) showed that trichomes protect plant tissues against damage by UV-B rays. The individuals of T. cinerea assessed in the present study were collected in cerrado rupestre, which has shallow sandy soils and high incidence of solar radiation, coupled with a long dry season during the year. Thus, the indumentum formed by the dense cover of non-glandular and glandular trichomes is likely to be important for water economy and protection of chlorophyllous tissues against high temperatures and intense solar radiation, improving CO 2 assimilation during unfavorable periods. Furthermore, the elimination of secondary metabolites by glandular trichomes may provide chemical protection against herbivores and pathogens (Werker 2000, Glas et al. 2012). The biseriate vesicular glandular trichomes of T. cinerea are similar to those found on the aerial organs of other Asteraceae species and several aspects of these structures have been investigated (Ascensão and Pais 1987, Figueiredo and Pais 1994, Afolayan and Meyer 1995, Pagni 1995, Castro et al. 1997, Ascensão et al. 2001, Heinrich et al. 2002, Andreucci et al. 2008, Oliveira et al. 2013, Trindade et al. 2014.
Uniseriate trichomes curved towards the epidermis occur in several species of the tribes Eupatorieae (Cortadi and Gatuso 1994, Castro et al. 1997, Trindade et al. 2014) and Heliantheae (Castro et al. 1997, Duarte and Empinotti 2012. The occurrence of curved glandular trichomes is shared by the tribes Eupatorieae and Heliantheae, and provides evidence of a possible affi nity between them (Castro et al. 1997). This type of trichome is also found in Eupatoruim macrocephalum Less., E. inulaefolium H.B.K., and E. subhastatum Hook et Arn (Cortadi and Gatuso 1994). Also, biseriate pedunculate glandular trichomes have been registered in several species of the tribe Eupatorieae (Castro et al. 1997, Trindade et al. 2014. As already mentioned, the species of Trichogonia can be classified into two groups, and T. cinerea, T. eupatorioides, T. hassleri, and T. prancei belong to the first one, with convex receptacles and glandular stipitate trichomes on the corolla tubes (Roque et al. 2012). The results of the present study showed that the stipitate trichomes that occur on the corolla in T. cinerea are biseriate pedunculate and that, in addition to these, tector trichomes were also observed. Further studies are necessary in order to characterize the glandular trichomes found on the corolla of the other species of this group.
In T. cinerea, biseriate glandular trichomes develop from a single protodermal cell that divides anticlinally. This development is similar, in several aspects, to that of Madia sativa Molina  (Afolayan and Meyer 1995).
Secretions from glandular trichomes of T. cinerea contain volatile oils and phenolic compounds, a chemical composition that is common in the Asteraceae (Aguilera et al. 2004, Fonseca et al. 2006. The content of volatile oil produced by T. cinerea was low and the major fraction of the constituents identifi ed was composed of sesquiterpenes. Sesquiterpenoids are typical metabolites of Asteraceae species (Seaman 1982) and are used in the chemotaxonomy of the family (Bohlmann et al. 1981, Seaman 1982, Zdero and Bohlmann 1990. It is worth to emphasize that some of the constituents identified in this study present therapeutic potential. Sesquiterpene bicyclogermacrene, for instance, exhibited antifungal activity when isolated (Silva et al. 2007), whereas (E)-caryophillene showed antibacterial activity (Garg andSiddiqui 1992, Formisano et al. 2006).

CONCLUSION
T. cinerea has two types of biseriate and one type of uniseriate glandular trichomes on the leaves, stems, and involucral bracts. In contrast, only biseriate glandular trichomes are found on the corolla. The three types of trichomes secrete volatile oils and phenolic compounds. The glandular trichomes differentiate at the stem apices from a protodermal cell that expands and acquires a voluminous aspect. In biseriate glandular trichomes, the fi rst division of the expanded protodermal cell is anticlinal, while in the multicellular uniseriate trichome this division is periclinal. Most of the components of the volatile oil from T. cinerea identifi ed in the present study are sesquiterpenes. Taking into consideration that 320 YANNE S. FERNANDES et al.
the morphology of glandular trichomes and the chemical composition of volatile oils is of great importance for the Asteraceae, the results obtained in this study may help further taxonomic studies of this family at different taxonomic levels.

ACKNOWLEDGMENTS
The authors would like to thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CNPq) for the financial support through a fellowship, the Laboratório Multiusuário de Microscopia de Alta Resolução (LabMic) of the Universidade Federal de Goiás (UFG) for the SEM analyses, and Dr. Aristônio Magalhães Teles for determining the species.