Anais da Academia Brasileira de Ciências (2018) 90(3): 2887-2901 (Annals of the Brazilian Academy of Sciences) Printed version ISSN 0001-3765 / Online version ISSN 1678-2690 http://dx.doi.org/10.1590/0001-3765201820170606 www.scielo.br/aabc | www.fb.com/aabcjournal Structural characterization of vegetative organs of the endangered Brazilian native species Hesperozygis ringens (Benth.) Epling CARLOS G. PINHEIRO1, JOÃO MARCELO S. DE OLIVEIRA2 and BERTA M. HEINZMANN1, 3 1Programa de Pós-Graduação em Engenharia Florestal, Centro de Ciências Rurais, Universidade Federal de Santa Maria, Prédio 44, Avenida Roraima, 1000, Camobi, 97105-900 Santa Maria, RS, Brazil 2Laboratório de Botânica Estrutural, Departamento de Biologia, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, Avenida Roraima, 1000, Prédio 16, Sala 3251, 97105-900 Santa Maria, RS, Brazil 3Departamento de Farmácia Industrial, Centro de Ciências da Saúde, Universidade Federal de Santa Maria, Prédio 26, Campus Universitário, Avenida Roraima, 1000, Camobi, 97105-900 Santa Maria, RS, Brazil Manuscript received on August 4, 2017; accepted for publication on March 5, 2018 ABSTRACT The aim of this study was to describe the structural characterization of Hesperozygis ringens (Benth.) Epling vegetative organs. For this purpose, leaves, stems and roots of the endangered Lamiaceae were collected from a population located in Santo Antão, Santa Maria municipality, Rio Grande do Sul, Brazil. Results demonstrated that the H. ringens leaf blade presents glandular and non-glandular trichomes as well as two morphs of diallelocytic stomata, which are usually found above the epidermis level. The petiole is concave on ventral face and convex on its dorsal face, containing glandular and non-glandular trichomes as well as stomata in the epidermis. These types of trichomes were also detected in the stem. In addition, the presence of intercellular spaces within the organ is highlighted. Stomata above epidermis level also occurred in the stem. Phenolic idioblasts were found in the cortical region of plant root and deposit of lipophilic substance was observed in phloem cells. Great amount of apparently two different crystals were detected in all organs. Information obtained in this study provides knowledge about the characterization of H. ringens, which may be used to distinguish characters of taxa and can help understand the species survival in its occurrence sites. Key words: Lamiaceae, leaf blade, petiole, root, stem. INTRODUCTION known as espanta-pulga, the species occurs sparsely in rocky fields of southeastern region of Hesperozygis ringens (Benth.) Epling is a woody Rio Grande do Sul, Brazil (Von Poser et al. 1996, herb native from Brazilian Pampa, which is Tropicos 2017). Hesperozygis Epling. is formed by currently on the list of endangered species (Governo shrubs or sub shrubs, seven species occurring in do Estado do Rio Grande do Sul 2014). Initially southern Brazil and one species in Mexico (Pereira called Glechon ringens Benth. and commonly and Pereira 1973, Harley et al. 2004, Bräuchler Correspondence to: Berta Maria Heinzmann et al. 2010). The genus belongs to Lamiaceae E-mail: [email protected] Martynov, 1820 and within this family, it is placed An Acad Bras Cienc (2018) 90 (3) 2888 CARLOS G. PINHEIRO, JOÃO MARCELO S. DE OLIVEIRA and BERTA M. HEINZMANN in the Nepetoideae subfamily, Mentheae tribe, and Although H. ringens essential oil has been Menthinae subtribe (Harley et al. 2004). A study on studied under different aspects in the past years, the molecular phylogeny of this subtribe classified there is a lack of information about the structural Hesperozygis as a member of the monophyletic characterization of vegetative organs of this group The New World, along with other 21 genera species in literature. Additionally, there are no (Bräuchler et al. 2010). reports on the production and storage structures of Lamiaceae is composed of 236 genera and the essential oil in Hesperozygis representatives. about 7173 species, which are annual, biennial and Since such information could contribute to provide perennial herbs, shrubs, sub shrubs or trees (Harley knowledge to be used to distinguish taxa characters et al. 2004). Its species may be used in different and help understand the survival of H. ringens in areas such as medicinal, culinary and perfumery its occurrence sites, the purpose of this study was (Naghibi et al. 2005). The aromatic species classified to perform structural characterization of its leaf, in this family are characterized by essential oil stem and root. production in glandular trichomes located on MATERIALS AND METHODS aerial organs (Werker 1993). H. ringens has a great potential for essential oil production (Pinheiro et PLANT MATERIAL al. 2016), it stands out among Lamiaceae species. Hesperozygis ringens has been threatened with According to Werker (1993), these extractives may extinction (Governo do Estado do Rio Grande present beneficial functions for producer plants as do Sul 2014), thus a legal authorization for protection against herbivores and pathogens or as scientific activities was obtained through Sistema attraction of pollinator agents. This way, they often de Autorização e Informação em Biodiversidade exhibit significant biological effects on different (SISBIO, number 44197-2). Hesperozygis ringens living organisms (Badawy and Abdelgaleil 2014, leaf blade, petioles, stems and roots were randomly Pinheiro et al. 2017). Confirming this premise, a gathered from a single population located in series of biological activities have already been Santo Antão (S 29° 37’; W 53° 52’), Santa Maria described for H. ringens essential oil. In addition, municipality, Rio Grande do Sul, Brazil, in April the oxygenated monoterpenoid pulegone has been 2016. Leaf blades, petioles and stem samples were reported as its major compound and different gathered from 10 individuals, while root samples authors attributed the activities detected for this were gathered from 3 individuals. A voucher extractive especially to this molecule (Von Poser specimen was deposited at the Herbarium of the et al. 1996, Ribeiro et al. 2010, Silva et al. 2014, Forest Science Department (HDCF 6720), UFSM, Toni et al. 2014, Pinheiro et al. 2016, 2017). Brazil. Plant material was analyzed in the Structural Studies regarding this species as well as other Botany Laboratory (Federal University of Santa Hesperozygis representatives usually focus on Maria). essential oil production, describing their chemical characteristics and possible activities for the plant PROCEDURES FOR HISTOLOGY AND HISTOCHEMISTRY extractives (Von Poser et al. 1996, González- Chavez et al. 2011, Martini et al. 2011, Castilho et Hesperozygis ringens vegetative organs were al. 2016). Despite the chemical importance of the analyzed and dissected under stereomicroscope. genus, little information is known on the structural Then, the materials were fixed in 1% glutaraldehyde characterization of its species. and 4% formaldehyde in sodium phosphate 0.1M An Acad Bras Cienc (2018) 90 (3) CHARACTERIZATION OF Hesperozygis ringens ORGANS 2889 pH 7.2 buffer (Gabriel 1982, McDowell and Trump by Sudan Red 7B (Brundrett et al. 1991). Sudan 1976). Thereafter, fixed materials were submitted Black B was also applied on stem sections for to vacuum, followed by the procedure of washing detecting lipophilic substances in the endoderm in sodium phosphate buffer 0.1M pH 7.2 for 15 (Pearse 1972). Iodine dissolved in potassium min, according to methodology adapted from iodide (lugol’s reagent) was applied for starch Gabriel (1982). Afterwards, materials were washed (Johansen 1940). Slide analyses were performed in distilled water for 15 min, immersed in Tween under a Leica DM2000 light microscope in bright 20 2 mL/L (adapted from Freudenstein et al. 2002), field and polarized light for crystals. The plant and submitted to rotation for 15 days to remove material dissection and photographic records were epicuticular waxes. In the next procedure, the performed by a Leica DM80 stereomicroscope. dehydration of plant materials was performed in Photographic records for this study were obtained ascending ethylic series (10, 30, 50, 70, 90, 100%) using a Leica DFC 295 digital capture system and for 15 min in each concentration, followed by pre- software LAS (LeicaTM), as well as Zeiss Axio infiltration into (2-hydroxyethyl) methacrylate Imager A2 microscope, Zeiss MCr digital capture (HEMA) and absolute ethanol, finalizing by a system and ZEN (ZeissTM) software. process of infiltration in HEMA. Materials were put in embedding moulds filled with HEMA until RESULTS polymerization, according to Gerrits and Smid LEAF BLADE (1983). The procedures for histology occurred at least 3 times for each plant material. Leaf epidermis on abaxial and adaxial surfaces Crystal characterization was performed presents one cell layer which is amphistomatic by tests in fresh and included material. Samples (Figure 1a) with two different types of diallelocytic were submitted to alcoholic phloroglucin for two stomata (Figure 1b, c). The guard cells are commonly minutes followed by mounting in conc. HCl for observed above the level of the epidermis, a structure inulin identification, based on the formation of that is promoted by the growth of the palisade cells stained precipitate (Gahan 1984). Inulin was surrounding the substomatic chamber (Figure 1a). also identified by fresh material dehydration in The vascular system consists of collateral vascular bundles