Microsoft Word - 15-Agra_14382.doc 655 Original Article Biosci. J., Uberlândia, v. 29, n. 3, p. 655-666, May/June 2013 ANATOMY, HISTOCHEMISTRY AND MICROMORPHOLOGY OF LEAVES OF Solanum granuloso-leprosum DUNAL ANATOMIA, HISTOQUÍMICA E MICROMORFOLOGIA DE FOLHAS DE Solanum granuloso-leprosum DUNAL Edgard Augusto de Toledo PICOLI¹; Rosy Mary dos Santos ISAIAS², Marília Contin VENTRELLA³, Rafaela Marques de MIRANDA 4 1. Professor Adjunto, Departamento de Biologia Vegetal, Universidade Federal de Viçosa - UFV, Viçosa, MG, Brasil. epicoli@ufv.br ; 2. Professora Adjunta, Instituto de Ciências Biológicas - ICB, Universidade Federal de Minas Gerais - UFMG, Belo Horizonte, MG, Brasil; 3. Professora Associada, Departamento de Biologia Vegetal – UFV, Viçosa MG, Brasil; 4. Bolsista de Iniciação Científica, Departamento de Biologia Vegetal, Departamento de Biologia - UFV, Viçosa, MG, Brasil. ABSTRACT: In the present work the anatomical, histochemical and micromorphological features of S. granuloso-leprosum leaves were approached in order to evaluate its characteristics associated with its pioneer role. Glandular and non-glandular trichomes were observed on both epidermal surfaces, although in greater number on the ab axial surface. Stellate trichomes presented a thick lignified cell wall. Leaves were amphiestomatic with a single palisade layer and a slightly smaller spongy parenchyma. The epidermal cells of the abaxial surface were shorter than the adaxial ones, both with stomata paracytic. Vascular bundles were bicolateral and idioblasts with conspicuous crystalliferous inclusions were observed in the mesophyll. Lipid drops were evidenced in the spongy parenchyma by Sudan III, Nile Blue, Nadi reagent and Sudan Black histochemical tests. Negative results for alkaloids and phenol compounds were observed. The evaluated anatomical and hystochemical data highlights mesophytic characteristics in accordance with S. granuloso-leprosum pioneer plant role. KEYWORDS: Pioneer plant. “Curvitinga”. Plant anatomy INTRODUCTION Solanum granuloso-leprosum Dunal. is a small tree, with simple and entire leaves which produces fleshy fruits with small seeds (PETENATTI et al., 1998, VÁLIO; SCARPA, 2001). This plant specie is known in Brazil as “cuvitinga” and “fumeiro”, the botanical synonymy Solanum verbacifolium L. is also acknowledged (International Plant Names Index 2007). S. granuloso-leprosum is considered a pioneer plant (VÁLIO; SCARPA, 2001, FERREIRA et al., 2009), distributed in the northeast of Argentina, Uruguay, Paraguay, and South and Southeast of Brazil, occurring in secondary forests, and colonizing disturbed areas and ecosystems (FERREIRA et al., 2001, CASTELLANI et al., 2008, FERREIRA et al., 2009). Fowler and Carpanezzi (1997) emphasized that the main interest associated with this species was the recovery of degraded ecosystems. Considered a pioneer species (PETENATTI et al., 1998, FERREIRA et al., 2001, FERREIRA et al., 2009), it was verified that S. granuloso-leprosum fruits attracted a variety of animals, which participated on its seed dispersion (CÁCERES; MOURA, 2003, ALMEIDA et al., 2005). On the other hand, S. granuloso-leprosum was considered one of the potential host of Miridae (Heteroptera), a taxon whose species may cause damages to cultivated plants and also may be predators in potential to be used in biological control (FERREIRA et al., 2001). This Solanaceae was also described as a medicinal plant (PETENATTI et al., 1998, SHYUR et al., 2005, JOUZIER, 2005, ARAMBARRI et al., 2006). In Argentina, together with other tree medicinal species, S. granuloso-leprosum is known by the same vernacular name “ambay”, and is used as anti-inflammatory and calmative (PETENATTI et al., 1998). The increase of the worldwide market of biotechnological and pharmaceutical products (SHYUR et al., 2005) evidenced the importance of the knowledge and conservation of the genetic material, besides the need to evaluate, explore and maintain the genetic diversity. In the present work, the anatomical, histochemical and micro morphological characterization of S. granuloso- leprosum leaves were approached. MATERIAL AND METHODS Plant Material Leaves of Solanum granuloso-leprosum Dunal. (Solanaceae) were collected in a secondary forest at the Federal University of Viçosa. The shoots with flowers and fruits were dried, and Received: 03/02/12 Accepted: 05/07/12 656 Anatomy, histochemistry... PICOLI, E. A. T. et al. Biosci. J., Uberlândia, v. 29, n. 3, p. 655-666, May/June 2013 exsiccates were deposited at the VIC Herbarium (Federal University of Viçosa), under the numbers 31357 and 31358. The identification was confirmed by Dr. João Renato Stehmann (UFMG), specialist in Solanaceae. Light microscopy: Study of leaf surface Samples of leaf surface with approximately 0.5 cm2 were bleached with chloral hydrate, until the tissues were completely translucent. The samples were washed with distilled water, stained for 30 min with Astra Blue and Safranin (9: 1 v/v) (ROESER, 1962), dehydrated and mounted with synthetic resin (Permount, Fisher). The density of stomata per area was determined in samples from five expanded and healthy leaves, and an average of 33 fields per leaf, and expressed by the number of stomata per mm2. Histological analysis S. granuloso-leprosum Dunal. leaves were fixed in FAA50 (formalin, acetic acid, ethanol 50%; 5: 5: 90 v/v), for 24 h. Subsequently, the samples were dehydrated in ethylic series and included in metacrylate (Historesin, Leica). The leaves were sectioned transversally (8 µm thick) in an automatic advance microtome (RM 2155, Leica). The sections were stained with Toluidine Blue pH 4.0 (O´BRIEN; MCCULLY, 1981) for 10 min, and mounted with synthetic resin (Permount, Fisher). Histochemical analysis The histochemical tests were performed with fresh tissue samples, sectioned with a Microtome LPC (“Rolemberg & Bhering Comércio e Importação Ltda”).The following histochemical tests were carried out: Sudan III (JOHANSEN, 1940) and Sudan Black (LISON, 1960), for lipids; Nile Blue, for neutral and acidic lipids (JENSEN, 1962); NADI Reagent, for essential oils and oleoresins (DAVID; CARDE, 1964); chlorine vanillin, for phenol compounds (MACE;HOWELL 1974); ferric chloride, for phenolic compounds (JOHANSEN, 1940); potassium dichromate, for tannins (GABE, 1968); Wagner reagent and Dittmar reagent, for alkaloids (FURR; MAHLBERG, 1981); XP, for proteins (VIDAL, 1977); floroglucinol, for lignin (JENSEN, 1962); and ruthenium red, for pectic substances (JOHANSEN, 1940). One treatment without any reagent or staining was mounted in water and used as blank test. All the photographic documentation for light microscopy were performed with a microscope (Olympus AX70) equipped with U-Photo system. Micromorphological analysis - Scanning electronic microscopy The micromorphological analysis on the basis of the Scanning Electron Microscopy was carried out with completely expanded leaf segments, fixed in Karnovsky mixture (KARNOVSKY, 1965). The samples were submitted to vacuum for 24 h and kept in a 0.05 M potassium phosphate buffer for 30 min. After that they were dehydrated in ethylic alcohol series. Leaf samples were dehydrated by critical point of CO2 (BOZZOLLA; RUSSEL, 1992) using the Balzer's Critical Point Dryer (CPD020, Bal-Tec, Balzers, Liechtenstein), and set on stubs previous to the metallization with gold in the Sputter Coater (Balzer FDU010, Bal-Tec, Balzers, Liechtenstein). After the metallization with a fine gold layer (20 nm), the material was photographed using the scanning electron microscope (Model 1430VP, LEO). RESULTS Solanum granuloso-leprosum leaves presented trichomes on the abaxial and adaxial surface, even though they were more frequent on the former and non-glandular trichomes varied from multi seriated and multi cellular to unicellular and glandular trichomes were observed as well (Figures 1 A to K and 2 A to G). The leaf surfaces of S. granuloso-leprosum (Figures 1 and 2) showed non-glandular trichomes with one cell (Figure 1A), one ramification and an evident basal cell (Figure 1B), two ramifications and an evident basal cell (Figure 1C), three ramifications and an evident basal cell (Figure 1D), four ramifications (Figure 1E), five ramifications (Figure 1F), six ramifications (Figure 1G), seven ramifications (Figure 1H), and more (Figures 1I, 1J and 1K). This may considered different developmental stages of the same trichomes. In addition to thick and lignified cell walls (Figure 1K, 5E and 5H), stellate trichomes may have own several ramifications (Figure 2A, 2B, 2C, and 2E) which may imprison or provide an appropriate environment to some minute arthropods (Figure 2F). The trichome density is higher on the ab axial surface (Figures 2A, 2B and 2C). A few glandular trichomes were also observed on both surfaces (Figures 2G and 6B), although the density of non-glandular trichomes caused difficulties for the observation of other epidermal cells. The paracytic stomata were at the same level of common epidermal cells (Figure 2H). 657 Anatomy, histochemistry... PICOLI, E. A. T. et al. Biosci. J., Uberlândia, v. 29, n. 3, p. 655-666, May/June 2013 Figure 1. Scanning Eletron Microscopy photograph of the leaf surface of Solanum granuloso-leprosum Dunal. A – Unicelular tector trichomes; B – tector trichome, note that there are projections at the trichome base; C – tector trichome, note that there is a larger projection/ramification at the trichome base; D – tector trichome, note that there are two larger projection/ramification at the trichome base; E – tector trichome, note that there are three larger projection/ramification at the trichome base; F – tector trichome, note that there are four larger projection/ramification at the trichome base; G – tector trichome, note that there are five larger projection/ramification at the trichome base; H – tector trichome, note that there are six larger projection/ramification at the trichome base; I – tector trichome, note that there are eight larger projection/ramification at the trichome base; J – another angle from the six ramification tector trichome; and K – multicelular and multisseriated tector trichome, note the thick secondary cell wall. Scale Bars = 20 µ m. Anatomy, histochemistry... PICOLI, E. A. T. et al. Biosci. J., Uberlândia, v. 29, n. 3, p. 655-666, May/June 2013 Figure 2. Scanning Eletron Microscopy photograph of the leaf surface of Solanum granuloso-leprosum Dunal. A – detail of the adaxial surface; B – front view of the leaf blade; C – detail of the abaxial surface, note the difference in trichome density; D – front view of the leaf blade, note a idioblast with cristaliferous sand; E – detail of a branched pluricelular tector trichome; F – detail of branched trichomes with small animals held; G – detail of a glandular trichome; and H – stomata ranging from diacytic to paracytic. Scale Bars in Figures A, B and C = 100 µ m, in Figure E = 30 µ m, in Figure F = 15 µ m, and in Figures D, G and H = 20 µ m. The anticlinal walls of epidermal cells were slightly sinuous (Figure 2H), the ab axial cells being shorter than the ad axial ones (Figures 3B and 3C). The epidermis presented only one stratum (Figure 4A) and dimensions for the cells of the superior and inferior surfaces were respectively 12.8±2.4 and 7.1±1.5 µ m (Figure 3B) with the corresponding CVs of 18.8 and 20.9%. A single palisade layer and a slightly smaller spongy parenchyma constituted the mesophyll (Figure 4A). Leaf blade thickness exhibited an average and standard deviation of 131.4±17.2 µ m, with a CV of 13.1%, whereas the dimensions for the palisade and spongy parenchyma’s were respectively 63.3±12.0 and 48.2±11.3 µ m (Figure 3B). The corresponding CVs were 18.8 and 20.9%. Except for the sub epidermal layers, the parenchyma in the mid vein was composed by larger cells compared to the mesophyll, some idioblasts with crystal sand and did not present chloroplasts (Figures 4B and 4C). The vascular bundles were bicolateral and the phloem almost surrounds xylem cells completely (Figures 4B and 4C). Some idioblasts in the palisade and spongy parenchyma, in the midrib parenchyma, and in the phloem contained conspicuous crystalliferous inclusions (Figures 4A, 4B and 4D) which may be depicted in polarized light (Figures 4C and 4E). These inclusions may occupy considerable space in these idioblasts (Figures 4D and 4E) Anatomy, histochemistry... PICOLI, E. A. T. et al. Biosci. J., Uberlândia, v. 29, n. 3, p. 655-666, May/June 2013 Figure 3. Histometry and stomata index of Solanum granuloso-leprosum Dunal. leaves. A – Percentage of abaxial and adaxial epidermis, and palisade and spongy parenchyma, in relation to the total leaf width. All values are given in percentage (%). Superior Epidermis Ratio (SER); Palisade Parenchyma Ratio (PPR); Spongy Parenchyma Ratio (SPR); and Inferior Epidermis Ratio (IER); B – Abaxial and adaxial epidermis, and palisade and spongy parenchyma heights; All values are given in micrometer (µ m). Superior Epidermis (SE); C – Stomata index of adaxial and abaxial epidermis. Palisade Parenchyma (PP); Spongy Parenchyma (SP); and Inferior Epidermis (IE). Averages were taken based on 411 samples each and a mean number of 5 measurements per field. The coefficient of variation (CV) were 21.5, 13.1, 17.3 and 23.0, SER, PPR, SPR and IER, respectively. Average and standard deviation of stomata index were based on average of 182 fields per each face and 36 fields per sampled leaf. The CV for the stomata index were 10.6 and 14.0% for the abaxial and adaxial faces, respectively. Anatomy, histochemistry... PICOLI, E. A. T. et al. Biosci. J., Uberlândia, v. 29, n. 3, p. 655-666, May/June 2013 Figure 4. Cross section in Solanum granuloso-leprosum Dunal. leaves. A – Leaf blade section stained with Toluidine Blue, detail of the mesophyll and adaxial and abaxial epidermis. Note the idioblasts with cristaliferous sand in spongy and palisade parenchyma. B – Detail of mid vein under light microscopy; C – detail of the same mid vein under polarized light. Note several idioblasts in the parenchyma and smaller ones associated with phloem. D – Detail of parenchyma cells and some idioblasts with cristaliferous sand under light microscopy; and E – Detail of the same parenchyma cells under polarized light. Scale Bars in Figure A = 100 µ m; in Figures B and C = 200 µ m; and in Figures D and E = 100 µ m. 661 Anatomy, histochemistry... PICOLI, E. A. T. et al. Biosci. J., Uberlândia, v. 29, n. 3, p. 655-666, May/June 2013 In the mid vein, a small protuberance in the ad axial surface and a bigger one in the ab axial surface, mostly constituted of collenchyma, parenchyma and vascular tissue (Figures 5A to 5C). Untreated tissues for histochemical tests were documented (Figures 5A, 5D and 5G), whereas the positive reaction for floroglucin (Figures 5B, 5E and 5H) and red ruthenium tests (Figures 5C, 5F and 5I) were observed. Attention is drawn to the xylem elements, lignified and fully differentiated (Figures 5B and 5H), whereas pectic substances were evidenced in parenchyma, collenchyma, and phloem cells (Figures 5E, 5F and 5I). Histochemical test with XP reagent revealed that there were no protein accumulation in a particular cell or location in S. granuloso-leprosum leaves (Figure 6A), although a stained background was observed. The midrib exhibited one sub epidermal chlorenchyma and several layers of angular collenchyma, approximately 12 and 5 cell layers in the ad axial and ab axial faces, respectively (Figures 5F and 5G). The grayish cells corresponded to the idioblasts with crystal sand (Figures 2D, 4, 5A, 5C and 5I). Occasional glandular trichomes were observed (Figure 6B). A weak reaction with potassium dichromate was observed suggesting the presence of phenolic substances in these trichomes. Contrarily to non-glandular trichomes, the base and head of glandular trichomes cell walls were thin and non-lignified (Figure 6A). Except for floroglucinol (Figure 6C), no other test were positive for the non glandular trichomes. Figure 5. Histochemical tests in Solanum granuloso-leprosum Dunal. leaves (transversal sections). A – Control treatment, detail of the mid vein; B – tissue treated with floroglucinol, detail of the mid vein; C – mid vein treated with ruthenium red; D – leaf blade control treatment; E – leaf blade treated with floroglucinol; F – mid vein treated with ruthenium red, detail of collenchyma tissue in the adaxial (upper figure) and in abaxial face (lower figure), note a layer of chlorophyll parenchyma above and beneath the collenchyma, respectively; G – non treated sub epidermal collenchyma layers in the abaxial face of the leaf mid vein; H – detail of xylem (mid vein) treated with floroglucinol; and I – detail of phloem in mid vein treated with ruthenium red. Scale Bars in Figures A, B and C = 500 µ m; in Figures D, E, G, H and I = 100 µ m; in Figure F= 100 µ m. 662 Anatomy, histochemistry... PICOLI, E. A. T. et al. Biosci. J., Uberlândia, v. 29, n. 3, p. 655-666, May/June 2013 Figure 6. Histochemical tests in Solanum granuloso-leprosum Dunal. leaves (transversal sections). A – leaf blade treated with XP reagent, note that the mesophyll, although stained, did not present a conspicuous cell or organelle marked in particular; B – detail of a glandular tricome; C – leaf blade treated with Sudan III, note that the spongy parenchyma exhibit lipid bodies stained with this reagent; D – detail of a trichome tector multiseriated, note the lignified secondary cell wall of the trichomes; E – leaf blade treated with Nile Blue reagent, note the lipid bodies stained; F – leaf blade treated with Nadi reagent, note the lipid bodies stained; G – leaf blade treated with Sudan Black, note the lipid bodies stained. Scale Bars in Figures A to G = 50 µ m; in Figures E, F and G details = 100 µ m. Sudan III (Figure 6D), Nile Blue (Figure 6E), Nadi reagent (Figure 6F) and Sudan Black (Figure 6G) tests indicated the presence of lipid bodies in the spongy parenchyma, which suggests the presence of a mixture of essential and or neutral oils and acidic lipids. Additionally, S. granuloso- leprosum cuticle was more evident when using Sudan III, Nile Blue and Nadi, and corresponded to one sixth to one tenth of the epidermis height, and was thicker in the ad axial surface. Negative results for the chlorine vanillin, ferric chloride, Wagner and Dittmar reagents suggest that phenol compounds and alkalioids were not stored in S. granuloso- leprosum leaves. 663 Anatomy, histochemistry... PICOLI, E. A. T. et al. Biosci. J., Uberlândia, v. 29, n. 3, p. 655-666, May/June 2013 DISCUSSION The different trichomes observed on the epidermal surfaces of S. granuloso-leprosum leaves are in accordance with the Solanaceae literature (SIDDIAS, 1980, D’ARCY et al., 2001, MAITI et al., 2002, ELIAS et al., 2003, ALIERO et al., 2006, ARAMBARI et al., 2006, ALVES et al., 2007). Following Metcalfe and Chalk’s (1979) classification of stellate trichomes, stellate multiangulate sessile and stalked over the surface were found in S. granulosum-leprosum. Considering the number of ramifications and the differences on trichome density, it is hypothesized that the trichome and this morphogenesis process started at different times, what would account for the differences observed. Alves et al. (2007) observed the occurrence of glandular and non-glandular trichomes in Solanum cernuum, although unicellular non- glandular trichome was not reported. Previous work on Solanum anatomy established that the trichomes are very diverse in shape and size (EDMONDS, 1982). A few glandular trichomes were also observed in the ad axial (Figure 2G) and on the abaxial epidermis, although the density of non- glandular trichomes made difficult the observation of glandular trichomes and stomata. This is especially useful when approaching its use as a medicinal plant, considered the different trichomes observed in S. granuloso-leprosum, and Solanum trichome diversity (EDMONDS, 1982, MAITI et al., 2002, ALIERO et al., 2005, ALVES et al., 2007). Although considered a pioneer plant, S. granuloso-leposum exhibited, in general, mesophytic characteristics. It displayed large leaves with one palisade layer, and a thinner leaf and epidermis, and a higher number of stomata per mm2 than Capparis spinosa, a plant adapted to prolonged summer drought (RHIZOPOULOU; PSARAS, 2003). Despite the great density of non-glandular trichomes on S. granuloso-leprosum leaves, it probably would not be as adapted to a drought condition as it is to high light intensities. Nevertheless, Chaves Filho and Staccianrini- Seraphin (2001) observed that the osmotic adjustment was one of the mechanisms of drought resistance detected in a related species S. lycocarpum. Additionally, in xeric conditions, the gaseous exchange may be favored by means of a greater stomatic area (MEDRI; LLERAS, 1980). Solanum granuloso-leprosum epidermis had only one stratum with slightly sinuous cell walls, what may be associated with an adaptation to reduce the excessive waist of water (MEDRI; LLERAS, 1980). Irregular epidermal cells with different sizes are observed, what also reflected on the coefficient of variation (CV) of the epidermis height in the histometry data (Figures 3B and 3C). The paracytic stomata were at the same level of common epidermal cells, and, even though the number of stomata per area was different, they were observed on both leaf epidermal surface. Contrarily, Pentenatti et al. (1998) observed only anisocytic stomata in S. granuloso-leprosum. Maiti et al. (2002) observed two types of stomata, anisocytic present in D. inoxia, D. stramonium, N. glauca, P. viscosa, S. americanum, and S. rostratum, and anomocytic in L. esculentum, S. nitida, S. eleagnifolium, S. erianthum, S. nigrescens. Micromorphology of S. pseudocapsicum reveled amphystomatic leaves presenting anisocytic stomata (ALIERO et al., 2005). S. lycocarpum, displayed the same variation in the types of stomata observed (D’ARCY et al., 2001; ELIAS et al., 2003) observed for S. granuloso-leprosum, similar number of stomata per area for the ab axial epidermis, although a higher number of stomata for the ad axial surface (ELIAS et al., 2003). As the abaxial epidermis displayed a higher number of stomata per area, the trichomes may be just protecting the leaf from losing too much water, what is considered to be a xerophytic feature (FAHN; CUTLER, 1992). Moreover, the trichomes may be also indirectly associated with the transpiration decrease, reducing the solar radiation that reaches the leaves (FAHN, 1986). S. granuloso-leprosum mesophyll was constituted of a single palisade layer, with a spongy parenchyma cell layers slightly smaller than the palisade, and the abaxial epidermis shorter than the adaxial (Figure 4A). Some idioblasts with cristaliferous sand were present in the parenchyma and in the phloem and may occupy considerable space in these cells (Figure 4). Prominent midrib and the presence of crystal sand in this region were reported for other Solanaceae, like Tubocapsicum anomalum (D’ARCY et al., 2001). Pentenatti et al. (1998) reported the same bifacial structure, although also mentioned the presence of lipids and mucilage in addition to the crystal sand. Occasional druses were also reported at spongy and palisade parenchyma (D’ARCY et al., 2001). Different forms of crystals were also observed for ten Solanum species, clustered in the form of sands in L. esculentum, N. glauca, P. viscosa, S. americanum, S. eleagnifolium, S. erianthum, S. rostratum and S. triquetrum; druses 664 Anatomy, histochemistry... PICOLI, E. A. T. et al. Biosci. J., Uberlândia, v. 29, n. 3, p. 655-666, May/June 2013 in D. inoxia, D. stramonium, Solandra nitida, S. orienthum, S. nigrescens, S. rostratum and S. triquetrum (MAITI et al., 2002). The mid vein of S. granuloso-leprosum leaves was evident in transverse sections, with a small protuberance in the ad axial surface and a bigger one in the ab axial surface (Figures 5A to 5C), mostly constituted of vascular tissue, parenchyma and collenchyma. Pectic substances were evidenced in fundamental tissues (Figures 5C, 5F and 5I) whereas stellate trichomes and xylem cells presented lignified cell walls (Figures 5B, 5H and 6C). Similarly to Arambarri et al. (2006), no sclrerenchymatic tissue was observed in S. granuloso-leprosum leaves (Figures 5B, 5E and 5H). Histochemical test with XP reagent did not detect protein accumulation in S. granuloso- leprosum leaves (Figure 6A). Despite the weak reaction with potassium dichromate in glandular trichomes, no other leaf part was stained. Contrarily, high tannin contents were reported for D. stramonium, N. glauca, Solandra nitida, S. americanum and S. triquetrum (MAITI et al., 2002). The positive results with Sudan III (Figure 6D), Nile Blue (Figure 6E), Nadi reagent (Figure 6F) and Sudan Black (Figure 6G) tests indicates a good characteristic to discriminate S. granuloso- leprosum, since only for Solanum nitida the presence of lipids had been reported (MAITI et al., 2002). Although Solanaceae is known for containing alkaloids (MAITI et al., 2002), negative results for the histochemical tests suggests that phenol compounds and alkalis were not stored in S. granuloso-leprosum leaves. Other substances, such as solanin, saponin and tannins were reported in Petenatti et al. (1998) review, nevertheless, it was not found in S. granuloso-leprosum leaves. It was hypo the sized that the seasonal or environmental influence on the production might have accounted for the absence of such substances in leaf tissues. Maiti et al. (2002) observed that the majority of the species studied presented variations on the alkaloid and protein content. The present histochemical and anatomical approach allowed us to verify that S. granuloso- leprosum presented mesophytic and xerophytic characteristics, features that are in accordance with its phytossociological role as a pioneer plant. A well differentiated dorsiventral mesophyll, amphiestomatic leaves, the presence of a great density of non-glandular trichomes on the epidermis, and idioblasts with crystal sand were observed. Curiously, substances such as alkaloids were not observed in leaves, although lipid drops were present in the chlorophyll parenchyma and may represent important features in an ecological sense, or for medicinal usage. The histochemical and micro morphological features also suggest that the non-glandular trichomes grant leaves with some protection role. ACKNOLEGMENT We are indebted with Dr. João Renato Stehmann (UFMG) for the identification of our excicates. The authors thank to Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) for Grant support. RESUMO: No presente trabalho foi abordada a anatomia, histoquímica e micromorfologia de folhas de S. granuloso-leprosum no intuito de se levantar características associadas a sua função de espécie pioneira. Tricomas glandulares e não glandulares foram observados em ambas as faces da epiderme, apesar de maior número na superfície abaxial. Tricomas estrelados apresentaram uma parede espessa e lignificada. As folhas são anfiestomáticas com uma única camada de paliçada e um parênquima lacunoso com células menores que as do paliçádico. As células da face abaxial da epiderme são menores, e ambas as faces apresentam estômatos paracíticos. Os feixes vasculares são bicolaterais e idioblastos com inclusões cristalinas conspícuas. Gotas de lipídeo foram evidenciadas no parênquima lacunoso com os testes histoquímicos de Sudan III, azul do Nilo, reagente de Nadi e Sudan Black. Foram observados resultados negativos para alcaloides e fenóis. Os dados anatômicos e histoquímicos avaliados permitiram verificar que S. granuloso-leprosum apresentam características mesófilas, os quais estão em conformidade com o seu papel fitossociológicos como pioneira. PALAVRAS - CHAVE: Plantas pioneiras. “Curvitinga”. Anatomia. REFERENCES ALIERO, A. A.; GRIESON, D. S.; AFOLAYAN, A. J. The foliar micromorphology of Solanum pseudo capsicum. Flora, Jena, v. 201, n. 4, p. 326-330, jun. 2006. 665 Anatomy, histochemistry... PICOLI, E. A. T. et al. Biosci. J., Uberlândia, v. 29, n. 3, p. 655-666, May/June 2013 ALMEIDA, C. G.; MORO, R. S.; ZANON, C. M. V. Feeding habits of two species of frugivorous bats (Chiroptera, Phyllostomidae) from urban disturbed forest patches in Ponta Grossa, PR, southern Brazil. Publ. UEPG Ci. Biol. Saúde, Ponta Grossa, v. 11, n. 3/4, p. 15-21, set./out. 2005. ALVES, T. M. A.; MARENGO, S.; MACHADO, C.; CALDEIRA, R.; CARVALHO, O.; ISAIAS, R. M. I. S.; STEHMANN, J. R.; ZANI, C. Morphological, anatomical, macro and micromolecular markers for Solanum cernuum identification. Rev. Bras. Farmacognos, Curitiba, v. 17, n. 4, p. 542-548, dec. 2007. ARAMBARRI, A. M.; FREIRE, S. E.; COLARES, M. N.; BAYÓN, N. D.; NOVOA, M. C.; MONTI, C.; STENGLEIN, S. A. Leaf anatomy of Medicinal Shrubs and Trees from Gallery Forests of the Paranaense Province (Argentina). Part 1. Bol. Soc. Argent. Bot., Córdoba, v. 41, n. 3-4, p. 233 – 268, dec. 2006. BOZZOLLA, J. J.; RUSSEL, L. D. Electron microscopy., 1st Ed., Jones and Bartlett Publishers. 1992, 670p. CÁCERES, N. C.; MOURA, M. O. Fruit Removal of a wild tomato, Solanum granuloso-leprosum Dunal (Solanaceae) by birds, bats, and non-flying mammals in an urban Brazilian environment. Rev. Bras. Zoo, Curitiba, v. 20, n. 3, p. 519-522, set. 2003. CASTELLANI, E. D.; DAMIÃO FILHO, C. F.; AGUIAR, I. B.; PAULA, R. C. Morfologia de frutos e sementes de espécies arbóreas do gênero Solanum L. Rev. Bras. Sem., Londrina, v. 30, n. 1, p. 102-113, 2008. CHAVES FILHO, J. T.; STACCIARINI-SERAPHIN, E. Alteração no potencial osmótico e teor de carboidratos solúveis em plantas de lobeira (Solanum lycocarpum St.Hil.) em resposta ao estresse hídrico. Rev. Bras. Bot., São Paulo, v. 24, n. 2, p. 199-204, jun. 2001. D’ARCY, W. G. D.; KEATING, R.C.; ZHANG, Z.-Y.; PENG, C.-I. The genus Tubocapsicum (Solanaceae). Bot. Bull. Acad. Sin., Taiwan, v. 42, n. 1, p. 67-84, jan. 2001. DAVID, R.; CARDE, J. P. Coloration différentielle des inclusions lipid iqueetterpeniques des pseudophyllies du Pin maritime au moyen du reactif Nadi. Comptes Rendus Hebdomadaires dês Séances de I' Academie dês Sciences Paris, Sér. D, v. 258, p. 1338-1340, 1964. DICKSON, W. C. Integrative Plant Anatomy. 1st ed., San Diego: Harcourt Academic Press, 2000. 533p. EDMONDS J. M. Epidermal hair morphology in Solanum L. section Solanum. Bot. J. Lin. Soc., Londres, v. 85, n. 3, p. 153-167, out. 1982. ELIAS, S. R. M.; ASSIS, R. M.; STACCIARINI-SERAPHIN, E.; REZENDE, M. H. Anatomia foliar em plantas jovens de Solanum lycocarpum A. St. –Hil. (Solanaceae). Rev. Bras. Bot., São Paulo, v. 26, n.2, p. 169-174, jun. 2003. FAHN, A. Structural and functional properties of trichomes of xeromorphic leaves. Ann. Bot., London, v. 57, p. 631-637, 1986. FAHN, A.; CUTLER, D. Xerophytes. Berlin: Gebruder Born traeger, 1992. 176p. FERREIRA, P. S. F.; SILVA, E. R.; COELHO, L. B. N. Miridae (heteroptera) fitófagos e predadores de Minas Gerais, Brasil, com ênfase em espécies com potencial econômico.Iheringia, Sér. Zool., Porto Alegre, v. 91, p. 159-169, nov. 2001. FERREIRA, W. C.; BOTELHO, S. A.; DAVIDE, A. C.; FARIA, J. M. R. Establishment of riparian forest at the margins of the reservoir of the Camargos hydro eletric plant, Minas Gerais. Ciên. Florest., Santa Maria, v. 19, n. 1, p. 69-81, Jan.-Mar. 2009. 666 Anatomy, histochemistry... PICOLI, E. A. T. et al. Biosci. J., Uberlândia, v. 29, n. 3, p. 655-666, May/June 2013 FURR, Y.; MAHLBERG, P. G. Histochemical analysis of laticifers and glandular trichomes in Cannabis sativa. J. Nat. Prod., v. 44, p. 153–159, 1981. GABE, M. Techniques histologiques. Paris: Masson & Cie.1968. 1113p. International Plant Names Index.http://www.ipni.org/index.html. Acesso em 24/07/2007. JENSEN, W. A. Botanical histochemistry: principles and practice. 1st ed., Berkley: W. H. Freeman & Co, 1962, 408p.. JOHANSEN, D. A. Plant microtechnique. New York/London: McGraw-Hill Book Co. Inc., 1940. 523p. KARNOVSKY, M. J. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol., v. 27, p. 137-138, 1965. LISON, L. A. Histochemie et cytochemie animales: principles et méthodes. Paris: Gaulthier Villars, 1960. MACE, M. E; HOWELL, C. R. Hystochemistry and identification of condensed tannin precursor in roots of cotton seedlings. Phytopatol., St. Paul, v. 64, p. 1297-1302, out. 1974. MAITI, R. K.; VILLARREAL, L. R.; TREVINO, V. A.; VALADES-CERDA, M. C. Some aspects on pharmacognosy of ten species of the family solanaceae utilized in traditional medicine. Caldasia, Bogotá, v. 24, n. 2, p. 317-321, 2002. MEDRI, M. E.; LLERAS, E. Aspectos da anatomia ecológica de folhas de Hevea brasiliensis Muell. Arg. Acta Amazon., Manaus, v. 10, p. 463-493, 1980. METCALFE, C. R.; CHALK, L. Anatomy of the Dicotyledons. 2nd. ed. Vol. 1.Oxiford: Clarendon Press., 1979. O´BRIEN, T. P.; McCULLY, M. E. The study of plant structure principles and select methods. Melbourne: Termarcarphi, Pty, Ltda, 1981. PETENATTI, E. M.; PETENATTI, M. E.; DEL VITTO, L. A. Medicamentos herbarios en el Centro-Oeste Argentino. “Ambay”: controle de calidad de la droga oficial y sus adulterantes. Acta Farm. Bonaerense, Buenos Aires, v. 17, n. 3, p. 197-212, 1998. RHIZOPOULOU, S.; PSARAS, G. K. Development and structure of drought-tolerant leaves of the Mediterranean shrub Capparis spinosa L. Ann. Bot., London, v. 92, n. 3, p. 377-383, set. 2003. ROESER, K. R. Die nadel der schwarzkiefer-massenprodukt und kunstwerk der natur., Mikrokosmos, Stuttgartv. 61, n. 2, p. 33-36, 1962. SIDDIQI, S. A.; AHMAD R.; SIDIQUI S. P. Studies on the structure and ontogeny of stomata and trichomes on the leaves of some Solanum species. Geophytol., v. 10, p. 188-192, 1980. VÁLIO, I. F. M.; SCARPA, F. M. Germination of seeds of tropical pioneer species under controlled and natural conditions. Rev. Bras. Bot., São Paulo, v. 24, n. 1, p. 79-84, mar. 2001. VIDAL, B. C. Acid glycosaminoglycans and endochondral ossification: microespectrophotometric evaluation and macromolecular orientation. Cell Mol. Biol., v. 22, p. 45-64, 1977.