Leaf Blade Anatomy and Ultrastructure of Six Simira Species (rubiaceae) from the Atlantic Rain Forest, Brazil Introduction

Simira is a predominantly woody Neotropical genus comprising 41 taxa, 16 of which occur in Brazil and eight of them in the southeastern region of Brazil. Leaf blades of Simira eliezeriana Peixoto, S. were collected in the southeastern region of Brazil and fixed according to usual methods for light and electron microscopy. The leaf blades show typical characteristics of the Rubiaceae family as dorsiventral mesophyll and paracytic stomata. The presence of two bundle sheaths that extend to the upper epidermal layer, prismatic crystal and crystal-sand, alkaloids in the mesophyll and the organization micromorphological of the outer periclinal wall are considered characteristics representative for the genus. This study also demonstrates some leaf blade characteristics that can be used to Simira species identification (leaf surface, domatia types, epicuticular wax types and patterns of epidermis anticlinal cell walls).


Introduction
The Atlantic Rain Forest constitutes the second most threatened ecosystem in the world, and has been reduced over the years (Myers et al., 2000).This biome presents a high diversity of plant families and Rubiaceae is one of the most important families in several remnant forests in Rio de Janeiro state/Brazil (Guedes-Bruni, 1998).The Rubiaceae family has ca.650 genera and 13,000 species with a wide geographical distribution in the tropical and subtropical areas (Struwe, 2002).Among these, Simira Aubl. is a predominantly woody Neotropical genus consisted of 41 species, 16 of which occur in Brazil, and eight from southeastern Brazil (Silva Neto, 2000).This genus is important due to its economic value, e.g.production of handmade pieces, urban landscaping and dyestuff.
The present study aims to characterize the leaf blade anatomy and micromorphology of six Simira species of Atlantic Rain Forest from Southeastern Brazil, and to contributing to the knowledge the implications in survival strategies and taxonomic importance below the genus level.We particularly want to identify anatomical and morphological variations, which may be useful in species identification as well as to test the validity of the sections currently recognized in Simira.(19º 06' -19º 18' S and 39º 45' -40º 19' W), in the Municipal District of Linhares, Espírito Santo state, Brazil,, in the Municipal District of Duque de Caxias, and at Reserva Biológica de , in the Municipal District of Duque de Caxias and Nova Iguaçu, in Rio de Janeiro state, in the southeastern region of Brazil.

Light microscopy
Leaf fragments were fixed in a solution of 2.5% glutaraldehyde and 4.0% paraformaldehyde in 0.05 M cacodylate buffer at pH 7.2, for two hours.Subsequently, the samples were rinsed three times with the buffer and post-fixed for two hours at room temperature with 1.0% osmium tetroxide in 0.05 M cacodylate buffer at pH 7.2.The post-fixed samples were dehydrated in a graded series of acetone solutions.Subsequently, the material was infiltrated and embedded in epoxi resin (Polybed).Microtome sections (1.0 μm) were cut and stained with toluidine blue (0.05% aqueous solution).The slides were sealed with Entellan ® (Merck) and examined with an Axioplan ZEISS microscope.For epidermis, dissociation was achieved using a solution 1:1 of 10% nitric acid and 10% chromium oxide (Jensen, 1962), and stained with 10% safranine.Epidermis outer periclinal cell walls visualization, images were obtained using an ANALYSIS SIS LINK/OXFORD -ZEISS.

Histochemistry
Histochemical tests were carried out on freehand sections of the collected material alkaloids were localized by the Dragendorff test described by Costa (1982); and crystals were tested by the insolubility in acetic acid and solubility in hydrochloric acid (McLean and Cook, 1958), and examined with an Axioplan ZEISS microscope.

Scanning electron microscopy
The fragments of leaf blades were fixed, post-fixed and dehydrated as for light microscopy.The samples were subsequently critically point dried in CO 2 , sputter coated with 20 nm gold, and observed with a digital scanning electron microscope (ZEISS DSEM 962).The authenticity of epicuticular wax was verified by washing leaves in chloroform for one minute and comparing to the unwashed specimens under SEM.

Transmission electron microscopy
The samples were fixed, post-fixed, dehydrated and embedded as described previously.The ultrathin sections (80 nm) were collected in copper grids (300 mesh), stained with acetate with 1.0% uranyl followed by 5.0% lead citrate for routine observation.Three other stains were used to elucidate the cytochemical of the outer periclinal cell walls.(1) The preservation and contrast of lipids were enhanced and observed using imidazolebuffered osmium tetroxide (Angermüller and Fahimi, 1982).(2) 0.5 mg.ml -1 Ruthenium red was used to detect negatively charged components of the outer periclinal cell walls (Luft, 1971).( 3) Periodic acidthiocarbohydrazide (THC)-silver proteinate (PATAg) was used to detect polysaccharides containing 1,2-glycol groups.For the latter technique, sections were stained with periodic acid and solutions not containing peri-odic acid and THC as control (Thiéry, 1967).Sections were observed at 80 kV using a transmission electron microscope (ZEISS EM 900).

Extraction and determination of leaf proteins
The protein extraction was performed as described by Granier (1988) with some modifications.About 5 g of leaf blades were dry crushed in a mortar cooled with liquid nitrogen.The dry powder was resuspended in 5 mL of extraction buffer containing 100 mM Tris-HCl, pH 8.7, 1 mM dithiothreitol (DTT), 1 mM ascorbic acid, ered and submitted to further electrophoresis.The protein determination was performed by the method of Bradford (1976), using bovine serum albumin as standard and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Laemmli (1970).Proteins utilized as molecular mass standards for PAGE-SDS were bovine serum albumin (66 kDa), ovalbumin (45 kDa), glyceraldehyde-3-phosphate dehydrogenase (36 kDa), carbonic anhydrase (29 kDa), trypsinogen (24 kDa), trypsin inhibitor (20 kDa) and β-lactoglobulin (14 kDa).

Results
The presence of domatia is observed in the abaxial surface between the midrib and secondary veins.Simira grazielae (Fig. 1) presented domatia in pockets, while the other species presented domatia in tufts of hairs (Fig. 2).Cuticular ornamentation is observed on both surfaces as in S. glaziovii (Fig. 3), except in S. pikia (Fig. 4), and is restricted near the stomata of the other species studied.The cuticle of epidermic cells is non-striated (Fig. 3) on both surfaces, except in S. pikia (Fig. 4).The cuticle ornamentations is observed only near the stomata, except in S. elizeriana.In S. grazielae (Fig. 5), these cuticular striations are parallel to the stomata axis, while in other species they are perpendicular (Fig. 6).Epicuticular wax is observed in all species studied.Simira glaziovii, S. pikia, S. rubra, and S. sampaioana present a fine film; however, a smooth layer is seen S. elizeriana (Fig. 7) and granules in S. grazielae (Fig. 8).
In a transverse section of the leaf blade, the adaxial and abaxial epidermis present a single layer covered with a fine cuticle in all species as can observed in S. rubra (Fig. 11, 12), and in S. pikia (Fig. 13).The Simira species studied have dorsiventral mesophyll.Palisade tissue comprises two layers in S. elizeriana and one or two layers in the other species; and the number of spongy tissue layers varies from three layers in S. sampaioana to six in S. glaziovii.The species present prismatic crys-tals and crystal-sand, identified by the insolubility in acetic acid and solubility in hydrochloric acid, as well as alkaloids, identified by Dragendorff reagent, in the vacuole of mesophyll cells.
Paracytic stomata slightly protrude above the level of the other epidermal cells.Stomata are not usually seen over primary and secondary veins.In transverse sections, the guard cell presents a parietal thickening that is uniform.
In epidermis, at both leaf surfaces, the pattern of anticlinal cell walls varies from straight to sinuous: straight anticlinal walls in S. sampaioana (Fig. 15); slightly undulating walls forming an "S" in the S. elizeriana, S. glaziovii and S. rubra; and S. grazielae and S. pikia (Fig. 16) exhibit a strong sinuous.Anticlinal walls of epidermal cells are thin or moderately thick.
The vascular system shows a collateral arrangement and two layers of the bundle sheath (Fig. 11).The inner sheath is constituted by bundle perivascular fibers presenting an interruption in the phloematic tissue and parenchymatic cells in the outer bundle.The parenchymatic cells of the S. pikia bundle sheath contain a large amount of prismatic crystals, forming a crystalliferous series (Fig. 13).The bundle sheaths are extended towards adaxial epidermis in all species (Fig. 11, 13).
In the TEM cross sections, the outer periclinal cell walls in both epidermis show a fine epicuticular wax and three distinct layers: the inner polysaccharide-rich layer, mainly composed of cellulose; the intermediate cuticular layer, with a tree-like polysaccharide-rich network, immersed in a matrix of cutine; and a cuticle proper layer (Fig. 17-24).The cuticle proper is homogenous and relatively electron lucent.The cuticular layer is formed by a reticulated network layer and composed of a cutine matrix and a tree-like layer composed of polysaccharides and pectins, as observed with cytochemistry stains.The polysaccharide portion of the cuticular layer reacts with PATAg for polysaccharides and with ruthenium red, for pectin (Fig. 19-21).In contrast, the lipid portion of the cuticular membrane, the cuticle proper and the matrix of the cuticular layer react with imidazole (Fig. 23, 24).The species studied present differences in cuticular layer organization showing the tree-like layers more prominent in the adaxial surface (Fig. 17,19,21,23).In S. pikia that presents a cuticular striated relief, the cuticular layer keeps up with a striated organization, mainly the tree-like layer (Fig. 20).
The preliminary analysis of the soluble proteins of Simira and Bathysa species shows the presence of several specific proteins between 66 and 14.5 kDa.Two bands of the same molecular weight of specific proteins between 66 and 45 kDa are observed for the Simira and Bathysa species (Fig. 25, arrows).Two other proteins close to 36 kDa are also detected in the Bathysa species (Fig. 25,arrows).A band of approximately 24 kDa is observed just in the lanes of the Simira species.Our study also demonstrated proteins with low molecu-lar masses, more specifically below 14.5 kDa, present in all species of the Bathysa and Simira species analysed.

Discussion
Anatomical and ultrastructural studies have indicated important characteristics capable of distinguishing taxa, as well as relating structural characteristics with the environment (Bredenkamp and Van Wyk, 2000;Kong, 2001).The characteristics observed may be useful for identifying characters of genera, and to distinguish species and possible implications in survival strategies in the Atlantic Rain Forest.
The presence of domatia in the abaxial surface of the leaf blades between the midrib and secondary veins occur in the six species studied, and Robbrecht (1988) relate that it is a common character in the Rubiaceae family.Domatia are structures that frequently shelter small predators or fungivore arthropods.These structures appear scattered among 277 families of angiosperms, occurring mostly in tropical and subtropical areas (Agrawal and Karban, 1997).The presence and type of domatia constitute a morphological character with useful systematic value to separate genera and species (Adâmoli Barros, 1959).The domatia in pockets are only found in Simira grazielae and is a good character to separate this species from the others.Lateral projections in the midrib vein were also observed forming wakes covered with hairs in S. rubra, described as a domatia type in Psychotria velloziana (Da Cunha and Vieira, 1993/97).The organisms that living in domatia, are indirectly beneficial for the plant because they reduce the herbivore arthropods or the pathogenic fungi that commonly inhabit the surface of the plant leaves (O'Dowd and Wilson, 1989;Agrawal et al., 2000).These structures are possibly associated with defense mechanisms against microorganisms in Atlantic Rain Forest.Further studies are required to determine the possible role of these structures in plant protection.
The leaf micromorphology aspects can sometimes reflect the adaptation of plants to their habitat (Juniper and Jeffree, 1983); however, several characters can also provide conclusive data for taxa separation (Barthlott, 1981).Only S. pikia exhibit cuticular ornamentation in both surfaces, distinguishing the others species studied.In the species studied, the adaxial surfaces are smooth, except in Simira pikia that exhibit cuticular ornamentation that distinguish this species from the others.The cuticle of the plants is covered with epicuticular waxes that possess considerable ultrastructural and chemical diversity.These waxes show variable shapes that can be used in taxonomy (Barthlott et al., 1998).In the species studied, three types of epicuticular waxes were observed according to Barthlott et al. (1998): a smooth layer in Simira elizeriana; a granular type in S. grazielae, and a fine film in the other species.These different types were a complementary characteristic for separating S. elizeriana and S. grazielae.
Structural aspects of the epidermis, such as the form of the anticlinal cell walls, seem to be related to the light intensity.According to Combes (1946), shaded leaves exhibit epidermis with sinuous anticlinal cell walls.The anticlinal cell wall of Simira sampaioana is straight in the adaxial epidermis, as in Psychotria nuda and P. leiocarpa (Vieira et al., 1992), and Coussarea meridionalis and C. graciliflora (Tavares and Vieira, 1994).Other species of Simira show variation in the sinuosity, such as S. grazielae and S. pikia, suggesting that others factors, besides luminosity, may be related to the expression of this characteristic.According to Barthlott (1981), the form of the anticlinal cell wall can be useful as a taxonomic value.The straight anticlinal cell wall of S. sampaioana was used to separate it from the other species.
The outer periclinal cell walls are organized forming layers of varied compositions that are observed by TEM.Few studies have utilized technical cytochemistry to identify these components (Lyshede, 1978;Tenberge, 1992;Barros and Miguens, 1998).In all species studied, three distinct layers were observed: the inner polysaccharide-rich layer mainly composed of cellulose, the intermediate cuticular layer, with a tree-like polysaccharic-rich network immersed in a matrix of cutine, and the cuticle proper in all species studied.Comparative analyses in Simira leaf blades have shown evidence of biochemical similarities that can be useful for taxonomy.
Rubiaceae trichomes have a relatively simple structure and present only minor diversity (Robbrecht, 1988).According to Metcalfe and Chalk (1950), they can be unicellular, uniseriate, in tuff or rarely stellate.In the species studied, trichomes were unicellular, pluricellular and stellate at both surfaces, except in Simira grazielae.Cuticular ornamentations were observed on the surface of the trichomes of S. pikia, S. rubra and S. sampaioana as described for others Rubiaceae, i.e.Bathysa australis and Psychotria suterella (Barros et al., 1997).
The vascular system in the Simira species shows a collateral arrangement described before in other rubiaceous species (Robbrecht, 1988).The presence of two layers of the bundle sheath extending until the adaxial epidermis observed in Simira species can constitute a diagnostic characteristic of the genus.The bundle sheath prevents the vascular terminations from being exposed to the air contained in the intercellular spaces; involved in the short distance transportation between the bundle and the mesophyll; the sheath extensions have facilitated this function, taking the product of the bundle to the epidermis cells (Dickison, 2000).The same author believes that these extensions provide an additional mechanical support for the leaf blade.
Several types of crystalline inclusions are found in Rubiaceae species and may be a taxonomic character.In the present investigation, prismatic crystals and crystal-sand were observed in the mesophyll cells corroborating with the occurrence of crystalline inclusions for this genus as described by Metcalfe and Chalk (1950).These crystal types have been already observed in wood of many genera of Cinchonoideae and Ixoroideae (Jansen et al., 2002).In S. pikia, prismatic crystals were also observed in the parenchymatic bundle sheath.The presence of crystals in plants has been attributed to a possible defense function against herbivory, ion balance role or tissue support (Franceschi and Horner Jr., 1980).Robbrecht (1988) reported the presence of indolic, quinolinic and isoquinolinic alkaloids in tribes of Rubiaceae.Some benziliquinolinic alkaloids identified have been mentioned as an important taxonomic character (Dahlgren, 1975;Cronquist, 1981).Chromatographic analyses have isolated alkaloids from the wood and bark extract of the S. glaziovii (Alves et al., 2001;Bastos et al., 2001), S. salvadorensis and S. rubra (Arnason et al., 1983).In the species studied, alkaloids were identified in mesophyll cells through the positive reaction to the Dragendorff reagent.Phytochemistry studies are necessary to determine their chemical composition.
The accumulation of specific proteins has a taxonomic value and can be used to additionally characterize plant taxa (Chen et al. 1997).Several studies have shown the importance of protein markers identification present in some families as: Poaceae (Chen et al., 1997), Brassicaceae (El Naggar, 2001;Marques et al., 1998), Cucurbitaceae (Pasha and Sen, 1991;Castro et al., 1999), Fabaceae (Misset and Fontenelle, 1992) and Leguminosae (=Fabaceae) (Niknam et al., 2004).These techniques have also been used in phylogenenetic studies in the Rubiaceae family.Through the preliminary analysis of the present soluble proteins in the Rubiaceae species, the presence of a great diversity of molecular masses of several proteins can be observed.Some bands seem to be characteristic of each species or each genus and may come to represent specific markers to be used for identification of Bathysa and Simira genus.
The results reported here as the presence of domatia, "rubiaceous" stomata and dorsiventral mesophyll in Simira leaf blades confirms the leaf characteristics for the Rubiaceae family (Metcalfe and Chalk, 1950;Robbrecht, 1988).The presence of two layers of the bundle sheath extending until the adaxial epidermis, as well as the presence of alkaloids may be taxonomic value in Simira genus, helping to segregate this genus.Micromorphological and anatomical aspects such as cuticle ornamentation, domatia type, epicuticular wax type, the pattern of anticlinal cell walls, and the presence or absence of trichomes, can be used to segregate the Simira species studied.The combination of anatomical and micromorphological aspects of the leaf blade may be helpful in the systematic of these species.However, it is necessary more information about others Simira species for the genus comparative study.