Anatomy and Histochemistry of the Vegetative Organs of Brachystele guayanensis (Lindl.) Schltr. (Orchidaceae), a Potential Medicinal Species

1. Introduction

Brachystele Schltr. comprises 20 species distributed from southern Mexico to northern Argentina [1]; 10 species occur in Brazil, of which three are endemic [2]. The taxon is terrestrial, herbaceous, with short rhizomes, fleshy roots, leaves arranged in rosettes, lateral racemes with small flowers, resupinate, usually indumented, with a bilobed stigma [1,2].

Anatomical descriptions of the vegetative organs of Orchidoideae Lindl., the subfamily in which Brachystele is included, are still incipient and have mainly focused on species not occurring in Brazil [e.g., 3–20]. Anatomical studies of Brachystele are currently limited to the work of Bernal et al. [18], who examined the root trichomes of representatives of Spiranthinae Lindl. ex Meisn. as well as B. widgrenii (Rchb.f.) Schltr. There have been no histochemical studies of any Brachystele species, although some medicinal properties of B. dilatata (Lindl.) Schltr. and B. unilateralis (Poir.) Schltr. were mentioned (e.g., their diuretic and carminative property) in ethnobotanical studies [21,22].

We provide here anatomical and histochemical characterizations of the vegetative organs of B. guayanensis (Lindl.) Schltr. species (Figure 1) to expand available knowledge of that diversity in Orchidaceae, especially in Brachystele, and subsidize phylogenetic and micromorphological studies of that genus.

2. Results and Discussion

2.1. Anatomical data

2.1.1. Leaf anatomy

In frontal view, the leaf epidermis of B. guayanensis is covered by a striated cuticle and is composed of polygonal cells with both straight and curved walls (Figure 2a, b) – characters that have also been reported for Microchilus arietinus (Rchb. f. & Warm.) Ormerod and Zeuxine strateumatica (L.) Schltr.) by Andreota et al. [17] and Bona et al. [20] and described for Aa paleacea (Kunth) Rchb. f. and Pterichis multiflora (Lindl.) Schltr. by Corredor and Arias [15]. Anomocytic, diacytic and tetracytic stomata were observed on both faces of the leaf blade of B. guayanensis (Figure 2a, b), as seen in other species of Orchidaceae [3,14,16,19].

In cross-section, the epidermis of B. guayanensis is covered by a thin, striated cuticle and is uniseriate and composed of rounded, oblong cells with slightly thickened external periclinal walls (Figure 2c, d) – aspects reported for other members of Orchidoideae [e.g., 6,9,10,13–15,17,20]. The leaves are amphistomatic (Figure 2c-e), with stomata at the same level as common cells of the epidermis. The substomatal chambers are wider than the suprastomatal chambers (Figure 2c, d) – characteristics related to reducing water losses and evapotranspiration [23,24]. Amphistomatic leaves are commonly observed in plants having both high photosynthetic capacities and high stomatal conductance, especially those that grow in open and sunny environments [25,26], as studied here.

The mesophyll of B. guayanensis is homogeneous and consists of 6–11 layers of rounded cells having varying dimensions (Figure 2e). Those cells are interspersed with crystalliferous idioblasts containing raphides (Figure 2f) – a pattern cited for different groups of Orchidaceae [e.g., 6–10,13–17,19,20,27–29]. Collateral vascular bundles surrounded by a parenchyma sheath were observed in the median portion of the mesophyll (Figure 2e). The bundle corresponding to the midrib is flat-convex (Figure 2g) and has the largest caliber; the elements of that vessel are arranged in a “V” (Figure 2h); smaller bundles (Figure 2i), with smaller calibers are interspersed. This same pattern has been reported in other taxa by the authors cited above. The leaf margins of B. guayanensis are straight and rounded (Figure 2j).

2.1.2. Rhizome anatomy

The anatomy of the rhizomes of B. guayanensis was observed to be similar to other representatives of Orchidoideae [e.g., 6,9,10,13–16,19] in having rounded outlines and variable calibers (Figure 3a, b), with cataphylls in nodal regions or protecting the buds (Figure 3b, c). The epidermis is covered by a smooth, thin cuticle, is uniseriate, and composed of oblong, or occasionally, rounded common cells with thin cell walls (Figure 3d). Stomata were only observed on exposed portions of the rhizome and were arranged at the same level as the other common cells, with only tiny substomatal and suprastomatal chambers (Figure 3d).

The cortex of B. guayanensis is surrounded by a ring of pericyclic fibers (Figure 3h) and consists of 16–22 layers of rounded parenchyma cells of varying sizes (Figure 3a, b) with small triangular intercellular spaces; some of the parenchyma cells contain raphides (Figure 3e) and starch grains (Figure 3f). The arrangement is similar to that described by Stern [16] and by Stern and Judd [27,28] for the aerial stems of other Orchidaceae groups (such as Vanilloideae [27,28]; Orchidoideae, tribes Diseae Dressler and Ochideae Small; and Epidendroideae Lindl., tribes Sobralieae Pfitzer and Triphoreae Dressler [16,29]), as well as other groups of Monocotyledons [e.g., 30]. The endodermis and/or pericycle, as described here (Figure 3b), may participate in the genesis of adventitious roots, and therefore exhibit meristematic activity [30].

Fungal hyphae and pelotons were observed to be concentrated mainly in the more peripheral portions of the cortical parenchyma (Figure 3g), as was reported by Pereira et al. [31] for the roots of Bulbophyllum sp., Campylocentrum organense (Rchb.f.) Rolfe and Gomesa crispa (Lindl.) Klotzsch ex Rchb. f. According to these authors, the strategic positions of those structures contribute to the maintenance and (re)colonization of internal tissues and thus constitute important sources of inoculum for adventitious roots that will extend from the rhizome, corroborating the findings of Bougoure et al. [32] for the terrestrial orchids Rhizanthella gardneri R.S. Rogers and Suetsugu et al. [33] and Oreorchis indica (Lindl.) Hook. f.

From 28 to 45 collateral vascular bundles can be observed internally, surrounded by a parenchyma sheath, in an atactostelic arrangement (Figure 3i) typical of Monocots [25,34,35]. Those bundles are observed in the central portion of the organ analyzed (Figure 3a, b, i), and have been observed in other groups of that family [e.g., 9,10,16,27–29]. The vessel elements of the xylem are arranged in “V” formations in the collateral vascular bundles (Figure 3j), consistent with the findings of Andreota [36] for the tribe Cranichideae.

2.1.3. Root anatomy

Brachystele guayanensis has transversely circular roots of variable calibers (Figure 4a) that are anatomically similar to those found in other taxa of Orchidoideae [e.g., 5,6,11,12,15–17], with uniseriate velamen of the “Spiranthes type”. The component cells are irregular, elliptic, and thin, with thin cell walls with helicoidal thickenings and small pores in the non-thickened regions (Figure 4b, c). Our observations were similar to those of Porembski and Barthlott [4] for Pelexia dolichorhiza Schltr. (= Pachygenium pteryganthum (Rchb. f. & Warm.) Szlach., R. González & Rutk.), Prescottia colorans Lindl. (= P. stachyoides (Sw.) Lindl.), and Sauroglossum elatum Lindl. Moreira and Isaias [12] reported that terrestrial orchids generally have thinner velamen than epiphytic or rupicolous species in terms of their numbers of layers. Pridgeon [37], Moreira and Isaias [12], and Chomicki et al. [38] noted that velamen protects the roots from high solar radiation levels and excessive evapotranspiration losses, helps prevent overheating, and provides mechanical support to internal tissues, among other functions.

Simple, unicellular trichomes, such as those observed here (Figure 4c), were reported for Brachystele widgrenii, Lankesterella caespitosa (Lindl.) Hoehne, L. ceracifolia (Barb. Rodr.) Mansf., Pelexia orthosepala (Rchb.f. & Warm.) Schltr, and Sacoila lanceolata (Aubl.) Garay. by Bernal et al. [18], for Cranichis candida (Barb.Rodr.) Cogn. by Andreota et al. [17], and for 11 species of Aspidogyne Garay and Microchilus C. Presl. by Bona et al. [20]. According to Stern et al. [6], Andreota et al. [17] and Bernal et al. [18], simple trichomes provide better attachment to the substrate and increase contact with that surface – thus facilitating the absorption of both water and mineral salts.

The root cortex is composed of 18–20 layers of rounded parenchyma cells of varying sizes with innumerable small, triangular intercellular spaces (Figure 4d); hyphae and pelotons are also observed, mainly concentrated in more peripheral portions and underlying the exodermis (Figure 4d, g, h). This pattern has been reported in the roots of other Orchidaceae taxa by Pereira et al. [31], who noted that the presence of such symbiotic microorganisms is of great importance to the germination of orchid seeds as they increase the surface area of the roots and thus facilitate water and nutrient absorption [31,39].

These characters are frequently observed in Orchidoideae [e.g., 11,15,16], with terrestrial members of the family (as was observed in the species studied here) generally having thick, fleshy roots with a more expressive cortex (in terms the numbers of layers) that is responsible for holding reserves of water, starch grains, and other nutrients, with a less expressive velamen. Some of the cells in the cortical parenchyma contain raphides (Figure 4e), as observed in different groups of Monocotyledons [e.g., 34], with polyhedral starch grains gathered in spiranthosomes (Figure 4f) – spherical specialized amyloplasts found in Cranichideae and interpreted as a synapomorphy of the tribe [See 5,17,40,41].

The exodermis (Figure 4b, c), endodermis and pericycle (Figure 4k) of B. guayanensis are uniseriate, and are composed of rounded, elliptic cells with thin walls; the cells of the exodermis are slightly thickened (Figure 4b, c), and endodermis cells have evident Casparian strips (Figure 4k). Those patterns are repeated in other terrestrial orchids, especially in the subfamily Orchidoideae [See 13,15,17,19,20]. Benzing et al. [42] and Sanford and Adanlawo [43] noted that the thickening of the exodermal cells, as well as presence of a Casparian strip, aid in minimizing water losses to the external environment and provide mechanical support to root tissues.

The vascular cylinders in the roots of B. guayanensis are of variable calibers, have circular or elliptic shapes, and 12–14 protoxylem poles (Figure 4i, j), categorizing those roots as polyarchs, which are common in Monocotyledons [25,35]. The xylem and phloem are interspersed (Figure 4i-k); the central portion of the vascular cylinder is composed of parenchyma cells of varying sizes and shapes with tiny triangular intercellular spaces (Figure 4i, j), similar to those observed in other groups of Orchidaceae [e.g., 11,13,15–17,19,20].

2.2. Histochemistry data

Coomassie blue and Xylidine Ponceau strongly stained the pelotons and hyphae, indicating their protein contents (Figure 5a, b), and Sudan IV staining confirm the lipidic nature of the waxy cuticle (Figure 5c); the raphides are composed of calcium oxalate. Lignin was identified in the walls of xylem vessel elements (Figure 5d, f, g) and pericyclic fibers (Figure 5e), as well as in Casparian strips (Figure 5g). Starch grains and alkaloids were identified within the stomata guard cells (Figure 5h, o), epidermal cells, mesophyll cells (especially in the vicinity of vascular bundles) (Figure 5i, j, p), the cortical parenchyma of rhizomes (and occasionally in the central portion) (Figure 5k, l, q, r), as well as in roots (where they were most abundant) (Figure 5m, n, s-u). The histochemical tests were negative for reducing sugars, phenolic compounds, and tannins.

The waxy cuticle performs several functions, including protection against solar radiation, overheating, and water losses from internal tissues; it confers protection against the entry and the attacks of pathogens and herbivores [44,45,46].

Calcium oxalate raphides that are found in different groups of Orchidaceae, as well as other Angiosperms [e.g., 16,47], serve as defenses against herbivores, act in osmoregulation and in other metabolic activities requiring calcium, and have a role in the detoxification of aluminum (an element very abundant savannas soils) [47,48].

The lignin commonly deposited in vessel elements, pericyclic fibers, and the Casparian strip of the vegetative organs of the studied species confer stability, rigidity and mechanical support to the cell walls and internal tissues (25, 35). The Casparian strip, together with suberin deposits (both hydrophobic substances), contribute to solute selectivity in the root endodermis and act as barriers against apoplast movement – therefore preventing the influx of ions from the vascular cylinder to the cortical region [49,50,51].

Starch grains, especially those found in the roots and rhizomes, constitute some of the principal plant reserves are used (among other purposes) for vegetative growth and propagation [52,53,54,55]. Starch grains inside the stomata guard cells of the species studied here have also been reported in Oxalis L. (Oxalidaceae), Sambucus australis Cham. & Schltdl. (Adoxaceae), and Socratea exorrhiza (Mart.) H. Wendl. (Arecaceae) by Nunes et al. [56], Kikuchi et al. [57] and Reis and Alvim [58]. According to Appezzato-da-Glória and Carmello-Guerreiro [35], potassium levels in those structures appear to be associated with starch hydrolysis, which provides those organic anions. There is also evidence that malate, the regulator responsible for guard cell movements, can be synthesized through starch degradation [59,60].

According to Bulpitt et al. [61] and Sut et al. [62], many of the secondary metabolic substances identified in representatives of the Orchidaceae (such as flavonoids, alkaloids, terpenes, glycosides) act in plant defenses against herbivores and pathogens, and show bioactive pharmacological effects [63,64]. We believe that the presence of alkaloids in the stomata guard cells and in the cortical parenchyma of the rhizomes and roots together with the raphides identified in B. guayanensis, act to defend the species against attacks by pathogens and herbivores, as mentioned by Franceschi and Nakata [47] and Vizzotto et al. [65] for plants in general. Similar to what has been postulated by Li et al. [66] for the vegetative organs of other orchids, the abundant presence of raphides and secondary metabolites (e.g., alkaloids) in B. guayanensis, especially in the reserve organs of that species (e.g., the roots and rhizomes), are responsible for the renewal of their aerial portions, and help prevent pathogens and herbivores from reaching the vascular system (which is usually found in more internal regions) and causing local and/or systemic damage.

Among the bioactive substances found in Orchidaceae species are alkaloids (e.g., dendrobin, nobilonin, dendroxin) and nitrogenous heterocyclic organic molecules derived from the secondary metabolism of amino acids (e.g., phenylalanine, lysine, arginine, tyrosine, tryptophan). More than 100 types of bioactive alkaloids have been identified in more than 2,000 orchid species [67,68]. Those alkaloids have been found to be effective for treating gastrointestinal disorders and cardiovascular diseases, and also evidence anti-inflammatory, diuretic, analgesic, antioxidant, immunomodulatory, antipyretic, and antitumor activities [69,70,71]. Within this context, the alkaloids found in B. guayanensis indicate it as a potential medicinal species. (Bio)phytochemical investigations could therefore be informative, with the subsequent isolation and testing of the toxicity of any bioactive substances, and the characterization of their chemical natures.

This study was designed to provide initial information concerning the anatomy and histochemistry of the vegetative organs of the genus Brachystele and its medicinal potential and indicates the importance of anatomical and histochemical studies focusing on neotropical Orchidaceae species, especially those that have been only poorly studied.

3. Materials and Methods

For the anatomical studies, samples of vegetative organs (e.g., the mid-portions of the leaf blades, rhizomes, and roots) of five adult individuals of B. guayanensis (Figure 1) were collected in open fields, close to Bosque Auguste Saint-Hilaire, at Campus II (Samambaia) of the Federal University of Goiás (UFG), Goiânia, GO, Brazil. Collections of botanical material followed the recommendations of Mori et al. [72], and voucher specimens were deposited in the UFG Herbarium (registration numbers: I.S. Santos 1160 and 1161).

The samples collected for anatomical studies were fixed in 70% FAA (glacial acetic acid, formaldehyde, and 70% ethyl alcohol, 1:1:18) in hermetically sealed containers for 48 hours; after that time, they were preserved in 70% ethyl alcohol [73]. For the anatomical descriptions, cross sections were cut using a razor blade and clarified in a 20% aqueous solution of sodium hypochlorite (NaClO) (v/v) [74], stained with Astra Blue and Safranin (9:1) [75], and mounted in aqueous Glycerol solution (1:1). For analysis of the leaf surface in frontal view, the epidermis was dissociated using the Jeffrey method [73]. For the procedures mentioned above, the slides were sealed with a colorless sealant and subsequently photomicrographed using a Leica ICC50 HD^® digital camera coupled to a Leica DM500^® microscope, using Motic 2.0 Image Plus Software.

For histochemical studies, in natura samples obtained the time of collection were stained with the following reagents: Coomassie Blue and Xylidine Ponceau [76,77] for detecting proteins; Ferric chloride for detecting phenolic compounds; acidified Phloroglucinol for lignin; Sudan IV [73] for total lipids; Dittmar [78] for alkaloids: Fehling [74] for reducing sugars, Lugol [79] for starch; and hydrochloric vanillin [80] to detect tannins. To verify the chemical constitution of the crystals, 10% hydrochloric acid was used, following Chamberlain [81]. The descriptions of the analyzed organs were based on the terminologies used in the specialized literature [15,17,18,20].