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Petaline nectaries in Swietenia macrophylla (Meliaceae): Distribution and structural aspects
Flora 206 (2011) 484–490
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Petaline nectaries in Swietenia macrophylla (Meliaceae): Distribution and structural aspects Elder Antônio Sousa Paiva ∗ Departamento de Botânica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil
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Article history: Received 6 July 2010 Accepted 16 September 2010 Keywords: Cell ultrastructure Extranuptial nectary Mahogany Meliaceae Nectar secretion
a b s t r a c t There are few anatomical studies of the reproductive organs of Swietenia macrophylla, despite its economic importance. This study aims to describe the structural and ultrastructural organization of the petaline nectaries in mahogany flowers. Flower buds and flowers at anthesis were collected, fixed, and processed for studies under light and electron microscopy. Nectaries occur in the median region of the petal, on the abaxial surface. Nectar is produced at all stages, from the very young buds until anthesis. The nectary presents a uniseriate epidermis, without stomata; intercellular spaces among the epidermal cells are frequent and contiguous to the subcuticular space. The secretory tissue consists of two to five layers of cells, which are rich in organelles. The nectaries lack vasculature, and the secretory tissue is isolated from the petaline mesophyll by an endodermoid layer. In the staminate flowers, the number of nectaries is less than that observed in pistillate ones. © 2011 Elsevier GmbH. All rights reserved.
Introduction Species of the genus Swietenia (Meliaceae) are known for their quality of wood (Pennington et al., 1981), especially Brazilian mahogany, whose intense exploitation has placed it on the list of endangered species (Barbosa, 1992). Mahogany (S. macrophylla King) is the only species of the genus that is naturally found in Brazil and is one of the most valuable species in South America. The identification of Swietenia species, by morphological characters, becomes even more difficult due to a large number of hybrids (Helgason et al., 1996). According to Helgason et al. (1996), although many of the morphological characters are well-known, future investigations may well reveal new and useful features, thus increasing the knowledge of interspecific relations that occur in this genus. According to Gouvêa et al. (2008), information on the reproductive biology of tropical forest trees is extremely scarce, even for the most common and most well-known species, mainly due to the fact that the flowers are frequently inaccessible and difficult to observe in the crowns of tall trees. Mahogany flowers are not well-known in their anatomical aspects and the morphological description appears to be restricted to findings from Pennington et al. (1981) and Gouvêa et al. (2008). This species shows both
∗ Corresponding author. Tel.: +55 31 34092683; fax: +55 31 34092671. E-mail address: [email protected] 0367-2530/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.flora.2010.09.009
staminate (male) and pistillate (female) flowers within the same plant, with only subtle differences between the sexes, restricted mainly to reduced anthers and aborted ovules in the pistillate and staminate flowers, respectively (Pennington et al., 1981). According to Gouvêa et al. (2008), mahogany male and female flowers can be distinguished by the naked eye as a result of differences in the dimensions of the gynoecium. Although contradictory to the initial definition proposed by Caspary (1848 apud Schmid, 1988), the term “extrafloral nectary” has been widely employed with the same meaning proposed by Delpino (1873) for extranuptial nectaries. Hence, the term “extrafloral” takes on a broader meaning and encompasses any nectary that is not directly involved in the process of pollination. In this manner, nectaries found in reproductive organs are considered to be extrafloral by various authors (see Elias and Gelband, 1976; Morellato and Oliveira, 1994), based on the great morphological and functional similarities, especially as regards the interaction of ants and their consequent role in the protection against herbivores (e.g. Nascimento and Del-Claro, 2010). In this work, extrafloral nectaries (sensu Caspary, 1848) and extranuptial nectaries (sensu Delpino, 1873) will be considered as homologous to allow comparisons with previous published data. This is appropriate because extrafloral and extranuptial nectaries have great morphological similarity. However, I wish to express my disagreement with the broad meaning of the term “extrafloral”, both due to historical reasons as well as to its coherence with the ethymology of the term itself; it appears to be obvious that the extrafloral nectary cannot be located within the flower.
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Fig. 1. Distribution and structure of the petaline nectaries (PTNs) of S. macrophylla. (A and B) General aspects of PTNs (arrows) under scanning electron microscopy, note the smooth cuticle and absence of stomata. (C) Diagram of the full expanded petal showing vasculature and PTNs distribution, note that the position of the PTNs appears to be separated from the sepal vasculature. (D–F) Transverse sections of a petal in the nectary region; note that cells of the secretory portion show dense cytoplasm, differing from those vacuolated cells of the endodermis-like layer (delimited by dotted line in E and F) (el – endodermic layer; st – secretory tissue; su – subglandular tissue; vb – vascular bundle).
Extrafloral nectaries in Meliaceae were first described for three species of Swietenia (Lersten and Rugenstein, 1982). In later literature, diverse extranuptial nectaries (sensu Delpino, 1873) were described for other Meliaceae genera (see Paiva et al., 2007), which can be useful as a taxonomic character. The relationship between sugar secreting glands and plant protection has been well-studied and appears to be a well-defined scientific fact. Several studies have demonstrated the beneficial effects of ant–plant interactions mediated by extrafloral nectaries (Nascimento and Del-Claro, 2010; Oliveira and Freitas, 2004). Considering the small number of studies concerning the structural organization of the floral organs of S. macrophylla, this study aimed to report, for the first time, the occurrence and distribution of nectaries at the corolla of functionally pistillate (female) and functionally staminate (male) flowers, highlighting their structural and ultrastructural aspects.
Material and methods Collection of plant material for anatomical and ultrastructural studies Flower buds and flowers at anthesis were collected from Swietenia macrophylla King plants growing on the Pampulha Campus of Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Minas Gerais, Brazil. After collection, the flowers and flower buds were dissected under a stereoscopic microscope to remove the petals, which were then processed using standard methods for plant anatomical studies. Light microscopy and histochemical tests The isolated petals were fixed in Karnovsky solution (Karnovsky, 1965) for 24 h, dehydrated in an ethanol series, and embedded in hydroxyethyl-methacrylate (Leica Microsystems Inc., Heidelberg, Germany). Cross-sectional and longitudinal (5 m) cuts were per-
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formed using a rotary microtome and were subsequently stained in 0.05% toluidine blue in phosphate buffer, pH 4.7 (O’Brien et al., 1964). Hand-cut sections of fresh material were obtained and submitted to the following histochemical tests: phloroglucinol + HCl to detect the lignified walls (Sass, 1951); Sudan IV to detect lipids; 10% ferrous chloride aqueous solution to detect phenolic compounds (Johansen, 1940); and Fehling reagent for reducing sugars (Sass, 1951). Aqueous safranin (1%) was used to detect the apoplastic pathway in inflorescences, as reported by Seago et al. (2000). To observe and count the nectaries, evaluate petal area, and determine the nectary density, petals were cleared according to Fuchs (1963) and analyzed under an Olympus BH-2 light microscope with a drawing attachment. Transmission electron microscopy For transmission electron microscopy (TEM), nectary samples were fixed in Karnovsky solution (Karnovsky, 1965) for 24 h, postfixed in osmium tetroxide (1%, in 0.1 M phosphate buffer, pH 7.2), and processed using standard techniques (Roland, 1978). Ultra-thin sections were contrasted using uranyl acetate and lead citrate, and were then examined under a Philips CM 100 transmission electron microscope at 60 kV. Scanning electron microscopy For scanning electron microscopy (SEM), floral buds were collected, fixed in 2.5% glutaraldehyde (0.1 M phosphate buffer, pH 7.2), dehydrated in an ethanol series, critical-point-dryed, and coated with 10 nm of gold (Robards, 1978). Preparations were examined using a Quanta 200 (Fei Company, Hillsboro, OR, USA) scanning electron microscope at 12–20 kV, and all images were processed digitally. Results Morphology and distribution The petaline nectaries (PTNs) of Swietenia macrophylla flowers commonly occur within the median region of the petal, on the abaxial surface (Fig. 1A–C). This distribution pattern and general aspects occur regardless of the flower type. Flower buds of the two sexual morphotypes show only subtle differences, with the pistillated being more globous. Both flower types contain a short calyx, which exposes the corolla at all stages, beginning with the young flower buds. The petals present a spiraled disposition, thus the nectaries occur only in exposed areas. In field observation, nectar secretion could be observed at all stages, beginning with the young flower buds, with the secretion period extending to anthesis. Due to the minimal volume, the nectar production could not be measured. Nevertheless, field observations allowed us to infer that there are no differences between sexes regarding the amount of nectar in each nectary. Sugars were detected by histochemical tests in fresh, hand-cut sections. The abaxial surface of the petal shows a single layered epidermis with a rather thick cuticle and scattered stomata. The vascular tissue does not modify its normal distribution in the nectary region, as these nectaries are non-vascularized (Fig. 1C and D). The position of the PTNs appears to be separated from the sepal vasculature (Fig. 1C and D). The number of nectaries per petal, as well as the nectary density (nectary.mm−2 ), is higher in functionally pistillate flowers, as compared to the functionally staminate flowers (Table 1). The structural and ultrastructural characteristics of PTNs were similar in both functionally pistillate and functionally staminate
Table 1 Petaline nectaries (PTNs) in Swietenia macrophylla staminate and pistillate flowers. Flower
Petal area, mm−2
Nectaries number by petal
Petaline nectaries cm−2
Staminate Pistillate
17.02a 16.29b
1.82a 3.07b
10.84a 19.02b
The means were compared by Tukey (p ≤ 0.05). Different letters within each column indicate significantly different means.
flowers, thus the description is based on both flower types and does not present any gender references. The PTNs are circular in frontal view, and vary from a slightly depressed position to that found at the epidermal level (Fig. 1A, E and F). These nectaries show a secretory tissue comprised of two to five cell layers subtended by an irregular sheath that is composed by one to two layer(s) of juxtaposed cells with thickened and lipid impregnated walls (Fig. 1E and F). The number of cell layers in secretory tissue does not determine if the nectary is depressed or at the epidermal level, since both morphotypes can contain from two to five layers. Ultrastructural aspects The petal’s epidermal cells show a thick cuticle on the abaxial side, which is relatively thin on the secretory surface of the nectaries (Fig. 1F), where large subcuticular and intercellular spaces develop (Fig. 2B and C). Cuticle ruptures cannot be seen on the secretory surface of these nectaries. Epidermal cells of the PTNs did not differ in general aspect when compared to those of the subjacent secretory layers; however, their intercellular spaces were found to be well-developed (Fig. 2A–C). The intercellular space observed among epidermal cells connects to the spaces observed at subjacent layers of secretory cells. This constitutes a large intercellular space system within which secretion possibly flows. The volume of intercellular space was not measured, but its reduction in the depth of secretory tissue could be observed. Stomata were absent on the nectary surface. The secretory cells of the nectary make up a group which extends from the epidermis to the mesophyll, subtended by a layer of cells which stands out due to its thickened cell walls and well-developed vacuome. Lipid substances, such as suberin, were detected at this lignin-free, endoderm-like layer. The secretory cells contain large nuclei and a cytoplasm that is characterized by high electron density, as compared to other parts of the nectary (Figs. 2A and 3A). Among cell organelles, the presence of mitochondria, dictyosomes and plastids stands out. The mitochondria are globose and present well-developed cristae (Fig. 2D–G). The vacuome consists of small vacuoles containing membrane remnants (Fig. 2A and G). The plastids contain a poorly developed membrane system, with dense stroma and few thylacoids (Fig. 2D). Reserve substances were not detected within these plastids. The rough endoplasmic reticulum (RER) appears disperse, but segments near the cell surface and those disposed parallel to the plasma membrane are frequent (Fig. 2F). Dictyosomes show few cisterns and contribute, as does RER, to vesicle production. All secretory cells are connected by plasmodesmata (Fig. 2E and F). The subtending layer forms the border between secretory and subglandular tissues (Figs. 1E, F and 3A, B). These cells extend from the epidermis on the nectary border and delimit the secretory tissue. At this subtending layer, cells show prominent nuclei (Fig. 3B) and plastids with osmiophilic droplets dispersed within the stroma matrix and accumulated starch grains (Fig. 3C). However, these cells show less mitochondria than do the other nectary
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Fig. 2. Ultrastructural aspects of secretory tissue of the petaline nectaries of S. macrophylla. (A) General view of secretory cells, showing dense protoplasts. (B and C) Epidermal layer in cross section showing subcuticular space and a well developed intercellular system. (D and E) Detail of the cytoplasm of a secretory cell showing prevalence of mitochondria and plastids; the small vacuoles seem to be the result of dictyosome-derived vesicles. (F) Detail of peripheral cytoplasm with dictyosomes and mitochondria; note the plasmodesmata connecting adjacent cells (arrows). (G) Detail of mitochondria with well developed cristae and active dictyosome (cu – cuticle; di – dictyosome; is – intercelullar space; mi – mitochondria; va – vacuole).
regions and prove to be of a composition similar to that of the subglandular region. Small oil droplets are frequent in the cytoplasm, many of which being located in the periplasmatic region. Although these cells show thicker walls, the primary pit field occurs more commonly on the periclinal surfaces, where also numerous plasmodesmata occur.
The endodermal layer constitutes an apoplastic barrier, which apoplastically isolates the PTN secretory tissue, as demonstrated by the application of aqueous safranin solution to demonstrate any apoplastic permeability: When the flowers and peduncles were immersed in 1% aqueous safranin, the solution penetrated vascular tissues and spread to the entire petal within only a few hours.
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Fig. 3. Ultrastructure of the deeper region of a petaline nectary of S. macrophylla, showing the endodermis-like layer and the subglandular tissue. (A) General view of the transition between secretory and subglandular regions, including the endodermic layer (delimited by dotted line). (B) Secretory cells with dense protoplasts and endodermic layer (lower part) showing cells with electron-lucent protoplasts. (C) Detail of an endodermic cell showing the thick cell wall and plastids with dense stroma. Note the prevalence of plastids, mitochondria and RER. (D) Detail of a subglandular parenchyma cell; note a plastid with well developed membrane system (el – endodermic layer; mi – mitochondria; nu – nucleus; pl – plastid; st – secretory tissue; su – subglandular tissue).
The solution, however, did not penetrate the secretory tissue, being restricted by the subtending layer. The subglandular parenchyma shows electron-lucent cytoplasm with mitochondria, endoplasmic reticulum, and chloroplasts. The chloroplasts of these cells differ from those of secretory tissue by their well-developed membrane system (Fig. 3D). Mitochondria are abundant and predominantly globous with welldeveloped cristae. Plasmodesmata are well-developed in these cells, and phenolic substances are commonly observed at the vacuoles. Discussion Morphology, distribution and secretory activity Until 1982 (see Lersten and Rugenstein, 1982), Meliaceae was described as a family with no extrafloral nectaries (EFNs). The present study describes a new type of nectary (PTNs), in addition to the foliar nectaries, in S. macrophylla. Thus, the nectaries in S. macrophylla show a distribution pattern that is similar to that observed
in Guarea macrophylla (Morellato and Oliveira, 1994), where the nectaries appear within both vegetative and reproductive organs. The present data regarding the PTNs of S. macrophylla, in addition to the EFNs that are dispersed throughout its leaf (Lersten and Rugenstein, 1982), allow the observation that these nectaries present an efficient distribution, given that, according to Morellato and Oliveira (1994), the occurrence of many randomly distributed nectaries most likely increases the area covered by nectar-collecting ants and thus augments the protection afforded to the plant. The nectar production distributed to several nectaries within the same organ, as observed in the S. macrophylla corolla, may be interpreted as a means that improves the efficiency of ant patrolling and their consequent protection against predation by herbivores, as suggested by experimental evidence in other cases (Elias and Gelband, 1976; Morellato and Oliveira, 1994; Nascimento and Del-Claro, 2010; Stephenson, 1982). Such a nectary distribution pattern has been observed also in other Meliaceae, such as Guarea macrophylla (Morellato and Oliveira, 1994) and Cedrela fissilis (Paiva et al., 2007), as well as in foliar EFNs of S. macrophylla (Lersten and Rugenstein, 1982).
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According to the findings it can be inferred that the PTNs act as extranuptial nectaries (sensu Delpino, 1873), since they did not appear to be related to pollination. Their disconnection from pollination is reinforced by the secretion time, which is not restricted to the anthesis, and their placement, which is exclusively located on the abaxial surface of the petals. According to Rosenzweig (2002) the secretion of extrafloral nectar is an attempt to distract insects from flowers. Since the secretion of nectar is an energy intensive process, Rosenzweig (2002) theorized that the cost of each extrafloral nectary divided by the cost of each flower must be less than the proportion of reproduction threatened by insect visits. Nectaries of the S. macrophylla corolla can be classified as embedded nectaries, according to Elias (1983), and can be characterized as immersed within the tissues of the organ in which they appear. The differential expansion of secretory cells can results in depressed nectaries or nectaries at the epidermal level, once the number of cell layers can be the same in both morphotypes. The intercellular spaces observed in the epidermal layer of PTNs, as well the spaces within secretory tissue, allow for the formation of nectar transport ways. With reference to Marginson et al. (1985), and Paiva and Machado (2006), the occurrence of intercellular spaces within epidermal cells must be assessed as unusual. The absence of stomata, as observed in the PTNs of S. macrophylla, is a common characteristic within EFNs and has been reported also for extrafloral nectaries in other Meliaceae (Paiva et al., 2007). In flowers, the presence of nectaries in protective verticils is a relatively common phenomenon. However, in S. macrophylla the calyx is actually the verticil on which these structures appear to occur. Presence of extranuptial nectaries on the abaxial surface of petals is quite rare. Likewise as in petaline nectarines of S. macrophylla also in the leaves no association exists of the vascular system with extrafloral nectaries that occur on them (Lersten and Rugenstein, 1982). Absence of vascular tissues in extrafloral nectaries is quite common; this appears to imply a smaller amount of nectar secretion.
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others, were reported already earlier as typical for nectaries; they appear to be indicative of high metabolic activity (Fahn, 1988; Paiva, 2009; Stpiczynska et al., 2005). The presence of RER is more restricted and the predominance of a smooth reticulum appears to be associated with lipid metabolism, as well as with transport of nectar precursors, as it was described for a number of nectaries of different plant species (Fahn, 1988, 2000; Paiva, 2009; Stpiczynska et al., 2005). Although details of the ontogenesis of these petaline nectaries are unknown, it should be emphasized that there is a structural similarity between the secretory cells in the epidermal stratum and the subjacent layers, which suggests a common ontogenetic origin, as it was described for foliar nectaries of Cedrela fissilis (Paiva et al., 2007). The greatest similarity observed among the extrafloral nectaries of different species of Meliaceae appears to be these wellconserved structures. They may be of importance when attempting to understand the phylogeny of the group. It should also be emphasized that the description of a new type of extranuptial nectaries in a relatively well-studied species such as mahogany, coupled with reports on the occurrence of nectaries in other species of Meliaceae (Paiva et al., 2007), reveal just how underestimated the occurrence and the distribution of these structures really are in this family. Information on the reproductive biology of Brazilian mahogany (Swietenia macrophylla) is still scarce and the present data can be useful to understand the mahogany flower biology. Details of the function of these petaline nectaries in plant-environment interactions must still be studied in order to clarify their role in plant protection and reproduction. Acknowledgements The author thanks the technical team of the Centro de Microscopia Eletrônica, Instituto de Biociências, UNESP Botucatu, for their help in preparing the samples and to CNPq for a research grant concession (302048/2008-1) and financial support (Proc. 471444/2008-1).
Ultrastructure and function The presence of plasmodesmata in all nectary regions, including cells of the subtending layer, indicates a symplastic transport of nectar precursors from the subglandular tissue, as suggested by Fahn (1979) with concern of similar secretory structures. Chloroplasts with well-developed membrane systems within subglandular tissue cells may be interpreted as indicating photosynthetic metabolism that provides carbohydrates for nectar secretion. The green color of young flower buds is caused by these chloroplasts. The layer subtending the secretory tissue in the PTNs of S. macrophylla may be interpreted as an endodermoid layer, given that its effect hindering apoplastic transport is analogous to the endoderm function. Presence of lipid substances on the radial surfaces of the cells of this layer, and the absence of intercellular spaces between them, as observed in the present study, may explain the restriction of apoplastic transport. In Hymenaea stigonocarpa (Fabaceae) Paiva and Machado (2006) demonstrated that such a layer is ontogenetically an endodermis. According to Paiva and Machado (2006) this layer blocks the apoplastic pathway and consequently avoids a secretion reflux; this interpretation can be backed by previous similar observations (Fahn, 1988, 2000; Lüttge, 1971; Owen and Lennon, 1999). As demonstrated by the aqueous safranin test, the endodermal layer constitutes a barrier which apoplastically isolates the PTN secretory tissue. Lipid droplets in the cells of the subtending layer are probably involved building the apoplastic barrier. Peculiar ultrastructural aspects of secretory cells of the PTNs, such as prominent nuclei, dense cytoplasm, well-developed endoplasmic reticulum, active dictyosomes, mitochondria, among
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Petaline nectaries in Swietenia macrophylla (Meliaceae): Distribution and structural aspects Elder Antônio Sousa Paiva ∗ Departamento de Botânica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil
a r t i c l e
i n f o
Article history: Received 6 July 2010 Accepted 16 September 2010 Keywords: Cell ultrastructure Extranuptial nectary Mahogany Meliaceae Nectar secretion
a b s t r a c t There are few anatomical studies of the reproductive organs of Swietenia macrophylla, despite its economic importance. This study aims to describe the structural and ultrastructural organization of the petaline nectaries in mahogany flowers. Flower buds and flowers at anthesis were collected, fixed, and processed for studies under light and electron microscopy. Nectaries occur in the median region of the petal, on the abaxial surface. Nectar is produced at all stages, from the very young buds until anthesis. The nectary presents a uniseriate epidermis, without stomata; intercellular spaces among the epidermal cells are frequent and contiguous to the subcuticular space. The secretory tissue consists of two to five layers of cells, which are rich in organelles. The nectaries lack vasculature, and the secretory tissue is isolated from the petaline mesophyll by an endodermoid layer. In the staminate flowers, the number of nectaries is less than that observed in pistillate ones. © 2011 Elsevier GmbH. All rights reserved.
Introduction Species of the genus Swietenia (Meliaceae) are known for their quality of wood (Pennington et al., 1981), especially Brazilian mahogany, whose intense exploitation has placed it on the list of endangered species (Barbosa, 1992). Mahogany (S. macrophylla King) is the only species of the genus that is naturally found in Brazil and is one of the most valuable species in South America. The identification of Swietenia species, by morphological characters, becomes even more difficult due to a large number of hybrids (Helgason et al., 1996). According to Helgason et al. (1996), although many of the morphological characters are well-known, future investigations may well reveal new and useful features, thus increasing the knowledge of interspecific relations that occur in this genus. According to Gouvêa et al. (2008), information on the reproductive biology of tropical forest trees is extremely scarce, even for the most common and most well-known species, mainly due to the fact that the flowers are frequently inaccessible and difficult to observe in the crowns of tall trees. Mahogany flowers are not well-known in their anatomical aspects and the morphological description appears to be restricted to findings from Pennington et al. (1981) and Gouvêa et al. (2008). This species shows both
∗ Corresponding author. Tel.: +55 31 34092683; fax: +55 31 34092671. E-mail address: [email protected] 0367-2530/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.flora.2010.09.009
staminate (male) and pistillate (female) flowers within the same plant, with only subtle differences between the sexes, restricted mainly to reduced anthers and aborted ovules in the pistillate and staminate flowers, respectively (Pennington et al., 1981). According to Gouvêa et al. (2008), mahogany male and female flowers can be distinguished by the naked eye as a result of differences in the dimensions of the gynoecium. Although contradictory to the initial definition proposed by Caspary (1848 apud Schmid, 1988), the term “extrafloral nectary” has been widely employed with the same meaning proposed by Delpino (1873) for extranuptial nectaries. Hence, the term “extrafloral” takes on a broader meaning and encompasses any nectary that is not directly involved in the process of pollination. In this manner, nectaries found in reproductive organs are considered to be extrafloral by various authors (see Elias and Gelband, 1976; Morellato and Oliveira, 1994), based on the great morphological and functional similarities, especially as regards the interaction of ants and their consequent role in the protection against herbivores (e.g. Nascimento and Del-Claro, 2010). In this work, extrafloral nectaries (sensu Caspary, 1848) and extranuptial nectaries (sensu Delpino, 1873) will be considered as homologous to allow comparisons with previous published data. This is appropriate because extrafloral and extranuptial nectaries have great morphological similarity. However, I wish to express my disagreement with the broad meaning of the term “extrafloral”, both due to historical reasons as well as to its coherence with the ethymology of the term itself; it appears to be obvious that the extrafloral nectary cannot be located within the flower.
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Fig. 1. Distribution and structure of the petaline nectaries (PTNs) of S. macrophylla. (A and B) General aspects of PTNs (arrows) under scanning electron microscopy, note the smooth cuticle and absence of stomata. (C) Diagram of the full expanded petal showing vasculature and PTNs distribution, note that the position of the PTNs appears to be separated from the sepal vasculature. (D–F) Transverse sections of a petal in the nectary region; note that cells of the secretory portion show dense cytoplasm, differing from those vacuolated cells of the endodermis-like layer (delimited by dotted line in E and F) (el – endodermic layer; st – secretory tissue; su – subglandular tissue; vb – vascular bundle).
Extrafloral nectaries in Meliaceae were first described for three species of Swietenia (Lersten and Rugenstein, 1982). In later literature, diverse extranuptial nectaries (sensu Delpino, 1873) were described for other Meliaceae genera (see Paiva et al., 2007), which can be useful as a taxonomic character. The relationship between sugar secreting glands and plant protection has been well-studied and appears to be a well-defined scientific fact. Several studies have demonstrated the beneficial effects of ant–plant interactions mediated by extrafloral nectaries (Nascimento and Del-Claro, 2010; Oliveira and Freitas, 2004). Considering the small number of studies concerning the structural organization of the floral organs of S. macrophylla, this study aimed to report, for the first time, the occurrence and distribution of nectaries at the corolla of functionally pistillate (female) and functionally staminate (male) flowers, highlighting their structural and ultrastructural aspects.
Material and methods Collection of plant material for anatomical and ultrastructural studies Flower buds and flowers at anthesis were collected from Swietenia macrophylla King plants growing on the Pampulha Campus of Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Minas Gerais, Brazil. After collection, the flowers and flower buds were dissected under a stereoscopic microscope to remove the petals, which were then processed using standard methods for plant anatomical studies. Light microscopy and histochemical tests The isolated petals were fixed in Karnovsky solution (Karnovsky, 1965) for 24 h, dehydrated in an ethanol series, and embedded in hydroxyethyl-methacrylate (Leica Microsystems Inc., Heidelberg, Germany). Cross-sectional and longitudinal (5 m) cuts were per-
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formed using a rotary microtome and were subsequently stained in 0.05% toluidine blue in phosphate buffer, pH 4.7 (O’Brien et al., 1964). Hand-cut sections of fresh material were obtained and submitted to the following histochemical tests: phloroglucinol + HCl to detect the lignified walls (Sass, 1951); Sudan IV to detect lipids; 10% ferrous chloride aqueous solution to detect phenolic compounds (Johansen, 1940); and Fehling reagent for reducing sugars (Sass, 1951). Aqueous safranin (1%) was used to detect the apoplastic pathway in inflorescences, as reported by Seago et al. (2000). To observe and count the nectaries, evaluate petal area, and determine the nectary density, petals were cleared according to Fuchs (1963) and analyzed under an Olympus BH-2 light microscope with a drawing attachment. Transmission electron microscopy For transmission electron microscopy (TEM), nectary samples were fixed in Karnovsky solution (Karnovsky, 1965) for 24 h, postfixed in osmium tetroxide (1%, in 0.1 M phosphate buffer, pH 7.2), and processed using standard techniques (Roland, 1978). Ultra-thin sections were contrasted using uranyl acetate and lead citrate, and were then examined under a Philips CM 100 transmission electron microscope at 60 kV. Scanning electron microscopy For scanning electron microscopy (SEM), floral buds were collected, fixed in 2.5% glutaraldehyde (0.1 M phosphate buffer, pH 7.2), dehydrated in an ethanol series, critical-point-dryed, and coated with 10 nm of gold (Robards, 1978). Preparations were examined using a Quanta 200 (Fei Company, Hillsboro, OR, USA) scanning electron microscope at 12–20 kV, and all images were processed digitally. Results Morphology and distribution The petaline nectaries (PTNs) of Swietenia macrophylla flowers commonly occur within the median region of the petal, on the abaxial surface (Fig. 1A–C). This distribution pattern and general aspects occur regardless of the flower type. Flower buds of the two sexual morphotypes show only subtle differences, with the pistillated being more globous. Both flower types contain a short calyx, which exposes the corolla at all stages, beginning with the young flower buds. The petals present a spiraled disposition, thus the nectaries occur only in exposed areas. In field observation, nectar secretion could be observed at all stages, beginning with the young flower buds, with the secretion period extending to anthesis. Due to the minimal volume, the nectar production could not be measured. Nevertheless, field observations allowed us to infer that there are no differences between sexes regarding the amount of nectar in each nectary. Sugars were detected by histochemical tests in fresh, hand-cut sections. The abaxial surface of the petal shows a single layered epidermis with a rather thick cuticle and scattered stomata. The vascular tissue does not modify its normal distribution in the nectary region, as these nectaries are non-vascularized (Fig. 1C and D). The position of the PTNs appears to be separated from the sepal vasculature (Fig. 1C and D). The number of nectaries per petal, as well as the nectary density (nectary.mm−2 ), is higher in functionally pistillate flowers, as compared to the functionally staminate flowers (Table 1). The structural and ultrastructural characteristics of PTNs were similar in both functionally pistillate and functionally staminate
Table 1 Petaline nectaries (PTNs) in Swietenia macrophylla staminate and pistillate flowers. Flower
Petal area, mm−2
Nectaries number by petal
Petaline nectaries cm−2
Staminate Pistillate
17.02a 16.29b
1.82a 3.07b
10.84a 19.02b
The means were compared by Tukey (p ≤ 0.05). Different letters within each column indicate significantly different means.
flowers, thus the description is based on both flower types and does not present any gender references. The PTNs are circular in frontal view, and vary from a slightly depressed position to that found at the epidermal level (Fig. 1A, E and F). These nectaries show a secretory tissue comprised of two to five cell layers subtended by an irregular sheath that is composed by one to two layer(s) of juxtaposed cells with thickened and lipid impregnated walls (Fig. 1E and F). The number of cell layers in secretory tissue does not determine if the nectary is depressed or at the epidermal level, since both morphotypes can contain from two to five layers. Ultrastructural aspects The petal’s epidermal cells show a thick cuticle on the abaxial side, which is relatively thin on the secretory surface of the nectaries (Fig. 1F), where large subcuticular and intercellular spaces develop (Fig. 2B and C). Cuticle ruptures cannot be seen on the secretory surface of these nectaries. Epidermal cells of the PTNs did not differ in general aspect when compared to those of the subjacent secretory layers; however, their intercellular spaces were found to be well-developed (Fig. 2A–C). The intercellular space observed among epidermal cells connects to the spaces observed at subjacent layers of secretory cells. This constitutes a large intercellular space system within which secretion possibly flows. The volume of intercellular space was not measured, but its reduction in the depth of secretory tissue could be observed. Stomata were absent on the nectary surface. The secretory cells of the nectary make up a group which extends from the epidermis to the mesophyll, subtended by a layer of cells which stands out due to its thickened cell walls and well-developed vacuome. Lipid substances, such as suberin, were detected at this lignin-free, endoderm-like layer. The secretory cells contain large nuclei and a cytoplasm that is characterized by high electron density, as compared to other parts of the nectary (Figs. 2A and 3A). Among cell organelles, the presence of mitochondria, dictyosomes and plastids stands out. The mitochondria are globose and present well-developed cristae (Fig. 2D–G). The vacuome consists of small vacuoles containing membrane remnants (Fig. 2A and G). The plastids contain a poorly developed membrane system, with dense stroma and few thylacoids (Fig. 2D). Reserve substances were not detected within these plastids. The rough endoplasmic reticulum (RER) appears disperse, but segments near the cell surface and those disposed parallel to the plasma membrane are frequent (Fig. 2F). Dictyosomes show few cisterns and contribute, as does RER, to vesicle production. All secretory cells are connected by plasmodesmata (Fig. 2E and F). The subtending layer forms the border between secretory and subglandular tissues (Figs. 1E, F and 3A, B). These cells extend from the epidermis on the nectary border and delimit the secretory tissue. At this subtending layer, cells show prominent nuclei (Fig. 3B) and plastids with osmiophilic droplets dispersed within the stroma matrix and accumulated starch grains (Fig. 3C). However, these cells show less mitochondria than do the other nectary
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Fig. 2. Ultrastructural aspects of secretory tissue of the petaline nectaries of S. macrophylla. (A) General view of secretory cells, showing dense protoplasts. (B and C) Epidermal layer in cross section showing subcuticular space and a well developed intercellular system. (D and E) Detail of the cytoplasm of a secretory cell showing prevalence of mitochondria and plastids; the small vacuoles seem to be the result of dictyosome-derived vesicles. (F) Detail of peripheral cytoplasm with dictyosomes and mitochondria; note the plasmodesmata connecting adjacent cells (arrows). (G) Detail of mitochondria with well developed cristae and active dictyosome (cu – cuticle; di – dictyosome; is – intercelullar space; mi – mitochondria; va – vacuole).
regions and prove to be of a composition similar to that of the subglandular region. Small oil droplets are frequent in the cytoplasm, many of which being located in the periplasmatic region. Although these cells show thicker walls, the primary pit field occurs more commonly on the periclinal surfaces, where also numerous plasmodesmata occur.
The endodermal layer constitutes an apoplastic barrier, which apoplastically isolates the PTN secretory tissue, as demonstrated by the application of aqueous safranin solution to demonstrate any apoplastic permeability: When the flowers and peduncles were immersed in 1% aqueous safranin, the solution penetrated vascular tissues and spread to the entire petal within only a few hours.
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Fig. 3. Ultrastructure of the deeper region of a petaline nectary of S. macrophylla, showing the endodermis-like layer and the subglandular tissue. (A) General view of the transition between secretory and subglandular regions, including the endodermic layer (delimited by dotted line). (B) Secretory cells with dense protoplasts and endodermic layer (lower part) showing cells with electron-lucent protoplasts. (C) Detail of an endodermic cell showing the thick cell wall and plastids with dense stroma. Note the prevalence of plastids, mitochondria and RER. (D) Detail of a subglandular parenchyma cell; note a plastid with well developed membrane system (el – endodermic layer; mi – mitochondria; nu – nucleus; pl – plastid; st – secretory tissue; su – subglandular tissue).
The solution, however, did not penetrate the secretory tissue, being restricted by the subtending layer. The subglandular parenchyma shows electron-lucent cytoplasm with mitochondria, endoplasmic reticulum, and chloroplasts. The chloroplasts of these cells differ from those of secretory tissue by their well-developed membrane system (Fig. 3D). Mitochondria are abundant and predominantly globous with welldeveloped cristae. Plasmodesmata are well-developed in these cells, and phenolic substances are commonly observed at the vacuoles. Discussion Morphology, distribution and secretory activity Until 1982 (see Lersten and Rugenstein, 1982), Meliaceae was described as a family with no extrafloral nectaries (EFNs). The present study describes a new type of nectary (PTNs), in addition to the foliar nectaries, in S. macrophylla. Thus, the nectaries in S. macrophylla show a distribution pattern that is similar to that observed
in Guarea macrophylla (Morellato and Oliveira, 1994), where the nectaries appear within both vegetative and reproductive organs. The present data regarding the PTNs of S. macrophylla, in addition to the EFNs that are dispersed throughout its leaf (Lersten and Rugenstein, 1982), allow the observation that these nectaries present an efficient distribution, given that, according to Morellato and Oliveira (1994), the occurrence of many randomly distributed nectaries most likely increases the area covered by nectar-collecting ants and thus augments the protection afforded to the plant. The nectar production distributed to several nectaries within the same organ, as observed in the S. macrophylla corolla, may be interpreted as a means that improves the efficiency of ant patrolling and their consequent protection against predation by herbivores, as suggested by experimental evidence in other cases (Elias and Gelband, 1976; Morellato and Oliveira, 1994; Nascimento and Del-Claro, 2010; Stephenson, 1982). Such a nectary distribution pattern has been observed also in other Meliaceae, such as Guarea macrophylla (Morellato and Oliveira, 1994) and Cedrela fissilis (Paiva et al., 2007), as well as in foliar EFNs of S. macrophylla (Lersten and Rugenstein, 1982).
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According to the findings it can be inferred that the PTNs act as extranuptial nectaries (sensu Delpino, 1873), since they did not appear to be related to pollination. Their disconnection from pollination is reinforced by the secretion time, which is not restricted to the anthesis, and their placement, which is exclusively located on the abaxial surface of the petals. According to Rosenzweig (2002) the secretion of extrafloral nectar is an attempt to distract insects from flowers. Since the secretion of nectar is an energy intensive process, Rosenzweig (2002) theorized that the cost of each extrafloral nectary divided by the cost of each flower must be less than the proportion of reproduction threatened by insect visits. Nectaries of the S. macrophylla corolla can be classified as embedded nectaries, according to Elias (1983), and can be characterized as immersed within the tissues of the organ in which they appear. The differential expansion of secretory cells can results in depressed nectaries or nectaries at the epidermal level, once the number of cell layers can be the same in both morphotypes. The intercellular spaces observed in the epidermal layer of PTNs, as well the spaces within secretory tissue, allow for the formation of nectar transport ways. With reference to Marginson et al. (1985), and Paiva and Machado (2006), the occurrence of intercellular spaces within epidermal cells must be assessed as unusual. The absence of stomata, as observed in the PTNs of S. macrophylla, is a common characteristic within EFNs and has been reported also for extrafloral nectaries in other Meliaceae (Paiva et al., 2007). In flowers, the presence of nectaries in protective verticils is a relatively common phenomenon. However, in S. macrophylla the calyx is actually the verticil on which these structures appear to occur. Presence of extranuptial nectaries on the abaxial surface of petals is quite rare. Likewise as in petaline nectarines of S. macrophylla also in the leaves no association exists of the vascular system with extrafloral nectaries that occur on them (Lersten and Rugenstein, 1982). Absence of vascular tissues in extrafloral nectaries is quite common; this appears to imply a smaller amount of nectar secretion.
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others, were reported already earlier as typical for nectaries; they appear to be indicative of high metabolic activity (Fahn, 1988; Paiva, 2009; Stpiczynska et al., 2005). The presence of RER is more restricted and the predominance of a smooth reticulum appears to be associated with lipid metabolism, as well as with transport of nectar precursors, as it was described for a number of nectaries of different plant species (Fahn, 1988, 2000; Paiva, 2009; Stpiczynska et al., 2005). Although details of the ontogenesis of these petaline nectaries are unknown, it should be emphasized that there is a structural similarity between the secretory cells in the epidermal stratum and the subjacent layers, which suggests a common ontogenetic origin, as it was described for foliar nectaries of Cedrela fissilis (Paiva et al., 2007). The greatest similarity observed among the extrafloral nectaries of different species of Meliaceae appears to be these wellconserved structures. They may be of importance when attempting to understand the phylogeny of the group. It should also be emphasized that the description of a new type of extranuptial nectaries in a relatively well-studied species such as mahogany, coupled with reports on the occurrence of nectaries in other species of Meliaceae (Paiva et al., 2007), reveal just how underestimated the occurrence and the distribution of these structures really are in this family. Information on the reproductive biology of Brazilian mahogany (Swietenia macrophylla) is still scarce and the present data can be useful to understand the mahogany flower biology. Details of the function of these petaline nectaries in plant-environment interactions must still be studied in order to clarify their role in plant protection and reproduction. Acknowledgements The author thanks the technical team of the Centro de Microscopia Eletrônica, Instituto de Biociências, UNESP Botucatu, for their help in preparing the samples and to CNPq for a research grant concession (302048/2008-1) and financial support (Proc. 471444/2008-1).
Ultrastructure and function The presence of plasmodesmata in all nectary regions, including cells of the subtending layer, indicates a symplastic transport of nectar precursors from the subglandular tissue, as suggested by Fahn (1979) with concern of similar secretory structures. Chloroplasts with well-developed membrane systems within subglandular tissue cells may be interpreted as indicating photosynthetic metabolism that provides carbohydrates for nectar secretion. The green color of young flower buds is caused by these chloroplasts. The layer subtending the secretory tissue in the PTNs of S. macrophylla may be interpreted as an endodermoid layer, given that its effect hindering apoplastic transport is analogous to the endoderm function. Presence of lipid substances on the radial surfaces of the cells of this layer, and the absence of intercellular spaces between them, as observed in the present study, may explain the restriction of apoplastic transport. In Hymenaea stigonocarpa (Fabaceae) Paiva and Machado (2006) demonstrated that such a layer is ontogenetically an endodermis. According to Paiva and Machado (2006) this layer blocks the apoplastic pathway and consequently avoids a secretion reflux; this interpretation can be backed by previous similar observations (Fahn, 1988, 2000; Lüttge, 1971; Owen and Lennon, 1999). As demonstrated by the aqueous safranin test, the endodermal layer constitutes a barrier which apoplastically isolates the PTN secretory tissue. Lipid droplets in the cells of the subtending layer are probably involved building the apoplastic barrier. Peculiar ultrastructural aspects of secretory cells of the PTNs, such as prominent nuclei, dense cytoplasm, well-developed endoplasmic reticulum, active dictyosomes, mitochondria, among
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