AOBPreview originally published online on April 15, 2008
Annals of Botany 2008 101(9):1341-1348; doi:10.1093/aob/mcn052
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Anatomy, Ultrastructure and Chemical Composition of Food Bodies of Hovenia dulcis (Rhamnaceae)
1 Departamento de Botânica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, 31270-901 – Belo Horizonte, MG, Brasil
2 Departamento de Produtos Farmacêuticos, Faculdade de Farmácia, Universidade Federal de Minas Gerais, 31270-901 – Belo Horizonte, MG, Brazil
* For correspondence. E-mail epaiva{at}icb.ufmg.br
Received: 6 February 2008 Returned for revision: 26 February 2008 Accepted: 10 March 2008 Published electronically: 15 April 2008
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ABSTRACT |
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Background and Aims: Food bodies (FBs) are structures that promote mutualism between plants and ants, which help protect them against herbivores. The present study aims to describe the anatomical organization, ultrastructure and chemical composition of the FBs in Hovenia dulcis, which represent the first structures of this type described in Rhamnaceae.
Methods: Leaves in various stages of development were collected and fixed for examination under light, transmission and scanning electron microscopy. Samples of FBs were subjected to chemical analysis using thin-layer chromatography and nuclear magnetic resonance of 1H and 13C.
Key Results: The FBs vary from globose to conical and are restricted to the abaxial leaf surface, having a mixed origin, including epidermis and parenchyma. The FB epidermis is uniseriate, slightly pilose and has a thin cuticle. The epidermal cells are vacuolated and pigments or food reserves are absent. The parenchyma cells of immature FBs have dense cytoplasm showing mitochondria, endoplasmic reticulum and plastids. Mature FB cells store oils, which are free in the cytosol and occupy a large portion of the cell lumen. In these cells the plastids accumulate starch.
Conclusions: The lipids present in FBs are glycerin esters characteristic of plant energy reserves. Ants were observed collecting these FBs, which allows us to infer that these structures mediate plant–ant interactions and can help protect the young plants against herbivores, as these structures are prevalent at this developmental stage.
Key words: Ant–plant interactions, cell ultrastructure, food body, Hovenia dulcis, lipid, myrmecophily, Rhamnaceae
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Food bodies (FBs) are nutrient-rich uni- to multicellular structures that can easily be removed by foraging ants (O'Dowd, 1982). FBs characteristically accumulate nutrient reserves, generally proteins, lipids and carbohydrates (Rickson, 1976, 1979; O'Dowd, 1980; Risch and Rickson, 1981). These structures may consist of uni- or multicellular trichomes, or be characterized as protuberances in which parenchyma occupies the central portions. FBs act in maintaining mutualistic relationships between the plant and ants, which benefit the plant by offering protection against herbivores (O'Dowd, 1980).
Myrmecophytes, or ant-plants, are defined as plants that are continuously inhabited by ants during major parts of their life cycle (Webber et al., 2007b) and that use these obligate ant mutualists as a constitutive indirect defence mechanism (Heil et al., 2004). On the other hand, non-myrmecophytes (also called myrmecophyles) can be defined as plants that offer food to ants, but not lodging (see Webber et al., 2007b).
According to Rickson (1980), all nutritional tissues contain proteins, carbohydrates and lipids, although the proportions of these compounds are variable and characteristic of each plant species. Rickson also noted that the selection pressures that cause one or another of these substances to predominate are unknown, but they are presumably related to the nutritional needs of the associated ant species. According to Heil et al. (1998) it is reasonable to suppose that the plant components produced in order to attract mutualistic animals will show adaptative traits by possessing specific contents that match the requirements of the consumers. Recent research has demonstrated that the chemical contents of FBs may undergo evolutionary change as an adaptation to their role in ant attraction (Rico-Gray and Oliveira, 2007).
Rickson (1980) described the ultrastructural characteristics of food bodies produced by species of Macaranga (Euphorbiaceae), and noted the accumulation of lipid droplets in the cytosol and the presence of large numbers of plastids with starch grains. These two storage products together accounted for approximately 80 % of the cell volume of fully differentiated FBs. However, immature FBs show ultrastructural characteristics typical of cells with high metabolic activities, including a dense cytoplasm and many mitochondria and dictyosomes (see Rickson, 1980).
There are few published reports of food storage structures in Rhamnaceae, or about corresponding cell ultrastructure, and no previous mention of FBs in this plant family. Hovenia dulcis or ‘Japanese raisin’ tree is a deciduous tree that can grow up to 25 m tall (Carvalho, 1994) and is native of Asia. It is an easy plant to cultivate and grow under controlled conditions, and the discovery of food bodies in H. dulcis presents the opportunity to use this species as a model for studies of such structures and to investigate the protection that is apparently conferred by ants against herbivores.
The present work examined the ontogenesis, morphology and ultrastructure of food bodies in H. dulcis, and investigated the formation and chemical composition of their reserve compounds.
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MATERIALS AND METHODS |
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Collection of plant material for morphological, anatomical and ultrastructural studies
Ten specimens of Hovenia dulcis Thunberg (five young plants and five adult individuals) were examined over 2 years. Material for morphological and anatomical studies were collected from young plants cultivated near the Pampulha campus of the Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais State, Brazil (19°52'7·0''S; 43°57'58·2''W), and the anatomical organization and ultrastructure of the food bodies were examined during different stages of leaf development. Patrolling ants were observed and collected to identification purposes.
Light microscopy
Leaves in different developmental stages were collected for ontogenetic and anatomical studies. The material was fixed in Karnovsky solution (Karnovsky, 1965) for 24 h, transferred to 70 % ethyl alcohol (Jensen, 1962), dehydrated in an ethanol series (Johansen, 1940), subjected to pre-infiltration, and subsequently embedded in synthetic resin (Leica historesin) according to standard procedures. Transverse and longitudinal sections (5 µm thick) were made using a microtome, mounted on slides and stained with 0·05 % toluidine blue at pH 4·7 (O'Brien et al., 1964).
Histochemical tests
Both fixed and fresh material were used for histochemical tests, as follows: Sudan Black B to detect lipids (Pearse, 1980); Lugol for the identification of starch (Johansen, 1940); 0·02 % aqueous solution of Ruthenium Red to detect pectic compounds (Jensen, 1962); and mercuric bromophenol blue to detect proteins (Mazia et al., 1953).
Transmission electron microscopy
Samples were fixed in a Karnovsky solution (Karnovsky, 1965) for 24 h, post-fixed 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 examined under a Philips CM 100 transmission electron microscope at 60 KV.
Scanning electron microscopy
Samples were fixed in 2·5 % glutaraldehyde (0·1 M phosphate buffer, pH 7·2), dehydrated in an ethanol series, subjected to critical-point drying, and coated with 10 nm of gold (Robards, 1978). Preparations were examined using a Quanta 200 (Fei Company) scanning electron microscope, and all images were processed digitally.
Chemical characterization of lipid compounds
Food bodies were collected and extracted with dichloromethane at room temperature. The extract was analysed by thin-layer silica gel chromatography (TLC; Wagner et al., 1984) and by nuclear magnetic resonance (NMR) of 1H and 13C (Silverstein et al., 1991). The NMR spectra were registered in a Bruker DRX400 spectrometer at 400 MHz for 1H and 100 MHz for 13C in deuterochloroform.
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RESULTS |
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Distribution and structural aspects
In the vegetative organs of H. dulcis, domatia or any nesting spaces for ants were absent. The FBs seem to be the unique food reward offered to ants.
The FBs occur exclusively on the abaxial face of the leaves, along the midrib as well on secondary and third-order veins, forming small, easily removable structures on the leaf surface. FBs occur predominantly on the lateral sides of the veins (Figs 1A–C and 2A); their distribution is irregular, but largely on the midrib.
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FBs occur H. dulcis plants from when they are young (20 cm high) up to tall trees approaching their reproductive phase. FBs were not observed on seedlings, nor are they commonly observed on mature individuals, except occasionally when new branches are formed with juvenile characteristics, for example after a particularly aggressive pruning. Ants (Camponotus and Crematogaster spp.) were observed actively collecting the FBs and transporting them. During the field observations, partially damaged mature FBs were observed, which suggests their use in situ by the worker ants.
Mature FBs were large, up to 1·2 mm long by 0·6 mm wide, their shape varied from globose to conical and they had slightly irregular surfaces (Fig. 1A). The epidermis of the FB is uniseriate, slightly papillose, composed of flattened cells covered by a cuticle and with straight, anticlinal walls. Subcuticular spaces or ruptured cuticles were not observed, nor was any release of contents due to ruptures (Fig. 1A). When mature, whole FBs can easily be detached from the leaf.
At the start of ontogenesis the FBs appear whitish-yellow, becoming more yellow as expansion and differentiation progress. In mature leaves, and in the absence of ants, the FBs become senescent, turning brown and dehydrated before finally abscising. Senescent FBs demonstrate progressive reduction of their lipid reserves, which can no longer be observed at the end of the senescence process.
The initial stages of FB development occur in young leaves in all phases of their development, including fully expanded leaves; different stages of development can be observed on the same leaf.
FBs begin to form through periclinal divisions in a small group of parenchyma cells situated in the first subabaxial layer, adjacent to the leaf epidermis, on the lateral face of the abaxial projection of the vein (Fig. 2B, C). These initial divisions result in the formation of a small protuberance, with the epidermal cells on the flanks of that protuberance dividing anticlinally. This initial stage is followed by divisions in the basal region of the young FB; divisions are predominantly periclinal in the central cells and anticlinal in the epidermal cells and result in the formation of a meristematic layer in the basal portion of the developing FB (Fig. 2D, E). Rapid cellular differentiation and expansion can be seen in the new cells that form the FB. These cells show accumulation of reserve compounds, which is concomitant with meristematic activity in the basal region. The mature FBs are composed of two distinct regions: an epidermal layer and the central cells.
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Stomata and trichomes are rare in the leaf veins, and are rarely associated with food bodies. However, when an FB begins to form in a region subjacent to stomata or trichomes, these structures will be present in the mature FB (Fig. 2B, C).
The outer subabaxial region of the leaf vein is composed of 3–4 layers of collenchyma that are interrupted at the site of the FB, where parenchyma cells are more prevalent (Fig. 2F). FBs are not vascularized and do not demonstrate any relationship with the leaf vascular bundles (Fig. 2A, B, F).
The epidermal cells of mature FBs are vacuolated and do not contain any reserve compounds or pigments; the central cells have lipids and starch reserves. Proteins seem to be restricted to the structural ones, and no protein reserves were detected.
Ultrastructural aspects
The central cells near the meristematic layer have dense cytoplasm and a globose nucleus (Fig. 3A). Cell organelles in the central cells include rough endoplasmic reticulum, plastids, mitochondria, free ribosomes and small vacuoles (Fig. 3A, B). The rough endoplasmic reticulum appears predominantly in the peripheral cytoplasm, near to the plasma membrane (Fig. 3C). The mitochondria are globose, with well-developed cristae, and are frequently observed near to plastids (Fig. 3B). The plastids are elongated and amoeboid, and have poorly developed membrane systems and dense stroma with few osmiophilic inclusions; starch grains are only infrequently observed (Fig. 3B). Starch grains are seen during all stages of FB development but are more numerous in mature FBs, occurring simultaneously with lipid reserves (see Fig. 3B, E). Cell walls are thin, and have a pectocellulosic composition. Plasmodesmata are frequently observed connecting adjacent cells (Fig. 3B, C). Protein reserves were not observed during any of the developmental stages of the FBs.
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As the differentiation process proceeds, lipidic compounds accumulate in the central region of the cells. These lipids are not delimited by a membrane and they push the nucleus and the cytoplasm towards the cell periphery (Fig. 3E, F). Changes in the composition of the cytoplasm can also be distinguished in this phase, as the cell matrix becomes less dense and organelle density decreases (Fig. 3F). The mitochondria and endoplasmic reticulum are notably less concentrated during the final phases of differentiation; plastids remain numerous although they undergo changes, becoming globe-shaped and containing larger numbers of starch grains (Fig. 3D, E).
At the final stage of development, the basal meristematic cells undergo differentiation and the FB reaches its final shape and size.
Chemical analysis of lipids
In the initial TLC analysis, dichloromethane extracts of the FBs were compared with eugenol, which is an allylphenol and the major component of the essential oils of Syzygium aromaticum (clove oil), as well as of copaiba oil (from Copaifera sp.), a turpentine. Development with anisaldehyde/sulfuric acid produced stained regions with colours varying from pink to purple, with intense staining of the FB extracts (Fig. 4A).
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The 1H and 13C NMR spectra of FB extracts showed characteristic signals of glycerin esters of fatty acids corresponding to the trans carbon–carbon double bond (
H, 5·34 to 5·40 p.p.m., m; C, 129·71 and 130·02 p.p.m.), the carbonyl ester group (C, 173·25 p.p.m.), methylene (H, 4·29 p.p.m., dd, J = 4·4 and 11·6 Hz; H, 4·14 p.p.m., dd, J = 5·6 and 11·6 Hz; C, 62·09 p.p.m.) and methine groups (H, 5·25 p.p.m., m; C, 68·89 p.p.m.) of glycerin, methyl groups (H, 0·88, t, J = 7·2 Hz and 0·89, t, J = 7·4 Hz), and methylene groups of lipid chains (H, 1·26 to 2·35 p.p.m., C, 22·6 to 34·19 p.p.m., 15 signals in the DEPT 135 spectrum). These results indicate the presence of lipids in the food reserves of the FBs. Comparison by TLC of FB extracts with oleic acid, after development with bromocresol blue, showed a yellow spot for oleic acid (as expected for a free fatty acid) while for the FB extracts an intense moss-green spot was observed (Fig. 4B). |
DISCUSSION |
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Distribution and ontogenesis of food bodies
Hovenia dulcis do not offer nesting space to ants, but produces lipid-rich food bodies. Thus this plant species can be considered as a non-myrmecophyte or a myrmecophyle, at least in the juvenile phase; although according Webber et al. (2007b) the current usage of these terms is not always consistent. The interactions of ants with H. dulcis need to be studied in more detail.
The occurrence of FBs exclusively on the abaxial surface of major leaf veins, as in H. dulcis, was also reported by Rickson (1980) in Macaranga hypoleuca, and by Webber et al. (2007a) in Ryparosa kurrangii. Parenchyma cells in the subabaxial region of veins appear to be essential for differentiation of FBs, and may explain the absence of FBs on small leaf veins: as these are immersed in the mesophyll, they do not have a subabaxial (cortex-like) region similar to that found in major veins. In relation to this, it is important to emphasize that the mesophyll cells are more differentiated and adapted to photosynthetic metabolism. The role of the tissues subjacent to the epidermis in the ontogenesis of the emergence that produces the FBs was also reported by Rickson (1976) in Cecropia peltata, and by Solano et al. (2005) in Cordia nodosa, both myrmecophytes.
The epidermis of the food bodies of H. dulcis do not show pigments that confer protection against UV radiation, such as anthocyanin. Their occurrence exclusively on abaxial side of leaf therefore seems to be a protection against this harmful radiation, as well as a means of avoiding the risk of desiccation, hence increasing their life span and availability to ants. In extrafloral nectaries, such positioning may contribute to reducing nectar loss by evaporation, which increases its availability for the patrolling ants (Paiva and Machado, 2006; Paiva et al., 2007).
The occurrence of FBs only in the juvenile phases of plant development, as observed in H. dulcis, has not been reported for other species bearing these structures. However, temporal restriction of food rewards is common in other plant–insect systems involving ants, such as in extrafloral nectaries where rewards are generally restricted to the juvenile phases of the leaves (Paiva and Machado, 2006). In these situations, the juvenile organs are protected by ants during the phases in which they have no fully developed chemical and/or mechanical defences (see Paiva and Machado, 2006). It is interesting to note that food rewards (i.e. FBs) are interrupted during the adult phase of H. dulcis, even though new leaves and branches may still be forming. It may be that the very large size of the adult individuals of this species, and the large biomass of their leaves, effectively reduce the potential for damage by herbivory that is presented by the lack of protection offered by ants. This pattern of production also supports theories of resource allocation that hypothesize that plant resources will be preferentially allocated to more vulnerable or valuable plant parts (McKey, 1979; Rhoades, 1979). In cases of facultative interactions, as occurs in H. dulcis, this situation is mainly apparent in patterns in the production of food rewards (Heil and McKey, 2003). According to Fischer et al. (2002), at least in myrmecophyte Piper species, only a minute part of the above-ground biomass is invested in food for ants; this can be more advantageous than investing in more costly constitutive defences.
Our observation of the presence of more than one ant species associated with consumption of food bodies in H. dulcis is in accordance with observations made by Fiala and Maschwitz (1992) in non-inhabited Macaranga species, where a variety of ants were observed collecting FBs with no evidence of a species-specific association. According to Echols (1966), lipids are mainly used by worker ant and larvae, which is compatible with our observations.
Structural aspects of food bodies
The presence of stomata in the area where FBs are formed, as previously reported by Rickson (1980) and Solano et al. (2005), was observed only in some of the FBs under formation in H. dulcis. The relationship between stomata and FBs appears to be random, since guard cell differentiation occurs before FB differentiation. As such, FB ontogenesis is apparently not linked to the proximity of stomata, and the same situation applies to trichomes. A lack of any association between the localities where FBs are formed and the presence of stomata was also reported by Webber et al. (2007a) in Ryparosa kurrangii.
In the initial stages of FB development the central cells possess dense cytoplasm that is rich in organelles, especially rough endoplasmic reticulum, plastids, mitochondria, free ribosomes and small vacuoles. The absence of chloroplasts indicates that these cells obtain energy resources from the neighbouring tissues. FBs thus appear to act as resource sinks, a hypothesis that is reinforced by the presence of plasmodesmata establishing symplastic connections with neighbouring cells. These ultrastructural characteristics are similar to those observed in plant cells that are involved in synthetic processes and that have heightened metabolic rates (Lüttge, 1971), and they persist during the entire cell expansion phase. A similar situation has been described by Rickson (1980) for FBs in Macaranga spp.
The absence of food reserves in the epidermal cells of the FBs of H. dulcis indicates that the primarily role of this tissue is to protect the food body, and it does not provide any significant nutritional benefits to the ants. A similar principal role of epidermal tissue was also observed in the ‘Müllerian bodies’ of Cecropia peltata (Rickson, 1976), which, like the structures found in H. dulcis, are also characteristic protuberances.
Food reserves
There was no indication of the direct participation of FB cell organelles in the synthesis of the lipids stored in the central cells of the FBs. Lipid drops were observed in the cytosol, similar to the situation reported by Rickson (1980) in the ‘Müllerian bodies’ of Cecropia peltata. In the seeds of Ricinus communis, lipid drops are formed in the cytosol near sites with significant concentrations of lipogenic enzymes (Harwood et al., 1971). It is reasonable to assume that a similar situation occurs in H. dulcis, although additional studies will be necessary in order to confirm the origin of these compounds.
The FBs of H. dulcis predominantly contain glycerin esters in the lipid fraction, and an accumulation of carbohydrates in the form of starch. This combination constitutes a high energy-value reward for the ants. As such, it is probable that the structural proteins available in the FBs are not sufficient to satisfy the nitrogen needs of these insects; this could stimulate the ants to search for other nitrogen sources and would result in an even greater stimulation of their predation on small herbivores.
Plant protection and ecological considerations
Physical and chemical defences (such as cellulose, lignin and tannins) would require more carbon allocation than the food bodies offered to ants in exchange for their defensive role (see Abe and Higashi, 1991). The FBs of H. dulcis may thus reduce carbon expenditures in the plant's defence against herbivores, leaving more metabolic resources available for use in its juvenile rapid-growth phase.
The FBs that occur in H. dulcis show a high energy value due to their lipid content. According to Heil et al. (1998), energy-rich lipidic reserves are uncommon in non-mymercophytes. Thus, the food resources offered by the non-mymercophyte H. dulcis could be important to the ants that harvest them.
Like similar structures described in other species (see Rickson, 1969, 1976; O'Dowd, 1980; Fiala and Maschwitz, 1992), the FBs produced in the leaves of H. dulcis appear to have an important role in interactions with ants, which can protect the plant against herbivores.
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ACKNOWLEDGEMENTS |
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The authors would like to thank Dr José Dias de Souza Filho, Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, for the NMR spectra; to Dr Denise Maria Trombert Oliveira for hepful comments; the team of the Centro de Microscopia Eletrônica, Instituto de Biociências, UNESP Botucatu, for preparing the electron microscope samples; and Igor Rismo Coelho for his help in identifying the ant species.
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