The secretory glands of Asphodelus aestivus flower

Various secretory glands are observed on Asphodelus aestivus flower, a common geophyte of Mediterranean type ecosystem. The floral nectary has the form of individual slits between the gynecium carpels (septal nectary). The septal slits extend downwards to the ascidiate zone of the carpels. The nectar is secreted by the epidermal cells of the slits, which differentiate into epithelial cells. The latter contain numerous organelles, among which endoplasmic reticulum elements and golgi bodies predominate. Nectar secretion results in an expansion of the space between the septa. The nectar becomes discharged through small holes on the ovary wall. Six closely packed stamens surround the ovary and bear numerous papillae at their basis. These papillae are actually osmophores, i.e. secretory structures responsible for the manufacture, secretion and dispersion of terpenic scent. A mucilage gland (obturator) exists between the lateral ovule and the ovary septa, giving a positive reaction with Schiff’s reagent. This gland secretes a mucoproteinaceous product to nourish the pollen tube and to facilitate its penetration into the ovary.


Introduction
Asphodelus aestivus Brot. (A. microcarpus Viv.), family of Asphodelaceae (Asparagales), is a common spring-flowering geophyte encountered on the Mediterranean coasts (Tutin et al. 1980). A. aestivus formations represent the last degradation stage of Mediterranean type ecosystems. These formations, often referred to as "asphodel deserts", result from frequent fires and overgrazing (Naveh 1973;Margaris 1984;Pantis & Margaris 1988). They are found on hill slopes interspaced among cultivated areas and are used as lowland pastures. A. aestivus, as other geophytes (cryptophytes), has a considerable distribution, since it has become the dominant life form in many degraded Mediterranean ecosystems.
The number of flowers per inflorescence varies from 400 to 600. In every cluster, the bottom flowers open first, followed by the above flowers. Every single flower remains in bloom for several days. The number of open flowers per inflorescence on the same day can be up to 30. The overproduction of flowers allows the plant to compensate for environmental variations and provides maternal chance in selective abortion of fruits and seeds (Stephenson 1981;Sutherland 1986;Lee 1988;Ehrlen 1991). The perianth of the actinomorphic flower of A. aestivus has a distinct calyx and corolla and the white petals have a dark stripe through the centre.
The entomophilous flower of A. aestivus secretes a considerable amount of nectar, which is involved in pollination. As in many other plants, nectar is used to reward insects, which in turn offer a beneficial relationship (Simpson 1993). The main pollinators are bumblebees and honeybees. Knuth (1899) provided general information about the floral biology of A. albus and Daumann (1970) described the morphology of the nectary. The release of the nectar onto the nectary surface occurs with a variety of mechanisms. Nectar may diffuse through the thin secretory cell walls or may accumulate beneath the cuticle of the nectary cells until the cuticle ruptures by the foraging vector releasing the nectar (Sawidis et al. 1987;Sawidis et al. 1989;Fahn 1990;Sawidis 1991).
Asphodel meadows had first been referred by Homer. According to his epics (Odyssey,XI,539,573,XXIV,13), the souls of the dead arrived in underground meadows (asphodelos leimon) on which only asphodels bloomed. The root tubers of A. aestivus, dried and boiled in water, yield mucilaginous matters which in some countries, when mixed with flour or potato, make the asphodel bread. In Spain and other countries they are used as cattle and especially as sheep fodder. In Persia, strong glue is made from the root tubers, which first become dried, pulverized and then mixed with cold water. Under the term "Tsinisse", the root tubers of Asphodelus bulbosus, were used in eastern countries as mucilage and to adulterate powdered salep. The ultrastructure and function of A. aestivus root tubers have been recently studied in order to explain the abundance of this plant in the Mediterranean region (Sawidis et al. 2005).  Because of the importance of A. aestivus as a consistent component of the Mediterranean vegetation and its dominance over wide areas, we have undertaken several studies, which are focused on various features and processes in order to identify mechanisms that contribute to its remarkable distribution in the Mediterranean region. The aim of the present study was to determine the morphology, anatomy and fine structure of the flower glands (nectary, osmophores and mucilage gland).

Material and methods
Plant material was taken from 1-year-old plants of A. aestivus collected from a hill about 25 km southwest of Larissa, Thessaly, Central Greece. Asphodel semi-deserts in Thessaly ( Fig. 1) occupy an area of about 10.000 ha which is gradually expanding due to overgrazing, frequent fires and soil erosion (Pantis & Margaris 1988).
Floral parts (Fig. 2) were fixed with 2.5% glutaraldehyde and 2% paraformaldehyde in 0.05 M cacodylate buffer for 3 h. After post-fixation in 2% osmium tetroxide and dehydration in an ethanol series, the tissue was embedded in Spurr's epoxy resin. Semi-thin sections of 0.5-1.0 µm thickness from resin embedded tissue were stained with 0.5% toluidine blue in 5% borax for preliminary light microscope (LM) observations. Ultrathin sections (0.08 µm) were obtained in a Reichert-Jung Ultracut E ultramicrotome and examined using a Zeiss 9 S-2 transmission electron microscope (TEM).
For scanning electron microscopy (SEM), specimens were fixed in cacodylate-buffered glutaraldehyde without any osmium post fixation. After dehydration in an ethanol series (10-100%), specimens were critical-point dried with liquid CO2 as an intermediate and coated with gold in a CS 100 Sputter Coater. Observations were made using a BS-340 Tesla scanning electron microscope at various accelerating potentials.
To stain lipophilic substances, semi-thin sections (1-2 µm) of fixed material or hand cut sections of fresh flowers were stained with 1% Sudan Black B (Bronner 1975) or 2% osmium tetroxide (Molisch 1923), respectively. For the identification of phenolic compounds which, as artifacts, also have a positive reaction to Sudan Black B, the following histochemical reagents were applied on fresh hand cut-sections. A) DMB reagent (0.5% solution of 3,4 dimethoxybenzaldehyde in 9% HCl). This forms a red reaction product with condensed tannin precursors (Mace & Howell 1974). B) Millon's reagents as modified by Bakker (1956). With this stain, coloured nitrosoderivatives of any phenols become evident (Sawidis 1991(Sawidis , 1998. For polysaccharide staining, semithin sections of fixed or fresh material were treated with the periodic acid-Schiff's reagent (PAS) according to Nevalainen et al. (1972). Ultrathin sections were treated with periodic acid-thiosemicarbazide silver proteinate (PA-TCH-SP), according to Thiery (1967).

Floral morphology
The flower of Asphodelus aestivus consists of a heavily sclerified calyx of six elongated white petals, six conspicuously long stamens with orange anthers and a central gynoecium with superior ovary (Fig. 2). The six fertile androecial members of the perianth (stamens) are organized into two whorls (3+3) and are free of each other. The outer line of stamens is slightly shorter than the inner one. The bases of the yellowish green stamen filaments are wide and coalesce to form a cavity where nectar accumulates. At this basal region, the epidermal cells become larger and stretch outwards forming numerous papillae (osmophores) (Figs. 3-4). The lower most and uppermost papillae often appear shrunk. The light green trilocular ovary has isomerous carpels, the lateral faces of which are united by fusion with one another (syncarpous gynoecium) (Fig. 5). In the middle of the ovary a hole connecting the septal slid and the outer space exists (Fig. 6).

Nectary
In the cross section of the syncarpous gynecium, the three carpels appear separated from each other by distinct septal slits. In these slits the floral nectary is developed (Fig. 7). The three septal slits proceed downwards entering the ascidiate zone of the carpels. The epider-Figs 3-6. 3 -Hand section at the base of the stamens. The margins of the filaments bear papillae (osmophore) ×300; 4 -Scanning electron micrograph of the osmophore papillae at the filament basis ×300; 5 -The ovary is formed by fussion of three carpels (syncarpous gynoecium). At the middle point of carpel fussion, a small hole occurs (arrow) from which the nectar is released ×90; 6 -Higher magnification of an ovary hole ×300. Hand section of the base of the stamens. Papillae located at the margins of the filament. mal cells (epithelial cells) of the septal nectary contain numerous organelles within a granular cytoplasm (Fig. 8). At the stage of maximal development, the secretory epithelial cells contain many mitochondria. The endoplasmic reticulum (ER) is well developed and occurs as long strands of parallel cisternae (Fig. 10). Golgi bodies are also prominent. The cuticle lining the secretory cells is uniformly thin and electron opaque.
The nectary is supported by the parenchymatic septal tissue, which consists of about 10 layers of relatively large parenchyma cells. Parenchyma cells are connected to the nectary cells with plasmodesmata grouped in pit fields (Fig. 10). Plastids with large starch grains, especially at the initial developmental stages of the nectary, are very common in the parenchymatic cells (Fig. 9). Later, during nectar secretion, starch content drastically diminishes. Two days after anthesis, the secretory cells of the nectary appear to have large vacuoles, many of which contain dark bodies. The nectar starts being secreted by the epithelial secretory cells and accumulates in a space between the fusioned ovary carpels (slits). At that space, a hole exists in the ovary wall through which the nectar is released to the outside (Fig. 6).

Obturator
In the ovary of A. aestivus there are several ovules per carpel and placentation is axile. Between the lateral ovule and the ovary septa a central protrusion devel-ops into a gland -the obturator (Figs. 11-12). It is a prominent ovary wall outgrowth of placental origin, which lies in close contact with the micropyle of each ovule. The obturator is a mucilage gland, which gives a weak reaction (Schiff's reagent) at the initial developmental stages when starch grains of parenchyma cells are intensively red (Fig. 11) and an intense reaction during pollination when starch has disappeared from the plastids (Fig. 12). In the secretory cells of the obturator, cisternal elements of ER in parallel arrangement are widespread occupying a considerable fraction of cell volume (Fig. 13). Among ER cisternae, large vesicles with granular content occur.
Golgi bodies are also prominent and consist of stacks of three to four cisternae. Mucilage is usually secreted by the Golgi apparatus and becomes processed by the ER (mucoprotein). The cells of the gland are internally surrounded by an extraplasmic space filled with the mucilage (Fig. 14). This space progressively increases in volume towards the centre of the cells. In EM, mucilage can be detected after polysaccharide staining with the Thiery-reaction. A fine granular silver deposition reveals the fibrillar nature of the mucilage (Fig. 15).

Discussion
Flowers of all species of Asphodelus are very attractive to pollinators since they produce large amounts of nectar (Cruden 1977;Diaz Lifante 1996). Some envi- ronmental factors, like drought, may affect the amount and viscosity of nectar influencing the visiting of pollinators (Harder 1986). One of the prominent features of the Mediterranean climate is its periodicity, to which A. aestivus is adapted by synchronizing its annual biological cycle (Pantis et al. 1994). In A. aestivus, nectar production is supported by carbohydrate storing at the below-ground parts (Sawidis et al. 2005). Changes of the nectary colour provide insects with a signal that no reward is offered by a particular flower, thus forcing the insect to seek reward elsewhere. The maximum offer takes place during the morning, in a day of normal activity of insect visitors. The "large bee-dish shaped blossom" morphology of the A. aestivus flowers, as defined by Kugler (1977), allows the access of insects of a very wide range of size.

Nectary
The septal nectary is the most common type of nectary in monocots (Vogel 1998;Staufer et al. 2002). The septal nectary of A. aestivus matches the common model of a triradiate cavity (lined by a secretory epithelium) formed by the incomplete fusion of the carpel flanks. The nectar is released to the outside through small holes (one hole per chamber) on the ovary walls and accumulates in the base of the perianth tube. As to the manner of cellular secretion, the general literary consideration is that the type of granulocrine secretion is characterized by an abundance of active ER, mitochondria and Golgi. Eccrine secretion, on the other hand, is characterized by relatively few ER elements and Golgi bodies but numerous plastids (Fahn 1990). In the nectary of A. aestivus, the activity of the extensive ER, the high number of mitochondria and the presence of numerous vesicles in the Golgi cisternae suggest that granulocrine secretion is the mode of nectar secretion in this species. Few starch grains are present in the plastids of the nectariferous cells, a fact implying that starch reserves contribute little to nectar secretion. Thus the bulk of nectar precursors come from the subnectarian parenchymatic tissue and the phloem of the vascular bundles.

Osmophores
The six closely packed stamens surrounding the ovary are in the proximity of the nectar-releasing area. At the stamen basis there are numerous papillae, which form a sort of "barrier" to the outer environment preventing water evaporation from the nectar (Loew & Kirschner 1911;Kugler 1977). Nectar water loss leads to an exponential increase in viscosity, which makes nectar collection by pollinators problematic (Manetas & Petropoulou 2000). The papillae, located at the bottom of the perianth tube, presumably belong to osmophores, which are floral organs for the manufacture, secretion Figs 11-15. 11 -Section of the obturator papillae at the bottom of the perianth tube. The subglandular tissue contains many starch grains stained with the Schiff's reagent at the time before nectar secretion ×250; 12 -Intense reaction of obturator papillae to the Schiff's reagent at the time after nectar secretion. Plastid starch is completely absent in comparison to Fig. 11, ×200; 13 -Tip region of an obturator papillae cell. ER cisternae and large vesicles occupy a considerable fraction of the cell volume. Vacuoles often contain myelin-like structures ×22000; 14 -Large mucilage accumulations in the extraplasmatic space between the plasmalemma and the cell wall (asterisk) of the obturator papillar cell ×15.000; 15 -Fibrillar mucilage material after polysaccharide staining with the Thiery-reaction ×20000. and dispersion of a scent (Vogel 1990;Nilson 2000). The various types of osmophore scents (terpenes) are highly volatile and their compounds are mainly short-chained aliphatic aldehydes and alcohols (Vogel 2000).

Obturator
The obturator (mucilage gland) located between the lateral ovule and the ovary septa, which gives a positive reaction after employing the Schiff's reagent, secretes a mucoproteinaceous product. Within the gland cells, many dictyosomes with large vesicles and prominent ER elements reveal a secretory activity. The obturator is a placental protuberance at the ovary entrance connecting the transmitting tissue with the ovarian cavity (Tilton & Horner 1980;Tilton et al. 1984;Herrero 1992). It contributes to the fertilization process by se-creting different components involved in the growth of the pollen tubes just before they penetrate the micropyle or the control of the direction of pollen tube growth (Tilton et al. 1984;Cheung 1996 ). Pollen tubes first stop at the obturator, which lines the pollen tube pathway towards the ovule (Herrero 2000). This placenta outgrowth forms a bridge between the micropyle and the pollen transmitting tissue. Pollen tubes must grow through the mucilage-filled intercellular spaces across the obturator before reaching the micropyle of an ovule (Webb & Williams 1988;Clifford & Sedgley 1993, Ciampolini et al. 1995Weber & Frosch 1995;Cheung 1996).
At the stage when the gland becomes functionally active in mucilage secretions numerous ER cisternae, many dictyosomes and mitochondria appear. Golgi bodies become associated with ER elements and bud off vesicles with a mucoproteinaceous content. The vesicles subsequently move to cell periphery, fuse with the plasmalemma and release their contents into the extraplasmic space between the cell wall and the plasmalemma. This space progressively increases in volume at the expense of the protoplast. The above pattern of secretion has been also observed in other mucilage glands (Lynch & Staechelin 1995;Western et al. 2000).