The Root-Tuber Anatomy of Asphodelus Aestivus$ Thomas Sawidisã, Soﬁa Kalyva, Stylianos Delivopoulos

ARTICLE IN PRESS

Flora 200 (2005) 332–338 www.elsevier.de/ﬂora

The root-tuber anatomy of Asphodelus aestivus$ Thomas SawidisÃ, Soﬁa Kalyva, Stylianos Delivopoulos

Department of Botany, University of Thessaloniki, GR-54006 Thessaloniki, Macedonia, Greece

Received 5 January 2004; accepted 8 October 2004

Abstract

The root tubers of Asphodelus aestivus consist mostly of enlarged fleshy storage tissue. They are bounded by a multiple-layered velamen, responsible for rapid water uptake, water loss reduction, osmotic and mechanical protection. In the cortex area, thin-walled idioblasts contain numerous raphides of calcium oxalate in their large vacuole with a distinctive tonoplast. Wide morphological variations are observed among the raphide cross sections. Electron-dense compounds penetrate the raphide surface and raphide groove. Raphides seem to be vital for the protection of the root tuber parenchyma from herbivores. The cells of uniseriate endodermis are heavily thickened possessing a few plasmodesmata. The vascular cylinder is 20–28-arch and the root xylem consists of vessels in short radial rows, alternating with clusters of phloem cells. The presence of cells, which contain soluble polysaccharides in their large vacuole, is conspicuous after employing the Schiff’s reagent. Exodermis cells and all cell walls, especially the thick ones of the endodermis, are also stained. Numerous parenchyma cells, especially those around vascular bundles are stained intensely, when exposed to Sudan Black B. These cells occurring as solitary idioblasts abundantly accumulate oil. Electron-dense remnants are evident within the vacuoles of the storage cells especially near the vascular tissue. The morphological features of A. aestivus root tubers and the major part of the total plant biomass are responsible for the species’ occurrence and frequent dominance in a wide array of arid environments. A. aestivus possessing root tubers is proved to be very efficient in storing water during the long summer drought, less susceptible to climatic stress and well synchronized with the climatic fluctuations of the Mediterranean environment. r 2005 Elsevier GmbH. All rights reserved.

Keywords: Mediterranean climate; Geophyte; Asphodelus aestivus; Root tubers; Raphides

Introduction the calcareous soils of pastures and grasslands, particu- larly abundant along road sides. It is found both in arid Asphodelus aestivus Brot. (A. microcarpus Viv.) is a and semi-arid Mediterranean ecosystems (Margaris, perennial tuberous geophyte of the family Asphodela- 1984) and in certain regions of North Africa (Le ceae (Asparagales), widely distributed over the Medi- Houerou, 1979; Naveh, 1973). These ecosystems, very terranean basin (Diaz Lifante, 1996; Polunin and often referred to as ‘‘asphodel deserts’’, are character- Huxley, 1965). It is a widespread and invasive weed of ized as the final degradation stages in the Mediterranean regions (Ayyad, 1976; Ayyad and Hilmy, 1974; Le Houerou, 1981). According to the epics of Homerus, the $Dedicated to Prof. Dr. Georg Heinrich, University of Graz, Institut fu¨ r Pflanzenphysiologie, on the occasion of his retirement. souls of the dead first arrived in underground meadows ÃCorresponding author. (Asphodelos leimon) on which only asphodels bloomed E-mail address: [email protected] (T. Sawidis). (Odyssey, XI, 539, 573, XXIV, 13).

A. aestivus, as is the case of other geophytes A. aestivus and other geophytes are fragmentary. The (cryptophytes), has a considerable importance for ultimate goal is to identify the fine structure and vegetation as it has become the dominant life form in function of these organs and thus to explain the some degraded Mediterranean ecosystems. These for- remarkable success and abundance of A. aestivus in mations are found on hill sites interspaced among the Mediterranean region. More specifically, the plant’s cultivated areas and are used as lowland pastures. In adaptive strategy in the arid environment, the efficiency Thessalia plain (Central Greece) ‘‘asphodel deserts’’ are in water and nutrient storing during the long summer dominated by A. aestivus, which participates in the drought and its defense mechanisms against herbivores floristic composition and covers 10,000 ha. There are shall be elucidated. indications that these areas are gradually increasing in size due to overgrazing, frequent fires and soil erosion Materials and methods (Pantis and Margaris, 1988; Pantis and Mardiris, 1992). A. aestivus has two major phenological phases within A. aestivus plants were collected from a hill about a year. An active one (autumn–late spring) from leaf 25 km south-west of Larissa, Thessalia, Central Greece. emergence to the senescence of the above-ground Root tuber segments were fixed in 2.5% glutaraldehyde structures (photosynthetic period) and an inactive and 2% paraformaldehyde in 0.05 M cacodylate buffer (summer) phase (dormancy), which lasts until the for 2 h. After dehydration in an ethanol series and post- leaves emerge (Aschmann, 1973; Pantis et al., 1994). In fixation in 1% osmium tetroxide, the tissue was the Mediterranean region, geophytes as represented embedded in Spurr’s epoxy resin. Semi-thin sections of A. aestivus in a prominent way by and become the 0.5–1.0 mm thickness from resin embedded tissue were dominant life form in areas degraded by overgrazing heat-fixed to glass slides and stained with 0.5% toluidine and fire. The ability of A. aestivus, a native floristic blue in 5% borax for preliminary light microscope (LM) element, to spread and dominate in all those areas, observation. Ultrathin sections (0.08 mm) were examined reflects its capacity to face not only the peculiarities using a Zeiss 9 S-2 transmission electron microscope of the Mediterranean climate, but also to resist these (TEM). most common disturbances in its habitat (Pantis and To stain lipophilic substances, thick sections (1–2 mm) Margaris, 1988). of fixed material or hand cut sections of fresh root A. aestivus The over-wintering root tubers of develop tubers were stained with 1% Sudan Black B (Bronner, flat leaves (40–90 cm long Â 2–4 cm width) from a shoot 1975) and 2% osmium tetroxide (Molisch, 1923), apex, in February–March and flowering stalks respectively. For the identification of phenolic com- (70–170 cm tall) bearing between 60 and 200 flowers in pounds which, as artifacts, also have a positive reaction April–May. The leaves are protected from herbivores by to Sudan Black B, the following histochemical reagents steroid saponins (Dahlgren et al., 1985) and senesce in were applied on fresh hand cut-sections: (a) DMB June, before fruit maturation. The root tubers show reagent (0.5% solution of 3,4 dimethoxybenzaldehyde in lateral growth, and vegetative propagation is frequent in 9% HCl). This forms a red reaction product with mature plants. However, most of the root tubers remain condensed tannin precursors (Mace and Howell, 1974). attached to the mother plant. A. aestivus is a sessile (b) Millon’s reagents as modified by Bakker (1956). organism reproducing by means of root tubers as well as With this stain, colored nitrosoderivatives of any by seeds. These facts are of considerable importance as phenols become evident (Sawidis, 1991, 1998). For far as maintenance and even dominance of A. aestivus polysaccharide staining, sections of fixed or fresh within degraded areas are concerned. material were treated with the periodic acid-Schiff’s The root tubers, dried and boiled in water, yield a reaction (PAS) according to Nevalainen et al. (1972) and mucilaginous matter which in some countries, is mixed examined with LM. with grain or potato to make Asphodel bread. In Spain and other countries, they are used as cattle and especially for sheep fodder. In Persia, a strong glue is made with the root tubers, which are first dried, Results pulverized and then mixed with cold water. Under the term ‘‘Tsinisse’’ the root tubers of Asphodelus bulbosus Root tuber morphology were used in eastern countries as a mucilage and to adulterate powdered salep. The root tubers of A. aestivus are up to 12 cm long Given the importance of A. aestivus as a consistent and 4 cm thick. They have unlimited growth upwards, component of the Mediterranean vegetation and its while the lower part breaks down. The age of the living dominance over wide areas, we have undertaken the part of a root tuber can be determined by the number present study focusing on features of the root tubers of thickenings. The above-ground structures (inflores- of the plant. Still, reports of root tuber anatomy in cence stalks and fleshy leaves) are completely dry by ARTICLE IN PRESS 334 T. Sawidis et al. / Flora 200 (2005) 332–338

peculiar membraneous configurations (Fig. 4). Electron- dense, amorphous material of dead fungi and fungal pelotons appear around the outer surface cells (Fig. 5). Exodermal cells, regularly thin-walled throughout, polygonal and isodiametric sometimes tend to be anticlinally orientated. Many of these cells are fre- quently smaller than the velamen cells and often nucleated. The root tuber cortex is more or less 30 cells wide at the central cross section. Cells are circular to oval, thin walled, with triangular intercellular spaces. Large water- storage cells are present. Cortical cells subjacent to the exodermis are smaller, polygonal, isodiametric and lack intercellular spaces. Unmodified, thin-walled crystal- liferous idioblasts containing raphide bundles are present among ordinary cortical cells. It is somewhat difficult to illustrate, but under polarized microscopy, the material of these cells reflects the light (Fig. 6). These crystals are stored rather passively in larger vacuoles with a well distinctive tonoplast (Fig. 7). Raphides appear in packs and wide morphological variations are observed among the cross sections (Fig. 8). Some electron-dense substances penetrate the raphide surface and raphide grooves. The cells of the uniseriate endodermis are periclinally orientated and the cell walls are heavily thickened. The thin wall faces the cortex side while the thick one the central cylinder (Fig. 9). These walls refract polarized light and differ from the neighboring thin-walled Fig. 1–5. Cell wall of storage tissue revealing densely packed parenchyma cells. Endodermis cells possess a few zig-zag-like folding after dehydration during the summer plasmodesmata showing a bulge on their inner side period ( Â 10,000). Thick-walled velamen epidermis cells with- (Fig. 10), especially at the stage of leaf emergence and out cuticle on the outer surface ( Â 2500). Root tuber cross inflorescence stalks. section showing the cortex area between velamen and central cylinder ( Â 160). Myelin-like structures in a velamen epidermis The pericycle is uniseriate, while cells are periclinally cell ( Â 14,000). Amorphous material on the extracellular space orientated and isodiametric. The root xylem of the of the velamen epidermis ( Â 20,000). vascular cylinder is 20–28 arch. It consists of vessels in short radial rows, alternating with broadly elliptical to variable shaped clusters of phloem cells. The vascular tissue is surrounded by sharply differentiated, somewhat June, and only the root tubers survive the dry summer thick-walled, polygonal parenchyma cells. Sclerenchyma (dormancy). In the morphology of dormant (summer) cells are also present (Fig. 11). Vessel end-walls are root tubers indications of intense shrinkage are clearly mostly simple or scalariform. The pith is parenchyma- evident macroscopically in comparison to those of tous comprising oval and almost circular, thin-walled photosynthetic period (autumn–spring). This shrinkage cells with triangular, square and rectancular intercellular is also visible at LM level for the reason that the cell spaces. walls of storage tissue strongly fall back (Fig. 1).

Histochemistry Root tuber anatomy After employing the Schiff’s reagent the presence of Root tubers are covered by a multiple-layered vela- cells with a polysaccharidic content is more conspic- men, an epidermal system 4–6 cells wide. The velamen uous. In hand sections, the red color intensity of the epidermis is usually uniseriate and the cells are usually cortex cells is variable and no layered appearance of the devoid of cuticle. Velamen cells are thick-walled and content has been observed (Fig. 12). These cells contain sometimes single-celled hairs occur (Figs. 2 and 3). The soluble polysaccharides in their large vacuole, which in epidermis cells contain myelin-like structures and other some cases is more clearly seen after being plasmolysed ARTICLE IN PRESS T. Sawidis et al. / Flora 200 (2005) 332–338 335

Fig. 6–11. Root tuber cross-section. Cortex idioblast contain- Fig. 12–17. Polysaccharide staining (red; dark in B&W ing densely packed raphides. Staining safranin (green color), reproduction) in root tuber hand section ( Â 160). Polysac- polarization optics ( Â 3500). Raphides stored in a large charitic content (electronic version: red/paper version: dark vacuole of a cortex idioblast ( Â 12,000). Raphides in cross grey) in a plasmolysed vacuole of a root tuber cortex cell section penetrated by electron-dense material ( Â 19,000). ( Â 250). Exodermis cells, positively reacted to Schiff’ s reagent Endodermis cell with heavily thickened cell walls at the side (electr. vers.: red–paper vers.: dark grey) ( Â 160). Stained cell of central cylinder ( Â 2600). Details of Fig. 9 ( Â 15,000). Root walls of root tuber cortex cells. Some of them tangentially cut tuber central cylinder. Sclerenchymatous tissue. Sudan Black B ( Â 250). Endodermis cell walls, stained intensely with Schiff’ s negative staining ( Â 400). reagent ( Â 3500). Staining of xylem vessels at the upper part of the root tuber (dark grey in paper version) ( Â 160).

(Fig. 13). All exodermis cells, below the velamen, intensely react to polysaccharide staining (Fig. 14). not differ greatly by their size from neighboring cells The extracellular space between the positively reacting (Figs. 18 and 19). In the central cylinder the oil cells are and neighboring cells is not penetrated. The positively located around vascular bundles and sometimes are reacting cells typically do not have a suberized wall associated with terminal veins. These oil cells accumu- layer. The cell walls are also stained which becomes late abundant oil insoluble in water. At the stage of leaf more visible when the cell walls are cut tangentially emergence (active phase) the oil cells are very large, oval (Fig. 15). The thick cell walls of the endodermis cells are to circular in sectional view. The oil idioblasts are more conspicuously stained (Fig. 16). Large vessels rounded, almost devoid of cytoplasmic content. Plastids especially near the upper part of the root tuber, where are intact, but most of them appear to have degenerated, the xylem is more concentrated, are also red stained presumably giving rise to vacuoles. All oil cells possess a (Fig. 17). few plasmodesmata showing a bulge on their inner side. When semithin or hand cut sections are exposed Fresh material tested in 2% osmium tetroxide, also to Sudan Black B, numerous parenchyma cells are show the typical reaction of lipophilic substances intensely stained brown to black. In cortex, oil cells whereas both polyphenolic stains applied to sections of occur sporadically as solitary idioblasts and they do fresh material react negatively (with the exception of the ARTICLE IN PRESS 336 T. Sawidis et al. / Flora 200 (2005) 332–338

air. When it rains, however, the velamen cells become filled with water acting as absorptive tissue. The root tubers of A. aestivus explore shallow soil horizons (10–20 cm in depth). The root system is likely to be vulnerable to dehydration, in the upper soil profile, as witnessed by a visible shrinkage of older portions of root tubers. Given the maintenance of root apical turgor, this shrinkage probably reflects a hydraulic effect with water moving (Matyssek et al., 1991). The presence of a velamen is an adaptive strategy in the arid Mediterra- nean region, aiming to use up short seasonal rainfalls. There are many anatomical similarities between root tubers of A. aestivus and storage roots of Orchidaceae (Stern and Judd, 2001). A well-known example of a multi-layered root epidermis is the velamen of perennat- ing storage organs (air roots) of some Orchidaceae (terrestrial, epiphytic or mycoparasitic) and epiphytic Araceae species (Burr and Barthlott, 1991; Dahlgren and Clifford, 1982; Porembski and Barthlott, 1988; Pridgeon and Chase, 1995). A root velamen (velamen radicum) has also been reported in other families such as Cyperaceae and Velloziaceae (Poremski and Barthlott, 1995). The velamen structure of some taxa is associated with mycorrhiza (Pridgeon and Chase, 1995). In the case of A. aestivus uptake of nitrogen by the root tubers during the summer period might be the result of a mutual plant–fungus interaction. Fig. 18–21. Oil cells in the root tuber cortex stained brown to black with Sudan Black B. Fig. 18 ( Â 160), Fig. 19 ( Â 400). Oil cells in the root tuber cortex stained brown to black with Water economy Sudan Black B. Fig. 18 ( Â 160), Fig. 19 ( Â 400). Large variety of vacuolar content in central cylinder parenchyma cells, One of the prominent features of the Mediterranean especially near the vascular tissue ( Â 250). Large variety of climate is its periodicity, to which A. aestivus has vacuolar content in central cylinder parenchyma cells, responded by synchronizing the annual development of especially near the vascular tissue ( Â 250). its biological cycle. As stated by Evans et al. (1992), survival during drought is ultimately dependent on the maintenance of cell turgor. Still the root system is outer epidermis of the velamen). After conventional likely to be vulnerable to dehydration, in the upper fixation, vacuoles of oil cells show a greater variety of soil profile (10 cm in depth), as witnessed by a visible deposits (Fig. 20). Storage oil cells display enhanced shrinkage of older portions of the dahlia-like tuberous electron-density. Sometimes a few osmiophilic droplets roots. Given the maintenance of root apical turgor, or translucent vacuoles occur. Electron-dense remnants this shrinkage probably reflects a hydraulic effect are clearly seen within the vacuoles of storage cells, with water moving from the non-growing (upper) especially near the vascular tissue (Fig. 21). Grouped oil to the growing (deeper) parts (Matyssek et al., 1991). droplets are also detected in the cytoplasm of oil cells. Long before senescence, photosynthetic products are translocated to the below ground system, where they are stored. A. aestivus proves to be very efficient in storing water Discussion during the long summer drought. When values of the water content in the upper part of the soil profile vary Velamen around zero, the root tubers remain hydrated and turgid with relative water content higher than 70% (Pantis, Velamen is a multiseriate epidermal system respon- 1987). The root tubers are less susceptible to climatic sible for rapid water uptake, water loss reduction, stress and constitute a rather energetically stable system. osmotic and mechanical protection (Guttenberg, 1968; When the above-ground structures (influorescence stalks Fahn, 1990). During dry weather the cells are filled with and leaves) are completely dry by June, only the root ARTICLE IN PRESS T. Sawidis et al. / Flora 200 (2005) 332–338 337 tubers survive the dry summer (dormancy). Hence, this Storage tissue adaptation synchronizes the plant’s phenological development with the seasonality of the unstable Mediterra- Root tuber content of starch, lipids and soluble sugars nean climate (Grime, 1979; Meletiou-Christou et al., varies considerably over the year. The amount of 1992; Muller, 1979; Pantis, 1993). Analogous variations polysaccharides as well as total sugar contents, are in the above adaptation are reported for other geophytic always higher in root tubers than in leaves. The highest perennials from Japan (Kawano, 1975; Kawano et al., values of soluble sugars appear in root tubers in late 1982). spring–early summer (Meletiou-Christou et al., 1992). Since A. aestivus root tuber biomass is 6- to 30-fold higher than that of the above ground plant parts (Pantis, Raphides 1993), it can be concluded that the below ground part consists of a rather stable energy reserve under Many plant cells contain crystalline inclusions of continuous replenishment. Indeed, in case of A. aestivus, different chemical composition and shape. Bundles of the greatest percentage of allocated biomass and needle-shaped crystals are termed raphides (from the nutrients is located in the tubers during the whole year greek raphis ¼ needle). Calcium oxalate needles in cells (Meletiou-Christou et al., 1992). Changes of below- and of A. aestivus root tubers are typical for raphide bundles above-ground biomass allocation synchronize the found in monocots. Chemical composition, degree of plant’s phenological development with the seasonality crystallization and formation of the raphid bundles are of the Mediterranean climate. variable. The observed differences in the morphological features of raphide cross sections are caused probably due to various crystallization degrees, stage of development and penetration depth of electron-dense sub- Conclusion stances. Such morphological differences of raphides could potentially influence the degree of acridity. A. aestivus root tubers play a most important role in Raphides store calcium oxalates as metabolic waste or storing and utilizing water masses and nutrients, by-product of plant tissues. Accumulation of oxalic acid protecting the plant from drought stress and environ- in tissues, which is not readily metabolized, may cause mental hazards as well. Furthermore, they constitute a osmotic problems. Therefore, precipitation of calcium means of the synchronization of the plant with the oxalate crystals seems to be an appropriate way for the seasonality of the Mediterranean climate. Structurally, plant to avoid these undesirable situations. The relation- these functions are based on: (a) the multiseriate ship between calcium ion absorption and oxalic acid epidermis (velamen) enabling quick gain of only synthesis in plants is most probably established for ionic transiently available soil water, (b) the large parenchy- balance in tissues to be maintained (Bosabalidis, 1987). ma water storing cells, (c) the cortex cells containing On the other hand, the calcium content in dormant root soluble sugars, (d) the oil cells containing lipid material tubers may be viewed as an osmoregulatory adaptation of possibly defense character and (e) the cell idioblasts to drought during the dry-warm summer period (El which contain raphide crystals. These anatomical Chonemy et al., 1978; Evans et al., 1992; Levitt, 1980). peculiarities of the A. aestivus root tubers, combined Raphides could also play a role in penetration and with the ability of A. aestivus to avoid grazing, fires and carrying acrid factors on their surfaces and in their the endurance of drought, may explain the species’ grooves, for instance, lipid substances impregnated with frequent dominance in a wide area of arid environments, oxalic acid. The high level of acridity in A. aestivus root from the Mediterranean to the desert. tubers has been associated partly with the sharpness and size of its raphides (Esau, 1965). Irritation by raphides is both mechanical and References chemical. When raphides come into contact with the tender tissues of soil living worms and other herbivores Aschmann, H., 1973. Distribution and peculiarity of Medi- these substances are injected, enter the wounds and can terranean ecosystem. 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