Morphology
Viscum album subsp. album is a semiparasite growing on the branches of deciduous trees such as Malus domestica (Suckow) Borkh. (Fig. 1a). It has several articulated, glabrous and green stems (Fig. 1b, 1c). The leaves are leathery, opposite, simple, oblong, with an obtuse apex, attenuated base, entire margins, with 3–5 parallel veins, green to yellowish-green and measuring 2–6 cm long and 0.3-2 cm wide (Fig. 1b, 1c). The fruit is a pseudo berry, globular, whitish, translucent, sessile, 0.8-1 cm in diameter (Fig. 1d, 1e, 1f), crowned by the remains of the dried stigma and remains of the perigone leaves (Fig. 1e); they contain a seed coated by a skinny endocarp and embedded in a very viscous translucent mesocarp. The berries ripen during autumn before winter solstice, with the exocarp changing from green to white or yellowish-white when ripe. Berry color supports the differentiation of V. album (European mistletoe) from the closely related Viscum coloratum (Kom.) Nakai (Korean mistletoe) of Eastern Asia37.
Leaf anatomy
The surface view of the clarified leaf evidences straight and thin anticlinal cell walls on both adaxial and abaxial epidermises (Fig. 2a,d), covered by a thick cuticle (Fig. 2e). Paracytic stomata (Fig. 2a,d) are located at the same level as the adjacent epidermal cells, and are found on both sides of the lamina (Fig. 2a,b,c,d,f). Epicuticular wax crystalloids were observed on both epidermises (Fig. 2f).
Anatomical features such as epidermis with straight anticlinal cell walls, amphistomatic leaves, the presence of paracytic stomata, epicuticular wax crystalloids and thick cuticle, are commonly observed in Viscum species38–44. However, anomocytic stomata were evidenced in V. album subsp. austriacum. This subspecies also evidenced glandular trichomes on the adaxial side of the leaves44; this feature was not observed in the present study. The type of stomata and trichomes are might be used as anatomical markers of V. album subsp. austriacum.
In cross-section, the leaf epidermis is unilayered with strongly cutinized external cell walls on both sides (Fig. 2e,i). Although the leaves are photosynthetic, the mesophyll is undifferentiated and comprises polygonal cells (Fig. 2g,h). Minor collateral vascular bundles are distributed in the mesophyll (Fig. 2h). A dorsiventral mesophyll was found in V. album subsp. album collected from a host plant Tilia cordata Mill.41 and V. album subsp. austriacum growing on Pinus sp.44.
Metcalfe and Chalk38 stated that the mesophyll of the biennial leaves of V. album is formed by isodiametric cells during the first year, yet a single layer of palisade parenchyma develops towards both sides in the second year. From May until August the shoots of V. album have both types of leaves: in the first and in the second year of growth, as can be seen in Fig. 1c.
The mesophyll cells have several starch grains (Fig. 2i,k). They are small, rounded or elongated, and found solitarily or in groups. The presence of starch grains has been reported in various species and subspecies of Viscum44. Also, different morphotypes of crystals are distributed in the mesophyll (Fig. 2i) and in the vascular bundle (Fig. 2j). They are rectangular prisms, druses (Fig. 2l), platy aggregations of cluster crystals (Fig. 2m), and cubic-shaped crystals (Fig. 2n). Under light microscopy, the druses have a central thick and black region surrounded by many polygonal small crystals (Fig. 2i,j). While cubical, prismatic and druse crystals were observed in the mesophyll of V. album subsp. album in the present study, only druses were reported in V. album subsp. golestanicum43. The presence of crystals is commonly reported in various species and subspecies of Viscum41,42,45. However, no crystals were reported in a previous study of V. album subsp. album by Khan et al.42. Also, the presence of platy aggregation cluster crystals in V. album subsp. album is reported here for the first time. The grouping of more than one crystal morphotype can be present as the feature of the subspecies, species, section, subgenus or genus, giving support to the taxonomy46.
The midrib is flat on both sides (Fig. 2g). The vascular system is represented by a central collateral vascular bundle (Fig. 2g). Fibers are abutting the xylem and phloem (Fig. 2g,j). Bicollateral vascular bundles were found in V. album and in V. cruciatum Sieber ex Boiss. In a study by Khan et al.42.
The strongly shortened and weakly differentiated petiole of V. album subsp. album, sectioned transversely at the medial portion, had a concave-convex shape with two wings on adaxial side (Fig. 3a). The epidermal cells are covered by a thick cuticle (Fig. 3b). The ground tissue is undifferentiated as in the lamina. The vascular system is collateral and was represented by five bundles organized in an open arc (Fig. 3a). A cap of perivascular fiber adjoins the xylem and phloem (Fig. 3c). The same crystal morphotypes previously described for lamina were observed in the ground parenchyma (Fig. 3b). However, Khan et al.42 have not reported any crystal in the petiole of V. album. Although the outline of the petiole is a general diagnostic feature for higher plants47, there are only few studies involving the anatomy of the petiole of Viscum species. The same features were found in V. album subsp. album and V. album subsp. golestanicum43. However, 6–7 vascular bundles were observed in V. album and V. cruciatum in the study by Khan et al.42.
Stem anatomy
The stem is circular in cross-section (Fig. 3d). The epidermis is unilayered with tangentially elongated cells (Fig. 3g). The external wall is particularly thick and fully cutinized (Fig. 3g). Over each external wall, in the middle of the cell, a projecting monticule or papilla, half-moon or elliptical shaped, could be observed (Fig. 3e,g). The cortex is discreetly collenchymatous and formed by several layers of parenchyma cells that increase in size towards the vascular system (Fig. 3e). The vascular system is represented by nine vascular bundles forming a ring and delimiting the pith (Fig. 3d). The row and number of cells in the xylem are higher than in the phloem (Fig. 3e). One cap of perivascular fibers is attached to the xylem and another to the phloem (Fig. 3d,e,f,i), the latter being more developed. The pith is at the center of the stem and is composed of parenchyma cells (Fig. 3d). Idioblasts containing brownish substances corresponding to phenolic compounds are present in the phloem (Fig. 3h). Cortical, vascular and medullary parenchyma are filled with starch grains (Fig. 3g,h,i,j,k) with the same features previously described for leaves; however, these are not present in the first layers of the cortex.
Druses (Fig. 3i,j,l), cubic and quadrangular prismatic (Fig. 3m) crystals are also spread in the stem tissues. Abundant crystals are present in the cortex and in the phloem of V. cruciatum, yet are absent in V. album in a study of Khan et al.42. Mehrvarz et al.43 have reported that the distribution and morphotype of the calcium oxalate crystals could provide valuable support in delimitation of subspecific taxa in V. album. The use of calcium oxalate crystals in solving taxonomic problems has been suggested in previous studies of other plant groups such as Baccharis46,48, Eucalyptus49 and Piper50.
Most characteristics observed in the present study have been described for Viscum species41–44. The vascular bundles showed poorly developed phloem but well-developed xylem. It means that they can resourcefully take up water and prepared inorganic and organic nutrients from the host plant51.
Berry anatomy
The berry, in frontal view, presents an epidermis with straight anticlinal cell walls. In cross-section, the exocarp is formed by an unilayered epidermis covered by a smooth cuticle and 3–4 layers of angular collenchyma (Fig. 4a,b). In the medial region, small collateral vascular bundles are present (Fig. 4a). The mesocarp, which surrounds the seed, contains an outer fleshy layer and an inner sticky viscin tissue (Fig. 4a,c). The fleshy layer is not sticky.
The viscin tissue is composed of strongly vacuolated globular cells (Fig. 4c,d,e) and highly viscous, sticky and extensible, long cells with spirally thickened walls located towards the endocarp (Fig. 4f). These cells contain druses and rectangular prism crystals, and a few starch grains. The endocarp is thin and encloses the seed. Grazi & Urech52 reported that there is no information of extensible filaments between the seeds and the outer mesocarp in V. album subsp. abietis and V. album subsp. austriacum growing on conifers.
Seed anatomy
The seed is green and does not have a seed coat. Heide-Jørgensen53 stated that in Viscum genus no seed coat is formed since the integuments are lacking. In cross-section, the seed has an unilayered epidermis covered by a cuticle (Fig. 4h). The epidermal cells have several starch grains (Fig. 4h). The endosperm cells are large, parenchymatous and contain chlorophyll and large amounts of starch grains (Fig. 4g,h,i,j). The seed usually has two embryos, each with two cotyledons and a hypocotyl (Fig. 4i,j,k,l). The number of embryos per berry varies in Viscum album. Commonly, V. album subsp. album has two embryos, sometimes only one embryo, and rarely three or even four embryos, while the percentage of monoembryonal berries is higher in subsp. abietis and V. album subsp. austriacum8,40.
Elemental chemical composition of crystals
The chemical composition of the crystals occurring in plants can be identified using EDS54,55. In the present study, four morphotypes of calcium oxalate crystals were found in various tissues of V. album subsp. album (Figs. 2i,j,l,m,n, 3b,i,j,l,m). The EDS spectra of druses (Fig. 5a) exhibited prominent peaks for calcium (Ca), carbon (C), and oxygen (O). However, in addition, other elements such as magnesium (Mg), phosphorous (P), and potassium (K) were also found in minor concentrations in the cubic (Fig. 5c) and rectangular prisms (Fig. 5b), and platy aggregation crystals (Fig. 5d). The most common minerals formed by plants are calcium oxalate, calcium carbonate and silica56. This is the first study of the elemental chemical composition of crystals of Viscum species using EDS.
Histochemical tests
Microchemical analyses using specific reagents and stains under light microscopy are helpful for the characterization of chemical compounds present in plant tissues. They can also be used to detect, within specific cells and tissues, the distribution and accumulation of the compounds or groups of secondary metabolites, such as lipophilic material, protein, mucilage, lignified elements, and phenolic compounds26–36. In the present study, reagents such as Dragendorff, Ellram, Wagner, Sudan III, Sudan black, Nile blue sulfate, potassium dichromate, ferric chloride, hydrochloric vanillin, phloroglucinol/HCl, iodine solution, ruthenium red, PAS, methylene blue, Xylidine Ponceau and Coomassie Brillant were used to evidence specific chemical compounds and groups of secondary metabolites and the results are summarized in Table 1. No histochemical tests were previously reported for V. album subsp. album.
Sudan III stains the lipids red or red-orange while Sudan black gives a dark blue or dark color in the microchemical test32. V. album subsp. album reacts positively with Sudan III and Sudan black, showing lipophilic compounds in cuticles of the leaves (lamina (Fig. 6a) and petiole (Fig. 6b)), stems (Fig. 6c), berries (Fig. 6d) and seeds (Fig. 6e). Oil bodies also react with this reagent46 and are found in the epidermis and mesophyll (Fig. 6a) of the lamina, in the epidermis (Fig. 6b) and ground parenchyma (Fig. 6b) of the petiole, and in the exocarp cells of berries (Fig. 6d). Oil bodies (Fig. 6f) stain with Nile blue as well, indicating that the bodies contain neutral fats. Nile blue is a basic dye in the oxazine group that stains neutral fats and fatty acids in red and blue, respectively57.
Phenolic compounds can be evidenced using different reagents: ferric chloride solution through iron precipitation produces dark brown or black coloration; potassium dichromate solution produces a brown or reddish-brown color; and vanillin-hydrochloric acid gives rise to a bright red vanillin-tannin condensate58. V. album subsp. album reacts positively with potassium dichromate (Fig. 6g,h) and ferric chloride (Fig. 6i,j) solutions, and the cells containing phenolic compounds were found in the vascular bundles of leaves and stems. However, no condensed tannins were found.
Lignified elements can be detected using phloroglucinol/HCl, which stains lignin in the cell walls pink to red. Lignin is a compound present in several or all the secondary wall layers that contribute to the lignification process resulting in the modification of cell wall properties49. In the present study, lignified elements were evidenced in fibers and vessel elements in the leaves and stems (Fig. 6k,l).
Plant polysaccharides are macromolecules comprised of several identical or different monosaccharides with α- or β-glycosidic bonds. In microchemical tests, Schiff reagent is frequently used to distinguish certain mucins and other carbohydrates in a staining sequence called the PAS (periodic acid-Schiff) test. Polysaccharides that comprehend a pair of adjacent hydroxyl groups can be oxidized to aldehydes by periodic acid. The aldehydes react with colorless Schiff reagent and the positive tissue sites become magenta. Neutral polysaccharides are spread in the cell walls in the epidermis, ground parenchyma in the midrib (Fig. 6m), and phloem and pith in the stem (Fig. 6n), exocarp and mesocarp of berry and all the cell walls in the seed, especially near the embryo.
Iodine solution is used to stain starch in dark blue to black. Almost all other structures stain yellow, but this color has no specific meaning. Starch is one of the main ergastic substances of the protoplast and contains a long chain of polysaccharides grouped around a hilum and forming characteristic granules. Starch grains are found in epidermis and mesophyll cells (Fig. 7a) of the lamina and ground parenchyma of the midrib. They are widespread in the cortex, medullary rays, and pith of the stem (Fig. 7b), in some mesocarp cells in the berry (Fig. 7c), and in the endosperm of the seed (Fig. 7d). They are small, rounded and found in groups.
Xylidine Ponceau and Coomassie Brilliant Blue react positively with V. album subsp. album. The presence of protein globular corpuscles is observed occasionally in the leaves (Fig. 7e), stems (Fig. 7f) and berries (Fig. 7g,i). However, they are commonly found in seeds, especially in the endosperm (Fig. 7h,j). The protein globular corpuscles are vacuolated structures that accumulate reserve protein in the seeds and are called protein bodies or aleurone grains.
Ruthenium red is a polycationic stain that reacts with pectic substances, mucilage, and gums59. These substances are detected in almost all cells in the leaves, stems, berries (Fig. 7k) and seeds, yet not in fibers and xylem elements. Azuma et al.60 analyzed the cellulose system of the viscous fibrous cellulosic polysaccharide (viscan) in the viscin tissue of V. album and reported that it is formed by cellulose and hemicellulose together with a minor amount of pectic substance. The viscin tissue assists in attaching the mistletoe berries to the host branches.
Dragendorff, Ellram and Wagner are reagents that stain alkaloids from orange to reddish-brown27,28. These secondary metabolites were not detected in any of the organs of the V. album subsp. album. Methylene blue was used in order to reveal mucilaginous content in the cells, which swell and exhibit a sharper blue color when positive26. In the present study, no mucilaginous contents were detected in V. album subsp. album.
Table 1
Results of microchemical tests with V. album subsp. album. ND: not detected.
Microchemical reagents | Reaction | Occurrence in plant organs |
Leaf | Stem | Berry | Seed |
Lamina | Midrib | Petiole |
Sudan III | Staining lipids red or red-orange | Cuticle and oil bodies (epidermis and mesophyll) | Cuticle and oil bodies (epidermis and ground parenchyma) | Cuticle | Cuticle and oil bodies in the exocarp cells | Cuticle |
Sudan black | Staining lipids black |
Nile blue | Stains neutral fats in red and fatty acids in blue | Oil bodies | Oil bodies | ND | Oil bodies | ND |
Potassium dichromate | Gives phenolics a brown or reddish-brown color | Minor vascular bundles | Vascular bundles | Cortex and vascular bundles | ND |
Ferric chloride | Turns phenolics to dark brown |
Vanillin/HCl | Gives rise to a bright red vanillin-tannin condensate | ND |
Phloroglucinol/HCl | Reveals lignified elements in pink to red color | Fibers and vessel elements | Fibers and vessel elements | Elongate cells | Endocarp cells |
PAS (periodic acid-Schiff) | Neutral polysaccharides become magenta | Epidermis and mesophyll cells | Epidermis, ground parenchyma and phloem | Phloem and pith | Exocarp and mesocarp | All the cell walls, especially near the embryo |
Iodine solution | Stain starch in dark blue to black | Epidermis and mesophyll cells | Ground parenchyma | ND | Epidermis, cortex and pith | Mesocarp (globular cells region) | Endosperm |
Xylidine Ponceau | Reveals protein bodies in red color | Protein globular corpuscles | Endosperm, hypocotyl and embryo |
Coomassie Brilliant Blue | Turns protein bodies blue | Protein globular corpuscles |
Ruthenium red | Reacts with pectins, mucilages and gums turning them pink to red | Epidermis, phloem and mesophyll cells | Epidermis, ground parenchyma and phloem | Epidermis and exocarp cells |
Methylene blue | Stain mucilage in blue | ND |
Dragendorff, Ellram and Wagner | Gives alkaloids an orange to reddish-brown color | ND |