MICROSCOPIC INVESTIGATION OF TRICHOMES OF WILD-GROWING SATUREJA MONTANA L

Considering the importance of S. montana L. as a medicinal plant, due to the essential oils produced in its glandular trichomes, and the lack of data about the leaf ultrastructure and micromorphology, we have carried out a comprehensive study of trichomes distributed in the vegetative and reproductive organs. Micromorphological and ultrastructural investigations of the glandular trichomes of Satureja montana L. and autofluorescence analyses of their secreted products were carried out using scanning electron microscopy (SEM), transmission electron microscopy (TEM), confocal laser scanning electron microscopy (CLSM) and light fluorescence microscope. The leaves, stem and calyx bear numerous nonglandular, unbranched trichomes and two types of glandular trichomes – peltate and capitate. Ultrastructural analyses showed the presence of mitochondria, plastids and proliferations of the granular endoplasmatic reticulum in secretory phases of the heads of glandular trichomes. Strong red autofluorescence of the lipophilic and hydrophilic secreted material was observed with CLSM. Green-yellow autofluorescence of secreted material were noticed in peltate and capitate trichomes observed with a fluorescence microscope.


INTRODUCTION
The Lamiaceae is a large family with wide distribution, particularly in the Mediterranean region.It is rich in aromatic species used as culinary herbs, folk medicines, fragrance, etc.Many species of the family possess essential oils secreted by glandular hairs distributed on their aerial vegetative organs and some on their reproductive organs.The glandular trichomes are the primary secretory organs of these plants, and their structure can vary widely among species (Werker et al., 1985).Numerous studies of the glandular trichomes of Lamiaceae have been carried out, comprising morphological, structural and histochemical analyses of trichomes and the chemical composition of the essential oils (Antunes and Sevinate-Pinto, 1991;Ascensão et al., 1995;Ascensao and Pais, 1998;Bosabalidis, 1990;Marin et al., 2008).
The genus Satureja L. comprises more than 200 species of herbs and shrubs, often aromatic, widely distributed in the Mediterranean region, Asia and boreal America (Satil and Kaya, 2007).Satureja species are used widely as a flavoring agent of food products and as a traditional herbal medicine for the treatment of gastrointestinal disorders.The investigations of the essential oil composition and their antimicrobial activities are numerous, and has been done in some species in Croatia (Bezić et al., 2001;Dunkić et al., 2007;Husain et al., 1990), in Greece (Bosabalidis, 1990), and in Turkey (Dousti et al., 2009;Hadian et al., 2010;Satil et al., 2002), but morphological and ultrastructural studies are rare.Satureja montana L. savory is an aromatic species, used as a spice and as a traditional medicinal plant.It is a shrub typical for the submediterranean region.Vegetative and reproductive organs of S. montana bear numerous glandular and non-glandular trichomes.S. montana has been used in traditional medicine as a general tonic for the body.It may produce strong psychological effects with its energizer and motivational qualities, helping the stimulation of the nervous system.S. montana also has antibacterial, antiviral, antifungal, antiparasitic and immune stimulating effects (Ciani et al., 2000;Ćavar et al., 2008).Due to the presence of phenolic compounds in the essential oil, S. montana is approved for a few pharmacological activities (Bezić et al., 2005).
Considering the significance of the glandular trichomes as the site of essential oil production, we focused our study on their morphological, ultrastructural, confocal and fluorescence investigations.The morphology and distribution of trichomes on the leaves, stem and calyx were investigated using scanning electron microscopy (SEM), while the inner cell structure was analyzed using transmission electron microscopy (TEM).The autofluorescence of the lipophilic and hydrophilic secreted material in the glandular trichomes was investigated with CLSM and fluorescence microscope.

Microscopic analyses
For the micromorphological study, small segments of the leaves, stem and calyx were fixed in glutaraldehyde (3% with buffer solution at pH 7.0).The pieces were subsequently dehydrated in a graded ethanol series and critical point dried, coated with a thin layer of gold (ion sputtering coating) in BALTEC-SCD 005 Sputtering Device, and observations were carried out on a JEOL JSM T 220 15kV scanning electron microscope.
The leaf samples for ultrastructural examination were sectioned in a drop of 2% glutaraldehyde and put into a fixative solution of 2% glutaraldehyde in phosphate buffer (0.1 M, pH =7.0), postfixed in 2% OsO4 and embedded in Araldite.Sections of 1 µm thickness were stained with uranyl and lead citrate and examined with a Philips transmission electron microscope 208 S.
Fresh leaves were examined with CLSM 510 Carl Zeiss with Axioskop FS2mot microscope.Plan-Apochromat 20x /0.75 objective lens were used, and the excitation wavelengths were 488 and 543 nm.
Fresh material was also used for fluorescence analyses using a Leica DM LS fluorescence microscope with a 13 BLU 450-490 nm filter.

Micromorphology
The vegetative and reproductive organs of S. montana are covered by an indumentum containing both nonglandular and glandular trichomes.In this study, we gave most emphasis to the type and distribution of glandular trichomes secreting essential oils.
The leaves of S. montana are covered with a thick cuticle.The unilayered epidermis on the abaxial and adaxial surfaces was observed.Non-glandular and two types of glandular trichomes, peltate and capitate, were found on both leaf sides, as well as on the stem and calyx (Fig. 1 A-E).
The peltate trichomes were located in epidermal depressions on the adaxial and abaxial leaf surfaces and on the outer surface of the calyx teeth (Fig. 1A, 1C, 1E).They consisted of one basal epidermal cell, a    wide stalk cell and a multicellular head consisting of twelve cells (Fig. 1B).
The capitate trichomes were composed of one basal cell, one stalk cell and a unicellular head, and they were more numerous than peltate trichomes (Fig. 1C).
The leaves, stem and calyx bear numerous unicellular papillae and elongated unbranched nonglandular trichomes.These elongated trichomes are especially densely distributed on the stem (Fig. 1D), on the margins of the leaves and inside the calyx (Fig. 1E).

Cell ultrastructure
Using TEM, it was observed in the secretory phase of glandular trichomes that cells contained dense cytoplasm, plastids, numerous mitochondria (M) and large vacuoles (V).The outer cell wall (CW) was covered with a thick cuticle (Cu).On one side, the cytoplasm was limited by the plasma membrane of the cell, and on the other by the tonoplast (T).Plastids and mitochondria were bound by a double membrane.The basic structural unit of the plastid was the thin flattened sacs or lamellar vesicles known as thylakoids (Fig. 2).

Confocal laser scanning electron microscopy
A strong red autofluorescence of the secreted substances could be seen both in the peltate and capitate trichomes, whereas light-green autofluorescence was obtained on the non-glandular trichomes observed with CLSM (Fig. 3).

Fluorescence microscopy
Greenish-yellow autofluorescence on the surface of the leaves and non-glandular trichomes indicated the presence of suberin or cutin.Secretion substances showed a bright green-yellow autofluorescence inside the head of peltate and capitate trichomes (Fig. 4).

DISCUSSION
Trichomes are unicellular or multicellular projections from plant surfaces that originate from the epidermal cells.They are very variable in size and structure and have a number of roles, including chemical defense from fungal, insect and vertebrate pathogens and pests, production of allelochemicals, seed dispersal, water and nutrient acquisition, alluring insects for pollination or insectivory, enzyme secretion, and salt excretion in the halophytes (Werker, 2000).The essential oils in the glandular trichomes play a role in guiding pollinators or in defense against excessive transpiration and solar radiation.The non-glandular trichomes probably collaborate together with the glandular trichomes in the mechanical defense against predators.
The glandular trichomes are thought to be valuable diagnostic characters in plant systematics.In the family Lamiaceae, the morphology, distribution and frequency of glandular trichomes are used as discriminative characters at a subfamily level.Peltate trichomes appear in all species but with a variable number of head cells, ranging from 8 in Mentha x piperita L. (Amelunxen et al., 1969), to 18 in Micromeria fruticosa L. (Werker et al., 1985).This type of gland on Satureja montana consisted of one basal epidermal cell, a stalk cell and a head of twelve secretory cells.
Capitate trichomes are also widespread in Lamiaceae, but they vary more in stalk length and head shape.They generally consist of one to two stalk cells and one to two cells forming a rounded to pearshaped secretory head (Antunes and Servinate-Pinto, 1991;Bosabalidis, 1990;Werker et al., 1985).In S. montana they are composed of one basal cell, a stalk with one cell and an ovate unicellular head.
As in plants of most Lamiaceae species, the surface of S. montana leaves carried glandular and unbranched non-glandular trichomes on both leaf sides, stem and calyx.On the adaxial and abaxial surface, the peltate glandular trichomes were densely distributed and the non-glandular ones were scattered among them.The capitate trichomes were more abundant than peltate trichomes on both leaf sides.Both non-glandular and glandular trichomes had a cuticular covering that was thicker than that of the epidermis, as was found in Salvia blepharophylla (Bisio et al., 1999).Peltate and capitate trichomes differ in their structure, mode and timing of secretion (Werker et al., 1993).Peltate trichomes are the sites of production and storage of essential oils, while capitate trichomes produce and contain polysaccharides and a small quantity of oils.
Results obtained during micromorphological analysis of some other Satureja species, such as Satureja cuneifolia (Bezić et al., 2001) and Satureja thymbra (Bosabalidis, 1990), are in accordance with our results.Similar results for trichome types and distribution were obtained for Satureja species by Husain et al. (1990).Satureja khuzistanica (Dousti et al., 2009) was found to be different in structure of peltate trichomes, which consisted of six to eight cells of head, and in addition to two types of capitate trichomes, conoidal trichomes were noticed.In the comparative study of leaf anatomy and hairs of the genus Satureja represented in Turkey by 15 species, Satil and Kaya (2007) concluded that leaf and trichome characteristics could be useful in taxonomical studies of the genus.
Ultrastructural analyses are important in trichome research because the relations between morphology and cytology, secretion processes and the chemical nature of secreted material can be established.Ultrastructural changes during the secretory phase of glandular trichomes were characterized by a proliferation of endomembrane system (Turner et al., 2000).The high number of mitochondria is connect-ed with the high metabolic activity of the cell (Dunkić et al., 2007).A smooth endoplasmic reticulum (ER), numerous leucoplasts without thylakoids containing starch grains and large osmophilic drops, are characteristic organelles involved in terpene production, while dictyosomes are responsible for polysaccharide production (Cheniclet and Carde, 1985;Dunkić et al., 2007;Huang et al., 2008).A rough ER placed around plastids is involved in the direct transport of essential oil from the plastids to the periplasmatic space (Ascensao and Pais, 1998;Huang et al., 2008) and further by exocytosis in the subcuticular space, while polysaccharides are transported by Golgi vesicles.Confocal and fluorescence analyses confirm that the secreted substances are produced in the peltate and capitate glandular trichomes.
These results could be applied in the studies of medicinal properties, but more detailed chemical investigation needs to be done.In addition, the use of micromorphological and ultrastructural analyses of the glandular trichomes and the constituents of the essential oils could provide useful diagnostic characters and contribute to the solution of the very complex taxonomical problems of the Satureja sensu lato group.

Fig. 3 .
Fig. 3. CLSM Autofluorescence of secreted materials of trichomes of S. montana.Strong red autofluorescence in the peltate and light red in capitate trichomes 40X.