An investigation of the glandular and non-glandular foliar trichomes of Orthosiphon labiatus N.E.Br. [Lamiaceae]

Abstract

Using a combination of light (stereo and compound) and scaning electron microscopy, this study investigates the distribution and morphology of foliar trichomes of Orthosiphon labiatus (Lamiaceae) at different stages of leaf development. Three types of trichomes were observed: two glandular types (large peltate and small capitate) and one non-glandular type. Results revealed that the development of peltate trichomes was initiated in the early stages of leaf development. However, a higher number of developed peltate trichomes was observed in mature leaves, indicating an increase with age. Capitate trichomes are, by contrast, rare and sparsely distributed on both leaf surfaces. The highest density of non-glandular trichomes was observed on the abaxial leaf surface, especially along the veins, with the density decreasing with leaf maturity. This paper contributes towards the body of knowledge being assembled on the family Lamiaceae as it manifests in southern Africa.

Keywords:

• glandular trichomes
• non-glandular trichomes
• oil glands
• hosiphon labiatus
• lamiaceae
• scanning electron microscopy

Introduction

A vast number of angiosperm leaves are characterized by having hair-covered surfaces (Metcalfe & Chalk 1950). Such leaf trichomes have been proposed to have a number of functions. Non-glandular hairs reflect solar radiation off the leaf surface (Liakoura et al. 1999) to prevent heat damage by lowering leaf temperature (Klich 2000). They also decrease rates of water loss and facilitate photosynthetic gas exchange (Schreuder et al. 2001). Glandular trichomes, by contrast, contain or secrete a mixture of primary and secondary metabolites (including terpenes) that have a wide array of applications in pesticides, pharmaceuticals, food flavouring and fragrances (Cousins 1994; Maffei & Codignola 1990). In addition to these industrial uses, glandular trichomes found on some crop species confer resistance against insect pests. They do this by forming physical obstacles and by exuding defensive chemicals (Peter et al. 1995).

The Lamiaceae are well known for their often densely haired aromatic leaves (Metcalfe & Chalk 1950). The presence of both glandular (peltate and capitate) and non-glandular trichomes is a characteristic feature of this family (Maffei & Codignola 1990). The glandular trichomes are the source of aromatic, volatile oils and terpenes. As a consequence, some members of the Lamiaceae, the mint family, have since ancient times played an important role as a source of medicinal and aromatic plants of commercial importance. Mentha species were mentioned in the Code of Hammurabi some 3780 years ago (Bowman & Jordan 2006) and their uses were referred to by ancient scholars such as Pliny (Simpson 2006) and Dioscorides (Barkley 1974). Mint was also used as a form of money in biblical times, and, along with lavender, was widely used in countries surrounding the Mediterranean (Fernie 1897). In modern times, the essential oils of Lamiaceae and the structures that produce them have become a focal point for research worldwide (Ascensão et al. 1999; Ascensão & Pais 1998; Bosabalidis 1990; Bosabalidis & Skoula 1998; Bourett et al. 1994; Combrinck et al. 2007; Corsi & Bottega 1999; Cousins 1994; Gairola et al. 2009; Gang et al. 2001; Kaya et al. 2007; Lakusic et al. 2005; Maffei et al. 1989; Sharma et al. 2003; Werker 2000; Werker et al. 1985 ,1993).

Because it is a genus of medicinal and commercial importance, Orthosiphon Benth. has been chosen for this study (Adam et al. 2009; Awale et al. 2003; Grosvenor et al. 1995; Hossain et al. 2008; Hsu et al. 2010; Hussein et al. 2007; Khamsah et al. 2006; Nguyen et al. 2004; Sriplang et al. 2007; Sumaryono et al. 1991; Yam et al. 2009). Orthosiphon stamineus Benth. [syn. O. aristatus (Blume) Miq.] from Asia is the most important ethnobotanical species in the genus and is commonly known as ‘Java Tea’, misai kuching or kumis kucing. This species is used in the treatment of various diseases, including nephritis, gout, diabetes, hypertension, rheumatism, dysuria and jaundice (Chin et al. 2008; Faizul et al. 2009; Han et al. 2009; Maheswari et al. 2008; Mariam et al. 1996; Ngamrojanavanich et al. 2006; Olah et al. 2003; Ong & Norzalina 1999).

Orthosiphon, a genus related to Ocimum L., encompasses > 40 species found throughout Africa and Asia (Retief 2000). Nine species of Orthosiphon have been reported from southern Africa (Codd 1964 ,1985) and include the African endemic Orthosiphon labiatus N.E.Br.; commonly known as ‘pink sage’. This variable species is a strongly aromatic, twiggy herb or subshrub restricted to, but widespread within, southern Africa (Codd 1985; Joffe 1993; Retief & Herman 2003). Except for a cursory mention in the taxonomic literature (Bremekamp 1933; Codd 1964, 1985; Verdoorn 1943), there has been no detailed investigation into the foliar trichomes of O. labiatus. Given that the glandular trichomes of this species may be the source of chemicals that have been shown to have some effect against Mycobacterium species in the Mycobacterium tuberculosis complex (the cause of tuberculosis) (McGaw et al. 2008), O. labiatus is a good candidate for further study at all levels. Studies such as the one here, which are aimed at elucidating the nature of microscopic structures (potentially the source of beneficial secretions) are particularly important.

With each new species of Lamiaceae studied, researchers are building a more complete picture of the complex micromorphology of this family. Within the published scientific literature, a comprehensive body of knowledge is being built on the vestiture of the Lamiaceae: its structure, function, ecological and evolutionary significance.

Materials and methods

The leaves of O. labiatus were collected during the flowering stage in April 2008 from wild-growing plants at the University of KwaZulu-Natal, Westville Campus, Durban, South Africa. The identification of this material was verified and a voucher specimen (Nicholas and Bhatt 2993) was deposited in the Ward Herbarium (herbarium acronym UDW). Leaves of different developmental stages were selected for the present study, i.e. Stage I – very young leaves from first node, Stage II – young leaves harvested from the second and third nodes, and Stage III – fully expanded/mature leaves harvested from the fourth and fifth nodes. Five plants of O. labiatus were sampled and five leaves from each plant which were at different developmental stages were used for study. Fresh leaves were examined and photographed using a stereomicroscope (Nikon AZ 100).

For the light-microscopy studies, semi-thin sections of leaf material embedded in Spurr's (1969) low-viscosity resin were used. Monitor sections ranging in thickness from 0.5 to 2 µm, were cut with glass knives and fixed by heat onto pre-cleaned glass slides with drops of distilled water. The sections were stained with 0.5% Toluidine Blue-0 made up in 0.1% sodium carbonate at pH 11.1 (Feder & O'Brien, 1968). Sections were stained for ~ 1 min over heat, washed briefly in distilled water and mounted in immersion oil. Slides were viewed and photographed with a Leitz light microscope.

For scanning electron microscopy, leaf segments at different developmental stages were prepared by rapid quenching in liquid nitrogen and freeze drying in an Edwards Modulyo Freeze dryer at -60 °C in a vacuum of 10^-2 Torr for ~ 48 h. Leaf segments were secured with carbon conductive tape on brass stubs which were sputter coated with gold for 4 min. Observations were carried out with a Jeol 6100 scanning electron microscope at 12 kV and a working distance of 15 mm.

Results

Stereomicroscopy and scanning electron microscopy studies revealed that leaves possess three types of trichome: non-glandular or acicular hairs, peltate trichomes and capitate trichomes.

Non-glandular or acicular hairs

Non-glandular or acicular hairs are sharply pointed and swept in the direction of the leaf apex. They appear to be especially abundant along the veins, particularly on the abaxial surface of very young leaves (Fig. 1A and Fig. 2A), and were also evident on the serrated leaf margins (Fig. 1A,B). Mature non-glandular trichomes were observed in the early stages of leaf development and consist of approximately three to five cells (Fig. 2C). These trichomes are supported by a cellular pedestal formed by a group of epidermal cells arranged in a circle around the bases (Fig. 2C). Wart-like structures were observed on the surface of these trichomes. In the present study, new trichome formation was not observed in the later stages of leaf development.

Fig. 1  Stereo-micrograph showing the distribution of trichomes on an Orthosiphon labiatus leaf. (A) Distribution of non-glandular trichomes on leaf vein (bar = 50 µm), (B) leaf margin (bar = 100 µm), (C) glandular trichomes (bar = 100 µm).

Fig. 2  Scanning electron micrographs of portions of Orthosiphon labiatus leaves showing the two types of glandular trichomes. (A) Leaf vein with numerous glandular peltate (P) and non-glandular (NG) trichomes (bar = 200 µm). (B) Sparse distribution of non-glandular trichomes of mature leaf (bar = 20 µm). (C) Fully developed non-glandular (NG) trichomes on leaf surface supported by cellular pedestal (CP) (bar = 20 µm). (D) Peltate (P), capitate (C) and degenerative peltate (DG) trichome. (E) Peltate glandular trichomes (P) showing four head cells (bar = 10 µm). (E) Mature peltate trichome (P) (bar = 10 µm). (F) Secretory material (SM) on peltate trichome (P) (bar = 10 µm).

Peltate trichomes

Peltate trichomes were observed on both the adaxial and abaxial leaf surfaces (Figs 1C and 2A). Studies of transverse sections showed the peltate trichomes to consist of a basal cell embedded in the epidermis, a short stalk cell and a large spherical secretory head (Fig. 3A). This head consists of four secretory cells arranged in a single layer (Fig. 2D), with an enlarged subcuticular chamber within which secretory material accumulates (Fig. 3B). Fully developed peltate trichomes were observed in very young leaves; however, their density was lowest at this developmental stage. Some degenerative peltate trichomes were also observed in all stages of leaf development. It appears that the peltate trichomes secrete their materials, and subsequently become shrivelled and degenerate (Fig. 2D). The peltate trichomes appear to become completely distended as they fill with the secretory material (oil); especially in the mature leaves (Fig. 3A). These mature peltate trichomes were particularly abundant on mature leaves (Fig. 2E). Exuded secretion on the peltate trichomes was observed with scanning electron microscopy (Fig. 2F). Observations with scanning electron microscopy indicated that a uniform distribution of peltate trichomes exists on both leaf surfaces and that they are mostly concentrated on the intervein areas.

Fig. 3  Light micrographs of transverse sections of Orthosiphon labiatus leaves. (A) Peltate trichome (P), (B) capitate trichome (C), (C) degenerative peltate trichome (DG) (bar = 50 µm).

Capitate trichomes

Capitate trichomes consist of a single basal cell which is implanted in the epidermal layer, a stalk cell and a more or less spherical secretory head (Figs 2B and 3B). The capitate trichomes were rare and sparsely distributed (Fig. 2D).

Discussion

Trichomes are unicellular or multicellular appendages that originate from cells of the aerial epidermis (Werker 2000). They vary considerably in morphology, location, ability to secrete and mode of secretion, and different types of trichomes can be produced by the same plant. Orthosiphon labiatus leaves possess one non-glandular and two glandular trichome types on both the adaxial and abaxial leaf surfaces – similar to other species of the family Lamiaceae (Gairola et al. 2009).

Studies of trichome type, size and density in the Lamiaceae have shown that although they can be variable within genera (Dinç et al. 2009), within species (especially in size and density) (Grubešic et al. 2007) and between different organs of the same plant (Kahraman et al. 2010), most differences are species specific in nature and can often be of taxonomic significance (Bini Maleci & Servettaz 1991; Navarro & El Oualidi 2000) even at the subspecific level (Giuliani et al. 2008).

Bosabalidis and Skoula (1998) reported that in Origanum intercedens Rech.f., also an aromatic lamiaceous species, the non-glandular trichomes are diverse in morphology, anatomy and microstructure. They may be unicellular or multicellular, branched or unbranched. In O. labiatus, non-glandular trichomes were single, uniseriate and multicellular, and more abundant on the leaf veins and margins; particularly on the abaxial surface of very young leaves. Similar observations have been made in Plectranthus laxiflorus Benth. (Bhatt et al. unpublished data). As in other species of Lamiaceae, non-glandular trichomes of O. labiatus were supported by a cellular pedestal formed by a group of epidermal cells. It has been suggested that this basal cellular pedestal provides mechanical support (Ascensão et al. 1999) and serves as a point of connection between the trichome and the epidermal layer (Payne 1978). The presence of warts on the trichomes is indicative of maturity (Werker 2000). In the present study, we observed warts on the trichomes of very young leaves and this therefore indicates that non-glandular trichomes mature at the early stages of leaf development. Warty trichomes are a characteristic of the Lamiaceae and might also be common in other families of the Asterid clade, as defined by the Angiosperm Phylogeny Group (2009); Naidoo et al. unpublished data). The higher density of non-glandular trichomes on the abaxial leaf surface suggests that they prevent water loss by shielding and trapping air over the stomata (Wagner 1991), and also reflect UV light and maintain leaf temperature (Lakusic et al. 2005). However, the density of non-glandular trichomes decreased with leaf maturity. A similar pattern of trichome distribution was observed in other species of the family Lamiaceae (Ascensão & Pais 1998, Ascensão et al. 1999; Corsi & Bottega 1999).

We also observed the presence of glandular trichomes (peltate and capitate) on both the adaxial and abaxial leaf surface of O. labiatus. Similar observations were made for Salvia officinalis L. (Corsi & Bottega 1999), Plectranthus ornatus Codd (Ascensão et al. 1999), Mentha arvensis L. (Sharma et al. 2003) and Tetradenia riparia (Gairola et al. 2009). Peltate trichomes were mostly found on the intervein laminar of the leaves of O. labiatus. A similar type of distribution of peltate trichomes was reported in Plectranthus madagascariensis Benth. (Ascensão & Pais 1998).

The voluminous appearance of the peltate trichomes (Fig. 2E) may be due to the accumulation of secretory materials, which mostly occurred in the latter stages of leaf development. Although, mature peltate trichomes were observed in very young leaves, they were fewer in number. This was also confirmed by light microscopy studies (Figs 2A,B) which clearly showed the large subcuticular space present in the peltate trichome. Our results are consistent with those of Gang et al. (2001) who reported a similar pattern of distribution of peltate trichomes in Ocimum basilicum L. (Sweet Basil). The peltate trichome head consists of four central secretory cells. This type of peltate trichome with four-headed cells has been reported in other species of the Lamiaceae (Bosabalidis 1990; Kaya et al. 2007; Werker et al. 1993). It is reported that peltate trichomes have a characteristic spherical head due to the development of a large subcuticular space where secretory products accumulate prior to secretion (Ascensão et al. 1999) As the cells fill with secretory material, the secretory cells are no longer visible. Our findings are in agreement with Bourett et al. (1994) who reported that mature oil-producing peltate trichomes were more common on the mature leaf surface of Nepeta racemosa Lam. These glands were sutureless due to the accumulation of secretory material in the subcuticular space. Similar observations are applicable to peltate trichomes of O. labiatus which have a spherical or slightly flattened head, possibly indicating that they accumulate secretory products. Degenerative peltate trichomes were observed on both leaf surfaces. It is believed that these degenerative peltate trichomes may indicate a loss of turgidity because of the release of accumulated secretory products. Similar degenerative peltate trichomes have also been reported in Lippia scaberrima Sond. (Combrinck et al. 2007).

In O. labiatus capitate trichomes were distributed on both leaf surfaces, however, they appear to be very rare and sparse. The capitate trichomes consist of a single basal cell which is implanted in the epidermis, a stalk cell and a rounded secretory head. These capitate trichomes are similar in structure and morphology to those described by Werker et al. (1985) in their study of selected species of Lamiaceae. The round glandular head of these capitate trichomes develops only a small subcuticular space and it is assumed that the secretion is probably discharged through micropores (Kaya et al. 2007). It has been suggested that the compounds secreted by capitate trichomes are mostly excreted to the immediate environment (Kaya et al. 2007).

Our study contributes to the knowledge of the morphology, distribution and type of trichomes in angiosperms (in particular Lamiaceae), and will lend support to further studies on the chemical constituents of the glandular trichomes of O. labiatus. However, detailed anatomical and ultrastructural studies may be helpful in interpreting the function of glandular and non-glandular trichomes.

Acknowledgements

This research was carried out with financial support from the National Research Foundation-Thuthuka, South Africa. We wish to thank Dr J-W. Smith, P. Maartens and S. Eggers of the Electron Microscope Unit, UKZN-Westville campus for technical assistance. Our special thanks to Mr A. Rajh for assistance with the photographic plates. Mr M. Ngwenya of the KwaZulu-Natal Herbarium (South African National Biodiversity Institute) is thanked for help with identifying the species used for this study.