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Glandular Trichomes on the Leaves and Flowers of Plectranthus ornatus: Morphology, Distribution and Histochemistry
Annals of Botany 84 : 437–447, 1999 Article No. anbo.1999.0937, available online at http:\\www.idealibrary.com on
Glandular Trichomes on the Leaves and Flowers of Plectranthus ornatus : Morphology, Distribution and Histochemistry L. A S C E N S A4 O*, L. M O T A and M. D E M. C A S T R O† Centro de Biotecnologia Vegetal, Departamento de Biologia Vegetal, Faculdade de CieV ncias de Lisboa, Bloco C2, 1749-016 Lisboa, Portugal Received : 30 March 1999
Returned for revision : 12 May 1999
Accepted : 11 June 1999
The types of glandular trichomes and their distribution on leaves and flowers of Plectranthus ornatus were investigated at different stages of their development. Five morphological types of glandular trichomes are described. Peltate trichomes, confined to the leaf abaxial surface, have, in io, an uncommon but characteristic orange to brownish colour. Capitate trichomes, uniformly distributed on both leaf surfaces, are divided into two types according to their structure and secretory processes. In long-stalked capitate trichomes, a heterogeneous secretion (a gumresin) is stored temporarily in a large subcuticular space, being released by cuticle rupture, whereas, in the short-stalked capitate trichomes, the secretion, mainly polysaccharidic, is probably exuded via micropores. On the leaves, digitiform trichomes, which do not show a clear distinction between the apical glandular cell and the subsidiary cells, occur with a similar distribution to the capitate trichomes. They do not develop a subcuticular space, and secrete small amounts of essential oils in association with polysaccharides. The reproductive organs, particularly the calyx and corolla, exhibit, in addition to the reported trichomes, unusual conoidal trichomes with long unicellular conical heads. A large apical pore, formed by tip disruption, releases the secretion (a gumresin) stored in a rostrum-like projection. On the stamens and carpels, digitiform, capitate and conoidal trichomes are absent, but peltate trichomes are numerous. They occur between the two anther lobes, on the basal portion of the style, and between the four lobes of the ovary. The results presented are compared with those of other studies on Lamiaceae glandular trichomes. # 1999 Annals of Botany Company Key words : Plectranthus ornatus Codd, Lamiaceae, glandular trichomes, morphology, histochemistry, essential oils and mucilage secretion.
I N T R O D U C T I ON Plectranthus L’Herit. is a large genus of the Lamiaceae family (in which Coleus Lour. and certain other genera are included) comprising about 300 species of evergreen perennials and subshrubs widely distributed in tropical regions of Africa, Asia and Australia (Codd, 1985). Several Plectranthus species are cultivated as ornamentals or as sources of essential oils, whereas others are used for their edible tubers, or as food flavouring. In folk medicine, they are employed for headaches, sores, burns, dermatitis and against nausea and scorpion stings (Riviera Nun4 ez and Obo! n de Castro, 1992 ; Bown, 1995). Plectranthus ornatus Codd (syn. : Coleus comosus Hochst. ex Guerke) is a perennial succulent herb, unpleasantly aromatic, that grows over rocks in semi-shade from Ethiopia to Tanzania, being nowadays cultivated and seminaturalized in Southern Africa (Codd, 1985). Many Lamiaceae species have been investigated because of their high content of essential oils, which are widely used in pharmaceutical preparations, perfumery and cosmetics. Although much phytochemical work has been done during * For correspondence. Fax j351 1 7500048. † Current address : Departamento de Bota# nica, Instituto de Biologia, Universidade Estadual de Campinas, Caixa Postal 6109, 13083-970 Campinas, SP, Brasil.
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the last 20 years, the glandular trichomes of only a limited number of species have been studied, concentrating on the leaves. Some data are, however, recently available for glandular trichomes on the reproductive organs (Werker, 1993 ; Ascensa4 o, Marques and Pais, 1995 ; Ascensa4 o et al., 1998). In the course of an investigation of Plectranthus madagascariensis (Ascensa4 o et al., 1998), it became apparent that detailed morphological studies of the glandular indumentum of other Plectranthus species were needed to evaluate the usefulness of this feature for systematic purposes, in spite of the exhaustive critical taxonomic discussion of the genus by Codd (1975, 1985). In this paper, the glandular trichomes of Plectranthus ornatus on both vegetative and reproductive organs are considered, in relation to morphology, distribution and the histochemistry of the main secretion compounds.
MATERIALS AND METHODS Plant material Small branches of Plectranthus ornatus Codd were collected from plants growing in the Botanical Garden of Lisbon. A voucher specimen has been deposited in the Herbarium of this Botanical Garden (LISU : 171087). # 1999 Annals of Botany Company
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F. 1. For legend see facing page.
Ascensag o et al.—Glandular Trichomes of Plectranthus Leaes and Flowers Scanning electron microscopy (SEM ) Leaves, bracts and flowers at different developmental stages were fixed with 3 % glutaraldehyde in 0n1 sodium phosphate buffer, pH 7n2, for 4 h at 4 mC. After washing in the same buffer, the material was dehydrated in a graded acetone series, critical point dried and coated with a thin layer of gold. Observations were carried out on a Jeol JSM T220 scanning electron microscope at 15 kV. Light microscopy (LM ) The main classes of metabolite in the secreted material were investigated in fresh sections, using the following histochemical tests : Sudan Black B (Lison, 1960) and Neutral Red under UV (Clark, 1981) for total lipids ; Nile Blue A (Jensen, 1962) for neutral and acidic lipids ; osmium tetroxide (OsO ) (Lison, 1960) for unsaturated lipids ; Nadi % reagent (David and Carde, 1964) for terpenoids ; Wagner and Dittmar reagents (Furr and Mahlberg, 1981) for alkaloids ; periodic acid-Schiff (PAS) reagent (Jensen, 1962) and periodic acid-fluorescent Schiff (F-PAS) reagent (Pearse, 1985) for polysaccharides ; Ruthenium Red (Johansen, 1940) for pectins ; and ferric trichloride (Johansen, 1940), vanillinhydrochloric acid (Gardner, 1975) and Fast Blue B (Ganter and Jolle' s, 1969) for phenolic compounds. Flavonoids were detected by induction of fluorescence with the fluorochromes, aluminium trichloride and the Wilson reagent (Charrie' re-Ladreix, 1973). Standard control procedures were carried out simultaneously. Observations were made with a Leitz microscope using Nomarsky optics and with a Leitz Dialux epifluorescence microscope equipped with a HBO 50W mercury vapour lamp and a filter block A (exciter filter BP 340-380, dichroic mirror 450, barrier filter LP-430). For each type of trichome the length and width (at widest point) were measured by micrometer using transverse fresh sections of randomly-selected young mature leaves and sepals. Data from 50 measurements per trichome were analyzed statistically. RESULTS Morphology and distribution of the trichomes Vegetatie organs. The densely-pubescent succulent leaves (tomentose indumentum of P. ornatus) bear numerous non-glandular and glandular trichomes on both surfaces. Non-glandular trichomes are single, uniseriate, multicellular, pointed, erect or leaning towards the leaf apex (Fig. 2 A–C and F). They vary in length between 130 and 350 µm, consist of three to seven cells, and are supported by a
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cellular pedestal formed by a group of six to twelve epidermal cells arranged in a circle around the base (Fig. 2 C, F). These trichomes tend to develop a warty surface character. The glandular trichomes are of three main types—peltate, capitate and digitiform. Peltate trichomes have, in io, a characteristic orange to brownish colour and a balloon shape. They consist of a basal epidermal cell, a very short stalk cell with cutinized lateral walls and a large round head (Fig. 2 D) of eight secretory cells arranged in a single disc (Fig. 4 L). In SEM micrographs, peltate glandular heads show a smooth or wrinkled surface (Fig. 2 C, D), indicating the close attachment of the cuticle to the secretory upper cell walls, which emphasizes the cell outlines, or reveals the presence of a large subcuticular space formed by detachment of the cuticle, together with an outer part of the cell wall. The secretion accumulated in this space remains trapped, giving the trichome a more or less spherical shape. SEM and LM indicate that cuticle rupture is rare. Nevertheless, cuticular disruption can occur along a predetermined line of apparent weakness in the equatorial plane of the head (not shown). A mature trichome at the secretory stage is about 60 µm (p5) in height and 70 µm (p10) in diameter at the head. Capitate trichomes, in P. ornatus, can be divided into two types according to the dimensions of the stalk, the morphology of the glandular head and the secretion process. Short-stalked capitate trichomes have one basal cell, a short stalk cell with thick cutinized lateral walls and a uni- or bicellular ovoid to globoid head (Figs 1 A–C, arrows and 4 I, J). The secretory product accumulates inside the apical cells (Fig. 4 I) and in a very small subcuticular space, probably being exuded through cuticle micropores. However, neither cuticle rupture nor pores were observed. Longstalked capitate trichomes possess one basal cell, a long two to three-celled stalk of variable length and a unicellular bulb-shaped head (Figs 1 A–D and 4 A–H). The lower stalk cells, conical in appearance, are thick-walled, exhibiting a smooth or warty surface (Fig. 1 A–C). The secretory head, supported by a neck-cell of narrow diameter and cutinized side walls, develops a large and spherical subcuticular space where the secretion accumulates temporarily (Figs 1 D and 4 A–D). Cuticle detachment occurs only at the globular region of the glandular cell. Towards the base, at the point which corresponds to the narrowest region of this cell, the cuticle adheres closely to the cell wall (Fig. 4 A–D and G). Therefore, the observed long neck comprises, in addition to the neck cell (nc), the narrow lower region of the glandular cell (gc) (Fig. 4 A–D). The secretion is released by the rupture of the cuticle along a line of weakness that occurs at the top of the narrow neck-like basal part of the glandular cell (Figs 1 B, C, arrowheads and 4 B, arrowheads). At
F. 1. SEM micrographs showing the morphology of glandular trichomes on vegetative (A–D) and reproductive organs (E–H) of Plectranthus ornatus. A–D, Short-stalked (arrows) and long-stalked capitate trichomes (stars). A, A very long and low frequency digitiform trichome. B and C, Mature long-stalked capitate trichomes with torn cuticles (arrowheads), disclosing the head cells ; note in C the cuticular warts on the stalk. D, Mature long-stalked capitate trichome with a large spherical head due to the accumulation of secretion within the subcuticular space. E and F, Mature conoidal trichomes with the characteristic long conical glandular cell supported by a short bicellular stalk. F, Large apical pore (arrow) facilitates the release of the secretion. G, Low-frequency long-stalked conoidal trichomes at two different ontogenic stages ; digitiform, longstalked capitate trichomes and non-glandular hairs are also visible in the background. H, Digitiform trichomes consisting of three to four cells in line ; there does not appear to be a clear distinction between the apical (head-like cell) and the other cells. Bars l 25 µm.
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F. 2. SEM micrographs showing distribution and types of trichomes on vegetative (A–D and F) and reproductive organs (E) of P. ornatus. A, Portion of a very young leaf covered with a tomentose indumentum. B, Cross-section of a young leaf with capitate trichomes (arrows) on both epidermal surfaces, and peltate trichomes (arrowheads) confined to the abaxial. C, Dense cover of glandular and non-glandular trichomes on the abaxial leaf surface ; capitate trichomes are uniformly-distributed on the lamina whereas peltate trichomes are concentrated at the periphery. Bars l 250 µm. D, Detail of a peltate trichome partially sunk in the epidermis of the abaxial leaf surface. E, Long-stalked capitate trichomes at different stages of development and non-glandular trichomes with warty surfaces. F, Digitiform trichomes with a very uniform morphology ; note the radial arrangement of the epidermal cells on the basal portion of the non-glandular trichome. Bars l 25 µm.
Ascensag o et al.—Glandular Trichomes of Plectranthus Leaes and Flowers
F. 3. For legend see overleaf.
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maturity, the short-stalked capitate trichomes are about 35 µm (p5) in height whereas long-stalked vary between 70 and 145 µm. The horizontal diameters of the heads of the trichomes are, respectively, 25 µm (p5) and 35 µm (p5). No significant differences in stalk size between upper and lower epidermal surfaces were noted. Digitiform trichomes consist of three to four cells, in line, of similar diameter and approximately equal length. In SEM images, there is no clear distinction between head and stalk cells (Fig. 2 F). However, the glandular apical cells have rounded tips, thin walls and are rich in cytoplasm, unlike the basal cells that are thick-walled and more vacuolated (Fig. 5 A–D). Therefore, these glandular trichomes are made up of one basal cell, one or two stalk cells and one apical secretory (head-like) cell, which does not develop a subcuticular space. They are about 7 µm (p2) in diameter and 35 µm (p15) in height, occasionally taller (Fig. 1 A). The non-glandular trichomes are mature at an early stage of leaf development, completely covering the glandular trichomes on very young leaves (Fig. 2 A). As the leaf expands, the density of the non-glandular trichomes decreases, although they remain abundant on both surfaces of mature leaves (Fig. 2 B), predominating on the margins and veins of the abaxial surface. Peltate trichomes are confined to the abaxial leaf surface, being in the mature leaves particularly concentrated at the periphery of the lamina, in irregular submarginal rows partially sunken in the epidemis (Fig. 2 C, D). Capitate trichomes (short- and long-stalked) occur together on both leaf surfaces, where they are abundant and uniformlydistributed (Fig. 2 B). The long-stalked capitate trichomes are arranged in several stratified layers reflecting the different heights of the stalk (Fig. 2 B, arrows). Digitiform trichomes have a similar distribution to that of the capitate, but are less abundant. On the stem, peltate trichomes are scarce, whereas non-glandular, capitate and digitiform trichomes are numerous. Observations with SEM show that the number of glandular trichomes per leaf varies as a function of the level of the foliar verticil. Since the trichomes increase in number until the sixth verticil, and decrease up to the tenth verticil, all the glandular trichomes are completely differentiated on fully-expanded leaves. Reproductie organs. A thick pubescent indumentum is present on the flowers of P. ornatus, involving the same types of trichomes that occur on the vegetative organs (Fig. 2 E). In addition, and typically restricted to the reproductive organs, an unusual type of conoidal glandular trichome occurs. It consists of four basal cells, a bicellular short stalk, and a characteristic long, pointed, glandular cell of more or less conical shape (Figs 1 E, F and 5 E–L). However,
conoidal trichomes with long stalks can occasionally be found (Fig. 1 G). A large apical pore, formed by tip disruption, permits the secretion stored in a rostrum-like projection to be released (Figs 1 F, arrow and 5 G). Its tip, easily broken off upon contact, releases an abundant secretory product. These trichomes are about 110 µm (p15) long and 60 µm (p5) wide at the widest basal region. Trichome distribution on the calyx, which is two-lipped with a large entire upper lip and a lower lip divided into four small teeth, is very peculiar. Non-glandular trichomes, shortand long-stalked capitate trichomes, digitiform and conoidal trichomes are clustered on the outer, abaxial calyx surface (Fig. 3 A). Peltate trichomes are scarce and the long non-glandular hairs are nearly restricted to the periphery of the lips. Digitiform and conoidal trichomes are the most conspicuous trichome types present on the inner, adaxial, calyx surface (Fig. 3 A, arrows and arrowheads, D). On its abaxial side, the floral bract has a pattern of glandular trichomes similar to that of the calyx, whereas its adaxial side is nearly devoid of trichomes (Fig. 3 B). The sparingly-pubescent corolla shows digitiform and conoidal trichomes on the outer abaxial side, mainly on the corolla lobes (Fig. 3 C–E). On the stamens and carpels, all types of trichome are absent, except for peltate trichomes, densely arranged, which occur between the two anther lobes (Fig. 3 F, arrowheads), on the pistil on the basal portion of the gynobasic style, and between the four lobes of the ovary (Fig. 3 G, H, arrowheads). Since glandular trichomes develop very early during floral ontogeny, the period of secretion finishes before the corolla is totally exserted from the calyx.
Histochemistry Long-stalked capitate and conoidal trichomes store, in their large subcuticular spaces, an abundant, hyaline and slightly-viscous secretory product with an emulsion-like appearance (Figs 4 A and 5 E), whereas peltate trichomes accumulate an orange-brownish secretion (Fig. 4 L). Digitiform and short-stalked capitate trichomes accumulate only small amounts of secreted material within the glandular cells (Fig. 5 A) or in the narrow subcuticular spaces (Fig. 4 I, J). The secretion of capitate and conoidal trichomes stained positively for lipophilic and hydrophilic substances : staining with Sudan Black B (Figs 4 B and 5 F), OsO (Figs 4 D, J and % 5 G). PAS and Ruthenium Red (Figs 4 H and 5 I) revealed lipids and polysaccharides. These results were also confirmed using F-PAS for polysaccharides (Fig. 5 N) and Neutral Red under UV for lipids (Fig. 5 L). Nile Blue stained the
F. 3. SEM micrographs showing distribution and types of trichomes on flowers of P. ornatus. A, Top view of a young flower bearing numerous trichomes on both calyx surfaces ; conoidal (arrowheads) and digitiform trichomes (arrows) are very abundant on the larger entire upper lip and on the abaxial sides of the lower lip teeth. B, On the floral bract the inner side is nearly devoid of trichomes whereas the outer side is covered with a pubescent indumentum. C–E, Top views of buds with most of the calyx removed to show different phases of corolla development. C, Early ontogenic stages of glandular trichomes. D and E, Digitiform and conoidal trichomes are evident on the outer side of corolla lobes. F, Top view of a young flower after removal of the calyx and corolla ; the stamens, apressed to the gynoecium, present peltate trichomes (arrowheads) between the two anther lobes. G and H, Lateral views of buds after partial removal of calyx and corolla ; peltate trichomes (arrowheads) occur on the basal portion of the gynobasic style and between the four lobes of the ovary ; note, in G, the numerous papillae along the stomia of anthers (arrows). Bars l 250 µm.
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F. 4. Histochemical characterization of the secretions of P. ornatus trichomes. A–H, Long-stalked capitate trichomes. A, In io showing the long bulb-shaped glandular cell and the large spherical subcuticular space ; cuticle detachment occurs only at the globoid region of the glandular cells (gc), and towards the base, the cuticle remains closely attached to the cell wall. B, Neck cell (nc) lateral walls and the secretion stained blue with Sudan Black B ; remains of the cuticle are clearly visible at the head’s narrowest region (arrowheads) after cuticular rupture. C, Secretion stained red with Nile Blue A filling the subcuticular space. D, OsO test showing black staining of secretory material in the subcuticular space ; some minor % staining is evident in the neck cell and in the lower portion of the glandular cell. E–G, Successive stages of subcuticular space development ; violet droplets of various sizes can be seen after staining with the Nadi reagent. H, Secretory material stained with Ruthenium Red. I and J, Short-stalked capitate trichomes. I, Nadi reaction ; stalk lateral walls stained blue, whereas the secretion inside a small vacuole shows a light violet staining. J, OsO test showing the apical cells stained black. K and L, Peltate trichomes. K, Black staining of secretion with OsO . L, In io, showing the % % characteristic orange to brownish colour. Bars l 15 µm.
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F. 5. For legend see facing page.
Ascensag o et al.—Glandular Trichomes of Plectranthus Leaes and Flowers secretion in the subcuticular space pink, whereas the glandular cells showed a blue staining (Figs 4 C and 5 J). The Nadi reaction resulted in an intense violet or blue-violet staining of the secretion contained in the subcuticular space of the long-stalked capitate and conoidal trichomes (Figs 4 E–G and 5 H) and a faint staining of the glandular cells of short-stalked capitate trichomes (Fig. 4 I). In contrast, peltate trichomes gave positive results for lipophilic and terpenoid compounds only ; the reaction with OsO is % shown (Fig. 4 K). Unlike peltate trichomes, digitiform trichomes gave positive reactions for hydrophilic substances (Fig. 5 A) and negative or weak reactions with the tests used for lipophilic compounds (Fig. 5 B, C). The histochemical tests carried out to detect alkaloids and phenolic compounds gave negative results, except for digitiform trichomes which reacted positively for phenols ; the test with ferric trichloride is shown (Fig. 5 D). Despite these results, the blue autofluorescence observed under UV light indicated the presence of phenols. The autofluorescence of conoidal and long-stalked capitate trichomes is shown (Fig. 5 K, M). The fluorochromes for flavonoid detection, such as aluminium trichloride and the Wilson reagent induced a light yellow secondary fluorescence (not shown). In all trichome types, the lateral walls of the stalk cell (peltate and short-stalked capitate trichomes), of the neck cell (conoidal and long-stalked capitate trichomes) and of the sub-apical cell (digitiform trichomes) were Sudanpositive (Figs 4 B and 5 B, F) and PAS- and F-PAS-negative (Fig. 5 A, N, O). The absence of chlorophyll fluorescence reveals that chloroplasts were not present in the trichomes. D I S C U S S I ON The presence of peltate and capitate glandular trichomes is a characteristic feature of Lamiaceae species. Unlike peltate trichomes, that have a rather uniform morphology, capitate trichomes differ in terms of morphological characters which reflect different secretory processes and, probably, distinct functions. Peltate trichomes have short one-celled stalks and large flattened heads, of about 60–90 µm in diameter. They are formed by four or eight cells in a single disc (Amelunxen, Wahlig and Arbeiter, 1969 ; Antunes and Sevinate-Pinto, 1991 ; Bourett et al., 1994 ; Ascensa4 o et al., 1995, 1998 ; Serrato-Valenti et al., 1997) or by 12–18 cells arranged in two concentric circles, four central and eight or more peripheral cells (Werker, Ravid and Putievsky, 1985 a, b ; Bosabalidis, 1990 ; Hanlidou et al., 1991 ; Telepova, Budantzev and Shavarda, 1992). By the time they have reached
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the secretory phase they have a characteristic sphericalshaped head, due to the development of large subcuticular spaces where secretory products accumulate. Capitate trichomes generally consist of rounded to pearshaped heads of one to two cells supported by stalks of variable length. A survey of trichome-types on vegetative and reproductive organs of several Lamiaceae species (Werker et al., 1985 a) listed three kinds of capitate trichomes according to their morphology and secretion processes. Recent reports indicate a much higher diversity of glandular trichomes : long and short-stalked capitate trichomes with four-celled heads and narrow subcuticular spaces were described in Siderites syriaca (Karousou, Bosabalidis and Kokkini, 1992), Leonotis leonurus (Ascensa4 o et al., 1995) and in several Teucrium species (Maleci and Servettaz, 1991). The present studies confirm that the diversity of the glandular trichomes has not yet been fully understood and properly classified. Plectranthus ornatus bears peltate, capitate and digitiform trichomes on its leaves. The peltate trichomes, with eightcelled heads, are similar to those reported for Mentha piperita (Amelunxen et al., 1969), Leonotis leonurus (Ascensa4 o et al., 1995) and Salia aurea (Serrato-Valenti et al., 1997). Cuticle rupture is rarely observed by SEM. The secretion seems to remain trapped in the intact subcuticular spaces unless external factors, such as extreme climatic conditions or mechanical damage (including grazing), cause their rupture. The short-stalked capitate trichomes in this work correspond to the capitate type I described by Werker et al. (1985). They are the commonest capitate trichomes of Labiatae, occurring in nearly all the species studied. Their globoid to ovoid uni- or bicellular glandular heads, of diameter about 20–25 µm, developed only small subcuticular spaces, and the secretion is probably discharged via micropores. However, in P. ornatus, as well as in other species, pore formation has not been observed by SEM. The long-stalked capitate trichomes bear some resemblance to Werker’s types II and III, but are quite distinct. They are long with a bulb-shaped glandular head whereas, in type II, they are usually short with an elongated cell head of the same diameter as the stalk cells. Despite these differences, the development of the subcuticular spaces seems to be analogous in both. Cuticle detachment occurs only in the upper region of the glandular cell, the cuticle remaining firmly attached to the cell wall in the basal portion. During the secretory phase, the long-stalked capitate trichomes, with their typical globular heads and
F. 5. Histochemical characterization of the secretions of P. ornatus trichomes. A–D, Digitiform trichomes. A, Content of the apical cell and trichome cell walls stained pink with PAS, with the exception of the sub-apical cell lateral walls that show no staining at all. B, Lateral walls of the sub-apical cell stained intensely with Sudan Black B. C, Nadi reaction, showing a drop of secreted material stained violet. D, Ferric trichloride test : a faint staining is observed. Bars l 10 µm. E–L, Conoidal trichomes. E, In io, showing a heterogeneous secretion product. F, Secretion stained blue with Sudan Black B. G, OsO test ; a large drop of secretion seems to escape through the apical pore. H, The essential oil within the % subcuticular space has reacted positively with the Nadi reagent. I, Reaction of secretion to Ruthenium Red. J, Secreted material in the subcuticular space stained pink, whereas that in the glandular cell shows a blue staining with Nile Blue A. K, Bright blue autofluorescence of secretion under UV light. L, Gold-yellow secondary fluorescence observed with Neutral Red under UV light. M and N, Long-stalked capitate trichomes under UV light. M, Autofluorescence. N, F-PAS. A weak secondary yellow-orange fluorescence can be seen in the subcuticular space and a strong orange fluorescence in the periclinal neck cell walls. O, Digitiform trichome stained by F-PAS showing yellow fluorescence of the apical cell content and blue fluorescence of the lateral walls of the sub-apical cell. Bars l 15 µm.
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elongated stalks, look like type III trichomes, but their heads have a larger diameter and the glandular apical cells do not become cup-shaped. The digitiform trichomes, with three to four cells in line, without a clear distinction between the apical cells and the others, cannot be considered to be capitate trichomes. They are similar in shape and size to the elongated glandular hairs of Calamintha menthifolia leaves which have been included in the group of type II trichomes (Hanlidou et al., 1991). These authors have studied only the ontogenic development of these hairs and do not refer to the secretory process. In P. ornatus digitiform trichomes, the absence of a true glandular head and of a subcuticular space seem to be reliable criteria for establishing distinctness from Werker’s type II trichomes. Observations of P. ornatus reproductive organs revealed the presence of an unusual kind of glandular trichome with an elongated conical-shaped head, reported in the Lamiaceae for the first time on the Plectranthus madagascariensis calyx (Ascensa4 o et al., 1998). Modenesi, Serrato-Valenti and Bruni (1984) have also described an uncommon type of club-bulbous trichome on flowers and leaf buds of Thymus ulgaris. Despite their apparent resemblance to conoidal trichomes, they have different secretory processes. Clubbed-bulbous trichomes release their secretory product following the crushing of the glandular head, whereas conoidal trichomes appear to discharge the secretion via an apical pore. There is insufficient information to assign a taxonomic significance to such glandular trichomes, which have been described only in the Plectranthus genus. Further research is needed to evaluate their usefulness for systematic studies. The present histochemical results indicate that the secretion of P. ornatus peltate trichomes is an oleoresin containing terpenoids (essential oils and resiniferous acids) and flavonoid aglycones. In addition to these secretory products, conoidal and long-stalked capitate trichomes also contain non-cellulosic polysaccharides, which confer an emulsion-like appearance on the secretion (a gumresin). The exudates of digitiform and short-stalked capitate trichomes contain polysaccharides and flavonoids, with small amounts of essential oils. These results are consistent with other phytochemical data for Lamiaceae. Terpenoids and flavonoids are widely distributed in this family, whereas tannins are absent and a few minor alkaloids occur (Richardson, 1992). It has been generally assumed that peltate trichomes contain the bulk of the essential oils produced by Lamiaceae (Maffei, Chialva and Sacco, 1989 ; Kokkini, Karousou and Vokou, 1994 ; Clark et al., 1997). The present study is not in full agreement with this suggestion, since conoidal and longstalked capitate trichomes do seem to produce significant amounts of essential oils. As in most glandular trichomes studied, the thickenings of the cuticle on the lateral walls of the stalk cell (or of the neck cell) probably prevent the blackflow of secreted products into mesophyll tissue (Fahn, 1988). Regarding the functional significance of the glandular trichomes it has been suggested that their secretions may be involved in the chemical defence of plants or may act as
floral rewards to pollinators. However, the specific function of each trichome type is not known. For instance, shortstalked capitate trichomes have been considered by several authors to be trichome-hydathodes (Schnepf, 1972 ; Heinrich, 1977), whereas the mucilaginous secretions of bulbous-clubbed, and Werker’s type III, trichomes may act as lubricants to facilitate leaf expansion. This investigation shows that better terminology is needed to describe Lamiaceae glandular trichomes, and that detailed anatomical and ultrastructural studies may aid in the interpretation of their functions. A C K N O W L E D G E M E N TS M. de M. Castro is grateful to FAPESP (Fundaça4 o de Amparo a' Pesquisa do Estado de Sa4 o Paulo) for a postdoctoral grant (97\05809-1). L I T E R A T U R E C I T ED Amelunxen F, Wahlig T, Arbeiter H. 1969. U$ ber den Nachweis des a$ therischen O$ ls in isolierten Dru$ senhaaren und Dru$ senschuppen von Mentha piperita L. Zeitschrift fuW r Pflanzenphysiologie 61 : 68–72. Antunes T, Sevinate-Pinto I. 1991. Glandular trichomes of Teucrium scorodonia L. Morphology and histochemistry. Flora 185 : 65–70. Ascensa4 o L, Marques N, Pais MS. 1995. Glandular trichomes on vegetative and reproductive organs of Leonotis leonurus (Lamiaceae). Annals of Botany 75 : 619–626. Ascensa4 o L, Figueiredo AC, Barroso JG, Pedro LG, Schripsema J, Deans SG, Scheffer JJC. 1998. Plectranthus madagascariensis : morphology of the glandular trichomes, essential oil composition, and its biological activity. International Journal of Plant Sciences 159 : 31–38. Bosabalidis AM. 1990. Glandular trichomes in Satureja thymbra leaves. Annals of Botany 65 : 71–78. Bourett TM, Howard RJ, O’Keefe DP, Hallahan DL. 1994. Gland development on leaf surfaces of Nepeta racemosa. International Journal of Plant Sciences 155 : 623–632. Bown D. 1995. Encyclopaedia of herbs and their uses. London, New York : Dorling Kindersley. Charrie' re-Ladreix Y. 1973. E! tude de la se! cre! tion flavonoidique des bourgeons de Populus nigra L. var. italica. Cine! tique du phe! nome' ne glandulaire, ultrastructure et e! volution du tissu glandulaire. Journal de Microscopie 17 : 299–316. Clark G. 1981. Staining procedures. 4th edn. London : Williams & Wilkins. Clark LJ, Hamilton JGG, Chapman JV, Rhodes MJC, Hallanhan DL. 1997. Analysis of monoterpenoids in glandular trichomes of catmint Nepeta racemosa. The Plant Journal 11 : 1387–1393. Codd LE. 1975. Plectranthus (Labiatae) and allied genera in Southern Africa. Bothalia 11 : 371–442. Codd LE. 1985. Lamiaceae. In : Leistner OA, ed. Flora of southern Africa. Pretoria : Botanical Research Institute Department of Agriculture and Water Supply, vol. 28, part 4, 137–172. David R, Carde JP. 1964. Coloration diffe! rentielle des inclusions lipidiques et terpe! niques des pseudophylles du pin maritime au moyen du re! actif Nadi. Comptes Rendus de l ’AcadeT mie des Sciences, Paris 258 : 1338–1340. Fahn A. 1988. Secretory tissues in vascular plants. New Phytologist 108 : 229–257. Furr Y, Mahlberg PG. 1981. Histochemical analysis of laticifers and glandular trichomes in Cannabis satia. Journal of Natural Products (Lloydia) 44 : 153–159. Ganter P, Jolle! s G. 1969. Histochemie normale et pathologique. Vol. I, II. Paris : Gauthier-Villars. Gardner RO. 1975. Vanillin-hydrochloric acid as a histochemical test for tannin. Stain Technology 50 : 315–317.
Ascensag o et al.—Glandular Trichomes of Plectranthus Leaes and Flowers Hanlidou E, Kokkini S, Bosabalidis AM, Bessie' re JM. 1991. Glandular trichomes and essential oil constituents of Calamintha menthifolia (Lamiaceae). Plant Systematics and Eolution 177 : 17–26. Heinrich G. 1977. Die Feinstruktur und das a$ therische O$ l eines Dru$ senhaares von Monarda fistulosa. Biochemie und Physiologie der Pflanzen 171 : 17–24. Jensen WA. 1962. Botanical histochemistry. San Francisco : Freeman. Johansen DA. 1940. Plant microtechnique. New York, London : McGraw-Hill. Karousou R, Bosabalidis AM, Kokkini S. 1992. Sideritis syriaca ssp. syriaca : Glandular trichome structure and development in relation to systematics. Nordic Journal of Botany 12 : 31–37. Kokkini S, Karousou R, Vokou D. 1994. Pattern of geographic variation of Origanum ulgare trichomes and essential oil content in Greece. Biochemical Systematics and Ecology 22 : 517–528. Lison L. 1960. Histochemie et cytochemie animales. Principes et me! thodes. Vols. 1, 2. Paris : Gauthier-Villars. Maffei M, Chialva F, Sacco T. 1989. Glandular trichomes and essential oils in developing peppermint leaves. New Phytologist 11 : 707–716. Maleci LB, Servettaz O. 1991. Morphology and distribution of trichomes in Italian species of Teucrium sect. Chamaedrys (Labiatae)—a taxonomical evaluation. Plant Systematics and Eolution 174 : 83–91. Modenesi P, Serrato-Valenti G, Bruni A. 1984. Development and secretion of clubbed trichomes in Thymus ulgaris L. Flora 175 : 211–219. Pearse AGE. 1985. Histochemistry theoretical and applied. 4th edn. London : Churchill Livingstone.
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Richardson PM. 1992. The chemistry of the Labiatae : an introduction and overview. In : Harley RM, Reynolds T., eds. Adances in labiate science. Kew : Royal Botanic Gardens, 291–297. Riviera Nun4 ez D, Obo! n de Castro C. 1992. The ethnobotany of Old World Labiateae. In : Harley RM, Reynolds T., eds. Adances in labiate science. Kew : Royal Botanic Gardens, 455–473. Schnepf E. 1972. Tubula$ res endoplasmatisches Reticulum in Dru$ sen mit lipophilen Ausscheidungen von Ficus, Ledum, Salia. Biochemie und Physiologie der Pflanzen 163 : 113–125. Serrato-Valenti G, Bisio A, Cornara L, Ciarallo G. 1997. Structural and histochemical investigation of the glandular trichomes of Salia aurea L. leaves, and chemical analysis of the essential oil. Annals of Botany 79 : 329–336. Telepova MN, Budantzev AL, Shavarda AL. 1992. E! tude comparative de la se! cre! tion des terpe' nes par les e! le! ments glandulaires foliaires chez diffe! rentes espe' ces du genre Dracocephalum L. (Labiatae). Bulletin de la SocieT teT Botanique de France 139 : 247–264. Werker E. 1993. Function of essential oil secreting glandular hairs in aromatic plants of the Lamiaceae—a review. Flaour and Fragrance Journal 8 : 249–255. Werker E, Ravid U, Putievsky E. 1985 a. Structure of glandular hairs and identification of the main components of their secreted material in some species of the Labiatae. Israel Journal of Botany 34 : 31–45. Werker E, Ravid U, Putievsky E. 1985 b. Glandular hairs and their secretion in the vegetative and reproductive organs of Salia sclarea and Salia dominica. Israel Journal of Botany 34 : 239–252.
Glandular Trichomes on the Leaves and Flowers of Plectranthus ornatus : Morphology, Distribution and Histochemistry L. A S C E N S A4 O*, L. M O T A and M. D E M. C A S T R O† Centro de Biotecnologia Vegetal, Departamento de Biologia Vegetal, Faculdade de CieV ncias de Lisboa, Bloco C2, 1749-016 Lisboa, Portugal Received : 30 March 1999
Returned for revision : 12 May 1999
Accepted : 11 June 1999
The types of glandular trichomes and their distribution on leaves and flowers of Plectranthus ornatus were investigated at different stages of their development. Five morphological types of glandular trichomes are described. Peltate trichomes, confined to the leaf abaxial surface, have, in io, an uncommon but characteristic orange to brownish colour. Capitate trichomes, uniformly distributed on both leaf surfaces, are divided into two types according to their structure and secretory processes. In long-stalked capitate trichomes, a heterogeneous secretion (a gumresin) is stored temporarily in a large subcuticular space, being released by cuticle rupture, whereas, in the short-stalked capitate trichomes, the secretion, mainly polysaccharidic, is probably exuded via micropores. On the leaves, digitiform trichomes, which do not show a clear distinction between the apical glandular cell and the subsidiary cells, occur with a similar distribution to the capitate trichomes. They do not develop a subcuticular space, and secrete small amounts of essential oils in association with polysaccharides. The reproductive organs, particularly the calyx and corolla, exhibit, in addition to the reported trichomes, unusual conoidal trichomes with long unicellular conical heads. A large apical pore, formed by tip disruption, releases the secretion (a gumresin) stored in a rostrum-like projection. On the stamens and carpels, digitiform, capitate and conoidal trichomes are absent, but peltate trichomes are numerous. They occur between the two anther lobes, on the basal portion of the style, and between the four lobes of the ovary. The results presented are compared with those of other studies on Lamiaceae glandular trichomes. # 1999 Annals of Botany Company Key words : Plectranthus ornatus Codd, Lamiaceae, glandular trichomes, morphology, histochemistry, essential oils and mucilage secretion.
I N T R O D U C T I ON Plectranthus L’Herit. is a large genus of the Lamiaceae family (in which Coleus Lour. and certain other genera are included) comprising about 300 species of evergreen perennials and subshrubs widely distributed in tropical regions of Africa, Asia and Australia (Codd, 1985). Several Plectranthus species are cultivated as ornamentals or as sources of essential oils, whereas others are used for their edible tubers, or as food flavouring. In folk medicine, they are employed for headaches, sores, burns, dermatitis and against nausea and scorpion stings (Riviera Nun4 ez and Obo! n de Castro, 1992 ; Bown, 1995). Plectranthus ornatus Codd (syn. : Coleus comosus Hochst. ex Guerke) is a perennial succulent herb, unpleasantly aromatic, that grows over rocks in semi-shade from Ethiopia to Tanzania, being nowadays cultivated and seminaturalized in Southern Africa (Codd, 1985). Many Lamiaceae species have been investigated because of their high content of essential oils, which are widely used in pharmaceutical preparations, perfumery and cosmetics. Although much phytochemical work has been done during * For correspondence. Fax j351 1 7500048. † Current address : Departamento de Bota# nica, Instituto de Biologia, Universidade Estadual de Campinas, Caixa Postal 6109, 13083-970 Campinas, SP, Brasil.
0305-7364\99\100437j10 $30.00\0
the last 20 years, the glandular trichomes of only a limited number of species have been studied, concentrating on the leaves. Some data are, however, recently available for glandular trichomes on the reproductive organs (Werker, 1993 ; Ascensa4 o, Marques and Pais, 1995 ; Ascensa4 o et al., 1998). In the course of an investigation of Plectranthus madagascariensis (Ascensa4 o et al., 1998), it became apparent that detailed morphological studies of the glandular indumentum of other Plectranthus species were needed to evaluate the usefulness of this feature for systematic purposes, in spite of the exhaustive critical taxonomic discussion of the genus by Codd (1975, 1985). In this paper, the glandular trichomes of Plectranthus ornatus on both vegetative and reproductive organs are considered, in relation to morphology, distribution and the histochemistry of the main secretion compounds.
MATERIALS AND METHODS Plant material Small branches of Plectranthus ornatus Codd were collected from plants growing in the Botanical Garden of Lisbon. A voucher specimen has been deposited in the Herbarium of this Botanical Garden (LISU : 171087). # 1999 Annals of Botany Company
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F. 1. For legend see facing page.
Ascensag o et al.—Glandular Trichomes of Plectranthus Leaes and Flowers Scanning electron microscopy (SEM ) Leaves, bracts and flowers at different developmental stages were fixed with 3 % glutaraldehyde in 0n1 sodium phosphate buffer, pH 7n2, for 4 h at 4 mC. After washing in the same buffer, the material was dehydrated in a graded acetone series, critical point dried and coated with a thin layer of gold. Observations were carried out on a Jeol JSM T220 scanning electron microscope at 15 kV. Light microscopy (LM ) The main classes of metabolite in the secreted material were investigated in fresh sections, using the following histochemical tests : Sudan Black B (Lison, 1960) and Neutral Red under UV (Clark, 1981) for total lipids ; Nile Blue A (Jensen, 1962) for neutral and acidic lipids ; osmium tetroxide (OsO ) (Lison, 1960) for unsaturated lipids ; Nadi % reagent (David and Carde, 1964) for terpenoids ; Wagner and Dittmar reagents (Furr and Mahlberg, 1981) for alkaloids ; periodic acid-Schiff (PAS) reagent (Jensen, 1962) and periodic acid-fluorescent Schiff (F-PAS) reagent (Pearse, 1985) for polysaccharides ; Ruthenium Red (Johansen, 1940) for pectins ; and ferric trichloride (Johansen, 1940), vanillinhydrochloric acid (Gardner, 1975) and Fast Blue B (Ganter and Jolle' s, 1969) for phenolic compounds. Flavonoids were detected by induction of fluorescence with the fluorochromes, aluminium trichloride and the Wilson reagent (Charrie' re-Ladreix, 1973). Standard control procedures were carried out simultaneously. Observations were made with a Leitz microscope using Nomarsky optics and with a Leitz Dialux epifluorescence microscope equipped with a HBO 50W mercury vapour lamp and a filter block A (exciter filter BP 340-380, dichroic mirror 450, barrier filter LP-430). For each type of trichome the length and width (at widest point) were measured by micrometer using transverse fresh sections of randomly-selected young mature leaves and sepals. Data from 50 measurements per trichome were analyzed statistically. RESULTS Morphology and distribution of the trichomes Vegetatie organs. The densely-pubescent succulent leaves (tomentose indumentum of P. ornatus) bear numerous non-glandular and glandular trichomes on both surfaces. Non-glandular trichomes are single, uniseriate, multicellular, pointed, erect or leaning towards the leaf apex (Fig. 2 A–C and F). They vary in length between 130 and 350 µm, consist of three to seven cells, and are supported by a
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cellular pedestal formed by a group of six to twelve epidermal cells arranged in a circle around the base (Fig. 2 C, F). These trichomes tend to develop a warty surface character. The glandular trichomes are of three main types—peltate, capitate and digitiform. Peltate trichomes have, in io, a characteristic orange to brownish colour and a balloon shape. They consist of a basal epidermal cell, a very short stalk cell with cutinized lateral walls and a large round head (Fig. 2 D) of eight secretory cells arranged in a single disc (Fig. 4 L). In SEM micrographs, peltate glandular heads show a smooth or wrinkled surface (Fig. 2 C, D), indicating the close attachment of the cuticle to the secretory upper cell walls, which emphasizes the cell outlines, or reveals the presence of a large subcuticular space formed by detachment of the cuticle, together with an outer part of the cell wall. The secretion accumulated in this space remains trapped, giving the trichome a more or less spherical shape. SEM and LM indicate that cuticle rupture is rare. Nevertheless, cuticular disruption can occur along a predetermined line of apparent weakness in the equatorial plane of the head (not shown). A mature trichome at the secretory stage is about 60 µm (p5) in height and 70 µm (p10) in diameter at the head. Capitate trichomes, in P. ornatus, can be divided into two types according to the dimensions of the stalk, the morphology of the glandular head and the secretion process. Short-stalked capitate trichomes have one basal cell, a short stalk cell with thick cutinized lateral walls and a uni- or bicellular ovoid to globoid head (Figs 1 A–C, arrows and 4 I, J). The secretory product accumulates inside the apical cells (Fig. 4 I) and in a very small subcuticular space, probably being exuded through cuticle micropores. However, neither cuticle rupture nor pores were observed. Longstalked capitate trichomes possess one basal cell, a long two to three-celled stalk of variable length and a unicellular bulb-shaped head (Figs 1 A–D and 4 A–H). The lower stalk cells, conical in appearance, are thick-walled, exhibiting a smooth or warty surface (Fig. 1 A–C). The secretory head, supported by a neck-cell of narrow diameter and cutinized side walls, develops a large and spherical subcuticular space where the secretion accumulates temporarily (Figs 1 D and 4 A–D). Cuticle detachment occurs only at the globular region of the glandular cell. Towards the base, at the point which corresponds to the narrowest region of this cell, the cuticle adheres closely to the cell wall (Fig. 4 A–D and G). Therefore, the observed long neck comprises, in addition to the neck cell (nc), the narrow lower region of the glandular cell (gc) (Fig. 4 A–D). The secretion is released by the rupture of the cuticle along a line of weakness that occurs at the top of the narrow neck-like basal part of the glandular cell (Figs 1 B, C, arrowheads and 4 B, arrowheads). At
F. 1. SEM micrographs showing the morphology of glandular trichomes on vegetative (A–D) and reproductive organs (E–H) of Plectranthus ornatus. A–D, Short-stalked (arrows) and long-stalked capitate trichomes (stars). A, A very long and low frequency digitiform trichome. B and C, Mature long-stalked capitate trichomes with torn cuticles (arrowheads), disclosing the head cells ; note in C the cuticular warts on the stalk. D, Mature long-stalked capitate trichome with a large spherical head due to the accumulation of secretion within the subcuticular space. E and F, Mature conoidal trichomes with the characteristic long conical glandular cell supported by a short bicellular stalk. F, Large apical pore (arrow) facilitates the release of the secretion. G, Low-frequency long-stalked conoidal trichomes at two different ontogenic stages ; digitiform, longstalked capitate trichomes and non-glandular hairs are also visible in the background. H, Digitiform trichomes consisting of three to four cells in line ; there does not appear to be a clear distinction between the apical (head-like cell) and the other cells. Bars l 25 µm.
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F. 2. SEM micrographs showing distribution and types of trichomes on vegetative (A–D and F) and reproductive organs (E) of P. ornatus. A, Portion of a very young leaf covered with a tomentose indumentum. B, Cross-section of a young leaf with capitate trichomes (arrows) on both epidermal surfaces, and peltate trichomes (arrowheads) confined to the abaxial. C, Dense cover of glandular and non-glandular trichomes on the abaxial leaf surface ; capitate trichomes are uniformly-distributed on the lamina whereas peltate trichomes are concentrated at the periphery. Bars l 250 µm. D, Detail of a peltate trichome partially sunk in the epidermis of the abaxial leaf surface. E, Long-stalked capitate trichomes at different stages of development and non-glandular trichomes with warty surfaces. F, Digitiform trichomes with a very uniform morphology ; note the radial arrangement of the epidermal cells on the basal portion of the non-glandular trichome. Bars l 25 µm.
Ascensag o et al.—Glandular Trichomes of Plectranthus Leaes and Flowers
F. 3. For legend see overleaf.
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maturity, the short-stalked capitate trichomes are about 35 µm (p5) in height whereas long-stalked vary between 70 and 145 µm. The horizontal diameters of the heads of the trichomes are, respectively, 25 µm (p5) and 35 µm (p5). No significant differences in stalk size between upper and lower epidermal surfaces were noted. Digitiform trichomes consist of three to four cells, in line, of similar diameter and approximately equal length. In SEM images, there is no clear distinction between head and stalk cells (Fig. 2 F). However, the glandular apical cells have rounded tips, thin walls and are rich in cytoplasm, unlike the basal cells that are thick-walled and more vacuolated (Fig. 5 A–D). Therefore, these glandular trichomes are made up of one basal cell, one or two stalk cells and one apical secretory (head-like) cell, which does not develop a subcuticular space. They are about 7 µm (p2) in diameter and 35 µm (p15) in height, occasionally taller (Fig. 1 A). The non-glandular trichomes are mature at an early stage of leaf development, completely covering the glandular trichomes on very young leaves (Fig. 2 A). As the leaf expands, the density of the non-glandular trichomes decreases, although they remain abundant on both surfaces of mature leaves (Fig. 2 B), predominating on the margins and veins of the abaxial surface. Peltate trichomes are confined to the abaxial leaf surface, being in the mature leaves particularly concentrated at the periphery of the lamina, in irregular submarginal rows partially sunken in the epidemis (Fig. 2 C, D). Capitate trichomes (short- and long-stalked) occur together on both leaf surfaces, where they are abundant and uniformlydistributed (Fig. 2 B). The long-stalked capitate trichomes are arranged in several stratified layers reflecting the different heights of the stalk (Fig. 2 B, arrows). Digitiform trichomes have a similar distribution to that of the capitate, but are less abundant. On the stem, peltate trichomes are scarce, whereas non-glandular, capitate and digitiform trichomes are numerous. Observations with SEM show that the number of glandular trichomes per leaf varies as a function of the level of the foliar verticil. Since the trichomes increase in number until the sixth verticil, and decrease up to the tenth verticil, all the glandular trichomes are completely differentiated on fully-expanded leaves. Reproductie organs. A thick pubescent indumentum is present on the flowers of P. ornatus, involving the same types of trichomes that occur on the vegetative organs (Fig. 2 E). In addition, and typically restricted to the reproductive organs, an unusual type of conoidal glandular trichome occurs. It consists of four basal cells, a bicellular short stalk, and a characteristic long, pointed, glandular cell of more or less conical shape (Figs 1 E, F and 5 E–L). However,
conoidal trichomes with long stalks can occasionally be found (Fig. 1 G). A large apical pore, formed by tip disruption, permits the secretion stored in a rostrum-like projection to be released (Figs 1 F, arrow and 5 G). Its tip, easily broken off upon contact, releases an abundant secretory product. These trichomes are about 110 µm (p15) long and 60 µm (p5) wide at the widest basal region. Trichome distribution on the calyx, which is two-lipped with a large entire upper lip and a lower lip divided into four small teeth, is very peculiar. Non-glandular trichomes, shortand long-stalked capitate trichomes, digitiform and conoidal trichomes are clustered on the outer, abaxial calyx surface (Fig. 3 A). Peltate trichomes are scarce and the long non-glandular hairs are nearly restricted to the periphery of the lips. Digitiform and conoidal trichomes are the most conspicuous trichome types present on the inner, adaxial, calyx surface (Fig. 3 A, arrows and arrowheads, D). On its abaxial side, the floral bract has a pattern of glandular trichomes similar to that of the calyx, whereas its adaxial side is nearly devoid of trichomes (Fig. 3 B). The sparingly-pubescent corolla shows digitiform and conoidal trichomes on the outer abaxial side, mainly on the corolla lobes (Fig. 3 C–E). On the stamens and carpels, all types of trichome are absent, except for peltate trichomes, densely arranged, which occur between the two anther lobes (Fig. 3 F, arrowheads), on the pistil on the basal portion of the gynobasic style, and between the four lobes of the ovary (Fig. 3 G, H, arrowheads). Since glandular trichomes develop very early during floral ontogeny, the period of secretion finishes before the corolla is totally exserted from the calyx.
Histochemistry Long-stalked capitate and conoidal trichomes store, in their large subcuticular spaces, an abundant, hyaline and slightly-viscous secretory product with an emulsion-like appearance (Figs 4 A and 5 E), whereas peltate trichomes accumulate an orange-brownish secretion (Fig. 4 L). Digitiform and short-stalked capitate trichomes accumulate only small amounts of secreted material within the glandular cells (Fig. 5 A) or in the narrow subcuticular spaces (Fig. 4 I, J). The secretion of capitate and conoidal trichomes stained positively for lipophilic and hydrophilic substances : staining with Sudan Black B (Figs 4 B and 5 F), OsO (Figs 4 D, J and % 5 G). PAS and Ruthenium Red (Figs 4 H and 5 I) revealed lipids and polysaccharides. These results were also confirmed using F-PAS for polysaccharides (Fig. 5 N) and Neutral Red under UV for lipids (Fig. 5 L). Nile Blue stained the
F. 3. SEM micrographs showing distribution and types of trichomes on flowers of P. ornatus. A, Top view of a young flower bearing numerous trichomes on both calyx surfaces ; conoidal (arrowheads) and digitiform trichomes (arrows) are very abundant on the larger entire upper lip and on the abaxial sides of the lower lip teeth. B, On the floral bract the inner side is nearly devoid of trichomes whereas the outer side is covered with a pubescent indumentum. C–E, Top views of buds with most of the calyx removed to show different phases of corolla development. C, Early ontogenic stages of glandular trichomes. D and E, Digitiform and conoidal trichomes are evident on the outer side of corolla lobes. F, Top view of a young flower after removal of the calyx and corolla ; the stamens, apressed to the gynoecium, present peltate trichomes (arrowheads) between the two anther lobes. G and H, Lateral views of buds after partial removal of calyx and corolla ; peltate trichomes (arrowheads) occur on the basal portion of the gynobasic style and between the four lobes of the ovary ; note, in G, the numerous papillae along the stomia of anthers (arrows). Bars l 250 µm.
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F. 4. Histochemical characterization of the secretions of P. ornatus trichomes. A–H, Long-stalked capitate trichomes. A, In io showing the long bulb-shaped glandular cell and the large spherical subcuticular space ; cuticle detachment occurs only at the globoid region of the glandular cells (gc), and towards the base, the cuticle remains closely attached to the cell wall. B, Neck cell (nc) lateral walls and the secretion stained blue with Sudan Black B ; remains of the cuticle are clearly visible at the head’s narrowest region (arrowheads) after cuticular rupture. C, Secretion stained red with Nile Blue A filling the subcuticular space. D, OsO test showing black staining of secretory material in the subcuticular space ; some minor % staining is evident in the neck cell and in the lower portion of the glandular cell. E–G, Successive stages of subcuticular space development ; violet droplets of various sizes can be seen after staining with the Nadi reagent. H, Secretory material stained with Ruthenium Red. I and J, Short-stalked capitate trichomes. I, Nadi reaction ; stalk lateral walls stained blue, whereas the secretion inside a small vacuole shows a light violet staining. J, OsO test showing the apical cells stained black. K and L, Peltate trichomes. K, Black staining of secretion with OsO . L, In io, showing the % % characteristic orange to brownish colour. Bars l 15 µm.
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F. 5. For legend see facing page.
Ascensag o et al.—Glandular Trichomes of Plectranthus Leaes and Flowers secretion in the subcuticular space pink, whereas the glandular cells showed a blue staining (Figs 4 C and 5 J). The Nadi reaction resulted in an intense violet or blue-violet staining of the secretion contained in the subcuticular space of the long-stalked capitate and conoidal trichomes (Figs 4 E–G and 5 H) and a faint staining of the glandular cells of short-stalked capitate trichomes (Fig. 4 I). In contrast, peltate trichomes gave positive results for lipophilic and terpenoid compounds only ; the reaction with OsO is % shown (Fig. 4 K). Unlike peltate trichomes, digitiform trichomes gave positive reactions for hydrophilic substances (Fig. 5 A) and negative or weak reactions with the tests used for lipophilic compounds (Fig. 5 B, C). The histochemical tests carried out to detect alkaloids and phenolic compounds gave negative results, except for digitiform trichomes which reacted positively for phenols ; the test with ferric trichloride is shown (Fig. 5 D). Despite these results, the blue autofluorescence observed under UV light indicated the presence of phenols. The autofluorescence of conoidal and long-stalked capitate trichomes is shown (Fig. 5 K, M). The fluorochromes for flavonoid detection, such as aluminium trichloride and the Wilson reagent induced a light yellow secondary fluorescence (not shown). In all trichome types, the lateral walls of the stalk cell (peltate and short-stalked capitate trichomes), of the neck cell (conoidal and long-stalked capitate trichomes) and of the sub-apical cell (digitiform trichomes) were Sudanpositive (Figs 4 B and 5 B, F) and PAS- and F-PAS-negative (Fig. 5 A, N, O). The absence of chlorophyll fluorescence reveals that chloroplasts were not present in the trichomes. D I S C U S S I ON The presence of peltate and capitate glandular trichomes is a characteristic feature of Lamiaceae species. Unlike peltate trichomes, that have a rather uniform morphology, capitate trichomes differ in terms of morphological characters which reflect different secretory processes and, probably, distinct functions. Peltate trichomes have short one-celled stalks and large flattened heads, of about 60–90 µm in diameter. They are formed by four or eight cells in a single disc (Amelunxen, Wahlig and Arbeiter, 1969 ; Antunes and Sevinate-Pinto, 1991 ; Bourett et al., 1994 ; Ascensa4 o et al., 1995, 1998 ; Serrato-Valenti et al., 1997) or by 12–18 cells arranged in two concentric circles, four central and eight or more peripheral cells (Werker, Ravid and Putievsky, 1985 a, b ; Bosabalidis, 1990 ; Hanlidou et al., 1991 ; Telepova, Budantzev and Shavarda, 1992). By the time they have reached
445
the secretory phase they have a characteristic sphericalshaped head, due to the development of large subcuticular spaces where secretory products accumulate. Capitate trichomes generally consist of rounded to pearshaped heads of one to two cells supported by stalks of variable length. A survey of trichome-types on vegetative and reproductive organs of several Lamiaceae species (Werker et al., 1985 a) listed three kinds of capitate trichomes according to their morphology and secretion processes. Recent reports indicate a much higher diversity of glandular trichomes : long and short-stalked capitate trichomes with four-celled heads and narrow subcuticular spaces were described in Siderites syriaca (Karousou, Bosabalidis and Kokkini, 1992), Leonotis leonurus (Ascensa4 o et al., 1995) and in several Teucrium species (Maleci and Servettaz, 1991). The present studies confirm that the diversity of the glandular trichomes has not yet been fully understood and properly classified. Plectranthus ornatus bears peltate, capitate and digitiform trichomes on its leaves. The peltate trichomes, with eightcelled heads, are similar to those reported for Mentha piperita (Amelunxen et al., 1969), Leonotis leonurus (Ascensa4 o et al., 1995) and Salia aurea (Serrato-Valenti et al., 1997). Cuticle rupture is rarely observed by SEM. The secretion seems to remain trapped in the intact subcuticular spaces unless external factors, such as extreme climatic conditions or mechanical damage (including grazing), cause their rupture. The short-stalked capitate trichomes in this work correspond to the capitate type I described by Werker et al. (1985). They are the commonest capitate trichomes of Labiatae, occurring in nearly all the species studied. Their globoid to ovoid uni- or bicellular glandular heads, of diameter about 20–25 µm, developed only small subcuticular spaces, and the secretion is probably discharged via micropores. However, in P. ornatus, as well as in other species, pore formation has not been observed by SEM. The long-stalked capitate trichomes bear some resemblance to Werker’s types II and III, but are quite distinct. They are long with a bulb-shaped glandular head whereas, in type II, they are usually short with an elongated cell head of the same diameter as the stalk cells. Despite these differences, the development of the subcuticular spaces seems to be analogous in both. Cuticle detachment occurs only in the upper region of the glandular cell, the cuticle remaining firmly attached to the cell wall in the basal portion. During the secretory phase, the long-stalked capitate trichomes, with their typical globular heads and
F. 5. Histochemical characterization of the secretions of P. ornatus trichomes. A–D, Digitiform trichomes. A, Content of the apical cell and trichome cell walls stained pink with PAS, with the exception of the sub-apical cell lateral walls that show no staining at all. B, Lateral walls of the sub-apical cell stained intensely with Sudan Black B. C, Nadi reaction, showing a drop of secreted material stained violet. D, Ferric trichloride test : a faint staining is observed. Bars l 10 µm. E–L, Conoidal trichomes. E, In io, showing a heterogeneous secretion product. F, Secretion stained blue with Sudan Black B. G, OsO test ; a large drop of secretion seems to escape through the apical pore. H, The essential oil within the % subcuticular space has reacted positively with the Nadi reagent. I, Reaction of secretion to Ruthenium Red. J, Secreted material in the subcuticular space stained pink, whereas that in the glandular cell shows a blue staining with Nile Blue A. K, Bright blue autofluorescence of secretion under UV light. L, Gold-yellow secondary fluorescence observed with Neutral Red under UV light. M and N, Long-stalked capitate trichomes under UV light. M, Autofluorescence. N, F-PAS. A weak secondary yellow-orange fluorescence can be seen in the subcuticular space and a strong orange fluorescence in the periclinal neck cell walls. O, Digitiform trichome stained by F-PAS showing yellow fluorescence of the apical cell content and blue fluorescence of the lateral walls of the sub-apical cell. Bars l 15 µm.
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Ascensag o et al.—Glandular Trichomes of Plectranthus Leaes and Flowers
elongated stalks, look like type III trichomes, but their heads have a larger diameter and the glandular apical cells do not become cup-shaped. The digitiform trichomes, with three to four cells in line, without a clear distinction between the apical cells and the others, cannot be considered to be capitate trichomes. They are similar in shape and size to the elongated glandular hairs of Calamintha menthifolia leaves which have been included in the group of type II trichomes (Hanlidou et al., 1991). These authors have studied only the ontogenic development of these hairs and do not refer to the secretory process. In P. ornatus digitiform trichomes, the absence of a true glandular head and of a subcuticular space seem to be reliable criteria for establishing distinctness from Werker’s type II trichomes. Observations of P. ornatus reproductive organs revealed the presence of an unusual kind of glandular trichome with an elongated conical-shaped head, reported in the Lamiaceae for the first time on the Plectranthus madagascariensis calyx (Ascensa4 o et al., 1998). Modenesi, Serrato-Valenti and Bruni (1984) have also described an uncommon type of club-bulbous trichome on flowers and leaf buds of Thymus ulgaris. Despite their apparent resemblance to conoidal trichomes, they have different secretory processes. Clubbed-bulbous trichomes release their secretory product following the crushing of the glandular head, whereas conoidal trichomes appear to discharge the secretion via an apical pore. There is insufficient information to assign a taxonomic significance to such glandular trichomes, which have been described only in the Plectranthus genus. Further research is needed to evaluate their usefulness for systematic studies. The present histochemical results indicate that the secretion of P. ornatus peltate trichomes is an oleoresin containing terpenoids (essential oils and resiniferous acids) and flavonoid aglycones. In addition to these secretory products, conoidal and long-stalked capitate trichomes also contain non-cellulosic polysaccharides, which confer an emulsion-like appearance on the secretion (a gumresin). The exudates of digitiform and short-stalked capitate trichomes contain polysaccharides and flavonoids, with small amounts of essential oils. These results are consistent with other phytochemical data for Lamiaceae. Terpenoids and flavonoids are widely distributed in this family, whereas tannins are absent and a few minor alkaloids occur (Richardson, 1992). It has been generally assumed that peltate trichomes contain the bulk of the essential oils produced by Lamiaceae (Maffei, Chialva and Sacco, 1989 ; Kokkini, Karousou and Vokou, 1994 ; Clark et al., 1997). The present study is not in full agreement with this suggestion, since conoidal and longstalked capitate trichomes do seem to produce significant amounts of essential oils. As in most glandular trichomes studied, the thickenings of the cuticle on the lateral walls of the stalk cell (or of the neck cell) probably prevent the blackflow of secreted products into mesophyll tissue (Fahn, 1988). Regarding the functional significance of the glandular trichomes it has been suggested that their secretions may be involved in the chemical defence of plants or may act as
floral rewards to pollinators. However, the specific function of each trichome type is not known. For instance, shortstalked capitate trichomes have been considered by several authors to be trichome-hydathodes (Schnepf, 1972 ; Heinrich, 1977), whereas the mucilaginous secretions of bulbous-clubbed, and Werker’s type III, trichomes may act as lubricants to facilitate leaf expansion. This investigation shows that better terminology is needed to describe Lamiaceae glandular trichomes, and that detailed anatomical and ultrastructural studies may aid in the interpretation of their functions. A C K N O W L E D G E M E N TS M. de M. Castro is grateful to FAPESP (Fundaça4 o de Amparo a' Pesquisa do Estado de Sa4 o Paulo) for a postdoctoral grant (97\05809-1). L I T E R A T U R E C I T ED Amelunxen F, Wahlig T, Arbeiter H. 1969. U$ ber den Nachweis des a$ therischen O$ ls in isolierten Dru$ senhaaren und Dru$ senschuppen von Mentha piperita L. Zeitschrift fuW r Pflanzenphysiologie 61 : 68–72. Antunes T, Sevinate-Pinto I. 1991. Glandular trichomes of Teucrium scorodonia L. Morphology and histochemistry. Flora 185 : 65–70. Ascensa4 o L, Marques N, Pais MS. 1995. Glandular trichomes on vegetative and reproductive organs of Leonotis leonurus (Lamiaceae). Annals of Botany 75 : 619–626. Ascensa4 o L, Figueiredo AC, Barroso JG, Pedro LG, Schripsema J, Deans SG, Scheffer JJC. 1998. Plectranthus madagascariensis : morphology of the glandular trichomes, essential oil composition, and its biological activity. International Journal of Plant Sciences 159 : 31–38. Bosabalidis AM. 1990. Glandular trichomes in Satureja thymbra leaves. Annals of Botany 65 : 71–78. Bourett TM, Howard RJ, O’Keefe DP, Hallahan DL. 1994. Gland development on leaf surfaces of Nepeta racemosa. International Journal of Plant Sciences 155 : 623–632. Bown D. 1995. Encyclopaedia of herbs and their uses. London, New York : Dorling Kindersley. Charrie' re-Ladreix Y. 1973. E! tude de la se! cre! tion flavonoidique des bourgeons de Populus nigra L. var. italica. Cine! tique du phe! nome' ne glandulaire, ultrastructure et e! volution du tissu glandulaire. Journal de Microscopie 17 : 299–316. Clark G. 1981. Staining procedures. 4th edn. London : Williams & Wilkins. Clark LJ, Hamilton JGG, Chapman JV, Rhodes MJC, Hallanhan DL. 1997. Analysis of monoterpenoids in glandular trichomes of catmint Nepeta racemosa. The Plant Journal 11 : 1387–1393. Codd LE. 1975. Plectranthus (Labiatae) and allied genera in Southern Africa. Bothalia 11 : 371–442. Codd LE. 1985. Lamiaceae. In : Leistner OA, ed. Flora of southern Africa. Pretoria : Botanical Research Institute Department of Agriculture and Water Supply, vol. 28, part 4, 137–172. David R, Carde JP. 1964. Coloration diffe! rentielle des inclusions lipidiques et terpe! niques des pseudophylles du pin maritime au moyen du re! actif Nadi. Comptes Rendus de l ’AcadeT mie des Sciences, Paris 258 : 1338–1340. Fahn A. 1988. Secretory tissues in vascular plants. New Phytologist 108 : 229–257. Furr Y, Mahlberg PG. 1981. Histochemical analysis of laticifers and glandular trichomes in Cannabis satia. Journal of Natural Products (Lloydia) 44 : 153–159. Ganter P, Jolle! s G. 1969. Histochemie normale et pathologique. Vol. I, II. Paris : Gauthier-Villars. Gardner RO. 1975. Vanillin-hydrochloric acid as a histochemical test for tannin. Stain Technology 50 : 315–317.
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