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Glandular Trichomes of Teucrium scorodonia L. Ultrastructure and Secretion
Flora (1991) 185: 207-213 Gustav Fischer Verlag lena
Glandular Trichomes of Teucrium scorodonia L. Ultrastructure and Secretion ISABEL SEVINATE-PINTO and TERESA ANTUNES Departamento de Biologia Vegetal, Faculdade de Ciencias de Lisboa, Lisboa, Portugal
Summary The secretory trichomes (type I) of Teucrium scorodonia were investigated using electron microscopy. During an active phase of secretion the glandular head cells are characterized by the development of two cell compartments: the plastidome and ER. The plastids are probably involved in the synthesis of essential oil and the smooth endoplasmic reticulum, which surround the plastids, is implicated in the transport from plastids to the periplasmic space. During the secretory process the secretion is accumulated in the sub-cuticular space forming an unusual morphological feature.
Introduction Investigation of plants of the Labiatae family has demonstrated the presence of different kinds of trichomes that show a diversity of morphology, distribution and of secretion mechanism. The occurrence, morphology and histochemistry of gland trichomes of Teucrium scorodonia have been considered in a previous work (ANTUNES & SEVINATE-PINTO 1989). The purpose of this report is the ultrastructural study of the secretory cells of type I trichomes (as we have designed in a previous work) in an attempt to understand the role of cellular organelles in the secretory process. The morphological aspects of the secretory products accumulated in the sub-cuticular space have not been described until now.
Material and Methods Leaves of Teucrium scorodonia, at various stages of development, were used for investigation of their glandular trichomes. Plants were collected in their natural habitat (Monserrate-Sintra). The methods used in this study for scanning electron microscopy have been described in a previous paper (ANTUNES & SEVINATE-PINTO 1989). For transmission electron microscopy small pieces were fixed during 3 h in 3 % glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.3 at 4 °C and post fixed in 1% OS04 in the same buffer at 4 °C for 1 h. Other samples were post-fixed in 2% OS04 plus 0.8% K3Fe (CN)6 in the same buffer for 1 h according to HEPLER (1981). A third fixation was used in 2% uranyl aquous solution during 1 h at room temperature after dehydration. Specimens were embedded in Epon-Araldite according to MOLLENHAUER (1964). After staining with lead citrate sections were examined with an Hitachi HU-12 electron microscope at 75 kV. Semi-thin sections were stained with Paragon (SPURLOCK et al. 1966).
Results While glands morphology, as well as the chemical identification of natural products, have been studied in some detail (ANTUNES & SEVINATE-PINTO 1989, MARCO et al. 1983), little has been done to relate gland ultrastructure to the production of secretion in plants of Teucrium scorodonia. From an anatomical and ultrastructural point of view there are three types of
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glandular trichomes on the leaves of these plants. In this work we emphasize one type of trichome that we have designed as type I (Figs. 1, 2, and 3). These trichomes present at maturity a multicellular head with four glandular cells. These cells are characterized by a dense cytoplasm containing voluminous and amoeboid nucleus (Fig. 3) a smooth proliferated ER frequently associated with plastids (Fig. 4). All this membranous system becomes more evident after addition of potassium ferricyanide to the fixative (Fig. 3). Dictyossomes, as well as dictyossomederived secretory vesicles, are very scarce. Between glandular and stalk cells there are notorious ultrastructural differences: the stalk cells are characterized by an increase in the vacuome and a considerable reduction of ER and plastids (Fig. 3). The plasmodesmata were never observed between the glandular and stalk cells (Fig. 3). As secretory activity begins, the plastids from glandular head cells undergo a striking increase in their number showing an amoeboid appearance and a close relation with ER cisternae (Figs. 4 and 9) that in some cases appears arranged parallel to the plastid surface giving rise to a complex network (Fig. 4). Intraplastidial tubular elements are common as are starch grains and osmiophilic material interpreted as secretory product (Figs. 5, 6). The wall of glandular cells shows a separation on two different layers. During the secretory process these two layers became separated forming an annular space which is filled with a flocculent material where structures like vesicles are disseminated (Fig. 8). These vesicles present material which shows an heterogenous aspect. In some cases this electron-dense material reveals a similar contrast to the observed in the periplasmic space (Fig. 8-12).
Discussion Teucrium scorodonia, as other plants of the Labiatae family, has more than one type of secretory trichome (WERKER et al. 1985; ECONOMOU-AMILLI et al. 1982; HEINRICH et al. 1983). The secretory head cells of trichomes are similar at an ultrastructural level to the generally reported lipophilic secretion structures. The structural features are more often characterized by the development of two cell compartments: the plastidome and the ER. The involvement of these compartments in the essential oil biosynthesis is demonstrated by different authors (WOODING & NORTHCOTE 1965; BOSABALIDIS & TSEKos 1982; HEINRICH & SCHULTZE 1985). During the secretory phase we observed the increase in number of plastids, the presence of a complex intraplastidial tubular elements and the accumulation of osmiophilic material in the stroma. In Citrus deliciosa plastids with tubular intraplastidial elements associated with osmiophilic
Fig. I. Light micrograph of a leaf transverse section, showing the distribution of trichomes. They are scarce on the adaxial side and abundant on the abaxial one. Marker bar 10 [tm. Fig. 2. SEM graph of a glandular trichome (type I). Marker bar 10 [tm. Fig. 3. TEM graph of a longitudinal section of a glandular trichome after fixation according to HEPLER (1981). The selective property of OsFeCN reaction is shown. The SER is distinctly contrasted as are the intraplastidial tubules and nuclear membranes. The difference between stalk and head cells is visible; Marker bar 1 [tm. Figs. 4-7. TEM graphs of glandular head cells during a secretory phase. Figs. 4-6 - Different aspects of plastids (P) are observed. Fig. 4 - The plastids are surrounded by SER which formes a complex network (arrows). Figs. 5 and 6 - The plastids are filled with intraplastidial tubules and osmiophilic material identified as secretion (arrows). Fig. 7 - The beginning of sub-cuticular space formation is observed (asterisc). Marker bars 1 [tm. Figs. 8-12. TEM graphs of glandular head cells of trichomes (type I). Figs. 8 and 10 - The sub-cuticular space is filled with a flocculent material where structures like vesicles with electron-dense material (arrows) are disseminated. The SER is very abundant. Fig. 9 - After fixation according to HEPLER (1981) SER is contrasted by an intense stain deposit. At the periplasmic space (asterisc) the electron-dense material is accumulated. Figs. 11 and 12 - The secretion observed at the annular space showes less electron density than the material present in the periplasmic space (asteriscs). Marker bars I [tm.
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material are implicated in essential oil production (BOSABALIDIS & TSEKOS 1982) also HEINRICH (1970) described the presence of intraplastidial tubules in the secretory cells of Rutaceae, and related it with the biogenesis of essential oil. On the other hand GLEIZES et al. (1983) show a direct involvement of plastids as monoterpene precursors in Citrofortunella using labelled IPP. Some enzimes of the mevalonic pathway in the plastid membranes and SER were also detected by cytochemical methods (CURRY 1987). Correlating these data with our ultrastructural observations we are led to the conclusion that in the secretory cells of the trichomes the production of essential oil probably takes place in the plastids. To several workers, the close association of the SER with plastids in the form of periplastidial sheath has suggested that the essential oil, elaborated in the plastids, was translocated via ER and discharged in the periplasmic space (CARDE & BERNARD-DAGAN 1982; HEINRICH & SCHULTZE 1985). In Teucrium scorodonia this association is very evident. The presence of electron-dense material in the lumen of SER and the preferential way that appears marked after treatment with potassium ferricyanide, suggests a role of the ER in the transport of osmiophilic material to the periplasmic space. According to CARDE et al. (1980) the transport of the material to the outside via SER is important to avoid damage to the cell because the compounds of the essential oil are toxic. Since only very few golgi bodies can be found, their participation in synthesis and transport of essential oil seems to be negligible. The material accumulated at the periplasmic space crosses the inner layer of the wall showing, in the sub-cuticular space, different morphological and osmiophilic characteristics. We suggest that the different electron-density be probably due to a chemical alteration at this level. Similar interpretation has been considered by VERMEER & PETERSON (1979) and BOSABALIDIS & TSEKOS (1982). According to these last authors the final mixture of the individual oil components takes place within sub-cuticular space. Although the material accumulated at the periplasmic space does not reveal an apparent membrane, the material accumulated in the sub-cuticular space and disseminated in a flocculent matrix, is involved by an evident membrane. We are unable, at this moment, to explain this situation, but we have reasons to think that this is not an artefact because we have obtained the same results with different fixative solutions and with other species of Teucrium like Teucrium abutiloides (unpublished). Conclusions: In the glandular trichomes of Teucrium scorodonia, plastids are probably involved in synthesis of essential oil. Smooth endoplasmic reticulum is involved in the transport of essential oil from the plastids to the periplasmic space. The electron dense material accumulated in the periplasmic space migrates to the sub-cuticular space crossing the inner layer of cellular wall, revealing a chemical and morphological alteration at this level.
Acknowledgements The financial support was obtained from Instituto Nacional de Investigao;:ao Cientifica (INIC).
References ANTUNES, T., & SEVIN ATE-PINTO, I. (1989): Glandular trichomes of Teucrium scorodonia L. Morphology and histochemistry. Flora 184: 389-394. BOSABALIDIS, A. M., & TSEKOS, I. (1982): Ultrastructure of the essential oil secretion in glandular scales of Origanum dictamnus L. leaves. In: MARGARIS, N., KOEDAM, A., & VOKOU, D. (eds.): Aromatic Plants: Basic and Applied Aspects. Proc. Int. Symp. Aromatic Plants. Martinus Nijhoff Pub!., The Hague, The Netherlands. 3-12. - (1982): Ultrastructural studies on the secretory cavities of Citrus deliciosa TEN. I. Early stages of gland cells. Protoplasma 112: 55-62. CARDE, J. P., & BERNARD-DAGAN C. (1982): Compartimentation de la syntMse terpenique chez Ie pin maritime. Bull. Soc. bot. France 129, Actua!. bot., (1): 53-70. - GLEIZES, M. (1980): Membrane systems involved in synthesis and transport of monoterpene hydrocarbons in pine leaves. In: MAZLIAK, P., BENVENISTE, c., COSTES, c., & DOUCE, R. (eds.): Biogenesis and Function of Plant Lipids. Elsevier, North-Holland Biomed. Press.
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CURRY, K. J. (1987): Initiation of terpenoid synthesis in osmophores of Stanhopea anfracta (Orchidaceae): A cytochemical study. Amer. J. Bot. 74: 1332-1338. ECONOMOU-AMILLI, A., VOKOU, D., ANAGNOSTIDIS, K., & MARGARIS, N. S. (1982): Leaf morphology of Thymus capitatus (Labiatae) by scanning electron microscopy. In: MARGARIS, N., KOEDAM, A., & VOKOU, D. (eds.): Aromatic Plants: Basic and Applied Aspects. Proc. Int. Symp. Aromatic Plants. Martinus Nijhoff Publ., The Hague, The Netherlands. GLEIZES, M., GINETTE, P., CARDE, J. P., MARPEAU, A., & BERNARD-DAGAN, C. (1983): Monoterpene hydrocarbon biosynthesis by isolated leucoplasts of Citrofortunella mitis P1anta 159: 373-381. HEINRICH, G. (1970): Elektronenmikroskopische Beobachtungen an den Driisenzellen von Poncirus trifoliata, zugleich ein Beitrag zur Wirkung atherischer Ole auf Pflanzenzellen und eine Methode zur Unterscheidung fliichtiger von nichtfliichtigen lipophi1en Komponenten. Protop1asma 69: 15-36. SCHULTZE, W., PFAB, I., & BOTTGER, M. (1983): The site of essentiaJ oil biosynthesis in Poncirus trifoliate and Monardafistulosa. Physiol. Veg. 21: 257-268. SCHULTZE, W. (1985): Composition and site of biosynthesis of the essential oil in fruits of Phellodendron amurense RUPR. (Rutaceae). Isr. J. Bot. 34: 205-217. HEPLER, P. K. (1981): The structure of the endoplasmic reticulum revealed by osmium tetroxide-potassium ferricyanide staining. Eur. J. Cell. BioI. 26: 102-110. MARCO, J. L., BENJAMIN, R., PASCUAL, c., SAVONA, G., & P!OZZI, F. (1983): Teuscorodin, teuscorodonin and 2-hydroxyteuscorolide, neo-clerodane diterpenoids from Teucrium scorodonia. Phytochemistry 22: 727-731. MOLLENHAUER, H. H. (1964): Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39: 111-114. SPURLOCK, B. 0., SKINNER, M. S., & KATTlNE, A. A. (1966): A simple rapid method of staining for epoxiembedded specimens for light microscopy with the polychromatic stain Paragon-l301. Am. J. Clin. Pathol. 46: 252-258. VERMEER, J., & PETERSON, R. L. (1979): Glandular trichomes on the inflorescence of Chrysanthemum morifolium cv. Dramatic (Compositae). II Ultrastructure and histochemistry. Can. J. Bot. 57: 714-729. WERKER, E., RAVID, U., & PUTIEVSKY, E. (1985): Glandular hairs and their secretions in the vegetative and reproductive organs of Salvia sclarea and S. dominica. Isf. J. Bot. 34: 239-252. WOODING, F. B. P., & NORTHCOTE, D. H. (1965): The fine structure of the mature resin canal cells of Pinus pinea. J. Ultrastruct. Res. 13: 233-244. Received February 12, 1990 Authors' address: ISABEL SEVINATE-P!NTO and TERESA ANTUNES, Departamento de Biologia Vegetal, Faculdade de Ciencias de Lisboa, BI. C2 Campo Grande, 1700 Lisboa, Portugal.
Glandular Trichomes of Teucrium scorodonia L. Ultrastructure and Secretion ISABEL SEVINATE-PINTO and TERESA ANTUNES Departamento de Biologia Vegetal, Faculdade de Ciencias de Lisboa, Lisboa, Portugal
Summary The secretory trichomes (type I) of Teucrium scorodonia were investigated using electron microscopy. During an active phase of secretion the glandular head cells are characterized by the development of two cell compartments: the plastidome and ER. The plastids are probably involved in the synthesis of essential oil and the smooth endoplasmic reticulum, which surround the plastids, is implicated in the transport from plastids to the periplasmic space. During the secretory process the secretion is accumulated in the sub-cuticular space forming an unusual morphological feature.
Introduction Investigation of plants of the Labiatae family has demonstrated the presence of different kinds of trichomes that show a diversity of morphology, distribution and of secretion mechanism. The occurrence, morphology and histochemistry of gland trichomes of Teucrium scorodonia have been considered in a previous work (ANTUNES & SEVINATE-PINTO 1989). The purpose of this report is the ultrastructural study of the secretory cells of type I trichomes (as we have designed in a previous work) in an attempt to understand the role of cellular organelles in the secretory process. The morphological aspects of the secretory products accumulated in the sub-cuticular space have not been described until now.
Material and Methods Leaves of Teucrium scorodonia, at various stages of development, were used for investigation of their glandular trichomes. Plants were collected in their natural habitat (Monserrate-Sintra). The methods used in this study for scanning electron microscopy have been described in a previous paper (ANTUNES & SEVINATE-PINTO 1989). For transmission electron microscopy small pieces were fixed during 3 h in 3 % glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.3 at 4 °C and post fixed in 1% OS04 in the same buffer at 4 °C for 1 h. Other samples were post-fixed in 2% OS04 plus 0.8% K3Fe (CN)6 in the same buffer for 1 h according to HEPLER (1981). A third fixation was used in 2% uranyl aquous solution during 1 h at room temperature after dehydration. Specimens were embedded in Epon-Araldite according to MOLLENHAUER (1964). After staining with lead citrate sections were examined with an Hitachi HU-12 electron microscope at 75 kV. Semi-thin sections were stained with Paragon (SPURLOCK et al. 1966).
Results While glands morphology, as well as the chemical identification of natural products, have been studied in some detail (ANTUNES & SEVINATE-PINTO 1989, MARCO et al. 1983), little has been done to relate gland ultrastructure to the production of secretion in plants of Teucrium scorodonia. From an anatomical and ultrastructural point of view there are three types of
208
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glandular trichomes on the leaves of these plants. In this work we emphasize one type of trichome that we have designed as type I (Figs. 1, 2, and 3). These trichomes present at maturity a multicellular head with four glandular cells. These cells are characterized by a dense cytoplasm containing voluminous and amoeboid nucleus (Fig. 3) a smooth proliferated ER frequently associated with plastids (Fig. 4). All this membranous system becomes more evident after addition of potassium ferricyanide to the fixative (Fig. 3). Dictyossomes, as well as dictyossomederived secretory vesicles, are very scarce. Between glandular and stalk cells there are notorious ultrastructural differences: the stalk cells are characterized by an increase in the vacuome and a considerable reduction of ER and plastids (Fig. 3). The plasmodesmata were never observed between the glandular and stalk cells (Fig. 3). As secretory activity begins, the plastids from glandular head cells undergo a striking increase in their number showing an amoeboid appearance and a close relation with ER cisternae (Figs. 4 and 9) that in some cases appears arranged parallel to the plastid surface giving rise to a complex network (Fig. 4). Intraplastidial tubular elements are common as are starch grains and osmiophilic material interpreted as secretory product (Figs. 5, 6). The wall of glandular cells shows a separation on two different layers. During the secretory process these two layers became separated forming an annular space which is filled with a flocculent material where structures like vesicles are disseminated (Fig. 8). These vesicles present material which shows an heterogenous aspect. In some cases this electron-dense material reveals a similar contrast to the observed in the periplasmic space (Fig. 8-12).
Discussion Teucrium scorodonia, as other plants of the Labiatae family, has more than one type of secretory trichome (WERKER et al. 1985; ECONOMOU-AMILLI et al. 1982; HEINRICH et al. 1983). The secretory head cells of trichomes are similar at an ultrastructural level to the generally reported lipophilic secretion structures. The structural features are more often characterized by the development of two cell compartments: the plastidome and the ER. The involvement of these compartments in the essential oil biosynthesis is demonstrated by different authors (WOODING & NORTHCOTE 1965; BOSABALIDIS & TSEKos 1982; HEINRICH & SCHULTZE 1985). During the secretory phase we observed the increase in number of plastids, the presence of a complex intraplastidial tubular elements and the accumulation of osmiophilic material in the stroma. In Citrus deliciosa plastids with tubular intraplastidial elements associated with osmiophilic
Fig. I. Light micrograph of a leaf transverse section, showing the distribution of trichomes. They are scarce on the adaxial side and abundant on the abaxial one. Marker bar 10 [tm. Fig. 2. SEM graph of a glandular trichome (type I). Marker bar 10 [tm. Fig. 3. TEM graph of a longitudinal section of a glandular trichome after fixation according to HEPLER (1981). The selective property of OsFeCN reaction is shown. The SER is distinctly contrasted as are the intraplastidial tubules and nuclear membranes. The difference between stalk and head cells is visible; Marker bar 1 [tm. Figs. 4-7. TEM graphs of glandular head cells during a secretory phase. Figs. 4-6 - Different aspects of plastids (P) are observed. Fig. 4 - The plastids are surrounded by SER which formes a complex network (arrows). Figs. 5 and 6 - The plastids are filled with intraplastidial tubules and osmiophilic material identified as secretion (arrows). Fig. 7 - The beginning of sub-cuticular space formation is observed (asterisc). Marker bars 1 [tm. Figs. 8-12. TEM graphs of glandular head cells of trichomes (type I). Figs. 8 and 10 - The sub-cuticular space is filled with a flocculent material where structures like vesicles with electron-dense material (arrows) are disseminated. The SER is very abundant. Fig. 9 - After fixation according to HEPLER (1981) SER is contrasted by an intense stain deposit. At the periplasmic space (asterisc) the electron-dense material is accumulated. Figs. 11 and 12 - The secretion observed at the annular space showes less electron density than the material present in the periplasmic space (asteriscs). Marker bars I [tm.
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Glandular Trichomes of Teucrium scorodonia L.
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material are implicated in essential oil production (BOSABALIDIS & TSEKOS 1982) also HEINRICH (1970) described the presence of intraplastidial tubules in the secretory cells of Rutaceae, and related it with the biogenesis of essential oil. On the other hand GLEIZES et al. (1983) show a direct involvement of plastids as monoterpene precursors in Citrofortunella using labelled IPP. Some enzimes of the mevalonic pathway in the plastid membranes and SER were also detected by cytochemical methods (CURRY 1987). Correlating these data with our ultrastructural observations we are led to the conclusion that in the secretory cells of the trichomes the production of essential oil probably takes place in the plastids. To several workers, the close association of the SER with plastids in the form of periplastidial sheath has suggested that the essential oil, elaborated in the plastids, was translocated via ER and discharged in the periplasmic space (CARDE & BERNARD-DAGAN 1982; HEINRICH & SCHULTZE 1985). In Teucrium scorodonia this association is very evident. The presence of electron-dense material in the lumen of SER and the preferential way that appears marked after treatment with potassium ferricyanide, suggests a role of the ER in the transport of osmiophilic material to the periplasmic space. According to CARDE et al. (1980) the transport of the material to the outside via SER is important to avoid damage to the cell because the compounds of the essential oil are toxic. Since only very few golgi bodies can be found, their participation in synthesis and transport of essential oil seems to be negligible. The material accumulated at the periplasmic space crosses the inner layer of the wall showing, in the sub-cuticular space, different morphological and osmiophilic characteristics. We suggest that the different electron-density be probably due to a chemical alteration at this level. Similar interpretation has been considered by VERMEER & PETERSON (1979) and BOSABALIDIS & TSEKOS (1982). According to these last authors the final mixture of the individual oil components takes place within sub-cuticular space. Although the material accumulated at the periplasmic space does not reveal an apparent membrane, the material accumulated in the sub-cuticular space and disseminated in a flocculent matrix, is involved by an evident membrane. We are unable, at this moment, to explain this situation, but we have reasons to think that this is not an artefact because we have obtained the same results with different fixative solutions and with other species of Teucrium like Teucrium abutiloides (unpublished). Conclusions: In the glandular trichomes of Teucrium scorodonia, plastids are probably involved in synthesis of essential oil. Smooth endoplasmic reticulum is involved in the transport of essential oil from the plastids to the periplasmic space. The electron dense material accumulated in the periplasmic space migrates to the sub-cuticular space crossing the inner layer of cellular wall, revealing a chemical and morphological alteration at this level.
Acknowledgements The financial support was obtained from Instituto Nacional de Investigao;:ao Cientifica (INIC).
References ANTUNES, T., & SEVIN ATE-PINTO, I. (1989): Glandular trichomes of Teucrium scorodonia L. Morphology and histochemistry. Flora 184: 389-394. BOSABALIDIS, A. M., & TSEKOS, I. (1982): Ultrastructure of the essential oil secretion in glandular scales of Origanum dictamnus L. leaves. In: MARGARIS, N., KOEDAM, A., & VOKOU, D. (eds.): Aromatic Plants: Basic and Applied Aspects. Proc. Int. Symp. Aromatic Plants. Martinus Nijhoff Pub!., The Hague, The Netherlands. 3-12. - (1982): Ultrastructural studies on the secretory cavities of Citrus deliciosa TEN. I. Early stages of gland cells. Protoplasma 112: 55-62. CARDE, J. P., & BERNARD-DAGAN C. (1982): Compartimentation de la syntMse terpenique chez Ie pin maritime. Bull. Soc. bot. France 129, Actua!. bot., (1): 53-70. - GLEIZES, M. (1980): Membrane systems involved in synthesis and transport of monoterpene hydrocarbons in pine leaves. In: MAZLIAK, P., BENVENISTE, c., COSTES, c., & DOUCE, R. (eds.): Biogenesis and Function of Plant Lipids. Elsevier, North-Holland Biomed. Press.
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CURRY, K. J. (1987): Initiation of terpenoid synthesis in osmophores of Stanhopea anfracta (Orchidaceae): A cytochemical study. Amer. J. Bot. 74: 1332-1338. ECONOMOU-AMILLI, A., VOKOU, D., ANAGNOSTIDIS, K., & MARGARIS, N. S. (1982): Leaf morphology of Thymus capitatus (Labiatae) by scanning electron microscopy. In: MARGARIS, N., KOEDAM, A., & VOKOU, D. (eds.): Aromatic Plants: Basic and Applied Aspects. Proc. Int. Symp. Aromatic Plants. Martinus Nijhoff Publ., The Hague, The Netherlands. GLEIZES, M., GINETTE, P., CARDE, J. P., MARPEAU, A., & BERNARD-DAGAN, C. (1983): Monoterpene hydrocarbon biosynthesis by isolated leucoplasts of Citrofortunella mitis P1anta 159: 373-381. HEINRICH, G. (1970): Elektronenmikroskopische Beobachtungen an den Driisenzellen von Poncirus trifoliata, zugleich ein Beitrag zur Wirkung atherischer Ole auf Pflanzenzellen und eine Methode zur Unterscheidung fliichtiger von nichtfliichtigen lipophi1en Komponenten. Protop1asma 69: 15-36. SCHULTZE, W., PFAB, I., & BOTTGER, M. (1983): The site of essentiaJ oil biosynthesis in Poncirus trifoliate and Monardafistulosa. Physiol. Veg. 21: 257-268. SCHULTZE, W. (1985): Composition and site of biosynthesis of the essential oil in fruits of Phellodendron amurense RUPR. (Rutaceae). Isr. J. Bot. 34: 205-217. HEPLER, P. K. (1981): The structure of the endoplasmic reticulum revealed by osmium tetroxide-potassium ferricyanide staining. Eur. J. Cell. BioI. 26: 102-110. MARCO, J. L., BENJAMIN, R., PASCUAL, c., SAVONA, G., & P!OZZI, F. (1983): Teuscorodin, teuscorodonin and 2-hydroxyteuscorolide, neo-clerodane diterpenoids from Teucrium scorodonia. Phytochemistry 22: 727-731. MOLLENHAUER, H. H. (1964): Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39: 111-114. SPURLOCK, B. 0., SKINNER, M. S., & KATTlNE, A. A. (1966): A simple rapid method of staining for epoxiembedded specimens for light microscopy with the polychromatic stain Paragon-l301. Am. J. Clin. Pathol. 46: 252-258. VERMEER, J., & PETERSON, R. L. (1979): Glandular trichomes on the inflorescence of Chrysanthemum morifolium cv. Dramatic (Compositae). II Ultrastructure and histochemistry. Can. J. Bot. 57: 714-729. WERKER, E., RAVID, U., & PUTIEVSKY, E. (1985): Glandular hairs and their secretions in the vegetative and reproductive organs of Salvia sclarea and S. dominica. Isf. J. Bot. 34: 239-252. WOODING, F. B. P., & NORTHCOTE, D. H. (1965): The fine structure of the mature resin canal cells of Pinus pinea. J. Ultrastruct. Res. 13: 233-244. Received February 12, 1990 Authors' address: ISABEL SEVINATE-P!NTO and TERESA ANTUNES, Departamento de Biologia Vegetal, Faculdade de Ciencias de Lisboa, BI. C2 Campo Grande, 1700 Lisboa, Portugal.