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Zinc iodide-osmium tetroxide (ZIO) reactive sites in the extrafloral nectary of Citharexylum mirianthum Cham. (Verbenaceae)
Tissue & Cell, 2001 33 (1) 72±77 ß 2001 Harcourt Publishers Ltd DOI: 10.1054/tice.2001.0157, available online at http://www.idealibrary.com
Tissue&Cell
Zinc iodide-osmium tetroxide (ZIO) reactive sites in the extrafloral nectary of Citharexylum mirianthum Cham. (Verbenaceae) S. R. Machado,1 E. A. GregoÂrio2 Abstract. This paper reports on a study of the zinc iodide-osmium tetroxide method (ZIO) applicability to formaldehyde-glutaraldehyde prefixed extrafloral nectary tissues of Citharexylum mirianthum Cham. (Verbenaceae). The ZIO solution impregnates the dictyosome stacks and adjacent vesicles, smooth endoplasmic reticulum, nuclear envelope, multivesicular bodies, and peroxisomes. The use of this method greatly facilitates the observation and recognition of organelles in each nectary region. It also allows the correlation between structure and function in nectariferous cells. ß 2001 Harcourt Publishers Ltd Keywords: ZIO method, ultrastructure, extrafloral nectary, Citharexylum mirianthum, Verbenaceae
Introduction Some authors (see McDade & Turner, 1997) have called attention to the structural complexity and diversity of the extra¯oral nectaries, especially in members of the Lamiales that include the families Acanthaceae, Bignoniaceae, and Verbenaceae. Citharexylum mirianthum Cham., a tall deciduous forest species of the Verbenaceae family, has nectaries located at the petiole and lower surface of the blade. The petiolar nectaries are responsible for the secretion of large quantities of nectar in young leaves. Previous work (Machado, 1999) has shown that these nectaries are composed of three regions, each with distinct morphological and functional features. These regions include a layer of elongated palisade-like epidermal cells, up to
1
Departamento de botaÃnica, Instituto de BiocieÃncias, UNESP, CaÃmpus de Botucatu, C.P.510, CEP 18618-000, Botucatu, SP, Brasil, 2Centro de Microscopia EletroÃnica, Instituto de BiocieÃncias, UNESP, CaÃmpus de Botucatu, C.P.510, CEP 18618-000, Botucatu, SP, Brasil Received 27 June 2000 Accepted 17 October 2000 Correspondence to: Silvia Rodrigues Machado. Tel.: 55 14 6802 6053; Fax: 55 14 6821 3744; E-mail: [email protected]
72
two layers of endodermoid sub-epidermal cells, and ®ve±seven layers of parenchyma cells. Studies on extra¯oral nectary structure and ultrastructure as well as on the relationship between the sub-cellular structures and possible mechanisms of the secretory process are numerous (see Fahn, 1979, 1988, 1990; Durkee, 1983). However, most of the ultrastructural investigations use conventional techniques, which do not always allow a clear characterization of the membranous organelles in the nectariferous cells. In order to study issues related to plant secretory systems and to correlate structure and function, cytochemical techniques associated with conventional ultrastructural investigations are essential (for review see Hall & Hawes, 1991). The zinc iodide-osmium tetroxide method (ZIO), which was ®rst introduced by Maillet (1962) for neurohistochemical studies, is suitable for general impregnation of the endomembrane system of many plant, algal, and fungal tissues (Hawes, 1991). However, the use of this method to study specialized plant cells is not common (Harris, 1979; Hawes, 1981). The objective of this work is to report on the applicability of the ZIO method to study sub-cellular compartments of the three nectary regions in Citharexylum mirianthum.
THE ZIO METHOD IN EXTRAFLORAL NECTARY 73
V
D3 D2
D
D1
2
1
T
3
4
Figs 1±4 Epidermal cells. 1. Stained prominent dictyosomes (D1 and D2); Dictyosome (D3) with non-stained cisternae. Bar 0.3 mm. 2. Stained endoplasmic reticulum near vacuole (V). Dictyosome (D). Bar 0.4 mm. 3. Stained vesicles of different sizes near the forming face of dictyosome. Bar 0.2 mm. 4. Heavily impregnated vesicles and a few endoplasmic reticulum elements associated with the tonoplast (T). Bar 0.2 mm.
74
MACHADO AND GREGORIO
W
5
6
W Mb
7
8
THE ZIO METHOD IN EXTRAFLORAL NECTARY 75
Figs 5±8 Sub-epidermal cells. 5. Stained extensive endoplasmic reticulum near the anticlinal cell wall (W). Bar 0.2 mm. 6. Vesicles with dense deposits are
incorporated into the vacuole. Note the incomplete tonoplast (arrows). Bar 0.2 mm. 7. Extensive endoplasmic reticulum near the numerous plasmodesmata. The arrows indicate the desmotubule. Cell wall (W). Bar 0.4 mm. 8. Stained multivesicular bodies (Mb) fragmented into vesicles. Note the stained nuclear membrane (arrow). Bar 0.4 mm.
Material and methods Petiolar nectaries samples were obtained from Citharexylum mirianthum (Verbenaceae) specimens, growing in a semi-deciduous forest at the Edgardia Reserve, SaÄo Paulo State (22836'37"), southeastern Brazil. Samples were ®xed for 12 h with 2.5% glutaraldehyde-4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.3, and incubated for 22 h at 108C in a solution containing Zn, I, TRISaminomethane, and OsO4 (Reinecke and Walther 1978). Samples were then dehydrated using a graded acetone series and embedded in Araldite resin at room temperature. Polymerization was performed at 608C for 48 h. Ultrathin sections were double-stained with uranyl acetate and lead citrate and observed in a Philips 100 EM electron microscope at 60 kV.
Results The ZIO method revealed sub-cellular structures in the three nectary regions. Epidermal cells These cells showed many well-structured dictyosomes with adjacent vesicles (Fig. 1). Dictyosome polarization, which comprises a forming face and a secretion face, was clearly visible. These well-structured dictyosomes were completely impregnated, and deposits of the reaction products were seen in the lumen of cisternae, in many of the vesicles at the ends of such cisternae, and in the vesicles and vacuoles associated with dictyosomes (Fig. 3). Some dictyosomes with a few un¯attened cisternae did not stain (Figs 1 & 2). Vesicles of different sizes could be seen near the dictyosome forming face and the endoplasmic reticulum (Fig. 3). These vesicles showed different ZIO impregnation intensities. However, the vesicles associated with the dictyosome secretion face were intensely stained (Figs 1 & 3). The membrane of some vacuoles showed heavy depositions of metal, and most of these vacuoles were associated with dictyosome cisternae (Fig. 4). The sub-epidermal cells In these cells, the smooth endoplasmic reticulum was abundant and heavily impregnated with metal (Figs 5 & 7). The vacuolar membranes (Fig. 6), nuclear envelope, and multivesicular bodies (Fig. 8) were also stained. These cells showed numerous plasmodesmata, but they were not stained by the ZIO reactive (Figs 6 & 7).
The parenchyma cells In these cells, the ZIO reactive stained the nuclear envelope (Fig. 9), plastid thylakoids (Fig. 10), dictyosomes (Figs 10 & 11), and showed structured peroxisomes (Fig. 12).
Discussion The ultrastructural differentiation of the three nectary regions in C. mirianthum was remarkably consistent using the ZIO method. The striking distinction of the epidermal cells is due to the heavy deposition of metal on the dictyosomes. The cytochemical af®nity of the ZIO for the Golgi apparatus and its vesicles have been widely demonstrated in several animal and plant tissues (Hall & Hawes, 1991). The numerous dictyosomes in the epidermal cells can be correlated with the synthesis and secretion of the hydrophilic components of exudate, as suggested by Fahn (1988). The presence of many secretory vesicles during the secretory stage has been related to nectar transport (Jian et al., 1997). Reaction variations between impregnated dictyosome cisternae and/or vesicle membranes in secretory systems are clear, yet the probable cause of the af®nity to ZIO is not known. However, these variations can result from modi®cations in the dictyosome membrane properties during their functioning (Hall & Hawes, 1991). The sub-epidermal cells show a large amount of smooth endoplasmic reticulum. This organelle can be related to the synthesis and secretion of the lipid, one of the main components of extra¯oral nectary exudate. This sub-cellular compartment is not easily seen in conventional nectariferous cell preparations. Abundant smooth endoplasmic reticulum and multivesicular bodies close to the plasmodesmata in the subepidermal cells might be associated with the transport of pre-nectar to the epidermal secretory cells. The endoplasmic reticulum involvement in translocation and/or temporary concentration of sugars in nectaries has been suggested by several authors (see Fahn, 1979; Durkee, 1983; Figueiredo & Pais, 1992; Jian et al., 1997). According to Mohan & Inamdar (1986), the presence of numerous plasmodesmata in cell walls between the epidermal and sub-epidermal cells clearly indicates the pre-nectar transport pathways in the symplast. The prominent well-structured peroxisomes and chloroplasts in the parenchyma cells could be related to the lipid biosynthesis (Hall et al., 1982). The characteristics compartments of these cells, which become more
76
MACHADO AND GREGORIO
9
10
11
12
Figs 9±12 Parenchyma cells. 9. Stained nuclear membrane. Note the nuclear pores (arrows). Bar 0.4 mm. 10. Stained plastid thylakoids. Bar 0.3 mm.
11. Stained prominent dictyosomes and vesicles. Note that the intensity of the reaction diminishes in the dictyosome median zone. Bar 0.2 mm. 12. Stained peroxisomes showing a system of tubules and a cuneiform inclusion. Bar 0.2 mm.
THE ZIO METHOD IN EXTRAFLORAL NECTARY 77
visible with the ZIO, suggest their participation on the nectar biosynthesis. The chemical nature of the material that is preferentially and heavily impregnated with metal by the ZIO method cannot be ascertained (Hall & Hawes, 1991). Some authors have postulated that the material, which is heavily impregnated after ®xation with ZIO, might be a lipid (Niebauer et al., 1969). According to Maillet (1962), the ZIO solution acts to uncouple lipid moieties from lipoprotein complexes, and the newly exposed lipid groups would then be available for increased metal deposition. It has also been suggested that in the ZIO procedure, Zn2 either binds to or displaces Ca2 sites on membranes and osmium is reduced to the form of electron-opaque zinc osmate deposits (Gilloteaux & Naud, 1979). Although the chemical basis for the ZIO reaction has not yet been elucidated, our results show that this reaction greatly facilitates the observation of membranes and helps to elucidated the role of nectary regions and subcellular compartments associated with nectar secretion. ACKNOWLEDGEMENT The authors wish to thank Mrs M.M.H. Moreno, Mrs D.C. Santos and Mrs M.E.L. Perez for technical assistance, Dr Denise M. T. Oliveira for useful suggestions, and Ms H.M.P. Toledo for English review. REFERENCES Durkee, L.T. 1983. The ultrastructure of floral and extra-floral nectaries. In: The biology of nectaries B.L. Bentley and T.S. Elias' (eds), Columbia University Press, New York, pp 4±29.
Fahn, A. 1979. Ultrastructure of nectaries in relation to nectar secretion. Am. J. Bot., 66, 977±985. Fahn, A. 1988. Secretory tissues in vascular plants. New Phytol., 108, 229±257. Fahn, A. 1990. Plant anatomy. Pergamon Press, Oxford, 463±465. Figueiredo, A.C.S. and Pais, M.S. 1992. Ultrastructural aspects of the nectary spur of Limodorun abortivum (L.) Sw. (Orchidaceae). Ann. Bot., 70, 325±331. Gilloteux, J. and Naud, J. 1979. The zinc iodide-osmium tetroxide staining-fixative of Maillet. Histochemistry, 63, 227±243. Hall, J.L., Flowers, T.J. and Roberts, R.M. 1982. Plant cell structure and metabolism. Longman, New York. Hall, J.L. and Hawes, C. 1991. Electron microscopy of plant cells. Academic Press, London. Harris, N. 1979. Endoplasmic reticulum in development seeds of Vicia faba. A high voltage electron microscope study. Planta, 146, 63±69. Hawes, C. 1981. Applications of high voltage electron microscopy to botanical ultrastructure. Micron, 12, 227±257. Hawes, C. 1991. Stereo-electron microscopy. In: Electron microscopy of plant cells. J.L. Hall and C. Hawes (eds), Academic Press, London, 67±84. Jian, Z., Zheng-hai, H. and MuÈller, M. 1997. Ultrastructure of the floral nectary of Arabidopsis thaliana L. prepared from high pressure freezing and freeze substitution. Acta Bot. Sin., 39, 289±295. Machado, S.R. 1999. Estrutura e desenvolvimento de nectaÂrios extraflorais de Citharexylum mirianthum Cham. (Verbenaceae). Thesis, UNESP, Botucatu, SaÄo Paulo, Brasil. Maillet, M. 1962. Le reactif au tetraoxyde d'osmium-iodure au zinc. Z. Zellforsch., 70, 397±406. McDade, L.A. and Turner, M.D. 1997. Structure and development of bracteal nectary glands in Aphelandra (Acanthaceae). Am. J. Bot., 84(1), 1±15. Mohan, J.S.S. and Inamdar, J.A. 1986. Ultrastructure and secretion of extrafloral nectaries of Plumeria rubra L. Ann. Bot., 57, 389±401. Niebauer, G., Krawczyk, W.S., Kidd, R.L. and Wilgran, G.F. 1969. Osmium zinc iodide reactive sites in the epidermal Langerhans cell. J. Cell Biol., 43, 80±89. Reinecke, M. and Walther, C. 1978. Aspects of turnover and biogenesis of synaptic vesicles at locust neuromuscular junctions as reveled by iodide-osmium tetroxide (ZIO) reacting with intravesicular sh-groups. J. Cell Biol., 21, 839±855.
Tissue&Cell
Zinc iodide-osmium tetroxide (ZIO) reactive sites in the extrafloral nectary of Citharexylum mirianthum Cham. (Verbenaceae) S. R. Machado,1 E. A. GregoÂrio2 Abstract. This paper reports on a study of the zinc iodide-osmium tetroxide method (ZIO) applicability to formaldehyde-glutaraldehyde prefixed extrafloral nectary tissues of Citharexylum mirianthum Cham. (Verbenaceae). The ZIO solution impregnates the dictyosome stacks and adjacent vesicles, smooth endoplasmic reticulum, nuclear envelope, multivesicular bodies, and peroxisomes. The use of this method greatly facilitates the observation and recognition of organelles in each nectary region. It also allows the correlation between structure and function in nectariferous cells. ß 2001 Harcourt Publishers Ltd Keywords: ZIO method, ultrastructure, extrafloral nectary, Citharexylum mirianthum, Verbenaceae
Introduction Some authors (see McDade & Turner, 1997) have called attention to the structural complexity and diversity of the extra¯oral nectaries, especially in members of the Lamiales that include the families Acanthaceae, Bignoniaceae, and Verbenaceae. Citharexylum mirianthum Cham., a tall deciduous forest species of the Verbenaceae family, has nectaries located at the petiole and lower surface of the blade. The petiolar nectaries are responsible for the secretion of large quantities of nectar in young leaves. Previous work (Machado, 1999) has shown that these nectaries are composed of three regions, each with distinct morphological and functional features. These regions include a layer of elongated palisade-like epidermal cells, up to
1
Departamento de botaÃnica, Instituto de BiocieÃncias, UNESP, CaÃmpus de Botucatu, C.P.510, CEP 18618-000, Botucatu, SP, Brasil, 2Centro de Microscopia EletroÃnica, Instituto de BiocieÃncias, UNESP, CaÃmpus de Botucatu, C.P.510, CEP 18618-000, Botucatu, SP, Brasil Received 27 June 2000 Accepted 17 October 2000 Correspondence to: Silvia Rodrigues Machado. Tel.: 55 14 6802 6053; Fax: 55 14 6821 3744; E-mail: [email protected]
72
two layers of endodermoid sub-epidermal cells, and ®ve±seven layers of parenchyma cells. Studies on extra¯oral nectary structure and ultrastructure as well as on the relationship between the sub-cellular structures and possible mechanisms of the secretory process are numerous (see Fahn, 1979, 1988, 1990; Durkee, 1983). However, most of the ultrastructural investigations use conventional techniques, which do not always allow a clear characterization of the membranous organelles in the nectariferous cells. In order to study issues related to plant secretory systems and to correlate structure and function, cytochemical techniques associated with conventional ultrastructural investigations are essential (for review see Hall & Hawes, 1991). The zinc iodide-osmium tetroxide method (ZIO), which was ®rst introduced by Maillet (1962) for neurohistochemical studies, is suitable for general impregnation of the endomembrane system of many plant, algal, and fungal tissues (Hawes, 1991). However, the use of this method to study specialized plant cells is not common (Harris, 1979; Hawes, 1981). The objective of this work is to report on the applicability of the ZIO method to study sub-cellular compartments of the three nectary regions in Citharexylum mirianthum.
THE ZIO METHOD IN EXTRAFLORAL NECTARY 73
V
D3 D2
D
D1
2
1
T
3
4
Figs 1±4 Epidermal cells. 1. Stained prominent dictyosomes (D1 and D2); Dictyosome (D3) with non-stained cisternae. Bar 0.3 mm. 2. Stained endoplasmic reticulum near vacuole (V). Dictyosome (D). Bar 0.4 mm. 3. Stained vesicles of different sizes near the forming face of dictyosome. Bar 0.2 mm. 4. Heavily impregnated vesicles and a few endoplasmic reticulum elements associated with the tonoplast (T). Bar 0.2 mm.
74
MACHADO AND GREGORIO
W
5
6
W Mb
7
8
THE ZIO METHOD IN EXTRAFLORAL NECTARY 75
Figs 5±8 Sub-epidermal cells. 5. Stained extensive endoplasmic reticulum near the anticlinal cell wall (W). Bar 0.2 mm. 6. Vesicles with dense deposits are
incorporated into the vacuole. Note the incomplete tonoplast (arrows). Bar 0.2 mm. 7. Extensive endoplasmic reticulum near the numerous plasmodesmata. The arrows indicate the desmotubule. Cell wall (W). Bar 0.4 mm. 8. Stained multivesicular bodies (Mb) fragmented into vesicles. Note the stained nuclear membrane (arrow). Bar 0.4 mm.
Material and methods Petiolar nectaries samples were obtained from Citharexylum mirianthum (Verbenaceae) specimens, growing in a semi-deciduous forest at the Edgardia Reserve, SaÄo Paulo State (22836'37"), southeastern Brazil. Samples were ®xed for 12 h with 2.5% glutaraldehyde-4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.3, and incubated for 22 h at 108C in a solution containing Zn, I, TRISaminomethane, and OsO4 (Reinecke and Walther 1978). Samples were then dehydrated using a graded acetone series and embedded in Araldite resin at room temperature. Polymerization was performed at 608C for 48 h. Ultrathin sections were double-stained with uranyl acetate and lead citrate and observed in a Philips 100 EM electron microscope at 60 kV.
Results The ZIO method revealed sub-cellular structures in the three nectary regions. Epidermal cells These cells showed many well-structured dictyosomes with adjacent vesicles (Fig. 1). Dictyosome polarization, which comprises a forming face and a secretion face, was clearly visible. These well-structured dictyosomes were completely impregnated, and deposits of the reaction products were seen in the lumen of cisternae, in many of the vesicles at the ends of such cisternae, and in the vesicles and vacuoles associated with dictyosomes (Fig. 3). Some dictyosomes with a few un¯attened cisternae did not stain (Figs 1 & 2). Vesicles of different sizes could be seen near the dictyosome forming face and the endoplasmic reticulum (Fig. 3). These vesicles showed different ZIO impregnation intensities. However, the vesicles associated with the dictyosome secretion face were intensely stained (Figs 1 & 3). The membrane of some vacuoles showed heavy depositions of metal, and most of these vacuoles were associated with dictyosome cisternae (Fig. 4). The sub-epidermal cells In these cells, the smooth endoplasmic reticulum was abundant and heavily impregnated with metal (Figs 5 & 7). The vacuolar membranes (Fig. 6), nuclear envelope, and multivesicular bodies (Fig. 8) were also stained. These cells showed numerous plasmodesmata, but they were not stained by the ZIO reactive (Figs 6 & 7).
The parenchyma cells In these cells, the ZIO reactive stained the nuclear envelope (Fig. 9), plastid thylakoids (Fig. 10), dictyosomes (Figs 10 & 11), and showed structured peroxisomes (Fig. 12).
Discussion The ultrastructural differentiation of the three nectary regions in C. mirianthum was remarkably consistent using the ZIO method. The striking distinction of the epidermal cells is due to the heavy deposition of metal on the dictyosomes. The cytochemical af®nity of the ZIO for the Golgi apparatus and its vesicles have been widely demonstrated in several animal and plant tissues (Hall & Hawes, 1991). The numerous dictyosomes in the epidermal cells can be correlated with the synthesis and secretion of the hydrophilic components of exudate, as suggested by Fahn (1988). The presence of many secretory vesicles during the secretory stage has been related to nectar transport (Jian et al., 1997). Reaction variations between impregnated dictyosome cisternae and/or vesicle membranes in secretory systems are clear, yet the probable cause of the af®nity to ZIO is not known. However, these variations can result from modi®cations in the dictyosome membrane properties during their functioning (Hall & Hawes, 1991). The sub-epidermal cells show a large amount of smooth endoplasmic reticulum. This organelle can be related to the synthesis and secretion of the lipid, one of the main components of extra¯oral nectary exudate. This sub-cellular compartment is not easily seen in conventional nectariferous cell preparations. Abundant smooth endoplasmic reticulum and multivesicular bodies close to the plasmodesmata in the subepidermal cells might be associated with the transport of pre-nectar to the epidermal secretory cells. The endoplasmic reticulum involvement in translocation and/or temporary concentration of sugars in nectaries has been suggested by several authors (see Fahn, 1979; Durkee, 1983; Figueiredo & Pais, 1992; Jian et al., 1997). According to Mohan & Inamdar (1986), the presence of numerous plasmodesmata in cell walls between the epidermal and sub-epidermal cells clearly indicates the pre-nectar transport pathways in the symplast. The prominent well-structured peroxisomes and chloroplasts in the parenchyma cells could be related to the lipid biosynthesis (Hall et al., 1982). The characteristics compartments of these cells, which become more
76
MACHADO AND GREGORIO
9
10
11
12
Figs 9±12 Parenchyma cells. 9. Stained nuclear membrane. Note the nuclear pores (arrows). Bar 0.4 mm. 10. Stained plastid thylakoids. Bar 0.3 mm.
11. Stained prominent dictyosomes and vesicles. Note that the intensity of the reaction diminishes in the dictyosome median zone. Bar 0.2 mm. 12. Stained peroxisomes showing a system of tubules and a cuneiform inclusion. Bar 0.2 mm.
THE ZIO METHOD IN EXTRAFLORAL NECTARY 77
visible with the ZIO, suggest their participation on the nectar biosynthesis. The chemical nature of the material that is preferentially and heavily impregnated with metal by the ZIO method cannot be ascertained (Hall & Hawes, 1991). Some authors have postulated that the material, which is heavily impregnated after ®xation with ZIO, might be a lipid (Niebauer et al., 1969). According to Maillet (1962), the ZIO solution acts to uncouple lipid moieties from lipoprotein complexes, and the newly exposed lipid groups would then be available for increased metal deposition. It has also been suggested that in the ZIO procedure, Zn2 either binds to or displaces Ca2 sites on membranes and osmium is reduced to the form of electron-opaque zinc osmate deposits (Gilloteaux & Naud, 1979). Although the chemical basis for the ZIO reaction has not yet been elucidated, our results show that this reaction greatly facilitates the observation of membranes and helps to elucidated the role of nectary regions and subcellular compartments associated with nectar secretion. ACKNOWLEDGEMENT The authors wish to thank Mrs M.M.H. Moreno, Mrs D.C. Santos and Mrs M.E.L. Perez for technical assistance, Dr Denise M. T. Oliveira for useful suggestions, and Ms H.M.P. Toledo for English review. REFERENCES Durkee, L.T. 1983. The ultrastructure of floral and extra-floral nectaries. In: The biology of nectaries B.L. Bentley and T.S. Elias' (eds), Columbia University Press, New York, pp 4±29.
Fahn, A. 1979. Ultrastructure of nectaries in relation to nectar secretion. Am. J. Bot., 66, 977±985. Fahn, A. 1988. Secretory tissues in vascular plants. New Phytol., 108, 229±257. Fahn, A. 1990. Plant anatomy. Pergamon Press, Oxford, 463±465. Figueiredo, A.C.S. and Pais, M.S. 1992. Ultrastructural aspects of the nectary spur of Limodorun abortivum (L.) Sw. (Orchidaceae). Ann. Bot., 70, 325±331. Gilloteux, J. and Naud, J. 1979. The zinc iodide-osmium tetroxide staining-fixative of Maillet. Histochemistry, 63, 227±243. Hall, J.L., Flowers, T.J. and Roberts, R.M. 1982. Plant cell structure and metabolism. Longman, New York. Hall, J.L. and Hawes, C. 1991. Electron microscopy of plant cells. Academic Press, London. Harris, N. 1979. Endoplasmic reticulum in development seeds of Vicia faba. A high voltage electron microscope study. Planta, 146, 63±69. Hawes, C. 1981. Applications of high voltage electron microscopy to botanical ultrastructure. Micron, 12, 227±257. Hawes, C. 1991. Stereo-electron microscopy. In: Electron microscopy of plant cells. J.L. Hall and C. Hawes (eds), Academic Press, London, 67±84. Jian, Z., Zheng-hai, H. and MuÈller, M. 1997. Ultrastructure of the floral nectary of Arabidopsis thaliana L. prepared from high pressure freezing and freeze substitution. Acta Bot. Sin., 39, 289±295. Machado, S.R. 1999. Estrutura e desenvolvimento de nectaÂrios extraflorais de Citharexylum mirianthum Cham. (Verbenaceae). Thesis, UNESP, Botucatu, SaÄo Paulo, Brasil. Maillet, M. 1962. Le reactif au tetraoxyde d'osmium-iodure au zinc. Z. Zellforsch., 70, 397±406. McDade, L.A. and Turner, M.D. 1997. Structure and development of bracteal nectary glands in Aphelandra (Acanthaceae). Am. J. Bot., 84(1), 1±15. Mohan, J.S.S. and Inamdar, J.A. 1986. Ultrastructure and secretion of extrafloral nectaries of Plumeria rubra L. Ann. Bot., 57, 389±401. Niebauer, G., Krawczyk, W.S., Kidd, R.L. and Wilgran, G.F. 1969. Osmium zinc iodide reactive sites in the epidermal Langerhans cell. J. Cell Biol., 43, 80±89. Reinecke, M. and Walther, C. 1978. Aspects of turnover and biogenesis of synaptic vesicles at locust neuromuscular junctions as reveled by iodide-osmium tetroxide (ZIO) reacting with intravesicular sh-groups. J. Cell Biol., 21, 839±855.