Acta Bot. Neerl. 46(4), December 1997, p.413-420 © 1997 Royal Botanical Society of The Netherlands Secretory tissues of the flower of Sanango racemosum (Gesneriaceae). I. Light microscopy Sara Maldonadoi* and Marisa Oteguiif SUMMARY Sanango racemosum (Ruiz & Pav.) Barringer has a dry stigma without a free-flowing secretion fluid but with a hydrated proteinaceous pellicle. The stigmatic surface is covered with unicellular, bottle-shaped papillae. At maturity, a viscous emulsion is accumulated between the cuticle and the pecto-cellulosic wall of the papillae, causing it to become detached from the surface of the papilla cell walls. The style has a central solid core of transmitting tissue. The cells of the transmitting tissue are rich in starch and exhibit thick lateral walls rich in pectic substance. The nectary disk is a ring elongated into a cup, with five lobes at the top. One of the most conspicuous histological features of the disk is the abundance of starch in the secretory cells. The disk is supplied only by phloem; the stomata are found in the top of the lobes. A fluid substance is produced just before anthesis and secreted through the stomata with no visible decline in starch level. During anthesis and after fertilization, a rapid decline in starch is observed. The hypothesis that the disk has other functions besides that of a nectary is discussed. Key-words: disk, nectary, osmophore, Sanango, stigma, transmitting tissue. INTRODUCTION * Institute de Recursos Bioldgicos, INTA 1712, Villa Udaondo, Castelar, Argentina; fFacultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, 1900 La Plata, Argentina The monotypic genus Sanango G. S. Bunting and J. A. Duke is related to the Scrophulariales but what the most satisfactory family assignment of the genus should be remains to be seen. On the basis of the anatomical, morphological, embryological and chemical evidence, S. racemosum (Ruiz & Pav.) Barringer appears to be closely related to the Gesneriaceae but also shares important embryological features with the Scrophulariaceae (Dickison 1994; Jensen 1994; Maldonadoet al. unpublished; Norman 1994; Wiehler 1994). Most families of the order Scrophulariales present an annular nectary-disk around the base of the ovary. Wiehler (1994) reported that the pronounced development of a nectary with the nectariferous tissue elongated into a cup as seen in the flowers of Sanango is not found in the Scrophulariaceae, nor in any other family of the Scrophulariales, except in the genus Anetanthus of the Gesneriaceae. In any case, at present, little is known about the secretory tissue of this annular nectary disk. The morphological characteristics of the stigma have scarcely been accorded any significance in taxonomic and phylogenetic studies of the Scrophulariales. It seems that they could not be added to the list of criteria to be taken into account when classifying 414 S. MALDONADO AND M. OTEGUI © 1997 Royal Botanical Society ofThe Netherlands, Acta Bot. Neerl. 46, 413-420 This is the first histological study of cup-shaped disk and stigmatic-transmitting tissues of S. racemosum. A taxonomic comparison of these structures is impossible at present because of a lack of analogous studies of genera in families to which Sanango may be related. Much remains to be known about the ultrastructure of the tissue and composition of the secretion. We propose to take up these topics in further papers. MATERIAL AND METHODS Source of material. Sanango racemosum: Ecuador, Napo: Jatun Sacha, 21 Sep 1990, Neill 9458 (DLF, MO. US). Specimen preparation. Flowers fixed in FAA (formalin acetic acid-ethylic alcohol) were embedded in paraffin and sectioned. Sections were cut at 8-10 pm and stained with iodine-potassium iodideand periodic acid-Schiff (PAS) (O’Brien & McCully 1981); fast green FCF (Fulcher et al. 1972); Sudan black (Bronner 1975); ruthenium red, Coomassie brilliant blue (Pearse 1985); acid fushsin and toluidine blue O (Feder & O'Brien 1968). RESULTS Secretory disk The disk is a ring elongated into a cup between stamen bases and ovary (Fig. la), and forming five lobes at the top. It consists of a specialized parenchymatous tissue covered by an epidermis without trichomes. The epidermis is composed of cells with dense cytoplasm and starch plastids. Starch was determinedwith iodine-potassium iodide and PAS. Stomata occur at the tops of the lobes and their guard cells also contain starch grains (Fig. lc,d). The secretory tissue is composed of small cells with thin walls, densely staining cytoplasm, small vacuoles and relatively large nuclei. Starch plastids are present in large amounts. Intercellular spaces are well defined in the parenchymatous tissue and a cuticular lining occurs along some intercellular spaces. Cell walls were stained with PAS, acid fuchsin and toluidine blue O and cutin was determinedwith Sudan black. Synthesis of starch grains occurs during flower development and their number increases dramatically with the approach of anthesis (Fig. le). A fluid substance is produced just before anthesis (Fig. lb) and is secreted through the stomata (Fig. lc,d) with no visible decline in starch level. During anthesis and after fertilization, a rapid decline in starch is observed. The disk is initially supplied by five traces composed entirely of phloem. The frequent branching of these results in the formation of a rich vascular system composed of a high number of bundles that extend acropetally into the disk. Phloem elements come into direct contact with the secretory cells (Fig. If). Stigmatic and transmitting tissues The stigma of Sanango is capitate with a bilobed head (Fig. 3a). The stigmatic surface is covered with unicellular bottle-shaped papillae, being the elongate tip of the receptive part of the papillae (Fig. 2b-e). the order Scrophulariales since wet and dry, papillate and non-papillate stigmas have been described in families of the order, including Scrophulariaceae and Gesneriaceae (Dumas 1975; Weber & Westphal 1978; Heslop-Harrison & Heslop-Harrison 1981). 415SECRETORY TISSUES OF THE FLOWER OF SANANGO RACEMOSUM © 1997 Royal Botanical Society ofThe Netherlands, Ada Hot. Neerl. 46, 413-420 Papillae are closely packed in young stigmas (Fig. 2a). During flower development, papillae separate and cuticle becomes demonstrable at the LM-level (Fig. 2b). A distinctive feature of these cells is the presence of numerous grains of starch. Such grains, single or compound, are iodine-potassium iodideand PAS positive. Cells of the subtending parenchyma also contain starch, but the grains are larger. In the papillate cells the cytoplasm stains densely with fast green and Coomassie blue. A relatively large vacuole is present at their base (Fig. 2b,c). At maturity a very thin lipidic-proteinaceous pellicle, detected only after staining, overlies the cuticle. The protein was detected by staining with Coomassie blue and fast green FCF, and lipid by Sudan black. Later, a viscous emulsion accumulates between the cuticle and the pecto-cellulosic walls of the papillae, causing it to become detached from the surface of the cell walls (Fig. 2c). The cuticle is retained during pollination (Fig. 2d,e), suffering only partial disruption. No polysaccharides, lipids or proteins were present in the emulsion, as determined by a lack of staining with PAS, Sudan black and fast green FCF. Fig. 1. (a) Secretory disk. Scale bar: 100 pm (b) Disk secreting a fluid substance just before anthesis (stained with PAS reaction and fast green FCF). Scale bar: 100 |im (c) and (d) Detail of the disk secreting the fluid substance through the stomata (arrows) (stained with PAS reaction and fast green FCF), Scale bar: 10 pm, (e) Secretory tissue of the disk with abundant starch (positive iodine-potassium iodide and PAS reactions). Scale bar: 50 pm. (f) Phloem bundles (arrows) in the secretory tissues of the disk. Scale bar: 10 pm. D, disk; S, semi-liquid secretion. 416 S. MALDONADO AND M. OTEGUI © 1997 Royal Botanical Society of The Netherlands, Ada 801. Neert 46, 413-420 The style has a centralsolid core of transmitting tissue. Cells of the transmitting tissue have some features in common with those of the stigma i.e. they have dense cytoplasm and are rich in starch (Fig. 3b). These cells exhibit thick lateral walls, rich in pectic substances, as determined with ruthenium red. The pollen tubes grow intercellularly through the outermost wall layer. The single strand of transmitting tissue is connected to the two placentae of the ovary constituting a compitum (Fig. 3c,d). (Latin: the open space where two or more ways meet, a crossing. According to Carr & Carr (1961), the eu-syncarpous gynoeceum is characterized by the possession of a compitum, a connec- tion between the carpels which allows the pollen tubes from grains germinating on any lobe of stigma to cross over to more than one loculus.) Pollen grains adhere to the stigmatic surface. Those that germinate produce tubes which penetrate the cuticle (Fig. 2d,e) and grow towards the base of the papilla in the space occupied by the viscous emulsion. At thebase of the papilla the tube continues to grow, moving intercellularly in the secretion produced by the subtending parenchyma and then in the transmitting tissue of the style. In the ovary, the pollen tubes go through the compitum then reach the secretory epidermis of the placenta. Pollen tubes are visible exiting the placenta, growing on the funiculus and penetrating the micropyle. DISCUSSION According to Wiehler (1994) the cup-shaped disk of Sanango is a nectary. However, histological features, such as the abundance of starch in the secretory cells of the disk might indicate that the disk has other functions besides that of a nectary. Even though Fig. 2. (a) Young stigma with closely packed papillae. Scale bar: 10 pm. (b) Separated papillae covered with a cuticle (arrow). Scale bar: 10 pm. (c) Mature stigma with the detached cuticle (arrows). A very thin lipidic proteinaceous pellicle overlies the cuticle (staining with Coomassie brilliant blue, fast green FCF and Sudan black). Large vacuoles are present in the papilla bases. Scale bar: 10pm. (d) and (e) Pollen grains adhered to the stigmatic surface producing tubes which penetrate the cuticle (arrows). Scale bar: 10 pm. G, pollen grains; P, papillae; V, vacuoles. 417SECRETORY TISSUES OF THE FLOWER OF SANANGO RACEMOSUM © 1997 Royal Botanical Society of The Netherlands, Acta Bot. Neeri 46, 413-420 in some plants, e.g. Passiflora (Dumas et al. 1981; Durkee 1983) and Rosmarinus officinalis (Zer & Fahn 1992), the amount of starch in the nectary of the flowers is very large and starts to decline as the nectary approaches the state of secretion, in almost all studies so far starch is present in a nectary only in small amounts that commonly disappear as secretion progresses (Durkee 1983). Conversely, cells rich in cytoplasm and filled with starch grains are associated with the osmophores which are restricted to certain areas ofthe flowerorgans, including nectaries(Fahn 1979; Roschina & Roschina 1993). Coincidentally, this statement is supported by an herbarium label (Dwyer 6228, from Tingo Maria, Peru) that notes that the flowers of Sanango are very fragrant Fig. 3. (a) Bilobed stigma, style and part of the ovary. Scale bar: 100 pm. (b) Central, solid core of transmittingtissue in the style. Transmittingcells with starch grains (arrows) and thick lateral walls. Scale bar: 10 pm. (c) and (d) The compitum connecting the two placentae (stained with PAS reaction and fast green FCF). Scale bar: 50 pm. C, compitum; O, ovary; S, stigma; St, style. 418 S. MALDONADO AND M. OTEGUI © 1997 Royal Botanical Society of The Netherlands, Acta Bot. Neerl. 46, 413-420 (Wiehler 1994). The chemical composition of the secretion has not yet been studied; if it were, it could perhaps reveal that the disk produces a combination of nectar and essential oils. The massive store of reserve material, which is utilized during emission, would provide the energy for the endothermic synthesis of the fragrance material as well as of building material. It should be emphasized that there are few direct experimental data confirming the existence of one or the other role of the disk in Scrophulariales. Few descriptions of the nectar vascular tissue are available. In almost all studies so far, the vascular bundles consist of both phloem and xylem. Nectaries supplied entirely by phloem have been reported by Fahn (1979) for flowers of different families of Dicotyledons: Aceraceae, Crassulaceae, Cruciferae, Fumariaceae, Oenotheraceae, Polemoniaceae, Rhamnaceae, Rutaceae, Salicaceae and Scrophulariaceae, by Daphni et al. (1988) for flowers of the Labiatae, and by Figier (1971) in the stipular nectary of Vicia faba. According to Vogel (1990) the most compact fragrance glands are characterized by an intensive, chiefly phloemic venation and a well developed inter- cellular system with abundant stomata. The intense respiration during the active phase is generally correlated with the development of a ventilation system. In Sanango racemosum, the disk is supplied by phloem in direct contact with starch-rich tissue with many intercellularspaces; stomata are found at the top of the disk. The style of Sanango accords to the DPU type in the Heslop-Harrison & Heslop- Harrison classification (1981) because; (a) stigma surface is dry; (b) receptive cells are concentrated in a head; (c) stigma surface is papillate; and (d) papillae are unicellular. According to Heslop-Harrison & Shivanna (1977), dry stigmas with papillate surfaces and unicellular papillae occur in genera belonging to Gesneriaceae (Aeschynanthus, Columnea, Fieldia and Saintpaulia) and Scrophulariaceae ( Anthirrinum , Chelone, Digi- talis, Gratiola, Hebe, Lathraea, Linaria, Pentstemon , Scrophularia, Torenia, Verbascum and Veronica) and multicellular papillae are present in Alonsoa (Scrophulariaceae). Nevertheless, it is impossible to assess the taxonomic significance of these characteristics because dry stigmas with non-papillate surfaces have been found in the genus Veronicastrum of Scrophulariaceae and wet stigmas with low to medium papillae have been reported in genera of those same families, i.e. Sinningia and Streptocarpus of Gesneriaceae and Calceolaria and Nemesia of Scrophulariaceae. The basic division into wet and dry types presents difficulties in some species where the stigma bears only a small amount of secretion (Heslop-Harrison 1981). The morphology of the papillae in Sanango is arguably an odd phenomenon in the order Scrophulariales. Notwithstanding the taxonomic remoteness between Sanango and Iridaceae, the structure of the papillae with a wide base and a long, narrow receptive point are quite similar to those observed in Crocus by Heslop-Harrison (1977). In S. racemosum the stigmatic cells secrete a viscous material beneath the cuticle, causing it to become detached from the outer surface of the stigmatic cell walls. This behaviour was observed in Brassica oleraceae, Arabidopsis thalianaand Papaver rhoeas by Elleman et al. (1992) and interpreted as one of the early stigmatic responses in species with dry stigmas to pollination. In Sanango, as in Brassica and Papaver, the pollen tubes then penetrate the cuticle and grow towards the base of the papilla in the space thus generated. A pellicle overlies the cuticle of the stigma papillae of S. racemosum. The pellicle was first found in Silene vulgaris, Brassica oleraceae and Raphanus sativus and its presence confirmed in some 80 other angiosperm families, indicating that it may well be universal in the dry stigma group (Heslop-Harrison 1977; Heslop-Harrison & Heslop-Harrison 419SECRETORY TISSUES OF THE FLOWER OF SANANGO RACEMOSUM © 1997 Royal Botanical Society of The Netherlands, Acta Bot. 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