Acta Botanica 1-2017 - za web.indd ACTA BOT. CROAT. 76 (1), 2017 41 Acta Bot. Croat. 76 (1), 41–48, 2017 CODEN: ABCRA 25 DOI: 10.1515/botcro-2016-0049 ISSN 0365-0588 eISSN 1847-8476 Structure of fl oral nectaries in Aesculus hippocastanum L. Elżbieta Weryszko-Chmielewska, Mirosława Chwil* University of Life Sciences in Lublin, Faculty of Horticulture and Landscape Architecture Department of Botany, Lublin, Poland Abstract – Representatives of the family Sapindaceae exhibit high morphological diversity of the nectary structure. The present paper shows for the fi rst time the results of micromorphological, anatomical, and ultra- structural analyses of fl oral nectaries in Aesculus hippocastanum. We have also described the forage and sig- nal attractants of these fl owers, which are important for the ecology of pollination. Using light, fl uorescence, and electron microscopy, we demonstrated that the A. hippocastanum nectary forming a lobed disc is histo- logically differentiated into the epidermis with stomata, nectariferous parenchyma, subglandular parenchyma, and vascular bundles reaching the basal part of the nectariferous parenchyma. The use of histochemical as- says revealed the presence of insoluble polysaccharides, lipids, terpenoids, and polyphenols including cou- marins in the nectary tissues. Nectar is exuded onto the nectary surface via stomata and the permeable cuticle. As indicated by the observation of the ultrastructure of the nectary cells, transport of pre-nectar into paren- chymal cells may proceed via the symplast and apoplast. We have also demonstrated that nectar transfer out- side the protoplasts of parenchymal cells has a character of granulocrine secretion. A. hippocastanum fl owers produce nectar abundantly; one fl ower secreted on average 2.64 mg of nectar and the concentration of sugars in the nectar was 33%. Keywords: Aesculus hippocastanum, anatomy, fl ower, histochemical assays, micromorphology, nectar, nec- tary, ultrastructure * Corresponding author, e-mail: miroslawa.chwil@up.lublin.pl Introduction The genus Aesculus L. (Sapindaceae) comprises twelve species distributed in the northern hemisphere, eleven of them in Asia and America while one is native in Europe (Xiang et al. 1998, Forest et al. 2001). A. hippocastanum is a Balkan endemic and Tertiary relict, present as an autoch- thonous species in Bulgaria, Albania, Greece, and Macedo- nia (Polunin 1997, Avtzis et al. 2007). For many years, it has been widespread in nearly all parts of Europe with the exception of its northern zone (Kremer 1995). Based on morphological phylogenetic analysis, Forest et al. (2001) suggested an American origin for Aesculus with a single migration to Eurasia via the Bering land bridge. Harrington et al. (2005) distinguished between four subfamilies within the Sapindaceae subfamily Hippocastanoideae as well as Acereae and Hippocastaneae tribes in this subfamily. The decorative values of A. hippocastanum include a regularly shaped crown, large, palmate-compound leaves, numerous white fl owers forming up to 30-cm long panicles, and attractive spiky fruits (Seneta and Dolatowski 2007). Chestnut leaves, bark, fl owers, and seeds are medicinal raw material containing fl avonoids, coumarins, and saponins (Matysik et al. 1994, Zhang et al. 2010, Dudek-Makuch and Matławska 2013). Zygomorphic fl owers of the species are polygamous (male and hermaphroditic). The perianth is formed by 4–5 petals exhibiting dimorphism and 5 fused sepals of different sizes. The generative elements comprise 5–8 stamens and one pistil with a three-loculed superior ovary. Hermaphrodite fl owers are protogynous. At the time of fl owering, fi laments change their position. Corolla petals bear nectar guides, which change colour at the time of fl ow- ering. Flowers of this species produce nectar and pollen; therefore, they are visited by bees and bumblebees (Mau- rizio and Grafl 1969, Weberling 1992). Pollen is also used as food by Syrphidae (Kugler 1970). The types of trichomes producing olfactory attractants in chestnut fl owers were de- scribed in our previous paper (Chwil et al. 2013). Nectaries in the fl owers of various plant species exhibit characteristic topography and structure, which constitute more or less permanent traits for genera and families (Ber- nardello 2007). The current knowledge of the distribution WERYSZKO-CHMIELEWSKA E., CHWIL M. 42 ACTA BOT. CROAT. 76 (1), 2017 and structure of fl oral nectaries in higher taxonomic groups of orders and superorders is incomplete (Vogel 1997, Smets at al. 2000, Bernardello 2007). Simultaneously, numerous investigations reveal a high degree of anatomical diversity in terms of fl oral nectaries in plants (Durkee 1983). The po- sition of the nectary within the fl ower may be a result of evolutionary selection taking place during the fl ower – pol- linator interaction (Proctor et al. 1996). The nectaries in Sapindales are receptacular (Cronquist 1981, Bernardello 2007). In the family Sapindaceae, ex- trastaminal annual nectaries placed between the perianth and stamens are widespread (Cronquist 1981, Cui et al. 2003). In Koelreuteria fl owers, the nectary is situated on the slope of the androgynophore between the petal bases and the stamen whorl as a massive crested protuberance (Ronse De Craene et al. 2000, 2002). In Cardiospermum, the nectary is located at the base of the androgynophore and consists of two horn-like lobes. In Urvillea, the nectary has four lobes of different sizes (Solís and Ferrucci 2009, Zini et al. 2014), and the nectary in Acer forms a regular ring (Weryszko-Chmielewska and Sulborska 2011). Maurizio and Grafl (1969) reported that the nectary in the A. hippocastanum fl ower had the shape of an irregular protuberance located between the corolla tube and stamens. Ronse De Craene et al. (2002) found that the nectary in this genus is unilaterally set between the petal bases and the sta- men whorl. Acevedo-Rodrigues et al. (2011) described the nectary in Aesculus fl owers as a 4-lobed unilateral disc. As reported by Zini et al. (2014), currently only a few data on nectaries and their evolutionary shifts are available for most taxa. Since we found no detailed data on the struc- ture of the A. hippocastanum nectary in the available literature, we undertook comprehensive studies of the morphological, anatomical, and ultrastructural traits of this nectariferous gland in order to fi ll the gap in this knowledge. We also used histochemical assays to reveal the content of some substances in nectary tissues that are also present in other A. hippocastanum organs. We have also presented the charac- teristics of other fl oral parts associated with the ecology of pollination. Materials and methods The fl owers were collected from 3 different trees of Aesculus hippocastanum in the Botanical Garden of Maria Curie–Skłodowska University in Lublin. The analyses carried out in 2014–2015 were focused on the micromorpho logy of the surface of the epidermis of selected fl oral elements as well as the anatomy and ultrastructure of the nectary. The observations were performed using stereoscopic (SM), fl u- orescence (FM), light (LM), scanning (SEM), and trans- mission electron microscopes (TEM). Floral nectaries were measured in 16 hermaphroditic and male fl owers. We also analysed the quantities of nectar and sugar content in the nectar. Fixation of the material Fragments of nectaries and fl oral elements sampled from fresh fl owers on fl owering day 1 were fi xed in 4% glutaraldehyde for 6 h at room temperature, and next in 0.01 M phosphate buffer, pH 7.0, at 4 ºC for 48 h. After be- ing rinsed in phosphate buffer, the samples were contrasted in a 1.5% solution of osmium tetroxide for 1.5 h. Next, the nectary fragments were dehydrated in ethyl alcohol series at concentrations of 15, 30, 50 70, 90, 96, and 99.8% for 15 min and twice in absolute alcohol. Subsequently, the mate- rial was embedded in Spurr low viscosity resin and poly- merised at a temperature of 60 ºC for 48 h. Light microscopy Longitudinal, semi-thin nectary sections were made from the fi xed material. 0.8–1 μm sections were cut with a glass knife using a Reichert Ultracut S microtome and stained with 1% toluidine blue and 1% azure II (1:1) at a temperature of 60 ºC for 5 min. Periodic acid Schiff (PAS) reaction was applied to lo- calise starch grains in the plastids and other polysaccharides (cellulose and pectins) in the nectary cells (Nevalainen et al. 1972). Comparative histochemical assays, i.e. Sudan IV (Pearse 1985) and Sudan red (Brundrett et al. 1991) indicat- ing the presence of lipid compounds in the cells as well as Nile blue A (Jensen 1962) staining acidic and neutral lipids, were carried out. We also used the Nadi-reagent (David and Carde 1964) for terpenoids, ferric trichloride for phenolic compounds, and Lugol’s solution (Johansen 1940) for cou- marins. Anthers were stained with Sudan red. Fluorescence microscopy Hand-cut nectary sections (longitudinal) and anther samples from fresh material were placed in a droplet of a fl uorochrome (0.01% auramine O) in order to analyse the cuticle layer (Wędzony 1996). The observations were car- ried out under a Nikon Eclipse 90i fl uorescence microscope equipped with a FITC (excitation light 465–495 nm) and barrier (515–555 nm wavelength) fi lter. Scanning electron microscopy Fixed fragments of the nectaries, corolla petals, sta- mens, and pistils were dehydrated in acetone series: 15, 30, 50, 70, 90, and 99.5%, and twice in absolute acetone. Next, they were critical-point dried in liquid CO2 using an Emi- tech K850 dryer and sputter-coated with gold using an Emi- tech K550X sputter-coater. The surface of the epidermis of the fl oral elements was observed under a Tescan Vega II LMU scanning electron microscope. Transmission electron microscopy Ultrathin 70-nm thick sections were cut from the fi xed and resin-embedded nectaries, which were stained with an 8% solution of uranyl acetate in 0.5% acetic acid for 40 min. After double rinsing of the sections with distilled wa- ter, Reynolds’ reagent was applied for 15 min (Reynolds 1963). After rinsing with water, the sections were dried. The ultrastructure of nectary epidermal and parenchymal cells in the full-secretion phase was observed under an FEI, USA Tecnai Spirit G2 transmission electron microscope. STRUCTURE OF NECTARIES IN AESCULUS HIPPOCASTANUM ACTA BOT. CROAT. 76 (1), 2017 43 Quantity of nectar secretion Nectar was sampled from fl owers between 9:30 and 10:30 AM with the pipette method developed by Jabłoński (2003). The sample contained nectar secreted throughout the fl owers’ life (within 3 days) and was collected from 6–12 fl owers. Sixteen nectar samples were collected. The percent content of sugars was estimated with an Abbe refractometer (RL-1 PZO). Qualitative and quantitative nectar content Quantitative and qualitative analysis of sugars in the nectar sampled throughout the fl owers’ life was performed with high-performance liquid chromatography (HPLC) ac- cording to Bogdanov et al. (1997) with modifi cations by Rybak-Chmielewska (2007). Statistical analyses Standard deviation (±SD) was calculated for the mea- surements of the fl oral nectar mass, nectar sugar concentra- tion, and nectar sugar mass using Excel 7.0 (Microsoft). Results Structure of the infl orescence and fl oral attractants The number of fl owers in the A. hippocastanum infl o- rescence was 155–240, with an average of 220. Among the polygamous fl owers, hermaphroditic fl owers (Fig. 1A), which on average accounted for 27% in the infl orescences, had a large ovary and a long style. This type of fl ower was found in the lower and middle part of the infl orescence. Male fl owers characterised by a small, cylindrical pistil with an underdeveloped ovary (Figs. 1B, H, I) and produc- ing no fruits represented the largest proportion (approx. 73%). Nectar and pollen constitute forage attractants for in- sects in A. hippocastanum fl owers. Signal attractants com- prise yellow and red nectar guides located on petals (Figs. 1A, B), red connective protrusions on both anther poles (Figs. 1C, 2B, C), red stigmas, and odour emitted by differ- ent parts of the fl ower. Trichomes located on petals (Fig. 2A) as well as trichomes and colleters present on the ovary (Figs. 2G, H) may serve the function of fl oral attractants for insects. On the surface of the connective protrusions, we observed fl uorescent secretion droplets (Figs. 1D, E). Treat- ment of these tissues with Sudan IV revealed the presence of lipid compounds in epidermal cells (Fig. 1F). Similarly, SEM images indicated the presence of the secretion under the cuticle convexities of the epidermal cells of these pro- trusions (Fig. 2 D). The stigma covered by papillae oc- cupied a small part of the apical zone of the pistil (Figs. 2E, F). Nectar secretion in A. hippocastanum fl owers com- mences in the bud-opening phase. Throughout its lifetime, one fl ower secreted on average 2.64 mg of nectar. The mean concentration of sugars in the nectar was 33%. The weight Fig. 1. Flowers and fl oral parts of Aesculus hippocastanum: (A, G) hermaphroditic fl ower; (B, H, I) male fl owers with a nectary (dou- ble-headed arrow denotes nectar droplet) and a reduced pistil; (C) anther with red connective protrusions (two arrows) at the poles; (D, E) fl uorescent microscopy photographs of anther parts stained by auramine O (asterisks denote fl uorescent secretion); (F) longi- tudinal section of connective protrusion stained by Sudan red (ar- rows denotes lipid compounds); n – nectary, o – ovary, p – pistil. Fig. 2. Micromorphology of the epidermis surface of selected fl ower parts of the Aesculus hippocastanum examined by scan- ning electron microscope (SEM): (A) corolla petal with trichomes at the apex; (B, C) anther with protrusions at the poles and tri- chomes on the surface; (D) – external surface of protrusion with cuticle convexities (arrows); (E, F) – fragments of the style; papil- lae visible on the stigma; (G, H) trichomes and colleters on the ovary surface; (I) nectary located between the bases of fi laments and portion of the petal. f – fi lament, k – colleters, n – nectary, p – petal, s – sepal. WERYSZKO-CHMIELEWSKA E., CHWIL M. 44 ACTA BOT. CROAT. 76 (1), 2017 of sugars was in the range of 0.31–1.39 mg per fl ower (Tab. 1). Sucrose, i.e. the dominant sugar in the nectar, accounted for 92% of all sugars. Nectary micromorphology The nectary in A. hippocastanum fl owers, characterised by a light creamy colour, is located in the receptacle at the ovary base (Figs. 1G–I). The longitudinal section revealed a visible hollow in the central part of the nectary (Fig. 2I). The nectary gland is present in two types of polygamous fl owers in this species but differs in size. It has a larger di- ameter in hermaphroditic fl owers (on average 3.88 mm) than in functionally male fl owers (on average 3.23 mm). Greater differences were noted for the nectary height in both fl ower types: hermaphroditic 1.01 mm, functionally male 0.61 mm. The nectary forms an undulated ring with 4 larger and 2–3 smaller lobes. The largest lobe is adjacent to the base of two lower petals (Figs. 3A, B). On the ovary side, there are 7 concavities with the basal parts of the fi laments (Figs. 3A–C). Stomata are situated in the hollow of the nectary concavities (Figs. 3D, E). At the site of the stomata, there are concavities surrounded by nonstomatal epidermal cells located above (Figs. 3E–G). Remnants of secretion were visible both on the surface of the stomata and in their vicin- ity. In nectary zones devoid of stomata, numerous vesicle- like cuticle convexities, probably containing secretion, and remnants of dried secretion were observed (Figs. 3H, I). Nectary anatomy The longitudinal nectary sections viewed under fl uores- cence and light microscopes showed the distribution of nec- tariferous cells in the central part of the nectary disc (Figs. 4A, B). The epidermis was formed by one layer of cells with relatively thin walls (Figs. 4E, G). A few stomata were observed in the sections (Fig. 4D). At the onset of the secre- tory activity, the cells of this tissue were already character- Tab. 1. Nectar production in Aesculus hippocastanum fl owers. Studied trait min.–max. mean±SD Nectar mass (mg per fl ower) 1–4.42 2.64±0.94 Nectar sugar (percentage, %) 25–40 33.05±5.04 Nectar sugar mass (mg per fl ower) 0.31–1.39 0.86±0.29 Fig. 3. Overall view and fragments of the Aesculus hippocasta- num nectary examined by SEM: (A–C) lobed nectary, visible dif- ferent-sized lobes; (D, E) nectary epidermis surface with stomata (arrows); (F, G) stomata between nonstomatal cells; (H, I) vesicle- like cuticle convexities in epidermal cells (double-headed arrows denote remnants of secretion); f – fi lament, l – lobes, n – nectary, o – ovary, p – petal, s – sepal. Fig. 4. Longitudinal sections of fragments of the Aesculus hippo- castanum nectary observed by fl uorescent microscope (A) and light microscope (B–F): (A) epidermal and nectariferous paren- chymal cells; (B) parenchymal cells in the marginal and central zones of the nectary; (C) nectary epidermal and parenchymal cells; (D) stoma in the nectary epidermis; (E–G) thick-walled epi- dermal cells and thin-walled parenchymal cells, visible plastids with starch grains and vascular bundles (F); (H) acidic lipids stained blue with Nile blue A; (I) positive reaction of coumarins (purple staining) with Lugol’s solution; (J) total lipids (red stain- ing with Sudan IV); (K) purple-stained (Nadi-reagent) terpenes in epidermal cells; (L) phenolic compounds stained with ferric tri- chloride; e – epidermis, np – nectary parenchyma, sg – subglandu- lar parenchyma, s – stoma, vb – vascular bundle; arrow in (F) de- notes granular structure in the vacuole. STRUCTURE OF NECTARIES IN AESCULUS HIPPOCASTANUM ACTA BOT. CROAT. 76 (1), 2017 45 ised by substantial vacuolation. Small-sized parenchymal cells of the nectary were mainly located in the concave part of the nectary ring. The marginal zones of this gland were formed by considerably larger parenchymal cells (Fig. 4B). In their shape and size, the thin-walled parenchymal cells of the nectary resembled meristematic tissue. In the observed activity phase, they were characterised by a high degree of vacuolation (Figs. 4C, E). Numerous branches of vascular bundles containing phloem elements were located primarily in the subglandular parenchyma layer and some of them reached the basal part of the nectary parenchyma (Figs. 4B, F). Histochemistry The histochemical assays used in the study revealed the presence of different substances in the nectary epidermis and parenchyma. Starch was detected in the nectariferous and subglandular parenchyma and in some epidermal cells with the use of the PAS-reagent (Figs. 4D–G). The treat- ment of nectary tissues with this reagent yielded pink stain- ing of the cell walls in the parenchyma and vascular bun- dles and red staining of the epidermal cell walls. Moreover, after treatment with the PAS-reagent, parenchymal cells ex- hibited light brown staining of an unknown substance con- tained in the vacuoles and forming granular clusters in many cells (Fig. 4F). The content of epidermal and parenchymal cells stained positively for acidic lipids with Nile blue A (Fig. 4H). Su- dan IV revealed lipids in the cell wall and cytoplasm of epi- dermal cells (Fig. 4J). Nadi-reagent stained terpenoids pur- ple in the same parts of these cells (Fig. 4K) and ferric trichloride gave positive reaction for phenolic compounds in epidermal cells (Fig. 4L). Lugol reagent revealed couma- rins stained purple and bright red in epidermal cells (Fig. 4I, Tab. 2). Nectary ultrastructure The epidermis cells had various shapes and overlapped at some sites. The outer wall was thicker than the radial walls and the inner tangential wall. The central part of the cell was occupied by a large vacuole (Fig. 5A) or a central cytoplasm band containing one large cell nucleus, numerous mitochon- dria, plastids, and ER separating smaller vacuoles. The vacu- oles of some nectary epidermal and parenchymal cells con- tained deposits of an osmophilic substance resembling deposits observed in the epidermal and parenchymal cell of the calyx (Figs. 5A, H). Small intercellular spaces were observed in nectary pa- renchyma (Figs. 5A–C). Numerous plasmodesmata were present in the cell walls (Fig. 5D). A substantial part of the protoplast was occupied by cell nuclei. A great number of mitochondria mainly characterised by tubular arrangement of inner membranes were observed in the electron-dense Fig. 5. Ultrastructure of Aesculus hippocastanum nectary cells ex- amined by transmission electron microscope: (A) epidermal and subepidermal cells (two arrows denote deposits of an osmophilic substance); (B) parenchymal cells with a centrally located nucleus and vacuoles of different size, electron-dense cytoplasm, numer- ous mitochondria, plastids, membranous structures in the vacuole; (C) cytoplasm with polymorphic plastids, rough endoplasmic re- ticulum (RER), Golgi apparatus, and transport vesicles (arrow) at the plasmalemma; (D) cell wall with plasmodesmata (double- headed arrows), plasmalemma invaginations, numerous parietal situated mitochondria, RER profi les; (E, G) polymorphic plastids with varied arrangement of inner membranes; (F) well-developed RER, numerous vesicles (arrows), mitochondria, and Golgi appa- ratus; (H) plastid from the cell of sepal; cw – cell wall, G – Golgi apparatus, n – nucleus, p – plastid, v – vacuole, m – mitochondrion. Tab. 2. Chemical compounds detected with histochemical assays in the epidermal cells in Aesculus hippocastanum nectar. PAS – periodic acid Schiff. Histochemical assay Compound group Cell staining Nectary epidermal cells Sudan IV fats red ++ Sudan red lipids, resins, oils, waxes red ++ Lugol reagent coumarins aesculin grey-purple red + Nile blue A acidic lipids blue ++ neutral lipids pink ‒ Nadi-reagent terpenoids purple-blue ++ Ferric trichloride polyphenols black + PAS-reagent polysaccharides red + Auramine O cutin, suberin green ++ WERYSZKO-CHMIELEWSKA E., CHWIL M. 46 ACTA BOT. CROAT. 76 (1), 2017 cytoplasm (Figs. 5C, D). Polymorphic plastids exhibited an electron-dense stroma and irregular arrangement of the sys- tem of internal membranes with bright content and plasto- globules therein (Figs. 5E, G). The structure of the nectary plastids was markedly different from the structure of chlo- roplasts with regularly distributed grana thylakoids origi- nating from the parenchyma of the sepals (Fig. 5D). The cytoplasm exhibited numerous profi les of rough ER form- ing characteristic confi gurations around the plastids and in the peripheral parts of the cytoplasm (Figs. 5C, F). Further- more, we observed Golgi apparatuses and numerous ribo- somes. Some intercellular spaces contained a grey substance with vesicle-like structures (Fig. 5C). Numerous vesicles formed clusters near the plasmalemma (Figs. 5C, F). Discussion High morphological diversity of nectaries can be found within the family Sapindaceae. A large (3–4 mm in diame- ter), lobed nectary is characteristic for A. hippocastanum. Our investigations partly confi rmed the fi ndings reported by Ronse De Craene (2002) and Acevedo-Rodríguez et al. (2011), who described the nectaries of the analysed species as lobed, unilateral discs. Zygomorphic A. hippocastanum fl owers exhibit bilateral symmetry of the nectary. The best- developed lobe of this gland is directed towards petals that have the lowest location. The fl owers in this species are horizontally arranged in the infl orescence. Abundant nectar accumulates in the lower part of the fl ower and numerous trichomes located on the petals, sepals, and ovary probably protect it against outfl owing. Dafni and Kevan (1996) showed that bilaterally sym- metrical nectar guides were a characteristic trait of zygo- morphic fl owers. This trait was found in the A. hippocasta- num fl owers as well. In terms of location in the fl ower and morphological traits, it can be noted that there is certain similarity of the A. hippocastanum nectary to the undulated nectary disc in Koelreuteria, a genus from the family Sap- indaceae presented in the paper of Ronse De Craene et al. (2000). As indicated by various authors, nectaries with bilateral symmetry are also present in plant species from other fami- lies producing zygomorphic fl owers, e.g. in Lamiaceae (Dafni et al. 1988, Weryszko-Chmielewska 2000) and in Ericaceae from the genus Rhododendron (Weryszko-Chmi e- lewska and Chwil 2005, 2007). Various authors report that nectar in Sapindaceae is ex- uded onto the nectary surface via stomata, as shown in A. hippocastanum in this study. Nectarostomata have been ob- served in fl oral nectaries in Acer platanoides (Weryszko- Chmielewska and Sulborska 2011), three Cardiospermum species, and Urvillea (Solís and Ferruci 2009, Zini et al. 2014). In contrast, Ronse De Craene et al. (2000) did not fi nd stomata in the nectary of Koelreuteria. Our study indi- cates that nectar in A. hippocastanum can be secreted via not only stomata but also the permeable cuticle of the nec- tary, which forms a thin layer and covers very numerous vesicles, probably containing the secretion. The possibility of existence of two pathways of nectar exudation onto the nectary surface is confi rmed by data provided for other plant species, e.g. Prunus (Radice and Galati 2003) and Cit- rus (Konarska and Weryszko-Chmielewska unpublished data). A. hippocastanum fl owers are characterised by abun- dant nectar secretion. In our paper, we have shown that the weight of nectar secreted by one chestnut fl ower was in the range of 1–4.42 mg, with an average of 2.64 mg. These val- ues are close to those reported by Maurizio and Grafl (1969) for this species. Our results concerning the sugar content in the nectar (25–40%) differ from the fi ndings shown by Maurizio and Grafl (1969), i.e. 60–76%. In our study, su- crose was the dominant sugar in the nectar of the analysed species, likewise in the earlier data provided by different authors (Maurizio 1959, Maurizio and Grafl 1969, Fahn 1979). A relationship between the type of nectar, form of the nectary, and type of visitors was noted. Nectar with high su- crose content is used by various pollinator insects, e.g. moths, butterfl ies, and long-tongued bees. The sugar content in nectar collected by bees is approximately 40% (Proctor et al. 1996, Nicolson et al. 2007). Besides, zygomorphy and yellow and blue or purple fl ower colours belong among pol- lination signals for bees (Proctor et al. 1996). Since several of these traits are exhibited by A. hippocastanum fl owers, the plants can be regarded as adjusted to bee pollination. Both anther poles bear red appendages, which not only play a role of visual attractants for insects but also may provide them with food, as they release lipid compounds. Our investigations indicate that some epidermal cells of the A. hippocastanum nectary produce phenolic compounds (including coumarins), which may protect the gland against being consumed by insects. As shown in the literature, phe- nolic compounds serve an important protective function by exerting an adverse effect on the biology of herbivorous in- sects (Oleszek et al. 2001). Cisowski (1983) demonstrated the presence of aesculin, i.e. a coumarin compound, in A. hip pocastanum fl owers. We additionally detected terpe- noids (Nadi-reagent) in the A. hippocastanum nectary. A positive reaction of the Ananas ananassoides nectary to this reagent was reported by Stahl et al. (2012). The results of these assays may indicate the presence of essential oils in the analysed nectaries. At the onset of nectar secretion, we found low numbers of starch grains in the plastids from the nectary parenchyma cells in the semithin sections. In ultrastructural analyses of the nectary, we did not demonstrate starch content in these cells, which must have been related to the more advanced phase of nectary activity. The plastids observed in our study on electronograms were characterised by a system of inner membranes similar to those present in tubular chromoplasts demonstrated by Woźny et al. (2001) and Evert (2006). Based on the ultrastructural traits of the A. hippocasta- num nectary, it is possible to defi ne the mode of nectar transfer outside the protoplast in the cells of the nectarifer- ous parenchyma. The great number of mitochondria, nu- merous RER profi les, Golgi structures, and very abundant vesicles located in close proximity to the plasmalemma and in the cell wall may indicate a granulocrine mode of nectar secretion. Granulocrine secretion has been demonstrated by authors of other studies on the nectary structure in a number of plant species (Fahn and Benouaiche 1979, Sawidis 1989, STRUCTURE OF NECTARIES IN AESCULUS HIPPOCASTANUM ACTA BOT. CROAT. 76 (1), 2017 47 O’Brien et al. 1996, Weryszko-Chmielewska et al. 2006, Wist and Davis 2006, Chwil and Chwil 2012, Kowalkowska et al. 2015). The numerous plasmodesmata present in the parenchy- mal cell walls in the A. hippocastanum nectary indicate symplastic transport of nectar, whereas the presence of the secretion in the intercellular spaces and transfer thereof to the stomata suggest a possibility of nectar transport via the apoplast. Two pathways of nectar transport within nectarif- erous tissues have been shown by Radice and Galati (2003) in Prunus as well as Chwil and Chwil (2012) in Polemonium. Many authors believe that the location and structure of fl oral nectaries are important plant taxonomic trait (Rudall et al. 2000, Smets et al. 2000, Bernardello 2007, Chwil and Weryszko-Chmielewska 2012). In the present study, we presented for the fi rst time the micromorphology, anatomy, and ultrastructure of the A. hippocastanum nectary, which expands the knowledge of the nectary structure and may complete the set of diagnostic traits used in taxonomy. Our research resulted in following main conclusions: 1. A ma- jority of fl owers (ca. 73%) in A. hippocastanum infl ores- cences are functionally male fl owers, which produce an under developed ovary. The other part is represented by her- maphroditic fl owers. 2. A. hippocastanum fl owers offer pol- linators various food attractants: abundantly secreted nec- tar, pollen, and colourful food bodies located on both anther poles. 3. Nectaries are present in both types of A. hippocas- tanum fl owers; despite the different sizes, they have the same structure. Nectar exudation onto the nectary surface proceeds in two ways: via stomata and the permeable cuti- cle. Nectary cells contain phenolic compounds and emit ar- omatic substances. 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