Annals of Botany 87: 27±33, 2001doi:10.1006/anbo.2000.1287, available online at http://www.idealibrary.com on

Nectary Structure and Ultrastructure of Unisexual Flowers of Ecballium elaterium (L.)A. Rich. (Cucurbitaceae) and their Presumptive Pollinators

ABRAHAM FAHN* and CARMELA SHIMONY

Department of Plant Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel

Received: 22 June 2000 Returned for revision: 3 August 2000 Accepted: 6 September 2000 Published electronically: 16 November 2000

during two

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The structure and ultrastructure of the nectaries of the monoecious species Ecballium elaterium were studied. Largedi�erences in size and structure of the nectaries were observed in the two genders of ¯owers, those of the staminate¯owers being much larger and more developed than those of the pistillate ¯owers. The latter do not secretemeasurable amounts of nectar. In the nectariferous cells, especially of the staminate ¯owers, numerous plasmo-desmata are present. The pre-nectar originating in the phloem is stored in the plastids of the nectariferous cellsprimarily as starch grains. The nectar appears to be exuded from the nectary via modi®ed stomata. Very small insectsof the order Hemiptera were found to dwell inside the ¯owers of the two genders, but in di�erent numbers; theirnumber in the staminate ¯owers was more than twice that in the pistillate ¯owers. These insects may take part in theprocess of pollination. # 2000 Annals of Botany Company

Key words: Ecballium elaterium, Cucurbitaceae, monoecious plant, nectaries, structure, ultrastructure, nectar

secretion, stomata, Hemiptera insects.

identity and possible role in pollination is discussed here.

INTRODUCTION

The Cucurbitaceae are monoecious plants, bearing bothpistillate and staminate ¯owers on the same individual. Thestudy of nectar secretion in Cucumis sativus, Cucurbita pepoand Cucurbita maxima showed that there is a di�erence inthe amount of nectar produced between the two types of¯owers, with pistillate ¯owers producing much more nectarper ¯ower than staminate ones (Fahn, 1949; Nepi et al.,1996b). A correlation was also found between the amountof secreted nectar and the volume of the nectariferous tissue(Fahn, 1949; Nepi et al., 1996b). Bahadur et al. (1986) didnot ®nd nectaries in the pistillate ¯owers of three of the ®vespecies of Cucurbitaceae they studied (Momordica char-antia, Lagenaria vulgaris and Lu�a acutangula). Theabsence of nectaries in the pistillate ¯owers of Lu�a wasalso reported by Haran Iyer et al. (1989). The present paperdeals with the structure and ultrastructure of the ¯oralnectaries of Ecballium elaterium (L.) A. Rich. (Cucurbita-ceae), a monotypic Mediterranean plant that grows onroadsides and waste ground and ¯owers between the endof April and December (Zohary and Feinbrun-Dothan,1978). It di�ers in several morphological and biologicalcharacteristics from the other Cucurbitaceae species by thelack of tendrils and the detaching of fruit from thepeduncle. Dukas (1987), who studied the frequency ofvisits of various bees to the pistillate and staminate ¯owersof E. elaterium, found that Apis mellifera discriminatesbetween the two genders of ¯owers and pays relatively fewervisits to the pistillate ¯owers which do not secrete nectar.The present authors, while carrying out observations

¯owering seasons on a large number of

1/010027+07 $35.00/00

spondence. Fax 00 972 26519590, e-mail

Ecballium plants, observed numerous small, dark, veryactive insects inside all ¯owers of both genders, whose

MATERIALS AND METHODS

Observations were made over 2 years on staminate andpistillate ¯owers of Ecballium elaterium plants growing invarious locations in Jerusalem. Hand-sections of fresh andepoxy-embedded basal parts of ¯owers were examinedunder the light microscope.

For transmission electron microscope (TEM) studies,¯ower portions containing the nectaries were ®xed over-night in 4% glutaraldehyde in potassium phosphate bu�er(0.1 M, pH 7.2) at 48C. The material was post®xed with 1 or0.5% OsO4 , in the same bu�er, for 1±1.5 h (Hayat, 1981).The ®xed material was then dehydrated in a graded series ofethanol solutions and embedded in epoxy resin (Spurr,1969). Ultrathin sections were cut with an LKB ultratomeIII, stained with a saturated solution of uranyl acetate in50% ethanol for 30±60 min followed by lead citrate for2±5 min (Reynolds, 1963) and viewed with a Philips 300transmission electron microscope.

Sections 2±4 mm thick were stained with 0.05% toluidineblue for 1±2 min, washed and dried and viewed under alight microscope, applying immersion oil directly to thesection itself.

For scanning electron microscopy (SEM), basal parts of¯owers ®xed with FAA were dehydrated in a graded seriesof ethanol solutions. The ¯ower portions were then criticalpoint dried with solvent substituted liquid carbon dioxide,coated with a thin layer of gold and examined with a JEOL

scanning electron microscope, 5410 LV.

# 2000 Annals of Botany Company

FIGS 1 and 2. Diagrams of longitudinally sectioned ¯owers. Fig. 1. Staminate ¯ower. Fig. 2. Pistillate ¯ower. Ne, nectary; Ov, ovary; Ps, pistil; S,stamen; Sd, staminodes. � 3.

28 Fahn and ShimonyÐNectary Structure and Ultrastructure of Unisexual Flowers

RESULTS

The ¯owers

Ecballium elaterium is a perennial or perennating procum-bent, monoecious herb, without tendrils. The ¯owers of thetwo sexes are the same size. The staminate ¯owers occur inracemes; the sepals are linear lanceolate; the corolla isyellow, campanulate and deeply divided; the stamens arefree (Figs 1 and 19). The pistillate ¯owers are solitary, oftenin the same leaf-axil as the staminate racemes. The corollaand sepals are similar to those of the staminate ¯owers.Around the pistil there are three to ®ve very smallstaminodes (Figs 2 and 9). Multicellular, uniseriate gland-ular trichomes are present on the inner surface of thecorolla of the ¯owers of both genders (Figs 6 and 9),increasing in density towards the base of the corolla tube.The cells of the trichomes contain dense granular material,which stains with Sudan IV and with Coomassie brilliantblue (Fisher, 1968) for lipophilic material and proteins,respectively.

The ¯owers, as observed on plants grown in a growth

room, remain open for about 4 d.

The nectary

Light and SEM microscopy. The nectary belongs to thetoral type, developing in the receptacle (Fahn, 1952). Instaminate ¯owers, the nectary is relatively well-developedand occupies the centre of the receptacle, surrounded by thestamens (Figs 3 and 7). In pistillate ¯owers, the nectary ismuch reduced and is situated at the base of the pistil (Figs 2and 5). The epidermis of the nectary is covered by a cuticle,which increases in thickness and becomes folded in thegroove occurring between the nectary and the stamens instaminate ¯owers and between the nectary and the corollain pistillate ¯owers (Figs 2, 3, 5, 10, 17 and 18). Modi®ed

stomata are present in the epidermis (Figs 4, 8 and 11). The

nectariferous cells di�er from those of the neighbouringtissues by their smaller size and denser cytoplasm. In thenectaries of staminate ¯owers, vascular bundles containingboth phloem and xylem are present inside the nectariferoustissue. However, in nectaries of pistillate ¯owers vascularbundles were observed only below the nectariferous tissue(Fig. 5). Measurements of nectar secretion made in theearly morning on ¯owers covered with gauze on thepreceding day, showed that staminate ¯owers secreteabout 0.25 ml nectar per ¯ower. No measurable nectarcould be detected in pistillate ¯owers.

Hand-sections were treated with IKI solution to studychanges in starch content. Before anthesis the whole nectaryof the ¯owers of both genders stained uniformly. Atanthesis, small unstained areas were observed in the nectaryof the staminate ¯owers, whereas in the pistillate ¯owers nochanges in staining could be distinguished.

Transmission electron microscopy (TEM). TEM micro-graphs of nectariferous cells of staminate ¯owers at theearly stage of development revealed that they are rich incytoplasm and their walls contain numerous plasmo-desmata. The nuclei are relatively large, and the numerousplastids contain starch grains and thylakoids. Numerousmitochondria with well developed cristae, dictyosomes andportions of rough endoplasmic reticulum (RER) arepresent. The ground cytoplasm is rich in ribosomes(Figs 12 and 13). At the stage of anthesis the cells increasein volume and the cytoplasm becomes restricted to arelatively narrow region around a large central vacuole.

In several nectariferous cells of staminate ¯owers, plastidscontaining numerous small electron-dense unidenti®edgranules, some with crystal-like outline, were observed atanthesis. Such granules were also seen bound to innertubular membranes of plastids (Figs 14 and 15). Innectariferous cells of pistillate ¯owers the density of the

cytoplasm and the quantity of cell organelles was much less

FIGS 3±6. Light microscope photographs. Figs 3 and 4. Nectaries of staminate ¯owers. Fig. 3. Nectary occupying the space between the stamens.Part of a stamen (S) is seen on the right. The cells of the nectariferous tissue are much smaller than those of the neighbouring tissues. VB, Vascularbundle; arrow points to the groove with the folded cuticle. Fig. 4. Portion of a hand-section of a nectary, showing a modi®ed stoma (St) and alarge substomatal chamber. Fig. 5. Nectary (Ne) of a pistillate ¯ower at the base of a pistil (Ps). Cr, Corolla. Arrow facing the groove with thefolded cuticle. Fig. 6. Glandular trichomes occurring on the inner surface of the lower part of the corolla. Bars � 100 mm (Figs 3, 5 and 6), 50 mm

.

Fahn and ShimonyÐNectary Structure and Ultrastructure of Unisexual Flowers 29

than those in nectariferous cells of staminate ¯owers.Relatively large intercellular spaces occurred in the

(Fig

nectariferous tissue of both genders (Figs 14 and 16).

the insects.

Heteropteran insects

In all ¯owers of the numerous plants examined, veryactive insects were present, their mean number (+s.e) was3.57+ 0.07 per staminate ¯ower (n � 35) and 1.60+ 0.43per pistillate ¯ower (n � 32). These insects were identi®edby Professor M. P. Penner (Hebrew University of Jerusa-lem) as belonging to the order Hemiptera, suborder

Heteroptera, division Geocoridae and possibly to the

family Anthocoridae (cf. Imms, 1960). Under the micro-scope, pollen grains were seen to adhere to the variousorgans of these insects (Fig. 20). In a dissecting microscope,the insects were seen pricking the epidermis of the nectaryand its neighbouring region with their proboscis. In SEMphotographs minor holes could be seen in the epidermis ofthis area (Fig. 11). These may represent punctures made by

4).

DISCUSSION

A big di�erence in size was observed between the nectaries

of the staminate and pistillate ¯owers of Ecballium

FIGS 7±11. SEM photographs. Figs 7 and 8. Nectaries of staminate ¯owers. Fig. 7. A ¯ower from which most of the upper part has been removedshowing the nectary surface (Ne). Fig. 8. Enlarged part of nectary surface, showing modi®ed stomata (St). Figs 9±11. Pistillate ¯owers. Fig. 9.Flower from which the upper part has been removed. Ne, Nectary; Sd, staminode; Ps, pistil. Fig. 10. Epidermis with folded cuticle at the groovebetween the nectary and corolla. Fig. 11. Nectary epidermis, showing stomata (St) and holes (Ho) apparently made by the insects trying to suck

food from inside the nectary. Bars � 500 mm (Figs 7 and 9), 10 mm (Figs 8, 10 and 11).

30 Fahn and ShimonyÐNectary Structure and Ultrastructure of Unisexual Flowers

elaterium, the former being much larger and more devel-oped than the latter. The amount of secreted nectar wasaround 0.25 ml per staminate ¯ower and not measurablein the pistillate ¯owers. Di�erences in the amount ofnectar secretion by the two genders in monoeciousplants have been observed in a number of plant species.In some plants such as Cucumis sativus, Cucurbita pepoand C. maxima, the volume of secreted nectar is larger inpistillate than in staminate ¯owers (Fahn, 1949; Nepi et al.,1996b), whereas in the banana (Fahn and Benouaiche,1979) and in E. elaterium (Dukas, 1987 and the presentauthors), staminate ¯owers secrete more nectar than

pistillate ¯owers. The present observations on Ecballium

support the view that there is a correlation between theamount of nectar secreted and the volume of thenectariferous tissue (Fahn, 1949; Nepi et al., 1996b). Acorrelation was also reported between the amount ofsecreted nectar and the volume of nectariferous tissue inclosely related species and varieties with bisexual ¯owerswith similarly structured nectaries, as for instance in theRutaceae, subfamily Aurantioideae (Fahn, 1949).

The nectaries of Ecballium possess modi®ed stomata intheir epidermis as do those of some other Cucurbitaceae(Caspary, 1848; Arcangeli, 1893; Collison andMartin, 1975;Okoli, 1989; Nepi et al., 1996a, b) and species of a number of

other families (Fahn, 1952; Durkee et al., 1981; Davis and

FIGS 12±15. TEM photographs of nectariferous cells of staminate ¯owers. Figs 12 and 13. Cells at early stage of nectary development containinga large nucleus (Nu), numerous plastids (Pl) with starch grains and thylakoids, mitochondria (M), dictyosomes (D) and numerous,plasmodesmata (Pd) traversing the cell walls. Figs 14 and 15. Cells at the stage of anthesis. Fig. 14. A plastid containing a starch grain andnumerous, as yet unidenti®ed, small electron-dense granules. Fig. 15. The small electron-dense granules (Gr) are bound to an inner tubular

u

Fahn and ShimonyÐNectary Structure and Ultrastructure of Unisexual Flowers 31

Gunning, 1992; Zer and Fahn, 1992; Ga�al et al., 1998).Electron micrographs of the nectariferous cells of

membrane of a plastid. N

E. elaterium show, in general, similarities to those of another

Cucurbitaceae species,Cucumis sativus, studied by Vassilijev(1972). The plastids are numerous and contain starch grains.

, Nucleus. Bars � 1 mm.

In nectariferous cells of several plant species, e.g. Passi¯ora

FIGS 16±18. TEM photographs. Fig. 16. Nectariferous cells of a pistillate ¯ower at a late stage of anthesis. IS, Intercellular space; V, vacuole.Fig. 17. Epidermal cells of a staminate ¯ower close to the groove between the nectary and stamen, showing the thick cuticle (FC), which starts to

become folded. Fig. 18. The thick folded cuticle (FC) in the groove between the nectary and corolla of a pistillate ¯ower. Bars � 1 mm.

32 Fahn and ShimonyÐNectary Structure and Ultrastructure of Unisexual Flowers

spp. (Durkee et al., 1981) and Rosmarinus o�cinalis (Zerand Fahn, 1992), the number of starch grains declinedtowards anthesis. It was suggested that the pre-nectaroriginating in the phloem is primarily stored in the plastidsas starch grains, which are hydrolysed to sugar towards thestage of nectar secretion. In the nectariferous cells ofEcballium numerous starch grains also occur, but thedi�erence in their density at various stages of ¯owerdevelopment was not su�ciently evident, possibly because

of the very small amount of secreted nectar. A most

interesting observation was the presence of Heteropteraninsects inside the basal part of the ¯ower. These insectsappeared in the ¯owers throughout the ¯owering season(April±December). The massive presence of the insects inthe ¯owers raises two questions: (1) what are they foraging inthe ¯owers, and (2) do they take part in the process ofpollination? Our observations suggest that these insects suckwhatever secreted nectar is available as well as the pre-nectaraccumulated inside the nectariferous cells; the latter by

pricking the cell walls.

University of Jerusalem for the identi®cation of the insects.

FIGS 19 and 20. Photographs of ¯owers and insects. Fig. 19. A ¯owerwith numerous insects. �2.5. (Photograph by Mr Uri Steinmetz.)Fig. 20. A light-microscopic photograph showing part of an insect and

Fahn and ShimonyÐNectary Structure a

While moving from ¯ower to ¯ower the insects maytransfer pollen grains from staminate ¯owers to the stigmaof pistillate ¯owers. During anthesis, the glandular tri-chomes occurring on the inner surface of the corolla arecovered with pollen grains, which are released from theopened anthers. Pollen grains were observed adhering to theinsects. Dukas (1987) found that the honey bee, Apismellifera, discriminates between the two genders of ¯owers,paying relatively fewer visits (about 30%) to the mimicpistillate ¯owers, which do not secrete nectar. In contrast,species of solitary bees visited pistillate ¯owers with nearlyequal frequencies at the beginning of each foraging day andstayed longer in these ¯owers. The number of insects inpistillate ¯owers is about one third of that found instaminate ¯owers. The insects apparently do not distinguishbetween the ¯owers of the two genders, visiting both types,but because of the shortage or lack of secreted nectar in themimic pistillate ¯owers, they appear to stay there for shorter

pollen grains adhering to it. Bar � 200 mm.

periods.

ACKNOWLEDGEMENTS

Thanks are due to Professor M. P. Penner of the Hebrew

nd Ultrastructure of Unisexual Flowers 33

LITERATURE CITED

Arcangeli G. 1893. Sull'impollinazione in varie Cucurbitaceae e sui loronettarii. In: Penzig O, ed. Atti del Congresso Botanico Inter-nazionale di Genova 1892. Genova: Tipogra®a del R. IstitutoSordo-Muti, 441±454.

Bahadur B, Chaturvedi A, Swamy NR. 1986. Floral nectaries in someCucurbitaceae. Journal of the Swamy Botanical Club 3: 149±156.

Caspary R. 1848. De Nectariis. PhD Thesis. Bonn: Elverfeldae.Collison CH, Martin EC. 1975. A scanning electron microscope study

of cucumber nectaries, Cucumis sativus. Journal of ApiculturalResearch 14: 79±84.

Davis AR, Gunning BES. 1992. The modi®ed stomata of the ¯oralnectary of Vicia faba L. 1. Development, anatomy and ultra-structure. Protoplasma 166: 134±152.

Dukas R. 1987. Foraging behavior of three bee species in a naturalmimicry system: female ¯owers with mimic male ¯owers inEcballium elaterium (Cucurbitaceae). Oecologia (Berlin) 74:256±263.

Durkee LT, Gaal DJ, Reisner WH. 1981. The ¯oral and extra¯oralnectaries of Passi¯ora. I. The ¯oral nectary. American Journal ofBotany 68: 453±462.

Fahn A. 1949. Studies in the ecology of nectar secretion. PalestineJournal of Botany, Jerusalem Series 4: 207±224.

Fahn A. 1952. On the structure of ¯oral nectaries. Botanical Gazette113: 464±470.

Fahn A, Benouaiche P. 1979.Ultrastructure, development and secretionin the nectary of banana ¯owers. Annals of Botany 44: 85±93.

Fisher DB. 1968. Protein staining for ribboned epon sections for lightmicroscopy. Histochemie 16: 92±96.

Ga�al KP, Heimler W, El-Gammal S. 1998. The ¯oral nectary ofDigitalis purpurea L. Annals of Botany 81: 251±262.

Haran Iyer KRP, Subramanian RB, Inamdarand JA. 1989. Structure,ontogeny and biology of nectaries in Lu�a acutangula (1.) Roxb.var. amara (Lam.) C1. Korean Journal of Botany 32: 101±107.

Hayat MA. 1981. Principles and techniques of electron microscopy:biological applications. London: Edward Arnold.

Imms AD. 1960. A general textbook of entomology, 9th edn. London:Methuen and Co Ltd.

Nepi M, Ciampolini F, Pacini E. 1996a. Development and ultrastruc-ture of Cucurbita pepo nectaries of male ¯owers. Annals of Botany78: 95±104.

Nepi M, Pacini E, Willemse MTM. 1996b. Nectary biology ofCucurbita pepo: ecophysiological aspects. Acta Botanica Neerlan-dica 45: 41±54.

Okoli BE. 1989. SEM study of surface characteristics of the vegetativeand reproductive organs of Telfairia (Cucurbitaceae). Phytomor-phology 39: 103±108.

Reynolds ES. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. Journal of Cell Biology 17:208±212.

Spurr AR. 1969. A low-viscosity epoxy embedding medium for electronmicroscopy. Journal of Ultrastructure Research 26: 31±43.

Vassilijev AE. 1972. The ultrastructure of the nectary cells of cucumberCucumis sativus L. Tsitologia (Akademia Nauk SSSR) 14:405±415 (in Russian).

Zer H, Fahn A. 1992. Floral nectaries of Rosmarinus o�cinalis L.Structure, ultrastructure and nectar secretion. Annals of Botany70: 391±397.

Zohary M, Feinbrun-Dothan N. 1978. Flora Palaestina. Jerusalem:

Israel Academy of Science and Humanities.