Diversity of the floral nectaries surface of four Crataegus L . species MIROS £

The performed studies focused on the surface structure of floral nectaries of four species from the following genus: C. coccinea L , C. crus-galli L, C. curvisepala Lindm and C. prunifolia (Poiret) Pers. The observations of the epidermis area were made in a scanning electron microscope (SEM). A nectary appears to be shaped like a slightly curved disk situated between the pistil style and the basal part of the stamens filaments. The nectary area of the studied species differed substantially as regards the cuticle sculpture and stomata number. The nectary secretion in Crataegus flowers proceeds through the stomata located below a level of the other epidermis cells, in the deep indents of the secretory tissue. The highest stomata number in 1 mm nectary epidermis was recorded in C. crus-galli, C. coccinea, C. prunifolia and finally, C. curvisepala. Analyzing the nectary cuticular structure in respect of its increasing complexity (absence or presence of stripes), the investigated taxons can be ordered as following: Crataegus curvisepala, C. coccinea, C. crus-galli and C. prunifolia.


INTRODUCTION
Species from the Crataegus L. genus (f.Rosaceae, subf.Pomoideae) are found most frequently in North America.The following species, among others, are often cultivated in Poland: C. coccinea L., C. curvisepala Lindm., C. crus-galli L. and C. prunifolia (Poiret) Pers (S e n e t a and D o l a t o w s k i , 1997).Many of them are used in urban plantings on account of their ornamental flowers, fruit and leaves, as well as their medicinal properties (R u t k o w s k i , 1998; A s g a r y et al., 1998; W a n g , 1998).Trees from the Crataegus genus are characterised by abundant nectar secretion and are included among valuable melliferous plants (L i p i ñ s k i , 1976; S e n e t a and D o l at o w s k i , 1997).Among honey types, hawthorn honey is also distinguished (R ob e r t s , 1994).Previously conducted studies show that within the Crataegus genus there are large differences in the amount of nectar secreted by flowers and the concentration of sugar in the nectar (J a b ³ o ñ s k i and K o ³ t o w s k i , 1996; W e r y s z k o -C h m i e l e w s k a et al., 1996).The sugar yield of several previously studied hawthorn species ranged between 0.33 and 4.22 mg of sugars from 10 flowers.The highest weight of sugars was found in C. crus-galli, while the lowest in C. coccinea (We r y s z k o -C h m i e l e w s k a et al., 1996, 2003b).Due to the content of particular types of sugars, the nectar of hawthorns was included in the Pyrus -type, in which little sucrose occurs and the amount of glucose significantly exceeds the content of fructose (M a u r i z i o and G r a f l, 1969).Hitherto, it has been observed that cells of the nectary epidermis demonstrate large differences in the surface, also between the genera within the Pomoideae subfamily (W e r y s z k o -C h m i e l e w s k a and K o n a r s k a , 1995, 1996; T o t h et al., 2000; W e r y s z k o -C h m i e l e w s k a et al., 2003a, 2004).The structure of the epidermis of the nectary gland is one of the factors affecting the amount of nectar secreted.The structural features of nectaries may also be of taxonomic value.
The aim of this study was to compare the surface structure of the secretory tissue of nectaries in four species from the Crataegus genus, with special attention to the cuticular ornamentation and the position of secretory stomata.The number of stomata found in mm 2 of the nectary epidermis was also determined.

MATERIALS AND METHOD
The studies conducted related to the structure of floral nectaries of Crataegus coccinea L., C. crus-galli L., C. curvisepala Lindm.and C. prunifolia (Poiret) Pers.Trees from which flowers were sampled grew in the Botanical Garden of the Maria Curie-Sk³odowska University in Lublin.
For the scanning electron microscopy (SEM) examination of the surface of the nectary epidermis, fragments of flowers were fixed in 4 % glutaraldehyde 4 and (0.1 M) phosphate buffer with pH 7.0 for 4 hours at room temperature.After dehydrating the specimens in ethanol series and acetone, they were dried at critical point with liquid CO 2 .Then the plant material was coated with gold by using the CS 100 Sputter Coater 100.Observations of the surface of the nectary epidermis in the aforementioned taxa were carried out by using the BS-300 Tesla microscope.
In the longitudinal sections, the thickness of the secretory tissue was compared based on semi-permanent slides from fresh plant material which were obtained by using the Vibratome 2000 (Technical Product International INC, USA).The 60 µm thick sections were embedded in glycerol-gelatine.
In order to prepare semi-thin sections, the plant material was postfixed with 1% osmium tetroxide, then dehydrated and embedded in Spurrs epoxy resin.Sections of 0.75 mm thickness were cut by using the Reichert Ultracut S ultramicrotome, then stained with 1% methylene blue with 1% azur II.The sections were mounted in synthetic resin Eukitt.

RESULTS
The flowering of the aforementioned species occurs in May and June.The length of this period in the climatic conditions of Lublin was about 14 days.Flowers of the taxa studied were characterised by a white corolla and bright red anthers, as well as a diverse structure of the pistil.C. coccinea, C. prunifolia and C. crus-galli flowers develop a distyloulos pistil, whereas in C. curvisepala a monostylous pistil occurs (Figs 1-4).figs 1, 2, 3, 4).
Around the style of the pistil in the four species studied, numerous, strongly elongated unicellular hairs were observed in the epidermis of the nectary tissue (Figs 2, 4-6).
Nectar in the analysed flowers of the Crataegus representatives is easily accessible for insects, since stamens and elements of the perianth, when opening during flowering, uncover the nectary gland, situated on the adaxial surface of the concave flower receptacle.The nectary was yellow coloured, and it covered the surface between the style of the pistil and the basal part of filaments (Figs 1-6).The secretory tissue was characterised by a several-layers of cells, and it made up half of the thickness of the flower receptacle in this region (Figs 5-7).Observed from above, the contour of the epidermis cells covering the nectary tissue in flowers of the taxa studied had a quadrangular shape 16,17).Nectar is secreted through stomata located below the level of the other cells of the nectary epidermis 16,17).As a result of a large depression, it was difficult to determine the aperture of the stomata, it was only found in C. prunifolia that they were open (Fig. 12).The modified stomata occurring in the epidermis of the nectaries of the observed species from the Crataegus genus were classified as anomocytic.They are characterised by the occurrence of a variable number of epidermis cells which surround the guard cells in a ring, and they do not differ from other cells of the epidermis 16).The studied species of hawthorn developed a large number (175-308) of stomata in mm 2 of the nectary epidermis.The largest number of stomata was recorded in the epidermis of C. crus-galli, a much smaller number in C. coccinea, C. prunifolia, and C. curvisepala (tab.1).The stomata were evenly distributed over the whole surface of the nectary (Figs 9,11,16).The observations in scanning electron microscopy show that the outer wall of the nectary epidermis was covered by a layer of the cuticle differing in the ornamentation in particular taxa.The cuticle on the surface of the epidermis cells of the C. curvisepala nectary was smooth with wax coating which formed in some places an irregular, reticulate structure (Figs 9,10).Some cells of the epidermis in C. coccinea and C. crus-galli showed clear striation of the cuticle, and other cells had a smooth surface (Figs 8,13 17).On the surface of the C. crus-galli nectary, cuticular striae were observed in some places, undulate and interleaved, forming an ornamentation partly concealing the contours of the epidermis cells (Figs 8).But cells of the epidermis covering the C. prunifolia nectary tissue had clear cuticular sculpture in the form of longitudinal, slightly undulate striae running parallel within particular cells and radially around the stomata (Figs 11,12).When analysing the cuticular sculpture of the nectary epidermis in terms of the increasing degree of complexity of structure, the taxa studied can be ranked as follows: Crataegus curvisepala, C. coccinea, C. crusgalli and C. prunifolia.

DISCUSSION
The nectaries in flowers of the taxa studied from the Crataegus genus form on the surface of the flower receptacle, a glandular layer which, according to S m e t s (1986), developed from the reduced stamen whorl in the Rosidae subclass.F r e y -W y s s l i n g (1955) found that the disc nectary belongs to highly specialised structures occurring within the abovementioned plant subclass.In terms of the form in the Pomoideae, the following types of nectaries were distinguished: automorphic (Crataegus, Pyrus, Chaenomeles), epimorphic (Cydonia) and transitoric (Malus) (We r y s zk o -C h m i e l e w s k a and K o n a r s k a , 1995; 1996).
Literature data show that nectaries of plants belonging to the Rosidae can be divided in terms of the presence or absence of modified stomata (Smets, 1988).Another division, relating to the tissue origin of nectaries, defines them as mesophilic (Vo g e l , 1977; S c h m i d , 1988).
In flowers of the studied taxa from the Crataegus genus the presence of the secretory tissue was found, secreting nectar through anomocytic stomata.The state of opening of the stomata in C. curvisepala, C. crus-galli and C. coccinea was difficult to determine due to their deep location.In the longitudinal sections of flowers, it was observed that the stomata in C. crus-galli and C. coccinea reach the second level of the glandular cells, which were characterised by a thick wall, numerous plastids with starch grains and a high degree Crataegus prunifolia is a species developed as a result of crossing of C. crus-galli and C. macracantha (Rutkowski, 1998).In spite of the very close affinity of C. crus-galli and C. prunifolia, significant differences were demonstrated in the structure of the surface of the nectary epidermis in these species.The cuticle was characterised by a different ornamentation, from partially striate in C. crus-galli to strongly striate over the whole surface in C. prunifolia.In the four Crataegus species studied, a total of 3 types of the nectary surface, related to the cuticular sculpture, were distinguished: smooth, striate in some places and striate over the whole surface.The striate sculpture of the cuticle of the nectary probably performs different functions, among others, protects nectar against drying and facilitates the distribution of the secretion over the surface of the nectary.According to H e j n o w i c z ( 2002), the rich cuticular sculpture more effectively repels from the epidermis rainwater drops which, when falling, do not flatten, and rolling over the surface, they enable it to self-clear.In its composition, the cuticle contains special carbohydrates, and on its surface waxes deflecting excessive UV radiation.It is particularly important, especially for the glandular tissue unprotected by stamens and the perianth.This layer also helps to limit the penetration of different chemical substances affecting plants.
At the same time, the cuticle, which is a complex compound made up of strongly polymerised fatty acids resistant to the action of water, is characterised by chemical stability (E s a u , 1973; H e j n o w i c z , 2002), thanks to which it does not decompose.Esau (1973) states that no microorganism decomposes the cuticle, since it does not have any enzymes which degrade the cutin.As a result of this, the cuticle of the nectary epidermis is an important mechanical barrier against unfavourable conditions of the environment, among others: infection, damage by the wind and insects.Significant differences, observed in this study, in the micromorphology of the nectaries of closely related species within the Crataegus genus may result from adaptations to different habitat conditions in which they occur in natural locations.According to H e y w o o d (1974), the varied cuticular ornamentation may be used as a taxonomic value.

CONCLUSIONS
1.In flowers of the studied species from the Crataegus genus, nectar is secreted through anomocytic stomata located in deep depressions of the secretory tissue.
2. The largest amount of stomata for one unit of surface area was found in the epidermis of the C. crus-galli nectaries, then in C. coccinea, C. prunifolia and C. curvisepala.
3. The epidermis covering the floral nectaries in the studied species from the Crataegus genus formed three types of cuticular ornamentation: smooth, partially striate and strongly striate.
of vacuolisation (W e r y s z k o -C h m i e l e w s k a et al., 2003b).Open stomata in the epidermis of the nectaries analysed in the present study of hawthorns were only visible in Crataegus prunifolia.However, earlier studies of W er y s z k o -C h m i e l e w s k a et al. (2003b) showed the presence of closed stomata in the secretory tissue of Crataegus crus-galli, in spite of the fact that observations were carried out during the secretion period, while in Malus (T o t h et al, 2000) the stomata were characterised by different apertures, what indicates their ability to open and close.These observations suggest the active functioning of stomata in the epidermis of nectaries of the abovementioned taxa, what is related to the rhythmic secretion of nectar (T o t h et al., 2000; W e r y s z k o -C h m i e l e w s k a et al, 2003b).Among the analysed species from the Crataegus genus, the largest number of stomata on the surface area of 1 mm 2 of the nectary epidermis was observed in C. crusgalli, and the lowest in C. curvisepala.In many plant species, a positive correlation between the number of stomata in the nectary epidermis and the abundance of nectar secretion was found (T e u b e r et al, 1980; M u r r e l l et al. 1982; D a v i s and G u n n i n g , 1991; W e r y s z k o -C h m i e l e w s k a et al., 1997).One can expect that a similar dependence applies to nectaries of all the Crataegus species studied, since it was previously demonstrated for C. crus-galli and C. coccinea (We r y s z k o -C h m i e l e w s k a et al., 2003b).
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Table 1
Number of stomata and kind of cuticle ornamentation of four species from Crataegus genus.