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Scanning and transmission electron microscopy related to immunochemical analysis of grass pollen G. Peltre , M.-Th. Cerceau-Larrival , M. Hideux , M. Abadie & B. David To cite this article: G. Peltre , M.-Th. Cerceau-Larrival , M. Hideux , M. Abadie & B. David (1987) Scanning and transmission electron microscopy related to immunochemical analysis of grass pollen, Grana, 26:2, 158-170, DOI: 10.1080/00173138709429945 To link to this article: http://dx.doi.org/10.1080/00173138709429945

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Date: 21 January 2017, At: 06:31

Grana 26: 158-170, 1987

Scanning and transmission electron microscopy related to immunochemical analysis of grass pollen G . PELTRE, M.-TH. CERCEAU-LARRIVAL, M. HIDEUX, M. ABADIE and B. DAVID

Peltre, G . , Cerceau-Larrival, h1.-Th., IIideux, M., Abadie, hl. 6: David, B. 1987. Scanning and transmission electron microscopy related t o immunochemical analysis of grass pollen. - Grana 26: 158-170, 1987. Upsala 25 June 1987. ISSN 0017-3134. T h e exine stereostructure (scanning electron microscopy) and ultrastructure (transmission electron microscopy) of pollen of seven grass species, is related to the allergens extracted from these pollen grains. The heterogeneity of the allergens was studied by the immunoprint technique and revealed by labelling the binding of grass pollcn sensitive patients IgE antibodies. Using patient sera recognizing a very restricted number of allergens, we showed that a group of pollcn had a great number of allergens in common (Dacrjlis, Agrosris, Fesritca, Loliitrri, Holcirs) and, in decreasing cross reactivities order, we found Averia and, finally, Zen mays. T h e tectum stereostructure shoivs presence of insulae in all pollen grains except in Zea rnajs which has small isolated spinules. These insulae are separated by very wide and deep interinsular spaces in Avena saliva with connections between insulae. I n the remaining species, no connections were seen between the insulae. These observations were in good correlation with the immunological cross-reactivity of the allergens present in the pollen. In all species, there are microperforations in the bottom of the interinsular spaces, which are the opening of the tectal microchannels.

G. Peltre & B . David, Unite d’lrrirriiirioallergie,Insritiit Posreiir, 25 rite dit Dr. Roitx, 75015 Paris, France. hl.-TIi. Cerceait-Larrival & h f . tlideiu, CNRS A1 0312S4, Laboratoire de Paljnologie, hfirsPiirn National d’tlistoire Nariirelle, 61 rite de Bitffon, 75005 Pnris, Frnnce. hl. Abadie, Laboratoire de Cryptoganiie Ultrastriictiirale, EPIIE, 12 rite de Bitffon, 75005 Paris, France. (hfaniucript received 8 Aiigitst 1956, revised version ncceprrd 12 Febriiary 1987)

Fundamental studies of grass pollen are very important for palynologists and allergologists. Palynologists are mainly interested in the function and structure of pollen grains. The allergologists wish to find out which structures are associated with the allergens present in the pollen grains. In our study, two groups have worked together on grass pollen to compare the results obtained by their respective research tools: light and electron microscopy on one hand, immunochemistry on the other. Previous studies carried out in the above manner are those of Heslop-Harrison et (1973), K~~~ et al. (1975)7 Knox (1979)’ Vithanage et (1980) and Singh 8r Knox (lgg5). The present investigationintroduces a new technique in this field; the nitrocellulose immunoprint technique (Peltre et al., 1982) which is a powerful means to study the heterogeneity Grana 26

of antigens and allergens in aqueous crude pollen extracts. The aim of this investigation is to compare‘the results of structural analysis of pollen of some grass species observed by light and electron (scanning and transmission) microscopy and those obtained by immunodetection of the allergen contents from the same species. MATERIAL AND METHODS Pollen was sampled by the immuno-allergy unit of the Pasteur Institute, Paris, defatted in acetone-ether, dessicated and refrigerated at 4’C. The following taxa were studied, and numbered according t o alDhabetic order of French vernacular names,

-

.

* 1. &rosris albn L.,Agrostis (Creeping bent) 2 . Avena saliva L.,Avoine (Common oat)

Electron riiicroscopy arid iriiriiirriochertiistry of grass polleri

159

Table I. I'roposal for a classification of soltie Graniirieae according to pollen charncters NO. of spinuledinsulae Loliuni perenne (1-6) 31-47; 42.6f1.26

Not well differentiated

Well differentiated

Isolated spinules

Generally grouped spinules

1

Zea ntays

95-105; 102f1.32

range; mean S.E. 3 3 4 0 ; 37.0520.92

Wide interinsulae spaces

1 With connections between insulae

1

Avena safiva (1-3)

5340; 56.75f I .O

* 3. Broniits anwtsis L., Brome (Field bromc) 4. Dacfjlis glonierarn L., Dactyle (Cocks foot)

5. Fesiiica prarensis Huds., Fituque (hleadow fescue) 6. AnfIioxant1iiitii odorafirni L., Flouve (Sweet vernal grass) * 7. Ifolciis lanaiits L., Iioulque (Yorkshire fog) * 8. Loliitrii perenne L., Ivraie (Rye-grass) * 9. Zea iiiays L., hlai's (Maize, corn) 10. Peririisefitrn fyphoi'des (Burm.) Stapf et IIubb., hlil (Pearl millet) 11. Ilordeioti sativitm Pers., Orge (hleadow barley) 12. Poa prafensis L., Paturin (Smooth meadow grass) 13. PIiIeirm praterise L., Phlkole (Timothy grass or Cat's tail) 14. Secale cereale L., Seigle (Rye) Cult. 15. Alopecrtrirspraferisis L., Vulpin (hkadow or common fox tail)

Only seven species (*) were investigated pollenmorphologjcally in this paper; all 15 species were examined in previous studies. The results of which confirm the present ones (Cerceau-Lamval et al. 1986). Imiit irtioclieiiiisfry

agarose IEF is carried out on a flat plastic sheet of Gelbond 11x11 cm (hlarine Colloids, Rockland, USA). The gel, 0.4 mm thick, consists of 1% agarose IEF (Pharmacia, Uppsala, Sweden), 12% sorbitol and 2% ampholytes. The crude extract of each pollen sample is made just before use by suspending 50 mg of pollen in 0.5 ml distilled water under agitation for 1 h at room temperature followed by centrifuging for 5 min at 1OooO g. 10 pl of each supernatant are applied on a cellulose paper strip (2x 10 mm) on the agarose gel 2 cm from the anode, side by side, on a line parallel to the electrodes. IEF is performed at 4°C at a constant power of 4 W for 90 min. The IEF equipment used was the FBE-3000 and the constant power supply apparatus, ECPS 3000/150 from Pharmacia.

Agarose isoelecrric focitsing.-The

-

No connections between insulae

-

% 5' "J 01

Fesrirca prafeiisis (1-7) 31-45; 41.03k1.16

P

Dacfylis glonierafa (1-5) 33-30; 37.0520.92

ij.

c

2.

r:

"I

Agrostis alba (3-7) 25-33; 30.3620.87

--- -

Holcics lariatics (2-7) 27-32; 30.25f 0.74

Intniitnoprinf on riirrorelliclose filrers.-A print of the agarose IEF separation is taken on a nitrocellulose filter in 15 mm by simply pressing as described earlier (Peltre et al. 1982). The allergens are recognized by IgE antibodies from patient sera sensitive to grass pollens. Patient sera were selected following the heterogeneity of the allergens (or allergen spectrotype) they were able to detect in Dactylis glomerata crude extract. The first group of patients (A) had a very restricted allergen spectrotype: 1 to 5 allergens recognized by approximately 20% of the patients that we have studied. Group B patients had a more complex and common allergen spectrotype (observed in 60% of our patients). Group C patients had the most heterogeneous spcctrotype (20% of our patients). Pollen rnorphology Light microscopy.-The \\'odehouse technique has been used for direct observation: pollen grains are mounted in fuchsin stained glycerol jelly (U'odehouse 1935). Some observations are made on acetolysed pollen grains heated at 100°C for less than a minute in an anhydric acetic acid and sulphuric acid mixture (9 : 1) following Erdtman's technique described in detail by Cerceau-Larrival 8r Hideux (1953). Semi-thin sections of Zen and Dacrylis pollen grains are made after embedding in Spurr resin (Spurr 1969). hleasurements are based o n 50 pollen grains per species (mounted according to "odehouse technique). The mean value and standard deviation are given inTable I, along with extreme values of the diameter (minimum and maximum).

Inditced jltiorescence of the pollen grains.-The pollen grains were fixed in a 4% p-formaldehyde solution, washed 12 h in phosphate buffer (0.1 hl, pH 8.0) containing 0.05 hl EDTA, then incubated for 18 h at 4°C in a 0.3%Thioflavine S solution adjusted at pII 9.0 by K2HP040.1 hl. The pollen was washed 3 times (5 min) in distilled water, incubated for 18 h at 4°C in 0.3% Phosphine 3R (Gurr) and 0.001% Rhodamine B (Gurr) solution and finally washed again 3 times (3 min) in distilled water. Crana 26

160

G. Peltre et al.

-_

5.9,

10.6.

The observations were carried out with a Leitz Orthomat microscope equipped with a Phloem Fluorescence attach. ment: a 150 W high pressure mercury lamp, a 2 mm UG 1 filter and a K 430 filter. The photographs were taken using Ilford FP4 film (125 ASA).

Ir #&-

Scanning electron

A

1.

1 2 3 4 .5 6 7 8 9 10 11 121314 15.

tnicroscopy (SEM).-The pollen grains were either untreated, or acetolysed and lyophilised. 40philisation was done by freezing under vacuum for 48 h at -50°C at lo-* Torr in Pearse tissue dryer. Specimens were then coated in an Edwards Sputter coater type S 15OA.The observations were made on a Jeol 35C microscope at the Geology Laboratory from the hluseum National d’Histoire Naturelle. Transmission electron microscopy (TEM).-For TEhl studies the method followed is that of Abadie 6( Hideux (1979). Photographs were taken using a IIitachi IIU A microscope at the hluseum National d’Histoire Naturelle.

RESULTS Itiitii iitiocliemistry Three representative experiments performed with three different patient sera are reported here (Fig.

1).

I

I

1Q6,

1 2 3 4 56789tO1112131415

Fig. 1. Immunochemical detection of allergens. Immunoprint patterns obtained with 3 representative types of grass pollen sensitive patient sera A, B and C. Fifteen different Grana 26

T h e first experiment was performed by using a serum, the Dactylis allergen spcctrotype (see Materials and Methods) of which is shared by 20% of the 2000 grass pollen sensitive patient sera we have screened. These patients recognize only a very limite d number of allergens, from Dactylis pollen, less than five bands. One band, the major allergen called Dae g 1, has an isoelectric point (PI) of 5.9 (Fig. 1 A). The other bands are basic, from PI 10 to 10.6, and not clearly defined. A group of pollen shows a rather similar allergen pattern. In decreasing order of recognition intensity, they are: Fcstiica, Lo!iiini, Agrostis, Dactylis and liolcin. A second group of pollen is less strongly recognized by the patients IgE antibodies to allergens. It contains: Brotiziis with an acid allergen, Hordeiitn with an intensely basic allergen, Poa with three distinct bands ranging from PI 7 to 10. Secale with weakly basic allergens and to a lesser extent, Avetza and Atitlioxaiztliuni. Among the non-allergic pollen for this group of patients are those of Pfileuni, Alopecimis and Pe~itiisetum. grass pollen extracts, numbered as in Material and Methods, were separated side by side by IEF in a large pH range. A print of the separation was taken on a nitrocellulose filter, incubated with one patient serum, and finally with radiolabelled anti IgE antibodies. After autoradiography the heterogeneity of the allergens were visualized.

Electron rtiicroscopy arid itiiriiziriochetiiistry of grass polleri The heterogeneity of the allergens revealed by the sera from the sccond group of patients (60% of them) (Fig. 1 B) shows a great similarity to the patterns expressed by Dactylis, Loliiuri and Phlezini and, to a lesser extent, by Hordezini. More than 10 allergen bands are recognized from pI.4 to 10.6. A second group of pollen exhibits common allergens in decreasing amounts, Agrostis, Astiicn and Secnlc. A third group is far less recognized as allergenic: Brorms, fiolczis and Pon. Finally, two pollen species, Zea and Alopeciirzis, are very weakly recognized as allergenic. Averin, Arirlioxarirliiirti and Perzrzisetzitti are not allergenic at all. T h e third type of allergen pattern is obtained from about 20% of the patients who are strongly reactive t o Dactylis as shown in Fig. 1C. This pattern diffcrs from the previous one (Fig. 1 B) by modifications in the intensity of the reactions. The first group consists of Dactylis, Fesrzica (which is even more strongly recognized than Dactylis); Lolizirn, Agrostis and Holciis which is very weakly recognized (Fig. 1 B). The second group is made up of Hordeiitn, Pon, Plzlezirii, Brotiiiis, Alopeciirzis, Secale and Aiwza (negative in A and B) and Aritlzoxarzihiiii which is very weak whereas Zea and Perzrziserzirti are not allegenic.

Polleti riiorpiiology Pollen grains of all the seven species are heteropolar, monoporate, operculate and more or less circular in outline (except Averin which rcsenibles a truncated cone). T h e pore which is bordered by a thick annulus and covered by an operculum is located very close to the distal pole (Fig. 2). Induced fluorescence observations were made and a good fluoroehrome fixation was obtaincd in an alkaline medium (Figs. 4 A , 5A,7A). Three species: Dactylis glottzerata (Fig. 4), Avetiu sativa (Fig. 7), Zea rmys (Figs. 5, 6 ) ) were studied in greater detail. The exine fluorescence was obvious. The insulae were clearly visible with dark spaces in between. The annulus and the operculum are highly fluorescent. The space between the pore and operculum is strikingly dark. In semithin sections (sub-equatorial), it is remarkable that exine and intine are thin compared to the pollen diameter (Figs. 4 B , 5C). The exine thickness ranges from 2 o r 3 pm. In non-defatted Zea mays pollen, the cytoplasm contains a great number of starch inclusions used as reserves o r stock material (Fig. 5C). In contrast, a similar section of Dactylis, which was defatted before fixation, shows a totally disorganised cytoplasm (Fig. 4 B). I 1-878362

161

Stereostriictzire Tectzitri (Fig. 3).-1n general, the tectum is complete with supratectal processes (spinules) either grouped on insulae which are distinctly separated by more o r less wide and deep grooves or incisions at the bottom of which microperforations can b e seen, as in Averza (Figs. 3F, 7B). T h e most obvious differences concern the insulae. Zea riznys is the only species which exhibits isolated spinules not grouped on insulae, with rather wide interspinular spaces (Fig. 5 D). Averzn sativn has elongated insulae with 1 to 3 spinules and is characterised by the presence of connections between adjacent insulae and by very clear microperforations in the deep interinsulae spaces (Fig. 7 B). T h e remaining species investigated are devoid of inter-insular connections. The pollen grains are distinguished only by'the shape Fig. 2. General view of pollen grains. (A) Agrosris albn, non acetolyzed pollen. SEhl ~ 2 6 0 0 .(B) Docijlis glorrierura, acetolyzed pollen. SEhl x2 600. (C) Fesrircu prafensis, acetolyzed pollen. SEhl X2 600.(D) Loliirrn pereririe, acetolyzed pollen. SEhl ~ 2 6 0 0 .(E) Averzn sariva, acetolyzed and freeze-dried pollen. SEhI X2600. (F) IIolczu lariaiiis, non acetolyzed pollen. SEhl X2600. Fig. 3. A-E Tectal surface of acetolyzed pollen grains. SEhl ~ 1 0 0 0 0 .(A) Docqlir glonierafa. Widely and deeply separated polygonal insulae bearing 1 t o 15 spinules, one or several are localized in the middle of the insulae, the remaining at the edge. No bridges between insulae. (B) Festrrca praferisis. Elongated or sometimes polygonal insulae of varying sizes, spaces between insulae k wide but not deep. Generally 1 to 7 spinules on each insula often at the edge. No bridges between insulae. (C) Lolirrnt pereritie. Elongated or rarely polygonal insulae of varying size, spaces between insulaekwide but not deep. Generally 1 t o 6 spinules on each insula often at the edge. No bridges between insulae. (D) Agrosris alba. Elongated to polygonal insulae of varying size, widely and deeply separated and beset with 3 or 7 spinules mostly at the edge, 1 or 2 in the middle. No bridges between insulae. (E) llolctu loriorus. Elongated to polygonal insulae of varying size, widely and deeply separated and beset with 2 t o 7 spinules mostly at the edge, 1 or 2 in the middle. No bridges between insulae. (F) Aveno sariva. Elongated insulae very widely separated with 1 t o 10 spinules mostly at the edge. Connections bet w w n insulae and large microperforations clearly visible.

Fig. 1.A-E. Dncrjlis glomerota. (A) Induced fluorescence. Lhl ~ 2 5 0 0 .(B) Section of an entire pollen grain. TEhl x5OOO. (C) Section through the apertural zone showing pore, operculum, annulus, and a thickened sole. T E h l ~ 1 5 0 0 0(D) . Part of exine showing teetum with widely and deeply separated insulae. Spinules at the edge of the insulae. Acetolyzed and freeze-dried. SEhl X20000. (E) Section showing tectum with microchannels and supporting columellae, and an underlying thick sole. T E h l X5OOOO. Grana 26

162

G . Peltrc

el a!.

Electrori microscopy arid ittiniirriochetiiistry of grass pollen

Fig. 3.

163

164

Fig. 4.

G. Peltre el 01.

Electroti microscopy

of the insulae and the depth of the inter-insular spaces. T h e insulae are of different sizes and their shape varies from elongated to polygonal. No deep inter-insular spaces are found. One to 15 spinules are generally located on the periphery of insulae (Fig. 3). Fesritca pratetzsis has elongated and sometimes polygonal insulae of different sizes; spinules are 1 to 7 in number (Fig. 3 B). In Loliztttz peretitie, no deep inter-insular spaces are seen, 1 to 6 spinules are seen on the periphery of each insula (Fig. 3C). Agrostis alba and HOICLIS latznfzs are very similar. Their elongated to polygonal insulae of different sizes are widely and deeply separated. Each insula bears 2 to 7 spinules most of which are on the periphery, only one or two in the middle (Fig. 3 D , E). Dactylis glotnerata has widely and well separated polygonal insulae, usually with 1 to 15 spinules a few of which are located in the middle (Figs. 3 A, 4 D). The above results are summarized inTable 1. A big variation of the pollen diameter can be seen. The biggest variation is encountered in Zen nzays and the smallest in Agrostis alba and H O ~ C laitatits. ~IS Aperture.-The apcrture consists of a pore with an operculum surrounded by a thick annulus. The striking thickness of the annulus can be seen in Fig. 2. The ratio between the diameters of the pore and the pollen grain is 1 : 10 (Fig. 2) except in Zea nzays which is about 1 :20 (Fig. 5 B). The stereostructure of the annulus and the operculum are similar to the one described for the tectum (Fig. 6A). Ultrastriictiire T h e pollen grain of 3 representative species were chosen for an ultrastructural study by TEM: Zea tnays, Avetia sativa and Dactylis glottierata.

Exitze stratificatiotz.-In general, the tectum is spinuliferous and traversed by microchannels. The infratectum is columcllar and the intercolumellar spaces are variable. No microchannels are seen in the columellae, but are present in the sole or foot layer (Figs. 4F, 6 C, 6 D, 7 D). Thc tectum seems to be thicker than the sole. The presence of endexine could not be ascertained because the pollen grains were defatted and not fixed in their natural condition. As a consequence no observations were possible on the relation between intine and cytoplasm. In Dactylis, the presence of well differentiated insulae with grouped spinules (Fig. 4C, E) confirms the tectum structure seen in SEM (Fig. 4D). The

atid

itiittiirtioclzettiistry of gross polkti

165

sole and tectum of Dactylis are thinncr than Arwza and Zen. Microchanncls arc seen in both the sole and the teetum (Fig. 4E). In Averza, the presence of insulae with grouped spinules (Fig. 7 D) is also seen in SEM (Fig. 7 B). Tectal microchannels are more frequent than in Dactylis. This is consistent with the fact that numerous microperforations are seen in the bottom of intcrinsular spaces, which are the openings of microchannels. The presence of isolated spinules on the tcctum of Zea niays (Fig. 6C, D) confirms what was seen by SEM in Fig. 5 D. A dense system of microchannels is seen in the tectum, both in cross section (Fig. 6C) and in sub-tangential plank (Fig. 6D). Apertitre itiorphology.--In the three species studied (Figs. 4C, 6 A , 6B, 7C) thc pore margin consists of a distinct annulus made u p of three strata. The innermost, the sole, is thick and lamellated. The pore itself is made up of operculum which shows the normal 3-layered structure of the exine. Below the operculum the layer is remarkably thickened.

DISCUSSION For this preliminary study we have observed the pollen grains already treated (not fixed, in living state) obtained from Institut Pasteur. Fig. 5. A-D. Zea tnajs. (A) Pollen grain showing cxinc and pore fluorescence. Induced fluorescence. Lhl X2500. (B) Acetolyzed and freeze-dried pollen grain, general view. SEht X I500. (C) Section of an entire pollen grain. Lht X2000. (D) Part of exine showing distribution of spinulcs. Note absence of insulac. Non-acetolyzed and freeze-dried. SEht ~ 2 0 0 0 0 .

Fig. 6. A-D. Zea nrays. (A) Non acetolyzed pollen grain showing details of a pore, operculum and annulus. SEht X10000. (B) Section showing details of thc apcrtural zone. Note pore, operculum and Z-layer. TEM X l S 0 0 0 . (C) Section showing tcctum with rnicrochannels, supporting colurnellac and a thick sole. TEhl X50000. (D) A tangential scction showing tectum, rnicrochannels, supporting colurnellac and sole. TEM XSOOOO. Fig. 7. A-D. Avena sarivn. (A) General view of a pollen grain showing exine and fluorescent pore. Lht ~ 2 5 0 0 (B) . Part of exine showing elongated, and widely and deeply separated insulac beset with 1-3 spinulcs. Note bridges between insulac and microperforations in spaccs between insulac. Non-acctolyzed. SEM ~ 2 0 0 0 0 .(C) Section through apcrtural zone showing pore, operculum, annulus and thickened sole. TEM ~ 1 5 0 0 0 .(D) Section of cxine showing microchannels, tectum ( r ) , columellae (c). sole (s), spinulcs and insulae. TEM X5OOOO. Grana 26

166

Fig. 5.

G. Peltre et 01.

168

G. Peltre et 01.

I

Fig. 7.

. ..

Elecrrori microscopy arid itrtt~ir~tiocliettiistry of grass pollen The techniques employed (Lhl, fluorescence, S E h l andTEM) enabled a detailed study of the main characters of Gramineae pollen grains. T h e pollen grains are: monoporate, with an operculum, annulus and a very thin spinuliferous exine (0.5 t o 2 pm). Ectexine is composed of tectum, columellae and sole. The above is in conformity with the results of Rowley (1960, 1964), Andersen & Bertelsen (1972), Damblon (1975), and Linder & Ferguson (1985).The endexine is not clearly seen and its presence in monocots and in the Gramineae is doubted. Skvarla & Larson (1966), Heslop-Hamson 6: Heslop-f iarrison (1980), Kress & Stone (1982), Zavada (1983) and El Ghazaly & Jensen (1986) have described a thin endexinous layer in wheat. However, the main differences between the grass pollen grains, besides the pollen size, are those of the tectum stereostructure (cf. Table I). One group of pollen comprises Dactylis, H o h i s , Agrosris, Festrrca and Loliiitn, the others Zea and finally Averin. Pollen studies of the 15 previously mentioned species (p. 158-159) confirm the isolated position of Zea and Avctin (Cerceau-Lamval e t al., 1986). Microchannels observed by S E M were confirmed by TEM. Similar microchannels were also described in Viciu fuba in SEhl and T E M by Cerceau-Lamval et al. (1982), as well as in other grasses by El Ghazaly & Jensen (1985, 1986) and Rowley, El Ghazaly & Rowley (1986: personal communication). Possibly these microchannels play a role in the exchange of fluids and material between the pollen grain and its microenvironment, the water exchanges (hydrodynamics) according to Heslop-Harrison (1979) which also implies the ZwischenkBrper (Heslop-Harrison & Heslop-Harrison, 1980), a s well a s in the storage and transit of the impregnation substances of sporophytic origin from the tapktum: glycoproteins, glycolipids, proteins and carotenoid pigments (Cerceau-Larrival e t al. 1980-81, Ilideux 1981). These above observations are in total agreement with the immunochemical analysis of the allergen present in the pollen grains. Most of the grass sensitive patients (60% represented by serum type B in Fig. 1plus 20% represented by the serum type C) did not recognize any allergen in Zea pollen; only a small amount in Averla and a lot in the remaining pollen group. By the immunoprint technique a sharper comparison of the allergens contained in each pollen species is possible both qualitatively and quantitatively. The present study is an example of the capacity of such a technique which can be in the taxo-

169

nomic comparison of grasses. The number of common allergen bands and their intcnsity seems t o correlate very well with the common stereo and ultrastructural aspects seen among different pollens. The fact that a limited number of patients (20% represented by serum type A in Fig. 1) recognized rare allergens in Zen as well as a restricted number of a!lergens in the main allergenic pollen group which suggests a possible identity between allergen bands of close isoelectrie points and even the presence of common allergenic epitopes on molecules of different isoelectric points (as seen in observations with mouse monoclonal antibodies t o allergens). According to the classification proposed by Gaussen et al. (1982), the Gramineae (Poaceae) belongs to the order Graminales or Poales of the subclass Commelinidae, class hlonocotyledonae. The family consists of about 620 genera and 10000 species, grouped in tribes. I n the old classification of Bonnier (1934) as well as in recent classifications of Hubbard (1980), Watson 6: Dallwitz (1981), Macfarlane & Watson (1982) and Hilu & Wright (1982), Averia and Zea are usually isolated and the other genera studied are grouped together, which is in total agreement with our results.

ACKNOWLEDGEhlENTS Special thanks are due to Dr Siwert Nilsson (Palynological Laboratory, Stockholm who has provided valuable advice throughout the preparation of the manuscript and for providing generous assistance in revising the English. We are grateful to Professor L. Leclaire (Geologic, hiuseurn, Paris, France) who kindly allowed us to use the Jeol microscope and L. Derouet (Palynologie, CNRS, hluseum, Pans, France) for operating the instruments, preparing materials, and SEhl observations.

REFERENCES Abadie, hl. & Hideux, hl. 1979. L'anthere de Saxvraga cymbalaria L. ssp. Iiuefiatia (Boiss.) Engl. et Irmsh. en microscopie ilectronique (M.E.B. et h.1.E.T.).1. Gin& ralitCs. OntoginPsc des orbicules. - Ann. Sci. Nat. Bot. ser. 13: 199-223. Andersen, S. T. & Bertclscn, E 1972. Scanning electron microscope studies of pollen of cereals and other grasses. - Grana 12: 79-86. Bonnier, G. 1933. nore complete illustrie en couleurs de France, Suisse et Belgique. - In: R . Douin: Gramineae, 11: 113-159, 12:5-76. - Libr. Gin. I'Enseignement, Paris. Cerceau-Larrival. hl.-Th., Abadie, hl., Albertini, L., Audran, J. C., Cornu, A., Cousin, hl.-Th., Dan DickoZafimahova, L., Duc, G., Ferguson, I. K., Hideux, hf., Grana 26

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