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Nature and distribution of the exine-heldmaterial in mature pollen grains of apiumnodiflorum l (apiaceae)Martina Weber aa Institute of Botany and Botanical Garden, University of Vienna, Rennweg,14, A-1030, Vienna, AustriaVersion of record first published: 01 Sep 2009.

To cite this article: Martina Weber (1992): Nature and distribution of the exine-held material in maturepollen grains of apium nodiflorum l (apiaceae), Grana, 31:1, 17-24

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G r a m 31: 17-24, 1992

Nature and distribution of the exineheld material in mature pollen grains of Apium nodiflorum L. (Apiaceae)

hlARTINA WERER

\Vcbcr, hf. 1992. Nature and distribution of the cxinc-hcld matcrinl in mature pollen crnins of Apitrnt riodiflortrrn L. (Apinccac). - Gmna 31: 17-21. 1992. Odcnsc. February 1992. ISSN 0017- 3131.

In Apiiirft riodiflorttni two kinds of exinc-held material are obscrvcd: pollcnkitt and residuals of the polysaccharidic primexine matrix. The latter is forming a loosc texture inbcttvccn thc tectum elements, while pllenkitt exists in spaces betwen bacules as ivcll as the outer cxinc surface. Furthermore the existance of an exinic outer membrane is discussed.

hlariirta Wcbcr, Institicre oJ Botany arid Botanical Gnrden, University OJ Vicma. Reniiweg 14. A-1030 Vieiiria, Airsfria.

(hfnriirscript received 3 April 1990; revised version accepted I0 Decernber 1990)

In mature pollen grains of many insect- as well as wind- pollinated species the most striking surface coat is pol- letikitt (see rcview Bhandari 1984). It is generally accepted, that pollenkitt is o f tapctal origin (Hcslop-Harrison 19686) and mainly comprises neutral lipids with carotenoid pig- ments (Dobson 1988). Sometimes it is mixed with a small amount of cytoplasmic remnants (tryphine) (Keijzer & Crcsti 1987). Besides pollenkitt, membrane-like compo- nents are found at the pollen surface in a few species. A membrane invests each mature pollen grain of Chrysarrtlre- I I I I I I I I (Brewer & Ilenstra 1970). In Gladioltis a thin os- miophilic layer is enveloping the pollen surface (Clarke & Knox 1978). Gaudc & Dumas (1953) demonstrate the pres- ence of an exinic outer layer in Brassica, which is covering the cxine surface, but inside the pollen coat. Some recent investigations rcporting n polysaccharidic cxinc-held mate- rial in the bacula cavities. This material, unlikely in an environment full of exoccllular enzymes, is interpreted as remnants of the primcxine matrix (Takahashi 1989), having no more function in pollen biology. The present paper rcports three different cxine-held materials in Apirun 110-

tlifloriirn L.

MATERIAL A N D METHODS Flo\vers of Apirrvi nodiflorrci~i L. were fixed in 3% glutaraldehydc (GA) in 0.1 hl phosphntc buffer (pH 7.2) for 21 hours at room tcmpcraturc. After rinsing in buffer and distilled water postfixa- tion was carried out either in 2% OsO, at 6°C for 2 hours or in 2% OsO, plus 0.8% phosphate buffered K,Fc(CN), (OsFeCN), in the ratio 1:1 at 6°C for 4 hours. The OsFeCN-fixation is a modification of the method dcscrihcd by Hepler (1981) and Cresti & Keijzcr (1985). After washing in distilled water the anthers were block stained with uranyl acetate (2% methanolic solution) for 6 hours. Samples were dehydrated in ethanol and embedded in Spurr's low viscosity epoxy resin (Spurr 1969).

Scctions were cut o n a Hcichcrt Ultracut. using a diamond knife.

Conventional staining was accomplished with uranyl acetate (45 minutes/30"C) followd by lead citratc ( 1 minutcROT) in the LKB UltroStaincr. Thc spccimens were examined in a Zciss Ehl 109 at 50 kV.

For localization of neutral polysaccharidcs sections collected on gold grids were treated with 1% pcriodic acid (PA) for 45 minutes, 0.2% thiocarbohydrazine (TCH) for 5 hours and 1% silver protei- nnte (SP) for 30 minutes (IliiCry 1967). Without PA oxidation the ThiCry-test was used for thc dctcction of unsaturated lipids in osmiuni fixed material (Howlcy 6: Dahl 1977). Some sections were treated with only 1% SP for staining native and exogenous ions (Rowley et al. 1981).

For scanning electron microscopy anthers wcrc fixed in 3% GA. dchydratcd in ethanol and FDA and critical point dried. Pollen grains were coated with gold and observed in n JcoI-T~OO.

RESULTS

The tricolporate pollen grains of Apiuni iiodiflorwi apper- tain t o the sub-rhomboidal pollen-type (Cerceau-Larrival & Roland-Heydacker 1976), with a polar axis of about 13 pm and an equatorial axis of 8 Iim (Fig. IA). The rugulate- striate exine is divided into ektcxinc, with tcctum, bacula, foot layer, and endcxine (Figs. lC, 3A), which becomes thickened at the oral zone of the aperture (Figs. lB, 4C; asterisks). Depending on the method of fixation the cndcx- ine stains in different ways. For example GI\- and Os- FeCN-fixation produces an electron-lucent endexine (Figs. l B , 3C; asterisks), while OsO, leads to an electron-dense contrast of the same kind :is the ektcxinc, whereby the two layers are separated by a distinct white line. Within the dark endexine electron-lucent spots are observed (Figs. 3A, 1B).

As a n insect-pollinated species, Apiluii tiodifloriiiii pro- duces a considerable amount of pollenkitt, which is charac- terized by small electron-dense spots within a homogene- ous material (Figs. 3A,R, 2C,D). It is commonly found deposited at the bottoni of the exine cavities and also in

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18 Martina Weber

Fig. 1. (A) Tricolporatc pollen grain with rugulate-striate exine pattern. Scale 1 pm. (I3)Pollen grain partly coated with pollenkitt dense to electrons. Note same stainability as the liqid droplets in the vegetative cell. and thickcncd cndcxinc at thc oral zonc of the aperture (asterisks). OsFeCN-Thiiry. Scale 1 pm. (C) Strongly labeled pollenkitt at the bottom of the exine cavities and on the tectum, a k r TCH-SP-staining for unsaturatcd lipids. Endcxinc (asterisks) electron-lucent. Scalc 0.5 Iim.

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Exitie-lield tnciterinl iti polleti gmitis of Apicicene 19

Fig. 2. (A, B) TCH-SP and (C. D) SP stains the pollenkitt (P), while the primexinc matrix (M) inbetween the tcctum clcmcnts remains unstained. Thc cxinic surface layer visible as a white line (orroirs). GA-OsO,. Scalcs 1 (im (A), 0.5 (im (B. C, D).

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20 hlartitin Weber

.Fig. 3. (A, B) Primexine matrix (M) and pollenkitt preserved by Os0,-fixation.Dark stained endexine ( E ) separated from the foot layer (F] by a distinct white line. Exinic outer layer marked (arrows). GA-OsO,. U + Pb. Scale 0.5 pm. (C) GA-fixation presewes the polysaccharidic primexine matrix (M). The endexine is transparent to electrons (asterirks); the exinic outcr laycr dcnse to electrons (urrows). GA-Thiiry. Scale 0.5 pn. (D) Tangential section. Tecturn perforations partly filled with primexinc matrix (Al ) . GA-Thihy. Scale 0.5 Iim.

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fiitie-held mnterial in polleti gruitis of Apiaceae 21

fig. 4. Young pollen grains in the loculus. Note the intact tapetum cells (TC). GA-ThiCry. Scale 10 pm. (B) Bacula arcades loosely filled with polysaccharidic matrix fibrills. GA-ThiCry. Scale 0.5 pm. (C) Pollen grain perhaps surrounded by primcxine residuals. GA-ThiCry. Scalc 1 pm. (D) Primexinc matrix extends outward from the bacula cavities through tecturn perforations (arrow). GA-ThiCry. Scale 0.5 lim.

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22 hlnrtitin M b e r

largc quantities at the surfacc of the pollen grain, some- times forming droplets (Figs. IB, 2A). Excellent prcscrva- tion is obtained using OsO, or OsFeCN as postfixatives, which intimates liqids as the dominant substances. In fact, pollenkitt is not preserved by GA-fixation alone. The com- mon chemical compounds detected are unsaturated lipids. This was observed by TCH-SP-stain following osmium fixa- tion (Fig. 2A, B). This reaction \vas particularly strong whcn OsFcCN \vas the postfixative (Fig. IC). Pollcnkitt as ivcll as the lipid droplets within the vegetative cell are strongly labeled (Figs. IB. 2A). Even SP stains the same lipids as TCH-SP (Fig. 2C, D), perhaps due to the presence of native cations, like calcium. The Thiery-test for neutral polysacchnrides was not significant for pollenkitt. But fol- lowing OsFcCN-fixation an excellent presentation of cell organcllcs is obtained (Fig. ID). This was not achieved by conventional uranylacctatc-lead-citratc staining.

Bcsidcs the pollcnkitt a fibrillar material is found, promi- nantly aggrcgatcd inbetween the bacula arcade close to the tcctum clcmcnts. forming a loosc tcxturc (Figs. 2D, 3A, C, D). It is prcscrvcd by GA-fixation alone. Labeling after the Thidry-test indicatcs polysaccharides as the dominating compounds (Fig. 3C, D). When OsO, or OsFcCN is part of thc postfixative the same results arc obtained. This fibrillar polysaccharidic matcrial is not considered as a pollenkitt, since i t is found in young pollen grains. charactcrizcd by many starch grains. and intact tapctal cells, where pol- lcnkitt is not yct produced (Fig. 4A, B). This indicates, that this material is a rcmnant of the primcxine matrix. Another intcrcsting fcature is (Fig. 4C) thc situation aftcr the dis- solution of the tapctum cells, whcre inbetwcen the pollen grains a fibrillar matrix is found. Since the ThiCry-tcst modcratcly stains this matcrial, the qucstion whether this is primexine matrix arises. On the other hand, Fig. 4D shows, that primexine matrix lcavcs the cxinc cavities, perhaps caused by cxpansion of the pollcn grain and consequently thc tcctum elcments.

Besides pollcnkitt and primcxinc matrix a very thin layer is found inside thc pollcn-coat, outside thc exine. It is clearly visible ;IS ;I dark linc aftcr Thiery-test (Fig. 3C, arrows) and as a white layer aftcr SP-stain (Fig. 2D, ar- rows). and O,O,-iir;inylacetatc-lcadcitratc-staining (Fig. 3A. B, arrows).

DISCUSSION

In mature pollcn grains of Apirriii IiodijloriritI two types of cxinc-held niatcrials arc clcarly distinguishcd, where one should bc dcfinitely namcd as pollcnkitt. Apiirrti produces a considcrablc amount of pollcnkitt, which is dcposited at the bottom of the cxinc-cavitics and on thc surfricc of thc tcctum. This kind of distribution is typical for inscct-polli- natcd plants (Ilcssc 1979 n, b; 1981). Usually more than onc pollcnkitt-type is found within one spccics, differing in structurc and/or electron-dcnsity, obviously both caused by

chcmical diversity. There are only few examplcs in litcra- turc reporting onc kind of pollenkitt, e.g. , Acer upnliu (Hcsse 1979 b) , Sringiiisorbn initlor (Hcsse 1979 a). Pol- lenkitt in Apiiriti is preserved only, when osmium is used as postfixative. This fact together with the strong labeling aftcr TCH-SP-staining certainly spccify unsaturated lipids as the main components. This corresponds with the results of the pollcnkitt analysis made by Dobson (1988), where ncutral lipids, as hydrocarbons, terpenoids, and carotcnoid pigments, are the dominating compounds of pollcnkitt in 69 species. Proteins, undoubtedly with enzymatic propcr- tics, are demonstrated by Abadic & Ilideux (1979) in Saxi- froga cyiubaloria and by Dobson (1988) in Brrissicn cniw pestris and RapIimiis sntivirs. So far, polysaccharidcs have been reported only in Compositae (Klungncss & Peng 1984), possibly attributed to the failure of most fixativcs used in electron microscopy to stabilize polysaccharidcs, unless they arc bound to proteins, polyphenols ctc. (Fostcr

It seems, that fixatives arc morc cffcctive in the prcscrva- tion of the structure of surface coatings than for other ccll components of a pollen grain. In mature Apiirm-pollen an cxccllent prcscrvation of the cell organcllcs, including lipid-droplets, is obtained even without OsO,. while pres- ervation of pollcnkitt is dependent on osmium. Grote (1989) demonstrated that, the two diffcrcnt kinds of elec- tron-dense material accociatcd with the birch pollen exine arc not prcscrvcd, although OsO, was used. Only the addi- tion of precipitating agents, as cuprolinic blue or cctyl- pyridinium chloride saved thc soluble surface components.

The relationship between different fixafivcs and the ul- trastructural appearence of pollcnkitt is shown by Elleman & Dickinson (1986). Elleman ct al. (1987). and Dunbar (1981). After vapour-fixation with OsO, the pollen coating of Rrssicn olerocerr is f electron-lucent, containing small electron-opaque bodies; conventional GA-OsO, fixation results in a vesicular electron-opaque pollcnkitt. Also the hydration-rate of the pollen grain as well as an intcraction with the stigma influences the structure and clcctron dcn- sity of the pollcnkitt (Elleman & Dickinson 1986, Ellcman ct (11. 1987). Using conventional GA-OsO, fixation. Ilesse reports granular pollcnkitt, c-g., in Acer (Hesse 1979 b) , fibrillar in Ririiwx (Hessc 1979 c). lamellar in Cnrex (Hcssc 1980). blistered with partly crystalline inclusioncs in Calnn- t h i i r (Hcsse 19SI), and homogeneous, c.g., in Poljgoiiiriii (Hcssc 1979 c).

As mentioncd before, the preservation of pollcnkitt in Apiirni is dcpcndcnt on osmium. Ilo\vcvcr. if fixed with GA alone, a fibrillar polysaccharidic matrix inbctwcn thc tcctum elements of mature pollen grains rcniains. Prob- ably, this material& a rcmnant o f thc primexinc matrix and probably 'did not originate from the tapctum cells. In other Apiaccac, c.g. At~gcl i~n, it is being obscrvcd in young microspores (Wcber. unpublishcd). Normally exinc forma- tion is accompmicd by the dcposition of a prinicxinc. Lit- erature highlights. that the primcxinc matrix is a micro-

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Exitie-held iiiaterial in polleti graitls of Apiaceae 23

of the exine (ed. I. K. Ferguson & J. hluller), pp. 481-498. - Lin. SOC. Symp. Ser. 1, Academic Press, London.

Clarke, A. E. & Knox. R. B. 1978. Cell rccognition in flowering plants. - 0. Rev. Biol. 53: 3-28.

Cresti, hl. & Keijzer. C. J. 1985. The structure of the endoplasmic reticulum in pollen grains and pollen tubes, after osmium tc- troxide-potassium ferricyanidc staining. - J. Submicrosc. Cytol. 17: 615-620.

Dickinson H. G. 1970. Ultrastructural aspects of primexine forma- tion in the microspore tetrad of Liliimr longiflontni. - Cytobiol- ogie 1: 43749.

Dohson, H. E. M. 1988. Suney of pollen and pollenkitt lipids - chemical cues to flower visitors. - Am. J. Bot. 75: 170-182.

Dunbar. A. 1981. The preservation of soluble material on the surface and in cavities of the pollen wall of Campanulaceac and Pentaphragmataceae. - Micron 12: 47-64,

Ellcman, C. J. & Dickinson, H. G. 1986. Pollen-stigma interac- tions in Brssica. 1V. Structural rcorganization in the pollen grains during hydration. - J. Cell Sci. 80: 141-157.

Elleman, C. J., Willson, C. E. & Dickinson, H. G. 1987. Fixation of Brussica oleracea pollen during hydration: a comparative study. - Pollen Spores 29: 273-290.

Fernindez, hi. C. & Rodriguez-Garcia, hi. I. 1988. Pollen wall development in OIea eitropaea L. - New Phytol. 108: 91-99.

Foster, R. C. 1981. Polysaccharides in soil fabrics. - Science 214: 665-667.

Gaudc, E. & Dumas, C. 1984. A membrane-like structure on the pollen wall surface in Brussicu. - Ann. not. 51: 821-825.

Grote, hl. 1989. Techniques to preserve soluble surface compo- nents in birch pollen wall: a scanning and transmission electron microscopic study. - J. Histochem. Cytochem. 37: 981-987.

Helper. P. K. 1981. The structure of the endoplasmic reticulum revealed by osmium tetroxide-potassium ferricyanide staining. - Eur. J. Cell Biol. 26: 102-110.

HeslopHarrison. J. 1963. An ultrastructural study of pollen wall ontogeny in Silerie petrditla. - Grana 4: 7-25.

Heslop-Harrison, J. 1968 a. Wall development within thcmicros- pore tetrad of Lilirini longiflorum. - Can. J. Bot. 46: 1185- 1192.

Heslop-Harrison, J. 1968 b. Tapetal origin of pollen-coat sub- stances in Liliittii. - New Phytol. 67: 779-786.

Hessc, hl. 1979 a. Entstehung und Auswirkungen der unterschie- dlichen Pollenklebrigkeit von Surigirisorba officinalis und S. minor. - Pollen Spores 21: 399413.

Hesse, M. 1979 b. Ultrastruktur und Verteilung des Pollenkitts in der insekten- und windblfitigen Gattung Acer (Aceraceac). -

Ilesse, hl. 1979 c. Entwicklungsgeschiclite und Ultrastruktur von Pollenkitt und Exine bei nahe venvandtcn entomo- und ane- mophilen Angiospermen: Polygonaccac. - Flora 168: 558-577.

Hessc, M. 1980. Entwicklungsgeschichte und Ultrastruktur von Pollenkitt und Exine bei nahe venvandtcn entomo- und am- mophilen Angiospermensippen der Alisrnataceae, Liliaccac, Juncaceae, Cyperaceac, Poaceae und Araceae. - PI. Syst. Evol. 134: 229-267.

Hcssc, hi. 1981. The fine structure of the exine in relation to the stickiness of angiospcrm pollen. - Rev. Paleobot. Palynol. 35:

Keijzer. C. J. & Crcsti, hl. 1987. A comparison of anther tissue development in male sterile Aloe w r n and male fcrtile Aloe ciliaris. - Ann. Bot. 59: 533-512.

Klungncss, L. hl..&P_eng Y-S. 1984. A histochemical study of pollen digestion in ttie alimentary canal of honey bees ( A p i ~ niellifera L.). - J. Insect. Physiol. 30: 511-521.

Rowley, J. R. 1981. Pollen wall characters with cmphasis upon applicability. - Nord. J. Bot I: 357-380.

Rowley, J. R. & Dahl, A. 0. 1977. Pollcn development in Arrerrii-

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81-92.

fibrillar layer, composed of cellulosic material, and syn- thesized by the protoplast of the microspore at the tetrad period (Heslop-Harrison 1963, 1968 n; Dickinson 1970, Audran & Batcho 1981, Skvarla & Rowley 1987). Informa- tion available is meagre as to indicate where the primexine matrix remains after exine formation. Heslop-Harrison (1963, 1968 a) referes to a disorganised fibrillar material in the usually empty bacula arcades of Siletie and Liliitni as remnants of the former primexinc wall, which is destroyed after tetrad disruption. It seems, that in Apiittn the narrow tectum elements (Fig. 1A) prevent their complete disap- pearence during pollen development (Fig. 4D). The rem- nants of the primexine matrix form a distinct layer in ma- ture pollen grains in Caesalpitiinjap~iiica (Takahashi 1989), and also in Olca eitropaea (Ferniindez & Rodriguez-Garcia 1988). While in Caesnlpitiia and Oleo the matrix rcsiduals are located at the bottom of the exine cavities, in Apiiwi they are found inbetween the tectum elements. However their loose texture may allow the entry of pollenkitt into the bacula cavities.

The thin layer covering the patterned exine in Apiiriii is quite similar to the exinic outer layer (EOL) reported by Gaude & Dumas (1984) in Brassica. According to this report, the trilamellar EOL, with characteristics of a typical biological membrane, is participating in the male-female recognition reaction. In Brassica and Apirtni the membra- nelike structure is covering the exine surface inside the pollen coat, whereas the membrane in C/irysatirheniitm is investigating the whole pollen grain, including the porus (Brewer & Henstra 1970). It is fixing the pollen grain to the stigma and forms a protective hood during germination. Finaly, the surface "membrane" in I'opitlils should be men- tioned, which is likely a part of the substructure of the exine (Rowley 1981).

A C K N O W L L E D G E M E N T S

The author is much indepted to Prof. Dr. M. Hesse for helpful discussions, and to Dr. K. Samuel for correcting the English text.

R E F E R E N C E S

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Audran, J-C. & Hatcho, hi. 1951. Cytochcmical and infnstructu- ral aspects of pollcn and tapctum ontogcny in Silore dioica (Caryophyllaccae). - Grana 20: 65-80.

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Drciver, J. G. gL Ilcnstra, S. 1970. A membrane investing mature individual pollcn grains of pyrethrum (C/irysnrri/ret~rio,r ciner- uriaefolirorr Vis.). - Euphytica 19: 121-124.

Cerccau-Larrival. hl. 1%. R: Roland-ffeydacker. E 1976. The evolutionary significance of the ultrastructure of the exinc in uiiibcllifcrous pollen grains. - In: The evolutionary significance

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