Taiwania, 48(3): 147-167, 2003 Comparative Study of the Pollen Wall Development in Illicium floridanum (Illiciaceae) and Schisandra chinensis (Schisandraceae) Nina I. Gabarayeva(1,2) and Valentina V. Grigorjeva(1) (Manuscript received 13 May, 2003; accepted 21 August, 2003) ABSTRACT: Pollen wall development of Illicium floridanum and Schisandra chinensis have been studied and compared. Both species have a similar reticulate exine, but their similarity occured to be only superficial. At early tetrad stage of both species the plasma membrane acquires a regularly invaginative profile, the distribution pattern of invaginations on the microspore surface corresponds to the future reticulate exine pattern. In the invaginative sites of the plasma membrane of both species fibrillar strands appear which are the auxiliary (phantom) structures. Further developmental process is different in both species. In Illicium sporopollenin accumulates around the auxiliary strands, localized in plasma membrane invaginations, resulting in the appearance of the reticulate sculpture of hollow tunnels on the surface of a microspore; this reticulate pattern becomes concave after lifting of the invaginated portions of the plasma membrane. In Schisandra, on the contrary, sporopollenin never accumulates in the location of the auxiliary fibrillar strands (these are sites of future lumina), but sporopollenin accumulations concentrate on the elements of the glycocalyx on the evaginated top (protruding sites) of plasma membrane. The latter are sites for columellae formation, and muri are constructed from the rows of columallae covered by tectum. Hence, the development of the exine in both species is different, and the inner structure of reticulate exine in Illicium differs from that of Schisandra - in spite of the very similar sculpture. KEY WORDS: Exine patterns, Auxiliary structures, Illicium floridanum, Schisandra chinensis. INTRODUCTION The group of so-called primitive angiosperms (Magnoliidae) is of special interest in many aspects. Nevertheless, only several studies have been dedicated to the modes of sporoderm development (Meyer, 1977; Zavada, 1984 – Austobaileya maculata (Austrobaileyaceae); Gabarayeva, 1986a, b – Michelia figo (Magnoliaceae), 1987a, b, c – Manglietia tenuipes (Magnoliaceae); Waha, 1987 – Asimina triloba (Annonaceae); Gabarayeva, 1991a – Magnolia delavayi (Magnoliaceae), 1991b – Magnoliaceae; Rowley and Flynn, 1990-1991 – Tambourissa (Monimiaceae); Zhang and Chen, 1992 – Magnolia denudata (Magnoliaceae); Gabarayeva, 1992, 1993a – Asimina triloba (Annonaceae); Rowley and Vasanthy, 1993 – Cinnamomum (Lauraceae); Takahashi, 1994 – Illicium religiosum (Illiciaceae); Gabarayeva and Rowley, 1994 – Nymphaea colorata (Nymphaeaceae); Gabarayeva, 1995 – Anaxagorea brevipes (Annonaceae), 1996 – Liriodendron chinense (Magnoliaceae); Gabarayeva and El-Ghazaly, 1997 – Nymphaea mexicana (Nymphaeaceae); Kreunen and Osborn, 1999 – Nelumbo (Nelumbonaceae); El-Ghazaly et al., 2000 – Magnolia (Magnoliaceae); Gabarayeva et al., 2001 – Nymphaea capensis (Nymphaeaceae); Tsou and Fu, 2002 – Annona (Annonaceae); Gabarayeva et al., in press – Cabomba aquatica (Cabombaceae)). ___________________________________________________________________________ 1. Komarov Botanical Institute, Popov st., 2, St.-Petersburg, 197376, Russia. 2. Corresponding author. Email: [email protected] 148 TAIWANIA Vol. 48, No. 3 Our studies on Magnoliaceae, cited above, have shown that it should be expected a range of different morphogenetic modes leading to the resolving of similar morphological goals. For instance, several different types of endoplasmic reticulum (smooth endoplasmic reticulum, chain-mail reticulum, zebra-reticulum) were found during tetrad period, involved into the process of the formation of similar patterns of exine in Magnoliaceae species (Gabarayeva, 1991b, 1996, 1997). By analogy, we suggested different modes of development in the two Illiciaceae species under our study which have very similar reticulate pattern of the exine. Our task in this study was to investigate in detail the sporoderm ontogeny in Illicium floridanum and Schisandra chinensis and to compare the both processes. Our aim was to show the possibility of the achievement of similar morphological structures in nature by different morphogenetic processes. MATERIALS AND METHODS Flower buds of Illicium floridanum Ellis and Schisandra chinensis (Turcz.) Baill. were collected from the glass-houses of the Komarov Botanical Institute (St.-Petersburg). The materials were fixed in 3% glutaraldehyde and 2.5% sucrose in O.1 M phosphate buffer (pH 7.3, 20J, 24 h) and post-fixed in 2% osmium tetroxide (pH 7.4, 20J, 1 h). After acetone dehydration the samples were embedded in mixture of Epon and Araldite. Ultrathin sections were stained with a saturated solution of alcoholic uranyl acetate and 0.2% aqueous lead citrate. Sections were examined with Hitachi H-600. Pollen grains for SEM study were air dried, transferred to stubs and sputter coated. Specimens were examined in Jeol JSM 35. RESULTS Sporoderm development in Illicium floridanum Mature pollen grains have a reticulate pattern of exine (Figs. 1 & 2). Pollen grains are isopolar and tricolpate with fused furrows at both poles. The developmental process of this structure is rather complex and needs detailed analysis. Early post-meiosis tetrad microspores are covered by a thick callose envelope, and their plasma membrane is rather even and lacked any signs of the glycocalyx (Fig. 3). Later the first generation of the glycocalyx appears on the surface of the plasma membrane as roundish dark contrasted units (Figs. 4 & 5). Dictyosomes and their vesicles, contained similar roundish units, are observed in the microspore cytoplasm. These first units of the glycocalyx are evidently excreted by Golgi vesicles. At the next developmental step this first generation of the glycocalyx disappers - probably has been engulfed in the process of endocytosis. In young tetrad microspores the plasma membrane acquires a periodically invaginated profile. Thin fibrillar strands appear in invaginated regions (Figs. 6 & 7). They are rather long if sectioned longitudinally (Figs. 6 & 7), and they are narrow in cross section (Fig. 10). Being first thin, they become more pronounced later (Figs. 8 & 9). These fibrillar strands are auxiliary structure and play an important role in establishing of exine pattern, but they disintegrate in the later developmental stages. September, 2003 Gabarayeva & Grigorjeva: Pollen wall development 149 Figs. 1-2. Scanning electron micrographs of the mature pollen grain of Illicium floridanum. 1. A survey of a pollen grain. Bar = 2 µm. 2. Sculpture of the reticulate exine. Bar = 0.5 µm. At middle tetrad stage the secondary generation of the glycocalyx appears between and around the fibrillar strands (Figs. 10-13). In Fig. 10 and in Figs. 13-15 the fibrillar strands are crossly sectioned, and the portions of the glycocalyx alternate with the auxiliary strands. In Figs. 11 & 12 the auxiliary fibrillar strands are longitudinelly sectioned. The microspore cytoplasm is abundant in Golgi vesicles. The auxiliary strands, as previously, are arranged to the invaginations of the plasma membrane and form a “network” around the microspore surface which corresponds to the future reticulate exine pattern (Figs. 6 & 8). Microfilaments are seen perpendicularly to the plasma membrane (Figs. 11 & 12). 150 TAIWANIA Vol. 48, No. 3 Figs. 3-5. Early tetrad microspores of Illicium floridanum. 3. Early post-meiosis tetrad microspore enveloped in the thick callose wall. There is no any signs of the glycocalyx on the surface of the plasma membrane. 4. The appearance of the roundish units of the glycocalyx (arrows) on the plasma membrane surface. 5. The magnified portion of fig. 4. (Ca: callose, D: dictyosome, GV: Golgi vesicles, LG: lipid globules, P: plasma membrane, Pl: plastid, SER: smooth endoplasmic reticulum). 3 & 4: Bar = 0.5 µm. 5: Bar = 0.25 µm. At late tetrad stage abundant clusters of a dark contrasted lipoid substance, which are probably a sporopollenin precursor, accumulates on the plasma membrane and in the periplasmic space (Figs. 14 & 15). This process starts at previous stage, when small droplets of this substance are observed on the surface of the plasma membrane (Fig. 13). Sporopollenin precursor begins to accumulate around the auxiliary fibrillar strands (Figs. 14 & 15). September, 2003 Gabarayeva & Grigorjeva: Pollen wall development 151 Figs. 6-7. Young tetrad microspore stage in Illicium floridanum. The appearance of long thin fibrillar strands alongside the plasma membrane, in its invaginations (arrows). The first generation of the glycocalyx disappeared. (AV: autophagic vacuole, Ca: callose, GV: Golgi vesicles, LG: lipid globule, N: nucleus, P: plasma membrane, SER: smooth endoplasmic reticulum). 6: Bar = 1 µm. 7: Bar = 0.5 µm. At young free microspore stage the auxiliary fibrillar strands disintegrate, and sporopollenin, which had been accumulated around these strands as around frame at previous stage, form a reticulate system of tunnels. These tunnels look as archs on cross sections (Figs. 16-19). These archs lean on the foot layer. At this stage the endexine is formed and consists of lamellae with white lines and granules of a fibrillar substance. 152 TAIWANIA Vol. 48, No. 3 Figs. 8-10. The transition from young to middle microspore tetrad stage in Illicium floridanum. 8-9. The alternation of the invaginations and evaginations of the plasma membrane. The fibrillar strands (arows) are located in the invaginations (longitudinal sections through the strands). 10. Middle tetrad microspore stage. Cross section through a fibrillar strand (arrow). The plasma membrane underneath the strand is covered with the secondary generation of the glycocalyx (G). (AV: autophagic vacuole, Ca: callose, D: dictyosome, EP: evagination of the plasma membrane, GV: Golgi vesicles, LG: lipid globule, M: mitochondrion. P: plasma membrane). 8 & 9: Bar = 0.5 µm. 10: Bar = 0.2 µm. At next stage the intine appears. Its inner profile is wavy. The archs of ectexine which are cross sections of hollow tunnels are very prominent (Fig. 19). September, 2003 Gabarayeva & Grigorjeva: Pollen wall development 153 Figs. 11-12. Middle tetrad microspore stage in Illicium floridanum. 11. The glycocalyx (G) appears above and around the fibrillar strands (arrows). Arrowheads: microfilaments. 12. The glycocalyx surrounds the fibrillar strands (FS). The discrete, radially oriented units of the glycocalyx – tufts – are shown with arrows. (AV: autophagic vacuoles, Ca: callose, D: active dictyosome, LG: lipid globule, MC: microspore cytoplasm, P: plasma membrane, SER: smooth endoplasmic reticulum). 11 & 12. Bar = 0.5 µm. Sporoderm development in Schisandra chinensis Mature pollen grains of Schisandra also have a reticulate pattern (Figs. 20 & 21), but, as we shall see below, the similarity is only superficial. 154 TAIWANIA Vol. 48, No. 3 Figs. 13-15. The transition from middle to late tetrad microspore stage in Illicium floridanum. 13. The appearance of a dark contrasted lipoid substance on the surface of the plasma membrane (arrows). 14-15. Late tetrad microspore stage. Vast deposition of the dark contrasted lipoid substance on the plasma membrane and in the periplasmic space (arrows). The fibrillar strands (FS) become covered with the dark contrasted lipoid substance. A spiral of a binder element of the glycocalyx tuft is shown by arrowhead. (Ca: callose, D: dictyosome, FS: fibrillar strand, G: glycocalyx, LG lipid globule, P: plasma membrane, PS: periplasmic space, SER: smooth endoplasmic reticulum). 13: Bar = 0.25 µm. 14 & 15: Bar = 0.5 µm. Young tetrad microspores have a wavy profile which resulted from exocytosis of many Golgi vesicles (Fig. 22). Somewhat later the plasma membrane forms deep invaginations in a way that invaginations and evaginations arranged alternately, and keeps this configuration (Figs. 23, 25, 26). A layer of the glycocalyx appears on the surface of the plasma membrane (Fig. 24). September, 2003 Gabarayeva & Grigorjeva: Pollen wall development 155 Figs. 16-17. Early free microspore stage in Illicium floridanum. Callose is disintegrated. The elements of the exine are seen on cross section as archs on the surface of the foot layer. These archs (ARCH) were formed around the fibrillar strands which are disintegrated now (place of disintegrated strands – PDS). The endexine is lamellated under the apertural site (A: aperture) and intermixed with granules in interapertural regions (END). (D: dictyosome, ECT: ectexine, FL: foot layer, LG: lipid globule, M: mitochondrion, MC: microspore cytoplasm, N: nucleus, NE: nucleus envelope, P: plasma membrane, RER: rough endoplasmic reticulum, V: vacuole) 16: Bar = 0.5 µm. 17: Bar = 0.25 µm. At middle tetrad stage fibrillar strands appear above those portions of the glycocalyx which are located in the invaginated regions of the plasma membrane (Figs. 25 & 26). They are absent at the evaginative points, where the glycocalyx initiates to accumulate sporopollenin. The tetrad microspore cytoplasm is abundant in active dictyosomes (Fig. 25). 156 TAIWANIA Vol. 48, No. 3 Figs. 18-19. Young free microspore (18) and the appearance of the intine (19) in Illicium floridanum. The elements of ectexine on cross section are seen as archs (ARCH). They are sections of hollow tunnels, the latter create reticulate pattern on the surface of pollen grain. (ECT: ectexine, END: endexine, FL: foot layer, Int: intine, MC: microspore cytoplasm, N: nucleus). 18: Bar = 2 µm. 19: Bar = 0.5 µm. At late tetrad stage the glycocalyx located on the evaginated portions of the plasma membrane becomes especially prominent by virtue of sporopollenin accumulation (Fig. 27). These are actual sites where the procolumellae begin to form. The fibrillar strands, spreaded by the glycocalyx, occupy the places only above the invaginations of the plasma membrane – the places form the future lumina (Figs. 26 & 27). Many Golgi vesicles, lipid globules and autophagic vacuoles are seen in the microspore cytoplasm (Fig. 27).