The unique features and ultrastructure of the microspore and pollen grain of the Antarctic dicotyledonous plant Colobanthus quitensis (Kunth) Barlt.

Irena Giełwanowska¹, Ewa Szczuka², Irena Agnieszka Pidek³, Aleksandra Seta², Marcin Domaciuk²

^1* Department of Plant Physiology and Biotechnology, University of Warmia and Mazury, Oczapowskiego 1A, 10-719 Olsztyn, Poland

^** Department of Antarctic Biology, Polish Academy of Science, Ustrzycka 10/12, Warsaw, Poland

² Department of Plant Anatomy and Cytology, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland, eszczuka@biotop.umcs.lublin.pl

³ Department of Physical Geography and Palaeogeography, Maria Curie- Skłodowska University, al. Kraśnicka 2 c/d, 20-718 Lublin, Poland

ABSTRACT

The formation, structure and ultrastructure of microspore and pollen grain of Colobanthus quitensis (Kunth) Bartl. (one dicotyledonous and one from the only two native flowering plants growing in Antarctica) were investigated. Plant material was collected in the vicinity of the Polish Antarctic Station. The studies of microspores and pollen grains were carried out by means both light and electron microscopes.

The development of microspores and pollen grains of C. quitensis takes place in chasmogamic or cleistogamic flowers. Both the chasmogamic and cleistogamic flowers of the investigated species usually form five stamina with short filaments. One stamina contains two elongated microsporangia. The microsporogenesis and male gametogenesis are carried out in the microsporangia enveloped with a three-layer anther wall (epidermis, endothecium and tapetum). In both processes microsporogenesis and male gametogenesis of the investigated plant species proceed in a way typical of other angiosperms. C. quitensis forms spherical, two-nuclear pollen grains enveloped by the thick polyporate sporoderm. Characteristically, there is no callose wall between generative and vegetative cells in the bicellular pollen grain.

INTRODUCTION

Colobanthus quitensis (Kunth) Bartl. (Caryophyllaceae) is one of the only two native flowering plants that grow in Antarctica and the only native dicotyedonous plant on the continent. Antarctica is considered to be the most distant, inhospitable, the coldest and the most inaccessible polar region of the Earth (Giełwanowska and Szczuka, 2005; Giełwanowska et al., 2005; Szczuka et al., 2007; Szczuka et al., 2008). The only possible period for vegetation is the summer period which lasts no more than 5 months, typically from November to March (in this case the length of day and night is taken into consideration). Actually, the plant growth season is shortened to no more than 2-3 months, because of the length of the snow-cover period in the areas of Antarctica that are uncovered by ice.

The mentioned above and other climatic events that result from the severe conditions of the Antarctic region make growing plants develop various adaptation mechanisms. In order to survive, C. quitensis, the Antarctic pearlwort have developed well functioning physiological and biochemical adaptations to adapt to rapidly fluctuating growth conditions. Proving this is the fact, that pearlwort flowers profusely and produces seeds almost each year (Giełwanowska et al., 2007; Giełwanowska et al., 2007). Additionally, unfortunately shy in number literature and data concerned the investigations of flower and seed development both Antarctic pearlwort and Antarctic hair grass is given in the paper of Giełwanowska et al., (2007).

On the other hand, the specificity of extremely severe climatic conditions of the Antarctic region induce scientists to investigate the possibility of plant growth and development in this area. C. quitensis can be considered a good plant model for analysis of various adaptation mechanisms to the both biotic and abiotic stress factors acting in Antarctica (Levi-Smith, 2003). The knowledge of the biology concerning flowering and reproduction of these species is still insufficient. Only a few papers report the results of the investigations concerning the generative male line development (Giełwanowska 2005; Giełwanowska et al., 2005 Giełwanowska et al., 2006. Therefore, the focus of this paper is on the features and ultrastructure of microspores and pollen grains of Antarctic flowering plant C. quitensis.

MATERIAL AND METHODS

Plant material

Flower buds of Colobanthus quitensis (Kunth) Bartl. (Caryophyllaceae) at various stages of development were collected from plants growing under natural conditions near H. Arctowski Antarctic Station (62º09.8' S, 58º28.5' W) in the area of SSSI (Site of Special Scientific Interest) No. 8. on King George Island (the South Shetland Islands) (see map in Fig. 1). Fresh flower buds of C. quitensis were picked up during the Antarctic summer, mainly in January 2004 during 26^th expedition to the Antarctic, organized by the Department of Antarctic Biology, Polish Academy of Sciences in Warsaw.

Light microscopy

Flower buds were fixed in 3.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.0) for 8 hours at room temperature. The fixed buds were washed in two changes of 0.1 M phosphate buffer and postfixed overnight in 2.5% OsO4. The material was then washed in buffer, dehydrated in a graded ethanol series, and transferred into mixtures with increasing ratios of Poly Bed 812 resin. Semi-thin sections (1-2 μm thick) were stained with toluidine blue and observed under the light microscope.

Electron microscopy

For transmission electron microscopy (TEM), small pieces of sporangia were fixed in 2% glutaraldehyde in 0.05 M sodium cacodylate buffer (pH 7.2). Specimens were washed three times in 0.05 M sodium cacodylate buffer and post-fixed in 1% osmium tetroxide. Samples were dehydrated in graded ethanol and aceton series followed by propylene oxide, infiltrated and embeded in Spurr’s resin (Spurr, 1969). The material was sectioned at 50 nm and stained with 2% uranyl acetate and lead citrate (Reynolds, 1963), for 30 min. Finally, thin sections were studied with the JEOL JEM 100S transmission electron microscope.

RESULTS

The Colobanthus quitensis is a small dicotyledonous plant. In the investigated area (vicinity of Henryk Arctowski Polish Antarctic Station on King George Island) (Fig. 2), the plants usually grow next to the Deschampsia antarctica (Monocotyledones), mosses and lichens, forming flat, dense mats (Fig. 3).

In natural conditions, C. quitensis, a hardy, perennial plant, forms cushions consisting of numerous multi-module individuals. The single, small, inconspicuous, and bisexual buds or flowers (Fig. 4) of this species may terminate module shoot or may be axillary. In the investigated species C. quitensis varies from the cleistogamic (closed) flowers typically formed in unfavorable conditions. In the case of the latter, the pollen grains are scatterred after anthesis in the vicinity of D. antarctica tussocks or C. quitensis tufts. In the investigated Anatarctic pearlwort, the stamens (usually 5) and the pistil coated with five elements of the undifferentiated perianth develop in closed flower buds hidden between the leaf blades. The number of perianth elements may vary, from four to six of them. Green sepals arranged in two verticils form the perianth.

The central part of the young microsporangium is occupied by archesporial tissue, as well as microsporocytes. After meiosis with simultaneous cytokinesis, tetrahedrally arranged microspores (tetrads of microspores) are formed from microsporocytes. The microspores released from the tetrad are enveloped by the sporoderm. Single microsporangium is circular in a transverse section. At the stage when microspores are in the loculus, the external layer (epidermis) envelops a single cell layer of endothecium, cellular tapetum, and microspores (Fig. 5).

After mitotic division of the microspores, two-nuclear pollen grains appear in the loculus. At the time when the pollen grains are formed the structure of the single microsporangium does not change in any significant way. During the previous stage of development with microspores in the loculus, the microsporangium in a transverse section is regular and circular in shape. Similarly to the previous stage, at the stage when pollen grains are in the anther loculus, the wall of the microsporangium is built out of three layers of cells: the external – epidermis, middle – endothecium, and the most internal – tapetum (Fig. 6).

A longitudinal section of a closed flower of C. quitensis shows its detailed anatomical structure with the ovules settled on the placenta and pollen grains in opened microsporangia or released from microsporangia (Fig. 7). Mature pollen grains of C. quitensis are rather spherical, two-nuclear, and enveloped by the polyporate sporoderm. A longitudinal section of a closed flower of C. quitensis allows observation of the germinating pollen grains in opened microsporangia or released from microsporangia. Even the very early stage of pollen grain germination shows two or more pollen tubes growing from a single pollen grain (Fig. 8). Nevertheless, on a dry, pulmose stigma only one pollen tube from single pollen grain wins the competition (Fig. 9).

The ultrastructural details of the microspore and pollen grain structure with special attention paid to the sporoderm are presented in the figures ten to twelve. The complex cell wall, at first sporoderm is formed on the surface of the microspore protoplast. Nevertheless, its structure is similar to the sporoderm that envelopes mature pollen grains (Figs. 10 and 11). In both cases of the microspore sporoderm and the pollen grain sporoderm consist of electron-dense sporopollenin material which is not present in apertures. In most pollen grains, the sporoderm is perforated in about a dozen apertural sites. The separating callose wall between generative and vegetative cells of pollen grain is not visible during observation through the TEM (transmission electron microscope) (Fig. 12).

DISCUSSION

Microsporogenesis of the investigated plant species Colobanthus quitensis proceeds in a way typical of other angiosperms. In C. quitensis, microsporogenesis ends with simultaneous cytokinesis. Simultaneous cytokinesis is often meet in dicotyledonus plants. The other type of cytokinesis - successive cytokinesis is characteristic of monocotyledones. As in the other angiosperms, the microspores at the tetrad stage of C. quitensis are surrounded with a thick callose wall (data not shown in any figure in this paper). This callosic wall accompanies the dividing microsporocyte (microspore mother cell) from the earlier first meiotic prophase. During meiosis the callose wall becomes thicker and fulfill the isolational functions until the tetrad stage begins (Giełwanowska at al., 2007). Callose begins to hydrolise when the construction of sporoderm starts (Heslop-Harrison et al., 1986).

The sporoderm enveloping the microspore and, later, the pollen grain of C. quitensis is relatively thin. The osmophilic material, in the form of proorbicules, moved from various areas of the tapetal cells towards pollen grains, and in the form of orbicules joined the developing sporoderm. The formation of the sporoderm is similar to this process of the other pollen grain sporoderm in different plant species (Raghavan, 2000).

Similarly to microsporogenesis, male gametogenesis is typical of other angiosperms (for detailed description of both processes in flowering plants see Raghavan, 2000). Characteristically, in C. quitensis, the separating calose wall is not formed between generative and vegetative cells. The existence of a cell wall separating the generative cell from the vegetative cell has been controversial. No doubt, that the generative cell is usually bound by the plasma membrane and a distinct cell wall. The cell wall often contains callose, e.g. in orchids (Heslop-Harrison, 1968a). The generative cell of Anatarctic pearlwort, immersed in the cytoplasm of the vegetative cell, was observed through a transmission electron microscope (Giełwanowska et al., 2007). Using the same technique, the male germ unit of C. quitensis was investigated. The morphological features of the generative cell and sperm cells of Anatarctic pearlwort are comparable to the features of cells in Plumabago zeylanica (Russel, 1984) or Brassica (Dumas et al., 1985; Charzyńska et al., 1989)

In closed flowers of C. quitensis microsporangia dehisce releasing pollen grains, which reach the stigma surface and germinate (Giełwanowska et al., 2005; Giełwanowska et al., 2005; Szczuka et al., 2006). The pollen grains of C. quitensis can germinate inside the closed microsporangium. Often pollen grains of this species germinate with two or more pollen tubes.

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LEGENDS

Fig. 1. Location map of King George Island in the South Shetland Islands Archipelago and Cockburn and Seymour Islands in the Antarctic Peninsula sector. Arrow shows the magnified area of main map.

Fig. 2. The vicinity of the Polish Polar Station on King George Island.

Fig. 3. Colobanthus quitensis and Deschampsia antarctica in a natural habitat in the vicinity of the Polish Polar Station on King George Island. Both species grow here together with mosses, and form flat, dense mats.

Fig. 4. Flower bud of Colobanthus quitensis.

Fig. 5. A magnified microsporangium (mi) of Colobanthus quitensis with microspores in the loculus surrounded with sepals (pe). A semi-thin, transverse section stained with toluidine blue. Visible epidermis (ep), endothecium (en) and disintegrated tapetum (ta). 2000 x.

Fig. 6. A magnified microsporangium (mi) of Colobanthus quitensis with pollen grains in the loculus . A semi-thin, transverse section stained with toluidine blue. Visible epidermis (ep), endothecium (en) and disintegrated tapetum (ta). 2000 x.

Fig. 7. A longitudinal section of a closed flower of Colobanthus quitensis with ovules settled on placenta (pl) and pollen grains in opened microsporangium (left arrow) or released from microsporangia. Pollen grains (arrow) on a dry, pulmose stigmas. A semi-thin section stained with toluidine blue. 1000x

Fig. 8. A longitudinal section of a closed flower of Colobanthus quitensis with germinating pollen grains in opened microsporangia or released from microsporangia. Early stage of pollen grain germination. Pollen grains (arrows) on dry, pulmose stigma. A semi-thin section stained with toluidine blue. 2500 x.

Fig. 9. A longitudinal section of a closed flower of Colobanthus quitensis with germinating pollen grains released from microsporangia. Later than in Figure eight stage of pollen grain germination. Pollen grains (arrows) on dry, pulmose stigma. A semi-thin section stained with toluidine blue. 2500 x.

Fig. 10. TEM. The ultrastructure of the microspore sporoderm of Colobanthus quitensis and neighbouring tissue of the microsporangium wall. Note disintegrated tapetum (ta). ap – aperture, ne – nexine, se – sexine, v – vacuole. 20000 x.

Fig. 11. TEM. The ultrastructure of the pollen grain sporoderm of Colobanthus quitensis. Note disintegrated tapetum with tapetal membrane (arrows). ne – nexine, se – sexine, v – vacuole. 20000 x.

Fig. 12. TEM. The ultrastructure of the pollen grain of Colobanthus quitensis. Arrow points the cell wall separating generative and vegetative cells. gc – generative cell, ne – nexine, se – sexine. 20000 x.