Structure and function of the tentpole in the reproductive process of Ginkgo biloba L.

Abstract

The tentpole is a unique structure of the female gametophyte in Ginkgo biloba; however, its exact functions in the reproductive process are unclear. In the present study, we used semi-thin sectioning and electron microscopy to study the structure and function of the tentpole during fertilization in G. biloba. The tentpole was always initiated between two or more deeply immersed archegonia. Before fertilization, the tentpole had developed into a column-like structure, protruding toward the archegonial chamber; cells at the periphery of tentpole were loosely ranged, and abundant lipid droplets and starch grains were accumulated in the tentpole cells. After fertilization, the tentpole degenerated, and some membranous debris was overlaid on its surface. In addition, there were significant decreases in the lipids and starch grains. These results suggested that the tentpole led to the degeneration of the megaspore membrane and then supported the pliable apex of the nucellar tissues. Importantly, the tentpole also contributed to supplying nutrition for fertilization and embryo development.

Keywords: :

• Ginkgo biloba L.
• anatomical feature
• fertilization
• morphological structure
• tentpole

Introduction

Ginkgo biloba L. is a unique living representative of the order Ginkgoales, which may have originated in the Permian approximately 150–200 million years ago.¹ The Ginkgoales once flourished in the early Mesozoic, but they were largely destroyed during the last ice age, with only one species, Ginkgo biloba L., surviving in China. Thus, G. biloba is believed to be one of the most primitive extant seed plants.² Many botanists have studied the primitive characteristics of G. biloba in terms of its morphology and anatomy. There are a number of characteristics of the reproductive process that are considered to be ancestral in G. biloba, including the structure of the archegonium, pollen chamber, tentpole, and haustorial pollen tubes.^3-7 There has been considerable interest in the gametophyte development and fertilization process,^4-7 while little attention has been paid to the tentpole, a unique structure in G. biloba.

The tentpole in G. biloba is commonly situated between two or more archegonia of the female gametophyte, and protrudes toward the archegonial chamber.⁸ Researchers have speculated that the tentpole functions as a secretory structure, producing a liquid favorable for the spermatozoid to swim via its flagella toward the archegonium just before fertilization.⁹ Our earlier research indicated that the cells of the tentpole accumulated abundant starch grains as the female gametophyte developed.¹⁰ Therefore, the tentpole may play an important role in the fertilization process in G. biloba. However, to date there has been little convincing evidence about the morphology of the tentpole and its specific function in the sexual reproduction process.

In the present study, we observed the shape, anatomical structure, and ultrastructure of the tentpole in G. biloba using resin sections and scanning and transmission electron microscopy (SEM and TEM) techniques. In addition, we compared the changes in the tentpole before and after fertilization. Our findings enable deeper insights into the primitive characteristics of G. biloba, and clarify the functions of the tentpole in the reproductive process.

Results

Morphological features of the tentpole

The female gametophyte of G. biloba usually reaches the cellularization stage in approximately mid-May, and then it begins to differentiate the archegonia at the micropylar end. In many cases, some cells between the two archegonia differentiate to form the tentpole.⁸ We observed the developmental process in detail via SEM. We observed that at approximately 55 d after pollination (Jun. 5), the center part of the micropylar end of the female gametophyte began to protrude upward to form a flat column-like structure, the early tentpole (Fig. 1A). Cells of the tentpole actively divided and protruded gradually, although they were smaller than the surrounding cells (Fig. 1B and C). Approximately 110 d after pollination, the tentpole was a cylindrical, 300-μm high structure. Cells at the base near the endosperm cells had a regular appearance (Fig. 1D), whereas the upper cells of the tentpole were wrinkled and irregularly arranged (Fig. 1E). At this stage, archegonia with swollen neck cells were located around the tentpole (Fig. 1F). The tentpole was located nearly in the center of three archegonia, with similar distances between the tentpole and each archegonium. Meanwhile the archegonia sunk down into the endosperm, while the tentpole and the surrounding cells protruded upward. This formed a pattern in which the tentpole and archegonia protruded in the center, while the area surrounding them showed a concave appearance (Fig. 1F). At approximately 120 d after pollination, the upper cells of the tentpole ceased to divide and began to enlarge (Fig. 1G). The loosely ranged outer cells were no longer wrinkled; in contrast, they protruded outwards and had a plump appearance (Fig. 1H). At approximately 130 d after pollination, the tentpole appeared to tilt toward one side (Fig. 1I). At this stage the upper cells were arranged loosely (Fig. 1J), whereas the lower cells were regularly arranged and had a wrinkled appearance (Fig. 1K and L). An interesting phenomenon that was observed during this period was that a membranous substance emerged on the surface of the tentpole. It occurred either as a type of debris (Fig. 1M–Q) or as a thinner but well-distributed layer (Fig. 1R and S). At approximately 150 d after pollination, at harvest time, the embryo was well developed. Generally only one of the two or more zygotes developed to form the embryo, while the other one(s) aborted. The tentpole showed a significant inclination toward the developing embryo (Fig. 1T). At that time, there was still a thin layer of the membranous substance overlaying the tentpole surface, but the outer cells had a wrinkled appearance (Fig. 1U).

Figure 1. Morphological structures of G. biloba tentpole during development and fertilization. (A) Micropylar end of female gametophyte at ~55 d after pollination. (B, C) Gradually protruding tentpole consisting of small cells. (D) Cylindrical tentpole at ~110 d after pollination. (E) Irregularly shaped cells of tentpole. (F) Distribution of tentpole and archegonia. (G, H) Loosely ranged superficial cells at ~120 d after pollination. (I) Tilted tentpole at ~130 d after pollination. (J) Upper cells of the tentpole at ~130 d after pollination. (K, L) Wrinkled cells of tentpole. (M-Q) Membranous debris on tentpole surface. (R, S) Layer of membranous substance on tentpole surface. (T) Tentpole and embryo at ~150 d after pollination. (U) Tentpole surface at ~150 d after pollination. A, Archegonium; E, embryo; T, tentpole.

Generally, one female gametophyte had only one well-developed tentpole (Fig. 1F). However, we observed some exceptions; Figures 2A and B show two tentpoles connected at the base that originated from one female gametophyte. Figures 2C and D show an over-developed tentpole in which nearly all the surface cells were highly specialized to form finger-like protrusions.

Figure 2. Abnormal tentpoles. (A) Two tentpoles initiated in one female gametophyte. (B) An extra tentpole originated from the base of a tentpole that had already formed. (C) Over-developed tentpole with highly specialized surface cells. (D) Magnified photo of (C), showing the finger-like surface cells. T, Tentpole.

Anatomical features of the tentpole during fertilization

At approximately 55 d after pollination, the cells between the archegonia started to differentiate. They were approximately square-shaped and closely arranged, with large nuclei and light-stained cytoplasm (Fig. 3A). There were no signs of nutrient accumulation, such as starch grains (Fig. 3A). Cells at the top of the tentpole were small, but showed vigorous cell division (Fig. 3A). At approximately 110 d after pollination, cells of the tentpole enlarged and began to accumulate starch grains. The cells had two different shapes: the inner and lower cells were elongated longitudinally, whereas the upper cells were not (Fig. 3B). At approximately 120 d after pollination, the outer cells of the tentpole protruded outward and accumulated more starch grains, but the cytoplasm was still lightly stained (Fig. 3C). The inner cells elongated significantly, but showed a decrease in the number of starch grains (Fig. 3D). At approximately 130 d after pollination (the crucial fertilization period, Aug. 20) the upper cells continued to accumulate starch grains, resulting in dense staining of the cytoplasm (Fig. 3E). The inner cells showed a reduction in starch grains, and many contained no starch grains at all, although the nuclei were still visible (Fig. 3E). After fertilization, starch grains were only present in the upper cells, and were almost completely consumed in the inner cells. Upper cells from different seeds showed different shapes; some had finger-like protrusions (Fig. 3F), and others were regularly arranged (Fig. 3G). Before harvest, starch grains gradually disappeared from the upper cells, while the nuclei, some vacuoles, and densely stained materials remained visible (Fig. 3H). At harvest time, cells had begun to collapse from the upper part of the tentpole, with only the nuclei remaining visible (Fig. 3I).

Figure 3. Anatomical structures of tentpole during development. (A) Cells at micropylar end of female gametophyte at ~55 d after pollination. (B) Tentpole at ~110 d after pollination, with starch grains beginning to accumulate. (C) Surface cells of tentpole at ~120 d after pollination. (D) Inner cells of tentpole at ~120 d after pollination. (E) Tentpole cells at ~130 d after pollination. (F) Finger-like surface cells of tentpole. (G) Regularly arranged surface cells of tentpole. (H) Tentpole cells after fertilization. (I) Tentpole cells at harvest time. N, Nuclei; S, Starch grain. Scale Bar = 20 μm.

Ultrastructure of tentpole during fertilization

At approximately 110 d after pollination, cells of the tentpole were approximately square with a prominent nucleus (Fig. 4A). They contained many spherical lipid droplets that were distributed densely around the vacuoles and the nucleus. At this time, starch grains began to accumulate in plastids, although these grains were very small (Fig. 4A and B). There were abundant mitochondria, Golgi complexes, and small vacuoles scattered in the cytoplasm (Fig. 4C and D), and the plasma membrane had a convex appearance (Fig. 4D). At approximately 120 d after pollination (the period just before fertilization), the starch grains enlarged (Fig. 4E), the number of vacuoles increased (Fig. 4F), and the plastids elongated (Fig. 4F). During fertilization, the spherical or elliptical starch grains reached their maximum volume and showed a significant hilum and layer vein. They were approximately 3 μm in diameter; therefore, they can be classified as ‘C-type’ (Fig. 4G). There were still many lipid drops around the vacuoles (Fig. 4H). The rough endoplasmic reticulum (RER) in the periplasmic region showed a parallel arrangement with the cell wall, with many small vacuoles and mitochondria scattered among the RER membranes (Fig. 4I). Vesicles were present in the peripheral cytoplasm and they were frequently joined to the plasma membrane (Fig. 4J). Moreover, there were some plasmodesmata on the cell wall (Fig. 4K), which were likely important for material transport. After fertilization, starch grains lost their central hilum and were surrounded by vacuoles (Fig. 4L and M). There was a decrease in the number and size of lipid droplets, especially near the vacuoles (Fig. 4M), and some material appeared on the surface of the outer cell wall (Fig. 4M and N). The nucleus began to degenerate (Fig. 4L), but the mitochondria and RER were still present (Fig. 4N). At harvest time, all organelles were difficult to distinguish, and only large vacuoles and some starch grains remained in cells. However, the layer vein of starch grains had completely disappeared (Fig. 4O).

Figure 4. Ultrastructures of tentpole cells before and after fertilization. (A-D) Tentpole cells at ~110 d after pollination containing many mitochondria, golgi complexes, lipid droplets, plastids, and vacuoles. (E, F) Cells at ~120 d after pollination with enlarged starch grains and vacuoles. (G-K) Cells before fertilization with starch grains at maximum size, numerous vesicles and RER distributed near boundary and plasmodesmata scattered along cell wall. (L-N) After fertilization, many starch grains surrounded by vacuoles and marked decrease in lipid droplets. (O) At harvest, only large vacuoles and a few starch grains remain in cells. Cw, Cell wall; Gi, Golgi complex; L, Lipid; M, mitochondria; N, nucleus; P, Plastid; Pm, Plasma membrane; RER, Rough endoplasmic reticulum; S, Starch; V, Vacuole; Ve, Vesicle. Scale bars: (A, O, L) = 5 μm; (B, F-H, M) = 2 μm; (C-D) = 1 μm; E = 10 μm; I = 0.2 μm; (J-K, N) = 0.5 μm.

Discussion

Morphological features of tentpole that favor fertilization

In plants, there are some essential morphological features in both male and female partners that facilitate fertilization and allow successful reproduction.^11,12 For example, in Ceratopteris thalictroides, a fertilization pore forms in the upper extra egg membrane that tightly surrounds the egg before insemination, and the spermatozoid subsequently enters through it.^13,14 As another example, the sperm of Ceratopteris richardii extend to their full length once they are released from the antheridium, and their width decreases by approximately one-half. This allows enough space for more than one sperm to swim side by side along the canal of the egg, so as to maximize the likelihood of fertilization.¹² Gymnosperms also have some special architectural features to enhance the rate of fertilization success. For instance, the archegonial chamber in Encephalartos villosus provides sufficient space for the spermatozoid to swim toward its target.¹⁵ The pollen tubes of Pinus monticola and Picea sitchensis may branch after arriving at the megagametophytes as a strategy to search for archegonia.¹⁶ In G. biloba, just before fertilization, the proximal unbranched portion of the male gametophyte that is anchored to the pollen chamber swells and takes on a saccate appearance, characterizing maturation of the spermatozoid.⁵ After release, the spermatozoid must swim from the pollen chamber and the archegonial chamber to the archegonium for successful fertilization.¹⁷ Previous studies showed that both the pollen chamber and the archegonial chamber may undergo programmed cell death to connect to each other before fertilization.^3,18 Therefore, the only barrier between the spermatozoid and the archegonium would be the layer of megaspore membrane that separates the nucellar tissues from the female gametophyte.⁶ In the present study, we found many morphological and structural characteristics of the tentpole that favored entry of the spermatozoid into the archegonium. First, inside the megaspore membrane, the tentpole protruded from between two or more archegonia due to the proliferation of apical cells of the female gametophyte (Fig. 5A). At maturity, the tentpole was approximately 300-μm high and protruded upwards (Fig. 5B). Except for those on the surface layer, the tentpole cells were arranged compactly, suggesting that the tentpole had a firm structure. During fertilization, there was usually some membranous debris overlaying the tentpole surface. We assumed that the tentpole initiated degeneration of the megaspore membrane (Figs. 5C and 6A–B), and consequently assisted the spermatozoid to enter the archegonium from the archegonial chamber to complete fertilization. Second, the tentpole was usually initiated in an area central to several archegonia on a female gametophyte, in other words, the archegonia were generally located around the tentpole. Furthermore, the base of the column-like tentpole slightly protruded from the adjacent endosperm cells, while the archegonium was embedded deeply in the female gametophyte, with the portion toward the tentpole more deeply embedded and the other part protruding. Therefore, we can conclude that after the rupture of the megaspore membrane, the tentpole probably exercised its fundamental function: to hold the pliable apex of the nucellar tissues and, therefore, protect both the male gametophyte and the archegonia and prevent them from contacting each other (Figs. 5D and 6C). It could also help the pollen chamber retain its shape, thereby providing enough space for swelling of the proximal unbranched portion of the male gametophyte (Fig. 6D).

Figure 5. Tentpole development. (A) Initiation of tentpole between archegonia. (B) Mature tentpole. (C) Tentpole is assumed to stimulate megaspore membrane. (D) Tentpole supporting apex of the nucellus. Ar, archegonium; Ac, archegonial chamber; E, egg cell; Mg, male gametophyte; Mm, megaspore membrane; Nu, nucellus; Pc, pollen chamber; T, tentpole.

Figure 6. Apex of nucellus just before fertilization. (A, B) Rupture of megaspore membrane before fertilization. (C) Pollen chamber and archegonial chamber. (D) Four swollen male gametophytes in pollen chamber. Ac, archegonial chamber; Mg, male gametophyte; Mm, megaspore membrane; Nu, nucellus; Pc, pollen chamber. Scale bars = 50 μm.

Anatomical features of the tentpole favoring fertilization

As the fertilization period approaches, some anatomical features emerge in the female gametophyte that indicate that the male and female gametes will contact each other. In siphonogamous gymnosperms such as Pseudotsuga menziesii and Picea glauca, numerous lipid droplets accumulate in the archegonial chamber at the exact site to which pollen tubes grow. This may be an analogous situation to the chemical signaling in gymnosperm pollen tubes.^19-21 Zoidogamous plants must produce fluid to form a pool around the archegonia. The male gamete is released into the pool and moves through it to fertilize the archegonium.^17,22 G. biloba is one of the most notable zoidogamous gymnosperms.^23,24 Studies on the sexual reproductive process in G. Biloba confirmed that when the spermatozoid is released from the pollen tube, the archegonial chamber is full of liquid, through which the spermatozoids swim.^7,17 It was thought that the liquid originated from the pollen tube, or from the degenerated nucellar tissues.⁷ In this research, we observed and analyzed the ultrastructure of the tentpole before and after fertilization. We observed that during fertilization, the loosely arranged upper cells of the tentpole protruded outwards or showed a finger-like appearance, similar to the stigma of many angiosperms during maturation.^25-27 The cytoplasm was very dense in the upper cells of the tentpole, and they contained abundant organelles such as vacuoles, plastids, mitochondria, Golgi complexes, and RER. The mitochondria could provide considerable energy for material synthesis and transportation; the Golgi complexes would produce vesicles to participate in exocytosis; and the RER would also be important for material synthesis. In addition, some plasmodesmata were scattered along the cell walls. The presence and distribution of those cellular structures and organelles indicated the intensive exchange of material and information among the tentpole cells. We also observed the accumulation of abundant nutrients in the tentpole before and during fertilization. In the early period of tentpole formation, there were abundant lipid droplets, which remained densely distributed in cells until the time approaching fertilization. After fertilization there was a marked decrease in the size and abundance of lipid droplets. This observation suggested that lipids, which are important substances synthesized in the tentpole, participated in the fertilization process. Starch grains were another important substance synthesized in the tentpole. The plastids in tentpole cells accumulated abundant, well-developed, small starch grains before fertilization. After fertilization, the hilum and layer vein disappeared, indicating that they were exhausted during fertilization. Therefore, we can conclude that the G. biloba tentpole cells have secretory characteristics. During fertilization, the tentpole can secrete some materials that facilitate successful fertilization.

Materials and methods

Plant material

Specimens were randomly collected from healthy female G. biloba trees every week from mid-April to early October in 2009 to 2011 at the Ginkgo Experimental Station in Yangzhou University, Yangzhou, China (32°20′N, 119°30′E). The tentpoles were located at the micropylar parts of the female gametophytes in the G. biloba ovules. Therefore, after removal of three layers of seed coats, the micropylar parts were carefully dissected out from the female gametophytes with a razor blade and a steel needle under a dissecting microscope.

Scanning electron microscopy

The samples were prepared for SEM observations as follows: samples were fixed in an improved FAA solution (50% alcohol:glacial-acetic-acid:formaldehyde = 89:6:5 v/v/v) and kept at 4°C prior to use. They were dehydrated in a graded alcohol series (30, 50, 70, 80, 90, 95, and 100% v/v, 15 min each step), and further dehydrated with acetone, then immersed in isoamyl acetate for at least 30 min. After drying at critical point, the samples were coated with a layer of gold, and observed under a S-4800 field-emission SEM (Hitachi) at 15 kV accelerating voltage.²⁸

Semi-thin sections

Semi-thin sections were prepared as follows. Samples were prefixed in 2.5% (v/v) glutaraldehyde for 20 h at 4°C. After being postfixed in 1% (w/v) osmium tetroxide for 6 h at room temperature, the samples were washed three times in 0.2 M phosphate buffer (pH 7.2), dehydrated in an ethanol series (30, 50, 70, 80, 90, and 100% v/v, 15 min each step), treated twice for 30 min with propylene oxide, and then infiltrated with 1:1 propylene oxide/resin in embedding capsules overnight before being embedded in Spurr’s resin. Sections (1-µm thick) were cut with glass knives and then stained with 1% (w/v) toluidine blue O dissolved in 1% (w/v) sodium borate.²⁹ Sections were observed under a microscope (Zeiss Axioskop 40, Carl Zeiss Shanghai Company Ltd.).

Transmission Electron Microscopy

Samples were embedded in Spurr’s resin as for semi-thin sections. The samples were cut into 70-nm thick sections with a diamond knife, and then stained with 1% (w/v) uranyl acetate and 1% (w/v) lead citrate.²⁹ The top and surface cells of the tentpole were observed and photographed under a Philips Tecnai 12 TEM (JEOL Ltd, Tokyo, Japan).

Authors’ contributions

BJ and LW designed the project; DW, YL and MZ participated in SEM and TEM observations and semi-thin sectioning experiments; BJ and LW drafted the manuscript; and all co-authors participated in its editing.

Acknowledgments

We are very grateful to Prof. Peng Chen and Prof. Zhong Wang (Yangzhou University) for technical advice. This work was financially supported by the Natural Science Found of Jiangsu Province No. BK2011444, and the National Natural Science Foundation of China No. 30870436.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.