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. 2006 Jan;97(1):71–77. doi: 10.1093/aob/mcj015

Mode of Pollen-Tube Growth in Pistils of Myrica rubra (Myricaceae): A Comparison with Related Families

AKIKO SOGO 1, HIROSHI TOBE 1,*
PMCID: PMC2803377  PMID: 16291781

Abstract

• Background and Aims It is generally known that fertilization is delayed for more than a few weeks after pollination in Fagales. Recent studies showed that, during that period, pollen tubes grew in pistils in close association with the development of the ovule in a five-step process in Casuarina (Casuarinaceae) and a four-step process in Alnus (Betulaceae). The number of pollen tubes was reduced from many to one, a fact suggesting that delayed fertilization plays a role for gametophyte selection. Myrica (Myricaceae) also shows delayed fertilization for >2 weeks after pollination, but nothing is known of how pollen tubes grow in the pistil during that period.

• Methods Pollen-tube growth and the development of the ovule in pistils was investigated by fluorescent and scanning electron microscopy and analysis of microtome sections of the pistils.

• Key Results Developmental study of the pollen-tube growth in the pistil of M. rubra showed that the tip of the pollen tube was branched or lay in a zigzag pattern in the upper space of the ovarian locule or near the tip of the integument, and subsequently was swollen on the nucellar surface. Such morphological changes indicate that the pollen-tube growth was temporarily arrested before fertilization. The pollen-tube growth in M. rubra can therefore be summarized as occurring in three steps: (1) from the stigma to the ovarian locule; (2) from the ovarian locule to the nucellar surface; and (3) from the nucellar surface to the embryo sac.

• Conclusion Myrica differs from other families in that the pollen tubes arrest their growth on the nucellar surface, probably digesting nutrient from nucellar cells. There is little information on five other families of Fagales. An extensive study is needed to better understand the diversity and function of the mode of pollen-tube growth within the order.

Keywords: Fagales, fertilization, micropyle, Myrica, Myricaceae, pollen-tube growth

INTRODUCTION

Fagales, comprising eight families (Betulaceae, Casuarinaceae, Fagaceae, Juglandaceae, Myricaceae, Nothofagaceae, Rhoipteleaceae and Ticodendraceae) (APG II, 2003), are mostly wind-pollinated, with strongly reduced, often unisexual flowers that are usually borne in catkins (Li et al., 2004). Through studies dating back more than a century, some distinctive features related to pollination and/or fertilization have been shown for those families. For instance, probably all families of Fagales show a delay in fertilization of 2 weeks to 15 months between pollination and fertilization (e.g. Benson, 1894; Swamy, 1948; Stairs, 1964; Boavida et al., 1999; Williams et al., 1999). Several families such as Betulaceae (e.g. Nawaschin, 1893; Benson, 1894; Finn, 1936), Casuarinaceae (e.g. Treub, 1891; Swamy, 1948; Maheshwari, 1950; Sogo et al., 2004a, b), and Juglandaceae (e.g. Nawaschin, 1895; Nast, 1935; Luza and Polito, 1991) show chalazogamy, a mode of fertilization where the pollen tube passes through the chalaza, instead of through the micropyle (porogamy). Several families, such as Betulaceae pro parte (Kubitzki, 1993a), Juglandaceae (Stone, 1993), Myricaceae (Kubitzki, 1993b), Nothofagaceae (Poole, 1952) and Ticodendraceae (Tobe, 1991) have unitegmic rather than bitegmic ovules. Juglandaceae (Langdon, 1934; Nast, 1935; Stone, 1993) and Myricaceae (Yen, 1950; Håkansson, 1955; Kubitzki, 1993b) have orthotropous rather than anatropous or hemitropous ovules.

Recently it was reported that fertilization is delayed for >1 month after pollination in Casuarina equisetifolia (Casuarinaceae) (Sogo et al., 2004a) and two species of Alnus (Betulaceae) (Sogo and Tobe, 2005) because the pistils are immature when pollen is delivered to the stigma. During that period, the pollen tube(s) grows discontinuously within the pistil in five steps in the former and in four steps in the latter. In the light of the aforementioned distinctive features in Fagales, these results have suggested the likelihood that diversity exists in the mode of growth of pollen tubes in pistils within the order.

This paper will document the growth of pollen tubes in the pistils of Myrica rubra. Unlike Casuarinaceae and Betulaceae, Myricaceae (three genera, 57–62 species) have a unitegmic orthotropous ovule (Kubitzki, 1993b) and show porogamy (i.e. pollen tube entry through the micropyle) (Treub, 1891; Yen, 1950; Håkansson, 1955). It is also known that fertilization after pollination is delayed for about 6–7 weeks in M. rubra (Yen, 1950) and for 4 or 5 weeks in M. gale (Håkansson, 1955), and that M. rubra has immature ovules at the time of pollination (Yen, 1950). Nothing, however, is known of how the pollen tube(s) grows from the stigma to the embryo sac as the pistil develops in those species.

MATERIALS AND METHODS

Plant materials

Female inflorescences of Myrica rubra Siebold et Zucc. from trees cultivated along the roadside in the city of Kyoto, Japan, were collected every 3 or 4 days from the middle of April to the middle of May in 2001–2003. They were fixed in FAA (stock formalin/glacial acetic acid/50 % ethanol; 5 : 5 : 90).

Observation of pollen-tube growth and ovule development

To observe the growth of pollen tubes within the pistils, the pistils were removed from the inflorescences and each of them cut longitudinally into halves in a solution of 50 % ethanol. They were cleared in a 0·01 % solution of sodium hypochlorite (NaClO) at room temperature overnight. After being rinsed two or three times in water, the pistils including the ovules were macerated in 1 n NaOH at 60 °C for 1 h. Pollen tubes within the pistils were stained with 0·5 % aniline blue in 0·1 n K3PO4 for 2–3 h and observed with a fluorescent microscope (Zeiss LSM410, Germany) using a UV filter set (model No. 01) with excitation filter (365 nm, band pass 12 nm), dichroic mirror (FT395) and barrier filter (LP397).

Pollen tubes around the apical surface of the ovule or nucellus were observed by scanning electron microscopy. The samples were dehydrated through an ethanol series, and then critical-point dried. After being coated with gold, the ovules and pollen tubes were observed with a JSM-5800LV microscope (JEOL DATUM, Japan). In some pistils, the ovary wall was removed to expose an ovule or the surface of the nucellus.

To observe the pollen tubes in the developing pistils, serial longitudinal microtome sections of the pistils were also prepared. For microtoming, some pistils were dehydrated through a t-butyl alcohol series, embedded in Paraplast, and sectioned to a thickness of 6 or 7 µm. Some of the sections were stained with Heidenhains haematoxylin, safranin and fast green FCF, and mounted in Entellan (Merck, Darmstadt, Germany) to observe ovule development; the others were stained with aniline blue to observe the pollen tubes.

Resin sections of some ovaries were also prepared to observe the fine structure of the embryo sac and pollen tubes in the nucellus. To make those sections, the ovaries were dehydrated through an acetone series, then embedded in Spurr's resin and sectioned using an ultra-microtome. Sections cut at 1 µm in thickness were stained with Toluidine blue O and observed with an Olympus BX-51 microscope.

RESULTS

Pollination in Myrica rubra, an evergreen, dioecious shrub, began in mid-April. Female inflorescences (catkins) bore 10–25 flowers and were about 8–15 mm long. The pistillate flower has two carpels terminating in two elongate stigmas at the time of pollination (Fig. 1A). When the stigmas receive pollen grains, the pistils are 3–3·5 mm long including the stigmas. Compared with the stigmas, the ovary is small, about 1 mm long, and the surface of the ovary is knobby.

Fig. 1.

Fig. 1.

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(A–F) Growth of pollen tube and development of ovule at the time of pollination in Myrica rubra. (B, C, E and I) Photographs taken using a fluorescent microscopy; (D and H) microtome sections. (A) Pistils at the time of pollination; (B) pollen tubes in part of stigma indicated by the rectangle with a one asterisk in (A); (C) pollen tubes in style indicated by rectangle with two asterisks in (A); (D) longitudinal section of ovary indicated by rectangle with three asterisks in (A), showing pollen tubes (arrows) growing in ovarian locule; (E) pollen tubes (arrows) in ovary indicated by rectangle with three asterisks in (A); (F) diagram reconstructed from (B–E), in which lighter lines show pollen tubes; (G) older pistil; (H) longitudinal section of ovule with a dyad of megaspores; (I) pollen tube (arrows) lying in zigzag position in ovarian locule—this ovule has a dyad of megaspores. Abbreviations: i, integument; md, megaspore dyad; mmc, megaspore mother cell; nu, nucellus; ova, ovary; ovu, ovule; sg, stigma; sy, style. Scale bars: A and G = 1 mm; B, C and E = 100 μm; D, H and I = 50 μm.

Many pollen grains landed on the stigma surface, and most of them (>20 pollen tubes per stigma) germinated and grew into the tissue of the stigma (Fig. 1B). The ovary (or ovule), however, was still immature, so that fertilization was delayed for 2 to <3 weeks. The pollen tubes thus stay within the pistil (ovary) for over 2 weeks until the ovule becomes mature enough for fertilization. Table 1 presents the number of pistils in which the tip of the pollen tube was found at particular positions, showing when and where the pollen tube lies within the growing pistil. During the 2 weeks after pollination, the tip of the pollen tube was mainly in the following three places: the upper space of the ovarian locule, the surface of the nucellus, and the embryo sac. Those positions are closely associated with the length of time after pollination and the developmental stages of the ovary (or ovule). This is explained in more detail below.

Table 1.

Position of the pollen-tube tip in developing pistils of Myrica rubra

Developmental stages of ovules
Position of pollen-tube tip(s) Megaspore mother cell stage Megaspore dyad stage Megaspore tetrad stage One-nucleate embryo sac stage Two-nucleate embryo sac stage Four-nucleate embryo sac stage Mature embryo sac stage
Upper space of the ovarian locule 27 2 3 1 – – –
Surface of the integument or the nucellus – 2 15 1 10 10 5
Embryo sac – – – – – – 17*
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Figures indicate the number of pistils observed.

*

Includes embryo sacs just after fertilization.

Before and during megasporogenesis

The ovary is bilocular, with one locule larger than the other. In the larger locule, a single unitegmic, orthotropous ovule, with the apex of the nucellus facing upward, developed from its base. In early stages, as the integument was initiated as a small emergence around the nucellus, many pollen tubes germinated in the stigmatic tissue and some of them grew downward in the style (Fig. 1C). In some such ovules the megaspore mother cell had differentiated (Fig. 1D), while in others the megaspore mother cell had not differentiated. In 27 pistils with the ovule at the megaspore mother cell stage, only one to three pollen tubes (mean = 1·43) reached the upper space of the ovarian locule (Table 1 and Fig. 1D–F). The pollen tubes were branched and/or lay in a zigzag pattern within the upper space of the locule. When the ovule was removed from the locule, the tips of the branched and/or zigzag pollen tubes remained on the inner surface of the ovary, indicating that the tip of the pollen tubes was free from the ovule.

By about 1 week after the pollen tubes had reached the locule, the stigmas started to wither from their tips and the ovary had enlarged to about 1·5 mm in width (Fig. 1G). While the megaspore mother cell underwent meiosis in the nucellus to form a dyad or tetrad of megaspores, the branched and/or zigzag pollen tubes remained within the locule (Table 1). At the same time, the integument developed to nearly the same height as the nucellar apex (Fig. 1H) and the tip of the pollen tubes shifted its position to the inside of the tip of the integument (Fig. 1I).

During megagametogenesis

In 16 of 20 pistils with the ovule at the megaspore tetrad stage or the one-nucleate embryo sac stage, one to three pollen tubes reached the surface of the nucellus (Table 1 and Fig. 2A–D), indicating that upon completion, or soon after, megasporogenesis, the tip of the pollen tubes moved to the surface of the nucellus. The pollen tubes that reached the nucellus did not immediately penetrate the nucellus, but remained on the nucellar surface while the embryo sac successively developed into the two- and four-nucleate stage within the nucellus (Fig. 2E and F and Table 1). During that period, the tips of the pollen tubes doubled or tripled in thickness (Fig. 2G–J). Microtome sections showed that such swollen pollen tube tips penetrated the tissue of nucellus slightly, but never reached the embryo sac. The sections also revealed that the cells of the nucellar apex were rich in starch grains, suggesting that the pollen tubes may utilize them for nutrients while adhering to the nucellar surface.

Fig. 2.

Fig. 2.

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Growth of pollen tube and development of ovule in older pistils of Myrica rubra: (A, B, E and F) Photographs taken using light microscopy; (C and G) photographs taekn using fluorescent microscopy; (H and I) photographs taken using scanning electron microscopy. (A) Longitudinal section of ovule with a tetrad of megaspores; (B) part of (A) indicated by rectangle, magnified to show a tetrad of megaspores—chalazal megaspore develops into embryo sac, while others (arrowheads) degenerate; (C) pollen tube (arrow) growing on surface of nucellus in ovule with a tetrad of megaspores; (D) a diagram reconstructed from (A–C), in which lighter lines show pollen tubes in ovule; (E) longitudinal section of ovule with four-nucleate embryo sac; (F) part of (E) indicated by rectangle with one asterisk, magnified to show four-nucleate embryo sac in nucellus; (G) thickened pollen tubes (arrows) on surface of nucellus indicated by rectangle with two asterisks in (E); (H and I) pollen tubes (arrows) on nucellus; (J) diagram reconstructed from (E–I), in which lighter lines show pollen tubes in ovule. Abbreviations: es, embryo sac; i, integument; n, nucleus; nu, nucellus. Scale bars: A and E = 100 μm; C, G, H and I = 50 μm; B and F = 20 μm.

Maturity of the embryo sac

When the ovary had developed to about 2–2·5 mm in thickness (Fig. 3A), the ovule inside had reached maturity and contained one mature embryo sac (Fig. 3B). In 17 of 22 such ovules (or pistils) examined, only a single pollen tube had reached the embryo sac, even though more than two pollen tubes were attached to the nucellar surface (Table 1 and Fig. 3C–F). By the time the pollen tube started growing toward the mature embryo sac, it had reverted to its original thickness (Fig. 3E and F). The ovary with a fertilized ovule then developed into a one-seeded fruit in the final stage.

Fig. 3.

Fig. 3.

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Growth of pollen tube and development of ovule in mature pistils of Myrica rubra: (B, D and E) microtome sections; (C) photograph taken using fluorescent microscopy. (A) Mature pistil; (B) longitudinal section of mature ovule; (C) pollen tube in nucellus indicated by rectangle with one asterisk in (B); (D and E) longitudinal section of mature ovule indicated by rectangle with two asterisks in (B); pollen tube (arrows) grows toward embryo sac through tissue of nucellus—one or two more pollen tubes appear in the subsequent sections of the ovule; (F) diagram reconstructed from (B–E), in which lighter lines show pollen tubes in ovule. Abbreviations: es, embryo sac; i, integument; nu, nucellus; ova, ovary. Scale bars: A = 1 mm; B and C = 100 μm; D and E = 50 μm.

DISCUSSION

Mode of pollen tube growth in Myrica rubra

As documented above, the ovary was still immature at the time of pollination in Myrica rubra, and >2 weeks passed between the germination of the pollen grains on the stigma and fertilization. During that period, the tip of the pollen tubes remained first within the space inside the locule and then on the surface of the nucellus, and showed notable changes in shape. The pollen tubes were branched or lay in a zigzag formation in the upper space of the ovarian locule or near the tip of the integument. Subsequently, they became swollen while on the nucellar surface. Each of the morphological changes gave the impression that pollen-tube growth was temporarily arrested, since some time was required for such morphological changes to take place. The growth of the pollen tube in the pistils of M. rubra can therefore be summarized as a three-step process: (1) from the stigma surface to the upper space of the locule of the ovary (although the tip of the pollen tube shifted to the inside of the apex of the developing integument); (2) from the locule to the surface of the nucellus; and (3) from the surface of the nucellus to the embryo sac (Fig. 4A). The number of pollen tubes was reduced from many to one to three in the first step, and from one to three to only one in the third step.

Fig. 4.

Fig. 4.

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Mode of pollen-tube growth in pistils. (A–C) Drawings illustrating how pollen tubes grow in pistils: (A) Myrica; (B) Casuarina (from Sogo et al., 2004a); (C) Alnus (from Sogo and Tobe, 2005). Note that the pollen tubes intermittently grow in three steps in Myrica, five steps in Casuarina, and four steps in Alnus. (D) Comparison of the growth of pollen tubes in pistils among Myrica, Casuarina and Alnus.

As in Casuarina (Casuarinaceae) (Sogo et al., 2004a, b) and Alnus (Betulaceae) (Sogo and Tobe, 2005), growth of the pollen tube proceeded in close association with the development of the ovule or embryo sac (female gametophyte) in Myrica rubra. The first step proceeded irrespective of the presence of a well-developed ovule and lasted for about 1 week until the ovule reached the one-nucleate embryo sac stage; the second step lasted for another week until a mature embryo sac developed within the ovule. When the pollen tubes reached the surface of the nucellus in the second step, the embryo sac was still immature and not yet ready for fertilization. This suggests that the pollen tubes are not dependent on a mature embryo sac to reach the nucellar surface. Only in the final or third step is the pollen tube probably guided by the mature embryo sac, or more precisely, by a synergid in the mature embryo sac, as occurs in Torenia (Higashiyama et al., 2001).

Comparisons with mode of pollen-tube growth in related families

A similar mode of pollen-tube growth is known in Casuarinaceae (Sogo et al., 2004a, b) and Betulaceae (Sogo and Tobe, 2005), both in the Fagales. Casuarina equisetifolia (Casuarinaceae) has two anatropous bitegmic ovules per ovary, while Alnus firma and A. sieboldiana (Betulaceae) have two anatropous unitegmic ovules per ovary. They all undergo chalazogamy instead of porogamy. In C. equisetifolia growth of the pollen tubes in the pistils can be summarized in five steps: (1) from the stigma to the style; (2) from the style to the upper region of the locule of the ovary; (3) from the upper region of the locule to the funicular surface; (4) from the funicular surface to the chalaza of the ovule; and (5) from the chalaza to the embryo sac (Fig. 4B). In Alnus, the pollen tubes grow in four steps within the pistils: (1) from the stigma to the style; (2) from the style to the upper space of the ovarian locule; (3) from the upper space of the ovarian locule to the chalaza of the ovule; and (4) from the chalaza to the embryo sac (Fig. 4C).

Compared with the mode of pollen-tube growth in Casuarina and Alnus, the first step in Myrica appears to correspond to the first and second steps in Casuarina and Alnus (Fig. 4D). In Myrica, the pollen tubes grow downward from the stigma to the locule of the ovary without stopping in the style. In contrast, in both Casuarina and Alnus the pollen tubes grow from the stigma to the style, then stop growing for a long period. Such arrested growth in the style was also reported in several other genera of Fagales, including Betula (Dahl and Fredrikson, 1996; Williams et al., 1999) and Quercus (Boavida et al., 1999). Whether a pollen tube arrests growth in the style appears to depend on the developmental stage of the ovary at the time of pollination. At the time of pollination in Myrica the ovules have already developed both the nucellus and the integument, whereas in the species of Alnus, Betula, Casuarina and Quercus examined, they were still primordial. In other words, by the time of pollination the source of a signal to guide the pollen tube toward the locule of the ovary has already developed in Myrica, but not in Alnus, Betula, Casuarina and Quercus.

Similarly, the second step in Myrica (and the third step in Alnus) correspond to the third and fourth step in Casuarina (Fig. 4D). The second step in Myrica has not been reported in other fagalean species that show porogamy. In Casuarina and Alnus the pollen tube stops at the chalaza, which is massive with ample vascular tissues, because the embryo sac was still immature when it reached there (Sogo et al., 2004a, b; Sogo and Tobe, 2005). Likewise in Myrica, since the embryo sac is still immature (one-nucleate stage) when the pollen tubes reaches the nucellar surface, they remain there for about 1 week until the embryo sac reaches maturity. During that time the tips of the pollen tubes adhere to the nucellar surface, are likely to digest starch in the nucellar cells for nutrition, and become swollen to varying degrees. The nucellar surface in Myrica appears to play the same role as the chalaza in Casuarina and Alnus in terms of probably providing the pollen tube with nutrition.

CONCLUSIONS

A study of intermittent growth of pollen tubes in pistils, which occurs in close association with the development of the female gametophyte(s), has just begun with Fagales (Sogo et al., 2004a; Sogo and Tobe, 2005). While it has an evolutionary meaning for gametophyte selection, it may provide an important clue for the study of pollen tube guidance (Sogo et al., 2004a; Sogo and Tobe, 2005). As has been reported, the mode of pollen-tube growth is partly consistent throughout the families examined (Casuarinaceae, Betulaceae, and Myricaceae), but differs in part among them. There is little information on the five additional families of Fagales. It is known that delayed fertilization is prevalent in the order. An extensive study is needed to better understand the diverse modes of pollen-tube growth.

Acknowledgments

We thank Hiroaki Setoguchi, Jung Sung Kim and Junko Noguchi for assistance in collecting material for our research, and David E. Boufford for valuable suggestions in completing the manuscript. The study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 14405012) and a grant for Biodiversity Research of the 21st Century COE (A14).

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