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Development of sterile ovules on bisexual cones of Gnetum gnemon (Gnetaceae)¹

Department of Biology, University of North Dakota, Grand Forks, North Dakota 58202 USA

Received for publication June 13, 2000. Accepted for publication December 22, 2000.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

 
Angiosperms and Gnetales (Ephedra, Gnetum, Welwitschia) represent the only seed plants that regularly produce bisexual cones. Unfortunately, the fertility and function of ovules formed on bisexual cones of Gnetales have remained unclear. Some reports indicate that the ovules are sterile while others indicate that they may develop into seeds. This study demonstrates three different developmental patterns of ovules formed on bisexual cones of Gnetum gnemon. Type I ovules did not develop at all after pollination and represented the majority of ovules on each cone. Type II ovules enlarged slightly after pollination due to the enlargement of nucellar tissue. Type III ovules were typically found on the terminal whorl and developed into seed-like structures. The enlargement was due to proliferation of megagametophyte tissue. Sectioned material revealed that megagametophytes show altered development compared to those found in functional female ovules. None of the ovules studied contained embryos, and thus all were sterile. Densitometry of 4',6-diamidino-2-phenylindole (DAPI)- stained sections revealed that megagametophyte nuclei formed in the sterile ovules are unreduced (diploid) and thus do not form viable female gametes.

Key Words: bisexual • DAPI • densitometry • diploidy • Gnetales • Gnetum • megagametophyte

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

 
Besides flowering plants, Gnetales (Ephedra, Gnetum, Welwitschia) represent the only extant seed plants that regularly produce bisexual cones or strobili as part of their normal reproductive pattern. Most members of the Gnetales produce bisexual (ovulate and staminate) and female (ovulate) cones on separate plants and are therefore classified as structurally gynodioecious. Although ovules are regularly produced on bisexual cones of Gnetales, the function of these ovules has remained unclear. In most accounts of Ephedra, Gnetum, and Welwitschia, the ovules on bisexual cones are reported to be sterile or abortive (Maheshwari and Vasil, 1961 ; Endress, 1996 ; Hufford, 1996 ). As a result, the bisexual cones are functionally male and members of Gnetales are typically categorized as functionally dioecious. Interestingly, the "male" ovules of Gnetum (ovules formed on structurally bisexual but functionally male cones) have been reported to occasionally enlarge and develop into mature seeds (Lata, 1960 ; Maheshwari and Vasil, 1961 ; Endress, 1996 ). Unfortunately, it has never been documented whether these "seeds" are indeed viable and represent true reproductive units. Reports by Vasil (1959) and Lata (1960) represent the only data available on the internal structure of the "male" ovules in Gnetum. The results consist only of camera-lucida drawings and do not provide comprehensive documentation of the events that transpire during the development of "male" ovules, in particular, those that appear to develop into seed-like structures. Their accounts indicate that megagametophyte-like structures form within the "male" ovules. The megagametophytes appear to be arrested at an early, free-nucleate stage of development. It is not clear whether they mature and become fertile, whether fertilization occurs in the "male" ovules, or whether the ovules are capable of developing into functional seeds complete with viable embryos.

Given the recent interest in the reproductive biology of Gnetales (Endress, 1996 ; Hufford, 1996 ; Friedman, 1998 ; Frohlich, 1999 ) and its implications for our understanding of the evolution of higher plant groups, a developmental analysis of the "male" ovules of Gnetum is warranted. The objectives of this study were to ascertain the fertility of ovules produced on bisexual cones and document the potential mechanism(s) of ovule sterility in Gnetum gnemon L. Development of the megagametophyte-like structures, identified previously by Lata (1960) , is a major focus of this study since sexual reproduction in seed plants is ultimately tied to the formation of functional gametophytes and gametes. It is hoped that knowledge of the development, internal structure, and function of the ovules formed on bisexual plants of Gnetum gnemon will ultimately increase our understanding of reproductive diversity within Gnetales.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

 
Plant conditions
Male and female plants of G. gnemon were maintained in greenhouses at the University of Georgia, Department of Botany, and the University of North Dakota, Department of Biology. Under greenhouse conditions, plants were fertile throughout the year and produced bisexual cones on a regular basis. Over the course of several years, ovules formed on bisexual cones ("male" ovules) were either collected at various stages of development before maturation (as evidenced by the presence of pollination droplets) or pollinated and collected at subsequent time intervals. Eight separate individual male plants were used for these studies. Over 100 self-pollinations (using pollen and ovules formed on the same plant) and 100 cross-pollinations (using pollen and ovules from separate plants) were performed by hand.

Microscopy
"Male" ovules and mature microsporangia were collected and processed for brightfield and fluorescence microscopy according to the methods of Carmichael and Friedman (1996) . For brightfield microscopy, samples were fixed in acrolein, dehydrated in ethanol, embedded in glycol methacrylate, serially sectioned into 5-µm thick ribbons, and the sections were stained in toluidine blue. For fluorescence microscopy, samples were fixed in 3:1 (V/V, ethanol:acetic acid), dehydrated, embedded, and sectioned as mentioned above. Sections were stained with 1.0 µg/mL 4',6-diamidino-2-phenylindole (DAPI) in 50 mmol/L Trizma buffer, pH 7.2. Trace amounts (0.1 mg/mL) of p-Phenylene-diamine were added to the DAPI solution to prevent fading. Slides were flooded with the DAPI solution for 5 min at room temperature in a dark environment. Excess DAPI solution was removed and a coverslip was placed on the slide. Nuclei were visualized on an Olympus BX 60 microscope (Olympus, Tokyo, Japan) equipped for UV epifluorescence with HBO 200 W burner (Ushio, Tokyo, Japan) and wide band UV filter cube (Olympus).

Computer imaging and nuclear DNA quantification
Color images of sections containing megagametophyte structures were captured through computer-assisted video microscopy using a Color CCTV Camera (Panasonic, model WV-CP410, Osaka, Japan). The camera was linked to an IBM-compatible computer equipped with Snappy Video Snapshot® (Play Incorporated, Rancho Cordova, California, USA). The images were captured at 640 x 480 pixels and stored for future reference.

DNA quantification techniques were used to study ploidy levels of megagametophytes formed within the "male" ovules. Images of DAPI-stained serial sections were captured through video microscopy and converted to grayscale prior to analysis. DNA levels of nuclei were quantified using ImageTool image analysis software, version 1.28, developed at the University of Texas Health Science Center, San Antonio, Texas, USA, available via anonymous FTP at ftp://maxrad6.uthscsa.edu. It was determined that nucellar and megagametophyte nuclei were 10 µm in diameter during interphase and therefore occupied two adjacent 5 µm thick sections. This was verified by careful observation of multiple serial sections through individual nuclei. One section of each nucleus measuring 10 µm in diameter was used for DNA/ploidy analysis. This procedure allowed the quantification of relatively large populations of nuclei. Nuclei to be analyzed were individually identified as objects on ImageTool by thresholding manually to a range that isolated nuclei from the background image. In order to insure that the relative fluorescence did not exceed the maximum detectable level, the brightest regions of nuclei were measured to ensure they fluoresced below 256 (the maximum grayscale level). Relative fluorescence units (RFU) were reported as integrated density, which represents the product of the area and the average fluorescence of the selected nucleus. Standardization of DNA/ploidy levels was performed by measuring the RFU of nucellar nuclei during interphase. By definition, sporophyte nuclei in interphase contain between the 2C and the 4C quantity of DNA depending on their stage in the cell cycle (assuming they are diploid). The approximate low value (in RFU) was therefore set equal to the 2C quantity of DNA and the high value, which was twice the low, set equal to the 4C quantity of DNA. These values were then used to calibrate the relative DNA/ploidy levels of megagametophyte nuclei, also in interphase. This calibration was tested by measuring the relative fluorescence of generative cell nuclei in mature pollen grains. These haploid nuclei, by definition, contain half the number of chromosomes of sporophyte (e.g., nucellus) nuclei and are therefore expected to exhibit one-half RFU compared to sporophyte nuclei.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

 
Previous studies have documented the general structure and morphology of mature bisexual cones in Gnetum gnemon (Maheshwari and Vasil, 1961 ; Endress, 1996 ; Hufford, 1996 ). Therefore, a description of basic cone morphology is not included in this report.

Prepollination megagametophyte development
Between one and five megasporocytes were found within each ovule (Fig. 1), each of which develops into a four-nucleate coenocytic megaspore. No cell walls or degenerate megaspores were observed and thus megasporogenesis is tetrasporic in nature. Each coenomegaspore enters a period of free nuclear development, the earliest stage of which is characterized by a scattered distribution of nuclei within the megagametophyte (Fig. 2). A vacuole is evident during early stages of development, but is usually not located centrally. Several megagametophytes are initiated in each ovule, each of which develops from a single coenomegaspore. The more chalazal megagametophyte becomes dominant and develops more fully than the others (Fig. 3). At the time of pollination the dominant megagametophyte is entirely free nuclear and measures 700 µm in length. It consists of an enlarged micropylar region and a constricted chalazal region. The micropylar region exhibits a central vacuole with nuclei positioned in a thin band of parietal cytoplasm. The chalazal region typically lacks a vacuole and contains free nuclei within cytoplasm.

View larger version (194K): [in this window] [in a new window]   Figs. 1–6. Development of megagametophytes and ovules on bisexual cones of Gnetum gnemon. 1. DAPI-stained section showing four megasporocytes (arrowheads). Bar = 20 µm. 2. Longitudinal section through a young coenocytic megagametophyte. Bar = 20 µm. 3. Coenocytic megagametophyte at the time of pollination. Note the numerous vacuoles and degenerate megagametophyte (arrowhead). Bar = 20 µm. 4. Longitudinal section through a pollinated ovule. Pollen tubes are found growing through the nucellus toward the megagametophyte. Bar = 50 µm. 5. High-magnification view showing a pollen tube with sperm nuclei entering the apex of a coenocytic megagametophyte. Sperm remained within pollen tubes and fertilization did not occur. Bar = 20 µm. 6. Bisexual cone 3 mo after pollination. All of the microsporangia have abscised by this point. The majority of ovules did not develop after pollination (Type I). Some ovules enlarged, but only slightly (Type II). Other ovules developed into large seed-like structures and turned red in color (Type III). Bar = 1 cm. Figure Abbreviations: MG, megagametophyte; N, nucellus; RFU, relative fluorescence units; SN, sperm nucleus; TN, tube nucleus; TI, type one ovule; TII, type two ovule; TIII, type three ovule.

Postpollination events
Within three days of pollination, pollen grains were withdrawn into the micropylar tube of "male" ovules. Shortly thereafter, pollen germinated and pollen tubes were found to grow through the nucellus and enter the micropylar region of the megagametophyte (Fig. 4). Two sperm nuclei and a tube nucleus were clearly visible within each pollen tube (Fig. 5). The pollen tubes appear to penetrate the megagametophyte wall and come in contact with the peripheral band of cytoplasm. However, in all of the sampled ovules, sperm nuclei were never found discharged from pollen tubes and zygotes and embryos were never encountered. Thus, although pollen tubes deliver sperm, fertilization was never observed and the megagametophyte was assumed to be infertile.

Three separate and distinct developmental patterns were observed in pollinated "male" ovules (Fig. 6). The most common pattern was characterized by ovules that did not enlarge or develop to any significant extent (Type I ovules). Other ovules enlarged to a small extent, but never developed into seed-like structures (Type II ovules). Some of the ovules, usually ones located on the terminal whorl, ultimately enlarged and developed into seed-like structures (Type III ovules). Sectioned material revealed that megagametophytes were initiated in all three types of ovules, although they sometimes aborted in Type I ovules by the time pollination droplets were evident. All of the Type III ovules sampled contained three fully developed integuments. This contrasts with Type I and Type II ovules in which the second, or middle, integument is aborted early in ovule development. The relationship between the middle integument and the enlargement of the ovules is unclear, but the two are highly correlated. The middle integument is critical for seed development in functional female ovules since this is the layer that ultimately develops into the sclerotesta during seed maturation. These three developmental patterns were observed on plants that were self-pollinated as well as those that were cross-pollinated.

Different developmental phenomena were responsible for the enlargement of Type II and Type III ovules. Type II ovules were filled primarily with nucellar tissue that had proliferated from its prepollination state. The "seeds" (Type III ovules) were filled entirely with cellular, starch-filled, megagametophyte tissue. It is believed that the chalazal region of the megagametophyte becomes cellular and develops into large starch-filled tissue as this is what transpires in normal, functional female ovules. It is unclear what mechanism triggers the cellularization and proliferation of the megagametophyte in Type III ovules, but it is likely associated with the presence of pollen tubes in the nucellus or megagametophyte. Despite the cellularization and proliferation of the megagametophyte into typical embryo-nourishing tissue, no embryos were found in the seed-like structures. This was true of self-pollinated as well as cross-pollinated plants. Thus, self-incompatibility is not apparent in G. gnemon and did not have any bearing on the infertility of "male" ovules.

Gametophyte DNA/ploidy levels
We hypothesized that ovule sterility may be due, in part, to an inability of megasporocytes to pass through meiosis. Instead of developing into a functional, haploid megagametophyte, this phenomenon would yield sterile, unreduced (diploid) gametophytes. Densitometry of DAPI-stained sections was used to ascertain the relative DNA/ploidy level of nuclei found in the megagametophytes (Fig. 7).

View larger version (15K): [in this window] [in a new window]   Fig. 7. DNA/ploidy levels of gametophytes formed in bisexual cones of G. gnemon based on in situ densitometry of DAPI-stained nuclei. (A) DNA levels of nucellar nuclei during interphase of the cell cycle (N = 104). These sporophyte nuclei contain between 2C and 4C levels of DNA depending on their phase of the cell cycle (assuming they are diploid). The relative fluorescence of nucellar nuclei was used to calibrate DNA/ploidy levels. (B) DNA levels of generative cell nuclei in interphase (N = 25). These gametophyte nuclei, by definition, contain one-half as much DNA as sporophyte nuclei and ultimately give rise to two sperm within pollen tubes. Fluorescence of generative cell nuclei indicate that they contain the haploid, 1C level of DNA and was used as a control to verify the calibration scale obtained in Fig. 7A . (C) DNA levels of megagametophyte nuclei in interphase during coenocytic stages of development (N = 106). The megagametophyte nuclei contain diploid quantities of DNA

Nuclei in megagametophytes from "male" ovules displayed a comparable range of fluorescence values, and thus comparable amounts of DNA, as the nucellar nuclei (Fig. 7C). Relative fluorescence units of megagametophyte nuclei ranged from 12 RFU (2C DNA) to 28 RFU (4C DNA). This range of values is likely because some of the sampled nuclei were in G1 phase of the cell cycle while others were in S-phase or G2. A few nuclei displayed RFU levels slightly above the 4C level. The reason for this is not clear, although it may be a result of endomitosis or simply an artifact of sampling a large number of nuclei. These results indicate that megagametophyte nuclei contain the diploid quantity of DNA and are likely formed by mitotic (not meiotic) divisions of the megasporocyte nucleus. The megasporocyte nucleus appears to enter directly into several rounds of mitosis to form a coenocytic megagametophyte by the time ovules are receptive to pollination.

The formation of sterile, diploid megagametophytes is not unique to Gnetum, but has also been documented in ovules found on bisexual cones of Ephedra (Mehra, 1950 ). Polyploidy in megagametophytes has also been reported in Ginkgo biloba (Avanzi and Cionini, 1971 ; Cionini, 1971 ) and Ephedra nevadensis (Friedman, 1990 ). However, in those cases polyploidy occurs only in accessory gametophyte tissue (not in gamete nuclei) and is due to fusion of two or more haploid nuclei. The formation of unreduced gametophytes is common among angiosperms that display diplosporic apomixis (Koltunow, 1993 ). Unlike apomictic angiosperms, however, the megagametophytes formed in "male" ovules of Gnetum showed no signs of apomictic embryo development.

Bisexuality in Gnetales
All three gnetalean genera display some level of bisexuality, and gynodioecy is the most common. Most species of Gnetum that have been studied display a condition of bisexuality similar to that of G. gnemon (Maheshwari and Vasil, 1961 ). However, bisexual cones are not characteristic of the entire genus as ovules do not appear to form on male cones of G. cuspidatum (Kato, Inoue, and Nagamitsu, 1995 ) and G. buchholzianum (Pearson, 1929 ). They are also rarely present on male cones of G. africanum (Maheshwari and Vasil, 1961 ). Ovules of Ephedra, complete with pollination droplets, are typically formed on the terminus of male cones (Endress, 1996 ). Like Gnetum, however, these ovules are generally considered sterile and Ephedra is also classified as functionally dioecious. The strobili formed on bisexual plants of Welwitschia are themselves bisexual (bisporangiate) and produce a single ovule with a pollination droplet surrounded by six microsporangia. Nevertheless, they are sterile as the inner integument forms a flattened micropylar disk that prevents entry of pollen into the ovule (Endress, 1996 ; Hufford, 1996 ).

The role of bisexual cones with sterile ovules in the breeding system of Gnetales is not entirely clear. The sterile ovules produce pollination droplets that may play a role in entomophily. Indeed, the pollination droplets of Ephedra (Moussel, 1980 ), Welwitschia (Pearson, 1929 ), and Gnetum (van der Pijl, 1953 ; Kato and Inoue, 1994 ; Kato, Inoue, and Nagamitsu, 1995 ), are rich in sugars that may attract insects. Whether or not the sterile ovules themselves are necessary for entomophily has remained uncertain. For example, Ephedra campylopoda is reported to produce nectar directly from the perianth of male flowers and from the bracts surrounding the flowers (Bino, Devente, and Meeuse, 1984 ). Gnetum gnemon var. tenerum is reported to form nectar on the collar surrounding each microsporangial node (Kato, Inoue, and Nagamitsu, 1995 ). In addition, E. aphylla (Bino, Devente, and Meeuse, 1984 ) and G. cuspidatum (Kato, Inoue, and Nagamitsu, 1995 ) appear to lack ovules on male cones and yet are regularly visited by insects that may be attracted to the odor of male cones. These observations are based on intact cones and the actual source of attractant is not entirely clear. More detailed studies are needed on the development of bisexual cones in other members of Gnetales in order to ascertain the fundamental role of sterile ovules and bisexual cones in the breeding system of Gnetales.

	FOOTNOTES

 
¹ The authors thank S. Pyle and C. Hughes for valuable discussions throughout this project. Support for this work was provided by a grant-in-aid of research by North Dakota EPSCoR to J.S.C. and a Grant for Graduate Student Research from the Biology Department, University of North Dakota to C.J.H.

² Author for reprint requests (Tel.: 701-777-4666; jeffrey_carmichael{at}und.nodak.edu ).

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

 
Avanzi S. P. G. Cionini 1971 A DNA cytophotometric investigation on the development of the female gametophyte of Ginkgo biloba. Caryologia 24: 105-116

Bino R. J. N. Devente A. D. J. Meeuse 1984 Entomophily in the dioecious gymnosperm Ephedra aphylla Forsk. (=E. alte C.A. Mey.), with some notes on E. campylopoda C.A. Mey. II. Pollination droplets, nectaries, and nectarial secretion in Ephedra. Proceedings—Nederlandse Akademie van Wetenschappen. Series C: Biological and medical sciences 87: 15-24

Carmichael J. S. W. E. Friedman 1995 Double fertilization in Gnetum gnemon: the relationship between the cell cycle and sexual reproduction. Plant Cell 7: 1-14[CrossRef][ISI]

———, and ———. 1996 Double fertilization in Gnetum gnemon (Gnetaceae): its bearing on the evolution of sexual reproduction within the anthophyte clade. American Journal of Botany 83: 767-780[CrossRef][ISI]

Cionini P. G. 1971 A DNA cytophotometric study on cell nuclei of the archegonial jacket in the female gametophyte of Ginkgo biloba. Caryologia 24: 493-499[ISI]

Coulter J. M. l908 The embryo sac and embryo of Gnetum gnemon. Botanical Gazette 46: 43-49

Endress P. K. 1996 Structure and function of female and bisexual organ complexes in Gnetales. International Journal of Plant Sciences 157: S113-S125[CrossRef]

Friedman W. E. 1990 Sexual reproduction in Ephedra nevadensis (Ephedraceae): further evidence of double fertilization in a nonflowering seed plant. American Journal of Botany 77: 1582-1598[CrossRef][ISI]

———. 1998 The evolution of double fertilization and endosperm: an "historical" perspective. Sexual Plant Reproduction 11: 6-16[CrossRef][ISI]

Frohlich M. W. 1999 MADS about gnetales. Proceedings of the National Academy of Sciences, USA 96: 8811-8813[Free Full Text]

Hufford L. 1996 The morphology and evolution of male reproductive structures of Gnetales. International Journal of Plant Sciences 157: S95-S112[CrossRef]

Kato M. T. Inoue 1994 Origin of insect pollination. Nature 368: 195[CrossRef]

———, ———, and T. Nagamitsu 1995 Pollination biology of Gnetum (Gnetaceae) in a lowland mixed dipterocarp forest in Sarawak. American Journal of Botany 82: 862-868[CrossRef][ISI]

Koltunow A. M. 1993 Apomixis: embryo sacs and embryos formed without meiosis or fertilization in ovules. Plant Cell 5: 1425-1437[Free Full Text]

Lata M. 1960 Morphology and embryology of Gnetum gnemon L. Part 1. Ph.D. dissertation, University of Delhi, Delhi, India

Lotsy J. 1899 Contributions to the life history of the genus Gnetum. I. The grosser morphology of reproduction of Gnetum gnemon. Annales du Jardin botanique de Buitenzorg 16: 46-114

Maheshwari P. V. Vasil 1961 Gnetum. Council of Scientific & Industrial Research, New Delhi, India

Mehra P. N. 1950 Occurrence of hermaphrodite flowers and development of female gametophyte in Ephedra intermedia Shrenk et Mey. Annals of Botany 14: 165-180[Free Full Text]

Moussel B. 1980 Gouttelette receptrice du pollen et pollinisation chez l’Ephedra distachya L.: observations sur le vivant et en microscopies photonique et electronique. Revue de cytologie et de biologie vegetales—le botaniste 3: 65-89

Negi V. M. Lata 1957 Male gametophyte and megasporogenesis in Gnetum. Phytomorphology 7: 230-236

Pearson H. H. W. 1929 Gnetales. Cambridge University Press, London, UK

van der Pijl L. 1953 On the flower biology of some plants from Java with general remarks on fly-traps (species of annona, Artocarpus, Typhonium, Gnetum, Arisaema and Abroma). Annales Bogorienses 1: 77-99

Vasil V. 1959 Morphology and embryology of Gnetum ula Brongns. Phytomorphology 9: 167-215

This Article Abstract Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via ISI Web of Science (1) Citing Articles via Google Scholar Google Scholar Articles by Haycraft, C. J. Articles by Carmichael, J. S. Search for Related Content PubMed PubMed Citation Articles by Haycraft, C. J. Articles by Carmichael, J. S. Agricola Articles by Haycraft, C. J. Articles by Carmichael, J. S.