Novel sporophyte-like plants are regenerated from protoplasts fused between sporophytic and gametophytic protoplasts of Bryopsis plumosa
Protoplasts of the marine coenocytic macrophyte Bryopsis plumosa (Hudson) C. Agardh. [Caulerpales] can easily be obtained by cutting gametophytes or sporophytes with sharp scissors. When a protoplast isolated from a gametophyte was fused with a protoplast isolated from a sporophyte of this alga, it germinated and developed into either one of two completely different forms. One plant form, named Type G, appeared quite similar to a gametophyte, and the other, named Type S, looked similar to a sporophyte. While the Type G plant contained many small nuclei of gametophyte origin together with a single giant nucleus of sporophyte origin, the Type S plant contained many large nuclei of uniform size. These large nuclei in the Type S plant had metamorphosed from the gametophytic nuclei, and were not formed through division of the giant nucleus of sporophyte origin. Fragments of the Type S plant, each having such a large nucleus, developed into creeping filaments that look very similar to sporophytes. While cell walls of gametophytes and Type G plants were stained by Congo-red, those of the thalli of regenerated Type S plants and sporophytes were not stained by the dye. This indicated that the large nuclei of the Type S plant did not express genes for xylan synthesis, which are characteristic of gametophytes. Two-dimensional gel electrophoretic analysis revealed that most of the proteins synthesized in the Type S plant were identical to those of sporophytes. These results strongly suggest that in the Type S plant, the gametophytic nuclei are transformed into sporophyte-like nuclei by an unknown factor(s) produced by the giant nucleus of sporophyte origin and that the transformed nuclei express the set of genes characteristic of sporophytes. Despite morphological similarity, however, the regenerated Type S plant could not produce zoospores, because its large nuclei did not divide normally. The transformed large nuclei of gametophyte origin still seemed to be in the haploid state.
KeywordsBryopsis Cell fusion Gametophyte Nuclear transformation Protoplast Sporophyte
Differential interference contrast
Provasoli’s enriched seawater
The marine green alga Bryopsis plumosa (Hudson) C. Agardh. has a heteromorphic biphasic life cycle, i.e., a macroscopic Bryopsis phase (gametophyte) and a microscopic, creeping filamentous phase (sporophyte) (Tatewaki 1973). The body of the gametophyte is composed of a large cylindrical coenocytic thallus with beautiful pinnae. In laboratory conditions, however, such a pinnate gametophyte can only be obtained when it is cultured with gentle aeration (Yamagishi et al. 2003). The dioecious gametophyte of B. plumosa contains many small haploid nuclei. When it matures, the pinnae become either male or female gametangia, and a number of male or female gametes are generated within these gametangia. By contrast, a sporophyte is composed of a small, creeping, sparsely branched cell, which contains a single, large diploid nucleus. When maturation of the sporophyte is induced either spontaneously or experimentally, the giant nucleus undergoes meiosis, produces many small nuclei by the following mitosis, and finally forms a number of zoospores (Yamagishi et al. 2003).
Such great differences in morphology and physiology between gametophytes and sporophytes could be a reflection of different sets of genes being expressed during the two alternating generations. Analysis of such generation-dependent gene expression seems to be impractical with seed plants, because sporophytic generation occupies most of the life cycle of seed plants and their gametophytes are too greatly reduced. We demonstrate in this article that B. plumosa is a very suitable material for such an approach.
It is well known that when the protoplasm of the gametophyte of B. plumosa is squeezed out, protoplasts are easily formed, and these easily regenerate cloned gametophytes (Tatewaki and Nagata 1970; Kobayashi and Kanaizuka 1985). In the present study, we applied this phenomenon and the usefulness of Bryopsis to the study of nuclear reprogramming. We fused the two protoplasts isolated from a gametophyte and a sporophyte of B. plumosa and traced the morphology and fate of the nuclei of the regenerated plants. In this article, we report that some intracellular factors produced by a nucleus can regulate the gene expression of different co-existing nuclei in a fused protoplast. To our knowledge, this is the first attempt at cell fusion between different generations.
Materials and methods
Male and female gametophytes of Bryopsis plumosa (Hudson) C. Agardh. were collected from the habitat at Murohama, Miyagi, Japan in July 1998. A unialgal culture of gametophytes was achieved according to a previously described method (Yamagishi et al. 2003). Gametophytes were cultured in Provasoli’s enriched seawater medium (PES; Provasoli 1966) at 23°C under continuous white light until use. Since B. plumosa is dioecious, male and female gametophytes were cultured separately.
Sexual reproduction of gametophytes was induced by aeration, as previously described (Yamagishi et al. 2003). Liberated male and female gametes were collected and fertilized by mixing them in a Petri dish, which was filled with PES medium. Within several days, zygotes germinated and started to grow as young sporophytes. In suitable culture conditions, i.e., at 23°C under long-day (14 h light:10 h dark regime) exposure to white light at an intensity of about 6 W m−2, the sporophytes reached a length of about 10 mm 1 month after germination. One-month-old sporophytes were stored at 23°C under dim white light (1 W m−2, 14 h light:10 h dark regime) until use. Although sporophytes never mature in these conditions, we used sporophytes within 4 months of storage.
Preparation of protoplasts from gametophytes and sporophytes
We prepared gametophytic protoplasts by placing upright thalli of gametophytes in a Petri dish, which was filled with PES medium, and squeezing protoplasm out of the cells. These protoplasmic masses coagulated into spherical protoplasts within several minutes. The protoplasts were divided into two groups according to size, i.e., 148–98 and 62–40 µm in diameter, by filtering through nylon mesh (Kyoushin Riko, Tokyo).
A sporophytic protoplast was obtained by cutting a sporophyte near the single giant nucleus and squeezing out the protoplasm, so that the protoplast always contained one giant nucleus. The sporophytic protoplasts were trimmed with fine tweezers so that they were about 70 µm in diameter. These protoplasts can easily be fused with each other by bringing one into contact with the other (Fig. 1a–c). In the present study, the term fused protoplasts always indicates that the protoplasts were the result of fusion between one gametophytic protoplast and one sporophytic protoplast. Fused protoplasts thus produced were cultured in PES medium at 23°C under continuous white light (approx. 6 W m−2).
Staining of nuclei with SYBR Green I
Nuclei were visualized with SYBR Green I (Molecular Probes, Eugene, OR, USA) staining, as previously described (Yamagishi et al. 2003). Another well-documented stain 4′,6-diamidino-2-phenylindole (DAPI) did not stain the giant nucleus at all. Nuclei were observed with an epifluorescence microscope (Axioplan 2 or Axioskop; Zeiss).
Extraction of proteins and two-dimensional gel electrophoresis
Plant materials (gametophytes, sporophytes or germlings from fused protoplasts) of 500 mg fresh weight were homogenized in a mortar with 2 ml of extraction buffer [50 mM Mes, 5 mM EGTA, 10 mM MgSO4 (pH 6.8)] on ice. The homogenate was centrifuged at 10,000 g for 10 min. Soluble proteins were precipitated with 50% saturated (NH4)2SO4 by keeping them at 0°C for 6 h. The precipitates were collected by centrifugation at 12,000 g for 20 min, and the pellet was resolved in a sample buffer [8 M urea, 1% (v/v) Triton X-100, 2% (v/v) Ampholine (pH 3.5–10), 5% (v/v) 2-mercaptoethanol].
Protein samples were analyzed by two-dimensional electrophoresis according to O’Farrell (1975). Isoelectric focusing (IEF)–PAGE was used for first-dimension separation and SDS–PAGE was used for second-dimension separation. The IEF–PAGE gel was composed of 8 M urea, 4% (v/v) polyacrylamide, 2% (v/v) Triton X-100, 1.6% (v/v) Ampholine (pH 3.5–10) and 0.4% (v/v) Ampholine (pH 3.5–5). The IEF–PAGE gels were run stepwise at 50, 100, 150 and 300 V for 1, 4, 9 and 1 h, respectively. The SDS–PAGE gel was composed of a resolving gel [13% acrylamide, 25% 1.5 M Tris–HCl (pH 8.8)] and a stacking gel [0.4% acrylamide, 25% 0.5 M Tris–HCl (pH 6.8)]. The stacking gel was run at 10 mA for 30 min and the resolving gel was run at 20 mA for 1 h. The electrophoresis gels were then stained with silver stain (2D-SILVER STAIN II “DAIICHI”, Daiichi-Kagaku, Tokyo).
Fusion of protoplasts prepared from gametophytes and sporophytes
Development of germlings
On the other hand, fusion between large sporophytic protoplasts and small gametophytic protoplasts resulted in a higher yield of Type S plants (Fig. 4b). In this experiment, the size of sporophytic protoplasts was fixed at a diameter of about 70 µm, while that of gametophytic protoplasts was set at a diameter of 40–62 µm or 98–148 µm. The volume-ratios of the pairs of protoplasts, i.e., Vsporophytic protoplast/Vgametophytic protoplast, were about 2.6 or 0.18, respectively. As shown in Fig. 4b, when sporophytic protoplasts were 2.6 times larger than gametophytic protoplasts (small Gp), the relative yield of Type S plants increased to about 50%, irrespective of the gender of the gametophytic partner. It should be noted, however, that the total regeneration rate decreased when larger sporophytic protoplasts were used.
Another clear difference between Type G and Type S plants is their time course of germination. As Fig. 4c shows, Type G plants germinated within 2 days after fusion. This is almost the same as in case of non-fused gametophytic protoplasts. Type S plants, however, did not germinate sooner than 9 days.
Fate of nuclei in Type G and Type S plants
What are the large nuclei in the Type S plants? Are they enlarged small nuclei of gametophyte origin or are they generated from the giant nucleus of sporophyte origin by nuclear division? To answer this question, nuclei of the young Type S plant were investigated. As Fig. 6 clearly demonstrates, a 7-day-old, Type S plant contains only a single giant nucleus 20 µm in diameter and several intermediate nuclei, i.e., 8–9 µm in diameter, but no small nuclei. This strongly suggests that the small nuclei of gametophyte origin in the Type S plant expanded under the influence of the co-existing giant nucleus of sporophyte origin. Division of the single giant nucleus is ruled out, because the giant nucleus remains for quite a long period without becoming emaciated.
Enlarged nuclei of gametophyte origin now express sporophyte-specific genes
Comparison of the protein spots expressed in Type S plants, gametophytes and sporophytes of Bryopsis plumosa, analyzed by two-dimensional gel electrophoresis
Total No. of spots
No. of spots in common with gametophyte
No. of spots in common with sporophyte
Type S plants cannot produce zoospores
The proportion of Type S plants to total regenerated protoplasts is dependent on the volume ratio of the sporophytic protoplast to the gametophytic protoplast, but not on the gender of the gametophyte (Fig. 4). Since there must be a clear correlation between number of nuclei and the size of a gametophytic protoplast, there may be a critical number of gametophytic nuclei, lower than that necessary for the development of Type S plants.
During the development of the Type S plant, the small nuclei of gametophyte origin gradually expand. When it is young, e.g., at 7 days after its germination (Fig. 6), the Type S plant contains one large nucleus of sporophyte origin, which is thought to be diploid (2n), and many intermediate-sized (i.e., 8–9 µm in diameter) nuclei. When the Type S plant matures, there are no longer small (5–6 µm in diameter) or intermediate-sized nuclei. Since all nuclei in the mature Type S plant are large and they have a transparent perinuclear cytoplasm, one cannot discriminate, even through SYBR-Green I staining, which is the original giant nucleus introduced from a sporophyte (Fig. 5e). This means that during development of the Type S plant, small nuclei of gametophyte origin (i.e., haploid, n) gradually expand up to the size of the giant nucleus of the sporophyte. The large nuclei in mature Type S plants are not formed through fusion of the small nuclei, because the number of small nuclei in the young Type S plant and that of large nuclei in the mature Type S plant are almost the same (data not shown).
It is well known that the cell wall of the gametophyte of Bryopsis is mainly composed of β-1,3-xylan as the skeletal polysaccharide, while that of the sporophyte is mainly composed of β-1,4-mannan (Huizing et al. 1979). This is also the case in Derbesia (Huizing et al. 1979). As Congo-red and chlor-zinc-iodide stain only the cell wall of the gametophyte, the different stainability reflects a difference in cell wall composition. Congo-red does not stain cell walls of the sporophyte or filaments of the Type S plants. Since the Type S plant has many large nuclei introduced from a gametophyte and one giant nucleus of sporophyte origin, the question arises of which nucleus(-i) directs formation of the mannan cellwall. By using regenerated Type S plants that have only one transformed large nucleus, we were able to confirm that the enlarged nuclei of gametophyte origin stopped expressing the genes for xylan synthesis (Fig. 7f). The cell wall of the regenerated Type S plant is very probably composed of β-1,4-mannan. This strongly suggests that instead of a set of genes characteristic of gametophytic generation, a new set of genes for sporophytic generation is expressed by the enlarged gametophytic nuclei.
This was further confirmed by the protein analysis (Fig. 8). The pattern of proteins synthesized in the Type S plant corresponds fairly well with that in the sporophyte. It should be noted, however, that regenerated Type S plants were not used in the protein analysis of Type S plants because obtaining a sufficient number of regenerated Type S plants was difficult. Nevertheless, this probably does not affect the results.
Our present results are explained in terms of nuclear reprogramming. Regeneration and development of the Type S plant from the protoplast fused between the gametophyte and sporophyte probably result from the activation of genes specific to sporophyte generation and simultaneous silencing of some other genes specific to gametophyte generation through the influence of cytoplasmic factors produced by the co-existing giant sporophytic nucleus. Also, in the Type G plant, gene expression of many gametophytic nuclei may supersede that of the sporophytic giant nucleus, because we found that the larger the gametophytic protoplast, the more Type G plants were regenerated (Fig. 4).
Division of the giant nucleus (2n) is the first noticeable sign of the sporophyte’s maturation in Bryopsis plumosa (Yamagishi et al. 2003). Meiosis of the giant nucleus and subsequent repetitive mitoses of the secondary small nuclei (n) are followed by development of numerous stephanokontic zoospores (Rietma 1971). That the division of the giant nucleus constitutes meiosis was recently confirmed electron microscopically by the detection of a synaptonemal complex (Minamikawa et al. 2002). Meiosis of the large nuclei of the Type S plant was not detected in the present study. Since abnormal nuclear division should be the result of unequal chromosome segregation during meiosis, the many large nuclei found in a Type S plant, except one true sporophytic nucleus, are considered to be in the haploid (n) stage. Normal meiosis is not accomplished in haploid Ulva (Hoxmark and Nordby 1974) and in many other haploid plants produced by anther culture, such as in wheat (Person 1955), oat (Nishiyama and Tabata 1964), pearl millet (Manga and Pantulu 1971) and barley (Sadasivaiah and Kasha 1971). Since chromosomes cannot be paired at pachytene, they are arbitrarily distributed to daughter cell, and hence there is no further growth.
We are grateful to Prof. Tomonobu Kusano of Tohoku University for his valuable discussion. Thanks are also due to the late Dr. Eiji Kamitsubo for the use of his personal Axioskop.
- Huizing HJ, Rietema H, Sietsma JH (1979) Cell wall constituents of several siphonaceous green algae in relation to morphology and taxonomy. Br Phycol J 14:25–32Google Scholar
- Manga V, Pantulu JV (1971) The meiotic behaviour of a haploid pearl millet. Genetica 42:319–328Google Scholar
- Minamikawa B, Yamagishi T, Hishinuma T, Ogawa S (2002) Division of giant primary nucleus in the coenocytic green alga Bryopsis. J Plant Res [Suppl] 115:329Google Scholar
- Nishiyama I, Tabata M (1964) Cytogenetic studies in Avena, XII. Meiotic chromosome behavior in a haploid cultivated oat. Jpn J Genet 38:311–316Google Scholar
- Person C (1955) An analytical study of chromosome behaviour in a wheat haploid. Can J Bot 33:11–30Google Scholar
- Provasoli L (1966) Media and prospects for the cultivation of marine algae. In: Watanabe A, Hattori A (eds) Cultures and collections of algae. Proceedings of the US–Japan conference held at Hakone, 12–15 Sept 1966. Jpn Soc Plant Physiol, pp 63–75Google Scholar
- Rietema H (1971) Life histories in Bryopsis hypnoides Lamx. from different points along the European coast. Acta Bot Neerl 20:291–298Google Scholar
- Sadasivaiah RS, Kasha KJ (1971) Meiosis in haploid barley—an interpretation of non-homologous chromosome associations. Chromosoma 35:247–263Google Scholar
- Tatewaki M (1973) Life history of Bryopsis plumosa (Huds.) and B. maxima Okam. (in Japanese). Bull Jpn Soc Phycol 21:125–129Google Scholar
- Tatewaki M, Nagata K (1970) Surviving protoplasts in vitro and their development in Bryopsis. J Phycol 6:401–403Google Scholar