Cytological Features of Gametophyte Development and Zygotic Formation in Senecio scandens Buch.-Ham. ex D.Don(Compositae)

XIE Xin QIAN Qiu-Bo-Yan WANG Lei WANG Qian-Xing PING Jun-Jiao HE Li-Fang QIAN Gang*

(1.Department of Cell Biology,Zunyi Medical University,Guizhou 563003； 2.Department of Fundamental Nursing,Qiannan Medical College for Nationalities,Duyun 558003； 3.Undergraduate of 2016,Department of Fundamental Nursing,Qiannan Medical College for Nationalities,Duyun 558003； 4.Third People Hospital of Zhongshan City,Zhongshan 528400)

Abstract Senecio scandens Buch.-Ham. ex D.Don(Compositae) is a crucial plant source of Chinese traditional medicine with antibacterial properties. To obtain basic knowledge for an insight into its cytology mechanism and further practical breeding application in this species, we focus on characterizing progress on gametophyte developments and zygote formation in this paper. Based on a chronological description of the male gametic development, two dimorphic sperm cells release from their separate position in the male germ unit. Moreover, mature embryo sac exhibits an ovoid to pyriform shape locating at wider micropylar extreme, by following the monosporic pattern on development of embryo sac in the current species. It is therefore reasonable to deduce that the origin of apico-basal polarity of zygotic embryo may be related to an unevenly spaced distribution of cytoplasm in zygotic embryo which results from fertilization between the dimorphic sperm cells and the polar ovule. As the distinguishable characteristics, these morphological features will be usefulness for the taxonomy studies in other Compositae species, including dimorphic sperm cells, polar embryo sac and zygotic polarity.

Key words Senecio scandens;gametophyte;double fertilization;zygote

In flowering plants, sexual reproduction occurs by a process of double fertilization in which one sperm fuses with an egg cell to form a zygote and a second sperm nucleus fuses with two or more polar nuclei to produce endosperm, a polyploidy embryo-nourishing tissue[1-2]. Embryological studies are fullness for further understanding of interaction factors in order to update the mechanism of taxonomy, in vitro fertilization and production of haploid plants, associated with the processes of embryo development[3]. In regard to this aspect, the male/female gametic formations play an important role in the crucial stage of sexual reproduction process such as the direction of pollen tube invasion and growth towards the egg cell[4], the transport of sperm nuclei through the central and egg cells during the fertilized process in the embryo sac[5]. Indeed, the double fertilization is unique to flowering plants among living organisms and permits the establishment of a new generation, from the zygote to the embryo included within the seed[6]. Therefore, clear description of these steps of gametophytic development and fusion, and especially the syngamy step, is largely useful for understanding both at the cytological and molecular levels[7].

SenecioscandensBuch.-Ham. ex D. Don, a crucial antibacterial source from Chinese traditional medicinal materials, belongs to the member of the Compositae family[8]. Owing to its important economical plant in Chinese traditional medicine, this species is commercially cultivated by asexual methods in order to reduce its genetic variability, thus maintaining growth superiority, antibacterial virtue and medicinal production, as well as increasing its vulnerability to environmental stress. The focused study of sexual reproduction is warranted to examine the structure of the gametes, document the fertilization process, and determine whether the double fertilization events do practically occur in the current species. Thus, studies of early embryo pattern formation may elucidate mechanisms through which plant cells are able to generate functional body patterns in the absence of pre-structuring multi cellular templates[9]. However, while basic aspects of gametophytic development are known in some species, female gametophyte structure and zygotic embryo development remains unclear in some seed plant species differ in many substantial ways[10-11]. Therefore, clear description of these steps of gametophytic development and fusion, and especially the syngamy step, is largely useful for understanding both at the cytological and molecular levels[7]. We executed observations on the developmental biology of the gametophytes and the early embryo formation to generate cytological knowledge for its usefulness of taxonomy and breeding application in the Compositae species.

Whereas, the information about male/female gametophytic formation, double fertilization and zygotic embryo development is limited in the sub-family Compositae, this investigation is aimed at characterizing key stage of the reproductive process and document changes in gametophytes, embryo sac, zygotic embryo and endosperm development inS.scandensBuch.-Ham. ex D. Don. The observations may be usefulness for in depth illumination on cytological mechanism by providing the morphological features of embryogenesis for unequivocal evidence of double fertilization process, zygote formation and early embryo development in the other Compositae species.

1 Materials and Methods

1.1 Experimental materials

In our previous study, we selected the differential antibacterial phenotypic individuals(parental lines) and their generation hybrids to develop molecular makers[8], using a series of standardization bacteria(S.aureus,P.aeruginosa,E.coli,S.paratyphi,S.flexneri,A.sobriaandE.tarda) as described by Shapiro and Baneyx[12]. Here, the parental lines that are utilized to conduct hand pollination in this work consisted of the elite individual(P1) with superior antibacterial characteristics and the general individual(P2) with poor quality antibacterial traits ofS.scandensBuch.-Ham. ex D. Don.

1.2 Experimental pollination

The plant flowers once per year with its anthesis occurring from November to December. As the perfectly self-incompatible flowering species, at the time of anthesis the whole inflorescences with flower buds are bagged with glassine paper to prevent uncontrolled pollination. Hand pollination of female accession(P1) and male accession(P2) is completed by brushing pollen directly onto the elongated stigmas of receptive female flowers on the subsequent days. In order to confirm various developmental stages of the male/female gametophytes, unpollinated pistils of different sizes from the inflorescences of plants are randomly harvested and fixed. Depending on the time of sample collection, and as a consequence of the acropetal pattern of pollination receptivity and ovule maturation, ovules could be found at various developmental stages after hand pollination. The unripe samples with different time after hand pollination are harvested to characterize the processes from fertilization to zygotic embryo development. The specimens with stigma and ovule are thus harvested and immediately fixed after hand pollination.

1.3 Chemical fixation

Hand-pollinated ovules and immature seeds are excised from the ovaries. Integuments as well as extraneous nucellar tissue surrounding the female gametophyte are carefully removed from each ovule before chemical fixation. The trimmed specimens are overnight expressed to the laboratory for immediate dissection and chemical fixation in FAA(10:5:50:35 formaldehyde: acetic acid: ethanol: distilled water) for 24 h at room temperature. After fixation, the sample is transferred to a 70% ethanol solution and stored at 4℃ for the following stain.

1.4 Mayer’s hemalum-methyl salicylate stain-clearing

The fixed samples are stained with Mayer’s hematoxyl in solution for 3 h at room temperature before they are treated with heat for 30 min in a water bath at 40℃. Later, the specimens are suffered from 2% acetic acid for 40 min at 40℃ and then treated with 0.5% acetic acid for 12 h. Next, the samples are washed with 0.1% sodiumbicarbonate until the solution is clear. Finally, the ovules are then rinsed three times in PIPES buffer, dehydrated through an ethanol series(10%, 20%, 30%, 50%, 75% for 30 min per step), and last 95% ethanol for 2 h. The clarification of the tissue of is completed, using a series of methyl salicylate(ethanol solutions of 3∶1, 1∶1, 1∶3) for 1 h each, at 4℃[13].

1.5 High-resolution light microscopy

The specimens are infiltrated for 3 weeks to ensure the complete displacement of ethanol with glycol methacrylate. Ovules were then embedded, and the embedding medium was polymerized in an oxygen-free environment by flushing nitrogen gas through a closed chamber. Embedded specimens are serially sectioned according to the methods of O’Brien and McCulley[14]. Each embedded ovule was serially sectioned on a Leica RM 2255 rotary microtome(Leica, Wetzlar, Germany) at thicknesses of 5 μm with a glass knife made from a microscope slide. And then, the ovules were mounted with 100% Leica immersion oil type F for microscopic illumination. Female gametophyte analysis was completed on a Leica TCS SPE RGBV confocal microscope with a 532 nm laser excitation and a detection window between 555 and 700 nm. Images were captured and treated with the LAS X® software(Leica Microsystems) with either 512×512 or 1 024×1 024 pixels. Image manipulations are restricted to operations that are applied to the entire image, except where are specifically noted in figure captions[15].

2 Results

2.1 Pollen tub germination

When pollen grain gets away from stamen, fluorescent protein tagged on the pollen exine presents the regular distributions inS.scandensBuch.-Ham. ex D. Don(Fig.1a). Suffered from dehydration, the shriveled pollen grain serves as unclear functional unit of its surface before it reaches to stigma(Fig.1b). At the initial time of pollination droplet adhesion to stigma, the stigma serves as a source of water and other factors required for this crucial hydration step. Thus, the pollen grain swells again and a spherical protrusion forms soon after the typical pollen germination occurs at the surface of stigmatic papilla cells(Fig.1c). The clear outline of pollen grain still maintains at initial process of the pollen tube invading into stigma(Fig.1d). However, entire structural pollen grain can no longer be clearly discerned during the pollen tube invades the stigma and grows in the intercellular space between papilla cells towards the transmitting tract(Fig.1e).

Fig.1 Pollen tub germination a.Mature pollen grain released from stamen; b.Dehydrated pollen grain; c.Germinated pollen grain at the surface of stigmatic papilla cells(red arrow); d.The profile of pollen grain(Astarte) in pollen tub(arrow); e.Successive extension of pollen tube(arrow) with disappeared pollen grain(Astarte)

Fig.2 Spermatogenesis a.Microspore at late bicellular stage:the vegetative cell nucleus(red arrow) and the generative cell nucleus(green arrow); b.The unit of binucleate sperm nuclei(red arrows) in pollen tube; c.Binucleate sperm cells after Pollen Mitosis Ⅱ(green arrows); d.Sperm cells(green arrows) and vegetative nucleus(red arrow) travel as male germ unit

Fig.3 Megasporogenesis a.Megaspore mother cell with the condensed nucleus(arrow); b.Megaspore mother cell locating at the micropylar extreme of the ovule(arrow); c.Diad resulting from MMC meiosis Ⅰ(arrows); d.“T” arrangement of a tetrad after meiosis Ⅱ; e.Linear arrangement of tetrad; f.The monosporic pattern of functional megaspore from the normal development of the tetrad(arrows); g.Triad(arrows)

Fig.4 Female gametophyte development a.Female gametophyte containing four meiotically derived megaspore nuclei(arrows); b.Functional megaspore with a monosporic pattern(arrows); c.Female gametophyte nuclei with haploid; d.Female gametophyte with coenocytic

Fig.5 Zygote formation a.Micropylar region including a large central vacuole; b.The mature embryo sacs with an egg cell(asterisk),three antipodal cells(red arrows) and two polar nuclei(green arrows); c.Unfertilized egg cell while the entry of two dimorphic sperm cells(green arrows); d.The zygote nucleus relocating to its position at the micropylar end(arrow)

2.2 Spermatogenesis

Unicellular microspores are released from tetrads to undergo asymmetric cell divisions known as Pollen Mitosis Ⅰ(PM Ⅰ). The small generative cell becomes covered in the cytoplasm of the large vegetative cell forming a cell-within-a-cell structure(Fig.2a). As in majority of flowing plants, mitosis without cytokinesis of the spermatogenous cell gives rise to the unit of binucleate sperm nuclei(SN1/2) within each pollen tube(Fig.2b). At the late bicellular stage, it is apparent that the binucleate sperm cells emerge after Pollen Mitosis Ⅱ(PMⅡ) of the sperm nucleus at both poles involved in separation of the twin spermatogenous cells(Fig.2c). At early tricellular stage, twin sperm cells(SC1/2) and vegetative nucleus travel as male sperm unit with common arrangement in parallel(Fig.2d).

2.3 Megasporogenesis

The megaspore mother cell(MMC) originates from the differentiation of an arquesporial cell inS.scandensBuch.-Ham. ex D. Don, having a larger size of nucleus than the surrounding cells(Fig.3a). The larger size of MMC appears and transfers toward the micropylar extreme of the ovule at the early stage of megasporogenesis, Due to its large, dense and well defined nucleus, the MMC is not difficult to be distinguished from the other cells which locates at the micropylar end of the ovule(Fig.3b). The diploid MMC formation is performed by meiosis generating in meiosis Ⅰ a dyad of haploid cells of similar size before the integuments start to be differentiated(Fig.3c). Subsequently, meiosis Ⅱ takes place and results in a tetrad of cells commonly arranged in a “T” shaped manner, where the two micropylar megaspores are observed one beside the other or in an intermediate type(Fig.3d). Locating near to the micropyle extreme, the two megaspores are separated by an oblique division instead of a fully cross division at this stage. As a common patter of the tetrad formation, the linear arrangement parallel is also observed in this investigation(Fig.3e). The female gametophyte of the current sample is tetrasporic in origin(Fig.3f). In addition, the formation of triads instead of tetrads appears in our observations, which is interpreted by its presence resulting from the possible non division of the megaspores of the dyad to enter meiosis Ⅱ(Fig.3g).

2.4 Female gametophyte development

At the initial stage of megagametogenesis, each female gametophyte is a single, coenocytic cell containing four meiotically derived megaspore nuclei(Fig.4a). While three megaspores of the closest to the micropylar extreme degenerate, the chalazal cell remains intact and becomes the functional megaspore(FM) with a monosporic pattern. At this point, one nucleus migrates to the chalazal region, while the other three nuclei remain at the central of embryo sac(Fig.4b). The chalazal portion of the female gametophyte is densely cytoplasmic, with free nuclei positioned roughly equidistant from each other. Throughout early stages of development, female gametophytic nuclei remain haploid and exhibit synchronous cell cycles(Fig.4c). Up to this stage of female development, the embryo sac generates an ovoid to pyriform shape with narrower chalazal extreme and wider micropylar end. Next, the first mitotic division of the FM undergoes results in forming a binucleate sac with the newly formed nuclei shifting towards the chalazal extreme(Fig.4d).

2.5 Zygote Formation

During the following stages of female gametophyte development, the second mitotic division generates a sac with four nuclei. The micropylar region includes a large central vacuole, which occupies the majority of the cell, and a parietal band of free nuclear cytoplasm(Fig.5a). After the nuclei undergo successive mitotic divisions, the third mitotic division results in forming an embryo sac with eight nuclei. The ovary displays mature embryo sacs with an egg cell composing a hooked egg apparatus at the micropylar end, two polar nuclei, and three antipodals at the chalazal end. The entire female gametophyte remains coenocytic through the time of fertilization, and it is within the micropylar region that fertilization eventually takes place(Fig.5b). The embryo develops from a fertilized egg cell, positioned within the embryo sac, which is itself embedded in the protective maternal tissue of the ovule until the entry of two dimorphic sperm cells. The entire female gametophyte remains coenocytic through the time of fertilization, and it is within the micropylar region that fertilization eventually takes place(Fig.5c). To complete fertilization, the pollen tube enters the ovule through the micropyle and delivers two haploid nuclei, one of which fuses with the nucleus of the egg cell to form a zygote, while another combines with the central cell. The zygotic nucleus is found to relocate its position at the micropylar region where the egg cell previously occupied at the stage of mature embryo sac(Fig.5d).

3 Discussions

Fertilization generally describes the fusion of haploid gametes to initiate the development of a new diploid organism[16]. Understanding the relationship between gametic behavior and sexual reproduction will accelerate a clearer interpretation of the unique process of double fertilization inS.scandensBuch.-Ham. ex D. Don. However, other than the current investigation, the limited reports of seed plants have documented the advantages of using ovules as an experimental tool to characterize the phases of fertilization progress including male/female gametophytic developments and zygote formation in the Compositae species. Based on these observations on a progress of double fertilization, we can thus capture a series of recordings of the gametic phase, allowing us to further elucidate the sequential events related to spermatogenesis(Fig.2), megasporogenesis(Fig.3), megagametogenesis(Fig.4) and zygote formation(Fig.5) in the current seed plant.

It is generally known that the occurrence of male gamete transport and delivery system, the pollen tube, which is considered as key innovation in the evolutionary success of the flowering plants[17]. Of the entire fertilization process in flowering plants, however, little information is available concerning the events in the gametic stage of double fertilization from pollen tube discharge to the initiation of the fusion between sperm nuclei and female gamete nuclei[18-19]. Here, morphological data recover the major stage of male development and spermatogenesis, similar to those described by Dresselhaus and Franklin-Tong[20], where pollen adheres(Fig.1a), hydrates(Fig.1b), and germinates(Fig.1c) on the stigma, pollen tube invades and travels into the stigma, pollen tube negotiates various layers of ovary tissues, and is guided to the micropyle end of the embryo sac(Fig.4).

Although the two sperm cells are isomorphic in most flowering plants, two dimorphic sperm cells from exceptionally asymmetrical generative cell division in some plant species[19,21]. From notably morphological differentiation in the two sperm cells(Fig.2:c-d), our results of spermatogenesis support the idea that the two functionally diverse sperm cells show a preference with regard to fertilization objectives based on their separate position in the male germ unit to fertilize predetermined targets(Fig.5c). As parallel with our results, Kliwer and Dresselhaus[17]also report that the pollen isoforms of maize(Zeamays) might possess different functions and/or perform physicochemical properties, respectively. Indeed, numerous experimental data testify the occurrence of preferential fertilization as a general appearance[22,6].

The normal development of embryo sac(Fig. 4) follows a monosporic pattern in the present species, in which the entire female gametophyte remains coenocytic through the time of fertilization and the fertilized event eventually occurs within the micropylar region. Once the double fertilization is completed, the relative functional genes expressed in the maternal tissue are initiated in every stage of the embryo and endosperm development[23,3]. As the initial investigation, the structural traits of this study will provide the morphological basis for the further screening of the relative functional genes in the Compositae species. It therefore seems reasonable to suggest that the ovule polarity(Fig.5a) impinges on the polar orientation of the embryo sac, the egg cell and thereby the zygotic embryo(Fig.5d). As a good confirmation with these findings, the polarity documented by the zygote of the current article is similar to the report on embryological illumination performed in model plants such asCapsellabursa-pastoris[24],Nicotianatabacum[25]andArabidopsisthaliana[26-27]. Confirming our results, Devic[28]provides similar evidence that the observation argues in favor of an influence of the fertilization process itself on the polarity of the zygote, based on the nucleus and most of the cytoplasm are located at the micropylar half of the egg cell prior to fertilization.