Angiosperms have a unique sexual reproduction system called “double fertilization.” One sperm cell fertilizes the egg and another sperm cell fertilizes the central cell. To date, plant gamete membrane dynamics during fertilization has been poorly understood. To analyze this unrevealed gamete subcellular behavior, live cell imaging analyses of Arabidopsis double fertilization were performed. We produced female gamete membrane marker lines in which fluorescent proteins conjugated with PIP2a finely visualized egg cell and central cell surfaces. Using those lines together with a sperm cell membrane marker line expressing GCS1-GFP, the double fertilization process was observed. As a result, after gamete fusion, putative sperm plasma membrane GFP signals were occasionally detected on the egg cell surface adjacent to the central cell. In addition, time-lapse imaging revealed that GCS1-GFP signals entered both the egg cell and the central cell in parallel with the sperm cell movement toward the female gametes during double fertilization. These findings suggested that the gamete fusion process based on membrane dynamics was composed of (1) plasma membrane fusion on male and female gamete surfaces, (2) entry of sperm internal membrane components into the female gametes, and (3) plasmogamy.
KeywordsArabidopsis Double fertilization GCS1 Membrane dynamics Plasma membrane intrinsic protein 2A (PIP2a)
Angiosperms possess a unique sexual reproduction system called “double fertilization.” Upon pollination, a pollen grain germinates and its pollen tube elongates through the pistil to deliver two immotile sperm cells to an embryo sac that contains two female gametes (the egg cell and the central cell). Once the pollen tube reaches one of the synergid cells in the embryo sac, a pair of sperm cells is discharged from the pollen tube (Sandaklie-Nikolova et al. 2007) to come face to face with the female gametes. One of the sperm cells fertilizes the egg cell to give rise to a zygote that develops into an embryo, whereas the other sperm cell fertilizes the central cell to produce a triploid endosperm that serves as the nurse tissue for embryo development. Thus, the two parallel fertilization events should be controlled precisely for normal seed formation.
To understand in detail the sexual reproduction process in angiosperms, histological observations of non-living cells with light and electron microscopes have been performed and both plasmogamy and karyogamy have been speculated in double fertilization for a long time (Faure 2001; Faure and Dumas 2001). Plant cell observation techniques have shown remarkable improvement with the emergence of fluorescent proteins and high-performance fluorescence microscopes. Arabidopsis fluorescent marker lines that visualize specifically the subcellular components of gametes and gametophytic cells with a fluorescent protein have enabled the observation of fertilization in living cells (Berger 2011). Furthermore, a semi in vitro pollen tube guidance assay system was established in Arabidopsis (Palanivelu and Preuss 2006; Sandaklie-Nikolova et al. 2007). This assay system has enabled the time-lapse imaging of double fertilization with labeled gametes (Hamamura et al. 2011; Ingouff et al. 2007; Kasahara et al. 2012; Matsushima et al. 2008). Hitherto reported studies of gamete dynamics during double fertilization have focused on gamete cytosol, nuclei or mitochondria (Hamamura et al. 2011; Ingouff et al. 2007; Matsushima et al. 2008).
There are few studies of gamete plasma membrane behavior during gamete fusion in plant (Russell 1980, 1983, 1992) even though the cell surface structure is critical for male and female gamete interactions in order to regulate double fertilization. As syngamy is completed within a short time, it is difficult to observe the behavior of such structures at fertilization. As far as we know, no one has tried to analyze membrane dynamics during gamete fusion in living plant tissues. In this study, Arabidopsis female gamete membrane marker lines were produced first to gain an insight into the cell membrane structures during double fertilization. By using these marker lines in addition to the sperm cell membrane marker line expressing GCS1-GFP (Mori et al. 2010) as the observation tools, sperm membrane behavior in the gamete fusion process was analyzed for the first time.
Materials and methods
Surface-sterilized Arabidopsis thaliana (ecotype Columbia 0) seeds were germinated on agarose plates and the seedlings were grown for 2–3 weeks followed by transfer to soil for acclimatization. Plants were grown at 22 °C under a 16/8 h light/dark cycle.
Plasmid construction and production of Arabidopsis transgenic plants
The synthesized oligonucleotide primers used for plasmid construction are listed in Table S1. DD45 promoter (ca. 1 kb of the 5′ untranslated region of At2g21740) (Steffen et al. 2007) and FWA promoter (ca. 2 kb of the 5′ untranslated region of At4g25530) (Kinoshita et al. 2004) were amplified by genomic PCR to add a SalI site to the 5′ terminus and a BamHI site to the 3′ terminus, respectively. A BamHI site and a KpnI site were, respectively, added to the 5′ and 3′ termini of sGFP and TagRFP by PCR. The open reading frame of PIP2a (At3g53420) was amplified by RT-PCR to add a KpnI site to the 5′ terminus and a XhoI site to the 3′ terminus. Each PCR product was, respectively, cloned into the pCR®-Blunt II-TOPO® vector (Life Technologies Japan, Ltd., Tokyo, Japan) and sequenced. Each of the DNA fragments cloned into the pCR®-Blunt II-TOPO® was excised with suitable restriction enzymes and then ligated to pENTR™3C (Life Technologies Japan, Ltd.) to construct “Promoter::fluorescent protein-PIP2a” expression cassettes. Each of the expression cassettes was transferred to the destination vector pGWB1 (Nakagawa et al. 2007) with the LR reaction (Gateway; Life Technologies Japan, Ltd.). Each of the produced constructs was introduced into wild type Arabidopsis plants. The resulting transformants were selected in kanamycin-containing media.
The sperm nucleus GFP marker line of +/gcs1 background was produced as follows. A genomic HTR10 sequence covering the putative promoter region (ca. 1.2 kb of the 5′ untranslated region of At1g19890) (Ingouff et al. 2007) was amplified by genomic PCR and cloned into the upstream site of sGFP cDNA conjugated with NOS terminator in the pPZP221 binary vector construct, produced in a previous study (Hirooka et al. 2009). The pHTR10::HTR10-GFP construct was introduced into +/gcs1 Arabidopsis plants. The resulting transformants were selected in kanamycin- and gentamycin-containing media.
The GPP-expressing line was derived from a previous study (Mori et al. 2010). The GPP line was crossed with the sperm nucleus RFP marker line (pHTR10::HTR10-mRFP) line (a gift from Dr. F. Berger) to obtain the double marker line expressing both sperm nucleus RFP and membrane GFP.
Microscopy and image analysis
A confocal laser scanning microscope (CLSM) (FV1000-D; Olympus Corp., Tokyo, Japan) and a two-photon laser scanning microscope (TPLSM) (FV1000-MPE; Olympus Corp.) were used for the acquisition of GFP/RFP and GFP images, respectively. An IR pulse laser that was attached to FV1000-MPE was set to 930 nm for excitation. FluoView software (Olympus Corp.) was used for image acquisition. Differential interference contrast and fluorescence images were captured with an epifluorescence microscope (BX51; Olympus Corp.) using an Olympus DP-72 digital camera and accompanying software (Olympus Corp.). Images were processed with Adobe Photoshop CS5 (Adobe Systems Inc., San Jose, CA, USA) or ImageJ software (http://rsbweb.nih.gov/ij/). Image stacking and production of movie files were performed with ImageJ software.
Observation of fluorescent markers during double fertilization
To control the timing of the in vivo double fertilization, mature flower buds of female marker lines were emasculated the day before the pollination. The emasculated female marker lines were hand-pollinated with male marker lines. For observation, the ovules were dissected after pollination for 7–8 h because double fertilization was frequently observed at this stage (Faure et al. 2002). When +/gcs1 male marker line was used for pollination, the ovules were dissected after pollination for more than 10 h.
For the time-lapse imaging of the double fertilization, flower buds of the female marker line expressing pDD45::TagRFP-PIP2a were emasculated 1 day before the experiment. The male marker line expressing GCS1-GFP (GPP) (Mori et al. 2010) was used for pollination to the emasculated female marker line. In vitro fertilization assay was performed as reported by Hamamura et al. (2011) with modification of the culture medium. The culture medium reported by Boavida and McCormick (2007) was used in this study. After the pollen tube reached the micropyle, GFP and RFP images were acquired at 5 min intervals by CLSM.
Fluorescent marker lines visualizing female gamete plasma membrane
Arabidopsis aquaporin (plasma membrane intrinsic protein 2A; PIP2a), a water channel protein, has been reported to uniformly label the plasma membrane along the surface of the cell (Cutler et al. 2000). To visualize Arabidopsis egg and central cell plasma membranes, GFP-PIP2a and RFP-PIP2a driven by promoters DD45 (Steffen et al. 2007) and FWA (Kinoshita et al. 2004), which allow egg-cell- and central-cell-specific expression, respectively, were constructed. All the Arabidopsis plants transformed with those constructs were fertile (data not shown).
Relative locations of male and female gametes before fertilization
Live cell imaging of sperm cell membrane during double fertilization
After being discharged from a pollen tube, the sperm nuclei remained at the boundary of the female gametes for a while and then resumed movement toward the egg cell or the central cell (Hamamura et al. 2011). The timing of resumption of sperm nucleus movement was considered to be a sign of plasmogamy between male and female gametes in Arabidopsis (Hamamura et al. 2011).
Arabidopsis PIP2a clearly visualized female gamete surfaces
The observation of the gamete fusion process in angiosperms has met with difficulty as the fertilization event occurs in an embryo sac enclosed with thick integumentary tissues. To date, ultrastructural observations of gamete membrane morphology during double fertilization have been conducted in only a few plant species (Russell 1980, 1983, 1992) and no investigations of living cells have been reported so far.
In order to enable the live cell imaging of gamete membrane structures during double fertilization, Arabidopsis gametic marker lines, where PIP2a conjugated with a fluorescent protein was expressed specifically in the female gametes, were produced (Fig. 1). The marker lines clearly showed the shape of the female gametes. In addition, fluorescent signals were detected in the cytoplasm, similar to tobacco protoplast expressing PIP2a-mCherry (Hildreth et al. 2011). Those signals probably reflected the membrane components of cytoplasmic vesicles. The characteristic egg cell figure projected in this study (Movie S1) was similar to the three-dimensional figure based on sequential ultrathin sections (Wang et al. 2010).
In this study, AtPIP2a driven by a sperm cell specific HTR10 promoter (Ingouff et al. 2007) did not clearly visualize the sperm cell membrane because of the low signal intensity for observation (data not shown). It could be due to the level of promoter activity or the limited amount of AtPIP2a to localize in the plasma membrane of tiny sperm cells. We therefore utilized a GCS1-GFP marker line (GPP line) for sperm cell membrane observation in further live cell imaging experiments, because GCS1 protein was proven to be localized in the sperm cell membrane components and GPP was found to be functional, in previous studies (Mori et al. 2006, 2010; von Besser et al. 2006).
Gamete morphology before gamete fusion
The surface morphology of the female gamete during interaction with the sperm cells before and after fertilization was observed in detail with TPLSM (Fig. 2). The flattened shape of gcs1 sperm nuclei was rarely observed (Fig. 2b), and it could be an indication that the two sperm cells were appressed to the female gametes, similar to the case of Plumbago zeylanica (Russell 1980, 1983, 1992). In addition, no significant changes were observed in the egg cell facing the gcs1 sperm cells. In a previous study of Nun orchid, female gametes were found to extend toward sperm cells at the gamete fusion step (Ye et al. 2002). In Chlamydomonas fertilization, male and female gametes formed membrane protrusions that enabled them to attach to each other before gamete fusion (Liu et al. 2008). Fusion was arrested in Chlamydomonas gcs1 male and wild type female gametes, although membrane protrusion mediated attachment occurred (Liu et al. 2008, 2010). In the present study, Arabidopsis gametes did not show such morphological changes before gamete fusion, although the situation was similar to Nun orchid gamete fusion and Chlamydomonas gcs1 mutant phenotype. This result suggests that morphological changes occurring in the female gamete immediately before gamete fusion are not general, at least in angiosperms. Further analyses of other plant species may yield a general model of gamete morphology in double fertilization.
Arabidopsis gamete fusion process during double fertilization
Electron microscopy of P. zeylanica showed that plasma membrane fusion between male and female gametes occurred (Russell 1980, 1983, 1992). On the other hand, it has been reported that the actin-labeled sperm cells of Nun orchid appeared to penetrate female gametes with cytoplasmic mass (Ye et al. 2002). The latter study suggested the possibility that membrane-intact whole sperm cells entered the female gametes, similar to Drosophila sperm that entered the egg without membrane fusion (Karr 1991; Wilson et al. 2006). To analyze Arabidopsis gamete membrane behavior during double fertilization, live cell imaging was performed.
From the observations in the present study (Fig. 3d–i), it was suggested that plasma membrane fusion between male and female gametes occurred in Arabidopsis. Similarly, remained signal of the sperm cell membrane was observed on the rice in vitro fertilized zygote (Nakajima et al. 2010; Okamoto 2010). Therefore, GFP signals remaining at the boundary of the female gametes probably reflected the putative gamete membrane fusion site (Fig. 3d–i). In addition, the remaining GFP signals seemed to be somewhat diffused. In P. zeylanica, the model that the male and female plasma membranes fused at several sites was proposed based on observations of longitudinal sections (Russell 1980, 1983, 1992). Although whether the remaining diffused GFP signals in Arabidopsis could be an indication of such several membrane fusion events is remained to be judged, plasma membrane fusion between male and female gametes may be a general phenomenon in angiosperms. No remaining GFP signal was detected at the boundary of the female gametes by time-lapse imaging (Fig. 3j–m, Movie S2), probably because the weak plasma membrane signal could not be captured by the limited focus range of the Z-axis in CLSM.
Apart from the GFP signals at the boundary of the female gametes, additional GFP signals entering both the egg cell and the central cell were observed by time-lapse imaging, suggesting that internal membrane components derived from the sperm cell entered the female gametes. The male internal membrane components could have undergone rapid diffusion or degradation in the fertilized female gametes. Similar to P. zeylanica, the process from membrane fusion to the initiation of karyogamy may have taken several minutes in Arabidopsis (Hamamura et al. 2011; Kasahara et al. 2012; Russell 1980, 1983, 1992), and this might have hindered the acquisition of signals of the male internal membrane components in the fertilized female gametes immediately after the gamete membrane fusion.
Liu et al. (2010) have demonstrated that Chlamydomonas GCS1 was rapidly degraded in the zygote immediately after gamete fusion, and hypothesized that the degradation of GCS1 prevented polygamy (Liu et al. 2010). On the other hand, in Arabidopsis, GFP signals, namely, intact GCS1 molecules, remained at the putative gamete membrane fusion site until karyogamy, which was characterized by sperm nucleus decondensation (Fig. 3g–i). This suggests that the persistent GCS1 remnant has no effect on normal double fertilization and that polyspermy blocking in double fertilization is not based on rapid GCS1 degradation, unlike the case of Chlamydomonas.
The present study provided hitherto undisclosed information regarding gamete membrane dynamics during double fertilization, using live gametic fluorescent marker lines. Membrane behavior in the gamete fusion process was monitored with the male marker line, GPP. Further investigations with other membrane markers and plant species would provide more insight into the gamete membrane dynamics of angiosperms.
We are grateful to Dr. T. Nakagawa (Shimane University, Japan) for the pGWB1 vector. TagRFP gene was kindly provided by Dr. N. Inada and Dr. Y. Moriyama (NAIST, Japan). Laser scanning microscopy images were acquired with Olympus FV1000-D and FV1000-MPE at RIKEN BSI-Olympus Collaboration Center. This study was supported by a Grant-in Aid for Scientific Research from NAIST supported by MEXT, KAKENHI (22112515) to T.I., a Grant-in-Aid for Scientific Research on Innovative Areas (JSPS), KAKENHI (22657017) to T.I., a Grant-in-Aid for Challenging Exploratory Research (JSPS), KAKENHI (21112008) to T.M., and a Grant-in-Aid for Scientific Research on Innovative Areas.