Name: live imaging of arabidopsis pollen tube reception and double fertilization using the semi-in vitro cum septum method
Uploaded: 2023-02-24T12:30:00
Description: Watch this Scientific Journal Video about Live Imaging of Arabidopsis Pollen Tube Reception and Double Fertilization Using the Semi-In Vitro Cum Septum Method at JoVE.com

We thank Sara Simonini and Stefano Bencivenga for donating the pFG:roGFP2-ORP1-NLS construct and Christof Eichenberger, Johann Almendinger, Vincent Sutter, and Celia Baroux for their advice on microscopy. We kindly acknowledge advice from Ravi Palanivelu, Philipp Denninger, Sharon Kessler, Mark Johnson, Tomokazu Kawashima, and everyone else at the International Conference on Sexual Plant Reproduction 2022 who showed interest in a protocol on SIV cum septum. This work was supported by the University of Zurich and grants from the Swiss National Science Foundation to U.G.

NOTE: See the Table of Materials for a list of the materials and equipment used in this protocol.
1. Considerations for designing the imaging experiment
<ol>
	<li>Predetermine the imaging duration and sampling frequency required to capture the desired phenomenon to be observed. For example, following Nyquist&#39;s sampling theorem, the sampling frequency must be greater than twice the highest frequency of the input sample. Therefore, to accurately capture sy...

<ol>
	<li>Huck, N., Moore, J. M., Federer, M., Grossniklaus, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arabidopsis+mutant+feronia+disrupts+the+female+gametophytic+control+of+pollen+tube+reception.">The Arabidopsis mutant feronia disrupts the female gametophytic control of pollen tube reception.</a> Development. 130 (10), 2149-2159 (2003).</li><li>Rotman, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Female+control+of+male+gamete+delivery+during+fertilization+in+Arabidopsis+thaliana.">Female control of male gamete delivery during fertilization in Arabidopsis thaliana.</a> Current Biology. 13 (5), 432-436 (2003).</li><li>Palanivelu, R., Preuss, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+short-range+ovule+signals+attract+or+repel+Arabidopsis+thaliana+pollen+tubes+in+vitro.">Distinct short-range ovule signals attract or repel Arabidopsis thaliana pollen tubes in vitro.</a> BMC Plant Biology. 6, 7 (2006).</li><li>Susaki, D., Maruyama, D., Yelagandula, R., Berger, F., Kawashima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+imaging+of+F-actin+dynamics+during+fertilization+in+Arabidopsis+thaliana.">Live-cell imaging of F-actin dynamics during fertilization in Arabidopsis thaliana.</a> Methods in Molecular Biology. 1669, 47-54 (2017).</li><li>Iwano, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+Ca2++changes+dynamically+during+the+interaction+of+the+pollen+tube+with+synergid+cells.">Cytoplasmic Ca2+ changes dynamically during the interaction of the pollen tube with synergid cells.</a> Development. 139 (22), 4202-4209 (2012).</li><li>Ngo, Q., Vogler, H., Lituiev, D., Nestorova, A., Grossniklaus, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+calcium+dialog+mediated+by+the+FERONIA+signal+transduction+pathway+controls+plant+sperm+delivery.">A calcium dialog mediated by the FERONIA signal transduction pathway controls plant sperm delivery.</a> Developmental Cell. 29 (4), 491-500 (2014).</li><li>Denninger, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Male-female+communication+triggers+calcium+signatures+during+fertilization+in+Arabidopsis.">Male-female communication triggers calcium signatures during fertilization in Arabidopsis.</a> Nature Communications. 5, 4645 (2014).</li><li>Hamamura, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+calcium+spikes+during+double+fertilization+in+Arabidopsis.">Live imaging of calcium spikes during double fertilization in Arabidopsis.</a> Nature Communications. 5, 4722 (2014).</li><li>Ponvert, N., Johnson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergid+calcium+ion+oscillations+define+a+new+feature+of+pollen+tube+reception+critical+for+blocking+interspecific+hybridization.">Synergid calcium ion oscillations define a new feature of pollen tube reception critical for blocking interspecific hybridization.</a> bioRxiv. , (2020).</li><li>Hamamura, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+imaging+reveals+the+dynamics+of+two+sperm+cells+during+double+fertilization+in+Arabidopsis+thaliana.">Live-cell imaging reveals the dynamics of two sperm cells during double fertilization in Arabidopsis thaliana.</a> Current Biology. 21 (6), 497-502 (2011).</li></ol>

This manuscript introduces an efficient protocol for the imaging of pollen tube reception and double fertilization in Arabidopsis. The improved method, SIV cum septum,&#160;greatly increases the percentage and total number of successful pollen tube reception events that are observable per imaging session. The representative results shown here demonstrate an imaging session with 41 successful pollen tube reception events and 10 ovules showing reception defects (~80% efficiency). This is over double the n...

The generation of genetically unique offspring in sexually reproducing organisms is dependent on the successful fusion of male and female gametes. In flowering plants, the interaction of two male gametes (sperm cells) with two female gametes (egg cell and central cell) during double fertilization depends on sperm release from the pollen tube (the male gametophyte). This process, called pollen tube reception, is largely controlled by the synergid cells that reside within the embryo sac (the female gametophyte)1,2. As pollen tube reception takes place deep inside the flower, a method allowing for live-cell imaging of the process, called semi-in vitro (SIV) pollen tube reception, has been established3. With this method, excised Arabidopsis ovules are placed on semi-liquid pollen germination medium and targeted by pollen tubes that grow through the stigma and style of a pistil severed at the style-transmitting tract junction3,4. Since the development of this technique, detailed observations have led to several discoveries surrounding pollen tube guidance, reception, and fertilization. Among others, these discoveries include the acquisition of pollen tube targeting competence by growth through the stigma3, the onset of intracellular calcium oscillations in the synergids upon pollen tube arrival5,6,7,8,9, and the dynamics of sperm cell release and fertilization upon pollen tube burst10. Nevertheless, because this technique relies on the excision of ovules, the observations of fertilization are limited in number, and pollen tube reception is often aberrant, resulting in the failure of pollen tube rupture (Video 1 and Supplementary File 1). Therefore, there is a need for a more efficient approach allowing for high-throughput analyses of pollen tube reception and fertilization.
In developing this protocol, several new approaches to analyze pollen tube reception, spanning from the most &#34;in vitro&#34; to the most &#34;in vivo&#34; methods, were tested, and an efficient technique based on the excision of the entire septum was settled upon, which allows for up to 40 observations of fertilization per day. Here, the nuances and critical points of the technique are outlined, including flower staging, dissection, medium preparation, and imaging settings. By following this protocol, research focusing on pollen tube guidance, pollen tube reception, and double fertilization should be facilitated. The higher sample sizes the method allows for are expected to bolster the scientific soundness of the conclusions drawn from live imaging experiments. The potential applications of this technique include, but are not limited to, performing observations of the molecular and physiological changes in cytosolic calcium concentrations ([Ca2+]cyt), pH, or H2O2 during gametophyte interactions through the use of genetically encoded biosensors. Furthermore, cytological changes, such as degeneration of the receptive synergid, sperm cell migration, or karyogamy, can be more easily observed using this improved method. Finally, the timing of the different stages of fertilization can be monitored under widefield microscopy, and then more detailed analyses using confocal laser scanning microscopy (CLSM) or two-photon excitation microscopy (2PEM) can be conducted for higher resolution and 3D reconstruction.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mm glass slide&#160;</td>
			<td>Epredia&#160;</td>
			<td>16211551</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm glass bottom dish (14 mm well)</td>
			<td>Mattek&#160;</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride&#160;</td>
			<td>Roth&#160;</td>
			<td>CN93.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Columbia (Col-0)&#160;</td>
			<td>Nottingham Arabidopsis Stock Centre (NASC)</td>
			<td></td>
			<td>stigma donor</td>
		</tr>
		<tr>
			<td>Dissecting Scope&#160;</td>
			<td>Olympus&#160;</td>
			<td>SZX2-ILLT</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin needle (0.3 G)</td>
			<td>BD</td>
			<td>304000</td>
			<td></td>
		</tr>
		<tr>
			<td>Landsberg erecta (Ler-0)&#160;</td>
			<td>Nottingham Arabidopsis Stock Centre (NASC)</td>
			<td></td>
			<td>septum donor</td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Merck</td>
			<td>5886</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Roth</td>
			<td>6781.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Beldura</td>
			<td>7026797</td>
			<td></td>
		</tr>
		<tr>
			<td>Scotch double sided tape&#160;</td>
			<td>Scotch</td>
			<td>768720</td>
			<td>Less thick and good for stigma dissection</td>
		</tr>
		<tr>
			<td>Sodium Chloride&#160;</td>
			<td>Roth</td>
			<td>3957.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>ITW reagents</td>
			<td>A2211,1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Tesa double sided tape&#160;</td>
			<td>Tesa</td>
			<td>05681-00018</td>
			<td>Very sticky and good for septum dissection</td>
		</tr>
		<tr>
			<td>Ultra low gelling temperature agarose</td>
			<td>FMC SeaPrep</td>
			<td>50302</td>
			<td></td>
		</tr>
	</tbody>
</table>

live imaging of arabidopsis pollen tube reception and double fertilization using the semi-in vitro cum septum method

In flowering plants, the growth and guidance of the pollen tube (male gametophyte) within the pistil and the reception of the pollen tube by the female gametophyte are essential for double fertilization and subsequent seed development. The interactions between male and female gametophytes during pollen tube reception culminate in pollen tube rupture and the release of two sperm cells to effect double fertilization. As pollen tube growth and double fertilization are deeply hidden within the tissues of the flower, this process is difficult to observe in vivo.
A semi-in vitro (SIV) method for the live-cell imaging of fertilization in the model plant Arabidopsis thaliana has been developed and implemented in several investigations. These studies have helped to elucidate the fundamental features of how the fertilization process occurs in flowering plants and which cellular and molecular changes occur during the interaction of the male and female gametophytes. However, because these live cell imaging experiments involve the excision of individual ovules, they are limited to a low number of observations per imaging session, making this approach tedious and very time-consuming. Among other technical difficulties, a failure of the pollen tubes to fertilize the ovules in vitro is&#160;often reported, which severely confounds such analyses.
Here, a detailed video protocol for the imaging of pollen tube reception and fertilization in an automated and high-throughput manner is provided, allowing for up to 40 observations of pollen tube reception and rupture per imaging session. Coupled with the use of genetically encoded biosensors and marker lines, this method enables the generation of large sample sizes with a reduced time investment. Nuances and critical points of the technique, including flower staging, dissection, medium preparation, and imaging, are clearly detailed in video format to facilitate future research on the dynamics of pollen tube guidance, reception, and double fertilization.

To assess the timing of nuclear degeneration in the receptive synergid with respect to pollen tube rupture in Arabidopsis, as well as to observe whether the left or right synergid is predestined to become the receptive synergid,&#160;the SIV cum septum method described here was employed using a female gametophyte nuclear marker stacked with a synergid cytosolic marker (pFG:roGFP2-ORP1-NLS, pMYB98:roGFP2-ORP1) as the septum donor and a male gametophyte marker (pLAT52:R-GECO) as the poll...

Watch this Scientific Journal Video about Live Imaging of Arabidopsis Pollen Tube Reception and Double Fertilization Using the Semi-In Vitro Cum Septum Method at JoVE.com

Live Imaging of Arabidopsis Pollen Tube Reception and Double Fertilization Using the Semi-In Vitro Cum Septum Method

Here, we describe an improvement of the semi-in vitro (SIV) method for observing pollen tube guidance and reception in Arabidopsis thaliana,&#160;which increases the receptivity of ovules. The high-throughput SIV cum septum method may be coupled with gametophyte marker lines and genetically encoded biosensors to monitor the dynamic process of fertilization.

Live Imaging of Arabidopsis Pollen Tube Reception and Double Fertilization Using the Semi-In Vitro Cum Septum Method

In flowering plants, the growth and guidance of the pollen tube (male gametophyte) within the pistil and the reception of the pollen tube by the female ...

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