Name: evaluation of fertilization state by tracing sperm nuclear morphology in arabidopsis double fertilization
Uploaded: 2019-08-29T14:00:00
Description: Watch this Scientific Journal Video about Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization at JoVE.com

This work was supported by Japan Society for the Promotion of Science KAKENHI grant (JP17H05832 to T. I.) and by funding from the Strategic Priority Research Promotion Program on Phytochemical Plant Molecular Sciences, Chiba University (Japan).

1. Artificial Pollination
NOTE: Before starting the process, a pair of No. 5 forceps is required.
<ol>
	<li>Grow A. thaliana (Col-0) at 22 &#176;C under a 16-h light/8-h dark cycle in a growth chamber. 
	NOTE: Cut and remove the first developed flower stalk with scissors to promote development of axillary buds. Vigorously growing plants (2-3 weeks after cutting of the first stalk; plant height about 25 cm) are suitable for analysis.</li>
	<li>T...

<ol>
	<li>Mori, T., Kawai-Toyooka, H., Igawa, T., Nozaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gamete+dialogs+in+green+lineages.">Gamete dialogs in green lineages.</a> Molecular Plant. 8, 1442-1454 (2015).</li><li>Dresselhaus, T., Sprunck, S., Wessel, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertilization+mechanisms+in+flowering+plants.">Fertilization mechanisms in flowering plants.</a> Current Biology. 26, R125-R139 (2016).</li><li>Mori, T., Kuroiwa, H., Higashiyama, T., Kuroiwa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GENERATIVE+CELL+SPECIFIC+1+is+essential+for+angiosperm+fertilization.">GENERATIVE CELL SPECIFIC 1 is essential for angiosperm fertilization.</a> Nature Cell Biology. 1, 64-71 (2006).</li><li>von Besser, K., Frank, A. C., Johnson, M. A., 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=Arabidopsis+HAP2+(GCS1)+is+a+sperm-specific+gene+required+for+pollen+tube+guidance+and+fertilization.">Arabidopsis HAP2 (GCS1) is a sperm-specific gene required for pollen tube guidance and fertilization.</a> Development. 133, 4761-4769 (2006).</li><li>Mori, T., Igawa, T., Tamiya, G., Miyagishima, S. Y., Berger, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gamete+attachment+requires+GEX2+for+successful+fertilization+in+Arabidopsis.">Gamete attachment requires GEX2 for successful fertilization in Arabidopsis.</a> Current Biology. 24, 170-175 (2014).</li><li>Takahashi, T., 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=The+male+gamete+membrane+protein+DMP9/DAU2+is+required+for+double+fertilization+in+flowering+plants.">The male gamete membrane protein DMP9/DAU2 is required for double fertilization in flowering plants.</a> Development. 145, dev170076 (2018).</li><li>Ingouff, M., Hamamura, Y., Gourgues, M., Higashiyama, T., Berger, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+dynamics+of+HISTONE3+variants+between+the+two+fertilization+products+in+plants.">Distinct dynamics of HISTONE3 variants between the two fertilization products in plants.</a> Current Biology. 17, 1032-1037 (2007).</li><li>Smyth, D. R., Bowman, J. L., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+flower+development+in+Arabidopsis.">Early flower development in Arabidopsis.</a> Plant Cell. 2, 755-767 (1990).</li><li>Kasahara, R. D., Maruyama, D., Higashiyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertilization+recovery+system+is+dependent+on+the+number+of+pollen+grains+for+efficient+reproduction+in+plants.">Fertilization recovery system is dependent on the number of pollen grains for efficient reproduction in plants.</a> Plant Signaling &amp; Behavior. 8, e23690 (2013).</li><li>Kasahara, R. D., 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=Fertilization+recovery+after+defective+sperm+cell+release+in+Arabidopsis.">Fertilization recovery after defective sperm cell release in Arabidopsis.</a> Current Biology. 22, 1084-1089 (2012).</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, 497-502 (2011).</li><li>Igawa, T., Yanagawa, Y., Miyagishima, S., Mori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+gamete+membrane+dynamics+during+double+fertilization+of+Arabidopsis.">Analysis of gamete membrane dynamics during double fertilization of Arabidopsis.</a> Journal of Plant Research. 126, 387-394 (2013).</li></ol>

HTR10-mRFP labels paternal chromatin (i.e., visualizes sperm cell nuclei), and the dynamics in double fertilization have been reported7. Immediately after release from a pollen tube, HTR10-mRFP-labeled sperm nuclei are still condensed. However, each of the sperm nuclei is decondensed upon merging with a fertilized female gamete nucleus at karyogamy three to four hours after gamete membrane fusion7. Unfertilized sperm cells remain condensed, as shown in an embryo sac in whic...

Flowering plants produce seeds through double fertilization, a process that is directly controlled by interactions between proteins localized on gamete plasma membrane1,2. Flowering plant male gametes, a pair of sperm cells, develop in pollen. A pollen tube that grows after pollination delivers a pair of sperm cells to female gametes, an egg cell and a central cell, which develop in an embryo sac. After the male and female gametes meet, proteins on the gamete surface promote recognition, attachment, and fusion to complete double fertilization. In previous studies, the male gamete membrane proteins GENERATIVE CELL SPECIFIC 1 (GCS1)/HAPLESS2 (HAP2)3,4 and GAMETE EXPRESSED 2 (GEX2)5 were identified as fertilization regulators involved in gamete fusion and attachment, respectively. We recently identified a male gamete-specific membrane protein, DUF679 DOMAIN MEMBRANE PROTEIN 9 (DMP9), as a fertilization regulator involved in gamete interaction. We found that a decrease of DMP9 expression results in significant inhibition of egg cell fertilization during double fertilization in A. thaliana6.
As double fertilization occurs in an embryo sac, which is embedded in an ovule that is further wrapped with ovary tissue, it is difficult to observe and analyze the states of double fertilization processes. For this reason, there are still many unclear points that hinder a complete understanding of the whole mechanism of double fertilization control. The establishment of observation techniques to trace the behavior of gametes during double fertilization under in vivo conditions is indispensable for the functional analysis of potential candidates for fertilization regulators. Recent studies have yielded marker lines where gamete subcellular structures are labeled with fluorescent proteins. In this article, we demonstrate a simple and quick protocol for observing double fertilization that has occurred in an embryo sac derived from artificially pollinated pistils. Using sperm cell nucleus marker line HTR10-mRFP7, the fertilization state of each female gamete can be discriminated on the basis of sperm nuclear signal morphology. Our protocol focusing on such a morphological change of the sperm nuclei at fertilization can efficiently obtain a sufficient amount of data for statistical proof. A DMP9-knockdown line with HTR10-mRFP background (DMP9KD/HTR10-mRFP) was used as male plants to show a single fertilization pattern. The protocol is also suitable for the functional analysis of other fertilization regulators.

<table>
	<tbody>
		<tr>
			<td>BX51</td>
			<td>Olympus</td>
			<td></td>
			<td>Epifluorescence microscope</td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Matsunami glass</td>
			<td>C018181</td>
			<td></td>
		</tr>
		<tr>
			<td>DMP9KD/HTR10-mRFP</td>
			<td></td>
			<td></td>
			<td>Arabidopsis thaliana, HTR10-mRFP background 
			Takahashi et al. (2018)6</td>
		</tr>
		<tr>
			<td>Double-sided tape</td>
			<td>Nichiban</td>
			<td>NW-15S</td>
			<td>15 mm width</td>
		</tr>
		<tr>
			<td>DP72</td>
			<td>Olympus</td>
			<td></td>
			<td>Degital camera</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Vigor</td>
			<td></td>
			<td>Any No. 5 forceps are available</td>
		</tr>
		<tr>
			<td>Growth chamber</td>
			<td>Nihonika</td>
			<td>LPH-411PFQDT-SP</td>
			<td></td>
		</tr>
		<tr>
			<td>HTR10-mRFP</td>
			<td></td>
			<td></td>
			<td>Arabidopsis thaliana, ecotype Columbia-0 (Col-0) background 
			Ingouff et al. (2007)7</td>
		</tr>
		<tr>
			<td>Injection needle</td>
			<td>Terumo</td>
			<td>NN-2719S</td>
			<td>27 gauge</td>
		</tr>
		<tr>
			<td>Slide glass</td>
			<td>Matsunami glass</td>
			<td>S9443</td>
			<td></td>
		</tr>
		<tr>
			<td>SZX9</td>
			<td>Olympus</td>
			<td></td>
			<td>Dissecting microscope</td>
		</tr>
		<tr>
			<td>U-MRFPHQ</td>
			<td>Olympus</td>
			<td></td>
			<td>Fluorescence Filter Cube (Excitation: BP535-555, Emission: BA570-625, Dichromatic mirror:DM565)</td>
		</tr>
		<tr>
			<td>UPlanFL N 40x</td>
			<td>Olympus</td>
			<td></td>
			<td>Objective lens (NA 1.3), oil-immersion</td>
		</tr>
		<tr>
			<td>UPlanSApo 20x</td>
			<td>Olympus</td>
			<td></td>
			<td>Objective lens (NA0.75), dry</td>
		</tr>
	</tbody>
</table>

evaluation of fertilization state by tracing sperm nuclear morphology in arabidopsis double fertilization

Flowering plants have a unique sexual reproduction system called &#8216;double fertilization&#39;,&#160;in which each of the sperm cells precisely fuses with an egg cell or a central cell. Thus, two independent fertilization events take place almost simultaneously. The fertilized egg cell and central cell develop into zygote and endosperm, respectively. Therefore, precise control of double fertilization is essential for the ensuing seed development. Double fertilization occurs in the female gametophyte (embryo sac), which is deeply hidden and covered with thick ovule and ovary tissues. This pistil tissue construction makes observation and analysis of double fertilization quite difficult and has created the present situation in which many questions regarding the mechanism of double fertilization remain unanswered. For the functional evaluation of a potential candidate for fertilization regulator, phenotypic analysis of fertilization is important. To judge the completion of fertilization in Arabidopsis thaliana, the shapes of fluorescence signals labeling sperm nuclei are used as indicators. A sperm cell that fails to fertilize is indicated by a condensed fluorescence signal outside of the female gametes, whereas a sperm cell that successfully fertilizes is indicated by a decondensed signal due to karyogamy with the female gametes&#8217; nucleus. The method described here provides a tool to determine successful or failed fertilization under in vivo conditions.

Ovules from a pistil pollinated with DMP9KD/HTR10-mRFP were collected at 7-8 HAP and observed.
Most ovules contained two decondensed mRFP-labeled sperm nuclei at the egg cell (micropylar side) and central cell (charazal end side) nucleus positions, respectively (Figure 3A), indicating successful double fertilization. In addition, ovules containing a decondensed mRFP-labeled sperm...

Watch this Scientific Journal Video about Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization at JoVE.com

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization

We demonstrate a method to determine successful or failed fertilization on the basis of sperm nuclear morphology in Arabidopsis double fertilization using an epifluorescence microscope.

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization

Flowering plants have a unique sexual reproduction system called &#8216;double fertilization&#39;,&#160;in which each of the sperm cells precisely fuses ...

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