Name: a seed coat bedding assay to genetically explore in vitro how the endosperm controls seed germination in arabidopsis thaliana
Uploaded: 2013-11-09T21:00:00
Description: Watch this Scientific Journal Video about A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana at JoVE.com

This work was supported by grants from the Swiss National Science Foundation and by the State of Geneva.

Once the SCBA is assembled, the growth of embryos is monitored over several days. Therefore, before the seed dissection procedure and assembly of the SCBA, one needs to sterilize seeds to avoid future contaminations that could prevent proper assessment of the effect of seed coat material on embryonic growth.
1. Seed Sterilization
<ol>
	<li>Pour 50-60 &#956;l of mature and dry Arabidopsis seeds in a 1.5 ml microcentrifuge tube and prepare a 70% ethanol solution.</li>
	<li>Add 1 ml of...

<ol>
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Natl. Acad. Sci. U.S.A. 107, 19108-19113 (2010).</li><li>Reed, J. W., Nagatani, A., Elich, T. D., Fagan, M., Chory, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phytochrome+A+and+Phytochrome+B+Have+Overlapping+but+Distinct+Functions+in+Arabidopsis+Development.">Phytochrome A and Phytochrome B Have Overlapping but Distinct Functions in Arabidopsis Development.</a> Plant Physiol. 104, 1139-1149 (1994).</li><li>Shinomura, 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=Action+spectra+for+phytochrome+A-+and+B-specific+photoinduction+of+seed+germination+in+Arabidopsis+thaliana.">Action spectra for phytochrome A- and B-specific photoinduction of seed germination in Arabidopsis thaliana.</a> Proc. Natl. Acad. Sci. U.S.A. 93, 8129-8133 (1996).</li><li>Lee, K. 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=Spatially+and+genetically+distinct+control+of+seed+germination+by+phytochromes+A+and+B.">Spatially and genetically distinct control of seed germination by phytochromes A and B.</a> Genes Dev. 26, 1984-1996 (2012).</li><li>Lee, K. P., Lopez-Molina, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+seed+germination+in+the+shade.">Control of seed germination in the shade.</a> Cell Cycle. 11, 4489-4490 (2012).</li><li>Piskurewicz, U., 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+gibberellic+acid+signaling+repressor+RGL2+inhibits+Arabidopsis+seed+germination+by+stimulating+abscisic+acid+synthesis+and+ABI5+activity.">The gibberellic acid signaling repressor RGL2 inhibits Arabidopsis seed germination by stimulating abscisic acid synthesis and ABI5 activity.</a> Plant Cell. 20, 2729-2745 (2008).</li><li>Piskurewicz, U., Tureckova, V., Lacombe, E., Lopez-Molina, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Far-red+light+inhibits+germination+through+DELLA-dependent+stimulation+of+ABA+synthesis+and+ABI3+activity.">Far-red light inhibits germination through DELLA-dependent stimulation of ABA synthesis and ABI3 activity.</a> EMBO J. 28, 2259-2271 (2009).</li><li>Debeaujon, I., Koornneef, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gibberellin+requirement+for+Arabidopsis+seed+germination+is+determined+both+by+testa+characteristics+and+embryonic+abscisic+acid.">Gibberellin requirement for Arabidopsis seed germination is determined both by testa characteristics and embryonic abscisic acid.</a> Plant Physiol. 122, 415-424 (2000).</li><li>Sun, T. P., Kamiya, Y. <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+GA1+locus+encodes+the+cyclase+ent-kaurene+synthetase+A+of+gibberellin+biosynthesis.">The Arabidopsis GA1 locus encodes the cyclase ent-kaurene synthetase A of gibberellin biosynthesis.</a> Plant Cell. 6, 1509-1518 (1994).</li><li>Alonso, J. 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The seed coat bedding assay (SCBA) procedure described here is in principle applicable to any circumstance where Arabidopsis seed germination is blocked (or delayed) and where the endosperm is suspected to implement this arrest. The latter can be evidenced by removing the seed coat (testa and endosperm) and observing that embryonic growth proceeds faster relative to that observed when embryos are surrounded by the seed coat. Germination may be blocked in response to particular environmental physical parameters (...

In Arabidopsis mature seeds, the seed coat is composed of the testa, an external layer of dead tissue of maternal origin, and the endosperm, a single cell layer of live tissue directly surrounding the embryo1. The endosperm and the embryo are derived from separate fertilization events: the endosperm is a triploid tissue with two maternal and one paternal genome whereas the embryo is a diploid tissue with one maternal and one paternal genome2.
The main function traditionally assigned to the endosperm is that of a nutritive tissue. However, it is becoming increasingly evident that the endosperm also plays a central role to control seed germination. This notion became first apparent in the case of dormancy, a trait exhibited by newly produced seeds. Dormant seeds fail to germinate despite the presence of favorable germination conditions. Seeds lose their dormancy after a ripening period and become nondormant, i.e. they will germinate when exposed to favorable germination conditions. In many plant species, including the model plant Arabidopsis, the seed coat is absolutely required to prevent the germination of dormant seeds since seed coat removal triggers embryonic growth and greening3,4. In Arabidopsis, Bethke et al. observed that germination remained repressed after removing the testa while maintaining the endosperm surrounding the endosperm5. These observations strongly indicated that the endosperm is the tissue within the seed coat exerting a repressive activity on the embryo. However, seed coat removal experiments do not necessarily help clarifying the nature of the germination repressive activity provided by the seed coat nor identifying the genes that implement it.
We recently introduced a seed coat bedding assay (SCBA) where seed coats and embryos are physically separated but kept in close proximity so that the germination repressive activity provided by the endosperm is maintained6. The SCBA allows the combinatorial use of dormant, nondormant, and genetically modified seed coat and embryonic materials. As a result, the genetic pathways controlling germination and specifically operating in the endosperm and embryo can be dissected. The SCBA was used in the context of dormancy to show that the endosperm releases the phytohormone abscisic acid (ABA) towards the embryo to repress its growth6. Furthermore we could use the SCBA to identify the signaling pathways operating in endosperm and embryonic tissues to promote dormancy.
The role of the endosperm to control germination was further strengthened by considering the case of nondormant seeds exposed to a pulse of far red (FR) light. Early upon seed imbibition a FR light pulse is known to inhibit germination7,8. When seed coats were removed from seeds a pulse of FR light was unable to inhibit germination, strongly suggesting that the endosperm can also repress the germination of nondormant seeds 9. Remarkably, the SCBA could also be used to recapitulate FR-dependent inhibition of germination. This allowed to show that that FR-dependent inhibition of seed germination is also a process involving ABA release from the endosperm9. Furthermore, the SCBA allowed identifying the different light-signaling pathways operating in the endosperm and the embryo to control nondormant seed germination in response to light cues9,10.
The SCBA appears therefore to be a reliable technique to explore the function of the endosperm in the context of the control of seed germination. It is also a powerful tool to assess in vitro whether genes suspected to control germination operate in the endosperm, the embryo or both tissues. Here we detail the various steps required to assemble a SCBA.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>To refer</td>
		</tr>
		<tr>
			<td>Thermomixer Comfort</td>
			<td>Eppendorf AG</td>
			<td>5355 000.011</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
		</tr>
		<tr>
			<td>Vacusafe Comfort</td>
			<td>INTEGRA Biosciences AG</td>
			<td>158 310</td>
			<td>Integra Biosciences AG, Zizers, Switzerland</td>
		</tr>
		<tr>
			<td>Petri dish plate (100 mm x 20 mm)</td>
			<td>Greiner Bio-One GmbH</td>
			<td>664 102</td>
			<td>Greiner Bio-One GmbH, Frickenhausen, Germany</td>
		</tr>
		<tr>
			<td>Murashige and Skoog</td>
			<td>Sigma-Aldrich</td>
			<td>M5524</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
		</tr>
		<tr>
			<td>MES</td>
			<td>Sigma-Aldrich</td>
			<td>M3671</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
		</tr>
		<tr>
			<td>Agar (plant agar)</td>
			<td>Duchefa Biochemie B.V.</td>
			<td>P1001</td>
			<td>Duchefa Biochemie, Haarlem, Netherlands</td>
		</tr>
		<tr>
			<td>Dumont forceps #5</td>
			<td>Fine Science Tools GmbH</td>
			<td>11251-10</td>
			<td>Fine Science Tools GmbH, Heidelberg Germany</td>
		</tr>
		<tr>
			<td>Syringe needle</td>
			<td>BD Micro-Fine</td>
			<td>324827</td>
			<td>BD, Franklin Lakes, NJ USA</td>
		</tr>
		<tr>
			<td>Nylon mesh (SEFAR NYTEX)</td>
			<td>SEFAR AG</td>
			<td>03-50/31</td>
			<td>Sefar AG, Heiden, Switzerland</td>
		</tr>
		<tr>
			<td>Growth chamber</td>
			<td>CLF Plant Climatics</td>
			<td>Percival I-30BLLX</td>
			<td>CLF plant Climatics, Wertingen, Germany</td>
		</tr>
		<tr>
			<td>Paclobutrazol</td>
			<td>Sigma-Aldrich</td>
			<td>46046</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
		</tr>
	</tbody>
</table>

a seed coat bedding assay to genetically explore in vitro how the endosperm controls seed germination in arabidopsis thaliana

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of dormant seeds or that of nondormant seeds irradiated by a far red (FR) light pulse. In order to further gain insight into the molecular genetic mechanisms underlying the germination repressive activity exerted by the endosperm, a &#34;seed coat bedding&#34; assay (SCBA) was devised. The SCBA is a dissection procedure physically separating seed coats and embryos from seeds, which allows monitoring the growth of embryos on an underlying layer of seed coats. Remarkably, the SCBA reconstitutes the germination repressive activities of the seed coat in the context of seed dormancy and FR-dependent control of seed germination. Since the SCBA allows the combinatorial use of dormant, nondormant and genetically modified seed coat and embryonic materials, the genetic pathways controlling germination and specifically operating in the endosperm and embryo can be dissected. Here we detail the procedure to assemble a SCBA.

Previous work showed that mutant seeds unable to synthesize GA were unable to germinate as a result of high ABA accumulation in seeds 11,12. However, inability to germinate requires the seed coat since its removal triggers embryonic growth13. This strongly indicated that the endosperm of seeds unable to synthesize GA is releasing ABA to block embryonic growth. We therefore expect seeds coats unable to synthesize GA to block the growth of embryos in a SCBA unlike seed coats unable to synthesize GA an...

Watch this Scientific Journal Video about A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana at JoVE.com

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

We present the procedure to assemble a seed coat bedding assay (SCBA) from Arabidopsis thaliana seeds. The SCBA was shown to be a powerful tool to explore genetically and in vitro how the endosperm controls seed germination in dormant seeds and in response to light cues. The SCBA is in principle applicable under any situation where the endosperm is suspected to influence embryonic growth.

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of ...

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