这项工作是由瑞士国家科学基金会资助和日内瓦州的支持。

一旦空气呼吸器被组装时，胚胎的生长被监控超过数天。因此，空气呼吸器的种子解剖过程和组装之前，需要消毒的种子，以避免将来的污染，可以防止种皮材料对胚胎生长的影响，正确的评估。 1。种子消毒<ol><li>倒入50-60微升成熟和拟南芥干种子在1.5 ml离心管中，并准备一个70％的乙醇溶液。 </li><li>加入1毫升70％的乙醇溶液至含有种子的微量离心管中，并在1,200 rpm下在涡流混合器摇动在室温下搅拌10分钟。 </li><li>离心机的离心管中，持续3秒在4000×g离心浓缩种子在管的底部。 </li><li>用真空吸嘴小心而使种子在微量离心管的底部吸出70％的乙醇溶液。 </li><li>加入1毫升无菌蒸馏水至吨<html>他离心管中仍含有的种子。 </li><li>摇在室温下在1200rpm 10分钟。 </li><li>离心机的离心管中，持续3秒在4000×g离心浓缩种子在管的底部。用真空吸嘴小心而使种子在管的底部抽吸水上清液。 </li><li>加入1毫升无菌水蒸馏水至仍含有种子的微量离心管中。确保水的流动均匀挂起管内的种子。这样做的目的是为了避免进一步的晃动，从而保持种子的完整性。 </li><li>让种子定下来通过重力在该管的底部由仍留在管30秒。用真空吸嘴小心而使种子在管的底部抽吸水上清液。 </li><li>重复四次步骤1.8 - 1.9。整体灭菌过程大约需要30分钟。 </li></ol> 2。种子电镀ontent"&gt;所有后续步骤都在层流柜中进行，以保持无菌条件。 <ol><li>准备陪替氏培养皿盘（100毫米直径20毫米高）制备通过高压灭菌含有4.3克/ L的Murashige和Skoog培养基中2.5毫米2的溶液中装有30ml萌发培养基 - （N-吗啉）乙磺酸（MES-KOH ; pH值5.7）和0.8克/升琼脂。设在50℃水浴中冷却液倒在培养皿板浇筑前。 </li><li>加入200μl的无菌蒸馏水到含种子的试管中。重悬的种子，并用标准移液管与1毫升尖萌发培养基的表面上传送。 </li><li>除去周围用真空吸嘴的萌发培养基的表面上的种子的水分。 </li><li>离开含种子没有它的盖培养皿中的层流柜内2小时为进一步干燥。关闭培养皿的盖子，让它经得起层下流柜约90分钟，使约4小时，自该种子灭菌过程（步骤1.2）的启动通过。 </li></ol> 3。种子解剖下面的步骤必要用立体显微镜放置在层流罩柜内工作。一个杜蒙钳＃5于截断（ 即钝）提示极大地方便了种子的处理（ 图1A）。 <ol><li>准备装有30ml萌发培养基的新培养皿板。这里的介质2并列的矩形灭菌的Whatman 3MM纸（1.5厘米×1.0厘米; 图1B，步骤1）的表面上。的Whatman 3MM纸被高压灭菌。 </li><li>传送上使用无菌镊子（ 图1B，步骤2）中的Whatman 3MM纸种子。 </li><li>通过使用截断钳的并列钝头（轻轻按压种子仇视的Whatman 3MM纸单粒种子<st荣&gt;图1B，步骤3）。 </li><li>切割用注射器针头种子的种皮（外种皮和胚乳）（ 如 U100胰岛素注射器+ 1ml注射器，0.33毫米（29 G）×12.7毫米）（ 图1B，步骤4）。 拟南芥种子的形状是椭圆形的，因此建议削减种皮沿尽可能接近的地方子叶接合到胚根（ 即远离基端部）的位置上最长的半主轴。这有助于在清扫过程的下一步骤中胚胎的安全释放。 </li><li>使用截短的镊子（ 图1B，步骤5）的并置的钝头推压的Whatman 3MM纸上的种子。此步骤会释放出胚种皮通过在步骤3.4（ 图1B，步骤6）中创建的开口。它建议申请在该处的子叶的顶端是在最靠近胚根的尖端处的种子（ 即相差fr处的推OM开放）。 </li></ol> 4。大会的种皮床品含量（SCBA） 为种皮和胚胎的数量的适当的选择看讨论。 <ol><li>准备装有30ml萌发培养基的新培养皿板。地方的介质的表面上的无菌一块长方形的尼龙网片（3.5厘米×5.0厘米）（ 图2，步骤1）。 </li><li>为避免损坏，胚胎和种皮可以通过使用注射器针头（ 图2，步骤2）轻轻推他们移动到钳子的金属边缘的表面上。转移的胚胎和种皮上的尼龙筛网（ 图2，步骤3）。 </li><li>组装种皮的使用钝钳和针确保种子外套是在尽可能多的靠近，尽可能地（ 图2，步骤4）的圆形和单层。尝试以暴露尽可能由所产生的种皮开口针向上。这一步是没有系统地测试。但是，它可能会增加空气呼吸器的效率，因为可扩散物质抑制了胚胎的生长预期直接扩散出来对胚胎而不是远离它。 </li><li>将胚胎作为组装在步骤4.3种皮床中心的环状单层（ 图2，步骤5）。确保胚胎是在尽可能多的更接近越好。 </li><li>在20-21℃下孵育下连续光照（40微摩尔/米2秒 EC）的SCBAs在帧中继脉冲用来逮捕发芽，组装空气呼吸器正常光线下，与实际情况相符的FR脉冲9，离开培养皿在黑暗的情况下。 </li></ol>

<ol>
	<li>Debeaujon, I., Leon-Kloosterziel, K. M., 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=Influence+of+the+testa+on+seed+dormancy,+germination,+and+longevity+in+Arabidopsis.">Influence of the testa on seed dormancy, germination, and longevity in Arabidopsis.</a> Plant Physiol. 122, 403-414 (2000).</li><li>Baroux, C., Spillane, C., 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=Evolutionary+origins+of+the+endosperm+in+flowering+plants.">Evolutionary origins of the endosperm in flowering plants.</a> Genome Biol. 3, reviews1026 (2002).</li><li>Debeaujon, I., Lepiniec, L., Pourcel, L., Routaboul, J. -. M., Bradford, K., Nonogaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Seed development, dormancy and germination. , 25-43 (2007).</li><li>Finch-Savage, W. E., Leubner-Metzger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seed+dormancy+and+the+control+of+germination.">Seed dormancy and the control of germination.</a> New Phytol. 171, 501-523 (2006).</li><li>Bethke, P. C., 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+Arabidopsis+aleurone+layer+responds+to+nitric+oxide,+gibberellin,+and+abscisic+acid+and+is+sufficient+and+necessary+for+seed+dormancy.">The Arabidopsis aleurone layer responds to nitric oxide, gibberellin, and abscisic acid and is sufficient and necessary for seed dormancy.</a> Plant Physiol. 143, 1173-1188 (2007).</li><li>Lee, K. P., Piskurewicz, U., Tureckova, V., Strnad, M., 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=A+seed+coat+bedding+assay+shows+that+RGL2-dependent+release+of+abscisic+acid+by+the+endosperm+controls+embryo+growth+in+Arabidopsis+dormant+seeds.">A seed coat bedding assay shows that RGL2-dependent release of abscisic acid by the endosperm controls embryo growth in Arabidopsis dormant seeds.</a> Proc. 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. 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=Genome-wide+insertional+mutagenesis+of+Arabidopsis+thaliana.">Genome-wide insertional mutagenesis of Arabidopsis thaliana.</a> Science. 301, 653-657 (2003).</li><li>Rook, F., 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=Impaired+sucrose-induction+mutants+reveal+the+modulation+of+sugar-induced+starch+biosynthetic+gene+expression+by+abscisic+acid+signalling.">Impaired sucrose-induction mutants reveal the modulation of sugar-induced starch biosynthetic gene expression by abscisic acid signalling.</a> Plant J. 26, 421-433 (2001).</li></ol>

作者宣称，他们有没有竞争的财务权益。

这里所描述的种皮床上用品法（SCBA）程序原则上适用于其中拟南芥种子发芽受阻（或延迟），并在那里胚乳被怀疑实施这一逮捕任何情况下。后者可通过除去种皮（外种皮和胚乳），并观察到胚胎生长前进速度相对于所观察到的，当胚胎是由种皮包围得以证实。发芽可能被阻止响应于特定环境的物理参数（ 例如 ，水或潜在的光质）或在特定的遗传背景（ 例如，不存在GA合成?...

在拟南芥成熟种子的种皮是由种皮，母体来源的死组织的外层，和胚乳，直接围绕胚胎1活组织的单细胞层。胚乳和胚胎均来自不同的施肥事件:胚乳是三倍体组织有两个产妇和一个父亲的基因组，而胚胎是一个产妇和一个父亲的基因组2二倍体组织。 传统上分配给该胚乳的主要功能是，营养组织。然而，它正变得越来越明显的是，胚乳也起着控制种子发芽的核心作用。这个概念在休眠的情况下成为了第一个明显的，由新产生的种子表现出的特质。休眠种子不能发芽，尽管有利发芽条件的存在。种子成熟期后失去他们的休眠，成为nondormanT， 即它们会发芽，当暴露在有利的条件下催芽。在许多植物物种，其中包括模式植物拟南芥 ，种皮是绝对必要的，以防止休眠种子的萌发，因为种皮去除触发胚胎生长和绿化3,4。在拟南芥中 ，贝思克等人观察到，除去种皮，同时保持周围的胚乳5胚乳萌发后仍然压抑。这些观察结果强烈表明，胚乳，种皮施加的镇压活动在胚胎内的组织。然而，种皮去除的实验并不一定有助于澄清发芽镇压活动由种皮所提供的性质，也不确定实现它的基因。 我们最近推出了种皮床上用品法（SCBA），其中种皮和胚在物理上分开，但保持着密切PROXIMITY使得由胚乳提供的发芽压制性的活性得以维持6。空气呼吸器允许组合使用休眠，nondormant，和转基因种皮和胚料。其结果是，在遗传途径控制发芽并在胚乳和胚具体操作可以被解剖。空气呼吸器被用于休眠的 ​​情况下，显示的胚乳释放激素脱落酸（ABA）对胚胎来抑制其生长6。此外，我们还可以使用空气呼吸器，以确定在胚乳操作的信号通路和胚胎组织，以促进休眠。 胚乳控制发芽的作用考虑承受远红（FR）的光脉冲nondormant种子的情况下得到进一步加强。在种子吸胀初期帧中继光脉冲是已知的抑制发芽7,8。当从种子中除去种皮FR光脉冲无法抑制发芽，强烈的建议，胚乳也可以抑制nondormant 9种子的萌发。值得注意的是，SCBA也可以用来概括发芽的FR-依赖性抑制。这可以表明，种子萌发的FR-依赖性抑制也有来自胚乳9阿坝释放的过程。此外，SCBA允许识别在胚乳和胚来控制nondormant种子萌发响应于光线索9,10操作不同的光信号转导途径。 因此，空气呼吸器似乎是一个可靠的技术来探索种子萌发的控制范围内胚乳的功能。这也是一个强大的工具， 在体外 ，以评估是否涉嫌基因控制发芽的胚乳，胚或两者的组织工作。下面我们详细组装一台空气呼吸器所需的各个步骤。

<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

拟南芥胚乳由周边的成熟胚和播放，以防止休眠种子的萌发，或由远红光（FR）的光脉冲照射nondormant种子的重要作用单细胞层。为了进一步洞察底层由胚乳施加的发芽压制性活性的分子遗传机制，一个"种皮寝具"测定法（SCBA）被设计。空气呼吸器是一种剥离的过程实际上从种子，因此可以监视胚胎的生长种皮的下层上分离种皮和胚。值得注意的是，空气呼吸器重新组成种皮发芽镇压活动在种子休眠和萌发的FR-依赖的控制的情况下。由于空气呼吸器允许组合使用休眠，nondormant和转基因种皮和胚材料的遗传途径控制的萌发和专门运erating在胚乳和胚可以被解剖。下面我们详细的程序来组装一个自给式呼吸器。

以前的工作表明，突变体种子无法合成GA无法发芽，作为高ABA积累在种子11,12的结果。但是，不能发芽，需要种皮，因为它的去除触发胚胎生长13。这有力地表明，种子不能合成赤霉素的胚乳被释放ABA阻止胚胎发育。因此，我们预计种子大衣不能合成GA阻止胚胎在呼吸器的增长不像种皮不能合成赤霉素和脱落酸。这里我们使用GA1-21（Salk的插入线Salk_109115）携带一个T-DNA插入破?...

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

我们目前的程序，从拟南芥种子组装种皮床上用品法（SCBA）。空气呼吸器被证明是一个强大的工具，基因和体外探索胚乳控制种子发芽的种子的休眠和反应轻线索如何。空气呼吸器是适用的原则，在胚乳被怀疑影响胚胎生长任何情况下。

种皮床品检测到基因探索<em&gt;体外</em&gt;如何控制胚乳种子萌发的<em&gt;拟南芥</em

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

a-seed-coat-bedding-assay-to-genetically-explore-vitro-how-endosperm

Research

JoVE Journal

Biology

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

13.4K 次观看.  Université de Genève. 我们目前的程序，从拟南芥种子组装种皮床上用品法（SCBA）。空气呼吸器被证明是一个强大的工具，基因和体外探索胚乳控制种子发芽的种子的休眠和反应轻线索如何。空气呼吸器是适用的原则，在胚乳被怀疑影响胚胎生长任何情况下。 

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

Use of Arabidopsis eceriferum Mutants to Explore Plant Cuticle Biosynthesis

The plant cuticle is a waxy outer covering on plants that has a primary role in water conservation, but is also an important barrier against the entry of pathogenic microorganisms.  The cuticle is made up of a tough crosslinked polymer called "cutin" and a protective wax layer that seals the plant surface.  The waxy layer of the cuticle is obvious on many plants, appearing as a shiny film on the ivy leaf or as a dusty outer covering on the surface of a grape or a cabbage leaf thanks to light scattering crystals present in the wax.  Because the cuticle is an essential adaptation of plants to a terrestrial environment, understanding the genes involved in plant cuticle formation has applications in both agriculture and forestry. Today, we'll show the analysis of plant cuticle mutants identified by forward and reverse genetics approaches.


The plant cuticle is a waxy outer covering on plants that has a primary role in water conservation but is also an important barrier against the entry of pathogenic microorganisms. In this video, we demonstrate the analysis of plant cuticle mutants identified by forward and reverse genetics approaches.

使用拟南芥eceriferum突变体探索植物角质层的生物合成

The plant cuticle is a waxy outer covering on plants that has a primary role in water conservation, but is also an important barrier against the entry of ...

科研

生物学

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA methyltransferases is viable, thus providing a system to study how loss of DNA methylation in a genome affects growth and development. Imprinting refers to differential expression of maternal and paternal alleles and plays an important role in reproduction development in both mammal and plants. DNA methylation is critical for determining whether the maternal or paternal alleles of an imprinted gene is expressed or silenced. In flowering plants, there is a double fertilization event in reproduction: one sperm cell fertilizes the egg cell to form embryo and a second sperm fuses with the central cell to give rise to endosperm. Endosperm is the tissue where imprinting occurs in plants. MEDEA, a SET domain Polycomb group gene, and FWA, a transcription factor regulating flowering, are the first two genes shown to be imprinted in endosperm and their expression is controlled by DNA methylation and demethylation in plants. In order to determine imprinting status of a gene and methylation pattern in endosperm, we need to be able to isolate endosperm first. Since seed is tiny in Arabidopsis, it remains challenging to isolate Arabidopsis endosperm and examine its methylation. In this video protocol, we report how to conduct a genetic cross, to isolate endosperm tissue from seeds, and to determine the methylation status by bisulfite sequencing.

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Imprinting is a phenomenon in plant and mammal reproduction. DNA methylation plays an important role in mechanisms of imprinting. Isolating endosperm and determining methylation status of imprinted genes in Arabidopsis can be difficult. In this protocol, we describe how to isolate endosperm and determine methylation by bisulfite sequencing.

印记基因的DNA甲基化的测定拟南芥胚乳

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA ...

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways1-3.
	Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus4-6. Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.

A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.

一个高含量的成像流程研究Grb2的信号复合体的表达克隆

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light conditions, which are preferably variable in quantity and quality so that studies under different setups can be conducted. These requirements are met by climatic chambers featuring light emitting diodes (LED) lights, which can &#8211; in contrast to fluorescent lights &#8211; be set to different wavelengths. LEDs are energy conserving and emit virtually no heat even at light intensities, which often constitutes a problem with other light sources. The presented protocol provides a step-by-step guidance of how to program a climatic chamber equipped with variable LED lights as well as describing several approaches for in depth analysis of growth phenotypes. Depending on the experimental set-up various characteristics of the growing plants can be observed and analyzed. Here we describe how to determine fresh weight, leaf area, photosynthetic activity, and stomatal density. We demonstrate that in order to obtain reliable data and draw valid conclusions it is mandatory to use a sufficient number of individuals for statistical evaluation. Taking too few plants for this kind of analysis results in high statistical errors and consequently in less clear interpretations of the data.

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Here, we present a protocol for studying plant growth behavior and especially phenotypes in a reproducible manner. We show how to provide variable and at the same time stable light conditions. Proper analyses depend on sufficient sample numbers and valid statistical evaluations.

不同光照条件下的拟南芥生长行为分析

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light ...

A Simple Dry Sectioning Method for Obtaining Whole-Seed-Sized Resin Section and Its Applications

The morphology, size and quantity of cells, starch granules&#160;and protein bodies in seed determine the weight and quality of seed. They are significantly different among different regions of seed. In order to view the morphologies of cells, starch granules and protein bodies clearly, and quantitatively analyze their morphology parameters accurately, the whole-seed-sized section is needed. Though the whole-seed-sized paraffin section can investigate the accumulation of storage materials in seeds, it is very difficult to quantitatively analyze the morphology parameters of cells and storage materials due to the low resolution of the thick section. The thin resin section has high resolution, but the routine resin sectioning method is not suitable to prepare the whole-seed-sized section of mature seeds with a large volume and high starch content. In this study, we present a simple dry sectioning method for preparing the whole-seed-sized resin section. The technique can prepare the cross and longitudinal whole-seed-sized sections of developing, mature, germinated, and cooked seeds embedded in LR White resin, even for large seeds with high starch content. The whole-seed-sized section can be stained with fluorescent brightener 28, iodine, and Coomassie brilliant blue R250 to specifically exhibit the morphology of cells, starch granules, and protein bodies clearly, respectively. The image obtained can also be analyzed quantitatively to show the morphology parameters of cells, starch granules, and protein bodies in different regions of seed.

This technique allows for the fast and simple preparation of whole-seed-sized resin section for the observation and analysis of cells, starch granules, and protein bodies in different regions of the seed.

获得全种子尺寸树脂部分的简单干截块方法及其应用

The morphology, size and quantity of cells, starch granules&#160;and protein bodies in seed determine the weight and quality of seed. They are ...

Analysis of Effect of Compound Salt Stress on Seed Germination and Salt Tolerance Analysis of Pepper (Capsicum annuum L.)

To determine the salt tolerance and physiological mechanism of pepper (Capsicum annuum L.) at the germination stage, the Hongtianhu 101 and Xinxiang 8 varieties, which have large differences in salt tolerance, are employed as the study materials. Six mixed salt concentrations of 0, 3, 5, 10, 15, and 20 g/L derived using equal molar ratios of Na2CO3, NaHCO3, NaCl, CaCl2, MgCl2, MgSO4, and Na2SO4 are used. To determine their effects, the related indexes of seed germination, seedling growth, and physiology are measured, and salt tolerance is comprehensively evaluated using membership function analysis. The results show that as the mixed salt concentration increases, the germination potential, germination index, germination rate, seed germination vigor index, root length, and root fresh weight of the two cultivars significantly decrease, whereas the relative salt rate gradually increases. The hypocotyl length and fresh weight aboveground increase first and then decrease, while the malondialdehyde (MDA), proline (Pro) content, catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) activity decrease and then increase. The germination potential, germination index, germination rate, seed germination vigor index, root length, root fresh weight, MDA and Pro content, and CAT activity of the Hongtianhu 101 seeds are higher than those of Xinxiang 8 for all salt concentrations employed here. However, hypocotyl length, fresh weight aboveground, and relative salt rate are lower in Hongtianhu 101 than in Xinxiang 8. The comprehensive evaluation of salt tolerance reveals that the total weighted values of the two membership function indexes increase first and then decrease as the mixed salt concentration increases. Compared with 5 g/L, which has the highest membership function value, the index under salt concentrations of 3 g/L, 10 g/L, and 15 g/L decreases by 4.7%-11.1%, 25.3%-28.3%, and 41.4%-45.1%, respectively. This study provides theoretical guidance for the breeding of salt-tolerant varieties of pepper and an analysis of the physiological mechanisms involved in salt tolerance and salt-tolerant cultivation.

Analysis of Effect of Compound Salt Stress on Seed Germination and Salt Tolerance Analysis of Pepper Capsicum annuum L.

The paper below presents a protocol for measuring seed germination, seedling growth, and physiological indexes of two pepper varieties with salinity tolerance differences in response to six mixed salt concentrations. This protocol can be used to evaluate the salt tolerance of pepper varieties.

复合盐胁迫对辣椒种子萌发的影响及耐盐性分析

To determine the salt tolerance and physiological mechanism of pepper (Capsicum annuum L.) at the germination stage, the Hongtianhu 101 and Xinxiang 8 ...

通过共聚焦成像对 ERL1 动力学进行时空研究

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

作者聚焦：研究气孔谱系细胞中膜运输事件的基于图像的方法  

Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup.

基于图像的方法研究气孔谱系细胞中的膜运输事件

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane ...

使用 FluoZin-3 测定研究哺乳动物细胞中的锌转运机制

Characterizing Mammalian Zinc Transporters Using an In Vitro Zinc Transport Assay

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured indirectly by determining the expression level of the transporter under different concentrations of Zn2+. This was done by utilizing immunohistochemistry, measuring mRNA in the tissue, or determining the cellular Zn2+ levels. With the development of intracellular Zn2+ sensors, the activities of zinc transporters are currently primarily determined by correlating changes in intracellular Zn2+, detected using fluorescent probes, with the expression of the Zn2+ transporters. However, even today, only a few labs monitor dynamic changes in intracellular Zn2+ and use it to measure the activity of zinc transporters directly. Part of the problem is that out of the 10 zinc transporters of the ZnT family, except for ZnT10 (transports manganese), only zinc transporter 1 (ZnT1) is localized at the plasma membrane. Therefore, linking the transport activity to changes in the intracellular Zn2+ concentration is hard. This article describes a direct way to determine the zinc transport kinetics using an assay based on a zinc-specific fluorescent dye, FluoZin-3. This dye is loaded into mammalian cells in its ester form and then trapped in the cytosol due to cellular di-esterase activity. The cells are loaded with Zn2+ by utilizing the Zn2+ ionophore pyrithione. The ZnT1 activity is assessed from the linear part of the reduction in fluorescence following the cell washout. The fluorescence measured at an excitation of 470 nm and emission of 520 nm is proportional to the free intracellular Zn2+. Selecting the cells expressing ZnT1 tagged with the mCherry fluorophore allows for monitoring only the cells expressing the transporter. This assay is used to investigate the contribution of different domains of ZnT1 protein to the transport mechanism of human ZnT1, a eukaryotic transmembrane protein that extrudes excess zinc from the cell.

作者聚焦:通过 ZnT1 功能探索细胞锌调节 

Zinc transport has proven challenging to measure due to the weak causal links to protein function and the low temporal resolution. This protocol describes a method for monitoring, with high temporal resolution, Zn2+ extrusion from living cells by utilizing a Zn2+ sensitive fluorescent dye, thus providing a direct measure of Zn2+ efflux.

使用 体外 锌转运测定表征哺乳动物锌转运蛋白

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured ...