我们感谢杰西卡 · 哈巴希博士批判性阅读的手稿。我们感谢成像中心，首都师范大学 （中国北京），生命科学学院博士白先勇盛执行dr5 的表达绿色荧光蛋白测定的定位。这项工作由从北京市政政府科学基金会赠款 (CIT &#38; TCD20150102) 和中国国家自然科学基金会 (31600248)。

注: 此协议有三个部分: 1) 观测模式形成在野生型拟南芥胚胎使用 DIC 显微镜;2） 表征胚胎模式形成通过观察标记蛋白的表达，使用激光共聚焦扫描显微镜;和 3） 确定特定基因 （使用naa10突变体为例） 的胚胎发生过程中的作用。
1.胚珠结算
<ol>
	<li>植物材料的制备 注意: 此处所述的议定书 》 是一个例子如何种植健康的植物;其他的方法来生长健康的植物也工作。<ol>
		<li>一 1.5 毫升管，添加 100 种子，然后添加 1 毫升的 1.5%次氯酸钠 （混合 15 毫升 10%氯与 85 毫升的水） 来消毒 5 分钟倒种子管几次，确保种子和溶液充分混合完全消毒表面的种子。</li>
		<li>用离心法为 30 到管底力种子在 1,200 x g s 除去上清，和在一条长凳，以去除残留的次氯酸钠洗的种子用无菌水四倍。</li>
		<li>4.4 g 的村蕃和斯库格 (MS) 的基底盐混合物和 10 克的蔗糖添加 1 L 的超纯水。搅动混合物溶解的盐类和蔗糖。调整到 pH 5.8 用 1 米岛，解 3 g 的凝胶剂，培养基，MS-蔗糖溶液，加上消毒 121 ° C，15 分钟，使 MS 培养基的解决方案。</li>
		<li>30 毫升的 MS 培养基倒入 12 厘米的培养皿，生成 MS 中厚板。</li>
		<li>在 MS 板上放置消毒的种子。将板转移到生长室及在长日条件 (16 h 在光和 18 ° C 8 h 在黑暗中为 22 ° C) 下的板后种子分层 （在 4 ° C，在黑暗中的 3 天）。</li>
		<li>经过 8 天的生长，将植物转移到土壤 （组合富含养分的土壤和蛭石在比例为 1:1） 在一个托盘，涵盖 5 天，以避免水的损失，白色，透明盖纸盒和留下了一个缺口，避免玻璃化冷冻保存的植物。</li>
		<li>把盖子揭开，托盘和培养下长日条件 （见 1.1.5） 步入式室内植物。开灯，在 6:00 上午和关闭在 10:00 下午。大约 20 天之后的增长在步入式会议厅，植物会有花和产生长角果。</li>
		<li>马克花芽与种子中的位置的线程在 8:00 下午茎秆在花序 （不要使用第一到第三的花蕾; 通常从这些花蕾生成的长角果开发几个流产的种子，和这可能会改变正常异常胚珠的比例）。时红的芽将打开，并在 8:00 上午第二天早上花定义授粉的胚珠作为的开始。</li>
	</ol>
	</li>
	<li>霍耶的溶液的制备
	<ol>
		<li>24 g 的水合氯醛 50 毫升管加包装管完全用铝箔 （水合氯醛的解决方案不是稳定在灯光下的）。</li>
		<li>接下来，向上述解决方案中添加 9 毫升纯水和甘油 3 毫升和摇管解散水合氯醛。为了使霍耶的解决方案，让管一夜之间站在室温，消除任何气泡。</li>
	</ol>
	</li>
	<li>胚珠分离和清
	<ol>
		<li>将一块双面压敏胶带附加到显微镜载玻片。放置角果生长所需数目的幻灯片与垂直于表面; pseudoseptum (DAP) 授粉后天数这将使它容易拆分心皮 （图 1A和1B）。</li>
		<li>用针头劈开 pseudoseptum 两边都心皮和附加两个解剖心皮到幻灯片，以便在角果胚珠都可见镜下获得立体 （数字 1和1 D）。</li>
		<li>免去 70 微升的玻片，霍耶的溶液，然后用来蘸滋润一把尖头镊子 Hoyer 解决方案一角。使用立体，进入胚珠霍耶的解决方案在幻灯片上用镊子清洁的胚胎。应用于幻灯片轻轻地以避免产生任何气泡的盖玻片。</li>
		<li>把成品的滑入一个框，在黑暗的房间里为 2 ~ 12 小时 （越成熟胚珠，将需要更多的时间） 在室温。(1-2 DAP) 2/4-细胞阶段的胚胎可以后 2 h，显微镜下观察到，虽然可以在 4 小时后观察胚胎八区和球形早期 (3 DAP) 之间。更成熟胚 (4-8 DAP) 可以在 12 小时后观察。</li>
		<li>检查使用的显微镜 DIC 函数清除胚珠。在低功耗领域 (10 X) 下找到胚胎，然后更改到一个大功率的字段 （100 X 油浸没透镜） 以观察中发达国家胚胎的细胞模式。</li>
		<li>检查胚胎发育不同阶段。</li>
	</ol>
	</li>
</ol>
2.胚胎模式和通过观察标记蛋白表达细胞身份的表征
<ol>
	<li>植物材料的制备
	<ol>
		<li>克隆启动子和编码序列的标记基因或激素反应元件，然后将其融合与荧光蛋白 （ GFP); 如接下来，插入 （如 pCambia1300) 的二进制向量的构造。在这里，生长素反应元件dr5 的表达是提出了一种作为示例10。</li>
		<li>引入野生型植物根瘤农杆菌介导产生GFP dr5 的表达质粒-介导转化11。选择使用 MS 中厚板含潮霉素的 t1 代植株 （50 毫克/毫升的母液稀释 1:1，000）。转基因植物会表现出长根和经过 8 天的生长 1.1.5 中所述的条件下生成两个莲座丛叶。</li>
		<li>将转基因植物转移到土壤和培养他们如 1.1.6 和 1.1.7 所示。</li>
		<li>从独立的 T1 线路收集 T2 种子。播下的种子在 MS 潮霉素板上，选择适宜的植物，携带一份dr5 的表达绿色荧光蛋白插入 （敏感植物的抗性植株的比例将大约 3:1），和将他们转移到土壤。</li>
		<li>培养 T2 植物如 1.1.6 和 1.1.7 所示。</li>
		<li>从 T2 植物收集 T3 种子。播下的种子在 MS 潮霉素板上，选择纯合的转基因植物 （所有的植物从一行将展示潮霉素抗性），并将他们转移到土壤。</li>
		<li>1.1.8 所示，将标记的纯合子的 T3 植物花蕾。</li>
	</ol>
	</li>
	<li>绿色荧光蛋白信号观察
	<ol>
		<li>用来生成 6%甘油溶液超纯水 47 毫升混合 3 毫升的甘油。</li>
		<li>将一块双面压敏胶带附加到玻片和修复角果 1.3.1 所示。</li>
		<li>用针头劈开 pseudoseptum 两边都心皮和附加两个解剖心皮向下立体幻灯片 1.3.2 所示。</li>
		<li>免除 30 微升的 6%甘油到玻璃幻灯片并将所有从解剖心皮胚珠放入使用尖头镊子的解。轻轻地将盖玻片放在幻灯片上。</li>
		<li>种皮和胚乳中每个胚珠可以防止观察 GFP 信号在胚胎中，提取胚胎从胚珠。点击幻灯片快速、 轻轻挤压胚胎从胚珠 （越成熟胚珠是较少力量应该应用到幻灯片）。</li>
		<li>检查为 GFP 荧光共焦激光扫描显微镜使用幻灯片。在低功耗领域 (10 X) 下找到离体的胚和记录他们的位置，然后更改到一个大功率的字段 （100 X 油浸没透镜） 以观察dr5 的表达绿色荧光蛋白表达在胚胎通过将激发光源设置为 488 nm 的荧光信号检测。</li>
		<li>在胚胎来选择合适的 T3 线作进一步的分析检测 GFP 表达。</li>
	</ol>
	</li>
</ol>
3.通过胚珠间隙和标记蛋白观察胚胎致死突变体胚胎模式形成的表征: Naa10为例
<ol>
	<li>胚胎阵列的naa10突变体植株
	<ol>
		<li>准备的植物材料，1.1.1-1.1.7 所示。</li>
		<li>确定naa10+ −由 PCR 基因特异性引物5，然后标记植物花蕾的naa10+ −植物 1.1.8 所示。</li>
		<li>执行胚珠间隙和镜检的naa10+ −胚胎如 1.2 和 1.3 所示。</li>
	</ol>
	</li>
	<li>使用naa10胚胎的标记蛋白质分析
	<ol>
		<li>获得使用 2.1-2.2 所述程序适合dr5 的表达绿色荧光蛋白转基因株系。</li>
		<li>越界纯合子dr5 的表达绿色荧光蛋白转基因与naa10+ −工厂获得naa10+ −植物是纯合型 GFP 荧光标记在 F2 代 （标识基因特异性聚合酶链反应） 来表征naa10+ −5和观察 GFP 信号使用纯合子DR5 GFP行显微镜下的。</li>
		<li>标记的naa10 花蕾+ − DR5 GFP 2.1.7 所示的纯合的植物。</li>
		<li>搜索DR5 GFP 2.2 所示的纯合的naa10胚胎的绿色荧光蛋白信号。</li>
	</ol>
	</li>
</ol>

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作者没有透露。

胚珠间隙是种有用的检测细胞分裂和评估拟南芥; 在胚胎发生过程中的形态学方法使用这种技术，可以直接在 DIC 显微镜5，12下观察到胚。胚珠间隙关键的一步是一步 1.3.4。胚珠间隙 1.3.4 步骤中所需的时间是变量。2/4-细胞阶段的胚胎可以清楚地观察后 2 h 在霍耶的解决方案中，，而在 12 小时后，他们看隐隐绰绰。因此，越成熟的胚珠是，清关所?...

胚胎发育是中高等植物发育最早的事件。成熟胚的形式从一个受精卵通过细胞分裂和分化在严格的遗传控制1，2下。拟南芥胚胎是一个有用的模型，研究形态发生的控制，因为胚胎发生过程中细胞分裂的顺序遵循预期的模式3，4。然而，包含一到多个单元格拟南芥胚是太小了，观察和分析。此外，某些基因的突变会导致胚胎致死5，指示这些基因在胚胎发生过程中的关键作用。在胚胎致死突变模式形成的表征为理解藉以必需基因调节胚胎发育的分子机制提供了依据。
详细的方法在这里描述为表征的模式植物拟南芥胚胎模式形成由直接的微观观察。该方法涉及然后微观观察的胚胎的胚珠间隙。此外介绍胚胎致死突变体的特性。
不同发育阶段的胚胎的形态学可以直接测定显微镜与霍耶的解决方案6，7使用已被清除的胚珠。这种胚珠表现出高的折射率;因此，这胚珠结算方法是特别有利的必须遵守微分干涉显微镜 (DIC) 的标本。
此外，基因在胚胎发育过程中都是在一个特定的细胞或细胞数量有限表示可以用作标记来识别中的胚胎细胞。因此，胚胎发生过程中的细胞标识规范可以注明通过监视使用共聚焦激光扫描显微镜的 GFP 标记的标记蛋白的表达。相反，观察未知功能基因GFP融合胚胎发生过程中的表达模式可提供胚胎模式与该基因的表达之间的联系及促进所需的拟南芥胚胎发育的新基因的鉴定。
描述表型的胚胎致命的突变体，并确定受影响的基因在胚胎发生在细胞水平上的影响，也可以使用这里介绍的方法。此外，结合比较的标记基因的表达模式之间野生型与突变型植物胚胎发生过程中，该方法可以产生分子机制和信号传导通路在胚胎发生8，9的线索。事实上，该方法被应用于naa10植物，它被发现的突变体中垂体异常分裂可能引起胚胎发生5年间，生长素分布的变化。

<table><tbody><tr><td>Chloral Hydrate</td><td>Sigma</td><td>15307</td><td></td></tr><tr><td>Murashige and Skoog Basal Medium</td><td>Sigma</td><td>M5519</td><td></td></tr><tr><td>Phytagel</td><td>Sigma</td><td>P8169</td><td></td></tr><tr><td>Hygromycin</td><td>Roche</td><td>10843555001</td><td></td></tr><tr><td>Growth chamber</td><td>Percival</td><td>CU36L5</td><td></td></tr><tr><td>Fluorescence microscope</td><td>Zeiss Oberkochen</td><td>Zeiss Image M2</td><td>camera: AxioCam 506 color</td></tr><tr><td>Confocal Laser Scanning Microscopy</td><td>Zeiss Oberkochen</td><td>Zeiss LSM 5</td><td>filters: BP 495 - 555 nm</td></tr><tr><td>Stereoscope</td><td>Zeiss Oberkochen</td><td>Axio Zoom V16</td><td>camera: AxioCam MRc5</td></tr><tr><td>nutrient-rich soil</td><td>KLASMANN, Germany</td><td></td><td></td></tr><tr><td>50-mL tube</td><td>Corning Inc.</td><td></td><td></td></tr><tr><td>1.5-mL tube</td><td>Corning Inc.</td><td></td><td></td></tr><tr><td>10% sodium hypochlorite solution</td><td></td><td></td><td>Domestic analytical reagent</td></tr><tr><td>sucrose</td><td></td><td></td><td>Domestic analytical reagent</td></tr><tr><td>KOH</td><td></td><td></td><td>Domestic analytical reagent</td></tr><tr><td>glycerol</td><td></td><td></td><td>Domestic analytical reagent</td></tr><tr><td>culture dish</td><td></td><td></td><td>Domestic supplies</td></tr><tr><td>vermiculite</td><td></td><td></td><td>Domestic supplies</td></tr><tr><td>glass slide</td><td></td><td></td><td>Domestic supplies</td></tr><tr><td>syringe needle</td><td></td><td></td><td>Domestic supplies</td></tr><tr><td>fine-tipped tweezers</td><td></td><td></td><td>Domestic supplies</td></tr><tr><td>super clean bench</td><td></td><td></td><td>Domestic equipment</td></tr></tbody></table>

a method for characterizing embryogenesis in arabidopsis

给出的发展高度可预测性，拟南芥胚胎有被用作模型研究植物的形态发生。然而，早期植物胚胎很小，包含几个细胞，使它们难以观察和分析。一种方法在这里描述中使用的模式生物拟南芥在显微镜下的植物胚胎模式形成。之后使用 Hoyer 解决方案的新鲜胚珠的间隙，可以观察胚胎形态学和细胞数目中的，和他们发育阶段可以由微分干涉相衬显微镜使用 100 X 油浸没透镜。此外，标记与绿色荧光蛋白 (GFP) 的特异性标记蛋白的表达被监视批注单元格标识规范期间胚胎模式通过共聚焦激光扫描显微镜。因此，可以使用此方法，观察野生型植物胚胎细胞和分子层面的模式形成和通过比较模式形成的胚胎从野生型植物和胚胎致死突变表征特定基因在胚胎模式中的作用。因此，该方法可以用于表征在拟南芥胚胎发生。

在本文中描述的方法可以用于观察直接在显微镜下的胚胎发生、 带有特定标记，注释胚胎发生过程中的细胞标识规范和胚胎发育过程描述特定基因的作用。
代表分析结果得到的胚胎模式 （从细长的受精卵阶段到成熟的手杖阶段） 在野生型拟南芥如图 2所示。胚珠过关后不同发育阶段的胚胎可分辨...

Watch this Scientific Journal Video about A Method for Characterizing Embryogenesis in Arabidopsis at JoVE.com

A Method for Characterizing Embryogenesis in Arabidopsis

本议定书概述了观察胚胎发生的但在拟南芥中通过依次检查在显微镜下胚胎模式形成的胚珠间隙的方法。

表征在拟南芥胚方法

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, ...

a-method-for-characterizing-embryogenesis-in-arabidopsis

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

A Method for Characterizing Embryogenesis in Arabidopsis

11.0K 次观看.  Capital Normal University. 本议定书概述了观察胚胎发生的但在拟南芥中通过依次检查在显微镜下胚胎模式形成的胚珠间隙的方法。

视频: A Method for Characterizing Embryogenesis in Arabidopsis

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by pre-globular, globular, heart, torpedo and cotyledon stages. This progressive development is under the tight regulation of a complex molecular network. Harvesting sufficient early embryos at a similar stage of development is essential for investigating the cellular and molecular regulation of early embryogenesis. This is not straightforward since early embryogenesis undergoes rapid morphogenesis in a short while e.g. 8 days for Medicago truncatula to reach the early cotyledon stage. Here, we address the issue by two approaches. The first one establishes a linkage between embryo development and pod morphology in helping indicate the stage of the zygotic embryo. This is particularly based on the number of pod spirals and development of the spines. An alternative way to complement the in vivo studies is via culturing leaf explants to produce somatic embryos. The medium includes an unusual hormone combination - an auxin (1-naphthaleneacetic acid), a cytokinin (6-benzylaminopurine), abscisic acid and gibberellic acid. The different stages can be discerned growing out of the callus without dissection.

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

The goal is to illustrate that the model legume Medicago truncatula can be readily utilized to investigate the regulation of early plant embryogenesis to complement the non-legume Arabidopsis model. Pod morphology is linked to zygotic embryogenesis stages and a protocol to collect embryos using tissue culture is also provided.

协议获得合子和体细胞胚胎研究早期胚胎发育的调控中的示范豆科<em&gt;苜蓿</em

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by ...

科研

发育生物学

Live Confocal Imaging of Developing Arabidopsis Flowers

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. Recent advances in confocal microscopy, combined with the development of numerous fluorophores, have overcome these issues and opened the possibility to study the expression of several genes simultaneously, with a good cellular resolution, in live samples. Live confocal imaging provides plant biologists with a powerful tool to study development, and has been extensively used to study root growth and the formation of lateral organs on the flanks of the shoot apical meristem. However, it has not been widely applied to the study of flower development, in part due to challenges that are specific to imaging flowers, such as the sepals that grow over the flower meristem, and filter out the fluorescence from underlying tissues. Here, we present a detailed protocol to perform live confocal imaging on live, developing Arabidopsis flower buds, using either an upright or an inverted microscope.

Live confocal imaging provides biologists with a powerful tool to study development. Here, we present a detailed protocol for the live confocal imaging of developing Arabidopsis flowers.

住发展拟南芥花的共焦成像

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

体外在拟南芥中受精卵分化和胚模式的活体细胞成像的胚珠培养

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

半高通量成像方法和数据可视化工具包，用于分析C. elegans胚胎发育

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome affinity purification (TRAP) method and consecutive optimized low-input library preparation.
As starting material, we employ plant lines that express GFP-tagged ribosomal protein RPL18 in a cell type-specific manner by use of adequate promoters. Prior to immunopurification and RNA extraction, the tissue is snap frozen, which preserves tissue integrity and simultaneously allows execution of time series studies with high temporal resolution. Notably, cell wall structures remain intact, which is a major drawback in alternative procedures such as fluorescence-activated cell sorting-based approaches that rely on tissue protoplasting to isolate distinct cell populations. Additionally, no tissue fixation is necessary as in laser capture microdissection-based techniques, which allows high-quality RNA to be obtained.
However, sampling from subpopulations of cells and only isolating polysome-associated RNA severely limits RNA yields. It is, therefore, necessary to apply sufficiently sensitive library preparation methods for successful data acquisition by RNA-seq.
TRAP offers an ideal tool for plant research as many developmental processes involve cell wall-related and mechanical signaling pathways. The use of promoters to target specific cell populations is bridging the gap between organ and single-cell level that in turn suffer from little resolution or very high costs. Here, we apply TRAP to study cell-cell communication in lateral root formation.

Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Translating ribosome affinity purification (TRAP) offers the possibility to dissect developmental programs with minimal processing of organs and tissues. The protocol yields high-quality RNA from cells targeted with a green fluorescent protein (GFP)-labeled ribosomal subunit. Downstream analysis tools, such as qRT-PCR or RNA-seq, reveal tissue and cell type-specific expression profiles.

翻译核糖体亲和纯化（TRAP）以研究细胞类型特异性尺度下阿拉伯生物基塔利亚纳根发育

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

调查组织和器官特定的光敏色素反应，使用流式细胞仪辅助细胞类型特定的表达谱拟南芥</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

生物学

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

从长期来看，高解析度激光共聚焦时间流逝成像<em&gt;拟南芥子叶</em&gt;表皮萌发过程中

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

高效，快速的早期胚胎分离<em&gt;拟南芥</em&gt;种子

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

定量，单细胞在全安装胚珠在染色质修饰和核体系结构分析的有效方法<em&gt;拟南芥</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant productivity and survival. As such, understanding the mechanisms by which stomata develop and pattern has tremendous agronomic value. This paper describes two phenotypic methods using Arabidopsis cotyledons that can be used to characterize the genes controlling stomatal development and patterning. Presented first are procedures for analyzing the stomatal phenotypes using toluidine blue O-stained cotyledons. This method is fast and reliable and does not require the use of epidermal peels, which are widely used for phenotypic analyses but require specialized training. Due to the presence of multiple cysteine residues, the identification and generation of bioactive EPF peptides that have a role in stomatal development have been challenging. Thus, presented second is a procedure used to identify stomatal ligands and monitor their biological activity by bioassays. The main advantage of this method is that it produces reproducible data relatively easily while reducing the amount of peptide solution and the time required to characterize the role of the peptides in controlling stomatal patterning and development. Overall, these well-designed protocols enhance the efficiency of studying the potential stomatal regulators, including cysteine-rich secretory peptides, which require highly complex structures for their activity.

Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring

This paper describes two phenotyping methods without the use of epidermal peels to characterize the genes controlling stomatal development. The first method demonstrates how to analyze a stomatal phenotype using a toluidine blue O-stained plant epidermis. The second method describes how to identify stomatal ligands and monitor their biological activities.

通过表皮表型评分 鉴定 参与气孔发育的基因

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant ...

表征在拟南芥胚方法

摘要

探索更多视频

此视频中的章节

协议获得合子和体细胞胚胎研究早期胚胎发育的调控中的示范豆科

住发展拟南芥花的共焦成像

体外在拟南芥中受精卵分化和胚模式的活体细胞成像的胚珠培养

半高通量成像方法和数据可视化工具包，用于分析C. elegans胚胎发育

翻译核糖体亲和纯化（TRAP）以研究细胞类型特异性尺度下阿拉伯生物基塔利亚纳根发育

调查组织和器官特定的光敏色素反应，使用流式细胞仪辅助细胞类型特定的表达谱拟南芥

从长期来看，高解析度激光共聚焦时间流逝成像

高效，快速的早期胚胎分离

定量，单细胞在全安装胚珠在染色质修饰和核体系结构分析的有效方法

通过表皮表型评分鉴定参与气孔发育的基因

表征在拟南芥胚方法

摘要

探索更多视频

此视频中的章节

协议获得合子和体细胞胚胎研究早期胚胎发育的调控中的示范豆科

住发展拟南芥花的共焦成像

体外在拟南芥中受精卵分化和胚模式的活体细胞成像的胚珠培养

半高通量成像方法和数据可视化工具包，用于分析C. elegans胚胎发育

翻译核糖体亲和纯化（TRAP）以研究细胞类型特异性尺度下阿拉伯生物基塔利亚纳根发育

调查组织和器官特定的光敏色素反应，使用流式细胞仪辅助细胞类型特定的表达谱拟南芥

从长期来看，高解析度激光共聚焦时间流逝成像

高效，快速的早期胚胎分离

定量，单细胞在全安装胚珠在染色质修饰和核体系结构分析的有效方法

通过表皮表型评分 鉴定 参与气孔发育的基因

通过表皮表型评分鉴定参与气孔发育的基因