蒙哥马利实验室工作，在植物中的光敏反应是支持由美国国家科学基金会（赠款。MCB - 0919100，以博莱霉素）和化学科学，地球科学和生物科学部，基础能源科学办公室，科学办公室，美国能源部能源（授予没有。DE FG02 91ER20021以博莱霉素）。我们感谢技术援助梅利莎惠特克在拍摄过程中，批判地阅读手稿，实验的援助，协助发展中国家和优化用共聚焦荧光激活细胞分选的援助拟南芥原生质体排序博士和梅林达框架协议的路易王博士斯蒂芬妮Costigan显微镜。我们感谢马琳卡梅伦图形设计援助和Karen鸟社论援助。

1。植物生长<ol><li>证实UAS -超视距X GAL4 - GFP增强陷阱线隔离4（对于总结见图1）和野生型或亲土壤播种，即〜2000年，每行消毒的种子。 </li><li>植物生长5周100μmolm-2 s - 1时的白色照明下对土壤在22℃，湿度70％ 。 </li></ol> 2。叶原生质体分离（改编自9 Denecke和Vitale的） <ol><li>为了分离原生质体，准备TEX的缓冲区。对于1公升TEX的缓冲区，掂量出了以下组件：3.1克Gamborg的B5的盐，0.5 G 2 - （N -吗啉）ethanesulfonic酸（MES，2.56 mM的），0.75克钙氯化物二水（氯化钙2 .2 H 2 Ø，6.75毫米），0.25克的硝酸铵（NH 4 NO 3，3.12毫米），136.9克的蔗糖（0.4M）。溶解组件完全〜900毫升的去离子水，蒸馏水（DDH 2）和带1至5.7的pH值中号氢氧化钾。使最终体积为1升和0.2微米瓶顶过滤连接真空泵过滤消毒。 </li><li>收集绿色，健康的植物的叶子（〜250毫升的树叶在烧杯中松散包装）〜40毫升DDH 2 O 4次冲洗，随后用无菌DDH 2 O冲洗两次使用＃20手术刀，切叶，切成细条，平分到两个50毫升的无菌塑料管组织。准备加入5毫升分装10X叶消化库存解决方案（10X叶消化库存解决方案的1X叶消化混合溶于2％W / V Macerozyme的R - 10，4％W / V“Onozuka”纤维素酶在TEX缓冲的R - 10，过滤消毒用0.2微米的过滤器和冻结5毫升分装于-80 ° C）45毫升新鲜的TEX缓冲区。新增〜25毫升1X叶，每50毫升的管覆盖所有叶片组织消化混合。 </li><li>真空渗透在开放的管叶组织，在消化的混合物在室温下用真空干燥器连接到一台水泵，在室温下孵育3小时上轻轻摇动摇杆为1小时。回报上限的叶组织1X叶消化混合管保持在铝箔包裹在此过程中，以防止暴露在光线下。 </li><li>经过3个小时，增加1〜2分钟释放原生质体的摇杆速度。原油原生质体悬浮液通过两层无菌纱布过滤，清除杂物，并在无菌的玻璃烧杯收集滤液。通过无菌100微米尼龙网过滤，滤液进入无菌培养皿。收集流过，并转移到一个新的无菌50毫升管。约15-20毫升新鲜TEX的缓冲区，使用洗无菌培养皿中，并收集任何原生质体坚持表面。 </li><li>离心机的流量，通过使用100xg摆动斗转子在10℃15分钟（6加速，减速0）彗星。 </li><li>删除〜25 - 30毫升液体低于浮动原生质体层，其中含有残留和颗粒碎片，用无菌9“玻璃巴斯德吸管连接互不干扰浮动原生质体层和离开〜10蠕动泵 - 15毫升量。 </li><li>补充新鲜TEX缓冲区的最后一个40毫升的量，一边轻轻resuspending的原生质体。 </li><li>重复步骤2.5至2.7的两倍，以去除尽可能多的细胞碎片。离心时间缩短至10分钟，在第一次重复，并在最后重复5分钟。 </li><li>削减，到一个新的15毫升管1毫升无菌移液管吸浮原生质体。直接进行排序或在黑暗的存储长达2个小时在4℃，直到排序彗星。 </li></ol> 3。原生质体的排序由荧光激活细胞分选（FACS） <ol><li>排序之前，检查原生质体激光共聚焦显微镜（CLSM）使用488纳米的激光激发确认原生质体的完整性和绿色荧光的原生质体池的存在。最小的碎片是必要的，以避免堵塞流式细胞仪排序喷嘴。 </li><li>排序分离出原生质体在TEX缓冲区通过流式细胞仪（BD FACSVantage SE，BD Biosciences公司）与适应协议10磅约9的系统压力，使用200微米的喷嘴事件的发生率在6000和15000之间的宏观排序头。 </li><li>野生型非GFP原生质体是用来确定的自体荧光阈值。 </li><li>为了收集GFP阳性的原生质体，排序使用488纳米氩线的空气冷却的氩激光（光谱物理模型177，新港公司，加利福尼亚州欧文）在100毫瓦经营，使用的三十分之五百三十频段，以确定GFP荧光的细胞通滤波器。 </li><li> GFP阳性原生质体的集合，检查CLSM详细步骤3.1排序原生质体。 </li><li>从排序使用Qiagen公司的RNeasy植物试剂盒的原生质体中提取总RNA。然后可以准备杂交基因Affymetrix基因芯片Expr之间ession分析技术手册“和”杂交ATH1 Affymetrix的拟南芥全基因组阵列。 </li></ol>代表性的成果我们发现了大量GFP阳性细胞，用流式细胞仪（图2）的绿色荧光蛋白通道。在使用GFP增强子陷阱家长，组成表达GFP的线，J0571，显示〜17％至24％的GFP阳性的原生质体，而与血管和皮肤的表达，J1071，〜1.4％GFP阳性的原生质体的优化分析（见表1）。 3 × 500μLJ0571的原生质体（〜1.5毫升的总原生质体悬浮）排序了1小时〜10万的GFP阳性的原生质体。 3 × 500μLJ1071的原生质体（1〜1.5毫升总原生质体悬浮液）的排序为1.5小时，给〜3000 GFP阳性的原生质体。共聚焦显微镜图像显示为J0571和J1071样品原生质体非常高的收益率，然后再排序进行了（图3C和3E），并排序分数中唯一明亮的绿色荧光蛋白荧光的原生质体（图3G和数据未显示）。这一发现证实了GFP阳性的，完整的原生质体可通过流式细胞仪进行排序。非GFP的原生质体，也可以进行排序和收集在一个单独的通道。非GFP的原生质体作为一个理想的后续芯片分析阴性对照检测后在基因表达的具体变化，减少holophytochrome水平。 RNA提取原生质体（1毫升）RNA的检测fluorospectrometry（图4）有足够数量的产量。分离出的RNA使用NanoDrop仪（Thermo Scientific的NanoDrop 1000）进行了评估和量化NanoDrop 3.7.1 RNA定量软件。 RNA的产量从预先排序C24的野生型原生质体或流式细胞仪分选GFP阳性增强陷阱原生质体超过使用所需的最低20吴标签RNA检测微阵列（见表2）。 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td>增强陷阱线</td><td> GFP阳性原生质体的％ </td><td>排序的GFP原生质体</td></tr><tr><td> J0571 </td><td> 17.18％〜24.06％ </td><td> 26400〜36000 </td></tr><tr><td> J1071 </td><td> 〜1.43％ </td><td> 1000〜1300 </td></tr></tbody></table> 表1。两个增强子陷阱排序和运行500原生质体悬浮液通过荧光激活细胞分选（FACSVantage，BD）收集的GFP阳性原生质体的数量前行的GFP阳性原生质体的百分比。 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td>厂线</td><td> RNA产量（吴） </td></tr><tr><td> C24的WT </td><td> 12486.5 </td></tr><tr><td> J0571 </td><td> 60 </td></tr><tr><td> J1071 </td><td> 71.5 </td></tr></tbody></table> 表2。产量从原生质体RNA隔离。预先排序的C24的野生型或排序从两个增强子陷阱线GFP阳性原生质体RNA的提取和量化。 <img src="/files/ftp_upload/1925/1925fig1.jpg" alt="图1" /> 图1。GAL4增强子陷阱的胆绿素还原酶（BVR）在转基因拟南芥植物中的表达诱导。 （一）。 GAL4基于增强陷阱线，其中包含一个GAL4反应GFP标记基因，从库中选择一个个人可以跨过一个含有GAL4反应的靶基因，诱导靶基因的表达行GAL4含细胞标记GFP荧光。从吉姆博士Haseloff（http://www.plantsci.cam.ac.uk/Haseloff/geneControl/GAL4Frame.html）图的基础上。 （b）生产的光敏色素生色，phytochromobilin（PΦB）和holophytochrome父行。 （c）留，分别由胆绿素还原酶（BVR）活动，BR和PΦR，胆绿素IXα（BVIXα）和PΦB减少。 PΦB枯竭超视距活动的结果，并导致在减少生产的光敏holophytochrome。右，超视距催化反应。 <img src="/files/ftp_upload/1925/1925fig2.jpg" alt="图2" /> 图2。荧光激活细胞分选（FACS）收购点图。 C24的野生型（A），J0571（B）和J1071（三）原生质体细胞的比较排序。 （a）收购非GFP的荧光C24野生型激发488 nm的氩离子激光和用于确定自体荧光门槛的原生质体的散点图。 （B）和（c）收购点图显示原生质体的488 nm激光的响应激发GFP的阳性比例。 R3的排序盖茨在B和C划定GFP阳性排序荧光激活细胞分选（FACSVantage，BD）和收集的目标。红色通道表示v叶绿素从原生质体的自体荧光和绿色通道alues​​表明GFP荧光的值。 <img src="/files/ftp_upload/1925/1925fig3.jpg" alt="图3" /> 图3：用于细胞分选的植物原生质体共聚焦显微镜。共聚焦激光扫描前（A，C，E），后（G）的排序通过荧光激活细胞分选（FACS）原生质体的图像。 B，D，F和H DIC的图像。 C24野生型（A，B），（C，D）的J1071和J0571（E，F，G，H）的原生质体。图片C，E和G是合并后的GFP荧光（BP 505纳米 - 575纳米）和自体荧光（LP 650纳米）从488 nm激光激发获得的图像。 A到H的平均4下63X石油扫描。酒吧= 10微米。 <img src="/files/ftp_upload/1925/1925fig4.jpg" alt="图4" /> 图4由fluorospectrometry原生质体分离出的RNA定量。 （一）C24的野生型，（二）J0571，和（C）J1071 RNA提取。 A表示预排序的野生型原生质体的RNA。 B和C表示从荧光激活细胞分选（FACS）排序GFP阳性原生质体的RNA。 RNA定量分析软件，NanoDrop 3.7.1（Thermo Scientific的NanoDrop 1000）。

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（1）通过微阵列基因表达分析表明，超过30％的基因在拟南芥幼苗中轻监管 11（2）已经确定了浩瀚的基因组编码的光信号转导有关的蛋白质的光敏色素信号级联12 ，13 。这样的实验表明，光诱导基因表达的快速和长期的变化。每个池的光敏色素，可控制的发展和适应性反应的一个子集。此外，它可能是下游的信号元件，在细胞和组织特异性的方式2，14，15中的光敏色素互...

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<tr>
<td>Anti-BVR antibody</td>
<td>&nbsp;</td>
<td>QED Bioscience Inc.</td>
<td>56257-100</td>
<td>&nbsp;</td>
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<td>Cellulase &#x201C;Onozuka&#x201D; R-10</td>
<td>&nbsp;</td>
<td>SERVA Electrophoresis GmbH, Crescent Chemical Company</td>
<td>MSPC 0930</td>
<td>&nbsp;</td>
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<td>Gamborg&#x2019;s B5 basal salt mixture</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G5768</td>
<td>&nbsp;</td>
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<td>Macerozyme R-10</td>
<td>&nbsp;</td>
<td>SERVA Electrophoresis GmbH, Crescent Chemical Company</td>
<td>PTC 001</td>
<td>&nbsp;</td>
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<td>MES, low moisture content</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M3671</td>
<td>&nbsp;</td>
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<td>Murashige and Skoog salts</td>
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<td>Caisson Laboratories</td>
<td>74904</td>
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<td>Phytablend</td>
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<td>Caisson Laboratories</td>
<td>28302</td>
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<td>RNeasy Plant Minikit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
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investigating tissue- and organ-specific phytochrome responses using facs-assisted cell-type specific expression profiling in arabidopsis thaliana

阵列的光介导植物的整个生命周期的发展和适应过程。植物利用光吸收分子，称为光感受器感知和适应光线。红/远红光的光吸收光敏感光器已被广泛的研究。光敏色素存在，在所有高等植物系统的独特和重叠的职能中，他们已经研究了蛋白质与家庭<sup&gt; 1</sup&gt;。光敏色素介导的光反应，从种子发芽到开花和衰老的范围，往往是本地化特定的植物组织或器官<sup&gt; 2</sup&gt;。尽管通过突变分析发现和澄清个人和冗余的光敏功能，photoperception和本地化池的光敏色素的分子机制不同地点的结论性报告，调解空间特定的光敏反应是有限的。我们设计实验的基础上的假设，调节组织器官特异性和光形态方面，而且本地化的光敏池从事细胞 - 细胞信号转导下游靶基因的不同的子集的特定网站的光敏photoperception。我们开发了一种生化方法选择性地减少在器官或组织特异性的方式在转基因植物的功能光敏。我们的研究是基于二部的增强子陷阱的方法，在上游激活序列（UAS）的元素的控制之下的一个基因的表达，反式转录激活GAL4<sup&gt; 3</sup&gt;。胆绿素还原酶（<em&gt;超视距</em&gt;默默地保持在GAL4式激活UAS -超视距母公司的情况下）根据无人机的控制基因<sup&gt; 4</sup&gt;。一个UAS超视距转线和一个GAL4 - GFP增强陷阱的具体表达结果之间的遗传杂交<em&gt;超视距</em&gt;在标记细胞的基因<em&gt;绿色荧光蛋白</em&gt;表达式<sup&gt; 4</sup&gt;。超视距积累在拟南芥植物光敏色素生色团缺乏的结果<em&gt;在植物</em<sup&gt; 5-7</sup&gt;。因此，我们制作了展品GAL4激活依赖的转基因植物<em&gt;超视距</em&gt;基因，导致光敏生化灭活，以及GAL4依赖<em&gt;绿色荧光蛋白</em&gt;表达式。光生物和分子遗传学分析<em&gt;超视距</em&gt;转基因株系产生洞察到组织和器官特异性的光敏色素介导，已与相应的网站photoperception相关的的反应<sup&gt; 4，7，8</sup&gt;。荧光激活细胞分选（FACS）GFP阳性，增强子陷阱诱导<em&gt;超视距</em&gt;表达植物原生质体细胞类型特异性基因通过表达谱芯片分析加上正在使用，以确定参与调解空间特定的光敏反应的假定下游靶基因。这项研究是扩大我们的网站光感的理解，通过光调控植物生长和发育的各种组织或器官的合作，推进复杂的光敏色素介导的分子清扫细胞 - 细胞信号传导机制。

Watch this Scientific Journal Video about Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana at JoVE.com

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

空间特定的光敏反应的分子基础正在调查中使用，表现出组织和器官特异性的光敏色素的不足的转基因植株。参展隔离特定的细胞芯片分析排序，是用来识别在特定空间的光敏反应有关的基因的荧光激活细胞诱导的光敏色素生色枯竭。

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 ...

investigating-tissue-organ-specific-phytochrome-responses-using-facs

Research

JoVE Journal

Biology

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

15.7K 次观看.  Michigan State University (MSU). 空间特定的光敏反应的分子基础正在调查中使用，表现出组织和器官特异性的光敏色素的不足的转基因植株。参展隔离特定的细胞芯片分析排序，是用来识别在特定空间的光敏反应有关的基因的荧光激活细胞诱导的光敏色素生色枯竭。

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

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

花香浸改造拟南芥检查 pTSO2：β-葡萄糖醛酸酶报告基因的表达

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

科研

生物学

Heterokaryon Technique for Analysis of Cell Type-specific Localization

A significant number of proteins are regulated by subcellular trafficking or nucleocytolasmic shuttling. These proteins display a diverse array of cellular functions including nuclear import/export of RNA and protein, transcriptional regulation, and apoptosis. Interestingly, major cellular reorganizations including cell division, differentiation and transformation, often involve such activities3,4,8,10. The detailed study of these proteins and their respective regulatory mechanisms can be challenging as the stimulation for these localization changes can be elusive, and the movements themselves can be quite dynamic and difficult to track. Studies involving cellular oncogenesis, for example, continue to benefit from understanding pathways and protein activities that differ between normal primary cells and transformed cells6,7,11,12. As many proteins show altered localization during transformation or as a result of transformation, methods to efficiently characterize these proteins and the pathways in which they participate stand to improve the understanding of oncogenesis and open new areas for drug targeting.
Here we present a method for the analysis of protein trafficking and shuttling activity between primary and transformed mammalian cells. This method combines the generation of heterokaryon fusions with fluorescence microscopy to provide a flexible protocol that can be used to detect steady-state or dynamic protein localizations. As shown in Figure 1, two separate cell types are transiently transfected with plasmid constructs bearing a fluoroprotein gene attached to the gene of interest. After expression, the cells are fused using polyethylene glycol, and protein localizations may then be imaged using a variety of methods. The protocol presented here is a fundamental approach to which specialized techniques may be added.

A flexible and efficient method for the characterization of cell type-specific protein localization and nucleocytoplasmic shuttling is described. This heterokaryon approach uses fluorescently-labeled fusion proteins to image protein localizations after cell fusion. The protocol is amenable to steady-state localizations or more dynamic determinations based on live cell imaging.

异核技术的细胞类型特异性定位分析

A significant number of proteins are regulated by subcellular trafficking or nucleocytolasmic shuttling.  These proteins display a diverse array of ...

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as phosphatidylcholine or phosphatidylethanolamine, form bilayer membranes which act as both boundaries and interfaces for chemical exchange between cells and their surroundings. Unlike animals, plant cells have a special organelle for photosynthesis, the chloroplast. The intricate membrane system of the chloroplast contains unique glycerolipids, namely glycolipids lacking phosphorus: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG)4. The roles of these lipids are beyond simply structural. These glycolipids and other glycerolipids were found in the crystal structures of photosystem I and II indicating the involvement of glycerolipids in photosynthesis8,11. During phosphate starvation, DGDG is transferred to extraplastidic membranes to compensate the loss of phospholipids9,12.
Much of our knowledge of the biosynthesis and function of these lipids has been derived from a combination of genetic and biochemical studies with Arabidopsis thaliana14. During these studies, a simple procedure for the analysis of polar lipids has been essential for the screening and analysis of lipid mutants and will be outlined in detail. A leaf lipid extract is first separated by thin layer chromatography (TLC) and glycerolipids are stained reversibly with iodine vapor. The individual lipids are scraped from the TLC plate and converted to fatty acyl methylesters (FAMEs), which are analyzed by gas-liquid chromatography coupled with flame ionization detection (FID-GLC) (Figure 1). This method has been proven to be a reliable tool for mutant screening. For example, the tgd1,2,3,4 endoplasmic reticulum-to-plastid lipid trafficking mutants were discovered based on the accumulation of an abnormal galactoglycerolipid: trigalactosyldiacylglycerol (TGDG) and a decrease in the relative amount of 18:3 (carbons : double bonds) fatty acyl groups in membrane lipids 3,13,18,20. This method is also applicable for determining enzymatic activities of proteins using lipids as substrate6.

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC

Composition of polar lipid extracts and the fatty acid composition of individual glycerolipids are determined in a simple and robust lipid profiling experiment. For this purpose, glycerolipids are isolated by thin layer chromatography and subjected to transmethylation of their acyl groups. Fatty acyl methylesters are quantified by gas-liquid chromatography.

拟南芥极地甘油脂分析薄层色谱（TLC）再加上气相色谱（GLC）

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

细胞特异性分析<em&gt;拟南芥</em&gt;叶用荧光激活细胞分选

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

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 ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

组织化学染色<em&gt;拟南芥</em&gt;次生壁元素

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

结晶纤维素积累高分辨率的量化<em&gt;拟南芥</em&gt;根监视特定组织细胞壁修改

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

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 ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

tchip-seq: 细胞专用表观体分析

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

小鼠肝脏中细胞类型特异性基因表达分析

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...

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

摘要

探索更多视频

此视频中的章节

花香浸改造拟南芥检查 pTSO2：β-葡萄糖醛酸酶报告基因的表达

异核技术的细胞类型特异性定位分析

拟南芥极地甘油脂分析薄层色谱（TLC）再加上气相色谱（GLC）

细胞特异性分析

高效，快速的早期胚胎分离

组织化学染色

结晶纤维素积累高分辨率的量化

不同光照条件下的拟南芥生长行为分析

tchip-seq: 细胞专用表观体分析

小鼠肝脏中细胞类型特异性基因表达分析