我们感谢布赫兹数字视频制作团队的丹尼尔. 陈, 协助编辑视频。该研究的气孔保护细胞, 蛋白质组学和新陈代谢在陈实验室得到了资助, 美国国家科学基金会 (0818051, 1158000 和 1412547)。文文香港获中国奖学金委员会支持。Qiuying 博士获得中国奖学金委员会和国家自然科学基金 (31570396) 的支持。

1. 种植植物
<ol><li>在灌封混合物中发芽种子。在生长室种植植物在一个光强度的140µmol 光子 m−2 s −1和光周期8小时在22°c 和 16 h 黑暗在18°c 2 星期。</li>
	<li>将类似大小的移栽苗单独放入4英寸直径的花盆中, 并在步骤1.1 所述相同条件下生长3周。 注: 自来水厂每周两次, 以保持土壤湿润。</li>
</ol>2. 制备富气孔
<ol><li>用手术刀把5周大的叶子移开.</li>
	<li>将每片叶子附着在两片透明的透明胶带上, 一条附着在背面 (下部) 一侧, 另一块附着在叶子正面 (上部) 一侧。把带子留在叶子上5秒。</li>
	<li>用食指和拇指分别将两片带子分开, 将背面与人行道、守卫细胞、正面与叶肉细胞分开。</li>
	<li>将果皮从叶背面的60毫升气孔开放缓冲器 (50 毫米氯化钾, 10 毫米 MES, 调整到 pH 6.2 与1米 koh) 在40毫米 x 12 毫米大小的培养皿, 因为他们收集。<ol>
<li>重复上述步骤, 直到收集所有样品皮。</li>
	</ol>
</li>
	<li>在步骤1.1 所述的光条件下, 将含有果皮的培养皿置于生长室中。把果皮放在光下2小时, 完全打开气孔。</li>
	<li>将果皮放入150毫米 x 20 毫米培养皿中, 含有50毫升细胞壁消化酶混合物 (0.7% 纤维素酶 R-10, 0.025% 离析酶 R-10, 0.1% (w/v) polyvinylpyrrolidone-40, 0.25% (w/v) 牛血清白蛋白55% 基本溶液 (0.55 米山梨醇, 0.5 毫米CaCl2, 0.5 毫米氯化镁2, 0.5 毫米抗坏血酸, 10 µM, 2PO4, 5 毫米 4-morpholineethanesulfonic 酸 (MES) 在 pH 5.7 调整与 1 M KOH)。</li>
	<li>在50转20分钟的培养皿中, 在一个相互的振动筛上摇动果皮。</li>
	<li>20分钟后用镊子从酶溶液中取出果皮. 将果皮转移到150毫米 x 20 毫米大小的培养皿中, 含50毫升水。</li>
	<li>使用转移吸管, 冲洗果皮十五年代两次与10毫升的水, 以消除任何残留酶溶液。</li>
	<li>使用镊子将果皮放置在150毫米 x 20 毫米尺寸的培养皿中, 50 毫升的新鲜气孔开启缓冲液在步骤2.4 中使用。在步骤1.1 中所述的光条件下孵育1小时, 以允许气孔恢复。</li>
</ol>3. 脱落酸 (ABA) 治疗及气孔运动测定
<ol><li>在150毫米 x 20 毫米大小的培养皿。在30°c 上孵育富气孔果皮15分钟, 在气孔开启缓冲器的50毫升或气孔开启缓冲器的50毫升内, 以10µM ABA 的最后浓度为适当的处理时间。</li>
	<li>将果皮放在 50 rpm 的振动筛上, 5, 15 和30分钟。每隔一段时间间隔后用镊子去除果皮, 然后按照步骤3.3 中的成像步骤操作。</li>
	<li>采用光镜观察 aba 治疗前的气孔图像和不同的时间点。<ol>
<li>把果皮放在玻璃显微镜滑梯上。</li>
		<li>将幻灯片放在显微镜的舞台上, 调整舞台, 使果皮的图像可以可视化。</li>
		<li>调整在显微镜上的精细和课程重点, 以获得一个清晰的形象的守卫细胞。</li>
		<li>以40X 倍放大倍数的多种气孔图像。</li>
	</ol>
</li>
	<li>使用 ImageJ8测量60气孔光圈。</li>
	<li>在60气孔孔径测量的 p 值 < 0.05 中计算标准误差和意义。</li>
</ol>4. 蛋白质提取和 SDS 分页分离
<ol><li>十五年代用足够的液氮研磨果皮, 用冷冻砂浆和杵盖住果皮。</li>
	<li>每50果皮, 添加3毫升的三饱和苯酚 (pH 8.8) 和3毫升蛋白质提取缓冲 (0.9 米蔗糖, 0.1 米三盐酸, 0.01 米 EDTA, 0.4% 2-巯基乙醇, 10 µL 的1毫米蛋白酶和磷酸酶抑制剂) 与一个吸管的砂浆, 然后研磨的果皮5此外在通风罩分钟。</li>
	<li>使用吸管和果皮用金属镊子将萃取物转移到欧克瑞奇离心管上, 并在 50 rpm 1 小时的振动筛上搅动4摄氏度。</li>
	<li>从欧克瑞奇离心管中取出果皮, 离心萃取物在 5000 x g 处10分钟。然后用吸管将顶酚相转移到新的清洁微离心管上。</li>
	<li>在100% 甲醇中加入5卷冰冷0.1 米醋酸铵, 沉淀苯酚提取蛋白。漩涡简要地和孵化隔夜在-20 °c。</li>
	<li>离心机在 2万 x g 在4°c 为20分钟, 醒酒在上清液慢慢地倾吐它从管子并且保留蛋白质药丸在管子里。</li>
	<li>用0.1 米醋酸铵在甲醇中两次洗涤蛋白颗粒, 然后两次用80% 丙酮, 一次用100% 冷丙酮。 注: 通过将10毫升的指定试剂添加到蛋白质管中进行洗涤。在 50 rpm 5 分钟的振动筛上搅拌, 其次为 1.5万 x g 的离心, 4 摄氏度为10分钟。然后将试剂倒入试管中, 只保留蛋白颗粒。</li>
	<li>加入1毫升100% 冷丙酮的颗粒, 并通过缓慢吹打的向上和向下重新暂停。</li>
	<li>通过吸管转移蛋白质悬浮液到2毫升微离心管, 然后离心机在 2万 x g 在4°c 5 分钟。</li>
	<li>将丙酮醒酒从管中取出, 在通风罩中干燥10分钟。</li>
	<li>用200µL 溶解缓冲液 (8 米尿素、0.5% SDS、30毫米三盐酸盐在 pH 值 8.5) 和涡旋为 30 x g 在2万摄氏度为15度, 在新管中收集上清, 将蛋白质溶解在微离心管中。</li>
	<li>根据蛋白质量化协议9, 量化蛋白质浓度。</li>
	<li>在10号议定书之后使用蛋白质样品进行蛋白质分离。</li>
</ol>

<ol><li>Acharya, B., Assmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hormone+interactions+in+stomatal+function%5BTitle%5D)+AND+%22Plant+Molecular+Biology%22%5BJournal%5D)">Hormone interactions in stomatal function.</a> Plant Molecular Biology. 69 (4), 451-462 (2009).</li>
<li>Schroeder, J. I., Allen, G. J., Hugouvieux, V., Kwak, J. M., Waner, D. <a target="_blank" href="https://www.annualreviews.org/doi/abs/10.1146/annurev.arplant.52.1.627">Guard cell signal transduction.</a> Annual Review of Plant Physiology and Plant Molecular Biology. 52, 627-658 (2001).</li>
<li>Obulareddy, N., Panchal, S., Melotto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Guard+Cell+Purification+and+RNA+Isolation+Suitable+for+High-Throughput+Transcriptional+Analysis+of+Cell-Type+Responses+to+Biotic+Stresses%5BTitle%5D)+AND+%22Molecular+Plant-Microbe+Interactions%22%5BJournal%5D)">Guard Cell Purification and RNA Isolation Suitable for High-Throughput Transcriptional Analysis of Cell-Type Responses to Biotic Stresses.</a> Molecular Plant-Microbe Interactions. 26 (8), 844-849 (2013).</li>
<li>Pandey, S., Wang, X. Q., Coursol, S. A., Assmann, S. M. <a target="_blank" href="https://nph.onlinelibrary.wiley.com/doi/abs/10.1046/j.0028-646X.2001.00329.x">Preparation and applications of Arabidopsis thaliana guard cell protoplasts.</a> New Phytologist. 153 (3), 517-526 (2002).</li>
<li>Schroeder, J. I., Raschke, K., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Voltage+Dependence+Of+K%2B+Channels+In+Guard-Cell+Protoplasts%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Voltage Dependence Of K+ Channels In Guard-Cell Protoplasts.</a> Proceedings of the National Academy of Sciences of the United States of America. 84 (12), 4108-4112 (1987).</li>
<li>Wu, F. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tape-Arabidopsis+Sandwich+-+a+simpler+Arabidopsis+protoplast+isolation+method%5BTitle%5D)+AND+%22Plant+Methods%22%5BJournal%5D)">Tape-Arabidopsis Sandwich - a simpler Arabidopsis protoplast isolation method.</a> Plant Methods. 5, 10(2009).</li>
<li>Svozil, J., Gruissem, W., Baerenfaller, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proteasome+targeting+of+proteins+in+Arabidopsis+leaf+mesophyll%2C+epidermal+and+vascular+tissues%5BTitle%5D)+AND+%22Frontiers+in+Plant+Science%22%5BJournal%5D)">Proteasome targeting of proteins in Arabidopsis leaf mesophyll, epidermal and vascular tissues.</a> Frontiers in Plant Science. 6, (2015).</li>
<li>Schneider, C., Rasband, W., Eliceiri, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NIH+Image+to+ImageJ%3A+25+years+of+image+analysis%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">NIH Image to ImageJ: 25 years of image analysis.</a> Nature Methods. 9 (7), 671-675 (2012).</li>
<li>Agnew, B., Murray, D., Patton, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+rapid+solid-phase+fluorescence-based+protein+assay+for+quantitation+of+protein+electrophoresis+samples+containing+detergents%2C+chaotropes%2C+dyes%2C+and+reducing+agents%5BTitle%5D)+AND+%22Electrophoresis%22%5BJournal%5D)">A rapid solid-phase fluorescence-based protein assay for quantitation of protein electrophoresis samples containing detergents, chaotropes, dyes, and reducing agents.</a> Electrophoresis. 25 (15), 2478-2485 (2004).</li>
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没有宣布利益冲突。

保护细胞是研究植物信号转导机制的模型系统, 重要的是要确保研究中使用的样品的制备最适合回答生物学问题。尽管植物研究界对保护细胞的兴趣越来越浓厚, 但对于如何制备气孔保护细胞, 使气孔运动和生理以及分子变化在一个研究中都没有普遍的方法系统。隔离它们的挑战在很大程度上归因于小规模、低丰度和独特的结构, 这可以使它们或多或少难以从整个树叶中移除。
...

气孔在植物的整体生理和健身中起着重要的作用。这些微小的植物特定的结构是由两个专门的表皮细胞组成的, 称为守卫细胞, 在叶的背面最丰富的发现。气孔是在植物和大气之间交换气体和通过蒸腾控制水分损失所必需的。保护细胞通过不同外部 (例如, 光, 湿度, 病原体接触, 二氧化碳水平) 和内部 (e., 内源激素) 的整合来调节气孔孔径,刺激 1,2。在过去的20年中, 这种细胞类型已经成为研究植物细胞信号过程的模型系统。这些细胞构成了叶中总细胞的一小部分;因此, 以单细胞的方式研究其独特的细胞、生物化学和分子特性, 就有必要将它们与其他叶细胞类型分离。在过去, 守卫细胞研究经常介入了卫兵细胞原生质体的准备 (GCP)3,4,5。这通常需要使用大量的细胞壁降解酶和或通过混合的机械干扰, 并证明是昂贵和耗时, 因为它通常需要很多小时来准备一个 GCP 样品, 往往是很少的产量。更重要的是, 由于保护细胞作为完全对调节气孔孔径, 使用 GCP 可以被视为一个人工系统, 研究保护细胞信号。
在这里, 我们开发了一种方法来制备气孔, 其中完整的保护细胞丰富和保持生理反应的刺激。这种方法的灵感来自于分离叶肉细胞原生质体6,7的方法。在我们的方法, 气孔是准备使用透明的透明胶带, 首先分离的大部分叶肉细胞的正面层的叶子从背面层包含路面和守卫细胞。这是通过贴一块胶带到叶子的两侧, 并剥离它们分开。在气孔开放的缓冲区中, 允许从背面层的细胞恢复到光下。后来, 其余的路面细胞被删除使用少量的细胞壁降解酶, 留下保护细胞, 丰富的磁带。
在这种简单的方法中, 气孔保护细胞既可行又反应灵敏, 可以快速有效地制备, 花不到一小时就能从50皮中获得丰富的气孔保护细胞, 成本最低。这里的方法对植物细胞的损伤比以前的原生质体制备方法少。此外, 从这种方法收集的材料产生理想的蛋白质量。最重要的是, 由于叶子不受混合或长时间的消化, 并且保护细胞保持不变, 研究结果将更接近于保护细胞的自然生物学。利用这种方法, 气孔运动可以与分子水平的变化实时相关。因此, 从研究中获得的知识使用这种制备方法, 对于更全面地了解防护细胞信号是非常重要的。
我们用植物拟南芥作为模型;但该协议可应用于其他植物中的气孔保护细胞的富集, 如甘蓝型油菜的消化时间小改性。作为概念的证明, 我们已经证明, 通过这种方法丰富的气孔保护细胞是可行的, 并响应的刺激, 如荧光素二乙酸 (FDA) 的研究, 并利用植物激素脱落酸 (ABA), 分别。此外, 我们已经证明, 高质量的蛋白质可以从这些守卫细胞分离。在这里, 我们描述了这个过程的详细协议。

<table><tbody><tr><td>Stainless Steel Surgical Scalpel</td><td>Feather&amp;nbsp;</td><td>&amp;nbsp;NC9999403</td><td></td></tr><tr><td>Scotch Tape (3M)</td><td>ULINE</td><td>S-9781</td><td></td></tr><tr><td>Petri Dish</td><td>Sigma-Aldrich</td><td>BR455701</td><td></td></tr><tr><td>Cellulase (Onozuka R-10)&amp;nbsp;</td><td>Yakult Pharmaceutical Industry Co., Ltd.</td><td>21560003-3</td><td></td></tr><tr><td>Macerozyme R-10</td><td>Yakult Pharmaceutical Industry Co., Ltd.</td><td>21560003-4</td><td></td></tr><tr><td>DM6000B Microscope&amp;nbsp;</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Protease and Phosphatase Inhibitor Cocktails</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>&amp;nbsp;PI78443</td><td></td></tr><tr><td>Oak Ridge High-Speed PPCO Centrifuge Tubes</td><td>Thermo Fisher Scientific</td><td>3119-0030</td><td></td></tr><tr><td>EZQ Protein Quantitation Kit</td><td>Thermo Fisher Scientific</td><td>R33200</td><td></td></tr><tr><td>Fluorescein Diacetate (FDA)</td><td>Thermo Fisher Scientific</td><td>F1303</td><td></td></tr><tr><td>Abscisic Acid</td><td>Sigma-Aldrich</td><td>A4906</td><td></td></tr><tr><td>Metro Mix 500</td><td>BWI Companies&amp;nbsp;</td><td>TX-500</td><td></td></tr><tr><td>Laemmli Sample Buffer</td><td>Bio-Rad</td><td>161-0737</td><td></td></tr><tr><td>Bio-Safe Comassie (G-250)</td><td>Bio-Rad</td><td>161-0786</td><td></td></tr><tr><td>Microcentrifuge Tube (2mL)</td><td>USA Scientific, Inc.</td><td>1620-2700</td><td></td></tr><tr><td>ImageJ software</td><td>National Institute of Health</td><td></td><td></td></tr><tr><td>Tweezers&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F4017-1EA</td><td></td></tr><tr><td>12% Mini-PROTEAN TGX Precast Gel</td><td>Bio-Rad</td><td>456-1043</td><td></td></tr><tr><td>Microscope slides</td><td>Fisherbrand</td><td>12-550-A3</td><td></td></tr></tbody></table>

stomata tape-peel: an improved method for guard cell sample preparation

保护细胞的研究对于了解这种细胞类型对植物整体功能的特殊贡献是至关重要的。然而, 通常很难分离, 因此研究它们往往会带来挑战。
本研究建立了气孔保护细胞的富集方法。该协议利用透明胶带分离出保护细胞并从细胞中提取蛋白质。该方法在隔离过程中提高了保护细胞的完整性和产量, 为细胞信号过程的研究以及它们与气孔运动表型的相关性提供了可靠的样本。在这种方法中, 植物叶被分成两部分的胶带和剥离分开, 以消除背面的一侧。本协议适用于拟南芥叶组织分离保护细胞。用荧光素双乙酸 (FDA) 对细胞进行治疗, 以确定活性和脱落酸 (aba), 以评估 aba 对气孔运动的反应。最后, 该协议对制备富含气孔保护细胞和分离蛋白质具有重要意义。这里获得的细胞和蛋白质的质量使生理学和其他生物研究得以实现。

图 1显示了拟南芥保护细胞在叶肉和表皮细胞消化前后的代表性图像。使用 FDA 测量酶活性和细胞膜完整性 (图 1B和1D) 可以观察到叶肉和表皮细胞去除前后的保护细胞活力。图 2说明拟南芥的气孔运动对 ABA 的治疗反应。与 ABA 治疗30分钟后气孔孔径减少50% 以上。图 2A是气?...

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Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation

本协议描述了一种制备富含气孔保护细胞的方法, 用于生理学和其他生物研究。

气孔带皮: 一种改进的防护细胞样品制备方法

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is ...

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14.9K 次观看.  University of Florida. 本协议描述了一种制备富含气孔保护细胞的方法, 用于生理学和其他生物研究。

视频: Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

评估气孔反应活细菌细胞，采用全叶成像

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

科研

免疫与感染

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.

Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.

肽衍生的方法在运输跨越植物细胞的基因和蛋白质和细胞器障碍

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as ...

遗传学

mRNA Interactome Capture from Plant Protoplasts

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional gene regulation (PTGR) of messenger RNAs (mRNAs). In the past few years, a number of mRNA-bound proteomes from yeast and mammalian cell lines have been successfully isolated through the use of a novel method called &#34;mRNA interactome capture,&#34; which allows for the identification of mRNA-binding proteins (mRBPs) directly from a physiological environment. The method is composed of in vivo ultraviolet (UV) crosslinking, pull-down and purification of messenger ribonucleoprotein complexes (mRNPs) by oligo(dT) beads, and the subsequent identification of the crosslinked proteins by mass spectrometry (MS). Very recently, by applying the same method, several plant mRNA-bound proteomes have been reported simultaneously from different Arabidopsis tissue sources: etiolated seedlings, leaf tissue, leaf mesophyll protoplasts, and cultured root cells. Here, we present the optimized mRNA interactome capture method for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type that serves as a versatile tool for experiments that include various cellular assays. The conditions for optimal protein yield include the amount of starting tissue and the duration of UV irradiation. In the mRNA-bound proteome obtained from a medium-scale experiment (107 cells), RBPs noted to have RNA-binding capacity were found to be overrepresented, and many novel RBPs were identified. The experiment can be scaled up (109 cells), and the optimized method can be applied to other plant cell types and species to broadly isolate, catalog, and compare mRNA-bound proteomes in plants.

Here, we present an interactome capture protocol applied to Arabidopsis thaliana leaf mesophyll protoplasts. This method critically relies on in vivo UV crosslinking and allows for the isolation and identification of plant mRNA-binding proteins from a physiological environment.

mRNA从植物原生质体捕获

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional ...

生物化学

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings

Stomatal movement mediates plant gas exchange, which is essential for photosynthesis and transpiration. Stomatal opening and closing are accomplished by a significant increase and decrease in guard cell volume, respectively. Because shuttle transport of ions and water occurs between guard cells and larger neighboring epidermal cells during stomatal movement, the spaced distribution of plant stomata is considered an optimal distribution for stomatal movement. Experimental systems for perturbing the spaced pattern of stomata are useful to examine the spacing pattern's significance. Several key genes associated with the spaced stomatal distribution have been identified, and clustered stomata can be experimentally induced by altering these genes. Alternatively, clustered stomata can be also induced by exogenous treatments without genetic modification. In this article, we describe a simple induction system for clustered stomata in Arabidopsis thaliana seedlings by immersion treatment with a sucrose-containing medium solution. Our method is easy and directly applicable to transgenic or mutant lines. Larger chloroplasts are presented as a cell biological hallmark of sucrose-induced clustered guard cells. In addition, a representative confocal microscopic image of cortical microtubules is shown as an example of intracellular observation of clustered guard cells. The radial orientation of cortical microtubules is maintained in clustered guard cells as in spaced guard cells in control conditions.

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings

The goal of this protocol is to demonstrate how to induce clustered stomata in cotyledons of Arabidopsis thaliana seedlings by immersion treatment with a sugar-containing medium solution and how to observe intracellular structures such as chloroplasts and microtubules in the clustered guard cells using confocal laser microscopy.

拟南芥幼苗糖溶液浸入治疗的聚集性植物诱导系统

Stomatal movement mediates plant gas exchange, which is essential for photosynthesis and transpiration. Stomatal opening and closing are accomplished by a ...

环境科学

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

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It contains sugars, amino acids, proteins, RNA, lipids and other metabolites. While there is a large interest in understanding the composition and function of the phloem, the role of many of these molecules and thus, their importance in plant development and stress response has yet to be determined. One barrier to phloem analysis lies in the fact that the phloem seals itself upon wounding. As a result, the number of plants from which phloem sap can be obtained is limited. One method that allows collection of phloem exudates from several plant species without added equipment is the EDTA-facilitated phloem exudate collection described here. While it is easy to use, it does lead to the wounding of cells and care has to be taken to remove contents of damaged cells. In addition, several controls to prove purity of the exudate are necessary. Because it is an exudation rather than a direct collection of the phloem sap (not possible in many species) only relative quantification of its contents can occur. The advantage of this method over others is that it can be used in many herbaceous or woody plant species (Perilla, Arabidopsis, poplar, etc.) and requires minimal equipment and training. It leads to reasonably large amounts of exudates that can be used for subsequent analysis of proteins, sugars, lipids, RNA, viruses and metabolites. It is simple enough that it can be used in both a research as well as in a teaching laboratory.

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

Knowledge of the composition of the phloem sap as well as the mechanism of its loading and long-distance transport is essential for the understanding of long-distance signaling in plant development and stress/pathogen response and of assimilate transport. This manuscript describes the collection of phloem exudates using the EDTA-facilitated method.

收集和分析<em&gt;拟南芥</em&gt;韧皮部分泌物使用EDTA促进方法

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It ...

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 ...

拟南芥中细菌入侵的气孔反应自动测量

Direct Observation and Automated Measurement of Stomatal Responses to Pseudomonas syringae pv. tomato DC3000 in Arabidopsis thaliana

Stomata are microscopic pores found in the plant leaf epidermis. Regulation of stomatal aperture is pivotal not only for balancing carbon dioxide uptake for photosynthesis and transpirational water loss but also for restricting bacterial invasion. While plants close stomata upon recognition of microbes, pathogenic bacteria, such as Pseudomonas syringae pv. tomato DC3000 (Pto), reopen the closed stomata to gain access into the leaf interior. In conventional assays for assessing stomatal responses to bacterial invasion, leaf epidermal peels, leaf discs, or detached leaves are floated on bacterial suspension, and then stomata are observed under a microscope followed by manual measurement of stomatal aperture. However, these assays are cumbersome and may not reflect stomatal responses to natural bacterial invasion in a leaf attached to the plant. Recently, a portable imaging device was developed that can observe stomata by pinching a leaf without detaching it from the plant, together with a deep learning-based image analysis pipeline designed to automatically measure stomatal aperture from leaf images captured by the device. Here, building on these technical advances, a new method to assess stomatal responses to bacterial invasion in Arabidopsis thaliana is introduced. This method consists of three simple steps: spray inoculation of Pto mimicking natural infection processes, direct observation of stomata on a leaf of the Pto-inoculated plant using the portable imaging device, and automated measurement of stomatal aperture by the image analysis pipeline. This method was successfully used to demonstrate stomatal closure and reopening during Pto invasion under conditions that closely mimic the natural plant-bacteria interaction.

作者聚焦：通过自动孔径测量推进气孔研究

Here, we present a simple method for direct observation and automated measurement of stomatal responses to bacterial invasion in Arabidopsis thaliana. This method leverages a portable stomatal imaging device, together with an image analysis pipeline designed for leaf images captured by the device.

直接观察和自动测量对丁香假单胞菌 pv 的气孔反应。番茄拟南芥中的DC3000

Stomata are microscopic pores found in the plant leaf epidermis. Regulation of stomatal aperture is pivotal not only for balancing carbon dioxide uptake ...

气孔带皮: 一种改进的防护细胞样品制备方法

摘要

探索更多视频

评估气孔反应活细菌细胞，采用全叶成像

肽衍生的方法在运输跨越植物细胞的基因和蛋白质和细胞器障碍

mRNA从植物原生质体捕获

拟南芥幼苗糖溶液浸入治疗的聚集性植物诱导系统

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

收集和分析

组织化学染色

直接观察和自动测量对丁香假单胞菌 pv 的气孔反应。番茄拟南芥中的DC3000

直接观察和自动测量对丁香假单胞菌 pv 的气孔反应。番茄拟南芥中的DC3000

直接观察和自动测量对丁香假单胞菌 pv 的气孔反应。番茄拟南芥中的DC3000