Name: a strategy to validate the role of callose-mediated plasmodesmal gating in the tropic response
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韩国国家研究基金会（NRF-2015R1A2A1A10053576），这项研究得到支持，从下一代BioGreen 21计划的资助（SSAC，授予PJ01137901），农村振兴厅，韩国。 RK，WS，ABB和DK经脑韩国21 Plus计划（BK21 +）的支持。

1突变体筛选与改变向光性和向重力响应<ol><li>制备1×Murashige和Skoog（MS）培养基，pH值5.7，用0.8％的琼脂一天实验之前。 <ol><li>加800毫升双蒸水至一个2升的锥形烧瓶中，并用磁力棒搅拌。 </li><li> 4.4克MS盐添加到锥形瓶中。 </li><li>加入0.5克2-（N-吗啉代）乙烷磺酸（MES），搅拌直到所有的盐的完全溶解。 </li><li>调整培养基至5.7用1M KOH的pH。 </li><li>介质传送到海量缸和使终体积到1L的DDH 2 O </li><li>倒回介质到2升锥形瓶中，加入8克植物琼脂。 </li><li>用铝箔紧紧包裹锥形烧瓶口。 </li><li>高压灭菌的MS培养基和DDH 2 O的15分钟，在121℃，15磅，和高压灭菌后，保持介质，在60℃培养箱和DDH 2 O的室温。 </li><li>冷却至60℃之后，倒入介质成125×125×20mm的3方板的层流长凳上（50毫升每块板）。 </li><li>允许琼脂通过保持所述板部分打开在RT固化约1小时。如果板被不立即使用，通过在冰箱用保鲜膜和储存在4℃下密封。 </li></ol></li><li>表面消毒用1.1％的次氯酸钠的种子点在1.1节制备的1倍MS琼脂平板上的种子。 注:在这里， 拟南芥 {Col-0中的种子，dsGSL8（Col-0中 ）} 15用于热带响应，胼胝质染色和HPTS装载拟南芥 {Col-0中和dsGSL8（Col-0中 ）EMS突变}种子（。未公布的）被用于在图2中嗜响应。 <ol><li>高压灭菌300毫升的DDH 2 O的瓶子。 </li><li>添加大约为一百种子为每个背景， 即突变体/过表达系与野生型（Col-0中 ）种子，到1.5ml微量离心管中。 </li><li>取出从4℃的冰箱1倍的MS琼脂平板上，在部分打开位置晾干层流工作台上。 </li><li>通过加入1ml的0.2×漂白溶液（1.1％次氯酸钠）的表面消毒的种子，并保持在微量离心管在摇床上以250rpm进行5分钟。 </li><li>让种子通过重力沉在底部，然后取出用微量漂白粉溶液。 </li><li>加入1毫升的新鲜的DDH 2 O（灭菌）的种子并颠倒离心管数次拌匀。让种子再次沉降，然后除去水分。 </li><li>重复步骤1.2.6 5-6倍。 </li><li>水双倍量加入到最终洗涤之后被留下的种子的体积。 </li><li>点上使用含有200微升的种子1毫升微量移液器尖端的半干燥1倍的MS琼脂平板上的消毒的种子（在cluding水）。 <ol><li>保持两个种子和​​至少两种行之间1.8厘米之间约0.1厘米的距离。圆点每平方板（125×125×20mm的3）五横行的种子。让水将盖到前盘稍干。 </li></ol></li><li>密封采用外科胶带板，堆栈中的所有板垂直，并用铝箔包裹。 </li><li>转移包裹板，以暗箱（没有进入光）。然后，保持暗箱在4℃的室温/冰箱3天。 </li><li>从该步骤开始，保持板不变的方向。经过冷处理3天，保持在22℃，植物培养室在接下来的3天暗箱。 3天后，检查在黑暗的房间在绿灯种子的发芽状态。 注:通常3日龄的Col-0黄化苗可长到1.6厘米长。 </li></ol></li><li>分析改变向光性一个在突变体或过度表达的特定基因的线十二次向地响应。在黑暗的房间中选择下绿灯只有同样大小和垂直生长的直苗。传送所选苗使用杀菌牙签的前端的新鲜的1X MS琼脂板上。 <ol><li>轻轻地选择自己的下胚轴的最低部分秧苗时不接触任何其他部分。确保所有的苗木具有相同的子叶/挂钩方向，转让后，即苗挂钩的方向保持不变。使用最少20苗​​每行中的每个背景和用于进一步分析每个时间点至少两个板组成。 注意:四种不同的背景可以在每个板（4行）进行比较。 可选:在使用地塞米松（DEX）诱导型的RNAi /过表情纹，上面的苗转移到1X MS与+ DEX MS板块沿（含有1x MS板，0.8％琼脂培养基中添加20μMDEX）FOR 3小时，并在暗箱保持垂直板。 </li><li>要检查趋光反应，垂直转移上述板到黑匣子只开了一边。包含板的黑盒单侧​​白光转移到植物生长室。以确保单侧光，将朝向光源（未前侧）的板的边缘。指设置在图1中。 </li><li>同样，检查向地反应，保持与平板转移苗垂直，并用铝箔覆盖。其次，改变垂直板方向90度，并保持它从四面八方覆盖的黑盒子里面。指设置在图1中。 </li><li>在给定的时间点后，（通常为1.5小时，3小时，6小时和12小时）扫描所述板用扫描器，并保存在JPEG格式的图片。 </li><li>测量使用ImageJ软件（http://imagej.nih.gov/ij/index.html）幼苗的弯曲角度（ 图URE 2）。 <ol><li>启动通过双击（ 图2A）的ImageJ的程序。 注意:在角度测量中使用的主要工具是图2B所示。 </li><li>点击"文件"选项后面的子选项"打开"ImageJ的工具栏，并保存在JPEG文件格式来衡量热带弯曲角度（ 图2C）打开图片。 </li><li>点击放大镜工具，通过点击鼠标左右键，分别如图2B所示放大或缩小图片。 </li><li>点击滚动工具拖动和放置图像（ 图2D）。接下来，单击角度工具来测量弯曲（ 图2E）的角度。 <ol><li>使原始胚轴生长方向点&#39;A&#39;，拖动鼠标弯曲起始点&#39;B&#39;（ 图2F），然后单击左屁股左键双击上绘制的第一行（ 图2G）。 </li><li>从b点鼠标拖动到弯曲结束点"C"，并单击左键绘制第二行（ 图2H）和形成固定<img alt="角度" src="/files/ftp_upload/53513/angle.jpg" /> A.注意:真正的弯曲角<img alt="角度" src="/files/ftp_upload/53513/angle.jpg" /> B，它等于180° - <img alt="角度" src="/files/ftp_upload/53513/angle.jpg" /> A.（ 图2F）。 </li><li>测量和记录<img alt="角度" src="/files/ftp_upload/53513/angle.jpg" />通过按下键盘上的&#39;M&#39;A。 如图2I结果文件将单独打开。测量和一个记录所有苗木之一。例如，第一测量对于所有从Col-0中的下胚轴的弯曲角度，然后为突变品系。 </li></ol></li></ol></li><li>从ImageJ的转移数据集电子表格文件，使AV弯曲每个后台的角度erage。 </li><li>进行定量分析，使用统计测试结果，包括标准误差棒和概率的值（参见图2）的柱状图图。 <ol><li>计算真实弯曲<img alt="角度" src="/files/ftp_upload/53513/angle.jpg" /> B，使用电子表格设计一个公式， 即: <img alt="角度" src="/files/ftp_upload/53513/angle.jpg" /> B = 180° - <img alt="角度" src="/files/ftp_upload/53513/angle.jpg" />一张纸条: <img alt="角度" src="/files/ftp_upload/53513/angle.jpg" /> A是一个从ImageJ的分析得出的值。 </li><li>测量平均弯曲角度<img alt="角度" src="/files/ftp_upload/53513/angle.jpg" /> B中各使用后台电子表格工具（ 图2J）。 </li><li>分析使用电子表格"公式"的工具（ 图2J）每次测量重复之间的标准偏差。 </li><li>测试SIG结果通过施加t-试验对平均弯曲角度和上述计算出的标准偏差值的着性。 </li><li>使用上述值来使用的电子表格"插入"工具（ 图2K）以图形方式表示在两个不同的背景之间的弯曲角度的定量差绘制一个直条图。 注:强有力的候选基因突变/过度表达线将显示方面明显的差异野生型，如无弯曲，弯曲快或慢弯曲。 </li></ol></li></ol></li></ol> 2.屏幕株系有缺陷的热带响应由于PD SEL变化具有改变PD胼胝质水平<ol><li> 8羟基芘- -1,3,6-三对顶部幼苗与HPTS琼脂糖块酸（HPTS）染料加载执行下胚轴加载分析，比较染料的突变体之间的运动/过表达线和Col-0中 。 <ol><li>准备HPTS琼胶SE块胚轴加载试验。 <ol><li>取10毫升DDH 2 O的在50ml锥形瓶中，并添加0.1克琼脂糖以制备1％琼脂糖凝胶。煮沸琼脂糖在微波炉1-2分钟间歇摇动，并让它冷却5分钟。 </li><li> 50毫克HPTS的可加10毫升1％琼脂糖溶液，搅拌均匀，直到溶液变为绿色。 </li><li>倒入上述混合物至10毫米培养皿，并允许它固化15分钟。 </li><li>用锋利的刀片解剖剪琼脂糖凝固HPTS做小琼脂糖块（0.5×2 平方厘米）。 </li></ol></li><li>准备植物样品的HPTS染料加载试验。 <ol><li>设置了HPTS染料负载测定的平台;上放置一个新的MS培养基平板的显微镜盖玻片（24×50毫米2）， 如图3A所示 。 </li><li>从钩的基地使用锋利手术剪消费为期3天的老黄化苗。 </li><li>立即传送上述秧苗（从每个背景最少5苗），以微观盖玻璃的表面中，使得只有0.5厘米处的切除位置胚轴的应保留在盖玻片上的方式，和胚轴的其余部分应该接触如在图3B中所示的介质。 </li><li>放置在胚轴的顶部HPTS琼脂糖块（带有一个切钩）中5分钟以这样的方式使切下的区域接触HPTS琼脂糖块。 </li><li> 5分钟装载后，从胚轴表面去除琼脂糖块中，下胚轴转移到含有DDH 2 O的10毫米的培养皿并在DDH 2 O洗胚轴与连续摇动15分钟。 </li></ol></li><li>准备共聚焦显微镜的样品。 <ol><li>从激光扫描共聚焦显微镜下的每个背景观察选择至少5苗。 </li><li>在激光信道，波长设置为特定EXC 488纳米itation和500至550nm为带通发射。 </li><li>在2台传送秧苗（一个的Col-0 +一个突变）到一个新的显微镜载玻片用的DDH 2 O洗涤后添加100-150微升双蒸水至滑动，并用盖玻片覆盖。移幻灯片以共焦显微镜的阶段。 </li><li>采用亮场（白光）照明聚焦下胚轴区域（具有20X 40X或物镜）。然后，扫描荧光的幻灯片。 </li><li>拍摄图像最少10套，并通过从装货点检查染料移动的范围的两个背景进行比较。 注意:突变/过表达系具有改变的共质体的运动将表现出与野生型相比Col-0中染料载荷不同的模式。 </li></ol></li></ol></li><li>分析胼胝质水平突变/过度表达线条显示修改过的向光性和向重力响应和显示鲜明HPTS染料运动&lt;br/&gt;比较教育署COL-0。 <ol><li>准备苯胺蓝染色胼胝质染色。 <ol><li>使1M甘氨酸，pH为9.5和0.1％（W / V）的苯胺蓝在高压灭菌的DDH 2 O </li><li> 3体积比:通过混合0.1％苯胺蓝和2 1M甘氨酸使染色缓冲液。例如，对于50毫升染色缓冲液中，取20毫升0.1N％苯胺蓝和30毫升的1M甘氨酸。注意:请染色缓冲最低使用和储存在黑暗中前48小时。 </li></ol></li><li>染色弯曲或无弯曲与特定的胼胝质苯胺蓝染色苗。 <ol><li>以在染色过程中抑制从头胼胝质合成，1.5mM的2-脱氧-D-葡萄糖（DDG）的最终体积加至50ml染色缓冲液中。 </li><li>就拿胼胝质染色（有或无响应热带）3日龄的黄化苗。 </li><li>切薄用手术剪从他们的钩地区的苗。 </li><li>转移苗小培养皿中含有ING用牙签的尖端2毫升染色缓冲液中。 </li><li>孵化用染色缓冲幼苗在黑暗中2小时连续摇动以30rpm。 </li><li>染色后，清洗与DDH 2 O的幼苗2分钟以除去过量的染色缓冲液中。 </li><li>选择一个最小的5苗的激光扫描共聚焦显微镜下每个工厂背景观察。 </li></ol></li><li>准备共聚焦显微镜的样品: <ol><li>转移苗组（单的Col-0 +一个突变）清洁显微镜的DDH 2 O.洗涤后滑</li><li>添加DDH 2 O的100-150微升，并用盖滑盖。移幻灯片以共焦显微镜的阶段。 </li><li>在激光信道，波长设置为特定激发405nm和500至550nm发射苯胺蓝荧光的成像。 </li><li>与10X和后期带来胚轴区成为关注的焦点，第一R，其中采用亮场（白光）照明20X 40X或物镜。 </li><li>然后，打开过滤光线，扫描幻灯片荧光，并保存图像。 </li><li>衡量ImageJ的软件（http://imagej.nih.gov/ij/index.html）胼胝质信号强度（ 见图4）。 <ol><li>改变从奥林巴斯图像二进制格式（* .oib）到JPEG文件格式共聚焦显微镜图像的格式。注意:只有JPEG格式是由ImageJ的软件支持。 </li><li>启动通过双击（ 图4A）的ImageJ的程序。注:箭头指示矩形工具在胼胝质强度测量（ 图4B）被使用。 </li><li>点击"文件"选项后面的子选项"打开"ImageJ的工具栏，然后打开显微镜照片中保存的JPEG文件格式来测量胼胝强度（ 图4C）。 </li><li>点击伊玛矩形图标GEJ工具栏（ 图4D）。 </li><li>分析信号强度，拖动光标在画面和选择区域进行检查（ 图4E） </li><li>测量，按"M"键盘（ 图4E）上录制的信号强度的数值。注:结果文件将开立单独的（ 图4F）。 </li><li>测量和记录的强度值逐一对所有从每个工厂背景的苗木。 注意:在ImageJ的，结果文件"平均"表示所选择的"区域"的信号强度。 </li></ol></li><li>平均值从ImageJ的结果文件的拷贝，并在定量分析的电子表格文件粘贴。 </li><li>进行定量分析，使用统计检验，包括标准误差棒和概率值（ 图4）的柱状图图。 <ol><li>测量的平均信号强度（从ImageJ的意思）使用电子表格"公式"工具（ 图4G）每个后台。 </li><li>分析使用电子表格"公式"的工具（ 图4G）用于每次测量重复之间的标准偏差。 </li><li>计算使用thespreadsheet"公式"工具的标准误差（SE）。 注意:SE = S /√n，其中（s）是总体的标准偏差，和（n）是该样品的尺寸（观测数）。 </li><li>通过施加t检验到的平均信号强度，并且如上所述计算出的标准误差值测试结果的意义。 </li><li>使用上述值来使用的电子表格"插入"工具（ 图4H）以图形方式表示在两个不同的背景之间的胼胝质水平的定量差绘制的柱状图图。 注:强有力的候选人的突变/过度表达线将小号与野生型胼胝质的水平，如无胼胝质，胼胝质很少或超PD胼胝质的积累有什么不同。 </li></ol></li></ol></li></ol></li></ol>

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S., Hoogner, K. R., Peer, W. A., Taiz, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification,+purification,+and+molecular+cloning+of+N-1-naphthylphthalmic+acid-binding+plasma+membrane-associated+aminopeptidases+from+Arabidopsis.">Identification, purification, and molecular cloning of N-1-naphthylphthalmic acid-binding plasma membrane-associated aminopeptidases from Arabidopsis.</a> Plant Physiol. 128 (3), 935-950 (2002).</li><li>Kleine-Vehn, J., Friml, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polar+targeting+and+endocytic+recycling+in+auxin-dependent+plant+development.">Polar targeting and endocytic recycling in auxin-dependent plant development.</a> Annu. Rev. Cell Dev. Biol. 24, 447-473 (2008).</li><li>Yang, Y., Hammes, U. Z., Taylor, C. G., Schachtman, D. P., Nielsen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-affinity+auxin+transport+by+the+AUX1+influx+carrier+protein.">High-affinity auxin transport by the AUX1 influx carrier protein.</a> Curr. Biol. 16 (11), 1123-1127 (2006).</li><li>Geisler, M., Murphy, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ABC+of+auxin+transport:+the+role+of+p-glycoproteins+in+plant+development.">The ABC of auxin transport: the role of p-glycoproteins in plant development.</a> FEBS Lett. 580 (4), 1094-1102 (2006).</li><li>Band, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Root+gravitropism+is+regulated+by+a+transient+lateral+auxin+gradient+controlled+by+a+tipping+point+mechanism.">Root gravitropism is regulated by a transient lateral auxin gradient controlled by a tipping point mechanism.</a> Proc. Natl. Acad. Sci.USA. 109 (12), 4668-4673 (2012).</li><li>Vanneste, S., Friml, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auxin:+a+trigger+for+change+in+plant+development.">Auxin: a trigger for change in plant development.</a> Cell. 136 (6), 1005-1016 (2009).</li><li>Han, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auxin-callose-mediated+plasmodesmal+gating+is+essential+for+tropic+auxin+gradient+formation+and+signaling.">Auxin-callose-mediated plasmodesmal gating is essential for tropic auxin gradient formation and signaling.</a> Dev. Cell. 28 (2), 132-146 (2014).</li><li>Kumar, R., Kumar, D., Hyun, T. K., Kim, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Players+at+plasmodesmal+nano-channels.">Players at plasmodesmal nano-channels.</a> J. Plant Biol. 58, 75-86 (2015).</li><li>Chen, X. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arabidopsis+callose+synthase+gene+GSL8+is+required+for+cytokinesis+and+cell+patterning.">The Arabidopsis callose synthase gene GSL8 is required for cytokinesis and cell patterning.</a> Plant Physiol. 150, 105-113 (2009).</li></ol>

作者没有披露。

在此手稿，一个策略来筛选由于改变的PD胼胝质，因此，PD SEL中详细描述的突变体/过表达系是在向光性和向地响应缺陷的。 PD胼胝质合成和降解主要由胼胝质合成酶和β-1,3-葡聚糖酶来实现，但这些酶的调节是通过许多上游因素控制。要搜索这样的上游因素或直接参与PD调控的候选人，我们已建立了筛选此方法。这种设置有一些限制，调节PD胼胝质合成（gsl8）的一些关键酶的突变体是致命的<...

植物，如固着活的生物体，已经开发了细胞 - 细胞信号传导的高度复杂的网络，以解决各种环境刺激。热带反应是由植物对环境刺激作出反应的现象之一。植物显示出两个主要的热带响应，向光性和向地。光合植物朝向光源通过弯曲向光性收获最大的能量。同样地，向重力使植物朝向重心生长。导致这些热带反应的基本机制涉及到植物激素生长素1的不对称形成梯度。当地生长素梯度的形成的行为被很好地表征;所涉及的这种机制的基因提供激素作用2-8的路线图。生长素流出载体，如个人识别码形成的（PIN）和P糖蛋白的特定位置，从细胞质到供体细胞9,10的细胞壁执行生长素的运动。 Furthermore，通过将活性的 H + / IAA同向转运生长素涌入载体，如AUX1 / LAX家族蛋白的活性，生长素最终递送到邻近的接收器单元2,11,12。生长素的这个方向的运动被称为极性生长素输送（PAT）。 PAT中各个发育阶段以及响应于不同的环境刺激13,14导致差分生长素分布。此外，在任何这些生长素流入或流出的载体的定位或表达的破坏导致的PAT严重改变，这会导致生长素梯度的破坏，导致发育缺陷。近日，韩寒等人 。报告说plasmodesmal调节也有必要保持生长素梯度15。迄今为止，已有30 plasmodesmal蛋白质已经确定了16个 。在这些蛋白质，AtGSL8已被报告为在胞间连丝（PD），用于胼胝质合成的关键酶，因此起着MAINT至关重要的作用癌宁对PD尺寸排阻极限（SEL）。压抑AtGSL8表达导致扭曲的生长素梯度图案导致在对比野生型幼苗15没有嗜响应。 在这份手稿，方法，探索参与PD调控提供了新的候选基因。 AtGSL8用作模型蛋白来测试这些方法，因为它是促进PD胼胝质的生物合成的关键酶。由于gsl8敲除突变体17秧苗杀伤力，地塞米松（DEX）诱导型的RNAi线按照使用了与以前公布的报告15。这里提供的策略可适于筛选了在用PD SEL受控胚轴嗜响应牵连的基因。

<table><tbody><tr><td>HPTS (8-Hydroxypyrene -1,3,6-trisulfonic acid trisodium salt)</td><td>Sigma</td><td>H1529-1G</td><td>Fluorescent dye as symplasmic tracer</td></tr><tr><td>LE Agarose</td><td>Dongin-Genomic</td><td>GEL001-500G</td><td>Used for HPTS agarose block</td></tr><tr><td>Microwave oven</td><td>LG-Goldstar</td><td></td><td>Machine for boiling agarose gel</td></tr><tr><td>100 ml glass conical flask</td><td>Dong Kwang</td><td>A0205</td><td>Used to boil HPTS agarose gel</td></tr><tr><td>Petri dish (35 mm x 10 mm)</td><td>SPL life sciences</td><td>SPL10035</td><td>Used to make HPTS agarose blocks and wash plant samples&amp;nbsp;</td></tr><tr><td>Microscope cover slides and glass slides (24 mm x 50 mm)</td><td>Marienfeld Laboratory Glassware</td><td>101222</td><td>Used for HPTS agarose blocks and microscopic sample preparation</td></tr><tr><td>MS medium plates 125 mm x 125 mm x 20 mm</td><td>SPL life sciences</td><td>SPL11125</td><td>Plates to make MS agar medium</td></tr><tr><td>Scissors</td><td>Germany Stainless</td><td>HSB 942-11</td><td>Used to excise hook region of plant samples</td></tr><tr><td>Murashige and Skoog (MS) basal salt mixture</td><td>Duchefa</td><td>P10453.01</td><td>MS medium including vitamins.</td></tr><tr><td>(N-morpholino) ethanesulfonic acid (MES) monohydrate</td><td>Bioshop</td><td>3G30212</td><td>To make MS media.</td></tr><tr><td>Plant agar</td><td>Duchefa</td><td>P1001.1000</td><td>To solidify MS media.</td></tr><tr><td>Autoclave</td><td>ALP</td><td>CL-40L</td><td></td></tr><tr><td>Shaker</td><td>Wise Mix</td><td>SHO-1D</td><td>To wash off the aniline blue staining buffer and HPTS dye in a placid way.</td></tr><tr><td>1 ml Blue tips</td><td>Sorenson</td><td>10040</td><td></td></tr><tr><td>1 ml pipette</td><td>BioPette</td><td>L-1101-2</td><td></td></tr><tr><td>Surgical tape</td><td>MIcropore</td><td>1530-0</td><td>To seal the MS plate</td></tr><tr><td>Aniline blue (Methyl blue)</td><td>Sigma</td><td>M5528-25G</td><td>Used to prepare aniline blue staining buffer.</td></tr><tr><td>Glycine</td><td>Bioshop</td><td>GLN001</td><td>Used to prepare aniline blue staining buffer.</td></tr><tr><td>DDG</td><td>Sigma</td><td>D8375-1G</td><td>Used for the inhibition of callose synthases.</td></tr><tr><td>Confocal microscope</td><td>Olympus</td><td>FV1000MPE SIM</td><td>To check aniline blue staining and HPTS dye loading result.</td></tr><tr><td>Stirrer&amp;nbsp;</td><td>I lab&amp;nbsp;</td><td>K400</td><td>To mix media solution.</td></tr><tr><td>Aluminium foil</td><td>SW cooking foil</td><td></td><td>To wrap plates in a dark condition.</td></tr><tr><td>Sodium hypochlorite</td><td>Samjin Industry</td><td></td><td>To surface-sterilized seeds</td></tr></tbody></table>

a strategy to validate the role of callose-mediated plasmodesmal gating in the tropic response

植物激素生长素起着许多生长和发育过程，包括对光和重力热带反应中起重要作用。生长素梯度的建立是导致向光性和向地关键事件。先前，生长素极性运输（PAT）的结果表明在植物中的不同蜂窝结构域建立生长素梯度。然而，韩等人最近证明为正确生长素梯度的形成，在相邻小区之间plasmodesmal胼胝质介导的共质体连通也是一个关键的因素。在此手稿，策略阐明特定基因，其中可通过改变通过调制plasmodesmal胼胝质合成的共质体连通影响向光性和向地的作用，进行了讨论。第一步是筛选从突变体或基因的过表达的行3天龄的黄化苗异常嗜应答与野生型一起。这初步筛选可导致一系列基因PAT功能或控制共质体连通的识别。第二次筛选涉及候选人，显示通过影响共质体连通改变热带响应的排序。为了解决这样的候选人，一个共质体示踪剂的运动和plasmodesmal胼胝质的沉积进行检测。探索新的候选基因可以在热带的反应和其他发育过程直接或间接调节共质体连通这一战略将是有益的。

在当前的设置，AtGSL8的地塞米松（DEX）诱导型的RNAi线[以下dsGSL8（+ DEX / -dex）]中使用，作为纯合子gsl8 T-DNA插入突变体幼苗致死18。 dsGSL8和野生型苗±DEX三日龄的黄化苗暴露于向光性和向地刺激。我们发现，dsGSL8（+ DEX）幼苗中向光性和向地15有缺陷。 图5清楚地表明，dsGSL8（+ DEX）呈现既向光性和向地的?...

Watch this Scientific Journal Video about A Strategy to Validate the Role of Callose-mediated Plasmodesmal Gating in the Tropic Response at JoVE.com

A Strategy to Validate the Role of Callose-mediated Plasmodesmal Gating in the Tropic Response

本文介绍了筛选基因控制的热带响应期间plasmodesmal渗透性，因此生长素梯度的方法。这包括在胚轴热带反应程度的测量 拟南芥和8羟基芘-1,3,6-三磺酸（HPTS）装载终于胼胝质水平评估检查plasmodesmal渗透性。

的策略，以验证胼胝质介导的Plasmodesmal浇注在热带反应中的作用

The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The ...

a-strategy-to-validate-role-callose-mediated-plasmodesmal-gating

Research

JoVE Journal

Biology

10.2K 次观看.  Gyeongsang National University. 本文介绍了筛选基因控制的热带响应期间plasmodesmal渗透性，因此生长素梯度的方法。这包括在胚轴热带反应程度的测量 拟南芥和8羟基芘-1,3,6-三磺酸（HPTS）装载终于胼胝质水平评估检查plasmodesmal渗透性。 

视频: A Strategy to Validate the Role of Callose-mediated Plasmodesmal Gating in the Tropic Response

Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma membrane1. To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex2,3. Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein&#39;s behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (KNa) channels by GPCRs.

Optical biosensor techniques can detect changes in mass near the plasma membrane in living cells and allow one to follow cellular responses in both individual cells and populations of cells. This protocol will describe detection of the modulation of potassium channels by G-protein coupled receptors in intact cells using this approach.

使用无标记光学生物传感器由G-蛋白偶联受体，以检测钾通道的调制

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma ...

科研

生物工程

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

蛋白质中 Plasmodesmal 定位序列的识别在底中

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

免疫与感染

Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels

Here, a new technique termed Tetrotoxin (TTX) Engineered Resistance for Probing Synapses (TERPS) is applied to test for monosynaptic connections between target neurons. The method relies on co-expression of a transgenic activator with the tetrodotoxin-resistant sodium channel, NaChBac, in a specific presynaptic neuron. Connections with putative post-synaptic partners are determined by whole-cell recordings in the presence of TTX, which blocks electrical activity in neurons that do not express NaChBac. This approach can be modified to work with any activator or calcium imaging as a reporter of connections. TERPS adds to the growing set of tools available for determining connectivity within networks. However, TERPS is unique in that it also reliably reports bulk or volume transmission and spillover transmission.

Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels

This article features a method to test the monosynaptic connections between neurons by employing tetrodotoxin and the tetrodotoxin-resistant sodium channel, NaChBac.

使用河豚毒素耐药性钠通道检查果蝇中的 Monosynaptic 连接

Here, a new technique termed Tetrotoxin (TTX) Engineered Resistance for Probing Synapses (TERPS) is applied to test for monosynaptic connections between ...

神经科学

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 ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

对急性机械刺激的网柄反应的评估

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...

生物学

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. Upon wounding of a leaf, an increase in cytosolic calcium ion concentration (Ca2+ signal) occurs at the wound site. This signal is rapidly transmitted to undamaged leaves, where defense responses are activated. Our recent research revealed that glutamate leaking from the wounded cells of the leaf into the apoplast around them serves as a wound signal. This glutamate activates glutamate receptor-like Ca2+ permeable channels, which then leads to long-distance Ca2+ signal propagation throughout the plant. The spatial and temporal characteristics of these events can be captured with real-time imaging of living plants expressing genetically encoded fluorescent biosensors. Here we introduce a plant-wide, real-time imaging method to monitor the dynamics of both the Ca2+ signals and changes in apoplastic glutamate that occur in response to wounding. This approach uses a wide-field fluorescence microscope and transgenic Arabidopsis plants expressing Green Fluorescent Protein (GFP)-based Ca2+ and glutamate biosensors. In addition, we present methodology to easily elicit wound-induced, glutamate-triggered rapid and long-distance Ca2+ signal propagation. This protocol can also be applied to studies on other plant stresses to help investigate how plant systemic signaling might be involved in their signaling and response networks.

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Extracellular glutamate-triggered systemic calcium signaling is critical for the induction of plant defense responses to mechanical wounding and herbivore attack in plants. This article describes a method to visualize the spatial and temporal dynamics of both these factors using Arabidopsis thaliana plants expressing calcium- and glutamate-sensitive fluorescent biosensors.

阿拉伯多普西斯局部和系统伤口信号的广域实时成像

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. ...

A Fluorescence-Based Assay of Membrane Potential for High-Throughput Functional Study of Two Endogenous Ion Channels in Two Epithelial Cell Lines

Fluorescence-based studies are suitable for high-throughput plate reader assays of cells in culture. They have been commonly employed for drug discovery campaigns targeting recombinant ion channel proteins overexpressed in cells such as HEK-293 cells. However, there is increasing emphasis on the use of tissue-relevant cell lines for studying the effects of small molecule interventions. The following protocol describes the adaptation of a fluorescence-based membrane potential assay for the study of ion channels endogenously expressed in epithelial cell lines. The membrane potential assay details a high-throughput assay for chloride channel activity of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in two commonly studied epithelial cell lines, Caco-2 and Calu-3. In addition, this paper describes a novel application of this system to measure the activity of the Epithelial Sodium Channel (ENaC) in a high-throughput format in the same epithelial cell lines. Together, these fluorescence-based assays provide a robust and flexible platform for studying small molecule modulators, targeting two epithelial channels in a relevant cellular context.

This protocol describes a method for the study of electrogenic membrane proteins by measuring changes in membrane potential. This assay provides a platform for the functional readout of multiple ion channels endogenously expressed in epithelial cell lines.

基于荧光的膜电位测定，用于两个上皮细胞系中两个内源性离子通道的高通量功能研究

Fluorescence-based studies are suitable for high-throughput plate reader assays of cells in culture. They have been commonly employed for drug discovery ...

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae

Infection of Brassica crops by the soilborne protist Plasmodiophora brassicae leads to gall formation on the underground organs. The formation of galls requires cellular reprogramming and changes in the metabolism of the infected plant. This is necessary to establish a pathogen-oriented physiological sink toward which the host nutrients are redirected. For a complete understanding of this particular plant-pathogen interaction and the mechanisms by which host growth and development are subverted and repatterned, it is essential to track and observe the internal changes accompanying gall formation with cellular resolution. Methods combining fluorescent stains and fluorescent proteins are often employed to study anatomical and physiological responses in plants. Unfortunately, the large size of galls and their low transparency act as major hurdles in performing whole-mount observations under the microscope. Moreover, low transparency limits the employment of fluorescence microscopy to study clubroot disease progression and gall formation. This article presents an optimized method for fixing and clearing galls to facilitate epifluorescence and confocal microscopy for inspecting P. brassicae-infected galls. A tissue-clearing protocol for rapid optical clearing was used followed by vibratome sectioning to detect anatomical changes and localize gene expression with promoter fusions and reporter lines tagged with fluorescent proteins. This method will prove useful for studying cellular and physiological responses in other pathogen-triggered structures in plants, such as nematode-induced syncytia and root knots, as well as leaf galls and deformations caused by insects.

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae

The present protocol describes an optimized method for the histological observation of galls induced by Plasmodiophora brassicae. Vibratome sections of hypocotyls are cleared before fluorescence imaging to study the involvement of transcription factors and phytohormones during disease progression. This protocol overcomes resin embedding limitations, enabling in planta visualization of fluorescent proteins.

结合透明和荧光显微镜，可视化基因表达的变化和对芸苔疟原虫的生理反应

Infection of Brassica crops by the soilborne protist Plasmodiophora brassicae leads to gall formation on the underground organs. The formation of galls ...

的策略，以验证胼胝质介导的Plasmodesmal浇注在热带反应中的作用

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使用无标记光学生物传感器由G-蛋白偶联受体，以检测钾通道的调制

蛋白质中 Plasmodesmal 定位序列的识别在底中

使用河豚毒素耐药性钠通道检查果蝇中的 Monosynaptic 连接

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

对急性机械刺激的网柄反应的评估

阿拉伯多普西斯局部和系统伤口信号的广域实时成像

基于荧光的膜电位测定，用于两个上皮细胞系中两个内源性离子通道的高通量功能研究

结合透明和荧光显微镜，可视化基因表达的变化和对芸苔疟原虫的生理反应