Name: measuring spatial and temporal ca2+ signals in arabidopsis plants
Uploaded: 2014-09-02T14:00:00.000+00:00
Duration: 10 min 12 s
Description: Watch this Scientific Journal Video about Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants at JoVE.com

We are grateful to B. Stevenson for technical assistance and Dr Marc R. Knight for providing Aequorin transgenic line. This work was funded by the National Institutes of Health Grant R01 GM059138.

1，基于水母发光蛋白钙成像使用FAS系统<ol><li>准备苗木发光成像。消毒拟南芥植物表达水母发光蛋白含0.01％的Triton-100 10％的漂白粉溶液的种子。播下含有全强度MS（Murashige和Skoog基础盐混合物），1％蔗糖和1.2％琼脂的无菌种子在方形板（10×10厘米的正方形培养皿格）。代替板垂直地在分层后培养室中于4℃下进行2天（ 图1A）。 </li><li>转移到苗木电影。在7-10天龄幼苗的生长板的顶部放置一个粘合膜（ 图1B）。轻轻地用手推片，以确保苗附着于膜（ 图1C）。剥离该膜轻轻使得幼苗保持粘附于膜（ 图1D和1E） </li><li>孵育辅苗。将在Adhered幼苗在含正方形板（10×10厘米）将15ml 2微克/毫升H-CTZ（腔肠素）的水。孵育秧苗在室温下搅拌4小时至过夜（ 图1F）。 </li><li>准备发光成像。就拿拍出来的H-CTZ的解决方案，并剪下来的中间，形成两片。将每一块膜用苗面对两种不同的板块。留在暗处将板5分钟。 </li><li>获得发光图像。在黑暗中，将两个板彼此相邻的发光成像系统（ 图1G）的阶段。后，立即同时加入20ml的刺激溶液于板获得图像。 </li><li>分析发光图像。选择相同的显示范围为所有发光图像。作物感兴趣区域（ROI）的区域，并生成图像，JPEG文件（ 图2A）。另外，导出图像作为固相萃取格式的文件，并将其导入到ImageJ的图像分析软件。设置测量投资回报率的平均灰度值的计算。选择ROI区域的大小相同，并测量平均灰度和本数据作为柱状图（ 图2B）。 </li></ol> 2，活细胞共聚焦钙成像<ol><li>准备苗木共聚焦成像。消毒拟南芥植物表达case12含0.01％的Triton 10％的漂白粉溶液的种子。母猪在含有全量MS盐类，1％蔗糖，1.2％琼脂平板无菌种子。代替板垂直地在分层后培养室中于4℃下进行2天。 </li><li>安装成像钱伯斯<ol><li>装配使用幻灯片和盖玻片（室）的成像室。将一块浸透水的脱脂棉上滑动的中间。然后从板传送一个或两个5天龄幼苗到水浸泡药棉的顶部。坚持小块粘土的盖玻片，在每一个角落d将盖玻片上的幼苗创造盖玻片和载玻片之间的间隙（室）（ 图3A，左图）的顶部。聚乙烯管（0.58mL毫米直径）的一端连接至1ml注射器和放置管紧邻所述腔室的另一端。用胶带（ 图3A，右图）握住管到位。 </li><li>装配用有机玻璃室（B室）成像室。 <ol><li>散布一层薄薄的硅脂围绕两个聚乙烯管（0.58mL毫米直径），然后按涂覆管放置在有机玻璃室的每一侧的信道（ 图3B左面板）。散布一层薄薄的硅脂到包含所述管槽的腔室的表面上，按盖玻片入润滑脂，以密封腔室开口（切出）的一侧。 </li><li>从板块转移日龄苗成室，并放置了一块脱脂棉用开水浸泡在顶部ØF表示苗子。传播硅脂到所述腔室的另一面，然后按上顶端以密封另一盖玻片。其中一个管的一端连接到1毫升注射器。离开其他管开放（ 图3B右面板）的端部。 </li></ol></li><li>采用腔盖玻璃（C室）准备成像室。消毒腔盖玻璃用70％乙醇和离开它引擎盖上直到干燥。添加0.6毫升或0.2含1％蔗糖和0.5％植物凝胶成2孔腔的每个孔或8孔腔室（ 图3C右侧面板），分别为全强度MS培养基中。种子消毒，直接在含有一层薄薄的透明凝胶的墓室盖玻璃播下的种子，让他们上下5天（ 图3C）成长。 </li></ol></li><li>掌握用共聚焦显微镜图像。施加压力刺激，如盐，寒冷或过氧化物，慢慢注入大约200微升的150 mM氯化钠，冰栏ð水或1毫米的H 2 O 2获取图像，或轻轻地加刺激液500微升或100微升至2或8孔室的很好，分别前夕溶液进入室（厅A或B） 。拍摄的图像将立即使用倒置尼康A1R共聚焦激光扫描显微镜20倍水浸泡镜片（数值孔径0.75）刺激后的解决方案。收集在4秒的间隔与488 nm和500的激发和发射波长的时间序列图像 - 550纳米，分别，并且在512×512像素的分辨率。 </li><li>图像分析 使用尼康元素，投资回报是吸引每一个感兴趣的单元格（或区域）周围。每个感兴趣区域内的总强度是使用时间测量对话框（ImageJ的可以用来代替）随时间测量。总强度测量输出和数据图进行处理。 钙尖峰幅度定义为峰值强度减去休息的强度，持续时间为次起始和尖峰完成后，并作为两个尖峰相邻尖峰峰值之间的时间间隔周期E之间的时间。 吨 -test用于比较的装置。 </li></ol>

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The authors have nothing to disclose.

我们已经展示了FAS系统记录拟南芥幼苗的时空钙离子的响应。这FAS 钙记录系统提供了一个可能被适用于测量通过钙除了在此处提出的非生物胁迫的刺激各种刺激引起离子动力简便，灵敏，强健的方法。使用这个系统，我们可以很容易地在整株水平比较组织或刺激特定的时空钙离子动态。 FAS的高灵敏度允许使用低强度刺激的用于检查的 Ca 2 +的反应，这是为了避免造成物理损伤细胞的工件时，高强度的应力时非常重要的。 7到10天龄的拟南芥幼苗表现出组织或刺激特异性的 Ca 2 +的反应，虽然苗在不同生理阶段可具有不同的敏感性和特异性响应于给定的刺激。因此，它是优选使用7-10天龄的幼苗，以增加吞吐量FAS测量的，其中约100个​​苗，可以粘附到一块薄膜。连同容易和简单的性能，高的灵敏度和重现性，该FAS系统也可适于用于筛选的EMS诱变的突变体改变的 Ca 2 +的响应。对于筛选目的，关键是要具有良好生长在平板上，并附着在薄膜上均匀的苗。薄膜粘附苗H-CTZ的四五个小时的潜伏期通常是足够的水母发光蛋白的重建。具有孔用粘接膜是优选的，因为apoaequorin和辅因子的反应需要氧气。对于组织或刺激特异性的 Ca 2 +响应的比较，使用幼苗组最初是从同一影片是至关重要的，因为苗和/或重组的条件可能影响功能的水母发光蛋白的总量的状态。当这些requiremeNTS都满足，发光响应的差异成比例的组织或刺激特异性的 Ca 2 +的响应。 水母发光蛋白的 Ca 2 +信号的基础的测量反映细胞群体的响应。但是，考虑到细胞类型和/或细胞在组织中，以刺激的不同的可访问性的异质性，水母发光蛋白的结果的基础的 Ca 2 +测量不等同于任何一种细胞的行为。了解钙离子动力在细胞水平要求，可与亚细胞分辨率检测的钙指标。荧光蛋白的活细胞的共聚焦成像（FP）为基础的 Ca 2 +的指标提供了一种先进的 Ca 2 +检测系统，用于记录细胞内Ca 2 +动力学与在期望的组织细胞的分辨率。在这里，用活细胞共聚焦成像和FP型钙离子的指标，我们记录了细胞内钙离子 </suP&gt;在两者同时细胞质和细胞核的瞬变。有两种类型的FP型钙指标具有不同特性的植物已经实现。相较于YCS，单绿色荧光蛋白为基础的钙指标具有一定的优势:1。Case12不需要YFP和CFP过滤器设置或许多假设和衡量FRET 22需要复杂的计算。 Case12具有单一的激发/发射最大值，并且可以使用标准的GFP过滤器组容易地检测到。 2 Case12是生理pH值下稳定，是一个比较小的蛋白质（46 kDa的）。当在植物中表达的，非靶向Case12出现在细胞质以及细胞核，所以Case12的转基因植物可用于同时跟踪胞质和核钙动力学。记者的一个缺点是，它是一种非比例的指标，所以荧光水平也受到那些无关的 Ca 2 +浓度的因素，例如表达水平Case12和当地的pH值。然而，Case12是足以确定细胞内Ca 2 +浓度为材料与不同的遗传背景之间在细胞质中的时空钙动力学和细胞器的相同的实验条件下进行比较。在未来，新的GFP基于钙指标具有更高的信号-噪声比，快的动力学和更大的反应，像GCaMP3 24和​​GCaMP5 25中的动物表现出，可以用于记录细胞是更好的指标或亚细胞的 Ca 2 +动力学中植物。 这里所描述的水母发光蛋白和Case12检测系统提供无损体内的方法时空钙离子动力学在整个苗期和亚细胞水平，分别测量。在这两种系统中，活体的状态或生长条件可能会影响钙的幼苗或细胞2 +的响应。如果物理学LLY损坏或无法在一个良好的状态生长，细胞可以具有弱甚至没有的 Ca 2 +的响应。此外，空间和时间的 Ca 2 +信号的持续时间也受到了幼苗在测量过程中的条件。发光或荧光信号可以随时间而减少由于刺激诱导的损伤，和/或激光诱导的光损伤在共焦成像的情况下。虽然不能避免刺激诱导的损伤，光毒性可以通过限制激光照射的强度和/或频率降低。此外，基于水母发光蛋白钙记录系统需要重建的步骤。该中断CTZ到细胞和/或apoaequorin与CTZ的反应扩散的因素会影响被直接相关的 Ca 2 +浓度的发光信号。因此CTZ的浓度，以及在持续时间和CTZ培养条件需要为最佳的灵敏度和reproducibil进行优化性。遗传编码的 Ca 2 +指示剂可以不准确地报告细胞的 Ca 2 +浓度为其它原因，包括非预期靶向于特定的亚细胞区室，温度敏感性，或记者与蜂窝内Ca2 +信号用机械23的干扰。我们发现，水母发光蛋白或Case12表达的植物都没有明显的形态或条件的表型，例如改变的抗逆性。因此，这是不可能的，这些基因编码的钙指标产生重大干扰与钙在植物体内的信号。 总之，我们在这里提出的使用2 的 Ca 2 +检测系统的详细说明同时在整个植物幼苗和亚细胞水平的测量空间和时间上的 Ca 2 +信号。这两个系统可以被用于确定组织（或细胞类型）或刺激特定的 Ca 2 +具有高灵敏度的动态模式和重现性。因此，它们可用于阐明背后的 Ca 2 +信号传导响应于各种环境线索的基因网络的有力工具。

植物细胞响应该坐标细胞动作经由信令环境。早期的细胞在应对环境刺激信号事件是一个短暂的钙增加。的图案，或一过性升高签名中游离的 Ca 2 +浓度的特征在于，其振幅，频率和持续时间。不同的时空钙签名规范不同的细胞活动1。特定刺激，如热，冷，盐，干旱，光，或植物激素，可以微调膜本地化的 Ca 2 +通道和/或转运的时空活性，导致具体的 Ca 2 +的签名。虽然钙离子转运都得到很好的特点，鲜为人知的是， 钙离子通道在植物1分子的身份和功能。对于突变体遗传筛选具有改变的 Ca 2 +响应强调刺激可能是一种有效APPRoach用于识别构成的 Ca 2 +的签名的组件。最近基于几个水母发光蛋白的 Ca 2 +检测系统已被开发，以促进遗传筛选的钙响应于病原体攻击和非生物胁迫2-4信令组件。 水母发光蛋白最早是用于检测钙在植物体内离子信号在90年代初5。从那时起，水母发光蛋白已经被定位到不同的细胞区室，如细 ​​胞质5，核6，叶绿体7，液泡膜8，线粒体9和基质10，以及不同的细胞类型中的根来监视特定的Ca 2细胞+信号11。基于水母发光蛋白钙离子的测量揭示细胞应力刺激人口的空间和时间的 Ca 2 +的响应。然而，在大多数情况下，单个细胞的钙离子的反应是unsynchroni捷思在响应组织4。因此，水母发光蛋白的 Ca 2 +的记录不一定报告在个体细胞中的钙信号。近年来，基因编码荧光蛋白（FP）为基础的 Ca 2 +的指标，如黄色卡默莱昂（YCS）12和CASEs12 13已被用于研究的 Ca 2 +具有高亚细胞分辨率的信号。 YCS是荧光共振能量转移（FRET）为基础的 Ca 2 +的指标，含有CFP和YFP的变体由钙挂钩2 +结合蛋白钙调蛋白和钙调蛋白结合肽M13。钙调蛋白发生构象变化，因为它结合的 Ca 2 +，由此带来的CFP和YFP更加靠近，从而增加了能量转移（FRET增强）。随着时间的荧光共振能量转移的水平，大约为YFP对CFP信号强度的比值计算，反映了细胞内钙离子动态。几个YC版本已被用于在植物中。 YC3.6为吨argeted到细胞质14,15，核16，线粒体17，和质膜18，和YC4.6和D4ER被定位到ER 15,19，和D3cpv被靶向过氧化物酶体20。转基因植物表达YCS允许不同类型的细胞不同的细胞内钙离子动态的活细胞成像。例（大概钙 lcium 本身 nsor）是单圆置换荧光蛋白（cpFPs）窝藏钙调蛋白和钙调蛋白结合肽M13。结合后的 Ca 2 +，情况发生构象变化，从而导致增加的荧光强度。该案例的荧光响应和Ca 2 +浓度之间的相关性使得细胞内钙离子动力学来进行定量测定。在钙 -饱和脂肪形式Case12变种12倍的增加荧光。N。 benthiminana植物瞬时EXPRessing Case12或拟南芥稳定表达病例12为材料，研究钙信号在国防和非生物胁迫4,21。异步时空钙离子振荡，细胞应对病原体的攻击，或脱水的压力已经显露了Case12基于钙离子成像。 在这里，我们提出的组织-和刺激基于水母发光蛋白发光成像特定的CA在拟南芥幼苗离子动力，并为细胞内的共焦成像与核钙的详细说明，在拟南芥根细胞离子的动态表达的FAS案例12发光成像可适于分析在完整植物或此处未描述的组织应力诱导的 Ca 2 +的动态，或筛选诱变的拟南芥属植物种群，对具有改变的应力诱导的 Ca 2 +信号的突变体。活细胞的 Ca 2 +成像设置可适于分析钙使用其他钙指标内的不同亚细胞区室或在不同类型的细胞离子动力。

<table><tbody><tr><td>10 cm x 10 cm square Petri dish</td><td>VWR</td><td>60872-310</td><td></td></tr><tr><td>Adhesive film</td><td>VWR</td><td>60941-062</td><td></td></tr><tr><td>Polyethylene tubing</td><td>PerkinElmer</td><td>9908265</td><td></td></tr><tr><td>1 ml syringe</td><td>VWR</td><td>53548-000</td><td></td></tr><tr><td>Silicone grease</td><td>Beckman</td><td>335148</td><td></td></tr><tr><td>2-well chambered cover glass&amp;nbsp;</td><td>Nalge Nunc international</td><td>155379</td><td></td></tr><tr><td>8-well chambered cover glass</td><td>Nalge Nunc international</td><td>155409</td><td></td></tr><tr><td>Luminescence imaging system</td><td>Princeton Instruments</td><td>N/A</td><td></td></tr><tr><td>Inverted confocal laser-scanning microscope</td><td>Nikon Instruments Inc.</td><td>N/A</td><td>Nikon A1R&amp;nbsp;</td></tr><tr><td>Imaging software</td><td>Nikon Instruments Inc.</td><td>N/A</td><td>Nikon Elements&amp;nbsp;</td></tr><tr><td>DataGraph</td><td>Visual Data Tools Inc</td><td>N/A</td><td>DataGraph 3.1.1 is the newest version</td></tr><tr><td>Coelenterazine</td><td>NanoLight Technolgies</td><td>#301B NF-BCTZ-FB</td><td></td></tr><tr><td>All purpose bleach</td><td>Any local store</td><td>N/A</td><td></td></tr><tr><td>Triton X-100</td><td>Fisher</td><td>BP151500</td><td></td></tr><tr><td>MS salt</td><td>Phytotechnology Labs</td><td>M524-50L</td><td></td></tr><tr><td>Sucrose</td><td>VWR</td><td>BDH8029-12KG</td><td></td></tr><tr><td>Agar</td><td>Sigma</td><td>A1296-5KG</td><td></td></tr><tr><td>Phytagel</td><td>Sigma</td><td>P8169-1KG</td><td></td></tr></tbody></table>

measuring spatial and temporal ca2+ signals in arabidopsis plants

发展与环境线索诱发的 Ca 2 +的波动在植物细胞中。刺激特定的空间-时间的 Ca 2 +的图案是由发起的 Ca 2 +信号传导级联的细胞的 Ca 2 +结合蛋白的检测。但是，我们仍然知之甚少如何刺激产生特定的钙信号。 à 钙离子信号的特殊性可能是由于钙离子通道和/或转运活动的复杂调控响应给定的刺激。识别这些细胞成分和理解它们的功能，关键的是要使用的系统，允许的 Ca 2 +信号的灵敏和鲁棒记录同时在组织和细胞水平。基因编码的定位到不同的细胞钙指标都规定了细胞钙信号活细胞共聚焦成像的平台。在这里，我们描述的指令S为使用两钙离子检测系统:基于水母发光蛋白FAS（薄膜粘合苗）发光钙成像和case12基于活细胞共聚焦荧光钙离子成像。使用FAS系统发光成像提供了一种简单，可靠和灵敏的检测的空间和时间的 Ca 2 +信号，在组织水平的，同时采用Case12活细胞的共聚焦成像提供同时检测胞质和核的 Ca 2 +信号中的较高的分辨率。

甘露糖醇，氯化钠和H 2 O 2被用作代理进行脱水，盐和氧化应激刺激，分别。检查，如果重金属离子铜离子协同作用与任何这三个应力刺激，我们比较了的 Ca 2 +响应于在铜离子的存在或不存在各刺激。如示于图2中 ，FAS发光成像显示，Arabidoposis苗反应不同脱水，盐和氧化应激。为刺激我们研究在本研究中的浓度，75mM氯化钠诱导的第40秒的曝光（ 图2A中间面板和图2B）的过程中在叶和根中的最强的 Ca 2 +的响应，而1 mM的H 2 O 2诱导的无论是在第一次和第二次40秒（ 图2A右面板）在叶和根的一个强有力的 Ca 2 +的响应。 400mM的甘露糖醇的溶液中仅诱导第二次40秒的过程中时，叶片的第一和40年代非常微弱的信号根钙反应。 铜离子强烈增强的振幅和通过NaCl和甘露醇（ 图2A左面板）来触发的 Ca 2 +信号的持续时间。它显示了Cu 2 +对H 2 O 2诱导的 Ca 2 +信号，减少幅度和持续时间（ 图2A右面板和2B）的抑制作用。 使用Case12表达拟南芥中，我们测得的钙在细胞浆离子动力学和核同步。 Case12的分子量为46 kDa的，所以转基因植物表达Case12有两种胞质溶胶中的GFP信号和叶的细胞核和根细胞（ 图4A）。定制（ 图3A和3B）或商会（ 图3C）被用于活细胞成像与激光共聚焦显微镜。施加150 mM氯化钠的幼苗在腔室中，我们在两种细胞溶胶和各单电池（;补充电影1 图4B和C）的核观察到荧光强度的变化。使用NIS-Elements的（图像处理软件，尼康仪器公司），我们测得的荧光强度在感兴趣区域（ROI），随着时间的推移地区。的振幅，持续时间和胞质和核的 Ca 2 +振荡的频率以图形方式在图4B中给出。的振幅和周期的Ca 2 +的尖峰在细胞质和细胞核变化很大不同细胞中的不同的根相同的根的细胞，间以及。的细胞质的 Ca 2 +尖峰振幅和周期分别为60.36±45.22 AU（任意单位）和58.24±15.70秒，分别。核钙尖峰的振幅和周期分别为316.26±75.24（非盟）和61.71±16.31秒，分别。与此相反，在的细胞质和细胞核钙尖峰时间分别为细胞，分别为17.43±3.67秒和17.33±2秒之间相似。的Ca 2 +的振幅峰值随时间降低，这可能是由于焦平面和/或响应的损失的变化要强调的，因为在体外条件不利的刺激。这些结果表明，盐胁迫触发的 Ca 2 +振荡同时在细胞质和细胞核中。 <img alt="图1" fo:content-width="6in" src="/files/ftp_upload/51945/51945fig1highres.jpg" width="600" /> 图1:基于水母发光蛋白FAS系统，用于测量时空钙离子动力响应应力刺激。 A）成长拟南芥幼苗垂直于含MS培养基，B中板，C），对苗木的最高层（C），D，E）发布粘接薄膜（B） </强&gt;将10天龄的幼苗上的粘合膜。F）的温育苗附着于薄膜用H-CTZ的2微克/毫升水中搅拌4小时。G）的切膜拦腰并把每一块成不同空盘。留在暗处将板5分钟。同时适用于控制和刺激方案（请务必在幼苗完全覆盖），并立即采集图像。比例尺为5厘米。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51945/51945fig2highres.jpg" width="600" /> 图2比较时空的Ca 2 +的第10天幼苗不同应力刺激的反应。 A）的时间序列苗发光图像进行400毫米的甘露醇和400毫米甘露醇加1毫氯化铜（一，左图），75毫米氯化钠和75毫米氯化钠加1毫氯化亚铜图2（A，中图），以及1毫米H 2 O 2或1毫米的H 2 O 2加1 mM的氯化铜（一，右图）。 A的上部和下部板是第一40秒和秒40秒，分别期间获得的发光图像。强度比例尺为从低（黑）增加发光信号的高电平（白）。B）的条形图苗的平均发光强度的响应于所指示的压力刺激。 <img alt="图3" fo:content-width="6in" src="/files/ftp_upload/51945/51945fig3highres.jpg" width="600" /> 图3设置活细胞共聚焦成像实验。 A）定制室建有幻灯片，盖玻片用四块粘土和脱脂棉。两个5天的幼苗放置在幻灯片。的聚乙烯管的一端被放在旁边的装配室和固定一个个的磁带，而另一端被连接到1ml注射器。 乙）定制的滑动用有机玻璃制成具有开放的腔室中的中间，并且被连接到信道的两侧的两个通道。一盖玻片放置在载玻片的顶部和两个聚乙烯管被放置在信道。盖玻片和管被固定在用硅润滑脂的位置。管中的一个的一端连接到1ml注射器和另一管的端部保持打开状态。 5天老苗置于室和coveslip放在苗的顶部和硅脂密封。与装配腔的plexigalss载玻片放置在显微镜的阶段。℃）的拟南芥幼苗生长在两口井（左）或8个孔（右）含有的植物凝胶为0.6％，在MS一层薄薄的腔盖玻璃的媒体。 <img alt="图4" fo:content-width="6英寸"SRC ="/文件/ ftp_upload / 51945 / 51945fig4highres.jpg"WIDTH ="600"/&gt; 图4胞质和核的 Ca 2 +振荡响应于盐胁迫。 A）的共聚焦图像显示，Case12表现在细胞质和根细胞（左图）和转基因植物表达Case12叶细胞（右图）的细胞核。右侧面板上的图像的叶绿体自发荧光红和Case12。 的B绿色荧光）的盐胁迫（150毫米氯化钠）引起的钙离子振荡在细胞质中（上图）和细胞核（下图）的合并图像根细胞。所测量的小区的位置由与上面板的数字的箭头表示。

Watch this Scientific Journal Video about Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants at JoVE.com

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

的 Ca 2 +信号调控植物中不同的生物过程。在这里，我们提出的方法用于监测非生物胁迫诱导的时空钙在拟南芥的细胞，并使用基因编码的钙指标水母发光蛋白或Case12组织2 +信号。

测量时空的Ca<sup&gt; 2 +</sup&gt;在拟南芥信号

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

measuring-spatial-and-temporal-ca2-signals-in-arabidopsis-plants

Nanyang Technological University

Research

JoVE Journal

Biology

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

12.0K 次观看.  Purdue University.  的 Ca 2 +信号调控植物中不同的生物过程。在这里，我们提出的方法用于监测非生物胁迫诱导的时空钙在拟南芥的细胞，并使用基因编码的钙指标水母发光蛋白或Case12组织2 +信号。 

视频: Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Visualizing Cellular Gibberellin Levels Using the nlsGPS1 F&#246;rster Resonance Energy Transfer (FRET) Biosensor

The phytohormone gibberellin (GA) is a small, mobile signaling molecule that plays a key role in seed germination, cellular elongation, and developmental transitions in plants. Gibberellin Perception Sensor 1 (GPS1) is the first F&#246;rster resonance energy transfer (FRET)-based biosensor that allows monitoring of cellular GA levels in vivo. By measuring a fluorescence emission ratio of nuclear localized-GPS1 (nlsGPS1), spatiotemporal mapping of endogenously and exogenously supplied GA gradients in different tissue types is feasible at a cellular scale. This protocol will describe how to image nlsGPS1 emission ratios in three example experiments: steady-state, before-and-after exogenous gibberellin A4 (GA4) treatments, and over a treatment time-course. We also provide methods to analyze nlsGPS1 emission ratios using both Fiji and a commercial three-dimensional (3-D) micrograph visualization and analysis software and explain the limitations and likely pitfalls of using nlsGPS1 to quantify gibberellin levels.

Gibberellin Perception Sensor 1 (GPS1) is the first F&#246;rster resonance energy transfer-based biosensor for measuring the cellular levels of gibberellin phytohormones with a high spatiotemporal resolution. This protocol reports on the method to visualize and quantify cellular gibberellin levels using the genetically encoded nlsGPS1 biosensor in Arabidopsis hypocotyls and root tips.

利用 nlsgps1 förster 共振能量转移 (fret) 生物传感器可视化细胞赤霉素水平

The phytohormone gibberellin (GA) is a small, mobile signaling molecule that plays a key role in seed germination, cellular elongation, and developmental ...

科研

发育生物学

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 ...

生物学

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

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

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

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

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

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

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 ...

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution.

实时在体记录了拟南芥钙信号在使用荧光传感器的昆虫饲喂时

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant ...

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires careful and in-depth analyses and quantification to capture as much information about the underlying events as possible. Spatial, temporal and intensity parameters intrinsic to Ca2+ signals such as frequency, duration, propagation, velocity and amplitude may provide some biological information required for intracellular signalling. High-resolution Ca2+ imaging typically results in the acquisition of large data files that are time consuming to process in terms of translating the imaging information into quantifiable data, and this process can be susceptible to human error and bias. Analysis of Ca2+ signals from cells in situ typically relies on simple intensity measurements from arbitrarily selected regions of interest (ROI) within a field of view (FOV). This approach ignores much of the important signaling information contained in the FOV. Thus, in order to maximize recovery of information from such high-resolution recordings obtained with Ca2+dyes or optogenetic Ca2+ imaging, appropriate spatial and temporal analysis of the Ca2+ signals is required. The protocols outlined in this paper will describe how a high volume of data can be obtained from Ca2+ imaging recordings to facilitate more complete analysis and quantification of Ca2+ signals recorded from cells using a combination of spatiotemporal map (STM)-based analysis and particle-based analysis. The protocols also describe how different patterns of Ca2+ signaling observed in different cell populations in situ can be analyzed appropriately. For illustration, the method will examine Ca2+ signaling in a specialized population of cells in the small intestine, interstitial cells of Cajal (ICC), using GECIs.

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Genetically encoded Ca2+ indicators (GECIs) have radically changed how in situ Ca2+ imaging is performed. To maximize data recovery from such recordings, appropriate analysis of Ca2+ signals is required. The protocols in this paper facilitate the quantification of Ca2+ signals recorded in situ using spatiotemporal mapping and particle-based analysis.

时空映射和粒子分析技术在细胞内 ca2 +原位信令定量中的应用

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires ...

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is regulated spatially and temporally. The use of a fluorescent gene under the control of an immunity-related promoter in combination with an automated fluorescence microscopy is a simple way to understand spatiotemporal regulation of plant immunity. In contrast to the root tissues that have been used for a number of various intravital fluorescent imaging experiments, there exist few fluorescent live-imaging examples for the leaf tissues that encounter an array of airborne microbial infections. Therefore, we developed a simple method to mount leaves of Arabidopsis thaliana plants for live-cell imaging over an extended period of time. We used transgenic Arabidopsis plants expressing the yellow fluorescent protein (YFP) genefused to the nuclear localization signal (NLS) under the control of the promoter of a defense-related marker gene, Pathogenesis-Related 1 (PR1). We infiltrated a transgenic leaf with Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) and performed in vivo time-lapse imaging of the YFP signal for a total of 40 h using an automated fluorescence stereomicroscope. This method can be utilized not only for studies on plant immune responses but also for analyses of various developmental events and environmental responses occurring in leaf tissues.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

一种用于阴南芥叶片安装的通用方法

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...

Forward Genetic Screen Using Transgenic Calcium Reporter Aequorin to Identify Novel Targets in Calcium Signaling

Forward genetic screens have been important tools in the unbiased identification of genetic components involved in several biological pathways. The basis of the screen is to generate a mutant population that can be screened with a phenotype of interest. EMS (ethyl methane sulfonate) is a commonly used alkylating agent for inducing random mutation in a classical forward genetic screen to identify multiple genes involved in any given process. Cytosolic calcium (Ca2+) elevation is a key early signaling pathway that is activated upon stress perception. However the identity of receptors, channels, pumps and transporters of Ca2+ is still elusive in many study systems. Aequorin is a cellular calcium reporter protein isolated from Aequorea victoria and stably expressed in Arabidopsis. Exploiting this, we designed a forward genetic screen in which we EMS-mutagenized the aequorin transgenic. The seeds from the mutant plants were collected (M1) and screening for the phenotype of interest was carried out in the segregating (M2) population. Using a 96-well high-throughput Ca2+ measurement protocol, several novel mutants can be identified that have a varying calcium response and are measured in real time. The mutants with the phenotype of interest are rescued and propagated till a homozygous mutant plant population is obtained. This protocol provides a method for forward genetic screens in Ca2+ reporter background and identify novel Ca2+ regulated targets.

A forward genetic screen based on Ca2+ elevation as a read-out leads to identification of genetic components involved in calcium dependent signaling pathways in plants.

使用转基因钙处理器 Aequorin 前向基因屏幕，以识别钙信号中的新目标

Forward genetic screens have been important tools in the unbiased identification of genetic components involved in several biological pathways. The basis ...

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. ...

拟南芥幼苗中叶绿素生物合成的高分辨率成像

Non-invasive Assay for Chlorophyll Biosynthesis Kinetics Determination during Early Stages of Arabidopsis De-etiolation

Chlorophyll biosynthesis is a hallmark of de-etiolation, one of the most dramatic stages in the plant life cycle. The tightly controlled and highly dynamic process of chlorophyll biosynthesis is triggered during the shift from the dark to the light in flowering plants. At the moment when etiolated seedlings are exposed to the first traces of sunlight, rapid (in order of seconds) conversion of protochlorophyllide into chlorophyllide is mediated by unique light-accepting protein complexes, leading via subsequent metabolic steps to the production of fully functional chlorophyll. Standard techniques for chlorophyll content analysis include pigment extraction from detached plant tissues, which does not apply to studying such fast processes. To investigate chlorophyll kinetics in vivo with high accuracy and spatiotemporal resolution in the first hours after light-induced de-etiolation, an instrument and protocol were developed. Here, we present a detailed procedure designed for statistically robust quantification of chlorophyll in the early stages of Arabidopsis de-etiolation.

作者聚焦:去硫醇化过程中叶绿素合成的无创高分辨率测量

Here, we describe an advanced tool designed for chlorophyll biosynthesis monitoring during the early stages of Arabidopsis seedling de-etiolation. The novel methodology provides non-invasive real-time chlorophyll fluorescence imaging at high spatial and temporal resolution.

拟南芥脱硫早期叶绿素生物合成动力学测定的非侵入性测定

Chlorophyll biosynthesis is a hallmark of de-etiolation, one of the most dramatic stages in the plant life cycle. The tightly controlled and highly ...

测量时空的Ca

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