这项工作是由美国国家科学基金会NSF-IOS补助＃1144391 SHB支持。

1。植物的制备<ol><li> 拟南芥种子可以发芽的土壤直接或MS板。 12小时光照（22°C，15°C夜14），种植的植物在生长室为四到六周。水厂一次到每周两次。使用底部托盘浇水;不水厂从上边让盘前干再浇水。确保植物在收获时，充足的水分。 </li></ol> 2。制备解决方案和菜<ol><li> K 2 EDTA溶液：100（MM）K 2 EDTA溶液原液，它可以存放在冰箱里。的集合中的天，稀溶液一到五（的一部分EDTA溶液，4份水），以取得的20mM K 2 EDTA溶液中。 </li><li>填补玻璃或培养皿（直径7-15厘米），20毫米K 2 EDTA溶液的一半。如果收集样本从不同的治疗方法（前充足的控制和盐胁迫的植物或不同基因型），准备不同的菜肴，每处理。 </li><li>填写所需数量的1.7 ml反应管的20mM K 2 EDTA溶液1.4毫升。填充相等数量的反应管1.4毫升高压灭菌去离子水或Millipore公司。 </li><li>把纸巾在板凳上设置植物，得到一个新的刀片，并戴上手套。 </li></ol> 3。收集的韧皮部分泌物（描绘了图1中的流程图） <ol><li>从四到六周龄的植物收获的莲座叶，叶柄接近的莲座的中心上面的基础上，通过切割刀片。 </li><li>立即将叶子菜含有20毫米K 2 EDTA。确保的叶柄的切断端浸没在溶液中（ 图2）。 </li><li>一旦收集15叶（约三至四个植物），轻轻地堆叠的顶部叶片等彼此吨帽子切叶柄彼此对准。重新酶切叶柄（约1毫米）的基础上，用的20mM K 2 EDTA溶液，并立即转移到一个反应管的叶子。叶子适合最简单的，如果他们微微卷起。避免挤压，因为这可能会导致细胞损伤，从而导致到叶片细胞含量的收集树叶。 </li><li>如果需要更多的材料，用湿纸巾轻轻覆盖样品。如果从不同的治疗方法或基因型收集样本使用了一套新的菜肴。 </li><li>一旦所有的样品被固定在反应管，在黑暗中或在光线收集分泌物。 </li><li>在黑暗中，地板用湿纸巾的大浅盘线的收集。叶填充的反应管中的菜，要么盖湿纸巾或将机架放入一个黑色的塑料背部曝气狭缝。将整个安装在机柜或黑暗的房间。 </li><li>对于收集的光，排队一个明确的有机玻璃容器底部用湿纸巾。叶填充的容器中的反应管放置的机架，并关闭它。 </li><li> 1小时后，捞出叶子轻轻地从容器和反应管，并用蒸馏水或去离子微孔水彻底清洗，除去所有EDTA。此步骤有三个目的：（1）它消除EDTA的损坏最小化的细胞，（2）它消除了干扰EDTA与SDS-PAGE和层析，和（3）消除了任何化合物累积产生的剪切/损坏细胞。立即转移到含有无菌水制备的反应管和恢复加湿收集渗出液收集容器。在这一点上，加入RNase抑制剂渗出液，如果需要被用于RNA提取。可选：如果需要蛋白质分析，可以添加蛋白酶抑制剂。 </li><li>拟集合时间后，拔出DISCA第三届叶子和冻结韧皮部流出物在液N 2中以供进一步使用。如果需要扩展存储，冻干样品并储存于-80°C。 </li><li>在拟南芥的情况下，代谢产物已经可以被检测到1小时后收集的流出物。然而，收集5-8小时为宜，如果分析较丰富的组件计划。对于较大的植物，如紫苏 ，收集时间较长（8小时）建议。 渗出液从15叶子收集通常是足够的SDS-PAGE凝胶（蛋白质），mRNA的提取，GC-MS（糖和代谢物）的一个车道。血脂分析2-3个样品汇集（渗液30-45叶）建议，并导致更清晰的结果。 </li></ol> 4。随后的分析（用于视频我们只看4.4） <ol><li>蛋白质分析，重悬样品15μl水和30微升Lämmli缓冲，加热至95℃，5分钟，快速降速任意precipitates到SDS-PAGE凝胶（45微升加载口袋）和加载整个样本。样品应与胶体考马斯亮蓝染色或其他敏感的污渍，因为蛋白质丰度很低。 </li><li>糖和小的代谢物分析，添加干燥后的样品，在文献[14]中所描述的衍生剂。 </li><li> mRNA可以使用RT-PCR法，微阵列或RNA测序分析进行分析。 </li><li>脂质分析紧接池两到三个样品和分区对氯仿：甲醇（1:1）在通风柜如下：对于添加等量的氯仿：甲醇向每个样品中，涡旋几秒钟，离心4分钟在400×g。收集与聚四氟乙烯套在玻璃管中，与顶端相3次以上的重复分区的底部（有机）相。下N 2气流干燥合并的有机相，并提交进一步分析（薄层色谱法：TLC 14，37;液相色谱-质谱：LC-MS 14）。 </li></ol>

我们什么都没有透露。

的EDTA促进韧皮部渗出物的收集是非常简单的，需要最少的设备，适用于大多数植物。总体而言，此方法扩展分析能力，韧皮部分泌物从更多的物种。 ，虽然渗出液稀释，很容易收集许多不同的样本，或从许多植物中，以扩展至大量的材料。这就允许检测的新的化合物在非常低丰度的，否则将被忽略。 此方法可用于多种植物物种和工作所描述的这个手稿中列出的那些。如果探索新的物种，可能需要修改的两个方面是所使用的叶片的数目和渗出的时间。在大多数情况下，一个五至八个小时渗出应该是合适的。确认使用这种方法的一个新的植物物种，需要收集渗出物和一个或多个随后的分析中所描述的协议部分4 N进行电火工品。根据观察到的信号的强度，采集量，可能需要扩大。在一般情况下，将需要更多的脂肪和蛋白质分析的材料比糖和代谢物的分析，因为前者是在韧皮部分泌物较少丰富。 EDTA-促进渗出物的收集，因为涉及切割面，以及一个潜在的破坏性的影响的EDTA，有几点必须考虑到：（1）含EDTA的一个小时的孵育后，叶柄彻底洗净。此删除任何受伤的细胞以及乙二胺四乙酸本身，这可能会损坏细胞或干扰萃取得到的化合物。 （2）阳性和阴性对照，以确保获得的数据均来自韧皮部汁液，需要包括受伤的细胞，而不是从（请参阅下面的关键步骤）。 （3）韧皮部汁液含有多种酶，这可能是活跃在渗出液收集。因此，在某些情况下可能是有用的，以收集FOŗ不同的时间。 （4）由于该方法涉及到水渗出，绝对定量的化合物是不可能的。 关键步骤：成功地使用这种方法的关键是使用谨慎处理样品的同时，不会伤害任何细胞，以及使用适当的控制（见参考文献14）。一个合适的控制是未经处理的收集韧皮部分泌物用EDTA。在这种情况下，韧皮部，将密封本身，并可以收集没有渗出液。任何污染的化合物来自受伤的细胞将被检测到。第二个控制将渗出液中糖分析。在拟南芥中，应该是蔗糖的主要代谢产物内韧皮部汁液，果糖与蔗糖比在1:4和1:8之间（参考8，14）。 提取RNA的RNA酶抑制剂的使用是必要的。此外，阳性对照先前describ的编韧皮部的本地化的mRNA（UBC9：泛素结合酶9，At4g27960，8，14）和作为阴性对照的叶绿体mRNA表达（Rubisco的LSU）例如是可取的。 由于渗出液含有活性酶，一个时间过程应确保韧皮部轮廓的变化，不只是由于集合中时间的变化。在某些情况下，它可能是有益的使用蛋白酶抑制剂。然而，调查人员需要牢记，将集中收集的渗出物和任何添加的化合物将主要集中。在协议的第二个关键步骤是再切下叶柄EDTA。这一步有两个目的：第一，它删除任何密封筛板，同时防止形成新的插头从而允许自由流动的韧皮部汁液。其次，它消除了空化泡在切割时产生的木质部，从而允许木质部运输。第二次切割的大小取决于上线使用的植物。在较大的叶柄植物，它应该是几毫米（可达1厘米）以上的首次降息， 拟南芥等植物中,1-2毫米就足够了。 使用这种方法可以发现在韧皮部的渗出液的新的化合物，可能有信号，发育或生物医学方面的影响导致。它已经引发了更深入的看看在长距离的的脂质信号，这是一个小研究植物科学领域内由于缺乏进入韧皮部。

植物不能移动摆脱不利条件。因此，他们不得不开发机制来检测环境压力，引起整个工厂和传输相关的信号，并相应地调整发展。两个运输系统存在的水，营养物和其他化合物（信令）的分布。首先是木质部;，通常输送水和矿物质，整个植物的根。第二个是韧皮部。韧皮部的图已经改变从一个简单的吸收运输系统的导管用于RNA，蛋白质，病毒，脂质和其它小分子。它起着重要的作用，吸收和养分运输，生物和非生物胁迫的响应，以及​​在植物生长和发育。它现在被称为“信息高速公路”的植物18。 韧皮部是由多种细胞类型：韧皮薄壁，以及专门的伴侣CELLS和筛分子。筛元素是长距离的移动网站。要允许纵向流动通畅，筛分子最缺少的细胞器，以及原子核和被认为含有充其量只是一种有限的平移机械21，33。据认为，伴随细胞合成蛋白质和其它化合物，在韧皮部流旅行。然后，这些化合物是通过胞间连丝运送到筛分子，可以作为长距离信号4,16。 可以发现在韧皮部的排泄物的几组化合物： <ul><li>糖是经常光合作用的产品信息，并传输携带能源“分子植物其他部分的存储，或作为构建块。然而，它们也可以作为防冻剂或作为信号化合物。 </li><li>蛋白质也可以发现在韧皮部分泌物。它们包括代谢酶，但也有信号功能。一个examp发育信号作为一种蛋白质，是乐开花轨迹Ť蛋白信号的感应以及季节性开花植物叶脱落。 </li><li>核酸是mRNA的，小型和微型的RNA病毒的RNA的形式存在于韧皮部渗出。他们似乎在很大程度上参与信号27，28，39。与此同时，rRNA和瓜类42韧皮部汁液中已经观察到几乎所有氨基酸的tRNA。他们似乎被选择性地转移到筛管系统。 tRNA的显示必要修改，转移的能力氨基酸平移装置。然而，而不是参与翻译，他们似乎抑制这个过程。或者，他们可以作为一个源细胞分裂素或信号厂房42的代谢状况。 </li><li>在韧皮部渗出物3，13，14，23，也已发现的脂肪酸，oxylipins，和其他脂质。 JasmoNIC酸，为应对病原体感染22，29，31，32的一部分通过韧皮部的oxylipin移动。虽然大多数韧皮部血脂的作用仍不清楚，其中一些可能有信号功能4。 </li></ul>植物韧皮部工作的挑战在于它的容量密封后伤人。有四个主要用来收集韧皮部分泌物的方法，但他们只能在选择物种： 1）在葫芦科植物，它是通过削减叶柄未能取得合理数量的韧皮部渗出。一旦最初的下降与损伤的细胞的污染被除去，这是可以取得相当大的量的纯韧皮部汁液1，15。然而，随着收集时间，这样的渗出物变稠，使其越来越不适合用于液相色谱的方法（未发表）。最近的出版物表明，韧皮部汁液取决于物种，这是源于要么r的束状（FP）的：韧皮部extrafascicular（EP），而它确实包含“移动”的韧皮部筛分子（FP），也容易污染其他类型的细胞，包括木质部40，41 。 2）第二种方法是通过茎或叶柄浅切口或穿刺获得韧皮部汁液。该方法已被成功地使用，羽扇豆17，25，36葫芦科， 甘蓝型油菜 12。这里的污染是最小的，从受伤的细胞的分泌物很纯。然而，植物需要是健康和浇灌的，因为它是非常具有挑战性的选择性穿刺只是筛分子。如果木质部缺口，所有被抽入木质部流渗出。这使得该方法不适合非常脆弱或高度木质化叶柄或茎的植物。 3）蚜虫stylectomy允许他们的短剑插入到筛分子蚜虫Ñ​​用激光消除蚜虫。韧皮部汁液渗出，通过剩余的短剑2，9，10，35，38。从理论上讲，这种方法可用于任何被感染的植物，可以由蚜虫。然而，大多数温室或生长室经理将不支持使用的病原体。此外，蚜虫介绍几种蛋白质进入韧皮部与他们的唾液19，33。这将导致一个有限的转录重编程30，有可能改变韧皮部组合 ​​物26。 4）这里介绍的方法是EDTA促进韧皮部汁液渗出。该方法采用EDTA的韧皮部20，以防止密封。 EDTA螯合的Ca 2 +离子，否则将参加密封韧皮部的进程。虽然EDTA可导致细胞的损伤30，几组都使用此方法，并没有观察到不利影响的10mM的EDTA的浓度从20mM的的细胞超微结构上phloe的上24米5，装载和运输。为了减少任何破坏性影响以及干扰EDTA与色谱和凝胶电泳，植物搬进水1小时后，才有了后来的渗出物的一部分用于14。因此，而非渗出物进入EDTA，韧皮部渗出水（EDTA促进渗出）。这是一个直接，成本低，技术含量低的方法收集韧皮部分泌物。韧皮部汁液这种方式获得的，可用于分析蛋白质，小分子，脂质，和RNA，并已成功地用于在许多植物中。虽然在单子叶植物11中已经执行了有限数量的实验方法，该方法似乎是更适合于双子叶植物（ 紫苏 17，20， 拟南芥 8，13，14， 白杨 7）。收集分泌物发生在潮湿的环境中，通过蒸腾作用的分泌物，以避免损失。根据工厂的一到两小时，在EDTA中温育足以防止密封韧皮部/筛分子。然后集合可以发生水。这有利于防止EDTA对细胞的结构和稳定性的负面影响。它也消除了干扰的方法，如HPLC或SDS-网页的EDTA。示出，因为它适用于拟南芥 。对于较大的植物，培养在EDTA和渗出物馆藏有扩大规模，并在烧杯中进行，而不是在1.7毫升的反应管。

<table border="0" cellpadding="0" cellspacing="0" width="638">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">K2-EDTA</td>
			<td style="width:152px;">Sigma-Aldrich</td>
			<td style="width:111px;">ED2P-500G</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:153px;">Shallow Glass or plastic Petri Dish (7-15 cm)</td>
			<td>PYREX or Corning</td>
			<td></td>
			<td>Any clean, shallow dish will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloroform</td>
			<td>EMD</td>
			<td>CX1054-1</td>
			<td style="width:223px;">Only open containers in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol</td>
			<td style="width:152px;">J.T.Baker</td>
			<td style="width:111px;">9070-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">Screw cap tubes</td>
			<td style="width:152px;">VWR International</td>
			<td style="width:111px;">53283-800</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">Screw cap tubes</td>
			<td style="width:152px;">Sun Sri</td>
			<td style="width:111px;">13-425</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">Eppendorf tubes</td>
			<td style="width:152px;">Denville</td>
			<td style="width:111px;">C2170</td>
			<td></td>
		</tr>
	</tbody>
</table>

collection and analysis of arabidopsis phloem exudates using the edta-facilitated method

植物韧皮部是必不可少的长途运输（照片）以及输送生物或非生物胁迫的信号同化。它含有糖，氨基酸，蛋白质，RNA，脂质和其他代谢产物。理解的组成和功能的韧皮部，许多这些分子的作用，从而虽然是一个大的兴趣，在植物发育和应力响应的重要性尚未确定。韧皮部分析的一个障碍在于韧皮部密封后伤人的事实。其结果是，从该植物韧皮部汁液可以得到的数量是有限的。的一种方法，该方法允许韧皮部的排泄物收集不增加设备的一些植物物种是这里所描述的EDTA-促进的韧皮部渗出物的收集。虽然很容易使用，它导致伤人的细胞和护理注意消除受损细胞的内容。此外，有几个控制，以证明渗出液的纯度是必要的。因为它是一个而不是直接收集韧皮部汁液（不可能在许多物种）相对定量其内容可以发生渗出。比其他的这种方法的优点是它可以被用在许多草本或木本植物物种（ 紫苏 ， 拟南芥 ， 杨树 ， 等 ），并需要最少的设备和培训。它会导致相当大的蛋白质，糖类，脂类，RNA，病毒和代谢物用于随后的分析，可用于大量的排泄物。这是很简单的，它可以用来在一个研究中，以及在一个教学实验室。

现在已经很清楚，在过去几年的韧皮部汁液的复杂性，可能是​​植物如何发送不同的发育和压力信号的最佳响应问题的答案。使用EDTA-促进的韧皮部渗出，可能提供的机会，以分析其他的方法是不适合的，但可能是经济的或生理的兴趣。从植物的韧皮部渗出。 此方法允许足够的韧皮部分泌物的收获找出韧皮部本地化的蛋白质，并开发或应力响应检测出自己的水平的变化（ 图3）。正如图中所示，是在非常低丰度蛋白质，但他们的水平足够高，后续蛋白质组学实验利用LC-MS/MS或Western blot分析。葫芦科植物中的研究结果表明，茎的切断导致的水势平衡的中断和随后的水流入和从质外体41的可能的污染物。然而，收集时间为一至八个小时不影响韧皮部蛋白/组合物在拟南芥 （只有绝对量变化Guelette的等 ），收集的蛋白质的量，并找到了蛋白质的图案不同物种之间（ 拟南芥紫苏 ，巷子里，我和NI）和治疗（ 紫苏生长在不同的日子长，巷子里，我和NI）。其结果是蛋白质的信号，可以可视化，确定和遵守。 mRNA和微的RNA，也可以识别在韧皮部流出物通过该方法。但是，RNA酶抑制剂治疗是必要的，以防止降解的mRNA在延长的渗出时间。 ，由于筛元素不包含功能叶绿体，Rubisco小或大亚基（红细胞或rbcL基因，分别）的mRNA可作为阴性对照，而公知的韧皮部泛素结合酶类似的本地化的mRNA，可以使用作为阳性对照（ 图4）。 同样， 糖或小的代谢产物可以使用GC-MS或LC-MS分析。这里应该提到，韧皮部流出物中含有大量的功能性酶，包括几乎整个糖酵解途径12。因此，用几个时间点，可能会发现渗出液收集在代谢过程。在图5中显示了一个示例：在所有的流出物，蔗糖是最丰富的代谢物，这是最明显的，1小时后收集。然而，在五个小时后蔗糖果糖的比例略有减少。果糖比蔗糖如果相同的渗出液收集一小时，并留在了板凳接下来的四个小时，减少到一个更大的范围内，这表明活性酶的韧皮部分泌物导致的劣化的蔗糖在室温更多的峰值诠释温度（见14）。这是进入筛元素伴细胞分子的韧皮部装载和运输过程中可能会继续渗出，直到系统失去其活力34的研究结果相一致。 血脂韧皮部分泌物中，通过扩展，远距离的脂质信号是一个相当新的领域植物科学的兴趣。由于其疏水性脂质只存在于低浓度，可以被绑定到其他分子的增溶。然而，EDTA，促进渗出物可收集足够的材料，可视化（TLC 图6）和识别（LC-MS 图7），韧皮部血脂几种植物物种。正如图中所示，它是能够识别和区分一些种脂质。 LC-MS可以识别不同种类的脂质和内各色的血脂谱变化进行监测NT基因型或治疗。这允许在植物生长发育和应激反应血脂的作用的研究。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51111/51111fig1.jpg" width="600px" /> 图1 拟南芥或紫苏韧皮部分泌物的采集流程图。不同的路径导致不同的高端产品，以蓝色显示。制备RNA，RNA酶抑制剂（100 U / ml的，Roche公司）被添加到其中的韧皮部渗出物被收集到水。 <a href="https://www.jove.com/files/ftp_upload/51111/51111fig1highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51111/51111fig2.jpg" width="600px" /> 图2。设置韧皮部渗出液收集材料根离子。设置显示所有协议的步骤3.1-3.4所需材料。 <a href="https://www.jove.com/files/ftp_upload/51111/51111fig2highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51111/51111fig3.jpg" width="600px" /> 韧皮部中的图3。蛋白分泌物两种不同的植物物种， 拟南芥 （在）， 紫苏 （I和NI不同天长度）;分子量：分子量标记物（泳道2）。使用10-20％的梯度SDS-PAGE分离蛋白质。 <a href="https://www.jove.com/files/ftp_upload/51111/51111fig3highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51111/51111fig4.jpg" width="600px" /> 图4分析。的Rubisco小和大亚单位的mRNA的存在（红细胞和rbcL，分别）和泛素结合酶（UBC）。收集mRNA的从拟南芥叶片（L），的叶柄（P），和韧皮部渗出液（pH值），反转录和可视化用PCR的特异性转录。数字修改从Guelette 等14。 <a href="https://www.jove.com/files/ftp_upload/51111/51111fig4highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51111/51111fig5.jpg" width="600px" /> 图5。韧皮部分泌物的GC-MS文件收集不同的时间量。请注意集合（A）1小时到5个小时的采集时间（B）的相对量蔗糖的变化。渗出液收集资料档后1小时，随后在室温的“孵化器”<html>四小时的温度（RT）（C）。叶代谢轮廓（D）。 <a href="https://www.jove.com/files/ftp_upload/51111/51111fig5highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图6" fo:content-width="5in" src="/files/ftp_upload/51111/51111fig6.jpg" width="600px" /> 图6。薄层色谱图比较脂质紫苏 （左）和拟南芥 （右）的叶和韧皮部分泌物。星号表示特定血脂韧皮部分泌物。的DGDG：digalactosyldiacylglycerol; PG：磷脂，MGDG monogalactosyldiacyglycerol;身影显示拟南芥血脂的一部分已被修改从Guelette 等14。 <a href="https://www.jove.com/files/ftp_upload/51111/51111fig6highres.jpg" target="_blank">点击这里查看大图</a> 。 NT“FO：保持together.within”页面=“总是”&gt; <img alt="图7" fo:content-width="5in" src="/files/ftp_upload/51111/51111fig7.jpg" width="600px" /> 图7。血脂韧皮部分泌物拟南芥 （氯仿阶段）用LC-MS /负离子模式。图中的顶端（突变体中的A）和底部（野生型中的B）示出在脂质的档案（由箭头表示）两种不同基因型之间的差异。 <a href="https://www.jove.com/files/ftp_upload/51111/51111fig7highres.jpg" target="_blank">点击这里查看大图</a> 。

Watch this Scientific Journal Video about Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method at JoVE.com

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

远距离信号指示的理解，在植物生长发育和应力/病原体的反应和吸收运输的韧皮部汁液的组合物，以及其加载和长途运输的机制的知识是至关重要的。此手稿描述的韧皮部分泌物使用EDTA促进方法的集合。

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 ...

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Unit 1

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

Complexometric Titration, Precipitation Titration, and Gravimetry

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24.2K 次观看.  Michigan State Universtiy. 远距离信号指示的理解，在植物生长发育和应力/病原体的反应和吸收运输的韧皮部汁液的组合物，以及其加载和长途运输的机制的知识是至关重要的。此手稿描述的韧皮部分泌物使用EDTA促进方法的集合。 

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

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

一个影像的简单方法拟南芥叶浸润成像介质中使用全氟萘烷

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

科研

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 ...

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

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

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

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

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

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

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently attach a tagged form of dUTP to 3&#8217; ends of double- and single-stranded DNA breaks in cells. It is a reliable and useful method to detect DNA damage and cell death in situ. This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method of semi-automated TUNEL signal quantitation. Inherent normal tissue features and tissue processing conditions affect the ability of the TdT enzyme to efficiently label DNA. Tissue processing may also add undesirable autofluorescence that will interfere with TUNEL signal detection. Therefore, it is important to empirically determine tissue processing and TUNEL labeling methods that will yield the optimal signal-to-noise ratio for subsequent quantitation. The fluorescence-based assay described here provides a way to exclude autofluorescent signal by digital channel subtraction. The TUNEL assay, used with appropriate tissue processing techniques and controls, is a relatively fast, reproducible, quantitative method for detecting apoptosis in tissue. It can be used to confirm DNA damage and apoptosis as pathological mechanisms, to identify affected cell types, and to assess the efficacy of therapeutic treatments in vivo.

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method for semi-automated analysis of TUNEL labeling.

检测和DNA损伤分析小鼠骨骼肌<em&gt;原位</em&gt;使用TUNEL法

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

拟南芥种子高通量灭菌的标准化方法

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...

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 ...

Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection

Rice stripe virus (RSV), which causes significant economic loss of agriculture in East Asia, entirely depends on insect vectors for its effective transmission among host rice. Laodelphax striatellus (small brown planthopper, SBPH) is the primary insect vector that horizontally transmits RSV while sucking sap from the phloem. Saliva plays a significant role in insects' feeding behavior. A convenient method that will be useful for research on insects' saliva with piercing-sucking feeding behavior is described here. In this method, insects were allowed to feed on an artificial diet sandwiched between two stretched paraffin film layers. The diet containing the saliva was collected each day, filtered, and concentrated for further analysis. Finally, the quality of collected saliva was examined by protein staining and immunoblotting. This method was exemplified by detecting the presence of RSV and a mucin-like protein in the saliva of SBPH. These artificial feeding and saliva collection method will lay a foundation for further research on factors in insect saliva related to feeding behavior and virus transmission.

Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection

The present protocol describes a method to collect sufficient saliva from piercing-sucking insects using an artificial medium. This is a convenient method for collecting insect saliva and studying salivary function on insect feeding behavior and vector-borne virus transmission.

用于 老德法纹 状体唾液采集的双层膜夹层法

Rice stripe virus (RSV), which causes significant economic loss of agriculture in East Asia, entirely depends on insect vectors for its effective ...

High-throughput, Robust and Highly Time-flexible Method for Surface Sterilization of Arabidopsis Seeds

Arabidopsis is by far the plant model species most widely used for functional studies. The surface sterilization of Arabidopsis seeds is a fundamental step required towards this end. Thus, it is paramount to establish high-throughput Arabidopsis seed surface sterilization methods to handle tens to hundreds of samples (e.g., transgenic lines, ecotypes, or mutants) at once. A seed surface sterilization method based on the efficient elimination of liquid in tubes with a homemade suction device constructed from a common vacuum pump is presented in this study. By dramatically reducing labor-intensive hands-on time with this method handling several hundreds of samples in one day is possible with little effort. Series time-course analyses further indicated a highly flexible time range of surface sterilization by maintaining high germination rates. This method could be easily adapted for surface sterilization of other kinds of small seeds with simple customization of the suction device according to the seed size, and the speed desired to eliminate the liquid.

A high-throughput protocol for the surface sterilization of Arabidopsisthaliana (Arabidopsis) seeds is provided, optimizing the liquid handling steps with a simple suction device constructed with a vacuum pump. Hundreds of seed samples can be surface-sterilized in one day.

拟南芥种子表面灭菌的高通量、稳健性和高时间灵活性方法

Arabidopsis is by far the plant model species most widely used for functional studies. The surface sterilization of Arabidopsis seeds is a fundamental ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

一种用于快速鉴定根系分泌物中根细菌化学引诱剂的改进趋化性测定

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...

Live Imaging of Arabidopsis Pollen Tube Reception and Double Fertilization Using the Semi-In Vitro Cum Septum Method

In flowering plants, the growth and guidance of the pollen tube (male gametophyte) within the pistil and the reception of the pollen tube by the female gametophyte are essential for double fertilization and subsequent seed development. The interactions between male and female gametophytes during pollen tube reception culminate in pollen tube rupture and the release of two sperm cells to effect double fertilization. As pollen tube growth and double fertilization are deeply hidden within the tissues of the flower, this process is difficult to observe in vivo.
A semi-in vitro (SIV) method for the live-cell imaging of fertilization in the model plant Arabidopsis thaliana has been developed and implemented in several investigations. These studies have helped to elucidate the fundamental features of how the fertilization process occurs in flowering plants and which cellular and molecular changes occur during the interaction of the male and female gametophytes. However, because these live cell imaging experiments involve the excision of individual ovules, they are limited to a low number of observations per imaging session, making this approach tedious and very time-consuming. Among other technical difficulties, a failure of the pollen tubes to fertilize the ovules in vitro is&#160;often reported, which severely confounds such analyses.
Here, a detailed video protocol for the imaging of pollen tube reception and fertilization in an automated and high-throughput manner is provided, allowing for up to 40 observations of pollen tube reception and rupture per imaging session. Coupled with the use of genetically encoded biosensors and marker lines, this method enables the generation of large sample sizes with a reduced time investment. Nuances and critical points of the technique, including flower staging, dissection, medium preparation, and imaging, are clearly detailed in video format to facilitate future research on the dynamics of pollen tube guidance, reception, and double fertilization.

Live Imaging of Arabidopsis Pollen Tube Reception and Double Fertilization Using the Semi-In Vitro Cum Septum Method

Here, we describe an improvement of the semi-in vitro (SIV) method for observing pollen tube guidance and reception in Arabidopsis thaliana,&#160;which increases the receptivity of ovules. The high-throughput SIV cum septum method may be coupled with gametophyte marker lines and genetically encoded biosensors to monitor the dynamic process of fertilization.

拟 南芥 花粉管接收和半体外隔膜法 双受精的实时成像

In flowering plants, the growth and guidance of the pollen tube (male gametophyte) within the pistil and the reception of the pollen tube by the female ...