We thank Dr. M. Rosenberg for her advice and help with anatomical sectioning. We also thank D. Eisler for his technical assistance. This research was supported by grants from FP7-PEOPLE-IRG-2008, Binational Agriculture research and Development (BARD; IS-4246-09), and Israel Science Foundation (ISF; 592/13).

1.植物生长<ol><li>表面消毒的种子。 <ol><li>通过湿法或干法消毒的种子（最多30毫克）。作为一个例子，为干热灭菌法，在一个封闭的干燥器在50毫升漂白加入1毫升冰盐酸37％，在玻璃烧杯中，至少4小时和最多过夜。执行在化学罩此步骤。 </li></ol></li><li>传播在0.5倍的Murashige＆Skoog培养基（MS）培养基，0.8％植物琼脂平板上无菌种子。包装用封口膜或多孔带板和铝箔覆盖。 </li><li>存储平板在4℃下1 - 4天推匀发芽。 </li><li>传送板，以适于生长的拟南芥幼苗的生长室。将平板垂直地维持在琼脂培养基的顶端的根生长，并防止渗透。 </li><li>转移苗发芽（DAG）后固定液7天。 </li></ol> 2.固定<ol><li>准备50毫升理查德森搜索解决方案通过在双蒸水1％硼砂（缓冲剂），1％亚甲基蓝和1％天青（组织学染料）等体积的结合Ñ。通过使用前驱动0.22微米的PVDF过滤单元注射器过滤。 </li><li>准备50毫升固定液:1.25％戊二醛在0.05M二甲胂酸钠（缓冲剂）。 注意:这些物质是有毒的，并且必须在化学通风橱中处理。 </li><li>加的理查森溶液100微升（见2.1）的固定溶液（见2.2），得到最终的固定溶液。 </li><li>标记与相应的样品名称的6孔板的每个孔中。 </li><li>放置2 - 3毫升最终固定溶液（见2.3）插入的孔中，从而使幼苗，一旦转移，将在液体中完全覆盖。 </li><li>小心转移，使用镊子，最多10苗每个孔中。使用密封封口膜板。储存在4℃下，在黑暗中，用于至少一个晚上，最多一周。 </li></ol> 3.脱水<ol> &lt;LI&gt;脱水苗。转移他们的新6孔平板的孔之间，含有增加的乙醇浓度，如下所示:10％乙醇15分钟; 30％乙醇15分钟; 50％乙醇15分钟; 70％乙醇15分钟; 85％乙醇15分钟; 95％乙醇15分钟;在4℃，过夜，以最低95％的乙醇。通过他们的子叶，最好用塑料镊子小心地抓住他们处理苗。 </li></ol> 4.渗透<ol><li>准备渗透网上平台。将1袋ヒ套件激活的内容与一个小玻璃瓶50毫升基本树脂液套件。用磁铁20分钟搅拌。浸润培养基可保持在4℃几周。 </li><li>标记与相应的样品名称的新6孔平板的每个孔中，并用2填充 - 3毫升浸润培养基。 </li><li>从95％的乙醇溶液，以使用镊子渗入介质传送苗。确保苗福由介质LLY覆盖。 </li><li>储存在4℃下至少4天，以确保最大的渗透到植物组织。 </li></ol> 5.块准备<ol><li>将小标签，铅笔标明样品名称，嵌入模具的每一个孔里。 </li><li>准备加入1 ml固化剂到15ml渗透介质（见4.1）嵌入介质。不要试图准备一个容量小于15毫升，以避免聚合的问题。 </li><li>填充一半各孔在用200μl包埋介质的模具和与切断模具形状的塔顶薄膜片覆盖，但以一个大小为1毫米的每个侧大。在室温下孵育至少2小时，以允许聚合。不要超过5小时。 </li><li>制备额外批次包埋培养基（见5.2），并保持在冰上，以避免聚合而根尖被布置在模具中。 </li><li>用手术刀切下解剖范围内的每个根尖和非常小心我的位置T:垂直的横截面或横向纵向部分，约2 - 从模具边缘3毫米。与封闭液，还包括与架空电影完全填满模具。注意，一旦它被聚合的封闭溶液将略有收缩。 </li><li>保持在室温下2小时，在最低限度。 </li><li>干块，放置在干燥硅胶的盒子，并在室温离开过夜，在最低限度。 </li></ol> 6切片<ol><li>从准备玻璃棒玻璃刀用刀子机。使用玻璃刀用于组织切片。切玻璃棒成方形，然后切正方形对角线产生两把刀，使用金刚石得分利器。 </li><li>节块分成2 - 3微米宽度切片，用切片机。在载玻片上地方切片，在蒸馏水液滴的顶部，并放置为低热量的热块上的滑动件（50 - 60℃），直到水蒸发。 </li></ol><ol><li>理查德森稀释液（见2.1），以原始溶液的0.2％及使用方法1毫升它覆盖片。放置5秒钟的加热块上，然后用蒸馏水冲洗。 </li><li>干热板上的幻灯片。观察下解剖范围和标记的幻灯片用标记在后面，以指示感兴趣的切片的位置，以便于在显微镜下它们的后面的识别。 </li><li>将100μl封固剂和覆盖的盖玻片。 </li></ol> 8.图像采集<ol><li>放置装有装有适于获得延迟信息和偏振器/干扰光滤光器（ 图1的AD）Polscope系统在光学显微镜下的根部分中的覆盖滑动。 </li><li>选择放大，允许整个样本的清晰的可视化（ 例如 ，40X在这项研究中）和聚焦显微镜E在包含块段，但没有根部分的空场。试着找一个干净的区域，没有瑕疵。使用此区域设置背景。 </li><li>设置成像系统和软件参数:设定范围到17纳米。应用"自动曝光"，并从空场获得背景图像（见图8.2）。一个背景图像被每个实验使用。 </li></ol> 9.纤维素微纤丝取向Polscope分析<ol><li>分析纵向部分。 <ol><li>将含在显微镜下纵段的幻灯片，并获得它的延迟的图像。专注于感兴趣的根部显微镜。提供一个新的文件名。 </li><li>通过"实时Abrio"窗口捕捉abrio图像（迟缓的信息图像）。一定要捕获包括细胞壁，这可以通过沿着电池的周边运行染色纤维素被识别， 如图2C的剖面。</li></ol></li><li>图像分析。 <ol><li>在图像堆栈相关的图像上双击。在"显示设置"选项卡中选择"方向伪彩"。 </li><li>为了估计纤维素微纤丝角，匹配细胞壁的色轮中相应的树荫的。 注意:在图2C的彩色轮反射光沿其指示沿着微纤维的长轴方向上的微纤维躺着的慢轴。纤维素微纤丝的角度是由颜色和单元的长轴指示的角度。例如，在嵌入的伸长细胞显示青色，约90°倾斜的细胞的长轴。 </li><li>为了量化在细胞壁的平均微纤丝角，从软件的工具栏中用"延迟和方位角印记"的工具。在选择工具，点一下就所获得的图像中感兴趣的区域。 注:此WIL升结果在度角（方位角）（ 图2C）的显示。 </li><li>在另一种开放获取偏振光图像分析软件，午餐"波尔分析仪"，从插件菜单插件。在新打开的窗口中，选择"双折射"选项卡，打开并保存背景文件和图像文件。 </li><li>在图像窗口中，选择"规格"。并调整"ROI像素尺寸&#39;（使用5个像素为这项研究）。中的"规格"。窗口中，选择"在结果列表中显示的值"。 </li><li>使用多边形工具，以纪念在图像窗口中感兴趣的区域。选择"方向线"，以获得纤维素微纤维的取向。 注意:这将导致在度角（方位角）的显示。 注意:用两种方法之一计算出的平均角度应然后标准化为沿着滑动根部的定位。为了校正偏差，从沿着滑动整根部的水平定位，量化细胞壁侧翼沿其长轴的小区的平均微纤丝角（即，垂直于该部分和沿着根生长轴， 图2C）。此角度为180°时根部准确沿着滑动地定位。为计算微纤丝角，从180°减去侧翼细胞壁的平均测微纤丝角。所得差添加到细胞壁的平均测微纤丝角，关于滑动平整。 </li></ol></li></ol>结晶纤维素积累10 Polscope分析<ol><li>使用横向（横）截面。注意，结晶纤维素积累只能小区之间进行比较时其纤维素微以类似的角度对准的（如在9确定）。 </li><li>在图像堆栈相关的图像上双击。选择"P"中的"显示设置"选项卡seudo颜色。 </li><li>为了测量延迟。放大-在图像上。选择"地区"，并标记感兴趣的所有区域。移动到"三围"选项卡和"复制到剪贴板"的所有值。 <ol><li>将数据传输到Excel和提取所需的信息（平均延迟的区域）。正常化的数据。 注意:为了考虑切片之间的厚度的差异，表示相对于在滑动一个特定细胞壁边缘的值。正常化，除以不同细胞壁的同一图像中的平均延迟对应于感兴趣的细胞壁的平均延迟。例如，外表皮细胞壁被归一化到在我们的研究中的内皮层细胞壁。 </li></ol></li><li>重复测量每根的至少3个部分，用最少的每个样品3根。 </li></ol>

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The authors have no conflicts of interest.

在这里，我们提出了一个方法，测定不同组织组成拟南芥根部结晶纤维素的积累，同时保持解剖信息。因此，它提供了对在一个蜂窝分辨率理解在植物生长过程的附加步骤。这种方法也可以应用于其他植物物种和器官的研究。 数点必须应用该方法时，必须考虑。在给定的根段第一，结晶纤维素积累可以组织之间进行比较，从不同基因型的部分之间和处理间，如果他们?...

植物细胞壁是一个动态的结构。生长的细胞是由初级细胞壁，组织其允许细胞扩大包围。细胞停止生长存款更刚性的次生壁，增强植物的机械支撑。两个单元壁是由嵌入在不同的结构（例如 ，半纤维素和果胶），该跨不同发育阶段变化和组织1,2的多糖的基质纤维素微纤维。纤维素被合成为被严格对齐，以形成晶体结构的微纤丝（1,4）-β-D-葡聚糖的链。无定形纤维素是指其中葡聚糖链较少有序的区域。该结晶和无定形结构域之间的比率是认为影响细胞壁的机械特性的一个参数，通过提供机械强度和粘弹性特性，分别为3。几种方法已经开发检测和定量的两种形式的纤维素排列的，其中的X射线衍射和交叉极化/魔角旋转固态核磁共振4。 X射线衍射可以被用来确定样品5中结晶与非晶纤维素域的比例。另一种方法使用的细胞壁含量分馏成酸不溶性和酸可溶材料，分别结晶和无定形纤维素或其它聚合物之间进行区分。在这种方法中，标记的葡萄糖（[14 C]葡萄糖）的掺入来量化纤维素6,7。这些方法需要大量的用于整个器官的分析，在最好的植物材料，因此，是在细胞壁结构组织特异性变异充分敏感。在细胞分辨率纤维素微的可视化可以在用荧光染料8,9-结合实时成像研究来实现，它可以识别在改变纤维素微纤维的取向。然而，这些染料不用于定量，它们不是特定于结晶纤维素和可以与细胞壁8的正常结构干扰。 Polscope是成像技术，其依赖于结晶纤维素分裂光束和延缓的光10的一部分的能力。光相位差是最强的为垂直骗光传播方向的微纤维。对于具有类似取向的微纤维，结晶度越高，越大的光延迟11。因此，polscope是用来研究两个相对水平和纤维素微纤维的取向。 根部呈现线性的增长，在此期间，细胞产生于干细胞小生境，在根的前端，经过一系列的细胞分裂，它们迅速扩大12之前。包括根细胞在单向（各向异性）展开方式，作为决定由小分子信令激素影响细胞壁13的属性。不同的反应激素，在时间和空间，提供了确保平衡的器官生长14的一种手段。因此，细胞壁结构的高分辨率分析可以提供更好的理解，以整个器官生长的细胞类型特异性应答之间的连接所需的重要信息。在这里，我们报告polscope，研究结晶纤维素的组织特异性的积累拟南芥根系在高品质的解剖观察部分的执行情况。这种方法最近发现响应于激素活性15的空间扰动的结晶纤维素的细胞类型特异性蓄积。

<table><tbody><tr><td>Murashige &amp;amp; Skoog (MS)</td><td>Duchefa</td><td>M0221</td><td></td></tr><tr><td>Borax</td><td>LOBA CHEMIE&amp;nbsp;</td><td>6038</td><td></td></tr><tr><td>Methylene blue</td><td>Sigma</td><td>M-9140</td><td></td></tr><tr><td>Azure</td><td>Sigma</td><td>861065</td><td></td></tr><tr><td>Syringe Driven 0.22 mm PVDF Filter&amp;nbsp;</td><td>MILLEX-GV</td><td></td><td></td></tr><tr><td>Glutaraldehyde</td><td>EMS</td><td>16220</td><td></td></tr><tr><td>Sodium Cacodylate</td><td>Sigma</td><td>C-0250</td><td></td></tr><tr><td>Leica kit historesin&amp;nbsp;</td><td>Leica</td><td>7022 18 500</td><td></td></tr><tr><td>embedding molds</td><td>Agar Scientific</td><td>AGG3530</td><td></td></tr><tr><td>film 100 &amp;micro; P.P.C.&amp;nbsp;</td><td>www.Jolybar.com</td><td></td><td>overhead film</td></tr><tr><td>Knivemaker</td><td>LKB</td><td>7800</td><td></td></tr><tr><td>Ultratome III</td><td>LKB</td><td>8800</td><td>Ultratome</td></tr><tr><td>Shandon immumount&amp;nbsp;</td><td>Thermo</td><td>9990402</td><td>mounting medium</td></tr><tr><td>light microscope</td><td>Nikon</td><td>Eclipse 80i</td><td></td></tr><tr><td>Abrio imaging system</td><td>CRI</td><td>Abrio imaging system</td><td></td></tr><tr><td>Abrio V2.2 software</td><td>CRI</td><td>Abrio V2.2 software</td><td></td></tr><tr><td>Open access polarized light image analysis software &amp;nbsp;</td><td>OPS</td><td>OpenPolScope</td><td>&lt;a href=&quot;http://www.openpolscope.org/&quot;&gt;http://www.openpolscope.org/&lt;/a&gt;</td></tr></tbody></table>

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.

我们研究的细胞类型特异性反应油菜素内酯（BRS）的影响，使用拟南芥根作为模型器官15-17。当BR受体BRI1是有针对性的，在BRI1突变体的背景下，对被称为非毛细胞表皮细胞的一个子集（ 图2A，B）中 ，它抑制相邻小区的单向电池膨胀，并完全根生长15。进行揭示这背后的抑制机制Polscope分析。从野生型和从非毛细胞表达BRI1线得到的...

Watch this Scientific Journal Video about High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications at JoVE.com

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 ...

high-resolution-quantification-crystalline-cellulose-accumulation

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

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

8.1K 次观看.  Technion-Israel Institute of Technology. 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.

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

Fluorescent Immunolocalization of Arabinogalactan Proteins and Pectins in the Cell Wall of Plant Tissues

Plant development involves constant adjustments of the cell wall composition and structure in response to both internal and external stimuli. Cell walls are composed of cellulose and non-cellulosic polysaccharides together with proteins, phenolic compounds and water. 90% of the cell wall is composed of&#160;polysaccharides (e.g., pectins) and arabinogalactan proteins (AGPs). The fluorescent immunolocalization of specific glycan epitopes in plant histological sections remains a key tool to uncover remodeling of wall polysaccharide networks, structure and components.
Here, we report an optimized fluorescent immunolocalization procedure to detect glycan epitopes from AGPs and pectins in plant tissues. Paraformaldehyde/glutaraldehyde fixation was used along with LR-White embedding of the plant samples, allowing for a better preservation of the tissue structure and composition. Thin sections of the embedded samples obtained with an ultra-microtome were used for immunolocalization with specific antibodies. This technique offers great resolution, high specificity, and the chance to detect multiple glycan epitopes in the same sample. This technique allows subcellular localization of glycans and detects their level of accumulation in the cell wall. It also permits the determination of spatio-temporal patterns of AGP and pectin distribution during developmental processes. The use of this tool may ultimately guide research directions and link glycans to specific functions in plants. Furthermore, the information obtained can complement biochemical and gene expression studies.

This protocol describes in detail how the plant material for immunolocalization of Arabinogalactan proteins and pectins is fixed, embedded in a hydrophilic acrylic resin, sectioned and mounted on glass slides. We show cell wall related epitopes will be detected with specific antibodies.

植物组织细胞壁中阿拉比诺格拉坦蛋白和皮克廷的荧光免疫化

Plant development involves constant adjustments of the cell wall composition and structure in response to both internal and external stimuli. Cell walls ...

科研

发育生物学

Label-free in situ Imaging of Lignification in Plant Cell Walls

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find cost-effective and sustainable solutions has become a topical and critical task. Lignocellulosic biomass is poised to become the primary source of biomass for the conversion to liquid biofuels1-6. However, the recalcitrance of these plant cell wall materials to cost-effective and efficient degradation presents a major impediment for their use in the production of biofuels and chemicals4. In particular, lignin, a complex and irregular poly-phenylpropanoid heteropolymer, becomes problematic to the postharvest deconstruction of lignocellulosic biomass. For example in biomass conversion for biofuels, it inhibits saccharification in processes aimed at producing simple sugars for fermentation7. The effective use of plant biomass for industrial purposes is in fact largely dependent on the extent to which the plant cell wall is lignified. The removal of lignin is a costly and limiting factor8 and lignin has therefore become a key plant breeding and genetic engineering target in order to improve cell wall conversion.
Analytical tools that permit the accurate rapid characterization of lignification of plant cell walls become increasingly important for evaluating a large number of breeding populations. Extractive procedures for the isolation of native components such as lignin are inevitably destructive, bringing about significant chemical and structural modifications9-11. Analytical chemical in situ methods are thus invaluable tools for the compositional and structural characterization of lignocellulosic materials. Raman microscopy is a technique that relies on inelastic or Raman scattering of monochromatic light, like that from a laser, where the shift in energy of the laser photons is related to molecular vibrations and presents an intrinsic label-free molecular "fingerprint" of the sample. Raman microscopy can afford non-destructive and comparatively inexpensive measurements with minimal sample preparation, giving insights into chemical composition and molecular structure in a close to native state. Chemical imaging by confocal Raman microscopy has been previously used for the visualization of the spatial distribution of cellulose and lignin in wood cell walls12-14. Based on these earlier results, we have recently adopted this method to compare lignification in wild type and lignin-deficient transgenic Populus trichocarpa (black cottonwood) stem wood15. Analyzing the lignin Raman bands16,17 in the spectral region between 1,600 and 1,700 cm-1, lignin signal intensity and localization were mapped in situ. Our approach visualized differences in lignin content, localization, and chemical composition. Most recently, we demonstrated Raman imaging of cell wall polymers in Arabidopsis thaliana with lateral resolution that is sub-&mu;m18. Here, this method is presented affording visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species without staining or labeling of the tissues.

Label-free in situ Imaging of Lignification in Plant Cell Walls

A method based on confocal Raman microscopy is presented that affords label-free visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species.

无标签原位成像在植物细胞壁的木质化

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find ...

生物学

Atomic Force Microscopy to Study the Physical Properties of Epidermal Cells of Live Arabidopsis Roots

A method is described here to characterize the physical properties of the cell wall of epidermal cells of living Arabidopsis roots through nanoindentations with an atomic force microscope (AFM) coupled with an optical inverted fluorescence microscope. The method consists of applying controlled forces to the sample while measuring its deformation, allowing quantifying parameters such as the apparent Young's modulus of cell walls at subcellular resolutions. It requires a careful mechanical immobilization of the sample and correct selection of indenters and indentation depths. Although it can be used only in external tissues, this method allows characterizing mechanical changes in plant cell walls during development and enables the correlation of these microscopic changes with the growth of an entire organ.

Atomic Force Microscopy to Study the Physical Properties of Epidermal Cells of Live Arabidopsis Roots

The atomic force microscopy indentation protocol offers the possibility to dissect the role of the physical properties of the cell wall of a particular cell of a tissue or organ during normal or constrained growth (i.e., under water deficit).

原子力显微镜研究活拟 南芥 根表皮细胞的物理性质

A method is described here to characterize the physical properties of the cell wall of epidermal cells of living Arabidopsis roots through ...

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 ...

Using Flatbed Scanners to Collect High-resolution Time-lapsed Images of the Arabidopsis Root Gravitropic Response

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories can face is the development and attainment of these methods. Observation of phenotypes in a process of interest is a typical objective of research labs studying gene function and this is often achieved through image capture. A particular process that is amenable to observation using imaging approaches is the corrective growth of a seedling root that has been displaced from alignment with the gravity vector. Imaging platforms used to measure the root gravitropic response can be expensive, relatively low in throughput, and/or labor intensive. These issues have been addressed by developing a high-throughput image capture method using inexpensive, yet high-resolution, flatbed scanners. Using this method, images can be captured every few minutes at 4,800 dpi. The current setup enables collection of 216 individual responses per day. The image data collected is of ample quality for image analysis applications.

This protocol describes a process for rapid collection of images of Arabidopsis seedlings responding to a gravity stimulus using commercially-available flatbed scanners. The method allows for inexpensive, high-volume capture of high-resolution images amenable for downstream analysis algorithms.

使用平板扫描仪，以收集拟南芥根向地反应的高分辨率时间失效图片

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

细胞壁的弹性性能的原子力显微镜为基础的映射:在组织，细胞，亚细胞和决议

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

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

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

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

组织化学染色<em&gt;拟南芥</em&gt;次生壁元素

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

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 ...

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 ...

结晶纤维素积累高分辨率的量化

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此视频中的章节

植物组织细胞壁中阿拉比诺格拉坦蛋白和皮克廷的荧光免疫化

无标签原位成像在植物细胞壁的木质化

原子力显微镜研究活拟南芥根表皮细胞的物理性质

从长期来看，高解析度激光共聚焦时间流逝成像

使用平板扫描仪，以收集拟南芥根向地反应的高分辨率时间失效图片

细胞壁的弹性性能的原子力显微镜为基础的映射:在组织，细胞，亚细胞和决议

组织化学染色

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

阿拉伯多普西斯局部和系统伤口信号的广域实时成像

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

结晶纤维素积累高分辨率的量化

摘要

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植物组织细胞壁中阿拉比诺格拉坦蛋白和皮克廷的荧光免疫化

无标签原位成像在植物细胞壁的木质化

原子力显微镜研究活拟 南芥 根表皮细胞的物理性质

从长期来看，高解析度激光共聚焦时间流逝成像

使用平板扫描仪，以收集拟南芥根向地反应的高分辨率时间失效图片

细胞壁的弹性性能的原子力显微镜为基础的映射:在组织，细胞，亚细胞和决议

组织化学染色

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

阿拉伯多普西斯局部和系统伤口信号的广域实时成像

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

原子力显微镜研究活拟南芥根表皮细胞的物理性质