IEM要感谢丹麦研究委员会（FTP和FNU）的资金。 ERL承认ARC DP授予的支持。 AB承认在细胞壁上授予优秀的ARC中心的支持。

1。组织收集和准备<ol><li>收集100毫克植物组织的鲜重（至少10 mg干重）的至少一式三份的每个组织的利益。以下步骤描述了从植物组织中的细胞壁物质的准备。在存储组织的情况下，不想要的淀粉酶进行提取细胞壁聚合物如先前所述13之前除去。 </li><li>均质化样品使用Qiagen公司的TissueLyser II，24管适配器集和3毫米的碳化钨珠（30赫兹，2×30秒），用液氮成细粉。或者，如果只有少数样本正在处理中，使用研钵和杵。 </li><li>匀浆转移到10 ml塑料锥形管。 </li><li>准备细胞壁材料洗涤匀浆在10毫升80％v / v乙醇在RT和涡旋大力，持续2分钟。 </li><li>在3500 XG离心样品，弃去上清液。重复的70％乙醇洗涤至少3次，或直到上清液是清楚的，特别是对于含叶绿素的组织。 </li><li>执行最终用100％丙酮洗涤和离开的粒料含有酒精不溶残留量（AIR）空气干燥过夜。 </li><li>筛为0.4毫米2目，实现了精细，均匀粉末，能够去除较大，不良一段时间保持匀浆中的颗粒的空气样品。 </li><li>称取10毫克AIR范例成微管，每个都包含一个3毫米的玻璃珠以帮助混合样品。 </li></ol> 2。细胞壁聚糖的提取<ol><li>从样品中提取的果胶，和与果胶的聚合物由向每个样品中加入500微升50μM的CDTA（pH为7.5）。 </li><li>简言之涡管，以确保溶剂是在与样品材料接触，然后混合3小时8赫兹TissueLyser使用。 </li><li>以12,000 xg离心样品，仔细ŕemove上清并储存在4°C </li><li>洗净粒料在1毫升的去离子水，以稀释和除去任何剩余的溶剂，旋涡和在13000×g下离心。重复此步骤，而不会干扰颗粒，并删除所有的液体从管，然后再进行下一个步骤。 </li><li>从其它细胞壁颗粒的交联聚糖中依次提取用500μl的4M的NaOH，用0.1％w / v的硼氢化钠，使用相同的程序中描述的步骤2.1 - 2.4的CDTA提取。加入NaBH 4被添加到减少的醛（或酮）基团的多糖的还原性末端的醇，从而防止基多糖的剥离。 </li><li>离心后，再次除去上清液，在4℃下储存，​​并继续到下一个提取之前，该颗粒在去离子水洗涤两次。 </li><li>作为一个可选步骤，剩余的聚合物，如纤维素，用500μlcadoxen（31％体积/体积1,2-diami萃取noethane与CdO的0.78米），使用相同的程序中描述的步骤2.1 - 2.4。或者，其它粒料绝对纤维素含量可确定使用乙酸/硝酸法（见讨论）。 </li></ol> 3。印刷微阵列<ol><li>在13000 XG离心上清液中提取的细胞壁聚合物，以去除任何颗粒物质。 </li><li>将每个样品的50微升到聚丙烯384孔微量滴定板使用一个预先设计的自定义布局根据组织类型和提取类型的样品被布置。 </li><li>在0，5和25×串行系列稀释液，用去离子水稀释的细胞壁聚合物样品。 </li><li>如针高度，收集和停留时间，和洗涤步骤的参数设置的软件控制的微阵列上。 </li><li>印刷室的湿度控制在60％，以防止样品的蒸发。 </li><li>被开始使用LabNEXT软件，打印作业re和一个程序，它对应于微阵列布局。 </li><li>机器人使用的毛细通道引脚，20×20厘米的硝酸纤维素膜在机器，其安装在一平板，从样品板到打印的解决方案。在阵列上的每个点包含15个NL解决方案，并打印一式三份。 </li><li>相同的微阵列上的膜和切成单个阵列彼此相邻的印刷后的打印作业完成后。 </li><li>在每个实验中的阵列可以被修改，以容纳更多或更少的样品，稀释或复制。 </li></ol> 4。聚糖微阵列技术探讨<ol><li>印刷后，阻止个别芯片在5％w / v的脱脂奶粉溶解于磷酸盐缓冲盐水（MPBS），在室温下2小时，以减少非特异性结合。 </li><li>与细胞壁聚糖抗原决定簇特异的单克隆抗体2小时，在MPBS探针微阵列。的大多数单克隆antib的对细胞壁聚糖odies是从三个公司市售; Biosupplies（ <a href="http://www.biosupplies.com.au" target="_blank">www.biosupplies.com.au</a> ），Carbosource服务（ <a href="http://www.carbosource.net" target="_blank">www.carbosource.net</a> ）和PlantProbes（ <a href="http://www.plantprobes.net" target="_blank">www.plantprobes.net</a> ）。 </li><li>包含一个阴性对照，仅MPBS没有主抗体孵育的微阵列。 </li><li>微阵列洗净3次，在磷酸盐缓冲盐水（PBS）5分钟，以除去非特异性结合。 </li><li>探测芯片与二次抗体结合到辣根过氧化物酶（HRP）在2小时的MPBS。对细胞壁聚糖需要大多数单克隆抗体抗小鼠或抗大鼠二次抗体。 </li><li>重复3次，用PBS缓冲液中5分钟的洗涤步骤，以除去非特异性结合。 </li><li>开发微阵列使用显色（3,3 - 二氨基联苯胺）或切米尔uminecent（鲁米诺）基板。 </li></ol> 5。量化<ol><li>经过发展，个人微阵列采用了高解析度（1200 dpi）的桌面扫描仪扫描并保存图像为负，16位TIFF文件（ 图3）。 </li><li>计算Xplore数据库图像处理软件（LabNEXT），配有自动网格工具使用的每一个点的积分强度。积分光斑强度来自周围各点的格子区域中的像素的总和。 </li><li>每个芯片的网格数据导出为txt文件，并可以手动导入到Excel电子表格进行分析。在线的工具（ <a href="http://arraytemplates.charliehart.com.au/" target="_blank">http://microarray.plantcell.unimelb.edu.au/</a> ）已被自动翻译和处理从单个txt文件中的数据。 </li><li>的积分光斑强度的平均印刷复制和稀释以获得一个“平均的点对每个样品的强度的值（ 图2）。可选地，对应于阵列上的值只是一个稀释的点信号是用来量化聚糖抗原表位相对丰度对每个样品。 </li><li>平均相对强度不同样品之间的点作为热图（ 图4），使用条件格式在Excel或网上的heatmapper的工具（ <a href="http://bar.utoronto.ca/welcome.htm" target="_blank">http://bar.utoronto.ca/welcome.htm</a> ）。对每个抗体类型的数据被校正到100，和5％的临界值被施加来去除背景信号和误报。 </li></ol>

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没有利益冲突的声明。

分析的聚糖组成的数百植物性样品在短短几天内，CoMPP是一种快速，灵敏的方法。此方法的补充已经可用的细菌或哺乳动物聚糖阵列平台与多糖结合的蛋白质，如外源凝集素，受体，和抗体16的碳水化合物的相互作用的高通量筛选。随着多样性的探头可用于检测细胞壁聚糖，它有可能获得糖链抗原表位属于多糖在植物细胞壁8,9的所有主要类别的详细信息。然而，这些已查明的8,9...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> 3毫米碳化钨珠</td><td> Qiagen公司</td><td> 69997 </td><td></td></tr><tr><td>收集管（1.2毫米） </td><td> Qiagen公司</td><td> 19560 </td><td> 1.5毫升微量离心管中，也可以使用</td></tr><tr><td> Qiagen公司的TissueLyser II </td><td> Qiagen公司</td><td> 85300 </td><td></td></tr><tr><td> 3毫米的玻璃珠</td><td> Sigma Aldrich公司</td><td> Z143928 </td><td></td></tr><tr><td> CDTA </td><td> Sigma Aldrich公司</td><td> 34588 </td><td></td></tr><tr><td>氧化镉</td><td> Sigma Aldrich公司</td><td> 202894 </td><td></td></tr><tr><td> 1,2 - 二氨基乙烷</td><td> Sigma Aldrich公司</td><td> 03550 </td><td></td></<html> TR&gt; <tr><td>硝酸纤维素膜（0.22微米孔径大小） </td><td> GE水处理及工艺过程技术</td><td> EP2HY00010 </td><td>不同孔径大小的膜适用于不同的引脚类型</td></tr><tr><td> XACT的II微阵列机器人</td><td> Labnext </td><td> 001A </td><td> XACT的II机器人装有一个自定义的20×20厘米陶瓷板附着的硝酸纤维素膜</td></tr><tr><td> XTEND RM芯片引脚</td><td> Labnext </td><td> 0037-350 </td><td>引脚必须适合用于探测细胞膜上的</td></tr><tr><td> 384孔微量滴定板（聚丙烯） </td><td>格雷纳</td><td> 781207 </td><td></td></tr><tr><td>反聚糖单克隆抗体</td><td>植物探头/ CarboSource / Biosupplies </td><td></td><td>网站PlantProbes（ <a href="http://www.plantprobes.net" target="_blank">www.plantprobes.net</a> ），Carbosource（ww.carbosource.net“目标=”_blank“&gt; www.carbosource.net）和Biosupplies（ <a href="http://www.biosupplies.com.au" target="_blank">www.biosupplies.com.au</a> ）。 </td></tr><tr><td>的抗大鼠IgG（整个分子） - 过氧化物酶的山羊生产的抗体。 </td><td>西格玛</td><td> A9037 </td><td>二次抗体的类型依赖于使用的初级抗体（ 例如大鼠，小鼠，山羊等中提出）。 </td></tr><tr><td> SIGMA FAST 3,3&#39; -二氨基片</td><td>西格玛</td><td> D4293 </td><td>显影试剂的类型，取决于使用的二次抗体和检测方法（色彩值，或chemiluminecent的的）。 </td></tr><tr><td> SuperSignal West Pico化学发光底物</td><td> Thermoscientific </td><td> 34080 </td><td>看到以上</td></tr><tr><td> Xplore数据库图像处理软件</td><td> LabNext </td><td> 008 </td><td>自动网格工具是许多软件类型微阵列点的像素值来衡量。 </td></tr><tr><td>植物多糖</td><td> SIGMA / Megazyme的</td><td></td><td></td></tr></tbody></table>

glycan profiling of plant cell wall polymers using microarrays

植物细胞壁复杂的异构聚糖在生理和植物的发展，并发挥了重要作用，提供原料，为人类社会（ 如木材，造纸，纺织，生物燃料行业）1,2矩阵。然而，这些成分的生物合成和功能的了解仍然充满挑战。 细胞壁聚糖的化学性质和构象不同的复杂的构建块，糖基。这些形式在多个位置之间的联系，并在环结构的不同，同分异构体或端基异构体的配置，此外，被取代的与非糖残基的一个数组。糖基组合物在不同的细胞和/或组织类型或什至单个单元格的壁3的子域变化。此外，它们的组合物1在开发过程中，或在响应于环境线索4也被修改。 在EX制程的2000基因植物细胞壁异构聚糖被预测要参与细胞壁聚糖的生物合成和修改在拟南芥 5是复杂的矩阵。然而，相对较少的生物合成基因功能特点4,5。反向遗传学方法是困难的，因为基因通常差异表达，往往在低的水平，细胞类型之间的6。此外，突变体的研究往往阻碍了的基因冗余或代偿机制，以确保适当的细胞壁功能是保持7。因此，新的方法，需要迅速表征各种不同的聚糖结构和功能基因组学的方法，以方便了解细胞壁的生物合成和修改。 单克隆抗体（mAb）8,9已成为确定在植物中的糖链结构和分布的重要工具。这些识别区INCT抗原表位存在于植物细胞壁聚糖的主要类别，包括的果胶，xyloglucans，木聚糖，甘露聚糖，葡聚糖和阿拉伯半乳聚糖。最近，他们的使用已经扩展到大规模筛选实验，以确定相对丰富的多聚糖广泛的厂房及组织类型，同时9,10,11。 在这里，我们提出了一种基于微阵列聚糖的检查方法，使多个样本的全面的微阵列名聚合物的分析（CoMPP）（ 图1和图2）10,11（100秒）进行筛选，使用小型化的芯片平台，减少了试剂和样品量。在微阵列上的点信号可以正式量化约聚糖抗原表位发生，得到半定量数据。这种方法非常适合于跟踪聚糖变化的复杂的生物系统12，并提供一个全球细胞壁的组合物的概述，特别是当先验知识òF这是不可用。

的相对丰度聚糖在烟草阿拉塔花6个类型的组织（花药的花丝，花粉，子房，花瓣，萼片和耻辱）被确定使用CoMPP。图3A示出了有代表性的微阵列已与mAb JIM5部分（低）的甲基酯化homogalacturonan（HG），果胶多糖14上发生的表位特异的探测。 CDTA提取物中检测到的所有花组织JIM5表位不过是最高的，在花粉和最低的柱头组织（ 图3A和4A）。有趣的是，JIM5表位也检测到在NaOH在花?...

Watch this Scientific Journal Video about Glycan Profiling of Plant Cell Wall Polymers using Microarrays at JoVE.com

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

一种称为<strong&gt; C</strong&gt; omprehensive<strong&gt; M</strong&gt; icroarray<strong&gt; P</strong&gt; olymer探查（CoMPP）植物细胞壁聚糖的特性进行说明。该方法结合了与一个微型芯片分析平台允许筛选聚糖发生在一个广泛的范围内的生物上下文定义的聚糖-抗原决定簇特异性的单克隆抗体。

糖基谱研究植物细胞壁聚合物，用微阵列

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

glycan-profiling-of-plant-cell-wall-polymers-using-microarrays

Research

JoVE Journal

Biology

14.5K 次观看.  University of Melbourne. 一种称为 C omprehensive M icroarray P olymer探查（CoMPP）植物细胞壁聚糖的特性进行说明。该方法结合了与一个微型芯片分析平台允许筛选聚糖发生在一个广泛的范围内的生物上下文定义的聚糖-抗原决定簇特异性的单克隆抗体。

视频: Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Bacterial Gene Expression Analysis Using Microarrays

使用微阵列的细菌基因表达分析

科研

生物学

Measuring Plant Cell Wall Extension (Creep) Induced by Acidic pH and by Alpha-Expansin

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The plant hormone auxin rapidly stimulates cell elongation in young stems and similar tissues at least in part by an acid-growth mechanism 2, 3. Auxin activates a H+ pump in the plasma membrane, causing acidification of the cell wall solution. Wall acidification activates expansins, which are endogenous cell wall-loosening proteins 4, causing the cell wall to yield to the wall tensions created by cell turgor pressure. As a result, the cell begins to enlarge rapidly. This 'acid growth' phenomenon is readily measured in isolated (nonliving) cell wall specimens. The ability of cell walls to undergo acid-induced extension is not simply the result of the structural arrangement of the cell wall polysaccharides (e.g. pectins), but depends on the activity of expansins 5. Expansins do not have any known enzymatic activity and the only way to assay for expansin activity is to measure their induction of cell wall extension. This video report details the sources and preparation techniques for obtaining suitable wall materials for expansin assays and goes on to show acid-induced extension and expansin-induced extension of wall samples prepared from growing cucumber hypocotyls.
 To obtain suitable cell wall samples, cucumber seedlings are grown in the dark, the hypocotyls are cut and frozen at -80 &deg;C. Frozen hypocotyls are abraded, flattened, and then clamped at constant tension in a special cuvette for extensometer measurements. To measure acid-induced extension, the walls are initially buffered at neutral pH, resulting in low activity of expansins that are components of the native cell walls. Upon buffer exchange to acidic pH, expansins are activated and the cell walls extend rapidly. We also demonstrate expansin activity in a reconstitution assay. For this part, we use a brief heat treatment to denature the native expansins in the cell wall samples. These inactivated cell walls do not extend even in acidic buffer, but addition of expansins to the cell walls rapidly restores their ability to extend.

Measuring Plant Cell Wall Extension Creep Induced by Acidic pH and by Alpha-Expansin

We demonstrate the use of a constant-force extensometer to measure long-term extension (creep) of plant cell wall specimens induced by acidic buffers and expansin protein.

酸性α-膨大诱导测量植物细胞壁扩展（蠕变）

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

调查组织和器官特定的光敏色素反应，使用流式细胞仪辅助细胞类型特定的表达谱拟南芥</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Genome-wide Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both prokaryotic and eukaryotic cells lead to translational regulation which is a major cellular mechanism of stress response. Familiar examples are the stringent response in E. coli and the Gcn2 stress response pathway in yeast ([2-6]). Recent work in E. coli and S. cerevisiae have shown that tRNA charging patterns are highly dynamic and depends on the type of stress experienced by cells [1, 6, 7]. The highly dynamic, variable nature of tRNA charging makes it essential to determine changes in tRNA charging levels at the genomic scale, in order to fully elucidate cellular response to environmental variations. In this review we present a method for simultaneously measuring the relative charging levels of all tRNAs in S. cerevisiae . While the protocol presented here is for yeast, this protocol has been successfully applied for determining relative charging levels in a wide variety of organisms including E. coli and human cell cultures[7, 8].

Genome-wide Analysis of Aminoacylation Charging Levels of tRNA Using Microarrays

We describe a method for microarray analysis to determine relative aminoacylation levels of all tRNAs from S. cerivisiae.

（充电）酰化tRNA的使用微阵列技术水平的全基因组分析

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both ...

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1 (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2, and is amenable to high-throughput analyses3. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13, xylan using an endo-&beta;-(1-4)-xylanase 6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9.

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

胞外多糖的OLIGO质谱分析（OLIMP）

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

利用同位素标记的质谱剖析巯基氧化还原蛋白质

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

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 ...

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 ...

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.

激光辅助显微切割（LAM）作为个体细胞类型的转录谱的工具

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

An Easy Method for Plant Polysome Profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

植物多聚核糖体剖面的简单方法

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the ...