这项工作是由德国科学基金会（DFG）和德国联邦政府和各州政府的卓越倡议。

1。植物材料的交联<ol><li>收获1 - 2G的拟南芥叶（6到10cm莲座直径）在50毫升的塑料管，填补了交联的缓冲区，以40ML。 </li><li>确保叶留沉浸例如，通过量身定制的过滤海绵权馅上述缓冲表面。然后放置在干燥器管。 </li><li>真空渗透为10分钟。每管加入2.5毫升2M甘氨酸停止交联，并反转管，以保证平等的甘氨酸分散。 </li><li>真空渗透另一个5分钟，并丢弃液体，同时收获叶筛子。 </li><li>洗叶两次1L水在玻璃烧杯中，然后用纸巾彻底干叶。 </li><li>收集在一个新的塑料管的叶子和液氮冻结。商店叶在-80 ° C直到染色质隔离。 </li></ol> 2。染色质隔离<ol><li>叶地面砂浆和雌蕊，在液氮中保存，直到使用。 </li><li>转让粉，50毫升的塑料管，并暂停在30ml提取缓冲液＃1。 </li><li>在4 ° C孵育15分钟上的开销摇床。 </li><li>滤液暂停通过四层的miracloth到一个新的50毫升的塑料管。 </li><li>在2800 XG 20分钟旋转4 ° C。 </li><li>去除上清液，并中止与油漆刷在1​​ML提取缓冲液＃2沉淀。首先添加一个缓冲区＃2，体积小，然后用画笔暂停颗粒，然后添加其余的缓冲区。 </li><li>转移悬浮在12000 XG到1.5ml离心管和旋转为10分钟，4 ° C。 </li><li>同时，准备液2ml离心管1.5ml的提取缓冲液＃3。 </li><li>从纺管（步骤2.7）去除上清液，并暂停在提取缓冲液＃3300μL沉淀。首先添加一个缓冲区＃3，体积小，暂停与画笔颗粒，然后添加剩余的缓冲区。 </li><li>仔细吸管的悬浮颗粒（步骤2.9）1.5 mL提取缓冲液中，在步骤2.8准备＃3顶部。确保两个阶段不会混合。上半年的16,000 XG旋转4 ° C。 </li><li>去除上清液，并中止与染色质颗粒在超声缓冲液300μL的油漆刷。 </li><li>超声sonotrode，或在超声波浴染色，一个约400bp的DNA大小。当超声，确保染色不会被加热以上约30 ° C保护热敏感的交联。需要通过实验确定，因为它变化显著不同超声设备的超声时间。为指导，提供两个不同的设备设置： </li></ol>对于超声一个Bandelin品牌sonotrode暴露在0.6mL离心管中染色质的5倍至20连发设置25％的电力，周期= 50。虽然这样做，在冰浴后，每20 次阵阵清凉样品。 超声与Diagenode Bioruptor超声波浴，填补水和冰的浴缸和超声设置“高”的30秒在1.5ml的离心管的10倍。每隔30秒后，再过30秒冷却样品。 <ol start="13"><li>旋转超声16,000克的样品为5min，4 ° C至沉淀不溶物质。上清可直接用于免疫。另外，样品可在液氮中冷冻，并保存于-80℃直至进一步的冲击。 </li></ol> 3。免疫沉淀<ol><li>准备2 ml离心管40μL蛋白 - 琼脂糖和抗体结合缓冲液1.8mL。染色准备加入200μL（步长2.13）。 </li><li>开销摇床孵育1h后管。 </li><li>放弃蛋白A琼脂糖珠和使用免疫上清。 </li><li>删除一个40μL等分，以确定染色质浓度（“输入”）。 </li><li>加入30μL蛋白一琼脂糖和修改特异性抗体，是在特定的试剂和仪器400μL超声染色质悬浮如下表所示金额。 </li><li> 2.5H或架空摇床过夜孵育4 ° C。 </li><li>自旋为2分钟，在440 × G.珠</li><li>洗珠颗粒，每个洗涤缓冲液900μL（见以下列表）。对于每一个缓冲区，然后在10分钟的缓冲开销摇床4℃，孵化珠为2分钟旋转440 XG，弃上清液，并添加下一个缓冲区。 <ol><li>低盐缓冲液洗</li><li>高盐缓冲液洗</li><li>氯化锂洗涤缓冲液</li><li> TE缓冲液洗</li></ol></li><li>最后一次洗涤后，彻底清除任何剩余的缓冲区。 </li></ol> 4。组蛋白和DNA的交联和沉淀DNA纯化<ol><li>将去交联缓冲区100μL琼脂糖颗粒和步骤3.4样品40μl的“输入”，在旋涡混合器混合5min后，旋转样品，并培育贝特在65 ° C过夜。 </li><li>商业的DNA或PCR纯化试剂盒纯化的DNA。 </li><li>通常情况下1:5稀释的最后一列洗脱液5μL足以执行特定常规轨迹实时定量RT - PCR（RT - qPCR的）。 </li></ol> 5。缓冲区表<table border="1"><tbody><tr><td colspan="2"> 交联的缓冲区 </td></tr><tr><td>丁酸钠</td><td> 10MM </td></tr><tr><td>蔗糖</td><td> 400MM </td></tr><tr><td>的Tris - HCl，pH值8.0 </td><td> 10MM </td></tr><tr><td> β-巯基乙醇</td><td> 5MM </td></tr><tr><td>甲醛</td><td> 3％（V / V） </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> 提取缓冲液＃1 </td></tr><tr><td>丁酸钠</td><td> 10MM </td></tr><tr><td>蔗糖</td><td> 400MM </td></tr><tr><td>的Tris - HCl，pH值8.0 </td><td> 10MM </td></tr><tr><td> β-巯基乙醇</td><td> 5MM </td></tr><tr><td>完整的蛋白酶抑制剂</td><td> 1X </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> 提取缓冲液＃2 </td></tr><tr><td>丁酸钠</td><td> 10MM </td></tr><tr><td>蔗糖</td><td> 250MM </td></tr><tr><td>的Tris - HCl，pH值8.0 </td><td> 10MM </td></tr><tr><td> β-巯基乙醇</td><td> 5MM </td></tr><tr><td> MgCl 2的 </td><td> 10MM </td></tr><tr><td> TRITON X - 100 </td><td> 1％（W / V） </td></tr><tr><td>完整的蛋白酶抑制剂</td><td> 1X </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> 提取缓冲液＃3 </td></tr><tr><td>丁酸钠</td><td> 10MM </td></tr><tr><td>蔗糖</td><td> 1.7M </td></tr><tr><td>的Tris - HCl，pH值8.0 </td><td> 10MM </td></tr><tr><td> β-巯基乙醇</td><td> 5MM </td></tr><tr><td> MgCl 2的 </td><td> 2MM </td></tr><tr><td> TRITON X - 100 </td><td> 0.15％（W / V） </td></tr><tr><td>完整的蛋白酶抑制剂</td><td> 1X </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> 超声缓冲 </td></tr><tr><td>的Tris - HCl，pH值8.0 </td><td> 25MM </td></tr><tr><td> EDTA - NaOH溶液，pH值8.0 </td><td> 5MM </td></tr><tr><td> SDS </td><td> 0.5％（W / V） </td></tr><tr><td>完整的蛋白酶抑制剂</td><td> 1X </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> 抗体结合缓冲液 </td></tr><tr><td>的Tris - HCl，pH值8.0 </td><td> 50MM </td></tr><tr><td> EDTA - NaOH溶液，pH值8.0 </td><td> 1MM </td></tr><tr><td>氯化钠</td><td> 150MM </td></tr><tr><td> TRITON X - 100 </td><td> 0.1％（W / V） </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> 低盐缓冲液洗 </td></tr><tr><td>氯化钠</td><td> 150MM </td></tr><tr><td> SDS </td><td> 0.1％（W / V） </td></tr><tr><td> TRITON X - 100 </td><td> 1％（W / V） </td></tr><tr><td> EDTA - NaOH溶液，pH值8.0 </td><td> 2MM </td></tr><tr><td>的Tris - HCl，pH值8.0 </td><td> 20MM </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> 高盐缓冲液洗 </td></tr><tr><td>氯化钠</td><td> 500MM </td></tr><tr><td> SDS </td><td> 0.1％（W / V） </td></tr><tr><td> TRITON X - 100 </td><td> 1％（W / V） </td></tr><tr><td> EDTA - NaOH溶液，pH值8.0 </td><td> 2MM </td></tr><tr><td>的Tris - HCl，pH值8.0 </td><td> 20MM </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> 氯化锂洗涤缓冲液</td></tr><tr><td>氯化锂</td><td> 250MM </td></tr><tr><td> Nonidet - P40 </td><td> 1％（V / V） </td></tr><tr><td>钠去氧胆</td><td> 1％（W / V） </td></tr><tr><td> EDTA - NaOH溶液，pH值8.0 </td><td> 1MM </td></tr><tr><td>的Tris - HCl，pH值8.0 </td><td> 20MM </td></tr></tbody></table><table border="1"><tbody><tr><td> TE缓冲液洗 </td></tr><tr><td> EDTA - NaOH溶液，pH值8.0 </td><td> 10MM </td></tr><tr><td>的Tris - HCl，pH值8.0 </td><td> 1MM </td></tr></tbody></table><table border="1"><tbody><tr><td> Decrosslinking缓冲区 </td></tr><tr><td>的Tris - HCl，pH值6.8 </td><td> 62.5mM </td></tr><tr><td>氯化钠</td><td> 200MM </td></tr><tr><td> SDS </td><td> 2％（W ​​/ V） </td></tr><tr><td>数码地面电视</td><td> 10MM </td></tr></tbody></table> 6。代表性的成果： 拟南芥PR2防御基因的表达，编码β -1,3 -葡聚糖酶，抗菌活性 12，苯并噻二唑（BTH）诱导，人工合成的植物激素水杨酸3模拟图1表明，激活 PR2的表达与组蛋白乙酰化赖氨酸残基上增加相关。 H3和H4 PR2的发起人。由于核小体密度降低在基因的激活13，组蛋白乙酰化值正常化不变区域的组蛋白3抗体获得的信号。 <img src="/files/ftp_upload/3096/3096fig1.jpg" alt="图1"/> 图1 BTH诱导PR2的发起人在拟南芥组蛋白乙酰化 。植物均采用100μm的BTH或可湿性粉剂控制。 72h后，叶收集和染色质隔离。乙酰特异性抗体（如图所示）沉淀后得到的信号是标准化，以获得一种抗体降水不变域的组蛋白H3的信号。沉淀染色质，定量RT - qPCR的。数据是在BTH处理的情况下组蛋白修饰水平的标准化。

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ordered+histone+modifications+are+associated+with+transcriptional+poising+and+activation+of+the+phaseolin+promoter.">Ordered histone modifications are associated with transcriptional poising and activation of the phaseolin promoter.</a> Plant Cell. 18, 119-132 (2006).</li><li>Offermann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Illumination+is+necessary+and+sufficient+to+induce+histone+acetylation+independent+of+transcriptional+activity+at+the+C4-specific+phosphoenolpyruvate+carboxylase+promoter+in+maize.">Illumination is necessary and sufficient to induce histone acetylation independent of transcriptional activity at the C4-specific phosphoenolpyruvate carboxylase promoter in maize.</a> Plant Physiol. 141, 1078-1088 (2006).</li><li>Deng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+the+histone+acetyltransferase+AtHAC1+in+the+regulation+of+flowering+time+via+repression+of+FLOWERING+LOCUS+C+in+Arabidopsis.">Involvement of the histone acetyltransferase AtHAC1 in the regulation of flowering time via repression of FLOWERING LOCUS C in Arabidopsis.</a> Plant Physiol. 143, 1660-1668 (2007).</li><li>Benhamed, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+GCN5,+HD1,+and+TAF1/HAF2+interact+to+regulate+histone+acetylation+required+for+light-responsive+gene+expression.">Arabidopsis GCN5, HD1, and TAF1/HAF2 interact to regulate histone acetylation required for light-responsive gene expression.</a> Plant Cell. 18, 2893-2903 (2006).</li><li>Bowler, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+techniques+for+plant+cells.">Chromatin techniques for plant cells.</a> Plant Journal. 39, 776-789 (2004).</li><li>Orlando, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+chromosomal+proteins+in+vivo+by+formaldehyde+decrosslinked-chromatin+immunoprecipitation.">Mapping chromosomal proteins in vivo by formaldehyde decrosslinked-chromatin immunoprecipitation.</a> Trends Biochem. 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没有利益冲突的声明。

在协议中最重要的步骤是DNA和组蛋白，叶片材料的类型和数量的交联，研磨材料，染色质的超声。对于这些问题的更详细的讨论，请参阅参考文献。 9日和10日。 超声和​​交联： 重要的是要破坏约400bp的片段，在超声染色。力竭超声破坏，破坏DNA和组蛋白的染色质，而超声不足会留下长期的染色质片段。这些影响的方法的特异性，因为从远端测试位点的组蛋白修饰，不能从本地修改歧视。超声效率是最好的测试，通过收集样品20μL前后超声，交联DNA和组蛋白，染色质隔离的DNA，并确定剪切DNA的琼脂糖凝胶电泳长度。应增加核糖核酸酶电泳前样品，染色质的准备，因为仍含有大量的RNA，在这个阶段净化零散的DNA的可视化，可能会干扰。样品是有用的染色质免疫沉淀，如果从非剪切染色质DNA大于10kbp，而从剪切染色质DNA形式的DNA大约400bp的最大信号强度一抹黑。 我们经常使用完整的叶片的染色质交联3％（V / V）的甲醛，但在大多数情况下有足够的1％（V / V）的甲醛可能。在我们的手中，低甲醛浓度允许超声时间较短和随之而来的PCR反应的背景信号。然而，甲醛不足，往往会造成PCR检测信号之间的独立实验重复性低。这可能是由于不足的渗透与甲醛在低浓度的叶。确保经常交换甲醛股票的复合趋于聚合，随着时间的推移。甲醛溶液是稳定的，只有约一个月，而这个时期是难以长期甲醇稳定。这是建议设立实验条件时，以测试不同的甲醛浓度。 植物材料和磨削的金额： 重要的是要在大量的缓冲渗透叶片材料的低量，以避免彼此的树叶粘。叶坚持结果往往不足的甲醛渗透。此外，过多的植物材料将阻塞毛孔的miracloth组织。研磨效率大大取决于使用迫击炮和杵完美契合。我们经常磨，用液氮冷却的杵和砂浆在没有额外的液态氮50秒，直到材料磨成细粉。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td>蛋白A -琼脂糖</td><td>罗氏公司</td><td> 11 134 515 001 </td><td>取决于抗体类</td></tr><tr><td>蛋白 - G -琼脂糖</td><td>罗氏公司</td><td> 11 243 233 001 </td><td>取决于抗体类</td></tr><tr><td>反hyperacetyl - H4 </td><td> Millipore公司</td><td> 06-946 </td><td>我们用5μL </td></tr><tr><td>抗-乙酰H4K5 </td><td> Millipore公司</td><td> 07-327 </td><td>我们用5μL </td></tr><tr><td>抗-乙酰H4K8 </td><td> Millipore公司</td><td> 07-328 </td><td>我们用5μL </td></tr><tr><td>抗-乙酰- H4K12 </td><td> Millipore公司</td><td> 07-595 </td><td>我们用5μL </td></tr><tr><td>抗-乙酰- H4K16 </td><td> Millipore公司</td><td> 07-329 </td><td>我们用5μL </td></tr><tr><td>抗-乙酰- H4K18 </td><td> Millipore公司</td><td> 07-354 </td><td>我们使用1μL </td></tr><tr><td>抗-乙酰H3K9 </td><td> Millipore公司</td><td> 07-352 </td><td>我们用5μL </td></tr><tr><td>抗-乙酰- H3K14 </td><td> Millipore公司</td><td> 07-353 </td><td>我们使用1μL </td></tr><tr><td>反H3K4三甲基- </td><td> Diagenode </td><td> PAB - 003 - 50 </td><td>我们用5μL </td></tr><tr><td>抗-二甲基- H3K4 </td><td> Millipore公司</td><td> 07-030 </td><td>我们用5μL </td></tr><tr><td>抗-甲基- H3K4 </td><td> Abcam公司</td><td> ab8895 </td><td> 2,5 - 10μL </td></tr><tr><td>反H3 </td><td> Abcam公司</td><td> ab1791 </td><td>我们使用1μL检查组蛋白密度</td></tr><tr><td> Bioruptor </td><td> Diagenode </td><td> UCD - 200到</td><td></td></tr><tr><td> Sonotrode MS72 </td><td> Bandelin </td><td></td><td></td></tr><tr><td> Miracloth </td><td> Calbiochem </td><td> 475855 </td><td></td></tr><tr><td>完整的蛋白酶抑制剂</td><td>罗氏公司</td><td> 11836145001 </td><td></td></tr></tbody></table>

detection of histone modifications in plant leaves

在真核生物中基因表达的调控，染色质结构是很重要的。在这个过程中，染色质重塑，DNA甲基化和共价修饰组蛋白H3和H4的氨基末端尾巴，发挥重要作用 1-2 。 H3和H4组蛋白修饰包括赖氨酸和精氨酸甲基化，乙酰赖氨酸和丝氨酸残基 1-2的磷酸化。这些修改相关的基因的激活，镇压，或支持适当的信号（微生物相关分子模式，光，激素等）3-7知觉后，更迅速和更强大的表达激活的基因，致敏状态。 在这里，我们提出了一个具体选定的植物基因的染色质修饰可靠，灵敏的检测方法。该技术是基于（修改），组蛋白与甲醛8,9，提取和修改特定抗体9,10超声染色，染色质免疫沉淀（ChIP）DNA的交联，组蛋白- DNA复合物的交联，特定基因实时定量PCR。该方法已被证明可用于检测与C 4光合作用玉米5,11和3在拟南芥中的全身免疫相关的特定组蛋白修饰。

Watch this Scientific Journal Video about Detection of Histone Modifications in Plant Leaves at JoVE.com

Detection of Histone Modifications in Plant Leaves

一个可靠的和有用的方法来检测特定的植物基因组蛋白修饰的描述。该方法结合了染色质免疫沉淀（ChIP）和实时定量PCR。它允许在不同的生理过程作用，对特定的基因组蛋白修饰的检测。

检测组蛋白修饰在植物叶片

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent ...

detection-of-histone-modifications-in-plant-leaves

Research

JoVE Journal

Biology

24.8K 次观看.  RWTH Aachen University. 一个可靠的和有用的方法来检测特定的植物基因组蛋白修饰的描述。该方法结合了染色质免疫沉淀（ChIP）和实时定量PCR。它允许在不同的生理过程作用，对特定的基因组蛋白修饰的检测。

视频: Detection of Histone Modifications in Plant Leaves

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

检测采用ELISA法的母语完整的核小体的翻译后修饰

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

科研

生物学

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

使用网关兼容双分子荧光互补（附设）系统在植物蛋白相互作用检测

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

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 ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

植物蛋白复合体的翻译后修饰的鉴定

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

渗透离心技术质外体中提取植物液使用叶<em&gt;菜豆</em&gt;为例

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

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 ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

高通量CRISPR载体的构建及DNA修饰的表征番茄毛状根的一代

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

完整的工作流程为组蛋白翻译后修饰的分析采用自下而上质谱法：从组蛋白提取数据分析

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water deficit or pathogens plays a crucial role. Identifying small molecules that regulate stomatal movement can therefore contribute to understanding the physiological basis by which plants adapt to their environment. Large-scale screening approaches that have been used to identify regulators of stomatal movement have potential limitations: some rely heavily on the abscisic acid (ABA) hormone signaling pathway, therefore excluding ABA-independent mechanisms, while others rely on the observation of indirect, long-term physiological effects such as plant growth and development. The screening method presented here allows the large-scale treatment of plants with a library of chemicals coupled with a direct quantification of their transpiration by thermal imaging. Since evaporation of water through transpiration results in leaf surface cooling, thermal imaging provides a non-invasive approach to investigate changes in stomatal conductance over time. In this protocol, Helianthus annuus seedlings are grown hydroponically and then treated by root feeding, in which the primary root is cut and dipped into the chemical being tested. Thermal imaging followed by statistical analysis of cotyledonary temperature changes over time allows for the identification of bioactive molecules modulating stomatal aperture. Our proof-of-concept experiments demonstrate that a chemical can be carried from the cut root to the cotyledon of the sunflower seedling within 10 minutes. In addition, when plants are treated with ABA as a positive control, an increase in leaf surface temperature can be detected within minutes. Our method thus allows the efficient and rapid identification of novel molecules regulating stomatal aperture.

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

赫连图斯安努斯大型热成像筛选装置新稳压器的鉴定

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water ...

Global Level Quantification of Histone Post-Translational Modifications in a 3D Cell Culture Model of Hepatic Tissue

Flat cultures of mammalian cells are a widely used in vitro approach for understanding cell physiology, but this system is limited in modeling solid tissues due to unnaturally rapid cell replication. This is particularly challenging when modeling mature chromatin, as fast replicating cells are frequently involved in DNA replication and have a heterogeneous polyploid population. Presented below is a workflow for modeling, treating, and analyzing quiescent chromatin modifications using a three-dimensional (3D) cell culture system. Using this protocol, hepatocellular carcinoma cell lines are grown as reproducible 3D spheroids in an incubator providing active nutrient diffusion and low shearing forces. Treatment with sodium butyrate and sodium succinate induced an increase in histone acetylation and succinylation, respectively. Increases in levels of histone acetylation and succinylation are associated with a more open chromatin state. Spheroids are then collected for isolation of cell nuclei, from which histone proteins are extracted for the analysis of their post-translational modifications. Histone analysis is performed via liquid chromatography coupled online with tandem mass spectrometry, followed by an in-house computational pipeline. Finally, examples of data representation to investigate the frequency and occurrence of combinatorial histone marks are shown.

This protocol outlines how a three-dimensional cell culture system can be used to model, treat, and analyze chromatin modifications in a near-physiological state.

肝组织3D细胞培养模型中组蛋白翻译后修饰的全球水平定量

Flat cultures of mammalian cells are a widely used in vitro approach for understanding cell physiology, but this system is limited in modeling solid ...