This work was supported by a Natural Sciences and Engineering Research (NSERC)-USRA grant to S.J., and by an NSERC-Discovery Grant to I.M. We thank Richard Bourgault, Meghan Rains, and Amanda Fluke for technical assistance. Seeds of att1-1 and att1-2 mutants were kindly provided by Dr. Jian-Min Zhou, Institute of Genetics and Developmental Biology, Beijing, China.

注：该协议是改编自博纳等人。 （2004），Molina等人。 （2006年），李等人。 （2013年）7,10,11。步骤1-5 总结于图1。 1.组织脱脂注意：一定要清洗所有的玻璃器皿和瓶盖用氯仿，让干在通风橱，使用前。 注意：执行组织均匀化，并在通风橱中的所有溶剂转移步骤;总是穿着白大褂，手套和防溅安全护目镜，避免与化学物质直接接触，并保护样品免受污染。 <ol><li>预热水浴加热块到85℃。 </li><li>称取约0.5克每叶样品放入预称重的20个毫米×125mm玻璃管试管用聚四氟乙烯（PTFE）-faced螺丝帽。包括每个样品四次重复。 </li><li>放置在一个锥形瓶中（大约125毫升2-丙醇;每克SAMP 25ml的乐）。添加2,6-二 - 叔丁基-4-甲基苯酚（也被称为丁基羟基甲苯，BHT），以0.01％的终浓度（重量/体积）。 注意：从5％添加BHT（重量/体积）储备溶液在甲醇（BHT有助于减少不饱和脂肪酸的氧化）。 </li><li>预热2-丙醇溶液至85℃在水浴中。 </li><li>加12个毫升热的2-丙醇溶剂向每个样品管中，并加热15分钟，在85℃下在加热块。此步骤失活可从破碎的细胞释放的脂肪酶。 </li><li>让管冷却至室温，直至获得均匀的悬浮液用均化器充分研磨组织。 </li><li>将样品中的定轨摇床并振摇在100rpm和室温下1-2小时。 </li><li>离心10分钟，以800×g离心并弃去上清液。 </li><li>添加2-丙醇等体积并摇动在室温下12小时。 </li><li>离心10分钟，以800×g离心并弃去上清液。 </li><li>添加12毫升CH氯3：CH 3 OH（2:1，体积/体积）至残基（每克样品25毫升）中，在100rpm和室温下振摇过夜。 </li><li>离心10分钟，以800×g离心并弃去上清液。 </li><li>加12个ml的氯仿：CH 3 OH（1：2，V / V）至残基和摇动过夜以100rpm和室温。 </li><li>离心在800×g离心10分钟并除去溶剂。 </li><li>让样品在通风橱干燥在室温下过夜。 </li><li>风干残余物，然后在真空干燥器放置在无水氯化钙或的CaSO 4，直到达到恒重（3-5天）。 </li></ol> 2.解聚：甲醇分解用甲醇钠（图2） 注意：执行步骤2.3 - 2.4，2.7 - 2.8，2.10 - 2.11 2.13 - 2.15和2.17 - 2.18下通风柜;总是穿着白大褂，手套和防溅护目镜。 <ol><li>预热加热块到60℃。 </li><li>称量装有干渣（用于进一步的计算很重要）的试管。 </li><li>添加内部标准，以每管：25ωL甲基十七烷（1毫克/毫升的库存）和25微升ω十五内酯（1毫克/毫升的库存）。 </li><li>添加0.9 ml的乙酸甲酯，1.5 ml的甲醇钠和3.6毫升甲醇到每个管和盖它们。另外，准备一个反应混合物用这三种试剂，并添加6毫升分装到每个样品。 </li><li>热样品，在60℃涡流周期性地每间隔15分钟2小时。 </li><li>让样品冷却到室温。 </li><li>加入10毫升二氯甲烷（CH 2 Cl 2中）和1.5冰醋酸毫升提取脂肪酸甲基酯。 </li><li>加入盐水溶液（0.5M氯化钠），填补每个管和帽。 </li><li>涡旋样品1分钟，离心，在800×g下10分钟。 </li><li>转移有机相（低级）清洁介质尺寸试管（16×125mm玻璃管的试管与聚四氟乙烯（PTFE）-faced螺丝帽）。 </li><li>加入盐水溶液（0.5M氯化钠），填补每个管和帽。 </li><li>涡旋样品1分钟，离心，在800×g下10分钟。 </li><li>除去水（上）阶段，并重复步骤2.11-2.12。 </li><li>删除所有的水（上）阶段。 </li><li>添加无水硫酸钠（ 用 Na 2 SO 4）的溶剂中，盖上管，并涡旋1分钟;样品现在可能留到第二天。 此时样品可通风柜下放置过夜。 </li><li>离心2分钟，在800×g离心压缩在底部的用 Na 2 SO 4盐。 </li><li>将有机相转移到一个小玻璃一次性管（13×100毫米的玻璃试管用聚四氟乙烯（PTFE）-faced螺丝帽）。 </li><li>蒸发溶剂至干氮气下，然后继续衍生步骤。如果不立即处理，商店蒸发样品在-20℃（样品可以是一个LSO存储在蒸发步骤之前）。 </li></ol> 3.准备作为气相色谱注意：执行步骤3.1.2和3.1.5 - 3.1.8在通风橱;总是穿着白大褂，手套和防溅护目镜。 <ol><li> 三甲基硅衍生物 <ol><li>预热加热块到100℃。 </li><li>加入100微升吡啶和100微升BSTFA（N，O-双 - 三甲基硅烷基三氟乙酰胺），以每管和盖它们。 </li><li>热样品在100℃下进行10分钟。 </li><li>让样品冷却到室温。 </li><li>蒸发氮气下样品在室温下。避免将热施加到样品中，单体是高挥发性的，在这个阶段。 </li><li>加入500微升1：1（体积/体积）的庚烷：甲苯。 </li><li>涡旋样品1分钟，离心，在800×g下2分钟。 </li><li>添加样品GC小瓶，然后继续GC / MS分析。 </li></ol></li><li> 乙酰衍生工具 注意：对于乙酰化，修改步骤3.1.1-3.1.3以上如下（以视频未示出）;步骤3.1.4-3.1.8是相同的： <ol><li>预热加热块到60℃。 </li><li>加入100微升吡啶和100微升乙酸酐（徒2 O）到管中。 </li><li>热样品在60℃下1小时。 </li></ol></li></ol> 4. GC / MS分析<ol><li>使用的HP-5毛细管柱（30米×0.25毫米×0.25微米膜厚度）或等效（即，5％二苯基，95％二甲基聚硅氧烷）。程序用氦载气流量设定为1.5毫升/分钟，烘箱温度为150编程，以300℃，3℃/分钟的气相色谱。 </li><li>使用分流进样（分流比1:10），并设置质谱仪扫描模式在40-600 AMU（电子碰撞电离）。 </li><li>创建序列表包括溶剂（空白），WT和突变体重复，每个使用指示的角质分析方法。 </li><li>负载溶剂并且在转盘样品瓶，如果有必要加己烷到注射器漂洗小瓶在自动进样器，和启动序列。的顺序结束后，总离子色谱的痕迹可用于所有样品。 </li></ol> 5.数据分析<ol><li>通过比较每个峰的质谱公布的质谱或通过搜索商业库（如果可用）确定脂质聚酯单体。 </li><li>在总离子流色谱图每个识别出的峰中，使用各自的保留时间上找到从GC / MS软件（ 参考图1）的积分结果表中的区域。 </li><li>对于每种单体（列AB）中，添加在积分表（ 参考图1，列D）中发现的面积值，以对每个相应列复制样品中的单体的Excel表格（ 参考文件2中，列CF），它是电子表格量化拟南芥角质TMSI衍生物。 使用补充文件3，如果乙酰化衍生物的制备代替。 </li><li>加的内部标准（IS）于IS柱（HK）的区域。对于那些没有衍生，即脂肪酸甲酯单体（脂肪酸甲酯），和二羧酸二甲酯（DCA测距仪），使用17：0 FAME（IS1）作为用于量化（紫色阴影细胞中的单体表; 补充文件2/3）。为羟基化的单体，包括伯醇，阿魏酸，ω羟基酸，使用的15：0 15羟基FAME（IS2），为是选择用于化合物定量（绿色- shadded细胞中的单体表; 参考文件2/3） 。 </li><li>添加干叶量为每复制到列AX-BA（辅助文件2）或AQ-AT（ 参考文件3）;另外，扫描叶计算表面积和单体表添加面积值来表示每单位单体负荷表面积。 </li></ol>

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No conflicts of interest declared.

不像其他的生物聚合物如DNA和蛋白质，植物脂质聚酯不是从模板制成。相反，他们的组合物依赖于存在于使这些细胞外聚合物组织中的酶的特异性。这样，化学组成的分析成分是关键的了解脂质聚酯组合物。 化学方法裂解的酯键包括皂化，氢解，酸催化transmethylation，和碱催化transmethylation 2。他们每个人都有优点和缺点。皂化产生游离脂肪酸羟基酸，可以进行二次反应。氢解用氢化铝锂（的LiAlH 4）16已被用于角质分析7。氢解降低官能碳为醇和原始结构需要由deuteriolysis锂铝氘（LiAlD 4）推断。该这种方法的缺点是高清晰度的GC / MS的要求来比较获得使它们的结构分配的脂肪多元醇的deuteriation的程度。酸催化的酯交换用甲醇三氟化硼（BF 3）已被常用于角质和软木脂depolymerizations 8,17,18，但该试剂具有有限的货架寿命并可能引入由于副反应15假象。硫酸甲醇也产生的单体，但具有较大的2-羟基脂肪酸的比例，这大概是不正确的脂质聚酯组分甲酯，相比其他方法10。 在这个协议中所描述的的NaOMe催化酯交换法产生由羟基甲硅烷基化衍生的脂肪酸甲酯，用于识别提供特性质谱，或通过乙酰化，以提供羟基更稳定的衍生物FO- [R量化。这种技术的一个缺点是水解竞争与酯交换当水存在于反应。水发生反应的NaOMe（催化剂），并产生氢氧化钠，这反过来水解脂肪酸甲酯，得到游离酸（图2D）。这是一种不希望的副反应，因为两个峰值将存在对于每个脂肪酸：甲基酯和TMSI酯衍生物，因此复杂的分析。用无水试剂并加入乙酸甲酯作为助溶剂与皂化竞争因此关键步骤，以防止水解（图2D）。 1和26％甘油4之间的角质和木栓质含有。但是，此单体不会被在这个协议中所描述的实验条件下进行检测。甘油是高度亲水性的并且，不同于脂肪酸甲酯单体，将在水性溶剂洗涤步骤被消除。这种限制也是一个pplies其他角质解聚方法，但甘油可以在使用酶方法酯交换后得到的水层来确定。可替代地，它可以使用较温和的条件（，0.05M的的NaOMe 当量）定量无需进一步水萃取，以检测所有的单体，包括甘油19,20。虽然对于甘油量化的目的是有用的，在温和的条件通常给角质的不完全解聚和木栓质。 如果耦合到一个火焰离子化检测器（FID）的GC一个是可用的，所有的重复可在该仪器进行定量目的被分析，有代表性的样品的峰已确定的GC / MS后。或者，在GC / FID痕迹单体可以识别，如果他们的保留指数是已知的。火焰离子化检测器具有特别高的灵敏度和宽范围相称，这是主要和次要样品组分定量临界在单次运行。此外，它是坚固和易于维护和操作15。 所描述的协议允许的可靠和可重复的分离，鉴定和定量植物脂质聚酯单体，允许其在不同的一种或多种脂质聚酯单体的组合物的突变体的化学特性。该过程是可伸缩的，它可以很容易地适用于处理小型和大批量的各种植物材料，包括根，种子，叶，茎和花。来自许多物种的脂质聚酯单体的质谱数据已经公布，例如 ，21-26和构成宝贵资源适应这个协议对其他组织和/或物种时识别未知单体。这种方法适用于生物合成，调节和高等植物分布脂质聚酯的调查。

维管束植物依靠充当植物组织和外部环境之间的防水屏障外层。这些亲脂性的细胞壁相关结构限制病原体感染和调节的气体，水和溶解的物质的被动转运进出植物组织1。这种障碍是植物表皮，一个synapomorphic结构独特的植物2，以及不同的含软木脂扩散壁垒。角质层是由表皮细胞经由果胶层上的细胞壁3-5的外侧合成并结合到它们的亲油性层。它包住高等植物的主天线器官，用作植物组织和环境之间的一个重要的接口。 角质，角质层的结构基体，和软木脂与溶剂萃取的蜡2,4相关联的两个不溶甘油脂聚酯。这些聚合物升ipids是由饱和和不饱和脂肪酸衍生物的且都是结构上和功能上类似。然而，它们是可区分由在化学组成和沉积点特性的差异。 软木脂是一种脂族聚酯位于形成次生壁一定的外部和内部组织的细胞壁内。 Suberized组织包括根periderms，块茎和树皮，根内皮层，种皮层和愈合伤口2。不像角质，所述木栓质聚酯通常含有醇，饱和的和单不饱和二羧酸，和相当大的比例非常长链的单体的（C≥20）。 角质是在维管植物6中最丰富的脂类聚酯，和由甘油和C 16 -C 18交换的脂肪酸衍生物，如羟基和羟基取代的环氧脂肪酸4。而角质聚合物的组合物跨越维管种类而异，最主要主单体为10，16二羟基16：0，18羟基-9,10-环氧18：0，和9,10,18三羟基18：0脂肪酸。有趣的是，拟南芥属叶和茎角质主要由18：2的二羧酸7,8。 植物表皮也厚度呈现出相当大的变化，从几纳米到几微米9。由于角质层隔离是费力和耗时的步骤，特别是对于非常薄的叶子表皮诸如拟南芥 8，绕过角质层分离方法已经开发和验证7,8。在这里，我们描述了一个详细的协议来研究角质的拟南芥叶片由甲醇钠（甲醇钠）催化的解聚和随后的气相色谱/质谱（GC / MS）分析该单体组合物。该协议提供了一个强大的方法测定共mposition在全脱脂组织的植物脂肪聚酯，并已被改编自先前报告协议7,10,11。整个组织样品是第一匀浆，彻底去脂，除去溶剂萃取的脂质包括表皮和epicuticular蜡，膜脂，和三酰基甘油。细胞壁富集残基，然后由甲醇钠催化的甲醇分解解聚成其组成甲酯单体。脂肪酸甲基酯被提取在酸化时，并衍生以得到其相应的三甲基甲硅烷基或乙酰基的衍生物。衍生的残基是高度挥发性的，并且可以从在适当温度下用气相色谱柱洗脱而不会在GC / MS分析改变其结构构象。

<table border="0" cellpadding="0" cellspacing="0" width="1105">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Chemicals</td>
			<td style="width:228px;"></td>
			<td style="width:153px;"></td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">2-propanol</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;">BPA451-4</td>
			<td style="width:301px;">Solvent for delipidation</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Anhydrous sodium sulfate</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;">S421500</td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Acetic anhydride</td>
			<td style="width:228px;">Sigma Aldrich</td>
			<td style="width:153px;">320102</td>
			<td style="width:301px;">Derivatization agent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">BSTFA (N,O-bis(trimethylsilyl)-trifluoroacetamide)</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:153px;">15222</td>
			<td style="width:301px;">Derivatization agent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Butylated hydroxytoluene (BHT)</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:153px;">101162</td>
			<td style="width:301px;">Antioxidant</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Calcium chloride, anhydrous</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;">C614-3</td>
			<td style="width:301px;">Desiccation agent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Calcium suflate, anhydrous (DRIERITE- 8 MESH with indicator)</td>
			<td style="width:228px;">Acros Organics</td>
			<td>219090020</td>
			<td style="width:301px;">Desiccation agent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Chloroform (Trichloromethane)</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;">C6074</td>
			<td style="width:301px;">Organic solvent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Glacial acetic acid</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;">BP2401212</td>
			<td style="width:301px;">Acidification agent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Helium carrier gas, compressed</td>
			<td style="width:228px;">Air Liquide</td>
			<td style="width:153px;">ALPHAGAZ1-UN1046</td>
			<td style="width:301px;">Carrier gas, GC/MS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Heptane</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;">H3501</td>
			<td style="width:301px;">Organic solvent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Hexanes</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;">H3024</td>
			<td style="width:301px;">Organic solvent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Methanol</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;">A4124</td>
			<td style="width:301px;">Organic solvent, transmethylation reactive</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Methyl acetate</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:153px;">296996</td>
			<td style="width:301px;">Organic solvent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Methyl heptadecanoate</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:153px;">H4515</td>
			<td style="width:301px;">Internal standard (1mg/mL stock)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Methylene dichloride (Dichloromethane)</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;">D374</td>
			<td style="width:301px;">Organic solvent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Nitrogen, compressed</td>
			<td style="width:228px;">Air Liquide</td>
			<td style="width:153px;">ALPHAGAZ1-UN1044</td>
			<td style="width:301px;">Carrier gas, GC-FID</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Pentadecanolactone</td>
			<td style="width:228px;">Fluka</td>
			<td style="width:153px;">76530</td>
			<td style="width:301px;">Internal standard (1 mg/mL stock)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Pyridine</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:153px;">270970</td>
			<td style="width:301px;">Co-solvent for derivatization</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Sodium chloride</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;">BP358212</td>
			<td style="width:301px;">Saline solution</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Sodium methoxide (25wt.%)</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:153px;">156256</td>
			<td style="width:301px;">Nucleophile</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Toluene</td>
			<td style="width:228px;">FIsher Scientific</td>
			<td style="width:153px;">T2904</td>
			<td style="width:301px;">Organic solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Plant Growth Supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:423px;">Pro-Mix PGX</td>
			<td>Premier Tech Horticulture Ltd</td>
			<td style="width:153px;"></td>
			<td style="width:301px;">Pro-Mix PGX is recommended to grow Arabidopsis plants (Eddy, R. and Hahn, D.T., 2012,http://docs.lib.purdue.edu/pmag/2) 
			Purdue Methods for Arabidopsis Growth.&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">PermaNest Humidity Dome&#160;</td>
			<td style="width:228px;">Grower&#39;s Solution, LLC, Cookeville, TN&#160;</td>
			<td style="width:153px;">GD2211-24</td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">Perma-Nest Plant Trays (22x11in)</td>
			<td style="width:228px;">Grower&#39;s Solution, LLC, Cookeville, TN&#160;</td>
			<td style="width:153px;">N/A</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">Square greenhouse pots, 3.5 inch&#160;</td>
			<td style="width:228px;">Grower&#39;s Solution, LLC, Cookeville, TN&#160;</td>
			<td style="width:153px;">P86</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:423px;">General Purpose Plant Fertilizer, Plant-Prod 20-20-20</td>
			<td style="width:228px;">Premier Tech Home and Garden In., Brantford, ON</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glassware</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:423px;">13 x 100 mm glass test tube with Teflon-faced screw cap</td>
			<td style="width:228px;">Kimble Chase Life Science and Research Products LLC</td>
			<td style="width:153px;">45066A-13100</td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:423px;">16 x 125 mm glass test tube with Teflon-faced screw cap</td>
			<td style="width:228px;">Kimble Chase Life Science and Research Products LLC</td>
			<td style="width:153px;">45066A-16125</td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:423px;">20 x 125 mm glass test tubes with Teflon-faced screw cap</td>
			<td style="width:228px;">Kimble Chase Life Science and Research Products LLC</td>
			<td style="width:153px;">45066A-20125</td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">GC vial caps</td>
			<td style="width:228px;">National Scientific</td>
			<td style="width:153px;">C400051A</td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">GC vial microinserts</td>
			<td style="width:228px;">National Scientific</td>
			<td style="width:153px;">C4011631</td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">GC vials</td>
			<td style="width:228px;">National Scientific</td>
			<td style="width:153px;">C40001</td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Disposable pasteur pipets</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;">1367820B</td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Flasks</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;"></td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Allegra X15R centrifuge</td>
			<td style="width:228px;">Beckman Coulter</td>
			<td style="width:153px;"></td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Analytic balance</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;"></td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:423px;">Belly dancer</td>
			<td style="width:228px;"></td>
			<td style="width:153px;"></td>
			<td style="width:301px;">A shaker can be used for this purpose if Belly Dancer not available</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">DB-5 Capillary GC column</td>
			<td style="width:228px;">J&#38;W Scientific, CA, USA;&#160;</td>
			<td style="width:153px;"></td>
			<td style="width:301px;">30 m x 0.25 mm x 0.25 &#956;m film thickness</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Desiccator</td>
			<td style="width:228px;"></td>
			<td style="width:153px;"></td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Isotemp 202 water bath</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;"></td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">ISQ LT single quadupole mass spectrometer</td>
			<td style="width:228px;">Thermo Scientific</td>
			<td style="width:153px;"></td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Heat block</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;"></td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Nitrogen evaporator</td>
			<td style="width:228px;"></td>
			<td style="width:153px;"></td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Polytron homogenizer</td>
			<td style="width:228px;">Birkmann</td>
			<td style="width:153px;"></td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Trace 1300 gas chromatograph</td>
			<td style="width:228px;">Thermo Scientific</td>
			<td style="width:153px;"></td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Two-stage regulator</td>
			<td style="width:228px;">Air Liquide</td>
			<td style="width:153px;">Q1-318B-580</td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Vacuum desiccator</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;"></td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Vortex mixer</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;"></td>
			<td style="width:301px;"></td>
		</tr>
	</tbody>
</table>

isolation and compositional analysis of plant cuticle lipid polyester monomers

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester of glycerol and modified fatty acids that constitutes the framework of the cuticle that covers aerial plant surfaces. Suberin is a related lipid polyester that is deposited on the cell walls of certain tissues, including the root endodermis and the periderm of tubers, tree bark and roots. These lipid polymers are highly variable in composition among plant species, and often differ among tissues within a single species. Here, we describe a detailed protocol to study the monomer composition of cutin in Arabidopsis thaliana leaves by sodium methoxide (NaOMe)-catalyzed depolymerisation, derivatization, and subsequent gas chromatography-mass spectrometry (GC/MS) analysis. This method can be used to investigate the monomers of insoluble polyesters isolated from whole delipidated plant tissues bearing either cutin or suberin. The method can by applied not only to characterize the composition of lipid polymers in species not previously analyzed, but also as an analytical tool in forward and reverse genetic approaches to assess candidate gene function.

在这个手稿中描述的协议设置为确定脂质聚酯单体，最小化的非角质脂质10的贡献图1表示的测定法，其中共花费之间8的概述（即角质或软木脂） - 10天（从初始组织收获到获得的GC数据），这取决于样品多久被允许干燥。 所选择的碱催化的甲醇分解（图2）的方法来解聚聚酯先前被验证为拟南芥的种子，其中同时包含角质和软木脂。组织被第一匀化和彻底去脂以除去溶剂萃取的脂质。萃取后的残留物的产量，作为初始鲜重的百分比，为A.通常为6％ 拟南芥 Col-0中的叶子。细胞壁富集残基被干燥在真空干燥器，然后通过解聚成其组成甲酯单体碱催化transmethylation。两小时培养被选择作为需要进行适当的解聚和脂质聚酯组分的回收的临界时间。较长的孵育时间导致增加2-羟基酸;这些潜在源于鞘脂膜10。 如果拟南芥野生型叶角质如图3的典型色谱， 办理 O -TMSi醚衍生物（ 图3A）和O -乙酰基衍生物（ 图3B）。每个峰通过比较质谱从文献7,8和公共数据库12。我们的视频协议示出了如何准备TMSI衍生物确定，但样品可替代地乙酰化衍生化的羟基。硅烷化衍生物是很好的用于标识目的，因为他们给的诊断质谱。然而，乙酰化衍生物更稳定，一个很好的替代硅烷化一旦单体已经确定了10个。为了帮助实现此协议中的实验室仅具有GC耦合 ​​到火焰离子化检测器（FID）的是，GC / FID迹线对应于WT叶角质单体和同源物一系列脂肪酸甲酯标准乙酰化衍生物也示（ 参考图4）。 此方法是定性的，并且检测样本的定量差异，突变分析，因此它的值。各单体的量是使用量化的内标法，使样品之间的单体丰度比较来确定。然而，应该明确的是，峰的大小（总离子计数）可能不反映在聚酯中单体的摩尔比。我们包括单体的可编辑的表来计算在拟南芥叶子角质单体量为脂肪酸甲基酯和TMSI衍生物（ 参考文件1），或王牌<html>醇的TYL衍生物（ 参考文件2）。这些表可能需要的，如果样本是从不同的器官或植物物种中提取进行调整。 作为一个例子，我们已经分析拟南芥塔利亚娜哥伦比亚（Col-0中）野生型叶片和CYP86A2 / ATT1基因，att1-1（M-1）和att1-2的两个先前表征的空突变等位基因（M-2 ）13,14。在CYP86A亚家族的细胞色素P450单加氧酶编码假定ω-oxydases并参与木栓质和角质单体合成。我们的研究结果（图4）证明显著减少三个主要脂质单体的载荷在突变叶片与WT相比叶子。一致的与酶的预测的功能，16：0，18：2和18：1的二羧酸酯在ATT1突变体特别受到影响。 “SRC =”/文件/ ftp_upload / 53386 / 53386fig1.jpg“/&gt; 图中的脂质聚酯分析1.概述。 <a href="https://www.jove.com/files/ftp_upload/53386/53386fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53386/53386fig2.jpg" /> 图中的NaOMe催化甲基转移反应的2.机制的亲核甲醇阴离子攻击脂质聚酯的cabonyl碳，以形成不稳定的四面体中间体（A），它容易离解成脂肪酸甲酯和醇盐阴离子（B）中 。这些醇盐是共轭碱，和用甲醇反应，再生催化活性甲醇阴离子，从而维持附加解聚反应（C）。如果水存在于系统，它会与甲醇钠反应以形成氢氧化钠，强碱不可逆地水解的酯，以产生不希望的游离脂肪酸。乙酸甲酯加入助溶剂15，除去少量的氢氧化系统（D）中的钠。 <a href="https://www.jove.com/files/ftp_upload/53386/53386fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53386/53386fig3.jpg" /> 图3。野生型拟南芥叶角质单体的代表总离子色谱图。（A）O三甲基甲硅烷（TMSI）醚和（B）乙羟基衍生物。 <a href="https://www.jove.com/files/ftp_upload/53386/53386fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> </一&gt; <img alt="图4" src="/files/ftp_upload/53386/53386fig4.jpg" /> 图4.角质单体的拟南芥野生型组合物和CYP86A2基因的两个无效突变体等位基因。（突变-1 = att1-1;突变-2 = att1-2）误差棒代表平均值的标准偏差 （n = 4） 。从13改编，以©Blackwell出版（2007）的权限。 <a href="https://www.jove.com/files/ftp_upload/53386/53386fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图1" src="/files/ftp_upload/53386/53386supfig1.jpg" /> 补充图1峰值积分结果表从GC / MS软件。对应于识别的单体和内部标准的峰由它们的retenti确定<html>时间（列C）和面积值列于列D. <a href="https://www.jove.com/files/ftp_upload/53386/Supplemental_Figure_1.tif" target="_blank">请点击此处下载该文件。</a> <img alt="图2" src="/files/ftp_upload/53386/53386supfig2.jpg" /> 补充文件2。表拟南芥角质单体（羟基脂肪酸甲酯三甲基硅醚衍生物）的。 <a href="https://www.jove.com/files/ftp_upload/53386/Supplemental_File_2_Cutin_template_TMSi.xls" target="_blank">请点击此处下载该文件。</a> <img alt="图3" src="/files/ftp_upload/53386/53386supfig3.jpg" /> 补充文件3.表拟南芥角质单体（羟基脂肪酸甲酯-O-乙酰衍生物）的。COM /文件/ ftp_upload / 53386 / Supplemental_File_3_Cutin_template_Acetylated_derivati​​ves.xlsx“目标=”_空白“&gt;请点击此处下载该文件。 <img alt="图4" src="/files/ftp_upload/53386/53386supfig4.jpg" /> 补充图4的GC / FID的痕迹（AB）的脂肪酸甲酯（FAME）的保留指数的标准（峰标记与每个饱和FAME链长）; 和 （C）乙酰A.拟南芥 WT叶角质单体。上峰编号对应：16：0的FAME（1），阿魏酸（2），18：3的FAME（3），18：1/18：2脂肪酸甲酯（4），18：0的FAME（5），芥子（6 ），16：0 DCA（7），16-OH 16：0的FAME（8），18：2 DCA（9），18：1 DCA（10）18：0 DCA（11），18-OH 18：2 FAME（12），18-OH 18：1的FAME（13），20：0的FAME（14），-10,16- diOH 16：0的FAME（15），24：0的FAME（16）。 DCA：二羧酸二甲酯; FAME：脂肪酸甲酯; IS1：内标1，17：0 FAME; IS2：内部ST昂达尔2，15-OH 15：0 FAME。 <a href="https://www.jove.com/files/ftp_upload/53386/Supplemental_Figure_4.tif" target="_blank">请点击此处下载该文件。</a>

Watch this Scientific Journal Video about Isolation and Compositional Analysis of Plant Cuticle Lipid Polyester Monomers at JoVE.com

Isolation and Compositional Analysis of Plant Cuticle Lipid Polyester Monomers

Lipid polyesters constitute the structural components of two cell wall modifications, the plant cuticle and suberin-containing diffusion barriers. In this video, we describe a method to depolymerize cutin from whole delipidated leaves. The method can be applied to investigating mutants compromised in either cutin or suberin biosynthesis.

植物表皮脂质聚酯单体的分离及成分分析

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester ...

isolation-compositional-analysis-plant-cuticle-lipid-polyester

Research

JoVE Journal

Biology

16.6K 次观看.  Algoma University. Lipid polyesters constitute the structural components of two cell wall modifications, the plant cuticle and suberin-containing diffusion barriers. In this video, we describe a method to depolymerize cutin from whole delipidated leaves. The method can be applied to investigating mutants compromised in either cutin or suberin biosynthesis.

视频: Isolation and Compositional Analysis of Plant Cuticle Lipid Polyester Monomers

Use of Arabidopsis eceriferum Mutants to Explore Plant Cuticle Biosynthesis

The plant cuticle is a waxy outer covering on plants that has a primary role in water conservation, but is also an important barrier against the entry of pathogenic microorganisms.  The cuticle is made up of a tough crosslinked polymer called "cutin" and a protective wax layer that seals the plant surface.  The waxy layer of the cuticle is obvious on many plants, appearing as a shiny film on the ivy leaf or as a dusty outer covering on the surface of a grape or a cabbage leaf thanks to light scattering crystals present in the wax.  Because the cuticle is an essential adaptation of plants to a terrestrial environment, understanding the genes involved in plant cuticle formation has applications in both agriculture and forestry. Today, we'll show the analysis of plant cuticle mutants identified by forward and reverse genetics approaches.


The plant cuticle is a waxy outer covering on plants that has a primary role in water conservation but is also an important barrier against the entry of pathogenic microorganisms. In this video, we demonstrate the analysis of plant cuticle mutants identified by forward and reverse genetics approaches.

使用拟南芥eceriferum突变体探索植物角质层的生物合成

The plant cuticle is a waxy outer covering on plants that has a primary role in water conservation, but is also an important barrier against the entry of ...

科研

生物学

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and genetics because of its simple life cycle, ease of growth and manipulation for genetic analysis, genomic resources, and transformability of the nucleus and both organelles. Mating strains is a common practice when genetic approaches are used in Chlamydomonass, to create vegetative diploids for analysis of dominance, or following tetrad dissection to ascertain nuclear vs. organellar inheritance, to test allelism, to analyze epistasy, or to generate populations for the purpose of map-based cloning. Additionally, genetic crosses are routinely used to combine organellar genotypes with particular nuclear genotypes. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad separation. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas. Key parameters and trouble spots are explained. Finally, resources for further information and alternative methods are provided.

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

Mating and tetrad separation are required for genetic analysis in Chlamydomonas reinhardtii. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad dissection. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas.

交配和四分体分离衣藻遗传分析

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and ...

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part I: Lignin

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation1 and other products such as biocomposite materials7. Plant biomass remains one of the greatest untapped reserves on the planet4. It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses (matrix polysaccharides, and the polyphenol lignin6 and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls provide tensile strength to cells and the entire plants, ward off pathogens, and allow water to be transported throughout the plant; in the case of trees up to more the 100 m above ground level. Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant4. Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerization by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis. In this first part we focus on the analysis of the polyphenol lignin (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its lignin content by acetylbromide solubilization3, and its lignin composition in terms of its syringyl, guaiacyl- and p-hydroxyphenyl units5. The protocol for analyzing the carbohydrates in lignocellulosic biomass including cellulose content and matrix polysaccharide composition is discussed in Part II2.

Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part I: Lignin

Plant biomass is a major carbon-neutral renewable resource that could be used for the production of biofuels. Plant biomass consists mainly of cell walls, a structurally complex composite material termed lignocellulosics. Here we describe a protocol for a comprehensive analysis of the content and composition of the polyphenolic lignin.

综合植物细胞壁成分分析（木质纤维素生物质）第一部分：木质素

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st ...

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part II: Carbohydrates

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation2 and other products such as biocomposite materials6. Plant biomass remains one of the greatest untapped reserves on the planet4. It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses, and the polyphenol lignin5 and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls contribute extensively to the strength and structural integrity of the entire plant. Despite its necessary rigidity, the cell wall is a highly dynamic entity that is metabolically active and plays crucial roles in numerous cell activities such as plant growth and differentiation5. Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant4. Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerisation by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its monosaccharide composition of the hemicelluloses and other matrix polysaccharides1, and its content of crystalline cellulose7. The protocol for analyzing the lignin components in lignocellulosic biomass is discussed in Part I3.

Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part II: Carbohydrates

Plant biomass is a major carbon-neutral renewable resource that could be used for the production of biofuels. Plant biomass consists mainly of cell walls, a structurally complex composite material termed lignocellulosics. Here we describe a protocol for a comprehensive analysis of the content and composition of wall derived carbohydrates.

综合植物细胞壁成分分析（木质纤维素生物质）第二部分：碳水化合物

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

高效，快速的早期胚胎分离<em&gt;拟南芥</em&gt;种子

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

代谢标记和膜分馏的比较蛋白质组学分析<em&gt;拟南芥</em&gt;悬浮细胞培养

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

多蛋白复合物的比较分析新方法基于<sup&gt; 15</sup&gt;Ñ代谢标记和定量质谱

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

鼠心肌细胞的分离及生理分析

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while maintaining transparency of the visual axis. Perturbations to this delicate balance cause blinding illnesses, such as diabetic retinopathy. Therefore, the understanding of energy metabolism changes in the retina during disease is imperative to the development of rational therapies for various causes of vison loss. The recent advent of commercially-available extracellular flux analyzers has made the study of retinal energy metabolism more accessible. This protocol describes the use of such an analyzer to measure contributions to retinal energy supply through its two principle arms - oxidative phosphorylation and glycolysis - by quantifying changes in oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as proxies for these pathways. This technique is readily performed in explanted retinal tissue, facilitating assessment of responses to multiple pharmacologic agents in a single experiment. Metabolic signatures in retinas from animals lacking rod photoreceptor signaling are compared to wild-type controls using this method. A major limitation in this technique is the lack of ability to discriminate between light-adapted and dark-adapted energy utilization, an important physiologic consideration in retinal tissue.

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

细胞外通量分析技术在视网膜组织外种中的能量代谢测定

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

Rapid Lipid Droplet Isolation Protocol Using a Well-established Organelle Isolation Kit

Lipid droplets (LDs) are bioactive organelles found within the cytosol of the most eukaryotic and some prokaryotic cells. LDs are composed of neutral lipids encased by a monolayer of phospholipids and proteins. Hepatic LD lipids, such as ceramides, and proteins are implicated in several diseases that cause hepatic steatosis. Although previous methods have been established for LD isolation, they require a time-consuming preparation of reagents and are not designed for the isolation of multiple subcellular compartments. We sought to establish a new protocol to enable the isolation of LDs, endoplasmic reticulum (ER), and lysosomes from a single mouse liver.
Further, all reagents used in the protocol presented here are commercially available and require minimal reagent preparation without sacrificing LD purity. Here we present data comparing this new protocol to a standard sucrose gradient protocol, demonstrating comparable purity, morphology, and yield. Additionally, we can isolate ER and lysosomes using the same sample, providing detailed insight into the formation and intracellular flux of lipids and their associated proteins.

This protocol establishes a novel method of lipid droplet isolation and purification from mouse livers, using a well-established endoplasmic reticulum isolation kit.

利用成熟的细胞隔离试剂盒快速分离脂滴器

Lipid droplets (LDs) are bioactive organelles found within the cytosol of the most eukaryotic and some prokaryotic cells. LDs are composed of neutral ...