This work was supported by grants BB/J016012/1 and BB/E007872/1 from the UK Biotechnology and Biological Sciences Research Council (BBSRC) to Gail Preston.

注意:起始叶材料和植物生长的条件标准化的选择是至关重要的，因为这些因素会大大影响质量，可变性和AWF的产率（ 图1）。参数考虑示于表1，并在更详细地讨论了讨论。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>参数</td><td colspan="3">例如标准化</td></tr><tr><td></td><td> 菜豆 </td><td> 龙lycopersicum </td><td> 拟南芥 </td></tr><tr><td>叶型</td><td>第一片真叶，完全展开，健康</td><td>从底部开始第 2 次和第3 次叶子，4最大的单张拍摄没有用于质外体提取单张顶</td><td>完全展开的莲座叶</td></tr><tr><td>株龄</td><td> 21天</td><td> 7 - 9周</tD&gt; <td> 7周</td></tr><tr><td>一天中的时间</td><td>光周期中间（12:00） </td><td>光周期中间（12:00） </td><td>光周期中间（12:00） </td></tr><tr><td>水化</td><td>所有的植物浇水以及1小时收获前</td><td>所有的植物浇水以及1小时收获前</td><td>所有的植物浇水以及1小时收获前</td></tr><tr><td>光</td><td> 16小时光照，400微摩尔米-2秒-1 </td><td> 16小时光照，400微摩尔米-2秒-1 </td><td>从第2周10小时光照（短天），400微摩尔米-2秒-1 </td></tr><tr><td>湿度</td><td> 70％ </td><td> 70％ </td><td> 70％ </td></tr><tr><td>温度</td><td> 22°C的光，18℃的黑暗</td><td> 22°C的光，18℃的黑暗</td><td> 21°C </td></tr></tbody></table> 表1.标准化帕拉梅例子TER值在AWF提取使用的叶子。 1.生成质外体洗涤液注:在本协议危险materila从病叶inculding病原微生物不应枉然;而正确的处置程序，应该使用。 <ol><li>选择基于叶片大小设备: <ol><li>使用无针注射器潜入小叶子（ 如拟南芥，大豆）。使用真空瓶渗透的叶子是太大，不适合在注射器。用保温瓶方法同时渗透多个叶子。 </li><li>划分的叶子是太大了可用渗透法成用刀片更小的部分。在蒸馏水中彻底冲洗干净切叶的部分在渗透之前删除从受损细胞胞浆散发出的污染物。 </li></ol></li><li>使用60毫升注射器叶浸润: <ol><li>分离从叶植物在使用刀片的叶柄。对于复叶，如西红柿，分离传单的小叶柄。 </li><li>冲洗通过浸没在新鲜切下叶中蒸馏水以除去叶表面污染物。用吸水纸或纸轻轻吸干干燥叶。 </li><li>衡量浸润前的叶重。 </li><li>小心移动和/或叶折入60ml的注射器中，并填充用蒸馏水的注射器（其他渗透流体也可以使用，见讨论）。弹出任何空气注射器内，同时降低柱塞约40亳升位置。 </li><li>覆盖注射器尖端与任一戴手套的手指或一块封口膜，然后，通过向外拉动柱塞至60毫升标记创建所述注射器内的负压。慢慢松开柱塞。 注意:快速释放的压力可能会导致细胞裂解和细胞质污染。 </li><li>拔出注射器尖端和弹出的空气，然后重新插入提示，按在柱塞进一步向下创建的正压适度量。 </li><li>重复步骤1.2.5 - 1.2.6，直到叶子完全渗透，看到的渗透领域的黑暗色彩。 </li><li>取下注射器叶。轻轻但彻底吸干用吸水纸叶去除表面的液体。 </li><li>测量渗透叶的重量。之前和浸润其中近似于渗透体积后计算的重量差。 </li></ol></li><li>叶浸润保温瓶: <ol><li>分离从植物的叶，在使用刀片的叶柄。 </li><li>冲洗通过浸没在新鲜切下叶中蒸馏水以除去叶表面污染物。用吸水纸轻轻吸干干燥叶。 </li><li>衡量浸润前的叶重。 </li><li>放置叶（或叶片如果浸润多个叶）在250毫升或更大的侧臂烧瓶共ntaining蒸馏水（其他渗透流体也可以使用，见讨论 ）。完全覆盖叶片与液体。 </li><li>施加真空烧瓶中。轻轻搅动，以从叶释放气泡。小心缓慢地释放真空。 注意:快速释放的真空可能会导致细胞裂解和细胞质污染。 </li><li>重复步骤1.3.5直到叶子完全渗透，所看到的渗透区的变暗颜色。 </li><li>取下烧瓶叶。轻轻但彻底吸干用吸水纸叶去除表面的液体。 </li><li>测量渗透叶的重量。之前和浸润其中近似于渗透体积后计算的重量差。 </li></ol></li><li>恢复AWF通过离心: <ol><li>将叶上的一块4英寸（10厘米）宽的封口膜。使用5ml的枪头（或类似的大小的对象），卷起的封口膜中的叶。 </li><li>插入卷起叶与枪头到20ml的注射器中。位置朝上任意裁剪叶边。将注射器放入干净50毫升聚乙烯或聚碳酸酯管。 </li><li>离心管内的注射器10分钟，在1000×g离心以吊桶式转子在4℃下。 注:以下离心叶片目视检查应表明，大多数的渗透液已被开除。深绿色的补丁指示需要额外离心时间。 </li><li>吸取回收的AWF到新的1.5mL管。 注:AWF的体积应约匹配的渗透量为叶;如果卷是小于额外离心时间或离心速度是必需的。 </li><li>离心AWF样品在15000×g离心5分钟，以除去任何细胞或颗粒物质。可替代地，通过AWF通过0.22微米的过滤器，以字符型已经细胞和颗粒物质。 </li><li>吸取上清到一个新的试管中。继续冰样品，直到存储。 </li><li>存储AWF样品在-80℃。 </li></ol></li></ol> 2，测定的细胞质污染<ol><li>置于冰上的AWF样本，以尽量减少酶反应（ 如水解），并进行化验后，立即离心步骤完成。 注:对于代谢物测定，样品可以立即提取后冷冻并储存在-80℃直至使用。 </li><li>对于一个标准的苹果酸脱氢酶（MDH）分析方案看15;否则使用市售的苹果酸脱氢酶检测试剂盒。 </li><li>对于标准葡萄糖-6-磷酸脱氢酶（G6PDH）分析方案看16;否则使用市售的G6PDH检测试剂盒。 </li><li>对于细胞质代谢产物如葡萄糖-6-磷酸（G-6-P），使用GC-MS进行定量，在步骤5中描述的</stroNG&gt;。否则，使用市售的套件G-6-P的直接检测。 </li></ol>质外体稀释因子3.计算<ol><li>准备两卷步骤1.2.4或1.3.4（例如，蒸馏水）上面所用的渗透液。添加靛蓝胭脂红粉末以1溶液至50μM的终浓度，没有添加到其他。 </li><li>测量吸光度在200微升用酶标仪的靛蓝胭脂红溶液浸润的610纳米（OD 610）。减去OD 610 200微升的渗透解决方案，而靛蓝胭脂红更正此值。替代这次修正值作为OD 610infiltrate到下面的等式。 </li><li>执行至少三次重复的叶的渗透与上述（ 步骤1.2或1.3）与两个浸润溶液（有和没有靛蓝胭脂红）。 </li><li>后浸润，R樱雪的叶子在蒸馏水中，以除去任何剩余的靛蓝胭脂红本的外表面上。 </li><li>用离心继续为上述（ 步骤1.4）。 </li><li>确定平均OD 610 200微升靛蓝胭脂红AWF提取的。更正此值减去200微升的靛蓝胭脂红免费AWF平均OD 610值。替代这次修正值作为OD 610AWF到下面的等式。 </li><li>从校正的吸光度值作为计算稀释因子（ 即，倍稀释）: 质外体稀释倍数= OD 610infiltrate /（OD 610infiltrate - OD 610AWF） </li><li>从AWF乘以稀释因子乘法浓度或活性值来估计所述浓度/活性在质外体的流体在植物 。 </li></ol> AWF 4.要集中力量全面通过冷冻干燥<ol><li>打开冷冻干燥器和一llow它在工作温度和压力下，以稳定。 </li><li>池的新鲜或存储AWF样品所需量。 </li><li>均匀分布的样本中2毫升离心管中。 </li><li>确定重建容积: 重构体积=前冷冻干燥/质外体稀释系数体积</li><li>用盖子封闭，冷冻在液氮中的管中。 </li><li>转移冷冻管到一个或多个激冷冷冻干燥容器。在传输管道，更换一个已经刺穿了尖锐的物体，使气体交换的盖子。 </li><li>附加容器到冷冻干燥器并完全冻干的样品。 </li><li>从机器中取出样品，并通过加入蒸馏水计算量重构AWF。 </li><li>更换未穿孔盖子和储存样品在-80℃下。 </li></ol> 5.代谢物分析的气相色谱质谱法17 &lt;OL&gt; <li>吸移管将200μlAWF到1.5ml试管</li><li>加入700微升甲醇中含10微克/毫升核糖醇作为内标。 </li><li>热，在70℃下进行10分钟。 </li><li>离心机在11000×g下10分钟。 </li><li>转移700微升上清液至新的1.5毫升管。 </li><li>添加375微升冷氯仿和涡持续10秒。 </li><li>加入500μl卫生署2 O，涡旋10秒。 </li><li>离心15分钟，在2200×g下。 </li><li>转移150微升上层水层的成新的1.5毫升管，然后完全干燥的试样在真空中浓缩无热量。 </li><li>溶解于30μl吡啶含20毫克/毫升的甲氧基胺盐酸盐的样品。 </li><li>孵育在70℃，同时摇晃剧烈搅拌2小时。 </li><li>添加50微升MSTFA（N-甲基-N-（三甲基硅基）三氟乙酰胺）的。 </li><li>孵育在37℃，同时摇晃30分钟。 </li><li>转移样品GC-MS补偿atible玻璃小瓶。 </li><li>执行样本的GC-MS分析。参见李赛克等人，2009年17，上GC-MS设备安装和性能细节。 </li></ol>

<ol>
	<li>Felle, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoplastic+pH+during+low-oxygen+stress+in+barley.">Apoplastic pH during low-oxygen stress in barley.</a> Annals of Botany. 98 (5), 1085-1093 (2006).</li><li>Schaarschmidt, S., Kopka, J., Ludwig-M&#252;ller, J., Hause, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+arbuscular+mycorrhization+by+apoplastic+invertases:+enhanced+invertase+activity+in+the+leaf+apoplast+affects+the+symbiotic+interaction.">Regulation of arbuscular mycorrhization by apoplastic invertases: enhanced invertase activity in the leaf apoplast affects the symbiotic interaction.</a> The Plant Journal. 51 (3), 390-405 (2007).</li><li>Outlaw, W. H., De Vlieghere-He, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transpiration+rate.+An+important+factor+controlling+the+sucrose+content+of+the+guard+cell+apoplast+of+broad+bean.">Transpiration rate. An important factor controlling the sucrose content of the guard cell apoplast of broad bean.</a> Plant Physiology. 126 (4), 1716-1724 (2001).</li><li>Rico, A., Jones, R., Preston, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+to+the+plant+apoplast+by+plant+pathogenic+bacteria.">Adaptation to the plant apoplast by plant pathogenic bacteria.</a> Plant Pathogenic Bacteria. , 63-89 (2009).</li><li>Rico, A., Preston, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+syringae+pv.+tomato+DC3000+uses+constitutive+and+apoplast-induced+nutrient+assimilation+pathways+to+catabolize+nutrients+that+are+abundant+in+the+tomato+apoplast.">Pseudomonas syringae pv. tomato DC3000 uses constitutive and apoplast-induced nutrient assimilation pathways to catabolize nutrients that are abundant in the tomato apoplast.</a> Molecular Plant-Microbe Interactions. 21 (2), 269-282 (2008).</li><li>Solomon, P., Tan, K., Oliver, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nutrient+supply+of+pathogenic+fungi;+a+fertile+field+for+study.">The nutrient supply of pathogenic fungi; a fertile field for study.</a> Molecular Plant Pathology. 4, 203-210 (2003).</li><li>Brunner, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immunity+in+plants+and+animals:+emerging+parallels+between+the+recognition+of+general+elicitors+and+pathogen-associated+molecular+patterns.">Innate immunity in plants and animals: emerging parallels between the recognition of general elicitors and pathogen-associated molecular patterns.</a> Current Opinion in Plant Biology. 5 (4), 318-324 (2002).</li><li>Fontaniella, B., Vicente, C., Armas, R., Legaz, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+leaf+scald+(Xanthomonas+albilineans)+on+polyamine+and+phenolic+acid+metabolism+of+two+sugarcane+cultivars.">Effect of leaf scald (Xanthomonas albilineans) on polyamine and phenolic acid metabolism of two sugarcane cultivars.</a> European Journal of Plant Pathology. 119 (4), 401-409 (2007).</li><li>Mill&#225;n, A. F., Morales, F., Abad&#237;a, A., Abad&#237;a, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+iron+deficiency+on+the+composition+of+the+leaf+apoplastic+fluid+and+xylem+sap+in+sugar+beet.+Implications+for+iron+and+carbon+transport.">Effects of iron deficiency on the composition of the leaf apoplastic fluid and xylem sap in sugar beet. Implications for iron and carbon transport.</a> Plant Physiology. 124 (2), 873-884 (2000).</li><li>Dragi&#353;i&#263; Maksimovi&#263;, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filter+strip+as+a+method+of+choice+for+apoplastic+fluid+extraction+from+maize+roots.">Filter strip as a method of choice for apoplastic fluid extraction from maize roots.</a> Plant Science. 223 (1), 49-58 (2014).</li><li>Baker, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+internal+standard+technique+for+improved+quantitative+analysis+of+apoplastic+metabolites+in+tomato+leaves.">An internal standard technique for improved quantitative analysis of apoplastic metabolites in tomato leaves.</a> Physiological and Molecular Plant Pathology. 78, 31-37 (2012).</li><li>Nouchi, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overcoming+the+difficulties+in+collecting+apoplastic+fluid+from+rice+leaves+by+the+infiltration-centrifugation+method.">Overcoming the difficulties in collecting apoplastic fluid from rice leaves by the infiltration-centrifugation method.</a> Plant &amp; Cell Physiology. 53 (9), 1659-1668 (2012).</li><li>Boudart, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+wall+proteins+in+apoplastic+fluids+of+Arabidopsis+thaliana+rosettes:+identification+by+mass+spectrometry+and+bioinformatics.">Cell wall proteins in apoplastic fluids of Arabidopsis thaliana rosettes: identification by mass spectrometry and bioinformatics.</a> Proteomics. 5 (1), 212-221 (2005).</li><li>Lohaus, G., Pennewiss, K., Sattelmacher, B., Hussmann, M., Hermann Muehling, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+the+infiltration-centrifugation+technique+appropriate+for+the+isolation+of+apoplastic+fluid?+A+critical+evaluation+with+different+plant+species.">Is the infiltration-centrifugation technique appropriate for the isolation of apoplastic fluid? A critical evaluation with different plant species.</a> Physiologia Plantarum. 111 (4), 457-465 (2001).</li><li>Bergmeyer, H. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malate+Dehydrogenase.">Malate Dehydrogenase.</a> Methods of Enzymatic Analysis. 2, 614 (1974).</li><li>Lohr, G. W., Waller, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose-6-phosphate+Dehydrogenase.">Glucose-6-phosphate Dehydrogenase.</a> Methods of Enzymatic Analysis. 2, 636-643 (1974).</li><li>Lisec, J., Schauer, N., Kopka, J., Willmitzer, L., Fernie, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas+chromatography+mass+spectrometry-based+metabolite+profiling+in+plants.">Gas chromatography mass spectrometry-based metabolite profiling in plants.</a> Nature Protocols. 1 (1), 387-396 (2006).</li><li>Li, Z. C., McClure, J. W., Hagerman, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+and+bound+apoplastic+activity+for+peroxidase,+beta-d-glucosidase,+malate+dehydrogenase,+and+nonspecific+arylesterase,+in+barley+(Hordeum+vulgare+L.)+and+oat+(Avena+sativa+L.)+primary+leaves.">Soluble and bound apoplastic activity for peroxidase, beta-d-glucosidase, malate dehydrogenase, and nonspecific arylesterase, in barley (Hordeum vulgare L.) and oat (Avena sativa L.) primary leaves.</a> Plant Physiology. 90 (1), 185-190 (1989).</li><li>Dani, V., Simon, W. J., Duranti, M., Croy, R. R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+tobacco+leaf+apoplast+proteome+in+response+to+salt+stress.">Changes in the tobacco leaf apoplast proteome in response to salt stress.</a> Proteomics. 5 (3), 737-745 (2005).</li><li>Shabab, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fungal+effector+protein+AVR2+targets+diversifying+defense-related+Cys+proteases+of+tomato.">Fungal effector protein AVR2 targets diversifying defense-related Cys proteases of tomato.</a> Plant Cell. 20 (4), 1169-1183 (2008).</li><li>Cosgrove, D. J., Cleland, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solutes+in+the+free+space+of+growing+stem+tissues.">Solutes in the free space of growing stem tissues.</a> Plant Physiology. 72 (2), 326-331 (1983).</li><li>Husted, S., Schjoerring, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoplastic+pH+and+ammonium+concentration+in+leaves+of+Brassica+napus.">Apoplastic pH and ammonium concentration in leaves of Brassica napus.</a> L. Plant Physiology. 109 (4), 1453-1460 (1995).</li><li>Kettle, A. J., Clark, B. M., Winterbourn, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+converts+indigo+carmine+to+isatin+sulfonic+acid.">Superoxide converts indigo carmine to isatin sulfonic acid.</a> The Journal of Biological Chemistry. 279 (18), 18521-18525 (2004).</li><li>Bournonville, C. F. G., D&#237;az-Ricci, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+determination+of+superoxide+in+plant+leaves+using+a+modified+NBT+staining+method.">Quantitative determination of superoxide in plant leaves using a modified NBT staining method.</a> Phytochemical Analysis. 22 (3), 268-271 (2011).</li><li>Speer, M., Kaiser, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+relations+of+symplastic+and+apoplastic+space+in+leaves+from+Spinacia+oleracea+L.+and+Pisum+sativum+L.+under+salinity.">Ion relations of symplastic and apoplastic space in leaves from Spinacia oleracea L. and Pisum sativum L. under salinity.</a> Plant Physiology. 97 (3), 990-997 (1991).</li></ol>

No conflicts of interest declared.

优化的植物组织源执行质外体提取时，生物技术和变化可以很大，因而高度标准化的工作流程是有益的，以增加整个实验的连续性（ 图1）。重要的是，植物组织的来源必须标准化，包括叶型，叶年龄，生长/环境条件和天（ 表1）的时间。存在于与不同叶子渗入和AWF随后通过离心回收的容易性大的差异;这些差异与相关的气孔数量，光圈大小和叶肉阻力12,14。即使P.不同品种间寻常还有在易用性和AWF提取过程的产量差异较大;对于这里使用的Tendergreen各种例如叶子更适合使用这种方法比加拿大奇迹品种AWF提取。内夏枯草第一片真叶是最大的，也最容易渗透，使他们为质外体提取的不二之选。质外体的空气和水的体积已被证明随叶龄在几个物种导致AWF萃取12,14的差异。在P.寻常 ，老叶变得基本上更难渗透和产生离心后少AWF;因此，当他们达到充分扩张叶子收获。叶这是很难渗透随后要求较高的离心速度恢复AWF。每个人都应该因此，在决定一个组织来源大规模AWF拔牙前，仔细甄别几种不同的叶类型和品种。 植物的生长条件，还必须标准化尽可能的实验的范围内。使用生长柜，优选，因为它们允许一致的湿度，温度和照明要保持强度吨军团。叶的收获应该总是发生在一天的同一时间，因为代谢物， 酶等的浓度变化在整个昼夜循环14。最后，为了确保该植物叶都具有类似的膨压，将植物应尽快（〜1小时）在收获前浇水。 叶浸润和离心优化所述AWF由于局部细胞裂解不可取细胞质污染将导致如果由所述细胞所遇到的机械应力是在渗透期间或离心步骤太高。因此，对于给定叶子类型的程序应该是最大化产量和最小化AWF细胞质污染之间的优化权衡。在所有情况下，应该使用最低离心速度，AWF可以回收，以避免叶细胞的可能的机械破坏。在centrifu优化gation速度应通过监测回收量和质外体污染在一定范围的离心速度凭经验确定每个叶型。已经观察到，标记酶的活动，如MDH，G6PDH和葡萄糖磷酸异构酶，保持低电平直到阈离心力超过，上述这些活动迅速增加，这可能是由于细胞质泄漏9,12,14。在离心分离步骤，用于支持使用封口膜，正如贝克等人的 2012 11，可以提高AWF提取的功效，并且可以最大限度地减少机械损伤引起的过度的折叠和压缩叶片。此外，使用封口膜离心后改善了叶片的可视化时，应该检查叶为AWF提取的损伤和完整性。 Nouchi 12描述AWF复苏的优化，从割稻叶的部分，这是ðifficult渗透和需要更高的速度离心收集，因为他们的小气孔开口AWF。改善了稻叶面上的润湿性，或者通过预浸泡的叶子在蒸馏水或添加表面活性剂以渗透流体的，促进了渗透过程。较高的离心速度（6000 XG），而监测质外体污染12也被使用。当使用切叶切片始终存在的风险是细胞质污染甚至会与伤口部位广泛洗涤更为普遍;切叶因此应该只在必要时使用。 对于许多研究蒸馏水被用作渗透流体11。然而，化合物可以被添加到渗透流体，例如盐或缓冲剂，以改善某些质外体化合物，尤其是蛋白质13的提取。 Lohaus 14评估的离子和渗透强度的效果Øn中回收AWF的组合物，并发现他们可以忽略不计。 pH变化的渗透介质的，但是，可能会影响到AWF组合物14。 适当的处理所提取的质外体流体和储存是重要的。 AWF已被证明含有丰富蛋白酶和其他酶的13,20，以及挥发性有机化合物。因此，为了减少改变AWF的恢复后，建议将样本保持在冰上或以其他方式储存在-80℃的组合物。此外，AWF的酶测定，应尽快进行萃取后，以限制酶失活是由于蛋白酶解，长时间稀释或冷冻解冻。 浸润的方法，稀释了质外体流体，这是常常需要确定该稀释的程度。离心步骤也可以稀释AWF与细胞内的隔间的水。稀释因子​​是需要当测量是在AWF做编辑估计在体内质外体化学品的浓度。的稀释因子，也需要集中AWF回全强度时，它被用作一个质外体模仿生长培养基为微生物-从而匹配体内代谢物浓度尽可能准确。几种变化在步骤4，通过测量加入到渗透液中的标志化合物的稀释液确定AWF稀释因子中记载的方法存在。对于所有方法AWF稀释计算假设渗透液没有明显吸收或AWF恢复过程中被稀释了叶细胞。这种假设先前已确认为渗透步骤14，但未经证实的离心步骤。标志化合物还必须不被吸收，转运或质外体的同时修改。靛蓝胭脂红是使用最广泛的和使用的染料的全面测试对于AWF稀释计算。靛蓝胭脂红显示低的吸收，以阳离子交换树脂和分离的细胞壁和被证明是适合AWF计算在欧洲油菜，豌豆，茄lycopersicum和大豆 11,21,22。然而，在一些叶类型， 例如，水稻，黄瓜，靛蓝胭脂红出现不能得到充分渗透，这将导致该AWF稀释因子12,21的低估后回收。靛蓝胭脂红的复苏的缺乏可能是由于它的敏感性裂解成靛红磺酸盐由超氧23，这是公知的，在质外体，以产生，尤其是在压力下24。裂解靛蓝胭脂红的成靛红磺酸盐将导致吸光度的损失在610 nm和增加在245纳米。是否该反应发生在质外体的明显程度，或是否该反应可以通过加入过氧化物清除剂，以infiltrat的抑制离子流体还没有调查最新的。蓝色葡聚糖已经用来代替靛蓝胭脂红用于水稻AWF稀释因子的量化叶12，尽管其在AWF稳定性和恢复尚未见报道。可替代地，可以用来代替染料和放射性的或测量并用于计算稀释因子以类似方式11,14,25浓度的降低放射性标记的化合物，例如[14 C]山梨糖醇或其他可量化的内部标准。 该技术的局限性几个注意事项的渗透离心AWF隔离法存在。首先，质外体的浸润过程中稀释可引发从植物的响应。如果周围的叶细胞检测减弱浓度为质外体流体的某些组件，它们可以通过进一步排泄的代谢物，扭曲代谢物浓度的解释作出响应。在此问题的评估，观察到既不浸润和离心也不在渗透流体的离子强度适中的差异之间的时间影响所提取AWF 14,22的组成。因此，从质外体稀释被认为造成任何文物是微乎其微。 第二个潜在的缺点是，AWF通过离心洗脱不捕获所有存在的质外体有以下几个原因的分子。有些化合物，特别是阳离子和蛋白质可与带负电的细胞壁被紧密地相关联，并且不与AWF 13洗脱。其它分子如蛋白质可能过大，以有效地洗脱出的质外体中的用于14的离心速度。活性氧是一类重要的，在质外体产生的化合物，但由于这些化合物的短命的性质，以及它们的生产未指定的要求，他们的公关esence不能很好地AWF提取抓获。目前还不知道如何AWF准确代表质外体流体的体内组合物，这可能物种之间变化。 几个实验存在确定细胞质污染，虽然没有被普遍接受。的主要细胞质酶测定法（ 例如，G6PDH，己糖磷酸异构酶，MDH）的特点，是容易进行的益处，但不能与细胞质泄漏14,18很好地相关。细胞质的代谢产物（ 如己糖磷酸盐，叶绿素）的评估或许更指示，但并没有很好的建立11。然而，任一类型的测定可用于样品之间的质外体污染的相对定量是有用的。理想情况下，多个独立的测量应用于验证AWF样品的完整性。 虽然承认其局限性，渗透，centrifugati在这里描述的技术仍然在质外体蛋白质，初级和次级代谢产物和无机离子的研究一种简单而强大的技术。

<html>植物质外体是细胞间隙包围的植物细胞。这是一个动态的环境中，许多代谢和运输过程中发生。质外体的主要结构部件是细胞壁，它被组装并通过位于质外体中的酶，结构蛋白和代谢物改性。在健康的植物细胞的质外体一般保持在酸性状态，允许氨基酸，糖和其它营养物可从质外体用H +同向转运1导入到细胞质中。期间蔗糖运输，从通过质外体和韧皮部进入脉管光合来源蔗糖移动;蔗糖然后通过由质外体间转化在水槽2的器官切割蔗糖保持了渗透势运输。糖和其他营养物质，也可以向上输送，并通过蒸腾流3积聚在气孔腔。 "&gt;质外体也代表了环境的利基，许多病原体建立自己的寄生生活。细菌性植物病原体有乘高密度的质外体，它们通过自然开口，如气孔或通过伤口进入4的能力，氨基酸的浓度酸和其它含氮化合物在番茄质外体已被证明是足够的，以支持的细菌和真菌病原体5,6的营养需求。朝微生物病原体主要防御反应也发生在质外体，即产生活性氧物种由胞外过氧化物酶以及氧化酶和细胞壁的加强，通过交联和胼胝质沉积7所述的植物细胞壁中含有丰富的次级代谢产物具有抗微生物活性，这些次级代谢产物的生化性质物种8之间变化。 如上述提及的结果编等工序，叶质外体液中含有的各种蛋白质，糖类，有机酸，氨基酸，次生代谢物，金属和其它阳离子（ 例如，镁离子 ， 钾离子 ， 钠离子 ， 钙离子，铁2/3的+）。溶质浓度芽的质外体是由质外体和木质部，韧皮部和细胞质9之间发生的运输过程的平衡很大程度上控制。然而，代谢反应和微生物的生长也消耗或产生质外体溶质。质外体的组合物是已知的植物物种和基因型之间响应于变化的环境条件包括光，营养和生物和非生物胁迫9不同。通过研究质外体的组成和它如何改变，包括例如氧化还原和渗透势，pH值，营养物/代谢物的可用性和酶活性的特性，可以得到新的见解植物如何重新有反应到他们的环境。理解或表征发生在质外体溶液中的分子的变化是复杂的，因为它是一个空间结构和动态隔室，其中代谢物和/或离子可以是挥发性的，短暂的或者与细胞壁和细胞膜相关联。此外，需要不同的分析技术来覆盖不同的化学类型的范围。 研究可溶性质外体的组合物中，流体通常需要从组织中提取。有几种方法提取质外体流体从各种组织，包括新提出的过滤带10的方法，但最悠久的提取方法叶子是渗透离心。这种技术已被评估之前11-13和Lohaus 等人，2001年14提供了许多方法的技术参数进行彻底的检查。所暗示的名称，渗透-centrifugation技术是基本上涉及到更换质外体空间的空气与水的渗透液，该混合与天然质外体液，接着通过所述叶片的温和离心回收的渗透/质外体的流体混合物的两步法。作为回收的流体稀释，并且不包含存在于质外体的所有化合物（见下文），该流体通常被称为质外体洗涤液（AWF），或有时间冲洗液，而不是质外体的流体。浸润离心技术是很容易可扩展的，允许将要生成，并进行了广泛的下游的生化和分析技术（足够的体积单或汇集AWF样品如蛋白质电泳，酶的活性测定，NMR许多类型色谱和质量法）。叶AWF也为质外体模仿生长培养基的研究植物微生物殖民机智的互动有用ħ他们的环境5。 在下面的协议，我们将介绍如何使用菜豆品种进行渗透离心技术。 Tendergreen叶，并提供下游分析的一些例子，重点放在代谢。重要的是，方法评估AWF质量沿着与建议提供给优化不同叶子类型的过程。

<table border="0" cellpadding="0" cellspacing="0" width="906">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Name of Material/ Equipment</td>
			<td style="width:291px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Eppendorf Microcentrifuge Tubes</td>
			<td style="width:291px;">Eppendorf</td>
			<td style="width:139px;">22364111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">razor blade</td>
			<td style="width:291px;">Fisher</td>
			<td>12-640&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">60 ml syringe</td>
			<td style="width:291px;">Becton Dickinson</td>
			<td style="width:139px;">300865</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">20 ml syringe</td>
			<td style="width:291px;">Becton Dickinson</td>
			<td style="width:139px;">300613</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">4 inch parafilm</td>
			<td style="width:291px;">Bemis</td>
			<td style="width:139px;">PM-996</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">side arm flask</td>
			<td>SciLabware</td>
			<td>12972831</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">vacuum source</td>
			<td style="width:291px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">5 ml pipette tips</td>
			<td style="width:291px;">Fisher</td>
			<td>50-813-28&#160;</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:309px;">centrifuge</td>
			<td style="width:291px;">Beckman Coulter</td>
			<td>392932</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Swinging bucket rotor</td>
			<td style="width:291px;">Beckman Coulter</td>
			<td>369702</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">indigo carmine</td>
			<td style="width:291px;">Sigma</td>
			<td style="width:139px;">I8130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">microplate reader</td>
			<td style="width:291px;">Tecan</td>
			<td style="width:139px;">Infinite 200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">96 well plates</td>
			<td style="width:291px;">Becton Dickinson</td>
			<td style="width:139px;">353072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">freeze dryer</td>
			<td style="width:291px;">SciQuip</td>
			<td style="width:139px;">Christ Alpha 2-4 LD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">microcentrifuge</td>
			<td style="width:291px;">biorad</td>
			<td>166-0612EDU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">oxaloacetic acid</td>
			<td style="width:291px;">Sigma</td>
			<td>O4126</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">D-glucose-6-phosphate</td>
			<td style="width:291px;">Sigma</td>
			<td>G7250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">NADH</td>
			<td style="width:291px;">Roche</td>
			<td>10128023001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">MDH assay kit</td>
			<td style="width:291px;">Biovision</td>
			<td>K654-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">G6PDH assay kit</td>
			<td style="width:291px;">Sigma</td>
			<td>MAK015-1KT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">G-6-P assay kit</td>
			<td style="width:291px;">Biovision</td>
			<td>K657-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">ribitol</td>
			<td style="width:291px;">Sigma</td>
			<td style="width:139px;">A5502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">methanol</td>
			<td style="width:291px;">Sigma</td>
			<td style="width:139px;">650471</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">chloroform</td>
			<td style="width:291px;">Sigma</td>
			<td style="width:139px;">472476</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">vacuum concentrator</td>
			<td style="width:291px;">Thermor Scientific</td>
			<td>SC250EXP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">methoxyamine hydrochloride</td>
			<td style="width:291px;">Sigma</td>
			<td style="width:139px;">226904</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-Methyl-N-(trimethylsilyl) trifluoroacetamide</td>
			<td style="width:291px;">Sigma</td>
			<td style="width:139px;">394866</td>
			<td></td>
		</tr>
	</tbody>
</table>

the infiltration-centrifugation technique for extraction of apoplastic fluid from plant leaves using phaseolus vulgaris as an example

质外体是一种独特的细胞外隔室中的植物组织即位于质膜外侧，并且包括细胞壁。叶的植物质外体隔室是几个重要的生物学过程，包括细胞壁的形成，细胞的营养物和水的吸收和出口，植物内生菌的相互作用和防御反应对病原体的部位。渗入离心法是公认的健壮技术关于各种植物品种的可溶性质外体组合物的分析。用这种方法获得的流体通常被称为质外体洗涤液（AWF）。以下协议描述为AWF提取菜豆 （芸豆）品种优化真空渗透和离心法。 Tendergreen叶。此方法和协议，用于其它植物物种的优化的局限性进行了讨论。回收AWF可以在宽的范围内的下游实验技术可用于TS寻求到质外体的组合物的特征，它响应于植物物种和基因型，植物发育和环境条件如何变化，或者确定微生物在质外体流体如何生长，并改变它的组合物反应。

对健康3周老P.进行了AWF提取典型结果寻常的简历。 Tendergreen 叶，使用蒸馏水作为渗透液示于表2。杨P.寻常的叶子是适合于渗透，并在这里使用的离心速度（1,000×g离心）除去大部分浸润流体通过离心可直接被视为叶片恢复为原来的绿色。P.寻常叶鲜重平均为1.1 XG渗透之前，并取得了0.5毫升每克鲜重AWF的。所述AWF的蛋白质浓度测定为0.18±0.08毫克/使用标准Bradford蛋白测定毫升。这些叶子，通过测定靛蓝胭脂红稀释计算稀释因子，为2.3±0.3倍。上述值可以物种和先导实验之间变化基本上是必需的，以确定AWF每克叶鲜我们屈服飞行，以及AWF蛋白质浓度和稀释因子可预期对于给定的叶型。 破坏细胞的完整性可以在渗透步骤中的离心分离步骤，如果离心力过大发生二者中，如果压力变化过于急剧，和。因此，有必要以测定AWF样品细胞质污染的标记;理想情况下，多个独立的测定法进行的。在这里，从三个可能测定的结果列于表2中 。在P的良好实施萃取寻常 AWF，G6PDH检测不到由标准的酶测定。这是在哪里G6PDH活性仅高于阈值离心力12成为检测的其他报告一致。与此相反，苹果酸脱氢酶活性（2.5单位/毫升）的基线水平是可检测的在各第使用标准的代谢测定寻常 AWF样品。增加了对离心CE以上1000 xg离心阈值导致增加的MDH活性（结果未显示）。来自其他物种的质外体是已知含有内源MDH活性18，在这里检测出的量被认为是真正的质外体活性和显著增加到高于该基准电平是指示污染。这被认为是主要的细胞质，如己糖-6-磷酸的代谢物，也可以用来评估AWF的污染。这里，GC-MS分析检测到的G-6-P的零或痕量水平AWF萃取。与此相反，在切割玉米根段，G-6-P的被AWF提取在所有速度下离心分离之后检测并以更高的速度增加浓度，表明细胞质污染10。 由P的GC-MS分析的代谢物寻常叶AWF通常产生40 - 60可识别的代谢物和大致相等数目的不可识别的化合物（ 图2）。或加尼奇酸，单糖和氨基酸代表了大部分的可识别的代谢物，但是，次级植物代谢物也被检测并从AWF 11定量。从这些不同的分子类几个例子峰标记为在图2中进一步下游的分析技术都适用于这些AWF样品来量化各种分子，例如:ICP-MS，NMR，HPLC，原子吸收光谱法，蛋白质质谱分析。 质外体的蛋白质成分也被表示在AWF， 如图3所示，在考马斯亮蓝染色的SDS-PAGE凝胶，其中的其他角色，质外体酶负责细胞壁的合成和创建胞反应的氧种。 AWF的来自不同物种的蛋白质组学研究已经确定几十个别蛋白质和表明的质外体中的蛋白质成分来响应ENVIROnmental压力13,19。理想地AWF提取应不受污染细胞质蛋白如Rubisco的;然而，在实践中，这是很难实现的。一的Rubisco蛋白带的〜53 kDa的SDS-PAGE后的存在下提供了进一步的定性分析为AW​​F样品的完整性。例如，在泳道2装入图3中的样品中含有较大量的Rubisco污染该第1道的。 <table border="0" cellpadding="0" cellspacing="0" class="jove_content" fo:keep-together.within-page="always"><tbody><tr><td></td><td>菜豆AWF </td></tr><tr><td> AWF量（毫升克-1叶FW） </td><td> 0.49±0.09 </td></tr><tr><td>蛋白浓度。 （毫克毫升-1） </td><td> 0.18±0.08 </td></tr><tr><td>稀释倍数</td><td> 2.3±0.3 </td></tr><tr><td> G6PDH活性（U毫升-1） </td><td&gt;无检测</td></tr><tr><td>葡萄糖-6-P（毫克毫升-1） </td><td>没有检测</td></tr><tr><td> MDH活性（U毫升-1） </td><td> 2.5±0.9 </td></tr></tbody></table> 表2.典型结果从P. AWF提取寻常的简历。 Tendergreen叶。 <img alt="图1" fo:content-width="6in" src="/files/ftp_upload/52113/52113fig1highres.jpg" /> 图1.标准质外体提取的工作流程。中的数字代表在该协议的步骤。 <img alt="图2" src="/files/ftp_upload/52113/52113fig2highres.jpg" /> 图2.一个例子P的GC-MS色谱图。 寻常品种。 。Tendergreen AWF编号的示例的代谢产物的峰是:1-丙二酸盐，2-磷酸盐，3-琥珀酸酯，4-未知，5-苹果酸盐，6天冬酰胺，7-核糖醇（内部ST昂达尔），8 - 柠檬酸盐，9-葡萄糖，10肌醇，11-咖啡酸，12-蔗糖。 <img alt="图3" fo:content-width="3in" src="/files/ftp_upload/52113/52113fig3highres.jpg" width="250" /> 图3.实例的SDS-PAGE考马斯染色的P的凝胶。 柏 AWF叶提取物。这种凝胶提供两个AWF提取的蛋白质成分是不同的细胞质污染量通过丰富质体酶的Rubisco之间的比较。以下AWF提取两个样品进行丙酮蛋白质沉淀在冷丙酮的4倍过量（体积/体积），并在十分之一原来的体积resolubilized水。泳道1和2含40微克蛋白质。对应于所述的Rubisco大链带（〜53 kDa）的用箭头表示。 Mr为:蛋白质分子量标记物。

Watch this Scientific Journal Video about The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example at JoVE.com

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 ...

the-infiltration-centrifugation-technique-for-extraction-apoplastic

Research

JoVE Journal

Biology

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

36.7K 次观看.  University of Oxford. This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

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

Testing the Physiological Barriers to Viral Transmission in Aphids Using Microinjection

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When an uninfected aphid feeds on an infected plant it contracts the virus through the plant phloem. Once ingested, the virus must pass from the insect gut to the hemolymph (the insect  blood ) and then must pass through the salivary gland, in order to be transmitted back to a new plant. An aphid may take up different viruses when munching on a plant, however only a small fraction will pass through the gut and salivary gland, the two main barriers for transmission to infect more plants. In the lab, we use physalis plants to study PLRV transmission. In this host, symptoms are characterized by stunting and interveinal chlorosis (yellowing of the leaves between the veins with the veins remaining green). The video that we present demonstrates a method for performing aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut is preventing viral transmission.
The video that we present demonstrates a method for performing Aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut or salivary gland is preventing viral transmission.

Aphids are effective transmitters of plant viruses. Aphid microinjection of virus, the procedure we will show you today, is a technique allowing researchers to inject virus directly into the hemocoel of the aphid, bypassing the gut, one of the 2 major barriers for virus transmission in a circulative manner. The same technique is also used to inject dsRNA for RNAi.

测试使用微量注射蚜虫病毒传播的生理障碍

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When ...

科研

生物学

Testing Nicotine Tolerance in Aphids Using an Artificial Diet Experiment

Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well know to have insecticidal properties. One response of tobacco plants to herbivory, or being gnawed upon by insects, is to increase the production of this neurotoxic alkaloid. Here, we will demonstrate how to set up an experiment to address this question of whether a tobacco-adapted strain of the green peach aphid, Myzus persicae, can tolerate higher levels of nicotine than the a strain of this insect that does not infest tobacco in the field.

In this video and assay is demonstrated that tests the tolerance of nicotine to two types of aphids one that infests tobacco plants in the field and one that does not.

使用人工饲料实验测试蚜虫的尼古丁公差

Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well ...

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse 

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid peptide (A&#946;) and intraneuronal accumulation of hyperphosphorylated tau protein. Because cerebrospinal fluid (CSF) is in direct contact with the extracellular space of the brain, it provides a reflection of the biochemical changes in the brain in response to pathological processes. CSF from AD patients shows a decrease in the 42 amino-acid form of A&#946; (A&#946;42), and increases in total tau and hyperphosphorylated tau, though the mechanisms responsible for these changes are still not fully understood. Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses. Here, we demonstrate a refined cisterna magna puncture technique for CSF sampling from the mouse. This extremely gentle sampling technique allows serial CSF samples to be obtained from the same mouse at 2-3 month intervals which greatly minimizes the confounding effect of between-mouse variability in A&#946; or tau levels, making it possible to detect subtle alterations over time. In combination with A&#946; and tau ELISA, this technique will be useful for studies designed to investigate the relationship between the levels of CSF A&#946;42 and tau, and their metabolism in the brain in AD mouse models. Studies in Tg mice could provide important validation as to the potential of CSF A&#946; or tau levels to be used as biological markers for monitoring disease progression, and to monitor the effect of therapeutic interventions. As the mice can be sacrificed and the brains can be examined for biochemical or histological changes, the mechanisms underlying the CSF changes can be better assessed. These data are likely to be informative for interpretation of human AD CSF changes.

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse

Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses in human patients. Here, we demonstrate a refined cisterna magna puncture technique for serial CSF sampling from the mouse.

一个串行收集脑脊液从池鼠标麦格纳技术

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid ...

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is avoided when the herbivore is able to select other plants2. Antibiosis is the degree to which the plant affects the fitness of the herbivore feeding on it1.Tolerance is the degree to which the plant can withstand or repair damage caused by the herbivore, without compromising the herbivore's growth and reproduction1. The durability of herbivore resistance in an agricultural setting depends to a great extent on the resistance mechanism favored during crop breeding efforts3.
We demonstrate a no-choice experiment designed to estimate the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria spp. Several species of African grasses of the genus Brachiaria are valuable forage and pasture plants in the Neotropics, but they can be severely challenged by several native species of spittlebugs (Hemiptera: Cercopidae)4.To assess their resistance to spittlebugs, plants are vegetatively-propagated by stem cuttings and allowed to grow for approximately one month, allowing the growth of superficial roots on which spittlebugs can feed. At that point, each test plant is individually challenged with six spittlebug eggs near hatching. Infestations are allowed to progress for one month before evaluating plant damage and insect survival. Scoring plant damage provides an estimate of tolerance while scoring insect survival provides an estimate of antibiosis. This protocol has facilitated our plant breeding objective to enhance spittlebug resistance in commercial brachiariagrases5.

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

This video explains mechanisms of host plant resistance to herbivory and demonstrates a no-choice test that estimates the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria spp.

表征草食动物的耐药机制:Spittlebugs臂形 SPP。为例

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is ...

Detection of Histone Modifications in Plant Leaves

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent modifications on the amino-terminal tails of histones H3 and H4 play essential roles1-2. H3 and H4 histone modifications include methylation of lysine and arginine, acetylation of lysine, and phosphorylation of serine residues1-2. These modifications are associated either with gene activation, repression, or a primed state of gene that supports more rapid and robust activation of expression after perception of appropriate signals (microbe-associated molecular patterns, light, hormones, etc.)3-7. 
Here, we present a method for the reliable and sensitive detection of specific chromatin modifications on selected plant genes. The technique is based on the crosslinking of (modified) histones and DNA with formaldehyde8,9, extraction and sonication of chromatin, chromatin immunoprecipitation (ChIP) with modification-specific antibodies9,10, de-crosslinking of histone-DNA complexes, and gene-specific real-time quantitative PCR. The approach has proven useful for detecting specific histone modifications associated with C4 photosynthesis in maize5,11 and systemic immunity in Arabidopsis3.

A reliable and useful approach to detect histone modifications on specific plant genes is described. The approach combines chromatin immunoprecipitation (ChIP) and real-time quantitative PCR. It allows detection of histone modifications on specific genes with a role in diverse physiological processes.

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 ...

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

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

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

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

一个影像的简单方法拟南芥叶浸润成像介质中使用全氟萘烷

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

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. These structures are almost always localized on plant surfaces. Among them are nectaries or glandular trichomes. The secreted compounds are often employed in interactions with the biotic environment, for example as attractants for pollinators or deterrents against herbivores.
Glands that are unique in several aspects can be found in carnivorous plants. In so-called pitcher plants of the genus Nepenthes, bifunctional glands inside the pitfall-trap on the one hand secrete the digestive fluid, including all enzymes necessary for prey digestion, and on the other hand take-up the released nutrients. Thus, these glands represent an ideal, specialized tissue predestinated to study the underlying molecular, biochemical, and physiological mechanisms of protein secretion and nutrient uptake in plants. Moreover, generally the biosynthesis of secondary compounds produced by many plants equipped with glandular structures could be investigated directly in glands.
In order to work on such specialized structures, they need to be isolated efficiently, fast, metabolically active, and without contamination with other tissues. Therefore, a mechanical micropreparation technique was developed and applied for studies on Nepenthes digestion fluid. Here, a protocol is presented that was used to successfully prepare single bifunctional glands from Nepenthes traps, based on a mechanized microsampling platform. The glands could be isolated and directly used further for gene expression analysis by PCR techniques after preparation of RNA.

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Plants glands are specialized structures responsible for the biosynthesis and secretion of many compounds involved in interactions with the biotic environment. To enable studies on their molecular and biochemical features, a mechanical micropreparation technique was established in order to isolate single metabolically active glands, here from the carnivorous plant Nepenthes.

从食虫植物组织分离存活的多细胞腺的，猪笼草</i

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

测量渗透水渗透系数（P<sub&gt; F</sub&gt;）球形细胞:隔离植物原生质体为例。

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to study the functions and evolution of bacterial symbionts in their host. As most symbionts cannot be cultured in vitro, methods to isolate an adequate quantity of bacteria for genome sequencing are very important. In the whitefly Bemisia tabaci, a number of endosymbionts have been identified and are predicted to be of importance in the development and reproduction of the pests through multiple approaches. However, the mechanism underpinning the associations remains largely unknown. The obstacle partially comes from the fact that the endosymbionts in whitefly, mostly restrained in bacteriocytes, are hard to separate from the host cells. Here we report a step-by-step protocol for the identification, extraction and purification of endosymbionts from the whitefly B. tabaci mainly by dissection and filtration. Endosymbiont samples prepared by this method, although still a mixture of different endosymbiont species, are suitable for subsequent genome sequencing and analysis of the possible roles of endosymbionts in B. tabaci. This method may also be used to isolate endosymbionts from other insects.

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Here, we present a protocol to isolate endosymbionts from the whitefly Bemisia tabaci through dissection and filtration. After amplification, the DNA samples are suitable for subsequent sequencing and study of the mutualism between endosymbionts and whitefly.

从白蛉提取内分泌物的方法<em&gt; Bemisia tabaci</em

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to ...

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ecologically-important endpoints, though they are rarely considered in toxicological studies. D. melanogaster is emerging as a toxicology model for the behavioral, neurological, and genetic impacts of toxicants, to name a few. More importantly, species in the genus Drosophila can be utilized as a model system for an integrative framework approach to incorporate emergent properties and answer ecologically-relevant questions in&#160;toxicology research. The aim of this paper is to provide a protocol for exposing species in the genus Drosophila to pollutants to be used as a model system for a range of phenotypic outputs and ecologically-relevant questions. More specifically, this protocol can be used to 1) link multiple biological levels of organization and understand the impact of toxicants on both individual- and population-level fitness; 2) test the impact of toxicants at different stages of developmental exposure; 3) test multigenerational and evolutionary implications of pollutants; and 4) test multiple contaminants and stressors simultaneously.

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

In this paper, we provide a detailed protocol for exposing species in the genus Drosophila to pollutants with the goal of studying the impact of exposure on a range of phenotypic outputs at different developmental stages and for more than one generation.