We thank Wendy Zhang, Austin Tom, Jacquie Janney, Erin Lemley, and Brittany Janney for assistance with plant growth and extractions, Dr. Anatoli Lygin for assistance with chromatographic analyses, and Syngenta Crop Protection for funding.

1.植物材料，生长条件和营养克隆注:三个waterhemp种群进行了调查，本研究:MCR（由McLean县，IL），ACR（从亚当斯县，IL），和WCS（从韦恩县，IL），（表1）。 <ol><li>收集并暂停waterhemp种子0.1克L- -1琼脂:水的溶液在4℃下至少30天以促进种子发芽。注意:有些waterhemp人口都处于休眠状态，但是这一步有助于克服休眠，使种子发芽更均匀。 </li><li>发芽每个waterhemp人口的种子（ 如表1）在12×12含在温室灌封介质厘米盘。 <ol><li>保持在二十二分之二十八℃，昼/夜中的16/8小时光照期12温室的温度条件。 </li><li>补充自然光与光汞卤化物灯温室提供了至少500微摩尔米 -2 </SUP&gt;在植物冠层水平秒 -1光子通量。 </li></ol></li><li>移植出现苗时，他们是2厘米高为80厘米3盆在温室中。 </li><li>移苗进950厘米3盆含3:1:1:盆栽组合的混合物:土:草炭:砂子时，苗4厘米高。加入5克缓释肥料颗粒，以每盆以及与此土壤混合物混合。 </li><li>剪切和删除拍摄含7厘米高的植物三个最年轻的叶子顶端分生组织，消除顶端优势。 注:此步骤可确保每个工厂将生产足够3厘米腋芽克隆。 </li><li>消费5腋生芽（3厘米长）时苗约14厘米高的从每个waterhemp人口（如表1）。去除大部分这些芽腋叶，以减少水分流失经进一步处理，但保留了两个最小的叶子，让移植后的进一步增长。 <br/&gt;注意:小心操作，尽量不拆除的叶子时，损坏的竹笋。标签每个工厂有股份。 </li><li>移植腋生芽到80厘米3盆 （每盆一株幼苗）插入灌封介质，它是用于萌发的相同培养基。灌封介质最初应饱和直到在生长室根形式，然后维持在生长室恒定湿度和地点盆7天以建立根。 注意:植物是在这一点上是非常敏感的，并且不应加以强调。 <ol><li>保增长室条件22分之28℃，昼/夜中的16/8小时光照12下。 </li><li>白炽灯和荧光灯泡的组合可用于提供光生长室，并应提供至少550微摩尔米 -2在植物冠层12秒-1光子通量。 </li></ol></li><li>再次移植到950厘米3罐 （每罐一株幼苗）Containing一个3:1:1:1混合灌封搭配:土:草炭:砂（也包括5克缓释肥料颗粒）时，克隆植物4厘米高，然后传回温室。 </li><li>通过使用上述相同的方法克隆植物的第二时间 （即，重复步骤1.5至1.8，然后继续到步骤2.1）。对于一个典型的时间过程的研究，产生克隆从每个waterhemp人口至少有六个独立的植物，并将其记录为线MCR 1-6 ACR 1-6和1-6 WCS。 <ol><li>为了重复实验和充分地代表每个waterhemp种群内的遗传变异建立至少六个不同"父"无性系。 </li><li>取决于有多少时间点进行分析，产生足够的亚克隆从每个"母"克隆系进行时间过程研究。 </li></ol></li></ol> 2，开展与离体Waterhem一个代谢研究p叶<ol><li>切除第三年轻叶（长度在2到3厘米;包括0.5厘米附叶柄）当克隆waterhemp植物10至12厘米高为除草剂代谢和DT 50分析。 </li><li>切叶叶柄每个waterhemp厂第二次（确保0.3厘米叶柄仍然）在水中，然后将这些切割端进入1.5毫升塑料瓶（每瓶一片叶子）15。 注意:切割下的水的第二时间可以防止形成气泡和堵塞木质部导管。 <ol><li>首先，部分1小时浸泡离体叶片与叶柄切在0.1米的Tris-HCl（pH值为6）预孵育缓冲液（200微升）。 </li><li>其次，移动每个叶含有的孵育缓冲液（200微升）的0.1M的Tris-盐酸（pH值为6）加上150μM的[URL- 14 C〕除草剂一个新的1.5 ml的小瓶中，并孵育在28℃下1小时，以允许除草剂吸收到树叶。 注意:两个14 C-硝磺草酮和<s向上&gt; 14 C-氟嘧磺隆-甲基均在约20毫居里毫摩尔具体活动用在我们的研究中-1。通过木质部蒸腾流扩散和摄取促进除草剂的每片叶子吸收的量。 注意:代谢抑制剂的除草剂代谢的影响可以通过修改步骤2.2.1和2.2.2以上来确定。添加100μM的抑制剂预温育缓冲液中，让孵育2小时，然后转移到培养缓冲液用除草剂和再次添加100μM的抑制剂。例如，马拉硫磷和tetcyclacis抑制植物中15-17某些细胞色素P450活性。 </li><li>洗切叶用离子交换水的浸入部分和切叶转移到四分之一强度Murashige和Skoog（MS）盐溶液（500微升）适用于0小时，5小时，11小时，23小时，或35小时，分别在生长室中的时间过程代谢研究。 </li></ol></li><li>嘉实除去休假使用的聚丙烯管（15毫升）和玻璃杵从在适当的时间点孵育溶液并手动小号研磨在液氮中的每个叶组织。 </li><li>提取放射性标记的除草剂，并且从每个叶用7ml 90％丙酮的用实验室组织匀浆器在与上述相同的聚丙烯管及其​​代谢产物。冲洗组织匀浆用另外7 ml的90％丙酮中，并继续均质化，直至组织被完全粉碎（通常约2分钟）。注:90％丙酮可用于大多数除草剂的从植物组织中提取。然而，如果这种方法是无法提取超过90％吸收的放射性标记化合物，然后90％甲醇或90％乙腈，可被取代的作为萃取溶剂。 </li><li>标签90％丙酮提取物（14 ml的总），并存储在-4℃下16小时，以允许额外的提取发生。 注:在此之后的提取处理的叶片，提取弧度量ioactivity通常为至少98％的处理过的叶子吸收放射性标记化合物。在我们以前的研究12不可萃取的放射活性（结合残基）中切下叶平均只有0.3在所有时间点％，但该量可根据所使用的除草剂和时间点检查而变化。 </li><li>在5000×g离心10分钟，离心的样品和在40℃下用旋转蒸发器浓缩上清液直至0.5 ml的终体积中获得。此卷转移到2.0毫升的塑料管。 </li><li>添加乙腈:水（1:1，体积比）以调节植物的最终体积中提取到1.25 ml和重新离心机以10,000rpm离心10分钟，以除去任何颗粒。 </li><li>测量总放射性（DPM微升-1）通过液体闪烁光谱法（LSS）12从每个样品中取等分试样并正常化的量[14 C标记]注射进行HPLC分析（例如化合物，10,000总DPM /样品/运行），因此y轴将结果作图时要样品中都采用统一。 </li></ol>除草剂代谢的切下的叶片3高效液相色谱分析注:解决共提取到的放射性使用下面的RP-HPLC条件12父除草剂和除草剂的代谢物。 <ol><li>与C 18柱（4.6×250毫米，5微米颗粒尺寸为分析型HPLC）以1ml min的流速进行反相高效液相色谱法-1为大多数非极性除草剂。 注意:C 4或C 12 RP-HPLC柱也可以被用来优化从它的代谢产物母体除草剂的分离。预柱过滤器和/或保护柱也可用于保护和延长RP柱的寿命。 <ol><li>使用下面的流动相为RP-​​HPLC:洗脱液A，在水中的0.1％甲酸（体积/体积），和洗脱液B 100％HPLC级乙腈。 注意:HPLC级甲醇也可以用来作为埃尔为解决更多的极性化合物和代谢物uent B，但要注意保留时间也可能会更改。 注:用于解决硝磺草酮或氟嘧磺隆，甲从极性代谢物在我们的研究12洗脱图:第1步，A:B（4:1，V / V）为A:B（3:2，V / V ）中的线性梯度（12分钟）;步骤2中，A:B（3:2，体积/体积），以A:B（3:7，V / V）中的线性梯度（5分钟）;步骤3，A:B（3:7，V / V），以A:B（1:9，V / V）在2分钟的线性梯度（包括共19分钟）。 注意:渐变和恒溶剂步骤可能需要基于该母材，以便优化分辨率的物理性质来进行调节。 </li><li>遵循与甲上述线性梯度步骤:B（1:9，V / V），以A:B（4:1，体积/体积）中的线性梯度（3分钟）和A:B（4:1，V / v）的至少2分钟等度步骤（）来注入下一个提取12之前重新平衡RP柱。 </li></ol></li><li>检测和可视化放射性标记的化合物与流Scintillation分析。记录母体除草剂其余各样品为总放射性的每一个样品中的百分比中的量（被检测，并根据制造商的说明由流动闪烁分析仪定量）的时间过程中，以确定在每个waterhemp人口除草剂代谢的速率。 </li></ol> 4.统计程序<ol><li>安排在一个完全随机设计（或用于统计分析其它合适的布置）的治疗方法。 </li><li>执行两个独立的实验，每个治疗，由三个生物学重复每个实验。两个独立的实验，包括共有6个生物学重复。如果实验效果是非显著（α= 0.05），从每个独立的实验结合和分析数据。如果实验效果显著，然后分别分析每个实验。 注:包括前三个生物学重复我ñ实验1和实验2接下来的三个生物复制每个生物复制代表一个独特的"家长"，从每个群体衍生的（MCR 1-6 ACR 1-6和WCS 1-6）无性系，使每个时间当然，分析利用经过基因相同的植物。在两个独立的实验包括六个生物复制总数，其允许的中值DT 50为每个waterhemp群体的遗传变异性和测定的足够的代表性。 </li><li>分析具有非线性最小二乘回归分析的数据，并用一个简单的一阶曲线拟合它们以估计的DT 50秒。下面的公式描述模型: <img alt="式（1）" src="/files/ftp_upload/53236/53236eq1.jpg" /></li><li>其中在该模型中，y表示母体除草剂残留在时间t的百分比，μi是对DT 50为每个waterhemp人口i和参数C 0是母体除草剂本在t = 0 12所估计的量。 </li></ol>

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The authors have nothing to disclose.

本文描述的切叶方法已在研究氟嘧磺隆代谢的玉米叶片15以前使用过，但我们的研究结果表明，该协议也是有效的，准确的，和可重复的测量除草剂代谢双子叶杂草12。与全株研究相比在切下的叶片技术的一个主要的优点是，一个切下的叶片是独立的芽后的全株易位型态，全身性除草剂或植物或人群中除草剂的吸收差异。此外，环境的变化被减小，因为切下的叶片测定法在生长室?...

在杂草除草剂抗性提出了一个严重的威胁到粮食和纤维1,2全球生产。目前，成千上万的抗性种群和生物型，从百余种杂草世界范围内已被记录在案并研究3。赋予植物除莠剂抗性的主要机制是除草剂靶位点的基因和蛋白质，包括影响除草剂-蛋白结合动力学或目标站点基因2的扩增的基因突变的改变。通过细胞色素P450单加氧酶（P450）或谷胱甘肽 S-转移酶（GST）酶升高活性代谢排毒赋予杂草除草剂抗性，这是明显的从目标网站为基础的机制2几种方式另一种机制。代谢型电阻具有显著后果为是否植物适合度代价（又名健身处罚），可能会导致从除草剂抗性机制及米，以及关于用于单个解毒机制赋予在杂草种群1,2,4交叉或多除草剂抗性的潜力。通常，在植物的除草剂代谢可分为三个不同的阶段5。第一阶段涉及除草剂转化或活化如芳环或烷基的P450介导的羟基化，或用 N -或O-脱烷基化反应，从而增加极性和部分除草剂解毒5,6。新引入的官能团的第一阶段通过的GSTs提供连接位点结合，以还原型谷胱甘肽或UDP相关的糖基转移酶在二5,7阶段为葡萄糖。例如，氟嘧磺隆-甲酯，在玉米中主要初始代谢物是羟基氟嘧磺隆-甲酯8，它可以被进一步代谢成羟基氟嘧磺隆-葡糖苷（第二阶段），然后输送到液泡长期贮存或进一步代谢亲cessing 5,6（第三期）。 Waterhemp（ 苋tuberculatus）是一种难以控制的，双子叶一年生杂草，阻碍生产玉米（ 玉米 ）， 大豆 （Glycine max），棉花（ 陆地棉 ）在美国物种。 waterhemp遗传多样性的高度是由它的雌雄异株的生物学和长途风授粉便利，和一个女waterhemp厂月产能可达一百万种子9。这些种子很小，很容易传播，这自然赋予waterhemp一个有效的分散机制。 Waterhemp显示在整个生长季节9连续发芽，而它的种子可以经过几年的休眠发芽。 Waterhemp是C 4植物具有较高的增长速度比耕地种植系统10个最阔叶杂草。此外，众多waterhemp种群抗多种FAM除草剂3 ilies。 从伊利诺伊waterhemp（指定MCR）的人口是耐4-羟基苯丙酮酸双加氧酶（HPPD）的除草剂-inhibiting 11，如甲基磺草酮，以及莠去津和乙酰乳酸合酶（ALS）-inhibiting除草剂，包括氟嘧磺隆-甲基由于非目标站点为基础的机制12,13。一种不同的人口waterhemp的指定的ACR 14，其是氟嘧磺隆甲基抗性（由于在ALS基因中的突变）和阿特拉津抗性但对硝磺草酮敏感，并指定WCS 14 waterhemp人口是敏感的氟嘧磺隆-甲基，硝磺草酮，莠去津和分别在我们以前的研究12和当前实验的MCR用于比较（总结于表1）。最初的研究未检测到的改变在所述 HPPD基因序列或表达水平，或降低的硝磺草酮的摄取，在MCR人口与硝磺草酮敏感的人群相比，12时。然而，随着整个植物代谢研究证明了亲硝磺草酮除草剂显著较低水平在MCR与ACR和WCS，这与以前的表型应答相关的硝磺草酮11,12相比。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Waterhemp人口 </td><td> 缩写 </td><td> 表型硝磺草酮 </td><td> 硝磺草酮耐药机制 </td><td> 表型氟嘧磺隆 </td><td> 氟嘧磺隆抗性机制 </td></tr><tr><td>麦克莱恩县耐</td><td> MCR </td><td>耐</td><td>代谢* </td><td>耐</td><td>代谢</td></tr><tr><td>亚当斯县，耐</td><td> ACR </td><td> SENSIT香港专业教育学院</td><td> - </td><td>耐</td><td>在ALS 14靶位点突变</td></tr><tr><td>韦恩县敏感</td><td> WCS </td><td>灵敏的</td><td> - </td><td>灵敏的</td><td> - </td></tr></tbody></table> *非目标抗性的机制，比加强新陈代谢等，也可以赋予硝磺草酮阻力在MCR人口12。 表1:从伊利诺伊waterhemp种群在该研究中使用的描述。 除了 ​​确定在完整waterhemp幼苗除草剂代谢速率，不同的实验方法，开发并在我们以前的研究采用通过使用切waterhemp叶测定法12，以及各种细胞色素P450 抑制剂 （如，tetcyclacis和马拉硫磷）调查代谢。这种方法特别适用于waterhemp从PREVI在切下玉米氟嘧磺隆-甲基代谢的OU调查叶15中，由于切下的叶片检测尚未被报道用于进行除草剂代谢研究在双子叶植物。所述organophophosate杀虫剂马拉硫磷已经经常用于体内和体外除草剂代谢研究，以指示参与的P450 16。例如，宽容和硝磺草酮的玉米快速代谢是由于P450催化环羟化，当马拉硫磷增加玉米的敏感性硝磺草酮17进行了验证。同样，马拉硫磷抑制的ALS抑制剂氟嘧磺隆，甲在切玉米叶片15新陈代谢。该切下的叶片技术的一个主要的优点是，所产生的数据是独立的全株易位图案，一个重要的因素，以评估全身，芽后除草剂代谢在植物时考虑。因此，这种方法允许定量和定性的代谢分析集中于单个处理过的叶子12。 甲植物克隆策略，与切下的叶片协议组合，先前在waterhemp用来进行代谢研究12。由于waterhemp（单独的男性和女性的植物），和大程度雌雄异株物种苋 9内的遗传多样性的异交性质，该协议确保了经过基因相同waterhemp秧苗的时程实验中进行分析。本文演示了切叶法测定双子叶杂草（waterhemp）除草剂代谢率的效用。母体除草剂的量剩余在每个时间点（图1）通过非线性最小二乘回归分析确定，并适合用一个简单的一阶曲线，以估计为吸收除草剂的50％降解的时间（ DT 50）。典型从反相高效液相色谱法（RP-HPLC）色谱图的显示为ALS抗性及敏感waterhemp人群，其中一个时间过程研究中表明父除草剂伴随生成极性代谢产物的消失（ 图2）。我们的本文的重点是描述和演示的切下的叶片的试验中组合的效用与植物人克隆方法，用于确定在双子叶植物的除草剂代谢的精确和可重复的速率，使用均匀环标记（URL- 14℃）除草剂在3 waterhemp种群的不同在其全植物对HPPD-和ALS抑制除草剂（ 表1）。

<table><tbody><tr><td>Agar</td><td>Sigma-Aldrich</td><td>A1296</td><td>for pre-germinating seeds</td></tr><tr><td>Potting medium</td><td>Sun Gro Horticulture</td><td>49040233</td><td>for plant growth</td></tr><tr><td>Nutricote</td><td>Agrivert&amp;nbsp;</td><td>TOTAL BLEND 13-13-13 T100</td><td>slow-release fertilizer</td></tr><tr><td>Growth chamber E15</td><td>Controlled Environments Limited</td><td>20207</td><td>plant culturing</td></tr><tr><td>Tris base</td><td>Fisher Scientific</td><td>BP152-500</td><td>buffer for excised leaves</td></tr><tr><td>HCl (concentrated)</td><td>Fisher Scientific</td><td>A144500</td><td>adjust pH of buffer</td></tr><tr><td>Murashige and Skoog (MS) salts&amp;nbsp;</td><td>Sigma-Aldrich</td><td>M0404</td><td>incubation of excised leaves</td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A452-4</td><td>leaf washes after incubation</td></tr><tr><td>Acetone</td><td>Sigma-Aldrich</td><td>179124</td><td>plant extractions</td></tr><tr><td>Acetonitrile (HPLC grade)</td><td>Macron Fine Chemicals</td><td>MKH07610</td><td>HPLC mobile phase</td></tr><tr><td>Formic acid&amp;nbsp;</td><td>Mallinckrodt Analytical</td><td>MK259205</td><td>acidify mobile phase pH</td></tr><tr><td>Micro-centrifuge</td><td>Eppendorf</td><td>5417R</td><td>1.5 or 2.0 ml tubes</td></tr><tr><td>Centrifuge (temperature controlled)</td><td>Eppendorf</td><td>5810R</td><td>15 or 50 ml tubes</td></tr><tr><td>Polypropylene centrifuge tube</td><td>Corning Inc.</td><td>430790</td><td>15 ml, sterile</td></tr><tr><td>Rotary evaporator</td><td>B&amp;Uuml;CHI</td><td>R200</td><td>concentrate plant samples</td></tr><tr><td>Liquid scintillation spectrometry (LSS)</td><td>Packard Instruments</td><td>104470</td><td>quantify &lt;sup&gt;14&lt;/sup&gt;C</td></tr><tr><td>High-performance liquid chromatography</td><td>Perkin Elmer</td><td>N2910401</td><td>resolve herbicide metabolites</td></tr><tr><td>Flow scintillation analyzer&amp;nbsp;</td><td>LabLogic System</td><td>1103303</td><td>for HPLC analysis of &lt;sup&gt;14&lt;/sup&gt;C</td></tr><tr><td>Hypersil Gold C&lt;sub&gt;18&lt;/sub&gt; column&amp;nbsp;</td><td>Thermo-Scientific</td><td>03-050-522&amp;nbsp;</td><td>reversed phase</td></tr><tr><td>Ultima-Flo M cocktail</td><td>Perkin Elmer</td><td>6013579</td><td>for Flow-scintillation analyzer</td></tr><tr><td>Scintillation Cocktail (ScintiVerse BD)</td><td>Fisher Scientific</td><td>SX18</td><td>for LSS; biodegradable</td></tr><tr><td>Laboratory homogenizer</td><td>Kinematica</td><td>CH-6010&amp;nbsp;</td><td>homogenize leaf samples</td></tr></tbody></table>

measuring rates of herbicide metabolism in dicot weeds with an excised leaf assay

In order to isolate and accurately determine rates of herbicide metabolism in an obligate-outcrossing dicot weed, waterhemp (Amaranthus tuberculatus), we developed an excised leaf assay combined with a vegetative cloning strategy to normalize herbicide uptake and remove translocation as contributing factors in herbicide-resistant (R) and &#8211;sensitive (S) waterhemp populations. Biokinetic analyses of organic pesticides in plants typically include the determination of uptake, translocation (delivery to the target site), metabolic fate, and interactions with the target site. Herbicide metabolism is an important parameter to measure in herbicide-resistant weeds and herbicide-tolerant crops, and is typically accomplished with whole-plant tests using radiolabeled herbicides. However, one difficulty with interpreting biokinetic parameters derived from whole-plant methods is that translocation is often affected by rates of herbicide metabolism, since polar metabolites are usually not mobile within the plant following herbicide detoxification reactions. Advantages of the protocol described in this manuscript include reproducible, accurate, and rapid determination of herbicide degradation rates in R and S populations, a substantial decrease in the amount of radiolabeled herbicide consumed, a large reduction in radiolabeled plant materials requiring further handling and disposal, and the ability to perform radiolabeled herbicide experiments in the lab or growth chamber instead of a greenhouse. As herbicide resistance continues to develop and spread in dicot weed populations worldwide, the excised leaf assay method developed and described herein will provide an invaluable technique for investigating non-target site-based resistance due to enhanced rates of herbicide metabolism and detoxification.

要么WCS或ACR和MCR（图1）之间进行检测大的差异在硝磺草酮的代谢速率。在每个时间点，MCR已经比二甲基磺草酮敏感的人群中，WCS和ACR，这与以前的全植物表型反应11更迅速代谢硝磺草酮。通过从每个群体一个亲本植物克隆足够的植物，除草剂代谢的时间过程的分析是一致的和可重复的，由于每次课程12内部缺乏遗传变异性。例如，该时间过程中克隆系5 12硝磺?...

Watch this Scientific Journal Video about Measuring Rates of Herbicide Metabolism in Dicot Weeds with an Excised Leaf Assay at JoVE.com

Measuring Rates of Herbicide Metabolism in Dicot Weeds with an Excised Leaf Assay

This manuscript describes how herbicide metabolism rates can be effectively quantified with excised leaves from a dicot weed, thereby reducing variability and removing any possible confounding effects of herbicide uptake or translocation typically observed in whole-plant assays.

测量除草剂代谢的双子叶杂草价格与切下的叶片分析

In order to isolate and accurately determine rates of herbicide metabolism in an obligate-outcrossing dicot weed, waterhemp (Amaranthus tuberculatus), we ...

measuring-rates-herbicide-metabolism-dicot-weeds-with-an-excised-leaf

Research

JoVE Journal

Environment

11.9K 次观看.  University of Illinois. This manuscript describes how herbicide metabolism rates can be effectively quantified with excised leaves from a dicot weed, thereby reducing variability and removing any possible confounding effects of herbicide uptake or translocation typically observed in whole-plant assays.

视频: Measuring Rates of Herbicide Metabolism in Dicot Weeds with an Excised Leaf Assay

Protocols for Robust Herbicide Resistance Testing in Different Weed Species

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented protocols, based on whole-plant bioassays performed in a greenhouse, can be readily adapted to a wide range of weed species and herbicides through appropriate variants. Seed samples from plants that survived a field herbicide treatment are collected and stored dry at low temperature until used. Germination methods differ according to weed species and seed dormancy type. Seedlings at similar growth stage are transplanted and maintained in the greenhouse under appropriate conditions until plants have reached the right growth stage for herbicide treatment. Accuracy is required to prepare the herbicide solution to avoid unverifiable mistakes. Other critical steps such as the application volume and spray speed are also evaluated. The advantages of this protocol, compared to others based on whole plant bioassays using one herbicide dose, are related to the higher reliability and the possibility of inferring the resistance level. Quicker and less expensive in vivo or in vitro diagnostic screening tests have been proposed (Petri dish bioassays, spectrophotometric tests), but they provide only qualitative information and their widespread use is hindered by the laborious set-up that some species may require. For routine resistance testing, the proposed whole plant bioassay can be applied at only one herbicide dose, so reducing the costs.

A robust and flexible approach to confirm herbicide resistance in weed populations is presented. This protocol allows the herbicide resistance levels to be inferred and applied to a wide range of weed species and herbicides with minor adaptations.

协议的鲁棒抗除草剂试验在不同杂草

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented ...

科研

环境科学

Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing plant metabolites because it provides molecular weights with high sensitivity and specificity. Leaf spray MS is an ambient ionization technique where plant tissue is used for direct chemical analysis via electrospray, eliminating chromatography from the process. This approach to sampling metabolites allows for a wide range of chemical classes to be detected simultaneously from intact plant tissues, minimizing the amount of sample preparation needed. When used with a high-resolution, accurate mass MS, leaf spray MS facilitates the rapid detection of metabolites of interest. It is also possible to collect tandem mass fragmentation data with this technique to facilitate a compound identification. The combination of accurate mass measurements and fragmentation is beneficial in confirming compound identities. The leaf spray MS technique requires only minor modifications to a nanospray ionization source and is a useful tool to further expand the capabilities of a mass spectrometer. Here, fresh leaf tissue from Sceletium tortuosum (Aizoaceae), a traditional medicinal plant from South Africa, is analyzed; numerous mesembrine alkaloids are detected with leaf spray MS.

Leaf spray mass spectrometry is a direct chemical analysis technique that minimizes the sample preparation and eliminates chromatography, allowing for the rapid detection of small molecules from plant tissues.

叶喷雾质谱: 一种快速的环境电离技术直接评价植物组织中的代谢产物

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing ...

生物化学

Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more streamlined herbivory protocols. Perturbations of chewing insects are usually studied in whole plant systems. While this whole organism strategy is popular, it is not necessary if similar observations can be replicated in a single detached leaf. The assumption is that basic elements required for signal transduction are present within the leaf itself. In the case of early events in signal transduction, cells need only to receive the signal from the perturbation and transmit that signal to neighboring cells which are assayed for gene expression.
The proposed method simply changes the timing of the detachment. In whole plant experiments, larvae are confined to a single leaf which is eventually detached from the plant and assayed for gene expression. If the order of excision is reversed, from last in whole plant studies, to first in the detached study, the feeding experiment is simplified.
Solanum tuberosum var. Kennebec is propagated by nodal transfer in a simple tissue culture medium and transferred to soil for further growth if desired. Leaves are excised from the parent plant and relocated to Petri dishes where the feeding assay is conducted with the larval stages of M. sexta. Damaged leaf tissue is assayed for the expression of relatively early events in signal transduction. Gene expression analysis identified infestation-specific Cys2-His2 (C2H2) transcription factors, confirming the success of using detached leaves in early response studies. The method is easier to perform than whole plant infestations and uses less space.

The presented method creates natural herbivore damaged plant tissue through the application of Manduca sexta larvae to detached leaves of potato. The plant tissue is assayed for expression of six transcription factor homologs involved in early responses to insect herbivory.

分离叶测定,以简化马铃薯中基因表达研究,在咀嚼昆虫曼杜卡性塔感染

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

从茶中提取的新建细胞悬浮培养基中六种系统杀虫剂的代谢研究(山茶西宁西斯L.叶

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Elucidating the Metabolism of 2,4-Dibromophenol in Plants

Crops can be extensively exposed to organic pollutants, since soil is a major sink for pollutants discarded into the environment. This creates potential human exposure through the consumption of pollutant-accumulated foods. Elucidating the uptake and metabolism of xenobiotics in crops is essential for the assessment of dietary exposure risk in humans. However, for such experiments, the use of intact plants requires long-term experiments and complex sample preparation protocols that can be affected by various factors. Plant callus cultures combined with high-resolution mass spectrometry (HRMS) may provide a solution for the accurate and time-saving identification of metabolites of xenobiotics in plants, as it can avoid interference from the microbial or fungal microenvironment, shorten the treatment duration, and simplify the matrix effect of intact plants. 2,4-dibromophenol, a typical flame retardant and endocrine disrupter, was chosen as the model substance due to its widespread occurrence in soil and its uptake potential by plants. Herein, plant callus was generated from asepsis seeds and exposed to sterile 2,4-dibromophenol-containing culture medium. The results showed that eight metabolites of 2,4-dibromophenol were identified in the plant callus tissues after 120 h of incubation. This indicates that 2,4-dibromophenol was rapidly metabolized in&#160;the plant callus tissues. Thus, the plant callus culture platform is an effective method to evaluate the uptake and metabolism of xenobiotics in plants.

The present protocol describes a simple and efficient method for the identification of 2,4-dibromophenol metabolites in plants.

阐明植物中2,4-二溴苯酚的代谢

Crops can be extensively exposed to organic pollutants, since soil is a major sink for pollutants discarded into the environment. This creates potential ...

<meta charset="utf8"></head><body>估计三唑类农药在植物中的整合代谢物细胞毒性

Assessing Cytotoxicity of Metabolites of Typical Triazole Pesticides in Plants

Various organic pollutants have been released into the environment because of anthropogenic activities. These pollutants can be taken up by crop plants, causing potential threats to the ecosystem and human health throughout the food chain. The biotransformation of pollutants in plants generates a number of metabolites that may be more toxic than their parent compounds, implying that the metabolites should be taken into account during the toxicity assessment. However, the metabolites of pollutants in plants are extremely complex, making it difficult to comprehensively obtain the toxicological information of all metabolites. This study proposed a strategy to assess the integral cytotoxicity of pollutant metabolites in plants by treating them as a whole during toxicological tests. Triazole pesticides, a class of broad-spectrum fungicides, have been widely applied in agricultural production. Their residue pollution in farmland has drawn increasing attention. Hence, four triazole pesticides, including flusilazole, diniconazole, tebuconazole, and propiconazole, were selected as the tested pollutants. The metabolites were generated by the treatment of carrot callus with tested triazole pesticides. After treatment of 72 h, the metabolites of pesticides in carrot callus were extracted, followed by toxicological tests using the Caco-2 cell line. The results showed that the metabolites of tested pesticides in carrot callus did not significantly inhibit the viability of Caco-2 cells (P>0.05), demonstrating no cytotoxicity of pesticide metabolites. This proposed method opens a new avenue to assess the cytotoxicity of pollutant metabolites in plants, which is expected to provide valuable data for precise toxicity assessment.

<meta charset="utf8"></head><body>作者聚焦:一种对植物中三唑类农药进行综合代谢物细胞毒性评估的新型高效方法

The protocol describes a new method to assess the integral cytotoxicity of metabolites of triazole pesticides in plants.

评估植物中典型三唑类农药代谢物的细胞毒性

Various organic pollutants have been released into the environment because of anthropogenic activities. These pollutants can be taken up by crop plants, ...

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend themselves against pathogen attack. The contribution of a specific Arabidopsis gene to the overall plant immune response can be specifically and quantitatively assessed by assaying the pathogen growth within the infected tissue. For over three decades, the hemibiotrophic bacterium Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) has been widely applied as the model pathogen to investigate the molecular mechanisms underlying the Arabidopsis immune response. To deliver pathogens into the leaf tissue, multiple inoculation methods have been established, e.g., syringe infiltration, dip inoculation, spray, vacuum infiltration, and flood inoculation. The following protocol describes an optimized syringe infiltration method to deliver virulent Psm ES4326 into leaves of adult soil-grown Arabidopsis plants and accurately screen for enhanced disease susceptibility (EDS) towards this pathogen. In addition, this protocol can be supplemented with multiple pre-treatments to further dissect specific immune defects within different layers of plant defense, including Salicylic Acid (SA)-Triggered Immunity (STI) and MAMP-Triggered Immunity (MTI).

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

细菌性叶渗透试验使用的植物防御反应精细表征<em&gt;拟南芥，丁香假单胞菌</em&gt;病害系统

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend ...

免疫与感染

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method (EFM)

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to achieve a high stomatal conductance (gs) to capture CO2 for photosynthesis, water is lost by transpiration1,2. Water evaporating from the airspaces is replaced from cell walls, in turn drawing water from the xylem of leaf veins, in turn drawing from xylem in the stems and roots. As water is pulled through the system, it experiences hydraulic resistance, creating tension throughout the system and a low leaf water potential (&Psi;leaf). The leaf itself is a critical bottleneck in the whole plant system, accounting for on average 30% of the plant hydraulic resistance3. Leaf hydraulic conductance (Kleaf = 1/ leaf hydraulic resistance) is the ratio of the water flow rate to the water potential gradient across the leaf, and summarizes the behavior of a complex system: water moves through the petiole and through several orders of veins, exits into the bundle sheath and passes through or around mesophyll cells before evaporating into the airspace and being transpired from the stomata. Kleaf is of strong interest as an important physiological trait to compare species, quantifying the effectiveness of the leaf structure and physiology for water transport, and a key variable to investigate for its relationship to variation in structure (e.g., in leaf venation architecture) and its impacts on photosynthetic gas exchange. Further, Kleaf responds strongly to the internal and external leaf environment3. Kleaf can increase dramatically with irradiance apparently due to changes in the expression and activation of aquaporins, the proteins involved in water transport through membranes4, and Kleaf declines strongly during drought, due to cavitation and/or collapse of xylem conduits, and/or loss of permeability in the extra-xylem tissues due to mesophyll and bundle sheath cell shrinkage or aquaporin deactivation5-10. Because Kleaf can constrain gs and photosynthetic rate across species in well watered conditions and during drought, and thus limit whole-plant performance they may possibly determine species distributions especially as droughts increase in frequency and severity11-14.
We present a simple method for simultaneous determination of Kleaf and gs on excised leaves. A transpiring leaf is connected by its petiole to tubing running to a water source on a balance. The loss of water from the balance is recorded to calculate the flow rate through the leaf. When steady state transpiration (E, mmol &bull; m-2 &bull; s-1) is reached, gs is determined by dividing by vapor pressure deficit, and Kleaf by dividing by the water potential driving force determined using a pressure chamber (Kleaf= E /- &Delta;&psi;leaf, MPa)15.
This method can be used to assess Kleaf responses to different irradiances and the vulnerability of Kleaf to dehydration14,16,17.

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method EFM

We describe a relatively rapid (30 min) and realistic method for simultaneously measurement of leaf hydraulic conductance (Kleaf) and stomatal conductance (gs) for transpiring excised leaves. The method can be modified to measure the light and dehydration responses of Kleaf and gs.

测量叶片导水率和气孔导度和他们的反应，辐射和脱水蒸发通量法（EFM）

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to ...

测量除草剂代谢的双子叶杂草价格与切下的叶片分析

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细菌性叶渗透试验使用的植物防御反应精细表征

测量叶片导水率和气孔导度和他们的反应，辐射和脱水蒸发通量法（EFM）