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 border="0" cellpadding="0" cellspacing="0">
	<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&#160;</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&#160;</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&#160;</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&#220;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 14C</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&#160;</td>
			<td>LabLogic System</td>
			<td>1103303</td>
			<td>for HPLC analysis of 14C</td>
		</tr>
		<tr>
			<td>Hypersil Gold C18 column&#160;</td>
			<td>Thermo-Scientific</td>
			<td>03-050-522&#160;</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&#160;</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

Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. Radioisotope tracing is a major technique used to measure such fluxes, both within plants, and between plants and their environments. Flux data obtained with radiotracer protocols can help elucidate the capacity, mechanism, regulation, and energetics of transport systems for specific mineral nutrients or toxicants, and can provide insight into compartmentation and turnover rates of subcellular mineral and metabolite pools. Here, we describe two major radioisotope protocols used in plant biology: direct influx (DI) and compartmental analysis by tracer efflux (CATE). We focus on flux measurement of potassium (K+) as a nutrient, and ammonia/ammonium (NH3/NH4+) as a toxicant, in intact seedlings of the model species barley (Hordeum vulgare L.). These protocols can be readily adapted to other experimental systems (e.g., different species, excised plant material, and other nutrients/toxicants). Advantages and limitations of these protocols are discussed.

In planta measurement of nutrient and toxicant fluxes is essential to the study of plant nutrition and toxicity. Here, we cover radiotracer protocols for influx and efflux determination in intact plant roots, using potassium (K+) and ammonia/ammonium (NH3/NH4+) fluxes as examples. Advantages and limitations of such techniques are discussed.

植物与放射性示踪剂测量矿质营养和毒物的通量

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. ...

科研

环境科学

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 ...

LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic pathways requires a model in which external factors can be well controlled, allowing for reproducible and meaningful results. Many studies employ transformed cellular models for these purposes; however, metabolic reprogramming that occurs in many cancer cell lines may introduce confounding variables. For this reason primary cells are desirable, though attaining adequate biomass for metabolic studies can be challenging. Here we show that human platelets can be utilized as a platform to carry out metabolic studies in combination with liquid chromatography-tandem mass spectrometry analysis. This approach is amenable to relative quantification and isotopic labeling to probe the activity of specific metabolic pathways. Availability of platelets from individual donors or from blood banks makes this model system applicable to clinical studies and feasible to scale up. Here we utilize isolated platelets to confirm previously identified compensatory metabolic shifts in response to the complex I inhibitor rotenone. More specifically, a decrease in glycolysis is accompanied by an increase in fatty acid oxidation to maintain acetyl-CoA levels. Our results show that platelets can be used as an easily accessible and medically relevant model to probe the effects of xenobiotics on cellular metabolism.

Here we show isolated human platelets can be used as an accessible ex vivo model to study metabolic adaptations in response to the complex I inhibitor rotenone. This approach employs isotopic tracing and relative quantification by liquid chromatography-mass spectrometry and can be applied to a variety of study designs.

人类血小板的LC-MS分析作为研究线粒体代谢的平台

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic ...

Laboratory and Field Protocol for Estimating Sheet Erosion Rates from Dendrogeomorphology

Sheet erosion is among the crucial drivers of soil degradation. Erosion is controlled by environmental factors and human activities, which often lead to severe environmental impacts. The understanding of sheet erosion is, consequently, a worldwide issue with implications for both environment and economies. However, the knowledge on how erosion evolves in space and time is still limited, as well as its effects on the environment. Below, we explain a new dendrogeomorphological protocol for deriving eroded soil thickness (Ex) by acquiring accurate microtopographic data using both terrestrial laser scanning (TLS) and microtopographic profile gauges. Additionally, standard dendrogeomorphic procedures, dependent on anatomical variations in root rings, are utilized to establish the timing of exposure. Both TLS and microtopographic profile gauges are used to obtain ground surface pro&#64257;les, from which Ex is estimated after the threshold distance (TD) is determined, i.e., the distance between the root and the sediment knickpoint, which allows de&#64257;ning the lowering of the ground surface caused by sheet erosion. For each profile, we measured the height between the topside of the root and a virtual plane tangential to the ground surface. In this way, we intended to avoid small-scale impacts of soil deformation, which may be due to pressures exerted by the root system, or by the arrangement of exposed roots. This may provoke small amounts of soil sedimentation or erosion depending on how they physically affect the surface runoff. We demonstrate that an adequate microtopographic characterization of exposed roots and their associated ground surface is very valuable to obtain accurate erosion rates. This finding could be utilized to develop the best management practices designed to eventually halt or perhaps, at least, lessen soil erosion, so that more sustainable management policies can be put into practice.

Characterizing erosion from dendrogeomorphology has usually focused on accurately finding the starting time of root exposure, by examining macroscopic or cell level changes caused by exposure. Here, we offer a detailed description of different novel techniques to obtain more precise erosion rates from highly accurate microtopographic data.

利用树形地貌估算板材侵蚀速率的实验室和现场协议

Sheet erosion is among the crucial drivers of soil degradation. Erosion is controlled by environmental factors and human activities, which often lead to ...

An Anaerobic Biosensor Assay for the Detection of Mercury and Cadmium

Mercury (Hg) bioavailability to microbes is a key step to toxic Hg biomagnification in food webs. Cadmium (Cd) transformations and bioavailability to bacteria control the amount that will accumulate in staple food crops. The bioavailability of these metals is dependent on their speciation in solution, but more particularly under anoxic conditions where Hg is methylated to toxic monomethylmercury (MeHg) and Cd persists in the rhizosphere. Whole-cell microbial biosensors give a quantifiable signal when a metal enters the cytosol and therefore are useful tools to assess metal bioavailability. Unfortunately, most biosensing efforts have so far been constrained to oxic environments due to the limited ability of existing reporter proteins to function in the absence of oxygen. In this study, we present our effort to develop and optimize a whole-cell biosensor assay capable of functioning anaerobically that can detect metals under anoxic condition in quasi-real time and within hours. We describe how the biosensor can help assess how chemical variables relevant to the environmental cycling of metals affect their bioavailability. The following protocol includes methods to (1) prepare Hg and Cd standards under anoxic conditions, (2) prepare the biosensor in the absence of oxygen, (3) design and execute an experiment to determine how a series of variable affects Hg or Cd bioavailability, and (4) to quantify and interpret biosensor data. We show the linear ranges of the biosensors and provide examples showing the method's ability to distinguish between metal bioavailability and toxicity by utilizing both metal-inducible and constitutive strains.

Here, we present a protocol to use an anaerobic whole-cell microbial biosensor to evaluate how different environmental variables affect the bioavailability of Hg and Cd to bacteria in anoxic environments.

一种用于检测汞和镉的厌氧生物传感器检测方法

Mercury (Hg) bioavailability to microbes is a key step to toxic Hg biomagnification in food webs. Cadmium (Cd) transformations and bioavailability to ...

Estimating Sediment Denitrification Rates Using Cores and N2O Microsensors

Denitrification is the primary biogeochemical process removing reactive nitrogen from the biosphere. The quantitative evaluation of this process has become particularly relevant for assessing the anthropogenic-altered global nitrogen cycle and the emission of greenhouse gases (i.e., N2O). Several methods are available for measuring denitrification, but none of them are completely satisfactory. Problems with existing methods include their insufficient sensitivity, and the need to modify the substrate levels or alter the physical configuration of the process using disturbed samples.This work describes a method for estimating sediment denitrification rates that combines coring, acetylene inhibition, and microsensor measurements of the accumulated N2O. The main advantages of this method are a low disturbance of the sediment structure and the collection of a continuous record of N2O accumulation; these enable estimates of reliable denitrification rates with minimum values up to 0.4-1 &#181;mol N2O m-2 h-1. The ability to manipulate key factors is an additional advantage for obtaining experimental insights. The protocol describes procedures for collecting the cores, calibrating the sensors, performing the acetylene inhibition, measuring the N2O accumulation, and calculating the denitrification rate. The method is appropriate for estimating denitrification rates in any aquatic system with retrievable sediment cores. If the N2O concentration is above the detection limit of the sensor, the acetylene inhibition step can be omitted to estimate the N2O emission instead of denitrification. We show how to estimate both actual and potential denitrification rates by increasing nitrate availability as well as the temperature dependence of the process. We illustrate the procedure using mountain lake sediments and discuss the advantages and weaknesses of the technique compared to other methods. This method can be modified for particular purposes; for instance, it can be combined with 15N tracers to assess nitrification and denitrification or field in situ measurements of denitrification rates.

Estimating Sediment Denitrification Rates Using Cores and N2O Microsensors

This method estimates sediment denitrification rates in sediment cores using the acetylene inhibition technique and microsensor measurements of the accumulated N2O. The protocol describes procedures for collecting the cores, calibrating the sensors, performing the acetylene inhibition, measuring the N2O accumulation, and calculating the denitrification rate.

利用岩体和 n 2o 微传感器估计沉积物脱氮率

Denitrification is the primary biogeochemical process removing reactive nitrogen from the biosphere. The quantitative evaluation of this process has ...

Measuring the Shape and Size of Activated Sludge Particles Immobilized in Agar with an Open Source Software Pipeline

Experimental bioreactors, such as those treating wastewater, contain particles whose size and shape are important parameters. For example, the size and shape of activated sludge flocs can indicate the conditions at the microscale, and also directly affect how well the sludge settles in a clarifier.
Particle size and shape are both misleadingly &#39;simple&#39; measurements. Many subtle issues, often unaddressed in informal protocols, can arise when sampling, imaging, and analyzing particles. Sampling methods may be biased or not provide enough statistical power. The samples themselves may be poorly preserved or undergo alteration during immobilization. Images may not be of sufficient quality; overlapping particles, depth of field, magnification level, and various noise can all produce poor results. Poorly specified analysis can introduce bias, such as that produced by manual image thresholding and segmentation.
Affordability and throughput are desirable alongside reproducibility. An affordable, high throughput method can enable more frequent particle measurement, producing many images containing thousands of particles. A method that uses inexpensive reagents, a common dissecting microscope, and freely-available open source analysis software allows repeatable, accessible, reproducible, and partially-automated experimental results. Further, the product of such a method can be well-formatted, well-defined, and easily understood by data analysis software, easing both within-lab analyses and data sharing between labs.
We present a protocol that details the steps needed to produce such a product, including: sampling, sample preparation and immobilization in agar, digital image acquisition, digital image analysis, and examples of experiment-specific figure generation from the analysis results. We have also included an open-source data analysis pipeline to support this protocol.

The size and shape of particles in activated sludge are important parameters that are measured using varying methods. Inaccuracies arise from non-representative sampling, suboptimal images, and subjective analysis parameters. To minimize these errors and ease measurement, we present a protocol specifying every step, including an open source software pipeline.

利用开源软件管道测量琼脂中固定活性污泥颗粒的形状和大小

Experimental bioreactors, such as those treating wastewater, contain particles whose size and shape are important parameters. For example, the size and ...

Cultivation of Green Microalgae in Bubble Column Photobioreactors and an Assay for Neutral Lipids

There is significant interest in the study of microalgae for engineering applications such as the production of biofuels, high value products, and for the treatment of wastes. As most new research efforts begin at laboratory scale, there is a need for cost-effective methods for culturing microalgae in a reproducible manner. Here, we communicate an effective approach to culture microalgae in laboratory-scale photobioreactors, and to measure the growth and neutral lipid content of that algae. Instructions are also included on how to set up the photobioreactor system. Although the example organisms are species of Chlorella and Auxenochlorella, this system can be adapted to cultivate a wide range of microalgae, including co-cultures of algae with non-algae species. Stock cultures are first grown in bottles to produce inoculum for the photobioreactor system. Algae inoculum is concentrated and transferred to photobioreactors for cultivation in batch mode. Samples are collected daily for the optical density readings. At the end of the batch culture, cells are harvested by centrifuge, washed, and freeze dried to obtain a final dry weight concentration. The final dry weight concentration is used to create a correlation between the optical density and the dry weight concentration. A modified Folch method is subsequently used to extract total lipids from the freeze-dried biomass and the extract is assayed for its neutral lipid content using a microplate assay. This assay has been published previously but protocol steps were included here to highlight critical steps in the procedure where errors frequently occur. The bioreactor system described here fills a niche between simple flask cultivation and fully-controlled commercial bioreactors. Even with only 3-4 biological replicates per treatment, our approach to culturing algae leads to tight standard deviations in the growth and lipid assays.

Here, we present a protocol to construct lab-scale bubble column photobioreactors and use them to culture microalgae. It also provides a method for the determination of the culture growth rate and neutral lipid content.

气泡柱光生物反应器中绿色微藻的培养及中性脂质的测定

There is significant interest in the study of microalgae for engineering applications such as the production of biofuels, high value products, and for the ...

Leaf Area Index Estimation Using Three Distinct Methods in Pure Deciduous Stands

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for describing the vegetation structure in the fields of ecology, forestry, and agriculture. Therefore, procedures of three commercially used methods (litter traps, needle technique, and a plant canopy analyzer) for performing LAI estimation were presented step-by-step. Specific methodological approaches were compared, and their current advantages, controversies, challenges, and future perspectives were discussed in this protocol. Litter traps are usually deemed as the reference level. Both the needle technique and the plant canopy analyzer (e.g., LAI-2000) frequently underestimate LAI values in comparison with the reference. The needle technique is easy to use in deciduous stands where the litter completely decomposes each year (e.g., oak and beech stands). However, calibration based on litter traps or direct destructive methods is necessary. The plant canopy analyzer is a commonly used device for performing LAI estimation in ecology, forestry, and agriculture, but is subject to potential error due to foliage clumping and the contribution of woody elements in the field of view (FOV) of the sensor. Eliminating these potential error sources was discussed. The plant canopy analyzer is a very suitable device for performing LAI estimations at the high spatial level, observing a seasonal LAI dynamic, and for long-term monitoring of LAI.

An accurate estimation of leaf area index (LAI) is crucial for many models of material and energy fluxes within plant ecosystems and between an ecosystem and the atmospheric boundary layer. Therefore, three methods (litter traps, needle technique, and PCA) for taking precise LAI measurements were in the presented protocol.

在纯落叶支架中使用三种截然不同的方法估算叶面积指数

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for ...

Investigation of Xenobiotics Metabolism In Salix alba Leaves via Mass Spectrometry Imaging

The method presented uses mass spectrometry imaging (MSI) to establish the metabolic profile of S. alba leaves when exposed to xenobiotics. Using a non-targeted approach, plant metabolites and xenobiotics of interest are identified and localized in plant tissues to uncover&#160;specific distribution patterns. Then, in silico prediction of potential metabolites (i.e., catabolites and conjugates) from the identified xenobiotics is performed. When a xenobiotic metabolite is located in the tissue, the type of enzyme involved in its alteration by the plant is recorded. These results were used to describe different types of biological reactions occurring in S. alba leaves in response to xenobiotic accumulation in the leaves. The metabolites were predicted in two generations, allowing the documentation of successive biological reactions to transform xenobiotics in the leaf tissues.

Investigation of Xenobiotics Metabolism In Salix alba Leaves via Mass Spectrometry Imaging

This method uses mass spectrometry imaging (MSI) to understand metabolic processes in S. alba leaves when exposed to xenobiotics. The method allows the spatial localization of compounds of interest and their predicted metabolites within specific, intact tissues.

通过质谱成像对 萨利克斯阿尔巴 叶中异种生物代谢的调查

The method presented uses mass spectrometry imaging (MSI) to establish the metabolic profile of S. alba leaves when exposed to xenobiotics. Using a ...