测量除草剂代谢的双子叶杂草价格与切下的叶片分析
Summary
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.
Abstract
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 –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.
Introduction
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。
| Waterhemp人口 | 缩写 | 表型硝磺草酮 | 硝磺草酮耐药机制 | 表型氟嘧磺隆 | 氟嘧磺隆抗性机制 |
| 麦克莱恩县耐 | MCR | 耐 | 代谢* | 耐 | 代谢 |
| 亚当斯县，耐 | ACR | SENSIT香港专业教育学院 | – | 耐 | 在ALS 14靶位点突变 |
| 韦恩县敏感 | WCS | 灵敏的 | – | 灵敏的 | – |
*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）。
Protocol
Representative Results
Discussion
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?…
Declarações
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Agar | Sigma-Aldrich | A1296 | for pre-germinating seeds |
| Potting medium | Sun Gro Horticulture | 49040233 | for plant growth |
| Nutricote | Agrivert | TOTAL BLEND 13-13-13 T100 | slow-release fertilizer |
| Growth chamber E15 | Controlled Environments Limited | 20207 | plant culturing |
| Tris base | Fisher Scientific | BP152-500 | buffer for excised leaves |
| HCl (concentrated) | Fisher Scientific | A144500 | adjust pH of buffer |
| Murashige and Skoog (MS) salts | Sigma-Aldrich | M0404 | incubation of excised leaves |
| Methanol | Fisher Scientific | A452-4 | leaf washes after incubation |
| Acetone | Sigma-Aldrich | 179124 | plant extractions |
| Acetonitrile (HPLC grade) | Macron Fine Chemicals | MKH07610 | HPLC mobile phase |
| Formic acid | Mallinckrodt Analytical | MK259205 | acidify mobile phase pH |
| Micro-centrifuge | Eppendorf | 5417R | 1.5 or 2.0 mL tubes |
| Centrifuge (temperature controlled) | Eppendorf | 5810R | 15 or 50 mL tubes |
| Polypropylene centrifuge tube | Corning Inc. | 430790 | 15 mL, sterile |
| Rotary evaporator | BÜCHI | R200 | concentrate plant samples |
| Liquid scintillation spectrometry (LSS) | Packard Instruments | 104470 | quantify 14C |
| High-performance liquid chromatography | Perkin Elmer | N2910401 | resolve herbicide metabolites |
| Flow scintillation analyzer | LabLogic System | 1103303 | for HPLC analysis of 14C |
| Hypersil Gold C18 column | Thermo-Scientific | 03-050-522 | reversed phase |
| Ultima-Flo M cocktail | Perkin Elmer | 6013579 | for Flow-scintillation analyzer |
| Scintillation Cocktail (ScintiVerse BD) | Fisher Scientific | SX18 | for LSS; biodegradable |
| Laboratory homogenizer | Kinematica | CH-6010 | homogenize leaf samples |
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