This work was supported by the Natural Sciences and Engineering Council of Canada (NSERC), the Canada Research Chair (CRC) program, and the Canadian Foundation for Innovation (CFI).

<html> 1，植物文化和准备<ol><li>大麦种植水培苗7天的气候控制生长室（详见10）。 注意：要考虑审查的植物在不同的发育阶段，因为营养需求会随着年龄的变化是很重要的。 </li><li>前一天的实验，捆绑了几个苗在一起，使一个单一的重复（每包3植物的DI，每束6植物CATE）。丛苗通过包装有2厘米件聚乙烯管的周围芽的基部，并确保管道用胶带来创建一个“围脖”。 注：每捆植物的数目可以根据实验条件10,13,14而有所不同。集束做是为了提高统计和测量精度，特别是当根质量和/或比活性也低。 </li></ol> 2，准备实验方案/材料内容“&gt;注：以下是典型的执行前1天的实验。 <ol><li>对于直接投资，备齐下列各项：预贴标，标签和解吸的解决方案（详见11），离心管（离心式脱水干燥的植物样品），和样品瓶（植物材料和比活度[S O;见下文]）。曝气和混合所有的解决方案。 </li><li>对于美食，备齐下列各项：嗯混合，加气的标签和洗脱解决方案（有关详细信息，请参阅第10），外排液漏斗，离心管（离心式脱水干燥的植物样品），和样品瓶（为洗脱液，植物样品，并确定S 0和稀释因子[D F;见下文]）。 </li></ol> 3，放射性示踪剂准备注意：以下的安全步骤，应先与放射性工作的措施。 <ol><li>确保radioac的要求略去材料许可被理解和遵守。穿戴适当的安全设备（ 如护目镜，手套，实验室外套，铅背心/领）和剂量计（ 例如 ，TLD环和徽章）。设置屏蔽（ 即有机玻璃和铅砖），并执行它后面的放射性工作。确保盖革 - 米勒计数存在以定期监测污染。 </li><li> 42 K的制备+ <ol><li>放置在天平上一个干净，干燥的烧杯中。零余额。 </li><li>从包装中取出示踪剂小瓶（42 K 2 CO 3 20毫居里，呈粉末状）和倒示踪剂放入烧杯中。注意质量的。 </li><li>吸取19.93毫升DH 2 O，随后0.07毫升H 2 SO 4，放入烧杯中。这将推动下列化学反应： 42，K 2 CO 3的 （S）+ H 2 SO 4（L）+ H 2 O（L）→42 </s达&gt; K 2 SO 4 （L）+ CO 2（V）+ 2H 2 O（L） </li><li>计算出的放射性原液的浓度，给定的K 2 CO 3的质量和分子量，并且体积（20毫升）中。 注：如果有13的NH 3月 13日 NH 4 +的示踪剂是产于通过水的氧原子的质子轰击回旋加速器工作（通常产生100-200毫居里活动;生产的详细信息，请参阅第12）。由于14 NH 十四分之三 NH + 4的量是非常低的，这些解决方案中，N浓度的储备溶液是可以忽略不计。 </li></ol></li></ol> 4，直接流入（DI）测量<ol><li>为42 K +，吸取放射性原液到达K +的所需最终浓度为标记溶液所需的量。 <ol><li>为13的NH13分之3NH + 4，吸管少量（&lt;0.5毫升）到标记溶液。允许的标签解决方案，以充分混匀（通过曝气）。 </li></ol></li><li>吸管标签解决方案1毫升子样品放入样品瓶（共4个样本）重复3次。 <ol><li>测量放射性小瓶（在“每分钟计数”，CPM），用γ计数器。确保该计数器进行编程，使得每分钟的读数被用于校正同位素衰变（这是针对这样短暂的示踪剂特别重要）。 </li><li>通过计算四个样本的计数（CPM毫升-1），通过底物的浓度在溶液中除（微摩尔毫升-1）计算S 0（表示为每分钟微摩尔-1）。 </li></ol></li><li>沉浸在预标记（非放射性）溶液根部5分钟，进行预平衡的植物在试验条件下（见例如 ，10,13,14为变化前的标签时）。 </li><li>沉浸在根标签（放射性）溶液中5分钟。 注：标注时间可以根据实验3,4,7-10而有所不同。 </li><li>传送根部到解吸溶液中5秒以去除大量的表面附着的放射性。转移的根放入的解吸液的第二烧杯5分钟以外示踪剂进一步明确根。 </li><li>解剖和独立笋，竹笋基部和根。 </li><li>在低速放置于离心管中，根和自旋的样品30秒，临床级离心机（〜5000 XG），以除去表面和间隙水。 </li><li>称取根（鲜重，FW）。 </li><li>放射性计数植物样品中（拍摄，拍摄基础和根本，见步骤4.2.1）。 </li><li>计算的通量。使用下面的公式计算出流入到植物 Φ= Q * / S 0重量Ł 其中Φ是通量（微摩尔克-1小时-1），Q *为示踪剂在组织中累积的量（CPM，通常在根，芽，和基底的拍摄，结合），S o为标记溶液（CPM微摩尔的比活性- 1），w是根鲜重（g），而t L是标记时间（hr）。 注：更复杂的计算可以由标签和解吸过程中考虑到从根部同时示踪剂流出的基础上，从CATE获得的参数（见下文;有关详细信息，请参阅第4）。 </li></ol> 5，房室分析示踪流出（CATE）测量<ol><li>准备标签解决方案和措施，S 0（见步骤4.1 - 4.2以上）。 </li><li>测量稀释因子（D F）。 注意：通常情况下，相对于所述检测器中的γ射线计数样品的位置可以影响量辐射测量。详情请参阅讨论。 <ol><li>测定S 0后，添加19为h毫升2 O到各样品（使得最终体积=洗脱液体积= 20毫升）中。放射性计数每20毫升样品中（见步骤4.2.1）。 </li><li>通过将1毫升样品的平均CPM由20毫升样品的平均CPM计算D F。 </li></ol></li><li>沉浸在标签解决方案的根为1小时。 </li><li>从标签解决方案和转让植物去除植物外排液漏斗中，确保所有根材料的漏斗内。要流出漏斗一侧轻轻的安全设备通过应用一小片胶带在塑料套。 </li><li>轻轻倒出所述第一洗脱液进入漏斗。启动定时器（计数）。 </li><li>打开该轴颈和后15秒收集在样品瓶的洗脱物（注：洗脱时间会有所不同，见下文）。关闭插口。轻轻倒出下一洗脱液进入漏斗。 </li><li> Repea吨步骤5.6的溶出系列，其中如下，从第一到最后的洗脱物的剩余部分：15秒（4次），20秒（3倍），30秒（两次），40秒（一次），50秒（一次），1分钟（25次），为29.5分钟的总洗脱时间 注：解吸系列可根据实验条件7-10,13,14而有所不同。 </li><li>一旦洗脱协议完成后，收获的植物（步骤4.6 - 4.8以上）。 </li><li>计数放射性洗脱液和植物样品中的伽马射线计数器（阅读每个洗脱液用D F相乘，见5.2）。 </li><li>情节示踪剂释放（CPM克（根FW）-1分-1）为洗脱时间的函数。对于稳态条件下，进行线性回归和通量计算，半衰期交流和池大小（详见6-9）。 </li></ol>

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The authors have nothing to disclose.

这表现在上面的例子中，放射性示踪方法是一个功能强大的测量营养素和有毒物质在植物中的单向磁通的装置， 图1示出的NH 3流入可超过225微摩尔克-1小时-1，这也许是达到最高善意的跨膜通量曾报道在植物中的系统13，但是，如果测定仅净通量这个磁通量的大小将是不可见的。这是因为，NH 3的大流出发生在同一时间涌入，在徒劳...

营养物质和有毒物质的吸收和分布强烈地影响植物生长。因此，底层传输过程对调查构成的植物生物学和研究农业科学1,2的一个主要领域，特别是在营养优化和环境压力的环境中（ 例如 ，盐胁迫，铵毒性）。对于通量植物的测量方法中，最主要是利用放射性同位素示踪物，这是显著在20世纪50年代开发的。（ 例如 ，3），一直持续到今天被广泛使用。其他方法，如养分耗竭的从根介质和/或积累在组织中，使用的离子选择性微电极的振动测量，如MIFE（微电极的离子通量的估计）和SIET（扫描离子选择性电极法），并使用离子选择性荧光染料，也被广泛应用，但在他们的检测净感能力是有限XES（ 即 ，流入和流出之间的差异）。使用放射性同位素的，另一方面，允许研究者的独特能力来分离和量化单向通量，这可以被用来解决动力学参数（ 例如 ，K M和V max）和洞察能力，热力学，机制的运输系统和调节。与放射性示踪剂制成单向磁通测量条件下在相反的方向上的磁通量是高下特别有用的，并且细胞内池的周转迅速4。此外，放射性示踪方法允许较高的底物浓度下进行测量时，与许多其它的技术（参见“讨论”，下同），因为所跟踪的同位素是针对相同的元件的另一同位素背景观察。 这里，我们提供的单向和n中的放射性同位素的测量的详细步骤矿质营养等通量和毒物的完整植株。重点是对钾（K +）通量测量，植物大量营养5，和氨/铵制成（NH 3 / NH 4 +），另一个常量营养素是，然而，中毒性时在高浓度下（ 例如本，1 10毫米）2。我们将用放射性同位素42的K +（叔2 = 12.36小时）和13 NH13分之3NH 4 +（T 2 = 9.98分钟），分别在该模型系统中的大麦的完整的苗（ 大麦 Ļ ），在两个关键协议的描述：通过示踪剂流出（CATE）直接流入（DI）和室分析。我们应该注意到，从本文简单介绍了需要执行的每个协议的步骤开始。每种技术在适当的时候，提供计算和理论的简要说明，但详细的论述，的背景和理论能够在这个问题4,6-9几个关键物品被发现。重要的是，这些协议大致上可转移到磁通的其他营养素/毒物分析（ 例如 ，24的Na +，22的Na +，86 Rb的+，13 NO 3 - ）和其它植物物种的，尽管有一些需要说明的（参见下文） 。我们还强调，放射性物质工作的所有研究者必须在通过自己的机构的电离辐射安全监管机构设置许可工作的重要性。

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:103px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gamma counter</td>
			<td style="width:103px;">Perkin Elmer</td>
			<td style="width:119px;"></td>
			<td>Model: Wallac 1480 Wizard 3&#34;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Geiger-M&#252;ller counter</td>
			<td style="width:103px;">Ludlum Measurements Inc.</td>
			<td style="width:119px;"></td>
			<td>Model 3 survey meter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">400-mL glass beakers</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">89000-206</td>
			<td>For pre-absorption, absorption, and desorption solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass funnel</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">89000-466</td>
			<td>For efflux funnel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Large tubing</td>
			<td style="width:103px;">VWR</td>
			<td align="right" style="width:119px;">529297</td>
			<td>For efflux funnel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Medium tubing</td>
			<td style="width:103px;">VWR</td>
			<td align="right" style="width:119px;">684783</td>
			<td>For bundling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Small tubing</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">63013-541</td>
			<td>For aeration</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Aeration manifold</td>
			<td style="width:103px;">Penn Plax Air Tech</td>
			<td style="width:119px;">vat 5.5</td>
			<td>To control/distribute pressurized air into solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass scintillation vials</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">66022-128</td>
			<td>For gamma counting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass centrifuge tubes</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">47729-576</td>
			<td>For spin-drying root samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Kimwipes</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">470173-504</td>
			<td>For spin-drying root samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dissecting scissors</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">470001-828</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Forceps</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">470005-496</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Low-speed clinical centrifuge</td>
			<td style="width:103px;">International Equipment Co.</td>
			<td style="width:119px;">76466M-4</td>
			<td>For spin-drying root samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1-mL pipette</td>
			<td style="width:103px;">Gilson</td>
			<td style="width:119px;">F144493</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10-mL pipette</td>
			<td style="width:103px;">Gilson</td>
			<td style="width:119px;">F144494</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1-mL pipette tips</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">89079-470</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10-mL pipette tips</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">89087-532</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Analytical balance</td>
			<td style="width:103px;">Mettler toledo</td>
			<td style="width:119px;">PB403-S/FACT</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

图1示出了使用DI技术（13 N）中的等温线，用于NH 3的流入，在高生长完好大麦幼苗的根部（10毫米）的NH 4 +，和任一低（0.02毫摩尔）或高（5毫）K +。的等温线显示米氏动力学时NH 3通量被绘制为外部的NH 3浓度（[NH 3]分机号码 ;可以通过改变调节溶液的pH 13）的函数。 NH 3通量分别为显著更高的低K +</su...

Watch this Scientific Journal Video about Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers at JoVE.com

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

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. ...

measuring-fluxes-mineral-nutrients-toxicants-plants-with-radioactive

Research

JoVE Journal

Environment

11.2K 次观看.  University of Toronto. 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.

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

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture organisms, such as hyphae-building fungi and anaerobic bacteria, has hastened the use of recombinant technologies in this field. Plant expression methods are a desirable system for large-scale production of enzymes and other industrially useful proteins. Herein, methods for the transient expression of a fungal endoglucanase, Trichoderma reesei Cel5A, in Nicotiana tabacum are demonstrated. Successful protein expression is shown, monitored by fluorescence using an mCherry-enzyme fusion protein. Additionally, a set of basic tests are used to examine the activity of transiently expressed T. reesei Cel5A, including SDS-PAGE, Western blotting, zymography, as well as&#160;fluorescence and dye-based substrate degradation assays. The system described here can be used to produce an active cellulase in a short time period, so as to assess the potential for further production in plants through constitutive or inducible expression systems.

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Tobacco plants were used to produce a fungal cellulase, TrCel5A, via a transient expression system. The expression could be monitored using a fluorescent fusion protein, and the protein activity was characterized post-expression.

重组纤维素酶Cel5A从表达<em&gt;里氏木霉</em&gt;在烟草

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture ...

科研

环境科学

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.

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 ...

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in nutrient and gas cycles. After cores with intact sediment-water interfaces are collected, they are submerged in incubation tanks and kept under aerobic conditions at in situ temperatures. To initiate a time course of overlying water chemistry, cores are sealed without bubbles using a top cap with a suspended stirrer. Time courses of 4-7 sample points are used to determine the rate of sediment water exchange. Artificial illumination simulates day-time conditions for shallow photosynthetic sediments, and in conjunction with dark incubations can provide net exchanges on a daily basis. The net measurement of N2 is made possible by sampling a time course of dissolved gas concentrations, with high precision mass spectrometric analysis of N2:Ar ratios providing a means to measure N2 concentrations. We have successfully applied this approach to lakes, reservoirs, estuaries, wetlands and storm water ponds, and with care, this approach provides valuable information on biogeochemical balances in aquatic ecosystems.

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

Here, we present small core incubations for the measurement of sediment-water gas and solute exchange. These will provide reliable measurements of sediment-water exchange that assess the role of sediment in influencing biological and biogeochemical processes in aquatic ecosystems.

Ø的底栖交易所<sub&gt; 2</sub&gt;，N<sub&gt; 2</sub&gt;和营养盐使用小的核心孵化

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in ...

Continuous Instream Monitoring of Nutrients and Sediment in Agricultural Watersheds

Pollutant concentrations and loads in watersheds vary considerably with time and space. Accurate and timely information on the magnitude of pollutants in water resources is a prerequisite for understanding the drivers of the pollutant loads and for making informed water resource management decisions. The commonly used &#34;grab sampling&#34; method provides the concentrations of pollutants at the time of sampling (i.e., a snapshot concentration) and may under- or overpredict the pollutant concentrations and loads. Continuous monitoring of nutrients and sediment has recently received more attention due to advances in computing, sensing technology, and storage devices. This protocol demonstrates the use of sensors, sondes, and instrumentation to continuously monitor in situ nitrate, ammonium, turbidity, pH, conductivity, temperature, and dissolved oxygen (DO) and to calculate the loads from two streams (ditches) in two agricultural watersheds. With the proper calibration, maintenance, and operation of sensors and sondes, good water quality data can be obtained by overcoming challenging conditions such as fouling and debris buildup. The method can also be used in watersheds of various sizes and characterized by agricultural, forested, and/or urban land.

With the advancement of technology and the rise in end-user expectations, the need and use of higher temporal resolution data for pollutant load estimation has increased. This protocol describes a method for continuous in situ water quality monitoring to obtain higher temporal resolution data for informed water resource management decisions.

农业流域营养盐和泥沙的连续河道监测

Pollutant concentrations and loads in watersheds vary considerably with time and space. Accurate and timely information on the magnitude of pollutants in ...

Assessment of Methane and Nitrous Oxide Fluxes from Paddy Field by Means of Static Closed Chambers Maintaining Plants Within Headspace

This protocol describes the measurement of greenhouse gas (GHG) emissions from paddy soils using the static closed chamber technique. This method is based on the diffusion theory. A known volume of air overlaying a defined soil area is enclosed within a parallelepiped cover (named &#34;chamber&#34;), for a defined period of time. During this enclosure period, gases (methane (CH4) and nitrous oxide (N2O)) move from soil pore air near their microbial source (i.e., methanogens, nitrifiers, denitrifiers) to the chamber headspace, following a natural concentration gradient. Fluxes are then estimated from chamber headspace concentration variations sampled at regular intervals throughout the enclosure and then analyzed with gas chromatography. Among the techniques available for GHG measurement, the static closed chamber method is suitable for plot experiments, as it does not require large homogenously treated soil areas. Furthermore, it is manageable with limited resources and can identify relationships among ecosystem properties, processes, and fluxes, especially when combined with GHG driving force measurements. Nevertheless, with respect to the micrometeorological method, it causes a minimal but still unavoidable soil disturbance, and allows a minor temporal resolution. Several phases are key to the method implementation: i) chamber design and deployment, ii) sample handling and analyses, and iii) flux estimation. Technique implementation success in paddy fields demands adjustments for field flooding during much of the cropping cycle, and for rice plant maintenance within the chamber headspace during measurements. Therefore, the additional elements to be considered with respect to the usual application of non-flooded agricultural soils consist of devices for: i) avoiding any unintended water disturbance that could overestimate fluxes, and ii) including rice plants within the chamber headspace to fully consider gases emitted through aerenchyma transportation.

The overall goal of this protocol is to measure greenhouse gas emissions from paddy fields using the static closed chamber technique. The measurement system needs specific adjustments due to the presence of both a permanent water layer in the field and of the plants within the chamber headspace.

用静止封闭室维持顶空装置对稻田甲烷和一氧化二氮通量的评价

This protocol describes the measurement of greenhouse gas (GHG) emissions from paddy soils using the static closed chamber technique. This method is based ...

Protocol for Producing Three-Dimensional Infrared Video of Freezing in Plants

Freezing in plants can be monitored using infrared (IR) thermography, because when water freezes, it gives off heat. However, problems with color contrast make 2-dimensions (2D) infrared images somewhat difficult to interpret. Viewing an IR image or the video of plants freezing in 3 dimensions (3D) would allow a more accurate identification of sites for ice nucleation as well as the progression of freezing. In this paper, we demonstrate a relatively simple means to produce a 3D infrared video of a strawberry plant freezing. Strawberry is an economically important crop that is subjected to unexpected spring freeze events in many areas of the world. An accurate understanding of the freezing in strawberry will provide both breeders and growers with more economical ways to prevent any damage to plants during freezing conditions.
The technique involves a positioning of two IR cameras at slightly different angles to film the strawberry freezing. The two video streams will be precisely synchronized using a screen capture software that records both cameras simultaneously. The recordings will then be imported into the imaging software and processed using an anaglyph technique. Using red-blue glasses, the 3D video will make it easier to determine the precise site of ice nucleation on leaf surfaces.

Here, we present a protocol to image a strawberry plant freezing in 3 dimensions. Two infrared cameras positioned at slightly different angles are used to produce a red-blue anaglyph video to observe the freezing of the plant in 3 dimensions.

植物冷冻三维红外视频制作协议

Freezing in plants can be monitored using infrared (IR) thermography, because when water freezes, it gives off heat. However, problems with color contrast ...

An Optimized Rhizobox Protocol to Visualize Root Growth and Responsiveness to Localized Nutrients

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive harvest or expensive equipment. We present a customizable and affordable rhizobox method that allows the non-destructive visualization of root growth over time and is particularly well-suited to studying root plasticity in response to localized resource patches. The method was validated by assessing maize genotypic variation in plasticity responses to patches containing 15N-labeled legume residue. Methods are described to obtain representative developmental measurements over time, measure root length density in resource-containing and control patches, calculate root growth rates, and determine 15N recovery by plant roots and shoots. Advantages, caveats, and potential future applications of the method are also discussed. Although care must be taken to ensure that experimental conditions do not bias root growth data, the rhizobox protocol presented here yields reliable results if carried out with sufficient attention to detail.

Visualizing and measuring root growth in situ is extremely challenging. We present a customizable rhizobox method to track root development and proliferation over time in response to nutrient enrichment. This method is used to analyze maize genotypic differences in root plasticity in response to an organic nitrogen source.

一种优化的 Rhizobox 协议, 用于可视化根系生长和对局部营养素的响应

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive ...

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 ...

Xylem Water Distribution in Woody Plants Visualized with a Cryo-scanning Electron Microscope

A scanning electron microscope installed cryo-unit (cryo-SEM) allows specimen observation at subzero temperatures and has been used for exploring water distribution in plant tissues in combination with freeze fixation techniques using liquid nitrogen (LN2). For woody species, however, preparations for observing the xylem transverse-cut surface involve some difficulties due to the orientation of wood fibers. Additionally, higher tension in the water column in xylem conduits can occasionally cause artifactual changes in water distribution, especially during sample fixation and collection. In this study, we demonstrate an efficient procedure to observe the water distribution within the xylem of woody plants in situ&#160;by using a cryostat and cryo-SEM. At first, during sample collection, measuring the xylem water potential should determine whether high tension is present in the xylem conduits. When the xylem water potential is low (&#60; ca. &#8722;0.5 MPa), a tension relaxation procedure is needed to facilitate better preservation of the water status in xylem conduits during sample freeze fixation. Next, a watertight collar is attached around the tree stem and filled with LN2&#160;for freeze fixation of the water status of xylem. After harvesting, care should be taken to ensure that the sample is preserved frozen while completing the procedures of sample preparation for observation. A cryostat is employed to clearly expose the xylem transverse-cut surface. In cryo-SEM observations, time adjustment for freeze-etching is required to remove frost dust and accentuate the edge of the cell walls on the viewing surface. Our results demonstrate the applicability of cryo-SEM techniques for the observation of water distribution within xylem at cellular and subcellular levels. The combination of cryo-SEM with non-destructive in situ observation techniques will profoundly improve the exploration of woody plant water flow dynamics.

Observing the water distribution within the xylem provides significant information regarding water flow dynamics in woody plants. In this study, we demonstrate the practical approach to observe xylem water distribution in situ&#160;by using a cryostat and cryo-SEM, which eliminates artifactual changes in the water status during sample preparation.

木本植物的赛莱默水分布,用低温扫描电子显微镜可视化

A scanning electron microscope installed cryo-unit (cryo-SEM) allows specimen observation at subzero temperatures and has been used for exploring water ...

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term effects are very important in judging their consequences in the environment and on human health. In demonstrating long-term influences, effects of chemicals over generations in recent studies provide new insight. Here, we describe protocols for studying effects of chemicals over multiple generations using free-living nematode Caenorhabditis elegans. Two aspects are presented: (1) trans-generational (TG) and (2) multi-generational effect studies, the latter of which is separated to multi-generational exposure (MGE) and multi-generational residual (MGR) effect studies. The TG effect study is robust with a simple purpose to determine whether chemical exposure to parents can result in any residual consequences on offspring. After the effects are measured on parents, sodium hypochlorite solutions are used to kill the parents and keep the offspring so as to facilitate effect measurement on the offspring. The TG effect study is used to determine whether the offspring are affected when their parent is exposed to the pollutants. The MGE and MGR effect study is systematical used to determine whether continuous generational exposure can result in adaptive responses in offspring over generations. Careful pick-up and transfer are used to distinguish generations to facilitate effect measurement on each generation. We also combined protocols to measure locomotion behavior, reproduction, lifespan, biochemical and gene expression changes. Some example experiments are also presented to illustrate the trans- and multi-generational effect studies.

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Trans- and multi-generational effects of persistent chemicals are essential in judging their long-term consequences in the environment and on the human health. We provide novel detailed methods for studying trans- and multi-generational effects using free-living nematode Caenorhabditis elegans.

使用卡内哈布迪炎酶研究有毒物质的跨代和多代效应

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term ...