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。

<table><tbody><tr><td>Gamma counter</td><td>Perkin Elmer</td><td></td><td>Model: Wallac 1480 Wizard 3&quot;</td></tr><tr><td>Geiger-M&amp;uuml;ller counter</td><td>Ludlum Measurements Inc.</td><td></td><td>Model 3 survey meter</td></tr><tr><td>400 ml glass beakers</td><td>VWR</td><td>89000-206</td><td>For pre-absorption, absorption, and desorption solutions</td></tr><tr><td>Glass funnel</td><td>VWR</td><td>89000-466</td><td>For efflux funnel</td></tr><tr><td>Large tubing</td><td>VWR</td><td>529297</td><td>For efflux funnel</td></tr><tr><td>Medium tubing</td><td>VWR</td><td>684783</td><td>For bundling</td></tr><tr><td>Small tubing</td><td>VWR</td><td>63013-541</td><td>For aeration</td></tr><tr><td>Aeration manifold</td><td>Penn Plax Air Tech</td><td>vat 5.5</td><td>To control/distribute pressurized air into solutions</td></tr><tr><td>Glass scintillation vials</td><td>VWR</td><td>66022-128</td><td>For gamma counting</td></tr><tr><td>Glass centrifuge tubes</td><td>VWR</td><td>47729-576</td><td>For spin-drying root samples</td></tr><tr><td>Kimwipes</td><td>VWR</td><td>470173-504</td><td>For spin-drying root samples</td></tr><tr><td>Dissecting scissors</td><td>VWR</td><td>470001-828</td><td></td></tr><tr><td>Forceps</td><td>VWR</td><td>470005-496</td><td></td></tr><tr><td>Low-speed clinical centrifuge</td><td>International Equipment Co.</td><td>76466M-4</td><td>For spin-drying root samples</td></tr><tr><td>1 ml pipette</td><td>Gilson</td><td>F144493</td><td></td></tr><tr><td>10 ml pipette</td><td>Gilson</td><td>F144494</td><td></td></tr><tr><td>1 ml pipette tips</td><td>VWR</td><td>89079-470</td><td></td></tr><tr><td>10 ml pipette tips</td><td>VWR</td><td>89087-532</td><td></td></tr><tr><td>Analytical balance</td><td>Mettler toledo</td><td>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

Universidad San Sebastian

Research

JoVE Journal

Environment

11.3K 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

Measuring Carbon-based Contaminant Mineralization Using Combined CO2 Flux and Radiocarbon Analyses

A method is described which uses the absence of radiocarbon in industrial chemicals and fuels made from petroleum feedstocks which frequently contaminate the environment. This radiocarbon signal &#8212; or rather the absence of signal &#8212; is evenly distributed throughout a contaminant source pool (unlike an added tracer) and is not impacted by biological, chemical or physical processes (e.g., the 14C radioactive decay rate is immutable). If the fossil-derived contaminant is fully degraded to CO2, a harmless end-product, that CO2 will contain no radiocarbon. CO2 derived from natural organic matter (NOM) degradation will reflect the NOM radiocarbon content (usually &#60;30,000 years old). Given a known radiocarbon content for NOM (a site background), a two end-member mixing model can be used to determine the CO2 derived from a fossil source in a given soil gas or groundwater sample. Coupling the percent CO2 derived from the contaminant with the CO2 respiration rate provides an estimate for the total amount of contaminant degraded per unit time. Finally, determining a zone of influence (ZOI) representing the volume from which site CO2 is collected allows determining the contaminant degradation per unit time and volume. Along with estimates for total contaminant mass, this can ultimately be used to calculate time-to-remediate or otherwise used by site managers for decision-making.

Measuring Carbon-based Contaminant Mineralization Using Combined CO2 Flux and Radiocarbon Analyses

A protocol is described wherein CO2 mineralized from organic contaminant (derived from petroleum feedstocks) biodegradation is trapped, quantified, and analyzed for 14C content. A model is developed to determine CO2 capture zone's spatial extent. Spatial and temporal measurements allow integrating contaminant mineralization rates for predicting remediation extent and time.

测量碳为基础的污染物矿化结合使用CO<sub&gt; 2</sub&gt;助焊剂和放射性碳分析

A method is described which uses the absence of radiocarbon in industrial chemicals and fuels made from petroleum feedstocks which frequently contaminate ...

科研

环境科学

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

测定植物铁含量的比色法

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

生物化学

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. A stable isotope approach is necessary to follow and quantify the fate of fertilizer derived N (FDN) through the soil-crop system. The purpose of this paper is to describe a small-plot research&#160;design utilizing non-confined 15N enriched microplots for multiple soil and plant sampling events over two growing seasons and provide sample collection, handling, and processing protocols for total 15N analysis. The methods were demonstrated using a replicated study from south-central Minnesota planted to corn (Zea mays L.). Each treatment consisted of six corn rows (76 cm row-spacing) 15.2 m long with a microplot (2.4 m by 3.8 m) embedded at one end. Fertilizer-grade urea was applied at 135 kg N&#8729;ha-1 at planting, while the microplot received urea enriched to 5 atom % 15N. Soil and plant samples were taken several times throughout the growing season, taking care to minimize cross-contamination by using separate tools and physically separating unenriched and enriched samples during all procedures. Soil and plant samples were dried, ground to pass through a 2 mm screen, and then ground to a flour-like consistency using a roller jar mill. Tracer studies require additional planning, sample processing time and manual labor, and incur higher costs for 15N enriched materials and sample analysis than traditional N studies. However, using the mass balance approach, tracer studies with multiple in-season sampling events allow the researcher to estimate FDN distribution through the soil-crop system and estimate unaccounted-for FDN from the system.

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

A microplot design for 15N tracer research is described to accommodate multiple in-season plant and soil sampling events. Soil and plant sample collection and processing procedures, including grinding and weighing protocols, for 15N analysis are put forth.

微图设计及植物和土壤样品制备，进行15次氮气分析

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. ...

Two-Dimensional Visualization and Quantification of Labile, Inorganic Plant Nutrients and Contaminants in Soil

We describe a method for two-dimensional (2D) visualization and quantification of the distribution of labile (i.e., reversibly adsorbed) inorganic nutrient (e.g., P, Fe, Mn) and contaminant (e.g., As, Cd, Pb) solute species in the soil adjacent to plant roots (the &#8216;rhizosphere&#8217;) at sub-millimeter (~100 &#181;m) spatial resolution. The method combines sink-based solute sampling by the diffusive gradients in thin films (DGT) technique with spatially resolved chemical analysis by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The DGT technique is based on thin hydrogels with homogeneously distributed analyte-selective binding phases. The variety of available binding phases allows for the preparation of different DGT gel types following simple gel fabrication procedures. For DGT gel deployment in the rhizosphere, plants are grown in flat, transparent growth containers (rhizotrons), which enable minimal invasive access to a soil-grown root system. After a pre-growth period, DGT gels are applied to selected regions of interest for in situ solute sampling in the rhizosphere. Afterwards, DGT gels are retrieved and prepared for subsequent chemical analysis of the bound solutes using LA-ICP-MS line-scan imaging. Application of internal normalization using 13C and external calibration using matrix-matched gel standards further allows for the quantification of the 2D solute fluxes. This method is unique in its capability to generate quantitative, sub-mm scale 2D images of multi-element solute fluxes in soil-plant environments, exceeding the achievable spatial resolution of other methods for measuring solute gradients in the rhizosphere substantially. We present the application and evaluation of the method for imaging multiple cationic and anionic solute species in the rhizosphere of terrestrial plants and highlight the possibility of combining this method with complementary solute imaging techniques.

This protocol presents a workflow for sub-mm 2D visualization of multiple labile inorganic nutrient and contaminant solute species using diffusive gradients in thin films (DGT) combined with mass spectrometry imaging. Solute sampling and high-resolution chemical analysis are described in detail for quantitative mapping of solutes in the rhizosphere of terrestrial plants.

土壤中实验室、无机植物养分和污染物的二维可视化和量化

We describe a method for two-dimensional (2D) visualization and quantification of the distribution of labile (i.e., reversibly adsorbed) inorganic ...

Automated 90Sr Separation and Preconcentration in a Lab-on-Valve System at Ppq Level

A quick, automated and portable system for the separation and determination of radiostrontium in aqueous samples, using Sr-resin and multi sequential flow injection analysis, has been developed. The concentrations of radioactive strontium were determined by flow scintillation counting, allowing for on-line and also on-site determination. The proposed system can determine radioactive strontium at industrial relevant levels without further modification using overall analysis time of less than 10 min per aqueous sample. The limit of the detection is 320 fg&#183;g-1 (1.7 Bq/g).

Automated 90Sr Separation and Preconcentration in a Lab-on-Valve System at Ppq Level

Here, we present the portable autoRAD platform for quick radiometric separation and determination of 90Sr, an important fission product highly pertinent to nuclear waste.

Ppq 级实验室系统中的自动90Sr 分离和预富集

A quick, automated and portable system for the separation and determination of radiostrontium in aqueous samples, using Sr-resin and multi sequential flow ...

化学

A Method to Preserve Wetland Roots and Rhizospheres for Elemental Imaging

Roots extensively interact with their soil environment but visualizing such interactions between roots and the surrounding rhizosphere is challenging. The rhizosphere chemistry of wetland plants is particularly challenging to capture because of steep oxygen gradients from the roots to the bulk soil. Here a protocol is described that effectively preserves root structure and rhizosphere chemistry of wetland plants through slam-freezing and freeze drying. Slam-freezing, where the sample is frozen between copper blocks pre-cooled with liquid nitrogen, minimizes root damage and sample distortion that can occur with flash-freezing while still minimizing chemical speciation changes. While sample distortion is still possible, the ability to obtain multiple samples quickly and with minimal cost increases the potential to obtain satisfactory samples and optimizes imaging time. The data show that this method is successful in preserving reduced arsenic species in rice roots and rhizospheres associated with iron plaques. This method can be adopted for studies of plant-soil relationships in a wide variety of wetland environments that span concentration ranges from trace-element cycling to phytoremediation applications.

We describe a protocol to sample, preserve, and section intact roots and the surrounding rhizosphere soil from wetland environments using rice (Oryza sativa L.) as a model species. Once preserved, the sample can be analyzed using elemental imaging techniques, such as synchrotron X-ray fluorescence (XRF) chemical speciation imaging.

元素成像保护湿地根和岩石圈的方法

Roots extensively interact with their soil environment but visualizing such interactions between roots and the surrounding rhizosphere is challenging. The ...

Workflow Based on the Combination of Isotopic Tracer Experiments to Investigate Microbial Metabolism of Multiple Nutrient Sources

Studies in the field of microbiology rely on the implementation of a wide range of methodologies. In particular, the development of appropriate methods substantially contributes to providing extensive knowledge of the metabolism of microorganisms growing in chemically defined media containing unique nitrogen and carbon sources. In contrast, the management through metabolism of multiple nutrient sources, despite their broad presence in natural or industrial environments, remains virtually unexplored. This situation is mainly due to the lack of suitable methodologies, which hinders investigations.
We report an experimental strategy to quantitatively and comprehensively explore how metabolism operates when a nutrient is provided as a mixture of different molecules, i.e., a complex resource. Here, we describe its application for assessing the partitioning of multiple nitrogen sources through the yeast metabolic network. The workflow combines information obtained during stable isotope tracer experiments using selected 13C- or 15N-labeled substrates. It first consists of parallel and reproducible fermentations in the same medium, which includes a mixture of N-containing molecules; however,a selected nitrogen source is labeled each time. A combination of analytical procedures (HPLC, GC-MS) is implemented to assess the labeling patterns of targeted compounds and to quantify the consumption and recovery of substrates in other metabolites. An integrated analysis of the complete dataset provides an overview of the fate of consumed substrates within cells. This approach requires an accurate protocol for the collection of samples&#8211;facilitated by a robot-assisted system for online monitoring of fermentations&#8211;and the achievement of numerous time-consuming analyses. Despite these constraints, it allowed understanding, for the first time, the partitioning of multiple nitrogen sources throughout the yeast metabolic network. We elucidated the redistribution of nitrogen from more abundant sources toward other N-compounds and determined the metabolic origins of volatile molecules and proteinogenic amino acids.

This protocol describes an experimental procedure to quantitatively and comprehensively investigate the metabolism of multiple nutrient sources. This workflow, based on a combination of isotopic tracer experiments and an analytical procedure, allows the fate of consumed nutrients and the metabolic origin of molecules synthetized by microorganisms to be determined.

基于同位素示踪实验结合的工作流研究多营养源的微生物代谢

Studies in the field of microbiology rely on the implementation of a wide range of methodologies. In particular, the development of appropriate methods ...

生物工程

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a fluorescently tagged-protein of interest is transiently expressed in plant tissue following biolistic delivery of its encoding DNA construct. The intra- and intercellular distribution of the tagged protein is then analyzed by confocal microscopy. We describe this technology in detail, providing step-by-step protocols to assay and evaluate the extent of symplastic protein transport in three plant species, Arabidopsis thaliana, Nicotiana benthamiana and N. tabacum (tobacco).

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Macromolecular trafficking between plant cells can be assessed by transiently expressing a fluorescently-tagged protein of interest and analyzing its intra- and intercellular distribution by confocal microscopy.

细胞的细胞大分子运输检测在植物利用基因枪法

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a ...

生物学

Hydroponics: A Versatile System to Study Nutrient Allocation and Plant Responses to Nutrient Availability and Exposure to Toxic Elements

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several crops, including lettuce and tomato. Within the plant research community, numerous hydroponic systems have been designed to study plant responses to biotic and abiotic stresses. Here we present a hydroponic protocol that can be easily implemented in laboratories interested in pursuing studies on plant mineral nutrition.
This protocol describes the hydroponic system set up in detail and the preparation of plant material for successful experiments. Most of the materials described in this protocol can be found outside scientific supply companies, making the set up for hydroponic experiments less expensive and convenient.
The use of a hydroponic growth system is most advantageous in situations where the nutrient media need to be well controlled and when intact roots need to be harvested for downstream applications. We also demonstrate how nutrient concentrations can be modified to induce plant responses to both essential nutrients and toxic non-essential elements.

Here, we present an easy-to-follow protocol to establish a successful hydroponic system for plant nutrition studies. This protocol has been extensively tested in Arabidopsis and can easily be adapted to other plant species to study specific nutritional requirements or the effect of non-essential elements on plant growth and development.

水培:一个多功能的系统来研究营养分配和植物响应养分有效性及暴露于有毒元素

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several ...

Neutron Radiography and Computed Tomography of Biological Systems at the Oak Ridge National Laboratory's High Flux Isotope Reactor

Neutrons have historically been used for a broad range of biological applications employing techniques such as small-angle neutron scattering, neutron spin echo, diffraction, and inelastic scattering. Unlike neutron scattering techniques that obtain information in reciprocal space, attenuation-based neutron imaging measures a signal in real space that is resolved on the order of tens of micrometers. The principle of neutron imaging follows the Beer-Lambert law and is based on the measurement of the bulk neutron attenuation through a sample. Greater attenuation is exhibited by some light elements (most notably, hydrogen), which are major components of biological samples. Contrast agents such as deuterium, gadolinium, or lithium compounds can be used to enhance contrast in a similar fashion as it is done in medical imaging, including techniques such as optical imaging, magnetic resonance imaging, X-ray, and positron emission tomography. For biological systems, neutron radiography and computed tomography have increasingly been used to investigate the complexity of the underground plant root network, its interaction with soils, and the dynamics of water flux in situ. Moreover, efforts to understand contrast details in animal samples, such as soft tissues and bones, have been explored. This manuscript focuses on the advances in neutron bioimaging such as sample preparation, instrumentation, data acquisition strategy, and data analysis using the High Flux Isotope Reactor CG-1D neutron imaging beamline. The aforementioned capabilities will be illustrated using a selection of examples in plant physiology&#160;(herbaceous plant/root/soil system)&#160;and biomedical applications&#160;(rat femur and mouse lung).

This manuscript describes a protocol for neutron radiography and computed tomography of biological samples using a High Flux Isotope Reactor (HFIR) CG-1D beamline to measure a metal implant in a rat femur, a mouse lung, and an herbaceous plant root/soil system.

橡树岭国家实验室高通量同位素反应堆的中子射线照相和生物系统计算机断层扫描

Neutrons have historically been used for a broad range of biological applications employing techniques such as small-angle neutron scattering, neutron ...

植物与放射性示踪剂测量矿质营养和毒物的通量

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测量碳为基础的污染物矿化结合使用CO