Name: measuring fluxes of mineral nutrients and toxicants in plants with radioactive tracers
Uploaded: 2014-08-22T14:00:00.000+00:00
Duration: 13 min 14 s
Description: Watch this Scientific Journal Video about Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers at JoVE.com

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工場の文化と準備<ol><li> （詳細は、10を参照）、気候制御された成長チャンバー内で7日間の大麦苗の水耕を育てる。 注：それは栄養の要件は年齢とともに変化するように、発達段階の多様で植物を調べて検討することが重要です。 </li><li>実験前のある日、一つの複製（DI用のバンドルにつき3工場、CATE用のバンドルにつき6工場）を作るために一緒にいくつかの苗をバンドル。シュートの基部の周りにタイゴンチューブの2cmのピースをラップし、「カラー」を作成するテープでチューブを固定することによって苗をバンドルします。 注：バンドルあたりの植物の数は、実験条件の10,13,14に基づいて異なる場合があります。バンドリングは、根量および/または比活性が低い場合は特に、統計、測定精度を向上させるために行われる。 </li></ol>実験的ソリューション/材料の作製コンテンツ &quot;&gt;注：以下は、一般的に実験前に1日に実行されます。 <ol><li> DIは、次の収集：プレ標識、標識、及び脱着溶液（詳細については、11を参照のこと）、（植物試料の脱水のための）遠心分離管、及び植物材料および特異的活性のためのサンプルバイアル（[S o を ;下記参照]）。曝気し、すべての溶液を混合。 </li><li> CATEは、以下収集し、溶出液を、植物試料について（よく混合、曝気ラベリングおよび溶出ソリューション（詳細については、10を参照）（植物試料の脱水のために）、排出漏斗、遠心分離管、及びサンプルバイアルとS oと希釈係数の決定[D fは 、下記参照]）。 </li></ol> 3放射性トレーサーを準備注意：以下の安全のステップが放射能で作業する前に注意する必要があります。 <ol><li> radioacの要件を確認してください電性材料のライセンスが理解され、続いている。適切な安全装置（ すなわち 、ゴーグル、手袋、白衣、鉛ベスト/襟）と線量計（ 例えば 、TLDリングとバッジ）を着用してください。シールドを設定し（ すなわち 、プレキシグラス、鉛レンガ）と、その背後にある放射性作業を行う。ガイガーミュラー計数が日常的汚染を監視するために存在していることを確認してください。 </li><li> 42 K +の調製<ol><li>バランスに清潔で乾燥したビーカーを置きます。バランスをゼロ。 </li><li>パッケージからトレーサーのバイアル（粉末状の42 の K 2 CO 3の20 mCiの）を外し、ビーカーにトレーサーを注ぐ。塊をメモしておいてください。 </li><li>ビーカーにH 2 SO 4の0.07ミリリットルに続くのdH 2 Oをピペットは19.93ミリリットル、、。これは、次の化学反応を駆動する。 42 K 2 CO 3 （複数の）+ H 2 SO 4（リットル）+ H 2 O（l）の42→ </sK 2&gt;アップSO 4 （リットル）+ CO 2（v）の+ 2H 2 O（l）の</li><li>放射原液の濃度を計算し、質量およびK 2 CO 3の分子量および体積（20ml）に与えられた。 NOTE：13 NH 3/13と作業NH 4 +トレーサー水の酸素原子の陽子衝撃を介して、サイクロトロンで生成される場合（典型的には100〜200 mCiの活性が得られる;製造の詳細については、12を参照のこと）。 14 NH 14分の 3 NH 4 +の量は、これらの溶液中の非常に低いため、原液のN濃度は無視できる。 </li></ol></li></ol> 4。直接流入（DI）の測定<ol><li> 42 K +のため、ピペット標識溶液にK +の所望の最終濃度に達するのに必要な放射性ストック溶液の量。 <ol><li> NH 3/13 13のためにNH 4 +、ピペット標識溶液に少量（&lt;0.5）を加えた。標識溶液は、（エアレーションを介して）完全に混合できるようにします。 </li></ol></li><li>サンプルバイアルに1ミリリットルの標識溶液のサブサンプルをピペットで3回（計4サンプル）を繰り返します。 <ol><li>ガンマカウンターを使用して、（「分あたりのカウント」、CPMでの）バイアル中の放射能を測定します。 （これは、そのような短命なトレーサーは特に重要です）カウンタがcpmの測定値は、同位体の崩壊について補正されるようにプログラムされていることを確認してください。 </li><li>四つのサンプルのカウント（CPMを加え-1）を平均し、（マイクロモル溶液-1）溶液中の基質の濃度で割ることにより（CPMのマイクロモル-1として表される）、S o を計算します。 </li></ol></li><li>参照（試験条件下で植物を事前に平衡化し、5分間プレ標識（非放射性）溶液中に根を浸す例えば 、前のラベル時間のばらつきのため10,13,14）。 </li><li> 5分間の標識（放射性）溶液中に根を浸し。 注：ラベリング時間は実験3,4,7-10に基づいて変更することができる。 </li><li>表面付着放射能の大部分を除去するために5秒間脱離液に根を転送します。外トレーサーの更なる明確なルーツに5分間脱離液の第二のビーカーに根を移す。 </li><li>シュート、基底シュート、および根を解剖し、独立した。 </li><li>表面と間隙水を除去するために、低速、臨床グレードの遠心分離機（〜5000×g）で30秒間遠心管とスピンサンプル中の根を置きます。 </li><li>根（生重量、FW）を秤量する。 </li><li>植物試料中の放射能カウント（シュート、基底シュート、および根を、ステップ4.2.1を参照のこと）。 </li><li>フラックスを計算します。の式を用いて植物に流入を計算 Φ= Q * / S O重量L Φはフラックスです（マイクロモルgの-1時間-1）、Q *は、組織（通常はrootでのcpmで、シュート、および基底シュート、組み合わせ）に蓄積されたトレーサの量である、S oは標識溶液（CPMのマイクロモルの比活性である- 1）、w は根の新鮮重量（g）であり、t Lは、標識化時間（hr）である。 注：より高度な計算がCATEから得られたパラメータに基づいて、ラベル付け、脱着時の根からの同時トレーサー流出を考慮して行うことができる（;詳細については、4を参照して以下を参照）。 </li></ol>トレーサー流出（CATE）測定による5。コンパートメント解析<ol><li> （ -上記、4.2の手順4.1を参照）標識溶液を準備し、S o を測定する。 </li><li>希釈倍率（D fで ）を測定します。 注：多くの場合、ガンマカウンターで検出器に対する試料の位置量に影響を与えることができる放射線を測定。詳細については、説明を参照してください。 <ol><li> S o を測定した後、各試料にH 2 Oの19ミリリットルを追加（ように最終容量=溶出液量= 20ミリリットル）。各20mlの試料中の放射能をカウントする（ステップ4.2.1を参照）。 </li><li> 20ミリリットルの試料の平均CPMによって1mlのサンプルの平均cpmで割ることによってD Fを計算します。 </li></ol></li><li> 1時間標識溶液中で根を浸し。 </li><li>すべてのルート材料が漏斗内にある保証し、流出漏斗に標識溶液と転送植物から植物を取り出します。プラスチックカラーの上にテープの小片を適用することにより、排出漏斗の側に静かに安全な植物。 </li><li>静かに漏斗に最初の溶出​​液を注ぐ。 （カウントアップ）タイマを起動します。 </li><li>スピゴットを開き、15秒後にサンプルバイアル内の溶出液を収集する（注：溶出時間が異なります。下記参照）。スピゴットを閉じます。静かに漏斗に次の溶出液を注ぐ。 </li><li> Repea最初から最後の溶出液に、以下の溶出シリーズの残りのtステップ5.6：15秒（4回）、20秒（3回）、30秒（2回）、40秒（1回）、50秒（1回）、1分（25回）、29.5分の総溶出期間 注：脱着シリーズは実験条件7-10,13,14に基づいて変化することができる。 </li><li>溶出プロトコルが完了すると、収穫植物は、（ - 上記4.8、4.6ステップ）。 </li><li> （5.2参照、D fで各溶出物の読み取りを乗じ）ガンマカウンターで溶出液および植物試料中の放射能をカウントします。 </li><li>溶出時間の関数としてプロットトレーサーリリース（CPM gを（ルートFW）-1分-1）。定常状態では、（詳細は6-9を参照）フラックス、交換の半減期は、プールサイズの線形回帰や計算を実行します。 </li></ol>

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The authors have nothing to disclose.

上記の例で示したように、放射性トレーサー法が植物体に栄養素や毒性物質の単方向のフラックスを測定する強力な手段である。 図1は 、NH 3流入は225マイクロモルgの-1時間-1、おそらくあるの過剰に達することができることを示していますこれまでプラントシステム13に報告された最高善意の膜貫通フラックスが、唯一のネットフラ?...

栄養素や毒物の取り込みと分布が強く、植物の成長に影響を与える。したがって、基本的な輸送プロセスの研究は、特に栄養の最適化と環境ストレス（ 例えば 、塩ストレス、アンモニウム毒性）の文脈で、植物生物学や農学1,2の研究の主要な領域を構成している。植物中のフラックスを測定するための方法の中で最高経営責任者は、1950年代に大幅に開発された放射性同位体トレーサーを使用することである（ 例えば 、3を参照）、今日広く使用され続けています。そのような根培地および/または組織への蓄積から栄養枯渇の測定などMIFE（微小電極イオン磁束推定）とSIET（イオン選択性電極法をスキャン）のようなイオン選択性振動マイクロ電極の使用、の使用などの他の方法は、イオン選択性蛍光色素は、広く適用されるが、正味のインフルエンザを検出する能力が制限されているXES（ すなわち 、流入と流出の差）。放射性同位体の使用は、一方で、研究者は動力学的パラメーターを解決するために使用することができる一方向性フラックスを分離し、定量化するユニークな能力を可能にする（ 例えば 、K MおよびV max）は 、容量への洞察を提供し、エネルギー論、交通システムのメカニズム、および規制、。放射性トレーサーで作られた単方向フラックス測定は反対方向に磁束が高く、細胞内プールの代謝回転が急速4である条件の下で特に有用である。トレースされた同位体は、同じ要素の別の同位体を背景に観察されているため、また、放射性トレーサーの方法は、（以下に、「ディスカッション」を参照）の測定は、多くの他の技術とは異なり、かなり高い基質濃度下で行うことを可能にする。 ここでは、単方向であり、nの放射性同位体を測定するための詳細な手順を提供完全な植物ミネラル栄養素や毒物のらフラックス。重点が存在する場合、高濃度（ 例えば 、1〜において、カリウム（K +）のフラックス測定に有毒植物主要栄養素5、アンモニア/アンモニア（NH 3 / NH 4 +）、しかし、別の主要栄養素を説明する10ミリモル）2。私たちは、モデル系の大麦（ オオムギ L の無傷の苗に、それぞれ、NH 4 +（T 1/2 = 9.98分）の放射性同位元素42 K +（T 1/2 = 12.36時間）と13 NH 3月 13日を使用します。）、2主要プロトコルの説明において：トレーサー流出（CATE）による直接流入（DI）とコンパートメント解析。私たちは、この記事は、単純に各プロトコルを実行するために必要な手順を説明し、最初から注意してください。計算と理論の適切な、簡潔な説明が提供される場合、各技術の詳細な博覧会の背景と理論は対象4,6-9上のいくつかの重要な論文に記載されています。重要なことは、これらのプロトコルは他の栄養素/毒物の分析をフラックスに広く譲渡する（ 例えば 、24のNa +、22 Na +の、86のRb +、13 NO 3 - ）および他の植物種に、いくつかの注意事項ではあるが（下記参照） 。また、放射性物質を扱うすべての研究者が機関の電離放射線安全·レギュレータを介して配置されたライセンスの下で働かなければならない重要性を強調。

<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.

図1は 、高で成長した無傷の大麦の苗の根に、NH 3の流入のために（13をN）DI技術を使用して見つかった等温線を示している（10 mM）を、NH 4 +、およびいずれか（0.02 mM）の低または高（5 mMの）K +。 （;溶液のpH 13の変化によって調整する[NH 3] EXT）NH 3のフラックスは、外部のNH 3濃度の関数としてプロットされた場合?...

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.

放射性トレーサーで植物にミネラル栄養素や毒性物質のフラックスを測定する

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

11.3K ビュー.  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.

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

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes and soil organic matter formation to small-scale stable-isotope biomarker probing. Coupling multiple stable isotopes such as 13C with 15N, 18O or&#160;2H has the potential to reveal even more information about complex stoichiometric relationships during biogeochemical transformations. Isotope labeled plant material has been used in various studies of litter decomposition and soil organic matter formation1-4. From these and other studies, however, it has become apparent that structural components of plant material behave differently than metabolic components (i.e. leachable low molecular weight compounds) in terms of microbial utilization and long-term carbon storage5-7. The ability to study structural and metabolic components separately provides a powerful new tool for advancing the forefront of ecosystem biogeochemical studies. Here we describe a method for producing 13C and 15N labeled plant material that is either uniformly labeled throughout the plant or differentially labeled in structural and metabolic plant components.
Here, we present the construction and operation of a continuous 13C and 15N labeling chamber that can be modified to meet various research needs. Uniformly labeled plant material is produced by continuous labeling from seedling to harvest, while differential labeling is achieved by removing the growing plants from the chamber weeks prior to harvest. Representative results from growing Andropogon gerardii Kaw demonstrate the system&#39;s ability to efficiently label plant material at the targeted levels. Through this method we have produced plant material with a 4.4 atom%13C and 6.7 atom%15N uniform plant label, or material that is differentially labeled by up to 1.29 atom%13C and 0.56 atom%15N in its metabolic and structural components (hot water extractable and hot water residual components, respectively). Challenges lie in maintaining proper temperature, humidity, CO2 concentration,&#160;and light levels in an airtight 13C-CO2 atmosphere for successful plant production. This chamber description represents a useful research tool to effectively produce uniformly or differentially multi-isotope labeled plant material for use in experiments on ecosystem biogeochemical cycling.

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

This method explains how to build and operate a continuous 13C and 15N isotope labeling chamber for uniform or differential plant tissue labeling. Representative results from metabolic and structural labeling of Andropogon gerardii are discussed.

連続の設計および動作<sup&gt; 13</sup&gt; Cと<sup&gt; 15</sup均一または差動、代謝および構造·&gt; N標識化会議所、工場同位体ラベル化

Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes ...

環境学

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.

複合COを用いたカーボンベースの汚染鉱化作用を測定します<sub&gt; 2</sub&gt;フラックスと放射性炭素分析

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

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

放射性核種蛍光レポーターの遺伝子齧歯動物腫瘍モデルで腫瘍の進行を追跡するイメージング

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

がん研究

Fluorescently Labeled Bacteria as a Tracer to Reveal Novel Pathways of Organic Carbon Flow in Aquatic Ecosystems

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial communities has many limitations, and determining a predator, prey, and predatory rates is often difficult. Presented here is an optimized method based on the addition of fluorescently labelled prey as a tracer, which allows for reliable quantitation of the grazing rates in aquatic predatory eukaryotes and estimation of nutrient transfer to higher trophic levels.

Presented here is a protocol for a single-cell, epifluorescence microscopy-based technique to quantify grazing rates in aquatic predatory eukaryotes with high precision and taxonomic resolution.

水生生態系における有機炭素流の新しい経路を明らかにするトレーサーとしての蛍光標識細菌

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial ...

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.

15窒素分析のためのマイクロプロット設計と植物および土壌サンプル調製

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.

土壌中の不安定な植物、無機植物の栄養素および汚染物質の二次元可視化と定量化

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.

自動90Sr の分離と濃縮 Ppq レベルで研究室のバルブ システムで

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

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.

複数の栄養源の微生物代謝を調査するため同位体トレーサー実験の組み合わせに基づいてワークフロー

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.

水耕：栄養アベイラビリティと毒性元素への曝露に栄養の割り当てと植物の応答を研究するための汎用性の高いシステム

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

生物学

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.

オークリッジ国立研究所の高フラックス同位体原子炉における中性子ラジオグラフィーと生体システムのコンピューター断層撮影

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

放射性トレーサーで植物にミネラル栄養素や毒性物質のフラックスを測定する

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連続の設計および動作

複合COを用いたカーボンベースの汚染鉱化作用を測定します

放射性核種蛍光レポーターの遺伝子齧歯動物腫瘍モデルで腫瘍の進行を追跡するイメージング

水生生態系における有機炭素流の新しい経路を明らかにするトレーサーとしての蛍光標識細菌

¹⁵窒素分析のためのマイクロプロット設計と植物および土壌サンプル調製

土壌中の不安定な植物、無機植物の栄養素および汚染物質の二次元可視化と定量化

自動⁹⁰Sr の分離と濃縮 Ppq レベルで研究室のバルブシステムで

複数の栄養源の微生物代謝を調査するため同位体トレーサー実験の組み合わせに基づいてワークフロー

水耕：栄養アベイラビリティと毒性元素への曝露に栄養の割り当てと植物の応答を研究するための汎用性の高いシステム

オークリッジ国立研究所の高フラックス同位体原子炉における中性子ラジオグラフィーと生体システムのコンピューター断層撮影

放射性トレーサーで植物にミネラル栄養素や毒性物質のフラックスを測定する

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連続の設計および動作

複合COを用いたカーボンベースの汚染鉱化作用を測定します

放射性核種蛍光レポーターの遺伝子齧歯動物腫瘍モデルで腫瘍の進行を追跡するイメージング

水生生態系における有機炭素流の新しい経路を明らかにするトレーサーとしての蛍光標識細菌

15窒素分析のためのマイクロプロット設計と植物および土壌サンプル調製

土壌中の不安定な植物、無機植物の栄養素および汚染物質の二次元可視化と定量化

自動90Sr の分離と濃縮 Ppq レベルで研究室のバルブ システムで

複数の栄養源の微生物代謝を調査するため同位体トレーサー実験の組み合わせに基づいてワークフロー

水耕：栄養アベイラビリティと毒性元素への曝露に栄養の割り当てと植物の応答を研究するための汎用性の高いシステム

オークリッジ国立研究所の高フラックス同位体原子炉における中性子ラジオグラフィーと生体システムのコンピューター断層撮影

¹⁵窒素分析のためのマイクロプロット設計と植物および土壌サンプル調製

自動⁹⁰Sr の分離と濃縮 Ppq レベルで研究室のバルブシステムで