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。

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			<td height="21" style="height:21px;width:216px;">Gamma counter</td>
			<td style="width:103px;">Perkin Elmer</td>
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			<td>Model: Wallac 1480 Wizard 3&#34;</td>
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			<td height="63" style="height:63px;width:216px;">Geiger-M&#252;ller counter</td>
			<td style="width:103px;">Ludlum Measurements Inc.</td>
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			<td>Model 3 survey meter</td>
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			<td height="21" style="height:21px;width:216px;">400-mL glass beakers</td>
			<td style="width:103px;">VWR</td>
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			<td>For efflux funnel</td>
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			<td height="21" style="height:21px;width:216px;">Large tubing</td>
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			<td>For efflux funnel</td>
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			<td style="width:103px;">VWR</td>
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			<td>For bundling</td>
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			<td style="width:103px;">VWR</td>
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			<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>
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			<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>
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			<td height="21" style="height:21px;width:216px;">Glass centrifuge tubes</td>
			<td style="width:103px;">VWR</td>
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			<td>For spin-drying root samples</td>
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			<td>For spin-drying root samples</td>
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			<td height="21" style="height:21px;width:216px;">Dissecting scissors</td>
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			<td style="width:119px;">470005-496</td>
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			<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>
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			<td height="21" style="height:21px;width:216px;">1-mL pipette</td>
			<td style="width:103px;">Gilson</td>
			<td style="width:119px;">F144493</td>
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			<td height="21" style="height:21px;width:216px;">10-mL pipette</td>
			<td style="width:103px;">Gilson</td>
			<td style="width:119px;">F144494</td>
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			<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>
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		<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>
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		<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>
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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

11.2K 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

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.

組換えセルラーゼCEL5Aの式から<em&gt;トリコデルマ·</em&gt;タバコ植物における

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

環境学

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.

摘出葉のアッセイで双子葉雑草除草剤代謝の速度を測定します

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.

Oの底生交流<sub&gt; 2</sub&gt;、N<sub&gt; 2</sub小さなコアインキュベーションを使用&gt;と溶存栄養素

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.

連続インストリームの栄養素と農業生態系流域における堆積物の監視

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.

メタンおよび亜酸化窒素フラックス静電気による水田からの評価閉鎖チャンバー ヘッド スペース内の植物を維持します。

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.

植物の凍結の三次元赤外線ビデオを制作するためのプロトコル

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.

根の生長およびローカライズされた栄養素に対する反応性を可視化する最適化された Rhizobox プロトコル

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.

オープン ソース ソフトウェア パイプラインによる寒天培地に固定化活性汚泥粒子径・形状を測定

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.

クライオ走査型電子顕微鏡で可視化された木質植物のキシレム水分布

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.

毒性物質のトランスおよび多世代効果の研究のためのカエナルハブ炎エレガンスの使用

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