We are grateful to B. Stevenson for technical assistance and Dr Marc R. Knight for providing Aequorin transgenic line. This work was funded by the National Institutes of Health Grant R01 GM059138.

FASシステムを用いた1イクオリンベースのCA 2 +イメージング<ol><li>発光イメージングのための苗木を準備します。 0.01％のTriton-100を含有する10％漂白溶液でエクオリンを発現するArabidopsis植物の種子を滅菌する。フル強度MS（ムラシゲ·スクーグ基礎塩混合物）、1％スクロース、1.2％寒天を含む角板（グリッド10×10cmの正方形のペトリ皿）上で無菌種をまく。 2日間（ 図1A）、4℃で層別化した後、成長チャンバー内に垂直に配置し、プレート。 </li><li>フィルム上に苗を移します。プレート上で生育7-10日齢の苗の上に接着フィルム（ 図1B）を配置します。ゆっくりと苗はフィルム（ 図1C）に準拠していることを確実にするために手でフィルムを押してください。苗がフィルムに付着したままになるように静かにフィルムをはがします（ 図1Dおよび1E） </li><li>補因子と苗をインキュベートします。 Aを配置水の中に2μg/ mlののH-CTZ（セレンテラジン）の15ミリリットルを含む正方形のプレート上dhered苗（10×10センチ）。一晩に4時間（ 図1F）を、室温で苗をインキュベートする。 </li><li>発光イメージングのための準備をします。 H-CTZ溶液からフィルムを取り出し、中央を、それを切り倒し、二枚を形成する。二つの異なるプレートで表向きに苗木を有するフィルムの各部分を配置します。 5分間、暗所でプレートにしておきます。 </li><li>発光画像を取得する。闇の中で、発光イメージングシステム（ 図1G）のステージ上に、互いの横に二つのプレートを配置。すぐに同時にプレートに刺激を溶液20mlを加える際に画像を取得する。 </li><li>発光画像を分析します。すべての発光画像に対して同じ表示範囲を選択します。関心領域（ROI）をクロップし、JPEGファイル（ 図2A）のような画像を生成する。別の方法として、SPE形式のファイルとして画像をエクスポートしにインポートImageJの画像解析ソフトウェア。 ROIの平均グレー値を計算するための測定値を設定します。 ROI領域の同じサイズを選択して、棒グラフ（ 図2B）、平均グレーと現在のデータを測定します。 </li></ol> 2生細胞共焦点のCa 2 +イメージング<ol><li>共焦点イメージングのための苗木を準備します。トリトン0.01％を含む10％の漂白剤溶液でCASE12を表現するシロイヌナズナ植物の種子を滅菌する。フル強度MS塩、1％スクロース、および1.2％寒天を含むプレート上で無菌種をまく。 2日間で4℃で層別化した後、成長チャンバー内に垂直に配置し、プレート。 </li><li>セットアップイメージングチェンバース<ol><li>スライドとカバーガラス（商工A）を用いたイメージングチャンバーを組み立てます。スライドの真ん中に水に浸した脱脂綿の作品を配置します。その後、水に浸した脱脂綿の上部にプレートから1つまたは2つの5日齢の苗を移す。カバースリップの各コーナーへの粘土の小片、スティックカバーガラスとスライドとの間のギャップ（室）（ 図3A、左パネル）を作成する苗の上のd位のカバースリップ。 1mlシリンジにポリエチレンチューブ（0.58ミリメートル直径）の一端を接続し、チャンバーに直接隣接するチューブのもう一方の端を置きます。テープ（ 図3A、右パネル）で所定の位置にチューブを保持します。 </li><li>プレキシガラス室（B室）を使用して画像化チャンバを組み立てます。 <ol><li>プレキシガラス室のそれぞれの側のチャネルにコーティングされた管を2ポリエチレン管（0.58ミリメートル径）の周りにシリコーングリースの薄層を広げ、押した（ 図3（b）は、左パネル）。チューブの溝が含まれているチャンバーの表面にシリコーングリースの薄層を広げ、チャンバ開口部（切り出す）の一方の側を密封するグリースにカバースリップを押してください。 </li><li>チャンバー内にプレートから5日齢の苗を移し、トップO上の水に浸した脱脂綿の一部を置く苗F。チャンバーの他方の面にシリコーングリースを広げ、かつシールするために上に別のカバースリップを押してください。 1ミリリットルの注射器にチューブの一方の端を接続します。他の管開放（ 図3B右パネル）の終わりにしておきます。 </li></ol></li><li>チャンバーカバーガラス（商工C）を用いた撮像室を準備します。 70％エタノールでチャンバーカバーガラスを殺菌し、乾燥するまでボンネットの上に残す。それぞれ、2ウェルチャンバーまたは8ウェルチャンバー（ 図3C右パネル）の各ウェルに1％スクロースおよび0.5％phytagelを含む完全な強MS培地の0.6ミリリットルまたは0.2ミリリットルを追加します。種子を殺菌し、透明なゲルの薄層を含むチャンバーカバーガラスに直接種をまく、それらを5日間（ 図3C）のために垂直方向に成長してみましょう。 </li></ol></li><li>共焦点顕微鏡を使用して画像を取得します。寒さや過酸化物などの塩などのストレス刺激を、適用するには、ゆっくりと150のNaCl、約200μlの、アイスコルを注入直前にそれぞれ、画像を取得するか、静かに2または8ウェルチャンバーのウェルに刺激溶液500μlまたは100μlのを追加し、チャンバ（チャンバAまたはB）へのd水または1mMのH 2 O 2溶液。直ちに20X浸水レンズ（開口数0.75）を用いて反転ニコンA1R共焦点レーザー走査顕微鏡を用いて刺激剤溶液を塗布した後に画像を取り込む。それぞれ550nmで、および512×512ピクセルの解像度で - 488 nmおよび500の励起波長および発光波長を持つ4秒間隔で画像の時系列を収集します。 </li><li>画像解析 ニコンの要素を使用して、ROIが対象とする各セル（又は面積）の周りに描かれた。各ROI内の総強度は、時間測定ダイアログボックス（ImageJの代わりに使用することができる）を用いて経時的に測定した。全強度測定は、データグラフでエクスポート処理した。のCa 2 +スパイク振幅は番目としてピーク強度マイナス休止強度、持続時間として定義した2スパイクの隣接するスパイクピーク間の時間間隔として開始およびスパイクの終了、及び期間の間の電子の時間が-test手段を比較するために使用したトン 。 </li></ol>

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The authors have nothing to disclose.

私たちは、シロイヌナズナの苗の時空間のCa 2 +応答を記録するためのFASシステムを実証している。このFASのCa 2 +記録方式がここに提示されている非生物的ストレス刺激に加えて、さまざまな刺激によって誘発のCa 2 +動態を測定するために適合させることができる、単純な感度かつロバストな手法を提供する。このシステムを用いて、簡単に植物全体レベルでの組織ま?...

植物細胞は、細胞の行動を調整するシグナリングを介して環境に応答する。環境刺激に応答してイベントをシグナリング初期の細胞は一過性のCa 2 +の増加である。遊離Ca 2 +濃度のパターン、または一過性の上昇の署名は、その振幅、周波数、および期間によって特徴付けられる。個別時空間のCa 2 +署名は異なる細胞活性1を調節する。例えば、熱、寒さ、塩、干ばつ、光、植物ホルモンなどの具体的な刺激は、微調整も膜局在のCa 2 +チャネルおよび/ ​​またはトランスポーターの時空間的アクティビティを、特定のCa 2 +の署名を得た。のCa 2 +輸送体が十分に特徴付けされているが、ほとんどの植物1におけるCa 2 +チャネルの分子アイデンティティおよび機能についてはほとんど知られていない。ストレス刺激するために、変化したのCa 2 +応答を持つ変異体についての遺伝子スクリーニングが効果的APPRかもしれのCa 2 +の署名を構成するコンポーネントを識別するためのoach。最近、いくつかのイクオリンベースのCa 2 +検出システムは、病原体の攻撃および非生物的ストレス2-4に反応してCaのための遺伝子スクリーニング2 +シグナル伝達成分を容易に開発されている。 イクオリンは、まず1990年代初期5植物におけるCa 2 +シグナルを検出した。それ以来、イクオリンは、特定のCa 2セルを監視するためにそのような細胞質5、核6などの異なる細胞区画に、葉緑体7、8、ミトコンドリア9、および間質10液胞膜と同様に、ルート内の異なる細胞型に標的化されています+シグナル11。エクオリンベースのCa 2 +測定は、ストレス刺激する細胞の集団の空間的および時間的のCa 2 +応答を明らかにする。しかし、ほとんどの場合、単一の細胞のCa 2 +応答はunsynchroniある応答組織4にゼット。そのため、イクオリンのCa 2 +記録は、必ずしも個別のセル内のCa 2 +シグナルを報告しません。近年では、遺伝的にコードされた蛍光タンパク質（FP）は、黄色カメレオンなどのCa 2 +指標（YCS）12をベースとCASEs12 13は、Caを研究するために使用されている2 +高細胞下解像度とシグナリング。 YCSある蛍光共鳴エネルギー移動（FRET）は、CFPを含む、カルシウム2 +指標をベースとYFPのCa 2 +の結合タンパク質のカルモジュリンとカルモジュリン結合ペプチドM13によって連結された変異体。それは、Ca 2 +に結合するようにカルモジュリン、それによって増加したエネルギー移動（FRET強化）で、その結果、より緊密CFPとYFPをもたらし、コンホメーション変化を受ける。 CFPシグナル強度に対するYFPの比として概算時間をかけてFRETレベルは、細胞内Ca 2 +動態を反映している。いくつかのYCバージョンが植物において使用されている。 YC3.6 tはあった細胞質ゾル14,15、核16、ミトコンドリア17、および原形質膜18、およびYC4.6とD4ERにargetedは、ER 15,19をターゲットとし、D3cpvはペルオキシソーム20を対象とした。 YCSを発現するトランスジェニック植物は、異なる細胞型の異なる細胞区画内のCa 2 +動態の生細胞イメージングを可能にする。ケース（おそらくカルシウム lcium SE nsor）がカルモジュリンとカルモジュリン結合ペプチドM13を保有する単一の円順列蛍光タンパク質（cpFPs）です。のCa 2 +に結合すると、ケースは、蛍光強度の増加をもたらす、コンフォメーション変化を受ける。 CASEの蛍光応答およびCa 2 +濃度との相関は、細胞内Ca 2 +動態を定量的に測定することができる。 CASE12変異体は、12倍のCa 2 + -飽和形で蛍光が増加している。N. benthiminanaプラント過渡的にはexprディエッサーCASE12または安定的にケース12を発現しているシロイヌナズナ植物が防御と非生物的ストレス4,21中のCa 2 +シグナル伝達を研究するために使用された。病原体の攻撃に、または乾燥ストレスに応答する細胞内のCa 2 +の非同期空間的および時間的な振動がCASE12ベースのCa 2 +イメージングで明らかにされている。 ここでは、シロイヌナズナの苗において、組織との刺激特定のCA 2 +動態のエクオリンに基づく発光イメージングのための詳細な手順を提示、およびFASのケース12発光イメージングを表現シロイヌナズナの根の細胞内の細胞質ゾルおよび核のCa 2 +動態の共焦点イメージングのためのストレス誘発性のCaここでは記載していない無傷の植物又は組織における2 +動態を分析するために適合させることができる、またはCa 2 +シグナルを誘導し、変更された応力を有する変異体のための突然変異誘発シロイヌナズナ植物集団をスクリーニングする。生細胞のCa 2 +イメージングセットアップは、Caを分析するために適合させることができる他のCa 2 +指標を使用して、異なるsubcelluarコンパートメント内または異なる細胞型において2 +動態。

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measuring spatial and temporal ca2+ signals in arabidopsis plants

発生および環境信号は、植物細胞内のCa 2 +の変動を誘発する。刺激固有の空間-時間のCa 2 +のCa 2 +のパターンは、シグナル伝達カスケードを開始し、携帯のCa 2 +結合タンパク質によって感知される。しかし、私たちはまだ、特定のCa 2 +シグナルが生成される刺激方法について少し知っている。のCa 2 +シグナルの特異性は、与えられた刺激に応答してのCa 2 +チャネルおよび/ ​​またはトランスポーターの活性の洗練されたレギュレーションに起因し得る。これらの細胞成分を同定し、その機能を理解するには、組織や細胞レベルの両方でのCa 2 +シグナルの高感度かつ堅牢な記録を可能にするシステムを使用することが重要です。異なる細胞区画を標的とする遺伝的にコードされたのCa 2 +指標は、携帯のCa 2 +シグナルの生細胞の共焦点イメージングのためのプラットフォームを提供してきた。ここでは、命令を記述する2のCa 2 +の検出システムを使用するためのsが：FAS（フィルム接着剤の苗）の発光のCa 2 +イメージングおよびCASE12ベースの生細胞共焦点蛍光カルシウム2 +イメージングベースのエクオリン。 CASE12を用いて生細胞の共焦点イメージングは、高解像度でのサイトゾルおよび核のCa 2 +シグナルの同時検出を提供しながら、FASシステムを用いた発光イメージングは、組織レベルでの空間的および時間的のCa 2 +シグナルの単純な堅牢かつ高感度な検出を提供する。

マンニトール、NaClおよびH 2 O 2は、それぞれ、脱水、塩および酸化ストレス刺激のためのプロキシとして使用した。重金属イオンのCu 2 +は、これら三つのストレス刺激のいずれかと相乗作用しているかどうかを確認するために、私たちは、Cu 2 +の存在下または非存在下でそれぞれの刺激に対するCa 2 +応答を比較した。 図2に示されるように...

Watch this Scientific Journal Video about Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants at JoVE.com

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

のCa 2 +シグナル伝達は、植物において、多様な生物学的プロセスを調節する。ここでは、遺伝的にコード化されたのCa 2 +指標エクオリンまたはCASE12を使用して、シロイヌナズナの細胞および組織内の空間的および時間的のCa 2 +シグナルを誘発した非生物的ストレスを監視するためのアプローチを提示します。

時空のCaを測定する<sup&gt; 2 +</supシロイヌナズナ植物における&gt;シグナル

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

measuring-spatial-and-temporal-ca2-signals-in-arabidopsis-plants

Research

JoVE Journal

Biology

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

12.0K ビュー.  Purdue University. のCa 2 +シグナル伝達は、植物において、多様な生物学的プロセスを調節する。ここでは、遺伝的にコード化されたのCa 2 +指標エクオリンまたはCASE12を使用して、シロイヌナズナの細胞および組織内の空間的および時間的のCa 2 +シグナルを誘発した非生物的ストレスを監視するためのアプローチを提示します。 

ビデオ: Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Modeling Biological Membranes with Circuit Boards and Measuring Electrical Signals in Axons: Student Laboratory Exercises

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology used in electrophysiology. The same equipment is used in the membrane model as on live preparations. Some properties of an isolated nerve cord are investigated: nerve action potentials, recruitment of neurons, and responsiveness of the nerve cord to environmental factors.

This is a demonstration of how biological membranes can be understood using electrical models. We also demonstrate procedures for recording action potentials from the ventral nerve cord of the crayfish for student orientated laboratories.

回路基板と生体膜のモデル化と軸索の電気信号の測定：学生研究室の演習を

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology ...

生物学

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

アイスキャップ：成長させる方法シロイヌナズナ</emハイスループットジェノタイピング用96ウェルプレートで>とトマト

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Spermatozoa are male reproductive cells especially designed to reach, recognize and fuse with the egg. To perform these tasks, sperm cells must be prepared to face a constantly changing environment and to overcome several physical barriers. Being in essence transcriptionally and translationally silent, these motile cells rely profoundly on diverse signaling mechanisms to orient themselves and swim in a directed fashion, and to contend with challenging environmental conditions during their journey to find the egg. In particular, Ca2+-mediated signaling is pivotal for several sperm functions: activation of motility, capacitation (a complex process that prepares sperm for the acrosome reaction) and the acrosome reaction (an exocytotic event that allows sperm-egg fusion). The use of fluorescent dyes to track intracellular fluctuations of this ion is of remarkable importance due to their ease of application, sensitivity, and versatility of detection. Using one single dye-loading protocol we utilize four different fluorometric techniques to monitor sperm Ca2+ dynamics. Each technique provides distinct information that enables spatial and/or temporal resolution, generating data both at single cell and cell population levels.

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.

細胞内Ca測定<sup&gt; 2 +</sup4つの方法を使用して人間の精子で&gt;変更：従来の蛍光測定、停止フロー蛍光法、フローサイトメトリーおよび単一細胞イメージング

Spermatozoa  are male reproductive cells especially designed to reach, recognize and fuse  with the egg. To perform these tasks, sperm cells must be ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

ダイナミックカルシウムの自動分析<sup&gt; 2 +</supイメージシーケンス内の&gt;の信号

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

イメージングローカルCA<sup&gt; 2+</sup&gt;培養哺乳動物細胞内シグナル

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution.

リアルタイム体内シロイヌナズナカルシウム信号の間に記録して蛍光バイオ センサーを用いた昆虫の餌

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant ...

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires careful and in-depth analyses and quantification to capture as much information about the underlying events as possible. Spatial, temporal and intensity parameters intrinsic to Ca2+ signals such as frequency, duration, propagation, velocity and amplitude may provide some biological information required for intracellular signalling. High-resolution Ca2+ imaging typically results in the acquisition of large data files that are time consuming to process in terms of translating the imaging information into quantifiable data, and this process can be susceptible to human error and bias. Analysis of Ca2+ signals from cells in situ typically relies on simple intensity measurements from arbitrarily selected regions of interest (ROI) within a field of view (FOV). This approach ignores much of the important signaling information contained in the FOV. Thus, in order to maximize recovery of information from such high-resolution recordings obtained with Ca2+dyes or optogenetic Ca2+ imaging, appropriate spatial and temporal analysis of the Ca2+ signals is required. The protocols outlined in this paper will describe how a high volume of data can be obtained from Ca2+ imaging recordings to facilitate more complete analysis and quantification of Ca2+ signals recorded from cells using a combination of spatiotemporal map (STM)-based analysis and particle-based analysis. The protocols also describe how different patterns of Ca2+ signaling observed in different cell populations in situ can be analyzed appropriately. For illustration, the method will examine Ca2+ signaling in a specialized population of cells in the small intestine, interstitial cells of Cajal (ICC), using GECIs.

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Genetically encoded Ca2+ indicators (GECIs) have radically changed how in situ Ca2+ imaging is performed. To maximize data recovery from such recordings, appropriate analysis of Ca2+ signals is required. The protocols in this paper facilitate the quantification of Ca2+ signals recorded in situ using spatiotemporal mapping and particle-based analysis.

その場で細胞内 Ca2 +シグナル伝達を定量化する時空マッピングと粒子分析技術の応用

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.

植物における空間的および時間的組織特異的な遺伝子発現解析のためのレーザー捕捉マイクロ解離RNAシーケンシング

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify ...

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. Upon wounding of a leaf, an increase in cytosolic calcium ion concentration (Ca2+ signal) occurs at the wound site. This signal is rapidly transmitted to undamaged leaves, where defense responses are activated. Our recent research revealed that glutamate leaking from the wounded cells of the leaf into the apoplast around them serves as a wound signal. This glutamate activates glutamate receptor-like Ca2+ permeable channels, which then leads to long-distance Ca2+ signal propagation throughout the plant. The spatial and temporal characteristics of these events can be captured with real-time imaging of living plants expressing genetically encoded fluorescent biosensors. Here we introduce a plant-wide, real-time imaging method to monitor the dynamics of both the Ca2+ signals and changes in apoplastic glutamate that occur in response to wounding. This approach uses a wide-field fluorescence microscope and transgenic Arabidopsis plants expressing Green Fluorescent Protein (GFP)-based Ca2+ and glutamate biosensors. In addition, we present methodology to easily elicit wound-induced, glutamate-triggered rapid and long-distance Ca2+ signal propagation. This protocol can also be applied to studies on other plant stresses to help investigate how plant systemic signaling might be involved in their signaling and response networks.

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Extracellular glutamate-triggered systemic calcium signaling is critical for the induction of plant defense responses to mechanical wounding and herbivore attack in plants. This article describes a method to visualize the spatial and temporal dynamics of both these factors using Arabidopsis thaliana plants expressing calcium- and glutamate-sensitive fluorescent biosensors.

シロイヌナズナにおける局所および全身創傷信号の広視野、リアルタイムイメージング

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. ...

An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in turn requires accurate determination of disease phenotypes of plants upon infection with a particular fungal pathogen. However, accurate disease phenotyping with unculturable biotrophic fungal pathogens such as powdery mildew is not easy to achieve and can be a rate-limiting step of a research project. Here, we have developed a safe, efficient, and easy-to-operate disease phenotyping system using the Arabidopsis-powdery mildew interaction as an example. This system mainly consists of three components: (i) a wooden inoculation box fitted with a removable lid mounted with a stainless steel or nylon mesh of ~50 &#181;m pores for inoculating a flat of plants with fungal spores, (ii) a transparent plastic chamber with a small front opening for minimizing spore escape while conducting inoculation inside, and (iii) a spore-dislodging and distribution method for even and effective inoculation. The protocols described here include the steps and parameters for making the inoculation box and the plastic chamber at a low cost, and a video demonstration of how to use the system to enable even inoculation with powdery mildew spores, thereby improving accuracy and reproducibility of disease phenotyping.

We present a protocol for constructing a simple spore-distribution system consisting of an inoculation box with a ~50 &#181;m mesh and a transparent plastic chamber. This can be used to evenly inoculate plants with powdery mildew spores, thereby enabling accurate and reproducible assessment of disease phenotypes of plants under study.

シロイヌナズナなどのうどんこ病感染表現型を正確に評価するための簡便かつ柔軟な接種法

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in ...