その技術支援の応用生物科学部、東洋大学、群馬県農業技術センターの谷口広大の村上彰氏に感謝しております。東洋大学、博士 Uma Maheswari 医教授庄作柏田へ彼らの提案に感謝しております。本研究は、生活と環境科学、東洋大学の研究センター部分で支えられました。

注: 内因性ホルモンの分析を容易にするため、トコン (C. トコン) はこの研究で使用されました。
1 トコンの不定芽を誘発する成長条件
<ol><li>PH 5.710、調整無料の植物ホルモンの B5 培地を準備し、0.2% ジェランガムを追加します。オートクレーブに入れることによって殺菌しなさい。</li>
	<li>オートクレーブの中の 25 mL を滅菌シャーレ (90 × 20 mm) に注ぐ。</li>
	<li>手術のメスを用いた滅菌アクリル板に刃号 22 トコン苗木の 8 mm 節セグメントを切り取り、媒体 (図 1)。</li>
	<li>15 µmol m− 2の− 1 5 週間 14 h/10 h の光暗いサイクルの光の下の 24 の ° C で植物ホルモン無料 B5 培地で培養。</li>
	<li>地域を識別する私 (根尖部) から IV (基底) の各セグメント (図 1B)。週に一度顕微鏡下で地域ごとに不定芽 > 0.3 mm 長さの数をカウントします。</li>
</ol>2. 抽出と植物ホルモンの精製
<ol><li>それぞれ 4 つの 2 mL のサンプル チューブの 5 mm ジルコニア ビーズを入れてください。</li>
	<li>セグメントを切る地域 IV (各 2 mm 長さ) 滅菌アクリル板に手術のメスを使用します。</li>
	<li>別のサンプル チューブ (10-30 mg の新鮮な重量) の各地域の 8 つのセグメントを収集します。</li>
	<li>量る、そして液体窒素でサンプルを凍結します。</li>
	<li>ビーズ ベースのホモジナイザーを用いた凍結試料を粉砕します。</li>
	<li>オーキシンの各内部標準の 500 pg 及び CKs (d5独立行政法人、d5tZ、d5最も息、6d iP、d6知的財産権) を含む 1 mL のアセトニ トリルに押しつぶされたサンプルを中断渦のミキサーを使用しています。</li>
	<li>室温で 5 分間 3,500 × gで遠心分離後 1 h、4 ° C で保持します。</li>
	<li>1% (v/v) 酢酸を含む 80% (v/v) アセトニ トリルでペレットを洗浄します。3,500 × gで再度遠心して、室温で 5 分間。(手順 2.7 および 2.8) から使い捨てのガラス管の培養上清を組み合わせます。</li>
	<li>追加 600 μ L 各複合培養上清に含まれる 1% (v/v) 酢酸を水し、真空濃縮を用いたアセトニ トリルを蒸発させます。</li>
	<li>アセトニ トリル、メタノール、および 1% (v/v) 酢酸を含む水の各 1 mL を適用することによって、親水性・親油性-バランス (HLB) 列カートリッジを平衡します。</li>
	<li>平衡の HLB カートリッジあたり 1 つのサンプル ソリューションを適用します。</li>
	<li>1% (v/v) 酢酸を含む水を 1 mL でカートリッジを洗浄します。</li>
	<li>ガラス管に 1% (v/v) 酢酸を含む 2 mL 80% (v/v) アセトニ トリルのすべてのホルモンを溶出します。</li>
	<li>取得真空濃縮を使用して 1% (v/v) 酢酸を含む水で抽出する溶出液にアセトニ トリルを蒸発させます。 注: は、これを完全に乾燥しないでください。</li>
	<li>混合モードでは、強い陽イオン交換 (MCX) 列カートリッジを 0.5 mL、1 mL のメタノール 1 mL のアセトニ トリルを適用することによって平衡 0.1 M HCl と 1% (v/v) 酢酸を含む水を 1 mL。</li>
	<li>平衡の MCX カートリッジあたり 1 つのサンプル ソリューションを適用します。</li>
	<li>1% (v/v) 酢酸を含む水を 1 mL でカートリッジを洗浄します。</li>
	<li>IAA をガラス管に 1% (v/v) 酢酸を含む 2 mL 30% (v/v) アセトニ トリルで溶出します。</li>
	<li>1% (v/v) 酢酸を含む 2 mL 80% (v/v) アセトニ トリルでカートリッジを洗浄します。</li>
	<li>2 mL の水と 5% アンモニア水を含む水を 1 mL のカートリッジを洗ってください。</li>
	<li>ガラス管にアンモニア水 5% を含む 2 mL 60% (v/v) アセトニ トリル、CKs を溶出します。</li>
	<li>真空濃縮を使用して各ホルモンの分画の溶剤を蒸発し、クロマトグラフィー-タンデム質量分析まで +30 ° C で保存します。</li>
</ol>3 独立行政法人及び CKs の LC ・ MS/MS 分析
<ol><li>メタノールの 600 μ L で管内各ホルモン エキスを溶解し、ソリューションをねじ首総復旧時バイアルに転送します。真空濃縮を使用して溶媒を蒸発させます。</li>
	<li>20 μ L 30% (v/v) アセトニ トリル中で IAA 分数と CK 分数スクリューバイアル首総復旧時の 1% (v/v) 酢酸を含む 20 μ L の水に溶解します。</li>
	<li>高速液体クロマトグラフィー システムを搭載したトリプル四重極 MS システムで肯定的なイオン モードでサンプルを分析します。 注: 我々 は、表 1に記載されている HPLC 条件を設定します。<ol>
<li>IAA 溶出の 5%-50% 溶剤 B 7 分以上のバイナリのグラデーションを使用し、98% 溶剤 B 増し、1 分間保持巣注入による 5% 溶剤 B で 2 分間、平衡します。</li>
		<li>CK 溶出 2-40% 溶剤 B 5 分以上 7 分で 40-70% 溶剤 B のバイナリのグラデーションを使用し、95% 溶剤 B 増し、1 分間保持によって 2% 溶剤 B で 2 分間、平衡注入を入れ子にします。</li>
		<li>エレクトロ スプレー イオン化 (ESI) を設定-MSの表 2に掲げるイオン源のパラメーター。</li>
	</ol>
</li>
	<li>複数の反応 (MRM) 遷移表3 各試料の定量化のための監視を使用します。</li>
	<li>重水素標識基準にラベルの比の標準曲線に対して内因性 IAA と CK のレベルを定量化します。</li>
</ol>

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著者は、利害の対立があるないことを宣言します。

植物ホルモンの分布を識別するために器官形成に関与することが重要です植物ホルモンを誘導するための植に外に適用されるため、器官形成植物ホルモン フリー培地で観察できる植物材料を使用枝や根、細胞分化と器官形成における植物の固有の可塑性を評価するが難しく、全体外植体に影響します。カーネーション・ l ・11、エグレ marmelos (l.) Corrêa<sup class="...

ゴットリーブ Haberlandt (1854-1945 年) は、「全能性」、ことができます細胞植物によって分割、区別し大株1で特定の細胞型に分化させ事前後も全植物体を再生の概念を提案しました。組織培養、植物体再生を誘起することまたはないかどうか、組み合わせと成長培地におけるジベレリンの植物ホルモンの濃度によって決まります。培とミラーは、不定根が低比2を含む培地に誘導することが一方 CKs とオーキシンの高い比率を含む培養液中でタバコ由来カルスから不定芽を誘導することが発見しました。その発見以来組織培養行われている遺伝子組換え植物3の再生、経済的に重要な作物の伝播のため。不定芽は、茎頂、葉、根、茎など以外の組織から誘導することができます。植物ホルモン処理は、ほとんどの植物種の不定芽の誘導に必要です。ただし、最適濃度との組み合わせは異なる種と組織外植片として使用中です。したがって、多くの努力は、最適な濃度と植物ホルモンの実験のための組み合わせを決定するのに入ります。
トコン(Brot。)L. アンダーソン (トコン) は、エメチンと根4を中心に、セファエリンなどのアルカロイドが含まれている薬用植物です。ルート エキス、amoebicide5嘔吐、去痰薬として使用されます。トコンは、ブラジルの熱帯雨林に自生、文化では、種子を設定するには消極的だし、寒い気候6を使って、日本の種子貯蔵中に発芽率が低下します。代わりに、組織培養からの伝播が、撮影する不定節の形成は最も効率的な方法7,8。興味深いことに、植物ホルモン治療8なしこの種の不定芽を誘起することができます。
不定芽は、基底部9ではなく親、なし節セグメントの根尖部に表皮に形成されます。この違いは、おそらく ∼ 規制下にある節セグメントで組織の極性を示します。トコン文化システムにより、不定芽形成の間に内生植物ホルモン レベルの変化を分析するユニークな機会です。ここで 1 つオーキシン (インドール-3-酢酸酢酸 (IAA)) と 4 つの CKs の内因性レベルの解析の手法を紹介 (イソペンテニル アデニン (iP)、イソペンテニル アデニン ゼアチンリボシド (iPR)、トランス-ゼアチン (tZ) とトランス-ゼアチンリボシド (最も息))LC ・ MS/MS を使用して節間のセグメント。

<table><tbody><tr><td>[2H5]indole-3-acetic acid</td><td>Olchemlm Ltd</td><td>031 1531</td><td>Internal standard for LC-MS/MS</td></tr><tr><td>[2H5]trans-zeatin</td><td>Olchemlm Ltd</td><td>030 0301</td><td>Internal standard for LC-MS/MS</td></tr><tr><td>[2H5]trans-zeatin riboside</td><td>Olchemlm Ltd</td><td>030 0311</td><td>Internal standard for LC-MS/MS</td></tr><tr><td>[2H6]N6-isopentenyl adenine</td><td>Olchemlm Ltd</td><td>030 0161</td><td>Internal standard for LC-MS/MS</td></tr><tr><td>[2H6]N6-isopentenyl adenosine</td><td>Olchemlm Ltd</td><td>030 0171</td><td>Internal standard for LC-MS/MS</td></tr><tr><td>indole-3-acetic acid</td><td>Wako</td><td>098 00181</td><td>standard for LC-MS/MS</td></tr><tr><td>trans-zeatin</td><td>SIGMA-ALDRICH</td><td>Z0876 5MG</td><td>standard for LC-MS/MS</td></tr><tr><td>trans-zeatin riboside</td><td>Wako</td><td>262 01081</td><td>standard for LC-MS/MS</td></tr><tr><td>N6-isopentenyl adenine</td><td>SIGMA-ALDRICH</td><td>D7674 1G</td><td>standard for LC-MS/MS</td></tr><tr><td>N6-isopentenyl adenosine</td><td>ACROS ORGANICS</td><td>22648 1000</td><td>standard for LC-MS/MS</td></tr><tr><td>acetonitrile hypergrade for LC-MS LiChrosolv</td><td>MERCK</td><td>1.00029.1000</td><td>solvent for LC-MS/MS</td></tr><tr><td>Water for chromatography LiChrosolv</td><td>MERCK</td><td>1.15333.1000</td><td>solvent for LC-MS/MS</td></tr><tr><td>HPLC</td><td>SHIMADZU</td><td>Prominence</td><td></td></tr><tr><td>MS</td><td>Sciex</td><td>3200QTRAP</td><td></td></tr><tr><td>Oasis HLB 30 mg/1 cc</td><td>Waters</td><td>WAT094225</td><td>cartridge column</td></tr><tr><td>Oasis MCX 30 mg/1 cc</td><td>Waters</td><td>186000252</td><td>cartridge column</td></tr><tr><td>screw neck total recovery vial</td><td>Waters</td><td>186002805</td><td></td></tr><tr><td>blue, 12 x 32mm screw neck cap and PTFE/silicone septum</td><td>Waters</td><td>186000274</td><td></td></tr><tr><td>Acquity UPLC BEH C18, 2.1x100 mm</td><td>Waters</td><td>186002350</td><td>UPLC column</td></tr><tr><td>Proshell 120 EC-C18, 2.1x50 mm</td><td>Agilent</td><td>699775-902</td><td>UPLC column</td></tr><tr><td>Digital microscope</td><td>Leica</td><td>DHS1000</td><td></td></tr><tr><td>TissueLyser II</td><td>QIAGEN</td><td>85300</td><td></td></tr><tr><td>Surgical blade</td><td>Feather</td><td>No. 22</td><td></td></tr><tr><td>Scalpel handle</td><td>Feather</td><td>No. 4</td><td></td></tr><tr><td>Savant SpeedVac/Refregerated vapor trap</td><td>Thermo Fisher Scientific</td><td>SPD111/RVT4104</td><td>vacuum concentrartor</td></tr><tr><td>Disposable glass tobe (13x100 mm)</td><td>IWAKI</td><td>9832-1310</td><td></td></tr><tr><td>Sterile petri dish</td><td>INA OPTICA</td><td>I-90-20</td><td></td></tr></tbody></table>

quantification of endogenous auxin and cytokinin during internode culture of ipecac

不定芽形成は経済的に重要な作物の普及のため、遺伝子組換え植物の再生に重要な手法です。植物ホルモン処理は、ほとんどの種の不定芽の誘導に必要です。オーキシンとサイトカイニン (CK) 間のバランスによって決定されます不定芽を誘発することができるかどうかのレベル。多くの努力は、最適な濃度と外植片として使用される各組織、各植物は植物ホルモンの組み合わせを決定するのに入ります。ただし、トコンは培植物ホルモン治療なしで節間のセグメントに不定芽を誘導することができます。これは評価する細胞分化のトコンの固有の可塑性をことができます。トコンの不定芽を誘導して、5 週間 0.2% ジェランガムで凝固した植物ホルモン無料 B5 培地で 14 h/10 h の光暗いサイクルで 15 µmol m− 2の− 1光の下の 24 の ° C で節間のセグメントを培養しました。不定芽形成過程における植物ホルモンの動態を調査するには、計測における内生インドール-3 - 酢酸及び CKs セグメント液体クロマトグラフィー-タンデム質量分析法クロマトグラフィー-タンデム質量による。このメソッドは、単純な方法で解析における内生インドール-3 - 酢酸の CKs レベルをできます。それは、他の植物の器官の中に内生オーキシンと CK のダイナミクスを調べるに適用できます。

1st週不定芽が形成ないです。2nd週間で小さな芽が登場しました。3rdと 4th週間、数シュート頂端領域 (I および II) を中心に増加 (図 2A)。5 週目で番号シュートの地域であった約 7 で私と 5 地域 II (図 2B)。対照的に、いくつかの撮影だけは III と IV の地域で形?...

Watch this Scientific Journal Video about Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac at JoVE.com

Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac

トコンの節間文化の中に内生オーキシンとサイトカイニンの定量化

不定芽は、植物ホルモン治療せずトコンの節間のセグメントに誘導できます。不定芽形成過程における植物ホルモンの動態を評価するには、MS LC/MS による節セグメントにおける内生オーキシンとサイトカイニンを測定しました。

Adventitious shoot formation is an important technique for the propagation of economically important crops and for the regeneration of transgenic plants. ...

quantification-endogenous-auxin-cytokinin-during-internode-culture

Universidad de Cartagena UDEC

Research

JoVE Journal

Environment

8.9K ビュー.  Toyo University. 不定芽は、植物ホルモン治療せずトコンの節間のセグメントに誘導できます。不定芽形成過程における植物ホルモンの動態を評価するには、MS LC/MS による節セグメントにおける内生オーキシンとサイトカイニンを測定しました。

ビデオ: Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac

Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ripening. Exogenous ethylene applied during early plant development triggers the triple response phenotype; a shorter and thicker hypocotyl with an exaggerated apical hook. Despite the intimate relationship between plants and bacteria, the effect of exogenous ethylene on bacteria has been greatly overlooked. This is partly due to the difficulty of controlling gaseous ethylene within the laboratory without specialized equipment. 2-Chloroethylphosphonic acid (CEPA) is a compound that decomposes into ethylene, chlorine, and phosphate in a 1:1:1:1 molar ratio when dissolved in an aqueous medium of pH 3.5 or greater. Here we describe the use of CEPA to produce in situ ethylene for the investigation of ethylene response in bacteria using the fruit-associated, cellulose-producing bacterium Komagataeibacter xylinus as a model organism. The protocols described herein include both the verification of ethylene production from CEPA via the Arabidopsis thaliana triple response assay and the effects of exogenous ethylene on K. xylinus cellulose production, pellicle properties and colonial morphology. These protocols can be adapted to examine the effect of ethylene on other microbes using appropriate growth media and phenotype analyses. The use of CEPA provides researchers with a simple and efficient alternative to pure ethylene gas for the routine determination of bacterial ethylene response.

The protocols outlined herein facilitate the convenient investigation of bacterial ethylene responses by utilizing 2-chloroethylphosphonic acid (CEPA). Ethylene is produced in situ through the decomposition of CEPA in an aqueous bacterial growth medium, circumventing the requirement for pure ethylene gas.

細菌でエチレン応答を研究するためのツールとして、エチレン放出化合物、2-クロロエチルホスホン酸を利用します

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ...

生化学

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

An efficient and stable transformation system is fundamental for gene function study and molecular breeding of plants. Here, we describe the use of an Agrobacterium rhizogenes mediated transformation system on pigeon pea. The stem is infected with A. rhizogenes carrying a binary vector, which induced callus after 7 days and adventitious roots 14 days later. The generated transgenic hairy root was identified by morphological analysis and a GFP reporter gene.To further illustrate the application range of this system, CcCIPK14 (Calcineurin B-like protein-interacting protein kinases) was transformed into pigeon pea using this transformation method. The transgenic plants were treated with jasmonic acid (JA) and abscisic acid (ABA), respectively, for the purpose of testing whether CcCIPK14 responds to those hormones. The results demonstrated that (1) exogenous hormones could significantly upregulate the expression levelof CcCIPK14, especially in CcCIPK14 over-expression (OE) plants; (2) the content of Genistein in CcCIPK14-OE lines was significantly higher than the control; (3) the expression level of two downstream key flavonoid synthase genes, CcHIDH1 and CcHIDH2, were up-regulated in the CcCIPK14-OE lines; and (4) the hairy root transgenic system can be used to study metabolically functional genes in non-model plants.

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

Here we present an efficient and stable transformation system for the functional analysis of the CcCIPK14 gene as an example, providing a technical basis for studying the metabolism of non-model plants.

An efficient and stable transformation system is fundamental for gene function study and molecular breeding of plants. Here, we describe the use of an ...

遺伝学

Extraction and Quantification of Soluble, Radiolabeled Inositol Polyphosphates from Different Plant Species using SAX-HPLC

The phosphate esters of myo-inositol, also termed inositol phosphates (InsPs), are a class of cellular regulators playing important roles in plant physiology. Due to their negative charge, low abundance and susceptibility to hydrolytic activities, the detection and quantification of these molecules is challenging. This is particularly the case for highly phosphorylated forms containing &#8216;high-energy&#8217; diphospho bonds, also termed inositol pyrophosphates (PP-InsPs). Due to its high sensitivity, strong anion exchange high-performance liquid chromatography (SAX-HPLC) of plants labeled with [3H]-myo-inositol is currently the method of choice to analyze these molecules. By using [3H]-myo-inositol to radiolabel plant seedlings, various InsP species including several non-enantiomeric isomers can be detected and discriminated with high sensitivity. Here, the setup of a suitable SAX-HPLC system is described, as well as the complete workflow from plant cultivation, radiolabeling and InsP extraction to the SAX-HPLC run and subsequent data analysis. The protocol presented here allows the discrimination and quantification of various InsP species, including several non-enantiomeric isomers and of the PP-InsPs, InsP7 and InsP8, and can be easily adapted to other plant species. As examples, SAX-HPLC analyses of Arabidopsis thaliana and Lotus japonicus seedlings are performed and complete InsP profiles are presented and discussed. The method described here represents a promising tool to better understand the biological roles of InsPs in plants.

Here we describe strong anion exchange high-performance liquid chromatography of [3H]-myo-inositol-labeled seedlings which is a highly sensitive method to detect and quantify inositol polyphosphates in plants.

SAX-HPLCを用いた異種植物由来の可溶性、放射性標識イノシトールポリリン酸塩の抽出と定量化

The phosphate esters of myo-inositol, also termed inositol phosphates (InsPs), are a class of cellular regulators playing important roles in plant ...

Tuning Degradation to Achieve Specific and Efficient Protein Depletion

The plant auxin binding receptor, TIR1, recognizes proteins containing a specific auxin-inducible degron (AID) motif in the presence of auxin, targeting them for degradation. This system is exploited in many non-plant eukaryotes, such that a target protein, tagged with the AID motif, is degraded upon auxin addition. The level of TIR1 expression is critical; excessive expression leads to degradation of the AID-tagged protein even in the absence of auxin, whereas low expression leads to slow depletion. A &#946;-estradiol-inducible AID system was created, with expression of TIR1 under the control of a &#946;-estradiol inducible promoter. The level of TIR1 is tunable by changing the time of incubation with &#946;-estradiol before auxin addition. This protocol describes how to rapidly deplete a target protein using the AID system. The appropriate &#946;-estradiol incubation time depends on the abundance of the target protein. Therefore, efficient depletion depends on optimal timing that also minimizes auxin-independent depletion.

Here, we present a protocol to effectively and specifically deplete a protein of interest in the yeast Saccharomyces cerevisiae using the &#946;-est AID system.

特定および効率的なタンパク質枯渇を達成するための分解性の調整

The plant auxin binding receptor, TIR1, recognizes proteins containing a specific auxin-inducible degron (AID) motif in the presence of auxin, targeting ...

免疫・感染学

Lateral Root Inducible System in Arabidopsis and Maize

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is however tedious, because it occurs only in a few cells inside the root and in an unpredictable manner. To circumvent this problem, a Lateral Root Inducible System (LRIS) has been developed. By treating seedlings consecutively with an auxin transport inhibitor and a synthetic auxin, highly controlled lateral root initiation occurs synchronously in the primary root, allowing abundant sampling of a desired developmental stage. The LRIS has first been developed for Arabidopsis thaliana, but can be applied to other plants as well. Accordingly, it has been adapted for use in maize (Zea mays). A detailed overview of the different steps of the LRIS in both plants is given. The combination of this system with comparative transcriptomics made it possible to identify functional homologs of Arabidopsis lateral root initiation genes in other species as illustrated here for the CYCLIN B1;1 (CYCB1;1) cell cycle gene in maize. Finally, the principles that need to be taken into account when an LRIS is developed for other plant species are discussed.

Lateral Root Inducible System in Arabidopsis and Maize

The Lateral Root Inducible System (LRIS) allows for synchronous induction of lateral roots and is presented for Arabidopsis thaliana and maize.

側根誘導系で<em&gt;シロイヌナズナ</em&gt;とトウモロコシ

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is ...

生物学

A Strategy to Validate the Role of Callose-mediated Plasmodesmal Gating in the Tropic Response

The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The establishment of an auxin gradient is a key event leading to phototropism and gravitropism. Previously, polar auxin transport (PAT) was shown to establish an auxin gradient in different cellular domains of plants. However, Han et al. recently demonstrated that for proper auxin gradient formation, plasmodesmal callose-mediated symplasmic connectivity between the adjacent cells is also a critical factor. In this manuscript, the strategy to elucidate the role of particular genes, which can affect phototropism and gravitropism by altering the symplasmic connectivity through modulating plasmodesmal callose synthesis, is discussed. The first step is to screen aberrant tropic responses from 3-day-old etiolated seedlings of mutants or over-expression lines of a gene along with the wild type. This preliminary screening can lead to the identification of a range of genes functioning in PAT or controlling symplasmic connectivity. The second screening involves the sorting of candidates that show altered tropic responses by affecting symplasmic connectivity. To address such candidates, the movement of a symplasmic tracer and the deposition of plasmodesmal callose were examined. This strategy would be useful to explore new candidate genes that can regulate symplasmic connectivity directly or indirectly during tropic responses and other developmental processes.

This article describes the methods for screening the genes controlling plasmodesmal permeability and hence auxin gradient during tropic response. This includes the measurement of the degree of tropic response in hypocotyl of Arabidopsis thaliana and checking plasmodesmal permeability by 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) loading and finally callose level assessment.

トロピック応答におけるカロース媒介Plasmodesmalゲーティングの役割を検証するための戦略

The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The ...

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. Arabidopsis thaliana-Agrobacterium tumefaciens provides an excellent model system to study bacterial pathogenesis and plant interactions. Previous studies of plant-Agrobacterium interactions have largely relied on plant cell suspension cultures, the artificial wounding of plants, or the artificial induction of microbial virulence factors or plant defenses by synthetic chemicals. However, these methods are distinct from the natural signaling in planta, where plants and microbes recognize and respond in spatial and temporal manners. This work presents a hydroponic cocultivation system where intact plants are supported by metal mesh screens and cocultivated with Agrobacterium. In this cocultivation system, no synthetic phytohormone or chemical that induces microbial virulence or plant defense is supplemented. The hydroponic cocultivation system closely resembles natural plant-microbe interactions and signaling homeostasis in planta. Plant roots can be separated from the medium containing Agrobacterium, and the signaling and responses of both the plant hosts and the interacting microbes can be investigated simultaneously and systematically. At any given timepoint/interval, plant tissues or bacteria can be harvested separately for various &#34;omics&#34; analyses, demonstrating the power and efficacy of this system. The hydroponic cocultivation system can be easily adapted to study: 1) the reciprocal signaling of diverse plant-microbe systems, 2) signaling between a plant host and multiple microbial species (i.e.&#160;microbial consortia or microbiomes), 3) how nutrients and chemicals are implicated in plant-microbe signaling, and 4) how microbes interact with plant hosts and contribute to plant tolerance to biotic or abiotic stresses.

The described hydroponic cocultivation system supports intact plants with metal mesh screens and cocultivates them with bacteria. Plant tissue, bacteria, and secreted molecules can then be separately harvested for downstream analyses, simultaneously allowing for the molecular responses of both plant hosts and interacting microbes or microbiomes to be investigated.

植物/微生物の分子相互作用およびシグナル伝達の同時および系統的分析のための水耕栽培共生システム

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated with specialization, development and increase in cellular size. In plants, endoreduplication seems to facilitate the growth and expansion of certain tissues and organs. Among them is the tuber of potato (Solanum tuberosum), which undergoes considerable cellular expansion in fulfilling its function of carbohydrate storage. Thus, endoreduplication may play an important role in how tubers are able to accommodate this abundance of carbon. However, the cellular debris resulting from crude nuclear isolation methods of tubers, methods that can be used effectively with leaves, precludes the estimation of the tuber endoreduplication index (EI). This article presents a technique for assessing tuber endoreduplication through the isolation of protoplasts while demonstrating representative results obtained from different genotypes and compartmentalized tuber tissues. The major limitations of the protocol are the time and reagent costs required for sample preparation as well as relatively short lifespan of samples after lysis of protoplasts. While the protocol is sensitive to technical variation, it represents an improvement over traditional methods of nuclear isolation from these large specialized cells. Possibilities for improvements to the protocol such as recycling enzyme, the use of fixatives, and other alterations are proposed.

The protocol described herein is a method for measuring endoreduplication within tubers of potato (Solanum tuberosum). It includes plasmolysis and protoplast extraction steps to decrease the noise and debris in downstream flow cytometric analysis.

孤立した塊茎プロトプ ラストのフローサイトメトリーによる表現を測定

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant productivity and survival. As such, understanding the mechanisms by which stomata develop and pattern has tremendous agronomic value. This paper describes two phenotypic methods using Arabidopsis cotyledons that can be used to characterize the genes controlling stomatal development and patterning. Presented first are procedures for analyzing the stomatal phenotypes using toluidine blue O-stained cotyledons. This method is fast and reliable and does not require the use of epidermal peels, which are widely used for phenotypic analyses but require specialized training. Due to the presence of multiple cysteine residues, the identification and generation of bioactive EPF peptides that have a role in stomatal development have been challenging. Thus, presented second is a procedure used to identify stomatal ligands and monitor their biological activity by bioassays. The main advantage of this method is that it produces reproducible data relatively easily while reducing the amount of peptide solution and the time required to characterize the role of the peptides in controlling stomatal patterning and development. Overall, these well-designed protocols enhance the efficiency of studying the potential stomatal regulators, including cysteine-rich secretory peptides, which require highly complex structures for their activity.

Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring

This paper describes two phenotyping methods without the use of epidermal peels to characterize the genes controlling stomatal development. The first method demonstrates how to analyze a stomatal phenotype using a toluidine blue O-stained plant epidermis. The second method describes how to identify stomatal ligands and monitor their biological activities.

表皮表現型スコアリング による 気孔発生に関与する遺伝子の同定

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant ...

シロイヌナズナ実生のクロロフィル生合成の高分解能イメージング

Non-invasive Assay for Chlorophyll Biosynthesis Kinetics Determination during Early Stages of Arabidopsis De-etiolation

Chlorophyll biosynthesis is a hallmark of de-etiolation, one of the most dramatic stages in the plant life cycle. The tightly controlled and highly dynamic process of chlorophyll biosynthesis is triggered during the shift from the dark to the light in flowering plants. At the moment when etiolated seedlings are exposed to the first traces of sunlight, rapid (in order of seconds) conversion of protochlorophyllide into chlorophyllide is mediated by unique light-accepting protein complexes, leading via subsequent metabolic steps to the production of fully functional chlorophyll. Standard techniques for chlorophyll content analysis include pigment extraction from detached plant tissues, which does not apply to studying such fast processes. To investigate chlorophyll kinetics in vivo with high accuracy and spatiotemporal resolution in the first hours after light-induced de-etiolation, an instrument and protocol were developed. Here, we present a detailed procedure designed for statistically robust quantification of chlorophyll in the early stages of Arabidopsis de-etiolation.

著者スポットライト:脱エチオレーション中のクロロフィル合成の非侵襲的高分解能測定

Here, we describe an advanced tool designed for chlorophyll biosynthesis monitoring during the early stages of Arabidopsis seedling de-etiolation. The novel methodology provides non-invasive real-time chlorophyll fluorescence imaging at high spatial and temporal resolution.

シロイヌナズナ脱etiolationの初期段階におけるクロロフィル生合成速度測定のための非侵襲的アッセイ

Chlorophyll biosynthesis is a hallmark of de-etiolation, one of the most dramatic stages in the plant life cycle. The tightly controlled and highly ...

トコンの節間文化の中に内生オーキシンとサイトカイニンの定量化

要約

さらに動画を探す

この動画の章

細菌でエチレン応答を研究するためのツールとして、エチレン放出化合物、2-クロロエチルホスホン酸を利用します

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

SAX-HPLCを用いた異種植物由来の可溶性、放射性標識イノシトールポリリン酸塩の抽出と定量化

特定および効率的なタンパク質枯渇を達成するための分解性の調整

側根誘導系で

トロピック応答におけるカロース媒介Plasmodesmalゲーティングの役割を検証するための戦略

植物/微生物の分子相互作用およびシグナル伝達の同時および系統的分析のための水耕栽培共生システム

孤立した塊茎プロトプラストのフローサイトメトリーによる表現を測定

表皮表現型スコアリングによる気孔発生に関与する遺伝子の同定

シロイヌナズナ脱etiolationの初期段階におけるクロロフィル生合成速度測定のための非侵襲的アッセイ

トコンの節間文化の中に内生オーキシンとサイトカイニンの定量化

要約

さらに動画を探す

この動画の章

細菌でエチレン応答を研究するためのツールとして、エチレン放出化合物、2-クロロエチルホスホン酸を利用します

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

SAX-HPLCを用いた異種植物由来の可溶性、放射性標識イノシトールポリリン酸塩の抽出と定量化

特定および効率的なタンパク質枯渇を達成するための分解性の調整

側根誘導系で

トロピック応答におけるカロース媒介Plasmodesmalゲーティングの役割を検証するための戦略

植物/微生物の分子相互作用およびシグナル伝達の同時および系統的分析のための水耕栽培共生システム

孤立した塊茎プロトプ ラストのフローサイトメトリーによる表現を測定

表皮表現型スコアリング による 気孔発生に関与する遺伝子の同定

シロイヌナズナ脱etiolationの初期段階におけるクロロフィル生合成速度測定のための非侵襲的アッセイ

孤立した塊茎プロトプラストのフローサイトメトリーによる表現を測定

表皮表現型スコアリングによる気孔発生に関与する遺伝子の同定