Siamo grati al signor Akira Murakami del dipartimento di scienze biologiche applicate, Università Toyo e signor Koudai Taniguchi del Gunma Agricultural Technology Center per la loro assistenza tecnica. Siamo anche grati al Professor Shosaku Kashiwada e Dr. Uma Maheswari Rajagopalan, Toyo University per i loro suggerimenti. Questo studio è stato sostenuto in parte dal centro di ricerca per la vita e scienze ambientali, Università Toyo.

Nota: Ipecac (c. ipecacuanha) è stato utilizzato in questo studio perché facilita l'analisi dei fitormoni endogeni.
1. crescita condizioni per indurre i germogli avventizi di Ipecac
<ol><li>Preparare privo di fitormoni B5 supporto regolata a pH 5.710e aggiungere 0,2% gellano. Sterilizzare in autoclave.</li>
	<li>Versare 25 mL di terreno sterilizzato nell'autoclave in una piastra Petri sterile (90 mm × 20 mm).</li>
	<li>Tagliare 8 mm segmenti internodale piantine di ipecacuana usando un bisturi chirurgico con una lama n. 22 su una lastra di acrilico sterile di e posto sul supporto (Figura 1).</li>
	<li>Cultura il medium di B5 senza fitormoni a 24 ° C sotto 15 µmol m− 2 s− 1 di luce in un ciclo di buio luce/10-h 14-h per 5 settimane.</li>
	<li>Identificare le aree ho (apicale) IV (basale) in ogni segmento (Figura 1B). Contare il numero di germogli avventizi > 0,3 mm di lunghezza in ogni regione sotto un microscopio una volta a settimana.</li>
</ol>2. estrazione e purificazione di fitormoni
<ol><li>Mettere una perlina di zirconia di 5 mm in ciascuna delle quattro provette 2 mL del campione.</li>
	<li>Tagliare i segmenti in regioni I a IV (ogni 2 mm di lunghezza) usando un bisturi chirurgico su una lastra di acrilico sterile.</li>
	<li>Raccogliere otto segmenti di ciascuna regione in provette separate (10-30 mg di peso fresco).</li>
	<li>Pesare e quindi bloccare i campioni in azoto liquido.</li>
	<li>Schiacciare i campioni congelati utilizzando un omogeneizzatore basati sul tallone.</li>
	<li>Sospendere i campioni schiacciati in 1 mL acetonitrile contenente 500 pg di ogni standard interno di auxina e CKs (d5-IAA, d5- tZ, d5- tZR, d6- iP, d6- iPR) utilizzando un miscelatore vortex.</li>
	<li>Mantenere a 4 ° C per 1 h, quindi centrifugare a 3.500 × g per 5 min a temperatura ambiente.</li>
	<li>Lavare la pallina in acetonitrile 80% (v/v) contenente acido acetico 1% (v/v). Centrifugare nuovamente a 3.500 × g per 5 min a temperatura ambiente. Combinare i surnatanti (da punti 2.7 e 2.8) in un tubo di vetro monouso.</li>
	<li>Aggiungere 600 µ l acqua che contengono l'acido acetico 1% (v/v) per ogni surnatante combinato ed evaporare l'acetonitrile utilizzando un concentratore a vuoto.</li>
	<li>Equilibrare le cartucce di colonna (HLB) idrofilo-lipofilo-bilanciato applicando 1 mL ciascuno di acetonitrile, metanolo e l'acqua contenente acido acetico 1% (v/v).</li>
	<li>Applicare una soluzione di esempio per cartuccia HLB equilibrato.</li>
	<li>Lavare le cartucce con 1 mL di acqua contenente acido acetico 1% (v/v).</li>
	<li>Eluire tutti gli ormoni con 2 mL 80% (v/v) acetonitrile contenente acido acetico 1% (v/v) in un tubo di vetro.</li>
	<li>Evaporare l'acetonitrile nell'eluito per ottenere l'estratto in acqua contenente acido acetico 1% (v/v) utilizzando un concentratore a vuoto. Nota: Non si asciughi completamente.</li>
	<li>Equilibrare le cartucce di colonna di scambio cationico misto, forte (MCX) applicando 1 mL di metanolo, acetonitrile 1 mL, 0,5 mL 0.1 M HCl e 1 mL di acqua contenente acido acetico 1% (v/v).</li>
	<li>Applicare una soluzione di esempio per cartuccia MCX equilibrato.</li>
	<li>Lavare le cartucce con 1 mL di acqua contenente acido acetico 1% (v/v).</li>
	<li>Eluire IAA con 2 mL 30% (v/v) acetonitrile contenente acido acetico 1% (v/v) in un tubo di vetro.</li>
	<li>Lavare le cartucce con 2 mL 80% (v/v) acetonitrile contenente acido acetico 1% (v/v).</li>
	<li>Lavare le cartucce con 2 mL di acqua e 1 mL di acqua contenente ammoniaca acquosa 5%.</li>
	<li>Eluire il CKs con acetonitrile 60% (v/v) di 2 mL contenente ammoniaca acquosa al 5% in un tubo di vetro.</li>
	<li>Far evaporare il solvente di ogni frazione di ormone utilizzando un concentratore a vuoto e conservare a − 30 ° C fino all'analisi LC-MS/MS.</li>
</ol>3. LC-MS/MS analisi di IAA e CKs
<ol><li>Sciogliere ogni ormone Estratto in una provetta con 600 µ l di metanolo e trasferire la soluzione in un flaconcino di recupero totale del collo a vite. Far evaporare il solvente utilizzando un concentratore a vuoto.</li>
	<li>Sciogliere la frazione di IAA in 20 µ l 30% (v/v) acetonitrile e le frazioni CK in acqua di 20 µ l contenente acido acetico 1% (v/v) in vite collo recupero totale fiale.</li>
	<li>Analizzare i campioni in modalità ioni positivi su un sistema di MS triplo quadrupolo equipaggiato con un sistema HPLC. Nota: Impostiamo le condizioni HPLC come elencato nella tabella 1.<ol>
<li>Per l'eluizione di IAA, usare un gradiente binario di 5% - 50% solvente B in 7 minuti, quindi aumentare di 98% solvente B premuto per 1 min e poi equilibrare per 2 min a solvente B al 5% per iniezione di nido.</li>
		<li>Per l'eluizione di CK, usare un gradiente binario di 2% - 40% solvente B in 5 minuti, 40% - 70% solvente B in 7 min, poi aumentare da 95% solvente B premuto per 1 min e poi equilibrare per 2 min a 2% solvente B di iniezione del nido.</li>
		<li>Impostare l'ionizzazione electrospray (ESI)-MS parametri di sorgente di ioni come elencato nella tabella 2.</li>
	</ol>
</li>
	<li>Utilizzare la reazione più monitoraggio transizione (MRM) per la quantificazione di ciascun analita riportato nella tabella 3.</li>
	<li>Quantificare i livelli endogeni di IAA e CK contro una curva standard del rapporto di adenoide deuterio-etichetta standard.</li>
</ol>

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Gli autori dichiarano di non avere nessun conflitto di interessi.

Per identificare la distribuzione dei fitormoni coinvolti nell'organogenesi, è importante utilizzare materiali vegetali in cui organogenesi può essere osservato su medium senza fitormoni, poiché quando fitormoni esogenicamente vengono applicati agli espianti per l'induzione germogli o radici, colpiscono l'espianto intero, rendendo difficile valutare la plasticità intrinseca delle piante nella differenziazione cellulare e l'organogenesi. Germogli avventizi possono essere indotta su terreni di coltura privo di fitormon...

Gottlieb Haberlandt (1854-1945) ha proposto il concetto di "totipotenza", da quale pianta cellule possono dividere, differenziare e rigenerare le piante intere anche dopo la loro prima differenziazione in tipi cellulari specifici in piante mature1. Nella coltura del tessuto, se pianta rigenerazione può essere indotta o non è determinata dalla combinazione e concentrazione di fitormoni esogenicamente applicate nel mezzo di crescita. Skoog e Miller trovato che germogli avventizi potrebbero essere indotto dal callo di tabacco su terreno di coltura contenente un alto rapporto di CKs di auxine, mentre radici avventizie potrebbero essere indotto su terreno contenente un rapporto basso2. Da tale constatazione, la coltura del tessuto è stato ampiamente utilizzata per la propagazione delle colture economicamente importanti e per la rigenerazione di piante transgeniche3. Germogli avventizi possono essere indotta da tessuti diversi meristema apicale di sparare, come foglie, radici e internodi. Trattamento di fitormoni è necessaria per l'induzione di germogli avventizi nella maggior parte delle specie di piante. Tuttavia, le concentrazioni ottimale e combinazioni differiscono da specie e tra tessuti utilizzati come espianti. Così, molto sforzo va a determinare le concentrazioni ottimale e le combinazioni di fitormoni per esperimenti.
Psychotria ipecacuanha (Brot.) L. Andersson (ipecac) è una pianta medicinale che contiene alcaloidi come la emetine e Cefelina, principalmente in radici4. Gli estratti della radice sono utilizzati come espettorante, emetico e un amoebicide5. Anche se ipecacuana cresce spontaneamente nelle foreste pluviali tropicali del Brasile, è riluttante a impostare semi nella cultura, e il tasso di germinazione diminuisce durante la conservazione del seme in Giappone, con il clima più freddo6. Invece, si propaga dalla coltura del tessuto, in cui avventizi spara formazione su internodi è il più efficiente metodo7,8. Interessante, i germogli avventizi possono essere indotta in questa specie senza fitormoni trattamento8.
Germogli avventizi si formano sull'epidermide della regione apicale della internodale segmenti senza forzatura, ma non nella regione basale9. Questa differenza indica la polarità di tessuto nei segmenti internodale, che è probabilmente ai sensi del regolamento phytohormonal. Il sistema di cultura ipecacuana permette un'occasione unica per analizzare i cambiamenti nei livelli endogeni fitormoni durante la formazione di sparo accidentale. Qui vi presentiamo il nostro metodo per l'analisi dei livelli endogeni di uno auxina (acido indolo-3-acetico (IAA)) e quattro CKs (isopentenyl adenina (iP), isopentenyl adenina riboside (iPR), trans-zeatina (tZ) e trans-zeatin riboside (tZR)) nei segmenti internodale attraverso l'uso di LC-MS/MS.

<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

Formazione di sparo accidentale è una tecnica importante per la propagazione delle colture economicamente importanti e per la rigenerazione di piante transgeniche. Trattamento di fitormoni è necessaria per l'induzione di germogli avventizi in maggior parte delle specie. Se possono essere indotta germogli avventizi è determinata dall'equilibrio tra auxine e citochinine (CK) livelli. Molto sforzo va a determinare le concentrazioni ottimale e combinazioni di fitormoni in ogni tessuto utilizzato come espianti e in ogni specie di pianta. In ipecac, tuttavia, germogli avventizi possono essere indotto sui segmenti internodale in terreno di coltura senza trattamento di fitormoni. Questo consente la plasticità intrinseca di ipecac per differenziazione delle cellule deve essere valutata. Per indurre i germogli avventizi in ipecac, abbiamo coltivato internodale segmenti a 24 ° C sotto 15 µmol m− 2 s− 1 di luce in un ciclo di buio luce/10-h 14-h il medium di B5 senza fitormoni solidificato con gomma di gellan 0,2% per 5 settimane. Per studiare dinamiche di fitormoni durante la formazione di sparo accidentale, abbiamo misurato acido di indole-3-acetic endogeno e CKs nei segmenti di liquida cromatografia-spettrometria di massa LC-MS/MS. Questo metodo permette l'analisi di acido di indole-3-acetic endogeno e livelli di CKs in modo semplice. Può essere applicato per studiare le dinamiche di auxina endogena e CK durante l'organogenesi in altre specie vegetali.

A 1 settimana dist , germogli avventizi non avevano formato. Alla settimana 2nd , piccoli germogli apparso. Presso i 3rd e 4 settimane dith , il numero di spara aumentato soprattutto nelle regioni apicale (I e II) (Figura 2A). Presso il 5° settimana, il numero di germogli era circa 7 in regione e 5 nella regione II (Figura 2B). Al contrario,...

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

Quantificazione di endogeno dell'auxina e citochinina durante la coltura di internodo di Ipecac

Germogli avventizi possono essere indotta su segmenti internodale di ipecac senza trattamento di fitormoni. Per valutare la dinamica di fitormoni durante la formazione di sparo accidentale, abbiamo misurato endogeno dell'auxina e citochinina in segmenti internodale mediante LC-MS/MS.

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

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9.0K Views.  Toyo University. Germogli avventizi possono essere indotta su segmenti internodale di ipecac senza trattamento di fitormoni. Per valutare la dinamica di fitormoni durante la formazione di sparo accidentale, abbiamo misurato endogeno dell'auxina e citochinina in segmenti internodale mediante LC-MS/MS.

Video: 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.

Utilizzando il Compound Etilene-releasing, acido 2-Chloroethylphosphonic, come strumento per studiare risposta di etilene in batteri

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

Ricerca

Biochimica

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 ...

Genetica

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.

Estrazione e quantificazione di Polifosfati Inostoti a inositolo radioeti da diverse specie vegetali che utilizzano SAX-HPLC

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.

Ottimizzazione della degradazione per ottenere un esaurimento specifico ed efficiente delle proteine

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

Immunologia e infezioni

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.

Laterale Root inducibile di sistema in<em&gt; Arabidopsis</em&gt; E mais

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

Biologia

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.

Una strategia per convalidare il ruolo di callose mediata Plasmodesmal gating nella risposta Tropic

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.

Un sistema di co-coltivazione idroponica per l&#39;analisi simultanea e sistematica delle interazioni molecolari e della segnalazione delle piante / microbe

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.

Endoreduplicazione tramite flusso Cytometry di tubero isolato protoplasti di misura

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.

Identificazione dei geni coinvolti nello sviluppo stomatale tramite punteggio del fenotipo epidermico

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 ...

Imaging ad alta risoluzione della biosintesi della clorofilla nella piantina di Arabidopsis

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.

Autore in primo piano: Misurazione non invasiva ad alta risoluzione della sintesi della clorofilla durante la de-eziolazione

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.

Test non invasivo per la determinazione della cinetica della biosintesi della clorofilla durante le prime fasi della deeziolazione dell'Arabidopsis

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

Quantificazione di endogeno dell'auxina e citochinina durante la coltura di internodo di Ipecac

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Capitoli in questo video

Utilizzando il Compound Etilene-releasing, acido 2-Chloroethylphosphonic, come strumento per studiare risposta di etilene in batteri

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

Estrazione e quantificazione di Polifosfati Inostoti a inositolo radioeti da diverse specie vegetali che utilizzano SAX-HPLC

Ottimizzazione della degradazione per ottenere un esaurimento specifico ed efficiente delle proteine

Laterale Root inducibile di sistema in

Una strategia per convalidare il ruolo di callose mediata Plasmodesmal gating nella risposta Tropic

Un sistema di co-coltivazione idroponica per l'analisi simultanea e sistematica delle interazioni molecolari e della segnalazione delle piante / microbe

Endoreduplicazione tramite flusso Cytometry di tubero isolato protoplasti di misura

Identificazione dei geni coinvolti nello sviluppo stomatale tramite punteggio del fenotipo epidermico

Test non invasivo per la determinazione della cinetica della biosintesi della clorofilla durante le prime fasi della deeziolazione dell'Arabidopsis