Questo lavoro è stato sostenuto da sovvenzioni dal National Science Foundation svizzero e dallo Stato di Ginevra.

Una volta che il SCBA è assemblato, la crescita di embrioni viene monitorato per diversi giorni. Pertanto, prima della procedura di dissezione seme e montaggio del autorespiratori, si ha la necessità di sterilizzare i semi per evitare contaminazioni future che potrebbero impedire la corretta valutazione dell&#39;effetto di materiale tegumento sulla crescita embrionale. 1. Seed Sterilizzazione <ol><li> Versare 50-60 ml di semi di Arabidopsis maturi e secchi in una provetta da 1,5 ml e preparare una soluzione di etanolo al 70%. </li><li> Aggiungere 1 ml di soluzione di etanolo al 70% per la provetta contenente i semi e agitare a temperatura ambiente per 10 min a 1200 rpm in un vortex. </li><li> Centrifugare provetta per 3 sec a 4.000 xg per concentrare sementi al fondo della provetta. </li><li> Aspirare la soluzione di etanolo al 70% con una punta di aspirazione vuoto, lasciando accuratamente i semi sul fondo della provetta. </li><li> Aggiungere 1 ml di acqua distillata sterile per t<html> egli provetta contenente ancora i semi. </li><li> Agitare a temperatura ambiente per 10 min a 1200 rpm. </li><li> Centrifugare provetta per 3 sec a 4.000 xg per concentrare sementi al fondo della provetta. Aspirare il surnatante acqua utilizzando una punta di aspirazione vuoto, lasciando accuratamente i semi sul fondo della provetta. </li><li> Aggiungere 1 ml di acqua distillata acqua sterile alla provetta contenente ancora i semi. Assicurarsi che il flusso di acqua sospende omogeneamente i semi all&#39;interno del tubo. L&#39;obiettivo è quello di evitare un ulteriore agitazione in modo da preservare l&#39;integrità dei semi. </li><li> Lasciare che i semi depositano per gravità sul fondo del tubo lasciando il tubo ancora per 30 sec. Aspirare il surnatante acqua utilizzando una punta di aspirazione vuoto, lasciando accuratamente i semi sul fondo della provetta. </li><li> Ripetere quattro volte i passaggi 1,8-1,9. Nel complesso la procedura di sterilizzazione dura circa 30 min. </li></ol> 2. Seed placcatura ONTENUTO &quot;&gt; Tutte le fasi successive vengono eseguite all&#39;interno di una cappa a flusso laminare per mantenere condizioni di sterilità. <ol><li> Preparare una piastra piastra di Petri (diametro 100 mm, altezza 20 mm) contenente 30 ml di mezzo di germinazione preparato autoclavando una soluzione contenente 4,3 g / L Murashige e Skoog in 2,5 mM 2 - (N-morfolino) acido etansolfonico (MES-KOH ; pH 5.7) e 0,8 g / L di agar. Lasciare raffreddare la soluzione in un bagno d&#39;acqua a 50 ° C prima di versarlo sulla piastra piastra Petri. </li><li> Aggiungere 200 ml di acqua distillata sterile per il tubo contenente i semi. Risospendere i semi e trasferirli sulla superficie del mezzo di germinazione con una pipetta standard con una punta di 1 ml. </li><li> Rimuovere l&#39;acqua che circonda i semi sulla superficie del mezzo di germinazione con una punta di aspirazione sotto vuoto. </li><li> Lasciare la piastra Petri contenente i semi senza coperchio all&#39;interno della cappa a flusso laminare per 2 ore per ulteriore essiccamento. Chiudere la capsula di Petri con il coperchio e lasciar riposare sotto il laminareflusso armadio per circa 90 minuti in modo che circa 4 ore sono passati da quando l&#39;avvio della procedura di sterilizzazione seme (fase 1.2). </li></ol> 3. Seed Dissection Le seguenti operazioni richiedono lavoro con uno stereomicroscopio posto all&#39;interno dell&#39;armadio cappa a flusso laminare. Un Dumont pinze # 5 con troncate consigli (cioè smussato) facilita notevolmente la manipolazione dei semi (Figura 1A). <ol><li> Preparare una nuova piastra capsula di Petri contenente 30 ml di terreno di germinazione. Posto sulla superficie delle medie due rettangolari sterili carte Whatman 3MM giustapposte (1,5 cm x 1,0 cm; Figura 1B, step 1). Whatman 3MM carta viene sterilizzato in autoclave. </li><li> Trasferire i semi delle carte Whatman 3MM utilizzando pinze sterili (Figura 1B, step 2). </li><li> Tenere un seme di persona contro la carta Whatman 3MM premendo delicatamente il seme con le punte smussate giustapposti della pinza troncati ( <strong&gt; Figura 1B, punto 3). </li><li> Tagliare il cappotto di seme (testa e endosperma) del seme utilizzando un ago della siringa (ad esempio U100 insulina siringa + 1 siringa ml, 0,33 millimetri (29 G) x 12,7 mm) (Figura 1B, punto 4). La forma del seme di Arabidopsis è ellissoidale e si consiglia di tagliare il cappotto di seme lungo la più lunga semi-asse principale più vicino possibile al luogo in cui i cotiledoni sono uniti alla radichetta (cioè lontano dalla punta radicale). Questo aiuta il rilascio sicuro dell&#39;embrione nella fase successiva del procedimento dissezione. </li><li> Spingere il seme contro la carta Whatman 3MM utilizzando le punte smussate giustapposti della pinza troncate (Figura 1B, punto 5). Questo passaggio rilascia l&#39;embrione fuori il cappotto di seme attraverso l&#39;apertura creata nel passaggio 3.4 (Figura 1B, punto 6). Si raccomanda di applicare la spinta sul seme in cui la punta dei cotiledoni sono in più vicino alla estremità radicolare (cioè lontano from l&#39;apertura). </li></ol> 4. Assemblea del cappotto di seme Bedding Assay (SCBA) Si veda la discussione per la corretta scelta del numero di cappotti di seme ed embrioni. <ol><li> Preparare una nuova piastra capsula di Petri contenente 30 ml di terreno di germinazione. Posto sulla superficie del terreno un pezzo rettangolare sterile (3.5 cm x 5,0 centimetri) di maglia di nylon (Figura 2, punto 1). </li><li> Per evitare danni, embrioni e tegumenti possono essere spostati alla superficie dei bordi metallici della pinza spingendo delicatamente utilizzando l&#39;ago della siringa (Figura 2, punto 2). Embrioni di trasferimento e tegumenti sulla maglia di nylon (Figura 2, punto 3). </li><li> Montare uno strato circolare e singola di tegumenti utilizzando le pinze smussate e l&#39;ago fare in modo che i semi cappotti sono in quanto una maggiore vicinanza possibile (Figura 2, punto 4). Prova di esporre il più possibile l&#39;apertura tegumento generato dall&#39;ago verso l&#39;alto. Questo passo non è stato sistematicamente testato. Tuttavia, potrebbe aumentare l&#39;efficienza del SCBA da sostanze diffusibili inibiscono la crescita dell&#39;embrione dovrebbero diffondere direttamente verso l&#39;embrione piuttosto che lontano da esso. </li><li> Mettere gli embrioni come un unico strato circolare al centro del letto tegumento assemblato in fase 4.3 (Figura 2, punto 5). Assicurarsi che gli embrioni sono tanto maggiore vicinanza possibile. </li><li> Incubare le autorespiratori sotto luce continua (40 micromol / m 2 s ec) a 20-21 ° C. Nel caso in cui un impulso FR viene utilizzato per arrestare la germinazione, montare il SCBA a luce normale, applica l&#39;impulso FR come descritto 9 e lasciare piastra di Petri nell&#39;oscurità. </li></ol>

<ol>
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Gli autori dichiarano di non avere interessi finanziari in competizione.

Procedura Il test biancheria cappotto di seme (SCBA) qui descritto è in linea di principio applicabile a qualsiasi circostanza in cui la germinazione del seme di Arabidopsis è bloccato (o ritardato) e dove l&#39;endosperma è sospettato di implementare questo arresto. Quest&#39;ultimo può essere provata togliendo il cappotto di seme (testa e endosperma) e osservando che i proventi di crescita embrionali più veloce rispetto a quella osservata quando gli embrioni sono circondati dal tegumento. La germinazione...

In Arabidopsis semi maturi, il cappotto di seme è composto dalla testa, uno strato esterno di tessuto morto di origine materna, e l&#39;endosperma, un singolo strato di cellule di tessuto vivo che circonda direttamente l&#39;embrione 1. L&#39;endosperma e l&#39;embrione sono derivati ​​da eventi fecondazione distinti: il endosperma è un tessuto triploide con due materna e un genoma paterno che l&#39;embrione è un tessuto diploide con una materna e un genoma paterno 2. La funzione principale tradizionalmente assegnato al endosperma è quello di un tessuto nutritivo. Tuttavia, è sempre più evidente che l&#39;endosperma svolge un ruolo centrale per controllare la germinazione dei semi. Questa nozione è diventato il primo evidente nel caso di dormienza, un tratto esibita da semi di nuova produzione. Semi dormienti non riescono a germinare nonostante la presenza di condizioni di germinazione favorevoli. I semi perdono la loro dormienza dopo un periodo di maturazione e diventano nondormant, cioè germineranno quando esposto a condizioni favorevoli di germinazione. In molte specie vegetali, tra cui la pianta modello Arabidopsis, il cappotto di seme è assolutamente necessaria per impedire la germinazione dei semi dormienti poiché la rimozione tegumento innesca la crescita embrionale e greening 3,4. In Arabidopsis, Bethke et al. Ha osservato che la germinazione è rimasta repressa dopo aver tolto la testa, pur mantenendo l&#39;endosperma che circonda l&#39;endosperma 5. Queste osservazioni hanno indicato con forza che l&#39;endosperma è il tessuto all&#39;interno del cappotto di seme esercitando un&#39;attività repressiva sull&#39;embrione. Tuttavia, seme esperimenti rimozione coat non necessariamente aiutano chiarire la natura della germinazione attività repressiva fornite dal tegumento né identificare i geni che implementano. Abbiamo recentemente introdotto un test di biancheria cappotto di seme (SCBA) dove cappotti di seme ed embrioni sono separati fisicamente, ma tenuti in prossimmità modo che l&#39;attività repressiva germinazione fornita dal endosperma viene mantenuta 6. L&#39;autorespiratore consente l&#39;uso combinatorio di dormienti, nondormant e geneticamente modificati tegumento e materiali embrionali. Come risultato, i percorsi genetici che controllano la germinazione e specificamente operante nel endosperma ed embrione può essere sezionato. La SCBA stato usato nel contesto della dormienza per dimostrare che l&#39;endosperma rilascia l&#39;acido abscissico phytohormone (ABA) verso l&#39;embrione di reprimere la sua crescita 6. Inoltre, potremmo usare l&#39;autorespiratore per identificare le vie di segnalazione che operano in endosperma e tessuti embrionali per promuovere la dormienza. Il ruolo del endosperma di controllare la germinazione è stata ulteriormente rafforzata considerando il caso delle sementi nondormant esposti a un impulso di rosso lontano (FR) della luce. Presto su imbibizione semi di un impulso luminoso FR è noto per inibire la germinazione 7,8. Quando tegumenti sono stati rimossi dai semi un impulso di luce FRera in grado di inibire la germinazione, suggerendo fortemente che l&#39;endosperma può anche reprimere la germinazione dei semi nondormant 9. Sorprendentemente, la SCBA potrebbe anche essere utilizzato per ricapitolare inibizione FR-dipendente di germinazione. Questo ha permesso di dimostrare che tale FR-dipendente inibizione della germinazione dei semi è anche un processo che coinvolge ABA liberazione dal endosperma 9. Inoltre, l&#39;autorespiratore permesso identificare le differenti vie di segnalazione luminosa che operano nel endosperma e l&#39;embrione di controllare la germinazione dei semi nondormant in risposta a stimoli luminosi 9,10. La SCBA sembra quindi essere una tecnica affidabile per esplorare la funzione del endosperma nell&#39;ambito del controllo di germinazione dei semi. E &#39;anche un potente strumento per valutare in vitro se i geni sospettati di controllare germinazione operare nel endosperma, l&#39;embrione o entrambi i tessuti. Qui abbiamo dettaglio le varie fasi necessarie per assemblare un autorespiratore.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>To refer</td>
		</tr>
		<tr>
			<td>Thermomixer Comfort</td>
			<td>Eppendorf AG</td>
			<td>5355 000.011</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
		</tr>
		<tr>
			<td>Vacusafe Comfort</td>
			<td>INTEGRA Biosciences AG</td>
			<td>158 310</td>
			<td>Integra Biosciences AG, Zizers, Switzerland</td>
		</tr>
		<tr>
			<td>Petri dish plate (100 mm x 20 mm)</td>
			<td>Greiner Bio-One GmbH</td>
			<td>664 102</td>
			<td>Greiner Bio-One GmbH, Frickenhausen, Germany</td>
		</tr>
		<tr>
			<td>Murashige and Skoog</td>
			<td>Sigma-Aldrich</td>
			<td>M5524</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
		</tr>
		<tr>
			<td>MES</td>
			<td>Sigma-Aldrich</td>
			<td>M3671</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
		</tr>
		<tr>
			<td>Agar (plant agar)</td>
			<td>Duchefa Biochemie B.V.</td>
			<td>P1001</td>
			<td>Duchefa Biochemie, Haarlem, Netherlands</td>
		</tr>
		<tr>
			<td>Dumont forceps #5</td>
			<td>Fine Science Tools GmbH</td>
			<td>11251-10</td>
			<td>Fine Science Tools GmbH, Heidelberg Germany</td>
		</tr>
		<tr>
			<td>Syringe needle</td>
			<td>BD Micro-Fine</td>
			<td>324827</td>
			<td>BD, Franklin Lakes, NJ USA</td>
		</tr>
		<tr>
			<td>Nylon mesh (SEFAR NYTEX)</td>
			<td>SEFAR AG</td>
			<td>03-50/31</td>
			<td>Sefar AG, Heiden, Switzerland</td>
		</tr>
		<tr>
			<td>Growth chamber</td>
			<td>CLF Plant Climatics</td>
			<td>Percival I-30BLLX</td>
			<td>CLF plant Climatics, Wertingen, Germany</td>
		</tr>
		<tr>
			<td>Paclobutrazol</td>
			<td>Sigma-Aldrich</td>
			<td>46046</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
		</tr>
	</tbody>
</table>

a seed coat bedding assay to genetically explore in vitro how the endosperm controls seed germination in arabidopsis thaliana

L&#39;endosperma Arabidopsis è costituito da un singolo strato di cellule che circonda l&#39;embrione maturo e gioca un ruolo essenziale per impedire la germinazione dei semi dormienti o di semi nondormant irradiati da un impulso di luce rosso lontano (FR). Al fine di ottenere ulteriori informazioni sui meccanismi genetici molecolari alla base della germinazione attività repressiva esercitata dal endosperma, un saggio di &quot;assestamento cappotto di seme&quot; (SCBA) è stato concepito. La SCBA è una procedura di dissezione separando fisicamente tegumenti ed embrioni da semi, che consente il monitoraggio della crescita di embrioni in uno strato sottostante di cappotti di seme. Sorprendentemente, l&#39;autorespiratore ricostituisce la germinazione attività repressive del cappotto di seme nel contesto della dormienza dei semi e di controllo FR-dipendente di germinazione dei semi. Poiché l&#39;autorespiratore consente l&#39;utilizzo combinatoria di dormienti, nondormant e geneticamente modificati tegumento e materiali embrionali, i percorsi genetici che controllano la germinazione e specificatamente opdendo per l&#39;endosperma ed embrione può essere sezionato. Qui ci dettaglio la procedura per assemblare un autorespiratore.

Lavoro precedente ha mostrato che i semi mutanti incapaci di sintetizzare GA erano in grado di germinare come risultato di accumulo alta ABA in semi 11,12. Tuttavia, l&#39;incapacità di germinare richiede il cappotto di seme fin dalla sua rimozione innesca embrionale 13 la crescita. Questo indica fortemente che l&#39;endosperma dei semi in grado di sintetizzare GA sta rilasciando ABA per bloccare la crescita embrionale. Ci aspettiamo quindi semi cappotti in grado di sintetizzare GA per bloccare la...

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A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

Vi presentiamo la procedura per assemblare un seme saggio di biancheria da letto di mano (SCBA) da Arabidopsis thaliana semi. La SCBA ha dimostrato di essere uno strumento potente per esplorare geneticamente e in vitro come il germinazione del seme controlli endosperma nei semi dormienti e in risposta a stimoli luminosi. L&#39;autorespiratore in linea di principio applicabile in qualsiasi situazione in cui l&#39;endosperma è sospettato di influenzare la crescita embrionale.

Un cappotto Seed Bedding Assay per esplorare geneticamente<em&gt; In Vitro</em&gt; Come la Germinazione Controlli endosperma del seme in<em&gt; Arabidopsis thaliana</em

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of ...

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Biology

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

13.4K Views.  Université de Genève.  Vi presentiamo la procedura per assemblare un seme saggio di biancheria da letto di mano (SCBA) da Arabidopsis thaliana semi. La SCBA ha dimostrato di essere uno strumento potente per esplorare geneticamente e in vitro come il germinazione del seme controlli endosperma nei semi dormienti e in risposta a stimoli luminosi. L'autorespiratore in linea di principio applicabile in qualsiasi situazione in cui l'endosperma è sospettato di influenzare l...

Video: A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

Use of Arabidopsis eceriferum Mutants to Explore Plant Cuticle Biosynthesis

The plant cuticle is a waxy outer covering on plants that has a primary role in water conservation, but is also an important barrier against the entry of pathogenic microorganisms.  The cuticle is made up of a tough crosslinked polymer called "cutin" and a protective wax layer that seals the plant surface.  The waxy layer of the cuticle is obvious on many plants, appearing as a shiny film on the ivy leaf or as a dusty outer covering on the surface of a grape or a cabbage leaf thanks to light scattering crystals present in the wax.  Because the cuticle is an essential adaptation of plants to a terrestrial environment, understanding the genes involved in plant cuticle formation has applications in both agriculture and forestry. Today, we'll show the analysis of plant cuticle mutants identified by forward and reverse genetics approaches.


The plant cuticle is a waxy outer covering on plants that has a primary role in water conservation but is also an important barrier against the entry of pathogenic microorganisms. In this video, we demonstrate the analysis of plant cuticle mutants identified by forward and reverse genetics approaches.

L&#39;uso di Arabidopsis Mutanti eceriferum da esplorare per cuticole Biosintesi impianto

The plant cuticle is a waxy outer covering on plants that has a primary role in water conservation, but is also an important barrier against the entry of ...

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Biologia

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Isolamento dei ribosomi Polipeptidi Bound nascenti<em&gt; In vitro</em&gt; Per identificare i siti di pausa traslazione lungo mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways1-3.
	Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus4-6. Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.

A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.

Un alto contenuto di flusso di lavoro di imaging per studiare GRB2 Centri di segnalazione di espressione clonazione

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

In vitro metilazione Assay per studiare proteine ​​Arginina Metilazione

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Immunoprecipitazione della cromatina Saggio per la identificazione di Arabidopsis interazioni proteina-DNA<em&gt; In Vivo</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light conditions, which are preferably variable in quantity and quality so that studies under different setups can be conducted. These requirements are met by climatic chambers featuring light emitting diodes (LED) lights, which can &#8211; in contrast to fluorescent lights &#8211; be set to different wavelengths. LEDs are energy conserving and emit virtually no heat even at light intensities, which often constitutes a problem with other light sources. The presented protocol provides a step-by-step guidance of how to program a climatic chamber equipped with variable LED lights as well as describing several approaches for in depth analysis of growth phenotypes. Depending on the experimental set-up various characteristics of the growing plants can be observed and analyzed. Here we describe how to determine fresh weight, leaf area, photosynthetic activity, and stomatal density. We demonstrate that in order to obtain reliable data and draw valid conclusions it is mandatory to use a sufficient number of individuals for statistical evaluation. Taking too few plants for this kind of analysis results in high statistical errors and consequently in less clear interpretations of the data.

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Here, we present a protocol for studying plant growth behavior and especially phenotypes in a reproducible manner. We show how to provide variable and at the same time stable light conditions. Proper analyses depend on sufficient sample numbers and valid statistical evaluations.

Analisi del comportamento di crescita di Arabidopsis thaliana in diverse qualità di luce

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light ...

A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana

Plants responding to both intracellular and extracellular mechanical stimulations (or force signals) and develop special morphological changes, a called thigmomorphogenesis. In past decades, several signaling components have been identified and reported for being involved in the mechanotransduction (e.g., calcium ion binding proteins and jasmonic acid biosynthesis enzymes). However, the relatively slow pace of research in the study of force signaling or thigmomorphogenesis is largely attributed to two reasons: the requirement for laborious human hand-manipulated touch induction of thigmomorphogenesis and the force strength errors associated with people&#8217;s hand-touch. To enhance the efficiency of external force loading on a plant organism, an automatic touch-force loading machine was built. This robotic arm-driven hair brush touches provide a labor-saving and easily repeatable touch-force simulation, unlimited rounds of touch repetition and adjustable touch strength. This hair touch-force loading machine can be used for both large scale screening of touch-force signaling mutants and the phenomics study of plant thigmomorphogenesis. In addition, touch materials such as human hair, can be replaced with other natural materials like animal hair, silk threads and cotton fibers. The automated moving arms on the machine may be equipped with water sprinkling nozzles and air blowers to mimic the natural forces of rain drops and wind, respectively. By using this automatic hair touch-force loading machine in combination with the hand-performed cotton swab touching, we have investigated the touch response of two force signaling mutants, MAP KINASE KINASE 1 (MKK1) and MKK2 plants. The phenomes of the touch-force loaded wild type plants and two mutants were evaluated statistically. They have exhibited significant differences in touch response.

A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana

A gentle touch-force loading machine is built from human hair brushes, robotic arms and a controller. The hair brushes are driven by robotic arms installed on the machine and move periodically to apply touch-force on plants. The strength of machine-driven hair touches is comparable to that of manually applied touches.

Un protocollo dello schermo mutante di segnalazione della forza di manodopera e ripetibile per lo studio della Thigmomorphogenesis di un modello di arabidopsis thaliana

Plants responding to both intracellular and extracellular mechanical stimulations (or force signals) and develop special morphological changes, a called ...

A Simple Dry Sectioning Method for Obtaining Whole-Seed-Sized Resin Section and Its Applications

The morphology, size and quantity of cells, starch granules&#160;and protein bodies in seed determine the weight and quality of seed. They are significantly different among different regions of seed. In order to view the morphologies of cells, starch granules and protein bodies clearly, and quantitatively analyze their morphology parameters accurately, the whole-seed-sized section is needed. Though the whole-seed-sized paraffin section can investigate the accumulation of storage materials in seeds, it is very difficult to quantitatively analyze the morphology parameters of cells and storage materials due to the low resolution of the thick section. The thin resin section has high resolution, but the routine resin sectioning method is not suitable to prepare the whole-seed-sized section of mature seeds with a large volume and high starch content. In this study, we present a simple dry sectioning method for preparing the whole-seed-sized resin section. The technique can prepare the cross and longitudinal whole-seed-sized sections of developing, mature, germinated, and cooked seeds embedded in LR White resin, even for large seeds with high starch content. The whole-seed-sized section can be stained with fluorescent brightener 28, iodine, and Coomassie brilliant blue R250 to specifically exhibit the morphology of cells, starch granules, and protein bodies clearly, respectively. The image obtained can also be analyzed quantitatively to show the morphology parameters of cells, starch granules, and protein bodies in different regions of seed.

This technique allows for the fast and simple preparation of whole-seed-sized resin section for the observation and analysis of cells, starch granules, and protein bodies in different regions of the seed.

Un semplice metodo di sessatura a secco per ottenere una sezione di resina di dimensioni intere e le sue applicazioni

The morphology, size and quantity of cells, starch granules&#160;and protein bodies in seed determine the weight and quality of seed. They are ...

Analysis of Effect of Compound Salt Stress on Seed Germination and Salt Tolerance Analysis of Pepper (Capsicum annuum L.)

To determine the salt tolerance and physiological mechanism of pepper (Capsicum annuum L.) at the germination stage, the Hongtianhu 101 and Xinxiang 8 varieties, which have large differences in salt tolerance, are employed as the study materials. Six mixed salt concentrations of 0, 3, 5, 10, 15, and 20 g/L derived using equal molar ratios of Na2CO3, NaHCO3, NaCl, CaCl2, MgCl2, MgSO4, and Na2SO4 are used. To determine their effects, the related indexes of seed germination, seedling growth, and physiology are measured, and salt tolerance is comprehensively evaluated using membership function analysis. The results show that as the mixed salt concentration increases, the germination potential, germination index, germination rate, seed germination vigor index, root length, and root fresh weight of the two cultivars significantly decrease, whereas the relative salt rate gradually increases. The hypocotyl length and fresh weight aboveground increase first and then decrease, while the malondialdehyde (MDA), proline (Pro) content, catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) activity decrease and then increase. The germination potential, germination index, germination rate, seed germination vigor index, root length, root fresh weight, MDA and Pro content, and CAT activity of the Hongtianhu 101 seeds are higher than those of Xinxiang 8 for all salt concentrations employed here. However, hypocotyl length, fresh weight aboveground, and relative salt rate are lower in Hongtianhu 101 than in Xinxiang 8. The comprehensive evaluation of salt tolerance reveals that the total weighted values of the two membership function indexes increase first and then decrease as the mixed salt concentration increases. Compared with 5 g/L, which has the highest membership function value, the index under salt concentrations of 3 g/L, 10 g/L, and 15 g/L decreases by 4.7%-11.1%, 25.3%-28.3%, and 41.4%-45.1%, respectively. This study provides theoretical guidance for the breeding of salt-tolerant varieties of pepper and an analysis of the physiological mechanisms involved in salt tolerance and salt-tolerant cultivation.

Analysis of Effect of Compound Salt Stress on Seed Germination and Salt Tolerance Analysis of Pepper Capsicum annuum L.

The paper below presents a protocol for measuring seed germination, seedling growth, and physiological indexes of two pepper varieties with salinity tolerance differences in response to six mixed salt concentrations. This protocol can be used to evaluate the salt tolerance of pepper varieties.

Analisi dell'effetto dello stress salino composto sulla germinazione dei semi e analisi della tolleranza al sale del pepe (Capsicum annuum L.)

To determine the salt tolerance and physiological mechanism of pepper (Capsicum annuum L.) at the germination stage, the Hongtianhu 101 and Xinxiang 8 ...

Indagine spaziotemporale della dinamica ERL1 tramite imaging confocale

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

Autore in primo piano: Metodi basati su immagini per studiare gli eventi di traffico di membrana nelle cellule del lignaggio stomatico  

Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup.

Metodi basati su immagini per studiare eventi di traffico di membrana in cellule di lignaggio stomatico

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane ...