Este trabalho foi financiado por concessões do National Science Foundation suíço e pelo Estado de Genebra.

Uma vez que o ARA está montado, o crescimento dos embriões é monitorizado ao longo de vários dias. Portanto, antes do procedimento de dissecção sementes e montagem do equipamento autônomo, é preciso esterilizar as sementes, para evitar contaminações futuras que poderiam impedir a adequada avaliação do efeito do material de revestimento da semente no crescimento embrionário. 1. Esterilização Semente <ol><li> Despeje 50-60 ul de sementes de Arabidopsis maduros e secos num tubo de microcentrífuga de 1,5 ml e se preparar uma solução de etanol a 70%. </li><li> Adicionar 1 ml de solução de etanol a 70% para o tubo de microcentrífuga contendo as sementes e agitar à temperatura ambiente durante 10 min a 1.200 rpm num agitador. </li><li> Tubo de microcentrífuga Centrifugar durante 3 segundos a 4000 xg para concentrar as sementes no fundo do tubo. </li><li> Aspirar a solução de etanol a 70%, utilizando uma ponta de sucção a vácuo deixando cuidadosamente as sementes no fundo do tubo de microcentrífuga. </li><li> Adicionar 1 ml de água destilada estéril para t<html> ele tubo de microcentrífuga contendo ainda as sementes. </li><li> Agita-se à temperatura ambiente durante 10 min a 1200 rpm. </li><li> Tubo de microcentrífuga Centrifugar durante 3 segundos a 4000 xg para concentrar as sementes no fundo do tubo. Aspirar o sobrenadante de água utilizando uma ponta de sucção a vácuo deixando cuidadosamente as sementes no fundo do tubo. </li><li> Adicionar 1 ml de água destilada, água estéril para o tubo de microcentrífuga contendo ainda as sementes. Certifique-se de que o fluxo de água de forma homogénea suspende as sementes no interior do tubo. O objectivo aqui é o de evitar a agitação adicional de modo a preservar a integridade das sementes. </li><li> Deixe as sementes assentar por gravidade no fundo do tubo, deixando o tubo ainda durante 30 segundos. Aspirar o sobrenadante de água utilizando uma ponta de sucção a vácuo deixando cuidadosamente as sementes no fundo do tubo. </li><li> Repita quatro vezes os passos de 1,8-1,9. Em geral, o procedimento de esterilização leva cerca de 30 minutos. </li></ol> 2. Semente chapeamento ONTEÚDO &quot;&gt; Todas as etapas subseqüentes são realizadas dentro de uma câmara de fluxo laminar para preservar condições estéreis. <ol><li> Prepara-se uma placa de Petri (100 mm de diâmetro, 20 mm de altura) contendo 30 ml de meio de germinação preparado por autoclavagem de uma solução contendo 4,3 g / L de meio de Murashige e Skoog a 2,5 mM de 2 - (N-morfolino) etanossulfónico (MES-KOH , pH 5,7) e 0,8 g / L de agar. Deixar a solução arrefecer num banho de água a 50 ° C antes de coloca-lo na placa de Petri. </li><li> Adicionar 200 mL de água destilada estéril para o tubo contendo as sementes. Ressuspender as sementes e transferi-los na superfície do meio de germinação usando uma pipeta de padrão com uma ponta de 1 ml. </li><li> Remover a água em torno das sementes na superfície do meio de germinação usando uma ponta de sucção a vácuo. </li><li> Deixe a placa de Petri contendo as sementes, sem a tampa dentro da câmara de fluxo laminar para 2 horas para posterior secagem. Feche a placa de Petri com a tampa e deixe repousar sob a laminarfluir gabinete por cerca de 90 min para que cerca de 4 horas se passaram desde o início do processo de esterilização de sementes (passo 1.2). </li></ol> 3. Dissecção Semente Os seguintes passos necessitam de trabalhar com um estereomicroscópio colocado no interior do armário de câmara de fluxo laminar. Uma pinça Dumont # 5 com pontas truncadas (isto é, sem corte) facilita grandemente o manuseamento das sementes (Figura 1A). <ol><li> Prepara-se uma nova placa de Petri contendo 30 ml de meio de germinação. Lugar na superfície do meio de duas estéreis papéis Whatman 3MM justapostas rectangulares (1.5 cm x 1,0 cm; Figura 1B, passo 1). Whatman 3MM papel é esterilizada em autoclave. </li><li> Transferência de sementes nos papéis Whatman 3MM usando uma pinça estéril (Figura 1B, Passo 2). </li><li> Segure uma semente indivíduo contra o papel Whatman 3MM, pressionando suavemente a semente usando as pontas sem corte justapostas dos fórceps truncadas ( <stRong&gt; Figura 1B, passo 3). </li><li> Cortar o revestimento de semente (testa e do endosperma) da semente, utilizando uma agulha de seringa (por exemplo, insulina U100 + seringa seringa de 1 ml, 0,33 mm (29 L) x 12,7 mm) (Figura 1B, passo 4). A forma de sementes de Arabidopsis é elipsoidal e recomenda-se para cortar a casca da semente ao longo das mais longas semi-eixo principais o mais próximo possível do local onde os cotilédones são unidas à radícula (ou seja, longe da ponta radical). Isto ajuda a libertação segura do embrião na fase seguinte do processo de esvaziamento. </li><li> Empurre a semente contra o papel Whatman 3MM usando as pontas sem corte justapostas dos fórceps truncadas (Figura 1B, passo 5). Este passo liberta o embrião para o revestimento da semente através da abertura criada na etapa 3,4 (Figura 1B, passo 6). Recomenda-se aplicar a pressão sobre a semente em que a ponta dos cotilédones são em mais próximo da ponta da radícula (isto é, distância from a abertura). </li></ol> 4. Assembleia da tegumento Cama Assay (SCBA) Veja a discussão para a escolha adequada do número de demãos de sementes e embriões. <ol><li> Prepara-se uma nova placa de Petri contendo 30 ml de meio de germinação. Lugar na superfície do meio de uma peça rectangular estéril (3.5 cm x 5,0 cm) de malha de nylon (Figura 2, passo 1). </li><li> Para evitar danos, os embriões e os revestimentos de sementes podem ser transferidos para a superfície das bordas metálicas da pinça por suavemente empurrando-os utilizando a agulha da seringa (Figura 2, passo 2). Transferência de embriões e casacos de sementes sobre a malha de nylon (Figura 2, Passo 3). </li><li> Montar uma camada circular e único de tegumento usando a pinça embotados e da agulha certificando-se de que o tegumento das sementes estão em proximidade, tanto mais perto possível (Figura 2, passo 4). Tente expor tanto quanto possível a abertura do revestimento de semente gerado pelaagulha para cima. Este passo não foi sistematicamente testada. No entanto, ele pode aumentar a eficiência da ARA desde são esperados substâncias difusíveis que inibem o crescimento do embrião de se difundir para fora directamente para o embrião em vez de fora a partir dele. </li><li> Colocar os embriões como uma camada única circular no centro do leito de revestimento de semente montados no passo 4.3 (Figura 2, passo 5). Certifique-se de que os embriões estão em proximidade, tanto mais perto possível. </li><li> Incubar as SCBAs sob luz contínua (40 mmol / m 2 s ec) a 20-21 ° C. No caso em que um impulso de RF é usado para prender a germinação, montar o ARA sob luz normal, aplica o impulso de RF, como descrito 9 e deixar uma placa de Petri em escuridão. </li></ol>

<ol>
	<li>Debeaujon, I., Leon-Kloosterziel, K. M., Koornneef, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+the+testa+on+seed+dormancy,+germination,+and+longevity+in+Arabidopsis.">Influence of the testa on seed dormancy, germination, and longevity in Arabidopsis.</a> Plant Physiol. 122, 403-414 (2000).</li><li>Baroux, C., Spillane, C., Grossniklaus, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+origins+of+the+endosperm+in+flowering+plants.">Evolutionary origins of the endosperm in flowering plants.</a> Genome Biol. 3, reviews1026 (2002).</li><li>Debeaujon, I., Lepiniec, L., Pourcel, L., Routaboul, J. -. M., Bradford, K., Nonogaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Seed development, dormancy and germination. , 25-43 (2007).</li><li>Finch-Savage, W. E., Leubner-Metzger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seed+dormancy+and+the+control+of+germination.">Seed dormancy and the control of germination.</a> New Phytol. 171, 501-523 (2006).</li><li>Bethke, P. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arabidopsis+aleurone+layer+responds+to+nitric+oxide,+gibberellin,+and+abscisic+acid+and+is+sufficient+and+necessary+for+seed+dormancy.">The Arabidopsis aleurone layer responds to nitric oxide, gibberellin, and abscisic acid and is sufficient and necessary for seed dormancy.</a> Plant Physiol. 143, 1173-1188 (2007).</li><li>Lee, K. P., Piskurewicz, U., Tureckova, V., Strnad, M., Lopez-Molina, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+seed+coat+bedding+assay+shows+that+RGL2-dependent+release+of+abscisic+acid+by+the+endosperm+controls+embryo+growth+in+Arabidopsis+dormant+seeds.">A seed coat bedding assay shows that RGL2-dependent release of abscisic acid by the endosperm controls embryo growth in Arabidopsis dormant seeds.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 19108-19113 (2010).</li><li>Reed, J. W., Nagatani, A., Elich, T. D., Fagan, M., Chory, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phytochrome+A+and+Phytochrome+B+Have+Overlapping+but+Distinct+Functions+in+Arabidopsis+Development.">Phytochrome A and Phytochrome B Have Overlapping but Distinct Functions in Arabidopsis Development.</a> Plant Physiol. 104, 1139-1149 (1994).</li><li>Shinomura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action+spectra+for+phytochrome+A-+and+B-specific+photoinduction+of+seed+germination+in+Arabidopsis+thaliana.">Action spectra for phytochrome A- and B-specific photoinduction of seed germination in Arabidopsis thaliana.</a> Proc. Natl. Acad. Sci. U.S.A. 93, 8129-8133 (1996).</li><li>Lee, K. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+and+genetically+distinct+control+of+seed+germination+by+phytochromes+A+and+B.">Spatially and genetically distinct control of seed germination by phytochromes A and B.</a> Genes Dev. 26, 1984-1996 (2012).</li><li>Lee, K. P., Lopez-Molina, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+seed+germination+in+the+shade.">Control of seed germination in the shade.</a> Cell Cycle. 11, 4489-4490 (2012).</li><li>Piskurewicz, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gibberellic+acid+signaling+repressor+RGL2+inhibits+Arabidopsis+seed+germination+by+stimulating+abscisic+acid+synthesis+and+ABI5+activity.">The gibberellic acid signaling repressor RGL2 inhibits Arabidopsis seed germination by stimulating abscisic acid synthesis and ABI5 activity.</a> Plant Cell. 20, 2729-2745 (2008).</li><li>Piskurewicz, U., Tureckova, V., Lacombe, E., Lopez-Molina, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Far-red+light+inhibits+germination+through+DELLA-dependent+stimulation+of+ABA+synthesis+and+ABI3+activity.">Far-red light inhibits germination through DELLA-dependent stimulation of ABA synthesis and ABI3 activity.</a> EMBO J. 28, 2259-2271 (2009).</li><li>Debeaujon, I., Koornneef, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gibberellin+requirement+for+Arabidopsis+seed+germination+is+determined+both+by+testa+characteristics+and+embryonic+abscisic+acid.">Gibberellin requirement for Arabidopsis seed germination is determined both by testa characteristics and embryonic abscisic acid.</a> Plant Physiol. 122, 415-424 (2000).</li><li>Sun, T. P., Kamiya, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arabidopsis+GA1+locus+encodes+the+cyclase+ent-kaurene+synthetase+A+of+gibberellin+biosynthesis.">The Arabidopsis GA1 locus encodes the cyclase ent-kaurene synthetase A of gibberellin biosynthesis.</a> Plant Cell. 6, 1509-1518 (1994).</li><li>Alonso, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+insertional+mutagenesis+of+Arabidopsis+thaliana.">Genome-wide insertional mutagenesis of Arabidopsis thaliana.</a> Science. 301, 653-657 (2003).</li><li>Rook, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+sucrose-induction+mutants+reveal+the+modulation+of+sugar-induced+starch+biosynthetic+gene+expression+by+abscisic+acid+signalling.">Impaired sucrose-induction mutants reveal the modulation of sugar-induced starch biosynthetic gene expression by abscisic acid signalling.</a> Plant J. 26, 421-433 (2001).</li></ol>

Os autores declaram que não têm interesses financeiros concorrentes.

Procedimento O ensaio da cama tegumento (SCBA) aqui descrito é, em princípio, aplicável a qualquer circunstância em que a germinação de sementes de Arabidopsis é bloqueado (ou atrasado) e onde o endosperma é suspeito de implementar esta prisão. Este último pode ser evidenciada através da remoção do revestimento de semente (testa e do endosperma) e observando-se que os rendimentos de crescimento mais rápidas embrionárias relativos à observada quando os embriões são rodeados pelo revestimento da...

Na Arabidopsis, as sementes maduras, o revestimento de semente é composto do testa, a camada externa do tecido morto de origem materna, e o endosperma, uma camada única de células de tecido vivo, que rodeia o embrião 1. O endosperma e o embrião são derivadas de eventos de fertilização separadas: o endosperma é um tecido com duas triplóides materno e um genoma paterno enquanto que o embrião é um tecido com um diplóide materno e um genoma paterno 2. A principal função tradicionalmente designado para o endosperma é a de um tecido nutritivo. No entanto, torna-se cada vez mais evidente que o endosperma também desempenha um papel central para controlar a germinação das sementes. Esta noção tornou-se aparente pela primeira no caso de dormência, uma característica exibida por sementes produzidos recentemente. Sementes dormentes não germinam apesar da presença de condições de germinação favoráveis. Sementes perdem sua dormência depois de um período de maturação e se tornar nondormant, ou seja, elas vão germinar quando expostos a condições favoráveis ​​de germinação. Em muitas espécies de plantas, incluindo a planta modelo Arabidopsis, o tegumento é absolutamente necessário para impedir a germinação de sementes dormentes desde remoção do tegumento desencadeia o crescimento embrionário e greening 3,4. Na Arabidopsis, Bethke et al. Observou-se que a germinação permaneceu reprimida após a remoção do tegumento, mantendo o endosperma envolvente do endosperma 5. Estas observações indicaram nitidamente que o endosperma é o tecido no interior do revestimento de semente de exercer uma actividade repressiva sobre o embrião. No entanto, as experiências de remoção do tegumento não necessariamente ajudar a esclarecer a natureza da atividade repressiva germinação fornecido pelo tegumento nem identificar os genes que a implementam. Recentemente, lançou um ensaio semente cama casaco (ARA), onde casacos de sementes e embriões são separados fisicamente, mas manteve em estreita proxidade, de modo que a actividade repressora germinação fornecida pelo endosperma é mantido 6. O SCBA permite o uso combinatória de tegumento dormente, nondormant e geneticamente modificada e materiais embrionárias. Como resultado, as vias genéticas que controlam a germinação e operando especificamente no endosperma e no embrião pode ser dissecado. O ARA foi utilizado no contexto de dormência para mostrar que o endosperma liberta o ácido abscísico fitohormona (ABA) em direcção ao embrião para reprimir o crescimento 6. Além disso, podemos usar a ARA para identificar as vias de sinalização que operam no endosperma e nos tecidos embrionários para promover a dormência. O papel do endosperma para controlar a germinação foi reforçada por considerar o caso das sementes nondormant expostas a um pulso de longe vermelho (FR) de luz. Cedo após a embebição das sementes um pulso de luz FR é conhecido por inibir a germinação 7,8. Quando tegumento foram removidos a partir de sementes de um pulso de luz FRfoi incapaz de inibir a germinação, sugerindo fortemente que o endosperma também pode reprimir a germinação das sementes nondormant 9. Notavelmente, o ARA também poderia ser usado para recapitular dependente da FR inibição da germinação. Isto permitiu demonstrar que a inibição dependente da FR de germinação das sementes é também um processo que envolve a libertação a partir do endosperma do ABA 9. Além disso, o ARA permitiu identificar as diferentes vias de sinalização luminosa que operam no endosperma e no embrião para controlar a germinação das sementes nondormant em resposta a sinais de luz 9,10. O ARA, por conseguinte, parece ser uma técnica de confiança para explorar a função do endosperma no âmbito do controlo de germinação de sementes. É também uma ferramenta poderosa para avaliar in vitro se os genes suspeitos de controlar a germinação operar no endosperma, o embrião ou de ambos os tecidos. Aqui detalhamos os vários passos necessários para montar um autônomo.

<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

O endosperma de Arabidopsis consiste de uma única camada celular em torno do embrião maduro e desempenha um papel essencial para prevenir a germinação de sementes dormentes ou que de sementes nondormant irradiados por um pulso de luz vermelho longínquo (FR). A fim de obter mais conhecimentos sobre os mecanismos genéticos moleculares subjacentes à atividade repressiva germinação exercida pelo endosperma, um ensaio de &quot;semente da cama casaco&quot; (ARA) foi inventado. O SCBA é um procedimento de dissecção separando fisicamente casacos de sementes e embriões de sementes, o que permite monitorar o crescimento de embriões em uma camada subjacente de tegumento. Notavelmente, o SCBA reconstitui a germinação atividades repressivas do tegumento da semente no contexto da dormência de sementes e controle de FR-dependente de germinação das sementes. Uma vez que o equipamento autônomo permite o uso combinatória de tegumento dormente, normais e sob geneticamente modificado e materiais embrionárias, os caminhos genéticos que controlam a germinação e, especificamente, operating no endosperma e embrião pode ser dissecado. Aqui detalhamos o procedimento para montar um autônomo.

Trabalhos anteriores mostraram que as sementes mutantes incapazes de sintetizar GA foram incapazes de germinar, como resultado da acumulação de alta ABA nas sementes 11,12. No entanto, a incapacidade de germinar requer a casca da semente, desde a sua remoção provoca embrionário 13 crescimento. Isto indica fortemente que o endosperma de sementes incapazes de sintetizar o GA está lançando ABA para bloquear o crescimento embrionário. Esperamos, portanto, o tegumento das sementes incapazes de s...

Watch this Scientific Journal Video about A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana at JoVE.com

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

Nós apresentamos o procedimento para montar um ensaio da cama tegumento (SCBA) de Arabidopsis thaliana sementes. O ARA foi demonstrado ser uma ferramenta poderosa para explorar geneticamente e in vitro como a germinação de sementes de controlo do endosperma de sementes de dormentes e em resposta a sinais de luz. O SCBA é, em princípio, aplicável em qualquer situação em que se suspeita que o endosperma para influenciar o crescimento embrionário.

A Brasão Cama Ensaio de Sementes para explorar Geneticamente<em&gt; In Vitro</em&gt; Como a germinação Controles do endosperma das sementes em<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 ...

a-seed-coat-bedding-assay-to-genetically-explore-vitro-how-endosperm

Research

JoVE Journal

Biology

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

13.4K visualizações.  Université de Genève.  Nós apresentamos o procedimento para montar um ensaio da cama tegumento (SCBA) de Arabidopsis thaliana sementes. O ARA foi demonstrado ser uma ferramenta poderosa para explorar geneticamente e in vitro como a germinação de sementes de controlo do endosperma de sementes de dormentes e em resposta a sinais de luz. O SCBA é, em princípio, aplicável em qualquer situação em que se suspeita que o endosperma para influenciar o crescimento embrionário....

Vídeo: 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.

Uso de Arabidopsis mutantes eceriferum para Explorar Biossíntese Cutícula Vegetal

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 ...

Biologia

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA methyltransferases is viable, thus providing a system to study how loss of DNA methylation in a genome affects growth and development. Imprinting refers to differential expression of maternal and paternal alleles and plays an important role in reproduction development in both mammal and plants. DNA methylation is critical for determining whether the maternal or paternal alleles of an imprinted gene is expressed or silenced. In flowering plants, there is a double fertilization event in reproduction: one sperm cell fertilizes the egg cell to form embryo and a second sperm fuses with the central cell to give rise to endosperm. Endosperm is the tissue where imprinting occurs in plants. MEDEA, a SET domain Polycomb group gene, and FWA, a transcription factor regulating flowering, are the first two genes shown to be imprinted in endosperm and their expression is controlled by DNA methylation and demethylation in plants. In order to determine imprinting status of a gene and methylation pattern in endosperm, we need to be able to isolate endosperm first. Since seed is tiny in Arabidopsis, it remains challenging to isolate Arabidopsis endosperm and examine its methylation. In this video protocol, we report how to conduct a genetic cross, to isolate endosperm tissue from seeds, and to determine the methylation status by bisulfite sequencing.

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Imprinting is a phenomenon in plant and mammal reproduction. DNA methylation plays an important role in mechanisms of imprinting. Isolating endosperm and determining methylation status of imprinted genes in Arabidopsis can be difficult. In this protocol, we describe how to isolate endosperm and determine methylation by bisulfite sequencing.

Determinação de metilação de DNA de genes marcados em Arabidopsis Endosperma

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA ...

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 de Ribossomos ligados a polipeptídeos nascentes<em&gt; In vitro</em&gt; Identificar Sites Pausa translacionais Junto 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.

Um fluxo de trabalho de imagem de alta de conteúdo para estudar Grb2 Complexos de Sinalização por clonagem de expressão

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

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

Coloração histoquímica de<em&gt; Arabidopsis thaliana</em&gt; Celular Secundária parede Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

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.

A metilação in vitro Ensaio de Proteína Estudo Arginina metilação

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.

Ensaio para imunoprecipitação da cromatina a identificação de Arabidopsis interações proteína-ADN<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.

Análise do comportamento de crescimento de Arabidopsis thaliana em diferentes qualidades de luz

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.

Um trabalho-economia e repetível Touch-Force sinalização mutante Screen Protocol para o estudo de Thigmomorphogenesis de uma planta modelo Arabidopsis 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.

A Brasão Cama Ensaio de Sementes para explorar Geneticamente

Resumo

Explore mais vídeos

Capítulos neste vídeo

Uso de Arabidopsis mutantes eceriferum para Explorar Biossíntese Cutícula Vegetal

Determinação de metilação de DNA de genes marcados em Arabidopsis Endosperma

Isolamento de Ribossomos ligados a polipeptídeos nascentes

Um fluxo de trabalho de imagem de alta de conteúdo para estudar Grb2 Complexos de Sinalização por clonagem de expressão

Coloração histoquímica de

A metilação in vitro Ensaio de Proteína Estudo Arginina metilação

Ensaio para imunoprecipitação da cromatina a identificação de Arabidopsis interações proteína-ADN

Análise do comportamento de crescimento de Arabidopsis thaliana em diferentes qualidades de luz

Um trabalho-economia e repetível Touch-Force sinalização mutante Screen Protocol para o estudo de Thigmomorphogenesis de uma planta modelo Arabidopsis Arabidopsis thaliana

Um método simples de secção seca para obter seção de resina de tamanho inteiro e suas aplicações