Este trabalho foi apoiado pela Agência Nacional de Investigação francesa (ANR-14-CE02-0010). Agradecemos Dr. Benjamin Campo e Dr. Elodie Lanet para a leitura crítica do manuscrito. Agradecemos ao Sr. Michel Terese por sua ajuda com edição de vídeo.

1. Preparação de 20 a 50% (p / v) de sacarose Gradientes Nota: Os gradientes são feitos de 4 camadas de sacarose (50%, 35% e 2 camadas de 20%) em 13,2 ml de um tubo de ultracentrífuga. Em nossa experiência, despejando a sacarose 20% em duas camadas separadas melhora muito a qualidade das preparações polissomas. <ol><li> Preparar as soluções de estoque. Certifique-se que todas as soluções são RNAse e DNAse livre. <ol><li> Prepare 10X Solução Salina: Tris-HCl 400 pH 8,4, KCl 200 mM e MgCl 2 100 mM. </li><li> Prepare M de sacarose Solução 2: Para 200 ml, dissolver 137 g de sacarose em 1X solução salina. </li></ol></li><li> Dilui-se a solução de sacarose em solução salina, como descrito na Tabela 1, para preparar os gradientes. Os volumes indicados são para seis gradientes. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Concentração de sacarose final </td><td> sacarose 2H (ml) </td><td> Sal solução 1X (ml) </td><td> Vol final. (ml) </td></tr><tr><td> 50% </td><td> 8,8 </td><td> 3.2 </td><td> 12 </td></tr><tr><td> 35% </td><td> 12,9 </td><td> 12.1 </td><td> 25 </td></tr><tr><td> 20% </td><td> 7,4 </td><td> 17,6 </td><td> 25 </td></tr><tr><td> 20% </td><td> 5.8 </td><td> 14.2 </td><td> 20 </td></tr></tbody></table> Tabela 1. As diluições da solução de sacarose para preparar seis gradientes. <ol start="3"><li> Despeje as camadas conforme Tabela 2. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> camada de sacarose </td><td> 50% </td><td> 35% </td><td> 20% </td><td> 20% </td></tr><tr><td> Vol. (Ml) </td><td> 1.85 </td><td> 3.65 </td><td> 3.65 </td><td> 1,35 </td></tr></tbody></table> Tabela 2. Volume de solução de sacarose por camada. <ol start="4"><li> Depois de verter cada camada, manter os tubos num -40 ° C ou -80 ° C até estar completa congelador de congelação antes da adição de sacarose a camada seguinte. A congelação é geralmente alcançada após 2 h a -40 ° C, mas de esperar cerca de 6 h antes de se verter a camada seguinte é recomendado. A última camada de 20% pode ser congelada ou adicionado de fresco no dia da experiência. Nota: Congelamento cada camada antes de adicionar o próximo impede a partir de qualquer mistura ou perturbação das camadas. Os gradientes podem ser mantidos numa -40 ° C ou -80 ° C congelador durante pelo menos seis meses. </li><li> Se ainda não tiver sido feito, adicionar a última camada de 20% para os gradientes no dia da experiência. Então, deixe-os gradientes descongelar em uma sala fria ou um frigorífico. </li></ol> 2. Preparação de Extratos citossólicos Nota: Nós recomendaçãacabar usando dois gradientes por amostra biológica. 300 mg é a quantidade óptima de material vegetal para preparar dois gradientes quando se trabalha com 6 dias de idade plântulas de Arabidopsis thaliana. Quando se trabalha com tecidos traducionalmente menos activos, a quantidade de material de planta pode ser aumentada até 600 mg. <ol><li> Colheita de seis dias da germinação cultivadas em ½ Murashige e Skoog 8 meio suplementado com 1% de sacarose ou mídia equivalente a-congelação rápida em azoto líquido </li><li> material vegetal moer num almofariz e pilão pré-arrefecida com azoto líquido. </li><li> Pesar 300 mg de material em pó em um prato de pesagem pré-arrefecido. Execute esta etapa rapidamente para evitar o descongelamento da amostra. Nota: Mantenha as amostras congeladas por mais de uma semana em um C congelador -80 °. </li><li> Adicionar 2,4 ml de tampão pré-arrefecida polissoma (Solução Salina 4X, EGTA 5,26 mM, 0,5% (v / v), octilfenoxi poli (etilenoxi) etanol, ramificadas, 50 μg.ml -1 ciclo-heximida, 50 1 μg.mlcloranfenicol) e homogeneizar por mistura com a ponta da pipeta. Transferir 1,5 ml para dois tubos. Trabalhar rapidamente para impedir que as amostras de aquecimento. </li><li> Centrifuga-se a 16000 xg durante 15 min a 4 ° C numa microcentrífuga para sedimentar os detritos. Nota: Para evitar a degradação do RNA, mantenha sempre as amostras a 4 ° C, utilize RNase / DNase soluções livres e trabalhar em RNase / DNase condições livres. A heparina pode ser adicionado ao tampão de polissoma, a uma concentração final de 300 μg.ml -1, para melhorar a protecção de ARN. No entanto, uma vez que a heparina pode interferir com a análise a jusante, que terá de ser removido durante o passo de precipitação de ARN através da realização de precipitação de cloreto de lítio em vez de precipitação com etanol (nota cf. 4.7). </li></ol> 3. polysome Profiling <ol><li> Cuidadosamente pipeta o sobrenadante sem perturbar o sedimento. Se fragmentos de plantas foram pipetados, repita o passo 2.5. Carregar o sobrenadante no topo do gradiente por pipettin suavementeg para a parede lateral do tubo num fluxo constante. Use um gradiente para cada tubo de 1,5 ml. </li><li> Transferência para o pré-arref ecido baldes e centrifugar a 175.000 xg durante 2 h 45 min a 4 ° C, em uma ultracentrífuga (por exemplo 32.000 rpm ao utilizar um rotor SW41). </li><li> Configurar o sistema de recolha de gradiente, tal como descrito na Figura 1. A cuveta UV tem um percurso de 1 mm. </li></ol><img alt="figura 1" src="/files/ftp_upload/54231/54231fig1.jpg" /> Figura 1. Sistema de recolha de inclinação. A cuvete de UV está ligado um tubo de cloreto de polivinilo a um tubo capilar de vidro, que desce para o fundo do gradiente. O gradiente progride através do sistema graças a uma bomba peristáltica. OD 260 é continuamente lido e 2 ml frações são coletadas. <a href="https://www.jove.com/files/ftp_upload/54231/54231fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior</a>esta figura. <ol start="4"><li> Ajustar o colector de fracções para a TA, e definir o carrossel, a uma velocidade que permita a recolha de fracções de 2 mL. Use tubos de coleta pré-arrefecido. </li><li> Recolher as fracções de baixo para cima utilizando um tubo capilar de vidro ligada a um tubo de cloreto de polivinilo para o sistema de recolha de gradiente. Usar um filme de parafina de plástico para assegurar a ligação entre o tubo de cloreto de polivinilo e o tubo capilar de vidro. </li><li> Ler a absorvância a 260 nm a partir do fundo para o topo do gradiente. Quando todo o gradiente é recolhido, coloque os tubos de coleta no gelo antes de extração de RNA. </li></ol> 4. Extracção de ARN <ol><li> Reunir as fracções recolhidas a partir de dois gradientes, em 50 ml tapados tubos de centrifugação, como segue: Polissomas: fracções de 1 a 3 (12 ml) Monosomes: valor correspondente a 4 (4 ml) Sobrenadante: fracções 5 e 6 (8 ml) </li><li> Para cada fracção, adicione 1 vol. Hidrocloreto de guanidina 8M,50 ug transportador acrílico e 1,5 vol. isopropanol. Nota: Acryl transportador é acrilamida linear, usado como um co-precipitante para melhorar a recuperação de ácidos nucleicos durante a precipitação com álcool. </li><li> Misturar por inversão dos tubos e precipitar O / N à temperatura de -20 ° C. </li><li> Centrifugar a 175.000 xg durante 1 h a 4 ° C, em uma ultracentrífuga (32.000 rpm num rotor SW32). Se o passo de precipitação é realizada de um tubo, o qual não é compatível com a ultracentrifugação, transferir para um tubo adequado. </li><li> Elimine o sobrenadante e dissolve-se o sedimento em 200 de tampão TE isento de ARNase ul (Tris-HCl 10 mM, pH 7,5-1 mM de EDTA). Transferir para um novo tubo de 1,5 ml. </li><li> Extrair ARN por adição de 1 vol. fenol saturado de água (pH 6,6) e 1 vol. clorofórmio: álcool isoamílico (24: 1). Misturar energicamente. Centrifuga-se a 15000 xg durante 20 min a 4 ° C. Transferir a fase aquosa para um novo tubo de 1,5 mL. Nota: ácido fenol (pH 4,5) pode ser utilizado a fim de minimizar a contaminação do ADN. </li><li>Precipitar o ARN por adição de 1/10 vol. M de acetato de sódio 3 e 3 vol. 100% de etanol. Permitir a precipitação a -80 ° C durante 20 min ou O / N à temperatura de -20 ° C. Centrifuga-se a 15000 xg durante 15 min a 4 ° C. Nota: Se estiver usando heparina no tampão polysome (nota cf. 2.5), execute um cloreto de lítio precipitação (LiCl) em vez de precipitação de etanol para remover adequadamente qualquer vestígio de heparina que possa interferir com a análise a jusante 9. Após extracção, adicionar LiCl até uma concentração final de 2,5 M. permitir a precipitação durante 30 minutos à temperatura de -20 ° C ou O / N a 4 ° C. Centrifuga-se a 15000 xg durante 15 min a 4 ° C. </li><li> Lavar o sedimento com 500 ul de etanol a 75%. Ar secar o sedimento e dissolvem-se em 30 ul de tampão TE. Avaliar a qualidade do ARN por electroforese num ARNase livre de 1,2% em gel de agarose (100V, 15 min) ou por métodos de electroforese capilar. </li></ol> Análise 5. Os dados <ol><li> Exportação de dados brutos para um software gráfico e de análise de dados. Nota: Como mostrado na Figura 2A e B, os perfis de matérias dar informação qualitativa: o número de picos polissomas que pode ser visto, a altura do pico monosome, e a presença de um ombro correspondente para as subunidades de ribossomas livres. </li><li> Mesclar perfis para permitir a comparação dos perfis entre as amostras. Utilizar a ferramenta de leitor de tela para determinar o valor de X do pico monosome para cada amostra e alinhar os picos monosome removendo pontos de dados adicionais no início das curvas. Identificar o ponto mais baixo da curva e determinar seu valor Y (utilizando a ferramenta de leitor de tela). Defina o valor mais baixo Y ponto a 0 usando a função &quot;valor definido colunas&quot;. </li><li> Determinar a proporção polissomas / monosomes por integração da área sob a curva de perfis em bruto (Figura 3C). Utilizar a ferramenta selector de dados para definir a fronteira da área a ser integrados. Em seguida, use a função de integrar (Análise-Matemática). </li><li> Normalizaras curvas para o pico monosome dividindo todos os pontos de dados de uma curva pelo valor Y da cimeira do pico monosome (determinado utilizando a ferramenta de leitor de tela). Selecione a coluna, então sob análise em Matemática, selecione Normalize e escolher os &quot;dividido por um valor especificado&quot; métodos. Este passo permite a comparação do nível relativo de polissomas (Figura 3D). </li></ol><img alt="Figura 2" src="/files/ftp_upload/54231/54231fig2.jpg" /> Figura 2. Perfis polissomas representativos. Arabidopsis thaliana de tipo selvagem (wt, ecotipo Col-0) e mudas mutantes foram cultivadas durante seis dias em ½ Murashige e Skoog sob fotoperíodos o dia (luz 16 horas, 8 horas escuro). A: perfis polissomas brutos de mudas wt. B: perfis polissomas brutos de mudas mutantes. C: Determinação da percentagem de polissomas e monosomes por integração da área sob a curva D: profi polissomales normalizados para o pico monosome. <a href="https://www.jove.com/files/ftp_upload/54231/54231fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

<ol>
	<li>Nelson, C. J., Millar, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+turnover+in+plant+biology.">Protein turnover in plant biology.</a> Nat Plants. 1 (3), 15017 (2015).</li><li>Warner, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+economics+of+ribosome+biosynthesis+in+yeast.">The economics of ribosome biosynthesis in yeast.</a> Trends Biochem Sci. 24 (11), 437-440 (1999).</li><li>Amthor, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+McCree-de+Wit-Penning+de+Vries-Thornley+Respiration+Paradigms:+30+Years+Later.">The McCree-de Wit-Penning de Vries-Thornley Respiration Paradigms: 30 Years Later.</a> Ann Bot. 86 (1), 1-20 (2000).</li><li>Preiss, T., W Hentze, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Starting+the+protein+synthesis+machine:+eukaryotic+translation+initiation.">Starting the protein synthesis machine: eukaryotic translation initiation.</a> BioEssays news and reviews in molecular, cellular and developmental biology. 25 (12), 1201-1211 (2003).</li><li>Vogel, C., Marcotte, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+the+regulation+of+protein+abundance+from+proteomic+and+transcriptomic+analyses.">Insights into the regulation of protein abundance from proteomic and transcriptomic analyses.</a> Nat Rev Genet. 13 (4), 227-232 (2013).</li><li>Baerenfaller, K., 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-scale+proteomics+reveals+Arabidopsis+thaliana+gene+models+and+proteome+dynamics.">Genome-scale proteomics reveals Arabidopsis thaliana gene models and proteome dynamics.</a> Science. 320 (5878), 938-941 (2008).</li><li>Zanetti, E., Chang, I., Gong, F., Galbraith, D. W., Bailey-Serres, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunopurification+of+Polyribosomal+Complexes+of+Arabidopsis+for+Global+Analysis+of+Gene+Expression.">Immunopurification of Polyribosomal Complexes of Arabidopsis for Global Analysis of Gene Expression.</a> Plant physiol. 138 (2), 624-635 (2005).</li><li>Murashige, T., Skoog, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+revised+medium+for+rapid+growth+and+bioassays+with+tobacco+tissue+cultures.">A revised medium for rapid growth and bioassays with tobacco tissue cultures.</a> Physiol plant. 15 (3), 473-497 (1962).</li><li>del Prete, M. J., Vernal, R., Dolznig, H., M&#252;llner, E. W., Garcia-Sanz, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+polysome-bound+mRNA+from+solid+tissues+amenable+for+RT-PCR+and+profiling+experiments.">Isolation of polysome-bound mRNA from solid tissues amenable for RT-PCR and profiling experiments.</a> RNA. 13 (3), 414-421 (2007).</li><li>Salinas, J., Sanchez-Serrano, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Arabidopsis protocols. , (2006).</li><li>Piques, 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=Ribosome+and+transcript+copy+numbers,+polysome+occupancy+and+enzyme+dynamics+in+Arabidopsis.">Ribosome and transcript copy numbers, polysome occupancy and enzyme dynamics in Arabidopsis.</a> Mol syst biol. 5 (314), 314 (2009).</li><li>Morita, M., Alain, T., Topisirovic, I., Sonenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+Profiling+Analysis.">Polysome Profiling Analysis.</a> bio-protocol. 3 (14), 3-8 (2013).</li><li>Y&#225;ng&#252;ez, E., Castro-Sanz, A. B., Fern&#225;ndez-Bautista, N., Oliveros, J. C., Castellano, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+genome-wide+changes+in+the+translatome+of+Arabidopsis+seedlings+subjected+to+heat+stress.">Analysis of genome-wide changes in the translatome of Arabidopsis seedlings subjected to heat stress.</a> PloS One. 8 (8), (2013).</li><li>Sormani, R., 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=Sublethal+cadmium+intoxication+in+Arabidopsis+thaliana+impacts+translation+at+multiple+levels.">Sublethal cadmium intoxication in Arabidopsis thaliana impacts translation at multiple levels.</a> Cell Physiol. 52 (2), 436-447 (2011).</li><li>Lanet, E., 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=Biochemical+evidence+for+translational+repression+by+Arabidopsis+microRNAs.">Biochemical evidence for translational repression by Arabidopsis microRNAs.</a> Plant cell. 21 (6), 1762-1768 (2009).</li><li>Jabnoune, M., Secco, D., Lecampion, C., Robaglia, C., Shu, Q., Poirier, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Rice+cis-Natural+Antisense+RNA+Acts+as+a+Translational+Enhancer+for+Its+Cognate+mRNA+and+Contributes+to+Phosphate+Homeostasis+and+Plant+Fitness.">A Rice cis-Natural Antisense RNA Acts as a Translational Enhancer for Its Cognate mRNA and Contributes to Phosphate Homeostasis and Plant Fitness.</a> Plant cell. 25 (10), 4166-4182 (2013).</li><li>Ingolia, N. T. <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+translational+profiling+by+ribosome+footprinting.">Genome-wide translational profiling by ribosome footprinting.</a> Methods Enzymol. 470 (10), (2010).</li><li>Juntawong, P., Girke, T., Bazin, J., Bailey-Serres, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+dynamics+revealed+by+genome-wide+profiling+of+ribosome+footprints+in+Arabidopsis.">Translational dynamics revealed by genome-wide profiling of ribosome footprints in Arabidopsis.</a> PNAS. 111 (1), 203-212 (2013).</li><li>Ingolia, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome+profiling:+new+views+of+translation,+from+single+codons+to+genome+scale.">Ribosome profiling: new views of translation, from single codons to genome scale.</a> Nat Rev Genet. 15 (3), 205-213 (2014).</li></ol>

The authors have nothing to disclose

The protocol we present here is an easy and cheap method for generating polysome profiles and isolating mRNAs associated with polysomes, single ribosomes or free of ribosomes. A wide range of different polysome fractionation methods is described in the literature. The method we have described here has been optimized to keep only the necessary compounds and has been adapted for plant material. In particular, we reduced the amount of detergent11 and added chloramphenicol to the buffer to fix the chloroplastic ri...

A síntese de proteínas é um processo essencial e dispendiosa energeticamente em todas as células 1. Em primeiro lugar, as células devem investir energia na produção da maquinaria de tradução, os ribossomas. Por exemplo, uma célula de levedura dividindo ativamente produz tanto quanto 2.000 ribossomas por minuto. Essa produção exige até 60% da actividade de transcrição total e até 90% da actividade total de splicing da célula 2. Além disso, é necessária energia para a síntese de aminoácidos, aminoacil-ARNt e ligações peptídicas. Nas plantas, a adição de um aminoácido a uma cadeia peptídica custos de 4,5 a 5,9 moléculas de ATP 3. Portanto, não é surpreendente que a tradução do mRNA para a proteína é um importante local de regulação, especialmente quando se trata de lidar com a mudança de condições ambientais. A etapa de iniciação da tradução, que é a associação de um mRNA com o ribossoma, é o principal alvo da regulamentação dosTradução 4. Como uma consequência da regulação da tradução, bem como outros passos de regulação pós-transcricional, apenas 40% das variações na concentração de proteína pode ser explicada por ARNm de abundância 5,6. Assim, o estudo de mRNA total fornece relativamente pouca informação sobre a abundância de proteínas. Por outro lado, a associação de mRNA com ribossomos dá melhor visão sobre a abundância de proteínas, dando acesso a esses mRNAs envolvidos na tradução. mRNAs activamente traduzida estão associados com vários ribossomas em estruturas chamadas polissomas. Por outro lado, mRNAs mal traduzidas será associado a apenas um ribossomo (monosome). Por conseguinte, o status translacional de um ARNm pode ser avaliado por monitorização da sua associação com ribossomas 7. Este protocolo descreve o isolamento de polissomas a partir de seis dias de idade plântulas de Arabidopsis thaliana, o subsequente isolamento do ARN e a análise dos resultados. Polysomes monosomes e são separadas através de um gradiente de densidade de sacarose. Gradientes são coletados em seis fracções. Algumas das fracções são reunidas para obter três fracções bem separados: polissomos, monosomes ea fração leve (doravante denominado sobrenadante), que contém os 60 e 40S subunidades ribossomais livres e mRNAs que não estão associados a ribossomas. actividade de tradução global pode ser estimada através da geração de uma relação de polissoma / monosome, que é determinada por integração da área sob a curva, e por comparação dos perfis de polissomas. ARNm e as proteínas são, em seguida, extraiu-se a partir das diferentes fracções e utilizado para análise por RT-PCR, qRT-PCR, Northern blot, microarray, Western blot ou proteómica. Este protocolo foi validado para outras plantas e tecidos. Os equipamentos necessários para realizar este protocolo são comumente encontrados na maioria dos laboratórios: Não há necessidade de uma máquina de inclinação. Congelação cada camada antes de adicionar o próximo prevenirs de qualquer mistura ou perturbação das camadas. Nenhuma perfurador tubo é utilizado para a recolha de inclinação que pode ser obtida por imersão de um tubo capilar de vidro no gradiente. Por conseguinte, os tubos de ultracentrífuga dispendiosos não ficam danificadas e podem ser re-utilizada várias vezes. Coletivamente, isso faz com que o presente protocolo de um método fácil e barato para profiling polysome.

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			<td height="40" style="height:40px;width:233px;">Ultracentrifuge tube, thinwall, polyallomer - 13.2 ml</td>
			<td style="width:109px;">Beckman Coulter</td>
			<td style="width:132px;">331372</td>
			<td style="width:261px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Ultracentrifuge tube, thinwall, polyallomer - 38.5 ml</td>
			<td style="width:109px;">Beckman Coulter</td>
			<td style="width:132px;">326823</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Glass capillary tube</td>
			<td style="width:109px;">Drummond Scientific</td>
			<td style="width:132px;">1-000-1000</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Ultracentrifuge&#160;</td>
			<td style="width:109px;">Beckman Coulter</td>
			<td style="width:132px;">Optima series</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Ultracentrifuge Rotor SW41</td>
			<td style="width:109px;">Beckman Coulter</td>
			<td style="width:132px;">331362</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Ultracentrifuge Rotor SW32</td>
			<td style="width:109px;">Beckman Coulter</td>
			<td style="width:132px;">369650</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Peristaltic pump</td>
			<td style="width:109px;">Any</td>
			<td style="width:132px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Tygon R3607 polyvinyl chloride tubing&#160;</td>
			<td>Fisher Scientific</td>
			<td>070534-22</td>
			<td>Polyvinyl chloride tubing, 2.29 mm&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Fraction collector Model 2110</td>
			<td style="width:109px;">Bio-Rad</td>
			<td style="width:132px;">731-8120</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">UV cuvette</td>
			<td style="width:109px;">Hellma</td>
			<td style="width:132px;">170.700-QS</td>
			<td>Quartz flow-through cuvette</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">UV Spectrophotometer</td>
			<td style="width:109px;">Varian</td>
			<td style="width:132px;">Cary50</td>
			<td>Read every 0.0125 sec</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">All chemicals</td>
			<td style="width:109px;">Any</td>
			<td style="width:132px;"></td>
			<td>Use only Molecular Biology Grade</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Murashige and Skoog Basal Salt Mixture (MS)</td>
			<td>Sigma-Aldrich</td>
			<td>M5524</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Rnase-Free water</td>
			<td style="width:109px;">Any</td>
			<td style="width:132px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Petri Dishes</td>
			<td>Fisher Scientific</td>
			<td>10083251&#160;</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:233px;">Octylphenoxy poly(ethyleneoxy)ethanol, branched (Nonidet P40)</td>
			<td style="width:109px;">Euromedex</td>
			<td style="width:132px;">UN3500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">Linear acrylamide (acryl carrier)</td>
			<td style="width:109px;">ThermoFischer scientific</td>
			<td style="width:132px;">AM9520</td>
			<td>RNA precipitation carrier</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">OriginPro 8</td>
			<td style="width:109px;">OriginLab</td>
			<td style="width:132px;"></td>
			<td>Analysis software</td>
		</tr>
	</tbody>
</table>

an easy method for plant polysome profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

Na literatura, os perfis de polissomas são mostrados frequentemente a partir da fracção de luz para a fracção pesada como um resultado da forma como os gradientes são recolhidos, ou seja, a partir do topo para o fundo. Uma vez que no protocolo aqui descrito os gradientes são recolhidos a partir da base para o topo, os perfis que mostram começar com a fração pesada (as polissomos) e vá para a fração de luz (subunidades de ribossomas livres e RNAs) (Figura 2A).</strong...

Watch this Scientific Journal Video about An Easy Method for Plant Polysome Profiling at JoVE.com

An Easy Method for Plant Polysome Profiling

This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

Um método fácil para a planta polysome Profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the ...

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Research

JoVE Journal

Biology

12.4K visualizações.  Aix-Marseille Université. This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

Vídeo: An Easy Method for Plant Polysome Profiling

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1 (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2, and is amenable to high-throughput analyses3. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13, xylan using an endo-&beta;-(1-4)-xylanase 6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9.

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

Oligo Profiling Massa (OLIMP) de polissacarídeos extracelulares

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

Biologia

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

Profiling Proteome Redox tiol Usando Isótopos Espectrometria de Massa Tagging

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Profiling glicana de Polímeros da planta da parede celular usando Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.

Here, we describe a method to analyze changes in the initiation of mRNA translation of eukaryotic cells in response to stress conditions. This method is based on the velocity separation on sucrose gradients of translating ribosomes from non-translating ribosomes.

Análise da Tradução Iniciação Durante condições de estresse por polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

Um método eficiente para Quantitativa, Análise de células Único de cromatina Modificação e Arquitetura Nuclear em Todo-mount óvulos em<em&gt; Arabidopsis</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Avaliação da Seletiva mRNA Tradução em células de mamíferos por polissoma Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.

assistida por laser Microdissecção (LAM) como ferramenta para transcricional Profiling de tipos de células individuais

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

Hybrid-Cut: Um Método Seccionamento melhorado para amostras de tecidos de plantas recalcitrante

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

Método padronizado para a esterilização da elevado-produção de sementes de Arabidopsis

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this scenario, the search for new antifungal drugs is necessary. The protocol reported here details a method to screen peptides for their antifungal properties. It is based on the broth microdilution susceptibility test from the Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines with modifications to suit the research of antimicrobial peptides as potential new antifungals. This protocol describes a functional assay to evaluate the activity of antifungal compounds and may be easily modified to suit any particular class of molecules under investigation. Since the assays are performed in 96-well plates using small volumes, a large-scale screening can be completed in a short amount of time, especially if carried out in an automation setting. This procedure illustrates how a standardized and adjustable clinical protocol can help the bench-work pursuit of new molecules to improve the therapy of fungal diseases.

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

An easy and adaptable broth microdilution method for screening antifungal compounds and extracts.