Este trabalho é apoiado por uma bolsa da National Science Foundation EUA para Christoph Benning.

1. Extração de lipídeos <ol><li> Começar a extração de lipídios colhendo 30 mg deixa quatro semanas de idade Arabidopsis partir de plantas cultivadas em ágar solidificado médio ou solo e transferi-los para 1,5 mL tubos de reacção de polipropileno. Folhas frescas podem ser flash congelado em nitrogênio líquido e armazenadas a -80 ° C. </li><li> Adicionar 300 mL de extração de solvente composto por metanol, clorofórmio e ácido fórmico (20:10:01, v / v / v) para cada amostra. Agite vigorosamente (usando um misturador de tinta ou similar) por 5 minutos. </li><li> Adicionar 150 mL de ácido fosfórico 0,2 M (H 3 PO 4), 1 M de cloreto de potássio (KCl) e brevemente vortex. </li><li> Centrifugar a 13.000 xg à temperatura ambiente por 1 minuto. Lipídios dissolvidos na fase de menor clorofórmio será manchado em placas de TLC. </li></ol> 2. Cromatografia em camada fina (TLC) 15 <ol><li> Para preparar as placas TLC, submerge um 20cmx20cm placa de sílica gel TLC revestido com carga strip por 30 segundos em 0,15 M de sulfato de amônio ((NH 4) 2 SO 4) solução, depois de submergir por 30 segundos, secar a placa por pelo menos dois dias no um recipiente coberto. Durante a ativação da sublimação de amônio deixa para trás ácido sulfúrico, que protonates fosfatidilglicerol necessário para a sua separação de glycerolipids outros. </li><li> No dia do experimento, ativar placas por TLC assando no forno a 120 ° C por 2,5 horas. </li><li> Após resfriamento das placas ativado à temperatura ambiente, use um lápis para desenhar uma linha reta (1,5 cm da borda da placa) em toda a placa na origem do cromatograma. </li><li> Em uma capela, lentamente entregar 3 X 20 mL do extrato lipídico na fase de menor clorofórmio, utilizando uma pipeta 20 mL com 200 mL amarelo dicas de plástico sob uma corrente lenta de N 2. Para esta finalidade, um tubo Tygon é conectado ao regulador do 2 N tanque. Mantenha o ponto inferior a 1 cm de diâmetro. Cada placa pode conter até 10 amostras (quando a análise subseqüente é planejada GLC). </li><li> Como o de lipídios spots completamente seco na capela, prepare o solvente de desenvolvimento composto de acetona, tolueno, água (91 mL: 30 mL: 7,5 mL). Se a umidade relativa do ar ambiente é alta, a separação poderia ser afetada. Neste caso, a água deve ser reduzida para dar (mL 91: 30 mL: 7,0 mL) para conseguir a separação desejada. </li><li> Despeje 80 ml solvente de desenvolvimento em um TLC selável desenvolvimento câmara (L: H: W = 27,0: 26,5: 7,0, cm / cm / cm) e colocar a placa para dentro do tanque com o fim da amostra virada para baixo. Vedação do tanque usando a braçadeira. O solvente irá subir a placa e lipídios serão separados. O tempo de desenvolvimento é de aproximadamente 50 minutos em temperatura ambiente. </li><li> Quando a frente do solvente tenha atingido 1 cm do topo da placa, remova cuidadosamente a placa da tina e secar completamente na capela por aproximadamente 10 minutos. </li><li> Lipídios separados por TLC pode ser reversível manchada brevemente com iodo para a análise quantitativa ou irreversivelmente manchado com ácido sulfúrico ou α-naftol. <ol><li> Sulfúrico carbonização ácido: pulverizar a placa com 50% de ácido sulfúrico em água em um frasco spray de vidro no extractor de fumo e leve ao forno a 120 ° C por 15 minutos (Figura 2A). </li><li> α-naftol coloração para glycolipids: pulverizar a placa com 2,4% (w / v) α-naftol em 10% (v / v) de ácido sulfúrico, 80% (v / v) etanol e leve ao forno a 120 ° C por 3-5 minutos, até bandas glicolipídeo estão manchadas rosa ou roxo (Figura 2B). Sobretratamento levará a carbonização de todos os lipídios devido à presença de ácido sulfúrico no reagente. </li><li> Coloração de iodo: em um exaustor, colocar a placa em um tanque de TLC fechado com cristais de iodo (em uma bandeja na parte inferior levando à saturação da atmosfera com vapor de iodo até lipídios são visíveis). Não expor a placa ao iodo muito tempo como o iodo pode modificar covalentemente de ácidos graxos poliinsaturados (Figura 2C). Como alternativa, para evitar a oxidação de lipídios, apenas pistas padrão intercaladas com faixas de amostra deve ser manchado com um a lã de vidro conectado Pasteur pipeta com cristais de iodo através do qual N 2 é soprado sobre pistas individuais padrão. </li></ol></li></ol> 3. Acil graxos metílicos Reaction (FAME) 16 <ol><li> Remove manchas de sílica em torno de lipídios identificados a partir da placa de TLC com uma lâmina de barbear. Raspe a sílica contendo lipídios e transferir o pó de sílica utilizando um funil para um tubo de vidro com tampa de rosca de teflon (PTFE) alinhadas. </li><li> Adicionar 1 mL de ácido clorídrico 1 N (HCl) em metanol anidro para cada amostra de vidro pipeta. </li><li> Adicionar 100 mL 50 mg mL -1 pentadecanoic ácido (15:00), utilizando 200 mL de pipeta para cada amostra como padrão interno, utilizando uma pipeta 200 mL com 200 mL ponta de plástico amarela. Mantenha um tubo com apenas ácido pentadecenoic em metanólico HCl como controle. Fechar os tubos de vidro firmemente com Teflon alinhadas caps. </li><li> Incubar glass tubos em um banho de água 80 ° C por 25 minutos. Tubos devem ser selados de forma que o solvente não evapore. </li><li> Depois de tubos têm arrefecido, adicione 1 mL de cloreto de sódio a 0,9%, seguido por uma hexano mL e vortex vigorosamente. Centrifugar amostras 1000xg por 3 minutos. </li><li> Na capela, remova a camada de hexano / superior da amostra com uma pipeta Pasteur e colocá-lo em um novo tubo de vidro 13x100 milímetros. </li><li> Evaporar hexano sob uma corrente lenta de N 2 sem secar completamente. </li><li> Dissolver o resultado graxos acil methylesters s em 60 mL de hexano. Transferência de amostras em frascos de amostrador automático e tampa. Amostras podem ser armazenadas a 4 ° C para curto prazo e -20 ° C por alguns dias. </li></ol> 4. Cromatografia gás-líquido (GLC) 10 <ol><li> Antes de iniciar GLC, Certifique-se que o hidrogênio, hélio e cilindros de ar são preenchidos. </li><li> Hexano suficiente deve ser adicionado ao reservatório de solvente e o recipiente de resíduos deve estar vazio. Para graxos separação methylesters acila, anexar um DB-23 da coluna para a máquina. </li><li> Frascos lugar para o amostrador automático. Inicie o software ChemStation para GLC no computador do sistema. </li><li> Definir a temperatura de entrada a 250 ° C com taxa de fluxo de hélio em 48,6 mL min -1 ea pressão em 21,93 psi. A razão de separação é de 30,0: 1. </li><li> A temperatura do forno é definido inicialmente a 140 ° C por 2 min e aumentada para 160 ° C a uma taxa de 25 ° C min -1. Em seguida, defina a temperatura aumente de 160 ° C a 250 ° C a uma taxa de 8 ° C min -1 e mantenha a 250 ° C por 4 min seguido por uma diminuição para 140 ° C a uma taxa de 38 ° C min - 1. Uma corrida dura aproximadamente 21 minutos. </li><li> A temperatura do detector de ionização de chama é de 270 ° C com uma taxa de fluxo de hidrogênio de 30,0 mL min -1, vazão de ar de 400 mL min -1 e taxa de fluxo de hélio em 30,0 mL min -1. </li><li> Digite o número de frascos e nomes de amostra na tabela de seqüência de execução. Definir o injector de 10 L para injetar 2 mL da amostra por frasco. </li><li> Quando o instrumento está pronto, iniciar a seqüência de execução. </li></ol> 5. Resultados representativos: Exemplos de coloração irreversível de TLC-separados lipídios a partir de mudas de quatro semanas de idade Arabidopsis são mostrados na Figura 2. Os lipídios ácido sulfúrico corado (Figura 2A) estão carbonizados e aparecem como manchas marrons. α-naftol é preferível a mancha glycolipids como MGDG, DGDG, SQDG etc Glicolipídeos corados com α-naftol levar uma cor rosa-púrpura, enquanto outros lipídeos polares mancha amarela (Figura 2B). A coloração de iodo é reversível e dá lipídios uma cor amarelada que desaparecerão ao longo de um curto período de tempo como evapora iodo (Figura 2C). Lipídios brevemente iodo manchadas podem ser submetidos à análise GLC embora imaculada lipídios são preferíveis para reduzir a quebra de lipídios. Se feito com sucesso, os sinais distintivos que representam diferentes Fatty acil metílicos será observado após GLC (Figura 3). Metílicos de acila graxos de cadeia curta de carbono e menos ligações duplas têm menor tempo de retenção usando o DB-23 coluna. Graxos acil perfil metílico é um instrumento sensível para identificar mutantes com composição lipídica alterada. Na Figura 4, o MGDG18: 3 proporção molar de ácidos graxos é reduzida no mutante tgd4-1 em relação ao tipo selvagem 18. Dividindo-se o moles de gordos acil metílicos para uma classe de lipídios com as toupeiras de todas as classes de lipídios, a relação molar de cada lipídios são calculados. Por exemplo, para calcular a razão molar de MGDG: (MGDG) mol% =% x100 Σ [FAMEs (MGDG)] / Σ [FAMEs (total)] As relações resultantes molar de cada classe de lipídeos de tanto o tipo selvagem eo mutante pode ser comparado. Por exemplo, o mutante tgd4-1 aumentou quantidades relativas de MGDG e PG, mas diminuiu quantidades de DGDG e PE (Figura 5) 18. <img src="/files/ftp_upload/2518/2518fig1.jpg" alt="Figura 1" /> Figura Fluxograma 1. Da análise de lipídios polar usando mudas de Arabidopsis. Lipídios totais são extraídos de mudas de quatro semanas de idade Arabidopsis e separados por TLC. Os lipídios podem ser separados raspadas TLC placa de transesterificação seguido de análise GLC. <img src="/files/ftp_upload/2518/2518fig2.jpg" alt="Figura 2" /> Figura 2. Separação de lipídios em placas de TLC. Extratos de lipídios de 35 mg (peso fresco) de mudas do tipo selvagem são separados por TLC e corados pelo ácido sulfúrico (A), α-naftol (B) ou iodo vapor (C). Três repetições são mostradas em cada método de coloração. DGDG, digalactosyldiacylglycerol; monogalactosyldiacylglycerol MGDG,; PC, fosfatidilcolina, PE, fosfatidiletanolamina, PG, phosphatidylglycerol; PI, fosfatidilinositol; SQDG, sulfoquinovosyldiacylglycerol. <img src="/files/ftp_upload/2518/2518fig3.jpg" alt="Figura 3" /> Figura 3. GLC análise de Methylesters ácidos graxos (FAMEs) derivado de MGDG do tipo selvagem. FAMEs são separados em uma coluna de 30 m capilar e detectado por ionização de chama. Pentadecanoic ácido (15:00) é usado como padrão interno. <img src="/files/ftp_upload/2518/2518fig4.jpg" alt="Figura 4" /> Figura 4. Perfil de ácidos graxos de MGDG no tipo selvagem Col2 (colunas brancas) e os tgd4-1 mutante (colunas pretas). Ácidos graxos são apresentados como o número de carbonos, seguido do número de ligações duplas. Três repetições são em média e desvios-padrão são mostrados. <img src="/files/ftp_upload/2518/2518fig5.jpg" alt="Figura 5" /> Figura 5. Composição de lipídios polares do tipo selvagem Col2 (colunas brancas) e os tgd4-1 mutante (colunas pretas). Três repetições são em média e desvios-padrão são mostrados pela barra de erro.

<ol>
	<li>Ajjawi, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+reverse+genetics+in+Arabidopsis:+case+studies+from+the+Chloroplast+2010+Project.">Large-scale reverse genetics in Arabidopsis: case studies from the Chloroplast 2010 Project.</a> Plant Physiol. 152, 529-529 (2010).</li><li>Andersson, M. X., Kjellberg, J. M., Sandelius, A. 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+involvement+of+cytosolic+lipases+in+converting+phosphatidyl+choline+to+substrate+for+galactolipid+synthesis+in+the+chloroplast+envelope.">The involvement of cytosolic lipases in converting phosphatidyl choline to substrate for galactolipid synthesis in the chloroplast envelope.</a> Biochim. Biophys. Acta. 1684, 46-46 (2004).</li><li>Awai, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phosphatidic+acid-binding+protein+of+the+chloroplast+inner+envelope+membrane+involved+in+lipid+trafficking.">A phosphatidic acid-binding protein of the chloroplast inner envelope membrane involved in lipid trafficking.</a> Proc. Natl. Acad. Sci. U. S. A. 103, e10817-e10817 (2006).</li><li>Benning, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+lipid+transport+involved+in+organelle+biogenesis+in+plant+cells.">Mechanisms of lipid transport involved in organelle biogenesis in plant cells.</a> Annu. Rev. Cell Dev. Biol. 25, 71-71 (2009).</li><li>Browse, J., McCourt, P. J., Somerville, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acid+composition+of+leaf+lipids+determined+after+combined+digestion+and+fatty+acid+methyl+ester+formation+from+fresh+tissue.">Fatty acid composition of leaf lipids determined after combined digestion and fatty acid methyl ester formation from fresh tissue.</a> Anal. Biochem. 152, 141-141 (1986).</li><li>Dormann, P., Balbo, I., Benning, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+galactolipid+biosynthesis+and+lipid+trafficking+mediated+by+DGD1.">Arabidopsis galactolipid biosynthesis and lipid trafficking mediated by DGD1.</a> Science. 284, 2181-2181 (1999).</li><li>Guskov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyanobacterial+photosystem+II+at+2.9-A+resolution+and+the+role+of+quinones,+lipids,+channels+and+chloride.">Cyanobacterial photosystem II at 2.9-A resolution and the role of quinones, lipids, channels and chloride.</a> 16, 334-334 (2009).</li><li>Hartel, H., Dormann, P., Benning, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DGD1-independent+biosynthesis+of+extraplastidic+galactolipids+after+phosphate+deprivation+in+Arabidopsis.">DGD1-independent biosynthesis of extraplastidic galactolipids after phosphate deprivation in Arabidopsis.</a> Proc. Natl. Acad. Sci. U. S. A. 97, 10649-10649 (2000).</li><li>James, A. T., MARTIN, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas-liquid+partition+chromatography;+the+separation+and+micro-estimation+of+volatile+fatty+acids+from+formic+acid+to+dodecanoic+acid.">Gas-liquid partition chromatography; the separation and micro-estimation of volatile fatty acids from formic acid to dodecanoic acid.</a> Biochem. J. 50, 679-679 (1952).</li><li>Jordan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+structure+of+cyanobacterial+photosystem+I+at+2.5+A+resolution.">Three-dimensional structure of cyanobacterial photosystem I at 2.5 A resolution.</a> Nature. 411, 909-909 (2001).</li><li>Kobayashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type-B+monogalactosyldiacylglycerol+synthases+are+involved+in+phosphate+starvation-induced+lipid+remodeling,+and+are+crucial+for+low-phosphate+adaptation.">Type-B monogalactosyldiacylglycerol synthases are involved in phosphate starvation-induced lipid remodeling, and are crucial for low-phosphate adaptation.</a> Plant J. 57, 322-322 (2009).</li><li>Lu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small+ATPase+protein+of+Arabidopsis,+TGD3,+involved+in+chloroplast+lipid+import.">A small ATPase protein of Arabidopsis, TGD3, involved in chloroplast lipid import.</a> J. Biol. Chem. 282, 35945-35945 (2007).</li><li>Ohlrogge, J., Browse, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+biosynthesis.">Lipid biosynthesis.</a> Plant Cell. 7, 957-957 (1995).</li><li>Stahl, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thin-layer+chromatography;+methods,+influencing+factors+and+an+example+of+its+use.">Thin-layer chromatography; methods, influencing factors and an example of its use.</a> Pharmazie. 11, 633-633 (1956).</li><li>Stoffel, W., INSULL, W., AHRENS, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas-liquid+chromatography+of+highly+unsaturated+fatty+acid+methyl+esters.">Gas-liquid chromatography of highly unsaturated fatty acid methyl esters.</a> Proc. Soc. Exp. Biol. Med. 99, 238-238 (1958).</li><li>Welti, R., Wang, X., Williams, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospray+ionization+tandem+mass+spectrometry+scan+modes+for+plant+chloroplast+lipids.">Electrospray ionization tandem mass spectrometry scan modes for plant chloroplast lipids.</a> Anal. Biochem. 314, 149-149 (2003).</li><li>Xu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+trafficking+between+the+endoplasmic+reticulum+and+the+plastid+in+Arabidopsis+requires+the+extraplastidic+TGD4+protein.">Lipid trafficking between the endoplasmic reticulum and the plastid in Arabidopsis requires the extraplastidic TGD4 protein.</a> Plant Cell. 20, 2190-2190 (2008).</li><li>Xu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutation+of+the+TGD1+chloroplast+envelope+protein+affects+phosphatidate+metabolism+in+Arabidopsis.">Mutation of the TGD1 chloroplast envelope protein affects phosphatidate metabolism in Arabidopsis.</a> Plant Cell. 17, 3094-3094 (2005).</li><li>Xu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+permease-like+protein+involved+in+ER+to+thylakoid+lipid+transfer+in+Arabidopsis.">A permease-like protein involved in ER to thylakoid lipid transfer in Arabidopsis.</a> EMBO J. 22, 2370-2370 (2003).</li></ol>

Não há conflitos de interesse declarados.

TLC juntamente com GLC fornece uma ferramenta robusta e rápida de análise quantitativa de lipídios polares em plantas. Pequenas mudanças na composição lipídica podem ser identificados e, portanto, este método tem sido utilizado para triagem em larga escala de mutantes deficientes nas vias metabólicas lipídicas polar 1,20. Este método também é amplamente utilizado para atividades de monitoramento de enzimas utilizando lipídeos polares como substrato. 2,6,7 Além de folhas, a composição lipídica de outros tecidos vegetais, como raízes e sementes ou frações subcelulares, como cloroplastos e mitocôndria também pode ser determinado da mesma maneira. O sistema de solvente (acetona, tolueno, água) aqui utilizado é otimizado para a separação de glicolipídios e fosfolipídios em plantas. No entanto, em tgd1, 2,3,4 mutantes e cloroplastos isolados, TGDG executado em conjunto com o PE, enquanto tetragalactosyldiacylglycerol funciona com PC. Neste caso, um sistema de solventes com clorofórmio, metanol, ácido acético e água (85: 20: 10: 4, v / v / v / v) é usado 13. Às vezes bidimensional TLC usando dois diferentes sistemas solventes é realizada para mais glycolipids separado e fosfolipídios 19. Além disso, tecidos vegetais podem ser diretamente submetidas à reação FAME seguido por GLC para determinar o perfil de ácidos graxos totais, sem separação inicial sobre TLC 5. Ao lado do TLC-GLC demonstrado sistema, outro método utilizado para o perfil lipídico é baseado em directo de massas com ionização electrospray em tandem espectrometria de 17. Neste método, a separação cromatográfica inicial de lipídios no extrato é omitido. No entanto, este método requer equipamentos caros e pessoal experiente, o que o torna menos útil para análises de rotina no laboratório ou para a seleção de mutantes.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>&alpha;-naphthol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>N1000</td>
<td>&nbsp;</td>
<tr>
<td>Methanolic HCL 3N</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>33050-U</td>
<td>Dilute to 1N by methanol</td>
</tr>
<tr>
<td>Si250-PA TLC plates</td>
<td>&nbsp;</td>
<td>J.T.Baker</td>
<td>7003-04</td>
<td>With pre-absorbent </td>
</tr>
<tr>
<td>TLC chamber</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>Z266000</td>
<td>&nbsp;</td>
<tr>
<td>Screw cap tubes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>53283-800</td>
<td>&nbsp;</td>
<tr>
<td>Scew caps</td>
<td>&nbsp;</td>
<td>Sun Sri</td>
<td>13-425</td>
<td>&nbsp;</td>
<tr>
<td>PTFE disk</td>
<td>&nbsp;</td>
<td>Sun Sri</td>
<td>200 608</td>
<td>&nbsp;</td>
<tr>
<td>GLC system</td>
<td>&nbsp;</td>
<td>Hewlett Packard</td>
<td>HP6890</td>
<td>&nbsp;</td>
<tr>
<td>DB-23 column</td>
<td>&nbsp;</td>
<td>J&amp;W Scientific</td>
<td>122-2332</td>
<td>&nbsp;</td>
<tr>
<td>GLC vials</td>
<td>&nbsp;</td>
<td>Sun Sri</td>
<td>500 132</td>
<td>&nbsp;</td>
<tr>
<td>Caps of GLC vials</td>
<td>&nbsp;</td>
<td>Sun Sri</td>
<td>201 828</td>
<td>&nbsp;</td>
<tr>
<td>Chemstation software</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>G2070AA</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

arabidopsis thaliana polar glycerolipid profiling by thin layer chromatography (tlc) coupled with gas-liquid chromatography (glc)

Biológica células membranas separado do ambiente. A partir de uma única célula de plantas e animais multicelulares, glycerolipids, como fosfatidilcolina ou fosfatidiletanolamina, membranas formam bicamada que atuam tanto como os limites e interfaces para permuta química entre as células e seus arredores. Ao contrário dos animais, células vegetais têm uma organela especial para a fotossíntese, o cloroplasto. O sistema de membrana intrincados do cloroplasto contém glycerolipids única, ou seja, glicolipídeos falta de fósforo: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG) 4. Os papéis destes lipídios são além de simplesmente estrutural. Estes glicolipídeos e glycerolipids outros foram encontrados na estrutura cristalina do fotossistema I e II, que indica o envolvimento de glycerolipids na fotossíntese 8,11. Durante a fome de fosfato, DGDG é transferido para membranas extraplastidic para compensar a perda de fosfolipídeos 9,12. Muito do nosso conhecimento da biossíntese e função desses lipídios foi derivado de uma combinação de estudos genéticos e bioquímicos com 14 Arabidopsis thaliana. Durante esses estudos, um procedimento simples para a análise de lipídios polares tem sido essencial para o rastreamento e análise de mutantes lipídico e será descrito em detalhes. Um extrato de lipídios folha é primeiro separados por cromatografia em camada delgada (TLC) e glycerolipids estão manchadas reversivelmente com vapor de iodo. Os lipídios individuais são raspadas da placa de TLC e convertidos em gordura methylesters acila (FAMEs), que são analisados ​​por cromatografia gás-líquido acoplado com detecção de ionização de chama (FID-GLC) (Figura 1). Este método tem provado ser uma ferramenta confiável para a seleção de mutantes. Por exemplo, o tgd1, 2,3,4 retículo endoplasmático-to-plastid mutantes tráfico de lipídios foram descobertos com base na acumulação de um galactoglycerolipid anormal: trigalactosyldiacylglycerol (TGDG) e uma diminuição na quantidade relativa de 18:03 (carbonos: double títulos) grupos acila graxos em lipídios de membrana 3,13,18,20. Este método também é aplicável para a determinação atividades enzimáticas de proteínas usando lipídios como substrato 6.

Watch this Scientific Journal Video about Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC at JoVE.com

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC

Composição de extratos de lipídios polares e da composição de ácidos graxos de glycerolipids individuais são determinados em um experimento simples e robusto perfil lipídico. Para este fim, glycerolipids são isolados por cromatografia em camada delgada e submetido a transmetilação de seus grupos acil. Graxos methylesters acila são quantificados por cromatografia gás-líquido.

Arabidopsis thaliana Profiling Glycerolipid Polar por cromatografia em camada delgada (TLC) Juntamente com Cromatografia gás-líquido (GLC)

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as ...

arabidopsis-thaliana-polar-glycerolipid-profiling-thin-layer

Universidad San Sebastian

Research

JoVE Journal

Biology

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)

36.6K visualizações.  Michigan State University. Composição de extratos de lipídios polares e da composição de ácidos graxos de glycerolipids individuais são determinados em um experimento simples e robusto perfil lipídico. Para este fim, glycerolipids são isolados por cromatografia em camada delgada e submetido a transmetilação de seus grupos acil. Graxos methylesters acila são quantificados por cromatografia gás-líquido.

Vídeo: Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI preparação da amostra: o método de Camada Ultra Thin

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

Biologia

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

Isolamento de Protoplastos de tecidos de 14 dias de idade, mudas de Arabidopsis thaliana</em

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

Perfil de anticorpos por sistemas de imunoprecipitação luciferase (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

Isolamento de proteínas lábeis Multi-Complexos por In vivo Controlled Cellular Cross-Linking and Imuno-magnética cromatografia de afinidade

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

Investigando Tecido e órgãos específicos Responses Fitocromo usando FACS assistida Célula tipo Profiling expressão específica no Arabidopsis thaliana</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

Floral-dip Transformação de Arabidopsis thaliana Examine a PTSO2:: β-glucuronidase Expressão Gênica Reporter

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

Automated interação hidrofóbica Seleção coluna de cromatografia para uso na purificação de proteínas

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

Isolamento eficiente e rápida de embriões em início de carreira a partir de<em&gt; Arabidopsis thaliana</em&gt; Sementes

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Metabólica Labeling e Membrana Fracionamento para Comparative Analysis proteômica de<em&gt; Arabidopsis thaliana</em&gt; Culturas suspensão de células

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

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 ...