This work was supported by a Natural Sciences and Engineering Research (NSERC)-USRA grant to S.J., and by an NSERC-Discovery Grant to I.M. We thank Richard Bourgault, Meghan Rains, and Amanda Fluke for technical assistance. Seeds of att1-1 and att1-2 mutants were kindly provided by Dr. Jian-Min Zhou, Institute of Genetics and Developmental Biology, Beijing, China.

Nota: Este protocolo foi adaptado de Bonaventure et al. (2004), Molina et al. (2006), Li et ai. (2013) 7,10,11. Passos 1-5 são sumariados na Figura 1. Deslipidação 1. Tissue Nota: Sempre lavar todos os vidros e tampas com clorofórmio, deixando secar sob exaustor, antes de usar. CUIDADO: Executar homogeneização do tecido e todas as etapas de transferência solvente sob exaustor; sempre usar jaleco, luvas e óculos de segurança inicial para evitar o contato direto com produtos químicos e para proteger as amostras de contaminação. <ol><li> Banho de água Pré-aqueça e bloco de calor a 85 ° C. </li><li> Pesar aproximadamente 0,5 g de cada amostra de folha em 20 mm x 125 mm tubos de ensaio de vidro previamente pesado com politetrafluoretileno (PTFE) tampas de rosca -faced. Incluem quatro repetições por amostra. </li><li> Coloque 2-propanol num balão de Erlenmeyer (cerca de 125 ml; 25 ml por g de SAMPle). Adicionar 2,6-di-terc-butil-4-metilfenol (também conhecido como hidroxitolueno butilado, BHT) para uma concentração final de 0,01% (w / v). Nota: Adicionar BHT a partir de um 5% (w / v) de solução de reserva em metanol (BHT ajuda a minimizar a oxidação de ácidos gordos insaturados). </li><li> Pré-aqueça solução de 2-propanol a 85 ° C em banho de água. </li><li> Adicionar 12 ml de solvente quente de 2-propanol para cada tubo de amostra e calor durante 15 min a 85 ° C em bloco de calor. Este passo inativa lipases que podem ser libertadas a partir de células rompidas. </li><li> Deixe os tubos arrefecer até à temperatura ambiente e triturar o tecido cuidadosamente com homogeneizador até obter uma suspensão homogénea. </li><li> Coloque as amostras num agitador orbital e agitar durante 1-2 horas a 100 rpm e temperatura ambiente. </li><li> Centrifugar durante 10 minutos a 800 xg e descartar o sobrenadante. </li><li> Adicionar um volume igual de 2-propanol e agita-se durante 12 horas à temperatura ambiente. </li><li> Centrifugar durante 10 minutos a 800 xg e descartar o sobrenadante. </li><li> Adicionar 12 ml de CHCl 3: CH3OH (2: 1, v / v) até ao resíduo (25 ml por g de amostra) e agitar durante a noite a 100 rpm e temperatura ambiente. </li><li> Centrifugar durante 10 minutos a 800 xg e descartar o sobrenadante. </li><li> Adicionar 12 ml de CHCl 3: CH 3 OH (1: 2, v / v) até ao resíduo e agitar durante a noite a 100 rpm e temperatura ambiente. </li><li> Centrifugar durante 10 minutos a 800 xg e remover o solvente. </li><li> Deixe secar amostras sob exaustor durante a noite a temperatura ambiente. </li><li> Secar o resíduo e, em seguida, colocar-se num exsicador de vácuo sobre CaCl2 anidro ou CaSO4 até peso constante (3-5 dias). </li></ol> 2. A despolimerização: metanólise com metóxido de sódio (Figura 2) ATENÇÃO: Siga as etapas 2,3-2,4, 2,7-2,8, 2,10-2,11, 2,13-2,15 e 2,17-2,18 sob exaustor; sempre usar jaleco, luvas e óculos de segurança inicial. <ol><li> Pré-aqueça o bloco de calor a 60 ° C. </li><li> Pesar tubos de ensaio contendo o resíduo seco (importante para cálculos posteriores). </li><li> Adicionar padrões internos a cada tubo: 25 ωL heptadecanoato de metilo (1 mg / ml) e 25 ul ω-pentadecalactone (1 mg / ml de stock). </li><li> Adicionar 0,9 ml de acetato de metilo, 1,5 mL de metóxido de sódio, e 3,6 ml de metanol a cada tubo e cap-los. Em alternativa, preparar uma mistura de reacção com estes três reagentes e adicionar alíquotas de 6 ml para cada amostra. </li><li> Amostras de calor durante 2 horas a 60 ° C e periodicamente vórtice em intervalos de 15 min. </li><li> Deixe as amostras arrefecer para a temperatura ambiente. </li><li> Adicionar 10 ml de dicloreto de metileno (CH 2 Cl 2) e 1,5 ml de ácido acético glacial para extrair os ésteres metílicos dos ácidos gordos. </li><li> Adicionar uma solução salina (NaCl 0,5 M) para encher cada tubo e a tampa. </li><li> Amostras Vortex durante 1 min e centrifuga-se durante 10 min a 800 x g. </li><li> Transferir a fase orgânica (inferior) para limpar tubos de tamanho médio (16 x 125 tubos de ensaio de vidro com poli mmtetrafluoroetileno (PTFE) tampa de rosca -faced). </li><li> Adicionar uma solução salina (NaCl 0,5 M) para encher cada tubo e a tampa. </li><li> Amostras Vortex durante 1 min e centrifuga-se durante 10 min a 800 x g. </li><li> Remover fase aquosa (superior) e repita os passos de 2,11-2,12. </li><li> Remova toda a fase aquosa (superior). </li><li> Adicionar sulfato de sio anidro (Na 2 SO 4) para o solvente, tampão, e os tubos de vórtice durante 1 min; amostras pode agora ser deixada até ao dia seguinte. A esta amostras ponto pode ser deixada durante a noite sob a coifa. </li><li> Centrifugar durante 2 minutos a 800 xg para compactar o Na 2 SO 4 sal na parte inferior. </li><li> Transferir a fase orgânica para um tubo de vidro descartável pequena (13 x 100 mm tubos de ensaio de vidro com politetrafluoroetileno (PTFE) com tampa de rosca -faced). </li><li> Evaporar o solvente à secura sob azoto e avançar para a etapa de derivação. Se não processado imediatamente, armazenar evaporada amostras a -20 ° C (amostras pode ser umlso armazenado antes do passo de evaporação). </li></ol> 3. Preparação de Derivados de Cromatografia Gasosa ATENÇÃO: Siga as etapas 3.1.2 e 3.1.5 - 3.1.8 sob um exaustor; sempre usar jaleco, luvas e óculos de segurança inicial. <ol><li> Derivados trimetilsilílicos <ol><li> Pré-aqueça o bloco de aquecimento a 100 ° C. </li><li> Adicionar 100 ul de piridina e 100 ml de BSTFA (N, O-bis-trimetilsilil-trif luoroacetamida) a cada tubo e cap-los. </li><li> Amostras de aquecer a 100 ° C durante 10 min. </li><li> Deixe as amostras arrefecer para a temperatura ambiente. </li><li> Evapora-se as amostras sob azoto à temperatura ambiente. Evitar a aplicação de calor para amostras, os monómeros são altamente voláteis, nesta fase. </li><li> Adicionar 500 ul de 1: 1 (v / v) de heptano: tolueno. </li><li> Amostras Vortex durante 1 min e centrifuga-se durante 2 min a 800 x g. </li><li> Adicionar amostras para frascos de GC e proceder à análise GC / MS. </li></ol></li><li> Acetilderivados Nota: Para acetilação, modificar passos 3.1.1-3.1.3 acima da seguinte forma (não mostrado na vídeo); os passos 3.1.4-3.1.8 são os mesmos: <ol><li> Pré-aqueça o bloco de calor a 60 ° C. </li><li> Adicionar 100 ul de piridina e 100 ul de anidrido acético (Ac2O) para os tubos. </li><li> Amostras de calor 1 hora a 60 ° C. </li></ol></li></ol> 4. GC / MS Análise <ol><li> Usar uma coluna HP-5 capilar (30 mx 0,25 mm de espessura x 0,25 uM filme) ou equivalente (isto é., 5% de difenilo, 95% de dimetil polisiloxano). Programa de GC com o fluxo de gás transportador hélio fixado em 1,5 ml / min e a temperatura do forno programado para 150 a 300 ° C a 3 ° C / min. </li><li> Use injeção de divisão (split proporção de 1:10) e definir espectrômetro de massa modo para digitalizar mais de 40-600 amu (elétron impacto de ionização). </li><li> Criar uma tabela de seqüência incluindo solvente (em branco), WT e mutantes repetições, cada um usando o método de análise cutin indicado. </li><li> Carga de solventee frascos de amostra sobre o carrossel, adicione-hexano para frascos de lavagem de seringas no amostrador automático, se necessário, e iniciar a sequência. Após a sequência estiver completa, dadas vestígios cromatograma iónico estão disponíveis para todas as amostras. </li></ol> Análise 5. Dados <ol><li> Identificar monómeros de poliéster lipídico comparando espectro de massa de cada pico de espectros de massa publicado ou pesquisando uma biblioteca comercial, se disponível. </li><li> Para cada um dos picos identificados no cromatograma total de corrente de iões, utilizar os seus respectivos tempos de retenção para encontrar as áreas na tabela de resultados de integração a partir do software GC / MS (Figura Suplementar 1). </li><li> Para cada monômero (colunas AB), adicione os valores de área encontrados na tabela de integração (Figura Suplementar 1, coluna D) para a coluna correspondente para cada replicar amostra na tabela Excel de monómeros (Supplemental Arquivo 2, colunas CF), que é uma folha de cálculo para quantificar Arabidopsisderivados cutin EAS. Use Suplementar Arquivo 3 se derivados acetilados são preparados em vez disso. </li><li> Adicione as áreas das normas internas (SI) para as colunas é (HK). Para os monómeros que não são derivatizados, ésteres metílicos ou seja, ácidos gordos (FAMEs), e os ésteres de dimetilo do ácido dicarboxilicos (DCA EMDs), utilizar 17: 0 FAME (IS1) como IS para a quantificação (células roxo-protegida na tabela de monómero; Suplementar Arquivos 2/3). Para monómeros hidroxilados, incluindo álcoois primários, ácido ferúlico e ácidos ω-hidroxi, utilizar 15: 0 15-hidroxi FAME (IS2), como é de escolha para a quantificação composto (células verdes-shadded na tabela de monómero; Suplementar Arquivos 2/3) . </li><li> Adicione peso folha seca para cada replicar para colunas AX-BA (Supplemental arquivo 2) ou AQ-AT (Supplemental Arquivo 3); alternativamente, digitalizar folhas de calcular áreas de superfície e adicionar valores de área para a mesa de monômero para expressar cargas de monómeros por unidade deárea de superfície. </li></ol>

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No conflicts of interest declared.

Ao contrário de outros biopolímeros, tais como proteínas e ADN, poliésteres planta de lípidos não são feitas a partir de um molde. Em vez disso, as composições dependem da especificidade das enzimas presentes nos tecidos que formam estes polímeros extracelulares. Como tais análises, química do componente de componentes são essenciais para compreender a composição de poliéster de lípidos. Os métodos químicos para clivar ligações éster incluem saponificação, hidrogenólise, transmetilação catalisada por ácido, e catalisada por base transmetilação 2. Cada um deles tem vantagens e desvantagens. Saponificação produz ácidos gordos hidroxi livres que podem sofrer reações secundárias. A hidrogenólise com hidreto de alumínio e lítio (LiAlH4) 16 foi utilizado para a análise cutina 7. A hidrogenólise reduz carbonos funcionalizados para álcoois e as estruturas originais precisam de ser inferida por deuteriolysis com deutereto de alumínio e lítio (LiAlD 4). odesvantagem desta abordagem é a exigência de alta resolução GC / MS para comparar o grau de deuteriation dos polióis gordos obtidos para fazer atribuições das suas estruturas. Transesterificação catalisada por ácido com metanol trifluoreto de boro (BF3) tem sido freqüentemente utilizada em cutina e suberina depolymerizations 8,17,18, mas o reagente tem uma vida útil limitada e pode introduzir artefactos devido a reacções secundárias 15. Ácido sulfúrico metanólico também produz ésteres metílicos dos monómeros mas com maiores proporções de ácidos gordos de 2-hidroxi, que presumivelmente não são verdadeiros componentes de poliéster lípido, em comparação com outros métodos 10. O método de transesterificação catalisada NaOMe descrito neste protocolo produz ésteres metílicos de ácidos gordos que são derivatizados por sililação de grupos hidroxilo, fornecendo espectros de massa característico para a identificação, ou por acetilação para proporcionar derivados mais estáveis ​​de grupos hidroxilo foR quantificação. Uma desvantagem desta técnica é que a hidrólise compete com a transesterif icação, quando a água está presente na reacção. A água reage com NaOMe (o catalisador) e produz NaOH, que por sua vez hidrolisa ésteres metílicos de ácidos gordos para se obter os ácidos livres (Figura 2D). Esta é uma reacção colateral indesejável porque dois picos irá estar presente para cada ácido gordo: um éster de metilo e um derivado de éster TMSi, complicando assim a análise. Usando reagentes anidros e a adição de acetato de metilo como um co-solvente para competir com saponificação são assim passos cruciais para prevenir a hidrólise (Figura 2D). Cutin e suberina conter entre 1 e 26% de glicerol 4. No entanto, este monómero não será detectado por as condições experimentais descritas neste protocolo. O glicerol é altamente hidrofílico e, ao contrário dos monómeros de éster de metilo de ácidos gordos, será eliminada durante os passos de solvente de lavagem aquosas. Esta limitação também umpplies a outros métodos de despolimerização cutina, glicerol, mas pode ser determinada na camada aquosa obtida após transesterificação utilizando um método enzimático. Alternativamente, ele pode ser quantificada usando condições mais suaves (por exemplo., 0,05 M NaOMe) sem nova extracção da água para detectar todos os monómeros, incluindo glicerol 19,20 .Embora útil para a finalidade de quantificação glicerol, condições suaves geralmente dar incompleto de despolimerização e cutina suberina. Se um GC acoplado a um detector de ionização de chama (FID) está disponível, todas as repetições podem ser analisados ​​no presente instrumento, para fins quantitativos, depois de picos de uma amostra representativa foram identificados por GC / MS. Como alternativa, os monômeros em GC / FID traços podem ser identificados, se os seus índices de retenção são conhecidos. O detector de ionização de chama tem especialmente elevada sensibilidade e uma ampla gama de proporcionalidade, que é crítica para a quantificação de componentes principais e secundárias amostrasem corridas individuais. Além disso, é robusto e fácil de manter e operar 15. O protocolo descrito permite a fiável e reprodutível isolamento, identificação e quantificação dos monómeros de poliéster de lípidos de plantas, permitindo a caracterização de mutantes química que diferem na composição de um ou mais monómeros de poliéster de lípidos. O procedimento é expansível, ele pode ser facilmente adaptada para processar tanto pequenas quantidades a granel e de diversos materiais de planta, incluindo sementes, raízes, folhas, caules e flores. Dados de espectrometria de massa de monómeros de poliéster de lipídios de muitas espécies têm sido publicados por ex., 21-26 e constituem recursos valiosos para identificar monómeros desconhecidos quando adaptar este protocolo para outros tecidos e / ou espécies. Este método é aplicável para a biossíntese de investigações, regulação e de distribuição de poliésteres de lípidos em plantas superiores.

As plantas vasculares confiar em camadas extracelulares, que funcionam como barreiras impermeáveis ​​entre os tecidos de plantas e o ambiente externo. Estas estruturas associadas da parede celular lipofílico restringir a infecção patogénica e regular o transporte passivo de gases, água e substâncias dissolvidas dentro e para fora dos tecidos da planta 1. Tais barreiras são a cutícula da planta, uma estrutura única para plantas sinapomórficas 2, e barreiras de difusão diferentes contendo suberina. A cutícula é uma camada oleofï¿½lico sintetizado por células epidérmicas e ligado a eles por meio de uma camada péctico no lado extracelular da parede celular 3-5. É encerra os órgãos aéreos primárias de plantas superiores, funcionando como uma interface vital entre tecidos da planta e do meio ambiente. Cutina, a matriz estrutural da cutícula, e suberina são dois poliésteres glicerolípido insolúveis associadas com ceras solvente 2,4-extraíveis. Estes l poliméricoIPIDs são compostas de derivados de ácidos gordos saturados e insaturados e de ambos são estruturalmente e funcionalmente semelhante. No entanto, eles são distinguíveis por diferentes características de sites de composição e deposição química. Suberina é um poliéster alifático localizado no interior das paredes das células de certos tecidos internos e externos que formam uma parede secundária. Tecidos suberizadas incluem periderms de raízes, tubérculos e casca de árvore, endoderme raiz, camadas de revestimento de sementes, e feridas curadas 2. Ao contrário cutina, o poliéster suberina tipicamente contém álcoois, ácidos dicarboxílicos saturados e mono-insaturados, e uma grande proporção de monómeros de cadeia muito longa (C≥20). Cutina é o mais abundante poliéster lípido em plantas vasculares 6, e é composto de glicerol e C16-C18 derivados de ácidos gordos interesterificada, tais como ácidos gordos hidroxi e hidroxi-4 epóxi substituído. Embora a composição de polímeros cutinavaria entre espécies tracheophyte, os monómeros primários mais predominantes são de 10, 16-di-hidroxi 16: 0, 18-hidroxi-9,10-epoxi-18: 0, e 9,10,18-tri-hidroxi-18: 0 ácidos gordos. Curiosamente, folha de Arabidopsis e cutina haste é composta principalmente de 18: 2 de ácido dicarboxílico 7,8. Cutículas vegetais também apresentam uma considerável variabilidade em espessura, variando de poucos nanómetros a vários micrómetros 9. Uma vez que o isolamento cutícula é uma etapa trabalhosa e demorada, especialmente para cutículas folha muito fina, tais como aqueles da Arabidopsis thaliana 8, os métodos que não utilizam o isolamento cutícula foram desenvolvidos e validados 7,8. Aqui, nós descrevemos um protocolo detalhado para o estudo da composição de monómero de cutina em folhas de Arabidopsis thaliana por metóxido de sódio (NaOMe) catalisada por despolimerização e subsequente cromatografia em fase gasosa / espectrometria de massa (GC / MS) análise. Este protocolo oferece um método robusto para ensaiar a composition de poliésteres planta de lipídios nos tecidos delipidadas inteiros, e foi adaptado a partir de protocolos previamente relatados 7,10,11. Amostras de tecido integrais são primeiro homogeneizado e exaustivamente delipidadas, removendo lipídios solventes extraível incluindo cuticular e ceras epicuticulares, lípidos da membrana, e triacilgliceróis. Enriquecida resíduos de parede celular são então despolimerizados em seus constituintes monómeros de éster de metilo por metanólise catalisada por metóxido de sódio. Ésteres metílicos dos ácidos gordos são extraídos por acidificação, e os seus derivados para obter trimetilsililo ou acetilo derivados correspondentes. Resíduos derivatizados são altamente voláteis, e podem ser eluídas a partir de uma coluna de cromatografia em fase gasosa a uma temperatura razoável, sem alterar a sua conformação estrutural durante a análise de GC / MS.

<table><tbody><tr><td>Chemicals</td><td></td><td></td><td></td></tr><tr><td>2-propanol</td><td>Fisher Scientific</td><td>BPA451-4</td><td>Solvent for delipidation</td></tr><tr><td>Anhydrous sodium sulfate</td><td>Fisher Scientific</td><td>S421500</td><td></td></tr><tr><td>Acetic anhydride</td><td>Sigma Aldrich</td><td>320102</td><td>Derivatization agent</td></tr><tr><td>BSTFA (N,O-bis(trimethylsilyl)-trifluoroacetamide)</td><td>Sigma-Aldrich</td><td>15222</td><td>Derivatization agent</td></tr><tr><td>Butylated hydroxytoluene (BHT)</td><td>Sigma-Aldrich</td><td>101162</td><td>Antioxidant</td></tr><tr><td>Calcium chloride, anhydrous</td><td>Fisher Scientific</td><td>C614-3</td><td>Desiccation agent</td></tr><tr><td>Calcium suflate, anhydrous (DRIERITE- 8 MESH with indicator)</td><td>Acros Organics</td><td>219090020</td><td>Desiccation agent</td></tr><tr><td>Chloroform (Trichloromethane)</td><td>Fisher Scientific</td><td>C6074</td><td>Organic solvent</td></tr><tr><td>Glacial acetic acid</td><td>Fisher Scientific</td><td>BP2401212</td><td>Acidification agent</td></tr><tr><td>Helium carrier gas, compressed</td><td>Air Liquide</td><td>ALPHAGAZ1-UN1046</td><td>Carrier gas, GC/MS</td></tr><tr><td>Heptane</td><td>Fisher Scientific</td><td>H3501</td><td>Organic solvent</td></tr><tr><td>Hexanes</td><td>Fisher Scientific</td><td>H3024</td><td>Organic solvent</td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A4124</td><td>Organic solvent, transmethylation reactive</td></tr><tr><td>Methyl acetate</td><td>Sigma-Aldrich</td><td>296996</td><td>Organic solvent</td></tr><tr><td>Methyl heptadecanoate</td><td>Sigma-Aldrich</td><td>H4515</td><td>Internal standard (1mg/mL stock)</td></tr><tr><td>Methylene dichloride (Dichloromethane)</td><td>Fisher Scientific</td><td>D374</td><td>Organic solvent</td></tr><tr><td>Nitrogen, compressed</td><td>Air Liquide</td><td>ALPHAGAZ1-UN1044</td><td>Carrier gas, GC-FID</td></tr><tr><td>Pentadecanolactone</td><td>Fluka</td><td>76530</td><td>Internal standard (1 mg/ml stock)</td></tr><tr><td>Pyridine</td><td>Sigma-Aldrich</td><td>270970</td><td>Co-solvent for derivatization</td></tr><tr><td>Sodium chloride</td><td>Fisher Scientific</td><td>BP358212</td><td>Saline solution</td></tr><tr><td>Sodium methoxide (25wt.%)</td><td>Sigma-Aldrich</td><td>156256</td><td>Nucleophile</td></tr><tr><td>Toluene</td><td>FIsher Scientific</td><td>T2904</td><td>Organic solvent</td></tr><tr><td>Plant Growth Supplies</td><td></td><td></td><td></td></tr><tr><td>Pro-Mix PGX</td><td>Premier Tech Horticulture Ltd</td><td></td><td>Pro-Mix PGX is recommended to grow Arabidopsis plants (Eddy, R. and Hahn, D.T., 2012,http://docs.lib.purdue.edu/pmag/2)&lt;br/&gt; Purdue Methods for Arabidopsis Growth.&amp;nbsp;</td></tr><tr><td>PermaNest Humidity Dome&amp;nbsp;</td><td>Grower&#039;s Solution, LLC, Cookeville, TN&amp;nbsp;</td><td>GD2211-24</td><td></td></tr><tr><td>Perma-Nest Plant Trays (22x11in)</td><td>Grower&#039;s Solution, LLC, Cookeville, TN&amp;nbsp;</td><td>N/A</td><td></td></tr><tr><td>Square greenhouse pots, 3.5 inch&amp;nbsp;</td><td>Grower&#039;s Solution, LLC, Cookeville, TN&amp;nbsp;</td><td>P86</td><td></td></tr><tr><td>General Purpose Plant Fertilizer, Plant-Prod 20-20-20</td><td>Premier Tech Home and Garden In., Brantford, ON</td><td>N/A</td><td></td></tr><tr><td>Glassware</td><td></td><td></td><td></td></tr><tr><td>13 x 100 mm glass test tube with Teflon-faced screw cap</td><td>Kimble Chase Life Science and Research Products LLC</td><td>45066A-13100</td><td></td></tr><tr><td>16 x 125 mm glass test tube with Teflon-faced screw cap</td><td>Kimble Chase Life Science and Research Products LLC</td><td>45066A-16125</td><td></td></tr><tr><td>20 x 125 mm glass test tubes with Teflon-faced screw cap</td><td>Kimble Chase Life Science and Research Products LLC</td><td>45066A-20125</td><td></td></tr><tr><td>GC vial caps</td><td>National Scientific</td><td>C400051A</td><td></td></tr><tr><td>GC vial microinserts</td><td>National Scientific</td><td>C4011631</td><td></td></tr><tr><td>GC vials</td><td>National Scientific</td><td>C40001</td><td></td></tr><tr><td>Disposable pasteur pipets</td><td>Fisher Scientific</td><td>1367820B</td><td></td></tr><tr><td>Flasks</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>Allegra X15R centrifuge</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Analytic balance</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Belly dancer</td><td></td><td></td><td>A shaker can be used for this purpose if Belly Dancer not available</td></tr><tr><td>DB-5 Capillary GC column</td><td>J&amp;amp;W Scientific, CA, USA;&amp;nbsp;</td><td></td><td>30 m x 0.25 mm x 0.25 &amp;mu;m film thickness</td></tr><tr><td>Desiccator</td><td></td><td></td><td></td></tr><tr><td>Isotemp 202 water bath</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>ISQ LT single quadupole mass spectrometer</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Heat block</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Nitrogen evaporator</td><td></td><td></td><td></td></tr><tr><td>Polytron homogenizer</td><td>Birkmann</td><td></td><td></td></tr><tr><td>Trace 1300 gas chromatograph</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Two-stage regulator</td><td>Air Liquide</td><td>Q1-318B-580</td><td></td></tr><tr><td>Vacuum desiccator</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Vortex mixer</td><td>Fisher Scientific</td><td></td><td></td></tr></tbody></table>

isolation and compositional analysis of plant cuticle lipid polyester monomers

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester of glycerol and modified fatty acids that constitutes the framework of the cuticle that covers aerial plant surfaces. Suberin is a related lipid polyester that is deposited on the cell walls of certain tissues, including the root endodermis and the periderm of tubers, tree bark and roots. These lipid polymers are highly variable in composition among plant species, and often differ among tissues within a single species. Here, we describe a detailed protocol to study the monomer composition of cutin in Arabidopsis thaliana leaves by sodium methoxide (NaOMe)-catalyzed depolymerisation, derivatization, and subsequent gas chromatography-mass spectrometry (GC/MS) analysis. This method can be used to investigate the monomers of insoluble polyesters isolated from whole delipidated plant tissues bearing either cutin or suberin. The method can by applied not only to characterize the composition of lipid polymers in species not previously analyzed, but also as an analytical tool in forward and reverse genetic approaches to assess candidate gene function.

O protocolo descrito no presente manuscrito está configurado para determinar monómeros de poliéster de lipídios, minimizando as contribuições de lípidos não Cutin 10 Figura 1 apresenta uma visão geral do ensaio, que toma por completo entre 8 (ie, cutin ou suberina.) -. 10 dias (a partir de colheita de tecido inicial a obtenção de dados de GC), dependendo de quanto tempo as amostras são deixadas a secar. A metanólise catalisada por base seleccionada (Figura 2) Método para despolimerizar poliésteres foi previamente validado para as sementes de Arabidopsis, que contêm tanto cutina e suberina. Os tecidos são homogeneizados e exaustivamente primeiro delipidada para remover lípidos de solventes extraíveis. O rendimento resíduo após a extração, como porcentagem do peso fresco inicial, é normalmente de 6% para A. thaliana Col-0 folhas. Enriquecida resíduos de parede celular são secos num exsicador de vácuo e, em seguida, nos seus constituintes despolimerizado monómeros de éster de metilo decatalisada por base transmetilação. Duas horas de incubação foi escolhido como o tempo necessário para a despolimerização crítica e recuperação de componentes de poliéster de lípidos adequada. Tempos de incubação mais longos resultou em aumento de ácidos 2-hidroxi; estes potencialmente derivar de esfingolípidos membrana 10. Um cromatograma típico de Arabidopsis do tipo selvagem se cutina folha é mostrada na Figura 3, para derivados S -TMSi éter (Figura 3A) e derivados de O-acetil (Figura 3B). Cada pico foi identificado por comparação com os espectros de massa a partir da literatura 7,8 e uma base de dados pública 12 .O nosso protocolo de vídeo mostra como preparar derivados de TMSI, mas as amostras podem, alternativamente, ser acetilado para derivatizar grupos hidroxilo. Derivados silanisados ​​são bons para fins de identificação, porque eles dão espectros de massa de diagnóstico. No entanto, os derivados acetilados são mais estáveis ​​e uma boa alternativa para a sililaçãomonômeros uma vez foram identificados 10. Para ajudar a implementar esse protocolo em laboratórios que têm apenas GC acoplado ao detector de ionização de chama (FID), GC / FID traços correspondentes a derivativos acetilados de WT monómeros folha cutina e para uma série homólogo de normas metílicos dos ácidos gordos éster são também mostrados (Figura Suplementar 4). Este método é qualitativa e detecta diferenças quantitativas entre amostras, daí o seu valor para a análise mutante. As quantidades de monómeros individuais são determinadas usando o método do padrão interno de quantificação, permitindo comparações de abundância monómero entre as amostras. Deve esclarecer-se, no entanto, que o tamanho de pico (contagens totais de iões) podem não coincidir com as razões molares dos monómeros do poliéster. Estamos incluindo tabelas editáveis ​​de monômeros para calcular os montantes de monômero em folha Arabidopsis cutin como ésteres de ácidos gordos e derivados metílicos EAS (Supplemental arquivo 1), ou ace<html> Tyl (Supplemental derivados de arquivo 2) de álcoois. Estas tabelas podem ter de ser adaptados se amostras são extraídas a partir de órgãos ou de diferentes espécies de plantas. Como exemplo, temos analisado Arabidopsis Thalia na Columbia (Col-0) folhas de tipo selvagem e dois alelos nulos-mutante anteriormente caracterizados do gene CYP86A2 / ATT1, att1-1 (m-1) e att1-2 (m-2 ) 13,14. Monooxigenases do citocromo P450 da subfamília CYP86A codificar supostos co-oxidases e participe de suberina e cutina biossíntese monômero. Os nossos resultados (Figura 4) demonstram as reduções significativas na quantidade de três principais monómeros lipídicas nas folhas mutantes em comparação com as folhas WT. Consistente com a função da enzima previsto, 16: 0, 18: 2, e 18: 1 dicarboxilatos foram especificamente afectados em mutantes ATT1. &quot;Src =&quot; / files / ftp_upload / 53386 / 53386fig1.jpg &quot;/&gt; Figura 1. Visão geral da análise de poliéster lipídico. <a href="https://www.jove.com/files/ftp_upload/53386/53386fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/53386/53386fig2.jpg" /> Figura 2. Mecanismo de transmetilação NaOMe a reacção catalisada. Os aniões metóxido nucleofílicos atacar o carbono cabonyl de poliésteres lipídicos para formar um intermediário tetraédrico instável (A), que dissocia-se facilmente em ésteres metílicos de ácidos gordos e de alcóxido aniões (B). Estes alcóxidos são bases conjugadas, e reagir com metanol, regenerando os aniões metóxido cataliticamente activas, sustentando assim as reacções de despolimerização adicionais (C). Se a água está presente nasistema, que irá reagir com metóxido de sódio para formar hidróxido de sódio, uma base forte que irreversivelmente hidrolisa para produzir ésteres de ácidos gordos livres indesejáveis. Acetato de metila é adicionado como um co-solvente 15 para remover pequenas quantidades de hidróxido de sódio dentro do sistema (D). <a href="https://www.jove.com/files/ftp_upload/53386/53386fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 3" src="/files/ftp_upload/53386/53386fig3.jpg" /> Figura 3. Representante cromatograma iónico total de tipo selvagem folha Arabidopsis thaliana monómeros cutina. (A) O trimetilsilílico (TMSi) éter e (B) derivados de hidroxila de etilo. <a href="https://www.jove.com/files/ftp_upload/53386/53386fig3large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> </a&gt; <img alt="Figura 4" src="/files/ftp_upload/53386/53386fig4.jpg" /> Figura 4. cutina composição de monómero de Arabidopsis thaliana WT e dois alelos mutantes nulos do gene CYP86A2. (Mutante-1 = att1-1; mutante-2 = att1-2) As barras de erro representam o desvio padrão da média (n = 4) . Adaptado a partir de 13, com a permissão de © Blackwell Publishing (2007). <a href="https://www.jove.com/files/ftp_upload/53386/53386fig4large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="figura 1" src="/files/ftp_upload/53386/53386supfig1.jpg" /> Figura Suplementar 1. Pico resultados de integração tabela do software de GC / MS. Os picos correspondentes a monómeros identificados e os padrões internos são identificados pela sua retenti<html> a tempo (coluna C) e valores de área estão tabulados na coluna D. <a href="https://www.jove.com/files/ftp_upload/53386/Supplemental_Figure_1.tif" target="_blank">Por favor, clique aqui para fazer o download deste arquivo.</a> <img alt="Figura 2" src="/files/ftp_upload/53386/53386supfig2.jpg" /> Suplementar Arquivo 2. Tabela de Arabidopsis monómeros Cutin (derivados trimetilsililico de ésteres metílicos dos ácidos gordos hidroxi). <a href="https://www.jove.com/files/ftp_upload/53386/Supplemental_File_2_Cutin_template_TMSi.xls" target="_blank">Por favor clique aqui para fazer o download deste arquivo.</a> <img alt="Figura 3" src="/files/ftp_upload/53386/53386supfig3.jpg" /> Suplementar Tabela 3. Ficheiro de Arabidopsis monómeros cutina (derivados de O-acetil de ésteres metílicos de ácidos hidroxi-gordos).com / arquivos / ftp_upload / 53386 / &quot;target =&quot; _ blank Supplemental_File_3_Cutin_template_Acetylated_derivatives.xlsx &quot;&gt; Por favor clique aqui para fazer o download deste arquivo. <img alt="Figura 4" src="/files/ftp_upload/53386/53386supfig4.jpg" /> Suplementar Figura 4. GC / FID vestígios de (AB) de éster metílico de ácido gordo (FAME) padrões de índice de retenção (picos são rotulados com cada comprimento de cadeia saturada FAME); e (C) A. acetilado folha WT thaliana monómeros cutina. Os números no pico correspondem a: 16: 0 FAME (1), ácido ferúlico (2), 18: 3 FAME (3), 18: 1/18: 2 FAMEs (4), 18: 0 FAME (5), sinapate (6 ), 16: 0 DCA (7), 16-OH 16: 0 FAME (8), 18: 2 DCA (9), 18: 1 DCA (10) 18: 0 DCA (11), 18-OH 18: 2 FAME (12), 18-OH 18: 1 FAME (13), 20: 0 FAME (14), 10,16-DIOH 16: 0 FAME (15), 24: 0 FAME (16). DCA: éster dimetílico do ácido dicarboxílico; FAME: éster metílico de ácidos gordos; IS1: padrão interno 1, 17: 0 FAME; IS2: st internoAndard 2, 15-OH 15: 0 FAME. <a href="https://www.jove.com/files/ftp_upload/53386/Supplemental_Figure_4.tif" target="_blank">Por favor clique aqui para fazer o download deste arquivo.</a>

Watch this Scientific Journal Video about Isolation and Compositional Analysis of Plant Cuticle Lipid Polyester Monomers at JoVE.com

Isolation and Compositional Analysis of Plant Cuticle Lipid Polyester Monomers

Lipid polyesters constitute the structural components of two cell wall modifications, the plant cuticle and suberin-containing diffusion barriers. In this video, we describe a method to depolymerize cutin from whole delipidated leaves. The method can be applied to investigating mutants compromised in either cutin or suberin biosynthesis.

Isolamento e composicional Análise da cutícula vegetal lipídicas poliéster Monómeros

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester ...

isolation-compositional-analysis-plant-cuticle-lipid-polyester

Research

JoVE Journal

Biology

16.7K visualizações.  Algoma University. Lipid polyesters constitute the structural components of two cell wall modifications, the plant cuticle and suberin-containing diffusion barriers. In this video, we describe a method to depolymerize cutin from whole delipidated leaves. The method can be applied to investigating mutants compromised in either cutin or suberin biosynthesis.

Vídeo: Isolation and Compositional Analysis of Plant Cuticle Lipid Polyester Monomers

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.

Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.

Isolamento e Caracterização química do lípido A de bactérias Gram-negativas

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

Química

Isolation and Preparation of Bacterial Cell Walls for Compositional Analysis by Ultra Performance Liquid Chromatography

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the face of turgor pressures several atmospheres in magnitude. Across the diverse shapes and sizes of the bacterial kingdom, the cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by short peptides. Peptidoglycan&#8217;s central importance to bacterial physiology underlies its use as an antibiotic target and has motivated genetic, structural, and cell biological studies of how it is robustly assembled during growth and division. Nonetheless, extensive investigations are still required to fully characterize the key enzymatic activities in peptidoglycan synthesis and the chemical composition of bacterial cell walls. High Performance Liquid Chromatography (HPLC) is a powerful analytical method for quantifying differences in the chemical composition of the walls of bacteria grown under a variety of environmental and genetic conditions, but its throughput is often limited. Here, we present a straightforward procedure for the isolation and preparation of bacterial cell walls for biological analyses of peptidoglycan via HPLC and Ultra Performance Liquid Chromatography (UPLC), an extension of HPLC that utilizes pumps to deliver ultra-high pressures of up to 15,000 psi, compared with 6,000 psi for HPLC. In combination with the preparation of bacterial cell walls presented here, the low-volume sample injectors, detectors with high sampling rates, smaller sample volumes, and shorter run times of UPLC will enable high resolution and throughput for novel discoveries of peptidoglycan composition and fundamental bacterial cell biology in most biological laboratories with access to an ultracentrifuge and UPLC.

The bacterial cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by peptides. Ultra Performance Liquid Chromatography provides high resolution and throughput for novel discoveries of peptidoglycan composition. We present a procedure for the isolation of cell walls (sacculi) and their subsequent preparation for analysis via UPLC.

Isolamento e Preparação de Paredes Celulares Bacterianas para Análise Composicional por Cromatografia Líquida de Ultra Desempenho

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the ...

Use of Arabidopsis eceriferum Mutants to Explore Plant Cuticle Biosynthesis

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


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

Uso de Arabidopsis mutantes eceriferum para Explorar Biossíntese Cutícula Vegetal

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

Biologia

Chemical Isolation, Quantification, and Separation of Skin Lipids from Reptiles

Reptiles signal to conspecifics using lipids in their skin, primarily to enable mate tracking and assessment. The isolation of these lipids has utility in research focused on evolutionary patterns and mechanisms of chemical communication, in addition to understanding the waterproofing role of lipids in the evolution of terrestrial life. In an applied approach, such skin-based cues have potential use for wildlife managers dealing with invasive species. The main steps for quantifying reptile skin lipids in the protocol presented here include extraction, total lipid determination, and fractionation via column chromatography, the latter process resulting in purified eluates of compounds which can then either be analyzed to assign compound identifications (e.g., gas chromatography-mass spectrometry [GC-MS]) and/or used directly in more refined bioassays. Skin lipids can be extracted from living skin, shed skin, or dead whole animals, using nonpolar organic solvents (e.g., hexane, benzene, toluene). Extraction solubilizes the lipids and, then, the solvent can be evaporated to yield a measurable lipid-only extract. Fractionation involves the separation of the total lipid extract into specific eluates via traditional column chromatography. The total lipid extract is first bound to a substrate-based column (e.g., alumina) and, then, individual eluates (&#34;fractions&#34;) of solvent at specific volumes are passed sequentially through the column to elute sets of compounds from the lipid mixture based on common polarity. The fractions progress in polarity at a standardized sequence by increasing the relative amount of polar solvent (e.g., diethyl ether) in nonpolar solvent. In this manuscript, we describe several methods for extracting skin lipids of reptiles and, then, provide a standard protocol for isolating different sets of compounds based on polarity, using traditional column chromatography. Whole lipid extracts or specific fractions can, then, be used in bioassays to determine any biological activity elicited by the compounds therein.

In reptiles, skin lipids from conspecifics are crucial for sexual signaling, with potential use in invasive species management. Here, we describe protocols for extracting skin lipids from shed skin or whole animals, determining and analyzing the total lipid mass, and separating the lipids using fractionation via column chromatography.

Químico isolamento, quantificação e separação dos lipídios da pele dos répteis

Reptiles signal to conspecifics using lipids in their skin, primarily to enable mate tracking and assessment. The isolation of these lipids has utility in ...

Ambiente

Extraction of Lignin with High &#946;-O-4 Content by Mild Ethanol Extraction and Its Effect on the Depolymerization Yield

Lignin valorization strategies are a key factor for achieving more economically competitive biorefineries based on lignocellulosic biomass. Most of the emerging elegant procedures to obtain specific aromatic products rely on the lignin substrate having a high content of the readily cleavable &#946;-O-4 linkage as present in the native lignin structure. This provides a miss-match with typical technical lignins that are highly degraded and therefore are low in &#946;-O-4 linkages. Therefore, the extraction yields, and the quality of the obtained lignin are of utmost importance to access new lignin valorization pathways. In this manuscript, a simple protocol is presented to obtain lignins with high &#946;-O-4 content by relatively mild ethanol extraction that can be applied to different lignocellulose sources. Furthermore, analysis procedures to determine the quality of the lignins are presented together with a depolymerization protocol that yields specific phenolic 2-arylmethyl-1,3-dioxolanes, which can be used to evaluate the obtained lignins. The presented results demonstrate the link between lignin quality and potential for the lignins to be depolymerized into specific monomeric aromatic chemicals. Overall, the extraction and depolymerization demonstrates a trade-off between the lignin extraction yield and the retention of the native aryl-ether structure and thus the potential of the lignin to be used as the substrate for the production of chemicals for higher-value applications.

Here, we present a protocol to perform ethanol extraction of lignin from several biomass sources. The effect of the extraction conditions on the lignin yield and &#946;-O-4 content are presented. Selective depolymerization is performed on the obtained lignins to obtain high aromatic monomer products.

Extração da lignina com alto teor de β-O-4 por extração de etanol suave e seu efeito sobre o rendimento de despolimerização

Lignin valorization strategies are a key factor for achieving more economically competitive biorefineries based on lignocellulosic biomass. Most of the ...

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part I: Lignin

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation1 and other products such as biocomposite materials7. Plant biomass remains one of the greatest untapped reserves on the planet4. It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses (matrix polysaccharides, and the polyphenol lignin6 and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls provide tensile strength to cells and the entire plants, ward off pathogens, and allow water to be transported throughout the plant; in the case of trees up to more the 100 m above ground level. Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant4. Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerization by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis. In this first part we focus on the analysis of the polyphenol lignin (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its lignin content by acetylbromide solubilization3, and its lignin composition in terms of its syringyl, guaiacyl- and p-hydroxyphenyl units5. The protocol for analyzing the carbohydrates in lignocellulosic biomass including cellulose content and matrix polysaccharide composition is discussed in Part II2.

Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part I: Lignin

Plant biomass is a major carbon-neutral renewable resource that could be used for the production of biofuels. Plant biomass consists mainly of cell walls, a structurally complex composite material termed lignocellulosics. Here we describe a protocol for a comprehensive analysis of the content and composition of the polyphenolic lignin.

Análise da composição completa de paredes celulares das plantas (biomassa lignocelulósica) Parte I: A lignina

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st ...

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part II: Carbohydrates

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation2 and other products such as biocomposite materials6. Plant biomass remains one of the greatest untapped reserves on the planet4. It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses, and the polyphenol lignin5 and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls contribute extensively to the strength and structural integrity of the entire plant. Despite its necessary rigidity, the cell wall is a highly dynamic entity that is metabolically active and plays crucial roles in numerous cell activities such as plant growth and differentiation5. Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant4. Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerisation by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its monosaccharide composition of the hemicelluloses and other matrix polysaccharides1, and its content of crystalline cellulose7. The protocol for analyzing the lignin components in lignocellulosic biomass is discussed in Part I3.

Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part II: Carbohydrates

Plant biomass is a major carbon-neutral renewable resource that could be used for the production of biofuels. Plant biomass consists mainly of cell walls, a structurally complex composite material termed lignocellulosics. Here we describe a protocol for a comprehensive analysis of the content and composition of wall derived carbohydrates.

Análise da composição completa de paredes celulares das plantas (biomassa lignocelulósica) Parte II: Os hidratos de carbono

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

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as phosphatidylcholine or phosphatidylethanolamine, form bilayer membranes which act as both boundaries and interfaces for chemical exchange between cells and their surroundings. Unlike animals, plant cells have a special organelle for photosynthesis, the chloroplast. The intricate membrane system of the chloroplast contains unique glycerolipids, namely glycolipids lacking phosphorus: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG)4. The roles of these lipids are beyond simply structural. These glycolipids and other glycerolipids were found in the crystal structures of photosystem I and II indicating the involvement of glycerolipids in photosynthesis8,11. During phosphate starvation, DGDG is transferred to extraplastidic membranes to compensate the loss of phospholipids9,12.
Much of our knowledge of the biosynthesis and function of these lipids has been derived from a combination of genetic and biochemical studies with Arabidopsis thaliana14. During these studies, a simple procedure for the analysis of polar lipids has been essential for the screening and analysis of lipid mutants and will be outlined in detail. A leaf lipid extract is first separated by thin layer chromatography (TLC) and glycerolipids are stained reversibly with iodine vapor. The individual lipids are scraped from the TLC plate and converted to fatty acyl methylesters (FAMEs), which are analyzed by gas-liquid chromatography coupled with flame ionization detection (FID-GLC) (Figure 1). This method has been proven to be a reliable tool for mutant screening. For example, the tgd1,2,3,4 endoplasmic reticulum-to-plastid lipid trafficking mutants were discovered based on the accumulation of an abnormal galactoglycerolipid: trigalactosyldiacylglycerol (TGDG) and a decrease in the relative amount of 18:3 (carbons : double bonds) fatty acyl groups in membrane lipids 3,13,18,20. This method is also applicable for determining enzymatic activities of proteins using lipids as substrate6.

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

Composition of polar lipid extracts and the fatty acid composition of individual glycerolipids are determined in a simple and robust lipid profiling experiment. For this purpose, glycerolipids are isolated by thin layer chromatography and subjected to transmethylation of their acyl groups. Fatty acyl methylesters are quantified by gas-liquid chromatography.

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 ...

Isolation and Biophysical Study of Fruit Cuticles

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and abiotic stresses and also regulates water flow from the external environment.1 A biopolyester (cutin) and long-chain fatty acids (waxes) form the principal structural framework of the cuticle; the functional integrity of the cuticular layer depends on the outer 'epicuticular' layer as well as the blend consisting of the cutin biopolymer and 'intracuticular' waxes.2 Herein, we describe a comprehensive protocol to extract waxes exhaustively from commercial tomato (Solanum lycopersicum) fruit cuticles or to remove epicuticular and intracuticular waxes sequentially and selectively from the cuticle composite. The method of Jetter and Sch&auml;ffer (2001) was adapted for the stepwise extraction of epicuticular and intracuticular waxes from the fruit cuticle.3,4 To monitor the process of sequential wax removal, solid-state cross-polarization magic-angle-spinning (CPMAS) 13C NMR spectroscopy was used in parallel with atomic force microscopy (AFM), providing molecular-level structural profiles of the bulk materials complemented by information on the microscale topography and roughness of the cuticular surfaces. To evaluate the cross-linking capabilities of dewaxed cuticles from cultivated wild-type and single-gene mutant tomato fruits, MAS 13C NMR was used to compare the relative proportions of oxygenated aliphatic (CHO and CH2O) chemical moieties.
Exhaustive dewaxing by stepwise Soxhlet extraction with a panel of solvents of varying polarity provides an effective means to isolate wax moieties based on the hydrophobic characteristics of their aliphatic and aromatic constituents, while preserving the chemical structure of the cutin biopolyester. The mechanical extraction of epicuticular waxes and selective removal of intracuticular waxes, when monitored by complementary physical methodologies, provides an unprecedented means to investigate the cuticle assembly: this approach reveals the supramolecular organization and structural integration of various types of waxes, the architecture of the cutin-wax matrix, and the chemical composition of each constituent. In addition, solid-state 13C NMR reveals differences in the relative numbers of CHO and CH2O chemical moieties for wild-type and mutant red ripe tomato fruits. The NMR techniques offer exceptional tools to fingerprint the molecular structure of cuticular materials that are insoluble, amorphous, and chemically heterogeneous. As a noninvasive surface-selective imaging technique, AFM furnishes an effective and direct means to probe the structural organization of the cuticular assembly on the nm-&mu;m length scale.

Aerial plant organs are protected by the cuticle, a supramolecular biopolyester-wax assembly. We present protocols to monitor selective removal of epi- and intracuticular waxes from tomato fruit cuticles on molecular and micro scales by solid-state NMR and atomic force microscopy, respectively, and to assess the cross-linking capacity of engineered cuticular biopolyesters.

Isolamento e Estudo de Biofísica de Cutículas Frutas

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and ...

Plastoglobule Lipid Droplet Isolation from Plant Leaf Tissue and Cyanobacteria

Plastoglobule lipid droplets are a dynamic sub-compartment of plant chloroplasts and cyanobacteria. Found ubiquitously among photosynthetic species, they are believed to serve a central role in the adaptation and remodeling of the thylakoid membrane under rapidly changing environmental conditions. The capacity to isolate plastoglobules of high purity has greatly facilitated their study through proteomic, lipidomic, and other methodologies. With plastoglobules of high purity and yield, it is possible to investigate their lipid and protein composition, enzymatic activity, and protein topology, among other possible molecular characteristics. This article presents a rapid and effective protocol for the isolation of plastoglobules from chloroplasts of plant leaf tissue and presents methodological variations for the isolation of plastoglobules and related lipid droplet structures from maize leaves, the desiccated leaf tissue of the resurrection plant, Eragrostis nindensis, and the cyanobacterium, Synechocystis sp. PCC 6803. Isolation relies on the low density of these lipid-rich particles, which facilitates their purification by sucrose density flotation. This methodology will prove valuable in the study of plastoglobules from diverse species.

A fast and efficient protocol is presented for the isolation of plastoglobule lipid droplets associated with various photosynthetic organisms. The successful preparation of isolated plastoglobules is a crucial first step that precedes detailed molecular investigations such as proteomic and lipidomic analyses.

Isolamento de gotículas lipídicas de platoglobulos do tecido foliar da planta e cianobactérias

Plastoglobule lipid droplets are a dynamic sub-compartment of plant chloroplasts and cyanobacteria. Found ubiquitously among photosynthetic species, they ...