Name: isolation and compositional analysis of plant cuticle lipid polyester monomers
Uploaded: 2015-11-22T14:00:00.000+00:00
Duration: 9 min 47 s
Description: Watch this Scientific Journal Video about Isolation and Compositional Analysis of Plant Cuticle Lipid Polyester Monomers at JoVE.com

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: Questo protocollo è stato adattato da Bonaventura et al. (2004), Molina et al. (2006), Li et al. (2013) 7,10,11. Passi 1-5 sono riassunti nella figura 1. 1. Tissue delipidizzazione Nota: Sciacquare sempre la vetreria e cappellini con cloroformio, lasciando asciugare sotto cappa, prima di utilizzare. ATTENZIONE: Eseguire omogeneizzazione dei tessuti e tutte le misure di trasferimento di solvente sotto cappa; indossare sempre camice da laboratorio, guanti e occhiali di sicurezza iniziale per evitare il contatto diretto con i prodotti chimici e per proteggere i campioni dalla contaminazione. <ol><li> Bagnomaria Preriscaldare e blocco termico a 85 ° C. </li><li> Pesare circa 0,5 g di ciascun campione in foglia 20 mm provette di vetro pre-pesato x 125 mm con politetrafluoroetilene (PTFE) tappi a vite -faced. Include quattro repliche per campione. </li><li> Mettere 2-propanolo in una beuta (circa 125 ml, 25 ml per g di sample). Aggiungere 2,6-di-tert-butil-4-metilfenolo (noto anche come idrossitoluene butilato, BHT) ad una concentrazione finale di 0,01% (w / v). Nota: Aggiungere BHT da un 5% (w / v) di soluzione stock in metanolo (BHT aiuta minimizzare l&#39;ossidazione degli acidi grassi insaturi). </li><li> Preriscaldamento soluzione 2-propanolo a 85 ° C a bagnomaria. </li><li> Aggiungere 12 ml calda 2-propanolo solvente ogni provetta e calore per 15 min a 85 ° C in blocco termico. Questo passaggio inattiva lipasi che possono essere rilasciati dalle cellule distrutte. </li><li> Lasciare raffreddare tubi a temperatura ambiente e macinare accuratamente tessuto con omogeneizzatore fino ad ottenere una sospensione omogenea. </li><li> Mettere i campioni in un agitatore orbitale e agitare per 1-2 ore a 100 giri al minuto e la temperatura ambiente. </li><li> Centrifugare per 10 minuti a 800 xg e scartare il surnatante. </li><li> Aggiungere un volume equivalente di 2-propanolo e agitare per 12 ore a temperatura ambiente. </li><li> Centrifugare per 10 minuti a 800 xg e scartare il surnatante. </li><li> Aggiungere 12 ml di CHCl 3: CH 3 OH (2: 1, v / v) a residuo (25 ml per g di materiale) e agitare per una notte a 100 rpm e la temperatura ambiente. </li><li> Centrifugare per 10 minuti a 800 xg e scartare il surnatante. </li><li> Aggiungere 12 ml CHCl 3: CH 3 OH (1: 2, v / v) a residuo e agitare per una notte a 100 rpm e la temperatura ambiente. </li><li> Centrifugare per 10 minuti a 800 xg e rimuovere il solvente. </li><li> Lasciate asciugare i campioni sotto cappa notte a temperatura ambiente. </li><li> Asciugare il residuo e poi posto in un essiccatore sotto vuoto ad anidro CaCl 2 o CaSO 4 fino a peso costante a raggiungere (3-5 giorni). </li></ol> 2. Depolimerizzazione: metanolisi con sodio Methoxide (Figura 2) ATTENZIONE: Eseguire i passaggi 2,3-2,4, 2,7-2,8, 2,10-2,11, 2,13-2,15 e 2,17-2,18 sotto cappa; indossare sempre camice da laboratorio, guanti e occhiali di protezione spruzzi. <ol><li> Blocco termico Preriscaldare a 60 ° C. </li><li> Pesare provette contenenti il ​​residuo secco (importante per ulteriori calcoli). </li><li> Aggiungi standard interni in ogni provetta: 25 ωL heptadecanoate metile (1 mg / ml azione) e 25 ml ω-pentadecalactone (1 mg / ml azione). </li><li> Aggiungere 0,9 ml di acetato di metile, 1,5 ml di sodio metilato, e 3,6 ml di metanolo per ciascuna provetta e tappo. In alternativa, preparare una miscela di reazione con questi tre reagenti e aggiungere aliquote 6 ml per ogni campione. </li><li> Campioni di calore per 2 ore a 60 ° C e vortice periodicamente a intervalli di 15 min. </li><li> Lasciate raffreddare i campioni a temperatura ambiente. </li><li> Aggiungere 10 ml di cloruro di metilene (CH 2 Cl 2) e 1,5 ml di acido acetico glaciale per estrarre gli esteri metilici degli acidi grassi. </li><li> Aggiungere la soluzione salina (0,5 M NaCl) per riempire ogni provetta e tappo. </li><li> Campioni vortex per 1 min e centrifugare per 10 min a 800 x g. </li><li> Trasferire la fase organica (inferiore) per pulire i tubi di medie dimensioni (16 x 125 mm, provette di vetro con politetrafluoroetilene (PTFE) tappo a vite -faced). </li><li> Aggiungere la soluzione salina (0,5 M NaCl) per riempire ogni provetta e tappo. </li><li> Campioni vortex per 1 min e centrifugare per 10 min a 800 x g. </li><li> Rimuovere fase acquosa (superiore) e ripetere i passaggi 2,11-2,12. </li><li> Rimuovere tutti i fase acquosa (superiore). </li><li> Aggiungere solfato di sodio anidro (Na 2 SO 4) al solvente, tappare i tubi e vortex per 1 min; campioni possono ora essere lasciati fino al giorno successivo. A questo punto i campioni possono essere lasciati durante la notte sotto la cappa. </li><li> Centrifugare per 2 min a 800 xg per compattare il Na 2 SO 4 sale nel fondo. </li><li> Trasferire la fase organica in una provetta monouso bicchiere piccolo (13 x 100 mm provette di vetro con politetrafluoroetilene (PTFE) tappo a vite -faced). </li><li> Far evaporare il solvente a secco sotto azoto e procedere con il passo derivatizzazione. Se non vengono trasformati subito, negozio evaporato a -20 ° C (campioni possono essereLSO immagazzinato prima della fase di evaporazione). </li></ol> 3. Preparazione dei derivati ​​per gascromatografia ATTENZIONE: Eseguire i passi 3.1.2 e 3.1.5 - 3.1.8 sotto una cappa; indossare sempre camice da laboratorio, guanti e occhiali di protezione spruzzi. <ol><li> Derivati ​​trimetilsilil <ol><li> Preriscaldare il blocco termico a 100 ° C. </li><li> Aggiungere 100 ml di piridina e 100 ml di BSTFA (N, O Bis-trimetilsilil-trifluoroacetammide) per ogni tubo e cappuccio. </li><li> Campioni di calore a 100 ° C per 10 min. </li><li> Lasciate raffreddare i campioni a temperatura ambiente. </li><li> Evaporare campioni sotto azoto a temperatura ambiente. Evitare l&#39;applicazione di calore per campioni, monomeri sono altamente volatili, in questa fase. </li><li> Aggiungere 500 ml di 1: 1 (v / v) eptano: toluene. </li><li> Campioni vortex per 1 minuto e centrifugare per 2 min a 800 x g. </li><li> Aggiungere esempi di fiale GC e procedere dell&#39;analisi GC / MS. </li></ol></li><li> Acetilderivati Nota: Per acetilazione, modificare passaggi 3.1.1-3.1.3 sopra come segue (non mostrato in video); passi 3.1.4-3.1.8 sono gli stessi: <ol><li> Blocco termico Preriscaldare a 60 ° C. </li><li> Aggiungere 100 ml di piridina e 100 ml di anidride acetica (At 2 O) ai tubi. </li><li> Campioni di calore 1 ora a 60 ° C. </li></ol></li></ol> 4. GC / MS Analysis <ol><li> Utilizzare una capillare colonna HP-5 (30 mx 0,25 mm x 0,25 di spessore micron film) o equivalenti (es., 5% difenil, il 95% dimetilpolisilossano). Programma il GC con gas di trasporto elio flusso fissato a 1,5 ml / min e la temperatura del forno programmata da 150 a 300 ° C a 3 ° C / min. </li><li> Utilizzare iniezione split (divisione rapporto 1:10) e impostare spettrometro di massa modalità per eseguire la scansione su 40-600 amu (impatto elettronico di ionizzazione). </li><li> Creare una tabella di sequenza inclusi solventi (in bianco), WT e repliche mutanti, ciascuno utilizzando il metodo di analisi cutina indicato. </li><li> Carico di solventee fiale di campione sulla giostra, aggiungere esano per fiale-siringa risciacquo nel campionatore automatico, se necessario, e avviare la sequenza. Dopo la sequenza è completata, tracce di ioni totali cromatogramma sono disponibili per tutti i campioni. </li></ol> Analisi 5. I dati <ol><li> Identificare monomeri lipidi poliestere confrontando spettro di massa di ogni picco di spettri di massa pubblicato oppure cercando una libreria commerciale, se disponibile. </li><li> Per ogni picco individuati nel totale cromatogramma corrente ionica, utilizzare i rispettivi tempi di ritenzione per trovare le aree sul tavolo risultati di integrazione dal software GC / MS (Supplemental Figura 1). </li><li> Per ogni monomero (colonne AB), aggiungere i valori dell&#39;area trovano nella tabella integrazione (riferimento figura 1, colonna D) alla colonna corrispondente per ogni replicare campione nella tabella Excel di monomeri (File di riferimento 2, colonne CF), che è un foglio di calcolo per quantificare Arabidopsisderivati ​​cutina TMSI. Utilizzare file supplementare 3 se derivati ​​acetilati si preparano invece. </li><li> Aggiungere le aree delle norme interne (IS) alle colonne IS (HK). Per quei monomeri che non sono derivatizzata, esteri metilici cioè grassi acidi (fames) ed esteri dell&#39;acido dimetilici dicarbossilici (DCA DME), utilizzare 17: 0 FAME (IS1) come IS per quantificazione (cellule viola-ombreggiata nella tabella monomero; Supplemental File 2/3). Ai monomeri idrossilati, tra cui alcoli primari, acido ferulico e acidi ω-idrossi, utilizzare 15: 0 FAME 15-idrossi (IS2) come IS di scelta per la quantificazione composto (cellule verdi shadded nella tabella monomero; Supplemental file 2/3) . </li><li> Aggiungere a secco peso foglia per ogni replica alle colonne AX-BA (File supplementare 2) o AQ-AT (File supplementare 3); in alternativa, eseguire la scansione di foglie di calcolare superfici e aggiungere i valori dell&#39;area alla tabella monomero di esprimere carichi monomeri per unità diarea di superficie. </li></ol>

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No conflicts of interest declared.

A differenza di altri biopolimeri come il DNA e le proteine, poliesteri pianta di lipidi non sono fatti da un modello. Invece, le loro composizioni dipendono dalla specificità degli enzimi presenti nei tessuti che rendono questi polimeri extracellulari. Come tale analisi, chimica del componente componenti sono fondamentali per capire composizione di poliestere lipidica. Metodi chimici di fendere le obbligazioni estere includono saponificazione, idrogenolisi, transmetilazione acido-catalizzata, e la base-catalizzata transmetilazione 2. Ciascuno di essi presenta vantaggi e svantaggi. Saponificazione produce grassi idrossi acidi liberi che possono subire reazioni secondarie. Idrogenolisi con litio alluminio idruro (LiAlH 4) 16 è stato utilizzato per l&#39;analisi cutina 7. Idrogenolisi riduce carboni funzionalizzati per alcoli e delle strutture originali devono essere dedotto da deuteriolysis con deuteruro litio alluminio (LiAlD 4). Ilsvantaggio di questo approccio è il requisito di alta risoluzione GC / MS per confrontare il grado di deuteriation dei polioli grassi ottenuti per fare assegnazioni delle loro strutture. Transesterificazione acido-catalizzata con metanolica trifluoruro di boro (BF 3) è stata spesso utilizzata in cutina e suberina depolymerizations 8,17,18, ma il reagente ha una durata limitata e può introdurre artefatti dovuti a lato reazioni 15. Acido solforico metanolica produce anche esteri metilici dei monomeri, ma con proporzioni maggiori di acidi grassi 2-idrossi, che presumibilmente non sono veri componenti lipidici poliestere, rispetto ad altri metodi 10. Il metodo transesterificazione catalizzata Naome descritto in questo protocollo produce esteri metilici di acidi grassi che sono derivatizzati da sililazione dei gruppi ossidrilici, fornendo caratteristiche spettri di massa per l&#39;identificazione o per acetilazione di fornire più derivati ​​stabili di gruppi ossidrilici for quantificazione. Uno svantaggio di questa tecnica è che compete con l&#39;idrolisi transesterificazione presenza di acqua nella reazione. Acqua reagisce con Naome (catalizzatore) e produce NaOH, che a sua volta idrolizza grassi esteri metilici acido per produrre acidi liberi (Figura 2D). Questa è una reazione collaterale indesiderabile perché due picchi saranno presenti per ogni acido grasso: un estere metilico e un estere derivato TMSI, complicando così l&#39;analisi. Utilizzando reagenti anidri e aggiungendo acetato di metile come co-solvente di competere con saponificazione sono passaggi così cruciali per prevenire l&#39;idrolisi (Figura 2D). Cutin e suberina contengono tra l&#39;1 e il 26% glicerolo 4. Tuttavia, questo monomero non viene rilevata dalle condizioni sperimentali descritte in questo protocollo. Il glicerolo è altamente idrofila e, a differenza degli acidi grassi monomeri di estere metilico, sarà eliminato durante le fasi acquose di lavaggio con solvente. Questa limitazione anchepplies ad altri metodi di depolimerizzazione cutina, ma glicerolo possono essere determinati nello strato acquoso ottenuto dopo transesterificazione utilizzando un metodo enzimatico. In alternativa, può essere quantificato utilizzando condizioni più blande (ad es., 0,05 M Naome) senza ulteriore estrazione dell&#39;acqua per rilevare tutti monomeri, compresi glicerolo 19,20 .Anche se utile ai fini della quantificazione glicerolo, condizioni blande solito danno depolimerizzazione incompleta di cutina e suberina. Se un GC accoppiato ad un rilevatore a ionizzazione di fiamma (FID) è disponibile, tutte le repliche possono essere analizzati in questo strumento a fini quantitativi, dopo i picchi di un campione rappresentativo sono stati identificati mediante GC / MS. In alternativa, i monomeri in GC / FID tracce possono essere identificati se i loro indici di ritenzione sono noti. Il rivelatore a ionizzazione di fiamma ha particolarmente elevata sensibilità ed una vasta gamma di proporzionalità, che è fondamentale per la quantificazione della maggiori e minori componenti di esempioin corse singole. Inoltre, è robusto e facile da mantenere e far funzionare 15. Il protocollo descritto consente l&#39;affidabile e riproducibile isolamento, l&#39;identificazione e la quantificazione degli impianti lipidici monomeri poliestere, permettendo la caratterizzazione chimica di mutanti che differiscono nella composizione di uno o più monomeri di poliestere lipidi. La procedura è scalabile, può essere facilmente adattato per elaborare sia piccole e sfusi quantità di vari materiali vegetali, tra radici, semi, foglie, steli e fiori. Dati spettrali di massa di lipidi monomeri poliestere da molte specie sono stati pubblicati ad es., 21-26 e costituiscono risorse preziose per identificare monomeri sconosciuti quando adattare questo protocollo ad altri tessuti e / o specie. Questo metodo è applicabile alle indagini della biosintesi, la regolamentazione e la distribuzione di poliesteri di lipidi nelle piante superiori.

Piante vascolari si basano su livelli extracellulari che fungono da barriera impermeabile tra tessuti vegetali e l&#39;ambiente esterno. Queste strutture associate parete cellulare lipofilo limitano infezione patogena e regolano il trasporto passivo di gas, acqua e sostanze disciolte in e fuori di tessuti vegetali 1. Tali ostacoli sono la cuticola impianto, una struttura unica per synapomorphic piante 2, e le diverse barriere di diffusione-suberina contenente. La cuticola è uno strato oleophilic sintetizzata dalle cellule epidermiche e legato a loro tramite uno strato pectinaceous sul lato extracellulare della parete cellulare 3-5. Esso racchiude gli organi aerei primarie di piante superiori, funzionando come interfaccia vitale tra tessuti vegetali e l&#39;ambiente. Cutin, la matrice strutturale della cuticola, e suberina sono due poliesteri glycerolipid insolubili associati con cere solventi estraibili 2,4. Questi polimerico lipids sono composti derivati ​​degli acidi grassi saturi e insaturi e sono strutturalmente e funzionalmente simili. Tuttavia, essi si distinguono per differenze caratteristiche nei siti composizione e deposizione chimica. Suberina è un poliestere alifatico situato all&#39;interno delle pareti cellulari di alcuni tessuti esterni ed interni formando una parete secondaria. Tessuti Suberized includono periderms di radici, tuberi e corteccia degli alberi, endodermis radice, strati cappotto di semi, e le ferite rimarginate 2. A differenza cutina, poliestere suberina genere contiene alcoli, saturi e acidi dicarbossilici monoinsaturi, e una gran parte di monomeri-molto-lunga catena (C≥20). Cutin è il più abbondante in poliestere lipidico piante vascolari 6, ed è composta da glicerolo e derivati ​​di acidi grassi C16-C18 interesterificato, come idrossi e idrossi-sostituito epossidiche acidi grassi 4. Mentre la composizione di polimeri cutinavaria tra le specie tracheophyte, i monomeri principali predominanti sono 10, 16-diidrossi 16: 0, 18-idrossi-9,10-epossi 18: 0, e 9,10,18-triidrossi 18: 0 acidi grassi. È interessante notare che, foglie e gambo Arabidopsis cutina è principalmente composto da 18: 2 dicarbossilico 7,8. Cuticole pianta presente anche una notevole variabilità di spessore, da pochi nanometri a diversi micrometri 9. Poiché isolamento cuticola è un passo laboriosa e che richiede tempo, particolarmente per cuticole foglia molto sottili, come quelli di Arabidopsis thaliana 8, metodi che non utilizzano isolamento cuticola sono stati sviluppati e validati 7,8. Qui, descriviamo un protocollo dettagliato per studiare la composizione monomero di cutina in foglie Arabidopsis thaliana da metilato sodico (Naome) depolimerizzazione -catalyzed e gascromatografia successiva / spettrometria di massa (GC / MS) analisi. Questo protocollo offre un metodo affidabile per saggiare il comPosition di poliesteri pianta di lipidi in interi tessuti delipidato, ed è stato adattato da protocolli precedentemente riportati 7,10,11. Campioni di tessuto integrali sono prima omogeneizzati ed esaustivo delipidato, eliminando i lipidi solventi estraibili, tra cui cuticolare e cere epicuticolari, lipidi di membrana, e trigliceridi. Residui arricchita di parete cellulare vengono poi depolimerizzati nei loro costituenti monomeri di esteri metilici da sodio metilato-catalizzata metanolisi. Grassi esteri metilici degli acidi vengono estratti su acidificazione, e derivatizzati per ottenere i loro derivati ​​trimetilsilileteri o acetil corrispondenti. Residui derivatizzati sono altamente volatili, e possono essere eluiti da una colonna gascromatografica ad una temperatura ragionevole senza alterarne la conformazione strutturale durante l&#39;analisi 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.

Il protocollo descritto in questo manoscritto è impostato per determinare lipidi monomeri poliestere, riducendo al minimo i contributi di lipidi non cutina 10 figura 1 presenta una panoramica del saggio, che prende tutto tra le 8 (cioè, cutina o suberina.) -. 10 giorni (dalla raccolta dei tessuti prima di ottenere dati GC), a seconda di quanto tempo i campioni sono lasciate asciugare. Il metanolisi selezionato base-catalizzata (Figura 2) metodo per depolimerizzano poliesteri è stato precedentemente convalidato per i semi di Arabidopsis, che contengono sia cutina e suberina. I tessuti vengono prima omogeneizzati ed esaustivo delipidato per rimuovere i lipidi solventi estraibili. La resa residui dopo l&#39;estrazione, come percentuale del peso fresco iniziale, è di solito 6% per A. thaliana Col-0 foglie. Residui arricchita di parete cellulare vengono essiccati in un essiccatore a vuoto e poi depolimerizzati nei loro costituenti monomeri di esteri metilici dabase-catalizzata transmetilazione. Due ore di incubazione è stato scelto come il tempo critico richiesto per il corretto depolimerizzazione e il recupero dei componenti lipidici poliestere. Tempi di incubazione più, determina aumento di acidi 2-idrossi; questi potenzialmente derivano da sfingolipidi di membrana 10. Un tipico cromatogramma se viene mostrato Arabidopsis tipo selvaggio del foglio cutina in figura 3, per i derivati ​​O -TMSi etere (Figura 3A) e derivati ​​O acetil (Figura 3B). Ogni picco è stato identificato dal confronto con spettri di massa dalla letteratura 7,8 e una banca dati pubblica 12 protocollo video .La nostra mostra come preparare derivati ​​TMSI, ma campioni possono in alternativa essere acetilato a derivatize gruppi idrossile. Derivati ​​sililati sono un bene per scopi di identificazione perché danno spettri di massa diagnostico. Tuttavia, i derivati ​​acetilati sono più stabili e una buona alternativa a sililazionemonomeri una volta sono stati identificati 10. Per contribuire all&#39;attuazione di questo protocollo in laboratori che solo hanno GC accoppiato a ionizzazione di fiamma (FID), GC / FID tracce corrispondenti a derivati ​​acetilati di WT monomeri foglia cutina e ad una serie omologa di norme esteri metilici di acidi grassi sono anche visualizzati (Figura supplementare 4). Questo metodo è qualitativo e rileva le differenze quantitative tra i campioni, quindi il suo valore per l&#39;analisi mutante. Gli importi dei singoli monomeri vengono determinati con il metodo standard interno di quantificazione, consentendo il confronto di monomero abbondanza tra i campioni. Va chiarito, tuttavia, che la dimensione di picco (conte totali di ioni) non può riflettere i rapporti molari dei monomeri del poliestere. Stiamo comprese le tabelle modificabili di monomeri per calcolare gli importi dei monomeri in foglia Arabidopsis cutina come esteri di acidi grassi e derivati ​​metilici TMSI (File di riferimento 1), o asso<html> derivati ​​Tyl (File di riferimento 2) di alcoli. Le tabelle possono dover essere adattati se i campioni sono estratti da diversi organi o specie vegetali. A titolo di esempio, abbiamo analizzato Arabidopsis Thalia na Columbia (Col-0) foglie wild type e due alleli nulli-mutante in precedenza caratterizzati del gene CYP86A2 / ATT1, att1-1 (m-1) e att1-2 (m-2 ) 13,14. Citocromo P450 monossigenasi della sottofamiglia CYP86A codificano putativi Ohm-oxydases e partecipa nella suberina e cutina monomero biosintesi. I nostri risultati (Figura 4) dimostrano una significativa riduzione dei carichi di tre principali monomeri lipidici nelle foglie mutante rispetto alle foglie WT. Coerentemente con la funzione dell&#39;enzima previsto, 16: 0, 18: 2, e 18: 1 dicarbossilati furono particolarmente colpite nei mutanti ATT1. &quot;Src =&quot; / files / ftp_upload / 53386 / 53386fig1.jpg &quot;/&gt; Figura 1. Panoramica di analisi dei lipidi in poliestere. <a href="https://www.jove.com/files/ftp_upload/53386/53386fig1large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 2" src="/files/ftp_upload/53386/53386fig2.jpg" /> Figura 2. Meccanismo della reazione transmetilazione Naome catalizzata. Gli anioni metossido nucleofili attaccano il carbonio cabonyl di poliesteri lipidi per formare un intermedio instabile tetraedrica (A), che si dissocia facilmente in esteri metilici di acidi grassi e anioni alcossido (B). Questi alcossidi sono basi coniugate, e reagiscono con metanolo, rigenerando gli anioni metossido cataliticamente attivi, sostenendo così reazioni di depolimerizzazione addizionali (C). Se l&#39;acqua è presente nelsistema, reagirà con metilato di sodio per formare idrossido di sodio, una base forte che idrolizza irreversibilmente esteri producendo acidi grassi liberi indesiderabili. Acetato di metile viene aggiunto come co-solvente 15 per rimuovere piccole quantità di idrossido di sodio all&#39;interno del sistema (D). <a href="https://www.jove.com/files/ftp_upload/53386/53386fig2large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 3" src="/files/ftp_upload/53386/53386fig3.jpg" /> Figura 3. Rappresentante cromatogramma ionico totale di wild-type foglia Arabidopsis thaliana monomeri cutina. (A) O -trimethylsilyl (TMSI) etere e (B) Derivati ​​ossidrile acetato. <a href="https://www.jove.com/files/ftp_upload/53386/53386fig3large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> </a&gt; <img alt="Figura 4" src="/files/ftp_upload/53386/53386fig4.jpg" /> Figura 4. Cutin monomero composizione della Arabidopsis thaliana WT e due nulli alleli mutanti del gene CYP86A2 (mutante-1 = att1-1; mutante-2 = att1-2) barre di errore rappresentano la deviazione standard della media (n = 4). . Adattato da 13, con il permesso di © Blackwell Publishing (2007). <a href="https://www.jove.com/files/ftp_upload/53386/53386fig4large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 1" src="/files/ftp_upload/53386/53386supfig1.jpg" /> Figura 1. supplementare Peak risultati di integrazione tavolo dal software GC / MS. Picchi corrispondenti ai monomeri identificati e standard interni sono identificati dal loro retenti<html> in tempo (colonna C) e valori dell&#39;area sono riportati nella colonna D. <a href="https://www.jove.com/files/ftp_upload/53386/Supplemental_Figure_1.tif" target="_blank">Clicca qui per scaricare il file.</a> <img alt="Figura 2" src="/files/ftp_upload/53386/53386supfig2.jpg" /> Supplemental file 2. Tabella di Arabidopsis monomeri cutina (derivati ​​trimetilsilil etere di esteri metilici degli acidi grassi idrossi). <a href="https://www.jove.com/files/ftp_upload/53386/Supplemental_File_2_Cutin_template_TMSi.xls" target="_blank">Cliccate qui per scaricare il file.</a> <img alt="Figura 3" src="/files/ftp_upload/53386/53386supfig3.jpg" /> Supplemental file 3. Tabella di Arabidopsis monomeri cutina (derivati ​​O acetil di esteri metilici degli acidi grassi idrossi).com / file / ftp_upload / 53386 / Supplemental_File_3_Cutin_template_Acetylated_derivatives.xlsx &quot;target =&quot; _ blank &quot;&gt; Clicca qui per scaricare il file. <img alt="Figura 4" src="/files/ftp_upload/53386/53386supfig4.jpg" /> Supplementare Figura 4. GC / FID tracce di (AB) estere metilico di acidi grassi (FAME) norme indice di conservazione (picchi sono etichettati con ogni FAME saturo lunghezza della catena); e (C) acetilato A. thaliana WT foglia monomeri cutina. Numeri sulla visiera corrispondono a: 16: 0 FAME (1), Ferulate (2), 18: 3 FAME (3), 18: 1/18: 2 FAME (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: dicarbossilico dimetil estere; FAME: grassi estere metilico; IS1: standard interno 1, 17: 0 FAME; IS2: interna stAndard 2, 15-OH 15: 0 FAME. <a href="https://www.jove.com/files/ftp_upload/53386/Supplemental_Figure_4.tif" target="_blank">Cliccate qui per scaricare il file.</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 analisi della composizione delle piante cuticola lipidi poliestere monomeri

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 Views.  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.

Video: 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 Caratterizzazione chimica del lipide A da batteri Gram-negativi

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

Ricerca

Chimica

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 preparazione delle pareti cellulari batteriche per l'analisi compositia mediante cromatografia liquida ad alte prestazioni

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.

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

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

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.

Isolamento chimico, quantificazione e separazione dei lipidi della pelle da rettili

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.

Estrazione della lignina con alto contenuto di β-O-4 da etanolo lieve estrazione ed il relativo effetto sul rendimento depolimerizzazione

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.

Completa l&#39;analisi della composizione delle pareti cellulari delle piante (biomassa lignocellulosica) Parte I: 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.

Completa l&#39;analisi della composizione delle pareti cellulari delle piante (biomassa lignocellulosica) Parte II: Carboidrati

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 Cromatografia su strato sottile (TLC) Accoppiato con cromatografia gas-liquido (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 studio della biofisica cuticole di frutta

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 di goccioline lipidiche plastoglobule dal tessuto fogliare vegetale e cianobatteri

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

Isolamento e analisi della composizione delle piante cuticola lipidi poliestere monomeri

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