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 border="0" cellpadding="0" cellspacing="0" width="1105">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Chemicals</td>
			<td style="width:228px;"></td>
			<td style="width:153px;"></td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">2-propanol</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;">BPA451-4</td>
			<td style="width:301px;">Solvent for delipidation</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Anhydrous sodium sulfate</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;">S421500</td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Acetic anhydride</td>
			<td style="width:228px;">Sigma Aldrich</td>
			<td style="width:153px;">320102</td>
			<td style="width:301px;">Derivatization agent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">BSTFA (N,O-bis(trimethylsilyl)-trifluoroacetamide)</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:153px;">15222</td>
			<td style="width:301px;">Derivatization agent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Butylated hydroxytoluene (BHT)</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:153px;">101162</td>
			<td style="width:301px;">Antioxidant</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Calcium chloride, anhydrous</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;">C614-3</td>
			<td style="width:301px;">Desiccation agent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Calcium suflate, anhydrous (DRIERITE- 8 MESH with indicator)</td>
			<td style="width:228px;">Acros Organics</td>
			<td>219090020</td>
			<td style="width:301px;">Desiccation agent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Chloroform (Trichloromethane)</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;">C6074</td>
			<td style="width:301px;">Organic solvent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Glacial acetic acid</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;">BP2401212</td>
			<td style="width:301px;">Acidification agent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Helium carrier gas, compressed</td>
			<td style="width:228px;">Air Liquide</td>
			<td style="width:153px;">ALPHAGAZ1-UN1046</td>
			<td style="width:301px;">Carrier gas, GC/MS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Heptane</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;">H3501</td>
			<td style="width:301px;">Organic solvent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Hexanes</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;">H3024</td>
			<td style="width:301px;">Organic solvent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Methanol</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;">A4124</td>
			<td style="width:301px;">Organic solvent, transmethylation reactive</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Methyl acetate</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:153px;">296996</td>
			<td style="width:301px;">Organic solvent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Methyl heptadecanoate</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:153px;">H4515</td>
			<td style="width:301px;">Internal standard (1mg/mL stock)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Methylene dichloride (Dichloromethane)</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;">D374</td>
			<td style="width:301px;">Organic solvent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Nitrogen, compressed</td>
			<td style="width:228px;">Air Liquide</td>
			<td style="width:153px;">ALPHAGAZ1-UN1044</td>
			<td style="width:301px;">Carrier gas, GC-FID</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Pentadecanolactone</td>
			<td style="width:228px;">Fluka</td>
			<td style="width:153px;">76530</td>
			<td style="width:301px;">Internal standard (1 mg/mL stock)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Pyridine</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:153px;">270970</td>
			<td style="width:301px;">Co-solvent for derivatization</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Sodium chloride</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;">BP358212</td>
			<td style="width:301px;">Saline solution</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Sodium methoxide (25wt.%)</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:153px;">156256</td>
			<td style="width:301px;">Nucleophile</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Toluene</td>
			<td style="width:228px;">FIsher Scientific</td>
			<td style="width:153px;">T2904</td>
			<td style="width:301px;">Organic solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Plant Growth Supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:423px;">Pro-Mix PGX</td>
			<td>Premier Tech Horticulture Ltd</td>
			<td style="width:153px;"></td>
			<td style="width:301px;">Pro-Mix PGX is recommended to grow Arabidopsis plants (Eddy, R. and Hahn, D.T., 2012,http://docs.lib.purdue.edu/pmag/2) 
			Purdue Methods for Arabidopsis Growth.&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">PermaNest Humidity Dome&#160;</td>
			<td style="width:228px;">Grower&#39;s Solution, LLC, Cookeville, TN&#160;</td>
			<td style="width:153px;">GD2211-24</td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">Perma-Nest Plant Trays (22x11in)</td>
			<td style="width:228px;">Grower&#39;s Solution, LLC, Cookeville, TN&#160;</td>
			<td style="width:153px;">N/A</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">Square greenhouse pots, 3.5 inch&#160;</td>
			<td style="width:228px;">Grower&#39;s Solution, LLC, Cookeville, TN&#160;</td>
			<td style="width:153px;">P86</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:423px;">General Purpose Plant Fertilizer, Plant-Prod 20-20-20</td>
			<td style="width:228px;">Premier Tech Home and Garden In., Brantford, ON</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glassware</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:423px;">13 x 100 mm glass test tube with Teflon-faced screw cap</td>
			<td style="width:228px;">Kimble Chase Life Science and Research Products LLC</td>
			<td style="width:153px;">45066A-13100</td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:423px;">16 x 125 mm glass test tube with Teflon-faced screw cap</td>
			<td style="width:228px;">Kimble Chase Life Science and Research Products LLC</td>
			<td style="width:153px;">45066A-16125</td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:423px;">20 x 125 mm glass test tubes with Teflon-faced screw cap</td>
			<td style="width:228px;">Kimble Chase Life Science and Research Products LLC</td>
			<td style="width:153px;">45066A-20125</td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">GC vial caps</td>
			<td style="width:228px;">National Scientific</td>
			<td style="width:153px;">C400051A</td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">GC vial microinserts</td>
			<td style="width:228px;">National Scientific</td>
			<td style="width:153px;">C4011631</td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">GC vials</td>
			<td style="width:228px;">National Scientific</td>
			<td style="width:153px;">C40001</td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Disposable pasteur pipets</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;">1367820B</td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Flasks</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;"></td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Allegra X15R centrifuge</td>
			<td style="width:228px;">Beckman Coulter</td>
			<td style="width:153px;"></td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Analytic balance</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;"></td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:423px;">Belly dancer</td>
			<td style="width:228px;"></td>
			<td style="width:153px;"></td>
			<td style="width:301px;">A shaker can be used for this purpose if Belly Dancer not available</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">DB-5 Capillary GC column</td>
			<td style="width:228px;">J&#38;W Scientific, CA, USA;&#160;</td>
			<td style="width:153px;"></td>
			<td style="width:301px;">30 m x 0.25 mm x 0.25 &#956;m film thickness</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Desiccator</td>
			<td style="width:228px;"></td>
			<td style="width:153px;"></td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Isotemp 202 water bath</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;"></td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">ISQ LT single quadupole mass spectrometer</td>
			<td style="width:228px;">Thermo Scientific</td>
			<td style="width:153px;"></td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Heat block</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;"></td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Nitrogen evaporator</td>
			<td style="width:228px;"></td>
			<td style="width:153px;"></td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Polytron homogenizer</td>
			<td style="width:228px;">Birkmann</td>
			<td style="width:153px;"></td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Trace 1300 gas chromatograph</td>
			<td style="width:228px;">Thermo Scientific</td>
			<td style="width:153px;"></td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Two-stage regulator</td>
			<td style="width:228px;">Air Liquide</td>
			<td style="width:153px;">Q1-318B-580</td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Vacuum desiccator</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;"></td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:423px;">Vortex mixer</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:153px;"></td>
			<td style="width:301px;"></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

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16.6K 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

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 ...

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Biologia

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and genetics because of its simple life cycle, ease of growth and manipulation for genetic analysis, genomic resources, and transformability of the nucleus and both organelles. Mating strains is a common practice when genetic approaches are used in Chlamydomonass, to create vegetative diploids for analysis of dominance, or following tetrad dissection to ascertain nuclear vs. organellar inheritance, to test allelism, to analyze epistasy, or to generate populations for the purpose of map-based cloning. Additionally, genetic crosses are routinely used to combine organellar genotypes with particular nuclear genotypes. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad separation. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas. Key parameters and trouble spots are explained. Finally, resources for further information and alternative methods are provided.

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

Mating and tetrad separation are required for genetic analysis in Chlamydomonas reinhardtii. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad dissection. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas.

Accoppiamento e di separazione Tetrade Chlamydomonas reinhardtii Per l&#39;analisi genetica

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and ...

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

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

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

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

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

Isolamento efficiente e rapida di embrioni nelle prime fasi di<em&gt; Arabidopsis thaliana</em&gt; Semi

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

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

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

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

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

Metabolic Etichettatura e con membrana frazionamento per proteomica comparativa Analisi del<em&gt; Arabidopsis thaliana</em&gt; Culture sospensione cellulare

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

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

Un nuovo approccio per l&#39;analisi comparativa dei multiproteico Complessi Basato su<sup&gt; 15</sup&gt; N metabolica Etichettatura e quantitativa Spettrometria di Massa

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

Isolamento e fisiologica Analisi di mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while maintaining transparency of the visual axis. Perturbations to this delicate balance cause blinding illnesses, such as diabetic retinopathy. Therefore, the understanding of energy metabolism changes in the retina during disease is imperative to the development of rational therapies for various causes of vison loss. The recent advent of commercially-available extracellular flux analyzers has made the study of retinal energy metabolism more accessible. This protocol describes the use of such an analyzer to measure contributions to retinal energy supply through its two principle arms - oxidative phosphorylation and glycolysis - by quantifying changes in oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as proxies for these pathways. This technique is readily performed in explanted retinal tissue, facilitating assessment of responses to multiple pharmacologic agents in a single experiment. Metabolic signatures in retinas from animals lacking rod photoreceptor signaling are compared to wild-type controls using this method. A major limitation in this technique is the lack of ability to discriminate between light-adapted and dark-adapted energy utilization, an important physiologic consideration in retinal tissue.

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

Misurazione del metabolismo energetico in tessuto retinico Explanted usando l'analisi di flusso extracellulare

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

Rapid Lipid Droplet Isolation Protocol Using a Well-established Organelle Isolation Kit

Lipid droplets (LDs) are bioactive organelles found within the cytosol of the most eukaryotic and some prokaryotic cells. LDs are composed of neutral lipids encased by a monolayer of phospholipids and proteins. Hepatic LD lipids, such as ceramides, and proteins are implicated in several diseases that cause hepatic steatosis. Although previous methods have been established for LD isolation, they require a time-consuming preparation of reagents and are not designed for the isolation of multiple subcellular compartments. We sought to establish a new protocol to enable the isolation of LDs, endoplasmic reticulum (ER), and lysosomes from a single mouse liver.
Further, all reagents used in the protocol presented here are commercially available and require minimal reagent preparation without sacrificing LD purity. Here we present data comparing this new protocol to a standard sucrose gradient protocol, demonstrating comparable purity, morphology, and yield. Additionally, we can isolate ER and lysosomes using the same sample, providing detailed insight into the formation and intracellular flux of lipids and their associated proteins.

This protocol establishes a novel method of lipid droplet isolation and purification from mouse livers, using a well-established endoplasmic reticulum isolation kit.

Protocollo di isolamento gocciolina del lipido rapida utilizzando un Kit di isolamento organello ben consolidate

Lipid droplets (LDs) are bioactive organelles found within the cytosol of the most eukaryotic and some prokaryotic cells. LDs are composed of neutral ...