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.

Note: This protocol was adapted from Bonaventure et al. (2004), Molina et al. (2006), Li et al. (2013)7,10,11. Steps 1-5 are summarized in Figure 1.
1. Tissue Delipidation
Note: Always rinse all glassware and caps with chloroform, letting dry under fume hood, before using.
CAUTION: Perform tissue homogenization and all solvent transfer steps under fume hood; always wear lab coat, gloves and splash safety goggles to avoid direct contact with chemicals and to protect the samples from contamination.
<ol>
	<li>Preheat water bath and heat block to 85 &#176;C.</li>
	<li>Weigh approximately 0.5 g of each leaf sample into pre-weighed 20 mm x 125 mm glass test tubes with polytetrafluoroethylene&#160;(PTFE)-faced screw caps. Include four replicates per sample.</li>
	<li>Place 2-propanol in an Erlenmeyer flask (approximately 125 ml; 25 ml per g of sample). Add 2,6-di-tert-butyl-4-methylphenol (also known as butylated hydroxytoluene, BHT) to a final concentration of 0.01% (w/v). 
	Note: Add BHT from a 5% (w/v) stock solution in methanol (BHT helps minimize oxidation of unsaturated fatty acids).</li>
	<li>Preheat 2-propanol solution to 85 &#176;C in water bath.</li>
	<li>Add 12 ml hot 2-propanol solvent to each sample tube and heat for 15 min at 85 &#176;C in heat block. This step inactivates lipases that may be released from disrupted cells.</li>
	<li>Let tubes cool down to room temperature and grind tissue thoroughly with homogenizer until&#160;a homogeneous suspension is obtained.</li>
	<li>Place the samples in an orbital shaker and shake for 1-2 hours at 100 rpm and room temperature.</li>
	<li>Centrifuge for 10 min at 800 x g and discard supernatant.</li>
	<li>Add an equal volume of 2-propanol and shake for 12 hours at room temperature.</li>
	<li>Centrifuge for 10 min at 800 x g and discard supernatant.</li>
	<li>Add 12 ml CHCl3:CH3OH (2:1, v/v) to residue (25 ml per g of sample) and shake overnight at 100 rpm and room temperature.</li>
	<li>Centrifuge for 10 min at 800 x g and discard supernatant.</li>
	<li>Add 12 ml CHCl3:CH3OH (1:2, v/v) to residue and shake overnight at 100 rpm and room temperature.</li>
	<li>Centrifuge for 10 min at 800 x g and remove solvent.</li>
	<li>Let samples dry under fume hood overnight at room temperature.</li>
	<li>Air-dry the residue and then place in a vacuum desiccator over anhydrous CaCl2 or CaSO4 until constant weight is reached (3-5 days).</li>
</ol>
2. Depolymerization: Methanolysis with Sodium Methoxide (Figure 2)
CAUTION: Perform steps 2.3 &#8211; 2.4, 2.7 &#8211; 2.8, 2.10 &#8211; 2.11, 2.13 &#8211; 2.15 and 2.17 &#8211; 2.18 under fume hood; always wear lab coat, gloves and splash safety goggles.
<ol>
	<li>Preheat heat block to 60 &#176;C.</li>
	<li>Weigh test tubes containing the dry residue (important for further calculations).</li>
	<li>Add internal standards to each tube: 25 &#969;L methyl heptadecanoate (1 mg/ml stock) and 25 &#181;l &#969;-pentadecalactone (1 mg/ml stock).</li>
	<li>Add 0.9 ml methyl acetate, 1.5 ml sodium methoxide, and 3.6 ml methanol to each tube and cap them. Alternatively, prepare a reaction mix with these three reagents and add 6-ml aliquots to each sample.</li>
	<li>Heat samples for 2 hours at 60 &#176;C and vortex periodically in 15 min intervals.</li>
	<li>Let samples cool down to room temperature.</li>
	<li>Add 10 ml of methylene dichloride (CH2Cl2) and 1.5 ml of glacial acetic acid to extract the fatty acid methyl esters.</li>
	<li>Add saline solution (0.5 M NaCl) to fill each tube and cap.</li>
	<li>Vortex samples for 1 min and centrifuge for 10 min at 800 x g.</li>
	<li>Transfer the organic phase (lower) to clean medium size tubes (16 x 125 mm glass test tubes with polytetrafluoroethylene&#160;(PTFE)-faced screw cap).</li>
	<li>Add saline solution (0.5 M NaCl) to fill each tube and cap.</li>
	<li>Vortex samples for 1 min and centrifuge for 10 min at 800 x g.</li>
	<li>Remove aqueous (upper) phase and repeat steps 2.11-2.12.</li>
	<li>Remove all aqueous (upper) phase.</li>
	<li>Add anhydrous sodium sulfate (Na2SO4) to the solvent, cap the tubes, and vortex for 1 min; samples may now be left until the following day. At this point samples can be left overnight under the fume hood.</li>
	<li>Centrifuge for 2 min at 800 x g to compact the Na2SO4 salt in the bottom.</li>
	<li>Transfer the organic phase to a small glass disposable tube (13 x 100 mm glass test tubes with polytetrafluoroethylene&#160;(PTFE)-faced screw cap).</li>
	<li>Evaporate the solvent to dryness under nitrogen and proceed to the derivatization step. If not processed immediately, store evaporated samples at -20 &#176;C (samples can be also stored before the evaporation step).</li>
</ol>
3. Preparation of Derivatives for Gas Chromatography
CAUTION: Perform steps 3.1.2 and 3.1.5 &#8211; 3.1.8 under a fume hood; always wear lab coat, gloves and splash safety goggles.
<ol>
	<li>Trimethylsilyl derivatives
	<ol>
		<li>Preheat heat block to 100 &#176;C.</li>
		<li>Add 100 &#181;l of pyridine and 100 &#181;l of BSTFA (N,O Bis-trimethylsilyl-trifluoroacetamide) to each tube and cap them.</li>
		<li>Heat samples at 100 &#176;C for 10 min.</li>
		<li>Let samples cool down to room temperature.</li>
		<li>Evaporate samples under nitrogen at room temperature. Avoid applying heat to samples, monomers are highly volatile at this stage.</li>
		<li>Add 500 &#181;l of 1:1 (v/v) heptane:toluene.</li>
		<li>Vortex samples for 1 min and centrifuge for 2 min at 800 x g.</li>
		<li>Add samples to GC vials and proceed to GC/MS analysis.</li>
	</ol>
	</li>
	<li>Acetyl derivatives 
	Note: For acetylation, modify steps 3.1.1-3.1.3 above as follows (not shown in video); steps 3.1.4-3.1.8 are the same:
	<ol>
		<li>Preheat heat block to 60 &#176;C.</li>
		<li>Add 100 &#181;l of pyridine and 100 &#181;l of acetic anhydride (Ac2O) to the tubes.</li>
		<li>Heat samples 1 hour at 60 &#176;C.</li>
	</ol>
	</li>
</ol>
4. GC/MS Analysis
<ol>
	<li>Use a HP-5 capillary column (30 m x 0.25 mm x 0.25 &#181;m film thickness) or equivalent (i.e., 5% diphenyl,&#160;95% dimethyl polysiloxane). Program the GC with helium carrier gas flow set at 1.5 ml/min and oven temperature programmed from 150 to 300 &#176;C at 3 &#176;C/min.</li>
	<li>Use split injection (split ratio 1:10) and set mass spectrometer to scan mode over 40-600 amu (electron impact ionization).</li>
	<li>Create a sequence table including solvent (blank), WT and mutant replicates, each using the indicated cutin analysis method.</li>
	<li>Load solvent and sample vials on the carrousel, add hexane to syringe-rinsing vials in the autosampler if necessary, and start the sequence. After the sequence is completed, total ion chromatogram traces are available for all samples.</li>
</ol>
5. Data Analysis
<ol>
	<li>Identify lipid polyester monomers by comparing each peak&#8217;s mass spectrum to published mass spectra or by searching a commercial library if available.</li>
	<li>For each identified peak in the total ion current chromatogram, use their respective retention times to find the areas on the integration results table from the GC/MS software (Supplemental Figure 1).</li>
	<li>For each monomer (columns A-B), add the area values found in the integration table (Supplemental Figure 1, column D) to the corresponding column for every replicate sample in the Excel table of monomers (Supplemental File 2, columns C-F), which is a spreadsheet to quantify Arabidopsis cutin TMSi derivatives. Use Supplemental File 3 if acetylated derivatives are prepared instead.</li>
	<li>Add the areas of the internal standards (IS) to the IS columns (H-K). For those monomers that are not derivatized, namely fatty acid methyl esters (FAMEs), and dicarboxylic acid dimethyl esters (DCA DMEs), use 17:0 FAME (IS1) as IS for quantification (purple-shaded cells in the monomer table; Supplemental Files 2/3). For hydroxylated monomers, including primary alcohols, ferulic acid and &#969;-hydroxy acids, use 15:0 15-hydroxy FAME (IS2) as IS of choice for compound quantification (green-shadded cells in the monomer table; Supplemental Files 2/3).</li>
	<li>Add dry leaf weight for each replicate to columns AX-BA (Supplemental File 2) or AQ-AT (Supplemental File 3); alternatively, scan leaves to calculate surface areas and add area values to the monomer table to express monomer loads per unit of surface area.</li>
</ol>

<ol>
	<li>Kolattukudy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biopolyester+membranes+of+plants:+cutin+and+suberin.">Biopolyester membranes of plants: cutin and suberin.</a> Science. 208 (4447), 990-1000 (1980).</li><li>Kolattukudy, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyesters+in+higher+plants.">Polyesters in higher plants.</a> Adv. Biochem. Eng. Biot. 71, 1-49 (2001).</li><li>Yeats, T. H., Rose, J. K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+formation+and+function+of+plant+cuticles.">The formation and function of plant cuticles.</a> Plant Physiol. 163 (1), 5-20 (2013).</li><li>Pollard, M., Beisson, F., Li, Y., Ohlrogge, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+lipid+barriers:+biosynthesis+of+cutin+and+suberin.">Building lipid barriers: biosynthesis of cutin and suberin.</a> Trends Plant Sci. 13 (5), 236-246 (2008).</li><li>Beisson, F., Li-Beisson, Y., Pollard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solving+the+puzzles+of+cutin+and+suberin+polymer+biosynthesis.">Solving the puzzles of cutin and suberin polymer biosynthesis.</a> Curr. Opin. Plant Biol. 15 (3), 329-337 (2012).</li><li>Heredia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+and+biochemical+characteristics+of+cutin,+a+plant+barrier+biopolymer.">Biophysical and biochemical characteristics of cutin, a plant barrier biopolymer.</a> Biochim. Biophys. Acta. 1620 (1-3), 1-7 (2003).</li><li>Bonaventure, G., Beisson, F., Ohlrogge, J., Pollard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+aliphatic+monomer+composition+of+polyesters+associated+with+Arabidopsis+epidermis:+occurrence+of+octadeca-cis-6,+cis-9-diene-1,+18-dioate+as+the+major+component.">Analysis of the aliphatic monomer composition of polyesters associated with Arabidopsis epidermis: occurrence of octadeca-cis-6, cis-9-diene-1, 18-dioate as the major component.</a> Plant J. 40 (6), 920-930 (2004).</li><li>Franke, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoplastic+polyesters+in+Arabidopsis+surface+tissues+-+A+typical+suberin+and+a+particular+cutin.">Apoplastic polyesters in Arabidopsis surface tissues - A typical suberin and a particular cutin.</a> Phytochemistry. 66 (22), 2643-2658 (2005).</li><li>Vogg, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tomato+fruit+cuticular+waxes+and+their+effects+on+transpiration+barrier+properties:+functional+characterization+of+a+mutant+deficient+in+a+very-long-chain+fatty+acid+-ketoacyl-CoA+synthase.">Tomato fruit cuticular waxes and their effects on transpiration barrier properties: functional characterization of a mutant deficient in a very-long-chain fatty acid -ketoacyl-CoA synthase.</a> J. Exp. Bot. 55 (401), 1401-1410 (2004).</li><li>Molina, I., Bonaventure, G., Ohlrogge, J., Pollard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lipid+polyester+composition+of+Arabidopsis+thaliana+and+Brassica+napus+seeds.">The lipid polyester composition of Arabidopsis thaliana and Brassica napus seeds.</a> Phytochemistry. 67 (23), 2597-2610 (2006).</li><li>Li-Beisson, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acyl-lipid+metabolism.">Acyl-lipid metabolism.</a> The Arabidopsis Book. 11, e0161 (2013).</li><li>Molina, I., Ohlrogge, J. B., Pollard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deposition+and+localization+of+lipid+polyester+in+developing+seeds+of+Brassica+napus+and+Arabidopsis+thaliana.">Deposition and localization of lipid polyester in developing seeds of Brassica napus and Arabidopsis thaliana.</a> Plant J. 53 (3), 437-449 (2008).</li><li>Xiao, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+CYP86A2+represses+Pseudomonas+syringae+type+III+genes+and+is+required+for+cuticle+development.">Arabidopsis CYP86A2 represses Pseudomonas syringae type III genes and is required for cuticle development.</a> EMBO J. 23 (14), 2903-2913 (2004).</li><li>Christie, W. W., Han, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lipid Analysis - Isolation, Separation, Identification and Lipidomic Analysis. , 446 (2010).</li><li>Walton, T. J., Kolattukudy, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+structures+of+cutin+monomers+by+a+novel+depolymerization+procedure+and+combined+gas+chromatography+and+mass+spectrometry.">Determination of the structures of cutin monomers by a novel depolymerization procedure and combined gas chromatography and mass spectrometry.</a> Biochemistry. 11 (10), 1885-1896 (1972).</li><li>Matzke, K., Riederer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+into+the+chemical+constitution+of+cutins+and+suberins+from+Picea+abies+(L.)+Karst.,+Quercus+robur+L.,+and+Fagus+sylvatica+L.">A comparative study into the chemical constitution of cutins and suberins from Picea abies (L.) Karst., Quercus robur L., and Fagus sylvatica L.</a> Planta. 185 (2), 233-245 (1991).</li><li>Riederer, M., Sch&#246;nherr, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+gas+chromatographic+analysis+of+methyl+esters+of+hydroxy+fatty+acids+derived+from+plant+cutin.">Quantitative gas chromatographic analysis of methyl esters of hydroxy fatty acids derived from plant cutin.</a> J. Chromatogr. 360, 151-161 (1986).</li><li>Moire, L., Schmutz, A., Buchala, A., Yan, B., Stark, R., Ryser, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycerol+Is+a+Suberin+Monomer.+New+Experimental+Evidence+for+an+Old+Hypothesis.">Glycerol Is a Suberin Monomer. New Experimental Evidence for an Old Hypothesis.</a> Plant Physiol. 119 (3), 1137-1146 (1999).</li><li>Gra&#231;a, J., Schreiber, L., Rodrigues, J., Pereira, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycerol+and+glyceryl+esters+of+omega-hydroxyacids+in+cutins.">Glycerol and glyceryl esters of omega-hydroxyacids in cutins.</a> Phytochemistry. 61 (2), 205-215 (2002).</li><li>Eglinton, G., Hunneman, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas+chromatographic-mass+spectrometric+studies+of+long+chain+hydroxy+acids-I:+The+constituent+cutin+acids+of+apple+cuticle.">Gas chromatographic-mass spectrometric studies of long chain hydroxy acids-I: The constituent cutin acids of apple cuticle.</a> Phytochemistry. 7 (2), 313-322 (1968).</li><li>Espelie, K. E., K&#246;ller, W., Kolattukudy, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=9,16-dihydroxy-10-oxo-hexadecanoic+acid,+a+novel+component+in+citrus+cutin.">9,16-dihydroxy-10-oxo-hexadecanoic acid, a novel component in citrus cutin.</a> Chem. Phys. Lipids. 32 (1), 13-26 (1983).</li><li>Holloway, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracuticular+lipids+of+spinach+leaves.">Intracuticular lipids of spinach leaves.</a> Phytochemistry. 13 (10), 2201-2207 (1974).</li><li>Holloway, P. J., Deas, A. H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epoxyoctadecanoic+acids+in+plant+cutins+and+suberins.">Epoxyoctadecanoic acids in plant cutins and suberins.</a> Phytochemistry. 12 (7), 1721-1735 (1973).</li><li>Holloway, P. J., Cutler, D. F., Alvin, K. L., Price, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The chemical constitution of plant cutins. , 45-85 (1982).</li><li>Croteau, R., Fagerson, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+constituent+cutin+acids+of+cranberry+cuticle.">The constituent cutin acids of cranberry cuticle.</a> Phytochemistry. 11 (1), 353-363 (1972).</li></ol>

No conflicts of interest declared.

Unlike other biopolymers such as DNA and proteins, plant lipid polyesters are not made from a template. Instead, their compositions depend on the specificity of the enzymes present in the tissues that make these extracellular polymers. As such, chemical analyses of the constituent components are critical to understand lipid polyester composition.
Chemical methods to cleave ester bonds include saponification, hydrogenolysis, acid-catalyzed transmethylation, and base-catalyzed transmethylation2. Each of them has advantages and disadvantages. Saponification produces free fatty hydroxy acids that can undergo secondary reactions. Hydrogenolysis with lithium aluminum hydride (LiAlH4)16 has been used for cutin analysis7. Hydrogenolysis reduces functionalized carbons to alcohols and the original structures need to be inferred by deuteriolysis with lithium aluminum deuteride (LiAlD4). The disadvantage of this approach is the requirement of high resolution GC/MS to compare the degree of deuteriation of the fatty polyols obtained to make assignments of their structures. Acid-catalyzed transesterification with methanolic boron trifluoride (BF3) has been frequently used in cutin and suberin depolymerizations8,17,18, but the reagent has a limited shelf life and may introduce artefacts due to side reactions15. Methanolic sulphuric acid also yields methyl esters of the monomers but with larger proportions of 2-hydroxy fatty acids, which presumably are not true lipid polyester components, compared to other methods10.
The NaOMe-catalyzed transesterification method described in this protocol produces fatty acid methyl esters that are derivatized by silylation of hydroxyl groups, providing characteristic mass spectra for identification, or by acetylation to provide more stable derivatives of hydroxyl groups for quantification. One drawback of this technique is that hydrolysis competes with transesterification when water is present in the reaction. Water reacts with NaOMe (the catalyst) and produces NaOH, which in turn hydrolyses fatty acid methyl esters to yield free acids (Figure 2D). This is an undesirable side reaction because two peaks will be present for each fatty acid: a methyl ester and a TMSi ester derivative, thus complicating the analysis. Using anhydrous reagents and adding methyl acetate as a co-solvent to compete with saponification are thus crucial steps to prevent hydrolysis (Figure 2D).
Cutin and suberin contain between 1 and 26% glycerol4. However, this monomer will not be detected by the experimental conditions described in this protocol. Glycerol is highly hydrophilic and, unlike the fatty acid methyl ester monomers, will be eliminated during the aqueous solvent-washing steps. This limitation also applies to other cutin depolymerization methods, but glycerol can be determined in the aqueous layer obtained after transesterification using an enzymatic method. Alternatively, it can be quantified using milder conditions (e.g., 0.05 M NaOMe) without further water extraction to detect all monomers, including glycerol19,20.Although useful for the purpose of glycerol quantification, mild conditions usually give incomplete depolymerization of cutin and suberin.
If a GC coupled to a flame ionization detector (FID) is available, all replicates can be analyzed in this instrument for quantitative purposes, after peaks of a representative sample have been identified by GC/MS. Alternatively, monomers in the GC/FID traces can be identified if their retention indices are known. The flame ionization detector has especially high sensitivity and a broad range of proportionality, which is critical for quantification of major and minor sample components in single runs. In addition, it is robust and easy to maintain and operate15.
The described protocol allows for the reliable and reproducible isolation, identification, and quantification of plant lipid polyester monomers, allowing the chemical characterization of mutants that differ in the composition of one or more lipid polyester monomers. The procedure is scalable, it can be easily adapted to process both small and bulk quantities of various plant materials, including roots, seeds, leaves, stems and flowers. Mass spectral data of lipid polyester monomers from many species have been publishede.g.,21-26 and constitute valuable resources to identify unknown monomers when adapting this protocol to other tissues and/or species. This method is applicable to investigations of the biosynthesis, regulation, and distribution of lipid polyesters in higher plants.

Vascular plants rely on extracellular layers that function as waterproof barriers between plant tissues and the external environment. These lipophilic cell wall-associated structures restrict pathogenic infection and regulate the passive transport of gases, water, and dissolved substances in and out of plant tissues1. Such barriers are the plant cuticle, a synapomorphic structure unique to plants2, and different suberin-containing diffusion barriers. The cuticle is an oleophilic layer synthesized by epidermal cells and bound to them via a pectinaceous layer on the extracellular side of the cell wall3-5. It encases the primary aerial organs of higher plants, functioning as a vital interface between plant tissues and the environment.
Cutin, the structural matrix of the cuticle, and suberin are two insoluble glycerolipid polyesters associated with solvent-extractable waxes2,4. These polymeric lipids are composed of saturated and unsaturated fatty acid derivatives and are both structurally and functionally similar. However, they are distinguishable by characteristic differences in chemical composition and deposition sites.
Suberin is an aliphatic polyester located inside the cell walls of certain external and internal tissues forming a secondary wall. Suberized tissues include periderms of roots, tubers and tree bark, root endodermis, seed coat layers, and healed wounds2. Unlike cutin, the suberin polyester typically contains alcohols, saturated and mono-unsaturated dicarboxylic acids, and a large proportion of very-long-chain monomers (C&#8805;20).
Cutin is the most abundant lipid polyester in vascular plants6, and is composed of glycerol and C16-C18 interesterified fatty acid derivatives, such as hydroxy and hydroxy-epoxy substituted fatty acids4. While the composition of cutin polymers varies across tracheophyte species, the most predominant primary monomers are 10, 16-dihydroxy 16:0, 18-hydroxy-9,10-epoxy&#160;18:0, and 9,10,18-trihydroxy 18:0 fatty acids. Interestingly, Arabidopsis leaf and stem cutin is mainly composed of 18:2 dicarboxylic acid 7,8.
Plant cuticles also present a considerable variability in thickness, ranging from a few nanometers to several micrometers9. Since cuticle isolation is a laborious and time-consuming step, particularly for very thin leaf cuticles such as those of Arabidopsis thaliana8, methods that bypass cuticle isolation have been developed and validated7,8. Here, we describe a detailed protocol to study the monomer composition of cutin in Arabidopsis thaliana leaves by sodium methoxide (NaOMe)-catalyzed depolymerization and subsequent gas chromatography/mass spectrometry (GC/MS) analysis. This protocol offers a robust method for assaying the composition of plant lipid polyesters in whole delipidated tissues, and has been adapted from previously reported protocols7,10,11. Whole tissue samples are first homogenized and exhaustively delipidated, removing solvent-extractable lipids including cuticular and epicuticular waxes, membrane lipids, and triacylglycerols. Cell wall-enriched residues are then depolymerized into their constituent methyl ester monomers by sodium methoxide-catalyzed methanolysis. Fatty acid methyl esters are extracted upon acidification, and derivatized to obtain their corresponding trimethylsilyl or acetyl derivatives. Derivatized residues are highly volatile, and can be eluted from a gas chromatography column at a reasonable temperature without altering their structural conformation during GC/MS analysis.

<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.

The protocol described in this manuscript is set up to determine lipid polyester monomers (i.e., cutin or suberin), minimizing the contributions of non-cutin lipids10. Figure 1 presents an overview of the assay, which altogether takes between 8 &#8211; 10 days (from initial tissue harvesting to obtaining GC data), depending on how long samples are allowed to dry.
The selected base-catalyzed methanolysis (Figure 2) method to depolymerize polyesters was previously validated for Arabidopsis seeds, which contain both cutin and suberin. Tissues are first homogenized and exhaustively delipidated to remove solvent-extractable lipids. The residue yield after extraction, as percentage of initial fresh weight, is usually 6% for A. thaliana Col-0 leaves. Cell wall-enriched residues are dried in a vacuum desiccator and then depolymerized into their constituent methyl ester monomers by base-catalyzed transmethylation. Two-hour incubation was chosen as the critical time required for proper depolymerization and recovery of lipid polyester components. Longer incubation times resulted in increase in 2-hydroxy acids; these potentially derive from membrane sphingolipids10.
A typical chromatogram if Arabidopsis wild type leaf cutin is shown in Figure 3, for O-TMSi ether derivatives (Figure 3A) and O-acetyl derivatives (Figure 3B). Each peak was identified by comparison to mass spectra from the literature7,8 and a public database12.Our video protocol shows how to prepare TMSi derivatives, but samples can alternatively be acetylated to derivatize hydroxyl groups. Silylated derivatives are good for identification purposes because they give diagnostic mass spectra. However, acetylated derivatives are more stable and a good alternative to silylation once monomers have been identified 10. To help implement this protocol in laboratories that only have GC coupled to flame ionization detector (FID), GC/FID traces corresponding to acetylated derivatives of WT leaf cutin monomers and to a homolog series of fatty acid methyl ester standards are also shown (Supplemental Figure 4).
This method is qualitative and detects quantitative differences between samples, hence its value for mutant analysis. The amounts of individual monomers are determined using the internal standard method of quantification, enabling comparisons of monomer abundance between samples. It should be clarified, however, that the peak size (total ion counts) may not reflect the molar ratios of the monomers in the polyester. We are including editable tables of monomers to calculate monomer amounts in Arabidopsis leaf cutin as fatty acid methyl esters and TMSi derivatives (Supplemental File 1), or acetyl derivatives (Supplemental File 2) of alcohols. These tables may need to be adapted if samples are extracted from different organs or plant species.
As an example, we have analyzed Arabidopsis thaliana Columbia (Col-0) wild type leaves and two previously characterized null-mutant alleles of the CYP86A2/ATT1 gene, att1-1 (m-1) and att1-2 (m-2)13,14. Cytochrome P450 monooxygenases of the CYP86A subfamily encode putative &#969;-oxydases and participate in suberin and cutin monomer biosynthesis. Our results (Figure 4) demonstrate significant reductions in the loads of three major lipid monomers in the mutant leaves compared to WT leaves. Consistent with the enzyme&#8217;s predicted function, 16:0, 18:2, and 18:1 dicarboxylates were specifically affected in att1 mutants.
<img alt="Figure 1" src="/files/ftp_upload/53386/53386fig1.jpg" /> 
Figure 1. Overview of lipid polyester analysis. <a href="https://www.jove.com/files/ftp_upload/53386/53386fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53386/53386fig2.jpg" /> 
Figure 2. Mechanism of the NaOMe-catalyzed transmethylation reaction. The nucleophilic methoxide anions attack the cabonyl carbon of lipid polyesters to form an unstable tetrahedral intermediate (A), which readily dissociates into fatty acid methyl esters and alkoxide anions (B). These alkoxides are conjugate bases, and react with methanol, regenerating the catalytically active methoxide anions, thereby sustaining additional depolymerization reactions (C). If water is present in the system, it will react with sodium methoxide to form sodium hydroxide, a strong base that irreversibly hydrolyses esters to produce undesirable free fatty acids. Methyl acetate is added as a co-solvent15 to remove small amounts of sodium hydroxide within the system (D). <a href="https://www.jove.com/files/ftp_upload/53386/53386fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53386/53386fig3.jpg" /> 
Figure 3. Representative total ion chromatogram of wild-type Arabidopsis thaliana leaf cutin monomers. (A) O-trimethylsilyl (TMSi) ether and (B) acetate hydroxyl derivatives. <a href="https://www.jove.com/files/ftp_upload/53386/53386fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53386/53386fig4.jpg" /> 
Figure 4. Cutin monomer composition of Arabidopsis thaliana WT and two null mutant alleles of the CYP86A2 gene (mutant-1=att1-1; mutant-2=att1-2). Error bars represent standard deviation of the mean (n = 4). Adapted from 13, with permission of &#169; Blackwell Publishing (2007). <a href="https://www.jove.com/files/ftp_upload/53386/53386fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 1" src="/files/ftp_upload/53386/53386supfig1.jpg" /> 
Supplemental Figure 1. Peak integration results table from the GC/MS software. Peaks corresponding to identified monomers and internal standards are identified by their retention time (column C) and area values are tabulated in column D. <a href="https://www.jove.com/files/ftp_upload/53386/Supplemental_Figure_1.tif" target="_blank">Please click here to download this file.</a>
<img alt="Figure 2" src="/files/ftp_upload/53386/53386supfig2.jpg" /> 
Supplemental File&#160;2. Table of Arabidopsis cutin monomers (trimethylsilyl ether derivatives of hydroxy fatty acid methyl esters). <a href="https://www.jove.com/files/ftp_upload/53386/Supplemental_File_2_Cutin_template_TMSi.xls" target="_blank">Please click here to download this file.</a>
<img alt="Figure 3" src="/files/ftp_upload/53386/53386supfig3.jpg" /> 
Supplemental File 3. Table of Arabidopsis cutin monomers (O-acetyl derivatives of hydroxy fatty acid methyl esters). <a href="https://www.jove.com/files/ftp_upload/53386/Supplemental_File_3_Cutin_template_Acetylated_derivatives.xlsx" target="_blank">Please click here to download this file.</a>
<img alt="Figure 4" src="/files/ftp_upload/53386/53386supfig4.jpg" /> 
Supplemental Figure 4. GC/FID traces of (A-B) fatty acid methyl ester (FAME) retention index standards (peaks are labeled with each saturated FAME chain length); and (C) acetylated A. thaliana WT leaf cutin monomers. Numbers on peak correspond to: 16:0 FAME (1), ferulate (2), 18:3 FAME (3), 18:1/18:2 FAMEs (4), 18:0 FAME (5), sinapate (6), 16:0 DCA (7), 16-OH 16:0 FAME (8), 18:2 DCA (9), 18:1 DCA (10) 18:0 DCA (11), 18-OH 18:2 FAME (12), 18-OH 18:1 FAME (13), 20:0 FAME (14), 10,16-diOH 16:0 FAME (15), 24:0 FAME (16). DCA: dicarboxylic acid dimethyl ester; FAME: fatty acid methyl ester; IS1: internal standard 1, 17:0 FAME; IS2: internal standard 2, 15-OH 15:0 FAME. <a href="https://www.jove.com/files/ftp_upload/53386/Supplemental_Figure_4.tif" target="_blank">Please click here to download this 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.

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

16.6K 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.

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 ...

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.

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.

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.

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.

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.

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.

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.

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.

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.

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