Isolatie en analyse van de samenstelling van de Plant Nagelriem Lipid Polyester Monomeren

Vascular plants rely on extracellular layers that function as waterproof barriers between plant tissues and the external environment. These lipophilic cell, while associated structures, restrict pathogenic infection and regulate the passive transport of gases, water and dissolved substances in and out of plant tissues. One important barrier is the plant cuticle a complex structure unique to plants.

Cutin is an insoluble glycerol lipid polyester that constitutes the structural matrix of the cuticle and is associated with solvent extractable waxes. Here we present a reliable technique to assay the composition of lipid polyesters and hold de lipid plant tissues. In this protocol, whole tissue samples are harvested, homogenized, and exhaustively, dilapidated by a series of solvent extractions to remove solvent soluble lipids, including cuticular and epi particular waxes.

Triglycerol in membrane lipids. Samples are then de polymerized into their component lipid monomers by sodium meth oxide catalyzed methyl lysis. Simulation reagents are added to trans methylate hydroxyl groups into their corresponding trimethyl cell derivatives suitable for gas chromatographic analysis.

Derivative residues are then transferred to GC vials and loaded into A-G-C-M-S for analysis, generating a chromatogram, characterizing the monomeric composition of bio polyesters. Present in extracted samples. Here we harvest whole leaf samples from wild type and two mutants of mature arabidopsis ANA plants.

For extraction and analysis, add 0.5 grams of tissue to clean glass tubes that were pre rinsed with chloroform. Place 100 milliliters of isopropanol in a flask and add butylated hydroxy toluene to a 0.01%Concentration BHT is an antioxidant added to protect double bonds and fatty acids from oxidation during solvent extractions. Preheat the solvent in a water bath to approximately 85 degrees.

Add 25 milliliters of heated isopropanol per gram of plant tissue and incubate the samples in the heat LOC at 85 degrees for 15 minutes. Allow tubes to cool down to room temperature and with ear and eye protection. Finally, grind samples with a homogenizer to achieve a homogeneous dispersion seal tubes with screw caps and shake at 100 RPM for one to two centrifuge samples at 800 G for 10 minutes.

To separate the organic phase from the higher density materials compacted into the pellet, remove and discard the supernatant using a Pasteur or propent. Be careful to avoid disturbing the pellet. Add an equal volume of isopropanol at room temperature and shake samples overnight.

At 100 RPM, centrifuge the samples and discard the supernatant. I add 25 milliliters of a two to one solution of chloroform, methanol, pergram of tissue, and shake the samples overnight centrifuge and discard the organic phase. Then repeat this process using a one to two chloroform methanol solution.

Dry the pellet overnight and transfer tubes from the fume hood to a vacuum desiccate containing anhydrous calcium sulfate to dehydrate samples over a period of at least three days or until constant mass is achieved. Once thoroughly dried samples are ready for defloration by sodium meth oxide catalyzed metalysis. Add two solutions of internal standards to tubes containing dry tissues using a glass syringe, beginning with 25 microliters of methyl HETO decanoate, and 25 microliters of omega pentec and lactone.

To initiate deep polymerization, add 0.9 milliliters of methyl acetate to samples, followed by 1.5 milliliters of sodium meth oxide catalyst and 3.6 milliliters of methanol. Incubate samples at 60 degrees for two hours with periodic vortexing within the reaction system, lipid polyesters react with nucleophilic meth oxide anions to form unstable tetrahedral intermediates, which readily dissociate into fatty acid, methyl esters, and a oxide anions. These a oxides are conjugate bases and react with methanol regenerating the cataly active meth IDE anions, thereby sustaining additional deep polymerization reactions.

If water is present in the system, it will react with sodium meth oxide to form sodium hydroxide. A strong base that irreversibly converts polyesters to undesirable free fatty acids. Instead of fas to prevent hydrolysis methyl acetate is added to remove any sodium hydroxide within the system.

Allow tubes to cool down to room temperature. Following incubation, extract the monomers by adding 10 milliliters of methylene dichloride to each tube. Then add 1.5 milliliters of glacial acidic acid to acidify the samples.

Fill the tubes with 0.5 molar sodium chloride solution, vortex thoroughly and centrifuge for minutes. Under the fume hood. Carefully transfer the lower organic phase by pipet into clean pre rinsed, medium sized tubes.

Wash samples again with saline solution vortex and centrifuge for 10 minutes. Remove the upper aqueous phase, then add anhydrous sodium sulfate to samples as a drying agent to remove trace amounts of water remaining in solution vortex samples centrifuge, and transfer the solvent to clean pre rinsed small size tubes, load tubes into a nitrogen evaporator to evaporate the solvent and obtain dry residues of monomers. Using a glass syringe, add 100 microliters of purine and 100 microliters of B-S-T-F-A to convert hydroxyl groups into volatile trimethyl cell derivatives.

Incubate samples at 100 degrees for 10 minutes. Allow tubes to cool down and evaporate under nitrogen gas. Add 500 microliters of a one-to-one HEPTANE toluene solution to red dissolve residues, which are then briefly centrifuged.

Transfer derivate samples to GC vials containing 400 microliter inserts. Enclose the vials load samples into a gas chromatograph, which injects them into a capillary column for analysis, eluate are then detected by quadruple mass spectrometer. Raw data acquired by GCMS analysis is processed and presented as a chromatogram for each sample.

Individual monomers corresponding to different chromatographic peaks are identified by their specific mass spectra. In GC retention times, the amounts of individual monomers are determined using the internal standard method of quantification, enabling comparisons of monomer abundance and composition to be made between samples. Shown here are overlay chromatograms from representative analyses of monomers, released from a rabbit abscess leaf tissue corresponding to wild type and two mutant alleles of a gene encoding a P four 50 mono oxygenase enzyme demonstrating distinct monomeric profiles.

Our results demonstrate significant differences, the abundance of three major cutin, monomers, and wild type and mutant arabidopsis plants. This protocol is a robust method for assay the composition of plant lipid polyesters by the isolation identification and quantification of their constituent monomers. The procedure is scalable and can be readily adapted to process bulk quantities of various plant materials, including roots, seeds, leaves, and other whole tissues.

This technique is applicable to studies investigating the biosynthesis regulation and distribution of cutin and suberin in higher plants.