Home › Microbiology › Isolation and Compositional Analysis of Plant Cuticle Lipid Polyester Monomers
Microbiology JoVE (Open Access) Citable · DOI

Isolation and Compositional Analysis of Plant Cuticle Lipid Polyester Monomers

DOI: 10.3791/53386-v
What you'll learn
  • ✓ Isolate and depolymerize cutin from plant leaf samples
  • ✓ Prepare trimethylsilyl derivatives for GC-MS compositional analysis
  • ✓ Identify lipid polyester monomers in cuticle and suberin barriers
  • ✓ Apply method to characterize cutin/suberin biosynthesis mutants
Protocol

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.

Difficulty
advanced
Total time
~4–6 hours per sample (including delipidation overnight, methanolysis, derivatization, and GC-MS run)
Model organism
Arabidopsis thaliana (or other vascular plants)
Biosafety
BSL-1

Steps

1
Delipidize plant leaf tissue with organic solvents

Remove surface and intracellular lipids from whole leaves by sequential extraction using chloroform/methanol or similar organic solvents to isolate the cuticle and suberin-containing barriers for subsequent depolymerization.

▶ 01:24
2
Depolymerize cutin via methanolysis with sodium methoxide

Treat delipidated tissue with sodium methoxide in methanol to cleave ester bonds in cutin and suberin polyesters, releasing monomeric lipid components for analysis.

▶ 03:52
3
Prepare trimethylsilyl derivatives for GC-MS

Convert hydroxyl and carboxyl groups in depolymerized monomers to trimethylsilyl ethers to enhance volatility and detectability by gas chromatography–mass spectrometry.

▶ 07:07
4
Perform GC-MS analysis of lipid monomer composition

Inject trimethylsilyl-derivatized samples into a gas chromatograph coupled to a mass spectrometer to identify and quantify individual cutin and suberin monomer species.

▶ 07:54
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