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Microbiology JoVE (Open Access) Citable · DOI

Isolation and Biophysical Study of Fruit Cuticles

DOI: 10.3791/3529-v
What you'll learn
  • ✓ Isolate intact fruit cuticles using enzymatic digestion and exhaustive dewaxing
  • ✓ Characterize cuticular polymer composition using solid-state NMR spectroscopy
  • ✓ Distinguish epicuticular and intracuticular wax layers by selective extraction
  • ✓ Map nanoscale surface topography and mechanical properties using atomic force microscopy
Protocol

Biopharma Insights Aerial plant organs are protected by the cuticle, a supramolecular biopolyester-wax assembly. We present protocols to monitor selective removal of epi- and intracuticular waxes from tomato fruit cuticles on molecular and micro scales by solid-state NMR and atomic force microscopy, respectively, and to assess the cross-linking capacity of engineered cuticular biopolyesters.

Difficulty
advanced
Total time
~3–5 days (includes enzymatic digestion, Soxhlet extraction, NMR analysis, AFM imaging)

Steps

1
Enzymatically isolate tomato fruit cuticles

Digest tomato fruit tissue with enzymatic solution to selectively remove cell wall material and obtain intact cuticle membranes.

▶ 02:50
2
Perform exhaustive dewaxing via Soxhlet extraction

Remove all surface and internal waxes from isolated cuticles using organic solvent in a Soxhlet apparatus to obtain pure cutin polymer.

▶ 03:44
3
Characterize cutin structure by solid-state NMR

Analyze molecular composition and cross-linking of dewaxed cutin using cross-polarization magic-angle spinning (CPMAS) solid-state NMR spectroscopy.

▶ 05:12
4
Selectively isolate epicuticular and intracuticular waxes

Use sequential solvent extraction protocols to separately recover surface waxes and internal wax fractions from intact cuticles.

▶ 08:08
5
Map cuticle surface topography with atomic force microscopy

Probe nanoscale surface morphology, roughness, and mechanical properties of selectively dewaxed cuticles using AFM in contact and force spectroscopy modes.

▶ 09:32
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