In situ analysis of lateral triterpenoid distribution in plant cuticular waxes using Raman microspectroscopy and non-linear optical imaging
Creators: name: Yu, Marcia Mei Lin
Publisher: name: University of British Columbia
Year: 2008

description: The above-ground organs of plants are covered by a protective cuticle, an extracellular membrane performing important physiological and ecological functions, that consists of cuticular wax and the fatty acid-derived polymer cutin. Until the past decade, the cuticular wax was thought to be a homogenous mixture. Hence, previous interpretations relating the chemical composition and biological functions of the cuticular wax were based on the total wax composition, an average taken over the entire area and depth of the cuticle. However, recent selective sampling experiments showed a heterogeneity of the chemical composition between different wax layers. The finding of this heterogeneity imposes the need for a more accurate description of the cuticle in order to understand how the chemical composition determines the biological function. This thesis is aimed at mapping the lateral patterns of cuticular waxes on Prunus laurocerasus leaf surfaces with microscopic resolution to provide spatially resolved chemical information in the interest of describing the cuticle more accurately. Firstly, this thesis examines the post-acquisitional data processing and analysis techniques followed by the investigation of the potential of Raman microspectroscopy for the simultaneous detection of structurally similar triterpenoids in plant cuticles. Relative composition analysis was performed on artificial triterpenoid mixtures and the resulting calculated triterpenoid ratios were consistent with the expected values. Qualitative and quantitative analysis of the linear near infrared (NIR) Raman, coherent anti-Stokes Raman scattering (CARS), and third harmonic generation (THG) spectra of isolated adaxial and abaxial P. laurocerasus cuticles demonstrated the in situ detectability of the triterpenoids using this approach. Raman maps of the adaxial cuticle showed that the triterpenoids accumulate to relatively high concentrations over the periclinal regions of the pavement cells, while the very long chain aliphatic wax constituents are distributed fairly evenly across the entire adaxial cuticle. In the analysis of the abaxial cuticles, the triterpenoids were found to accumulate in greater amounts over the guard cells relative to the pavement cells. The very long chain aliphatic compounds accumulated in the cuticle above the anticlinal cell walls of the pavement cells, and were found at low concentration above the periclinals and the guard cells. The main research contributions include evaluating various data processing techniques as candidates for automated implementation and applying different imaging techniques to obtain chemical information about the lateral concentration gradient of the triterpenoid components in the cuticles, with high spatial resolution. This thesis also provides the first direct (i.e. in situ) evidence for lateral spatial heterogeneity of triterpenoids in the cuticle of a model species, P. laurocerasus. This work is expected to impel further structure-function investigation of the cuticular membranes of plants.