Application of GC-(TOF)MS based metabolite profiling techniques for compositional analysis of leaf cuticular waxes from potato

Tom Shepherd, C. Cognat, D. W. Griffiths, G. Dobson, H. V. Davies and D. Stewart

SCRI, Invergowrie, Dundee DD2 5DA, UK

A thin microcrystalline wax layer covers the cuticles of plant leaves, providing protection against the adverse effects of drought, disease and UV radiation (1).  Such waxes consist mainly of complex mixtures of long-chain aliphatic compounds, including acids, alcohols, aldehydes, alkanes, ketones and esters, in addition to compounds such as sterols and other terpenes.

Biosynthesis of leaf wax components occurs within or close to epidermal cells and the sites of photosynthesis.  Individual steps in the biosynthesis, including molecular activation, bond formation and functional group transformations, are sensitive to genetic and environmental factors.  This can lead to major variation in the quantity and distribution of the different structural classes of wax component, and also in the distribution of individual homologues within each class.  Consequently, wax composition is very species-specific, and within a species may provide some indication of the general metabolic state.

Metabolite profiling is a useful technique for investigation of biodiversity in Solanum tuberosum and the related species S. phureja, for example, by investigation of the metabolite content of potato tubers (2, 3).  However, such investigations use relatively complex and time-consuming procedures for sample extraction and preparation prior to analysis.  In contrast, pure samples of waxes are readily isolated by dipping excised leaves in solvents such as dichloromethane. 

We have carried out a survey of the cuticular wax chemistry of a wide range of cultivated potato varieties and landraces.  In addition, we have included several potato lines and controls genetically modified for changes in metabolism having no known relationship with wax biosynthesis.  The object of the work was to establish the natural variability within the wax composition and to determine whether GM procedures produce any unintended effects in wax composition.

The main constituents of potato wax were straight chain (n-) and iso (i-) and anteiso (a-) branched-chain (br-) hydrocarbons.  Smaller quantities of n-, i- and a- isomers of fatty acids, alkan-1-ols and alkan-2-ols were also found along with several terpenoids.  The main inter- and intra-species differences observed were related to the distribution of n- and br- alkanes, and within the br- compounds to the distribution of i- and a- isomers.  In addition, variations in the homologue chain-length distribution were seen within individual compound classes. These findings are consistent with the presence of separate but parallel systems for the biosynthesis of the different n- and br- cuticular wax components, which may be subject to differential genetic control.  In addition there were some differences in the distribution of the terpene components within the wax. 

Shepherd, T. & Griffiths, D. W. (2006). The effects of stress on plant cuticular waxes.  New Phytologist 171: 469-499.

Dobson, G., Shepherd, T., Marshall, R., Verrall, S. R., Connor, S., Griffiths, D. W., McNicol, J. W., Stewart, D. & Davies, H. V. (2007).  Application of metabolite and flavour volatile profiling to studies of biodiversity in solanum species.  In Nikolau B. L & Wurtele, E.S. (eds) Concepts in Plant Metabolomics, Springer, Dordrecht, The Netherlands, 259-268.

Shepherd, T., Dobson, G., Verrall, S. R., Conner, S., Griffiths, D. W., McNicol, J. W.,  Davies, H. V. & Stewart D. (2007). Potato metabolomics by GC-MS: what are the limiting factors?  Metabolomics, 3, 475-488.