Our metabolite profiling falls into two activities: metabolite profiling for discovery of chemical defence mechanisms with controlled greenhouse experiments, and global metabolite profiling in the QTL study under the Conifer Comparative Genomics program. For further information, please contact Joerg Bohlmann.
Discovery of Chemical Defence Mechanisms
This project involves replicated time-course experiments of Sitka spruce response to weevil attack and the examination of differences between weevil-resistant and susceptible Sitka spruce provenances. We examine hundreds of structurally diverse terpenoid and phenolic secondary metabolites. To this end, we have optimized a protocol for the simultaneous qualitative and quantitative analysis of monoterpenes, sesquiterpenes and diterpene resin acids. The improved protocol is superior to previously-published methods.
We have also conducted a greenhouse experiment to catalogue phenolic metabolites and identify those changing in abundance following weevil feeding. Analysis of extracts using LC-MS revealed few changes in the abundance of metabolites within the first 4 days after weevil feeding but revealed substantial changes in metabolite abundance by 16 days. Following MS/MS analysis of 30 purified compounds identified as peaks in the LC-MS analysis, these 30 samples were shipped to the Max-Planck Institute for NMR analysis by Dr. Bernd Schneider. Structural identification using NMR was obtained for highly abundant compounds (e.g. piceid, astringin). A second set of extracts was shipped for direct HPLC-NMR analysis. This new approach provided a larger number of structural identification including identification of moderate and low abundance compounds. The following compounds were identified: astringin, piceid, p-coumaroyl glucoside, feruloyl glucoside, catechin, p-coumaric acid, dihydroquercetin glucoside (additional monomeric and dimeric compounds have been tentatively identified).
Comparative metabolite profiling of weevil-resistant (H898) and susceptible (QI903) Sitka spruce genotypes identified the monoterpene 3-carene as a possible chemical marker for resistance. The initial analysis was extended to 111 genotypes (and 4 trees per genotype), which contains resistant and susceptible Sitka spruce trees. The specific objectives of this activity are to:
1) Determine the relationship between levels of 3-carene and resistance rating over a range of Sitka spruce genotypes (i.e., to determine whether resistance rating can be predicted from levels of 3-carene), and
2) Identify other potential terpenoid compounds or groups of compounds that correlate with resistance rating over a range of Sitka spruce genotypes. Sampling was conducted in May 2006. Weevils emerge, select their hosts and oviposit in the spring and so the metabolite profiles of the rated trees obtained at this time of year will ensure that our information is applicable to a period of weevil host selection. Terpene extraction and identification will be conducted next.
Global Metabolite Profiling
Global metabolite profiling methods have been successfully used within the QTL study under the Comparative Genomics program. We evaluated progeny of 6 controlled crosses for 327 trees sampled in 2006 for metabolite profiling evaluation, and metabolite profiles of stem tissue being evaluated by GC-MS, HPLC and LC-MS. Based on retention time similarity and Pearson correlations between peak intensities, the m/z peaks originating from the same metabolite were grouped and employed for statistical analyses, and we found hundreds of distinct compounds, of which many were positively correlated with current weevil attack, and some negatively correlated. We have identified some of these compounds and by comparison of their mass spectra with available library data, these compounds include carbohydrates, phenylpropanoids and amino acids. Furthermore, close to one hundred compounds significantly increased in correlation to current and past weevil attack, while some compounds decreased significantly (p<0.05). Identification of these compounds is underway. Mapping of “mQTLs” from the results of this Global Metabolic Profiling is underway and a paper should be submitted by early 2008.
We are also profiling phenolics in these samples by evaluating the polar fraction of the liquid-liquid extracts of bark-phloem samples by reversed phase HPLC. The UV/Vis absorbance spectra were recorded between 210 and 450 nm by photodiode array detector, and the peaks integrated at 280nm. We detected many compounds, some of which were negatively correlated with weevil attack, while none of the peaks were positively correlated with current weevil attack (p<0.05). Some compounds were also positively correlated with current and past weevil attack; however, most of these peaks were extremely small and often not completely pure. This analysis will be performed again on new bark extracts via LC-MS. The peaks will be resolved via XCMS and identified via their mass spectrum and UV/Vis spectrum.