Identification of transcription factors regulating plant secondary metabolism through the integration of functional genomics and metabolomics

Plants produce a very broad array of metabolites, which are not essential for growth of plant cells, but which are used by plants to provide protection against stress and pathogens, to attact pollinators and dispersal agents and as signals for development. These are often referred to as 'secondary metabolites' but are known more generally as plant 'natural products'. Natural products have recently become recognised as important components of the diet, offering protection against cardiovascular diseases, certain cancers and age-related degenerative diseases. They are also important components of beauty products and natural remedies for diseases. Plants often accumulate their natural products to relatively low levels, so there is a lot of interest in breeding or engineering plants that produce higher levels. It has been shown that the most effective way to increase the accumulation of secondary metabolites is to increase the activity of genes that regulate the activity of the biosynthetic pathways that make different natural products. Regulatory genes of this type encode proteins called transcription factors. The biggest bottleneck in using this strategy to develop plants that accumulate significantly higher levels of important natural products is that not many transcription factors regulating secondary metabolism have yet been identified at the molecular level. This proposal aims to identify transcription factors from the model plant, Arabidopsis thaliana, that control different branches of secondary metabolism. This will be achieved by studying a selected group of genes that probably encode transcription factors (identified from preliminary evidence as likely regulators). The activity of each of 38 such genes will be increased and the effects on metabolism will be measured by physico-chemical techniques that can identify major differences in the levels of small organic molecules (metabolites) within plant extracts. It is likely that several of the selected genes encode transcription factors that have the same regulatory activity. These genes will be identified because increasing their activity will result in exactly the same changes in metabolites in plants. The aim will be to identify all the proteins that have the same regulatory activities and then to choose one example from each distinct activity group to establish the effects of loss of that activity on metabolite levels. This will be done using sensitive techniques of separation coupled with quantitative identification by mass spectrometry. The combination of metabolic fingerprinting and metabolite profiling should allow us to identify new genes encoding transcription factors controlling secondary metabolism that can be used as tools for engineering natural product accumulation. This project will also make an important contribution to method development for broad range identification and comparison of metabolite profiles in plants.

The aim of this proposal is to identify new genes encoding transcription factors regulating secondary metabolism in plants. Because of the exceptional resources associated with the model plant, Arabidopsis thaliana, this work will focus on identification of the function of Arabidopsis transcription factors. While Arabidopsis can not provide the genes that regulate the most important branches of plant secondary metabolism (from a commercial or therapeutic point of view) it can provide the insight, through identification of genes regulating related pathways, ultimately to identify such regulators. Transcription factors offer exceptionally powerful tools to manipulate plant metabolism, either through genetic engineering or through marker (genes encoding transcription factors) assisted breeding. The identification of new regulatory activities will provide key tools for the improvement of production of important secondary metabolites in plants, and lead to the identification of new regulatory activities in species associated with the production of important specialised metabolites. The key anticipated deliverables of this project will be: 1. The identification of new transcriptional regulators of secondary metabolism in Arabidopsis. 2. Characterisation of the metabolic consequences of over- expressing specific transcription factors. This will provide the tools and engineering strategies for improving natural product accumulation by genetic engineering or marker-assisted breeding. 3. New methods for relatively holistic profiling of plant metabolism using NMR. 4. New methods for metabolite identification in LC/MS and NMR (and possibly GC/MS) through the concerted use of LC/SPE/NMR/MS. New reference spectra will be produced for mixture analysis, method calibration and standardisation. The prime beneficiaries will be members of the research community who will benefit from the method development and reference spectra that will be produced during this project. Other researchers will benefit from the identification of key genes controlling secondary metabolic pathways. If correct, the hypothesis that some R2R3MYB transcription factors regulate plant hormone levels will benefit those working on comprehending plant development. Ultimately this work should allow new strategies for engineering plant secondary metabolism, new tools for such engineering strategies and new insight for the future identification of regulators of accumulation of plant natural products.