The functional analysis component of Arborea aims to innovate in transcriptome analysis by applying transcript profiling within a reverse genetic experimental framework. We believe this approach will enable to fully exploit the breadth and depth of information which may be derived from genome-wide expression data. From the onset, we made transcriptome analysis a project focus, recognizing its potential for immediate application on a truly genomic scale. Transcript profiling methods have been applied to non model organisms but most often in descriptive or exploratory studies, which provide little insight into gene functions, regulatory networks, or biological processes. Our more systematic experimental approach targets candidate regulators predicted to control gene expression in processes linked to wood formation and defence response. Current thinking suggests that genetic variation in regulatory factors underlie many important adaptive traits and thus focus on functional analysis of transcription factors (TFs).

Phase 1 - Mis-expression of regulatory genes in transgenic trees

We established a functional genomics analysis pipeline that integrates gain-of-function/loss-of-function studies of TF genes with microarray transcript profiling. Mis-expression and manipulative physiology strategies are being used to assess the biological role of these regulatory genes, and gain insight into transcriptome modulation and regulation by these genes. This pipeline is highly integrated with other activities of the Arborea project.

In this project, mostly gain-of-function and a few loss-of-function studies were done for a total of 40 different conifer and hardwood genes. We have obtained stable transgenic spruce and poplar plants expressing candidate genes, all of which have been screened for transgene expression using QPCR. Transgenic trees have been grown in the greenhouse, with several displaying phenotypes. Detailed phenotypic characterization of transgenics and complementary studies of endogene expression are part of tailored graduate and postdoctoral research projects.

Phase 2 - High throughput functional analysis of transcription factors

Based on our established functional genomics pipeline we now propose to extend these analyses by implementing a HT assay approach to more efficiently screen a greater number of TFs. For this purpose, we will develop transient cellular transformation assays enabling HT testing of TF function based upon the ability of transcription factors to induce the expression of reporter genes driven by promoters germane to wood formation and seasonal growth. The TF functional assays will comprise a portfolio of approximately 20 promoters from a number of selected genes. Three complementary approaches will be used to assemble this portfolio that will cover a wide range of DNA binding domains hypothesized to be important to wood formation and seasonal growth, such as auxin-responsive or xylem-specific elements. We will first search for and isolate promoter and cis elements from genes with a well characterize function or that exhibit an expression profile relevant to growth or wood formation. We also plan to utilize promoters that have been characterized in other systems, including synthetic promoters. The proposed work will help to validate in conifers, the function of promoters from distantly related herbaceous angiosperm plants like Arabidopsis. In parallel we are currently identifying a broad collection of transcription factors from conifer EST resources using an array of bioinformatics tools such as TRANSFAC. Coding regions will be mobilized into the GATEWAY cloning vector (Invitrogen), enabling their rapid transfer to expression vectors. The choice of TFs and their priority in the analysis pipeline will be guided by results from Arborea as well as new results obtained in conjunction with association mapping objectives. Finally, transient HT will be based upon reporter gene activation following transient transformation of the promoter:reporter cell lines with a given transcription factor. This HT approach will enable testing of several hundred TF – reporter line combinations in a relatively short period of time. The use of fluorescent reporter genes and the development of cell lines harbouring several reporter constructs will ensure system efficiency and throughput. The specificity of positive TF - promoter interactions identified in transient assays will be verified with stable transformation of the same transgenic cells harbouring the reporter construct (see figure below).