A Functional Genomics Approach to Wood DevelopmentHertzberg, Magnus
Wood is widely used for various purposes, e.g. for pulp and paper, construction material and heating. Thus it is surprising that the cellular and molecular regulation of wood formation remains poorly characterised. This study therefore addresses the process of wood formation in order to elucidate factors controlling this process at the molecular level. Two different approaches were taken, using novel technologies that have become available in recent years.
First, a new group of homeobox transcription factors were identified, denoted the PALE class due to a five amino acid insertion between helix 1 and helix 2 in the homeodomain. All identified members of this class in hybrid aspen have an expression pattern suggesting a role in wood formation. This group of transcription factors were also identified in Arabidopsis thaliana, the model plant for plant molecular biology. Function of the PALE-class of homeobox genes was investigated using, transgenic hybrid aspen and transgenic Arabidopsis plants, expressing different PALE class members in sense and anti-sense direction under the control of the CaMV 35S promotor. Transactivation properties of the PttHB 1 protein was analysed in yeast, and it was demonstrated that a region in PttHB 1, which show homology to the strong transactivation domain of VP 16, can function as an activator of transcription in yeast.
In the second approach, anEST sequencing project was initiated where 4809 EST’s, originating from the cambial region of hybrid aspen, were sequenced. These EST’s corresponded to 2988 genes, based on cluster analysis. 745 different proteins were identified and annotated (not counting iso-enzymes). Using these EST’s as a base, a cDNA microarray was constructed representing the 2988 genes. The microarray was used for detailed transcriptprofiling of the wood-differentiation process, initiating in the meristematic cambium cells and terminating in mature xylem cells. In order to enable this experiment, a high fidelity target amplification method was developed, which is based on a 3’-prime tagged, PCR amplification protocol. This method allows transcript profiling with cDNA microarrays using minute amounts of starting material (-O.lrng of plant tissue). In practical use, two-fold expression changes observed with this technology is significant with 99% confidence. Using this technology, a transcriptional roadmap to xylem formation was produced. This data set describes unique, hitherto, unknown expression patterns for thousands of genes during xylem formation. We also demonstrate the utility of this expression data as a platform to rapidly perform functional analysis of genes involved in xylem formation, using Arabidopsis thaliana mutant collections.
Keywordshomeobox genes; PALE genes; wood development; transcript profiling; target amplification; genomics; EST sequencing
Published inActa Universitatis Agriculturae Sueciae. Silvestria
2001, number: 173
Publisher: Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences
UKÄ Subject classification
URI (permanent link to this page)