Activation Tagging in Poplar for Gene Function Discovery

Activation tagging is an approach in which a T-DNA fragment containing an engineered tetramer of the CaMV35S enhancer randomly integrates into the plant genome to activate the transcription of flanking genes. A total of 1164 activation tagged lines of hybrid Populus (P. tremula x P. alba) were grown for four years to monitor appearance of specific phenotypes. Close to 100 mutants were identified with a large proportion showing dwarfism while others showed early or late bud break, winter marcescent foliage and changes in tree or leaf architecture phenotype. In December 2010, all trees were cut and 727 stem sections were collected for wood chemistry analysis. A total of 18 lines were selected (from 400 analysed) based on significant variation of cell wall chemistry (e.g. lignin percentage, S/G ratio).

T-DNA insertions in the poplar genome were identified and genes located within the 15 kb region flanking the T-DNA insertion site were selected for further investigation. For each mutant, information was gathered on gene annotation in order to link observed phenotypes to specific candidate genes. Expression profiling was performed using RT-qPCR analysis to evaluate about 80 genes potential candidate genes in various tissues, including developing wood, leaves, and roots. For a select number of candidate genes we also compared expression profiles between wild-type plant and mutants, looking for up regulation in the mutants. Overall we have been able to identify candidate gene such as glucanase, hydrolase, F-box protein and HRGP, which are likely to be involved in the modified wood chemistry observed in specific mutants.We are continuing the characterization of these genes and their related proteins in order to improve our understanding of their roles in key physiological processes (eg. wood formation).

Funding support: This work is supported by the Genomics R & D Initiative of Canada and the Arborea project (www.arborea.ca funded by Genome Canada and Genome Quebec). Support from the United States Department of Energy (DOE) BioEnergy Science Center project is also acknowledged. The BioEnergy Science Center is a Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science.