Tree Physiology Advance Access published online on April 14, 2009
Tree Physiology, doi:10.1093/treephys/tpp023
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SABATH methyltransferases from white spruce (Picea glauca): gene cloning, functional characterization and structural analysis
1 Department of Plant Sciences, University of Tennessee, Knoxville, TN 37996, USA
2 Arborea and Centre d’Étude de la Forêt, Universite Laval, Pavillon Charles-Eugene-Marchand, Québec, Québec G1V 0A6, Canada
3 Natural Resources Canada, Canadian Forest Service, 1055 Rue du P.E.P.S., Québec City, Québec G1V 4C7, Canada
4 Institut de Biologie Structurale, Commissariat à l’Energie Atomique, Centre National de la Recherche Scientifique, Université Joseph Fourier, 38027 Grenoble cedex 1, France
5 Crop Diseases, Pests and Genetics, USDA-ARS, 9611 S. Riverbend Avenue, Parlier, CA 93648, USA
6 Corresponding author (fengc{at}utk.edu)
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Abstract |
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Known members of the plant SABATH family of methyltransferases have important biological functions by methylating hormones, signalling molecules and other metabolites. While all previously characterized SABATH genes were isolated from angiosperms, in this article, we report on the isolation and functional characterization of SABATH genes from white spruce (Picea glauca [Moench] Voss), a gymnosperm. Through EST database search, three genes that encode proteins significantly homologous to known SABATH proteins were identified from white spruce. They were named PgSABATH1, PgSABATH2 and PgSABATH3, respectively. Full length cDNAs of these three genes were cloned and expressed in Escherichia coli. The E. coli-expressed recombinant proteins were tested for methyltransferase activity with a large number of compounds. While no activity was detected for PgSABATH2 and PgSABATH3, PgSABATH1 displayed the highest level of catalytic activity with indole-3-acetic acid (IAA). PgSABATH1 was, therefore, renamed PgIAMT1. Under steady-state conditions, PgIAMT1 exhibited apparent Km values of 18.2 µM for IAA. Homology-based structural modelling of PgIAMT1 revealed that the active site of PgIAMT1 is highly similar to other characterized IAMTs from angiosperms. PgIAMT1 showed expression in multiple tissues, with the highest level of expression detected in embryonic tissues. During somatic embryo maturation, a significant reduction in PgIAMT1 transcript levels was observed when developing cotyledons become apparent which is indicative of mature embryos. The biological roles of white spruce SABATH genes, especially those of PgIAMT1, and the evolution of the SABATH family are discussed.
Keywords: gene expression, high-throughput enzyme assay, indole-3-acetic acid, somatic embryogenesis
Received October 30, 2008; Accepted March 13, 2009