Location: National Peanut Research LaboratoryTitle: Characterization of a higher plant herbicide-resistant phytoene desaturase and its use as a selectable marker
Submitted to: Plant Biotechnology Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/27/2005
Publication Date: 1/25/2006
Citation: Arias De Ares, R.S., Dayan, F.E., Albrecht, M., Howell, J., Scheffler, B.E. 2006. Characterization of a higher plant herbicide-resistant phytoene desaturase and its use as a selectable marker. Plant Biotechnology Journal. 4:263-273.
Interpretive Summary: The aquatic weed Hydrilla verticillata developed natural resistance to the herbicide Fluridone, the only one approved for use in water bodies in the United States. At USDA/ARS we determined that the cause of such resistance was several mutations on the phytoene-desaturase gene of Hydrilla. In the present work, we analyzed each of those mutations by genetically transforming other plant, Arabidopsis thaliana, and testing its herbicide resistance for several generations. We determined that 3 of the natural mutations conferred herbicide resistance to the transgenic plants, that the mutations conferred resistance to other phytoene-desaturase inhibitor herbicides (Fluridone, Norflurazon, Flurochloridone, Flurtamone), but resulted in higher susceptibility to other group of phytoene-desaturase inhibitor herbicides (Diflufenican, Picolinafen, Beflubutamid). We proposed in this work the possible use of the mutations in this gene as potential selectable markers for biotechnology.
Technical Abstract: Three natural somatic mutations at codon 304 of the phytoene desaturase gene (pds) of Hydrilla verticillata ( L. f. Royle) have been reported to provide resistance to the herbicide fluridone. We substituted the arginine 304 present in the wild-type H. verticillata phytoene desaturase (PDS) with all 19 other natural amino acids and tested PDS against fluridone. In in vitro assays, the threonine ( Thr), cysteine (Cys), alanine (Ala) and glutamine (Gln) mutations imparted the highest resistance to fluridone. Thr, the three natural mutations [Cys, serine (Ser), histidine (His)] and the wild-type PDS protein were tested in vitro against seven inhibitors of PDS representing several classes of herbicides. These mutations conferred cross-resistance to norflurazon and overall negative cross-resistance to beflubutamid, picolinafen and diflufenican. The T3 generation of transgenic Arabidopsis thaliana plants harbouring the four selected mutations and wild-type pds had similar patterns of cross-resistance to the herbicides as observed in the in vitro assays. The Thr304 Hydrilla pds mutant proved to be an excellent marker for the selection of transgenic plants. Seedlings harbouring Thr304 pds had a maximum resistance to sensitivity (R/ S) ratio of 57 and 14 times higher than that of the wild-type for treatments with norflurazon and fluridone, respectively. These plants exhibited normal growth and development, even after long-term exposure to herbicide. As Thr304 pds is of plant origin, it could become more acceptable than other selectable markers for use in genetically modified food.