|Howell, J Lynn|
Submitted to: Plant Biotechnology Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/27/2005
Publication Date: 3/1/2006
Citation: Arias, R.S., Dayan, F.E., Michel, A., 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: Hydrilla is an invasive aquatic weed that is a problem in many lakes in the southern United States. While this weed is normally controlled easily with the herbicide fluridone, new populations with increased herbicide resistance have emerged. We have discovered that the gene encoding for the enzyme targeted by the herbicide has mutated and the resulting mutated enzyme is much more resistant to the herbicide. This study shows that of all 20 possible amino acid mutations, the one with Threonine provided the greatest herbicide resistance. Therefore, the genes encoding for the herbicide resistant enzyme were put in Arabidopsis plants. The transformed plants became more herbicide resistant. This new technology can be useful to develop new biotechnological tools such as selectable markers and may even lead to a new class of herbicide resistant crops.
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 Arg304 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 Thr, Cys, Ala and Gln mutations imparted the highest resistance to fluridone. There was an inverse correlation between the residue volume of the amino acid replacing Arg304 in PDS and the increase in resistance to fluridone. Threonine, the three natural mutations (Cys, Ser, 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 the in vitro assays. The Thr304 hydrilla pds mutant proved to be an excellent marker for selection of transgenic plants. Seedlings harbouring Thr304 pds had maximum resistance to sensitivity ratio (R/S) of 57 and 14 times higher than the wild-type for treatments with norflurazon and fluridone, respectively. These plants exhibited normal growth and development even after long-term exposure to herbicide. Since Thr304 pds is from plant origin, it could become more acceptable for its use in genetically modified food than other selectable markers.