Abstract
The green alga Chlamydomonas reinhardtii provides a tractable genetic model to study herbicide mode of action using forward genetics. The herbicide norflurazon inhibits phytoene desaturase, which is required for carotenoid synthesis. Locating amino acid substitutions in mutant phytoene desaturases conferring norflurazon resistance provides a genetic approach to map the herbicide binding site. We isolated a UV-induced mutant able to grow in very high concentrations of norflurazon (150 µM). The phytoene desaturase gene in the mutant strain contained the first resistance mutation to be localised to the dinucleotide-binding Rossmann-likedomain. A highly conserved phenylalanine amino acid at position 131 of the 564 amino acid precursor protein was changed to a valine in the mutant protein. F131, and two other amino acids whose substitution confers norflurazon resistance in homologous phytoene desaturase proteins, map to distant regions in the primary sequence of the C. reinhardtii protein (V472, L505) but in tertiary models these residues cluster together to a region close to the predicted FAD binding site. The mutant gene allowed direct 5 µM norflurazon based selection of transformants, which were tolerant to other bleaching herbicides including fluridone, flurtamone, and diflufenican but were more sensitive to beflubutamid than wild type cells. Norflurazon resistance and beflubutamid sensitivity allow either positive or negative selection against transformants expressing the mutant phytoene desaturase gene.
Highlights
Chlamydomonas reinhardtii provides an attractive model organism to dissect processes unique to photosynthetic eukaryotes [1]
Strain Norflurazon resistant mutant strain 1 (NFR1) grew to similar densities in the presence or absence of 5 mM norflurazon after four days growth (Fig. 1B)
The NFR1 culture densities were highest at four days when exposed to herbicide and gradually declined
Summary
Chlamydomonas reinhardtii provides an attractive model organism to dissect processes unique to photosynthetic eukaryotes [1]. Forward genetic screens for gain-of-function mutations that confer herbicide resistance provide a powerful approach to investigate herbicide mode of action [2]. These studies proceed rapidly using algal and plant models containing herbicide target sites, which are often absent in animals and fungi. Resistance resulting from a mutation in a target protein can identify an herbicide’s binding site, which is normally revealed by identifying and sequencing the corresponding mutant gene. Forward genetics provided a powerful tool to locate the binding sites of triazine herbicides in the D1 protein of photosystem II by sequencing mutant psbA genes encoding D1 from herbicide tolerant C. reinhardtii strains [5,6,7]. The locations of the amino acid substitutions in the D1 protein identified the herbicide target site which overlapped with the QB quinone binding site in photosystem II [8,9]
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