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A new F131V mutation in Chlamydomonas phytoene desaturase locates a cluster of norflurazon resistance mutations near the FAD-binding site in 3D protein models.

Suarez JV, Banks S, Thomas PG, Day A - PLoS ONE (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom.

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.

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The influence of increasing norflurazon concentration on growth of C. reinhardtii.Cultures were inoculated to a density of 1×105 cells/ml on day 0 and optical density at 750 nm used to estimate cell density. (A) WT progenitor PDS1 strain. (B) NFR1 mutant pds1-nfr1d strain. The mean values of three experiments were plotted. Standard error bars are shown.
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pone-0099894-g001: The influence of increasing norflurazon concentration on growth of C. reinhardtii.Cultures were inoculated to a density of 1×105 cells/ml on day 0 and optical density at 750 nm used to estimate cell density. (A) WT progenitor PDS1 strain. (B) NFR1 mutant pds1-nfr1d strain. The mean values of three experiments were plotted. Standard error bars are shown.

Mentions: After exposure of C. reinhardtii nic7 arg7 y1 mt+ cells to high energy UV light (254 nm), colonies were selected by their ability to grow on TAP medium containing 33 µM norflurazon. Norflurazon resistant mutant strain 1 (NFR1) was chosen for further analysis because it grew much more strongly under 33 µM herbicide selection. The parent nic7 arg7 y1 mt+ strain giving rise to NFR1 was sensitive to norflurazon and henceforth will be referred to as wild type (WT). The lowest concentration of norflurazon tested (1 µM) reduced WT cell densities by about 70% after four days of growth in liquid TAP medium (Fig. 1A). Growth of the WT strain was prevented by 5 µM norflurazon. The effective concentration of norflurazon inhibiting WT growth by 50% (Ec50) after a four day period was 0.7±0.1 µM. In contrast, strain NFR1 grew to similar densities in the presence or absence of 5 µM norflurazon after four days growth (Fig. 1B). The NFR1 culture densities were highest at four days when exposed to herbicide and then gradually declined. The Ec50 for the NFR1 strain was calculated to be 108 µM norflurazon at four days, which is over 150-fold higher than the WT Ec50. NFR1 grew in media containing 150 µM norflurazon, the highest concentration tested, and after four days reached cell densities that were about 30% of those reached in the absence of herbicide (Fig. 1B). Coloured carotenoid content was measured by absorbance at 470 nm. The coloured carotenoid content of WT cells was reduced by 79% and 99% following exposure to 2 µM and 10 µM norflurazon, respectively (Fig. 2A). By comparison 2 µM and 10 µM norflurazon reduced coloured carotenoids in NFR1 cells by 13% and 44%, respectively (Fig. 2B).


A new F131V mutation in Chlamydomonas phytoene desaturase locates a cluster of norflurazon resistance mutations near the FAD-binding site in 3D protein models.

Suarez JV, Banks S, Thomas PG, Day A - PLoS ONE (2014)

The influence of increasing norflurazon concentration on growth of C. reinhardtii.Cultures were inoculated to a density of 1×105 cells/ml on day 0 and optical density at 750 nm used to estimate cell density. (A) WT progenitor PDS1 strain. (B) NFR1 mutant pds1-nfr1d strain. The mean values of three experiments were plotted. Standard error bars are shown.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4061028&req=5

pone-0099894-g001: The influence of increasing norflurazon concentration on growth of C. reinhardtii.Cultures were inoculated to a density of 1×105 cells/ml on day 0 and optical density at 750 nm used to estimate cell density. (A) WT progenitor PDS1 strain. (B) NFR1 mutant pds1-nfr1d strain. The mean values of three experiments were plotted. Standard error bars are shown.
Mentions: After exposure of C. reinhardtii nic7 arg7 y1 mt+ cells to high energy UV light (254 nm), colonies were selected by their ability to grow on TAP medium containing 33 µM norflurazon. Norflurazon resistant mutant strain 1 (NFR1) was chosen for further analysis because it grew much more strongly under 33 µM herbicide selection. The parent nic7 arg7 y1 mt+ strain giving rise to NFR1 was sensitive to norflurazon and henceforth will be referred to as wild type (WT). The lowest concentration of norflurazon tested (1 µM) reduced WT cell densities by about 70% after four days of growth in liquid TAP medium (Fig. 1A). Growth of the WT strain was prevented by 5 µM norflurazon. The effective concentration of norflurazon inhibiting WT growth by 50% (Ec50) after a four day period was 0.7±0.1 µM. In contrast, strain NFR1 grew to similar densities in the presence or absence of 5 µM norflurazon after four days growth (Fig. 1B). The NFR1 culture densities were highest at four days when exposed to herbicide and then gradually declined. The Ec50 for the NFR1 strain was calculated to be 108 µM norflurazon at four days, which is over 150-fold higher than the WT Ec50. NFR1 grew in media containing 150 µM norflurazon, the highest concentration tested, and after four days reached cell densities that were about 30% of those reached in the absence of herbicide (Fig. 1B). Coloured carotenoid content was measured by absorbance at 470 nm. The coloured carotenoid content of WT cells was reduced by 79% and 99% following exposure to 2 µM and 10 µM norflurazon, respectively (Fig. 2A). By comparison 2 µM and 10 µM norflurazon reduced coloured carotenoids in NFR1 cells by 13% and 44%, respectively (Fig. 2B).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom.

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.

Show MeSH
Related in: MedlinePlus