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1.
FIG. 7.

FIG. 7. From: Adaptation of Saccharomyces cerevisiae to the Herbicide 2,4-Dichlorophenoxyacetic Acid, Mediated by Msn2p- and Msn4p-Regulated Genes: Important Role of SPI1.

Time course variation in the accumulation of 14C-labeled 2,4-D in cells of the wild-type (•) and the Δspi1 mutant (○) strains, in the absence of glucose (− Glu) and its subsequent expulsion after a glucose pulse (+ Glu) (arrow). Cells were previously adapted to the herbicide and were harvested at mid-exponential phase (culture OD600 = 1 ± 0.1) of cultivation in minimal medium supplemented with 0.3 mM 2,4-D at pH 3.5. The corresponding growth curves are shown in Fig. 3. Accumulation values are representative of the several experiments.

T. Simões, et al. Appl Environ Microbiol. 2003 July;69(7):4019-4028.
2.
FIG. 3.

FIG. 3. From: Adaptation of Saccharomyces cerevisiae to the Herbicide 2,4-Dichlorophenoxyacetic Acid, Mediated by Msn2p- and Msn4p-Regulated Genes: Important Role of SPI1.

The SPI1 gene is required for S. cerevisiae adaptation and growth under 2,4-D stress as indicated by a comparison of susceptibilities by spot assays or growth curves (A) and cell viabilities (B) of wild-type (WT; •, ▪) and Δspi1 (○, □) strains in the absence (•, ○) or presence (▪, □) of 2,4-D (0.45 mM; pH 3.5). Values are representative of the many growth experiments carried out. Cell suspensions used to prepare the spots or to inoculate the liquid media were cultivated in the absence of herbicide until a standardized OD600 of 0.5 ± 0.05 was attained, in mid-exponential phase. The cell suspensions used to prepare the spots in lanes B were 1/4 dilutions of the cell suspensions (OD600 = 0.05 ± 0.005) in lanes A.

T. Simões, et al. Appl Environ Microbiol. 2003 July;69(7):4019-4028.
3.
FIG. 4.

FIG. 4. From: Adaptation of Saccharomyces cerevisiae to the Herbicide 2,4-Dichlorophenoxyacetic Acid, Mediated by Msn2p- and Msn4p-Regulated Genes: Important Role of SPI1.

Results from Northern blot hybridization experiments using SPI1 as probe and the actin-encoding gene, ACT1, as the internal control. Total RNA was extracted from cells of S. cerevisiae BY4741 (WT) harvested during cultivation under 2,4-D stress (0.3 mM) (A). Growth was monitored by measuring culture OD600. Relative SPI1 mRNA/ACT1 mRNA ratios were obtained by densitometry of the autoradiograms (B) and are average values from three experiments. The relative mRNA ratio immediately before cell exposure to the herbicide (0 h) was set as 1.

T. Simões, et al. Appl Environ Microbiol. 2003 July;69(7):4019-4028.
4.
FIG. 5.

FIG. 5. From: Adaptation of Saccharomyces cerevisiae to the Herbicide 2,4-Dichlorophenoxyacetic Acid, Mediated by Msn2p- and Msn4p-Regulated Genes: Important Role of SPI1.

Relative SPI1 mRNA/ACT1 mRNA ratios for the wild type (WT) and Δmsn2 and Δmsn4 mutants 1 h following yeast cell population exposure to 2,4-D (0.3 mM, pH 3.5). Relative SPI1 mRNA/ACT1 mRNA ratios (A) were obtained by densitometry of the autoradiograms (B) and are average values from three experiments. The relative mRNA ratio for the wild type immediately before exposure to the herbicide (control [c]) was set as 1. (C) Corresponding growth curves for the wild-type BY4741 strain (•) and Δspi1 (○), Δmsn2 (▵), and Δmsn4 (□) deletion mutants. Arrows, cell samples harvested for Northern blot assays.

T. Simões, et al. Appl Environ Microbiol. 2003 July;69(7):4019-4028.
5.
FIG. 2.

FIG. 2. From: Adaptation of Saccharomyces cerevisiae to the Herbicide 2,4-Dichlorophenoxyacetic Acid, Mediated by Msn2p- and Msn4p-Regulated Genes: Important Role of SPI1.

Effect of the expression of Msn2p- and Msn4p-regulated genes, encoding antioxidant enzymes and heat shock proteins, on yeast resistance to 2,4D. Growth susceptibilities to 2,4-D for wild-type (wt) strain BY4741 and 12 mutants with the indicated genes deleted were tested by comparing their growth curves in the absence (0 mM) or presence (0.62 mM) of 2,4-D. The growth curves shown are average values from at least three independent growth experiments. Cell suspensions used to prepare the inocula to obtain an initial OD600 of 0.1 ± 0.01 were cultivated in the absence of herbicide until a standardized OD600 of 0.5 ± 0.05 was attained, in mid-exponential phase.

T. Simões, et al. Appl Environ Microbiol. 2003 July;69(7):4019-4028.
6.
FIG. 8.

FIG. 8. From: Adaptation of Saccharomyces cerevisiae to the Herbicide 2,4-Dichlorophenoxyacetic Acid, Mediated by Msn2p- and Msn4p-Regulated Genes: Important Role of SPI1.

(A) Distribution of cells of S. cerevisiae BY4741 (WT) and Δspi1 deletion strains with different pHi values during cultivation under 2,4-D stress. WT, wild type. (B) Average pHi values for wild-type (▪) and Δspi1 (□) 2,4-D-stressed cell populations. Cells were cultivated in the presence of 0.3 mM 2,4-D and harvested at time zero (0 h), after 2.5 h, and at the exponential phase (OD600 = 1 ± 0.1; EXP). Representative growth curves (B, top) for wild-type (•) and Δspi1 (○) strains are shown. Cells were labeled with 5 (6)-CFDA,SE, and pHi was measured by fluorescence imaging. In vivo calibration curves were used to convert measurements of fluorescence to pHi values.

T. Simões, et al. Appl Environ Microbiol. 2003 July;69(7):4019-4028.
7.
FIG. 1.

FIG. 1. From: Adaptation of Saccharomyces cerevisiae to the Herbicide 2,4-Dichlorophenoxyacetic Acid, Mediated by Msn2p- and Msn4p-Regulated Genes: Important Role of SPI1.

MSN2 and MSN4 genes are required for S. cerevisiae adaptation and growth under 2,4-D stress. Susceptibility assays were based on spot assays (agarized medium at pH 4.5) or on comparison of the growth curves (at pH 3.5), in the same basal medium supplemented or not with 2,4-D (0.45 mM), of wild-type (WT) strain BY4741 (□) and the Δmsn2 (⋄) and Δmsn4 (○) deletion mutants. The growth curves show average values from at least three independent growth experiments. Cells used to prepare the spots or to inoculate the liquid media were grown in the absence of herbicide, until a standardized OD600 of 0.5 ± 0.05 was attained, during exponential phase. The cell suspensions used to prepare the spots in lanes B were 1/4 dilutions of the suspensions (OD600 = 0.05 ± 0.005) in lanes A.

T. Simões, et al. Appl Environ Microbiol. 2003 July;69(7):4019-4028.
8.
FIG. 6.

FIG. 6. From: Adaptation of Saccharomyces cerevisiae to the Herbicide 2,4-Dichlorophenoxyacetic Acid, Mediated by Msn2p- and Msn4p-Regulated Genes: Important Role of SPI1.

Lyticase sensitivities of cells of wild type (A and C) and Δspi1 (B and D) strains that were grown in the absence of 2,4-D and then incubated in growth medium unsupplemented (A and B) or supplemented with 0.3 mM 2,4-D (C and D) and harvested during the growth represented by the growth curves shown in Fig. 3, specifically, at time zero, after 2.5 h of incubation, and at the exponential phase when culture OD600 reached 1 (Exp). The different cell populations were washed with water and resuspended in 0.1 M sodium phosphate buffer, pH 7.5. After addition of 10 μg of lyticase (Sigma) per ml, the decrease in the OD600 of the cell suspension was measured periodically. Data are the means ± standard deviations of at least three independent experiments.

T. Simões, et al. Appl Environ Microbiol. 2003 July;69(7):4019-4028.

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