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Establishing a Low Redox Potential in Giant Yeast Colonies: Effects of Media and Rotation

Holly H. Birdsall
Holly H. Birdsall
, 
Patricia L. Allen
Patricia L. Allen
, 
Jeffrey S. Hammond
Jeffrey S. Hammond
, 
Margaret A. Gunter
Margaret A. Gunter
 et 
Timothy G. Hammond
Timothy G. Hammond
   | 17 juil. 2020
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Article Category: Research Article
Publié en ligne: 17 juil. 2020
Pages: 27 - 38
DOI: https://doi.org/10.2478/gsr-2016-0003
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© 2016 Holly H. Birdsall et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Figure 1

Experimental Design. Yeast were spotted onto yeast extract (YE) or yeast peptone dextrose (YPD) agar at staggered intervals 1, 7, 14, 21, or 28 days before the assay. Liquid cultures in YE or YPD broth were inoculated 18 hours before assay as a day-one control. This design allows all the time points to be harvested and assayed together.
Experimental Design. Yeast were spotted onto yeast extract (YE) or yeast peptone dextrose (YPD) agar at staggered intervals 1, 7, 14, 21, or 28 days before the assay. Liquid cultures in YE or YPD broth were inoculated 18 hours before assay as a day-one control. This design allows all the time points to be harvested and assayed together.

Figure 2

Effect of Culture Cell Death. Yeast were spotted on YPD agar impregnated with propidium iodide (PI) and imaged by fluorescence microscopy at 7 days (Panel A), 14 days (Panel B), and 28 days (Panel C). Green fluorescence is due to expression of eGFP linked to TRR1. Red fluorescence reflects cell death due to uptake of PI.
Effect of Culture Cell Death. Yeast were spotted on YPD agar impregnated with propidium iodide (PI) and imaged by fluorescence microscopy at 7 days (Panel A), 14 days (Panel B), and 28 days (Panel C). Green fluorescence is due to expression of eGFP linked to TRR1. Red fluorescence reflects cell death due to uptake of PI.

Figure 3

Effect of Media and Rotation and Media on Cell Death. Graphs show cell death in colonies harvested after 1, 7, 14, 21, and 28 days of growth on YE or YPD agar under static or rotating conditions, or in liquid YE or YPD media for one day. Values shown are the average percent of PI-positive cells as assessed by flow cytometry; error bars indicate +/− standard error of the mean (SEM) of six replicates from three separate experiments. Asterisks indicate time points where the Sok2 deletion clone (red line, squares) had significantly more cell death (p<0.0001), than either wild type (WT) (blue line, open circle), the Sfp1 deletion clone (purple lines), or the Msn4 deletion clone (green lines).
Effect of Media and Rotation and Media on Cell Death. Graphs show cell death in colonies harvested after 1, 7, 14, 21, and 28 days of growth on YE or YPD agar under static or rotating conditions, or in liquid YE or YPD media for one day. Values shown are the average percent of PI-positive cells as assessed by flow cytometry; error bars indicate +/− standard error of the mean (SEM) of six replicates from three separate experiments. Asterisks indicate time points where the Sok2 deletion clone (red line, squares) had significantly more cell death (p<0.0001), than either wild type (WT) (blue line, open circle), the Sfp1 deletion clone (purple lines), or the Msn4 deletion clone (green lines).

Figure 4

Effect of Media and Rotation on Reactive Oxygen Species. Graphs show the amount of ROS—measured by DC-FDA fluorescence—in giant yeast colonies cultured on YE or YPD agar for 1, 7, 14, 21, or 28 days. Rotated samples are shown with filled red squares and a solid line; static samples are shown with open circles and a blue line. Results from one day liquid cultures are shown with an X. Values shown are the net fluorescence in relative units, normalized to the amount of yeast as determined by absorbance at 620 nm; error bars indicate +/− SEM of 18 WT replicates, or 6 Sok2 deletion replicates taken from three separate experiments. Double-headed arrow indicates the time point at which rotated samples were significantly different from static samples (p=0.0001 for Sok2 deletion clones on YE media at 21 days).
Effect of Media and Rotation on Reactive Oxygen Species. Graphs show the amount of ROS—measured by DC-FDA fluorescence—in giant yeast colonies cultured on YE or YPD agar for 1, 7, 14, 21, or 28 days. Rotated samples are shown with filled red squares and a solid line; static samples are shown with open circles and a blue line. Results from one day liquid cultures are shown with an X. Values shown are the net fluorescence in relative units, normalized to the amount of yeast as determined by absorbance at 620 nm; error bars indicate +/− SEM of 18 WT replicates, or 6 Sok2 deletion replicates taken from three separate experiments. Double-headed arrow indicates the time point at which rotated samples were significantly different from static samples (p=0.0001 for Sok2 deletion clones on YE media at 21 days).

Figure 5

Effect of Media and Rotation on Glutathione. Graphs show the amount of glutathione—measured by monochlorobimane (mBCL) fluorescence—in giant yeast colonies cultured on YE or YPD agar for 1, 7, 14, 21, or 28 days. Rotated samples are shown with filled red squares and a solid line; static samples are shown with open circles and a blue line. Results from one day liquid cultures are shown with an X. Values shown are the net fluorescence in relative units, normalized to the amount of yeast as determined by absorbance at 620 nm; error bars indicate +/− SEM of 18 WT replicates, or 6 Sok2 deletion replicates taken from three separate experiments.
Effect of Media and Rotation on Glutathione. Graphs show the amount of glutathione—measured by monochlorobimane (mBCL) fluorescence—in giant yeast colonies cultured on YE or YPD agar for 1, 7, 14, 21, or 28 days. Rotated samples are shown with filled red squares and a solid line; static samples are shown with open circles and a blue line. Results from one day liquid cultures are shown with an X. Values shown are the net fluorescence in relative units, normalized to the amount of yeast as determined by absorbance at 620 nm; error bars indicate +/− SEM of 18 WT replicates, or 6 Sok2 deletion replicates taken from three separate experiments.

Figure 6

Effect of Rotation and Media on ATP. Graphs show the amount of ATP—measured by luminescence assay—in giant yeast colonies cultured on YE or YPD agar for 1, 7, 14, 21, or 28 days. Rotated samples are shown with filled red squares and a solid line; static samples are shown with open circles and a blue line. Results from one day liquid cultures are shown with an X. Values shown are the net luminescence in relative units, normalized to the amount of yeast as determined by absorbance at 620 nm; error bars error bars indicate +/− SEM of 18 WT replicates, or 6 Sok2 deletion replicates taken from three separate experiments.
Effect of Rotation and Media on ATP. Graphs show the amount of ATP—measured by luminescence assay—in giant yeast colonies cultured on YE or YPD agar for 1, 7, 14, 21, or 28 days. Rotated samples are shown with filled red squares and a solid line; static samples are shown with open circles and a blue line. Results from one day liquid cultures are shown with an X. Values shown are the net luminescence in relative units, normalized to the amount of yeast as determined by absorbance at 620 nm; error bars error bars indicate +/− SEM of 18 WT replicates, or 6 Sok2 deletion replicates taken from three separate experiments.
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