Fig 2: Arabidopsis VDACs differentially complement ?por1 yeast in complementation assay. (A) ?por1 transformed with different VDACs were tested in a drop-serial dilution assay. Cells were plated on media containing 2% glucose (YPD) or 3% glycerol (YPG) as the sole carbon source and incubated at 30°C and 37°C for 5 days and then their growth was documented. VDAC1, VDAC2 and VDAC4 enable ?por1 to grow on glucose and glycerol at 37°C. A partial recovery is also observed in the case of VDAC3. (B) Growth kinetics of the ?por1 transformed with different VDACs in YPD and YPG at different temperatures. The data shown are the mean of three independent biological replicates ± SD. (C) Western blot analysis of mitochondrial lysates of ?por1 yeast strain expressing (empty) pGV8 and Arabidopsis VDAC1, VDAC2, VDAC3, and VDAC4. The antibody anti-AtVDAC1 (1:5,000, Agrisera) was used to identify VDAC1, VDAC2, VDAC3, and VDAC4 and anti-Mtg2p (1:2,000) yeast antibody was used as a mitochondrial marker. Arabidopsis VDAC1, VDAC2, VDAC3, and VDAC4 proteins were expressed and directed to mitochondria of ?por1 yeast cells.

Fig 3: Functional diversity of Arabidopsis VDACs depending on their ability to complement ?por1. VDAC1, VDAC2, and VDAC4 can complement the lack of ScVDAC1 in ?por1 and maintain a stable mitochondrial membrane potential and also as a consequence maintain reactive oxygen species homeostasis. They can also survive under salt stress (as evidenced by our NaCl experiments). VDAC3 complemented ?por1 only show subtle complementation. When these transformants were analysed in oxidative stress (as evidenced by our acetic acid or methyl viologen tolerance experiments) the VDAC3 transformants show comparable results like the rest (VDAC1, VDAC2, and VDAC4) indicating a possible role of this protein in reactive oxygen species signaling/homeostasis.

Fig 4: Reactive oxygen species (ROS) measurement in yeast samples. (A) Confocal microscopy of M3 and ?por1 cells. ?por1 shows higher uptake of DHR123 than M3. White scale bar in 5 µm. (B) Confocal microscopy of ?por1 cells complemented with VDACs. White scale bar in 5 µm. (C) Confocal microscopy of M3 cells overexpressing VDACs. White scale bar in 5 µm. (D) Total ROS measurement in ?por1 cells transformed with VDAC (1, 2, 3, and 4) grown in 3% YPG. The ?por1 shows a higher ROS content than M3 when grown in YPG, probably due to poorly active mitochondria. Complementation of VDACs (1, 2, 3, and 4) in ?por1 lowers the ROS level. The data shown are the mean of three independent biological replicates ± SD. Student's t-test (**) P < 0.001 related to the ?por1 pGV8. (E) Total ROS measurement in M3 cells transformed with VDAC (1, 2, 3, and 4) grown in 3% YPG. Overexpression of VDAC (1, 2, 3, and 4) in M3 further lowers the ROS level. The data shown are the mean of three independent biological replicates ± SD. Student's t-test (*) P < 0.001 related to the M3pGV8.

Fig 5: Expression of Arabidopsis VDACs in wild type M3 cells. (A) M3 transformed with different VDACs were tested in a drop-serial dilution assay. Cells were plated on media containing 2% glucose (YPD) or 3% glycerol (YPG) as the sole carbon source and incubated at 30 and 37°C for 5 days and then their growth was documented. (B) Growth kinetics of the wild type M3 transformed with different VDACs in YPD and YPG at different temperatures. The data shown are the mean of three independent biological replicates ± SD. (C) Western blot analysis of mitochondrial lysates of M3 yeast strain expressing (empty) pGV8 and Arabidopsis VDAC1, VDAC2, VDAC3, and VDAC4. The antibody anti-AtVDAC1 (1:5000, Agrisera) was used to identify VDAC1, VDAC2, VDAC3, and VDAC4 and anti-Mtg2p (1:2,000) yeast antibody was used as a mitochondrial marker. Arabidopsis VDAC1, VDAC2, VDAC3, and VDAC4 proteins were expressed and directed to mitochondria of M3 yeast cells.