FIG. 4.

Deletion of aquaporin-encoding genes reduces freeze tolerance. (A and B) The effects of freezing on glucose consumption were measured in nonfermenting and fermenting cells of aquaporin single- and double-deletion mutants in the 10560-6B background. IGC, FGC, and RGC were determined for the wild-type strain, the aqy1Δ strain, the aqy2Δ strain, and the aqy1Δ aqy2Δ strain. The cells were either frozen (for 1 day at −30°C) (FGC) or not frozen (i.e., cooled on ice) (IGC) 30 min after resuspension of stationary-phase cells in YP (nonfermenting cells) (A) or 40 min after the subsequent addition of 200 mM glucose (fermenting cells) (B). After thawing, glucose consumption was measured for 4 h to assess residual yeast activity. RGC is calculated as (FGC/IGC) × 100. Representative results are shown. Compared to the wild-type strain 10560-6B, Aqy1-1, Aqy2-1, and double-deletion strains showed RGCs that were 0.7 (±0.1), 1.1 (±0.2), and 0.3 (±0.1) times higher, respectively, for nonfermenting cells and 0.6 (±0.1), 0.4 (±0.1), and 0.2 (±0.1) times higher, respectively, for fermenting cells. (C) Northern analysis of AQY1 and AQY2 expression in nonfermenting and fermenting wild-type 10560-6B cells. ACT1 was used as a loading control.