Overexpression of aquaporin-encoding genes improves freeze tolerance without affecting growth and initial fermentation rates. (A) IGC, FGC, and RGC were determined for S47 and AT25 overexpressing AQY1-1, S47 and AT25 overexpressing AQY2-1, and, as a control, S47 and AT25 with an integrated empty plasmid. The cells were either frozen (for 1 day at −30°C) (FGC) or not frozen (i.e., cooled on ice) (IGC) 30 min after the onset of fermentation by addition of 100 mM glucose. After thawing, glucose consumption was measured for 2.5 h to assess residual yeast activity. RGC is calculated as (FGC/IGC) × 100. Representative results are shown. Compared to AT25 containing an empty plasmid, AT25 AQY1-1 and AQY2-1 overexpression strains showed 1.5 (±0.1)- and 1.4 (±0.1)-times-higher RGCs, respectively. Compared to S47 containing an empty plasmid, S47 AQY1-1 and AQY2-1 overexpression strains showed 9.8 (±0.8)- and 9.0 (±1.2)-times-higher RGCs, respectively. (B) Growth of the same strains in YPD medium (Bioscreen measurements).