Antifreeze Proteins Modify the Freezing Process In Planta

A, Freezing in nonacclimated (N), ABA-treated (A), ethephon-treated (E), and cold-acclimated (C) winter rye leaves observed by IRVT as leaves were cooled 0.05°C min⁻¹ over a period of 38 min 30 s. Each image shows eight winter rye leaves oriented vertically with a droplet of ice⁺ bacteria placed midleaf. After freezing, the droplet was colder than the ambient temperature due to sublimation of water. The temperature scale and range are shown below each image. a and b, ABA-treated leaves froze first, followed quickly by nonacclimated leaves. c, Initiation of freezing (arrows) in cold-acclimated leaves. d, Initiation of freezing (arrows) in ethephon-treated leaves. Magnification bar in a represents 0.5 cm. B, Freezing in nonacclimated (NA), cold-acclimated (CA), and ethephon-treated (Eth) winter rye leaves. Leaves were cooled at 0.10°C min⁻¹ over a period of 16 min 40 s, and freezing was observed by IRVT. A droplet containing ice⁺ bacteria was placed midleaf. After freezing, the droplet was colder than the ambient temperature due to sublimation of water. The temperature range and time that the image was taken are shown below each image. a, Arrows indicate two freezing exotherms in nonacclimated leaves that were not observed in either cold-acclimated or ethephon-treated leaves. b, Nonacclimated, cold-acclimated, and ethephon-treated leaves froze within a 1-min interval. c, The exotherm dissipated in the cold-acclimated leaf as the temperature rose by 0.4°C. d, The cold-acclimated leaf refroze upon cooling. Magnification bar in d represents 0.5 cm.

Effect of apoplastic proteins from cold-acclimated winter rye leaves on the rate of migration of ice through solution-saturated filter paper at −2.5°C. The samples were AE (apoplastic extract; 0.2 mg protein mL⁻¹), and 2× AE (apoplastic extract concentrated 2-fold by ultrafiltration). Controls included water; Suc at a concentration of 40 mOs, which was the same osmotic concentration as AE and 2× AE; and BSA at a concentration of 5 mg protein mL⁻¹. Data are presented as mean ice migration rates ± sem; n = 4.

Recrystallization of ice in the presence of apoplastic proteins from nonacclimated (C and D) and cold-acclimated (E and F) winter rye. Apoplastic extracts dialyzed against 5 mm EDTA were adjusted to a final concentration of 0.14 mg protein mL⁻¹ and Suc was added to a concentration of 26% (w/w). The samples were frozen on a cold stage as follows: cooled at 30°C min⁻¹ to −50°C, warmed at 10°C min⁻¹ to −10°C, held 10 min, warmed at 1°C min⁻¹ to −7°C, cooled at 1°C min⁻¹ to −8°C, warmed to −7°C, and held isothermally. Samples were photographed at 3 min (A, C, and E) and 53 min (B, D, and F). A solution of 26% Suc was used as a control (A and B). Magnification bar = 100 μm.

Quantification of ice recrystallization in the presence of apoplastic proteins from nonacclimated (NA) and cold-acclimated (CA) winter rye. Ice crystals that formed in nonacclimated and cold-acclimated apoplastic extracts containing (26%, w/w) Suc were measured after holding at −7°C for 3 to 53 min to allow recrystallization. The blank (B) was a solution of 26% Suc. Data were calculated as equivalent diameter and as aspect ratio (maximum diameter/minimum diameter) and are presented as means ± se; n = 12 for the blank; n = 12 for nonacclimated samples; and n = 6 for cold-acclimated samples.

Cryoprotection of LDH by rye apoplastic proteins and BSA. LDH was frozen and thawed eight times in the presence of either BSA or apoplastic proteins isolated from cold-acclimated winter rye leaves. LDH activity was measured spectrophotometrically as the oxidation of NADH and calculated as a percentage of the unfrozen control. The data are presented as means ± se; n = 4.