Expression of Two Self-enhancing Antifreeze Proteins from the Beetle Dendroides canadensis in Arabidopsis thaliana

Abstract

Antifreeze proteins depress the non-equilibrium freezing point of aqueous solutions, but only have a small effect on the equilibrium melting point. This difference between the freezing and melting points has been termed thermal hysteresis activity (THA). THA identifies the presence and relative activity of antifreeze proteins. Two antifreeze protein cDNAs, dafp-1 and dafp-4, encoding two self-enhancing (have a synergistic effect on THA) antifreeze proteins (DAFPs) from the beetle Dendroides canadensis, were introduced into the genome of Arabidopsis thaliana via Agrobacterium-mediated floral dip transformation. Southern blot analysis indicated multiple insertions of transgenes. Both DAFP-1 and/or DAFP-4 were expressed in transgenic A. thaliana as shown by RT-PCR and Western blot. Apoplastic fluid from T 3 DAFP-1+DAFP-4-producing transgenic A. thaliana exhibited THA in the range of 1.2-1.35°C (using the capillary method to determine THA), demonstrating the presence of functioning antifreeze proteins (with signal peptides for extracellular secretion). The freezing temperature of DAFP-1+DAFP-4-producing transgenic A. thaliana was lowered by approximately 2-3°C compared with the wild type.

Advances in Antifreeze Molecules: From Design and Mechanisms to Applications

“…For instance, overexpression of cAFPs in tomatoes could enhance their tolerance for chilly temperatures . Introduction of two D. canadensis AFP genes into the genome of Arabidopsis thaliana resulted in TH activity of 1.2–1.35 °C . Besides, tomato seeds treated by 100 μg/L AFP I could germinate earlier with a higher percentage .…”

“…178 Introduction of two D. canadensis AFP genes into the genome of Arabidopsis thaliana resulted in TH activity of 1.2−1.35 °C. 179 Besides, tomato seeds treated by 100 μg/L AFP I could germinate earlier with a higher percentage. 180 In conclusion, antifreeze molecules, especially AFPs, represent a valuable strategy for food industry and agriculture, aiming at improving the quality of frozen products and assisting crops to survival under cold stress.…”

Microfluidic Cold-Finger Device for the Investigation of Ice-Binding Proteins

“…The ability of IBPs to prevent freezing and freeze injury in nature has current and potential uses. These include applications in the food industry (15); cryopreserva-tion and chill-storage of cells (16)(17)(18), tissues, and organs for transplantation (19); cryosurgery; preventing frost damage to plants (20); and anti-icing (21).…”