Grants and Contributions:

Grant or Award spanning more than one fiscal year. (2017-2018 to 2022-2023)

Survival at low temperatures in some overwintering organisms such as certain fish, plants and insects, is enhanced by the expression of a number of genes including those for the unique antifreeze proteins (AFPs). AFPs bind to forming ice crystals and this patchy cover prevents ice growth. In freeze-susceptible species when temperatures drop further, AFPs can no longer ‘hold’ ice crystals and explosive growth follows, leading to freezing and then death. In contrast, freeze-tolerant species freeze at higher sub-zero temperatures with AFPs keeping individual ice crystals small and offering cell protection. Ironically, considering the wealth of biochemical data on AFPs, the actual contribution of these proteins to freeze survival has not been established. We are now the first to do this by producing genetically-engineered brome grass with the AFPs ‘knocked down’. We must optimize this system in order to more fully explore the importance of these proteins. Our research suggests that AFPs from freeze-tolerant grasses (f-tAFPs) contribute to a complex web of low temperature, pathogen and immune interactions. We therefore postulate that f-tAFPs have multiple roles for: (i) protection of cell integrity by organizing ice, (ii) ‘spoiling’ the nucleation of ice by bacteria, and (iii) a more general immune response against low-temperature tolerant bacteria. Innovatively, we expect that freeze-tolerant insects and microbial communities will show that other f-tAFPs also evolved as part of an ‘immune response’. However, f-tAFPs are not well known, likely because they are less abundant than their evolutionary-distinct freeze-susceptible counterparts. Thus we will use modern techniques including ‘omic’ strategies to overcome this challenge in order to isolate, and subsequently characterize, novel f-tAFPs from several organisms. These include plants other than grasses, an insect and bacteria from an Arctic community.

We submit that these species contain ‘recipes’ that can be applied to increase freeze tolerance in crops. This is important since freeze-thaw events have increased in frequency over the last few decades due to climate change. These proteins will also have utility for the cryopreservation of animal tissues. Short and long-term goals will utilize our cloned f-tAFP sequences in order to understand their interactions with ice as well as crystalline gas hydrates. In the future, these f-tAFPs will find utility in a number of applied problems of global interest. Overall, our program of research establishes a new foundation for experimentation and hypothesis testing, with these enquiries encouraging diverse students to an exciting program of discovery.