Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/20/2005
Publication Date: 3/15/2005
Citation: Griffith, M., Lumb, C., Wiseman, S., Wisniewski, M.E. 2005. Antifreeze proteins modify the freezing process in planta. Plant Physiology. 138: 330-340. Interpretive Summary: In order to survive frost and low winter temperatures, plants undergo cold acclimation. During this process, numerous biochemical changes occur which allow a plant to tolerate low temperatures and the presence of ice in their tissues. One such change is the production of antifreeze proteins (AFPs). These unique proteins have the ability to lower the temperature at which ice forms within a plant and modify the shape and size of growing ice crystals. The degree of importance of these proteins in allowing plants to survive freezing temperatures, however, is not clearly understood. The goal of this research was to determine if AFPs can act as freeze protectors and determine if their presence modifies the process of freezing. Biochemical assays were used to assess freeze protecting activity and infrared thermography was used to observe the freezing process. Results indicated that AFPs did not have freeze protecting activity in rye plants but were able to modify the freezing process by significantly lowering the freezing temperature and slowing down the rate of ice propagation. This was the first direct evidence that AFPs play a role in the freezing process and can affect plant survival. This information will be used to develop a better understanding of the biochemical basis for cold tolerance and to develop novel approaches to improving cold hardiness in sensitive plants.
Technical Abstract: During cold acclimation, winter rye plants secrete antifreeze proteins (AFPs) into the apoplast that reduce freezing injury. The goal of this study was to determine whether AFPs increase survival by modifying the freezing process or by acting as cryoprotectants. In order to inhibit the growth of ice, AFPs must be mobile so that they can bind to specific sites on the ice crystal lattice. Guttate obtained from cold-acclimated winter rye leaves exhibited antifreeze activity, indicating that the AFPs are free in solution. Infrared video thermography was used to observe freezing in winter rye leaves. In the absence of an ice nucleator, AFPs had no effect on the supercooling temperature of the leaves. However, in the presence of an ice nucleator, AFPs lowered the temperature at which the leaves froze by 0.3 to 1.2EC. Apoplastic proteins extracted from cold-acclimated winter rye leaves also slowed the rate of migration of ice through solution-saturated filter paper. In addition, we examined the possible role of winter rye AFPs in cryoprotection of proteins and membranes. Lactate dehydrogenase activity was higher after freezing in the presence of AFPs compared with buffer, but the same effect was obtained by adding bovine serum albumin. AFPs had no effect on unstacked thylakoid volume after freezing, but did inhibit stacking of the thylakoids, thus indicating a loss of thylakoid function. We conclude that rye AFPs have no cryoprotective activity, rather they interact directly with ice in planta and may reduce freezing injury by slowing the growth of ice.