Characterization of cryoprotective activity of thermal hysteresis protein in evergreen plants

Thermal hysteresis proteins (THPs) inhibit the growth of ice by binding to the surface of ice crystals, preventing the addition of water molecules to cause a local depression of the freezing point. Recent findings demonstrate that apoplastic THPs from Norway spruce show a cryoprotective and also anti-ice nucleation activity (Jarz¹bek et al. 2008). Here, we have focused on the most active THPs from five frost-hardy conifers of North American and Eurasian boreal forest: Abies grandis (Douglas ex D.Don)Lindl., Picea pungens Engelm., Pinus nigra J.F.Arnold, Pinus sylvestris (L.), and Tsuga canadensis (L.) Carriere, and additionally from one alpine (2100 m a.s.l.) evergreen shrub Loiseleuria procumbens (L.). The objective of this study was to determine whether these thermal hysteresis proteins of frost-hardy plants (THPs) influence survival at subzero temperatures by modifying the freezing process and/or by acting as cryoprotectants. Apoplastic extracts were obtained by vacuum infiltration of leaves with 5 mM ascorbic acid, and the extracts were concentrated by using a 10 kDa cutoff Ultrafree centrifugal filter device (Millipore). Proteins were separated by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Leammli 1970). Cryoprotective activity of apoplastic proteins was determined with the use of the freeze/thaw inactivation, by four cycles in liquid nitrogen (-196oC) and room temperature while the assay of lactate dehydrogenase (LDH) was performed as described by Wisniewski et al. (1999). Antifreeze activity was determined by using the droplet freezing assay (Vali 1971).