INSTYTUT DENDROLOGII

Antifreeze proteins (AFPs), have been identified in various plants and animals tolerant to subzero temperatures. In plants, AFP proteins usually are located in the apoplast, i.e. at the place where ice crystals are first formed in the plant. In the presence of AFPs, the freezing temperature (Tf) of extracts decreases, but no difference in melting temperature (Tm) is observed. The difference between the melting and freezing temperatures is termed thermal hysteresis (TH), and is the major characteristic of AFPs. The aim of this study was to check if AFPs are present also in needles of evergreen Picea species. For this study, we selected apoplast proteins from needles of two spruce species: Picea abies and P. pungens, which differ in sensitivity to low temperatures. The concentration of apoplastic proteins per 1 g (fresh weight) of needles varied between months and depended on the stage of frost hardening of the trees. The highest concentration was recorded for fully hardened needles: 4.9 µg/g in P. abies and 4.5 µg/g in P. pungens. The concentration of apoplastic proteins in needles collected in summer was 3-fold lower. Electrophoretic separation of apoplastic proteins from P. abies and P. pungens showed that their molecular mass was from 7 to 80 kDa, and two fractions were distinguished: AP-1 (16-80 kDa) and AP-2 (<20 kDa). In the period of the highest freezing tolerance, the most distinct were the protein bands of ~16, ~27 and ~42 kDa. For apoplastic proteins of fully hardened spruce needles, TH values were high and significantly correlated with their concentration in aqueous solutions, e.g. at a concentration of 400 µg/ml, TH = 2.19°C for P. abies and TH = 2.02°C for P. pungens. High TH values were associated with frost hardening of spruces, because apoplast needles from needles collected in summer displayed 4-fold lower TH values. Measurements of the basic physical parameters of freezing, i.e. Tf, Tm, and calculation of TH values in the presence of apoplastic proteins of spruces, enabled classification of those proteins as AFPs. The spruce AFPs also showed in vitro a strong cryoprotective activity of cold-labile enzyme, lactate dehydrogenase (LDH) as compared to bovine serum albumin (BSA). Proteins of the low-molecular fraction AP-2 (<20 kDa) were the most active. At a low concentration of those proteins (25 µg/ml), the activity of LDH after freezing at  196°C, was maintained at a level of 87% in P. abies, and 91% in P. pungens. For apoplastic proteins from needles of P. abies and P. pungens, cryoprotective activity was first reported here. The similarity of N-terminal sequences of spruce apoplastic proteins of 27 kDa to chitinases of other plant species, may indicate another function of those proteins, i.e. as pathogenesis-related proteins.