Accumulation of pathogenesis‐related (PR) 10/Bet v 1 protein homologues in mulberry (Morus bombycis Koidz.) tree during winter

ABSTRACT Seasonal evaluation of total soluble protein fractions extracted from cortical parenchyma cells of mulberry (Morus bombycis Koidz.) tree identified a predominant 18 kDa protein that was directly correlated to periods of cold acclimation. The 18 kDa protein, designated as WAP18 (winter accumulating 18 kDa proteins) increased from September to December and then gradually decreased until June. The maximum levels of WAP18 were detected in mid‐winter, which corresponds to the maximum freeze tolerance in cortical parenchyma cells of mulberry tree. Two‐dimensional gel electrophoresis confirmed that WAP18 consists of at least three proteins that range between an isoelectric point of 5.0 and 6.0. All three proteins reacted with anti‐WAP18 antibodies, thereby suggesting that they represent individual isoforms. Furthermore, N‐terminal amino acid sequence analysis demonstrated that all three proteins contain high sequence similarity to each other and high homology to pathogenesis‐related (PR) −10/Bet v 1 protein families. The purified WAP18 exhibited in vitro cryoprotective activity for the freeze labile l‐lactate dehydrogenase (LDH) enzyme. These results suggest that WAP18 may function in the freezing tolerance mechanism of cortical parenchyma cells of mulberry tree during winter.