Cold-tolerant plants may endure subzero temperatures partially by inhibiting the development of ice crystals in the intercellular spaces and the xylem through the accumulation of antifreeze proteins (AFP) and the extra production of carbohydrates. Certain proteins associated with pathogen resistance in plants have the ability to bind and alter the growth of ice crystals. In this study, the accumulation of pathogenesis-related (PR) proteins and the development of freezing tolerance in seedlings of two latitudinal distinct Norway spruce (Picea abies L. Karst.) ecotypes were investigated. Despite freezing tolerance difference, timing of growth cessation and bud set variations, our results showed that there is no significant difference in the concentration of soluble carbohydrates between the two ecotypes. Immunoblots showed the presence of several β-1,3-glucanase and thaumatin PR proteins in the apoplastic fluid and the enzymatic assay showed an extra accumulation of several isoforms of PR chitinases in cold-treated Norway spruce needles. In addition to PR proteins, a presence of de novo protein in cold-treated needles was noticed. In contrary to mature plants, total proteins isolated from freezing-tolerant Norway spruce seedling did not show antifreeze activity. Our results suggest that the activity of the PR proteins and the accumulation of soluble carbohydrates that increased during cold acclimation may have an indirect impact on the freezing tolerance in Norway spruce, however, deciphering the direct mechanism behind freezing tolerance in Norway spruce seedlings growing under controlled environmental conditions require further investigation.
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